Adejumo B. I. (Medical Laboratory Sciences Department, University of Benin, Benin City, Nigeria.)

Nwobu G. O. (Medical Laboratory Sciences Department, Igbinedion University, Okada, Nigeria.)

Ewenighi C. O. (Medical Laboratory Sciences Department, Ebonyi State University, Abakaliki, Nigeria)

Dimkpa U. (Physiology Department, Nnewi Campus, Nnamdi Azikiwe University, Awka, Nigeria)

Odionyema U. C. (Department of Medical Laboratory Science, College of Health Technology, Akure, Nigeria)

Ezimokhai T. J. P . (Lulli Diagnostics Limited, Benin City, Nigeria.) 

All correspondence to: Adejumo B.I., Medical Laboratory Sciences Department, University of Benin,
Benin City, Nigeria. Email: babatunde.adejumo@uniben.edu, bigadejumo@yahoo.com

ABSTRACT
Aim: This study aimed at determining the prevalence of Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) in Edo State, Nigeria. Method: Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) levels were estimated using ELISA reagents from Biomerica Inc. Other parameters such as fasting blood sugar, urine glucose, and urine protein were assessed using standard colorimetric and biochemical techniques. A total number of four hundred and fifty five (455) subjects, 211 (46%) males, and 244 (54%) females with ages ranging between 5 – 76 years were assessed. Subjects were free from long standing illness, endocrine disorder and infectious diseases. Most of the subjects were those attending diabetes clinics in secondary and tertiary Institutions in Edo State, relatives of type I diabetics and volunteered controls. A structured questionnaire was used to collect sociodemographic data. Sociodemographic data included age at diagnosis, age at inclusion, gender, family history of diabetes, drinking and smoking habits, and the duration of diabetes. In cases of patient uncertainty the duration was calculated from the documented date of diagnosis. Other information was obtained from the patients’ files, including the mode of presentation, type of treatment, and course of the disease.
RESULTS: The prevalence of IAA in the study population is 100% in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 1.6% in non-diabetic control, while 11. 9% represents the prevalence of IAA in the general population. ICA also has 100% prevalence in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 2% in non-diabetic controls, and 12.1% in the general population.
CONCLUSION: The seroprevalence of ICA and IAA is high in this study, public enlightenment, change of life style and early routine check is advocated to prevent diabetes development, complications and reduce mortality rate.

Key words: Diabetes mellitus, IAA, ICA, Prevalence, Edo state

INTRODUCTION
Diabetes mellitus is a genetically and clinically heterogeneous group of disorders (1). It is a disease of ß cells of the Islets of Langerhans or disease resulting from defective secretion and utilization of insulin (2). The hyperglycaemia of diabetes mellitus results from abnormalities of insulin secretion (2). The secretion can be delayed, deficient or inappropriate for the blood glucose homeostasis, or its action being affected by circulating antagonists, e.g. antibody or abnormalities of its tissue receptor (3). Diabetes mellitus is broadly classified into two main groups, Insulin Dependent Diabetes Mellitus (IDDM), Type 1 and Non Insulin Dependent Diabetes Mellitus (NIDDM), Type 2. Type 1 is caused by auto destruction of ß cells of Islets of Langerhans

while the Type 2 results from non-production of insulin or insufficient circulating insulin.

Insulin autoantibodies (IAA) are one of several markers for Type 1 (autoimmune) diabetes. Unlike the other markers, their legend is unique to the beta cell (4). IAAs are the first markers to appear during the symptomless period which precedes diabetes and they are present in the vast majority of young children destined to develop diabetes (4). They are present at titres which relates inversely to age at onset of IDDM (5) (6), and are believed to mark for the rapidity of progression to the disease. Thus, IAA are rare in IDDM of adult onset, which is generally of slow progression, but present almost universally in children who develop diabetes below the age of five years (6), where progression is typically rapid. The origins of IAA, and the timing of their appearance in the circulation, are however unknown. Nevertheless, the rare description of congenital IDDM where the infant, but not the mother, is seropositive for islet related autoantibodies including IAA (7) indicate that insulitis can begin during gestation.

Recent studies in humans and in animal models of spontaneous insulin-dependent diabetes have demonstrated the presence of islet cell cytoplasmic antibodies (ICA), islet cell surface antibodies (ICSA) and insulin autoantibodies (IAA) during a prolonged but relatively asymptomatic pre-diabetic period (8)(9)(10)(11)(12)(13)(14). Most studies evaluating immune markers for prediction of Type 1 diabetes mellitus have focused on first degree relatives, although only 10% of newly diagnosed patients have affected first degree relatives. A previous study (15) has reported a significant association of IgG IAA with ICA – positive in first-degree relatives of Type 1 diabetics. Several attempts have been made to determine the prevalence of diabetes by surveys of community. Most surveys have shown that there are about as many unknown diabetes as known cases.
There is paucity of studies in the prevalence of IAA and ICA in Nigerian population. We therefore aimed at determining the prevalence of both IAA and ICA in diabetics compared with their non-diabetic controls. In addition, we determined the prevalence of IAA and ICA in the general population and the relatives of type 1 diabetics. An early detection of circulating IAA and ICA is important in order to identify the
individuals in the general population, the siblings, and families of IDDM patients, who are at a high risk of developing this disease because of their genetic predisposition to diabetes hence the need for this study.

Materials and Methods
Subjects
This study was conducted at Central Hospital, Benin City. The subjects included 55 diabetic patients on appointment visit to diabetic clinic and those on admission; 150 relations of the type 1 diabetic patients; 250 volunteer controls and a general population of 455 participants. Participants’ consents were obtained before participation in the study. Ethical clearance was obtained from the Ethical Committee of Central Hospital, Benin City, Edo State, Nigeria.

Classification of diabetes
Patients were classified as Type 1 or Type 2 according to the clinical criteria recommended in the 1997 World Health Organisation(16). Classifications were based on age at diagnosis, mode of onset (acute versus insidious presentation), duration of disease, current treatment, BMI, waist-to-hip ratio, blood pressure, random or fasting glucose, HbA1c and urine ketones. A classification of Type 1 diabetes was defined by the following criteria: onset in patients aged 30 years or less, presentation of acute classical symptoms, and that required insulin therapy to control hyperglycaemia. A classification of Type 1 diabetes was also designated to patients older than 30 years that required insulin treatment, lacked metabolic control, and were determined to be underweight. A classification of Type 2 diabetes was designated to patients who were older than 30 years at diagnosis and did not need insulin for metabolic control. A Type 2 classification was also designated to patients younger than 30 years who were obese and had diabetes for a long duration without requiring insulin treatment. Patients that did not clearly exhibit the clinical features of either Type were classified as undetermined and excluded from the present analysis.

Sample Collection
Ten millilitres of blood was collected intravenously, five millilitres was dispensed into a plain container, and the other five millilitres glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Data Analysis
Statistics was done using Statistical Package for Social Sciences program (SPSS) version 16.0. Values obtained in this research were represented as mean ± Standard error of mean (SEM) for continuous variables and percentages for categorical variables. Analysis of variance (ANOVA) was used to compare data at 95% confidence interval (p<0.05).
was dispensed into a fluoride oxalate container. The blood samples were spun at 1500rpm for 10minutes and the supernatant serum/plasma were separated into separate tubes. . The serum/plasma samples were stored at -200C for up to 2weeks prior to the analysis of fasting blood glucose and antibodies. IAA and ICA were determined using ELISA reagents from Biomerica Inc., U.S.A. Blood glucose was determined colorimetrically using Randox kits from United Kingdom. Fresh urine samples were voided into clean sterile containers, glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Tables 1 and 2 show the general prevalence of IAA (n = 54) in the general population (n = 455) to be 11.9% while the prevalence of ICA (n = 55) in the general population is (n = 455) is 12.1%. However, in diabetic Type 1 population (n = 5), the prevalence of ICA and IAA is 100%. In Type 2 diabetic population, the prevalence of IAA (n = 50) is 10%, while ICA (n = 50), is 10% (n = 50) respectively. Diabetic relatives population of IAA and ICA (n = 150) have a combined general prevalence of 26.7% (n = 40). However, the general population prevalence of IAA (n = 250) and ICA (n = 250), are 1.6% (n = 4) and 2% (n = 5) respectively.

DISCUSSION
Most studies on the role of autoimmune markers in the pathogenesis of type 1 (insulin – dependent) diabetes mellitus have focused on first degree relative. Although, the risk for type-1 diabetes is increased among relatives, it is likely many of them may have a reduced acute insulin response to glucose (17, 18). The present study indicated 26.7% prevalence of both IAA and ICA in relatives of type 1 diabetic patients. Another study, (19) in London among English school children found prevalence of 2.8% and 6.8% respectively in the population and siblings of the diabetic patients. The work carried out by a researcher (20), at Malmon, Sweden in a study of a heterogenous group of high risk subjects (non-diabetic patients genetically at higher risk for IDDM including discordant monozygotic twins and ICA positive first degree relatives), IAA were detected in 31% of ICA positive individuals.
The present study indicated 100% prevalence of IAA and ICA in the diabetic type 1 patients and 10% prevalence in type 2 diabetics for the two antibodies respectively. In addition, we observed 1.6% prevalence of IAA and 2.0% of ICA in non-diabetic controls. Our finding is not in agreement with some workers (21), who found the prevalence of IAA in 18% of newly diagnosed IDDM patients. But with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due difference in the methodology. Similarly, our study did not agree with a previous study (22) which found 2 out of 68 diabetics and 1 out of 68 controls among Nigerian diabetics have ICA. Some workers (23), (24), (25), (26), (27), (28), have also reported prevalence of 20 – 50% among newly diagnosed IDDM patients in Pasco Country, Florida, USA. However, with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due differences in methodologies. The 100% prevalence observed in IAA and ICA in this study may be due to the fact that the patients have all commence the administration of exogenous insulin.
Our data also indicated 11.9% prevalence of IAA in the general population. This is at variance with the work of some researchers (29), who reported the prevalence of IAA to be 4% among 1031 populace in Sweden. Also the prevalence of ICA in the general population (n = 455) is 12.1%. This is at variance with the work of (30) who found the prevalence of ICA to be 6.8% among Tanzanian Africans. Another previous study (29) also found 1.4% prevalence of ICA in a Swedish population.
It is believed that the variations in prevalence in this work relative to other previous works may be due to differences between the Africans and Caucasians in terms of diet, climate, method of diagnosis and other etiological factors that trigger the onset of the diabetes mellitus. However, to the best of our knowledge, this is the first documented work to evaluate combined IAA and ICA measurements among African populations either with diabetes or without diabetes.

Conclusion
The seroprevalence of ICA and IAA are high in this study and their occurrence are almost at the same rate in all the groups, it shows that categorising diabetes into type 1 or 2 on the basis of ICA may generally not fit the pattern of diabetes as seen in Nigeria and possibly other parts of the tropics. Though there is no plan for follow up on this work, if those positive IAA and ICA individuals are followed up, majority of them may eventually develop the disease, which brings us to the need to advocate for early routine check up and vigorous public enlightenment to avoid development of diabetes mellitus, which may lead to complications and subsequently mortality.

