Kudi A.A., Saminu C.M., Hassan M.D., Manga M.M:

Department of Medical Microbiology & Immunology Federal Teaching Hospital, Gombe
Deborah B., Madina E.K.,

Centre for Integrated Health Programme (CIHP), Gombe Regional Office.
Sa’idu A.

Department of Medicine, Federal Teaching Hospital, Gombe

All correspondence to: kudiayuba@gmail.com

ABSTRACT
A success story at the Federal Teaching Hospital, Gombe (FTHG) a 500-bed capacity tertiary institution situated at the heart of the North-eastern Nigeria. A manually operated PCR facility was first installed by a supporting partner – the Institute of Human Virology-Nigeria (IHVN) in 2009. The FTHG in collaboration with Centre for Integrated Health Programme (CIHP) and Centre for Disease Control (CDC) – Nigeria up-graded the facility to an Automated PCR machine (COBAS TAQMANN96) in 2015. It was a good response to partnership that demonstrates programme commitment and sustainability. In that spirit, human and material resources were committed towards the actualization of space and infrastructure with quality finishing/furnishing of the PCR Laboratory. Staff were highly motivated and the dearth in Laboratory infrastructure was greatly addressed. Timely Supply of reagents and consumables was also sustained. The resultant benefit of this partnership was quality improvement to services with increased in patients’ access to HIV and Hepatitis B and C diagnosis and treatment monitoring. Our Centre remains ever grateful to the PEPFAR HIV programme, our supporting partners and the Federal Ministry of Health, Nigeria.

Key Words: PCR, PEPFAR HIV and AIDS Programme, Partnership.

INTRODUCTION

Gombe, a State located in the Northeast region of Nigeria within the expansive savannah. It shares border with Yobe, Taraba, Bauchi, Borno and Adamawa States. The State has an area of about 20, 265 square kilometres and a population of around 2,353,000 people as at 2006 census.1 Gombe has 2 distinct climatic conditions, the dry season (November to March) and the raining season (April to October) with an average rain fall of 850mm.1

The Federal Teaching Hospital Gombe (FTHG) is located in Gombe, a tertiary 500-bed capacity health institution situated in the heart of the North-eastern Nigeria, relatively equipped with states of the arts facilities in Diagnostic Laboratory, addresses wide range of investigations. Before the coming of the Centre for International Health Programme (CIHP) as a supporting partner in the field of HIV investigations and treatment monitoring, the Laboratory could only boast of a manually operated PCR equipment installed by the Institute of Human Virology, Nigeria (IHVN)] in 2009, under the Presidential Emergency Plan for AIDS Relief (PEPFAR), a programme of the United States of America launched in 2003 to combat
global HIV/AIDS.2,3 For us at the Federal Teaching Hospital Gombe (FTHG), Nigeria, the up-grading of the PCR Laboratory to automation by the CIHP/CDC (Centre for Disease Control) – Nigeria in partnership with FTHG management was a success story. The Hospital Management, staff and people of Gombe State will ever be grateful to PEPFAR.

Team work and Positive Response to Partnership
The News to install an automated PCR Machine (COBAS TAQMANN96) came to us (Laboratory Management) as an institution, and was observed that our supporting partner, CIHP were serious, as ever; they placed the card on the table that; PCR automated equipment could be installed in our facility to boost the Centre’s capacity in HIV and AIDS investigations. What was required then of the FTHG Management to put in place were space (infrastructure) and few other logistics. The laboratory Team saw the possibility because of the close working relationship, support and co-operation being received at all times from the Hospital management.

In high Spirit, the Laboratory leadership Team led by the Director Laboratory Services, approached the Hospital management with a proposal to that effect, and sooner than expected, discussion with the management for theconstruction of a brand new PCR Laboratory was concluded and approved. The Chief Medical Director (CMD), Dr. Abubakar Sa’idu assured the leadership of CIHP that construction of the PCR Laboratory, furnishing and any other necessary item(s) needed according to specification to enable the centre have the automated PCR equipment installed will be put in place. He equally made a strong commitment towards the deadline given by CIHP/NCDC- Nigeria for the space to be ready. To his words, within 3 months (April to July, 2015), all were set for PCR equipment installation. On 1st October, 2015, PCR equipment with all the necessary accessories for Early Infant Diagnosis (EID) and Viral quantification (viral load) reagents arrived the FTHG. PCR Equipment (COBAS TAQMANN96) and all the accompanied accessories were received by the Director Laboratory services in good deadline given by CIHP/NCDC- Nigeria for the space to be ready. To his words, within 3 months (April to July, 2015), all were set for PCR equipment installation. On 1st October, 2015, PCR equipment with all the necessary accessories for Early Infant Diagnosis (EID) and Viral quantification (viral load) reagents arrived the FTHG. PCR Equipment (COBAS TAQMANN96) and all the accompanied accessories were received by the Director Laboratory services in good condition and according to the specifications earlier given.

On 13th October, 2015, installation and on-site training of staff were completed by a Team of Roche Engineers with the support of the CIHP Gombe Laboratory Team, led by the Senior Laboratory Supervisor, Mrs. Deborah Bulus. The arrival of -20oC and -80oC deep freezers from the CIHP supporting partners, and the installation of 20Kva inverter by same (CIHP) later, climaxed the infrastructural up-grading of the PCR Laboratory at the FTHG Centre. The now fully equipped PCR Laboratory was commissioned by the Hon. Minister of Health, Prof. Isaac F. Adewole on 11th January 2016. This has greatly addressed the dearth in Laboratory infrastructure, improves the quality of services in the Centre and increased access; staff were also highly motivated to quality issues.

To further galvanized its commitment to quality and demonstrate a kind of strong partnership, aside from EID and HIV (viral load) investigations fully supported by the CIHP/CDC-Nigeria, the FTHG management added Hepatitis B and Hepatitis C viral quantification to the menu of investigations using the installed PCR machine. So far, from the date the PCR machine was installed to the time of writing this report, more than 36,840 samples were received from Kaduna, Benue and Gombe States for HIV viral load, 5,157 for EID, while 242 for Hepatitis B virus
and 85 for Hepatitis C virus were analysed. The addition of these tests to the HIV investigations have greatly enhanced quality of patient care and reduced the hardship hitherto been experienced by the patients.
CONCLUSION
The FTHG Management and staff have not only demonstrated good response and sensitivity to partnership, but also understood the principle and importance of programme sustainability. This was clearly seen in the resources being committed, both human and material towards the actualization of space and infrastructure with quality finishing/furnishing of the PCR Laboratory. This is our success story. We remain ever grateful to our programme partners and the Federal Ministry of Health. We look forward to more of supports and such types of collaboration from well-meaning groups and organisations in and outside Nigeria.

REFERENCES

1. Gombe State action plan of His Excellency, 29th May, 2011 to 29th May, 2015. An official document of the Gombe State Government.

2. Partnering to Achieve Epidemic Control in Nigeria. www.pepfar.gov

3. PEPFAR HIV and AIDS programme on the Nigeria

Ojih M.A
Department, of Medical Microbiology Ahmadu Bello University Teaching Hospital Zaria.
Amadi Y.H
Department of Parasitology and Entomology Ahmadu Bello University Zaria.
Akpulu S.P
Department of Human Anatomy Ahmadu Bello University Zaria.
Yaro E.G
Department of Human Anatomy, Ahmadu Bello University, Zaria, Nigeria

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

ABSTRACT
Background: Intestinal parasites and helminthic infection has posed medical changes to the developing word. There is therefore need to source and develop local drugs or herbs to combat this menace. Aims and Objectives: This study therefore examined the anthelminthic activity of ethanolic leaf extract of combretum molle in vitro on the ova of Hookworm and Haemonchus contortus. Materials and Methods: One drop of the extract mixed 5 drops of distilled water was added to 6 drops of faecal samples on a microtiter well and incubated for 14 days at 260C, using water as control. The activity was assessed by comparing the number of ova present after a period of 2 weeks for both the extract and water. The result showed that there was only one egg (non -embryonated) of hookworm observed in the well with the extract. For the Haemonchus contortus in the well with extract, there as one egg,12 unembryonated eggs, 13 dead larvae. For the control(water), there was no positive effect on the hookworm and the Haemonchus contortus. Conclusion: Result from this study shows that ethanolic leaf extract of combretum molle was effective against the ova of the hookworm and the Haemonchus contortus and can be a possible anthelmentic agent.

Keywords: helminthic, Haemonchus contortus. combretum molle, Hookorm

Introduction

Intestinal parasites and helminthic infection has posed medical changes to the developing word. Resistance to antibiotics is becoming a serious problem worldwide. The discovery of new and effective antimicrobials and/or resistance modulators is necessary to tackle the activity of spread of helminths. Infections with helminths are among the most widespread diseases worldwide and the reason for considerable morbidity, mainly in populations from less developed countries of Sub-Saharan Africa, the Americas, and Southeast Asia (Hotez et al.,2008). Recent investigations estimated the number of people infected with the most common parasitic nematodes Ascaris lumbricoides, Trichuris trichiura, and hookworms (Ancylostoma duodenale and Necator americanus) to be more than 1.7 billion worldwide, including 100 million infections in West Africa (Pullan et al., 2010). Extensive measures have been undertaken by the WHO to provide effective and low-cost treatments for the inhabitants in endemic areas, which have successively led to a decrease in the morbidity and prevalence of intestinal helminthiases (Humphries et al.,2012). In most cases, albendazole is the deworming drug of choice, however, mebendazole, pyrantel, and levamisole are also classified as essential medicines (WHO,2013). Nevertheless, the number of approved anthelmintic drugs the societies are relying on is very limited and especially large-scale preventive applications bear the risk of resistances to emerge (Vercruysse et al.,2011). In veterinary medicine, this is already the case where worldwide resistances of parasites of livestock have increased to a dramatic degree within the last years (Kaplan and Vidyashankar,2012). Despite the availability of conventional drugs, traditional medicine, often based on medicinal plants, plays a crucial role in the treatment of helminthic infections. As indicated by a recent field study, many African patients prefer to consult traditional healers or herbal practitioners for this indication (Agyare et al.,2009). More than 300 species of plants have been reportedly used in traditional control of human and animal helminthosis in Africa (Nwude et al.,1998). Combretum molle of the combretacae family is used to treat human ailments including abdominal discomfort, body pains, respiratory discomfort, cods and fevers, ear and eye ailments, schistosomiasis, hookworms, dysmenorrhea and infertility in women, leprosy, syphilis, microbial infections and general body weakness (Ademola and Eloff, 2010). Helminthiasis is a major limitation to the livestock industry in Africa. Haemonchus contortus is the singular most important helminth responsible for major economic losses in small ruminants. The high cost of anthelmintics to small farmers, resistance to available anthelmintics and residue problems in meat and milk consumed by humans further complicates matters.

Haemonchus contortus is the single most important constraint to sheep production in South Africa (Vatta et al.,2001) with estimated direct and indirect economic losses of US $45 million (I.G. Horak personal communication quoted by Waller, (2006). The cost of controlling helminth infestation is also very high globally with chemotherapy remaining the most widely used method of treatment. Unfortunately, the excessive use of these drugs when not necessary, in addition to their use at incorrect doses, has resulted in the wide scale emergence of resistance in this parasite. The use of plants and plant extracts as a possible source of new anthelmintic has received more interest in the last decade. This study therefore aim to study the anthelminthic activity of Combretum molle (velvet bush willow) against ova of hookworm and Haemonchus contortus.

Materials and methods
This study was conducted in the department of parasitology and entomology, faculty of veterinary medicine Ahmad Bello University, Zaria. Positive stool sample was collected from Ahmad Bello University Teaching Hosital Zaria. Plant collection. Five-kilogram leaves of Combretum molle was collected in Zaria and identified in the biological science department of Ahmad Bello University, plant extraction. The leaves were air dried (290C) under shade and grinded sing pestle and mortar into fine powder as described by Onyeyili et al.,2001. 150g of the powdered sample was extracted with 2.5L of 96% ethanol using cold maceration (percolation) method. The filtrate was evaporated and the extract obtained used. One part of Acacia was mixed with 5 parts of the extract.one drop of the mixture as introduced into microtiter well for culture and left for two weeks using water as control. 0.1ml distilled water was added to 1.2ml positive faecal sample and 0.2ml extract. Culture was observed for ova microscopically on day 7, 10 and 14 post incubation respectively.

Discussion
The results from this study shows that at the end of 14 days, the extract at 0.2ml had an adverse effect on the ova of Hookworm and the egg, and larvae of Haemonchus contortuus. This was observed by the absence and dead of the ova and larva. which implies that the extract has anthelminthic effect. While in day 7 and 10, the extract did not reduce the number of the ova and larvae significantly the control only with water did not show any anthelminthic effect rather, the ova hatched to larvae.

These findings are similar with previous investigations on lethal anthelmintic effects against a variety of nematode species, including adult C. elegans Mohamed et al (2000) and L3 larvae of Toxocara canis Kiuchi et al (2000). Also, the activity against C. elegans (L4 larvae/ young adults) of a plant extract from Combretum mucronatum by Schum. & Thonn. (Combretaceae) leaves has been confirmed in vitro (adult worms) and in vivo against Trichuris muris and against adult stages of A. ceylanicum, Spiegler et al., (2015) while the same extract was inactive against third stage larvae of the hookworm Kone et al., (2012). This inactivity is probably caused by the sheath protecting the free-living L3 larva persevering in soil, which is unlikely to be penetrated by large polar molecules such as proanthocyanidins. T. et al., (2012). Similarly, Adamu et al (2013) reported that the plants extract generally had better inhibition activity on the eggs than on the larva of H. contortus based on the EC50 values recorded. This is contrary to report by Ademola where they had better activity for the larval inhibition compared to the egg hatch assay.

