Journal of Immunoassay and Immunochemistry, 36:343–358, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.952442
SERUM HSV-1 AND -2 IgM IN PREGNANT WOMEN IN PORT HARCOURT, NIGERIA
I.O. Okonko, T.I. Cookey, P.O. Okerentugba, and N. Frank-Peterside Medical Microbiology Unit, Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria
The present study was undertaken for the purpose of finding IgM antibodies against HSV-1 and 2 infections among pregnant women and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. A total of 180 pregnant women attending antenatal clinic at Braithwaite Memorial Specialist Hospital (BMSH) in Port Harcourt, Nigeria were consecutively recruited, after they had given consents to participate in the study. Serum of each sample was assayed for HSV1&2 IgM antibody using a commercial ELISA. Five (2.8%) of the pregnant women were positive for IgM antibody against HSV-1&2. Marital status mainly correlated (χ 2 = 221.5, P < 0.05) with HSV-2 infection and HSV-1/HSV-2 co-infection. Age, educational level, occupation, and gestation were not consistently associated (P>0.05) with HSV-1/HSV-2 infection and co-infection. We also observed a high overall anti-HSV-1&2 IgM seronegativity of 97.2% among these pregnant women. Group-specific seronegativity was also high ranging from 93.3–100%. Although the age-groups significantly differed, none of their variables showed statistical association with the seronegativity. This represents the first analysis of HSV IgM antibody reported in Port Harcourt, Nigeria and has important public health implications, particularly for pregnant women. Consideration of this information would benefit physicians providing primary gynecological and obstetric care to this population of women. Keywords HSV, prevalence, seropositivity, seronegativity, IgM, ELISA, BMSH
INTRODUCTION Herpes simplex virus types 1 and 2 (HSV-1 and -2) are significant human pathogens causing clinically indistinguishable facial and genital lesions.[1] There are two types of HSVs. HSV-1 has traditionally been associated primarily with oral-facial infections, whereas HSV-2 infection is the primary Address correspondence to I.O. Okonko, Medical Microbiology Unit, Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Choba, East-West Road, Port Harcourt, Rivers State, 234084, Nigeria. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ljii.
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cause of genital herpes and is one of the most prevalent sexually transmitted infections (STIs) worldwide.[1–5] The greatest incidence of HSV infections have been found to occur in women of reproductive age and the risk associated with maternal transmission of the virus to the fetus or neonate has become a major health concern.[6–8] Genital HSV infection in pregnant women is of particular concern because of the risk to the fetus and newborn caused by HSV-1 and HSV-2.[9] Over 95% of infected babies are born to women who are unaware that they have genital herpes. Even though genital herpes infection in pregnant women is common and rarely serious, the risk of vertical transmission to the infant when the mother develops a primary infection during the third trimester is high and this risk increases the closer to the time of delivery.[10] The highest risk of intrauterine infection has been observed in pregnant women (about 50%) who developed disseminated HSV infections and 90% of those were related to HSV-2.[11] The risk of neonatal infection varies from 30–50% for HSV infections that onset in late pregnancy (last trimester), whereas early pregnancy infection carries a risk of about 1%.[12] Moreover, studies in HIV infected pregnant women show that coinfection with HSV increases significantly the risk of perinatal HIV transmission above all in women who had a clinical diagnosis of genital herpes during pregnancy.[13–15] Currently, there is no licensed vaccine or cure for HSV.[16,17] As HSVs are significant human pathogens, rapid detection and diagnosis of these viruses is imperative.[1] Studies done in the past have suggested that genital HSV infection acquired during pregnancy is associated with preterm labor, intrauterine growth retardation, and spontaneous abortion.[18,19] The most severely affected population is neonates, who acquire HSV infection after exposure to the virus during birth.[19] Intrauterine HSV infection accounts for 5% of HSV infections in neonates. A percentage of 70–85% of neonatal HSV infections is caused by HSV-2, whereas the remaining cases are due to HSV-1. The HSV-2 infection carries a graver prognosis than that caused by HSV-1.[15] The consequences of neonatal infection are catastrophic resulting in permanent neurological damage for many infants despite appropriate antiviral therapy and the perinatal mortality is 50%.[13] Because HSV causes a lifelong infection with unpredictable reactivation and transmission, detecting antibodies to HSV plays an important role in identifying carriers of this infection.[9] Seroprevalence estimates of HSV infections allow a better understanding of the burden of infection and how this is evolving with time. This will help in monitoring prevalence of the virus in pregnant women to allow the identification of women at a higher risk of HIV and of neonatal transmission. Therefore, the early diagnosis of pregnant women with genital herpes infection is necessary for the protection of babies from the risk of neonatal herpes. Since screening tests are not