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A.A Sadeeq; A.O Ibegbu; J. A Timbuak; M, Tanko; S.P Akpulu; H.R Bello; S.A Musa
Department of Human Anatomy; Faculty of Medicine. Ahmadu Bello University, Zaria. Nigeria

L. H. Adamu: Department of Anatomy; Faculty of Medicine. Bayero University, Kano. Nigeria 

I.S El-Ladan: Department of Anatomy; Faculty of Medicine. Umaru Musa Yar’addua University, Katsina Nigeria

H.O Kwanashie: Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences. Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: Abubakar Sadeeq Adamu; aasadeeq@abu.edu.ng

ABSTRACT
Mercury is a wide spread environmental pollutant that occurs naturally and has been reported to cause some health problems worldwide. Calcium is an essential neurotrace elements that is required for nerve cells to release neurotransmitters. The present work study the effects of mercury toxicity on memory and level of calcium (ca2+) using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats. Twenty four (24) adults Wistar of both sexes were used for the studies and randomly divided into four groups of six rats per group. Group 1 was the Control and received normal saline, while Groups 2, 3 and 4 received 12.45mg/kg, 28.9mg/kg and 49.8mg/kg body weight of mercuric Chloride respectively for twenty one (21) days through oral route. Animals were trained for spatial learning and memory using Morris water maze method and latency time to find the flat form was recorded. The animals were anesthetized and humanely sacrificed using chloroform. The brain was fixed in Bouin’s fluid and processed histologically using H and E stain. Moreover, some of the tissues were crushed by using mortar and pestle after oven drying at 100oC for short-live irradiation method which was exposed to characteristic Gamma-ray to reveal the presence of Ca2+ ion. The photomicrograph reveals that, there was distortion of the pyramidal cells, congestion, necrosis and sparse distribution of hippocampal cells. The spatial memory test showed a decreased in latency time among the control group while the treated groups showed an increased in latency time that was statistically significant (P=0.05). NAA-1 shows an increased level of calcium concentration among mercury treated animals (P=0.01) when compared to the control animals in group I. It was concluded from this studies that; mercuric chloride exposure has effects on the cyto-archetecture of the hippocampus and alters the level of neurotrace element (calcium ion) which in turn affects spatial learning and memory in adults Wistar rats.

Keywords: NAA-1, short-live analysis, Mercury, memory, calcium ion.

INTRODUCTION
Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which are transferred to man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury can exist either as elemental, organic and inorganic mercury (Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer and coal combustion (Akagi, 1995; Bjomberg et al., 2011). Natural source of Mercury such as Mercury chloride that is found in higher densities in rocks and volcanic activities can give half off of the mercury present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (Vupputuri et al., 2005: Berlin, 2006). 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; Rao, 2001; Valera et al., 2008;). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (ATDRS, 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employed by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2005). Calcium is especially important element for signal transduction in cells. For this reason, it is sometimes called a second messenger (Khachaturiazn et al., 1989). Coupled with the fact that calcium is necessary for neurons to release neurotransmitters in the brain, calcium plays an important role in the areas of the brain responsible for storing and retrieving memory (Khachaturiazn et al., 1989). The present work, study the effects of mercury toxicity on memory and level of ca2+ using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats.

MATERIALS AND METHOD
Experimental Animals
Twenty four Wistar rats of average weight of 210g was used for this study and were acclimatized for three weeks in the animal house of the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria. The 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 drinking bottle and animals were allowed to feed and drink ad-bilitum. The animals were divided into four (4) groups with six animals per group.
Chemical substances
Mercuric chloride (May and Bakers limited, Dagenham England) with batch number XN202 was obtained from Steve Moore chemicals limited Samaru, Zaria. Kaduna State, Nigeria.
Administration of Mercury Chloride
The LD50 of Mercuric chloride was adopted from the Manufacturer (as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the LD50 per kg body weight according to the manufacturer’s instruction. Animals in Group I served as the Control and were given normal saline, while animals in Group II, III and IV were orally administered with mercuric chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg body weight respectively of the LD50 per kg body weight. The administration of mercuric chloride lasted for 21 days through oral route.

Neurobehavioral test
Spatial learning and memory test using Morris water maze
Morris water maze test was used to develop and test spatial learning and memory in animals according to the methods of Morris (1981) which was further developed by Liu et al (2011). Animals were placed in a circular pool of clear transparent water which was partition into four quadrants. According to this method, a platform was submerged beneath the surface of the maze pool; the animal task is to find the hidden platform. The animal starting point was changed from time to time so as to build a cohesive spatial representation of the pool in order to find the platform and the latency time was recorded both during the training and experimental periods.
Animal Sacrifices
After the administration, the animals were humanely sacrificed and incision was made through the skin and muscle of the skull. The skull was opened through a mid-sagittal incision and the brain was removed and fixed in Bouin’s fluid. The tissues were routinely processed and stained using haematoxylin and eosin. And some tissue were processed for short-lived irradiation for neurotrace elements detection.

Tissues Preparation for Neuro-trace Element Analysis Method
Trace element analysis of the brain tissue was carried out with the aid of Nuclear Research Reactor (NNIR-1) using NAA-1 method at the Center for Energy Research and Training Ahmadu Bello University, Zaria, Kaduna, Nigeria according to IAEA (2004). The brain tissue samples were Oven dried at 100oC using Memmert DINI ovum-280 model-KA. After successful drying, tissues were crushed in Agate mortar and piston at zero contaminant level in order to have the tissues in powdered form which were then put in sealed polyethene bag and weighed using Metter AE 240 Electronic Digital balance to prepare tissue for nuclear irradiation. In NAA-1 short-lived irradiation method used, each sample was irradiated for 5 minutes and exposed to a semi-conductor detector for characteristic gamma-rays detection for 10 minutes followed by another 10 minutes for 3 hours after the first count. This is to allow safe handling of the radioactive nuclei formed (Akaho and Nyako, 2002).The short lived analysis revealed the presence of neurotrace element calcium (Ca2+). The samples were counted for short –lived irradiation using Maestro software attached to an Analog to Digital Converter (ADC) card and Multi-channel Analyzer card (MCA) according to method of Jonah (2005).

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 between and within the groups. P-value less than 0.05 was considered 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. The Trace elements present in the tissue were analyzed using WIN SPAN 2004 software for peak analysis and efficient calibration and calculation of elements detected.

RESULTS
The effect of mercury chloride exposure on Morris water maze test.
The results on mercuric chloride exposure on spatial learning and memory test, showed a decreased in the meantime taken for the animals to complete Morris water maze task among the control group throughout the period of administration as shown in Table 1. While animals in group II and III had an increased in latency time to find an escape route during Morris water maze activity, though the increase is between weeks 3 and weeks 2 respectively which was significant (P=0.05). And animals in group IV showed an increased that was statistically significant (P=0.01) throughout the period of administration as shown in table 1 below.

Table 1: Effect of mercury chloride ingestion on spatial learning and memory using Morris water maze test

the hippocampus of group III animals, with degeneration of the pyramidal cell layer, loss of some of the pyramidal cells and clumping of pyramidal cell nuclei. Group IV animals show disorientation of the pyramidal cell layer and degeneration of some pyramidal cells with the pyramidal cells appearing to be smaller than normal as shown in Plate D.

Short-lived analysis for calcium (Ca2+) level in the brain
The results of Neuro-trace element analyses indicates the presence of Calcium (ca2+) in the brain tissues of the animals which showed a significant increase (P =0.01) in concentration of Calcium in Group III and IV animals when compared to the control. While Groups II animals showed an increased in calcium concentration that is not significant as shown in figure 1

DISCUSSION
The pyramidal cells in the hippocampus manifested some changes like necrosis degeneration and loss of neuronal cell fiber compared to the control group which could be as a result of the exposure of mercuric chloride. This implies that the activity of the hippocampus in memory formation and learning will be impaired and the role of the hippocampus that involved storage and retrieval of information will also be lost. The findings in this study agree with the studies of Wolf et al., (2003), who reported that rats exposed to high concentration of mercury vapor, showed neurodegenerative changes in the hippocampus which was responsible for memory deficit in such animals.
Result from the present study shows that, there was an increased in time taken by the experimental rats to find the hidden platform in Morris water maze test which was significant. Conversely, the pyramidal cell layer of the hippocampus appears to be damaged with dead cells, and vacuolated spaces and distortion in the general morphology of the pyramidal cells. These alterations can consequently result to memory impairments which could be as a result of neuronal degeneration. The destruction of the pyramidal cells implies that activity from the brain region that projects into the pyramidal layer of the hippocampus will also be lost such as memory and learning ability (Wolf, 2009; Quirino, 2012). Mutter (2010) had reported that short term occupational exposure to high levels of mercury induced slight cognitive deficits. A memory deficit among animals exposed to methyl mercury was not significant in latency time or swim length between the different groups of animals according to Olson (2005).
The findings of the present study revealed a high level of calcium in the brain due to mercury intoxication. Increased in calcium level among groups that receive medium and high doses of mercuric chloride implies that, the role of calcium as a second messenger in signal transmission between cells will be loss, which is required for nerve cells to release neurotransmitters in the central nervous system. This finding agrees with Bandtlowc (1993) which explained that excess calcium at the surface of nerve cells causes them to ‘fire’ spontaneously sending messages around the brain for no purpose except confusion and cause a ‘short circuit’ allowing messages sent to one destination to get misrouted to another which causes memory loss.

CONCLUSION
It was concluded from the present study that oral administration of mercuric chloride has effects on spatial learning and memory and can cause histopathological assault in the hippocampus like necrosis, loss of nerve fibers of the pyramidal cells and neuronal clumping. The short-lived analysis for neurotrace elements revealed the presence of calcium in the brain which was high in concentration due to mercury exposure.

ACKNOWLEDGMENT
I appreciate the support of Mal. Shehu Shika of Center for Energy Research and Training. Ahmadu Bello University Zaria, Nigeria for a bench space in his Laboratory and Mal Ado Garba of A. B.U for his financial support.

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Toryila J.E, Amadi K, Odeh S.A, Egesie U.G.: Human Physiology Department, Faculty of Medicine, ABU Zaria.

Adelaiye A.B. Achie L.N: Human Physiology Department, Faculty of Medical Sciences Unijos.

All correspondents to:  Toryila J.E Human Physiology Department, Faculty of Medicine, ABU Zaria. 

ABSTRACT
BACKGROUND: Contraception is an important health issue in preventive medicine because it protects women globally from the effects of unwanted pregnancy and allows them to integrate into society. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.
Method: Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four days treatment with one-day break. A haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT). Prothrombin time (PT) and Activated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit (Karmiya Biomedical company, USA).
Results: There were significant decrease in Hb, PCV, RBC and WBC counts, lymphocytes (L), PT, IL-6 and IL-11 in all treated groups compared to controls (P<0.005). There was significant increase in alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), sodium and potassium in all treated groups compared to controls(P<0.05). There were however no significant changes in activated thromboplastin time (APTT) and erythropoietin (EPO). The lowest values of Hb, PCV, RBC count, Lymphocytes, PT, IL-6 and Fibrinogen and the highest values of ALT,ALP and AST were observed in long
term used (72 days).Long term used of combined oral contraceptives may leads to more complications than short time use. Combined oral contraceptives users should be monitored for some Physiological parameters.

KEY WORDS: Combined Oral Contraceptives, Physiological Parameters, Wistar rats.

INTRODUCTION
Birth control is a major factor in public health and welfare, preserving the general and reproductive health of women and allowing them to choose the movement of a planned pregnancy

[1]. The World Health organization (WHO) and other global organizations are seeking ways to increase the amount of information and access people have to contraception and other resources related to family planning all round the world.
Oral contraceptives had been reported to have beneficial effects in reducing the incidence of pelvic inflammatory disease, decrease risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, protection against osteoporosis and rheumatoid arthritis among the users

[2]. Oral contraceptives are sometimes used to treat heavy or irregular menstruation and endometriosis. Oral contraceptive agents can also be used in hormonal replacement therapy, and in the emergency post-coital contraception

[3]. Oral contraceptive decreases the risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, and offer protection against osteoporosis and rheumatoid arthritis

[4]. Despite the general acceptability and the obvious advantages that have been attributed to oral contraceptives use, some serious side effects have been reported in women taking them. Studies have indicated a relationship of oral contraceptives use and cardiovascular disease, altered levels of coagulation factors, thrombosis, platelet changes, atherosclerosis and multiple sclerosis. Estrogen has been known to have prothrombin effects and elevates cardiovascular and venous thromboembolism risk

[5]. There is little or no data on the effects of Combined Oral Contraceptives (DUOFEM) on Haematological parameters, especially the growth factors such as the cytokines or interlukin-11 and 6, and erythropoietin. Also little or no data have been established in PT, APTT and Liver Function Test using animal model. It is hoped that the result might throw some light on the need of monitoring coagulopathy in women taking these pills. The world population is now seven billion. Nigeria population is estimated to be 176 million and will reach 400 million by the year 2050 [6]. Rapid population growths would have a detrimental effect on socioeconomic development of Nigeria.
There is a concern over population explosion in Nigeria and the drive to control it is leading to indiscriminate used of oral contraceptives. Unintended pregnancy leads to induced abortion which is not legalized in Nigeria, except to save the woman`s life. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.The knowledge might provide useful interventions towards solving the problem(s). Thus the safety in the contraceptives use and improvement in the health of the user are assured.