In comparison to work undertaken using extracts of other plant species, the activity shown by the three plants with the best activity were in the same order of activities found by Bizimenyera et al., (2006) using Peltophorum africanum leaf, bark and roots as well as extracts of Coriandum sativum on eggs and larval development of H. contortus Eguale et al.,(2007). Bizimenyera et al., (2006) recorded an EC50 of 0.62 mg/ml and 0.72 mg/ml for the leaf acetone extract for the EHA and LDT assays respectively. This is in the same order with values of 0.62 and 0.64 mg/ml recorded in the EHA and LDT for H. trifoliata in the current study. The EC50 of H. trifoliata was lower than the acetone leaf extracts of Combretum molle 0.87 mg/ml and 0.60 mg/ml for the EHA and LDT respectively (Eloff McGaw and Eloff, 2008). The weak activity recorded by M. azedarach (EC50 = 10.96 mg/ml) agrees with Maciel et al., (2006) in Brazil where they reported an LC50 of 9.18 mg/ml with the leaf ethanol extract of the plant. It is encouraging that despite the difference in geographical location and organic extractants used, similar bioactivity was obtained for related species.

Conclusion
This finding shows that Combretum Molle has an Anthelminthic activity on the Ova of Hookworm and Haemonchus 

Acknowledgment
We are grateful to the Management and Laboratory staff Ahmadu Bello University Teaching Hospital Zaria, and Department of Parasitology and Entomology, Faculty of Veterinary Medicine Ahmadu Bello University for permission, support and cooperation during the work.

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Akande T,

            Dept. of Chemical Pathology, Bingham University, Jos Campus

Olusanya T, Mamza Y.

            Dept. of Medical Laboratory Science, University of Maiduguri

All correspondence to: Akande T. Dept. of Chemical Pathology, Bingham University, Jos Campus

ABSTRACT

INTRODUCTION

Uric acid is the metabolic acid product of Purine metabolism. Previous studies have demonstrated a strong relationship between serum uric acid level and coronary heart disease (CHD) and some studies suggested that uric acid may be an independent risk factor for cardiovascular disease [1-9]. Recently, a meta-analysis showed that hyperuricemia may increase the risk of CHD events, independently of traditional CHD risk factors [10]. However, the nature of relationship between uric acid and cardiovascular disease remain a subject of debate [11-13].

Epidemiological studies have reported relationship between serum uric acid level and a wide variety of cardiovascular conditions, including hypertension metabolic syndrome [14] Coronary artery disease [15]. Cardiovascular disease is the number one global cause of death. An estimated 17.3 million people died from cardiovascular disease CVDs in 2008; representing 30% of all global death [16] CVDs are projected to remain the single leading cause of death [17]. Presently there is paucity of epidemiological data on serum uric acid level in cardiovascular disease in this environment and even with long standing awareness of this association, little attention has been paid to its potential significance, hence the need for this study.

Materials and Method

The study group comprised 100 consecutive cardiovascular disease patients attending the medical clinic at the university of Maiduguri teaching hospital, a referral center for North-eastern states of Nigeria and neighbouring countries e.g. Chad and Cameroon.

The patient recruited were seen in the medical clinics, reviewed by the physicians and provisionally diagnosed of cardiovascular disease. Control subjects, 50 apparently healthy individuals without signs of cardiovascular disorder were recruited. Patients with associated cases of kidney dysfunction and gout were excluded.

Data Collection

A standard questionnaire was used to collect information such as age, sex, medications, education level. The history of chronic disease and current use of medication were recorded.

In all study patents, a complete clinical work was done including height, weight and body mass index. The body mass index was calculated and expressed as Kg/m2. The blood pressure was recorded in the right upper arm in the sitting posture after a five-minute rest. Patient were categorized as being hypertensive if they were on antihypertensive treatment or if they had a systolic blood pressure >140mmHgand /or diastolic blood pressure >90mmHg [18].

Biochemical Measurements

Peripheral various blood samples were collected after an overnight fast and the following investigations were done: plasma glucose, serum cholesterol, and serum triglyceride high density lipoprotein-cholesterol and serum uric acid. Biochemical analyses were done spectrophotometrically using reagents supplied by Randox UK). Serum uric acid using Uricase enzymatic method, were estimated in all samples.

Statistical analysis

Statistical analysis was performed with the SPSS statistical package (version 11.0). Student’t’ test was used to compare the means of variables. The level of significance was set at p-value of <0.05.

Results

The study included a total population of 150 subjects, 100 of which were patients with confirmed cases of cardiovascular disease and 50 control individuals. Among the test individual’s females represented 66% of the total population while males represented a portion of 34%.

Table 1. Shows the mean distribution of the age, blood pressure, and body mass index of test and control individuals by sex. There was a significant difference (P<0.05) between the test and control for all characteristics. The total mean age for test individuals was 45.9±15.4 with the males (mean=54.7±14.3) averagely older than the female patients (mean=41.5±14.2). Table 2 shows a mean distribution of biochemical parameters.

Table 3: represent the percentage distribution of serum uric acid levels stratified by age group and sex. The total percentage incidence of hyperuricemia in the CVD patients was 57% among all the age groups and gender. There was a significant level of occurrence of hyperuricemia among all the age and sex. Few of the patients fell between the age group 0 – 19 (4%) and highest was 40 – 59 (54%) having an incidence of 56%.

Table 4: shows the relationship that exists between serum uric acid levels and cardiovascular disease in the presence and absence of individual CVD risk factors. The result revealed that hyperuricemia still persisted even in the absence of individual CVD risk factors. It shows that 54.5% of patients with normal BMI were hyperuricemic as well as 58% of non diabetic patients, 62% of patients with normal serum cholesterol levels and 63% of patients with normal blood pressure all of which were significantly higher than those of patients with one or more CVD risk factors.Table 5: shows the association of serum uric acid level and CVD after categorizing the test individually into diuretic users. The result shows a frequency of 39% diabetics as against 61% of those whose indication does not include diuretic. The incidence of hyperuricemic was substantially higher among persons on diuretic use (64%). However, the association of hyperuricemia with CVD was still maintained as a significant 52.5% of the non-diuretic

DISCUSSION

In this representative general population, hyperuricemia bore a continuous, independent, specific, and significant positive relationship to cardiovascular disease. This association was true for both men and women. The impact of hyperuricemia on cardiovascular disease was positive at all age groups but this influence was strongly observed from those aged 20-59 and above 80.

These data are consistent with a variety of other studies suggesting that an elevated serum uric acid level is an independent risk factor for cardiovascular disease [19, 20, 21, 22].

Furthermore, after controlling for potential risk factors, it was observed that the presence or absence of individual CVD risk factors did not generally alter the positive relation of hyperuricemia to cardiovascular disease as the association of increased serum uric acid level still persisted even among subjects with low cardiovascular risk (i.e. those without an increased cholesterol level, hypertension, diabetes, or obesity). On other hand, hyperuricemia was not strongly associated with CVD in diabetic and patients with elevated cholesterol. In adult, serum uric acid levels vary with height, body weight, blood pressure, alcohol intake and diuretic use [23, 24, 25]. The influence of diuretic therapy on serum uric acid is of particular note and potential importance [26, 27]. To check this influence on the study subjects, patients were stratified based on diuretic and non-diuretic users. In this group, diuretic therapy did appear to modestly increase serum uric acid level. However, the serum uric acid to CVD relation was independently of the effect of diuretics as a higher percentage of the non-diuretic users still had a hyperuricemic state.

The results of this study are not in agreement with that of Culleton etal [11]which reported that an elevated serum uric acid level at baseline was not independently associated with increased risk of cardiovascular mortality. They concluded that the apparent association of serum uric acid to cardiovascular events was probably due to the effect of other cardiovascular risk factors, particularly by diuretic use. The different results seen in these two studies may be due to difference in the population studied (i.e. population from their study was almost exclusively white while those in this study were all black). But considering the vast reports that favor the results of this study, it suggested that theirs may however be an exception, rather than the rule.

The vital question is whether increased serum uric acid is a casual factor for cardiovascular disease. Observational data alone cannot answer that question. It is not possible to determine here whether hyperuricemia is a marker, a co- morbid or intervening factor, or a direct cause of CVD.

In any event, however, hyperuricemia has been shown to meet conventional criteria for a cardiovascular risk factor. The association of increased serum uric acid to cardiovascular disease events is significant, is independent of other known CVD risk factors, is specific and has substantial effect size.

Conclusion

Hyperuricemia was associated with cardiovascular disease independently of conventional cardiovascular disease risk factors and may be considered a risk factor for cardiovascular disease.

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Omosigho O.P., Odedina E.O. EmumwenE.G.

Medical Microbiology Department, Federal Medical Centre Bida. Niger State, Nigeria.

Emumwen E.F

Department of Biological Sciences, The Federal Polytechnic Bida. Niger State, Nigeria.

All correspondence to: Omosigho Omoruyi Pius Medical Microbiology Department,

Federal Medical Centre Bida. Niger State .Nigeria Email –omosighoop@gmail.com.

ABSTRACT

Cytomegalovirus (CMV) is a common opportunistic infection in immunocompromised individuals particularly HIV/AIDS. Avidity is the strength with which the IgG attached to antigen; it measures the length of time after primary infection. CMV IgG avidity assay seems to be one of the most accessible tools to differentiate between primary and non-primary CMV infection.  This study was conducted to evaluate the significance of the utility of the relationship of IgG Avidity Index and IgM levels for differential diagnosis of primary Cytomegalovirus infection from non-primary infection in HIV/AIDS patients in Federal Medical Centre Bida. Blood samples were collected from HIV positive patients on Highly Active Anti-Retroviral Therapy for IgM Enzyme Linked Immonosorbent Assay, all positive IgM samples were tested for IgG Avidity. Out of the three hundred and eighty five (385) HIV positive subjects, an overall prevalence of 19.8% IgM was found among patients with HIV in Bida. Out of the 77 IgM positive samples tested for IgG Avidity index, 12(15.6%) had high avidity while 65(84.4%) had low avidity.  Low avidity indices indicate low maturation of IgG antibodies in blood caused by recent or acute primary CMV infection There was a significant difference (P=0.001) between the utility of IgG avidity index and IgM in the differential diagnosis of primary Cytomegalovirus infection. Primary CMV infections by IgM ELISA alone was significantly overestimated as only 65(84.4%) out of the 77 CMV IgM positive are actually primary infection while the rest 12 (15.6%) are non-primary infections. The utility of IgG avidity ELISA significantly reduced the reported prevalence of primary CMV infection with IgM ELISA method in this study. IgG avidity index is a sensitive, specific and less expensive method for identification of recent CMV infection in HIV positive patients compared to more sophisticated and expensive molecular methods.

Keywords: IgG Avidity, Cytomegalovirus, HIV/AIDS, Primary infection, Bida, Nigeria.

INTRODUCTION

Cytomegalovirus  a member of the human Herpes family of viruses, transmissible through blood component, transfusion is an important cause for concern worldwide this is because the majority of adults have serological evidence of previous infection . (Zhang et al., 1995). The prevalence of seropositivity of CMV  in the adult population over 40 years of age worldwide are reported to be 60-100%, due to transmission through sexual contact, spread from   children and breastfeeding (Helkeret al., 2004).

Like most other Heperviruses, CMV is latent in their host after primary infection and persists for life (Drobyskinet al., 1993). Nevertheless, reactivation in

immunosuppressive individuals leads to end organ disease such as retinitis, encephalitis and interstitial pneumonitis (Manezet al., 1993). Acute infection of CMV leads to serious morbidity and mortality (Hillyeret al., 1990). CMV co- infection is a major cause of morbidity and mortality in individuals with depressed cell mediated immunity associated with Acquired Immunodeficiency Syndrome AIDS (Macheret al., 1983).

CMV is one of the most successful of human pathogens, since it can be transmitted both vertically and horizontally, usually with little effect on the host. CMV is shed in body fluids (e.g., saliva, urine, semen, breast milk). Transmission usually occurs from person to person, including through the intrauterine route, but it can also be spread during transfusion or organ transplantation.

Globally, between 60 and 90% of the general population is infected with CMV, with generally higher rates in developing countries (Rowshaniet al., 2005).

The functional binding affinity of anti-CMV IgG antibodies increases progressively over time after immunity by infection; it is otherwise referred to as maturation of the humoral immune response Low IgG  avidity indices may indicate primary infection whereas high avidity indices indicate non-primary infection (Bonalumiet al.,2011).

The CMV-IgG avidity ELISA has also been successfully used to diagnose CMV in children. Vilibic-Cavleket al in 2011 evaluated the value of IgG avidity in diagnosis of CMV infection in newborns and infants. Their analysis of 40 serum samples from under 1 year old infants with suspected CMV infection. 13 (32.5%) of the subjects were seropositive to anti-CMV IgM, 3 (7.5%) had equivocal IgM antibodies result, and 24 (60.0%) patients had IgG antibodies only. With IgGavidity ELISA, CMV infections (with low avidity indices) were reported in 61.5% IgM positive and 54.2% IgM negative infants. IgG avidity distribution across age of infants demonstrated recent primary CMV infection in 58.8% patients younger than 3 months compared with 91.7% and 81.8% in 3–6 and 6–12 months old infants respectively. The study showed that IgG avidity ELISA results in children older than 3 months of age was significantly useful for CMV diagnosis regardless of their IgM result.(Vilibic-Cavleket al.,2011).

