Olufemi S.S. and Ehinola E. O

Department of Nutrition and Dietetics, Rufus Giwa Polytechnic, P.M.B. 1019, Owo, Ondo State, Nigeria.

Gbadamosi O.F

Department of Nutrition & Dietetics, Federal Polytechnic, Bida, P.M.B 55, Bida, Niger State.

All Correspondences to: Olufemi S.S. Department of Nutrition and Dietetics, Rufus Giwa Polytechnic, P.M.B. 1019, Owo, Ondo State, Nigeria.

ABSTRACT

The purpose of this study was to assess the dietary intake, blood glucose level and nutritional status of type II diabetic patients attending State Specialist Hospital, Akure. Sixty two (62) diabetic patients were randomly selected from the total number of (102) patients that registered with the hospital. A structured and semi-structured questionnaire was designed and administered to elicit information on social demographic, clinical and dietary intake of the subjects. Dietary intake of the subjects was assessed using 24-hour dietary recall procedure nutritional status was assessed using body mass index (BMI). The data obtained were analyzed using SPSS version 20 to determine the mean, frequency and percentage while chi-square was used to determine association between variables. The result shows 61.3% of the subjects were females while 38.7% were males. 6.4% were between the age range of 34-39 years, 59.5% were between 40-59 years while 30.0% were 60 years and above. The results also showed that 64.0% of the subjects have been suffering from diabetes for 6-12 months and 16% had been battling with it since 5-6 years. Thirty eight subjects (61.3%) usually consumed fried foods while 33.9% did not, with 69.4% of subjects eating three times daily. Mean daily carbohydrate and energy intake were 154.0g and 1778.0 403.0 kcal respectively. Daily intake of fat was 116.0g while protein was 36.0g. The mean fasting blood glucose level of the subjects was 9.06 2.539 (MMo1/l). The Body mass index shows that 38.4% of the subjects were overweight while 30.4% were obese. The authors concluded that the mean daily energy intake of the subjects was high for the sedentary and elderly subjects. There was excessive fat consumption and poor glycemic control amongst the subjects. Improvement of dietary intake and proper glycemic control through adequate meal planning and life style modification of diabetics is recommended.

INTRODUCTION

Diabetes in an ancient disease: the records of the ancient civilization of Egypt, Indian, Japan, Greece and Rome describe the symptoms of the disease and usually include recommendation for treatment.

By the first century, the Greek physician, Aretaus of Capadocia who lived between AD30 and 90 describe an illness in which the body “ ate its own flesh” and gave off large quantities of urine. He named the disease Diabetes after the word dia –bainein from the Greek word meaning to siphon or pass through and the term Mellitus which means honey like, was added later by a London physician. It is a chronic disease that has affected mankind through the word. The disease ranks third as a cause of blindness and eight as a cause of death worldwide (Albert, 1995). The World Health Organization defines diabetes mellitus as a metabolic disorder which occur as a result of defect in insulin secretion or action or both and is characterized by chronic hyperglycemia (increase in blood glucose level above normal), resulting into long term damage, dysfunction and failure of various organs. It is a disease characterized by high level of blood glucose