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performed routinely for the detection of genital herpes in asymptomatic pregnant women in Nigeria, there is inadequate available data on the prevalence of HSV IgM antibodies in these women in order to ascertain those at risk for neonatal transmission. The present study was thus undertaken for finding IgM antibodies against HSV-1 and 2 infections in pregnant women in Port Harcourt, Nigeria and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. Information on demographic variables-specific prevalence of HSV-1 and 2 infections is essential to optimize genital herpes control strategies, which increase in importance because accumulating data indicate that HSV-2 infection may increase acquisition and transmission of HIV.[3] MATERIALS AND METHODS Study Area The study was conducted from February to July, 2013 in Rivers State, South-South Nigeria and was limited to pregnant women attending antenatal clinic in Braithwaite Memorial Specialist Hospital (BMSH). BMSH is a Rivers State government owned specialist health institution which was initially established as a nursing home for senior civil servants in March 1925. It is located in the old Government Reserved Area (G.R.A.) of Port Harcourt city. Study Design A hospital-based cross-sectional survey was adopted for the present study which seeks to determine the prevalence of HSV-1&2 IgM antibody among pregnant women in Port Harcourt, Rivers State, South-South, Nigeria. The occurrence of HSV-1&2 IgM antibody among the pregnant women was investigated using commercially available ELISA. The influence of the age, marital status, occupation, educational status, and gestation period on the prevalence of HSV-1&2 IgM antibody was also considered. Study Population The target population constituted all pregnant women attending antenatal clinic for a routine antenatal check-up in Braithwaite Memorial Specialist Hospital (BMSH) from February to July, 2013. From the study population, a total sample size of 180 pregnant women were randomly selected and enrolled into the study. The demographic details relevant to the study were obtained from the clinic records, as shown in Table 1.
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TABLE 1 Socio-demographic data and seropositivity outcomes of pregnant women tested for IgM antibodies against HSV-1 and -2 Total Variables Age ≤25 26–30 31–35 ≥36 Marital status Married Singles Education Primary Secondary Post-secondary Tertiary Occupation Student Unemployed Self employed Employed Gestation 1st trimester 2nd trimester 3rd trimester Total
HSV 1&2 IgM
No.
%
Positives
%
Negatives
%
Statistics
30 73 57 20
16.7 40.6 31.7 11.1
2 3 0 0
6.7 4.1 0.0 0.0
28 70 57 20
93.3 95.9 100.0 100.0
χ 2 = 4.359, df = 3, P > 0.05
177 3
98.3 1.7
4 1
2.3 33.3
173 2
97.7 66.7
χ 2 = 221.5, df = 1, P < 0.05
1 66 12 101
0.6 36.7 6.7 56.1
0 2 1 2
0.0 3.0 8.3 2.0
1 64 11 99
100.0 97.0 91.7 98.0
χ 2 = 1.653491, df = 3, P > 0.05
24 47 68 41
13.3 26.1 37.8 22.8
2 1 1 1
8.3 2.1 1.5 2.4
22 46 67 40
91.7 97.9 98.5 97.6
χ 2 = 3.264087, df = 3, P > 0.05
28 93 59 180
15.6 51.7 32.8 100.0
0 1 4 5
0.0 1.1 6.8 2.8
28 92 55 175
100.0 98.9 93.2 97.2
χ 2 = 5.296946, df = 2, P > 0.05
Sample Collection, Preparation, and Storage Specimen of 5 mL venous blood was aseptically drawn from 180 pregnant women who were HSV-1&2 seropositive into sterile plain tubes. The blood was centrifuged and the serum aspirated into sterile eppendorf tubes. Samples were clearly identified with codes in order to avoid misinterpretation of results. Haemolysed and visibly hyperlipemic samples were discarded as they could generate false results. Samples containing residues of fibrin or heavy particles were also discarded as they could give rise to false results. Sera were stored at +2◦ − 8◦ C for up to five days after collection. For longer storage periods, samples were stored frozen at −20◦ C. Serological Analysis Serum from each samples were assayed for HSV-1&2 IgM using a commercially available ELISA kit (DIA.PRO Diagnostic Bioprobes Srl Via G. Carducci n◦ 27 20099 Sesto San Giovanni (Milano) – Italy) according to the manufacturer’s specifications.