Primary CMV infection is likely to occur when there is seroconversion from IgM negativity to IgM positivity in combination with low IgG avidityindex. The anti-cytomegalovirus IgG avidity ELISA is presently the most reliable investigation used to identify primary CMV infection in pregnant women. The IgG avidity test is highly specific (100%) and sensitive (94.3%). The degree of antibody avidity slowly and progressively increases reflecting the maturation of humoral immuneresponse over time (Lazzarottoet al.,2002, Egger et al.,2000 ). Low avidity indices indicate low maturation of IgG antibodies in blood caused by recent or acute primary CMV infection (Lazzarottoet al.,1997). Low avidity indices are encountered 18–20 weeks after the onset of symptoms in apparently healthy individuals (Egger et al.,2000).

The aim of this work was to establish the significance of IgG Avidity Index in the differential diagnosis of primary Cytomegalovirus infection from non-primary infection in patients attending Anti-Retroviral Therapy Clinic in FMC Bida Niger State.

Materials and Methods

This study was a cross sectional study of patients with HIV infection attending the Anti-Retroviral Therapy (ART) Clinic of Federal Medical Centre Bida, Niger State. The study was carried out in the Anti-Retroviral Therapy (ART) Clinic of Federal Medical Centre Bida, located in Bida Local Government Area of Niger State, North Central Nigeria.

Approval for this study was obtained from the Ethical Review Committee of FMC, Bida. The sample collection was explained to the subjects using the information sheet

prepared by the researcher. Each subject was required to give a written informed consent before being eligible to participate in the study. The minimum sample size for this study was determined using the Fischer formula. (Fisheret al., 1998)

Subjects:

Five milliliters of whole blood was drawn from the median cubital vein of every volunteer into a plain tube, allowed to clot; specimen was processed into serum by room temperature centrifugation at 3000 revolution per minutes for 10 minutes and stored at -200c. The frozen serum were thawed at room temperatures for 45 mins. Enzyme Linked Immonosorbent Assay (ELISA) IgM DIA Source (KAPRCVG01) CMV IgM kit was used following manufacturer instruction. All samples that were IgM positive were tested for IgG Avidity to differentiate primary from non-primary CVM infection.

IgG Avidity ELISA

Principle of the Test

Measurement of CMV –specific IgG avidity has proved to be a powerful tool for distinguishing primary from non-primary CMV infection. Defined as the strength with which the IgG attaches to antigen .IgG avidity measures the length of time following primary infection. Thus IgG produced within the first few months following primary infection exhibits low avidity, whereas IgG produced several months or years exhibits high avidity. However detection of CMV –specific IgG of high avidity is actually more informative from clinical standpoint as the presence of high avidity IgG essentially excludes the possibility that infection occurred within the previous four months.

Description of Methods

Cytomegalovirus IgG avidity ELISA utilizes urea, an agent that disrupts hydrogen bonds, to differentiate low avidity from high avidity antibodies. Following attachment to immobilized antigen, low avidity IgG readily dissociates from the antigen in the presence of urea whereas high avidity IgG does not. Thus, avidity was assessed by performing duplicate sets of the routine ELISA for CMV-specific IgG. The only difference was that after the initial incubation, one set was washed with buffer containing urea and the other set was washed with buffer lacking urea.

The CMV –specific IgG signal (optical density) of the set washed with urea buffer was divided by the CMV-specific IgG signal of the set washed with non- urea buffer providing the avidity index (AI). Avidity Index values lesser or equal to 0.50 indicates low avidity, values of 0.51-0.59 indicate intermediate avidity and values equal or greater than 0.60 indicate high avidity.

Assay Procedure IgG ELISA

1. The required number of cavities was placed in the frame and a protocol sheet was prepared.
2. Samples were diluted 1:101, distributing ten microliter of sample into one milliliter of sample diluents.
3. One hundred microliters of each diluted sample and calibrators (0, 1, 2 and 3) were pipetted to the appropriate wells. One well was left for the substrate blank.
4. The micro plate was incubated for 45 mins at 370C.
5. The micro plate was aspirated and each well was washed four times for 30 seconds with washing solution manually using a dispenser. Blotted and dried by inverting plate on absorbent material.
6. One hundred microliters of each Enzyme –Labeled 2nd Antibody was added into each well.
7. The micro plate was incubated for 45 mins at 370C.
8. Each well was aspirated and washed four times for 30 seconds with washing solution manually using a dispenser. Blotted and dried by inverting plate on absorbent material.
9. One hundred microliters of TMB Chromatogen solution was added to each of the well using a dispenser.
10. Incubated for 15 mins at room temperature. Exposure to direct sunlight was avoided.
11. One hundred microliters of stop solution was added to each well using a dispenser.
12. The absorbance of the solution in the wells was read using a microplate reader set to 450 nm. If wavelength correction was necessary, the instrument was set to dual wavelength measurement at 450nm with background wavelength correction set at 600 or 620nm.

Note:

(i)  Avidity was assessed by performing duplicate sets of      the routine ELISA for CMV-specific IgG.

(ii) One set was washed with buffer containing urea and     the other set was washed with buffer lacking urea

Statistical Analysis

Generated data from the study was entered into and analysed with SPSS version 20.0. Data was summarized in frequency tables. Relationships and interactions among variables were determined using Chi-square crosstabs. Statistical significance tests were based on the 95 % Confidence Interval (CI), P value of less than 0.05.

RESULTS

Three hundred and eighty five (385) blood samples were collected among patients with HIV/AIDS on Highly Active Anti-Retroviral Therapy attending Anti-Retroviral Therapy clinic in Federal Medical Centre Bida.

The prevalence of CMV IgM study report among HIV subjects in Bida is presented in Table 1. Out of the total three hundred and eighty five (385) HIV seropositive subjects, 77(19.8%) were positive for anti-CMV IgM antibodies.

IgG Avidity Index and IgM relationship in the differential diagnosis of primary Cytomegalovirus infection from non-primary infection in HIV/AIDS subjects is presented in Table 2 .Out of the 77 IgM positive samples tested for IgG Avidity index, 12(15.6%) were high avid while 65(84.4%) were low avid . There was a significant difference (p= 0.001) between the prevalence of primary infection from non-primary infection with the use of IgG avidity index ELISA.

Discussion

The specific CMV IgM antibodies prevalence was found to be 77(19.8%), this prevalence reported in Bida was one of the highest reported among HIV patients in recent times compared to other reports in other developing countries. The prevalence of  9.52% was reported by Basawarajuet al., (2011) from HIV seropositive patients in Khammam  India, while Ojide et al., (2013) in Benin City reported 7.0%,  Musa et al., (2003) in  Kano reported 13.0% and Akinbami et al.,(2010) in Lagos reported 6.6%.  All reported a lower prevalence compared to our findings in Bida.

Out of the 77 IgM positive sample this study reported 65(84.4%) low avidity and 12(15.6%) high avidity among the subjects,  this agreed well with the report among pregnant women in Kenya (Mainngi and Nyamache, 2014) who reported 11 (20.37%) high avidity and 44(79.63) low avidity out of the 54 IgM positive women.

This study found a statistical difference (P=0.001) between IgG Avidity index and IgM levels for differential diagnosis of CMV primary from non-primary infection among HIV subjects in Bida. However, Chakravaritet al., (2009) observed that CMV active infection might be a marker of extremely severe immunosuppression, which may ultimately lead to fatal outcome in HIV patients and the presence of IgM antibodies may be due to primary infection, reactivation or re-infection by CMV.

Our study have convincingly demonstrates that measurement of CMV IgG avidity is both sensitive and specific method for identifying patients with recent primary CMV infection which is also a less expensive techniques compared to polymerase chain reaction (PCR) for confirmation. Low CMV IgG avidity is an accurate indicator of primary infection of CMV within the preceding 3-4 months while high avidity excludes primary infection (Prince and Nixon, 2014).

The utility of IgM avidity ELISA significantly reduced the prevalence of primary CMV infection with IgM ELISA in this study, in other words primary CMV infections by IgM

ELISA alone was significantly overestimated as only 84.4% CMV IgM are actually primary infection while the rest 15.4%  are non-primary infections.

In conclusion, CMV IgG avidity assay is an accessible method in the differential diagnosis of primary Cytomegalovirus from non-primary CMV infection in HIV patients , this technique is not expensive compared to the highly sophiscated Polymerase Chain Reaction it is highly recommended for use in the confirmation of primary CMV infection.

REFERENCES:

1. Akinbami, A.A, Akamu A.S., Adeyemo T.A., Wright K.O., Dada M.O and Dosunmu A.O. (2010).Cytomegalovirus antibodies Amongst Immunocompromised (HIV) patients at Lagos University Teaching Hospital (LUTH) Idi- Araba, Lagos. Journal of Medicine 11:151-154.
2. Basawaraju, A., Mane, P. M. and Vijayadurga, S. (2011). The reactivation of Cytomegalovirus infection in HIV infected patients. Journal of Clinical Diagnostic Research. 5(4): 749-751.
3. Bonalumi, S.; Trapanese, A.; Santamaria, A.; D’Emidio, L.; Mobili, L.(2011)
4. Cytomegalovirus infection in pregnancy: Review of the literature. J. Prenat. Med. 5, 1–8.
5. Charravarti, A., Kashyap, B. and Matlani, M. (2009). Cytomegalovirus infections; An Indian Perspective. Indian Journal of Medical Microbiology 27: 3-11.
6. Drobyski, W., Dume, W., Burd, E., Knox, K., Ash, R. and Horowitz, M. (1993). Human herpes virus 6 (HHV) Infection in allogeneic bone marrow suppressive role for HHV 6. Infectious Disease 167: 735-739.
7. Egger, S.M. Bade, R.U. (2000)Ender, S.G. Combination of microneutralization and avidity    assays: Improved diagnosis of recent primary human cytomegalovirus infection in single serum sample of second trimester pregnancy. J. Med. Virol.  60, 324–330.
8. Fisher, A. A., Lang J. E., Stoeekel, J. E. and Townsend, J. W. (1998). Handbook for family planning operations research design. Population council.Pp 117-118.
9. Helker, M., Qiu D., Martin M., Linder P., Irish W., Torre –Cisneros J.(2004). Continuous Cytomegalovirus sero-conversion in a large group of healthy blood donors. Voxsanguinis86: 41-44.
10. Hillyer, C. D., Snydman D. R. and Berkman E. M. (1990). The risk of Cytomegalovirus infection in Solid organ and bone marrow recipients. Transfusion.30:659-660.
11. Lazzarotto, T., Spezzacatena, P., Pradelli, P., Abate, D.A., Varani, S.,Landini, M.P (1997).
12. Avidity of immunoglobulin G directed against human cytomegalovirus during primary and secondary infections in immunocompetent and immunocompromised subjects. Clin. Diagn. Lab. Immunol. 4, 469–473.
13. Lazzarotto, T., Gabrielli, L., Lanari, M.; Guerra, B., Bellucci, T., Sassi, M.,Landini, M.P.(2004) Congenital cytomegalovirus infection: recent  advances in the diagnosis of maternal infection. Hum. Immunol.  65:  410–415.
14. Macher, A. M., Reichert C. M., Straus, S. E., Longo D. L., Parrilo J. and Lane H.C. (1983).Death in the AIDS patients: Role of  Cytomegalovirus. New England Journal of Medicine 309:14-54.
15. Manez, R., Kusne, S., Martin, M., Linden, P., Irish, W., Torre-Cisneros, J. (1993).The impact of blood transfusion on the occurrence of Pneumonitis in primary Cytomegalovirus infection after liver transplant. Transplantation 30: 594-597.
16. Maingi Z. and Nyamachi A.K. (2014). Seroprevalence of Cytomegalovirus (CMV) among pregnant women in Thuka, Kenya. BMC Research Notes 7:794- 798.
17. Musa, A.M., Taura D. W., Mukhtar M. D., Koki Y. A. and Adamu S. (2014). Studies on cytomegalovirus among HIV Positive Patients attending Infectious Disease Hospital, Kano State Nigeria. Greener Journal of Epidemiology and Public Health.2(1): 32-36.
18. Ojide, C. K., Kalu, E. I., Nwadike, V. U., Ogbaini-Emovon E. I. and Omoti, C. (2013). Seroprevalence of Cytomegalovirus among HIV– Infected Adult Patients on HAART. International Journal of Tropical Disease and Health 3 (3): 233-241.
19. Prince H.E and Nixon M.L (2014). Role of CMV IgG Avidity testing in diagnosing primary CMV infection during pregnancy .Clinical Vaccine Immunology .22(10); 1377- 1384.
20. Rowshani, AT., Bemelman F.J., Van-Leeuwen E.M., Van-Lier R.A. and TenBerge I.J. (2005). Clinical and immunologic aspects of Cytomegalovirus infection in solid organ transplant recipients. Transplantation79(4):381-386.
21. Vilibic-Cavlek, T.; Ljubin-Sternak, S.; Vojnovic, G.; Sviben, M.; Mlinar-Galinovic, G. The Role of IgG Avidity in Diagnosis of Cytomegalovirus Infection in Newborns and Infants. Coll. Antropol. 2012, 36, 297–300.
22. Zhang, L., Hanff, P., Rutherford, C., Chinchull, C. and Crumpacker, L.(1996). Detection of human Cytomegalovirus DNA, RNA and Antibodies in normal blood donors. Journal of Infectious Disease 171:1002-1006.

Abdullahi Umar

Dermatology Unit, Department of Medicine, Ahmadu Bello University and Teaching Hospital, Zaria.

Adeiza Mukhtar Abdulmajid.

Infectious Disease Unit, Department of Medicine, Ahmadu Bello University and Teaching Hospital, Zaria.

Correspondences to: Dr. Abdullahi Umar, Department of Medicine P.M.B 06, Ahmadu Bello University Teaching Hospital, Shika-Zaria. Email: drumarabdallah@gmail.com

Abstract:

Key-words: *Neurofibromatosis type 1 *malignant peripheral nerve sheath tumours (MPNST) *Autosomal dominant disorder *Paraparesis

Introduction

Neurofibromatosis type 1 was first described by Frederic von Recklinghausen in 1882. It is a relatively common inherited disorder that affects about 1 in 2500 to 1 in 3000 people worldwide with no sex or ethnic variation (1).