concentration due to absence deficiency or infectiveness of insulin. It affects the metabolism, not only of carbohydrates but also fats and proteins, this lead to lose of glucose in the urine. Most time someone who has diabetic complains of persistent thirst and water consumption because utilized blood glucose is wastefully excreted in the urine. The inability to utilized glucose energy eventually leads to weight loss, tiredness. The most common types of diabetes are known as Non-Insulin dependent diabetes mellitus (NNDDM) or maturity onset diabetes. In Nigeria, half of those that are being afflicted with the ailment are not aware of the affliction. It usually occurs in adult over 40years of age, but many people with type II diabetes do not initially know they have it. Type II diabetes mellitus made up about 90% of the diabetic population in Nigeria and their relevant abnormalities include chronic hyperglycemia, dyslipideamia and insulin resistance (Albert, 1995). An apparent increase in the incidences of types II has been observed in association with the adoption of a “western lifestyle” among the urban class. The prevalence of types II diabetes mellitus is increasing worldwide (King, 1998). Population surveys using various diagnostic criteria for diabetes show a lower prevalence in developing countries than in developed countries. No one has found a convincing causal asocial between malnutrition and diabetes (Peter, 1983). However, the most dramatic increase with 94% compared to a 40% and 25% for South American and the Us and Canada respectively (WHO, 2004). Although, these figures represent diabetes of all types, the majority of persons (90-95%) 20 years of age and older have type II diabetes. Metabolic, genetic and environmental factors may play a role in the development of diabetes and its complication. It has been documented that the risk of developing type II diabetes increase with age, obesity, sedentary, life style, family history of diabetes and low high density lipoprotein (HDL) or high triglyceride concentrations (HTC). People with diabetes are subjected to acute and long-term complication (El-kebbi im, 1996). Types II diabetes mellitus (formerly known as non Insulin dependent diabetes mellitus-NIDDM) poses a major health threat worldwide, base on 1994 extra population from prevalence studies, there are now about (2million person with diabetes in America and Caribbean). Type ii diabetes mellitus account for approximately 9% of all diabetes (Albert, 1995). Untreated or inadequately treated types II diabetes patients may develop the same complication as that of types I or insulin dependent diabetes mellitus (IDDM) including eye, nerve and kidney damage. Types II diabetes also accounts for 25% of the world total population suffering from diabetes thus 28million estimate for person with diabetes in the Americas is projected to increase by about 45% by the year 2010, with Latin American and the Caribbean surpassing the US and Canada according to projections Diabetes is the most common cause of end-stage renal diabetes, and the leading cause of blindness in adult diabetes rank sixth as a primary cause of death in the US, and when its complication are considered, it rank third. The estimated economic impact of diabetes is considerable to 24 percent in the small number of subject above 40 years of age.

Diabetes people are routinely advised to adopt a healthful diet, dietary changes including modification in food habits and meal pattern on a lifelong basis. Self monitoring of blood glucose (SMBG) is recommended for all patients with diabetes. The distinction among the type of diabetes is the frequency of testing for patient with diabetes that is treated with insulin, more frequent testing is recommended, most often before meals and bed. With consistency in self care management and blood sugar levels, testing times to achieve a better a day, possibly alternating time to achieve a better overall picture of glycemic trends, for those on oral agents, testing range from one to four time per day as needed until glycemic treads are established.

MATERIAL AND METHOD Study design

This study is descriptive and cross-sectional in design, involving out-patients diabetics attending State Specialist Hospital, Akure

Study Area

The State Specialist Hospital, Akure is situated in Akure, the State capital of Ondo State. The study was carried out at the diabetic clinic of the hospital. The clinic offers a

comprehensive diabetes testing, drug and insulin therapy, dietary counseling, treatment and management.

Research Subjects:

A total of 68 out-patient diabetics which were 38 females and 24 males known diabetes patients, aged 34-90 years were included in the study. The subjects were non-insulin dependent diabetes mellitus patients (NIDDM). The inclusion criteria were already diagnosed diabetics. The subjects must have been confirmed positive through blood glucose testing, not earlier than six months before the date of the commencement of the study and must be registered with the diabetic clinic of the hospital.

Ethical consideration

Approval was sought and obtained from ethical research committee of the hospital.

Informed consent was obtained from individual subjects Inclusion criteria

• Consenting participants who suffered from

diabetes mellitius

Exclusion criteria

• Individuals without diabetes
• Non-consenting individuals

SAMPLE POPULATION

The population for this study was out-patients non-insulin dependent diabetics (NIDDM) attending Medicine Out-patient Department (MOPD) of the State Specialist Hospital, Akure.

SAMPLE SIZE

Sixty two (62) diabetes patients were selected for the study. The sample size for the study was determined based on three factors: the estimated prevalence of the variable of interest– diabetes patients in this instance, the desired level of confidence and the acceptable margin of error.

n= t² x p(1-p)

SAMPING TECHNIQUES

Simple random sampling method was used to select the required number of subject from the 102 diabetic patients that registered with the diabetes clinic. The subjects were selected randomly during the diabetes clinic sessions. Thirty two (32) diabetic patients were randomly selected at the first visit to the clinic while the remaining 30 subjects were selected during the second clinic. The total number of sample selected was sixty two (62), which were used for the study.

METHOD OF DATA COLLECTION Questionnaire

A structured and semi- structured questionnaire was used to collect the necessary information on socio demographic data, medical history, dietary history and food consumption pattern of the patient. The questionnaires were randomly distributed to the patients and they were completed and returned.

