Tunji Akande

Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

All Correspondences to:Tunji Akande Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

ABSTRACT

Background: Heart disease is a common and delibilitating condition that affects millions of patients globally. Measurement of troponin assays has been a tremendous boon to clinical diagnosis. Objective: To evaluate the clinical utility and the biochemical characteristics of serum troponin as an early and accurate cardiac marker for diagnosis of acute myocardial infarction. Method: a review of serum troponin as choice biomarker for diagnosis of Acute Myocardial Infarction (AMI). Result: Troponin T allows for early and late diagnosis of AMI. Troponin is only found in the myocardium in adults making it extremely specific for cardiac disease. Troponin I or T is replacing total CK and CK-MB detection as the marker of choice for cardiac dysfunction as seen in AMI. Conclusion: There is value in detection myocardial damage early after its onset. Clinical laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of AMI.

Key Words: AMI, Troponin, Cardiac Marker, Early diagnosis.

INTRODUCTION

Cardiovascular diseases are the leading cause of death globally. Cardiovascular disease includes coronary artery disease (CAD) such as angina and myocardial infarction commonly known as a heart attack The World Health Organization has established three criteria for the diagnosis of acute myocardial infarction (AMI) including a history of chest pain, evolutionary changes on the ECG, and elevations of serial cardiac markers [2] however it was rare for a diagnosis of AMI to be made in the absence of biochemical evidence of myocardial injury. [3] .Numerous biomarkers have been monitored to assess myocardial injury. Most are myocardial proteins and differ in their location within the myocyte, release kinetics after damage and clearance from the circulation [4] . The European Society of Cardiology/American College of Cardiology (ESC/ACC) has published consensus guidelines for the diagnosis of AMI [5,6 7]. A Corner stone of the consensus document is predicated on cardiac biomarker especially cTnI or CTnT [8,9]. Because of its dire consequence, great efforts have been made to determine the best tools for the early and accurate diagnosis of AMI. Therefore this article aimed at assessing some cardiac markers that could provide an early and accurate diagnosis for acute myocardial infarction

Myocardial infarction (MI)

Acute myocardial infarction is one of the largest killers globally. Its diagnosis is usually made on the clinical presentation and electrocardiagraphic (ECG) findings and confirmed by the characteristic changes in plasma enzyme activities or troponin levels Symptoms and ECG abnormalities however may be absent or non specific. Thus, the diagnosis of an acute MI has increasingly depended upon evaluation of Cardiac biomarkers particularly troponins [10,11]. Acute myocardial infarction is defined as an imbalance between myocardial oxygen supply and demand resulting in injury and the eventual death of myocyte. When the blood supply to the heart is interrupted, gross necrosis of myocardium results. Such extensive necrosis is most often associated with a thrombotic occlusion superimposed on coronary atherosclerosis. Myocardial infarction is seen as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction; this stratification is based upon cardiac markers reflecting ischemic damage. [12,13]. Myocardial ischemia and infarction are usually segmental diseases. The American College of Cardiology and European Society of cardiology have defined acute myocardial infarction as a typical rise and fall of biochemical marker, for example plasma creatine kinase isoenzyme MB (CK-MB) or troponin, with at least one of the following: Ischemic symptoms, new pathological Q waves, on electro cardiogram (ECG), ischemic ECG changes (ST.depression or elevation) and coronary artery intervention [14]

The patients previously classified as having unstable angina or minor myocardial injury are now reclassified as having non-ST segment elevation myocardial infarction (NSTEMI), therefore myocardial infarction is now regarded as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction. Acute myocardial infarction and NSTEMI have a common pathophysiological pathway (15)

Cardiac markers

Cardiac markers are biomarkers measured to evaluate heart function. They are clinical laboratory tests useful for detecting AMI or minor myocardial injury. They are most useful when patients have non-diagnostic ECG tracings.

Most efforts to date have been placed on the development of an ideal cardiac marker for the early and accurate diagnosis of AMI. Many factors must be considered in the selection of the most clinically diagnostic effective, cost effective and cost efficient laboratory tests for patients with chest pain which include

1. the time that has elapsed after onset of chest pain;
2. Any concomitant diseases;
3. The possibility of skeletal muscle injury
4. The ease of measurement and turn around time for results.
5. Assay specificity, sensitivity and interferences. (17, 18)

Serum enzymes such as aspartate aminotransferase (AST), Creatine Kinase (CK), Lactate dehydrogenase (LD) and their iso enzymes have all been used as biomarkers of AMI. Decades ago they were thought to be sensitive indicators of myocardial necrosis and could be used to correlate with other signs and symptoms such as abnormalities in ECG pattern [19,20] All three of these enzymes, however are found in other tissues as well, making them less specific to myocardial damage. AST for example is also found in skeletal muscle, liver parenchyma cells, and erythrocytes, while CK is found in skeletal muscles, brain tissue and embryonic and malignant tissue. LD is the least specific of these three enzymes in that it is found in virtually all tissues and is associated with damage to liver, skeletal muscle, cardiac muscle, erythrocytes renal cells and many other tissues as well as ovarian and testicular tumors. [21] Historically myocardial infarction was detected by looking for the CK Iso enzyme CK-MB. This marker is released into circulation from necrotic heart muscle. As the heart muscle becomes damaged this CK Isoenzyme is released into the blood stream and may be detected.

Cardiac proteins

Several proteins may be monitored in suspected cases of AMI to give significant diagnostic information myoglobin an oxygen-binding protein is rapidly released from striated muscles (both skeletal and cardiac muscle0 when damaged. However because of its small size, myoglobin is rapidly cleared by the kidney, making it an unreliable long-term marker of cardiac damage. [22] Myoglobin is significantly more sensitive them CK and CK-MB activities during the first hours after chest pain onset. It starts to rise within 1-4hrs and is detectable in essentially all AMI patients between 6 and 9 hours from chest pain onset returning to baseline levels within 18-24 hours. If myoglobin concentration remains within the reference range 8 hours after onset of chest pain, AMI can essentially be ruled out [23] Myoglobin is released into circulation with any damage to muscle tissue, including myocardial necrosis. Since skeletal muscle contains myoglobin, this measurement is quite non-specific for myocardial infarctions. The benefit lays in the fact that a detectable increase is seen only 30 minutes after injury occurs, unlike

troponin and creatine Kinase which can take 3-4 hours [24,25,26] Troponin I and troponin T are normal proteins important in the contractile apparatus of the cardiac myocyte. They are released into the circulation about 3-4 hours after myocardial infarction and are still detectable for 10 days afterwards. The long half-life allows for the late diagnosis of myocardial infarction [27,28]. All of the initial cardiac markers were enzymes, so the earliest techniques measured the catalytic activity of the marker. Immunoassay techniques measure the mass of a marker and they are the predominant methodology used in clinical laboratory practice today. Analytically, they offer lower limits of detection, improved precision, and faster assay time on both highly automated central laboratory Platforms and Point of Care Testing (POCT). [29,30] Over the past 15 years numerous manufacturers have described the development of monoclonal anthody –based diagnostic immunoassays for the measurement of troponins (CTnI and CTnT) in serum. Troponin assays has been a tremendous boon to clinical diagnosis. Troponins released from heart muscle remains in the blood stream for up to 10-14 days after onset of AMI, making them preferred marker for detection of an AMI [31]. Troponins as cardiac markers appear to have many advantages primarily due to their quick release following heart muscle damage.

Recommendations

The National Academy of clinical Biochemistry recommends that two biochemical markers be used for routine diagnosis of AMI: an early marker that is reliably increased within 6 hours after onset of symptoms and a definitive marker that remains increased after 6-9 hours but has high sensitivity and specificity for myocardial injury and remains abnormal for several days. [33]. To assist in differentiating patients with AMI from non – AMI, the European Society of cardiology/American College of cardiology (ESC/ACC) has published consensus guidelines for redefinition of AMI [34]. A cornerstone of this is predicated on cardiac biomarkers, especially CTnI or CTnT. The cardiology recommendations imply that for clinical laboratories that cannot move as rapidly as others to implement the new cardiac troponin standard, CK-MB (Preferable mass should be used. Although it is suggested that CK-MB be used together with cardiac troponin for assisting in timing the onset of myocardial injury, infarct sizing or determination of reinfarction at present there is no strong evidence to support dual testing for cTn and CK-MB.

Conclusion

The diagnosis of acute myocardial infarction can be difficult, but it is important to make prompt diagnosis as thrombolysis needs to be given early. The diagnosis of an acute myocardial infarction has increasingly depended upon evaluation of cardiac biomarker particularly troponins. Troponins are more specific cardiac marker than the CK. Also, plasma troponin stays elevated for longer than CK after an infarct. Troponins are therefore useful for the late diagnosis of myocardial infarction. Clinical Laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of myocardial infarction.

Table I. The time sequence of changes in plasma cardiac markers after myocardial infarction

Cardiac marker	Starts to rise	Time after Infarction	Duration of
	(hours)	for peak rise (hours)	rise (days)
CK (total)	4-6	24-48	3-5
AST	6-8	24-48	4-6
LDH/HBD	12-24	48-72	7-12
MYOGLOBIN	2-4	12-24	2-4
TROPONIN	4-6	12-24	7-10

CK= Creatine kinase; AST= aspartate transaminase

LDH= lactate dehydrogenase; HBD= Hydroxybutyrate dehydrogenase

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Kevin E. Aghatise

Department of Medical Laboratory Science, Igbinedion University Okada

Ivie Osula and Anslem O. Ajugwo

Department of Medical Laboratory Science, Madonna University Nigeria

All Correspondences to: Kevin E. Aghatise, Department of Medical Laboratory Science, Igbinedion University Okada

ABSTRACT

Anaemia constitutes a serious health problem in many tropical countries including Nigeria because of the prevalence of malaria and other parasitic infections which possibly leads to decrease of hemoglobin. Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been reported to be very good in building the constituents of the blood and also replacing them. This study was designed to investigate the effect of fluted pumpkin, bitter leaf and utazi leaf extracts on haematolgical parameters of anaemia induced albino wistar rats. Fifteen Wistar albino rats with weight range from (122-190g) obtained from the animal house of Madonna University, Elele Nigeria were separated into three groups of five rats each for the study. Rats in group B and C were injected 400mg/kg 2,4 dinitrophenylhydrazine for five days to induce anaemia, while rats in group A were fed with grower’s mash and water throughout the experiment. All analysis were done using the standard methods. The result of this study show that, there was a significant (p<0.05) difference when the PCV was compared among the Groups, the control groups had 41.0+2.65% anemia induced group had 25.3+6.8% and the treatment group recorded 33.6+4.9%. The anemia induce group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). There was also a significant difference when the Haemoglobin concentration was compared among the groups, the control group had 14.1+0.7g/dl, anaemia-induced group has 8.3+1.9g/dl and the treatment group had 10.+2.15g/dl. There was no significant difference (p>0.05) in all other parameters estimated. The result of this research indicate that aqueous leaf extract of fluted pumpkin, bitter leaf and utazi leaf could elevate the packed cell volume, red blood cells and hemoglobin concentration in anaemia-induced rats. It can be concluded that aqueous leaf extract of the combination of fluted pumpkin, bitter leaf and utazi leaf are a potential blood booster.

Keywords: anaemia-induced, blood booster, haematological, dinitrophenylhydrazine.

INTRODUCTION

The use of herbal products for medicinal benefits has an important role in nearly every culture on earth. Herbal medicine was practiced by ancient people of Africa, Asia, Europe and the Americas (Abosi and Raseroka, 2003). Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs of the pharmaceutical industry. The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, or cure of disease because plants have long served as a useful and rational source of therapeutic agents (babalolaet al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiochemical and nutritional constituent (Hunter and Fletcher, 2002).

Green leafy vegetables are particularly important in

promoting health because of their rich sources of nutrients (Gupta and Prakash, 2009). Telfairia occidentalis (fluted pumpkin) is a common tropical green leafy vegetables native to many African countries especially Eastern Nigeria (Burkett, 1968). It thrives in humid climate and well drained soils and is usually cultivated in garden and family farms around homes. Telfairia occidentalis is traditionally used by an estimated 30 to 35 million people indigenous people in Nigeria, including the Efik, Ibibio, and Urhobo (Akoroda, 1990). However, it is predominantly used by the Igbo tribe, who continue to cultivate it for food sources and traditional medicines (Okoli and Mgbeogu, 1983). A reoccurring subject in the Igbo’s folklore, the fluted gourd is noted to have healing properties and was used as a blood tonic, to be administered to the weak or ill (Akoroda, 1990). Bitter leaf is a medicinal plant of the family Asterceae. It is a small perennial shrub that grows in tropical Africa. Vernonia amygdalina is commonly called bitter leaf because of its bitter taste and it can be propagated either by cutting or seedling. Today, the plant is widely known throughout the continent and nearly 85% of Nigerians cultivates the plant due to its nutritional and medicinal values. It is locally called ‘ewuro; in Yoruba land (Abosi and Raseroka, 2003). The leaves of the plant may be consumed either as a vegetable (macerated leaves in soup) or aqueous extract as tonic for the treatment of various illnesses (Abosi and Reseroka, 2003).

Gongrone malatifolium also called utazi belongs to class of medicinal plants that are beneficial in preventing and treating certain disease and ailments that are detrimental to human health. Gongrone malatifolium leaf, which can be chewed infused or used for cooking is mainly used in the western part of America for nutritional and medicinal reason. Pharmacological studies suggest that utazi has both analgesic, antimicrobial, antibacterial, anti ulcer and antioxidant properties (Eguyoni et al., 2009). Utazi plant is highly medicinal in nature, which suggest why its health benefits cannot be overemphasized. Researchers agree that the fundamental ingredients used for medical purposes are stored in the various parts of the utazi plants such as leaves, fruits, roots etc (Eguyoni et al., 2009). The vital medicinal ingredients stored in the various parts of the utazi leaf can be extracted either through blending the fresh leaves, chewing the utazi seed, leaves or fruits, infusing either the dry or fresh utazi leaves and decoction. However these various methods of preparing and using utazi leaves for medical purpose mainly depends on the part of utazi plants where the active medicinal ingredient are present (Eguyoni et al., 2009).

Anaemia is decrease in the amount of red blood cells (RBCs) or hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen (Rodak and Bernadtte, 2007). When anaemia sets in slowly, the symptoms are often vague and may include: feeling tired, weakness, shortness of breath or a poor ability to exercise. Anaemia that comes on quickly often has greater symptoms, which may include; confusion, feeling like one is going to pass out, loss of consciousness, or increased thirst. Anaemia must be significant before a person becomes noticeably pale (Rodak and Bernadette, 2007).Use of plants has long been known to be used as local remedy for treatment of anaemia especially pumpkin leaf which is used in villages to treat anaemia especially when quality drugs cannot be accessed. This work was carried out to determine the effects of combination of herbal plants in anaemia-induced condition using haematological parameters.

Materials and Methods

Animal Model/Experimental design

Fifteen (15) Wistar strain albino rats with weight range of

(122 – 190g) obtained from the animal house, Madonna University Elele, Nigeria were used for the study. The rats were housed in wire meshed cage under standard conditions (temperature 25 – 29°C, 12 hours light and 12 hours darkness cycles) and fed with standard rat pelleted diet and water. They were housed in 3 meshed cages containing five

1. rats in each cage. The rats were made to acclimatize for 1 week before the experiment began. They were allowed to feed on standard feed and water freely throughout the period the experiment lasted.

Ethical Approval

The research was approved by the ethical committee of the institution. The standard, rules and regulations of use of animal for research purposes was strictly adhered to as approved by the committee.

Order of Placement

Group1: The rats in this group served as control and were

administered with feed and water only. Group2: The rats in this group served as test and were administered with feed, water and 1 ml of phenyl hydrazine for five days. Group3: The rats in this group served as treatment group and were administered with feed, water, 1 ml of phenyl hydrazine for five days and subsequently treated with 0.5 ml of a blend of the local plants for another five days. Preparation and Administration of the plant blend

Large quantities of fresh leaves of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) were purchased from the Central Market Elele. Identification and authentication of the plants was done by Mr Eze of the Faculty of Science. The leaves were washed and dried air-dried for eight days until there was no sign of moisture and grinded into powder. The powder was dissolved in 100 ml of deionized water and administered to the treatment group 0.5 ml in the morning and evening for 5 days.

Sample Collection

2ml of blood sample was collected by ocular puncture from each of the animal model using capillary tube and was dispensed into commercially prepared concentrations of ethylene diamine tetra acetic acid containers.

Sample Analysis

Blood samples collected were analyzed within six hours of collection for Hb, PCV, RBC count, Total WBC and differential analysis using standard manual methods (Cheesebrough, 2000)

RESULTS

Mean±S.D of hematological parameters of the control group (A), test group (B) and treatment group ©

PARAMETERS	GROUP A	GROUP B	GROUP C	P-VALUE
PCV (%)	41.0+2.65	25.3+6.8	33.6+4.9	P<0.05
HB (g/dl)	14.1+0.7	8.3+1.9	10.5+2.15	P<0.05
RBC (X1012/l)	6.0+0.56	4.4+0.62	5.2+1.0	p>0.05
TWBC X109/1)	9.3+1.9	13.7+5.4	11.7+2.11	p>0.05
Platelet (x109/1)	272+24.0	261+39.5	302+39.2	p>0.05
NEUTROPHIL (%)	56.7+3.51	63.7+6.11	55.7+5.86	p>0.05
LYMPHOCYTE (%)	34+4.0	29.7+7.23	33.7+11.06	p>0.05
EOSINOPHIL (%)	6.0+1.0	4.0+1.0	4.3+1.53	p>0.05
MONOCYTE (%)	3.3+1.15	3.3+1.52	3.3+1.15	p>0.05

20

 

DISCUSSION

The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, and or cure of diseases because plants have long served as a useful and rational sources of therapeutic agents (Babalola et al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiological and nutritional constituents (Hunter and Fletcher, 2002).

From the research work there was a significant (p<0.05) difference when the PCV was compared among the groups, the control group has 41.0±2.65%, anaemia induced group has 25.3±6.8% and the treatment group recorded 33.6±4.9%. The anaemia induced group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). This definitely is attributable to the local blend of plants. Previous work have shown that pumpkin is capable of improving haematological parameters (Alada,2000; Adias et al., 2013).

There was also a significant difference when the Hb was compared among the group, the control group had 14.1±0.7g/dl, anaemic induced group had 8.3±1.9g/dl and the treatment group had 10.5±2.15g/dl, the anaemic group recorded a reduction 8.3+1.9/dl but was elevated in the group given the extract. The reason for the significant increase in these haematological parameters could be related to the chemical composition of the leaves used in this research work. The chemical composition of Telfairia occidentals includes protein, fat, carbohydrates, calcium, iron, vitamin A, thiamine and riboflavin(Tindal, 1968; Adisa et al., 2014). Most of these constituents have positive impact on blood production (Akube, 1980). For instance, iron is a well established haemopoetic factor and deficiency of if produces anaemia (Ganong, 1997). There was no significant (p>0.05) difference in the RBC, the control group had 6.0±0.56×1012/1, the anaemic group had 4.4±062×1012/1, while the treatment group had 5.2±1.0×1012/l. The extract increased the RBC count of the anaemia-induced group from 4.4+062×1012/1 to 5.2+1.0×1012/1 but this was not significant. For TWBC there was no significant (p>0.05) difference when the test groups B (13.7±5.4×109/1) was compared to the control group A (9.3±1×109/1) although the TWBC was increase in the anaemic group (13.7±5.4109/1) and reduced in the treatment group (11.7±2.11×109/1).

There was also no significant (p>0.05) difference in platelets count when the groups B (261±39.5×109/1) and C (302±39.2×109/1) was compared with control group

(272±24.0×109/1) although the platelets count decreased in the anaemic group and increased in the treatment group but this was not significant (p>0.05). No significant difference was seen in all the differential count parameters. In conclusion, the combination of the extract of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been shown to improve blood parameters (PCV and haemoglobin concentration). This extract can be used in management of anaemia especially when the conventional blood builders are not readily available.

Conflict of interest

The Authors declare that we have no conflict of interest.

REFERENCES

• Abosi, A. O. and Raserok, B. H. ( 2003) “In vivo antimalarial activity of Vernonia amygdalina” British Journal of Biomedical Science. 8 (60) 89 – 91.
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• Eguyoni, A., Moody, J. O. and Eletu, O. M. (2009). “Anti-sickling activities of two ethnomedicinal plant recipes used for the management of sickle cell anaemia in Ibadan, Nigeria” African Journal of Biotechnology. 8 (1): 20 – 25.
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• Tindal, H. D. (1968). Commercial vegetable growing. Oxford Press, London. 69

 

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) Sold in Jos Metropolis

Ejinaka, OR; ; Lote-Nwaru IE; Agbalaka P.I; Ajik, H.

Federal School of Medical Laboratory Science, Jos-Nigeria

Jwanse, R.I

Health and Development Support Programme (Hands), Jos, Nigeria

All correspondence to: Obeta M.U. E-mail: uchejesoobeta@gmail.com;

ABSTRACT

The assessment of antibiotic susceptibility pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) sold in Jos was carried out in Jos Metropolis. This study was aimed at the occurrence of staphylococcus aureus in suya and nunu and its sensitivity to antibiotics. A total of 15 Suya and 15 Nunu sample were collected, the sample were cultured and identified by routine bacteriological method. Prevalence of S.aureus in Nunu was 40% and Suya was 33.3%. The susceptibility profile of staphylococcus aureus isolates from Suya (roasted meat) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (80%), Rifampin (80%) Norfloxacin (80%) and had high resistance to Chloramphericol (80%), Ciprofloxacin (80%), Amoxil (80%), Ampiclox (80%) and Erythromycin (60%). The susceptibility profile of staphylococcus aureus isolates from Nunu (milk) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (83.3%), Norfloxacin (83.3%), Erythromycin and Ciprofloxacin (66.7%) respectively, Rifampin and Levofloxacin 50% respectively and had high resistance to Chloramphericol, Amoxil and Ampiclox (66.7%) respectively; Rifampin and Levofloxacin 50% respectively. The S. aureus was present in Suya (Roasted Meat) and Nunu

(Milk) sold in Jos and resistant to some commonly used antibiotics in Jos.

Keywords: Staphylococcus aureus, Antibiotic Susceptibility, Suya, Nunu, Jos.

INTRODUCTION
Staphylococcus aureus is one of the major food borne abdominal cramping, with or without diarrhea. The disease is usually self-limiting and typically resolves within 24 – 48h after onset. Occasionally it can be severe enough to pathogens, frequently causing disease globally as a warrant hospitalization, particularly when infants, elderly result of food ingestion contaminated with or debilitated people are concerned (Argud´ın et al., 2012;
staphylococcal endotoxin (Mendoza, et al., 2010). S.
Mendoza et al, 2013).
S. aureus can cause wide spectrum of infections, ranging from superficial skin infections to severe, and potentially fatal, Jhalka et al., (2014) suggests the contributing factors for invasive disease due to combination of toxin-mediated the low incidence of SFD to include misdiagnosis, virulence, invasiveness, and antibiotic resistance. An improper sample collection in laboratory during important impediment to the control of the Staphylococcus examination, lack of seeking medical attention by the aureus infection is the tendency to gain resistance to almost affected persons, and lack of routine surveillance of all classes of antimicrobial agent (Jhalka et al., 2014).
clinical for S. aureus or its enterotoxins and unavailability
Staphylococcus aureus does not compete well with
of implicated foods for confirmation of laboratory testing
indigenous microbial in raw foods, contamination is
at the time of outbreak.
mainly associated with improper handling of cooked or
processed foods. However, S. aureus is also present in food,
Food handlers such as Suya meat and Nunu vendors are
animals, and dairy cattle, sheep and goats (Larkin et al.,
carrying enterotoxin-producing S. aureus in their noses or
2009). The S. aureus bacteria were then termed methicillin-
their hands which are regarded as the main source of food
resistant S. aureus (MRSA) usually show resistant to many
contamination, via manual contact or through respiratory
antibiotic (Ray et al., 2013). Staphylococcal food
secretions thereby contaminating raw foods, as a result of
poisoning (SFP) is an intoxication that results from the
improper handling of cooked or processed foods, followed
consumption of foods containing sufficient amounts of one
by storage under condition which allows growth of S.
(or more) preformed enterotoxin symptoms of SFP have a
aureus in food and animals. Food that have been frequently
rapid onset (2-8h), and include nausea, violet vomiting,
incriminated in staphylococcus intoxication include meat

and meat products, poultry and egg products, milk and dairy products, salads, bakery products, particularly cream-filled pastries and cakes, and sandwich filling. Salted food products (SFP) such as ham, have also been implicated according to the capacity of S. aureus to grow at relatively low water activity.

S. aureus have been found to survive in materials, clothes, hand nails, ATMs, mobile phones aside skins and nasal areas of the body (Oviasogie et al., 2014; Ekrakene & Igeleke, 2007; Adetona & Olabisio, 2010). It is noteworthy that Suya and Nunu hawkers come in contact with clothes, phones, ATMs and other materials while doing the merchandise without any consideration of washing before handling the food.