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Principle of the Test Immunoglobulin –M (IgM) antibodies against HSV-1 and HSV-2 were detected by a commercially available enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein M of HSV-1 or HSV-2. The assay was based on the principle of IgM capture where IgM class antibodies in the samples are first captured by the solid phase coated with anti hIgM antibody. After washing out all the other components of the samples and in particular IgG antibodies, the specific IgM captured on the solid phase were detected by the addition of a purified preparation of inactivated HSV-1&2, labeled with a specific antibody conjugated with peroxidase (HRP). After incubation, microwells are washed to remove unbound conjugate and then the chromogen/substrate is added. In the presence of bound conjugate, the colorless substrate is hydrolyzed to a colored end-product, whose optical density may be detected and is proportional to the amount of IgM antibodies to HSV-1&2 present in the sample. Results, Calculation, and Interpretation Results were interpreted according to the manufacturer‘s specifications. The test results were calculated by means of the mean OD450 nm value of the negative control (NC) and a mathematical calculation, in order to define the following cut-off formulation: Cut-Off = NC + 0.250. The values found were used to interpret the results as the ratio of the sample OD450nm and the cut off value (or S/Co) as follows: 1.2 (positive). Data Analysis The data were recorded in and analyzed using Microsoft Excel spreadsheet (Microsoft Corporation). The seroprevalence was calculated as the number of serologically positive samples divided by the total number of samples tested. The Chi-square test was used to determine associations between seropositivity and socio-demographic variables. The level of statistical significance was set at P ≤ 0.05. RESULTS Patient Characteristics The total number of pregnant women included in this study was 180. The socio-demographic data for these samples were stratified and shown in Table 1.
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Prevalence of HSV-1&2 IgM Antibody Of the 180 specimens tested, only 5 (2.8%) were seropositive for HSV-1&2 IgM antibody while most of the specimens 175 (97.2%) were seronegative (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Age Figure 1 shows the rate of seropositivity of HSV-1&2 IgM antibody according to age. In relation to age, only two groups out of the four age groups were reactive. Two (6.7%) in the age group ≤25 years and 3 (4.1%) in the age group 26–30 were found to be seropositive. The other age groups showed no presence of HSV-1&2 IgM antibody. There was no significant relationship between the age groups and prevalence of IgM antibody against HSV-1&2 (χ 2 = 4.359, df = 3, P >0.05) (Table 1).
7
6.7
6
Frequency and Percentage
5 4.1 4 3
3
2
2
1
0
0 ≤25
26–30
0.0
0
31–35
0.0 ≥36
Age (Years) HSV 1&2 IgM +ve frequency
HSV 1&2 IgM +ve2 %
FIGURE 1 The rate of seropositivity of HSV 1&2 IgM antibody according to age.
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Prevalence of HSV-1&2 IgM Antibody in Relation to Marital Status As shown in Figure 2, a higher seroprevalence of HSV-1&2 IgM antibody of 33.1% was observed for the pregnant women that were single which was greater than 2.3% observed for those that were married. Statistically, marital status was significantly associated with prevalence of IgM antibody against HSV-1&2 (χ 2 = 221.5, df = 1, P < 0.05) (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Educational Background While none of the pregnant women with primary education tested positive, 2 (3.0%), 2 (2.0%), and 1 (8.3%) with secondary, tertiary and post-secondary respectively were found to be seropositive, as shown in Figure 3. The level of education of the pregnant women attending antenatal care had no significant relationship with the prevalence of IgM antibody against HSV-1&2 (χ 2 = 1.653491, df = 3, P > 0.05) (Table 1).
35
33.3
30
Frequency and Percentage
25
20
15
10
5
4 2.3 1
0
MARRIED
SINGLE Marital status
HSV 1&2 IgM +ve frequency
HSV 1&2 IgM +ve %
FIGURE 2 The rate of seropositivity of HSV 1&2 IgM antibody according to marital status.
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Frequency and Percentage
7 6 5 4 3.0 3 2
2
2.0
1
1 0
2
0
0.0
PRIMARY
SECONDARY POST SECONDARY Educational status
HSV 1&2 IgM +ve frequency
TERTIARY
HSV 1&2 IgM +ve %
FIGURE 3 The rate of seropositivity of HSV 1&2 IgM antibody according to educational status.
