Seropositivity of Anti-CMV IgM Antibody among Women of Child-Bearing Age Attending Selected Hospitals in Kaduna State, Nigeria

Human Cytomegalovirus is one of the most common cause of congenital viral infections. The study was conducted to determine the seroprevalence of Human Cytomegalovirus among women of child-bearing age attending selected hospitals in Kaduna State, Nigeria. A total of 228 blood samples were obtained from the women and processed serologically using Enzyme Linked Immunosorbent Assay (ELISA). In addition, structured questionnaire was used to determine socio-demographic and risk factors associated with Human Cytomegalovirus infection. Out of the 228 women, 215 (94.3%) were positive for anti-CMV IgM. The highest seroprevalence was observed among women belonging to age group 15-24 (98.8%), those with secondary level of education (98%), those living in the urban areas (95%), the civil servants (100%), the married (94.2%) and those of medium socioeconomic status (94.9%). A statistical significant difference was observed between the age groups only (p = 0.000). With respect to risk factors, female patients; with no sex partners (100%), with history of blood transfusion (96.7%), who do not wash their hands with soap after changing baby’s diaper (95.5%), who wash their hands after contact with children’s urine (94.7%) and those in close contact with children had highest seroprevalence (94.4%). No statistical significant association was observed between Human Cytomegalovirus infection and the risk factors considered. Female patients should be educated on the transmission routes as well as preventive measures of Human cytomegalovirus infections.


Introduction
Congenital infections are significant cause of perinatal morbidity and mortality estimated to be responsible for up to 50% of stillbirths in low and middle income countries and 10-25% in high income countries [1].Human Cytomegalovirus (HCMV), an ubiquitous herpes virus with the highest morbidity and mortality compared to other herpes viruses [2] is one of the most common cause of congenital anomaly.The incidence of congenital Human Cytomegalovirus infection has increased by nearly 300% since the early 2000s [3].
Maternal HCMV infection could result to bad pregnancy outcome including abortion, premature delivery, stillbirth and congenital malformation [4,5].Sexual activity and contact with young children are the main sources of primary maternal HCMV infection (Fowler and Pass, 2006).Primary HCMV infection occurs in 0.7-4.1% of all pregnancies [6].About 32% of women with primary infection during pregnancy, transmit the virus across the placenta to produce intrauterine infection [7].
Human Cytomegalovirus is the most common infectious cause of fetal malformations [8].Congenital HCMV affects one in every 150 live births globally [9].Approximately 7 to 10% of infants with congenital HCMV develop clinical manifestations.These include petechiae, Management of the respective study hospitals.In addition, informed consent of the patients was sought for participation in the study.

Sample size determination
The sample size of the study was determined using a previous prevalence of 94.8% [16] for Human Cytomegalovirus in Kaduna state.The formula described by Naing, et al. [17] was employed.Seventy-six samples were collected from each of the study hospital.

Questionnaire administration and sample collection
Structured questionnaire was administered to the patients to determine socio-demographic and risk factors associated with Human Cytomegalovirus infection.Two milliliters of blood were collected from the patients using a sterile syringe and transferred into plain tubes.The blood samples were transported in insulated cooler containing ice packs to the Laboratory of Department of Microbiology, Ahmadu Bello University, Zaria for analysis.The blood samples were processed according to the method described by Umeh, et al. [18].Sera from the blood samples were separated by allowing the blood to clot at room temperature and centrifuged at 2000 rpm for 10 minutes.The sera were transferred into cryovials using clean Pasteur pipettes and stored at -20 °C until required for further analysis.

Detection of human anti-cytomegalovirus IgG using enzyme linked immunosorbent assay (ELISA)
All patients' sera were tested for anti-CMV IgM using commercially available ELISA kits (Diagnostic Automation, USA).The procedure was carried out according to manufacturer's instructions.Briefly, 1:40 dilutions were prepared by adding 5 μL of the test samples, negative control, positive control, and calibrators to 200 µL of sample diluent.It was mixed well.Hundred microliter (100 μL) of diluted sera, calibrator, and controls were dispensed into appropriate wells.Hundred microliter (100 μL) of sample diluent was added to the reagent blank well.The plate was incubated at room temperature for 30 minutes.The liquid was removed from all wells by decanting.This was followed by washing the wells with washing buffer and the washing was repeated three times.Hundred microliter (100 μL) of enzyme conjugate was added to each well and incubated for 30 minutes at room temperature.Enzyme conjugate was removed from all wells by decanting and washing was repeated three times with washing buffer.Hundred microliter (100 μL) of tetramethylbenzidine (TMB) chromogenic substrate was added to each well and incubated for 15 minutes at room temperature.This was followed by adding 100 μL of stop solution to stop the reaction.The optical density of each well was read at 450 nm with a microwell reader jaundice, hepatosplenomegaly, chorioretinitis as well as neurological deficits, which consists of physical and mental retardation, deafness, and even death in about 10% of patients [10][11][12].
Factors associated with Human Cytomegalovirus infection include age, socioeconomic status, exposure to young children, race/ethnicity [8,[13][14][15].In addition, increasing number of live births, early sexual debut, ≥ 10 life time sexual partners, and herpes type II seropositivity also predispose one to HCMV infection [15].
Despite the fact that most women of reproductive age in developing countries are assumed to have immunity against HCMV because of the high seroprevalence, the prevalence of congenital HCMV is still higher in developing countries.Most previous studies on HCMV were conducted on pregnant women and that necessitate the need to include non-pregnant women of reproductive age since maternal HCMV infection can occur prior to or during pregnancy.