SFP is common disease whose real incidence is probably underestimated for a number of reasons, which include misdiagnosis, unreported minor outbreaks, improper sample collection and improper laboratory examination. (Argud´ın et al., 2010)

Nowhere has this issue been of great concern than with the gram-positive bacteria pneumococci, enterococci and staphylococci where multidrug resistance has become a norm among these pathogens. S. aureus is perhaps the pathogen of great concern because of its intrinsic virulence its ability to cause a diverse array of life-threatening infection, and its capacity to adapt to different environment conditions (Argud´ın et al., 2012, Hennekinne et al., 2012). The mortality of S. aureus bacteremia remains approximately 20-40% despite the availability of effective antimicrobial agents (Hassen et al., 2006). S. aureus is now the leading overall cause of nosocomial infections and, as more patients are treated outside the hospital setting is an increasing concern in the community as it is popular practice in Jos where drug vendors and Chemists are patronized without diagnosis or medical advice and such could also lead to increasing resistance to a great number of antimicrobial agents. Inevitably this has left fewer effective bactericidal antibiotics to treat these often life-threatening infections. As rapidly as new antibiotics are introduced, staphylococci have developed efficient mechanism to neutralize them.

In Jos metropolis, most infection are been treated with available drugs in the clinic or just by visiting drug vendors otherwise called Chemists where they are given antibiotics without recourse to medical laboratory diagnosis to identify the disease causing organism and possible susceptible drugs for the disease. Such common antibiotics have been found to be resistant to S. aureus as reported by Wang et al., (2017).

This study is aimed to determine the presence and antibiotics susceptibility profile of pathogenic staphylococcus aureus in Nunu and and Suya meat sold in Jos metropolis.

Materials and Methods

Study Area

The study was carried out in Jos Metropolis, Jos North Local Government Area in locations: Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park. Sample Size / Collection

A total of 15 roasted meats (Suya) and 15 raw milk (Nunu) were collected from the select areas in and around Jos metropolis which includes: Hamaz, Gangare, Railway, Yan Taya and Plateau Riders Park. The Suya (roasted meat) and Nunu samples were obtained 3 each from every point of sales in Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park into a sterilized universal bottle, packed into a polythene bags, and transported into the medical laboratory for analysis. While the Nunu sample were also collected in sterile polythene bags and transported in an icebox and transported to the Federal School Research Laboratory, Jos for analysis within 3 hours.

Isolation of Staphylococcus Aureus in Nunu and Suya 1mL of each sample was transferred to flask containing 9ml of peptone water for enrichment. According to ISO 6888, the plates were incubated under aerobic conditions at 370C for 24hr and those that yielded growth were subjected to another 24hr incubation for susceptibility assessment.

The total counts were carried out using nutrient agar. In serial dilution preparation, 1.0g of sample was aseptically, transferred into 9.0ml of diluted water and homogenized by vortex. Subsequent serial dilutions up to 10-5 were made in line with Kalalou et al. (2004). The enumeration of microorganisms in the samples was by the pour plate technique. At the end of the incubation, resultant microbial colonies (staphylococcus aureus) were counted.

Identification of Staphylococcus Aureus from Nunu and Suya

Pure isolates of microorganism grown on the different chocolate agar plates were further identified biochemically using Cowan and Steel (1974) technique using oxidase, coagulase, maltose, glucose and gram’s staining. The staphilococcus aureus were the only organisms isolated from suya meat and nunu samples obtained from the food vendors in Jos metropolis as identified from the samples.

Antibiotic Susceptibility Test of the Isolates of Staphilococcus Aureus

All S. aureus isolates were screened for multi-drug-resistance using disc diffusion in line with Bauer et al., (1966). This was performed on nutrient agar plates. Furthermore, the antibiotic susceptibility pattern of S. aureus strains was determined by disc diffusion method for Ciprofloxacin (10mcg), Gentamycin (10mcg), Norfloxacin (10mcg), Erythromycin (30mcg), Streptomycin (30mcg), Levofloxacin (20mcg), Chloramphenicol (30mcg), Amoxil, Ampiclox and Rifampin. Chloramphenical, Erythromycin and Norfloxacin susceptibility was determined by the e-test according to the manufacturers’ guidelines (Biometriux, France).

The diameter of the zone of inhibition produced each antibiotic disc was measured, recorded and the isolates were classified as “resistant”, “intermediate” and “sensitive” based on the standard interpretative chart updated according to the CLSI (2015). Multi-resistance was defined by resistance of the strain to at least three antibiotic agents.

15

 

RESULTS

Prevalence of Staphylococcus aureus from suya (roasted meat) sample

S/N	Meat sample	S. aureus x(y)	Cell count	Percentage
				prevalence (%)
1.	A	2(3)	7 x 1010	66.7
			8 x 1010
2.	B	1(3)	4 x 1010	33.3
3.	C	1(2)	9 x 1010	50.0
4.	D	0(2)	–	0.0
5.	E	1(5)	5 x 1010	40.0
Total	5(15)			33.3

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Prevalence of Staphylococcus aureus from Nunu in Jos Metropolis

S/N	Meat sample	S. aureus x(y)	Cell count	Prevalence (%)
1.	A	2(3)	6x 1010	66.7
			8 x 1010
2.	B	1(2)	4 x 1010	50.0
3.	C	0(5)	–	0.0
4.	D	2(3)	4 x 1010	66.7
			7 x 1010
5.	E	1(2)	3 x 1010	50.0
Total	6(15)			40.0

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Antibiotics	Disc t			Samples			Percentage	Percentage
	Conten	A2	B1	C1	D3	E1	Resistance	Sensitive
	(ng)						(%)	(%)
Norfloxacin (NB)	10	R	S	S	S	S	20	80
Chloramphenicol (CH)	30	R	S	R	R	R	80	20
Ciprofloxacin (CPF)	10	R	R	S	R	R	80	20
Erythromycin (E)	30	S	R	R	R	S	60	40
Levofloxacin (LV)	20	R	S	S	S	S	20	80
Ampiclox (AML)	20	R	R	S	R	R	80	20
Amoxil (AML)	20	R	R	S	R	R	80	20
Gentamycin (CN)	10	R	S	S	S	S	20	80
Rifampin (RD)	5	S	S	R	S	S	20	80

Key: S = Sensitive, R = Resistance

16

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus…

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Antibiotics	Disc t			Samples				Percentage	Percentage
	Conten	A2	B1	D2	E1	B3	E2	Resistance	Sensitive
	(ng)							(%)	(%)
Norfloxacin (NB)	10	S	S	R	S	S	S	16.67	83.34
Chloramphenicol (CH)	30	R	R	S	S	R	R	66.67	33.34
Ciprofloxacin (CPF)	10	S	R	S	S	S	R	33.34	66.67
Erythromycin (E)	30	S	R	S	S	S	R	33.34	66.667
Levofloxacin (LV)	20	S	R	R	S	R	S	50.00	50.00
Ampiclox (AML)	20	R	S	R	R	S	R	66.67	33.34
Amoxil (AML)	20	R	R	R	R	S	S	66.67	33.34
Gentamycin (CN)	10	S	S	R	S	S	S	16.67	83.34
Rifampin (RD)	5	S	S	R	R	R	S	50.00	50.00

Key: S = Sensitive, R = Resistance

Discussion

Staphylococcus aureus is an important health care and community acquired infection in every region of the world (Wang et al., 2017). This studies shows that Staphylococcus aureus is prevalent in Suya (33.3%) and Nunu (40%) and could possess a potential health hazard (Sofos, 2008) to consumers in Jos Metropolis. Studies on isolation and antibiotic susceptibility frequency of isolation of S. aureus isolates from meat and milk may be due to the possible susceptibility of S. aureus isolates from Suya and Nunu to Levofloxacin, Erythromycin, Norfloxacin, Gentamycin and Rifampin as observed in this study. However, this report was not different from other studies reported by Waters et al. (2011). The high susceptibility of S. aureus to these antibiotics may be due to high costly or injectable forms and the possibility of abuse of such antibiotics may be low. The low susceptibility of chloramphenicol, amoxil, ampiclox observed in this study was not different from the other studies reported by Le et al., (2003). The low susceptibility of S. aureus to antibiotics mentioned may be due to inappropriate use of antibiotics without doctor’s prescription and inclusion of antibiotics in animal feeds as growth promoter (Le et al., 2003). From this study, it was however observed that the frequency of isolation of S. aureus observed in this study was high and isolates were more susceptible to flouroquinolones and aminoglycosides. In view of this, such drugs may be useful for treatment of staphylococcal infections, but there is a need to conduct appropriate medical laboratory diagnosis to find out the causative organism S. aureus for example before treatment especially in Jos Metropolis. The indiscriminate use of antibiotics/antimicrobials agents for prophylactic as well as other therapeutic purpose could be the reasons for increased antimicrobial resistance of S. aureus in animals and humans.

Conclusion

Staphylococcal food poisoning (SFP) is a major concern in public health programs worldwide. S. aureus may be present in cow milk as a result of milk collection from the animal suffering from disease condition and excreting S. aureus in milk or due to unhygienic conditions during production, processing, storage and handling of milk products, which are the main causes of food borne

diseases. The result of this study clearly indicated that some Nunu and Suya available in the Jos metropolis were contaminated with S. aureus, which could pose a risk of food poisoning to Jos metropolis. Thus, more hygienic preventive measures are required to reduce the bacterial contamination, so as to increase the wholesomeness of these Nunu and Suya. The study also revealed common antibiotics used in Jos which are resistant to S. aureus. This study highlights the need for continuous surveillance of antibiotic sensitivity pattern of staphylococcus aureus with a view to selecting appropriate therapy. This study also recommends that:

1. All Suya meat and Nunu vendors should imbibe adequate hygiene measures while handling the food products
2. The Suya and Nunu vendors should use nose masks while processing handling their products
3. The Suya and Nunu vendors in Jos metropolis should avoid using dirty rags to clean their hands while processing and dispensing their products to the public

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Matthias Pilecky, Anita Schildberger, Viktoria Weber

Center for Biomedical Technology, Department for Health Sciences and Biomedicine, Danube University Krems,

Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

Viktoria Weber

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

Ludwig Knabl and Dorothea Orth-Höller

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstraße 41, A-6020 Innsbruck, Austria.

Viktoria Weber

Department for Biomedical Research, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

All Correspondences to: Viktoria Weber E-mail: viktoria.weber@donau-uni.ac.at

ABSTRACT

Background: Early pathogen detection and identification are crucial for an effective and targeted antibiotic therapy in patients suffering from blood stream infection. Moleculardiagnostic methods can accelerate pathogen identification as compared to blood culture, but frequently suffer from the inhibition of polymerase chain reation (PCR) by sample matrix components, such as host DNA, anticoagulants, or plasma proteins. To overcome this limitation, molecular diagnostic methods commonly rely on pathogen enrichment by selective lysis of blood cells and pelleting of intact pathogens prior to analysis. Results: Here, we investigated the impact of antibiotic treatment on the recovery of pathogen DNA using an established pathogen enrichment protocol. Based on the hypothesis that induction of bacterial cell wall disintegration following antibiotic administration leads to incomplete pelleting of pathogen DNA, S. aureus was grown in human whole blood with or without addition of cell wall active (vancomycin, piperacillin) or non cell wall active (ciprofloxacin, clindamycin) antibiotics at clinically relevant concentrations. Pathogen detection remained unaffected by non cell wall active antibiotics or even increased in the presence of cell wall active antibiotics, indicating improved accessibility of pathogen DNA. Likewise, mechanical lysis of S. aureus prior to pathogen enrichment resulted in increased recovery of pathogen DNA. Quantification of pathogen and human DNA after selective lysis of blood cells and pathogen enrichment confirmed partial depletion of human DNA, leading to a net enrichment of pathogen DNA over human DNA. Conclusion: Concurrent antibiotic administration does not reduce the recovery of pathogen DNA during pathogen enrichment by selective lysis and centrifugation. Leads to a 10-fold human DNA depletion as compared to pathogen DNA. Moreover, we confirm that the recovery of pathogen DNA after pathogen enrichment is not negatively

influenced by concurrent antibiotic administration.

Keywords: Molecular diagnostics, Pathogen detection,Blood stream infection, DNA extraction, Selective lysis, Antibiotics.

BACKGROUND	management of sepsis [10] recommend immediate
. aureus is an opportunistic pathogen with the	antibiotic therapy as well as pathogen identification, since
S	early and targeted antibiotic treatment can significantly
potential to cause community-associated and
nosocomial infections [1]. It is the most common	improve the survival of sepsis patients [11–14]. Blood
gram-positive pathogen associated with sepsis, with a	culture is the current reference method for the
prevalence of up to 20% of all blood culture positive sepsis	identification of pathogens and for the characterization of
cases [2–4]. This high prevalence results from its ability to	their antibiotic susceptibility. It may, however, fail to detect
adapt to its environment by modulating the host immune	slow-growing or intracellular pathogens and yield
response [5], to switch from a highly proliferative	inconclusive results due to concurrent antibiotic treatment.
disseminative state into a slow growing, biofilm producing	Moreover, definitive results of culture are usually not
state [6], to grow intracellularly [7, 8], and to acquire	available before 48 h [11, 13, 15]. Molecular diagnostic
antibiotic resistance [9]. Clinical guidelines for the	methods provide a time-to-result of 4–7 h, compatibility

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 57

Influence of antibiotic treatment on the…

with antibiotic treatment, and do not depend on pre-selecting culture steps [13, 16, 17]. Still, the direct detection of pathogen DNA in blood samples (e.g. SeptiFast, Roche, Basel, Switzerland) is prone to interference of matrix components, such as host DNA [18,

19], heparin [20, 21], or plasma proteins [22–24]. To avoid

inhibition of PCR and to increase the sensitivity of pathogen detection, virtually all current molecular diagnostic systems, including FAST ID BSI (QVella, Richmond Hill, Canada), Hybcell Pathogen Array (CubeDX, St. Valentin, Austria), T2 Bacteria (T2 Biosystems, Lexington, MA), SepsiTest (Molzym, Bremen, Germany), as well as Magicplex Sepsis Test (Seegene, Seoul, Korea), rely on pre-analytical pathogen enrichment [25]. Common to all pathogen enrichment protocols is the selective lysis of blood cells by addition of a hypotonic detergent solution. Pathogens withstand the osmotic pressure and are enriched by subsequent centrifugation prior to DNA quantification. We hypothesized that antibiotic treatment, by affecting bacterial cell wall integrity, might lead to incomplete pelleting of pathogens after selective lysis of the blood cells and centrifugation, and, consequently, result in a loss of pathogen DNA prior to analysis. Therefore, we investigated the impact of both, cell wall active and non cell wall active antibiotics on the pre-analytical enrichment of DNA of different S. aureus strains from human whole blood.

Methods

Bacteria and reagents

Antibiotics (Table 1), 4 – (2 – hydroxyethyl) – 1 – piperazineethanesulfonic acid buffer (HEPES, pH 7.0, cell culture grade) and adenine were purchased from Sigma-Aldrich (St. Louis, MO). D-Glucose was obtained from Merck

(Darmstadt, Germany). S. aureus culture strains (ATCC 12600, 29213, and 29737) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Wild-type strains (WT32217, 32237, and 32248) were isolated from patient material at the Division of Hygiene and Medical Microbiology, Medical University

of Innsbruck, Austria. Strains were cultivated on lysogeny broth (LB, Lennox formulation) agar plates (Carl Roth, Karlsruhe, Germany) at 37 °C. Overnight cultures were obtained by inoculating single colonies into LB medium. To determine the minimal inhibitory concentration for individual antibiotics used in this study, overnight cultures were diluted 1:5,000 in LB medium in 96-well polystyrene microwell plates (CELLSTAR®, Greiner Bio-One GmbH, Frickenhausen, Germany), serial dilutions of antibiotics were added, and incubation was performed for 24 h at 37 °C.

Human whole blood

Venous human whole blood was collected from healthy

adult volunteers into tubes (Vacuette, Greiner Bio-One,

Kremsmuenster, Austria) containing sodium heparin or

EDTA. Blood collection was approved by the Ethical

Review Board of Danube University Krems, and written

informed consent was obtained from all donors.

Cultivation of S. aureus in human whole blood Freshly

drawn human whole blood anticoagulated with heparin

was buffered with 1/50 volume of 1M HEPES and

supplemented with 2 mg/L glucose and 48 μg/L adenine

per hour. Overnight cultures of each strain were diluted

1:1000 in LB medium, spiked into supplemented whole

blood (typically 4 mL) at a ratio of 1:2560 (ATCC 12600),

1:205 (ATCC 29213), 1:1700 (ATCC 29737), 1: 730

(WT32217), 1:1700 (WT32237), and 1:128

(Wt32248), and incubated at 37 °C with gentle shaking.

These spiking ratios were chosen based on growth curves

for the individual strains to obtain pathogen concentrations

of approximately 5000 colony forming units (CFU) per mL

after 4 h. For each strain, growth was assessed in whole

blood from six donors (n = 3 per donor) for up to 8 h.

Antibiotic pretreatment of S. aureus

To investigate whether weakening or disruption of bacterial cell walls by antibiotic pretreatment would influence subsequent pathogen enrichment and, consequently, PCR-based quantification of pathogen DNA, S. aureus was spiked into whole blood and incubated for 4 h as described above to ensure logarithmic growth. Subsequently, final concentrations of 15 μg/mL

Table 1 Antibiotics used in this study and their mechanism of action

Antibiotic	Cell Wall Active	Mechanism of Action
Vancomycin (VAN)	+	interferes with cell wall synthesis by preventing formation and crosslinking of peptidoglycan strands [26–28]
Piperacillin (PIP)	+	Interferes with cell wall synthesis by binding to enzymes required for the extracytoplasmatic stage of
		cell wall formation [28, 29]
Ciprofloxacin (CIP)	–	prevents replication of bacterial DNA by inhibiting DNA gyrase [30, 31]
Clindamycin (CLI)	–	inhibits bacterial protein synthesis by binding to 50S ribosomal subunits [32]

vancomycin (VAN), 20 μg/mL piperacillin (PIP), 1 μg/mL ciprofloxacin (CIP), or 2 μg/mL clindamycin (CLI), respectively (Table 1), were added, and incubation was continued for another 90 min at 37 °C with gentle agitation. Subsequent pathogen enrichment and DNA extraction were performed as described below. Spiked blood without antibiotic treatment served as control. Strain ATCC 29213 was additionally grown in the presence of CIP and VAN for up to 72 h to investigate potential differences between blood culture based and qPCR based pathogen detection.

Mechanical lysis of S. aureus

To achieve complete pathogen disintegration, fresh S. aureus overnight cultures (ATCC 29213) were diluted 1:5000 in LB medium, incubated for 2 h at 37 °C, mixed with an equal volume of 0.1mm zirconium beads (Biozym, Hessisch Oldendorf, Germany) in a 0.3 mL PCR tube (Bio-Rad, Hercules, CA), and vortexed using a regular benchtop vortex (VortexGenie2, Carl Roth) at maximum speed for up to 120 min. Samples drawn after 30 s, 5 min, and 120 min were characterized by scanning electron microscopy,

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and bacterial lysis was confirmed by quantification of viable bacteria (CFU) as described below. Suspensions of lysed bacteria obtained at the indicated time points were spiked into freshly drawn whole blood anticoagulated with EDTA at a ratio of 1:100, and pathogen enrichment as well as isolation and quantification of pathogen DNA were performed as described below.

Quantification of viable bacteria

To quantify viable bacteria, spiked blood samples were diluted 1:5 in 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany), and 100 μL of the dilutions were plated onto LB agar (Carl Roth, n = 3). Plates were incubated overnight at 37 °C, and colonies were counted manually on the next day. Pathogen enrichment and extraction of pathogen DNA Pathogen DNA was isolated from spiked blood samples using a commercial pathogen enrichment system (GINA Pathogen Enrichment, CubeDX, St. Valentin, Austria) according to the instructions of the manufacturer. The enrichment protocol relies on the selective lysis of blood cells by detergent buffer, followed by centrifugation to pellet intact pathogens, lysis of pelleted pathogens by heating in alkaline buffer, and extraction of pathogen DNA using MiniSpin columns. For comparison, total DNA was extracted without previous pathogen enrichment using a total DNA extraction kit (MagMAX DNA Multi-Sample Kit, Thermo Fisher Scientific, Waltham, MA) according to the protocol of the manufacturer.

Quantification of DNA

qPCR was performed using Staphylococcus spp. specific 16S rRNA gene primers and primers specific for the beta-actin gene as control for human DNA. All primers are specified in Additional file 1: Table S1. Samples were mixed with an equal volume of SsoAdvanced SYBR Green Master Mix (Bio-Rad, Hercules, CA) containing 0.5 μM of each primer to yield a total reaction volume of 20 μL. qPCR was performed using a Roche LightCycler 96 (Basel, Switzerland) and comprised a 2 min preheating step at 94 °C, followed by 45 cycles of denaturation at 94 °C for 5 s, annealing at 53 °C for 10 s, and extension at 72 °C for 15 s. Correct amplification was verified by melting curve analysis. cT values were calculated using the Roche LightCycler96 Software version 1.1.0.1320.

Scanning electron microscopy

Sample preparation for scanning electron microscopy included filtration of bacterial suspensions through 0.22 μm polycarbonate isopore membranes (Merck Millipore, Darmstadt, Germany), followed by fixation with 2.5% glutaraldehyde (Carl-Roth) in 0.9% NaCl for 2 h at room temperature and dehydration in a graded alcohol series (30–100%). Samples were sputtered with gold using a Q150R (Quorum Technologies Ltd., Laughton, UK) at 30 kV in DC mode for 60 s, and images were acquired using a FlexSEM 1000 scanning electron microscope (Hitachi, Mannheim, Germany) at an accelerated voltage of 20 kV.

Statistical analysis

Statistical analysis was performed using SigmaPlot 13.0 (Systat Software, Erkrath, Germany) and graphs were plotted using GraphPad Prism version 7.02 (La Jolla, CA). At least three replicates were performed for each

Viktoria Weber

experiment. Data are presented as mean ± standard deviation or as mean and 95% confidence intervals (CI). The significance of differences was calculated using paired t-test for comparison of a sample to a reference value, or, in case of multiple time points, by Friedman repeated measures analysis of variance on ranks, followed by multiple comparisons versus reference using Dunnett’s Method. P-values < 0.05 were considered as statistically significant.

RESULTS

Growth of S. aureus in human whole blood and antibiotic susceptibility The experimental setup of this study is outlined in Fig. 1. All S. aureus strains exhibited logarithmic growth in human whole blood supplemented with glucose and adenine after a lag phase of 2–4 h (Fig. 2). All strains were susceptible to VAN, PIP, CIP and CLI. Minimal inhibitory concentrations for individual

Fig. 1 Background and outline of the study. a Blood-borne pathogens can be detected using blood culture (detection of viable pathogens) or by molecular diagnostic methods, such as PCR (detection of pathogen DNA). PCR is either performed after extraction of total (host and pathogen) DNA or following pathogen enrichment to deplete human DNA as well as blood-borne inhibitors of PCR. We hypothesized that antibiotic treatment might

induce disintegration and, consequently, incomplete pelleting of pathogens during sample processing with established pathogen enrichment protocols, resulting in partial loss of pathogen DNA. b Commonly used pathogen enrichment protocols comprise the selective lysis of blood cells by detergent treatment, pelleting of intact pathogens, and extraction of pathogen DNA

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using spin columns. c To assess the influence of antibiotic treatment on PCRbased pathogen detection following pathogen enrichment, S. aureus was spiked into human whole blood and grown for 4 h. Thereafter, spiked samples were incubated without further treatment (control) or received antibiotic treatment as described in Materials and Methods. For comparison, S. aureus was mechanically lysed using zirconium beads to achieve complete disintegration as described in Materials and Methods, spiked into whole blood, and processed in parallel to untreated and antibiotic-treated samples. CFU counts (viable pathogens) were determined in all samples, and pathogen DNA was quantified after pathogen enrichment as shown in panel b

antibiotics are listed in Additional file 1: Table S2. Influence of antibiotic treatment on the recovery of pathogen DNA

To assess whether the presence of antibiotics would influence the recovery of S. aureus during pathogen enrichment, spiked whole blood was treated with either cell wall active (VAN, PIP) or non cell wall active (CIP, CLI) antibiotics (Table 1). cT values remained constant for CIP and CLI (ΔcT -0.5 ± 0.9 and 0.4 ± 0.9 as compared to the reference taken prior to the addition of antibiotics), or decreased significantly for the cell wall active antibiotics PIP and VAN (ΔcT -2.9 ± 1.4 and − 2.4 ± 1.3) (Fig. 3a), suggesting enhanced accessibility of pathogen DNA in the presence of cell wall active antibiotics. VAN, PIP, and CIP, which exhibit bactericidal activity [33–35], induced a reduction of CFU counts as compared to the reference, while CLI exerted bacteriostatic effects with stable CFU counts (Fig. 3b). No significant differences were detected between individual S.aureus strains (data not shown). Upon incubation with VAN and CIP for up to 72 h, pathogen DNA remained constant in the presence of VAN or even increased in the presence of CIP, providing further

evidence that antibiotic treatment does not induce a loss of pathogen DNA during pathogen enrichment (Fig. 3c). CFU counts decreased continuosly over time, and no viable bacteria were detected beyond 48 h (Fig. 3d). Scanning electron microscopy did not provide evidence for cell wall degradation or reduced density of culture (Fig. 3e). I

nfluence of mechanical lysis on the recovery of

pathogen DNA

Next, we performed mechanical lysis of S.aureus and spiked the lysates into whole blood to assess the impact of pathogen disintegration on the recovery of pathogen DNA during subsequent pathogen enrichment. Culture as well as scanning electron microscopy confirmed the efficient lysis of S. aureus (Fig. 4a). The recovery of pathogen DNA increased with progressing pathogen disintegration, confirming the efficient enrichment of pathogen DNA despite the absence of intact bacteria (Fig. 4b).