Anthropometric Assessments

Weight:

The subjects’ weight was measured using bathroom weighing scale. The scale was placed on a flat surface and the subject was made to stand uprightly on it barefooted. The readings were done in duplicates to the nearest 0.1kg and the average weight was constantly checked for accuracy (Caterson 1998).

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 67

Height:

Height was measured to the nearest 0.1m using a standiometer, measurement was taken with subjects standing barefooted and erect with feet parallel and heels put together, in line with the method of Jelifel (1996). BMI

was calculated as weight in kg/ divided by height in (m²).

Dietary assessment

Information on the patient dietary intake was collected using 24hours dietary recall. The full and appropriate description of all food eaten including drinks was recorded and converted to nutrient using the food exchange list for commonly eaten food in Nigeria.

Assessment of Blood glucose

A secondary data were used to obtain the blood glucose of the subjects. The case notes of the diabetics were obtained at the clinic and their fasting blood glucose in MMol/L was documented from the records.

METHOD OF DATA ANALYSIS

Data collected from this research work were analyzed through the use of descriptive statistical tools such as the use of percentage, mean, frequency e.t.c. T-test was used to determine the significance difference between the variable. Descriptive and inferential analysis technique was used for data analysis using Statistical Package for the Social Sciences (SPSS) software version 17. T-test and Chi-square test were performed to determine significant difference and relationship among variables.

RESULTS

Table 1: Distribution of subjects by Sex and Age

Table 2: Socio-economic characteristics of the subjects

Is your income enough to feed you?

Table 3: Distribution of subjects by Medical history

Apart from diabetes, what other disease do you suffer from?

..

Table 4: Distribution of subjects by dietary pattern

Variables s Frequency Percentage

How many times do you eat daily?

How many times do you eat fruit per day?

Table 6:

Distribution of Subjects by their fat consumption

Table 7: Attitude of Subjects to prescribed diet

Table 8: Distribution of subjects by their BMI

Table 9: Mean nutrient and clinical profile of diabetic subjects

DISCUSSION

The study reveals that majority of the diabetics in the study area were females (61.3%) and the mean age of the diabetics was (58.10%). In fact, this reflects the same pattern observed in the study of Sunday et’al, 2012, involving diabetic patients within the tertiary health centres in Nigeria where 60.5% of the diabetics were female and their mean age was 57.1years. The educational qualification of the subjects shows that majority of them had either primary or secondary education and majority of the patients (81%) claimed that their income was enough to feed them. The patients that had enough income may have money to supply the foods and medication needed. Some studies revealed that diabetic patients especially the female ones, though, may earn low income but would always receive financial support from family and loved ones in order to have access to medical treatment (Sunday et’al, 2012). However, most times, the economic status of a patient has a lot of influence not only on the food intake but also on the ability to have access to drugs and medication which determines the effectiveness of diabetes management. WHO reported that about 90.2% of Nigerians are below the poverty level of 2 dollars per day (WHO, 2004). Therefore, accessing health care is challenging for diabetes patients in Nigeria. Nineteen (19) 30.6% of the subjects were seen with hypertension, a figure so close to 36.5% reported in a similar study (Alebiosu, 2007). Prevalence of different diabetes complications like kidney problem, eye defect and heart disease is similar to figures from other studies in Nigeria. These complications are as a result of late intervention in most of the hospitals (Sunday et’al, 2012). Majority of the subjects have been living with diabetes for the past 1-2 years (37.3%) while as high as 16% have been living with the disease for the past 5-8 years. This is a reflection of lower life expectancy of Nigeria diabetic patients. The study reveals that the disease could be genetically link to the parents since it can be documented from the study that 26% of the diabetics’ parents also suffered from diabetes. This study shows that (72%) of the patients were either overweight or obese. Overweight/obesity contributes to cardiovascular risk of diabetic patients. The result shows that average energy intake of the subject was 1778403kcal per day which was below the RDA for non- elderly subject of (65.9%), while the average energy intakes was ideal for elderly RDA of about (33.6%). Mean fat intake of the subjects was higher than the RDA for fat (116.63g±11.542). The high fat consumption seen in the study may be due to high palm oil and other vegetable oil used by the patients in cooking. High fat intake generally is a contributory factor to cardiovascular risk (Gordon, 2002). There is no significance difference between blood glucose level and macronutrient intake (Protein, Carbohydrate and Fat) of the patients at 0.05 levels. However there is positive correlation between blood glucose and protein intake (0.183). The mean fasting blood glucose (FBG) level of the subjects was 9.06MMo1/l 2.539, which is an indication of poor glucose control among the patients. The deficient energy intake observed in 35% of the patients may be as a result of non-adherence to prescribed diet. The low calorie intake may also be linked to the fact that the majority of the subject (64.5%) ate 3times daily. In the recent management of diabetes, food consumption should be based on small frequent meals at interval to avoid glucose overloading (WHO, 2004). Poor glycemic control among Nigerian diabetic patients is non compliance to dietary prescription. Nigerian diabetics continue to look after permanent cure for the disease which results to poor glycemic control in the long run (Sunday, 2012). Therefore, the management of diabetes is patient education and lifestyle modification. Many diabetic patients are yet to realize that diabetes is incurable and needs long-life management.