Objectives of the Study
1. To determine the seroprevalence of anti-CMV IgM using Enzyme Linked Immunosorbent Assay (ELISA).
2. To determine socio-demographic and risk factors associated with Human Cytomegalovirus infection using structured questionnaire.

Methodology Study area
The

Study design
The study is a hospital-based, cross-sectional and descriptive research aimed at women of child-bearing age between the ages of 15-50 years attending Hajiya Gambo Sawaba General Hospital Zaria, Yusuf Dantsoho memorial Hospital, Kaduna and Sir Patrick Ibrahim Yakowa General Hospital, Kafanchan.

Ethical approval and informed consent
Ethical clearance was obtained from the Research Ethics committee of Kaduna State Ministry of Health.Permission to conduct research was sought from the (95.2%) and those without disabled child (94.2%) (Table 3).Patients having no sex partners, those with history of blood transfusion, women who do not wash their hands after changing baby's diaper, those who wash their hands after coming in contact with children's urine and those in close contact with children had higher seroprevalence with 100%, 96.7%, 95.5%, 94.7%, 94.4% respectively (Table 4).

Discussion
A seroprevalence of 94.3% was obtained for anti-CMV IgM among women of reproductive age attending selected hospitals in Kaduna State, Nigeria.Our finding is similar to 94.3% IgM seroprevalence reported by Nahla, et al. [19] among neonates in Sudan.The high IgM seroprevalence observed in the study is a suggestion that an epidemic might have occurred and interpreted.Samples having CMV G index ≥ 1 were considered positive while those with CMV G index ≤ 0.9 were considered negative.

Data analysis
Odds ratio and Chi-square were used at 95% confidence interval to determine association and significant difference between the variables respectively.P-values ≤ 0.05 were considered as statistically significant.

Results
The overall seroprevalence of Human Cytomegalovirus was 94.3% (Figure 1).Out of the 228 patients examined in the three study hospitals, 215(94.3)were positive for anti-CMV IgM.The highest seroprevalence was observed in HGSGH with 100% (Table 1).Female patients in age-group 15-24 years, those with secondary level of education, those living in the urban areas, the civil servants and the middle class patients had the highest seroprevalence with 98.8%, 98%, 95%, 100% and 94.9% respectively (Table 2).The highest seroprevalence was observed in nonpregnant women (95.2%), pregnant women in their second trimester of pregnancy (95.1%), those with no child and one child (100%), women who had no history of miscarriage (94.7%), those with history of still-birth (100%), women with history of premature delivery   The findings contradicts the reports of Khairi, et al. [22], Pathmavathy, et al. [29] and Yusuf, et al. [28] who observed highest seroprevalence among ≥ 40 years, 25-30 years and 21-30 years respectively.Most of the women in that age group are married and it may be as a result of increased sexual activity.
Women who had no congenitally infected child had higher seroprevalence than those with congenitally infected child.This shows that the child's anomaly could be as a result of other agents responsible for congenital abnormalities.

Conclusion and Recommendation
A seroprevalence of 94.3% was obtained for anti-CMV IgM among women of reproductive age attending selected hospitals in Kaduna State, Nigeria.Women in age group 15-24 years had the highest seroprevalence.Female patients should be educated on the transmission routes as well as preventive measures of cytomegalovirus infections.
during the time of the study.As symptoms of Human Cytomegalovirus infections mimic those of other viral infections, the outbreak might have gone unnoticed particularly in developing countries like Nigeria where outbreaks may not always be recognized.
Since studies have demonstrated high congenital Human Cytomegalovirus infections in population with high seroprevalence, the implications of this finding is that there may be a corresponding increase in congenital Human Cytomegalovirus infections among infants in the state.This may lead to rise in HCMVrelated hearing and vision loss in children.Maternal HCMV infection is regarded as a predisposing factor for congenital HCMV infection and HCMV-associated hearing loss in children [20].
A sample positive for anti-CMV IgM is not always indicative of recent infection.It may be due to reactivation as a result of the same virus or reinfection with a new or different strain of HCMV.In addition, specific IgM antibody may still be detected for several months or years in low titers after primary infection [21].
In relation to study hospitals, female patients who attended Hajiya Gambo Sawaba General Hospital (HGSGH) had the highest seroprevalence for anti-CMV IgM 100%.This may be as a result of low socioeconomic status and poor hygiene of the women.This is similar to the findings of Yusuf, et al. [28] who observed difference in relation to location.
Women in age group 15-24 years had the highest IgM seroprevalence which is comparable to the findings of Aliyu, et al. [27] who reported highest seroprevalence among women between 16-20 years.
study was conducted in Kaduna State, which is found in Northern part of Nigeria.Kaduna State is a cosmopolitan state owing to the various educational institutions, research institutes and other governmental and non-governmental parastatals situated which attract people from different parts of the country.The state is divided into three senatorial zones; North zone, Central zone and South zone.The hospitals selected include: Hajiya Gambo Sawaba General Hospital in Zaria from North zone, Yusuf Dantsoho Memorial Hospital in Kaduna metropolis from Central zone and Sir Patrick Ibrahim Yakowa General Hospital Kafanchan from South zone.

Table 1 :
Distribution of Human Cytomegalovirus in relation to study hospitals.

Table 2 :
Seroprevalence of Human Cytomegalovirus with respect to socio-demographic factors.

Table 3 :
Seroprevalence of Human Cytomegalovirus with respect to reproductive history.

Table 4 :
Seroprevalence of Human Cytomegalovirus with respect to risk factors.