Selectivity of DNA isolation: pathogen versus host DNA

Based on the enhanced recovery of pathogen DNA following pathogen disintegration by mechanical lysis, we went on to assess the ability of the pathogen enrichment protocol to selectively isolate S. aureus DNA, i.e. to deplete human DNA. S.aureus DNA and human DNA were quantified following DNA isolation from spiked whole blood using (i) pathogen enrichment (protocol A) or (ii) total DNA extraction without previous pathogen enrichment (protocol B). qPCR (Table 2)

Fig. 2 Growth of S. aureus in supplemented human whole blood. a S. aureus strains obtained from ATCC and b wild-type strains isolated from patient material reached exponential growth (log phase) after 2–4 h of incubation in whole blood supplemented with glucose and adenine as described in Materials and Methods. CFU counts are indicated relative to the initial spiking concentration of 103–104 CFU/mL (n = 6)

revealed the presence of residual human DNA in samples isolated with the pathogen enrichment protocol, however, samples obtained by total DNA extraction contained significantly higher amounts of human DNA (ΔcT 6.1 ± 0.3). Pathogen DNA was nearly equally well enriched with both protocols (ΔcT 2.1 ± 0.4), resulting in an approximately 16-fold enrichment of pathogen DNA over human DNA using the pathogen enrichment protocol.

DISCUSSION

Early adequate antimicrobial therapy is crucial in the management of blood stream infection, where broadspectrum antibiotics are administered upon the first suspicion of infection [10], followed by targeted antimicrobial treatment after identification of the causative pathogens by blood culture or molecular diagnostic methods [36, 37]. While there is ample evidence that antimicrobial treatment can interfere with blood culture

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[38, 39], the impact of antibiotic therapy on molecular diagnostic pathogen detection remains incompletely defined. The only prospective study published to date in this context revealed a significantly higher rate of positive results with SeptiFast multiplex PCR as compared to blood culture in children under concurrent antimicrobial therapy

1. Since PCR-based pathogen detection in whole blood is, however, susceptible to inhibition by blood-borne factors and host DNA, pathogens are commonly enriched from whole blood prior to analysis. Established enrichment

protocols rely on the selective lysis of blood cells in hypotonic detergent and subsequent pelleting of intact pathogens, followed by extraction of pathogen DNA and PCR. We hypothesized that antimicrobial therapy, in particular administration of cell wall active antibiotics, might result in a release of DNA from damaged bacteria and, consequently, in decreased DNA recovery during pathogen enrichment. To test this hypothesis, we chose S. aureus as model pathogen, since it is a leading cause of blood stream infection worldwide.

After an adaptation phase of several hours, both, S.aureus culture and wild-type strains reached stable growth in supplemented heparinized whole blood under our experimental conditions, while earlier studies with MRSA had observed rapid uptake of spiked S. aureus by neutrophils [7], with constant or decreasing CFU counts

Viktoria Weber

over time. The use of a methicillin-susceptible strain in our study could explain these divergent findings, since carriage of antibiotic resistance genes can reduce bacterial growth rates [41, 42]. Furthermore, we performed experiments for up to 72 h, after 4 h of adaptation of S.aureus to its environment. This required buffering as well as supplementation of whole blood with glucose and adenine, as commonly used for the storage of blood products [43]. Since wild type strains required a lag phase of 4 h for adaptation, we chose to incubate all strains for 4 h in supplemented whole blood prior to antibiotic administration, and we adjusted initial spiking concentrations to achieve a density of about 5000 CFU/mL after this adaptation phase. While this bacterial load was clearly higher than in septic patients, where pathogen loads of typically 1–10 CFU/mL have been reported [13], it allowed for the quantification of S. aureus using both, microbiological cultivation and qPCR and enabled the reliable determination of changes in qPCR signal intensity, which would not have been feasible at lower CFU counts.

To assess the influence of concurrent antibiotic treatment on pathogen enrichment, we used PIP and VAN according to clinical guidelines for the treatment of S.aureus infections [44, 45]. For comparison, we chose CIP, which is widely prescribed by general practitioners for the treatment of pneumonia, suspected gastrointestinal infections, or infections of the genitourinary tract [46, 47],

Fig. 3: Influence of antibiotic treatment on the recovery of pathogen DNA following pathogen enrichment. a-b S. aureus was grown in human whole blood, followed by a 90 min incubation in the presence of antibiotics, and CFU counts as well as pathogen DNA were quantified as described in Materials and Methods. Spiked whole blood without adsorbent treatment served as control (ctrl.). ΔcT values and CFU counts are given relative to the reference (sample taken prior to the addition of antibiotics). Recovery of pathogen DNA was not influenced by CIP and CLI, while DNA recovery was increased after treatment with the cell wall active antibiotics VAN and PIP. CFU counts were significantly reduced for PIP, VAN, and CIP (bactericidal), or remained constant for CLI (bacteriostatic), confirming efficacy of antibiotic treatment. Data are presented as mean ± standard deviation (n = 18; paired t-test). c-d Upon incubation of spiked blood samples with VAN and CIP over 72 h, the recovery of pathogen DNA remained stable (VAN) or even increased (CIP), while no viable bacteria were detectable beyond 48 h (n = 4; Friedman repeated measures and Dunnett’s Method). e Scanning electron micrographs of S. aureus incubated for 24 h in the presence of VAN and CIP failed to provide evidence for cell wall degradation or reduced culture density, as compared to the untreated control. Scale bar, 5 μm. Data are given as mean ± standard deviation.

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as well as CLI, which is commonly used for the treatment of skin and soft tissue infections in outpatient care [48]. All antibiotics were applied to achieve concentrations equivalent to mean plasma levels reported in the literature [26, 27, 29–32]. We used an established manual pathogen enrichment kit, since it allows for intervention at multiple steps of the pre-analytical protocol,

while protocols provided by other suppliers are fully integrated systems, which process blood samples automatically and do not allow for pathogen quantification at individual stages of the pre-analytical protocol. Still, our findings are likely to be valid for most other current pathogen enrichment protocols, as these systems differ

only insignificantly regarding the composition of lysis reagents.

Regardless of the antibiotic used, S. aureus DNA was not lost during the pre-analytical pathogen enrichment and was still detectable after 3 days of incubation in supplemented whole blood. Scanning electron microscopy revealed that S. aureus stayed largely intact over the course of the experiment despite antibiotic treatment, suggesting that pathogen DNA remained encapsulated by the bacterial cell wall and was protected from bloodborne degradation factors such as DNase [49]. Notably, the administration of cell wall active antibiotics resulted in a significant increase in the qPCR signal. It is highly probable that this effect was

Fig. 4 Influence of mechanical lysis on the viability and recovery of S. aureus DNA after pathogen enrichment. a S. aureus was mechanically lysed using zirconium beads and spiked into human whole blood as described in Materials and Methods. Culture confirmed the absence of viable bacteria after 5 min of lysis. Fragmentation of bacteria was confirmed by scanning electron microscopy. b Lysed S. aureus was spiked into whole blood, and DNA was quantified after pathogen enrichment as described in Materials and Methods. DNA recovery increased in parallel with pathogen disintegration. Data are given as mean ± standard deviation (n = 3; Friedman repeated measures and Dunnett’s Method). Scale bar, 5 μm

not related to the process of pathogen enrichment as such, but was rather due to incomplete lysis of gram-positive bacteria in alkaline solution after the actual pathogen enrichment step, and we assume that cell-wall active antibiotics support the release of pathogen DNA by reducing the thickness of the peptidoglycan layer of gram-positive bacteria [50]. Even mechanical disintegration of S. aureus prior to pathogen enrichment resulted in enhanced detection of pathogen DNA. This further confirms previous reports on the incomplete solubilization of bacterial DNA, which was particularly reported for

Table 2 Selective depletion of human DNA by pathogen enrichment

qPCR after	cT value
	Human DNA	S. aureus DNA
spiked whole blood protocol A	23.15 ± 0.38	25.98 ± 0.75
protocol B	17.06 ± 0.08	23.85 ± 0.64

Human whole blood was spiked with S. aureus and processed in parallel with an estalished pathogen enrichment protocol (protocol A) and by total (host and pathogen) DNA extraction with magnetic beads (protocol B) as described in Materials and Methods. qPCR revealed the presence of residual human DNA (positive PCR for beta-actin) in samples isolated with the pathogen enrichment protocol, but samples obtained by total DNA extraction contained significantly higher amounts of human DNA. Pathogen DNA was nearly equally well enriched with both protocols, resulting in a net enrichment of pathogen DNA over host DNA using the pathogen enrichment protocol. Data are presented as mean ± standard error of the mean (n = 4)

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gram-positive bacteria [51], most likely due to its association with the cell walls [45].

While a comparison of pathogen DNA recovery from whole blood, either after selective lysis and pathogen enrichment or after extraction of total DNA, revealed that pre-analytical pathogen enrichment was not associated with enhanced detection of pathogen DNA, human DNA was depleted by 4–5 cT values, which might be relevant for the pre-analytical depletion of blood-borne PCR inhibitors. Likewise, the reduction of human DNA background may be crucial for emerging analytical approaches including next-generation sequencing [52].

Conclusion

In conclusion, our study provides evidence that concurrent antibiotic administration is not associated with decreased recovery of pathogen DNA after pathogen enrichment by selective lysis and centrifugation. On the contrary, cell wall active antibiotics seem to increase the yield of pathogen DNA, presumably by supporting preanalytical DNA solubilization. Moreover, we confirmed a depletion of human DNA as compared to pathogen DNA by at least a factor of 10 during pathogen enrichment.

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1. Gudiol C, Cuervo G, Shaw E, Pujol M, Carratala J. Pharmacotherapeutic options for treating Staphylococcus aureus bacteremia. Expert Opin P h a r m a c o t h e r . 2 0 1 7 ; 1 8 ( 1 8 ) : 1 9 4 7 – 6 3 https://doi.org/10.1080/14656566.2017.1403585.
2. Rafalsky V, Andreeva I, Rjabkova E. Quinolones for uncomplicated acutecystitis in women. Cochrane D a t a b a s e S y s t R e v. 2 0 0 6 ; ( 3 ) : C D 0 0 3 5 9 7 https://doi.org/10.1002/14651858.CD003597.pub2.
3. Rothberg MB, Pekow PS, Lahti M, Brody O, Skiest DJ, Lindenauer PK. Comparative effectiveness of macrolides and quinolones for patients hospitalized with acute exacerbations of chronic obstructive pulmonary disease (AECOPD). J Hosp Med. 2010;5(5):261–7 https://doi.org/10.1002/jhm.628.
4. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJ, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of America. Clin Infect Dis. 2014;59(2):147– 59 https://doi.org/10.1093/cid/ciu296.
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Rykova EY, Pyshnyi DV, Vlassov VV, Laktionov PP. Circulating DNA and DNase activity in human blood. A n n N Y A c a d S c i . 2 0 0 6 ; 1 0 7 5 : 1 9 1 – 6 https://doi.org/10.1196/annals.1368.026.

1. Chakraborty SP, Sahu SK, Pramanik P, Roy S. In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus. Int J P h a r m . 2 0 1 2 ; 4 3 6 ( 1 – 2 ) : 6 5 9 – 7 6 https://doi.org/10.1016/j.ijpharm.2012.07.033.
2. Rantakokko-Jalava K, Jalava J. Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR. J Clin Microbiol. 2002;40(11):4211–7 https://doi.org/10.1128/ jcm.40.11.4211-4217.2002.
3. Hasan MR, Rawat A, Tang P, Jithesh PV, Thomas E, Tan R, Tilley P. Depletion of human DNA in spiked clinical specimens for improvement of sensitivity of pathogen detection by next-generation sequencing. J C l i n M i c r o b i o l . 2 0 1 6 ; 5 4 ( 4 ) : 9 1 9 – 2 7 https://doi.org/10.1128/JCM.03050-15.

 

Bertille Elodie Edinga-Melenge Suzanne Belinga, Eric Minkala, Prisca Armel Noudjeu, Michel Ondhoua, Samuel Walter Kokola, and Catherine Bilong

Department of Biochemistry, Centre Pasteur of Cameroon, Yaoundé, Cameroon.

Bertille Elodie Edinga-Melenge and Vicky Joceline Ama Moor

Department of Physiological Sciences and Biochemistry, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon.

Adrienne Tchapmi Yakam

Ebebda District Hospital, Centre Regional Delegation, Ministry of Public Health, Ebebda, Cameroon.

Jobert Richie Nansseu

Department for the Control of Disease, Epidemics and Pandemics, Ministry of Public Health, Yaoundé, Cameroon.

Jobert Richie Nansseu

Department of Public Health, Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I, PO Box 1364, Yaoundé, Cameroon.

Vicky Joceline Ama Moor

Laboratory of Biochemistry, Yaoundé University Teaching Hospital, Yaoundé, Cameroon.

Gloria Ashuntantang

Cardiology and Nephrology Unit, Yaoundé General Hospital, Yaoundé, Cameroon.

Gloria Ashuntantang

Department of Internal Medicine and Specialties, Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I, Yaoundé, Cameroon.

All Correspondences to: Jobert Richie Nansseu E-mail: jobertrichie_nansseu@yahoo.fr

ABSTRACT

Background: Serum cystatin C (SCysC) and serum creatinine (SCr) are two biomarkers used in common practice to estimate the glomerular filtration rate (GFR). For SCysC and SCr to be used in a given population, normal values need to be determined to better assess patients. This study aimed to determine SCysC and SCr reference intervals (RIs) in a Cameroonian adult population and factors susceptible of influencing them. Methods: We carried-out a cross-sectional study from November 2016 to May 2017 in Yaoundé, Cameroon. Participants were Cameroonians aged 18 years and above, residing inside the country and found in good health at study inclusion. SCysC and SCr were determined by particle-enhanced turbidimetric immunoassay standardized against the ERM-DA471/IFCC reference material and by the IDMS reference modified Jaffe kinetic method, respectively. RIs were determined using the 2.5th and 97.5th percentiles and their respective 90% confidence intervals (CIs). The quantile regression served to identify potential factors likely influencing SCysC and SCr values. Results: We included 381 subjects comprising 49.1% females.. RIs for SCysC varied between 0.57 (90%CI: 0.50–0.60) and 1.03 mg/L (90%CI: 1.00–1.10) for females, and from 0.70 (90%CI: 0.60–0.70) to 1.10 mg/L (90%CI: 1.10–1.20) for males. Concerning SCr, its RIs ranged from 0.58 (90%CI: 0.54–0.61) to 1.08 mg/dL (90%CI: 1.02–1.21) for females, and from 0.74 (90%CI: 0.70–0.80) to 1.36 mg/dL (90%CI: 1.30–1.45) for males. Men had significantly higher SCysC and SCr values than women (p < 0.001). Likewise, subjects aged 50 years and above had higher SCysC values in comparison to younger age groups (p < 0.001), which was not the case for SCr values (p = 0.491). Moreover, there was a positive and significant correlation between SCysC and SCr in women (ρ = 0.55, p < 0.001), in men (ρ = 0.39, p < 0.001) and globally (ρ = 0.58; p < 0.001). Furthermore, the sex influenced both biomarkers’ values across all quantile regression models while age and body surface area (BSA) influenced them inconsistently. Conclusion: This study has determined serum cystatin C and serum creatinine reference intervals in an adult Cameroonian population, whose interpretations might take into account the patient’s sex

and to a certain extent, his/her age and/or BSA.

Keywords: Reference interval, Cystatin C, Creatinine, Yaoundé, Cameroon.

BACKGROUND

Glomerularfiltrationrate(GFR)iswidelyacceptedas the mostusefuloverallindexofkidneyfunctionin healthand disease(1).It is best evaluated by clearance measurement ofexogenousmarkerssuchasinuline,but thecomplex proceduresofthesemeasureslimittheir routineuse(2,3). GFR is therefore commonly estimated fromserumlevelof endogenousfiltrationmarkers.The mostwidelyusedand recommendedendogenousmarker forinitialassessment ofGFRisserumcreatinine(4).

Despitethecheapestcostandthesimpleuseof creatinine-basedmeasurementsofGFR,estimationof thelevelof

renalfunctionobtainedisquiteimprecise.

Indeed, the steady-state serum creatinine level is determined byfactorsthatincludeleantissuemass; hence, it mayvarywithsex,age,weightandheight(3,5,6).

Asaresultoftheselimitations,alternativeendogenous markersforGFRsuchasserumcystatinChavebeen proposed.CystatinCisabiomarkerformedataconstant rate by all nucleated cells of the body which do not correlate with lean tissue mass (5). Evidence has demonstrated improved accuracy and sensitivity of cystatinC comparedtocreatinine(7).

For an accurate interpretation of biomarkers levels,

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Reference intervals for serum cystatin C…

referenceintervalsspecifictoapopulationneedtobe established.Intriguinglyandalthoughserumcreatinine is widely used in Cameroon, no previous study had yet focused at determining its reference intervals, interpretations relying on western countries’ data. Moreover consideringthegrowingimportanceofcystatin Casa prospectivemarkertoassesstherenalfunction,itis obvious thatthismarkerwouldbeintroducedinroutine clinicalpracticeinCameroonverysoon.Therefore,we conductedthepresentstudytodeterminethereference valuesofserumcreatinineandcystatinCinahealthy adult Cameroonianpopulationlivinginsidethecountry. Besides, weaimedtoidentifypotentialfactorslikely influencing thesereferenceintervals.

Methods

Studydesignandsetting

This was a population based cross-sectional study conducted betweenNovember2016andMay2017in Yaounde’thecapitalcityofCameroon.Participantswere recruitedfromthe4mostpopulatedhealthdistrictsout of the6thatconstitutesthecity,namely:Yaounde’1,2,4 and 7

(8). Biological analyses were performed at the Centre PasteurofCameroun.

Descriptionofthestudypopulation

ParticipantswereadultCamerooniansresidinginsidethe country,aged18yearsandabove,foundingoodhealthat studyinclusion-afterageneralexaminationincludinga briefmedicalinterview,urinalysisandmeasurementof bloodpressureandglycaemia-withnoevidenceofany acuteorchronicillnesssusceptibleofaffectingcreatinine orcystatinClevels.Weexcludedknownorsuspected hypertensives,thosewithanimpairedglucosemetabolism

(pre-diabetes or diabetes mellitus) or an abnormal dipstick urinetest.Pregnantandbreastfeedingwomenwerealso excluded, as well as drug users. No special dietary recommendations were required. Participants were consecutively recruited during the study period and a minimumof 120 participantswasrequiredforeachsex group,inline withtheInternationalFederationofClinical Chemistry’s (IFCC) recommendations (9).

Datacollection

Participants were mostly recruited in churches, sc hools/universities/collegesandmosques.Onthedaysof recruitment,eachpotentialparticipantwasrequiredto sign aconsentformasthetestimonyofhis/hervolunteering participation. Subsequently, he/she underwent a brief interview using a preconceived, standardized and pre-testedquestionnaire(Additionalfile 1);then a summary physicalexaminationwasconducted,duringwhich blood pressurewasmeasured.Weusedthesimplified calculation procedurefromMostellerRDtoderiveeach participant’s bodysurfacearea(BSA)(10).In addition, a urinesample wascollectedfordipstickurineanalysis andacapillary glycaemiawasperformedusinga OneTouch*analyzer.

Biochemicalassays

Ten milliliters of venous blood were collected by

venipuncture in 2 dry tubes of 5ml each. Serum was separatedbycentrifugationat3000rpmwithin10min. Biochemical assays were conducted using the autoanalyzer CobasC501/6000,RocheDiagnostics,USA. Serum cystatinCwasmeasuredinincrementsof0.1mg/L by particle-enhanced turbidimetric immunoassay using Tina-quant*CystatinCreagentkits(RocheDiagnostics, USA).Themethodappliedwasstandardizedagainstthe ERM-DA471/IFCCreferencematerial.Meanwhile, serum creatininewasdeterminedbytheIsotopeDilution Mass Spectrometry (IDMS) reference modified Jaffe kinetic methodusingCreatinineJaffeCobas*reagentkits (Roche

Diagnostics, USA).

Statisticalanalysis

DatawerecodedandenteredusingtheCensusand Survey ProcessingSystemversion7.1.Statisticalanalysis was performed using the Statistical Package for Social Sciencesversion23.0(IBMSPSSInc.,Chicago,Illinois, USA) and STATA version 12.0(STATACORP, Texas, USA).Categoricalvariablesarepresentedusingfrequency (percentage) while continuous variables are summarized withtheirmedian(interquartilerange,[IQR].

Edinga-Melengeetal.BMCClinicalPathology (2019) 19:4Page2 of 9

TheKolmogorovSmirnovtestwasusedtoassessthe normality of continuous variables’ distributions. Reference intervals(RIs) were determined by the nonparametric methodasdescribedintheIFCCguidelines (11).

This method was used to determine the 2.5 and 97.5 percentilesandtherespective90%confidenceintervals (CI) around these estimates. The Mann–WhitneyU-test and the Kruskal-Wallis H-test were used for bivariate analyses, to compare the distributions of continuous variables, consideringthatthesevariablesdidnotfollowa

Gaussianshape.Forthesamereason,itistheSpearman correlationtest(withitsrho(ρ)coefficient) that was used

to investigate existence of any correlation between continuousvariablesincludingserumcystatinC,serum creatinineandage.Furthermore,weuseda25th,50th and 75thpercentilequantileregressionanalysistoidentify any factor likely influencing serum cystatin C or serum creatininereferenceintervalsinamodelincluding theage, sex,andBSA.Statisticalsignificancewasset atap-value

lower than 0.05.

Results

Atotalof 485healthysubjectswerescreenedofwhom 104 wereexcludedbecauseofunderlyingdiabetesmellitus, pre-diabetes,hypertensionorabnormaldipstick urinetest. Thereferencepopulationcomprised 381 healthyadults (including49.1% females)agedbetween 18and71years oldwithamedianageof28years[IQR 23-40].Therewere nodifferencesinthedistributionof agebetweenmaleand femaleparticipants(p=0.290).By contrast,maleshad significantlyhigherBSAvaluesthan females:p=0.002 (Table 1).

Thenon-parametricreferenceintervalsforserum cystatin Cwere0.57-1.03mg/Lforwomenand0.70-1.10mg/Lfor

50 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Jobert Richie Nansseu

men; the reference intervals for the whole studypopulation were0.60 -1.10mg/L(Table 1).For serumcreatinine,these intervalswere0.58-1.08mg/dL forwomen,0.74-1.36 mg/dLformen,and0.61-1.30 mg/dLforallsubjects (Table1).As compared to women,menhadsignificantly highertitersofserum cystatinC(median0.90vs.0.80 mg/L; p<0.001;Table 1) thanwomen,exceptforthose aged50yearsandabove (p=0.125;Table 2).Similarly, men had significantly higher serum creatinine values (median1.06vs.0.79mg/dL; p<0.001;Table 1)than women, this tendency being thesameinallagegroups (Table3).

Additionally, serum cystatin C levels were higher in persons aged 50 years and above compared to their counterparts agedlessthan50yearsold(p<0.001;Table 2);on thecontrary,thisdifferencewasnotobservedwith serum creatininevalues(p=0.491;Table 3).Moreover, we found apositiveandsignificantcorrelationbetweenserum cystatinCandserumcreatininebothinfemales(p = 0.55; p

<0.001),in males(p =0.39 < 0.001) and in the total study population(p = 0.58;p<0.001).