CONCLUSION AND RECOMMENDATION

The dietary intake of diabetic patients at State Specialist Hospital was below recommended intake. Most diabetics engaged in wrong dietary habits and poor glycemic control. Improvement of dietary intake and proper glycemic control through adequate meal planning and life style modification of diabetic patients is recommended.

Olufemi S.S.

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The post Dietary Intake, Blood Glucose Level and Nutritional Status of Type II Diabetes Patients Attending State Specialist Hospital, Akure. appeared first on Nigerian Biomedical Science Journal.

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.

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

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,

58 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

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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Influence of antibiotic treatment on the…

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

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)

62 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

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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The post Influence of antibiotic treatment on the detection of S. aureus in whole blood following pathogen enrichment appeared first on Nigerian Biomedical Science Journal.

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

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

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

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

54 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

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The post Reference intervals for serum cystatin C and serum creatinine in an adult sub-Saharan African population appeared first on Nigerian Biomedical Science Journal.

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.

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).

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%.

46 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

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.

REFERENCES

1. Unitaid V (2014) Malaria Diagnostics Technology and Market Landscape (2nd Edn).
2. Dal-Bianco MP, Köster KB, Kombila UD (2007) High prevalence of asymptomatic Plasmodium falciparum infection in Gabonese adults. Am J Trop Med Hyg 77: 939-942.
3. Klein EY, Smith DL, Boni MF, Laxminarayan R (2008) Clinically immune hosts as a refuge for drug-sensitive malaria parasites. Malar J 7: 67.
4. Kiszewski A, Johns B, Schapira A (2007) Estimated

global resources needed to attain international malaria control goals. Bull World Health Organ 85: 623-630.

1. ACTwatch.
2. Cohen JM, Woolsey AM, Sabot OJ, Gething PW, Tatem AJ, et al. (2012) Public health. Optimizing investments in malaria treatment and diagnosis. Science 338: 612-614.
3. Yan J, Li N, Wei X (2013) Performance of two rapid diagnostic tests for malaria diagnosis at the China-Myanmar border area. Malar J 12: 73.
4. Arya SC, Agarwal N (2013) Laboratory tests for malaria: a diagnostic conundrum. S Afr Med J 103: 701.
5. (2012) Relevance Of Economic Field Microscope In Remote Rural Regions For Concurrent Observation Of Malaria & Inflammation, Advances In Infectious Diseases (AID).
6. Harchut K, Standley C, Dobson A (2013) Over-diagnosis of malaria by microscopy in the Kilombero Valley, Southern Tanzania: an evaluation of the utility and cost-effectiveness of rapid diagnostic tests. Malar J 12: 159.
7. Fançony C, Sebastião YV, Pires JE, Gamboa D, Nery SV (2013) Performance of microscopy and RDTs in the context of a malaria prevalence survey in Angola: a comparison using PCR as the gold standard. Malar J 12: 284.
8. Bhattacharya D, Bhuyan BM, Pradhan PK, Nayak DK (2013) Transmission Blocking of Year Round Resistant Malaria in Koraput (India) by OMARIA– A New Antimalarial Phytotherapy. British Journal of Pharmaceutical Research 3: 54-77.
9. Reyburn H, Mbakilwa H, Mwangi R, Mwerinde O, Olomi R, et al. (2007) Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: randomised trial. BMJ 334: 403.
10. Ansah EK, Narh-Bana S, Epokor M (2010) Rapid testing for malaria in settings where microscopy is available and peripheral clinics where only presumptive treatment is available: a randomised controlled trial in Ghana. BMJ 340: c930.
11. Kumar A, Chery L, Biswas C, Dubhashi N, Dutta P, et al. (2012) Malaria in South Asia: prevalence and control. Acta Trop 121: 246-255.
12. Mouatcho JC, Goldring JP (2013) Malaria rapid diagnostic tests: challenges and prospects. J Med Microbiol 62: 1491-1505.
13. Alonso PL, Tanner M (2013) Public health challenges and prospects for malaria control and elimination. Nat