Materials and methods
DRUGS: The combined oral contraceptive used is DUEFEM®. They were obtained from family clinic, Ahmadu Bello University Teaching Hospital, Shika-Zaria, and from the Society for Family Health (SFH) Abuja, Nigeria. COCs DUOFEM® tablets which combined ethinyl estradiol and Norgestrel were manufactured by Wyeth Ayerst (USA) and packed and marketed by the Society for Family Health, Lagos, Nigeria. DUOFEM® is a child spacing pill containing ferrous fumarate tablets. Each DUOFEM cycle contains 28 pills; each white tablet contains 0.3mg Norgestrel and 0.03mg Ethinglestradiol and each brown tablet contains 75mg ferrous fumarate. DUOFEM® has a molecular weight of 312.4458g/mol [7].
Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four-days treatment with one-day break). The COC was given intragastically in 5-day cycles (4-day treatment with 1-day break). All controls were given fresh water ad libitum daily for the period of the experiment. Experimental animals in the study were treated in accordance with the National Protection Laws of Animal Welfare [8]. Ethical clearance was obtained from the Ahmadu Bello University Animal Ethical Committee.
Haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) . Prothrombin time (PT) andActivated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit ( Karmiya Biomedical company, USA). Fibrinogen was estimated by Clauss Assay [9]

Statistical Analysis: The result obtained from this study was analyzed using SPSS version 20 for windows. Analysis of Variance (ANOVA) was used to compare means, and values were compared at P < 0.05. Post Hoc multiple comparisons for significant differences between groups were established by Turkey’s HSD. All the data are expressed as Mean ± Standard Error of Mean (SEM).

RESULTS:
Table 1: The effects of combined oral contraceptives on some haematological parameters in female wistar rats

A, b, c, and d- significant, N- No significant

Lowest values of Hb, PCV and RBC count were observed in treated group D (72days)

Table 2: The effects of combined oral contraceptives on some haemostatic parameters and cytokines in female wistar rats

A, b, c, and d – significant, N- No significant

The lowest values of PT, IL-6 and Fibrinogen were observed in the treated group D (72 Days).

Figure 1: The effects of combined oral contraceptives on AST in female wistar rats 

 Short and Long-Term Effects of Combined Oral…

DISCUSSION
In this study the effect of COC on haematological and biochemical parameters in female wistar rats were investigated. The result shows a reduction in haemoglobin(Hb), Packed cell volume(PCV), Red blood cell (RBC), White blood cell (WBC) counts and lymphocytes in all the groups that were given COC, compared to the controls in each group (P<0-05). The finding of lower Hb, PCV, RBC, WBC and Lymphocytes agrees with the finding of [10].The findings were however, contrary to that of other investigators like Bulur [11], Babatunde [12], Abdalla [13], who reported no changes in full blood count in women on COC. The least values of Hb, PCV and RBC were found in group D (72 days). It could be that the use of COC for a longer period may lead to anaemia. The finding of lower Hb, PCV and RBC counts in COC treated groups compared to controls may be as a result of the haemodilution effect of estrogen and the effect of estrogen on the cytokines like IL-11 and IL-6. The findings of lower values of WBC counts in COC treated groups suggest that the use of COC may alter immune response.
The findings of lower WBC count in COC treated groups was in agreement with that of Sajida [10], and contrary to the findings of Surasak et al [14] who observed no changes in WBC count and that of Araz et al[15] who observed increase in WBC count in COC treated female wistar rats. The difference may be as a result of the use of different COC with different concentration of estrogen and progesterone and the duration of use. There was no significant changes observed in COC treated groups in platelet counts compared to the controls. This finding agrees with that of Surasak et al. [14], Peter [16] Bulur et al [11] and Babatunde et al [12]. In general, the use of COC in female wistar rats suppressed haemopoiesis in this study resulting in lowering of Hb, PCV, RBC and WBC counts.
There was no significant change in serum erythropoietin level in all the controls. This finding is contrary to that of Prechile et al.[17] who found that estradiol benzoate inhibited the production of erythropoietin (EPO). Erythropoietin promotes the survival, proliferation and differentiation of erythrocytic progenitors. It appears COC has little or no effect in EPO production. Decreased RBC count in this study may not be as a result of COC effect on EPO. There was decrease fibrinogen levels in all the COC treated groups compared to the control. This finding differs from that of Eliana et al [18] and Peter et al. [16] who reported increase in fibrinogen level in women taking COC. The finding is also contrary to that of Akhigbe et al [19] who found no significant change in fibrinogen level in female wistar rats treated with COC. Fibrinogen functions as a messenger molecule that coordinates and regulates the body response to inflammation. The association between increase in plasma fibrinogen and thrombosis and the risk of myocardial infarction, atherosclerosis and other cardiovascular diseases are well established. Higher level of fibrinogen raises the risk of stroke [13] .Low fibrinogen levels is associated with low risk of cardiovascular diseases. Effect of COC on the liver as observed in this study may be the cause of decreased in fibrinogen level by a third generation COC (DUOFEM).
Prothrombin Time (PT) significantly reduce in all the COC treated groups compare to the controls (P<0.001). There was no significant reduction in Activated Prothrombin Time (APTT). This finding agrees with that of Abdalla 13] ,in Sudan found significant reduction in PT and APTT in women taking COC. Babatunde [12] found that there was no significant change in the level of APTT in Nigerian women taking COC for three months. This is in agreement with the finding of this study. Also Eliana [18] found reduced PT and APTT in Albanian women taking COC. Reduction in prothrombin time may be as a result of reduced serum level of fibrinogen. Estrogen is also said to increase coagulation factors leading to decreased prothrombin time. The finding of this study is also in agreement with that of Ahmed [20] and Nasir [21] who reported a significant reduction in APTT and PT in women taking COC. The exogenous estrogen in the COC has been shown to accumulate in the liver stimulate the release of procoagulants (Factors II, VII and X) from the degenerating liver cells. Significant decreased in PT observed in this study is possible due to enhanced coagulation process as a result of increased plasma levels of coagulation factors of extrinsic pathway.
There was a significant decrease in serum level of antithrombin (AT), protein C (PC) and protein S (PS) in COC treated groups compared to the controls. Proteins C and S and antithrombin are components of the anticoagulant system. In haemostasis, the procoagulant
system is in balance with the anticoagulant and fibrinolytic system [22]. It is important to emphasize that a disturbance in normal hemostasis may result in thrombosis. This finding is in agreement with that of Soare [23] and John [24] who observed lower values of PC in women using oral contraceptives. Decreased AT, PC and PS may be responsible for the changes observed in haemostasis. AT, PC and PS are vitamin K-dependent. Effect of COC on the liver may be responsible for the reduction of serum level of AT, PC and PS in COC treated groups.
There was a significant decrease in serum level of interleukin-6 (IL-6) and a slight decrease in serum interleukin-11 (IL-11). Estrogen is able to decrease IL-6 expression by blocking the estroblast’s synthesis of IL-6 receptors [25]. IL-11 and IL-6 are haemopoiesis-promoting factors capable of enhancing the growth of myeloid, erythroid and megakaryocytic progenitor cells. They are capable of mediating a complex array of pro- and anti-inflammatory effects. Reduction in IL-11 and IL-6 may be responsible for decreased RBC, PCV, WBC and platelet counts in this study. IL-6 produces C – reactive protein (CRP) which leads to cardiovascular risk. Experimental studies have shown strong correlations between the risk of cardiovascular diseases and inflammatory markers such as CRP and tissue neurosis factor-a (TNF a) [26].
IL-6 is a pleotropic cytokine which stimulates B-lymphocyte and T-lymphocyte differentiation, and activates macrophages and natural killer cells (NK). IL-6 promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions [27]. The finding of reduced serum IL-6 and11 is in agreement with other finding [28] who reported decrease serum level of IL-6 and 11 in menopausal women taking hormonal replacement therapy.
There were increased values of alkaline phosphate (ALP), Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) in all COC treated groups compared to controls (P<0.001). The liver plays a central role in the metabolism of estrogens and progesterones. COC acts directly or indirectly on the liver to produce a variety of biological effects which have both physiological and pathological significance [28]. Higher levels of liver enzymes in the blood streams are prime indicators of liver damage.
The use of COCs has been rarely associated with liver tumours, both benign (hepaticadeomas and nodular hyperplasia) and malignant (hepatocellular carcinoma). The finding of increased liver enzymes is in agreement with that of Dickerson [29] and contrary to that of Surasak [14] who found no changes in liver enzymes in women on COC. Raised serum level of ALP, AST and ALT in treated groups may be due to functional alterations involving the hepatic excretory mechanism. The effect of the COC (DUOFEM) on the liver may be the cause for haemostatic disorder, since PC, PS and AT were reduced.
There was a significant increase in serum sodium level in group D (72 days, P<0.044) compared to the control. Potassium was significantly increased in groups C (60 days) and D (72 days). Sodium retention has been reported in women taking COC [19]. The result in this study is contrary to that of Taneepanichskul [30] who reported no significant changes in electrolytes in women taking COC.
Hypertension occasionally occurs in women receiving estrogen-progesterone combination for contraception. Estrogen raises the plasma level of rennin and increases the production rate of aldosterone which leads to water and sodium retention [31, 32] reported reduced potassium level and increased sodium level in female rats. There was no significant change in bicarbonate and chloride in treated groups compared to the controls. Body electrolyte balances are critical for normal cellular function and maintaining adequate blood and plasma volume and osmolality.

CONCLUSION
This study confirms findings of previous studies which also reported an increased risk for venous thromboembolism with third generation COC. This research work has confirmed the effect of COC (DUOFEM) on haemopoiesis. There is a potential risk of long term immune suppression in female taking COC (Duofem). Estrogen has shown to have long term effect on liver cell which may affect liver enzymes production and functions. There is the tendency for COC to be used earlier in life and for longer period of time, the number of women entering the high risk group for venous thromboembolism and liver cell adenomas maybe on the increase. Therefore, we recommend that women on COC should be regularly monitored for Physiological parameters.

ACKNOWLEDGMENT
I want to appreciate all staff of Haematology, Immunology, Chemical Pathology departments and Histopatology Dept. of ABUTH Zaria for their assistance in the analysis of the samples. Dr. Olayemi of Society for Family Health Abuja, for supplying the COC (DUOFEM) and all staff of Human Physiology Department, University of Jos.

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17. Prechile, C., Ira, A., Rappaport, L. F. S., Mario, C., and Albert, S. G. (1972). The role of Estrogen in the regulation of Erythropoietin. Endocrinology. Obstetrics and Gynecology 180(6), 5375-5382.renal and liver function. J Med Assoc Thai.; 90: 426 – 31.

19. Akhigbe, R. E., Azeez, M. O., Ige, S. F., Oyeyipo, L. P., Ajao, F. O., and Alade A. O. (2008). Haematological effects of long-term administration of combined oral contraceptive in rats. International Journal of Pharmacology 4(5), 403-406..

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Oyewopo AO., Lawal IA., Olawepo A., Enaibe BU., Ajao MS

Department of Anatomy, Faculty of Basic Medical Sciences, Colleges of Health Sciences, University of Ilorin, Kwara State, Nigeria.

Oyewopo C.I.: Department of Anaesthesia, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

Afolabi O.O.: Department of Morbid Anatomy, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

All correspondence to: Dr. Oyewopo AO, E-mail: wolesake@yahoo.com.

