Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq DOI: https://doi.org/10.31351/vol30iss2pp106-112 106 Seroprevalence of CMV in Women with Bad Obstetric History in Babil/Iraq Maha Diekan Abbas*,1 and Solomon Egbe** * Department of Biotechnology, College of Biotechnology, Al-Qasim Green University, Al-Qasim, Iraq. ** Epidemiology department, Edo State Health Management Board, Edo State, Nigeria. Abstract Placental dysfunction and fetal central nervous system infestation caused by Human cytomegalovirus (HCMV) is the leading cause of congenital non-genetic neuro-developmental problems of the newborn worldwide. Although the highest rates of congenital infection and CMV seroprevalence occurs in developing countries like Iraq, there remains a paucity of data from that part of the world. This descriptive case control study was undertaken in Babylon/ Iraq to determine the local seroprevalence of CMV in women of child bearing age and to identify the socio-demographic factors associated with it. This study found a seropositivity peak amongst the 26-35 yr olds which declined in the 36 – 45 yr olds. However, the evidence of current infection was stable at 25% among the 26-35 yr olds and the 36 – 45 yr old women. Overall, seropositivity was at 77.32%, a susceptibility rate was at 22.68%, and seropositivity for IgG was highest among the educated, those living in overcrowded settings, and those with poor obstetric histories. Our study concludes that CMV screening of women in the Al Hamza district in Babylon/Iraq and the availability of advice on how to prevent the infection can be beneficial for health outcomes. Keywords: CMV, Cytomegalovirus, women, ELIZA, poor obstetric history, Iraq, Babil االنتشار المصلي للفيروس المضخم للخاليا لدى النساء ذوات تاريخ الوالدة السيئ في بابل / العراق **سليمان إغبي و 1* ، مها ديكان عباس .العراق ، القاسمقسم التقنيات االحيائيه، كلية التقنيات االحيائيه ، جامعة القاسم الخضراء ، * .قسم الوبائيات ، مجلس إدارة الصحة بوالية إيدو ، والية إيدو ، نيجيريا** الخالصة السبب الرئيسي (HCMV) المضخم للخاليا البشرييعد الخلل الوظيفي في المشيمة و / أو إصابة الجهاز العصبي المركزي للجنين بسبب الفيروس المصلي لمشاكل النمو العصبية الخلقية غير الوراثية للمواليد في جميع أنحاء العالم على الرغم من أن أعلى معدالت العدوى الخلقية واالنتشار البيانات من هذا الجزء من العالم. أجريت هذه الدراسة للفيروس المضخم للخاليا تحدث في البلدان النامية مثل العراق ، ال يزال هناك ندرة في في النساء في سن اإلنجاب ، وللتعرف على العوامل االجتماعية CMV الوصفية للشواهد في بابل / العراق لتحديد االنتشار المصلي المحلي لفيروس عاًما والتي انخفضت 35و 26شخاص الذين تتراوح أعمارهم بين والديموغرافية المرتبطة به. وجدت هذه الدراسة ذروة اإليجابية المصلية بين األ سنة. 45 - 36سنة والنساء بين 35-26٪ بين 25عاًما. ومع ذلك ، فإن األدلة على اإلصابة الحالية كانت مستقرة عند 45-36في الفئة العمرية كانت األعلى بين المتعلمين ، والذين IgG ، واإليجابية المصلية لـ٪ 22.68٪ ، ومعدل الحساسية 77.32كانت اإليجابية المصلية اإلجمالية عند يعيشون في ظروف مزدحمة ، وأولئك الذين لديهم تاريخ توليدي ضعيف. خلصت دراستنا إلى أن فحص الفيروس المضخم للخاليا للنساء في منطقة .مكن أن يكون مفيدًا للنتائج الصحيةالحمزة في بابل / العراق وتوافر النصائح حول كيفية الوقاية من العدوى ي الكلمات المفتاحية: الفيروس المضخم للخاليا ، النساء ، اليزا ، تاريخ الوالدة الضعيف ، العراق ، بابل Introduction Worldwide, 0.1-0.2% of all pregnancies result in permanent disabilities of varying degree because of active HCMV infection. Disabilities caused by HCMV in the newborn include blindness, deafness, microcephaly, cerebral palsy, intellectual disability, developmental delay, and in rare cases death (1). Such disabilities may be lifelong and / or severe. HCMV is the leading cause of infectious (congenital) neurologic handicap in the newborn (2). Globally, 0.5 – 1.1% of all pregnancies occur in women with active HCMV infection. Some of these infections are a reawakening of dormant historic episodes. 90% of these pregnancies result in the birth of asymptomatic babies, however, 5-10% of babies who are asymptomatic at birth eventually die of HCMV related defects (3). 