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Med 19: 150-155.

1. Hopkins H, González IJ, Polley SD (2013) Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. J Infect Dis 208: 645-652.
2. Coleman RE, Sattabongkot J, Promstaporm S, Maneechai N, Tippayachai B, et al. (2006) Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand. Malar J 5: 121.
3. Kaewkamnerd S, Uthaipibull C, Intarapanich A, Pannarut M, Chaotheing S, et al. (2012) An automatic device for detection and classification of malaria parasite species in thick blood film. BMC Bioinformatics 17: S18.
4. Vink JP, Laubscher M, Vlutters R, Silamut K, Maude RJ, et al. (2013) An automatic vision-based malaria diagnosis system. J Microsc 250: 166-178.
5. Srivastava B, Anvikar AR, Ghosh SK (2015) Computer-vision-based technology for fast, accurate and cost effective diagnosis of malaria. Malar J. 14: 526.
6. Bereczky S, Mårtensson A, Gil JP, Färnert A (2005) Short report: Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum. Am J Trop Med Hyg 72: 249-251.
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.

The post An Enhanced Computer Vision Platform for Clinical Diagnosis of Malaria appeared first on Nigerian Biomedical Science Journal.

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

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)

Internal Standard t-butanol

Cerilliant® calibrators (water)

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/

The post Quantification bias in blood alcohol determination by headspace gas chromatography appeared first on Nigerian Biomedical Science Journal.

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

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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.

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.

The post Valuable and Cost Effective Combination of CD123 and CD22 for Basophil Discrimination appeared first on Nigerian Biomedical Science Journal.

, 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.

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.

REFERENCES:

1. Alves, R.R. and I.L. Rosa, Why study the use of animal products in traditional medicines? Journal of ethnobiology and ethnomedicine, 2005. 1(1): p. 5.
2. Maroyi, A., An ethnobotanical survey of medicinal

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.
6. Golebiewska, E.M. and A.W. Poole, Platelet secretion: From haemostasis to wound healing and beyond. Blood reviews, 2015. 29(3): p. 153-162.
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

30

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.

1. Wongkrajang, P., W. Chinswangwatanakul, and P. Tientadakul, Whole Blood Clotting Time: Variation of

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.
2. de Andrade Luz, L., et al., Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters. International journal of biological macromolecules, 2013. 58: p. 31-36.

The post Investigating the effect of Ethanolic extract of moringa Oleifera seed on bleeding time and whole blood clotting time of Wistar Albino Rats appeared first on Nigerian Biomedical Science Journal.

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

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

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.

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

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

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

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.

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

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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The post Prevalence of Unsatisfactory Pap Smear and Associated Clinical History and Diagnosis in a Tertiary Teaching Hospital in Ghana appeared first on Nigerian Biomedical Science Journal.

An assessment of the availability of Medical Laboratory Services in primary health care centres in Ogbia LGA, Bayelsa State, Nigeria.

Atiegha C and Suama P

Department of Medical Laboratory Sciences, College of Health Technology Otuogidi-Ogbia, Bayelsa State.

Stella U. K.

Department of Medical Laboratory Science, Rivers State University, Port-Harcout.

Gbarebe B

Department of Health Information Management, College of Health Technology, Otuogidi-Ogbia, Bayelsa State.

All correspondence to: E-mail: achristo40@gmail.com

ABSTRACT

Services at the Primary Health Care (PHC) are the first set of health services that are provided at the grass-root or rural level. A medical laboratory is a place for examination of materials derived from human body for the purposes of providing information on diagnosis of diseases.