ABSTRACT
Introduction: The objectives of this study is to determine the effects of cryptorchidism and hyperthermia on the testicular cytoarchitecture of adult male rats, quality and quantity of semen and the level of oxidative stress in the testes before and after intervention of Alpha-tocopherol (Vitamin E).
Methods: Twenty four (24) adult Wistar rats were used for this experiment and they were divided into seven groups (n=4). Experimental cryptorchidism was carried out in some animals while others were subjected to hyperthermia from two sources (hot oven and light bulb) for 28 days; followed by intervention of 100mg/kg alpha-tocopherol for fourteen (14) days.
All animals were then euthanized under anaethesia. The testes were excised and fixed and processed using haematoxylin and eosin technique. Plasma was assayed for glutathione, superoxide dismutase (SOD) and malondialdehyde (MDA).
Discussion: Cryptorchidism and hyperthermia caused in cycto-architectural distortion in the testicular structures of exposed animals and significant reductions in caudal epididymal sperm count and motility, morphology and life and death ratio. Plasma SOD and GLU levels also reduced significantly in exposed animals while MDA levels increased significantly, indicating peroxidation of testicular tissue. Alpha-tocopherol due to its anti-oidative properties impeded lipoperoxidation of the membrane, conserving cell union and increasing the amount of germ cells in the seminiferous epithelium.
Conclusion: At doses and durations tested, alpha-tocopherol (aT) reverted to a significant extent the effects of cryptorchidism and hyperthermia by reducing apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

Keywords: Cryptorchidism, hyperthermia and Alpha-tocopherol.

INTRODUCTION
The testes function in production of the male gametes or spermatozoa and male sexual hormone, (hence referred to as double glands) which stimulates the accessory male sexual organs and causes the development of the male sex characteristics (Mescher, 2010; Lutz, 2009). In most mammals, the temperature around the testicular region is lower than the core body temperature. In man, scrotal temperature is 2-30C lower than rectal temperature and the optimum temperature. Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility (Thundathi et al., 2012). Oyewopo and Togun in 2005 reported that a small increase in the temperature of the testis does not destroy the germinal epithelium; however, it reduces testis weight and sperm production and brings a greater incidence of morphologically abnormal spermatids and spermatozoa (Oyewopo and Togun, 2005; Bedford, 1991). Occupational exposure to high temperatures adversely affects testicular function causing partial or complete spermatogenic arrest, also causes deterioration in sperm morphology and impairs motility. This leads to oligoasthenoteratozoospermia (OAT) and azoospermia (Dada et al., 2001). Cryptorchidism (undescended testes); the absence of one or both testes from the scrotum happens to be the most common birth defect of the male genitalia, which if persists could result in reduced fertility and increased risk of testicular germ cell tumors (Wood and Elder, 2009). Cryptorchidism has been implicated to cause oxidative stress in animals that the process was performed on, it was reported that Cryptorchid rats had lower testicular weight, sperm count, germ cell count, testicular superoxide dismutase (SOD) concentration, testicular total protein and higher testicular malondialdehyde (MDA) concentration compared to animals in control group (Ayobami et al., 2013; Dutta et al., 2013). Alpha-Tocopherol (aT) is an important antioxidant that localizes to cell membranes. The primary function of vitamin E is as an antioxidant; because it helps reduce oxidation of lipid membranes and the unsaturated fatty acids and prevents the breakdown of other nutrients by oxygen. This protective, nutritional antioxidant function is also performed and enhanced by other antioxidants, such as vitamin C, beta-carotene, glutathione (L-cysteine), and the mineral selenium (Laila and Sahar 2009; Aybek et al., 2008; Traber and Kayden 1987). A significant reduction of lipoperoxidation was observed in the cryptorchid group treated with a-Tocopherol compared to the untreated cryptorchid group, at long term, an increase in the area and maturation of the seminiferous epithelium, a decrease in apoptosis and histological alterations and an increase in fertility was observed in animals treated with alpha-Tocopherol (Saalu et al., 2014; Rosa et al., 2011).
The broad aim of this study investigate the protective properties of alpha tocopherol (vitamin E) on cryptorchidic and heat (hyperthermia) induced testicular injury.

MATERIALS AND METHODS
The experiment was performed in conformity with the Rules and Guidelines of the Animal Ethics Committee of University of Ilorin. The experiments were conducted at the Department of Anatomy, College of Health Science, University of Ilorin.
Animals
Twenty four (24) male Wistar rats (150-210g) were purchased from Banky livestock company, Oke-Ose, Ilorin, Kwara State. The animals where kept in the animal house of the Faculty of Clinical Science, University of Ilorin, under light and dark cycle at room temperature. Proper aeration maintained by the use of wire gauze wooden cage. The rats were fed on growers’ feed from Bendel feeds Nigeria Limited, Ilorin, and distilled water throughout the duration of the experiment. The rats were arranged randomly into groups A-F, kept in different compartments of the cage. The rats were left under the above stated condition for two weeks so as to acclimatize with the condition of their new environment.
Grouping of animals
Group A- (n=4) control animals; these experimental animals were given pelleted feed and tap water ad libitum throughout the period of the experiment;
Group B- (n=4) experimental cryptorchidism was carried out this group;
Group C- (n=4) experimental cryptorchidism was carried out and then 100mg alpha tocopherol for 14days;
Group D- (n=4) animals were exposed to heat from hot oven for 28 days;
Group E- (n=4) animals were exposed to heat from hot oven for 28 days then 100mg/kg vitamin E for 14days;
group F- (n=4) animals were exposed to heat from light bulb for 28 days, group G- animals were exposed to heat from light bulb for 28 days then 100mg/kg vitamin E for 14days.
Animal sacrifice and tissue collection
At the end of the substance administration, the rats were sacrificed 24hrs after the last day of the exposure using ketamine (0.2ml). The animals were pinned on the dissecting board. Dissection was done on the anterior abdominal wall (thoraco-abdominal sagittal incision) using the necessary surgical equipment/instruments. Blood was first collected via the apex of the heart with a hypodermic syringe, and the blood was centrifuged, the epididymis was also excised and placed in normal saline for semen analysis. The testis was removed and then fixed Bouin’s fluid.
Semen analysis
The testes from each rat were carefully exposed and removed. They were trimmed free of the epididymides and adjoining tissues. Epididymal sperm concentration: Spermatozoa in the right epididymis were counted by a modified method of Yokoi and Mayi, (2003). Briefly, the epididymis was minced with anatomic scizzors in 5mL physiologic saline, placed in a rocker
for 10 minutes, and allowed to incubate at room temperature for 2 minutes. After incubation, the supernatant fluid was diluted 1:100 with solution containing 5 g sodium bicarbonate and 1mL formalin (35%). Total sperm number was determined by using the new improved Neuber`s counting chamber (haemocytometer). Approximately 10µL of the diluted sperm suspension was transferred to each counting chamber of the haemocytometer and was allowed to stand for 5 minutes. This chamber was then placed under a binocular light microscope using an adjustable light source. The ruled part of the chamber was then focused and the number of spermatozoa counted in five 16-celled squares. The sperm concentration was the calculated multiplied by 5 and expressed as [X] x 106 /ml, where [X] is the number of spermatozoa in a 16-celled square (Oyewopo et al., 2010).
Sperm progressive motility: This was evaluated by an earlier method by Sonmez et al, (2005). The fluid obtained from the left cauda epididymis with a pipette was diluted to 0.5 mL with Tris buffer solution. A slide was placed on light microscope with heater table, an aliqout of this solution was on the slide, and percentage motility was evaluated visually at a magnification of x 400. Motility estimates were performed from three different fields in each sample. The mean of the three estimations was used as the final motility score. Samples for motility evaluation were stored at 350c. Sperm morphology: The sperm cells were evaluated with the aid of light microscope at x 400 magnification. Caudal sperm were taken from the original dilution for motility and diluted 1:20 with 10% neutral buffered formalin (Sigma-Aldrich, Oakville, ON, Canada). Five hundred sperm from the sample were scored for morphological abnormalities (Atessahin et al., 2006). Briefly, in wet preparations using phase-contrast optics, spermatozoa were categorized. In this study a spermatozoon was considered abnormal morphologically if it had one or more of the following features: rudimentary tail, round head and detached head and was expressed as a percentage of morphologically normal sperm (Oyewopo et al., 2010).

Estimation of lipid peroxidation (Malondialdehyde)
Lipid peroxidation in the testicular tissue was estimated calorimetrically by thiobarbituric acid reactive substances TBARS method of Buege and Aust (1 978). A principle component of TBARS being malondialdehyde (MDA), a product of lipid peroxidation. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 1 5% TCA) and placed in water bath for 1 5 min, cooled. The absorbance of clear supernatant was measured against reference blank at 535nm. Concentration was calculated using the molar absorptivity of malondialdehyde which is 1 .56 x1 05 M-1 cm-1 and expressed as nmol/mg protein (Oyewopo et al., 2010).

Estimation of Glutathione level
Total GSH was estimated in various tissues by the method of Sedlak and Lindsay. Briefly, 5% tissue homogenates were prepared in 20 mM EDTA, pH 4.7, and 100 µl of the homogenate or pure GSH was added to 0.2 M Tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 µl of Ellman’s reagent (10 mmol/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm in a Beckman DU-640 spectrophotometer. Samples were centrifuged before the absorbance of the supernatants was measured (Sedlak and Lindsay, 1968).

Estimation of Superoxide dismutase (SOD) level
Testicular tissues were transferred into 5ml ice-cold sucrose solution (0.25M) and homogenized. The homogenates were further centrifuged at 3000 rpm for 15 min to obtain the supernatant, which was then aspirated with Pasteur pipette into sample bottle, stored overnight at 4°C before being used for assays. Tissue activities of superoxide dismutase (SOD) were determined by the method of Marklund and Marklund, (1974).
Statistical analysis
Data collected were analysed using Microsoft Excel and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (HSD) with the aid of SPSS V20. Data were presented as means ± SEM (standard error of mean) P value less than 0.05 (p<0.05) was considered statistically significant.

RESULTS
HISTOLOGICAL OBSERVATION
The micrograph from control group (group A) showed normal population of germinal epithelium, intact structure and arrangement of seminiferous tubules, presence of leydig cell, and well vascularized. Micrograph from group B (cryptorchidic animals) showed degeneration of spermatogenic cells and absence of spermatozoa in the lumen. Micrograph from Group C (cryptorchidic animals that were given alpha tocopherol) showed regeneration of spermatogenic cells and evidence of spermatogenesis due to presence of spermatozoa in the lumen. Micrograph from animals exposed to heat from hot oven (Group D), showed degeneration of basement membrane and clustering of spermatogonia cells, but Regeneration of of spermatogenic cells and normal spermatogenesis was evident in animals exposed to heat from hot oven followed by alpha-tocopherol (group E). Micrograph of animals exposed to heat from light bulb (Group D), showed distorted interstitium and cellular hyperplasia, which was reversed in animals that was given alpha-tocopherol.

Figure (A-G): photomicrographs showing the cyto-architecture of the testes across all experimental groups. Stain- Haematoxylin and Eosin (H & E). Mag- 400.
A- Control, B- cryptorchidism, C- crypt + vit E, D- hot oven, E- hot oven + vit E, F- light bulb, G- light bulb + vit E.
L-Lumen, White arrows- Spermatogenic cells, Black arrow- Basement membrane

Semen analysis
Table 1: percentage sperm count, sperm motility, life and ratio and sperm morphology across all groups.

Biochemical analysis
Table 2: malondialdehyde (MDA), glutathione (GLU) and superoxide dismutase (SOD) levels across all groups.