Furthermore,thecorrelationbetweenserumcystatin C logarithmically-transformedvaluesandagewasweak and non-significantinmales(p=- 0.006,p=0.930;Fig. 1 a),but becamesignificantinfemales(p= 0.265, p< 0.001;Fig. 1b. Contrariwise,thecorrelationbetween serumcreatinine logarithmically-transformed values and age was significantinmales(p = 0.162, p=0.023;Fig. 2a),but insignificantinfemales(p = 0.127, p=0.082; Fig.2b).On theotherhand,resultsofthequantile regressionwhichare presentedinTable4showed thatacrossthevariousmodels, thesexremainedthe onlyfactorlikelyinfluencingboth serumcystatinC andserumcreatininevalues.Theage seemedtocontribute inexplainingserumcystatinCvalues inthe 75thpercentilequantileregressionmodel,which was identicalforserumcreatininevalues.TheBSAwas contributiveinexplainingserumcreatininevalues onlyin the50thpercentilequantileregressionmodel (Table 4).

Table 1 Reference intervals for serum cystatin C and serum creatinine according to sex

Parameter	All (n = 381)		Males (n = 194)		Females (n = 187)		p*
Age (years)	28 [23–40]		28 [24–40]		26 [22–43]		0.290
BSA (m2)	1.68	[1.55–1.79]	1.70	[1.81]	1.65	[1.52–1.76]	0.002*
Serum cystatin C (mg/L)
Median [IQR]	0.80	[0.70–0.90]	0.90	[0.80–1.00]	0.80	[0.70–0.90]	< 0.001
2.5th percentile (90%CI)	0.60	(0.60–0.61)	0.70	(0.60–0.70)	0.57	(0.50–0.60)
97.5th percentile (90%CI)	1.10	(1.10–1.11)	1.10	(1.10–1.20)	1.03	(1.00–1.10)
Serum creatinine (mg/dL)
Median [IQR]	0.92	[0.77–1.06]	1.06	[0.96–1.14]	0.79	[0.71–0.88]	< 0.001
2.5th percentile (90% CI)	0.61	(0.59–0.64)	0.74	(0.70–0.80)	0.58	(0.54–0.61)
97.5th percentile (90% CI)	1.30	(1.28–1.35)	1.36	(1.30–1.45)	1.08	(1.02–1.21)

BSA body surface area, CI confidence interval, IQR interquartile range, SCysC serum cystatin C, SCr serum creatinine; †The Mann-Whitney U-test was used for variable comparisons; *p < 0.05

Table 2 Reference intervals for serum cystatin C by age and sex

Age	N=381	Serum cystatin C (mg/L)
(years)		Median (IQR)			2.5th percentile (90%CI)	97.5th percentile (90%CI)		p„©
< 20	Male:	9 0.9 (0.85.1.05)			0.7 (0.70.0.90)	1.1		0.003
	Female:	14	0.75 (0.70.0.80)		0.6 (0.60.0.70)	0.9
	All: 23	0.8		(0.70.0.90) 0.6	(0.60.0.70)	1.1
[20.30]	Male: 95	0.9		(0.80.1.0)	0.6 (0.60.0.70)	1.1	(1.10.1.10)	< 0.001
	Female:	89	0.7 (0.70.0.80)		0.6 (0.50.0.60)	1.0	(0.90.1.00)
	All: 184	0.8		(0.70.0.90)	0.6 (0.60.0.60)	1.1	(1.10.1.10)
[30.40]	Male: 40	0.9		(0.80.0.90)	0.7 (0.70.0.80)	1.0	(1.00.1.00)	0.001
	Female:	31	0.8 (0.60.0.80)		0.5 (0.50.0.50)	1.0
	All: 71	0.8		(0.80.0.90)	0.5 (0.50.0.58)	1.0	(1.00.1.00)
[40.50]	Male: 32	0.9		(0.80.0.98)	0.7 (0.70.0.70)	1.1	0.002
	Female:	21	0.8 (0.70.0.90)		0.6 (0.60.0.70)	1.0
	All: 53	0.8		(0.80.0.90)	0.6 (0.60.0.70)	1.0	(1.00.1.00)
50	Male: 18	1.0		(0.88.1.0)	0.7 (0.70.0.80)	1.2		0.125
	Female:	32	0.9 (0.80.0.90)		0.6 (0.60.0.80)	1.1
	All: 50	0.9		(0.80.1.00)	0.7 (0.60.0.73)	1.2	(1.10.1.20)

CI confidence interval, IQR interquartile range. Some 90% confidence intervals are not presented due to the small number of participants in corresponding age groups. ┼ The Mann-Whitney U-test was used to compare the distribution of serum cystatin C values between males and females. The difference between agegroups was significant when using the Kruskal-Wallis H-test (p < 0.001)

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Reference intervals for serum cystatin C…

Discussion

In agreement with IFCC recommendations [11], the reference intervals for serum cystatin C and serum creatinine were determined in the present study among a healthy Cameroonian adult population. Our results revealed that the reference intervals for serum cystatin C

varied between 0.6 and 1.1 mg/L, with men having higher values than women (p < 0.001), except in the 50+ years age group. Concerning serum creatinine, the reference intervals ranged from 0.6 to 1.3 mg/dL; similarly, men had significantly higher levels than women (p < 0.001) across all age groups. Participants

Table 3 Reference intervals for serum creatinine by age and sex

Age	N=381	Serum creatinine (mg/dL)
(years)		Median (IQR)	2.5th percentile (90%CI) 97.5th percentile (90%CI)		p„©
< 20	Male: 9	1.09 (1.00.1.15)	0.80 (0.80.1.04)	1.18	< 0.00
	Female: 14	0.76 (0.72.0.79)	0.66 (0.66.0.70)	0.88
	All: 23	0.79 (0.75.1.08)	0.66 (0.66.0.70)	1.18
[20.30]	Male: 95	1.03 (0.94.1.12)	0.80 (0.70.0.85)	1.30 (1.29.1.35)	< 0.001
	Female: 89	0.79 (0.71.0.87)	0.60 (0.54.0.61)	1.07 (1.01.1.28)
	All: 184	0.91 (0.78.1.05)	0.61 (0.59.0.65)	1.30 (1.26.1.33)
[30.40]	Male: 40	1.06 (0.95.1.13)	0.75 (0.69.0.81)	1.29	< 0.001
	Female: 31	0.73 (0.65.0.83)	0.57 (0.57.0.60)	1.26
	All: 71	0.94 (0.74.1.08)	0.58 (0.57.0.62)	1.27 (1.22.1.32)
[40.50]	Male: 32	1.08 (0.99.1.17)	0.68 (0.68.0.74)	1.45	< 0.001
	Female: 21	0.74 (0.69.0.90)	0.51 (0.51.0.66)	1.02
	All: 53	0.99 (0.74.1.10)	0.61 (0.51.0.66)	1.38 (1.24.1.45)
≥50	Male: 18	1.17 (0.99.1.30)	0.79 (0.79.0.96)	1.52	< 0.001
	Female: 32	0.85 (0.79.0.93)	0.60 (0.60.0.65)	1.05
	All: 50	0.93 (0.81.1.06)	0.64 (0.60.0.66)	1.43 (1.33.1.52)

CI confidence interval, IQR interquartile range. Some 90% confidence intervals are not presented due to the small number of participants in corresponding age groups. ┼ The Mann-Whitney U-test was used to compare males and females; the difference in the distribution of serum creatinine values between age-groups was not significant with the Kruskal-Wallis H-test (p = 0.491)

Fig. 1 a Relationship between serum cystatin C (log) values and age in males [(n = 194); y = 0.0009x – 0.0114, ρ = 0.162 (p = 0.024)]. b Relationship between serum cystatin C (log) values and age in females [(n = 187); y = 0.0016x – 0.164, ρ = 0.265 (p < 0.001)]

52 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

aged 50 years and above had higher serum cystatin C values than those aged less than 50 years (p < 0.001), which was not the case for serum creatinine values (p = 0.491). Moreover, the correlation between serum cystatin C and serum creatinine was positive and significant (ρ = 0.58; p < 0.001) and the quantile regression pointed mostly the sex, and to a certain extent the age and BSA as independent factors susceptible of influencing serum cystatin C and/or serum creatinine values. Reference intervals for serum cystatin C obtained in this study (0.60–1.10 mg/L) are in compliance with those from previous studies which have also used turbidimetric assay. For instance, Köttgen et al. recorded in a US population a reference interval varying between 0.61– 1.04 mg/L; Okonkwo et al. in a Nigerian population recorded a reference interval ranging between 0.64–1.12mg/L and Li et al. in a Chinese population recorded a reference interval varying from 0.60 to 1.08 mg/L [12–14]. By contrast, the reference intervals for serum creatinine obtained in this study (0.61–1.3 mg/dL)

Jobert Richie Nansseu

seem to differ from that of Caucasians. Indeed, Pottel et al. found reference intervals around 0.48–0.93 mg/dL in women and 0.63–1.16 mg/dL in men within a healthy adult Caucasian population [15]. These intervals concur with those of Ceriotti et al. obtained in a multicenter analysis of three studies based on Caucasian adults. In this study indeed, the reference intervals for serum creatinine varied between 0.45–0.92mg/dL in women and 0.59–1.05mg/dL in men [16]. These differences could be explained by the fact that the measurement of serum creatinine used enzymatic methods in the two studies just cited, which could give slightly lower values than colorimetric assays that were used in our study. Additionally, evidence has accumulated that black people have a more important lean tissue mass and a lower GFR compared to Caucasians [3, 17]. However, our results corroborate those from other African authors such as Sakande et al. in Burkina Faso and Dosoo et al. in Ghana. Indeed, Sakande et al. reported reference intervals ranging between 0.63–1.41 mg/dL in

Fig. 2 a Relationship between serum creatinine (log) values and age in males [(n = 194); y = 0.0009x – 0.0114, ρ = 0.162 (p = 0.024)]. b Relationship between serum creatinine (log) values and age in females [(n = 187); y = 0.0007x – 0.129, ρ = 0.127 (p = 0.082)]

men and 0.45–1.24 mg/dL in women; reference intervals obtained by Dosoo et al. were 0.63–1.35 mg/dL in men and 0.60–1.20mg/dL in women [18, 19]. Furthermore, Lim et al. conducted a study among afro-Americans and found similar results with men having serum creatinine reference intervals around 0.73–1.45 mg/dL and women, around 0.52–1.15 mg/dL [20]. The sex-related differences in the non-parametric reference intervals for serum creatinine are in line with previous studies and reinforced by results of our quantile regression analysis indicating that the sex influenced serum creatinine values across all models, while adjusting for age and BSA. Indeed, muscular mass is

higher in men compared to women [3, 5, 6, 21]. Concurring with previous findings, our results indicate that serum cystatin C levels seem to be slightly affected by factors such as sex and age [22–24]. Pottel et al. showed for instance that cystatin C increases with age, after the age of 70 years old [24]. The influence of sex on serum cystatin C levels is still unclear. In fact, some studies have reported that serum cystatin C levels are independent of sex unlike other studies have claimed that sex influences significantly serum cystatin C values [13, 23, 25–28]. In our study for instance, we found that the sex constituted one independent explanatory factor for serum cystatin C values, whatever

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Reference intervals for serum cystatin C…

Table 4 Regression coefficients and p-values for the 25th, 50th and 75th percentiles quantile regression models

		Serum cystatin C		Serum creatinine
		25th	50th	75th		25th	50th	75th
	Sex	-0.1 (< 0.001)*	– 0.1 (< 0.001)*	-0.09 (< 0.001)*		-0.24 (< 0.001)*	-0.28 (< 0.001)*	-0.27 (< 0.001)*
	Age	7.67e-19 (1.000)	– 3.36e- 18 (1.000)	0.003 (0.001)*		0.0007 (0.171)	0.001 (0.162)	0.002 (0.031)*
	BSA	9.39e-17 (1.000)	7.35e-16 (1.000)	0.038 (0.475)		0.08 (0.072)	0.15 (0.001)*	0.127 (0.073)
	*p < 0.05
the quantile regression model considered; additionally,					perhaps because they used the Pearson correlation test and
serum cystatin C levels were 11% higher in men than in					rescaled their biomarkers. The inconsistent influence of
women (0.90 mg/L vs 0.80 mg/L; p < 0.001). These results					age on both serum cystatin C and serum creatinine values
corroborate those from Köttgen et al. in the US who					was observed after applying the quantile regression
reported a difference of 8% between males and females					analysis. Indeed, we found that age influenced
[12]. However, Al Wakeel et al. in a Saudi adult population					significantly both serum cystatin C and serum creatinine
reported lower serum cystatin C levels in men compared to					values only at the 75th percentile quantile regression
women (0.72 mg/L vs 0.77 mg/L; p < 0.001) as well as Li et					model, the estimator being insignificant at the 25th and
al. in China (0.84 mg/L vs 0.85 mg/L; p < 0.05) [14, 29]. In					50th percentile models. We need further well-designed
the Saudi study, women had higher body mass index that					studies to better investigate the influence of age (and BSA)
men and the positive correlation between serum cystatin C					on serum cystatin C and serum creatinine values in our
and body mass index could have explained the higher					context. However, our findings need to be interpreted in the
serum cystatin C levels in women [13, 29, 30]. In Li et al.’s					context of some limitations, mainly occurring from the
study, the sex difference was observed only between 30					non-random sampling method used and single
and 60 years [14]. Likewise, we found in our study that					measurement of serum cystatin C and serum creatinine. In
from 50 years old and beyond, differences of serum					fact, the representativeness of our study population and
cystatin C levels between men and women became non-					generalization of our results to the entire Cameroonian
significant (median 1.00 vs 0.90 mg/L; p = 0.125) while the					population would have been better obtained with
difference persisted for serum creatinine levels (median					randomization. Nevertheless, we selected the most
1.17 vs 0.85 mg/dL; p < 0.001). Actually, the influence of					populated health districts among the 6 that compose
sex on serum cystatin C levels seems non-significant with					Yaoundé, the cosmopolitan capital city of Cameroon. On
increasing age, suggesting a physiological or pathological					the other hand, participants were selected on the basis of
condition which should be more investigated in elderly.					their normal renal function which could be attested only by
Further studies are warranted in this respect. On the other					measurement of GFR by the gold standard (inuline).
hand, subjects aged 50 years and over had 11% higher					Nonetheless, the absence of risk factors for kidney disease
serum cystatin C levels compared to lower age groups					and the normal clinical and biological tests performed
(0.90 vs. 0.80; p < 0.001). Concurring with these results,					among our participants could be some indirect indicators
several other studies have demonstrated an increase in					of normal kidney function. Furthermore, we used rigorous
cystatin C values above a threshold age varying from 40 to					statistical procedures and applied the IFCC guidelines to
70 years [12, 14, 24, 29–32]. The higher levels of serum					depict our estimates. Notwithstanding and to the very best
cystatin C in older subjects could be due to the					of our knowledge, this is the first
physiological decrease in GFR which starts from 40 years					study providing the reference values for serum cystatin C
[33]. Serum creatinine levels are also expected to increase					and serum creatinine in Cameroon, which could be
around the same age (≥50 years); however, we observed					translatable to similar sub Saharan African populations.
that the distributions of serum creatinine values were					Conclusion
similar across the various age groups (p = 0.491).
Likewise, Pottel et al. using a Caucasian population					This study depicted serum cystatin C and serum creatinine
noticed that between 20 and 70 years old, the mean serum					reference intervals in a healthy adult Cameroonian
creatinine level was stable [24]. This could be explained by					population. Men had significantly higher levels of both
the drop in creatinine rate production due to reduction in					biomarkers compared to women. Subjects aged 50 years
the muscle mass which appears concomitantly with the					old and above had significantly higher serum cystatin C
decrease in GFR [33]. The physiological increase in					values than those aged less than 50 years old. Therefore,
creatinine levels will be therefore lately observed around					the interpretation of both biomarkers should probably take
65–70 years [3, 15]. We found a positive and significant					into account the patient’s sex and to a certain extent, his/her
correlation between serum cystatin C and serum					age (and/or body surface area) for an appropriate diagnosis
creatinine, both in males (ρ = 0.39, p < 0.001), in females (ρ					of a renal disease. Moreover, it is hoped that our data
= 0.55, p < 0.001) and in the total population (ρ = 0.58; p <					stimulate further research on a larger population that will
0.001). These findings mirror those from Pottel et al. who					be more representative of the whole country’s diversity.
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54 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

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Arnon Houri-Yafin, Yochay Eshel, Natalie Lezmy, Sarah Levy-Schreier,

Caitlin Lee Cohen, Joseph Joel Pollak1 and Seth J. Salpeter

Sight Diagnostics Ltd., Israel

Benedicta Larbi and Emma Wypkema

Department of Clinical Hematology Lancet Laboratories, Lancet Corner, South Africa

Veena Dayan

Department of Parisitology, City Hospital Mangalore, India

All Correspondences to: Seth Salpeter, Sight Diagnostics Ltd., Jerusalem Technology Park, Jerusalem 96951, Israel, E-mail: seth@sightdx.com

ABSTRACT

Accurate malaria diagnosis is necessary to prevent unnecessary deaths and curb malaria drug resistance related to unnecessary treatment. While numerous diagnostic assays exist, the need for a low-cost, rapid and highly accurate malaria test remains. Here we evaluate the diagnostic performance of a computer vision platform, the Sight Diagnostic P2 device for malaria diagnosis, speciation and parasite quantification. The trial was conducted at two centers on Plasmodium falciparum and Plasmodium vivax samples, using different testing protocols: 374 samples were collected at City Hospital Mangalore India and 167 samples were collected at Lancet Laboratories Johannesburg South Africa. At City Hospital, the device diagnoses were compared to RT-PCR results while at Lancet Laboratories the device diagnoses were compared to a panel of tests provided by the clinic. For identification of malaria, the device demonstrated a sensitivity of 97% and a specificity of 99.5% at City Hospital India, and a sensitivity of 97.8% and a specificity of 97.5% at Lancet Laboratories Johannesburg. For speciation, the device correctly identified 87.5% for Plasmodium Vivax and 93.5% for Plasmodium Falciparum at City Hospital India. Lastly, comparing the device parasite count with that of trained microscopes, produced an average pearsons correlation of 0.87.

Keywords: Malaria; Diagnostic; Computer vision; Machine Learninga

Introduction

ccurate diagnosis of malaria is imperative to Areduce morbidity, prevent resistance to anti-malarials, and limit the number of adverse treatment effects from unnecessary use [1]. Furthermore, many studies show that infectious malaria carriers maintain a very low parasitemia, making sensitive detection technologies imperative for treatment targeted at epidemiologic control [2,3]. Many governmental health organizations now require patients to undergo malaria testing before receiving any anti-malarial medicine. As a result, the WHO forecasts an increase in global demand for malaria tests from 500 million in 2012 to nearly 1 billion

tests by 2020 [1,4].

Due to increased demand for malaria tests, reliable, simple and highly accurate malaria diagnostic is needed. Globally, only 77% of suspected cases in the public sector are tested, while in Africa only 47% of cases are assayed [5]. A recent report showed that in some regions 81% of people taking ACT therapy are not infected, while only 31% of the positive cases received the treatment [6]. This glaring disparity not only leaves the needy untreated but encourages the further development of drug resistant malaria strains.

While new diagnostic modalities for malaria have emerged in recent years, none have the ideal set of test characteristics. According to the World Health Organization, an ideal test would be inexpensive,

consistent, highly-sensitive, adequately specific, quantitative, and species-differentiating. Microscopy remains the gold standard malaria test worldwide [7,8], as it supports direct parasite identification and also provides monitoring of systemic inflammation and its response to therapy [9]. However, microscopy can be very inaccurate, needs extensive analysis, and requires highly trained staff [10,11]. Notably, malaria is associated with systemic spiraling of innate inflammation and additional blood abnormalities, further complicating microscopy examination [12]. Numerous reports have also shown inconsistent sensitivity of microscopist due to the high volume of tests and varied level of skill among malaria technicians [13,14]. Rapid diagnostics tests (RDTs) continue to increase in popularity and market share as they have significantly improved the diagnosis of malaria in remote, inaccessible areas [15]. However, RDTs have significant limitations that make many practitioners wary of their use, including decreased sensitivity at low parasitemia, inability to quantitate parasite burden, and inconsistencies between brands in their ability to detect and differentiate different malaria species [16,17]. Recent improvements to malaria diagnostic technologies include Polymerase Chain Reaction (PCR) and loop-mediated isothermal amplification (LAMP), which offer superior sensitivity, speciation and parasitemia but are impractical for the vast majority malaria-endemic areas [18,19].

Malaria diagnosis using computer vision offers a potential solution to the shortcomings of other technologies. An

40 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An Enhanced Computer Vision Platform…

automated microscopist maintains the advantages of a microscopist with significant improvements in speed, cost, and consistency. Previous attempts at a creating a computer microscopist have not surpassed the development stage [20,21]. A recent report from our group, described a clinically available automated microscopist which was tested with the National Institute of Malaria Research India

1. The device, the SightDx P1 malaria platform, was tested on 431 patients and demonstrated a sensitivity of 97.05%, and a specificity of 96.33% when compared with PCR. Furthermore, the device was able to accurately speciate 73.3% of the PCR Plasmodium falciparum and 91.4% of the PCR Plasmodium vivax samples, and showed a parasitemia correlation with microscopists of 0.89.

Here, we present an enhanced version of the Sight Diagnostic malaria device, the SightDx P2 platform for malaria detection. The device is intended for laboratories performing high volumes of malaria tests as it is capable of scanning a sample in 4 minutes and can hold up to 30 tests. The system has dimensions of 45 x 50 x 58 cm (DxWxH) and can easily fit onto a standard laboratory bench top with minimal installation requirements.

In the following report, we describe the results of clinical studies performed in Mangalore India, and Johannesburg South Africa to evaluate the sensitivity, specificity, speciation and parasite count calculation as compared to standard diagnostic procedures.

Methods

Study design

The study was a double center, prospective, non-randomized, non blinded study conducted at City Hospital, Mangalore India with 374 blood samples from clinically-suspected malaria patients, and at Lancet Laboratories Johannesburg South Africa on 167 clinically suspected malaria patients.

Study Procedures

City Hospital, Mangalore India: Determination of eligibility for malaria treatment was solely based on the clinic’s standard diagnosis protocol and the patients course of treatment and was not altered due to the study or the SightDx diagnostic device. In most cases blood was scanned by the device within 24 hours of sampling. Samples more than 48 hours old were not included in the study. In addition, 100 μL of blood was collected on GE Healthcare FTA Whatman filter paper spots for RT-PCR evaluation. RT-PCR results were considered the standard of comparison for determining the sensitivity, specificity and speciation of the various methods. Lancet Laboratories, Johannesburg South Africa: Samples were provided from malaria tests performed at Lancet Laboratories Johannesburg and at surrounding Lancet Laboratory clinics in South Africa. Samples were tested on the Sight Diagnostic device within 1 week of drawing. RDT and microscopy were performed on all samples. Discrepancies between these tests were evaluated by PCR. Positive samples which were not Plasmodium falciparum or had a parasitemia under 1000 parasites/μL also underwent PCR. All negative samples were reviewed with QBC.

Laboratory Methods

Sight Diagnostic Device Analysis

In all locations digital imaging scanning was carried out onsite. To begin sample diagnosis, 5 μL of patient blood was mixed with a fluorescent dye solution that stained intracellular DNA and RNA. The sample was then loaded into a plastic cartridge and incubated for 5 minutes, during which time the cells formed a monolayer. The stained cells were then excited using 3 different LED light sources (370 nm, 475 nm and 530 nm) after which the imaging system recorded 600 images analyzing ~1.8 million cells. The total scan time per sample was 4 minutes and the device held up to 30 samples which can be loaded in batch. Samples which registered an error on the device due to incorrect user preparation were repeated. Computer vision and statistical models were used to detect the malaria parasites. Using statistical models, the device determined infection status, parasitemia levels, and species.

Parasitemia

Parasitemia counts were performed on 24 positive samples at Lancet laboratories Johannesburg. An expert microscopist analyzed 10 fields at 100X with approximately 100 RBCs counted per field. Parasitemia was calculated as a ratio of infected RBCs to total RBCs.

Real Time PCR Analysis

For PCR experiments performed on samples from City Hospital India, a whole punch was removed from the blood spot on the GE FTA Whatman paper and eluted as previously reported [23]. Real time PCR was performed with Fast Syber Green Master Mix at a volume of 10 μL (Applied Biosystems) using previously published primer sequences [24] for identifying falciparum, vivax and for general Plasmodium (Plu). All reactions were performed in 384 well qPCR plates (Bio-Rad) on a CFX384 real time PCR machine from Bio-rad.

Results

The Sight Diagnostic P2 malaria scanning device is a desktop system for computerized malaria diagnostics (Figures 1A and 1B). The stained blood is loaded into a cartridge which holds five patients samples. To evaluate device performance in a clinical setting, 374 samples were collected and scanned at City Hospital Mangalore and 167 samples were collected and scanned at Lancet Laboratories Johannesburg.

Figure 1: The SightDx Malaria Platform. (A) The P2 malaria scanning device. (B) The loading cartridge holds 5 patient samples.