Even though it is a benign tumour but malignant peripheral nerve sheath tumours (MPNST) may occur with an incidence of 0.16% in patients with neurofibromatosis type 1, compared to incidence of 0.001% in the general population (2,3). Patients are also at risk of developing other associated tumours such as; optic pathway glioma, glioblastoma, gastrointestinal stromal tumour, breast cancer, leukaemia, phaeochromocytoma etc.1.

Both the peripheral and central nervous system are affected in NF-1. Plexiform neurofibromas affects large nerves, plexi, spinal roots, sympathetic nerves, or small peripheral nerve fibres. This may results in pain and erosion of the neural foramen or cord compression at multiple levels (4).

In NF-1, the thorax and lungs can be affected in several ways such as cutaneous and subcutaneous neurofibromas on the chest wall, kyphoscoliosis, ribbon deformity of the ribs, Intrathoracic neoplasms and interstitial lung disease (5).

This case is reported because of the rarity of MPNST in

association with NF-1 presenting with Intrathoracic tumour and cord compression syndrome.

Case History:

A 33 year old woman with NF-1 on follow up at the neurology and plastic surgery clinic presented with a four months history of progressive paraparesis, associated numbness, paraesthesia and low back pain with no history of trauma to the back. She had associated history of both faecal and urinary incontinence but no history of headache, convulsions or loss of consciousness. There was no visual or hearing abnormalities and there was no family history of neurofibromatosis.

A month prior to presentation, she developed insidious onset of cough, productive of whitish mucoid, non-bloody sputum with associated right sided dull aching chest pain and progressive breathlessness with reduced exercise tolerance. There was no orthopnoea or Paroxysmal nocturnal dyspnoea, no leg swellings and no palpitations. No history of contact with a known open pulmonary tuberculosis case and was never treated for pulmonary tuberculosis before. She had history of high grade intermittent fever with chills and rigors noticed two weeks before presentation. There was no drenching night sweat but she had significant weight loss. No history of cigarette smoking or exposure to organic and inorganic dust. Review of gastrointestinal tract system essentially unremarkable. No family history of neurofibromatosis.

She was not diabetic or hypertensive and there was no risk factor for HIV infection and no previous exposure to ionizing radiation.

Physical examination revealed an acute on chronically ill-looking young woman in respiratory distress (tachypnoeic, oxygen saturation of 88% on room air). She was pale, not cyanosed and had no significant peripheral lymphadenopathy.Multiple cutaneous neurofibromas, cafe´-au-lait macules, and skinfold freckling with plexiform neurofibromas were seen. [Figure 1]

Her pulse rate was 120 per minute regular, full volume, blood pressure was 100/70mmHg supine with distended external jugular veins. Apex beat was displaced at 6th LICS lateral to mid-clavicular line, non-heaving and heart sounds were normal first and second with no murmurs. Respiratory rate was 32 cycles per minute with reduced chest movement on the right hemi thorax and trachea deviated to the left. Percussion note was stony dull over the right hemi thorax where breath sound was vesicular with reduced intensity. Coarse crepitations were heard over the left infra-clavicular and infra-axillary areas.

On examining the central nervous system she had spastic paraparesis with hypereflexia and ankle clonus. There was loss of sensation to both fine and crude touch and a sensory level at T4/T5. She has non tender hepatomegaly with no ascites.

Laboratory results showed normal blood sugar and electrolytes. HIV antibodies were negative and full blood count was normal. Pulmonary tuberculosis screen were negative (sputum AFB, gene xpert/RIF). Chest x-ray showed right sided pleural effusion with a mass on the right hemi thorax. Chest CT scan could not be done for financial reasons. Abdominal/chest ultrasound scan done showed hepatomegaly of 16.2cm with a well-defined right lower lung mass, right sided pleural effusion and anterior abdominal wall subcutaneous nodules. [Figure 2]

Diagnostic pleural fluid tap showed a haemorrhagic and exudative pleural effusion. A biopsy of the Intrathoracic mass was taken and histology report showed a cellular tumour growing in sheets with vague fascicular pattern, it was composed of markedly pleomorphic round to spindle cells including giant forms with oval to wavy nuclei, prominent nucleoli, coarse chromatin pattern and scanty to moderate cytoplasm. Mitoses (9p10hpf) and foci of necrosis were noted. The stroma was scanty to moderate and fibromyxoid. These findings were suggestive of malignant peripheral nerve sheath tumour (MPNST). [Figure 3] With these findings, a diagnosis of neurofibromatosis type 1 associated with malignant peripheral nerve sheath tumour of the thoracic spinal nerves was made complicated by a malignant pleural effusion and superior vena caval obstruction.

She had supplemental oxygen therapy and intercostal tube drainage of the effusion but died of progressive respiratory failure while awaiting the histology report.

Comment

Format of referencing 

Havard or APHA style?

Discussion:

Neurofibromatosis type 1 or von Recklinghausen?s disease is an autosomal dominant disorder of the ectoderm and mesoderm characterized mainly by the presence of neurofibromas, cafe-au-lait spots, and pigmented hamartomas in the iris (Lisch nodules) (6). According to national institute of health (NIH) consensus criteria for diagnosis of neurofibromatosis type 1 , Two or more of the following clinical features are sufficient toestablish a diagnosis of neurofibromatosis type 1:? Six or more cafe-au-lait macules (>0.5 cm at largestDiameter in a prepubertal child or >1.5 cm in post-pubertalIndividuals), ? Axillary freckling or freckling in inguinal regions, ? Two or more neurofibromas of any type or one or more plexiform neurofibromas, ? Two or more Lisch nodules (iris hamartomas), ? A distinctive osseous lesion (sphenoid wing dysplasia, long-bone dysplasia), ? an optic pathway glioma, ? A first-degree relative with neurofibromatosis type 1 diagnosed by the above criteria (1).

The NF-1 gene is located on chromosome 17q11 which functions as tumour suppressor gene and encodes for the protein Neurofibrin (6), which is predominantly expressed in neurons, Schwann cells and astrocytes.

Oncogenic mutations in Ras genes or inactivation of NF-1 gene leads to a permanentstimulation of a cascade of signals and excessive cell division, leading to the formation of tumours (7). The mutation of tumour suppressor NF-1 gene increases the risk for the development of other malignancies mainly derived from the neural crest such as malignant schwannoma, neurofibrosarcoma, intracranial glioma, and phaeochromocytoma in patients with NF-1 (6).

Malignant peripheral nerve sheath tumour (MPNST) as seen is this case is a rare and aggressive sarcoma that arises from the nerve sheath or shows features of nerve sheath differentiation, with an incidence of 0.001% in the general population as against incidence of 0.16% in patients with neurofibromatosis type 1 (2, 3).These tumours represent about 3?10% of all soft-tissue sarcomas, with majority of cases seen in individuals with neurofibromatosis type 1 (1). MPNST can arise anywhere within the body but more commonly seen in the extremities, head, and neck (2, 3). The index case had Intrathoracic and spinal involvement though in the literature, Intrathoracic MPNST is uncommon (8).

The cumulative lifetime risk of developing MPNST in a patient with NF-1 is about 8?13%9. The risk of developing this severe form of sarcoma is increased 20-fold in the area of an existing internal plexiform neurofibroma (1), which was noticed in this case.

Other risk factors for development of MPNST include previous radiation therapy which our patient has never had, and large germline mutations encompassing the entire NF1 gene (micro deletions) (1).

The index case met 4 of the 6 major diagnostic criteria for NF-1 though only 2 are needed namely; >6 cafe´ -au-lait macules, skinfold freckling, 1 plexiform neurofibromas and >2 cutaneous neurofibromas. She didn?t have osseous dysplasia, iris hamartomas and optic glioma. On the other hand, neurofibromatosis type 2 (NF-2) is characterized by bilateral vestibular schwannoma. Both conditions have high de novo mutation rates and carry a high risk of tumour formation, though NF-1 carries a higher risk of high-grade malignancy (10).

The median age for sporadic MPNST is between 30 and 60 years, and that for NF-1-associated MPNST is seen in much younger age group between 20 and 40 years (11), as seen in this case at 33 years.Patients with MPNST presents

with a rapidly enlarging mass that may be painful or cause local neurological symptoms such as weakness or paraesthesia. This case presented with progressive bilateral lower limb weakness that may be related to spinal cord compression by MPNST, but a dumb bell neurofibroma may also compress on the spinal nerves.

Exertional dyspnoea, cough and chest pain that may be related to the rapidly enlarging Intrathoracic tumour with pleural effusion, mediastinal shift and superior vena cava obstruction as shown in the above index case are obvious manifestations of Intrathoracic MPNST associated with NF-1.

Magnetic resonance imaging is considered the most useful imaging modality which helps to characterize the anatomical extent of the tumour for subsequent surgical intervention. Flourodeoxyglucose-positron emission tomography (FDG-PET) has been employed to differentiate between benign neurofibromas from MPNST in patients with NF-1, with relatively high specificity (12). However these advanced imaging modalities were not available or affordable in our centre.

Histology remains the most readily available diagnostic tool for MPNST, even though the histologic features may rather be non-specific (13). Histological features of MPNST may composed of monotonous spindle cells arranged in intersecting fascicles. Pleomorphic variants also exist as seen in the index case. Compared to benign neurofibromas, MPNST usually shows a marked increase in tumour cellularity, pleomorphism, and mitotic activity and have a more organized cellular growth pattern, with less extracellular matrix material (13).

The most effective treatment for MPNST requires complete surgical excision. Even with surgical excision, 5-year overall survival rates are poor and MPNST represents a substantial cause of mortality in individuals with NF-1 (1). Radiotherapy may delay recurrence but has little effect on long-term survival (14). Doxorubicin can be used as single-agent standard chemotherapy for soft tissue sarcoma in adults, but has a poor response rate of 12% (15). Furthermore, the presence of NF-1 is one of the most significant poor prognostic factors with use of chemotherapy against MPNST (16).

The late presentation of this case and the extensive nature of the disease at presentation contributed to the poor outcome.

References:

1. Angela C H, David H G. Neurofibromatosis type 1: a multidisciplinary approach to care. Lancet Neurol2014; 13: 83443.

2. Yamaguchi, U., Hasegawa, T., Hirose, T., Chuman, H., Kawai, A.et al. Low grade malignant peripheral nerve sheath tumour: varied cytological and histological patterns. J ClinPathol 2003; 56: 826?830.

3. Rekhi B, Abhijeet I, Rajiv K, Maria AD, Rajesh D, Nirmala A. J. Malignant peripheral nerve sheath tumors: Clinicopathological profile of 63 cases diagnosed at a tertiary cancer referral center in Mumbai, India. Indian J PatholMicrobiol. 2010; 53: 611-618.

Daniel, L.A.

Principal Education Officer, Department of Biology, Government Science Secondary School, Kaltungo,Gombe State Nigeria

Kahya, S.S.

Department of Molecular Biology Root Crop Research Institute Vom, Plateau State Nigeria.

Lasekum, E.P.

Assistant Chief Medical Laboratory Scientist, School of Medical LaboratoryScience, Gombe State College of Health Sciences and Technology,Kaltungo.

Yaro, M.B.

Department of Biology, School of Sciences, College of Education Hong Adamawa State Nigeria

All correspondence to: elasekum@gmail.com

Abstracts

KEY WORDS: Prevalence, Urinary Schistosomiasis, Community, Shonge,Shongom, Gombe-Nigeria.

INTRODUCTION

Urinary Schistosomiasis is caused by Schistosoma haematobium whose intermediate host is the freshwater snail of the genus Bulinus(WHO, 2013).Usually called bilharzia which rank second to malaria in terms of socio-economic and public health significance in tropical and sub-tropical areas. It is the most prevalent of the water-borne diseases, with a very great risk on the health of rural populations (Biu et al., 2000). Previous studies on the incidence and surveys of Schistosomiasis in Nigeria, described the prevalence in some towns and communities varies between 32% to 98% according to Adewumiet al., (1990); Emejulu et al., (1994); Edungbola et al., (1998); Adeyeba and Ojeaga, (2002).The geographical distribution of the infection and of severe morbidity is restricted to specific areas. Several studies indicate that a lot of research work need to be carried out to determine new endemic areas and to harness the predictive potential of schistosomiasis to enhance cheaper community diagnosis and preventive measures(Mbata et. al., 2009). The disease transmission occurs where freshwater snail intermediate host, the genus Bulinus is present and where there is contact between the human population and infested water (Uwaezuoke, 2005). Subjects who lived close to bodies of water are more exposed and therefore, more vulnerable to Schistosoma haematobium infection than those who lived further from the water (Ugbomoiko, et al., 2010). A study conducted in Ebonyi state revealed that 235(26.8%) people were infected with Schistosoma haematobium (Chigozie, et al., 2007). A study conducted in Zuru, Kebbi State, Nigeria (Daniel 2001) reported that prevalence rate was highest in males (67.5%) and (52.2%) in females. Nale et al (2002) reported a prevalence rate 19.4% in males, which was significantly higher than prevalence observed in females 3.11%. A study of prevalence and intensity of urinary Schistosomiasis conducted in Bauchi, showed that infection rate were higher among children under 15 years than adult, and males (25.2%) had higher infection rate than females (21.2%) (Elekeh, 2006). In another study Biu et al., (2000) reported on the incidence of the disease in north-eastern Nigeria but there was little data available in Shonge area, Shongom Local Government Area.

This study was to investigate the prevalence of urinary tract Schistosomiasis in Shonge, Shongom Local Government Area, Nigeria, and to determine the predisposing factors of the disease, thus to identify methods of preventing and controlling the disease.