1Corresponding author E-mail: mehaalzubaidy@yahoo.com Received: 23/1/2021 Accepted: 15/3 /2021 Published Online First: 2021-12-11 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol30iss2pp106-112 Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq 107 Epidemiology Globally, there is epidemiologic variation in HCMV infection in women of childbearing age; varying from 30.4% (in Ireland) to 98.9% (in Turkey) (4, 5). The seroprevalence rate in women with a bad obstetric history (BOH) varies from 14.2% (Iran) to 91.05% (India) (6, 7). Amongst Arab countries, seroprevalence in pregnant women varies from 77.8% (Iraq) to 88% (Jordan) (8, 9). For Arab women with BOH, it varies from 4.8% (Iraq) to 95% (Jordan) (10). Study aims and objectives Although there is an accumulation of information on HCMV seroprevalence worldwide, there is however little data informing the seroprevalence of HCMV local levels in Iraq. The objective of the current study is to investigate the seroprevalence of CMV in women of child bearing age with a BOH in the Al Hamza district in Babylon/Iraq and the socio-demographic variables which might affect seropositivity. Materials and Methods Study design This is a descriptive case—control study. The total study population involves (n=115) women of childbearing age (15-45 years old). To ensure a good spread of the subjects, women with bad obstetric history were identified and recruited from the private labs and the Primary Healthcare Centers situated in urban and rural areas in the Al Hamza district in Babylon Governorates. Twent-five of the women were excluded as they did not meet the study criteria and had missing data. Of the remaining (n=90) members of the study population, 54 (60%) had BOH and considered as study group subjects while the 36 (40%) had average to good obstetric history and considered as control group subjects. Collection of data Members of the project team visited primary health care centers and the private labs in the region on a regular basis. Possible candidates were identified, approached, interviewed and were suitable, included in the study population once informed consent and permission had been sought and obtained. Four groups of patients were recruited. Group 1 – Pregnant women with average to good obstetric history. Group 2- Pregnant women with bad obstetric history. Group 3 – Non-pregnant women with average to good obstetric history. Group 4- Non-pregnant women with bad obstetric history. Subjects were interviewed to exclude those whose BOH was due to other causes e.g. uterine or cervical abnormalities (e.g. cervical incompetence), chromosomal abnormalities in either spouse, poorly controlled hyperthyroidism, diabetes mellitus, hypertension, hypothyroidism, renal disorders, a history of foetal chromosomal or genetic anomalies. Collection of blood samples: Blood samples were collected from January 2018 until December 2019. Around 5—10 mL of blood was collected from vein for each case and placed in a sterile container with strict aseptic conditions. The serum was then separated and stored in aliquots at −20 ◦C until further processing. ELISA Essay Serum samples collected from the study and control groups subjects were diagnosed for CMV IgM and IgG antibodies using Cyto Megalo Virus ELISA kits which are commercially available. The kit was purchased from CALBIOTECH, CM028M and CM028G for IgM and IgG respectively. ELISA test was performed according to the manufacturer’s instructions (CALBIOTECH). The results were processed using a microwell ELISA reader and read at an optical density 450 nm. Data analysis Using Microsoft excel spreadsheet, the collected data were compiled, assembled and the odds ratios computed for suitable analysis. Using chi squared test via SPSS (V.24), the association between categorical data was examined. Accuracy, especially in instances of smaller sample sizes, was ensured using power analysis. Association between variables was determined using bivariate regression- line analysis to compute odds ratio. CMV infection determinants were ascertained through regression line analysis and odds ratio, while standardising for confounding factors such as age, residential or educational status. Results A total of (n=115) women were identified and pooled for this study. Incomplete data were recorded for 25 of the women in the study sample. The women in the sample were categorized into further subgroups using different factors such as education level, pregnant or not pregnant, residential status (overcrowding or not overcrowded), bad obstetric history or normal obstetric history and whether or not they aborted (Table 1). Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq 108 Table 1. Sample population with recorded versus missing IgG and IgM results Data Summary Valid Missing TOTAL N Percent N Percent N Percen t Sample population with recorded v missing CMV_IgM result. 90 78.3% 25 21.7% 115 100% Sample population with recorded v missing CMV_IgG result. 90 78.3% 25 21.7% 115 100% Of the 90 women whose data was complete, 34 were in the 15-25 yr old age group, 48 in the 26-35 yr old age group, and 8 in the 36 – 45 yr age group. 23 of the 34 (67%) of the 15-25 yr age group were positive for IgG, 40 of the 48 (83%) of the 26-35 yr age group were positive for IgG, and 6 of the 8 (75%) of the 36-45 yr age group were positive for IgG (Table 2). Table 2. Prevalence of CMV IgG among different age groups For IgM, 7 of the 34 (20.59%) of the 15-25 yr age group were positive for IgM, 12 of the 48 (25%) of the 26-35 yr age group were positive for IgM, and 2 of the 8 (25%) of the 36-45 yr age group were positive for IgM (Table 3). Table 3. Prevalence of CMV IgM among different age group There were age disparities in the distribution of IgG and IgM seroprevalence among the study population. Starting at 33.33% in the 15-25 yr old, IgG seroprevalence peaked at 57.97% in the 26- 35 yr olds before declining to 8.7% in the 36-45 yr old. A similar pattern is seen for IgM starting at 33.33% amongst the 15-25 yr old, peaks at 57.14% in the 26-35 yr old and declining to 9.52% in the 36-45 yr old. Age range CMV_IgG Total Negative Positive 15-25 yr old women 11.00 23.00 34.00 32.35% 67.65% 100.00% 26-35 yr old Women 8.00 40.00 48.00 16.67% 83.33% 100.00% 36-45 yr old Women 2.00 6.00 8.00 25.00% 75.00% 100.00% Total 21.00 69.00 90.00 23.33% 76.67% 100.00% Age range CMV_IgM Total Negative Positive 15-25 yr old women 27.00 7.00 34.00 79.41% 20.59% 100.00% 26-35 yr old Women 36.00 12.00 48.00 75.00% 25.00% 100.00% 36-45 yr old Women 6.00 2.00 8.00 75.00% 25.00% 100.00% Total 69.00 21.00 90.00 76.67% 23.33% 100.00% Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq 109 Table 4. Seroprevalence of CMV IgG and IgM in regards to pregnancy, abortion, education, BOH and residential status Age Groups BOH Pregnancy status Residential status Education status Aborted 15-25 yr. old women 26-35 yr. old women 36-45 yr. old women Normal pregnanc y BOH Not Pregnant Pregnant no over- crowding Over- crowding Un- Educated Educated Not Aborted Aborted CMV_IgM NEGATIVE 27.00 36.00 6.00 34.00 39.00 27.00 44.00 8.00 63.00 11.00 52.00 35.00 39.00 39.13% 52.17% 8.70% 46.58% 53.42% 38.03% 61.97% 11.27% 88.73% 17.46% 82.54% 47.3% 52.7% CMV_IgM POSITIVE 7.00 12.00 2.00 5.00 18.00 13.00 9.00 1.00 20.00 2.00 19.00 5.00 18.00 33.33% 57.14% 9.52% 21.74% 78.26% 59.09% 40.91% 4.76% 95.24% 9.52% 90.48% 21.74% 78.26% Pearson x2 value 0.23 4.47 3.04 0.78 0.76 0.65 P value >0.05 0.034 >0.05 >0.05 >0.05 >0.05 CMV_IgG NEGATIVE 11.00 8.00 2.00 15.00 6.00 11.00 10.00 2.00 20.00 4.00 13.00 16.00 6.00 52.38% 38.10% 9.52% 71.43% 28.57% 52.38% 47.62% 9.09% 90.91% 23.53% 76.47% 72.73% 27.27% CMV_IgG POSITIVE 23.00 40.00 6.00 24.00 51.00 29.00 43.00 7.00 63.00 9.00 58.00 24.00 51.00 33.33% 57.97% 8.70% 32.00% 68.00% 40.28% 59.72% 10.00% 90.00% 13.43% 86.57% 32.00% 68.00% Pearson x2 value 2.75 10.57 0.97 0.02 1.06 11.64 P value >0.05 0.001 >0.05 >0.05 >0.05 0.001 Bold values indicated a significant difference Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq 110 Overall, the seroprevalence of