The objective of this study is to assess the level of provision of medical laboratory services in primary health care centers in Ogbia local government area of Bayelsa-State, Nigeria. This study was carried out in 20 functional primary health facilities. A descriptive survey design involving questionnaire and a check list was used to assess information on the issue. The results reveal that only 20 primary health facilities operate in the LGA of over 55 communities. Among them only 9 health facilities are offering skeletal medical laboratory services. It further revealed a grossly under staffing of medical laboratory personnel. Medical laboratory services in the health centers in Ogbia Local Government of Bayelsa State, Nigeria are yet to be fully developed. We recommend therefore that policy makers should focus their attention in providing the needed manpower and equipments to boost health care services in these health care facilities.

Key words: Laboratory, PHC, Diagnosis, Scientist, Technician, Grass root.

INTRODUCTION

n India the National Rural Health Mission (NRHM) Itaught wise to look at the ways in which the missions and goals of Primary Health Care (PHC) had been strengthened in the rural settings. This process is carried out in order to explore the avenues for more upgrading. As part of the commitment of the NRHM to improve public health care services, one of its core strategies had been to strengthen PHCs to meet the level of the countries Public

Health Standards, IPHS (1).

However, the World Health Organization (WHO) had defined health as a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity. Health can also be seen as the ability to adapt and manage physical, mental and social challenges throughout life. ‘Health for All’ is a programming goal of the WHO, which envisions securing the health and well- being of people around the world that has been popularized since the 1970s. It is the basis for the WHO’s primary health care strategy to promote health, human dignity and enhanced quality of life (2).

Furthermore, health service is seen as the essential part of a Nation’s social security in which the state is compulsorily obligated by its constitutional provisions to direct its policies toward ensuring that there are adequate medical services for all persons, including management, prevention and treatment of diseases. Health care services

in Nigeria are rendered as an integrated services. However, it was further reorganized into three tiers by the Federal Civil Service in the year 1988 under the auspices of the federal ministry of health. The tiers are the primary, secondary and tertiary health care’s (3).

Services at the PHC are the first set of health services that are provided at the grass-root or rural level. Health care (services) here requires contact with the community health facilities. This level of health care service identifies the health chalenges of the inhabitants and resolve them using appropriate technologies. Referring much serious and complicated cases to the secondary or tertiary health care service delivery. It is the cornerstone of the rural health services (4).

These days quality health care of patients has become more dependent on findings of medical laboratory investigations (5).

Good Laboratory policies are the decisions which are taken in consultation with other medical staff to enable the laboratory to operate reliably and efficiently in accord with other departments (6).

Medical laboratory can be defined in several ways by different authors: a specialized laboratory for carrying out investigations (routine or research) on human specimen sin order to generate accurate and reliable result; where tests are carried out on clinical specimens in order to get information about the health of a patient pertaining to the diagnosis, treatment and prevention of diseases; a place for

14 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An assessment of the availability of Medical…

measurement and examination of materials derived from human body (fluids, tissues, excreta, cells, etc.) for the purposes of providing information on diagnosis, prognosis, prevention or treatment of diseases; a branch of medical science that plays a vital role in public health and disease control; is where clinical pathology tests are carried out on clinical specimens to obtain information about the health of a patient and to aid in diagnosis, treatment and prevention of diseases( 7,8).

Medical laboratory science as a multidisciplinary field encompassed medical microbiology, haematology, clinical chemistry and histopathology. The discovery of microscope by Antony Van L., a German Scientist revolutionized the field of medical sciences (9). The place of medical laboratory science in medicine and public health is very fundamental. Medical laboratory science is key in disease identification, disease control and surveillance and in treatment of patients. Yet this very important services is still under provided or utilized (10).

Medical Laboratory personnel includes multiple categories of Medical laboratory science practitioners who have different levels of education and training ranging from medical laboratory assistants, medical laboratory technicians and medical laboratory scientists with their particular functions accordingly (11).

There are mainly two types of medical laboratories that process the majority of medical specimens. They are the hospital based and private based laboratories. Hospital based laboratories are medical laboratories that are located within the hospital that perform tests on patient whereas private laboratories are located outside the hospital that also perform tests on patients (12,13). Although specific routine tests are not limited to few, it is expedient that medical laboratory facility at the primary health care level perform at least these routine tests such as urinalysis, pregnancy test, blood grouping/Rhesus typing, Haemoglobin genotyping, haemoglobinestimation, tuberculosis test and serological assays (4).