Prevalence of HSV-1&2 IgM Antibody in Relation to Occupational Status Figure 4 shows the prevalence rates of HSV 1&2 IgM antibodies according to occupational status which were found to be 8.3% (2), 2.1% (1), 1.5% (1), and 2.4% (1) for students, unemployed, self-employed, and employed women, respectively. Statistically, there was no significant relationship between occupation and prevalence of IgM antibody against HSV-1&2 (χ 2 = 3.264087, df = 3, P > 0.05) (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Gestation Period Four (6.8%) of the pregnant women that were seropositive for HSV1&2 IgM antibodies were in their third trimester and 1 (1.1%) was in her second trimester, as highlighted in Figure 5. However, no significant association was found between the gestation period and the prevalence of IgM antibody against HSV 1&2 (χ 2 = 5.296946, df = 2, P > 0.05) (Table 1).
351
HSV-1 and -2 IgM in Pregnant Women 9 8.3 8
Frequency and Percentage
7 6 5 4 3 2
1.5 1
1 0
2.4
2.1
2
STUDENT
1
UNEMPLOYED SELF EMPLOYED Occupation
HSV 1&2 IgM +ve frequency
1
EMPLOYED
HSV 1&2 IgM +ve %
FIGURE 4 The rate of seropositivity of HSV 1&2 IgM antibody according to occupation.
DISCUSSION The present study was thus undertaken for finding IgM antibodies against HSV-1 and 2 infections among pregnant women and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. As HSVs are significant human pathogens, rapid detection and diagnosis of these viruses is imperative.[1] Immunoglobulin–M (IgM) antibodies against HSV-1 and HSV-2 were detected by a commercially available enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein M of HSV-1 or HSV-2.[20] HSV-1 and HSV-2 are highly homologous genetically and antigenically. This results in an antigen-sharing profile for the two serotypes.[20] Seroprevalence estimates of HSV infections allow a better understanding of the burden of infection and how this is evolving with time.[3] The present study showed that of the 180 pregnant women tested, only 5 (2.8%) were seropositive for HSV-1&2 IgM antibody while 175 (97.2%) were seronegative. Detectable IgM antibodies in HSV infection usually reflect primary or recurrent infection. HSV-1&2 IgM, as an indicator of current infection was demonstrated in 2.8% of the pregnant women studied. Like Iraqi women,[21–23] little is known about the seroprevalence of HSV-1&2 in
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7
Frequency and Percentage
6
5 4
4
3
2 1
1
0
0
1.1
0.0
1ST TRIMESTER
2ND TRIMESTER Gestation Period (Months)
HSV 1&2 IgM +ve frequency
3RD TRIMESTER
HSV 1&2 IgM +ve %
FIGURE 5 The rate of seropositivity of HSV 1&2 IgM antibody according to gestation period.
Nigerian pregnant women. These three studies reported a wide range of HSV-2 seropositivity ranging from 8.1–73.9% for HSV IgM in Iraq,[24] which are higher than the 2.8% reported in our present study. This low seropositivity rate (2.8%) recorded in this study is in agreement with the low rates reported by previous authors.[1, 24–26] Alzahrani et al.[27] reported 0.5% for HSV-2 IgM in Arab countries. Ozdemir et al.[25] reported a seropositivity of 0.0% and 1.4% for HSV type 1 and 2 respectively in Turkey. Sadik et al.[26] reported 1.69% seropositivity in their study. Peña et al.[1] reported a 0.15% seropositivity for both HSV-1 and HSV-2 in women. Zainab et al.[24] demonstrated HSV-2 IgM seropositivity 3.2% of pregnant women. Our 2.8% HSV IgM seropositivity reported in these pregnant women in this study was higher than that reported the 0.0% reported by Ozdemir et al.[28] in Turkey; the 0.5% reported by Alzahrani et al.[25] in Saudi Arabia; the 1.2% reported by Vilibik-Cavlek et al.[29] in Croatia and the 1.8% reported by Nabi et al.[30] in Bangladesh. All these figures correlated favourably with our present findings. Thus, 2.8% current infection with HSV-1&2 reported in this study was lower to that reported in other studies elsewhere. It is lower than the 5.8% HSV IgM seroprevalence reported by Zainab et al.[24] in pregnant women