DISCUSSION
Cryptorchidism and hyperthermia caused desquamation and degeneration of germ cells which might be due to the destabilization of Sertoli cell membranes caused by lipoperoxidation, irregular shape of the seminiferous tubule, and degeneration of the interstitial tissues; this was in accordance to the report of Dutta et al., in 2013; Oyewopo and Togun in 2005 and Lee and Coughlin, in 2001. In cryptorchidic and hyperthermic animals exposed to alpha tocopherol (aT), lipoperoxidation of the membrane was impeded,
conserving cell union and increasing the amount of germ cells in the seminiferous epithelium (Shikone et al., 1994).
The reduction in sperm parameters analysed as seen in figures 4.2 to 4.6 (i.e. sperm counts, motility, life and death ratio and morphology) in both cryptorchidic and heat treated animals might be due oxidative stress action that cryptorchidism and hyperthermia cause to testicular tissues. Oyewopo & Togun in 2005 and Lee & Coughlin in 2001 also reported a significant reduction in sperm parameters of cryptorchidic and heat treated animals. But the effects of cryptorchidism and hyperthermia (heat) was reversed in animals treated with alpha tocopherol (Vitamin E) by not only returning sperm parameters to normalcy but also enhance the rate of spermatogenesis in the animals. My finding was corroborated by works of Saalu et al., in 2014 and Rosa et al., in 2011.
Cryptorchidism and hyperthermia cause a decrease in antioxidant enzyme activity or an increase in the production of ROS, (i.e. superoxide anion, hydroxyl radical, nitric oxide and hydrogen peroxide) which stimulates lipoperoxidation which results in increase in malondialdehyde and reduced glutathione and superoxide dismutase levels in exposed animals. The work of Saalu et al., 2014; Rosa et al., 2011; Ishii et al., 2005 corroborate my findings. However administration of alpha tocopherol to the treated groups showed increase in cell’s endogenous antioxidant defence system, inhibiting ROS production and impede lipoperoxidation (as seen in figures 4.8 to 4.10). This result was corroborated by work of Saalu et al., 2014; Ayobami et al., 2013; Rosa et al., 2011 and Laila and Sahar in 2009.
CONCLUSION
Cryptorchidism and hyperthermia resulted in oxidative stress in exposed animals which is evident in distortion seen in the histological slides, reduced sperm parameters and antioxidant activities. This work showed that aT reduced apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

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Isah, R.T.; Mohammed, I.
Department of Histopathology, Faculty of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto, Nigeria.
Avwioro, O.G.
Faculty of Science, Delta State University, Nigeria.
Abdullahi, K.
Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.
Bello, M.B.
Department of Surgery, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.

All correspondence to: Isah R.T. (tsamiyarilly@gmail.com)

ABSTRACT

The male breast in spite of being rudimentary is subject to the full spectrum of disease that affects the female breast. Male breast cancer is a rare condition globally, accounting for only 1% of all breast cancer. However, there is a wider increase in prevalence of this cancer in Africa approximately 5% – 15% as compared to developed nations. In Nigeria, several studies have shown prevalence in the range of 3.7% to 9.0% of all breast cancer cases. Mutation of BRCA 2 gene accounted for most of the cases of male breast cancer occurrence; other genetic conditions are BRCA 1 mutation, Klinefelter’s syndrome and Cowden’s disease. In addition, other risk factors associated with the disease are radiation exposure, hyperestrogenism, occupational and environmental exposure. Diagnostic methods include fairly specific techniques like routine light microscopic examination of haematoxylin and eosin stained sections of formalin fixed paraffin embedded biopsies, immunohistochemistry, fluorescent in situ hybridization and gene expression profiling. There is need for more public awareness program which focuses on behavioral modulation of diet, regular self-breast examination especially for those at risk and avoidance of exposure to environmental carcinogens.

KEY WORDS: Male breast, gynaecomastia, carcinoma, risk factors, diagnostic methods and prevention.

INTRODUCTION

Cancer can develop in any part of the body including breast of both male and females. Male breast cancer is rare compared to that of the female but they have similarities with few differences, as a result of which their diagnosis and treatment are almost similar [1, 2]. Several risk factors has been associated with development of male breast cancer which include family disposition, hyperestrogenism which may be caused by Klinefelter’s syndrome and liver cirrhosis, radiation exposure most especially at the chest region, testicular disorders, dietary source, aging and obesity [3].

Some of the signs and symptoms of breast cancer in males comprise of firm, non-painful mass located just below the nipple, skin dimpling around the breast region, nipple retraction, redness of the nipple or breast skin, bloody
discharge from the nipple and ulceration of the skin in advanced cases [4]. Pathogenesis of breast cancer in both sexes is essentially similar; much of this is linked to exposure to sex steroids (estrogen and progesterone) and activity of receptor tyrosine kinases (RTK) located on the surface of the breast parenchymal cells [5, 6].

Invasive ductal carcinoma (Not Otherwise Specified – NOS) is the most common histopathological type of breast cancer in males [7]. At least 8 out of 10 male breast cancers are invasive ductal carcinomas alone or mixed with other types of invasive or in situ breast cancer [8].

METHODOLOGY
This review paper involved gathering of already published articles and literature documents on male breast cancer. They were then extensively analyzed and presented as it affect African populace most especially Nigeria.

EPIDEMIOLOGY
Male breast cancer is a rare disease. It accounts for only about 1% of all breast cancer [9]. It was estimated that in 2013, about 2,240 new cases of breast cancer in men would be diagnosed and that breast cancer would cause approximately 410 deaths of men in United States [8]. Approximately 350-400 new cases of breast cancer in males are diagnosed annually in the United Kingdom [10]. A man’s lifetime risk of developing breast cancer is 1 in 1000, it can occur at any age, but it is mostly detected in the 60-70 years age group in developed countries [8].
The incidence of breast cancer in men has been increasing globally; one report suggested that incidence has increased 26 percent over the past 25 years [11]. In Africa, various researches conducted in the region have shown a wider increase in male breast cancer occurrence and mortality rate as compared to the developed countries. Incidence of male breast cancer is much higher in sub-Saharan Africa, approximately 5%-15% [12]. When they present, the tumours are often at advanced stages with poorer
prognoses [7]. A meta-analytical study on male breast cancer in African males indicated that the average age of occurrence was 54.6 years, which is 7 years older than the female counterparts [13]
Reasons for the differences in mortality rates between developed countries and African countries include advanced stages at presentation, worse biologic behavior, poor treatment facilities and poor patient acceptance of recommended treatments, which has been attributed to ignorance, superstition, self-denial, fear of mastectomy and unavailability of treatment facilities [14, 15].
In Tanzania for example and areas of central Africa, breast cancer accounts for up to 6 percent of cancers in men [16]. Similar researches in Uganda and Zambia Showed 5% and 15% respectively [17, 18]
Male breast cancer in Nigeria represents 3.7-8.6% of all breast cancers; this is higher than the 1% recorded from other parts of the world [19]. Majority are invasive ductal carcinoma which is characterized by late presentation at advanced stage with attendant poor prognosis [9].

RISK FACTORS
The exact cause of male breast cancer is not known but there are risk factors that make someone susceptible to develop the disease over a period of time. They include:

A. Genetic: Familial disposition just like that of the women breast cancer increases the risk of developing male breast cancer. Men that have first degree relatives with history of breast cancer tend to be susceptible to have the disease in the future. Basham et al [25] stated that 15-20% of male breast cancer cases originate from family history. Men can have mutation on breast cancer gene BRCA1 and BRCA2 but the latter is more common in this sex [26]. A male with BRCA2 mutation carries an increase 6% life time risk of having the disease than 0.1% in the normal population. Other genetic condition that increases susceptibility to the disease is Klinefelter’s syndrome [27].

B. Radiation Exposure: Frequent exposure to ionizing radiation has been associated with an increase risk of developing breast cancer in both men and women [7]. Men with history of undergoing chest x-ray or radiation therapy frequently have a greater chance of having the disease [28]. Therefore, prolong exposure to radiographs may be
harmful to individuals. Even workers that expose themselves to electromagnetic waves and other radiations daily without adequate protection are at risk [29].
C. Hyperestrogenism: Certain conditions can result in abnormally high levels of estrogen in men thereby increasing the risk of developing breast cancer. About 80-90% of male breast cancer has estrogen receptor meaning they are ER positive. Klinefelter’s syndrome and cirrhosis of the liver have been found to cause increase estrogen level in men [30]. Other conditions affecting estrogen in relation to androgen levels are taking exogenous estrogen as medication, mumps ochitis and testicular dysfunction [8]. Obesity also increases risk of breast cancer due to conversion of androgen hormone by the fat cells into estrogen; this means that obese men have higher level of estrogens in their body which subsequently may cause breast cancer [31].

DIAGNOSIS
A number of diagnostic methods are available. They include:
1. Medical history and physical examination: The medical history may give some clues about the cause of any symptom on the patient and tendency of having increase

risk factor(s) of developing breast cancer [8]. A thorough clinical breast examination will assist in locating any lumps or suspicious areas and assess the texture, size, and relationship of the breast to the skin and muscle tissue [32].

2. Biopsy: this involves removal of tissue sample from the body for examination under the microscope. There are different ways of obtaining tissue biopsy namely fine needle aspiration, core needle biopsy and surgical biopsy [33]. Majority of male breast cancers are invasive ductal carcinoma (80-90%) while ductal carcinoma in situ accounted for 10% [34, 35]. Cancer of lobular origin made up of only 1% due to lack of abundant lobules in male breast, others include Paget’s disease (1%), mucinous (1%), medullary and invasive papillary has 2% of occurrence each [36, 37, 38]. Special techniques are employed to further diagnosed breast tissue histopathologically as below:

1. Immunohistochemistry (IHC): This is a method of employing specialized antibodies against specific antigens on the cell membrane or nuclear region of the breast cancer cells. The standard tests include estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2) and Ki67 tests for invasive breast cancers [37]. About 90% of male breast cancers are ER positive and 16% of the cases over-expressed HER 2 [39].

2. Fluorescent in situ hybridization (FISH): This test uses fluorescent pieces of DNA that specifically stick to copies of the HER2/neu gene in cells, which can then be counted using a fluorescent microscope. Many breast cancer specialists think the FISH test gives more accurate results than IHC, but it is more expensive and takes longer to get the results. When IHC result is 2+, the HER2 status of the tumor is not clear and the tumor is then tested with FISH for confirmation [8, 40].

3. Genetic Testing: Genes are tested on the sample so as to understand the biology of the tumour. Some cancers are fast growing while others are not depending on the type of individual genes they possess. Examples of gene testing methods include Oncotype Dx™ and Mammaprint™ [38].

4. Imaging Tests: Different imaging techniques are use in assisting diagnosis of breast cancer, the methods are diagmostic mammography, ultrasound, magnetic resonance imaging [38]

5. Other Tests: There are several other tests use in assisting diagnosis of breast cancer namely nipple discharge examination, blood tests, X-ray, bone scan, computed tomography and positron emission tomography [8].

PREVENTION
There is need for men to take necessary steps in preventing themselves from having breast cancer by carrying out and observing the following steps:
1. Regular self examination: A person’s best chance of surviving breast cancer is early detection through regular self-examinations. Men should be familiar with the normal feel of their breast tissue so that they can bring any lump or change to the hospital for proper attention.

2. Active participation in exercise activities: It has been realized that increased physical activity is associated with decreased breast cancer risk. This may be because exercise lowers hormone levels, boosts the immune system, and changes metabolism while lack of exercise contributes to obesity.

3. Making healthier food choices: Several researches has shown immense important of particular food substances in preventing development of breast cancer and many other types of cancers. They contained antioxidants and other essential elements that repair and prevent cells from the damaging effects of free radicals and other carcinogens. Examples include tomatoes (lycopene), green vegetables (beta-carotene, folate, Vitamin C, E and K), berries (anthocyanins), onion (quercetin) and sweet potatoes (beta-carotene and Vitamin C).

4. Optimizing Vitamin D source: Vitamin D influences virtually every cell in the body and is one of nature’s most potent cancer fighters. This can be done by appropriate sun exposure or by using Vitamin D supplements.

5. Avoidance of unnecessary exposure to radiation/Environmental pollution: Getting medical imaging studies only when they are necessarily needed and avoidance of environmental pollutions help in cancer prevention.

6. Quitting smoking and sleeping well: Researches has shown cigarette smoking is associated with cancer development because it is injurious to the overall health of an individual. Avoidance of smoking is an essential mean of preventing cancer and having enough sleep maintain someone hormonal balance for a healthy living [41, 42, 43].

CONCLUSION:
Although cancer of the male breast is rare, when it occurs, it presents at an advanced stage especially in resource poor settings. This underscores the need for use of simple and painless preventive measures.

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Omosigho O P
Medical Microbiology Department, Federal Medical Centre Bida. Niger State .Nigeria

Emumwen E.G, Inyinbor H.E
Medical Microbiology Dept., Federal Medical Centre Bida. Niger State Nigeria.