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 45

 

Sensitivity and specificity

Sensitivity and specificity were analyzed for all trials (Table 1). For samples scanned at City Hospital India device results were compared to qPCR while for samples scanned at Lancet Johannesburg device results were compared to a final diagnosis based on a combination of several malaria diagnostic assays (Table 1). At City Hospital Mangalore sensitivity was calculated as 97%

Seth J. Salpeter

(167/172) and specificity was calculated as 99.5%

(201/202). For samples scanned at Lancet Laboratories Johannesburg sensitivity was 97.8% (46/47) while specificity was 97.5% (117/120). Positive predictive values (PPV) were 99.4% at City Hospital and 93.8% at Lancet Laboratories, and negative predictive values (NPV) were 97.5% at City Hospital and 99.1% at Lancet Laboratories.

		Sensitivity			Specificity
	Percent	Ratio	95% CI	Percent	Ratio	95% CI
City Hospital India	97%	167/172	0.934-0.988	99.50%	201/202	0.972-0.999
Lancet Labs South Africa	97.80%	46/47	0.843-0.994	97.50%	117/120	0.929-0.991

Speciation

Speciation studies were conducted on samples provided at City Hospital Mangalore (Table 2). At City Hospital, the device distinguished between P.v (Plasmodium vivax ) and P.f (Plasmodium falciparum ) and results were compared to qPCR analysis. A total of 167 samples were identified as positive by the device and were analyzed for species type. The device correctly identified samples with Plasmodium vivax at 87.5% sensitivity (119/136) and Plasmodium falciparum at 93.5% sensitivity (29/31).

	City Hospital (India)
	Percent	Ratio	95% CI
Plasmodium Vivax	87.50%	119/136	0.809-0.92
Plasmodium Falciparum	93.50%	29/31	0.793-0.982

Table 2: Speciation accuracy divided according to treatment groups. Speciation percentages of the trials from City Hospital India are presented in the table, as well as the specific number of patients and confidence Index.

Parasitemia

For cases diagnosed at Lancet Laboratories with thin smear microscopy, parasitemia was provided and compared to values from the device (Figure 2).

Figure 2: Micrscopist compared to the device. At Lancet Laboratories a trained microscopist analyzed 24 slides and the results were compared to the parasitemia reported by the device. The correlation between the two produced a Pearsons correlation coefficient of 0.87.

A comparison of the percentage of infected RBC determined by the microscopist and the device yields a Pearsons correlation coefficient of 0.87. The microscopist calculated parasitemia by analyzing the number of infected red blood cells out of the total number of blood cells.

Discussion

This study evaluated the SightDx P2 malaria detection platform, an enhanced computer vision platform for rapid and automated malaria diagnostics. Previous attempts to develop vision based malaria detection devices have had varying levels of success [21,25-28]. While a specific report showed high sensitivity and specificity [29], others demonstrated relatively low performance numbers. Notably, these papers describe development stage technologies showing initial device construction or preliminary algorithm designs for malaria detection. Previous studies showed problems in cartridge design and focus mechanisms, yielding slow scanning times and poor results. While these reports used complicated microfluidics systems, our study presents an easy to use plastic cartridge which fills quickly upon loading using capillary forces activated by mixing the blood with our stain solution. Moreover, we have solved image focus difficulties, by implementing unique algorithms which allow the scanning system to quickly autofocus on each new field allowing for high quality images of all cells scanned. In a previous study [22] we presented the first clinically available computer vision based reader for malaria diagnostics. The P1 device showed a sensitivity of 97.05%, specificity of 96.33% and speciation of 73.3% Plasmodium falciparum and 91.4% for Plasmodium vivax

• The P2 device features many functional and performance based improvements over the earlier system. While the P1 device holds only 5 patient samples, the P2 machine holds 30 samples and is capable of asynchronous batch loading. Moreover, the P1 device requires 8 minutes to scan a sample while the P2 device requires only 4 minutes, allowing for the rapid scanning of large volumes of specimens. On a performance level, the device showed a similar sensitivity at an average of 97.4% but a significantly improved specificity at an average of 98.5%. Speciation of Plasmodium vivax was comparable to the previous study at 87.5% while speciation of Plasmodium falciparum was significantly improved at 93.5%.

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An Enhanced Computer Vision Platform…

Importantly, in contrast to the study on P1 which was performed only in India, the current trial was conducted on samples from both continental Africa and India. Numerous studies have shown the ability of strains of malaria to develop mutations causing significant difficulties in diagnosis [1,30,31]. In particular, it has been shown that RDTs which identify HRP-2 from Plasmodium falciparum can yield false negatives due to specific antigen mutations

1. Specific regions are known to develop unique genetic variants of even the most standard species of Plasmodium. Our results confirm the devices ability to detect strains of malaria in variety of geographical regions. In the current study, the device maintains a limit of detection of 50 parasites/ μL. While the system is capable of identifying as few as 5 parasites/ μL the current algorithm only identifies a positive sample if it detects more than 50 objects identified as malaria parasites. This limitation was evident from the six cases of false negatives where the parasitemia was found to be under 50 parasites/ μL, explaining the misdiagnoses. Decreasing the current limit of detection causes an increase of false positives reported by the system. False positives have been found to be caused by particles that have flourescence morphology similar to the stained malaria within the RBC. The four false positives found in our study were determined to result from Howell Jolly bodies which are malaria-like DNA/RNA fragments found in RBCs. By collecting larger libraries of samples, as well as samples with Howell Jolly bodies, we will be able to apply machine learning to improve the accuracy of the algorithm classification and overall diagnosis. Additional data collection and algorithm design work will be necessary to further improve the differentiation between malaria and these objects to lower the limit of detection. Several device improvements are currently under development to strengthen diagnostic performance and provide additional clinical information to assist in patient treatment. Speciation for P.v was calculated at 87.5% and P.f at 93.5%, leaving room for increased accuracy. As the device speciation is based on a machine learning algorithm which improves with an increased database, the collection of additional scanned samples of both P.v and P.f should significantly improve speciation results. Moreover, to expand speciation capabilities to Plasmodium ovale , Plasmodium malarie and mixed infection a large library of P.o and P.m and mixed infection samples will need to be collected and analyzed. Additional studies will also be necessary which feature completely blinded data collection as well as PCR ground truth for all samples.

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7. Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF (2013) Multiplex qPCR for detection and absolute quantification of malaria. PLoS One 8: e71539.
8. Xie L, Li Q, Johnson J, Zhang J, Milhous W, et al.

Seth J. Salpeter

(2007) Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria. Parasitology 134:1151-1162.

1. Prescott WR, Jordan RG, Grobusch MP, Chinchilli VM, Kleinschmidt I, et al. (2012) Performance of a malaria microscopy image analysis slide reading device. Malar J 11: 155.
2. Frean JA (2009) Reliable enumeration of malaria parasites in thick blood films using digital image analysis 8: 218.
3. Purwar Y, Shah SL, Clarke G, Almugairi A, Muehlenbachs A (2011) Automated and unsupervised detection of malarial parasites in microscopic images. Malar J 10: 364.
4. May ZAS, Salamat R (2013) Automated quantification and classification of malaria parasites in thin blood smears. Signal and Image Processing Applications (ICSIPA), IEEE International Conference 8-10.
5. Atroosh WM, Al-Mekhlafi HM, Mahdy MA, Surin J (2012) The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia. Malar J 11: 251.
6. Pillai DR, Lau R, Khairnar K (2012) Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections. Malar J 11: 131.
7. Kumar N, Singh JP, Pande V (2012) Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity of malaria rapid diagnostic tests. Malar J 11:298.

 

Quantification bias in blood alcohol determination by headspace gas chromatography

Elia Mattarucchi, Carlo Peruzzo, Ramona Consuelo Maio, Ursula Andreotta, Marco Mario Ferrario

Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

Marco Mario Ferrario

Università dell’Insubria, Research Centre in Epidemiology and Preventive Medicine (EPIMED), Varese, Italy

All Correspondences to: Elia Mattarucchi Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

ABSTRACT

The quantification of blood alcohol concentration is routinely performed in many forensic laboratories by gas chromatography. In this short report, the influence of the matrix miss-match between samples and calibrators (e.g. the use of aqueous standards to quantify blood samples) and the actual efficiencies of different internal standards used to normalise the ethanol signal were considered. Test samples in blood and water were prepared from a certified reference standard of ethanol. The samples were analysed by gas chromatography using n-propanol and t-butanol as internal standards. The collected data provided evidences that a sub-optimal setting of the considered variables can be responsible for a quantification bias up to 15%. These issues are of practical relevance in the medico-legal sector and should be considered during the process of

method development.

KEY WORDS: blood alcohol concentration, calibrator, internal standard, gas chromatography, bias.

INTRODUCTION:

uman blood samples are the primary type of Hknown^{2, 3}. Method development should start from evidence received for medico-legal testing of tuning of the most important parameters; in this alcohol

concentration by gas chromatography. context we feel that further attention should be paid Whole blood is the sample of choice, since plasma typically entails an overestimation¹. For the purpose of forensic testing, different thresholds of alcohol concentration are set before a person is charged with a fine or crime. The consequent need to produce medico-legal reports underlines the importance of the careful development and validation of the analytical methods applied. The literature on this argument is vast and many possible influencing factors are to the choice of the internal standard and calibrators. The quantification of unknown samples is achieved by interpolation of a calibration curve. For this reason, reference materials at known concentrations of ethanol are widely available on the marketplace⁴. The majority of these materials are aqueous solutions produced according to ISO Guide 34 and satisfy the regulatory needs posed by ISO 17025. However, the opportunity to use aqueous calibrators to quantify blood samples should be carefully evaluated, even if an internal standard is used to correct for the differences among samples and calibrators^{5, 6}. The actual efficacy of the internal standard needs to be experimentally verified. A “good” internal standard is c h e m i c a l l y s i m i l a r t o e t h a n o l w i t h o u t a n y chromatographic overlap with the target substance or other volatiles that could be present in the samples (e.g., acetone, methanol or acetaldehyde). n-propanol is commonly considered the best suited substance for this purpose, even if t-butanol is also used^5,6. The samples can also be diluted

with water up to practically correct for the matrix mismatch (usually a 1:10 dilution is sufficient)⁷. However, this procedure inevitably increases the limit of quantification with evident constraints in dealing with the “zero tolerance” threshold posed for young drivers or commercial vehicle operators.

MATERIALS AND METHODS:

In this report we present some data retrieved from the development of the analytical procedure adopted at our laboratory. The method was arranged starting from the protocol suggested by the producer of the available equipment (i.e. a PerkinElmer Clarus 500 gas chromatographer equipped with a Turbomatrix40 headspace autosampler). The original parameters were: sample equilibration: 30 min 70 °C, vial pressurisation: 15 psi for 3 min, transfer line temperature: 110 °C, injection: 200 °C, oven: 40 °C isothermal, column: Elite-BAC1 (PerkinElmer Cod. N9316579), carrier gas: N₂.n-propanol and t-butanol were both tested as internal standard. Samples were mixed with ammonium sulphate in order to increase the partition coefficient of alcohol in the gas phase and amplify the analytical response⁸. A fixed volume of sample (1 ml) was added to 0.9 ml of 1 M ammonium sulphate and to 0.1 ml of 8 g/L t-butanol or n-propanol (internal standard).

Two type of test samples were prepared: an intermediate series of sample at 0.30, 0.75, 1.50, 3.75, 7.50 g/L of ethanol in water were produced independently from a mother solution 15 g/L. All the samples (including the mother solution) were further diluted 1:5 in water and blood (previously checked for the absence of ethanol). At the end of the process, 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L in blood and water were obtained.

32 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Quantification bias in blood alcohol…

This dilution scheme ensured that each concentration was independent, and the matrix composition uniform within each series. The second type of samples consists of 4 solutions at the nominal concentrations of 0.5 and 2 g/L in water and blood. These samples were prepared by adding 100 and 400 µl of a 0.5 g/L ethanol standard to a final volume of 1 ml of water or blood.

RESULTS:

The first type of test samples described in the “materials and method” session (i.e. 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L of ethanol in blood and water) were prepared and analysed independently 10 times. For each repetition, blood and water samples were analysed within the same analytical session. For each concentration, it was expected that the average analytical response (i.e. the ethanol/internal standard area ratio) of the blood and water samples would be similar due to normalisation with the internal standard. However, a progressive response difference was observed (Table 1). The disparity becomes clearly significant among samples normalised by t-butanol, while it is not supported by a statistical evidence when n-propanol is used. In order to quantify the possible bias, test samples at the nominal concentrations of 0.5 and 2 g/L in water and blood were used (i.e. the second type of test samples described in the “Materials and methods” section). The 2 couples of samples were produced and analysed independently 10 times using a quantitative method calibrated by commercial certified reference materials based on water solutions. As reported in Table 2, the average error ranges from 2% at 0.5 g/L with n-propanol up to 16% at 2 g/L with t-butanol. The error is indeed negligible, if the analytical samples are matrix matched with the calibrators (i.e. water samples quantified against water calibrators).

DISCUSSION:

The presented data show how a matrix difference between samples and calibrators and/or the actual efficacy of the internal standard can bias the outcome of the alcohol test by gas-chromatography. These factors should be carefully considered during method development. Our data point out the superiority of npropanol for BAC determination. However, a few additional aspects should be considered, especially if t-butanol is selected as internal standard. As the error seems to increase with the amount of ethanol, the use of a fixed correction factor becomes difficult. On the other hand, the practice of making up “in-house” standards by spiking known concentrations of ethanol into “blank” blood samples can lead to regulatory problems associated with the medico-legal sector. The whole process should be carefully controlled and documented, even though it would be unrealistic for a medical laboratory to have enough resources to produce certified reference materials. A possible solution could be the use of commercial blood standards that are industrially manufactured by spiking ethanol into a blood matrix. However, the availability of such materials is still limited and the assigned concentration is usually an average value derived from a number of independent certified laboratories (usually these standards don’t comply with the ISO guide 34 requirements).

CONCLUSIONS:

In conclusion, this report offers some helpful thoughts on the calibration of the analytical methods used to assess blood alcohol concentration for medico-legal purposes. These considerations may seem trivial, but a recent comment by Schug⁹ shows that these analytical issues are still highly relevant. In our practice, the guidelines of the Italian Forensic Toxicologist Group don’t report any specific indication about the internal standard and the calibrators used for alcohol tests¹⁰ and the only way to control the risk of possible bias is to asses experimentally the real efficacy of such measures adopted to minimize the matrix mismatch and to participate periodically to suitable proficiency tests on blood samples.

Table 1. Analytical response of water and blood samples

t-BUTANOL

Blood samples Water samples

0.06	0.0096	0.0009	0.0130	0.0016	-0.0034	3.E-02
0.15	0.0301	0.0031	0.0323	0.0036	-0.0021	5.E-01
0.30	0.0746	0.0020	0.0712	0.0038	0.0034	2.E-01
0.75	0.2281	0.0022	0.1994	0.0034	0.0286	3.E-04
1.50	0.4736	0.0121	0.4073	0.0065	0.0663	1.E-03
3.00	0.9754	0.0407	0.8224	0.0148	0.1530	4.E-03
		t-PROPANOL
	Blood samples		Water samples

0.06	0.0197	0.0012	0.0288	0.0006	-0.0091	1.E-02
0.15	0.0613	0.0034	0.0718	0.0017	-0.0105	6.E-02
0.30	0.1557	0.0136	0.1669	0.0159	-0.0112	5.E-01
0.75	0.4880	0.0287	0.4788	0.0245	0.0093	8.E-01
1.50	0.9824	0.0599	0.9699	0.0424	0.0124	8.E-01
3.00	2.0218	0.0969	1.9658	0.0551	0.0560	6.E-01

Average analytical response (ethanol/internal standard area ratio) of blood and water samples at different concentrations (0.06-3.00 g/L of ethanol) analysed using n-propanol or t-butanol as internal standards (n=10).

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 33

 

Table 2. Ethanol quantification in water and blood samples

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal	Av. Quant.	Av. Quant.	Error on blood
Conc. g/L	Water	Blood samples	samples %
	samples
0.50	0.50	0.56	12%
2.00	2.01	2.32	16%

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal	Av. Quant.	Av. Quant.	Error on blood
Conc. g/L	Water	Blood samples	samples %
	samples
0.50	0.50	0.51	2%
2.00	2.01	2.11	5%

Average alcohol quantification of 0.5 and 2 g/L ethanol spikes in blood and water analysed using n-propanol or tbutanol as internal standard and Cerilliant® reference materials (water solutions) as calibrators (n = 10).

REFERENCES:

1. Rainey PM. Relation between serum and wholeblood ethanol concentrations. Clin Chem 1993; 39:2288–92.
2. Sklerov JH, Couper FJ. Calculation and verification of blood ethanol measurement uncertainty for headspace gas chromatography. J Anal Toxicol 2011;35:402–10.
3. Scientific Working Group for Forensic Toxicology. SWGTOX standard practices for method validation in forensic toxicology. J Anal Toxicol 2013;37:452–74.
4. Archer M, Vos J, S Visser M. The preparation, assay

Elia Mattarucchi

and certification of aqueous ethanol reference solutions. Accreditation Qual Assur 2007; 12:188–93.

1. Strassnig S, Lankmayr EP. Elimination of matrix effects for static headspace analysis of ethanol. J Chromatogr A 1999;849:629–36.
2. A. Boswell H, Dorman F. Uncertainty of Blood Alcohol Concentration (BAC) Results as Related to Instrumental Conditions: Optimization and Robustness of BAC Analysis Headspace Parameters. Chromatography 2015;2:691–708.
3. Karch S, editor. Forensic Issues in Alcohol Testing. Boca Raton: CRC Press; 2007.30–3.
4. Christmore DS, Kelly RC, Doshier LA. Improved recovery and stability of ethanol in automated headspace analysis. J Forensic Sci 1984;29:1038–44.
5. Schug K. An Indisputable Case of Matrix Effects in Blood Alcohol Determinations. Available at: http://www.chromatographyonline.com/indisputab le-case-matrix-effects-blood-alcoholdeterminations. Accessed: 16 Oct 2018
6. (2017) Guidelines for institutions equipped with laboratories for the detection of substances of abuse for toxicological-forensic and medico-legal purposes on biological samples taken from living subjects – Rev. N° 5. GTFI guidelines. Available at: http://www.gtfi.it/linee-guida/

 

AL Marshad Ibrahim, Hassanein Nagwa, Balakrishnan Bargavi

King Fahad Specialist hospital, Qassim, KSA.

Hassanein Nagwa

Clinical pathology department, Faculty of medicine for girls, Al-Azhar University, Cairo, Egypt.

All Correspondences to: Hassanein Nagwa, King Fahad Specialist hospital, Qassim, KSA.

ABSTRACT

Although the phenotypic features of basophils have been highlighted in literatures, yet some labs generate confusion while discriminating blasts from basophil gate in flow cytometry analysis. This is sometimes due to the overlap of different population on CD45 side scatter plot, and in other situations due to lack of markers which highlight basophils due to financial

jeopardy. Aim of the study: Selection of a proper combination of markers with unique expression on basophils to clearly highlight basophils especially in underresourced labs with limited budget. Methods: Comprehensive basophil phenotyping applied on 40newly diagnosed cases of CML and 20 cases of regenerating marrow post chemotherapy for Precursor B lymphoblastic leukemia with no evidence of abnormal cellsas control. Flow cytometry markers used were CD45, CD38, CD71, CD33, CD22, CD123, HLA-DR, CD34, CD117, CD3, CD4, CD8, CD56, CD11b, HLADR and CD15. Results: Basophils from patients and control samples were clearly discriminated from blasts and lymphocytes by using CD123 and CD22, a unique pattern of expression with perfect back-gating on CD45 side scatter plot which proved the discrimination. Conclusion: CD123 and CD22 will clearly discriminate basophils from different types of population which may occupy the window area as myeloblast, precursor B cells (hematogones), abnormal B cells (lymphoblasts) and mature B cells. This combination should therefore be part of the screening panel used as first line along with CD45in labs with limited budget.

KEY WORDS: Basophil, CML, CD123, CD22.

INTRODUCTION:

low Cytometry has clearly and quickly emerged to Fbe the most helpful technique in hematology lab with predominant role in the diagnose of hematological and non hematological malignancy. Also it can be used for accurate enumeration of bone marrow cells in an objective analytical method¹. Basophil is a unique type of granular myeloid offspring; its granules have high solubility in water, which results in its location in de-granulated cells with low side scatter properties, this criterion make it stand close to lymphocytes and occupy part of blasts window in CD45 side scattered plot. Basophilia is manifested in different condition of benign and malignant diseases. Myeloproliferative malignancies, typically chronic myeloid leukemia (CML) characterize by basophilia, as well as polycythemia rubravera, myelofibrosis and rarely essential thrombocythemia. Basophilia is also seen in benign condition such as hypothyroidism, hypersensitivity reaction, autoimmune disease as

rheumatoid arthritis and ulcerative colitis.²

Many markers have been used to highlight basophil in both normal and diseased condition as CD9, CD13,CD22,CD25,CD33,CD36,CD38,CD45 and CD123,294 and 69 ³

CD22 is one of the Pan B cell markers, however it is not restricted to that lineage as it has been reported as one of basophil marker with a different pattern of expression. In spite of being bright on B cells,it tends to be dimmer on Basophiles,this difference in pattern related to the structure difference of the isoform ligand. ⁴

CD123 is an Interleukin-3 receptor alpha ,it is highly expressed on plasmacytoid dendritic cells and basophils and to lesser degree on monocytes, eosinophil, myeloid and dendritic. It is also expressed on myeloblasts, mature subpopulation of hematogones and mature B cell in certain malignancy such as Hairy cell leukemia.^5,6.

CD25 is an interleukin 2 receptor alpha, its dim expression on basophils had been reported. Its expression on malignancy myeloidprecursor cellsis associated with bad prognostic molecular markers such as FLT3 and DNMT genes.⁷

CD117 is a c- kit marker heavily expressed on Mast cells and also detected on the surface normal myeloblast and promyelocytes, however its expression on normal basophil has not been detected.⁸

Although many markers have been proven to be

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 35

Valuable and Cost Effective Combination…

expressed on basophil, yet a cost effective specific combination has not been discussed for accurate robust identification. This combination could be used in every panel used for identification of WBC, leukemia cells, MRD cells. This combination will discriminate Basophil from myeloblast and also B cell population.

SUBJECT AND METHODS:

40 newly diagnosed cases labeled as CML,18 female and 22 males age range from 22 to 76 years old, 38 cases were in chronic phase of CML and 2 cases were labeled as accelerated phase based on the WHO criteria published 2008. The CML cases were collected over a 4 years period, from January 2014 to December 2018. 20 regenerating phenotypically normal marrows with no residual abnormal cells were assessed for Basophil population as control. 12 pediatric patients (less than 16 years) and 8 adult patient (more than 16 years old). The study was

approved by the research ethical committee of Al Qassimgovernorate.

Flow cytometry analysis:

B o n e m a r r o w a s p i r a t e ( B M A ) s a m p l e s anticoagulated with sodium heparin were used, clotted samples were rejected. Samples were processed within twenty –four hours of extraction. Samples were prepared using gentle processing techniques. Briefly, Antibodies for CML panel were prepared using a four color panel ,which are listed as FITC/PE/PERCP/APC for each tube as follows : 38/33/45/71, 20/10/45/19, 3 / 1 6 + 5 6 / 4 5 / 1 9 , 45/22/123/HLA DR, 15/117/45/34, 25/4/45/11b. The MoAB were used in different combination of flurochrome as fluorescein isothiocyanate (FITC), phycoerythrin (PE), Peridinin Chlorophyll protein complex (per cp), APC (Allophycocyanin). All the antibodies used to analyse basophils were obtained from BD biosciences. Different combination of MoAb used are, CD38(HB7), CD3 (SK7), CD20 (L27), CD15 (MMA), CD25 (2A3), CD45(2D1) were conjugated with FITC, CD33(P67.6), CD10 (MEM-78), CD16 (B73.1), CD56 (NCAM 16.2), CD22 (S-HCL-1), CD117 (104 D2), CD4 (SK3) were conjugated with PE, CD71(L01.1), CD19 (SJ25C1), CD34 (8G12), CD11B (D12), HLA DR (L243) were conjugated with APC , CD123 (7G3) conjugated with PER CP CY5.5 and CD45 (2D1) PER CP was used as a gating marker in all the tubes. The amounts of antibodies added were according to the laboratory titration.

1X10⁶ White Blood cells were added to each tube and incubated at room temperature (RT) in a dark area for 15-20mins. Red cells were lysed using 1x RBC lysing reagent (Becton Dickinson USA)followed by washing cells with cell wash buffer PH 7.0-7.4 (BD) with 1%BSA(bovine serum albumin) as a blocking agent to minimize non-specific binding. In certain

cases when there is non specific binding, cells were incubated for 10 minutes with 50ul of human IgG (immunoglobulin G) for blocking Fc-receptors on the cells. Finally, cells were re-suspended in a cell wash buffer with 1%bovine serum albumin(BSA) for acquisition in flow cytometer.