MATERIALS AND METHODS

The study was conducted in Shonge, Shongom Local Government Area, Gombe State -Nigeria, which comprises of three (3) communities-Burak, Bangunji and Kushi. The relief of the area provides uplands, hills and mountains that give rise to streams and vegetation of the savannah. It occupies a fertile land, which has much rainfall and used for agricultural purposes, several streams, ponds, and few earth dams mostly used for dry season farming. However, the streams overflow their banks during the rainy season and dries up during the dry season. The populace make use of streams, ponds and dams as their source of water for domestic purposes which as a result become infected and re-infected.

The General Hospital Kaltungo, Kaltungo LGA Gombe State, Nigeria approved the procedures for sample collection. The information on age, sex, community, occupation, and source of water supplies was obtained from the subjects.

A total of 300 urine samples comprising samples from adult males and adult femaleseach were collected randomly and examined from January to June 2015. The urine samples were collected in 50ml universal sample bottles between 10am and 2pm (WHO, 2003) and taken to General Hospital Kaltungo laboratory in a box of Ice pack for analysis within 1 hour. Each urine sample was observed for evidence of turbidity. Urinalysis was carried out with reagent strip meditest combi-9 manufactured by Machery-Nagel according to themanufacturer’s instructions to detect haematuria and proteinuria in the urine samplesand subsequent color change compared with the standard color scale provided by the manufacturer (Nnoruka, 2000).

The samples were analyzed by centrifugation and sedimentation techniques (WHO, 1991).Twenty milliliters (20ml)of urine samples were centrifuged for 2 minutes at 2000rpm and the deposit of each sample bottle collected while the other samples were allow to sediment for about 1hour. The deposit were separately examined microscopically with x10 and x40 objectives to identify Schistosoma haematobium ova or egg(s) which appears as golden yellowish and elliptical in shape with a terminal spine. The urine samples containing ova or egg(s) of Schistosoma haematobium and without eggs were recorded. The data obtained were analyzed using Chi-Square statistic.

Water reservoirs, streams and rivers were visited in all the three communities to search for intermediate host snails in these areas. Snails found were handpicked and put in small plastic bucket, transportedimmediately to the laboratory

anddissectedfor sporocystafter exposure to sunlight for about 3hours (Emejuluet al., 1994).

Results

The result of this study shows that out of the 300 urine samples examined from three wards in Shonge, Shongom LGA, Gombe State, the overall number of people infected with Schistosoma haematobiumeggs was 84 (28.0%)

The prevalence of Schistosoma haematobium in the urine samples among the communities, 100 urine samples were examined in each community. Table 1 indicates that Bangunji had the highest infection rate of 37 (12.3%) followed by Burak with rate of 30(10.0%) while Kushi 17 (6.0%) was found to be the least infected. Chi square analysis indicated that there was significant differencein infection betweenthe communities (p< 0.05).

Table 2 shows the prevalence of infections in relation to gender, which indicated that 58(19.3%) were males while 26(8.7%) were females and there was a significant difference in infection between sexes (p<0.05).

The highest prevalence rate of infection was recorded among subjects belonged to age group 30-35, 36(12.0%) years old, while the least rate of infection was observed in those within age bracket 40-above, 2(0.6%) years old. Others were 18-23 years old with prevalence rate 14 (4.7%), 24-29 years old had the prevalence infection rate

Table 1: Incidence of Schistosoma haematobium among Communities in the Study Area

Table 2: Prevalence of Urinary Schistosomiasis in relation to Gender

(66.7%) in age group 30-35 years old in males and females respectively (Table 3).

Table 4 showed the distribution of urinary schistosomiasis in relation to occupation which revealed that farmers 57(19.0%) were mostly infected by Housewives 18(6.0%).Civil servants 5(1.7%) and others 4(1.3%) who did not specified their occupations had the least incidence rate of infection due to Schistosoma haematobium. There wassignificant difference in infection between the inhabitant occupation (p<0.05).

The prevalence of infection in relation to sources of water supply (Table 5) indicated those that used Dam 42(14.0%)

Table 3: Distribution of Urinary Schistosomiasis among study subjects in relation to Age group

21(7.0%), 36-41 years old with 11(3.7%)years old (Table 3). Statistical analysis shows that there was significant difference in infection between age group (p <0.05).

The distribution of age group and gender among inhabitants of the study area shows that high infection rate (47.4%) was found in age group 36-41 years old and had the highest infection rate followed by those that used stream 40(13.3%) and Borehole 2(0.6%); those that used pipe borne had no infection. There was a significant difference in infection between sources of water supply (p<0.05).

Table 4: Urinary schistosomiasis among inhabitant in the study area in relation to occupation

Table 5: Urinary Schistosomiasis in relation to sources of Water Supply

Bangunji had (12.3%) infection rate this could be due to their closeness to streams and other water bodies that came from the highlands in the area. Subjects who lived close to bodies of water or irrigation canals were more exposed and therefore, more vulnerable to urinary schistosomiasis than those who lived further from the water (Ugbomoikoet al., 2010).

Infection rates in the area varied with their ages; age group 30-35 years had the highest incidence rate (12.3%). This age group formed part of the workforce in the study area which was farming. This might be associated with frequent water contact since ages engaged in activities that involve

Discussion and conclusion

The results of this research indicated Schistosomahaematobiuminfection was prevalent in the study area with an overall incidence rate(28.0%), males(19.3%) were more infected than females(8.7%), this result agrees with Naleet al., (2001) who reported a prevalence of 19.4% in males which was significantly higher than prevalence observed  in females (3.11%). The study supports a number of previous reports that have consistently shown Schistosoma heamatobium infection endemicity in Nigeria is on the increase particularly in rural areas (Okoli, et al., 2006).

Bangunji had (12.3%) infection rate this could be due to their closeness to streams and other water bodies that came from the highlands in the area. Subjects who lived close to bodies of water or irrigation canals were more exposed and therefore, more vulnerable to urinary schistosomiasis than those who lived further from the water (Ugbomoikoet al., 2010).

Infection rates in the area varied with their ages; age group 30-35 years had the highest incidence rate (12.3%). This age group formed part of the workforce in the study area which was farming. This might be associated with frequent water contact since ages engaged in activities that involve contact with water. Prevalence rate in age group 42-above (0.6%) was low in the study area this could be likely that they were ageing and would venture going near such water bodies to get infected with Schistosoma haematobium. The low prevalence in age people may be due to progressive increase in the level of natural acquired immunity against Schistosoma haematobiumless water contact (Dawetet al,.2012).The high incidence (47.4%) in age group 36-41 years (66.7%) in 30-35yeares were found in males and females respectively, this does not agree with (Ombugadu, 2012).

The occupation of the inhabitants of the three communities showed greater influence on the prevalence of infection, farmers and housewives had (19.0%) and (6.0%) respectively. This could partly be due to the constant exposure to contaminatedwater bodies on their farmlands at work or on their ways to farm as they cross the streams .Also housewives go out to fetch water and wash household utensils in the slow moving stream which might expose birth to infection withSchistosoma haematobium.This does not agree with Olusegunetal., (2011) who reported the heights prevalence of 0.7% in artisan which there was noinfections among household in HIV positive patient attending university of Benin teaching hospital Benin City Edo state, Nigeria.

The incident rate of e infection in civil servant study area was low (1.7%), The low infection rate among civil servant could associated with fact that neither go to farms nor streams because they have alternative means of livelihood. This contradicts (Pukumaet al., 2006) who reported a high prevalence 38.9% among civil servants or shelleng town Adamawa state, Nigeria.

Those that used stream (13.3%) were more infected, followed by those that used dam (14.0%) astheir sources of water supply, this could be that the inhabitant of the community used stream water and dam for domestic activities such as drinking and washing of pots and dishes or engaged in agricultural and recreational activities, thereby had contact with infested water bodies (Chigozieet al., 2007).

Conclusion

In conclusion, the study has indicated high prevalence of Schistosoma haematobium infection among the inhabitant in the study area. The high infection recorded could probably be due to reasons like unhealthy environment, socio-cultural practices, poverty, that the inhabitants in study area were ignorant of the mode of transmission of this disease and improper sanitation increase contamination of area and so infecting the available snail host.

The overall number of people infected with schistosomahaematobium ova was (28.0%) in the study area. Infection may be possibly higher because it was only a single sample obtained from each person and very light infections might have been missed. In a study (Engels, 1997) suggested duplicate samples on three or more days increase the chances of finding ova.

Prevention of Schistosoma haematobuimincludes basically the destruction of intermediate host snail; infective stage with molluscicides and larvicides respectively (Nwagu, 1998). The control of snail intermediate host would reduce the rate of transmission, thereby reduction in prevalence of infection in the study area. Subjects in the study area should be educated by the Local government health care workers on proper means of waste disposal, and construction of sanitary latrines in their homes so as to reduce the act of urinating or defecating in the open surroundings. The communities in the study area should embark on routine environmental sanitation.

REFERENCES

1. Adewumi, G. O., Furu. P., Christensen, N. O. &Olorunmola, F. (1991). Prevalence ofUrinaryschitosomiasis in North-western Nigeria. Journal of Tropical Medicine and Parasitology, 42 (4), 332- 334

2. Adeyeba O.A. and Ojeaga S.G. (2002): Urinary Schistosomiasis and Concomitant urinary tract pathogens among school children in Ibadan, Nigeria. African Journal of Biomedical Research.5:103-107.

3. Biu, A. A., Nwosu, C. O. &Akuta, A. (2000). The incidence of humanschistosomiasisin Maiduguri, Northern Nigeria. Bioscience Res. Comm., 12(1), 9-11

4. Chigozie, J., Patrick, G., Cletus, D.C., Reuben, O. (2007) Urinary schistosomiasis amongschool children in Ebonyi state Nigeria. The international Journal of LaboratoryMedicine
5. Dawet, A., Benjamin, C.B., Yakubu, D.P (2012) Prevalence and intensity of Schistosoma haematobium among residents of Gwong and Kabong in Jos in North Local Government Area, Plateau state, Nigeria. International Journal of Tropical Medicine7(2) 69-73

6. Emejulu,A.C., Alabaronye F.F., Ezenwaje, H.M.G and Okafor F.C. (1994)Investigation into the Prevalence of Urinary Schistosomiasis in the Agulu Lake Area of Anambra State, Nigeria. Journal of Helminthology. 68: 119-123.

7. Edungbola, L.D, Asolu S.O, Omonisi, M.K. and Ayedun R.A. (1998): Schistosomahaematobium Infection among school children in the Babana District Kwara State.African Journal of Medicine.17: 187-193.

8. Mbata, T.I., Orji M.U and Oguoma V.M.(2009). High Prevalence of Urinary Schistosomiasis in a Nigerian CommunityAfrican Journal of Biomedical Research,Volume 12,

9. Nale, V., Galadima, M. and Yakubu, S.E., (2001) Index of potential contamination of urinaryschistosomiasis in five settlement near River Kubanni in Zaria, Nigeria.NigerianJournal of Parasitology 24:95-101

10. Nnoruka V.N. (2000). Epidemiological study of urinary Schistosomiasis and the physico-chemical characteristics of the transmission sites in Imo State, Nigeria.Nigerian Journal of Parasitology.21:21-32

11. Okoli,C.G., Anosike, J.C., and Iwuala, M.O.E. (2006) Prevalence and distribution of urinaryschist-osomiasisinOhaji/Egbema Local Government Area Imo state NigeriaJournal Am. Science 2:46-49

12. Olusegun, A.F., Ehis, O.C. and Richard, O. (2011) Proportion of urinary schistosomiasisamong HIV infected subjects in Benin City Nigeria Omen Medical Journal26:175-177.

13. Ombugadu, R.J. (2001) A study of Schistosoma haematobium among the inhabitant ofUdege community in Nasarawa state Nigeria Biol., Environ.Scien., Journal of TropicalMedicine8:81-84

14. Ugbomoiko, U.S., Ofoezie, I.E., Okoye, I.C and Heukelback, J. (2010) Factors associated with urinary schistosomiasis in two peri-urban communities in South-western Nigeria Annal Tropical Medical Parasitology 104:409 419

15. World Health Organisation (2003): Manual of Basic Techniques for a Health Laboratory.2nd edition World Health Organization, Geneva

16. World Health Organisation (1991): Sedimentation Method. In Basic Laboratory Methods in Medical Parasitology, World Health Organization Geneva. Pp. 33-35.

17. World Health Organisation, (2013): Schistosomiasis, Fact Sheet No 115. Available atwww.who.int/ mediacentre/facesheet

Christian, Serekara Gideon; Koate, Baribefe Daniel

Rivers State University, Nkpolu-Oroworukwo, Port Harcourt, Nigeria.

All correspondence to: serekara.christian1@ust.edu.ng or christian.serekara@yahoo.com

ABSTRACT

KEYWORDS: Menorrhagia, Issue of Blood, von Willebrand factor

1.1       BACKGROUND INFORMATION

The woman with the issue of blood as recorded in the Holy Bible was diseased with an issue of blood twelve years (Matthew 9:20). She probably had suffered a lot in the hands of many physicians and had spent all that she had, but could not get her condition better of, instead it rather grew worse (Mark 5: 26). She had heard of Jesus Christ and when she saw He was near she joined in the throng of people following Him as He made His way to Jarius’ house. She said within her, “If I may but touch his garment, I shall be whole.” (Matthew 9: 21). She came behind Him and touched the hem of His garment (Matthew 9:20). As soon as she touched Him “the fountain of her blood was dried up” and she “felt in her body that she was healed of that plague” (Mark 5: 29). As soon as she touched Him, Jesus Christ immediately knowing in himself that virtue had gone out of him (Mark 5:30) stopped and asked “Who touched me?” Everyone around Him denied it and Peter asked him “Master, the multitudes throng thee and press thee and sayest thou, who touched me?” (Luke 8: 45). When the woman saw that she was unable to hide from the Lord she came trembling and falling down before him, she declared unto him before all the people for what cause she had touched him, and how she was healed immediately (Luke 8: 47). Christ looked on her and told her, “Daughter be of good comfort: thy faith hath made thee whole; go in peace” (Luke 8:48).