All correspondents to: Medical Microbiology Department, Federal Medical Centre Bida. Niger State, Nigeria. Email –omosighoop@gmail.com

ABSTRACT

Male urogenital tract infection is an important factor in the management of infertility. This study was carried out to evaluate the prevalence of bacteriospermia, antibiotic susceptibility pattern and its effect on the quality of spermatozoa in male infertility in Bida. Five hundred and six semen samples cultured in the medical microbiology laboratory for three years were analyzed. This study showed a prevalence of bacteriospermia of 39.9% with Staphylococcus aureus having the highest frequency of 30.4%. Bacterial infection has remained one of the important factor in the management of infertility ,this study showed high rate of bacteriospermia in primary infertility 27.7% which is statistically significant (P= 0.000). The prevalence of bacteriospermia in Bida was more pronounced in oligospermia 22.7%. This study found a significant statistical difference between bacteriospermia and age (P=0.000) .In conclusion, the prevalence of bacteriospermia is high in Bida, it is therefore advocated that attention should be given to the treatment of urogenital infection in the management of male factor infertility.

KEY WORDS-Bacteriospermia, Staphylococcus aureus, Antibiotic susceptibility, Male factor, Infertility.

INTRODUCTION

According to World Health Organization (WHO), seminal fluid infection was define as the presence of significant bacteriospermia (= 103 bacterial/ml ejaculate), detection of Nisseria gonorrhoae, Chlamydia trachomatis, Ureaplasma urealyticum, significant leucocytospermia (WHO 1999).
The isolation of microorganisms in seminal fluid especially of infertile men has been widely reported, while the exact role of microbial infection in the etiology of infertility is not very certain owing to the limitation in diagnostic criteria and asymptomatic nature of infection, as some possible effect on the properties of seminal fluid associated with fertility has been suggested (Gregoriov et al .,1989, Merino et al., 1995, Villanueva-Diaz et al., 1999, Purvis and Christiansen1993, Buhharin et a.,l 2000 and Rodin et al., 2003 ).
There is disagreement as to the influence of certain microbial infection on male infertility, several investigation have reported different types of organisms in seminal fluid specimens depending on the method of examination (Macleod and Gold 1951). It was reported that detection of bacteria in semen does not necessary suggest
infection since bacteria isolates in seminal fluid may represent contamination, colonization of urethral orifice or infection. Opportunistic microorganism cause classical infection of urogenital tract and subclinical reproductive tract infection, these infections of the seminal fluid leads to decrease in number of spermatozoa, the suppression of their motility, changes their morphology and fertility capacity (Buhharin et al., 2000).
This study was carried out to evaluate the prevalence of bacteriospermia, antibiotic susceptibility patterns and its effect on the quality of spermatozoa in male infertility in Bida, Niger State.

MATERIALS AND METHOD
Five hundred and six (506) seminal fluids from men investigated for infertility over a period of three years were analyzed. These were seminal fluid of patient referred to the Laboratory from the Gynecology Clinic of Federal Medical Centre, Bida.
The semen was collected after the patient had abstained from sex at least three days. Samples were collected either by self or assisted masturbation into sterile bottle. Patients were educated on proper sample collection to reduce contamination and submitted to the Laboratory within one hour of production. The semen were cultured on Blood, Chocolate and MacConkey agar media and incubated for 24 hours at 37oC while Chocolate agar were incubated under 5% Co2. Emergent colonies were identified according to standard bacteriological method.
The samples were analyzed within one hour of collection or as soon as liquefaction occurred using manual method.
Initial microscopy examination of the appearance, viscosity and volume estimation was done, after which microscopy was carried out to estimate the sperm concentration, motility and morphology according to WHO guideline (WHO 2010).

DISCUSSION

Male genital tract infection is an important etiological factor leading to deterioration of spermatogenesis, impairment of sperm function and/or obstruction of seminal tract (Owolabi et al., 2013). The prevalence of bacteriospermia among male partners of infertile couples in this study is 39.9% with Staphylococcus aureus having the highest frequency of 154(30.4%) followed by Eschericia coli 34(6.7%) and Pseudomonas aeruginosa 7(1.2%) which is in agreement with other studies in Nigeria (Ibekwe and Mbazor 2002 in Abakeliki , Ugboma et al., 2012 in Port Harcout and Emokpae et al ., 2009 in Kano).
Generally ,the risk of infertility increases by age, in this study a high bacteriospermia prevalence rate was obtained among age 36-40 years (12.4%) followed by age 26-30 years (9.2%) and 31-35 years (8.8%) this findings is statistically significant (p=0.000) and agrees with the findings in Port Harcourt Nigeria (Olutimilehin et al .,2012).

Infection has remained one of the important factors in infertility our findings showed a higher rate of bacteriospermia in primary infertile couples 27.7% compared to 12.3% in secondary infertility and it is found to be statistically significant(p=0.000) though, in contrast to the findings of Olujubu et al., (2013)who reported a higher prevalence in secondary infertility.

Bacteriospermia was more pronounced in couples with oligospermia 22.7% followed by normospermia 10.5% and azoospermia 6.7% similar to the findings in Kano by Emokpae et al .,2009, while Owolabi et al ., in Ile ife reported 50.2% seminal infection in normospermia ,20.3% in oligospermia and 4.4% in azoospermia.
Antibiotic susceptibility pattern of bacteriospermia in Bida from our study shows that all isolates are 70-90% susceptible to Levofloxacin, Ciprofloxacin, Pefloxacin and Azithromycin while many are resistant to Gentamycin , Cefuroxime, Ceftazidime and Cotrimoxazole.

In conclusion, the prevalence of bacteria in semen may affect fertility in several ways including damage of spermatozoa, hampering their motility, altering the chemical composition of fluid (Mogra et al., 1981). Bacterial infection in this study is high and we advocate that proper attention should be given to the treatment of bacteriospermia in the management of male factor cause of infertility.

Bakari AG
Department Of Medicine Ahmadu Bello University Teaching Hospital, Zaria
Lawal N; Akuyam SA ; Anaja PO
Department of Chemical Pathology Ahmadu Bello University Teaching Hospital, Zaria

All correspondence to: Lawal N nasiruacademy@gmail.com

ABSTRACT

Type 2 diabetic patients are at an increased risk of developing liver diseases owing to the nature of the disease and its inherent complications. Elevated serum activities of the liver enzymes are the most frequent indicators of liver disease. The purpose of this study was to determine the pattern of abnormal serum liver enzymes activities in type 2 diabetic individuals. The study comprised of 170 type 2 diabetic patients attending Medical Outpatients Department of Ahmadu Bello University Teaching Hospital, Zaria. Diabetes mellitus (DM) was confirmed according to the new diagnostic criteria based on 2 fasting or 2 random plasma glucose levels of more than 7.0 mmol/L and 11.1 mmol/L respectively. A concise history of the patients, physical examination and laboratory findings were recorded on a proforma. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the kinetic method of IFCC. Serum gamma glutamyl transferase (GGT) activities were measured using the kinetic method of SZASZ. Serum alkaline phosphatase (ALP) activities were measured using the colorimetric method of King and Amstrong. The concentrations of serum FBG and RBG were measured using glucose oxidase method of Trinder.
One hundred and eighteen (69.4 %), 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. In addition 42 (24.5 %) patients had both mild increases of AST-ALT and 10 (5.9%) had mild increases of all the liver enzymes activities. It can be concluded from the findings of the present study that there is chronic mild increases in serum liver enzymes activities in diabetic patients therefore, liver function tests (LFTs) be included into routine laboratory investigations of DM in Nigerian hospitals.

KEY WORDS: Diabetic, Serum, Liver Enzymes

INTRODUCTION

Diabetes mellitus (DM) is a systemic disease caused by absolute or relative deficiency of insulin and is manifested by disorders of carbohydrates, lipid and protein metabolism1. The prevalence of diabetes is high in patients who have liver disease such as non alcoholic fatty liver disease (NAFLD), chronic viral hepatitis, haemochromatosis alcoholic liver disease and cirrhosis. Similar studies have shown that DM plays a significant role in the initiation and progression of liver injury (Hickman IJ and MacDonald GA, 2007).
Onyemelukwe and Bakari (1998) observed that chronic liver disease was responsible for secondary diabetes mellitus in 15 cases (2% of total and 36 % of secondary diabetes mellitus). Of this number, 10 were secondary to liver cirrhosis, 3 with schistosomal liver fibrosis and 1 case each secondary to chronic active
hepatitis and chronic persistent hepatitis. All cases except Schistosomal liver fibrosis tested positive to serum hepatitis B surface antigen.
The hallmark of the disease is fasting hyperglycemia (WHO; Geneva, 1999) and studies have shown that liver plays a critical role in carbohydrate homeostasis and insulin degradation therefore, it is not surprising that it’s function may be affected by DM (Hanley et al. 2004). Association exists between DM and liver injury (Meltzer A and Everhart JE 1997). There are evidences have revealed that patients with type 2 DM have two times the risk of developing liver diseases than their healthy counterparts (Karen Barrow, 2005). Hsiao et al;2007 reported that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were associated with insulin resistance (IR) as glycaemic status progresses in the impaired fasting glucose group.

Liver disorders among diabetics is similar to that of alcoholic liver disease including fatty liver (steatosis), steatohepatitis, fibrosis and cirrhosis. Elevated serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase(ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase(GGT) are the most frequent indicators of liver disease and occur in diabetics more frequently than in healthy individuals Hsiao et al; 2007.The aim of the present study was to determine the pattern of abnormal liver enzyme activities in type II diabetic patients in Zaria , Northern Nigeria.

MATERIALS AND METHODS
The study was conducted in Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. This study was approved by the ethical committee of the ABUTH, Zaria in accordance with the declaration of Helsinki. A total of 170 diabetic patients attending Medical-outpatients Department (MOPD) and 80 apparently healthy individuals were studied. The criteria for diagnosis of type 2 DM was the American Diabetes Association Criteria (2004), fasting blood glucose of 7.0 mmol/L on two occasions or random blood glucose of 11.1 mmol/L with diabetic symptoms. The diabetic patients were provided with conventional diabetes care/control measures. At the MOPD, arrangement was made with the Physicians whereby subjects who satisfy the study inclusion criteria were selected. Informed consent for inclusion into the study was obtained from the subjects. The nature of the study was explained to the subjects by using an appropriate language. A full medical history was obtained from the subjects by the Physician followed by clinical examination. The findings were documented in the Proforma. Blood specimens were taken into plain tubes,
using sterile technique. The blood was centrifuged and the serum was carefully drawn into sample bottles and then stored frozen at -200C until the time for analysis. The samples were analyzed for plasma glycatedheamoglobin (GHbA1c) using the method of Triveli et al; 971 and Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) using the method of Trinder 1964..
Statistical analysis was performed using statistical package for social sciences (SPSS) for Windows, version 15.0. Data were presented as Mean±SEM. plasma glycated heamoglobin (GHbA1c) levels and serum Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) levels were compared with those of the apparently individuals using two tailed student t-test. A p-value of equal to or less than 0.05 (p=0.05) was considered as statistically significant.

RESULTS
The results of clinical parameters are presented in table I. The differences in weight and body mass index (BMI) between diabetic subjects and controls were statistically significant. Plasma GHbA1c and serum FBG as well as RBG in diabetic patients and controls are presented in table II. The mean values of plasma GHbA1c and serum FBG as well as RBG were significantly higher in diabetic patients than the control subjects (p<0.01).

The result of pattern of abnormal liver enzymes activities are presented in Table II. One hundred and eighteen (69.4 %) , 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. Similarly, in the control subjects, 33 (41.3 %), 1 (1.3 %) 27 (36 %) 11 (13.8 %) individuals also had mild increase in serum levels of AST, ALT, GGT and ALP respectively.

DISCUSSION

The results obtained in the present study showed that the percentage prevalence of abnormal serum liver enzymes activities were significantly higher in diabetic patients than in control subjects. The finding of the present study was similar with those of Salmela et al; 1984 and Erbey et al; 2000 as seen in (table II). Even though there were variations in the values of percentage prevalence reported by the above mention authors as well as the present study however, the values were higher in diabetic patients than in control subjects.