CMV within the study sample was 77.32%. IgG levels were highest among those living in overcrowded situations (90%), those who had abortions (68%), and the educated (86.57%). Interestingly, IgG levels were highest (68%) among those with a poor obstetric history (study group), compared to 32% for those with moderate to good obstetric history (control group). Evidence of current infection, i.e. positive IgM levels was highest amongst those in overcrowded situations (95.24%), those with a poor obstetric history (78.26%), those who had abortions (78.26%), and the educated (90.48%). Contrary to other studies the seroprevalence of IgM amongst pregnant women (40.91%) was lower than in the women who were not pregnant (59.09%). The converse was true of IgG levels being 59.72% (pregnant) and 40.28% (not pregnant) (Table 4). The chi squared values and p-values indicate that x2 figures for women with a bad obstetric history, both for IgG and IgM was significant at a p value of below 0.05 at 95% confidence interval (CI). This was also true of those women with aborted fetus. This association was also confirmed by the odds ratio, the x2 BOH * CMV_IgM was 4.47 (p value = 0.034) while the x2 of BOH * CMV_IgG was 10.57 (P value=0.001) and the x2 of CMV_IgG * Abortion was 11.64 with (P value=0.001). There is however no significant correlation (P value>0.05) found between CMV_IgG/ IgM and Residency, Educational level, age group and pregnancy status. Discussion According to the WHO (11), the seroprevalence of HCMV in women of child bearing age in the European region is 63-76%, this is lower than that in the East Mediterranean region (88-95%), the region of the Americas (69-87%), the west Pacific region (86-94%), the African region (82- 94%), and the South east Asian regions (82-94%) (12). Other studies undertaken in other parts of Iraq have found seroprevalence rates in the region of 95.7% in Kirkuk (13). The overall seroprevalence of CMV within this study sample was 77.32%, this study found HCMV seroprevalence highest amongst 26-35 yr old women but declining with age. This study found a susceptibility rate of 22.68%, which is higher than the average for the country. The study found an association of current infection with abortion and BOH (Control group). Other findings of this study include the higher rates of seroprevalence amongst the educated and among those living in overcrowded households. The higher seroprevalence amongst 26-35 yr old is aligned with previous studies which have attributed this to the exposure of these women to school age children, especially as their own children begin to attend school (14). The finding of higher rates amongst the educated and amongst those living in overcrowded households is also in alignment with previous studies (15). The overall prevalence of 77.32% and the susceptibility rate of 22.68% highlights the need for routine screening in antenatal clinics. This study shows that while the average rates of CMV seroprevalence in Iraq may be over 95% (12), there are regional variations which require local adjustment of national policies on CMV infection prevention and management. Scholars have argued against routine screening because of high levels of seroprevalence, cost, extensive viral strain diversification, and the inconsistent and ineffective treatment options available for HCMV (16). However, the arguments against routine screening, awareness promotions, prevention campaigns, and treatment may be predicated on a general underestimation or a lack of understanding of the enormity of the challenge posed by HCMV infection as a disease burden. HCMV is highly adapted to its human host, it is a member of the HSV group. Primary infection with the virus results in an initial period of viral replication and shedding in body fluids including saliva, breast milk, urine, and in genital secretions. This is followed by a viraemia and, in some people, an infectious mononucleosis phase. Finally, the host develops a generalized immune response involving the entire immune