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with bad obstetrics history (BOH). It was lower than the seropositivity rates reported for India,[31–34] Baghdad, Iraq,[21] Waset, Iraq,[23] and Mosul, Iraq.[22] The overall Serum HSV-1 and 2 IgM seroprevalence was 15.66% in the study by Tada and and Khandelwal.[35] Al-Marzoqi et al.[36] reported seroprevalence of 28.9% HSV-2 IgM in Iraq pregnant women. Other studies in Arab countries reported a range of 0.5–7.6% for HSV2 IgM[24,37] and 6.5% to 27.1%[24, 27, 38] for HSV-2 IgG in pregnant women. Our 2.8% rate is also lower than the 6.25% reported by Dordevic;[39] the 28.9% reported by Al-Marzoqi et al.[36] in Babylon, Iraq; the 13.8% reported by Duran et al.[40] in Turkey and the 7.6% reported by Abu-Madi et al.[37] in Qatar. It is also lower than the 14.0% reported by Peña et al.[1] for either HSV-1 alone, HSV-2 alone, or both HSV-1 and HSV-2. It is also lower than the 48.0% seropositivity reported for HSV-1 alone, 7.0% for HSV-2 alone and 12.0% for co-infections with HSV-1 and HSV-2.[41] In a previous small-cohort analysis by Adelson et al.,[42] an 11.0% HSV positivity rates for HSV-1 and/or HSV-2 was reported. This 2.8% HSV-1&2 seropositivity value is much lower than 28.0% HSV-1 positivity rate found in a previous study reported by Pena et al. in 2005. However, our present study therefore suggests a very low acute HSV infection in the population of study. Now a days, serum HSV-1&2 IgM positive in patients of non herpetic clinical manifestations is quite common. This has also been reported by many authors outside Nigeria.[35, 43–45] At first glance, our 2.8% seropositivity value from this first analysis in Port Harcourt, Nigeria seems comparable to the results of a focused study which reported a 16.0% positivity rate.[1, 46] However, contrary to the study by Peña et al.,[1] our study was specifically derived from specimens from pregnant women who were not known to have genital herpes or any clinical lesions. Our study population focused exclusively on pregnant women who were not known to have genital HSV disease. This shows that these pregnant women may have either simultaneous co-infection with HSV-1 and/or 2, although the virus is not clinically manifesting the herpetic symptoms and patients show asymptomatic seroconversion or HSV-1 and/or -2 IgM reactivation.[35,47] Seropositivity of HSV-1&2 IgM antibody and its association with sociodemographic variables showed that marital status was the main correlate of HSV-1&2 IgM seropositivity. HSV IgM seropositivity was significantly higher in singles compared to married women. Whereas age, level of education, occupation, and gestational period were not consistently associated with HSV-1/HSV-2 infection and co-infection. This association to marital status can be as a result of the married women being infected by their husbands, thereby leading to an acute HSV infection. This differs from the findings of Beydoun et al.[41] which reported that age, sex, race, and level of education independently predicted all three outcomes, lifetime sexual activity, as well as use of tobacco products and recreational drugs mainly correlated
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with HSV-2 infection and HSV-1/HSV-2 co-infection, whereas lifetime use of alcohol was not consistently associated with HSV-1/HSV-2 infection and co-infection.[41] HSV-1&2 IgM was not detected in women with age of greater than 30 years, however, the higher seropositivity rate (6.7%) was observed in pregnant women with age of less than 25 years and 4.1% in ages 26–30 years. This deviates from what was reported by Pena et al.[1] Pena et al.[1] reported a zero seropositivity of HSV-2 IgM in women with ages less than 20 years and higher seroprevalence rate (5.5%) in women with age of 20–29 years. Peña et al.[41] also reported that percentages of genital HSV-1 positivity vary with age. In their study, samples which were positive for both HSV-1 and HSV2 were detected in all age groups.[41] In variance with our present finding, Zainab et al.[24] found both HSV-2 IgG and IgM to significantly vary with age groups, with trends of increasing with older ages. In this study, level of education was not consistently associated with HSV infection. HSV-1&2 IgM was not detected in women with primary education, however, the higher seropositivity rate (8.3%) was observed in pregnant women with post-secondary education, 3.0% in secondary education, and 2.0% in women with tertiary education. In disagreement with our finding, significant association was found between HSV-2 seroprevalence and education levels.