Acquisition was done after machine calibration by CST beads and optimization of the machine setting. Acquisition of cells was performed on BD FACS Canto2, and analysis was performed using FACS Diva V6 software (Becton Dickinson USA). The cells were gated based onCD45, side scatter allocation of abnormal cells and the expression of markers.

RESULTS:

We examined forty newly diagnosed CML cases 18 female and twenty two males age range from (22-76 years old) 38 diagnosed at the chronic phase and two cases diagnosed in the accelerated phase, also 20 control samples from regenerating normal marrow, originally diagnosed as precursor B lymphoblastic leukemia 12 pediatric patient (less than 16 y) and 8 adult patient (more than 16 years old). We found that certain markers as CD33, Cd38. CD22, C D 1 2 3 , CD11b are invariably expressed for every basophil benign or Malignant (Figure 1, 2). HLA-DR was variably expressed on basophil from CML and control cases in the proportion 60% and 50% respectively. CD117 was not detected on normal basophils as well as CD34 and CD25 on normal basophils while CML in accelerated phase show positivity for CD117 (Figure 3). CD11c were found on normal basophil and abnormal basophils (dim expression).

Seven cases of the control Marrow detected hematogones at different proportion (1-9%) with the characteristic pattern of hematogones. Spectrum of maturation located from the negative CD45 to the brightest location of normal mature B lymphocytes with their side scatter were lower than basophils. CD123 expression was dimmer on hematogones in the seven cases (100% ) than on the basophil, while CD22 versus HLA-Dr was overlapping (Figure 4).

Table.1 show the pattern of expression of different markers on basophils in cases and control.

Marker	CML number (40)	Control samples
		(20) number
CD33	Mod	Mod
CD38	Bright	Bright
CD123	Bright	Bright
CD22	Dim	Dim
CD11b	Dim	Dim
HLA-DR	Dim 24(60%)	Dim 10(50%)
CD117	Positive in
	accelerated phase
	(2/2) 100%	Negative
CD25	Negative	Negative

36 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Figure. 3 show one CML (accelerated phase) case with positive expression of CD117 on basophil while the blast (blue population) positive for both CD34 and CD117 mean while B cell(red population negative for both.

Hassanein Nagwa

Figure. 4 A control case (regenerating marrow ) show the phenotypic difference between hematogones and Basophil regard CD123, CD22, HLA-DR. Basophils are Magenta in color while hematogones are red.

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 37

Valuable and Cost Effective Combination…

Figure. 5 One CML case show an example of basophil (magenta )negative for HLA-DR while positive for CD123 (brightly) and for CD22 dimly. The B cell appears in red and carry HLA-DR and CD22 brightly, while dim for CD123.

Figure. 6 show basophil (magenta color population

negative for CD25 while positive for CD11b.

DISCUSSION:

Although automatic gating has now been suggested now to be the routine practice, still the manual gate is still the main method for analysis especially in the low budget countries. CD45, side scatter is the standard back gating scatter gram for hematological malignancy’s immunophenotyping. The area of interest is where blast could locate, it is the window area or the whole area, and normally it is occupied by normal myeloblasts and basophils mainly. In the pathological condition, it could be occupied by myeloblasts, lymphoblasts or lymphoma cells as Burkett’s lymphoma.

Accurate determination of occupying population is mandatory for accurate conclusion. Also in MRD, overlap between two populations could lead to confusion and mislabeling of populations detected^9,10. Furthermore, the identification of basophilic

38 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

precursors in cases of acute basophilic leukemia and blast crises of CML with basophilic differentiation rely mainly on the knowledge of the robust markers usually used for discriminating this basophils ¹¹.

Basophil is a minor population of mature myeloid cells, sharing myeloid markers as CD33, CD13, CD11b, as well as other markers as CD38, CD123

and CD22. This spectrum of markers expressed, could lead to the improper separation if no unique expression pattern of robust markers is specified. We examined different markers expressed on basophil in different combination and conclude that the combination of CD22 and CD123 will discriminate basophil and separate it accurately from different population located in the blast gate. The extent of CD123 on basophil is unique since it is the brightest marker detected on this cell. This strong expression of CD123 correlates it with the major role in the proliferation and differentiation of basophilis^{12, 13}.

CD22 dimmer expression will isolate it from mature B cells. Although, the argument about CD22 expression on precursor B lymphocytes or lymphoblast may express dim CD22, yet bright CD123 will clearly determine if it is B cell or basophil. In addition, hematogones has very low side scatter comparative to basophils and myeloblasts. Furthermore, lack of CD19 on the basophil which is a fundamental marker used in every lab.since the expression pattern of CD123 at some maturation stages of hematogones and in leukemic condition is far less than that of Basophil. Furthermore, relying on back-gating on CD45, side scatter will clearly discriminate mature B cells with b r i g h t CD45 expression.

Hematogones present with heterogonous spectrum of maturation from negative to moderate CD45 and very low side scatter as well⁵.

Basophil expresses bright CD38 as well as B cell precursor (hematogones) is positive for CD 38 at the same level. However both are less bright than plasma cells which typically locate at the same area on CD45,side scatter and express unique pattern for CD38 ¹². Gating basophil using CD38 versus side scatter was not helpful but the combination of CD38 versus CD33 was able to separate basophils in control group as well as in CML cases¹³.

The expression of CD11b was invariably detected on basophils in both normal and malignant condition which stand to discriminate it from myeloblast which is typically negative forCD11b¹⁴.

Other markers as HLA-DR were recorded in 60% of the cases and around 50% of the gated basophil in the control bone marrow samples. The pattern of expression was dimmer than B cells, however comparable to myeloblasts. Very few cases does not express it, this finding is alarming since scientist use the CD123 positive /HLA-DR negative to gate basophil may lead to losing part of basophil. It is

Hassanein Nagwa

possible that DR expression is caused by cytokine activation or related to the disease itself because basophil is one of the cytokine sensitive cells^{13, 15, 16}.

CONCLUSION:

To conclude, the accurate quantification of different population located in the blast gate is a requirement for proper reporting at diagnosis and follow up of different hematological malignancies. The combination of CD22 and CD123 along with CD45 were definitely and precisely able to highlight and discriminate basophils from other type of cells occupy the blast gate. Identification of a single specific marker with stable expression on basophil is recommended to be investigated in the future to minimize the panel used.

REFERENCES:

1. Jacob MC, Souvignet A, Pont J, Solly 7. F, MondetJ, KesrS, PernolletM, Dumestre PerardC, CamposL, CesbronJY. One tube with eight antibdies for 14-part bone marrow leukocyte differential using flowc ytometry. Cytometry B Clin Cytom 2017 Jul; 92(4): 299-309. doi: 10.1002/cyto.b.21369. Epub 2016 May 6.
2. SProven D,Singel C,Baglin T, Dokal I,2015. White blood cells abnormalities.in Oxford Handbook of clinical hematology. 8. Fourth edition, chapter 13, Oxyford University Press, Great clarend on street, oxford,OX26DP,UK,page 116.
3. Han X, Jorgensen JL, Brahmandam A, Schlette E,

H u h Y O , S h i Y, Aw a g u S , C h e n W. Immunophenotypic study of basophils by multiparameter flow cytometry. Arch Pathol Lab Med. 2008 9. May; 132(5):813-9. doi: 10.1043/1543-10.1186/s13601-016-0100-4. E Collection 2016.

1. Gönen, M., Sun, Z., Figueroa, M. E., Patel, J. P., Abdel-Wahab, O., Racevskis, J., Ketterling, R. P., Fernandez, H., Rowe, J. M., Tallman, M. S., Melnick, A., Levine, R. L. Paietta, E. CD25 expression status improves prognostic risk classification in AML independent of established biomarkers: ECOG phase 3 trial, E1900. Blood,(2012). 120(11), 2297306.
2. Agis, H., Füreder, W., Bankl, H. C., Kundi, M., Sperr, W. R., Willheim, M., Boltz-Nitulescu, G., Butterfield,

J. H., Kishi, K., Lechner, K., …Valent, P. (Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes. Immunology (1996)., 87(4), 535-43.

1. Saksena A, Gautam P, and Singh TSide scatter versus CD45 flow cytometric plot 2165(2008)132 (813:ISOBBM) 2.0.CO;2. can distinguish acute leukaemia subtypes.

 

, Oto-Obong V. Idah and Bitrus N. Lekshak

Department of Haematology and Blood Transfusion, University of Jos, Nigeria

Ahmed M. Sabo and Oto-Obong V. Idah

Department of Human Physiology, University of Jos, Nigeria.

Thomas P. Yakubu

Department of Pharmacognosy University of Jos, Nigeria

Moses D. Lugos

Department of Medical Laboratory Science, Faculty of Health Sciences & Technology, University of Jos, Nigeria.

All Correspondences to: Changjul L. Singnap

, Department of Haematology and Blood Transfusion, University of Jos, Nigeria

ABSTRACT

Background: It is estimated that over 80% of people still depend mainly on the traditional use of parts of plants and herbs to treat ailments. The medicinal properties of these plants and herbs are linked to the presence of a variety of phytochemicals and their elemental composition. Justification: Every year, 1 in 4 people die of conditions related to thrombosis, with many never knowing their risk for the condition. Prevention of intravascular thrombosis, however, has a narrow therapeutic window, bleeding risk, the incidence of resistance, and unwanted drug interactions, hence the need for anti-thrombotic drugs that deliver more effective prevention of intravascular thrombosis. Aim: The research sought to investigate the effect of Moringaoleifera ethanolic seed extract on Bleeding Time (BT) and Clotting Time (CT), in Wistar albino rats. Materials and Methods: Forty (40)Wistar rats were weighed and randomly divided into two groups, group I=15 rats for BT, group II=15 rats for CT and 10 rats for control tests. After the administration of ethanolic extract of M. oleifera seed at the dose of 100mg/kg, 200mg/kg and 400mg/kg (administered to a group of 5 rats per dose) for 28 days, BT and CT were determined using Tail transection and Lee & White method respectively. The data were analysed using GraphPadPrism (7.03). Result: There was a statistically significant increase at P <0.0001 of BT and CT compared to the control (administered with only distilled water). Conclusion: The prolonged BT and CT indicate that the seed extract of M. oleifera could pose an antagonising effect on both the primary (platelets) and secondary haemostatic activities, a property that can be explored in the management of

thrombotic diseases.

Keywords: Moringaoleifera, Bleeding Time, Clotting Time, Thrombosis.

INTRODUCTION

or thousands of years, plants have been a vital Fsource of medicine. Even today, the World Health Organization (WHO) has reported that up to 80% of people still depend essentially on traditional medications such as the use of herbs to treat ailments.^1,2 Diverse plants phytochemicals and their elemental composition have been

shown to possess medicinal values.^3,4

A growing concern in the development and evaluation of natural antioxidants from medicinal plants mainly in the field of preventive health care and the food industry is reported. Among those herbs, one promising species is Moringa oleifera Lam (Moringa or drumstick tree), which is native to the sub-Himalayan regions of Northwest India.⁴Moringa oleifera (Moringaceae) is a well-known plant in North Eastern,

Nigeria.⁵Lam seed; is a highly valued plant, with an impressive range of medicinal uses and high nutritional value.⁶A good number of traditional medicine references attest to its therapeutic potential, and scientific validation of theseplantsis developing to support at least some of the claims.⁷ Historically, ancient Romans, Greeks and Egyptians, as well as Asian communities, have been using the plant for various medicinal and nutritional purposes.⁸ The plant is used locally for various medicinal purposes by traditionalists and herbalists in Maiduguri, North Eastern Nigeria.⁵ M. oleifera roots and seeds are prescribed for the treatment of snake bites, and scorpion stings and the plant contains a mixture of several hydrolytic enzymes, in which proteases are the key enzymes responsible for the observed pharmacological actions.^9,10 Ajibade et al., 2011 also reported that the seed could reduce the number of platelets, hence the need to look into its effect on haemostatic

 

Investigating the effect of ethanolic extract…

activities in detail.^11,12 Platelets, also called “thrombocytes”, are a component of blood whose function (along with the coagulation factors) is to stop bleeding by clumping and clogging blood vessel injuries. Platelets release a variety of substances that promote blood 1clotting, which causes haemostasis. The normal blood platelets count is between 150 x 10⁹/L and 450 x 10⁹/L (150,000 – 450,000/ mm³). Platelets have no cell nucleus: they are produced by budding-off of the cytoplasm of megakaryocytes.¹³ Formation of this platelets plug (primary haemostasis) is associated with activation of the coagulation cascade; which results in fibrin formation and crosslinking (secondary haemostasis).They also play an important role in the conversion of prothrombin to thrombin because much of the prothrombin first attaches to prothrombin receptors on the platelets already bound to the damaged tissue.14Intravascular thromboses such as deep vein thrombosis (DVT), a major cause of myocardial infarction, stroke and pulmonary embolism, can be caused by abnormal platelet activation, augmented clotting, vascular dysfunction and shear stress due to interrupted blood flow and atherosclerosis.^15,16 This research was therefore designed to investigate the effect ofMoringa oleifera seed extract on bleeding time (BT) and clotting time (CT).

MATERIALS AND METHODS:

Seed Collection and Authentication

Dried seeds of Moringa oleifera were obtained from Lamingo Jos-North local government Area of Plateau State Nigeria.

Preparation of Seed Extract

Good quality seeds were selected for this study. The coat and wings of the seeds were removed and the kernel collected. The kernel was dried for 5 days at room temperature (25 – 38°C) in the Department of Pharmacognosy. Then we produced a fine powder of the seeds by using coffee mill attachment of Moulinex domestic food blender (Moulinex Genuine Blender 1.25 Liter, 400 Watt and White – LM2421EG made in France). The seeds were weighed before and after blending. The quantity after blending was soaked in 4 litres of 70% ethanol at room temperature for 3 days and filtered; the bulked filtrate was evaporated using hot air oven at 41°C for 24 hours. Until needed, the powder produced was stored at room temperature. A fresh solution of the seed extract was prepared when needed. The test solution was prepared by dissolving the fine powder of Moringa oleifera seed in distilled water in the ratio of 10g to 100ml (to give 100mg/ml concentration) and stirred for two minutes before intubation.¹⁷

Experimental Animals

A total of forty (40) male and female Wistar rats weighing between 150g to 250g were used for the research. The rats were kept in a plastic cage at room temperature with twelve hours a night/dark cycle. They had access to their feed and a hygienic environment maintained to prevent infection. The rats were weighed using an electric beam balance at the commencement of the experiments and weekly throughout the duration of the experiment.

Experimental design

A total of 40 Wistar rats were used for this study to determine the BT and CT; 15 rats wereused per test by grouping them into three (5 rats/each dose) 100 mg/kg (low

dose), 200 mg/kg (medium dose) and 400 mg/kg (high dose) of the seed extract in that order. We used 5 rats to control for BT and 5 rats were used to control for the CT. The oro-gastric method of intubation was adapted for the administration of seed extract for experimental rats while distilled water was administered to control groups, these were done in the morning hours before feeding the animals. The rats were intubated daily throughout the period of the experiment (for 28 days).

Sample Collection

Animals were euthanized 24 hours after the last doses administered to them. The rats were weighed and then anaesthetized by placing them in a closed jar containing cotton wool sucked with chloroform and euthanized by cervical dislocation and collected from jugular vein into aodium-citrated sample bottle (at the ratio of 4.5 to 0.5) and the content properly mixed by gentle rolling of the bottle for haematological analysis. The blood parameters analyzed included Bleeding Time (Duke’s method) and Clotting Time (Lee and White method).

Laboratory Methods

Bleeding Time (Tail transection; a modified Duke’s

method)

After 24 hours of the final oral administration of Moringa oleifera seed extract, the bleeding time was measured in rats by modified Duke’s method as described briefly; the tail of the anaesthetized rat was cut 1 cm from the end using a sharp pair of surgical scissors and then the tail lesion blotted with a clean Whatman filter paper every 15 seconds. The interval, from the time the tail incision was made until the time blood was no longer apparently transferred to the filter paper, was recorded as the bleeding time in seconds.¹⁵

Whole blood clotting time (Lee White method)

Three test tubes were placed in a 37°C water bath and 3ml of blood sample was collected from the rats by the jugular method. As soon as blood appeared in the syringe, the stop watch was started. To each of the pre-warmed glass test tubes, 1ml of the blood was added. After the initial 3 minutes of incubation, the three tubes were simultaneously tilted to observe for clot formation at 30 seconds intervals. This procedure was repeated until the blood was clotted. The time taken for the first blood clot to appear was recorded in seconds. Subsequently, the process of tilting was repeated for the remaining 2 tubes until clotting was observed. The average value of clotting time of the three tubes was reported as clotting time of the rat in seconds.

Statistical Analysis

Data collected were expressed as mean ± S.D and analysed using the SPSS software program (Graph PadPrism, 7.03). One Way analysis of variance (ANOVA) was used to determine the level of significance of confidence at 95%, (pvalue< 0.05 was considered significant).¹⁸

RESULTS:

A total of 40 rats were used for the experiment to determine the BT and CT; 5 rats/each dose of: 100 mg/kg (low dose), 200 mg/kg (medium dose) and 400 mg/kg (high dose) of the seed extract in that order. We used 5 rats to control for BT, and5 rats were used to control for the CT. The result showed that the mean value of BT and CT in control groups fall within the normal range of 60-420s and 240-540s respectively. At 100mg/ml of the extract, the mean value of

 

 

BT and CT were 1059.60s and 253.30 seconds respectively, at 200mg/ml BT=

1773.00s, while CT= 346.40s, and at 400mg/ml,

BT= 1917.20s, while CT= 392.00s (Table 1 The effect of ethanolic extract of M. oleifera seed as presented in figures 1 & 2 was seen to be accompanied by significant increase in both BT and CT with increasing concentration of the dosage of extract in the order of 100mg/ml, 200mg/ml, and 400mg/ml (P<0.0001).

Table 1: Mean values of Bleeding Time (BT) & Clotting Time (CT) of experimental ratsfed on daily doses of 100mg/ml, 200mg/ml, and 400mg/ml of ethanolic extract of m. oleiferaseed and a control group on distilled water.

Dose of m.	No. of	Mean BT	Mean CT
oleifera extract	Rats	(Secs)	(Secs)
Control	5	173.20 ± 25.19	240.60	± 12.60
100mg/ml	5	1059.60 ± 52.05	253.30	± 9.32
200mg/ml	5	1773.00 ± 64.43	346.40	± 9.43
400mg/ml	5	1917.20 ± 47.32	392.00	± 9.62

Figure 2: Bar charts showing the dose effect of ethanolic extract of M. oleifera seed onwhole blood Clotting Time of experimental and control rats.

DISCUSSION:

Footnote: The mean and standard deviation of bleeding time and whole blood clotting time ofexperimental and control rats are reported in seconds. While the total is the average values forthe total means of BT and CT.

Figure 1: Bar charts showing the dose effect of ethanolic extract of M. oleifera seed on Bleeding Time of experimental and control rats.

In this work, we investigated the effect of Moringa oleifera on haemostatic profile particularly BT and whole blood CT in vivo using Wistar albino rats.

M. Oleiferawas described as a plant with many medicinal values. The different parts of the plant such as the leaves, seeds, fruits, flowers and back act as cardiac and circulatory stimulants and antihypertensive among others.7 Also, the coagulant property of the seed has been shown to be useful in water purification and treatment processes.¹⁹

We, therefore, sought to investigate the effect of the Moringa oleifera seed extract on blood coagulation in rats. The study showed that Moringa oleifera seed extract might be responsible for the prolonged bleeding and whole blood clotting time in Wistar albino rats as reported in the results section. The average BT of the control (173.20 ± 25 seconds) falls within the normal range of between 120 – 420 seconds (2-7 minutes).

However, at 100mg/kg concentration of the seed extract, a significant increase in the average BT of 1060.0 ± 52.05 seconds (17minutes ± 52.05s) was observed (p ≤0.0001). The result further showed a dosedependent increase with the highest at 1917.20 ± 47.32 seconds (about 33 minutes) at the dose of 400mg/kg. Also, the CT of the control group at 240.60 ± 12.06 seconds (4.10 minute) falls within the normal range of 240 – 540 seconds (4-9 minutes). However, there was no significant difference in the CT at 200mg/ml concentration (346.40 ± 9.43 seconds) of M. oleifera seed extract compared to mean value at 100mg/ml (253.80 ± 9.32 seconds). A statistically significant increase in the mean CT value of 392.00 ± 9.62 seconds was observed compared to CT mean value of 253.80 ± 9.32 seconds at 100mg/ml (P ≤ 0.005).Data revealed that a higher dose of M. oleifera extract seed may prolong both

 

Investigating the effect of ethanolic extract…

bleeding and whole blood clotting time.

The delay in BT and CT may be due to the seed’s ability to cause platelets aggregation hence reducing the number of platelets receptor sites needed for adhesion (the initial stage of bleeding arrest). Our work supported the findings of Luz et al., which reported that a coagulant isolated from M. oleifera seed called Lectin significantly prolongs activated partial thromboplastin time (aPTT) and prothrombin time (PT).²⁰ The research of Luz et al., 2017 was done in vitro using human blood, but we investigated whole blood CT in vivo using rats.

Analysis of variance was performed to compare the mean of extract doses and concentrations which reveals significantly (p≤ 0.05) that all the doses (100mg/ml, 200mg/ml, and 400mg/ml) resulted in increased coagulation (haemostatic) time in rats. The higher the dose the longer it takes for clotting to take place. This means that hypercoagulability (one of the causes of thrombotic events) can be delayed with the right doses of Moringa oleifera seed extract. This findings could be the reason why M. oleifera seeds are used as anti-scorpion, antisnake bites.10

CONCLUSION:

The results of this study showed that M. oleifera seed could delay haemostatic activity for as long as the administered dose lasts, usually within twenty-four hours (24hrs.). Since M. oleifera works similarly to heparin and aspirin; it may be a suitable replacement or another alternative of blood thinners. The seed extract of M. oleifera has the advantage of being a nutritional supplement meaning it has far less side effect if any.

RECOMMENDATIONS:

1. It is necessary to look into this wonderful gift (M. oleifera) to mankind with the aim of developing the seeds as a nutritional supplement or even as a drug which could be recommended in the management of thrombotic events such as deep vein thrombosis (DVT) or atherosclerosis in order to minimize ischaemic heart attack or even stroke since normal haemostasis cannot be possible without the activation of platelets.
2. Further research on the effect of M. oleifera seed tract on other haemostatic profiles such as PTT, PT, clotting factors & co-factors, vWf, and platelets satelitism may be able to reveal more on the efficacy of this plant seed in arresting thrombotic activities.
3. When used as nutritive supplement at the right dose, it can help in the prevention of unnecessary activation of platelets leading to thrombosis; this is because the extract of M. oleifera acts efficiently at both the primary and secondary stages of haemostasis.

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plants used by the people in Nhema communal area, Zimbabwe. Journal of ethnopharmacology, 2011. 136(2): p. 347-354.

1. Ferreira, P.M.P., et al., Safety and Efficacy of Moringa oleifera Lamarck (1785)— Therapeutic and Toxicological Properties, in Pharmacology and Therapeutics. 2014, InTech.
2. Valdez-Solana, M.A., et al., Nutritional content and elemental and phytochemical analyses of Moringa oleifera grown in Mexico. Journal of Chemistry, 2015. 2015.
3. Zaku, S., et al., Moringa oleifera: An underutilized

tree in Nigeria with amazing versatility: A review.

African Journal of Food Science, 2015. 9(9): p. 456-461.

1. Gopalakrishnan, L., K. Doriya, and D.S. Kumar, Moringa oleifera: A review on nutritive importance and its medicinal application. Food Science and Human Wellness, 2016. 5(2): p. 49-56.
2. Anwar, F., et al., Moringa oleifera: a food plant with

multiple medicinal uses. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 2007. 21(1): p. 17-25.

1. Petrovska, B.B., Historical review of medicinal plants’ usage. Pharmacognosy reviews, 2012. 6(11): p. 1.
2. Ogbunugafor, H., et al., In vitro and in vivo evaluation of antioxidant properties of Moringa Oleifera ethanolic leaves extract and effect on serum lipid indices in rat. Macedonian Journal of Medical Sciences, 2012. 5(4): p. 397-403.
3. Satish, A., et al., Moringa oleifera Lam.: Protease activity against blood coagulation cascade. Pharmacognosy research, 2012. 4(1): p. 44.
4. Ajayi, I.A., O. Ajibade, and R. Oderinde, Preliminary phytochemical analysis of some plant seeds. Res. J. Chem. Sci, 2011. 1(3): p. 58-62.
5. Day, I.S.C.f.W.T., et al., Thrombosis: a major contributor to the global disease burden. Journal of Thrombosis and Haemostasis, 2014. 12(10): p. 1580-1590.
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7. Eyarefe, O.D., A. Idowu, and J.M. Afolabi, Healing potentials of oral Moringa oleifera leaves extract and tetracycline on methicillin resistant Staphylococcus aureus infected wounds of Wistar rats. Nigerian Journal of Physiological Sciences, 2015. 30(1-2): p. 73-78.
8. Furie, B. and B.C. Furie, Molecular and cellular

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biology of blood coagulation. New England Journal of Medicine, 1992. 326(12): p. 800-806.