1.2 SPECULATIONS ABOUT THE WOMAN WITH THE ISSUE OF BLOOD

Under Mosaic laws a woman with an issue of blood (referring to menstrual or postpartum bleeding) was considered unclean and was “put apart” for 7days. During this time anything she lay on or sat on was considered “unclean” meaning that if anyone touched one of those

things he would have to wash his clothes and bathe in water to become clean again. Also during this time if any man was sexually intimate with her he was also unclean for 7days and must adhere to the same sort of “setting apart” as a woman (and actually the guidelines for a man who has an issue of blood, or who was unclean were more strict than for women, Leviticus 15:1-30).

The Mosaic law also specified that if a woman had an issue of blood that lasted longer than 7days that all the days of her issue were considered unclean and she must be treated as such (Leviticus 15: 25). This means that this woman had probably been unclean for 12years and that she had to live “put apart” from others for all that time. If she had been married her husband probably would have divorced her as she would have been unable to care for her children or for others without making them all unclean. Her unclean status would also have meant that she was probably unable to attend the temple or other worship services.

1.3 THE PROBABLE CAUSE

One of the probable causes of the issue of blood could be “prolonged menstruation” and today this woman probably would have been diagnosed with menorrhagia, which is an abnormally heavy and long menstruation that causes enough cramping and blood loss, such that it makes normal daily activities impossible. It may also be right in calling it a “hemorrhage” because the amount of blood lost is significant, such that it is enough to fill a maxi pad at least every hour for several weeks (not to mention 12 years!).

In the Holy Bible, it was reported that the woman spent all of her living upon Clinicians who could not help her solve her problem. Today, Clinicians are usually able to treat or control menorrhagia with hormone pills but in severe cases hysterectomy (removal of the uterus) and endometrial ablation or resection (which permanently destroys the entire lining of the uterus) are used to treat it. It is incredible that Jesus Christ was able to heal this woman with only by her touching the hem of His garment!

Abnormal menstrual bleeding in adolescents can be caused by a number of conditions: the most common cause is immaturity of the hypothalamic pituitary ovarian axis, bleeding disorders are another. Assessment of each case with thorough history, physical examination, and laboratory investigations are crucial in reaching the diagnosis. Once a proper diagnosis is made, counselling of the patient and her parents is necessary, follow up and long term therapy in some cases is required (Gillani and Mohammad, 2012).

Phillip et al., 2005, reported an incidence of abnormal platelet aggregation in 45% of women with bleeding disorder. In our study 3 patients (8.6%) had idiopathic thrombocytopenic purpura. Acute idiopathic thrombocytopenic purpura is most commonly seen in the young and is immunological, caused by immune complex containing viral antigens that bind to platelet Fc receptors or by antibodies produced against viral antigens that cross react with platelets. The majority of studies in the West report von Willebrand disease as the most common inherited bleeding disorder leading to menorrhagia whereas studies in South East Asia have found platelet function disorders as the leading inherited bleeding disorder in women with menorrhagia.

Endocrine disorders can cause anovulation producing an environment of unopposed oestrogen. In the absence of progesterone, the endometrium eventually breaks down causing menorrhagia (Foster, 1995). The menorrhagia associated with hypothyroidism responds to thyroid replacement therapy in doses sufficient to correct the other manifestations of the condition.

1.4 THE HAEMATOLOGY OF MENORRHAGIA

Menorrhagia is a common clinical problem, reported by 5–10% of women of reproductive age (Vessey et al, 1992). The investigation of menorrhagia has traditionally focused on the organic pathology of the uterus itself (Oehler and Rees, 2003). Women with menorrhagia frequently undergo intensive investigation, yet in approximately 50% of cases, the cause may not be identi?ed (Oehler & Rees, 2003). However, in the past decade, studies in women with a normal gynaecological examination have shown that a sizable proportion may have an underlying haemostatic defect. In a systematic review of 11 studies involving 988 women, the overall prevalence of von Willebrand disease (vWD) has been reported to be 13% (Shankar et al, 2004). Additionally, an estimated 50% of women presenting with menorrhagia could have a platelet function disorder (PFD) (Philipp et al, 2003).

The reported prevalence of von Willebrand’s disease (vWD) is increased in women with menorrhagia, with current estimates ranging from 5% to 20%. The consistent results of multiple studies suggests that testing for von Willebrand’s factor level should be included in the evaluation of patients with menorrhagia, especially in unexplained cases and prior to any surgical intervention. Although a cyclic variation in von Willebrand’s factor levels has not yet been confirmed, several studies suggest lower levels during menses and the early follicular phase.

Menorrhagia is one of the most common bleeding manifestations of von Willebrand’s disease, as reported by 60–95% of women afflicted with this bleeding disorder. Menorrhagia is typically severe, often resulting in anemia and interfering with quality of life. Despite the frequency of menorrhagia, there is no consensus on optimal management. Although oral contraceptives are frequently prescribed, there are no studies confirming their efficacy using objective measures of response. Desmopressin was associated with an 80–92% response rate in several uncontrolled studies relying on patient assessment of efficacy. However, a small, randomized trial found no significant reduction in menstrual blood flow compared with a placebo. There are anecdotal reports of the successful use of antifibrinolytic agents alone and in combination with other therapies. There are no studies comparing the relative efficacy and safety of the available medical therapies for von Willebrand’s disease associated menorrhagia. Until these studies are completed, treatment should be individualized based on von Willebrand’s disease subtype, patient age, contraceptive needs, and personal preference (Kujovic, 2005).

1.5 LABORATORY TESTING

Haemostatic testing can be performed on specimens collected during the first 4days of the menstrual cycle, based on demonstration of a statistically signi?cant decrease in von Willebrand factor (VWF) levels during that time (Miller et al., 2002). Other laboratory testing should include coagulation factors level and clot screens (prothrombin time, activated partial thromboplastin time, thrombin time, euglobulin clot lysis time, factors II, V, VII, VIII, IX, X, XI, and XIII) as described (Dilley et al, 2001; Miller et al, 2003) and ?brinolytic factors like plasminogen activator inhibitor (PAI)-1 activity and alpha-2-antiplasmin.

1.6 TREATMENT

In patients with severe bleeding associated with haemodynamic changes, blood transfusions are indicated with administration of intravenous conjugated equine oestrogen, 25 mg I/V every 4 hours for up to 24hours. Bleeding usually decreases within 24 hours and then oral oestrogen can be substituted, progesterone is also usually added. In Polycystic ovarian disease, ovaries typically have multiple follicular cysts less than 10mm in size and increased stroma. It is associated with chronic anovulation and hyper androgenism. The primary menstrual irregularity in these patients is oligomenorrhoea, although about 5% may demonstrate polymenorrhoea. This may be temporary in adolescents or may eventually progress to advanced polycystic ovarian disease. Diagnosis is confirmed with altered cycle day-2 luteinizing hormone and follicle stimulating hormone ratio. These patients are given prescribed hormonal pills (Gillani and Mohammad, 2012).

REFERENCES

1. Foster, P.A. (1995). The reproductive health of women with von willebrand disease Unresponsive to DDAVD: result of an international survey. On behalf of the subcommittee on von willebrand factor of the scientific and standardization committee of ISTH Thromb. Haemostat, 74, 784-90.

2. Gillani, S. & Mohammad, S. (2012) Puberty Menorrhagia Causes and Treatment. Journal of Medical Science (Peshawar, Print), 20(1): 15-18.

3. Kujovich, J. L. (2005) von Willebrand’s Disease and Menorrhagia: Prevalence, Diagnosis, and Management. American Journal of Haematology, 79, 220–228.

4. Miller, C.H., Dilley, A., Drews, C., Richardson, L. & Evatt, B. (2002) Changes in von Willebrand factor and Factor VIII levels during the menstrual cycle [letter]. Thrombosis and Haemostasis, 87, 1082–1083

5. Miller, C.H., Haff, E., Platt, S.J., Rawlins, P., Drews, C.D., Dilley, A.B. & Evatt, B. (2003) Measurement of von Willebrand factor activity: relative effects of ABO blood type and race. Journal of Thrombosis and Haemostasis, 1, 2191–2197.

6. Oehler, M.K. & Rees, M.C. (2003) Menorrhagia: an update. Acta Obstetricia et Gynecologica Scandinavica, 82, 405–422.

7. Philipp, C.S., Dilley, A., Miller, C.H., Evatt, B., Baranwal, A., Schwartz, R., Bachmann, G. & Saidi, P. (2003) Platelet functional defects in women with unexplained menorrhagia. Journal of Thrombosis and Haemostasis, 1, 477–484.

8. Phillipp, C. S., Faiz, A., Dowling, N., Dilley, A. & Micheals, L.A. (2005) Age and prevalence of bleeding disorders in women with menorrhagia. Obstetric gynaecology, 105, 61-68.

9. Shankar, M., Lee, C.A., Sabin, C.A., Economides, D.L. & Kadir, R.A. (2004) von Willebrand disease in women with menorrhagia: a systematic review. British Journal of Obstetrics and Gynaecology, 111, 734–740.

10. Vessey, M.P., Villard-Mackintosh, L., McPherson, K., Coulter, A. & Yeates, D. (1992) The epidemiology of hysterectomy: Findings in a large cohort study. British Journal of Obstetrics and Gynaecology, 99, 402–407.

Adamu. L.H.: Department of Human Anatomy, Faculty of Medicine, Bayero University, kano. Nigeria
A.A. Sadeeq; A.O. Ibegbu; S.P Akpulu;: Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University. Zaria. Nigeria.
A.R. Hadiza I.S El-ladan: Department of Human Anatomy, Faculty of Medicine, Kaduna State University. Nigeria.
H.O Kwanashie: Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences.Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: A.A. Sadeeq Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University. Zaria. Nigeria.

ABSTRACT
Mercury is a widespread toxic environmental and industrial pollutant. The present study
was carried out to investigate the possible effect of mercury chloride (HgCl2) on exploratory motor activity (EMA) of adult wistar rats. Twenty four Wistar rats of both
sexes weighing (190-220 gm) were administered with HgCl2 orally for twenty one days at
concentrations of; 12.45mg/kg, 24.9mg/kg and 49.8mg/kg b.wt to group II, III, and IV respectively. While group I served as control which was administered normal saline. Exploratory motor activity (EMA) was evaluated using Montoya staircase test. Brain tissue specimens, representing all groups, were fixed in a Bouin’s fluid and taken for histopathological examination. Complex motor behavior (motor movements coordination) was significantly impaired due to mercury intoxication. Furthermore, histopathological evaluation revealed distinct neurodegenerative changes of nerve cells of cerebellar cortex. In conclusion, these results suggest that intoxication with mercury chloride has potentially deleterious effects on brain as reflected in exploratory motor activity and histopathological observations of the cerebellar cortex.
Keywords; Mercury, motor activity, wistar rats, cerebellar cortex, intoxication.

INTRODUCTION
Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which is transferred in man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Mercury can exist either as elemental, organic and inorganic mercury (WHO, 2003; ATDRS, 1995; Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer, coal combustion (Akagi, 1995; Bjomberg et al., 2011). And majorly from Natural source such as Mercury chloride that is found in higher densities in rocks and volcanic activities which can give half of the HgCl 2 present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (EHD, 2002; Vupputuri et al.,2005: WHO, 2005; Mohan et al., 2011; Berlin, 2006; Nardofy et al., 2000). Mercury and its compounds have been shown to also have effects on the growth, weight, renal system, liver, enzymes, memory and psychological disturbances to mention but a few (Kosan et al., 2001; WHO, 2003; Oliveri, 2000; Valera et al., 2008; Rao, 2001). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (Amin- zaki et al., 1974; WHO, 2005; ATDRS, 2007 and 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employ by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2003 and 2005). The aim of the study is to evaluate the effects of Mercury exposure on exploratory Motor activity (EMA) of adult Wistar rats.

Materials And Method

Experimental Designed
Twenty four (24) adult Wistar rats of both sexes were obtained from the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. Nigeria. The weights of the animals were between 190 – 220g and were housed in polyester cages with wire gauze covering. The animals were allowed to acclimatize for two (2) weeks in the animal house of the Department of Human Anatomy, Ahmadu Bello University, Zaria. Nigeria. Animals were fed with Grower’s mesh brand of the Animal feed which was prepared in pellet form to reduce spillages, the animals were fed 3 times daily while clean water was provided in plastic

Administration of Mercury Chloride
The LD of Mercury chloride was adopted from (ATDRS, 50 (2011). as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the standard LD per kg body weight 50 according to the method. Animals in group I served as control and was given normal saline, while animals in group II, III and IV were administered with mercury chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg respectively of the LD per kg body weight. 50
Animal Sacrifices
At the end of the 21 days of mercury chloride administration, animals were anasthesize using chloroform and the brain tissues were removed by opening through the sutures of the skull using a brain opener. The brain tissue were then transferred into specimen bottle, containing Bouin’s fluid.
Tissue Processing
The brain specimens were extracted placed in Bouin’s fluid for 48 hrs for proper fixation and was processed routinely by embedding in paraffin. Tissue sections (4-5 μm) were stained with haematoxylin-eosin for general features and examined under light microscope (Celestron).
Statistical analysis
Data obtained was expressed as Mean ± SEM (Standard error of Mean). One Way Analysis of Variance (ANOVA) was used to compare the Means difference between and within the groups and a P-value less than 0.05 was considered to be statistically significant. Statistical analysis was performed using EZanalyze v3.0 and a post hoc test of Bonferroni was applied. Chart was produce using Microsoft(R) Excel 2007 for windows.