system. Thereafter the infection enters a latent phase when viral levels are either low or even absent to detection but present in CD14+ and also in the CD33+ & 34+ cells of the mononuclear cells of the peripheral blood and bone marrow respectively. These sites then serve as reservoirs for future reactivation at opportune moments (17). This cycle perpetuated by the virus accounts for the delayed onset of symptoms in congenitally infected babies, the initial asymptomatic infection in many, and therefore, the presumption in many quarters that babies born to mothers with prior HCMV antibodies have normal outcomes. Yet nothing could be further from the truth. The diversification of the virus creates a potential new threat to those who are already seropositive. The fact that the source of such diversity has been found to be endogenous makes those who are seropositive or living in a high seroprevalence environment particularly at risk (18). It is not surprising therefore, that the greatest burden of HCMV burden is found in developing countries where there is also a high level of seropositivity. High viral diversification continues to present challenges to the development of effective vaccines. A challenge which is amplified by the fact that large proportions of congenitally infected babies are the product of mothers with demonstrable HCMV antibodies (19). The consistent finding of an association between poorer socioeconomic conditions such as overcrowding, breast milk transmission and high HCMV seroprevalence offers an opportunity to Iraqi J Pharm Sci, Vol.30(2) 2021 Seroprevalence of CMV in women with bad obstetric history in Babil/Iraq 111 address this problem while an effective vaccine is awaited (15). Contrary to previous conception that higher seroprevalence confers immunity, the high viral diversity of HCMV predisposes the already seronegative women to reinfection with a different strain from the community or from endogenous viral reactivation. Thus, higher seroprevalence may actually predispose to higher likelihood of infection. Therefore, the higher seroprevalence of HCMV in the rural and/or urban areas of Babylon should not occasion complacency to prevention and treatment but rather is the more reason for intervention. Educating women and children about HCMV preventive measures is known to be effective (20). Basic preventive measures like: Teaching people to wash their hands regularly, catch their sneezes and body fluids and dispose of this in the appropriate way, avoiding the sharing of cutleries, plates and cups, educating mothers not to put babies’ pacifiers in their mouths if it has been in a child’s mouth, or advising mothers to avoid kissing children on the lips, have been shown to be feasible practices observable by mothers when adopted and effective in limiting the spread of infection. Other measures include the introduction and reinforcement of these messages in antenatal clinics, children’s clinics, and hospitals using relevant staff holding classes and demonstrations with women and their husbands. There is also an opportunity to use billboards or posters in the hospital to adorn the walls of the waiting rooms or consulting rooms and wards where women are likely to see them. All such posters, classes, advice, and discussions should take place and employ cultural and language sensitive media, observing the complexities and subtleties of local customs and practices to drive home the message. Conclusion This is the first study into the seroprevalence of HCMV amongst women of childbearing age in Al-Hamza district in Babylon/Iraq. The study found that a significant number of women were susceptible to HCMV infection, this finding contrasts the average of over 95% seroprevalence rate for the country. This study highlights the need, as the search for an effective vaccine and the debate over the cost benefit of routine screening continues, for seroprevalence data of HCMV in local populations to be researched and documented. 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