1. Bain, B.J., I. Bates, and M.A. Laffan, Dacie and Lewis Practical Haematology EBook. 2016: Elsevier Health

Sciences.

17. J a j a – C h i m e d z a , A . , e t a l . , B i o c h e m i c a l characterization and anti-inflammatory properties of an isothiocyanate-enriched moringa (Moringa oleifera) seed extract. PloS one, 2017. 12(8): p. e0182658.

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Practice in Coagulation Laboratory, Members of Thailand National External Quality Assessment Scheme. Siriraj Medical Journal, 2017. 63(3): p. 8184.

1. Camacho, F.P., et al., The use of Moringa oleifera as a natural coagulant in surface water treatment. Chemical Engineering Journal, 2017. 313: p. 226-237.
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Maxwell Hubert Antwi

Department of Molecular Medicine, School of Medical Sciences, College of Health Sciences,

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana.

Seth Christopher Yaw Appiah

Department of Sociology and Social Work, Faculty of Humanities and Social Sciences,

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Centre for International Health (CIH), University of Munich Medical School, Ludwig-Maximilians-Universitate of Munchen, Munchen, Germany

All correspondence to: Seth Christopher Yaw Appiah, sychrist2007@gmal.com

ABSTRACT

Background: A major limitation of cervical cytology is the unsuitability of proportion of  smears submitted for analysis and for cytological assessment (unsatisfactory). This study examines the prevalence of unsatisfactory Pap smear, clinical history and diagnosis in the Department of Pathology, Korle-Bu Teaching Hospital (KBTH), Ghana. Materials and Methods: A retrospective review of 15,290 cases spanning 12 years (2005-2016) was carried out at the cytology unit of the Pathology Department of the KBTH. Out of the 15,290 Pap smear records retrieved, 2347 reports were excluded leaving 12,943 for the study. All unsatisfactory smear cases were analyzed and categorized using the Bethesda 2001 System. Results are presented using descriptive statistics. Results: The overall prevalence of unsatisfactory Pap smear was 402 (3.1%). Routine screening smear accounted for 115 (0.9%); reports without clinical history and diagnosis gave 21 (0.2%) and cases with clinical history and diagnosis were 287 (2.2%). The common cause of unsatisfactory Pap smear was scanty cellularity 222 (1.72%). Patient’s history accounted for the least cause of unsatisfactory Pap smear 2 (0.02%). Conclusion: Pap smear results reported as unsatisfactory could

harbor cancer malignancy. Samples should be taken by well-trained persons.

INTRODUCTION

Cancer is a global health concern. Millions of individuals have been diagnosed and several affected people have lost their lives. Cancer of the uterine cervix is a leading public health issue globally and

is the commonest cancer in the female genital tract [1, 2]. Over the past two decades, the incidence of the disease has shown a decline in the developed countries [3, 4], yet in poor resource settings like Ghana, there has not been significant change in both prevalence and incidence. [1, 5]

A report by Ghana health service rated cervical cancer as the topmost cancer affecting women in Ghana, and attributed 50.5% of the condition to human papillomavirus (HPV) types 16 and 18 with 16% as cause of death attributable to cancer [6].

Estimates by the World Health Organization (WHO) posit that cervical cancer case in Ghana will exceed 5000 with mortality of 3300 annually by 2025 if the trend persists [7]. It has become difficult to relegate the condition in the country to the background without attention. Larger percentage of women of reproductive age stand at increased risk of having cancer of the uterine cervix since there is no national screening programme or management readily available [6, 8].

Screening of cervical cancer is uncommon in Ghana as the test is done in few public and private health centers in the country and is lowly patronized by the target group (women) [8, 9]. Nevertheless, risk of dying from cervical

cancer can be reduced with routine Pap test [1, 4]. It has been reported that, the annual global death rates attributable to cervical cancer has declined by 2% ever since Pap smear test was introduced with overall death rate by 74% [10].

The Pap test results indicate various changes relating to clinical observation such as; unsatisfactory, normal, inflammation, benign cellular changes, Atypical Squamous cell of undetermined significance (ASCUS), High Grade Squamous Intraepithelial Lesion (HGSIL) and cervical cancer [2, 11]. Despite its outstanding success, the Pap smear is not 100% perfectly accurate [12-14]. Problems occur at every level from failure of women to get regular Pap smear test in the first place to sampling and interpretation error which is likely to give unsatisfactory results that would need a clinical follow-up [12-14].

The number of false negative reports is of great concern because some are reported as a result of unsatisfactoriness [2, 15]. One of the limitations of cervical cytology is that a proportion of smears submitted for analysis in the light microscope are unsuitable for reliable cytological assessment (3, 4). This category of limitation is considered as unsatisfactory Pap smears [11]. Pap smear according to the Bethseda system can be categorized as follows; Sampling error; this error can be due to slide lacking an adequate number of well-preserved and well-visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap or 5000 for Liquid-

base pap, thus scanty cellularity) [11, 14].

Processing error includes poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material being too thick or multilayering smear [11, 14]. Obscuring error includes cells being too atrophic, blood and inflammation obscuring over 75% of the epithelial cells, excessive cytolysis [11, 14]. A broken slide which affects smear for cytologic examination is technically unaccepted. Lack of pertinent patient information and clinical history are as cause of unsatisfactory Pap smear. Any of the causes under these errors can be unsatisfactory.

A large prospective study done in Norway found that unsatisfactory Pap smear test results indicated a 1.6 to 4.0 times high risk of harbouring CIN 2/3 or invasive cervical cancer compared with woman with a normal Pap test result [16]. Unsatisfactory category constitutes 1% to 2% of all Pap test [17-19]. There are however little information and knowledge on its prevalence in Ghana and particularly at the nation’s biggest teaching hospital, Korle-Bu Teaching Hospital.

The essence of this study was to provide adequate data and knowledge on unsatisfactory Pap smears’ prevalence and causes in line with their respective clinical history and diagnosis and to help health-care practitioners with measures to minimize any error that could lead to unsatisfactory Pap smear. This study provides baseline data for comparison with similar reviews in future since there is currently no known scientific documented evidence on unsatisfactory Pap smear prevalence in Ghana.

METHODS

Setting

The study was conducted at the Korle-Bu Teaching hospital. The hospital is the largest health facility and also the premier teaching hospital in Ghana. The Korle-Bu Teaching hospital has a bed capacity of over 2000 and offering medical training for medical doctors, nurses and other health professionals. There are 17 clinical and diagnostic departments/units in the hospital. The hospital is host to the National Centre for Radiotherapy and Nuclear Medicine which functions as a referral centre for the management of cancer supporting other specialized services such as renal transplantation, DNA investigations and brachy therapy for the treatment of prostate cancer. The study was carried out using data from the cytology unit of the Pathology Department of the KBTH.

Study Design and Sampling

The study was retrospective review of all Pap smear registry data of women who underwent Pap smear at the cytology unit of the Korle-Bu hospital spanning a period of 12 years (2005-2016). The study used a hospital based registry which has a catchment that covers the entire southern part of Ghana and beyond without any well-defined population. However, under the period the studied, 15,290 conventional Pap smear test reports were retrieved. The cytology unit actively collects data on all Pap smear cases presenting to the hospital for possible diagnosis of cervical cancer. The data sources are often from either women regular visits to the various clinics and wards of admission or by referral to the unit from within

the hospital or outside the hospital. A special case folder is created (folders) within the unit to abstract needed information with succinct case definition. Information on the clinical history of patients was extracted from the medical folders of each client through a careful sifting of the records.

Analysis

The reports taken from the archives had their covers cleaned and the total Pap smear reports were noted as 15,290. Out of the 15,290 reports, 2347 (both missing reports and lack of TZ components reports) were excluded so 12,943 reports were left for the study. The criteria for the unsatisfactory diagnosis were according to the Bethesda System 2001 and their causes categorized as; sampling error due to inadequate number of well preserved and well visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap smear)—thus scant cellularity (<10% of slide covered by interpretable squamous cells), processing error due to poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material too thick or multilayering smear, presence of obscuring error of the test due to cells being too atrophic with blood and or inflammation obscuring over 75% of the epithelial cells, excessive cytolysis, broken slide, and lack of patient’s history. The 12,943 reports were reviewed for unsatisfactory diagnosis in line with their respective clinical history and diagnosis viz. age, and their causes were noted, counted and grouped into the various categories. The frequencies of the various categories and what constitute them were determined. The prevalence of the unsatisfactory Pap smears in percentages annually were determined and the overall prevalence within that time frame was also known. The prevalence in percentage was calculated as; (total unsatisfactory Pap smears/total Pap smears) multiply by 100. The data was exported into Microsoft Excel and SPSS version 16 for windows and further analysis. Ethics approval was sought from the University of Ghana Ethics approval committee

RESULTS

Population Characteristics

A total number of Pap smears and smears rejected as unsatisfactory reviewed over the twelve year period (Table 1). A total of 15,290 conventional Pap smear test reports were retrieved for review from the archives at the cytology unit of the department of pathology, KBTH over a twelve-year period. Out of the 15,290 Pap smears reports, 529 were missing in the archives, 1818 were limited by lack of transformation zone component and so both (2347 reports) were excluded from this study. The entire Pap smear reports for 2011 could not be found from archives and were excluded. The total Pap smear over the period was 12,943 and unsatisfactory Pap smear was also

1. The average age and age range of women who attended the unit to do Pap smear test were 39.5 and 19 – 80 years respectively. Smears rejected as unsatisfactory were assessed in line with their respective clinical history and diagnosis and their percentages recorded as follows. Unsatisfactory rate with history as routine screening was
2. (0.9%), with clinical history and diagnosis was 287 (2.2%), rate in reports without clinical diagnosis gave 21

(0.2%) and the overall rate in all diagnosis and routine		unsatisfactory categories were presented according to each
screening was 402 (3.1%).		year. In 2005, total unsatisfactory Pap smear was 63 (4.2%),
Causes of Unsatisfactory Pap Smear and Associated		2006 presented 23 (3.7%), 2007 gave 54 (5.3%), 2008 was
		38 (3.7%), 2009 gave 38 (2.9%), 2010 was 47 (3.7%), 2012
Diagnosis and Clinical History		gave 33 (2.7%), 2013 was 39 (2.8%), 2014 gave 40 (2.5%),
Table 2 shows the various causes of unsatisfactory Pap		2015 was 16 (1.2%), 2016 gave 11 (1.6%) and the overall
smears in line with clinical history and diagnosis. Scanty		annual percentage was 402 (3.1%).
cellularity 77 (0.6%) gave higher number for almost all the		The unsatisfactory categories based on their causes
clinical history and diagnosis with routine screening. This		were reported. Sampling error gave total unsatisfactory
was followed by uterine fibroid 52 (0.4%), infertility 27		rate of 222 (1.72%), processing error gave 46 (0.36%),
(0.2%) (amenorrhea, dysmenorrhea, menorrhagia,		inherent error of the test was 132 (1.02%) and patient
dysuria, leiomyoma), cancer 20 (0.2%) (cervical cancer,		history was 2 (0.02%).
endometrial cancer). Accordingly, the least factors that		Year to Cause Categorization of Unsatisfactory Pap
accounted for the unsatisfactory Pap smear were post-		Smear
menopausal bleeding, bleeding in urine) and cytolysis 2		Table 4 shows the various categorization of
(0.02%)		unsatisfactory Pap smear. Scanty cellularity was the
Cause Specific Categorization of Unsatisfactory Pap		highest recording 222 (1.72%) followed by obscured
Smear		inflammation 69 (0.53%), blood obscurance 56 (0.43%),
The results presented in Table 3 show the frequency and		thick smear 17 (0.13%), with the east being cytolysis 7
annual percentages of the unsatisfactory categories based		(0.05%) and patient history 2 (0.02%).
on their causes. The total and annual percentages of the
	Table 1. Study population characteristics.
	Data		N (%)
	Actual number of conventional Pap smear retrieved		1520 (100%)
	Missing cases in the Archive		529 (3.5%)
	Pap smear data limited by transformation zone component		1818 (10.9%)
	Total number of Pap smears reviewed		12,943
	Total unsatisfactory Pap smears reviewed		401
	Age of women (range) years		9 – 80 yrs
	Mean age (yrs)		39.5
	Unsatisfactory rate in routine screening		115 (0.9%)
	Unsatisfactory rate in clinical history and diagnosis		287 (2.2%)
	Unsatisfactory rate in reports without clinical diagnosis		21 (0.2%)
	Unsatisfactory rate in all diagnosis and routine screening		402 (3.1%)

Table 2. Causes of unsatisfactory Pap smear and associated diagnosis and clinical history.

	Clinical History and Diagnosis
Reason for	RS No. ND No. UF No. Hys/Myo Inf No. Ca PB No. Total
unsatisfactory	%		%	%	%	%	%	%
Scanty cells	77	(0.6)	11 (0.08)	52 (0.4)	20 (0.2)	27 (0.2)	20 (0.2)	15 (0.1)	222 (1.7)
Obscuring
inflammation	18	(0.1)	4 (0.03)	3 (0.02)	17 (0.1)	7 (0.05)	11 (0.08)	9 (0.07)	69 (0.5)
Obscuring blood	10	(0.08)	2 (0.02)	4 (0.03)	4 (0.03)	11 (0.08)	12 (0.09)	13 (0.1)	56 (0.4)
Thick smear	4 (0.03)		1 (0.01)	2 (0.02)	3 (0.02)	3 (0.02)	1 (0.01)	3 (0.02)	17 (0.1)
Drying artifact	3 (0.02)		1 (0.01)	3 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	15 (0.1)
Poor fixation	1 (0.01)		2 (0.02)	3 (0.02)	3 (0.02)	2 (0.02)	1 (0.01)	2 (0.02)	14 (0.1)
Cytolysis	2 (0.02)		0	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	7 (0.05)

RS-Routine screening, ND-No diagnosis, UF-Uterine fibroid, Hys/Myo-Hysterectomy/Myomectomy, Inf-Infertility (amenorrhea, dysmenorrhea, menorrhagia, dysuria, leiomyoma), Ca-(cervical cancer, Endometiral cancer), PB-(Post coital bleeding, Post-menopausal bleeding, bleeding in urine).

 

Table 3. Categorization of unsatisfactory Pap smear based on their causes.

Years	Sampling Processing			Patient	Broken	Total Unsat.	Total Pap	%
	Inherent error error			history	slide	Pap smears	smears
	error of the test
2005	34	8	21	0	0	63	1488	4.2
2006	14	1	8	0	0	23	621	3.7
2007	35	9	9	1	0	54	1011	5.3
2008	26	4	8	0	0	38	1023	3.7
2009	25	3	9	1	0	38	1298	2.9
2010	35	3	9	0	0	47	1274	3.7
2012	13	4	16	0	0	33	1215	2.7
2013	17	9	13	0	0	39	1382	2.8
2014	14	5	21	0	0	40	1594	2.5
2015	6	0	10	0	0	16	1336	1.2
2016	3	0	8	0	0	11	701	1.6
	222	46	132	2	0	402	12943	3.1
Total %	1.72	0.36	1.02	0.02	0.00	3.12	–	–

Table 4. Year-to year causes and categorization of unsatisfactory Pap smear.

	Years	cellularity Scant		smear Thick fixationPoor			dryingAir-	obscureBlood Cytolysis inflammation
		Obscured		Patient history Broken slide Unsat papTotal
2005		34	0	5	3	8	2	11	0	0	63
2006		14	0	1	0	1	0	7	0	0	23
2007		35	5	1	3	5	2	2	1	0	54
2008		26	2	1	1	6	0	2	0	0	38
2009		25	2	1	0	5	0	4	1	0	38
2010		35	0	0	3	5	0	4	0	0	47
2012		13	3	0	1	3	1	12	0	0	33
2013		17	1	5	3	5	0	8	0	0	39
2014		14	4	0	1	13	0	8	0	0	40
2015		6	0	0	0	2	2	6	0	0	16
2016		3	0	0	0	3	0	5	0	0	11
	Total	222	17	14	15	56	7	69	2	0	402
	Total Pap
	Smears	12943 %	1.72	0.13	0.11	0.12	0.43	0.05	0.53	0.02	0 3.1

Prevalence to Year Specific Rates of Unsatisfactory Pap Smear

The graph presented in Figure 1 shows that over the years, the highest number of sampling error cases was recorded in 2007 with 35 (5.3%) cases. Highest processing error related cases were recorded in the years 2007 and 2013 (n = 9). No case of broken slide was recorded during the 12 years period. Despite the decline in recorded cases resulting from inherent error of the test from 21 cases in 2005 to a stable 8 and 9 recorded cases over a five year period up to 2010, it increased again in 2012 to 16 recorded cases, fell to 13, increased again to 21 in 2014 and begun to record lower cases in the last two years preceding the end point year for this study (2016).

DISCUSSION

The purpose of this study was to determine the prevalence of unsatisfactory Pap smear in the department of pathology, KBTH and to know the various causes in line with their respective clinical history and diagnosis. The overall prevalence of unsatisfactory Pap smear in this study was 3.1 percent. This estimate is lower when compared to reports from studies done in India, Italy and Taiwan but on the rise when compared to previous studies by Ransdell et al., McGaraghan and Smith-McCune in United States and Netherlands [17, 19-22]. Though the annual prevalence of unsatisfactory Pap smear demonstrates no consistent pattern, year on year prevalence appears to be on the decline. From the annual prevalence in percentages of the unsatisfactory Pap smears, the year 2007 recorded the highest prevalence of 5.3 percent. There was however no

 

suggestive trend or pattern to conclude that the higher the total number of Pap smears or unsatisfactory Pap smear, the higher the prevalence in percentage of unsatisfactory Pap smear to demonstrate any association.

Despite, the study’s inability to determine specific cause attribution to the Pap smear decline, the increasing training and clinical experiences of laboratory scientist at the Cytology Unit of the Korle-Bu Teaching Hospital (KBTH) might play a role.

Though the current study had a lower rate of unsatisfactory Pap smears compared to previously reported findings in India, Italy and Taiwan, the rate (3.1%) remains high because unsatisfactory Pap smear rate of all categories according to reference books should be between 1% to 2% This is against the background that

longitudinal studies of women with unsatisfactory Pap tests have reported an increased risk of epithelial abnormalities [18, 23]. The higher prevalence could be attributed to the conventional approach of Pap smear preparation done at the department but not the liquid-base technology that is able to significantly reduce unsatisfactory rate [14, 24] at the time of the data period. The common reason for unsatisfactory Pap smear in this study was scanty cellularity followed by obscuring inflammation and blood. Scanty cellularity as a cause of unsatisfactory Pap smear has been confirmed in previous studies [17, 20, 23]. It is not surprising to emerge as the most common cause of unsatisfactory Pap smear.

Figure 1. A line plot of graph of unsatisfactory Pap smear prevalence against annual years.

The increasing contribution of scanty cellularity to unsatisfactory Pap smear rates may be explained within the context of its relationship with the technique of sampling. Thus, sample taking by well-trained persons might contribute to reducing the overall rate of unsatisfactory Pap smear. In the current study, though lack of Transformation Zone components in a smear was not considered as unsatisfactory, it is related to sampling technique giving about 1818 excluded reports in this study. The type of sampling device used can also influence specimen adequacy and smear quality (thus present of TZ components) and necessitating the adoption of proper sampling device is encouraged [3, 25]. As reported in previous studies elsewhere [3], scraping the cervix with the extended tip of the spatula followed by using a cytobrush gives adequate samples far better results [3, 25].

Adopting this approach will be key to enhancing the quality of the sampling. In terms of frequency in the current study, obscuring inflammation and blood were the next most frequent causes of unsatisfactory Pap smear and were

related to the obscuring error of the test. Consistent with previously studies by McGaraghan and Smith-McCune in 2000 and by Owens and colleagues in 2013, Obscurance by blood or inflammation as a cause of unsatisfactory Pap smear [14, 20] results from obscuring error of the test. It is the recognition of this error that led to the bringing on board of the liquid-based sampling method that is able to correct the anomaly and generally decreases obscuring problems [24].

This current study had significant rate from this error because, the department of cytology still uses the conventional method of Pap smear preparation. Improved patient preparation or clinician technique (thus not sampling during the patient’s menstrual period) may correct or reduce this cause of the unsatisfactory by obscured Pap [12, 26]. Beyond improving patient preparation, careful attention to transferring of cells onto slide (thus smear preparation), immediate and proper fixation can address thick smear and air-drying problems [3, 25] since these contributed to the causes of unsatisfactory Pap smear under

 

processing error in this study. Better processing of Pap smears therefore yields quality or satisfactory smears for cytological diagnosis [3, 25].

In our present study, patient’s history was labeled as unsatisfactory though the number was not high. Care should be taken to avoid such clerical mistakes. There is little extensive data on how patient history accounts for unsatisfactory Pap smear. However, we found patient history potentially resulting from clerical errors to be responsible for 2 (0.02%) cases of unsatisfactory Pap smear, a development which rarely occurs. None was labeled as unsatisfactory due to technically broken slide and this quality of handling of Pap smear slides should be maintained.

Following a classical definition of unsatisfactory Pap smear as an unreliability in the detection of cervical epithelial abnormalities [18, 27], the definition is relevant in patient management especially within the context of this studies and its findings.

As suggested in previous studies, considering unsatisfactory Pap smears as negative is problematic since negative means absence of disease (SIL or malignancy) and may not prompt adequate follow-up measures [17, 24]. Evidence exists on the possible outcomes when unsatisfactory Pap smears are considered as negative because strong association between false-negatives and unsatisfactory specimens has been amply documented in retrospective studies [17, 18]. Thus, a clear operational definition would need to comply with, for which clinical significance of such smears would need to be determined as well.

The recommended management for unsatisfactory Pap test is to repeat within 2 to 4 months, but this is hardly done in the present study setting due to the patient’s inability to comply usually resulting in missed screening for these women. Similar observations have been reported in low resource settings particularly in different parts of Ghana where infrequent screening opportunities [8, 9] exist. Due to the difficulty with the management of unsatisfactory Pap smear, some studies have posited the need for re-screening though re-screening of previous false-negative Pap smears in patients with current CIN 3 and cancer appears difficult in resource limited settings

1. Notwithstanding, Davey and others study are reassuring in terms of how retrospective re-screening of previously false-negative patients with current CIN 3 and cancer was proven be unsatisfactory after review [13], a finding consistent with earlier studies [16-18]. This calls for the urgent need to have repeated screening whiles minimizing the quick disposition to consider screening results as false-negative.

Unsatisfactory causes were also reviewed in line with their respective clinical history and diagnosis for which unsatisfactory rate from routine screening accounted for 0.86%, whilst and unsatisfactory rate in reports without diagnosis and clinical history was 0.17%.

The clinical history and diagnosis is mostly the primary reason propelling women to do Pap smear in the study setting. The indications as reflected in this current study suggest that patronage by women to do medical check-up for Pap smear is not on the rise because possible predictions could be made from such perspective on the number of women who do Pap smear screening. Mostly, sampling from women with clinical history and diagnosis

of any kind especially malignancy and some benign is a bit challenging and could lead to unsatisfactory smear. This observation carries some veracity particularly when it has been established that additional clinical evaluation, thus inflammation and bleeding associated with CIN 3/carcinoma and some benign cases may cause partially obscured or unsatisfactory Pap tests [16] despite the suggested guidelines [13, 16, 28] on having additional clinical evaluation on women with symptoms and abnormal physical findings [16]. The adoption of continuous quality improvement measures and giving of feedback on quality indicators is critical to decreasing unsatisfactory Pap smears as it has been confirmed in different settings so that CIN 2/3 and invasive carcinoma, are not harboured [15, 24, 28, 29].

LIMITATIONS

The major limitation of this study is the possibility for data entry errors from the source data. The study relied on secondary data over the years. However this is the best available data from the cytology unit that has been documented. Our inability to access background characteristic of the women limited the authors from establishing some association with the profiles of the women. Due to the fact that this study was informed by data from a single facility, making generalization of findings difficult. However this study would serve as a baseline for further research to compare all the regional and teaching hospitals to better understand the national prevalence of unsatisfactory Pap smear.

CONCLUSIONS

This study to the best of our knowledge, is the first ever study on unsatisfactory Pap smear in Ghana.

The study identified scanty cellularity as the main cause of unsatisfactorily Pap smear. The 12-year prevalence compared to other studies is higher though incidence appears to be on the decline year on year. It is expected that the cytology unit adopts the liquid base preparation to get a monolayer smear and to lyse any blood obscurance to avoid unsatisfactory Pap smear.

The study has also provided evidence for the need to train staffs who take the Pap smears to increase their confidence in taking the smear due to high number of scanty cellularity’s reports and lack of TZ component whilst encouraging the use of the recommendations that the right sampling tools be used. Computerized based archives should be implemented at the department since there was significant loss of reports and moreover the entire 2011 reports could not be found from archives.