NEUROBEHAVIORAL STUDIES

Montoya Staircase Test Method
The Montoya staircase test method was used to study the locomotor function and skills in motor co-ordination. This method helps in determining the effects of a test substance on motor function abilities (Montoya et al., 1990 and 1991). The tasks involved forepaw reaching for food pellets located at various steps along the staircase and the time taking to explore the activity cage (Abrous et
al.,1994; Whishaw et al.,, 1986 and 2000). The test was carried out as a measure of Motor coordination shown by muscle action in climbing and exploring the staircase and motor and locomotor function shown by the independent forelimb ability.
Training Time
At the outset of the test, animals were first trained to recognize that there are food pellets along the wells in Montoya staircase for three consecutive days. The animals were able to familiarize themselves with the Montoya staircase and then the animals were deprived of food for 18 hours in order to facilitate their search for food. During the test, each animal was placed on the foot of the staircase which will enable them to climb the stairs in order to reach out, grasp, or retrieve the food pellets and each test session lasted for 5 min, at the end of which the time taken to climb up and down from the basement was recorded as exploratory motor activity (EMA).

RESULT

Motor Activity Test Using Montoya Staircase Test
The results of exploratory motor activity test using Montoya staircase method were shown in Table 1. This showed that there was a non-significant decrease in the mean time taken for the animals to reach the top of the six stairs throughout the 3 weeks of administration. While the animals in group II that received the minimum concentration (12.45mg/kg b.w) of mercury chloride had the mean time taken to reach the stairs increased (P ≥0.05) between the 1st and 2nd week of administration. The increase in the mean time taken was statistically significant (P≤ 0.05) between the 1st and 3rd week, and between the 2nd and 3rd week, respectively as shown in Table 1. The results showed that the time taken for the animals to reach the top of the stairs was significantly increased in both groups III and IV animals. There was significant increase in time taken by the animals between week 1 and 2, between week 1 and 3 and between week 2 and 3, respectively in group III and IV animals.

Table 1: The mean time taken to explore Montoya staircase used for testing motor activity

Histological Observation
The cerebellum of the animals control group (group I) showed cytoarchitecture of the Molecular,
Granular and Purkinje cell layer appearing normal as shown in plate 1.While group II animals showed similar cellular architecture with cells and layers looking normal in plate 2. The histological observations for the group III animals as shown in plate 3 depicts normal molecular and granular layer but there was degeneration of the purkinje cells within the Purkinje cell layer. The same pattern of histological changes was observed in group IV for the purkinje cells with cells showing mark degeneration as shown in plate 4.

Plate 1; A section of the cerebellum of the control group (G1) showing normal orientation of the Molecular layer (ML), Granular layer (GCL), and Purkinje cell layer (PCL) with normal Purkinje cells (PC) (H&E Stain, ×250)

Plate 2; A section of the cerebellum of the group II showing normal orientation of the Molecular layer (ML), Granular layer (GL), and the Purkinje cell layer (PCL) with normal Purkinje cells (PC) (H&E Stain, ×250)

Plate 3; A section of the cerebellum of the Animals in group III showing the Molecular layer (ML), Purkinje cell layer (PCL) with degenerating Purkinje cells (DPC) and some densely populated areas in granular layer (DPA) in the Granular layer (GL) (H&E Stain,×250).

Plate 4; A section of the cerebellum of the animals in group IV showing the normal Molecular layer (ML), with degeneration of the Purkinje cell layer (DPL) and degeneration of the Purkinje cells (DPC) while the Granular layer (GL) showed sparsely granular cells (SGC) (H&E Stain, ×250)

Discussion

The results in the present study showed a significant decrease in motor activity which was shown by the increase in the time taken to reach the maximum attainable distance in the Montoya staircase method. This could be attributed to various concentrations mercury administered. Mercury intoxication was reported to cause a decrease in essentials trace elements copper; that modulates the activity of the neurotransmitter, dopamine which is important in motor function activity. (Gagelli et al., 2011) had reported an association between copper and dopamine concentration that was positive in patients with Parkinson’s disease which was characterized by motor function disorders. The decrease in the level of dopamine was observed in patients with Parkinson’s disease, in which there was loss of smooth and controlled movements (Rasia, 2005; Madsen, 2007). Histomorphological changes in the cerebellum were observed, which can in turn interfere with the motor ability of animals in the experimental group. It has been shown that ingestion of mercury of 220ml affected muscle coordination and physical movement which caused fatigue and tremor. (Dolbec, 2000). It has been shown that neurobehavioral manifestation, affected motor functions which was associated with high and medium dose of mercury exposure especially in movements, grip and balance (Mutter, 2010). Studies also shown that exposure to high concentration of mercury vapor causes tremor initially affecting the hand and then spread to the other part of the body (Dolbec et al.,2000; Yoshida et al.,2011). The cerebellum showed evidence of mercury chloride exposure by manifesting some histopathological changes and these changes include the destruction of the Purkinje cell layer, infiltration of cells in the granular cell layer and the loss of the architecture of the Purkinje cell layers. There was sparse distribution of the Purkinje cells of the Purkinje cell layer. These could result in interference with the motor activity and other motor functions such as loss of fine movement, lost of grasping, maintenance of equilibrium and loss of regulation of muscle tone which are modulated by the spinal cord and brain stem mechanisms involved in postural control. Moreover, it has been shown that neuronal degeneration in the cerebellum can affect the level of copper concentration in the cerebellum and this can affect the action of the neurotransmitter dopamine which is very crucial in motor activity. This is because copper serve as a modulator for the neurotransmitter, dopamine activity. Mutter et al., 2010 had reported decrease in dopamine concentration in the cerebellum of mice ingested with methy mercury chloride for a week which resulted in motor function disorder. It has been shown that mercury intoxication does not show any significant changes in the granular layer of chicken treated with mercury chloride in their food (Wolf et al., 2009; Quirino et al., 2012). This is not in agreement with the findings of the present study which showed infiltration of granular cells of the granular layer into the molecular layer.

Conclusion

Mercury chloride intoxication causes cytoalterations of the cerebellar cortex and also impaired motor activity (exploratory motor activity) in adult Wistar rats.

ACKNOWLEDGMENT

I appreciate Mal. Ado Garba of Ahmadu Bello
University,Zaria. Nigeria for his financial support and to
the technical assistance of the laboratory staff of the
Department of Pathology and Morbid Anatomy, Ahmadu
Bello University Teaching Hospital, Shika, Zaria, Nigeria.

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Ankuma S.J, Joshua A R and Opaluwa S A
Department, of Medical Microbiology Ahmadu Bello University Teaching Hospital Shika- Zaria.
Mary S.
Department of Nursing Services, Ahmadu Bello University Teaching Hospital, Shika-Zaria.
Akpulu S.P.
Department of Human Anatomy Ahmadu Bello University Zaria.

Correspondences to: Ankuma S.J. Department, of Medical Microbiology Ahmadu Bello University Teaching Hospital Shika- Zaria.

ABSTRACT

The study was conducted with the view to determining the occurrence of Chlamydia
trachomatis (C.trachomatis) infection among pregnant women attending antenatal clinic in Sir Yahaya Memorial Birnin-Kebbi, Kebbi state, Nigeria. In the study, Diaspot Chlamydia Rapid Diagnostically swabs test kit which is a rapid chromatographic immunoassay for the qualitative detection of Chlamidia antigens from female cervical swabs was used. Out of 100 subjects recruited for the study, two women within the age group 25-30 tested positive for C.trachomatis antigens, giving a prevalence rate of 2%. The study found that the infection was not endemic in the study area.

Key Word: Chlamydia trachomatis, infection, and pregnant women.

INTRODUCTION

Chlamydiae are small obligate intracellular parasites that were formerly considered to be viruses. However, since they contain DNA, RNA, ribosomes and make their own proteins and nucleic acids,
they are now considered to be true bacteria. They possess an inner and outer membrane similar to Gram-negative bacteria and a lipopolysaccharide but do not have a peptidoglycan layer. Although they synthesize most of their metabolic intermediates, they are unable to make their own ATP and thus are energy parasites (1). Chlamydiae derived their name from the Greek word “Chlamys” meaning “cloak draped around the shoulder”, which describes how the intracytoplasmic inclusions causes by the bacterium are “draped” around the infected cell’s nucleus (2). The family Chlamydiaceae consists of two
genera. One species of Chlamydia and two species of Chlamydophila which are important in causing disease in humans. C. trachomatis causes urogenital infections, trachoma, conjunctivitis, pneumonia and lymphogranuloma venereum (LGV). Chlamydophila pneumoniae causes bronchitis, sinusitis, pneumonia and possibly atherosclerosis, while Chlamydophila psittaci, causes Psittacosis (1). Of the three species, C. trachomatis is the species of interest. It is a sexually transmitted microorganism responsible for a wide spectrum of diseases that include cervicitis, salpingitis, endometritis, urethritis, epididymitis, conjunctivitis and neonatal pneumonia (3), Fitz-Hugh-Curtis syndrome (inflammation of the liver capsule), and trachoma, the world’s leading cause of acquired blindness which is spread from eye-to-hand-to-eye and cause by serotypes A, B and C (4). According to (5), C. trachomatis is one of the most common sexually transmitted bacterial pathogen in the world, causing serious adverse events on women’s reproductive health including complication of pregnancy, pelvic inflammatory disease and infertility. The Centre for Disease Control and Prevention (6) also reported Chlamydia as one of the most
frequently bacterial sexually transmitted disease with over three million new cases estimated to occur annually in the United States.
Infection during pregnancy is associated with adverse outcomes, including fetal loss, premature rupture of membranes, preterm labor, low birth weight, infant mortality, neonatal infection and postpartum endometritis (7, 8). Chlamydia can be transmitted during vaginal, anal, or oral sex and any sexually active person can be infected with the disease. The greater the number of sex partners, the greater the risk of infection. Because the cervix (opening to uterus) of teenage girls and young women is not fully matured and is probably more susceptible to the infection, they are particularly high risk for infection if
sexually active (6). The infection is most prevalent in persons aged 15-24 years (4). Chlamydia is also known as a “silent” disease because the majority of infected people (up to 70%) have no symptoms. If symptoms do occur, they usually appear within 1 to 3 weeks after exposure. Even though symptoms of Chlamydia are usually mild or absent, serious complications that cause irreversible damage, including infertility, can occur “silently” before a woman ever recognizes a problem. Chlamydia also can cause discharge from the penis of an infected man (6, 9). In women, the bacteria initially infect the cervix and the urethral. Women who have symptoms might have an abnormal vaginal discharge or a burning sensation when urinating. If the infection spreads from the cervix to the fallopian tubes, some women still have no signs or symptoms; others have lower abdominal pain, low back pain, nausea, fever, pain during intercourse, or bleeding between menstrual periods. Men with signs or symptoms might have a discharge from their penis or a burning sensation when urinating. Men might also have burning and itching around the opening of the penis (6). According to (4), deaths due to C. trachomatis infection are rare and are caused by progression to salpingitis and tuboovarian abscess with rupture and peritonitis.The most significant morbidity occurs when repeated episodes of Chlamydia lead to obstruction and scarring of the fallopian tubes, resulting in partial or total sterility. Chlamydia is an indirect cause of mortality from ectopic pregnancies. Mortality due to ectopic pregnancy is probably more common than death due to tuboovarian abscess. Chlamydia may also increase the chances of becoming infected with HIV, if exposed (6). Furthermore, Chlamydia death statistics for various regions worldwide as reported by the (10) shows that about 1,000 deaths from Chlamydia in Africa, 8,000 deaths in South East Asia, and about 1,000 deaths in Eastern Mediterranian.

Materials and method

Diaspot Chlamydia Rapid Diagnostic test kit was used mainly for its sensitivity (88.5%), specificity (96.7%) and accuracy (93.7%) relative to Polymerase Chain Reaction (PCR) when used on female cervical swabs. It is also said to detect all known Chlamydia serovars (Package insert, 2015). In this test, antibody specific to the Chlamydia antigen is coated on the test line region of the device. The extracted antigen solution reacts with an antibody to Chlamydia that is coated onto particles. The mixture then
migrates up to react with the antibody to Chlamydia on the membrane and generates a colored line in the test line region. The presence of colored line indicates a positive test while its absence indicates a negative test. To serve as a procedural control, a colored line will always appear in the control line region, indicating that proper volume of specimen has been added and membrane wicking has occurred (Package insert, 2015).

Table 1: Showing occurrence of C. trachomatis infection in relation to number of subjects.

Table 2: In relation to age, all the 2-positive subjects were within the age group 25-30 years

Furthermore, all the subjects of the study were married women (Table-3).

Table 3: Showing occurrence of C. trachomatis infection in relation to marital status.

Specimen collection: Endocervical swabs (ECS) were collected at the maternity unit of Sir Yahaya Memorial Hospital in Birnin Kebbi. As soon as they were collected, they were labeled appropriately and taken to the laboratory for immediate processing.
Results: One hundred women attending antenatal clinic in Sir Yahaya Memorial Hospital Birnin Kebbi were recruited for the study, out of which 2-women were tested positive for C. trachomatis antigen, giving 2% disease prevalence (table-1).