The high percentage prevalence of elevated liver enzyme activities in type II diabetic patients seen in the present study suggest that the liver is diseased. It was reported that elevation of serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase (GGT) are the most frequent indicators of liver disease (Monica et al 2005 and Meltzer A, Everhart JE 1997) therefore, the high percentage prevalence of elevated liver enzyme activities in type II diabetic patients as well as the higher mean level liver enzyme activities in type II DM than control subjects seen in the present study further confirm the abnormalities of liver function in type II DM. The hallmark of type 2 DM is hyperglycaemia and it leads to fat accumulation in the liver14. Fat accumulation is known to be directly toxic to hepatocytes . An elevation of liver enzymes such as ALT and GGT even within the normal range, reflects deposition of excess fat in the liver (Meybodi et al 2008 and Maryam et al 2008). This may be responsible for the increased serum activities of transaminases seen in the present study. Zachary, 2007stated that fat accumulation in the liver also stimulates the release of inflammatory cytokines such as tumor necrosis factor-α (TNF) and interleukine-6 (IL-6) which may contribute to hepatocellular injury. This might also result to increased serum activities of transaminases in the present study.

Hyperglycemia leads to myoinositol depletion in several tissues susceptible to diabetic complications (for example liver), although the exact mechanism is unclear. The fall in myoinositol levels in the cell membrane alters the functioning of Na+/K+ ATPase pump and the integrity of the cell membrane is impaired (Malnick et al; 2003). Clinical observations and experimental studies have shown that subtle changes of cell membrane are sufficient to allow passage of intracellular enzymes such as AST and ALT to the extracellular space with subsequent elevation of their levels in plasma (Malnick et al; 2003). This could be another reason for increased serum activities of transaminases in the present study.

Monica et al; 2005 reported that ALT, ALP and GGT were associated with prevalent type 2 DM and AST with prevalent IGT). NAFLD is a complication in 32%-78% of patients with type 2 DM and 50% of these patients may have NASH. Some studies demonstrated that hyperglycemia and hyperinsulinaemia can promote fatty infiltration of the liver (De Marco et al 1999).
CONCLUSION
It can be concluded from the findings of the present study that there were high prevalence abnormal liver enzyme activities in diabetic patients therefore, liver function tests (LFTs) should be included into routine laboratory investigations of DM in Nigerian hospitals.

ACKNOWLEDGMENT
We acknowledged the assistance of University Board of Research of Ahmadu Bello University, Zaria. We thank Dr IS Aliyu, Head of Chemical Pathology Department of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria for his constant advice and encouragement during this study. We are also grateful to Dr Muazu Babura of the Department Medicine of ABUTH, Zaria for his assistance during the course of this work. We also thank Mal. MA Auwal and of the Department of Chemical Pathology ABUTH, Zaria for his assistance in analysis and other logistics.

Narendranath Epperla, Patrick Foy

Division of Hematology and Oncology, Medical College of Wisconsin, 9200 W Wisconsin Avenue, Milwaukee, WI, USA

Erika Peterson
Division of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI, USA

Correspondences to: Narendranath Epperla · ·nepperla@mcw.edu

ABSTRACT
Background: Heterotaxy is a relatively uncommon congenital anomaly that is usually diagnosed incidentally on imaging studies in adults. We present an unusual case of venous thromboembolism in a 26 year old pregnant female with Heterotaxy syndrome.

Case presentation: A 26 year-old pregnant female at 13 weeks gestation suffered cardiac arrest with successful cardiac resuscitation and return of spontaneous circulation. The cardiac arrest was secondary to massive pulmonary embolism requiring thrombolytic therapy and stabilization of hemodynamics. She had extensive evaluation to determine the etiology for the pulmonary embolism and was noted to have an anatomic variation consistent with heterotaxy syndrome on imaging studies. After thrombolysis the patient was treated with UFH and then switched to enoxaparin without complication until 25 weeks of gestation when she experienced worsening abdominal pain with associated headaches, lightheadedness and elevated blood pressures needing elective induction of labor. The infant died shortly after delivery. The anticoagulation was continued for additional 3 months and she was subsequently placed on low dose aspirin to prevent recurrent venous thromboembolic episodes. She is currently stable on low dose aspirin and is into her third year after the venous thromboembolism without any recurrence.

Conclusion: To our knowledge, this is the first reported case of venous thromboembolism in pregnancy associated with heterotaxy syndrome. A discussion on pathophysiology of venous thromboembolism in pregnancy and heterotaxy syndrome has been undertaken along with treatment approach in such situations.

Keywords: Venous thromboembolism; Inferior vena cava; Low molecular weight heparin

BACKGROUND

Heterotaxy is an uncommon polymalformative syndrome characterized by congenital anomalies that arise from disorderly arrangement of asymmetric thoracic and abdominal viscera and blood vessels [1]. The estimated prevalence is approximately 1 in 10,000 live births. Morbidity and mortality rates are high approaching nearly 70 % due to major cardiac malformations (especially in heterotaxy with asplenia patients). Five to ten percent of patients with minor or no cardiac anomalies will survive to the adulthood. Some will remain asymptomatic and can be diagnosed incidentally on imaging studies. Heterotaxy has been reported to be associated with VTE and is felt to be related to abnormal lower extremity venous system. Infact in the last few years some authors considered it as a VTE risk factor. However VTE in pregnancy with heterotaxy syndrome has not been
reported.
Herein we present a case of 26 year-old pregnant female who suffered cardiac arrest related to massive pulmonary embolism requiring cardiac resuscitation. Imaging studies incidentally discovered anatomic variation consistent with heterotaxy syndrome. She was anticoagulated for 6 months with low molecular weight heparin (LMWH) and subsequently placed on low dose aspirin to prevent recurrent VTE. A detailed discussion of the pathophysiology of VTE in pregnancy and heterotaxy syndrome has been undertaken with a focus on treatment approach in such situations.

Case presentation
A 26 year old otherwise healthy, G1P0 female at 13 weeks gestation was admitted to medical intensive care unit after she experienced cardiac arrest at home. She had reported shortness of breath earlier that day and then collapsed.

During initial paramedic evaluation, she was pulseless and found to be in pulse less electrical activity (PEA). She was successfully resuscitated with return of spontaneous circulation (ROSC) in approximately 7 min and was brought to the hospital. Emergency department (ED) evaluation revealed the patient to be nonresponsive, and she was immediately intubated. Her exam was remarkable for cold and cyanotic extremities, without palpable distal pulses but strong femoral pulse. Shortly after her arrival to ED, the patient became pulse less again needing resuscitation and ROSC on 2 occasions.

Initial blood work was remarkable for severe metabolic acidosis (HCO3 of 10) and elevated troponin I (2.77 ng/ml, normal range 0–0.34 ng/ml). EKG showed right bundle branch block while bedside 2 D echocardiogram demonstrated right heart strain pattern. Bedside ultrasound (USG) showed no significant free fluid with live intrauterine gestation. Bilateral lower extremity compression venous dopplers of the entire venous system were negative for any evidence of deep venous thrombosis. She underwent computerized tomography (CT) of the chest which revealed a nearly occlusive thrombus in the bilateral lower lobar arteries extending into the segmental arteries (Fig. 1). The main pulmonary arterial trunk was noted to be enlarged with evidence of right heart strain (Fig. 2). Head CT scan was negative for any acute intracranial abnormalities. She was administered intravenous tissue plasminogen activator (TPA) (50 mg after ROSC the second time) in the ED with improvement in her hemodynamics. Hypothermia protocol was concurrently initiated. She was subsequently transferred to medical intensive care unit on unfractionated heparin (UFH), bicarbonate and norepinephrine drips. The patient was extubated two days following and was gradually weaned off blood pressure support. UFH drip was titrated based on aPTT and 6 days later she was transitioned to LMWH (dalteparin 10,000 units subcutaneously once daily in view of her insurance issues). She had aggressive supportive care and after a 2 week hospital stay, she was discharged to rehabilitation facility on Dalteparin. A month later dalteparin was changed to enoxaparin 55 mg (1 mg/kg) s/q twice daily (monitored with anti Xa levels).
At approximately 20 week’s gestation, the patient had a fetal ultrasound that revealed singleton gestation with massive hydrops, fetal contractures and ventriculomegaly/hydranencephaly. It was presumed that these findings were secondary to early hypoxic injury and she was counseled about poor prognosis. During her 25th week gestation, the patient noted worsening abdominal pain with associated headaches, lightheadedness and weakness. She had elevated blood pressures and was suspected to have mirror syndrome. Enoxaparin was discontinued 24 h prior to the planned procedure with initiation of UFH drip. The patient underwent elective induction of labor with palliative care services. The infant died shortly after delivery. The patient experienced increased vaginal bleeding secondary to retained products of conception and underwent dilatation and curettage with achievement of hemostasis. Enoxaparin at previous dosage (55 mg s/q twice daily) was restarted 24 h after the delivery without any major or untoward complication. Platelet count and anti-Xa levels were checked while the patient was on enoxaparin. Anti-Xa levels were in therapeutic range (0.8–1, goal 0.6–1.0 IU/ml) and platelet counts remained within normal limits.
In order to identify the cause of her VTE the patient had further work up including thrombophilia testing and CT chest, abdomen/pelvis. Thrombophilia testing revealed the patient to be wild type for Factor V and Prothrombin gene. Protein C and S antigen levels were normal. Antithrombin activity was initially low (71, normal range 75–125 % ACT) at the time of initial VTE but subsequently normalized (107). Antiphospholipid antibody testing including lupus anticoagulant, anti-cardiolipin antibodies and beta 2 glycoprotein I antibodies was negative. CT chest, abdomen and pelvis showed constellation of imaging findings compatible with heterotaxy syndrome. These included left-sided superior vena cava (SVC) draining into the left coronary sinus, hemiazygous vein drains into left SVC, absent azygous vein, absent (interrupted) inferior vena cava (IVC) below the hepatic IVC with persistent left IVC which drains into left coronary sinus, polysplenia, bilateral left lung and benign hepatic hemangioma (Fig. 3A-C).

The patient completed 6 month duration of anticoagulation with LMWH and was subsequently placed on ASA 81 mg. Prior to discontinuation of anticoagulation therapy the patient had CT pulmonary angiogram which showed normal diameter of the main pulmonary artery without any evidence of acute or chronic pulmonary emboli. She is currently stable on low dose ASA and is into her third year after the VTE without any recurrence. She has had no further pregnancies.