[24] In this study, occupation was not consistently associated with HSV infection. HSV-1&2 IgM was lower in women who are self-employed (1.5%), unemployed (2.1%) and employed (2.4%) however, higher seropositivity rate (8.3%) was observed in pregnant women who were students. This also deviated from the previous reports. Zainab et al.[24] suggested occupation as a correlate of HSV seropositivity. Their study found HSV-2 infection to be statistically significantly higher in working women as compared to those who were housewives.[24] Of importance to note is the higher seropositivity rate of HSV-1&2 IgM observed in pregnant women that were in their third trimester (6.8%) than those in their second trimester (1.1%), however, HSV-1&2 IgM was not detected in pregnant women in their first trimester. This was not statistically associated with HSV infection. It suggests that these women in their third trimester are undergoing a primary or recurrent infection and are therefore at a higher risk of neonatal transmission. This is because when primary HSV infection occurs during late pregnancy, there is no adequate time to develop antibodies needed to suppress viral replication before labor. Previous studies have also shown that genital HSV infection acquired during pregnancy is associated with preterm labor, intrauterine growth retardation, and spontaneous abortion.[18,19] The risk of neonatal infection varies from 30–50% for HSV infections that onset in late pregnancy (third trimester).[12] Even though genital herpes infection in
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pregnant women is common and rarely serious, the risk of vertical transmission to the infant when the mother develops a primary infection during the third trimester is high and this risk increases the closer to the time of delivery.[10] We observed a high overall anti-HSV-1&2 IgM seronegativity of 97.2% among these pregnant women. Group-specific seronegativity was also high ranging from 93.3–100%. Though the age-groups (
SERUM HSV-1 AND -2 IgM IN PREGNANT WOMEN IN PORT HARCOURT, NIGERIA
I.O. Okonko, T.I. Cookey, P.O. Okerentugba, and N. Frank-Peterside Medical Microbiology Unit, Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria
The present study was undertaken for the purpose of finding IgM antibodies against HSV-1 and 2 infections among pregnant women and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. A total of 180 pregnant women attending antenatal clinic at Braithwaite Memorial Specialist Hospital (BMSH) in Port Harcourt, Nigeria were consecutively recruited, after they had given consents to participate in the study. Serum of each sample was assayed for HSV1&2 IgM antibody using a commercial ELISA. Five (2.8%) of the pregnant women were positive for IgM antibody against HSV-1&2. Marital status mainly correlated (χ 2 = 221.5, P < 0.05) with HSV-2 infection and HSV-1/HSV-2 co-infection. Age, educational level, occupation, and gestation were not consistently associated (P>0.05) with HSV-1/HSV-2 infection and co-infection. We also observed a high overall anti-HSV-1&2 IgM seronegativity of 97.2% among these pregnant women. Group-specific seronegativity was also high ranging from 93.3–100%. Although the age-groups significantly differed, none of their variables showed statistical association with the seronegativity. This represents the first analysis of HSV IgM antibody reported in Port Harcourt, Nigeria and has important public health implications, particularly for pregnant women. Consideration of this information would benefit physicians providing primary gynecological and obstetric care to this population of women. Keywords HSV, prevalence, seropositivity, seronegativity, IgM, ELISA, BMSH
INTRODUCTION Herpes simplex virus types 1 and 2 (HSV-1 and -2) are significant human pathogens causing clinically indistinguishable facial and genital lesions.[1] There are two types of HSVs. HSV-1 has traditionally been associated primarily with oral-facial infections, whereas HSV-2 infection is the primary Address correspondence to I.O. Okonko, Medical Microbiology Unit, Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Choba, East-West Road, Port Harcourt, Rivers State, 234084, Nigeria. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ljii.