DISCUSSION

The prevalence of C. trachomatis infection as found in this study is 2.0%. The prevalence is much lower than the 5.9% earlier reported by (11) in Gonder, North-West Ethiopia, 5.2% in Dhaka, Bangladesh by (12), 12.2% in Nanjing China, 6.4% in Lima, Peru, 10.4% in Moscow, Russia, 2.5% in Harare, Zimbabwe as reported by (5, 8), and also, 4.7% prevalence in Baltimore, USA. The prevalence is however much higher than the 0.1% reported by (5) in Chennai India. Even though the level of infection as found
by this study appears to be very low (2%), one thing that was evident is the fact that many people have not even heard of the infection before. And because the symptoms if there is, resembles that of gonorrhea, many people may have unknowingly presumed any infection giving such symptoms to be gonorrhea and may have suffered silent morbidity as a result of such ignorance. With respect to age
group, all the positive subjects were in the age group 25-30 years. This finding is slightly at variance with the findings of (13, 14) and several other workers who reported that age group 19-24 years is most at risk of Chlamydia infection. The study however agrees with (8) and who are of the view that factors other than age may impact upon the diagnosis of C. trachomatis in pregnant women and that more comprehensive testing strategy should be considered. On whether untreated C. trachomatis infection could lead to miscarriage, one of the positive subjects, a 28 years old woman who previously had 4-successful births just had a miscarriage in her first trimester, the reason for she came to the hospital. This finding confirms earlier reports by (15, 7 & 16), all linking infection with Chlamydia during pregnancy to early miscarriage or premature birth of the baby.

CONCLUSION

The 2% prevalence obtained in this study suggests that C.trachomatis infection is not endemic in Birnin-Kebbi, the study area. This finding is however not conclusive as factors such as specimen collection may have play a role in the low prevalence rate, as the quality of specimen obtained is of extreme importance. Even though guidelines for specimen collection were given, the rigorous and thorough collection technique that provides cellular material rather than just body fluid may not have been strictly followed. Also, it should be noted that all the women screened were married which has reduced the possibility of having multiple sex partners, and consequently could have influenced the low prevalence rate. A follow up to this study is therefore recommended.

ACKNOWLEDGMENT

The authors sincerely thank the management and staff especially the maternity unit of Sir Yahaya Memorial Hospital, Birnin Kebbi, for permission and support during this work.

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Sheila West, Jonathan Zenilma (2008).
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infection.
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2011). Chlamydia prevention: Challenges and
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Ezeiruaku, F. C. & Baro, J. O.: Department of Medical Laboratory Science, Faculty of Basic Medical Sciences,
College of Health Sciences, Niger Delta University, Bayelsa State, Nigeria.
Okeke, C.U.:Department of Prosthetics and Orthotics, School of Health Technology, Federal University of Technology, Owerri, Imo State, Nigeria.

All correspondence to: Ezeiruaku, F. C., Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Bayelsa State, Nigeria.

ABSTRACT:

Dehyroepiandrosterone (DHEA) and its sulphate (DHEA-S) are the most abundant sex steroid hormones in humans. It is present in the blood of both men and women and can be metabolized by the body into testosterone and androstenedione, or can be changed into the female hormone, oestrogen. Measurement of serum DHEA (S) is a useful marker of adrenal androgen synthesis and its level in the serum of both men and women has been helpful in the diagnosis and treatment of adrenal androgen dysfunction and infertility. Establishment of laboratory reference range is a useful tool for the interpretation of individuals result. This study was designed to establish the normal level of this androgen hormone (DHEA-S) in the serum of apparently health adult persons in Bayelsa state, Niger Delta region of Nigeria with respect to age and gender. This is with the aim to establish the DHEAS hormone ranges and if there is any significant difference in the level of the hormone with respect to age and gender. Enzymes linked immunosorbent Assay (ELISA) method was used to estimate the levels of the hormone. From the result, the level of the serum DHEA-S showed no significant difference (P<0.05) in the gender, but there was a significant decrease with increase in age. The graph slope from the study showed that DHEAS in men decline faster than that of the female with inclining age. The results indicate adrenal insufficiency with increase in age for both male and female.

Key Words: Dehydroepiandrosterone, Hormones, Steroids, Adrenal gland, Androgens, Aging, Reference.

INTRODUCTION
Dehyroepiandrosterone and its sulphate (DHEA-S) is the most important human C-19 steroid (Buttler et al., 2013) and it’s a precursor for androgens and estrogens (Longcope, 1996). DHEA is secreted by the
adrenal gland with small fraction from brain and production is at least partly controlled by
adrenocorticotropic hormone (ACTH). DHEAS is the most abundant steroid in humans with serum concentrations 250 – 500 times higher than DHEA, 100 – 500 times higher than testosterone and 1000 – 10,000 times higher than estradiol (Labine et al., 2004). DHEA-S is desulphated enzymatically to produce DHEA, which is in turn converted into various estrogenic and androgenic compounds. According to Hammer et al., (2005), because the two forms are easily inter converted, it is hard to discuss one without the other and it’s common to refer to them as DHEA (S). Both hormones are albumin bound, but binding of DHEA-S is much higher (Gao et al., 2005). In gonads and several other tissues, most notably skin, steroid sulfatases can convert DHEA-S back to DHEA, which can then be metabolised to stronger androgens and to estrogens (Maeda et al., 2002). However, only the non-sulphated (free) DHEA shows biological activity. Dehyroepiandrosterone (DHEA) and its sulphates are
synthesized and secreted primarily by the Zona veticularis of the adrenal cortex (Chadwick et al., 2005). The adrenal gland is the sole source of these steroids in women whereas in men, the testes secrete 5% of DHEAS and 10-25% of DHEA (Bird et al., 1984). DHEA and DHEAS serve as the precursors to approximately 50% of androgens in adult men, 75% of active estrogens in premenopausal women and 100% of active estrogens in post menopausal women (Longcope, 1996). Circulating levels of DHEA(S) decline after until about the age of five, and then start to rise a few years before sexual maturation begins. Levels peaks around the age of 20 to 30 years and then decline to only 20-30% of peak levels by the age of 70 to 80 years (Kahonen et al., 2000). Because of the coincidence between the natural decline of DHEA(S) levels with age and the onset of diseases associated with aging process, a great deal of research has been directed into the examination of the roles that both hormones play in the body, and to the possibility of supplementary levels to slow or reverse the aging process. These studies have severally reported widespread effect for the hormones, but a molecular mechanism of action has not yet been isolated for either DHEA or its sulphate form. It remains unclear therefore, whether either compound has a physiological role other than serving as a precursor molecule (Perrini et al., 2005). Recent views point out that this lack of understanding about the mode of action of the two hormones along with the problem in the design and analysis of certain studies; have led to discrepancies among the findings.
The test for DHEA(S) is performed widely these days to check the function of the two adrenal glands. This is done in women who show signs of having excess male hormones. Some of these signs are male body changes, excess hair growth, irregular periods and problems associated with becoming pregnant, low Libido and not having sex satisfaction (Perrini et al., 2005). Serum DHEA levels may be increased in the following conditions, polycystic ovary syndrome (PCOS), Androgen Secreting Adrenal Adenomas, Adrenal insufficiency and post Menopausal Vulvovaginal atrophy (Genges, 2004). DHEAS has appeared to be useful in the retardation of the aging process in humans. DHEA levels can become a
standard diagnostic tool in the prediction of disease, mortality and life span. Therefore DHEA levels are directly related to the probability of dying in humans. This has led to its widespread use in so many countries (Leowattana, 2004). Many methods apply in the laboratory measurement of DHEAS in this part of the world. These include competitive binding assays (Lewis et al., 1996), chemiluminescence immunoassays (Elmlinger et al., 2002), Radioimmunoassay (Remer et al., 1994), measurement of serum or plasma DHEAS using the enzyme immunoassay (Dhar et al., 1985) and the Chromatography systems (Nakajima et al., 1998). All the methods however have its drawbacks and in this study of DHEA-S, the Enzyme linked immunosorbent assay (ELISA) method was used because of its cost effectiveness and availability. It is a good idea to be a partner in the management of our hormonal health. The more we know about it, the better off we will be; one thing that can be of enormous assistance in understanding our hormonal treatment and hormonal drug administration is to simply know what the normal reference ranges are for the various hormones. It becomes necessary for every laboratory and regions with regard to race to establish its own normal reference ranges, utilizing a particular method of assay. This makes effective interpretation of patient’s results, cum management of various hormonal disorders.

Materials/methods
Study area:
The study was carried out in Bayelsa State, Niger Delta region of Nigeria. The state covered an area of about 8,150 sq meters and with a population of about 1.7 million people (Nigeria census, 2006) of which 43% are adults. Blood samples were collected from subjects and volunteers from the Niger Delta University environment, Southern Ijaw Local Government area, Yenagoa municipal area and its environs.
Study population:
A total of one thousand, five hundred (1500) subjects were used for this study. This was distributed into four age groups of 18-30 , 31-50 , 51-70 with 200 subjects each and >70 years with 150 subjects for both male and female. The sex and age of the subjects were obtained through the administered questionnaire. The individuals excluded from the study were subjects identified with different endocrine dysfunction (Stephen et al., 2008) and those taking DHEA drugs and vitamins or supplements that contain DHEA/DHEAS for certain reasons. The apparently healthy subjects were selected for the study Sample collection and preparation: About 5.0ml of blood were collected from each subject using the standard venepuncture technique. The samples were discharged into a clot activator tube and centrifuged for 10minutes at 3,000 rpm. The serum collected was stored frozen at -200c and analysis was done within 10 days of collection. The subject’s age and gender were noted after collection with history of having endocrine disorder and or on vitamins/supplements containing DHEA(S). The essence and details of the study were explained to the subjects and informed consent was obtained from them before sample collection. Ethical approval was gotten from the ethical committee of the University for this Study.
Sampling method:
To achieve this reference range establishment, a nonrandom sampling of the different subjects from the
population was used with respect to age and gender (Lumsden and Mullen, 1978; Alex et al., 2010; Mikael, 2014). The questionnaire administered took into consideration the subjects with various endocrine dysfunctions as established. The study was carried out between April, 2012 and May, 2015.
Method of assay:
The study was carried out with a method that has been established to give a precise range of results in hormonal assay (Amballi et al., 2007). The enzyme linked immunosorbent assay (ELISA) method was used for the Dehydroepiandrosterone sulphate hormone estimation (Dhar et al., 1985). The Elisa test is used on the principle of solid phase enzyme linked immunosorbent assay, where the antibody to be measured is incubated with specific antigen coupled to a solid phase (Uotila et al., 1951., Peter et al., 2001). The product kit was acquired from Micro Well Laboratories, USA.
The components of the ELISA were specifically designed to analyse the DHEAS hormone. It applies to in-vitro quantitative determination of the hormone concentration in serum (Georges, 2014). The DHEA-S measurement was preferred because of its stability in the blood and a better marker in clinical practise because it has a longer half life with no circadian variety.
Statistical analysis:
In order to establish the range of reference values for this study, the Gaussian techniques was employed. The data are expressed as mean + standard deviation and normal ranges. Correlation between the groups studied was tested using the tool of ANOVA. The standard t-test; pair two samples for mean was used. 95% confidence level (P<0.05) were used and considered significant.

RESULTS

The results of the Serum DHEA-S levels in the adults male and female in Yenagoa, Niger Delta region of Nigeria is as Presented in the tables and graphs below.

Table 1: The Mean+ S.D serum DHEA-S in the adult female and their ranges with respect to age groups.

Table 2: The Mean+ S.D serum DHEA-S in the adult male and their ranges with respect to age groups.

DISCUSSION

Dehydroepiandrosterone sulphate (DHEAS) has been described as a fountain of youth in that it appears to be the hormone that retards the aging process in humans (Leowattana, 2004; Chadwick et al., 2005). This has lead to uncontrolled use of the DHEA-S all over the world, particularly by the aged. This study was carried out to establish the normal ranges in adult men and women in Yenagoa, Bayelsa State, Niger Delta Region of Nigeria because of its clinical implication in fertility treatment. Most often the test is performed to check the function of the two adrenal glands which is the major sources of androgens in humans. The interpretation of medical laboratory data is an example of decision making by comparison. Therefore, reference values are needed for all tests performed in the clinical laboratory, not only from healthy individuals but from patients with relevant diseases. This is because a patient’s laboratory result simply is not medically useful if appropriate data for comparison are lacking. Data collected during the medical interview, clinical examination and supplementary investigations according to Mikael, 2014,
must be interpreted by comparison with reference data, which might be based on reasoning using advanced knowledge of normal and pathologic anatomy, physiology, biochemistry and other areas of medical sciences. The results obtained from the study (tables 1, 2,) showed that the serum DHEA-S levels declines with inclining age. The statistical test of significance at (P<0.05), 95% confidence level showed no significance difference between the age bracket 18-30 and 31-50. There is a significant decrease with this two age groups and 51-70 and greater than 70 years. The serum DHEA-S values for men were statistically not significantly different from that obtained for women at this confidence level when the age groups were matched. The graph slope from the study showed that DHEAS in men decreases faster than that of the female. This study is in comparison with the work done by Nafziger et al., 1998; Kahonen et al., 2000 and Perrini et al., 2005 that showed the declining levels if DHEA-S with age. The decrease in DHEA-S with age might be as a result of decrease muscle mass, bone mass and adrenal insufficiency (Elmlinger et al., 2003). A gradual decline throughout life result from “adrenopause” contributing few percentage (Nakamoto, 2006) (Etieme-Emile et al., 2000). The skin which is also known to produce DHEA-S becomes elastic (skin elasticity) as one age leading to diminished DHEAS levels (Nafziger et al., 1998).

CONCLUSION

Thanks to the goodwill of the subjects and volunteers used for this reference study. The subjects in this study eager to collaborate in this effort of public health importance, were in such good condition, particularly the subjects over 70 years by giving out their blood sample. The study established the ranges of DHEA-S in the serum of male and female adults in Yenagoa, Bayelsa State, Nigeria with the levels declining as the subject’s ages and differences not statistically significant in the gender, but differs
significantly in the age groups. The results indicate adrenal insufficiency with increase in age for both male and female.

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