Number of medical laboratory analysis had greatly impacted on the operations of PHC. Medical Laboratory diagnosis as the branch of diagnostic activities of PHC is saddled with the duty of producing laboratory tests results needed for the effective treatment of diseases (8,10).

It is clear that the role of medical laboratory in medicine and the society at large can and will never be over-emphasized. Though, there had been series of complains concerning the services in the laboratory at PHC level; they provide the physicians and other health care givers the information to detect disease, predisposition to disease, establish prognosis, guide patients management, monitor efficacy of treatment, confirm or refute a diagnosis. Reports obtained revealed that patients got inadequate satisfaction in regard to primary health care service delivery. The low turn up could be attributed to the non-equipment in the laboratory and bad information management system. It is apparent that patients do not effectively assess the services of medical laboratory at the primary health care level, possibly due to its non-establishment at the level of primary health care provider or simply due to lack of political will to make it functional (14, 15, 16). Despite these great functions and benefits of medical laboratory services, it had been reported that this service had been under assessed in general health practices at the PHC level (17).

However in the practice of modern Medicine, medical services had proven to be comprehensive and of global health standard only with the support of basic laboratory facilities to serve as the important determinant influencing the utilization of health services (18).

It is therefore clear that primary goal of PHC entail the involvement of ‘appropriate technology’ which is designed to equip the health facilities to render effective and quality medical care (19).

The ‘Bedrock” of modern medicine they say is the Medical laboratory. It plays a role that cannot be quantified in the three tiers of health services and the society at large. Therefore, this study is aimed at assessing the medical laboratory services in primary health care centres in Ogbia local government area of Bayelsa-State, Nigeria.

MATERIALS AND METHODS

This study was carried out in 20 functional primary health care facilities in Ogbia Local Government Area of Bayelsa State. A descriptive survey designed was used to assess the Medical Laboratory Services in the region. Subjects

The study subjects were the head of the Health facilities. This instrument was further divided into two sections: section ‘A’ includes names of community, health facilities and staff cadre in the laboratory, while section ‘B’ includes type of tests conducted. Ethical approval was obtained from the officers in charge from the respective health facilities and the committee of ethics and man power development, college of Health Technology, Otuogidi, Ogbia-Town, Bayelsa state.

PROCEDURE

An interview guide/checklist was used as method of data collection. The data were analyzed and results were presented in tables.

RESULT

From the data generated, only 20 communities had health centres, of which 9(45.0%) operate medical laboratory services while 11(55.0%) had no medical laboratory services (figure 1).

Out of the 9 available medical laboratories, the staff manning them are 5(55.6%) medical laboratory technicians, 1(11.1%) are medical laboratory assistants and 3(33.3%) are community health extension workers (Table 1).

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Table 1: Personnel in charge of the laboratory

Of the 9 functional medical laboratories, the quality of services are 4(44.4%) good, 5(55.6%) are fair and 0(0.0%) and poor (Table 2).

Table 2: Quality of MLS

Of these 9 functional medical laboratories, 3(33.3%) offer regular services while 6(66.7%) have irregular services (figure 2).

Figure 2 Regularity of laboratory services provided

Table 3 gives the laboratory tests carried out in each of the health centres. In these 9 functional health facilities, 8(88.9%) parameters:Blood grouping, Hb-genotype, Hb-estimation, Hepatitis B, urinalysis, malaria parasites test, pregnancy test and HIV screening are carried out Otuogidi health facility, 6(66.7%) parameters: Blood grouping, Hb-estimation, urinalysis, malaria parasites test, pregnancy test and HIV screening are done in Otuoke health facility, 5(55.6%) parameters: HIV screening, Hb-estimation, urinalysis, malaria parasites test and pregnancy test are done in Otuakeme health facility, 4(44.4%) parameters: HIV screening, urinalysis, malaria parasites test and pregnancy test are carried out in Emeyal health facility, 3(33.3%) parameters: HIV screening, urinalysis, malaria parasites test and pregnancy test are done in Otuobagi health facility, 2(22.2%) parameters: HIV screening and malaria parasites test are done in Kolo health facility, while 3(33.3%) parameters: HIV screening, malaria parasites test and pregnancy test are carried out in Okodi, Oloibiri and Ewoi health facilities respectively (Table 3).