DISCUSSION
Heterotaxy or situs ambiguous refers to malposition and dysmorphism of viscera and blood vessels, often with indeterminate atrial arrangement. This abnormal arrangement of body organs is different from the orderly arrangement seen in situs solitus or situs inversus. Heterotaxy syndrome can be associated with either asplenia or polysplenia. Our patient had heterotaxy with polysplenia and hence will limit our discussion to this abnormality and hereon will be referred to as heterotaxy.
Heterotaxy implies that the patients have bilateral bilobed lungs, bilateral pulmonary atria, a centrally located liver, a stomach in indeterminate position, interrupted IVC with azygous continuation and multiple spleens. After polysplenia, the most frequent finding in heterotaxy is the hypoplasia of the IVC with absence of the intrahepatic segment and direct continuation with the azygous venous system. Heterotaxy commonly occurs as a sporadic condition but it can be inherited.
Varied timing of embryological causative factors during the development of fetus can explain the wide range of anomalies in the heterotaxy syndrome. Cardiac anomalies are mostly the result of abnormal embryological development around day 28 of embryogenesis, as it is during this period that connection between primitive heart and venous channels occurs. The failure of fusion of fetal lobules leads to individualization of multiple splenic nodules, which are located along the greater curvature of the stomach, as the spleen develops in the mesogastric region. IVC is a complex vascular structure that is developed during weeks 6–8 of embryogenesis. During this period three pairs of primitive venous channels (posterior cardinal, subcardinal and supracardinal veins) form the mature venous system (IVC) through a complex sequential process. Various malformations including partial or even complete absence of IVC can result from the failure in the developmental steps related to embryonic dysontogenesis that can affect separate segments or even the entire IVC. Thus mutations in genes that control left-right patterning and teratogenic exposures in early embryonic period underlie majority of heterotaxy cases.
The occurrence of DVT seems to be higher in patients with heterotaxy compared to the general population. It is postulated that stasis related to the abnormal venous drainage of the lower extremity venous system due to interrupted IVC, and increased platelet aggregation related to the anomalous drainage of the splenic venous system into the azygous system seems to be the possible culprits for heightened risk of pulmonary thromboembolism in these patients.
VTE is 5 times more frequent in pregnant women than in non-pregnant women of similar age. This is because pregnancy induces a state of venous stasis and
Fig. 3. A. CT scan of the chest with contrast.
Coronal section showing bilateral bilobed lungs
(yellow arrows depict the major fissure). B. CT
scan of the abdomen. The arrow points to multiple
splenic lobules suggestive of polysplenia.
C. CT scan of the abdomen. Coronal section
showing azygous continuation of inferior vena cava

hormonal and hematological changes leading to an increased risk for VTE. Venous stasis occurs from progesterone mediated increased venous distension in the first trimester and the compressive effect by the enlarging uterus on the common iliac vein in the late second and third trimesters. In addition there is an imbalance between the procoagulant (increased circulating levels of fibrinogen, von Willebrand factor, VII, VIII, IX, X and XII and increased generation of fibrin) and anticoagulant factors (decreased Protein S levels and fibrinolysis) through the pregnancy. Though pregnancy is considered to be a relative contraindication to systemic thrombolysis (recombinant TPA or streptokinase) in hemodynamically unstable patients with pulmonary embolism, the risk of complications for pregnant females treated with thrombolytic agents may be similar to that in the non-pregnant population.

In our case, the exact etiology for VTE is unclear. It may be related to pregnancy alone or mechanical factors from distorted venous system anatomy or a combination of both. Because the patient’s VTE occurred early in pregnancy, uterine enlargement is unlikely to have contributed to venous thrombosis. Effects of pregnancy in heterotaxy have been poorly described.

Challenges to the clinical management of our patient include the duration of anticoagulation and future pregnancy. Based on the 2012 American College of Chest Physicians (ACCP) guidelines, anticoagulation with LMWH is recommended for at least 3 months from the initial pulmonary embolism and 6 weeks postpartum. In our case, the patient was into her 3rd month of anticoagulation when she had induction of labor and delivery; hence the anticoagulation was continued for additional 3 months for a total of 6 months. Subsequently she was placed on low dose aspirin (81 mg) to prevent recurrent VTE (especially given her heterotaxy syndrome) based on the Warfarin and Aspirin (WARFASA) and Aspirin to Prevent Recurrent Venous Thromboembolism (ASPIRE) trials as well as the individual patient data analysis of WARFASA and ASPIRE trials performed by the INSPIRE Collaboration. Currently there is no evidence about the risk of VTE recurrence and anticoagulation duration in similar patients. According to the 2012 ACCP guidelines the decision regarding the duration of anticoagulation in a patient should be made after careful evaluation of both the risk of VTE recurrence and bleeding risk. Given the congenital prothrombotic risk factor (interrupted IVC related to heterotaxy syndrome), severity of the presentation and apparently low bleeding risk extended anticoagulation treatment with targeted oral anticoagulation maybe a suitable alternative to aspirin with periodic assessment of the bleeding risk.

Our patient had a massive pulmonary embolism with cardiac arrest that created a great sense of apprehension both in the patient and her family regarding future pregnancy. Unfortunately, there is paucity of data to either support or refute future pregnancy associated with her condition. Previous VTE alone is not a contraindication to future pregnancy provided anticoagulation is available. However patients with heterotaxy syndrome and VTE are undoubtedly at increased risk of VTE compared to other pregnant women. Hence future pregnancies in this patient group should be considered high-risk, and requires multi-disciplinary management. We recommend full intensity anticoagulation with LMWH (likely enoxaparin 1 mg/kg two times daily) with regular monitoring of anti-Xa levels prior to her pregnancy. It is important to measure anti-Xa levels 4 h after the last dose and be aware of the different targets based on which LMWH regimen is used (once-daily [goal 1.0–2.0 IU/mL] or twice-daily [goal 0.6–1 IU/mL]). In addition we recommend that there is a detailed discussion between the physician and the patient regarding the role of thrombolytic therapy in the event of recurrent VTE and possible termination of pregnancy in the worst case scenario.

CONCLUSION
To our knowledge this is the first reported case in the English literature that shows pregnancy associated VTE in a person with heterotaxy. After completion of anticoagulation therapy for the initial thrombotic event, the patient needs secondary prevention for VTE recurrence and low dose aspirin was the chosen treatment in this case. Although there are no published guidelines we believe that there are no contraindications for future pregnancy in this special group provided the patient is on anticoagulation prior to the pregnancy.

Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

Atiegha C. Igoni M , Victor I.,
Department Of Medical Laboratory Sciences, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Ayaowei I.T.
Department Of Health Information Management, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Eseimokumo Me.
Department Of Pharmarcy Technician Studies, College Of Health Technology Otuogidi-ogbia , Bayelsa State.

All correspondence to: IGONI M ; Bayelsa State College of Health Technology: e mail; achristo40@gmail.com.

ABSTRACT
The treatment of tuberculosis has evolved from streptomycin to the DOTS strategy .Even as at that it is a serious challenge. This retrospective study was carried out to evaluate the effectiveness of the method. A total of sixty seven(67) Tuberculosis cases were evaluated at the Tuberculosis Treatment centre Nembe comprehensive Health Centre, Bayelsa state , it covered a period of four (4) years, from 2009 to 2012. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. It is therefore recommended for community and family based treatment.

Key words: DOTS, Cured, treatment completed and treatment failure.

INTRODUCTION
Tuberculosis, a major health challenge and leading infectious killer is caused mainly in man by Mycobacterium tuberculosis. The bacilli enters the body by inhalation of droplets or dust particles containing it. It occurs as pulmonary tuberculosis or non- pulmonary tuberculosis. In pulmonary tuberculosis the pulmonary aveoli and surrounding lymph glands are lodged by the bacilli resulting in lesion characterized by acute inflammatory reactions with accumulation of fluid and white blood cells around the aveoli, while the non pulmonary tuberculosis includes the renal and urogenital tuberculosis, military tuberculosis and tuberculosis meningitis with varying symptoms1.
In sub-saharan Africa, cases of tuberculosis have increased dramatically, overwhelming control programs 2. This could be as a result of the fact that tuberculosis infection control interventions are not routinely implemented in contrast to what happens in high income countries with low prevalence of tuberculosis where infection control policy is routinely observed3. However the launch of directly observed treatment shortcourse (DOTS) in 1995 by the world health organization (WHO) has shown to be an effective intervention that will lead to reduced tuberculosis transmission and decreasing number of tuberculosis cases4,5. Also it is among the most cost-effective global health interventions available today6,7.
The DOTS strategy is based around a short-course treatment regimen for a minimum of six months of four drugs in combination (2 months of Isoniazide, Rifampicin ,Pyrazinamide and Ethambutol), a good management practice, sputum smear microscopy for diagnosis and the direct observation of doses to ensure adherence. Treatment success under DOTS concerns two outcomes- cured and treatment completed: “cured” if patient finish the treatment regimen with negative bacteriology at the end of the treatment whereas “treatment completed” refers to patients who have finished the regiment in full without showing evidence of treatment failure and without negative bacteriology. This treatment regiment however use to suffer from a number of drawbacks, with the combination of available drugs, the duration of treatment cannot be reduced below six months without a significance in relapses although re-infection with a different strain of mycobacterium tuberculosis can cause tuberculosis recurrence, which is considered to be an important measure of efficacy of tuberculosis treatment8,9. Also when treatment is given under sub-optimal conditions, regiments are associated with high rate of patients non-adherence specially in low income countries which harbor high burden of diseases10. The consequence of this is increased mortality and creation of clinic infections, drug resistant cases. The World Health Organization estimates in 2004, 424,203 cases of multidrug resistant tuberculosis globally among which 181,408 cases occurred in patients who had already been treated with standard (first line) therapy11.
This study was therefore carried out to determine the effectiveness of DOTS (Directly Observed Treatment Short course) on tuberculosis in the Nembe Local Government Area of Bayelsa state south-south Nigeria.

MATERIALS AND METHODS
Setting Comprehensive Health Centre Nembe, is situated in Nembe, the headquarters of Nembe local government area, about eighty kilometers south-east of Yenagoa the capital of Bayelsa State, Nigeria.

Subjects
The subjects of this report are those patients that reported to the TB treatment centre and those patients who were got through surveillance in the surrounding communities. A total of sixty seven positive cases were involved in the study. These cases were got over a four year period (from 2009 – 2012)

Laboratory analysis
Patients were given sterile wide mouthed plastic containers which they voided sputum into. Three specimens were collected from them over a two days period (spot, early morning and spot). Sputum analysis was done using the Ziehl-Neelson technique as described by cheesbrough12.

Method of drug administration
The strategy adopted here is the short course regiment that lasts for six months. Upon visit to the treatment center, Zeihl-Neelson(AFB) test is carried out. Treatment commences on patients who are newly diagnosed i.e. their sputum show smear positive. After the initial test, a second test (Zeihl- Neelson) is conducted upon revisit at the end of the second month of chemotherapy. The same thing applies
upon a second and third revisit which is at the end of the fifth and sixth months respectively.
For category 2 patients i.e. those who defaulted treatment or had a relapse, treatment is for seven months. Treatment on the intensive phase is for three months after which the second AFB test is done. The third and fourth AFB tests are done at the end of the fifth and seventh month of chemotherapy. Patients that absconded during treatment were not included in this work.

In 2009 eighteen new cases of sputum positives were recorded (six scanty and twelve +s). Upon first revisit i.e. after intensive phase, all eighteen persons were tested smear (AFB) negative. On second revisit the same feet was recorded. In the last revisit seventeen tested negative while one patient was AFB positive: table 1.

In 2010, seventeen persons were tested positive with varying degree of intensity of infection ( one scanty, eighty +s, four ++s and four +++s). sixteen persons tested smear negative at the end of the first revisit while person was tested positive. At the end of the fifth month of chemotherapy all fifteen persons tested smear negative (second revisit). After six months of chemotherapy one patient was tested smear positive while the other sixteen persons that continued their treatment to the end were tested negative. Conclusively, eleven persons were cured while one person was termed a “Treatment Failure”: table 2
Table 3 represents the summary of the 2011 treatment outcome. There were ten sputum smear positive cases in
2011. Eight of them were graded +, while one patient had 2+ and another 3+. Seven persons tested AFB negative upon the first revisit and one patients sputum was positive. In the second and third revisits, all eight patients had there AFB test negative. i.e. in 2011 eight persons were cured of tuberculosis in the treatment center.
Two scanty cases, twelve +s and eight cases of 2+s were recorded in the year 2012, twenty two cases in all. Three scanty cases were recorded in the first revisit stage, while nineteen tests were negative. In the second and third revisit all twenty two tests were smear negative. in 2012 twenty two persons were cured of tuberculosis in the treatment center. Table 4

Discussion
In this four years under study, the success of this method of treatment has been so tremendous. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. When the result was subjected to the chi-square test, it shows that there is a significant difference between those that are cured of TB by the dots strategy in the five years under study and those that were not cured by the strategy p<0.05, (234.845, df 3, assymp. Sig (2-sided) 0.000

Conclusion:
Since with the DOTS strategy it is very difficult for patients to skip treatment, (which in most times is the cause of resistance), the researcher recommends that this strategy should be adopted, be it community based or family based)

Acknowledgment
We wish to express our heartfelt thanks to God almighty for his abundance grace to carry out this research and the strength to stand it all, without him nothing would be done. We also greatly appreciate the provost of the Bayelsa State College of Health technology, Dr Adias T. Charles for his inspiration and motivation. Also, the research and manpower development department of the College was involved with the funding. Finally we thank the staffs of the comprehensive health centre Nembe, Bayelsa State.