ACKNOWLEDGEMENTS

We acknowledge the Department of Pathology, Cytology Unit, Korle-Bu Teaching Hospital, Accra-Ghana for allowing us to use access data from the unit for the conduct of this study.

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Prevalence of Unsatisfactory Pap Smear and Associated Clinical History and Diagnosis in a Tertiary Teaching Hospital in Ghana

Maxwell Hubert Antwi

Department of Molecular Medicine, School of Medical Sciences, College of Health Sciences,

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana.

Seth Christopher Yaw Appiah

Department of Sociology and Social Work, Faculty of Humanities and Social Sciences,

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Centre for International Health (CIH), University of Munich Medical School, Ludwig-Maximilians-Universitate of Munchen, Munchen, Germany

All correspondence to: Seth Christopher Yaw Appiah, sychrist2007@gmal.com

ABSTRACT

ackground: A major limitation of cervical cytology is the unsuitability of proportion of Bsmears submitted for analysis and for cytological assessment (unsatisfactory). This study examines the prevalence of unsatisfactory Pap smear, clinical history and diagnosis in the Department of Pathology, Korle-Bu Teaching Hospital (KBTH), Ghana. Materials and Methods: A retrospective review of 15,290 cases spanning 12 years (2005-2016) was carried out at the cytology unit of the Pathology Department of the KBTH. Out of the 15,290 Pap smear records retrieved, 2347 reports were excluded leaving 12,943 for the study. All unsatisfactory smear cases were analyzed and categorized using the Bethesda 2001 System. Results are presented using descriptive statistics. Results: The overall prevalence of unsatisfactory Pap smear was 402 (3.1%). Routine screening smear accounted for 115 (0.9%); reports without clinical history and diagnosis gave 21 (0.2%) and cases with clinical history and diagnosis were 287 (2.2%). The common cause of unsatisfactory Pap smear was scanty cellularity 222 (1.72%). Patient’s history accounted for the least cause of unsatisfactory Pap smear 2 (0.02%). Conclusion: Pap smear results reported as unsatisfactory could

harbor cancer malignancy. Samples should be taken by well-trained persons.

INTRODUCTION

ancer is a global health concern. Millions of Cindividuals have been diagnosed and several affected people have lost their lives. Cancer of the uterine cervix is a leading public health issue globally and

is the commonest cancer in the female genital tract [1, 2]. Over the past two decades, the incidence of the disease has shown a decline in the developed countries [3, 4], yet in poor resource settings like Ghana, there has not been significant change in both prevalence and incidence. [1, 5]

A report by Ghana health service rated cervical cancer as the topmost cancer affecting women in Ghana, and attributed 50.5% of the condition to human papillomavirus (HPV) types 16 and 18 with 16% as cause of death attributable to cancer [6].

Estimates by the World Health Organization (WHO) posit that cervical cancer case in Ghana will exceed 5000 with mortality of 3300 annually by 2025 if the trend persists [7]. It has become difficult to relegate the condition in the country to the background without attention. Larger percentage of women of reproductive age stand at increased risk of having cancer of the uterine cervix since there is no national screening programme or management readily available [6, 8].

Screening of cervical cancer is uncommon in Ghana as the test is done in few public and private health centers in the country and is lowly patronized by the target group (women) [8, 9]. Nevertheless, risk of dying from cervical

cancer can be reduced with routine Pap test [1, 4]. It has been reported that, the annual global death rates attributable to cervical cancer has declined by 2% ever since Pap smear test was introduced with overall death rate by 74% [10].

The Pap test results indicate various changes relating to clinical observation such as; unsatisfactory, normal, inflammation, benign cellular changes, Atypical Squamous cell of undetermined significance (ASCUS), High Grade Squamous Intraepithelial Lesion (HGSIL) and cervical cancer [2, 11]. Despite its outstanding success, the Pap smear is not 100% perfectly accurate [12-14]. Problems occur at every level from failure of women to get regular Pap smear test in the first place to sampling and interpretation error which is likely to give unsatisfactory results that would need a clinical follow-up [12-14].

The number of false negative reports is of great concern because some are reported as a result of unsatisfactoriness [2, 15]. One of the limitations of cervical cytology is that a proportion of smears submitted for analysis in the light microscope are unsuitable for reliable cytological assessment (3, 4). This category of limitation is considered as unsatisfactory Pap smears [11]. Pap smear according to the Bethseda system can be categorized as follows; Sampling error; this error can be due to slide lacking an adequate number of well-preserved and well-visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap or 5000 for Liquid-

base pap, thus scanty cellularity) [11, 14].

Processing error includes poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material being too thick or multilayering smear [11, 14]. Obscuring error includes cells being too atrophic, blood and inflammation obscuring over 75% of the epithelial cells, excessive cytolysis [11, 14]. A broken slide which affects smear for cytologic examination is technically unaccepted. Lack of pertinent patient information and clinical history are as cause of unsatisfactory Pap smear. Any of the causes under these errors can be unsatisfactory.

A large prospective study done in Norway found that unsatisfactory Pap smear test results indicated a 1.6 to 4.0 times high risk of harbouring CIN 2/3 or invasive cervical cancer compared with woman with a normal Pap test result [16]. Unsatisfactory category constitutes 1% to 2% of all Pap test [17-19]. There are however little information and knowledge on its prevalence in Ghana and particularly at the nation’s biggest teaching hospital, Korle-Bu Teaching Hospital.

The essence of this study was to provide adequate data and knowledge on unsatisfactory Pap smears’ prevalence and causes in line with their respective clinical history and diagnosis and to help health-care practitioners with measures to minimize any error that could lead to unsatisfactory Pap smear. This study provides baseline data for comparison with similar reviews in future since there is currently no known scientific documented evidence on unsatisfactory Pap smear prevalence in Ghana.

METHODS

Setting

The study was conducted at the Korle-Bu Teaching hospital. The hospital is the largest health facility and also the premier teaching hospital in Ghana. The Korle-Bu Teaching hospital has a bed capacity of over 2000 and offering medical training for medical doctors, nurses and other health professionals. There are 17 clinical and diagnostic departments/units in the hospital. The hospital is host to the National Centre for Radiotherapy and Nuclear Medicine which functions as a referral centre for the management of cancer supporting other specialized services such as renal transplantation, DNA investigations and brachy therapy for the treatment of prostate cancer. The study was carried out using data from the cytology unit of the Pathology Department of the KBTH.

Study Design and Sampling

The study was retrospective review of all Pap smear registry data of women who underwent Pap smear at the cytology unit of the Korle-Bu hospital spanning a period of 12 years (2005-2016). The study used a hospital based registry which has a catchment that covers the entire southern part of Ghana and beyond without any well-defined population. However, under the period the studied, 15,290 conventional Pap smear test reports were retrieved. The cytology unit actively collects data on all Pap smear cases presenting to the hospital for possible diagnosis of cervical cancer. The data sources are often from either women regular visits to the various clinics and wards of admission or by referral to the unit from within

the hospital or outside the hospital. A special case folder is created (folders) within the unit to abstract needed information with succinct case definition. Information on the clinical history of patients was extracted from the medical folders of each client through a careful sifting of the records.

Analysis

The reports taken from the archives had their covers cleaned and the total Pap smear reports were noted as 15,290. Out of the 15,290 reports, 2347 (both missing reports and lack of TZ components reports) were excluded so 12,943 reports were left for the study. The criteria for the unsatisfactory diagnosis were according to the Bethesda System 2001 and their causes categorized as; sampling error due to inadequate number of well preserved and well visualized squamous epithelial cells (minimum of 8000 – 12,000 for conventional Pap smear)—thus scant cellularity (<10% of slide covered by interpretable squamous cells), processing error due to poor fixation, air-drying artifact or contaminants obscuring over 75% of the epithelial cells and cellular material too thick or multilayering smear, presence of obscuring error of the test due to cells being too atrophic with blood and or inflammation obscuring over 75% of the epithelial cells, excessive cytolysis, broken slide, and lack of patient’s history. The 12,943 reports were reviewed for unsatisfactory diagnosis in line with their respective clinical history and diagnosis viz. age, and their causes were noted, counted and grouped into the various categories. The frequencies of the various categories and what constitute them were determined. The prevalence of the unsatisfactory Pap smears in percentages annually were determined and the overall prevalence within that time frame was also known. The prevalence in percentage was calculated as; (total unsatisfactory Pap smears/total Pap smears) multiply by 100. The data was exported into Microsoft Excel and SPSS version 16 for windows and further analysis. Ethics approval was sought from the University of Ghana Ethics approval committee

RESULTS

Population Characteristics

A total number of Pap smears and smears rejected as unsatisfactory reviewed over the twelve year period (Table 1). A total of 15,290 conventional Pap smear test reports were retrieved for review from the archives at the cytology unit of the department of pathology, KBTH over a twelve-year period. Out of the 15,290 Pap smears reports, 529 were missing in the archives, 1818 were limited by lack of transformation zone component and so both (2347 reports) were excluded from this study. The entire Pap smear reports for 2011 could not be found from archives and were excluded. The total Pap smear over the period was 12,943 and unsatisfactory Pap smear was also

1. The average age and age range of women who attended the unit to do Pap smear test were 39.5 and 19 – 80 years respectively. Smears rejected as unsatisfactory were assessed in line with their respective clinical history and diagnosis and their percentages recorded as follows. Unsatisfactory rate with history as routine screening was
2. (0.9%), with clinical history and diagnosis was 287 (2.2%), rate in reports without clinical diagnosis gave 21

(0.2%) and the overall rate in all diagnosis and routine		unsatisfactory categories were presented according to each
screening was 402 (3.1%).		year. In 2005, total unsatisfactory Pap smear was 63 (4.2%),
Causes of Unsatisfactory Pap Smear and Associated		2006 presented 23 (3.7%), 2007 gave 54 (5.3%), 2008 was
		38 (3.7%), 2009 gave 38 (2.9%), 2010 was 47 (3.7%), 2012
Diagnosis and Clinical History		gave 33 (2.7%), 2013 was 39 (2.8%), 2014 gave 40 (2.5%),
Table 2 shows the various causes of unsatisfactory Pap		2015 was 16 (1.2%), 2016 gave 11 (1.6%) and the overall
smears in line with clinical history and diagnosis. Scanty		annual percentage was 402 (3.1%).
cellularity 77 (0.6%) gave higher number for almost all the		The unsatisfactory categories based on their causes
clinical history and diagnosis with routine screening. This		were reported. Sampling error gave total unsatisfactory
was followed by uterine fibroid 52 (0.4%), infertility 27		rate of 222 (1.72%), processing error gave 46 (0.36%),
(0.2%) (amenorrhea, dysmenorrhea, menorrhagia,		inherent error of the test was 132 (1.02%) and patient
dysuria, leiomyoma), cancer 20 (0.2%) (cervical cancer,		history was 2 (0.02%).
endometrial cancer). Accordingly, the least factors that		Year to Cause Categorization of Unsatisfactory Pap
accounted for the unsatisfactory Pap smear were post-		Smear
menopausal bleeding, bleeding in urine) and cytolysis 2		Table 4 shows the various categorization of
(0.02%)		unsatisfactory Pap smear. Scanty cellularity was the
Cause Specific Categorization of Unsatisfactory Pap		highest recording 222 (1.72%) followed by obscured
Smear		inflammation 69 (0.53%), blood obscurance 56 (0.43%),
The results presented in Table 3 show the frequency and		thick smear 17 (0.13%), with the east being cytolysis 7
annual percentages of the unsatisfactory categories based		(0.05%) and patient history 2 (0.02%).
on their causes. The total and annual percentages of the
	Table 1. Study population characteristics.
	Data		N (%)
	Actual number of conventional Pap smear retrieved		1520 (100%)
	Missing cases in the Archive		529 (3.5%)
	Pap smear data limited by transformation zone component		1818 (10.9%)
	Total number of Pap smears reviewed		12,943
	Total unsatisfactory Pap smears reviewed		401
	Age of women (range) years		9 – 80 yrs
	Mean age (yrs)		39.5
	Unsatisfactory rate in routine screening		115 (0.9%)
	Unsatisfactory rate in clinical history and diagnosis		287 (2.2%)
	Unsatisfactory rate in reports without clinical diagnosis		21 (0.2%)
	Unsatisfactory rate in all diagnosis and routine screening		402 (3.1%)

Table 2. Causes of unsatisfactory Pap smear and associated diagnosis and clinical history.

	Clinical History and Diagnosis
Reason for	RS No. ND No. UF No. Hys/Myo Inf No. Ca PB No. Total
unsatisfactory	%		%	%	%	%	%	%
Scanty cells	77	(0.6)	11 (0.08)	52 (0.4)	20 (0.2)	27 (0.2)	20 (0.2)	15 (0.1)	222 (1.7)
Obscuring
inflammation	18	(0.1)	4 (0.03)	3 (0.02)	17 (0.1)	7 (0.05)	11 (0.08)	9 (0.07)	69 (0.5)
Obscuring blood	10	(0.08)	2 (0.02)	4 (0.03)	4 (0.03)	11 (0.08)	12 (0.09)	13 (0.1)	56 (0.4)
Thick smear	4 (0.03)		1 (0.01)	2 (0.02)	3 (0.02)	3 (0.02)	1 (0.01)	3 (0.02)	17 (0.1)
Drying artifact	3 (0.02)		1 (0.01)	3 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	2 (0.02)	15 (0.1)
Poor fixation	1 (0.01)		2 (0.02)	3 (0.02)	3 (0.02)	2 (0.02)	1 (0.01)	2 (0.02)	14 (0.1)
Cytolysis	2 (0.02)		0	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	1 (0.01)	7 (0.05)

RS-Routine screening, ND-No diagnosis, UF-Uterine fibroid, Hys/Myo-Hysterectomy/Myomectomy, Inf-Infertility (amenorrhea, dysmenorrhea, menorrhagia, dysuria, leiomyoma), Ca-(cervical cancer, Endometiral cancer), PB-(Post coital bleeding, Post-menopausal bleeding, bleeding in urine).

 

Table 3. Categorization of unsatisfactory Pap smear based on their causes.

Years	Sampling Processing			Patient	Broken	Total Unsat.	Total Pap	%
	Inherent error error			history	slide	Pap smears	smears
	error of the test
2005	34	8	21	0	0	63	1488	4.2
2006	14	1	8	0	0	23	621	3.7
2007	35	9	9	1	0	54	1011	5.3
2008	26	4	8	0	0	38	1023	3.7
2009	25	3	9	1	0	38	1298	2.9
2010	35	3	9	0	0	47	1274	3.7
2012	13	4	16	0	0	33	1215	2.7
2013	17	9	13	0	0	39	1382	2.8
2014	14	5	21	0	0	40	1594	2.5
2015	6	0	10	0	0	16	1336	1.2
2016	3	0	8	0	0	11	701	1.6
	222	46	132	2	0	402	12943	3.1
Total %	1.72	0.36	1.02	0.02	0.00	3.12	–	–

Table 4. Year-to year causes and categorization of unsatisfactory Pap smear.

	Years	cellularity Scant		smear Thick fixationPoor			dryingAir-	obscureBlood Cytolysis inflammation
		Obscured		Patient history Broken slide Unsat papTotal
2005		34	0	5	3	8	2	11	0	0	63
2006		14	0	1	0	1	0	7	0	0	23
2007		35	5	1	3	5	2	2	1	0	54
2008		26	2	1	1	6	0	2	0	0	38
2009		25	2	1	0	5	0	4	1	0	38
2010		35	0	0	3	5	0	4	0	0	47
2012		13	3	0	1	3	1	12	0	0	33
2013		17	1	5	3	5	0	8	0	0	39
2014		14	4	0	1	13	0	8	0	0	40
2015		6	0	0	0	2	2	6	0	0	16
2016		3	0	0	0	3	0	5	0	0	11
	Total	222	17	14	15	56	7	69	2	0	402
	Total Pap
	Smears	12943 %	1.72	0.13	0.11	0.12	0.43	0.05	0.53	0.02	0 3.1

Prevalence to Year Specific Rates of Unsatisfactory Pap Smear

The graph presented in Figure 1 shows that over the years, the highest number of sampling error cases was recorded in 2007 with 35 (5.3%) cases. Highest processing error related cases were recorded in the years 2007 and 2013 (n = 9). No case of broken slide was recorded during the 12 years period. Despite the decline in recorded cases resulting from inherent error of the test from 21 cases in 2005 to a stable 8 and 9 recorded cases over a five year period up to 2010, it increased again in 2012 to 16 recorded cases, fell to 13, increased again to 21 in 2014 and begun to record lower cases in the last two years preceding the end point year for this study (2016).

DISCUSSION

The purpose of this study was to determine the prevalence of unsatisfactory Pap smear in the department of pathology, KBTH and to know the various causes in line with their respective clinical history and diagnosis. The overall prevalence of unsatisfactory Pap smear in this study was 3.1 percent. This estimate is lower when compared to reports from studies done in India, Italy and Taiwan but on the rise when compared to previous studies by Ransdell et al., McGaraghan and Smith-McCune in United States and Netherlands [17, 19-22]. Though the annual prevalence of unsatisfactory Pap smear demonstrates no consistent pattern, year on year prevalence appears to be on the decline. From the annual prevalence in percentages of the unsatisfactory Pap smears, the year 2007 recorded the highest prevalence of 5.3 percent. There was however no

 

suggestive trend or pattern to conclude that the higher the total number of Pap smears or unsatisfactory Pap smear, the higher the prevalence in percentage of unsatisfactory Pap smear to demonstrate any association.

Despite, the study’s inability to determine specific cause attribution to the Pap smear decline, the increasing training and clinical experiences of laboratory scientist at the Cytology Unit of the Korle-Bu Teaching Hospital (KBTH) might play a role.

Though the current study had a lower rate of unsatisfactory Pap smears compared to previously reported findings in India, Italy and Taiwan, the rate (3.1%) remains high because unsatisfactory Pap smear rate of all categories according to reference books should be between 1% to 2% This is against the background that

longitudinal studies of women with unsatisfactory Pap tests have reported an increased risk of epithelial abnormalities [18, 23]. The higher prevalence could be attributed to the conventional approach of Pap smear preparation done at the department but not the liquid-base technology that is able to significantly reduce unsatisfactory rate [14, 24] at the time of the data period. The common reason for unsatisfactory Pap smear in this study was scanty cellularity followed by obscuring inflammation and blood. Scanty cellularity as a cause of unsatisfactory Pap smear has been confirmed in previous studies [17, 20, 23]. It is not surprising to emerge as the most common cause of unsatisfactory Pap smear.

Figure 1. A line plot of graph of unsatisfactory Pap smear prevalence against annual years.

The increasing contribution of scanty cellularity to unsatisfactory Pap smear rates may be explained within the context of its relationship with the technique of sampling. Thus, sample taking by well-trained persons might contribute to reducing the overall rate of unsatisfactory Pap smear. In the current study, though lack of Transformation Zone components in a smear was not considered as unsatisfactory, it is related to sampling technique giving about 1818 excluded reports in this study. The type of sampling device used can also influence specimen adequacy and smear quality (thus present of TZ components) and necessitating the adoption of proper sampling device is encouraged [3, 25]. As reported in previous studies elsewhere [3], scraping the cervix with the extended tip of the spatula followed by using a cytobrush gives adequate samples far better results [3, 25].

Adopting this approach will be key to enhancing the quality of the sampling. In terms of frequency in the current study, obscuring inflammation and blood were the next most frequent causes of unsatisfactory Pap smear and were

related to the obscuring error of the test. Consistent with previously studies by McGaraghan and Smith-McCune in 2000 and by Owens and colleagues in 2013, Obscurance by blood or inflammation as a cause of unsatisfactory Pap smear [14, 20] results from obscuring error of the test. It is the recognition of this error that led to the bringing on board of the liquid-based sampling method that is able to correct the anomaly and generally decreases obscuring problems [24].

This current study had significant rate from this error because, the department of cytology still uses the conventional method of Pap smear preparation. Improved patient preparation or clinician technique (thus not sampling during the patient’s menstrual period) may correct or reduce this cause of the unsatisfactory by obscured Pap [12, 26]. Beyond improving patient preparation, careful attention to transferring of cells onto slide (thus smear preparation), immediate and proper fixation can address thick smear and air-drying problems [3, 25] since these contributed to the causes of unsatisfactory Pap smear under

 

processing error in this study. Better processing of Pap smears therefore yields quality or satisfactory smears for cytological diagnosis [3, 25].

In our present study, patient’s history was labeled as unsatisfactory though the number was not high. Care should be taken to avoid such clerical mistakes. There is little extensive data on how patient history accounts for unsatisfactory Pap smear. However, we found patient history potentially resulting from clerical errors to be responsible for 2 (0.02%) cases of unsatisfactory Pap smear, a development which rarely occurs. None was labeled as unsatisfactory due to technically broken slide and this quality of handling of Pap smear slides should be maintained.

Following a classical definition of unsatisfactory Pap smear as an unreliability in the detection of cervical epithelial abnormalities [18, 27], the definition is relevant in patient management especially within the context of this studies and its findings.

As suggested in previous studies, considering unsatisfactory Pap smears as negative is problematic since negative means absence of disease (SIL or malignancy) and may not prompt adequate follow-up measures [17, 24]. Evidence exists on the possible outcomes when unsatisfactory Pap smears are considered as negative because strong association between false-negatives and unsatisfactory specimens has been amply documented in retrospective studies [17, 18]. Thus, a clear operational definition would need to comply with, for which clinical significance of such smears would need to be determined as well.

The recommended management for unsatisfactory Pap test is to repeat within 2 to 4 months, but this is hardly done in the present study setting due to the patient’s inability to comply usually resulting in missed screening for these women. Similar observations have been reported in low resource settings particularly in different parts of Ghana where infrequent screening opportunities [8, 9] exist. Due to the difficulty with the management of unsatisfactory Pap smear, some studies have posited the need for re-screening though re-screening of previous false-negative Pap smears in patients with current CIN 3 and cancer appears difficult in resource limited settings

1. Notwithstanding, Davey and others study are reassuring in terms of how retrospective re-screening of previously false-negative patients with current CIN 3 and cancer was proven be unsatisfactory after review [13], a finding consistent with earlier studies [16-18]. This calls for the urgent need to have repeated screening whiles minimizing the quick disposition to consider screening results as false-negative.

Unsatisfactory causes were also reviewed in line with their respective clinical history and diagnosis for which unsatisfactory rate from routine screening accounted for 0.86%, whilst and unsatisfactory rate in reports without diagnosis and clinical history was 0.17%.

The clinical history and diagnosis is mostly the primary reason propelling women to do Pap smear in the study setting. The indications as reflected in this current study suggest that patronage by women to do medical check-up for Pap smear is not on the rise because possible predictions could be made from such perspective on the number of women who do Pap smear screening. Mostly, sampling from women with clinical history and diagnosis

of any kind especially malignancy and some benign is a bit challenging and could lead to unsatisfactory smear. This observation carries some veracity particularly when it has been established that additional clinical evaluation, thus inflammation and bleeding associated with CIN 3/carcinoma and some benign cases may cause partially obscured or unsatisfactory Pap tests [16] despite the suggested guidelines [13, 16, 28] on having additional clinical evaluation on women with symptoms and abnormal physical findings [16]. The adoption of continuous quality improvement measures and giving of feedback on quality indicators is critical to decreasing unsatisfactory Pap smears as it has been confirmed in different settings so that CIN 2/3 and invasive carcinoma, are not harboured [15, 24, 28, 29].

LIMITATIONS

The major limitation of this study is the possibility for data entry errors from the source data. The study relied on secondary data over the years. However this is the best available data from the cytology unit that has been documented. Our inability to access background characteristic of the women limited the authors from establishing some association with the profiles of the women. Due to the fact that this study was informed by data from a single facility, making generalization of findings difficult. However this study would serve as a baseline for further research to compare all the regional and teaching hospitals to better understand the national prevalence of unsatisfactory Pap smear.

CONCLUSIONS

This study to the best of our knowledge, is the first ever study on unsatisfactory Pap smear in Ghana.

The study identified scanty cellularity as the main cause of unsatisfactorily Pap smear. The 12-year prevalence compared to other studies is higher though incidence appears to be on the decline year on year. It is expected that the cytology unit adopts the liquid base preparation to get a monolayer smear and to lyse any blood obscurance to avoid unsatisfactory Pap smear.

The study has also provided evidence for the need to train staffs who take the Pap smears to increase their confidence in taking the smear due to high number of scanty cellularity’s reports and lack of TZ component whilst encouraging the use of the recommendations that the right sampling tools be used. Computerized based archives should be implemented at the department since there was significant loss of reports and moreover the entire 2011 reports could not be found from archives.

ACKNOWLEDGEMENTS

We acknowledge the Department of Pathology, Cytology Unit, Korle-Bu Teaching Hospital, Accra-Ghana for allowing us to use access data from the unit for the conduct of this study.

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