16 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An assessment of the availability of Medical…

Table 3: Panel of Tests done in MLS

DISCUSSION

Our study revealed that there are only 20 established primary health care centres in Ogbia Local Government Area of Bayelsa-State (which has over 55 communities) that are providing health services to the people in the communities. This finding is not in tandem with NRHM, 2005 which has the commitment of improving public health care service deliveries as one of its core strategies to strengthen PHCs to meet the level of Public Health Standards (1).

With this limited number of health facility, it is obvious that most of the persons residing in most communities within the rural areas in the local government had no access to quality health services. This issue also negates what Dibia, (2002) pointed out. He said health service is an essential part of a Nation’s social security which mandated the state to direct its policies toward ensuring adequate medical services for all persons (3).

The fact that of the twenty existing Health facilities only 9 are offering medical laboratory services which indicates that the quality of health service delivery rendered to the people is grossly inefficient and as such, undermined the role of medical laboratory services. This finding are also in contrast with the agreement of the World Health Assembly, that said WHO advocates for basic laboratory services and using appropriate technologies to support and strengthen clinical and public health activities at the PHC level (19,20).

The study further revealed an inappropriate staff strength and cadre working in the medical laboratory department that may not be able to face more rigorous analysis. There are also high tendencies of releasing wrong or falsified tests results because there are no qualified and trained medical laboratory scientists to verify and ascertain the validity of the generated tests results. We identified the presence of untrained, incompetent, non-qualified and non-licensed laboratory workers in all the laboratories. Invariably, the facilities are operated by quacks. It is therefore obvious that the tests results generated in these facilities may not be accurate.

This study also revealed a massive substandard, poorly functional and a dilapidated medical laboratory system with series of complains of dissatisfaction from patients. This had really hampered the progress to patients’ treatment. This findings are in agreement with that of Coulter & Ellim, 2006, attributing a poor functional medical laboratory and monitoring scheme majorly to the lack of political will, thereby causing health set back during treatment (15).

The quality of medical laboratory services depend greatly on the personnel involve in the diagnostic processes. Poor staffing posed an impediment to patients’ satisfaction to service deliveries. Our findings are in consonant with Sambo et al., 2010, whose record revealed that patients returns home with insufficient satisfaction owing to poor service delivery at the primary health care level (16).

The type of laboratory tests done is so skeletal and inconsequential. These array of tests varies significantly from one facility to the other that are managing to provide laboratory services. This findings negates that of Azad, 2012, who highlighted some of the essential laboratory services that should be provide at the primary health care level (4).

CONCLUSION

World Health Organization (WHO) believed in ‘Health for all’. However, this declaration had not been achieved because so many persons or patients are still deprived from accessing quality health services. These services are carried by trained, certified and license medical laboratory scientists only and as such, the services of medical laboratory scientists are often demanded by the requesting officers (physicians) or the patients concern. Medical laboratory science is the bed rock of modern medicine hence, when medical scientists are not integrated in patients’ management, the patients had been denied to accessing quality health service delivery. The role of medical laboratory scientists is so pivotal in health services and the society at large. Ironically, there are only 20 primary health care facilities that offered health services in Ogbia Local Government Area of Bayelsa State, of which

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only 9 are offering a skeletal medical laboratory services in other to attend to the health need of the people which is grossly inadequate. The medical laboratories are not furnished, no equipment or reagents. The staff strength is totally inadequate characterized with unqualified and non-licensed personnel. Common routine clinical tests that cut across all the units in the laboratory are not comprehensively done. Results generated are not reliable and reproducible consequent upon, detrimental to patients’ treatment. Lack of political will by the ruling class is another impediment that had resulted in the dilapidated nature of the health industry hence, hampered the WHO programme of ‘Health for All’, since patients cannot and are not accessing quality medical laboratory services at the third tier of health services. We therefore recommend that the authorities ( state and local government as well as federal agencies) should commence the equipmenting, staffing and training of all cadre of laboratory personnel as well as the provision of new laboratries for the health facilities that do not have them in order to enhance quality and efficient health care for the inhabitants.

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