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cause of genital herpes and is one of the most prevalent sexually transmitted infections (STIs) worldwide.[1–5] The greatest incidence of HSV infections have been found to occur in women of reproductive age and the risk associated with maternal transmission of the virus to the fetus or neonate has become a major health concern.[6–8] Genital HSV infection in pregnant women is of particular concern because of the risk to the fetus and newborn caused by HSV-1 and HSV-2.[9] Over 95% of infected babies are born to women who are unaware that they have genital herpes. Even though genital herpes infection in pregnant women is common and rarely serious, the risk of vertical transmission to the infant when the mother develops a primary infection during the third trimester is high and this risk increases the closer to the time of delivery.[10] The highest risk of intrauterine infection has been observed in pregnant women (about 50%) who developed disseminated HSV infections and 90% of those were related to HSV-2.[11] The risk of neonatal infection varies from 30–50% for HSV infections that onset in late pregnancy (last trimester), whereas early pregnancy infection carries a risk of about 1%.[12] Moreover, studies in HIV infected pregnant women show that coinfection with HSV increases significantly the risk of perinatal HIV transmission above all in women who had a clinical diagnosis of genital herpes during pregnancy.[13–15] Currently, there is no licensed vaccine or cure for HSV.[16,17] As HSVs are significant human pathogens, rapid detection and diagnosis of these viruses is imperative.[1] Studies done in the past have suggested that genital HSV infection acquired during pregnancy is associated with preterm labor, intrauterine growth retardation, and spontaneous abortion.[18,19] The most severely affected population is neonates, who acquire HSV infection after exposure to the virus during birth.[19] Intrauterine HSV infection accounts for 5% of HSV infections in neonates. A percentage of 70–85% of neonatal HSV infections is caused by HSV-2, whereas the remaining cases are due to HSV-1. The HSV-2 infection carries a graver prognosis than that caused by HSV-1.[15] The consequences of neonatal infection are catastrophic resulting in permanent neurological damage for many infants despite appropriate antiviral therapy and the perinatal mortality is 50%.[13] Because HSV causes a lifelong infection with unpredictable reactivation and transmission, detecting antibodies to HSV plays an important role in identifying carriers of this infection.[9] Seroprevalence estimates of HSV infections allow a better understanding of the burden of infection and how this is evolving with time. This will help in monitoring prevalence of the virus in pregnant women to allow the identification of women at a higher risk of HIV and of neonatal transmission. Therefore, the early diagnosis of pregnant women with genital herpes infection is necessary for the protection of babies from the risk of neonatal herpes. Since screening tests are not
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345
performed routinely for the detection of genital herpes in asymptomatic pregnant women in Nigeria, there is inadequate available data on the prevalence of HSV IgM antibodies in these women in order to ascertain those at risk for neonatal transmission. The present study was thus undertaken for finding IgM antibodies against HSV-1 and 2 infections in pregnant women in Port Harcourt, Nigeria and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. Information on demographic variables-specific prevalence of HSV-1 and 2 infections is essential to optimize genital herpes control strategies, which increase in importance because accumulating data indicate that HSV-2 infection may increase acquisition and transmission of HIV.[3] MATERIALS AND METHODS Study Area The study was conducted from February to July, 2013 in Rivers State, South-South Nigeria and was limited to pregnant women attending antenatal clinic in Braithwaite Memorial Specialist Hospital (BMSH). BMSH is a Rivers State government owned specialist health institution which was initially established as a nursing home for senior civil servants in March 1925. It is located in the old Government Reserved Area (G.R.A.) of Port Harcourt city. Study Design A hospital-based cross-sectional survey was adopted for the present study which seeks to determine the prevalence of HSV-1&2 IgM antibody among pregnant women in Port Harcourt, Rivers State, South-South, Nigeria. The occurrence of HSV-1&2 IgM antibody among the pregnant women was investigated using commercially available ELISA. The influence of the age, marital status, occupation, educational status, and gestation period on the prevalence of HSV-1&2 IgM antibody was also considered. Study Population The target population constituted all pregnant women attending antenatal clinic for a routine antenatal check-up in Braithwaite Memorial Specialist Hospital (BMSH) from February to July, 2013. From the study population, a total sample size of 180 pregnant women were randomly selected and enrolled into the study. The demographic details relevant to the study were obtained from the clinic records, as shown in Table 1.
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TABLE 1 Socio-demographic data and seropositivity outcomes of pregnant women tested for IgM antibodies against HSV-1 and -2 Total Variables Age ≤25 26–30 31–35 ≥36 Marital status Married Singles Education Primary Secondary Post-secondary Tertiary Occupation Student Unemployed Self employed Employed Gestation 1st trimester 2nd trimester 3rd trimester Total
HSV 1&2 IgM
No.
%
Positives
%
Negatives
%
Statistics
30 73 57 20
16.7 40.6 31.7 11.1
2 3 0 0
6.7 4.1 0.0 0.0
28 70 57 20
93.3 95.9 100.0 100.0
χ 2 = 4.359, df = 3, P > 0.05
177 3
98.3 1.7
4 1
2.3 33.3
173 2
97.7 66.7
χ 2 = 221.5, df = 1, P < 0.05
1 66 12 101
0.6 36.7 6.7 56.1
0 2 1 2
0.0 3.0 8.3 2.0
1 64 11 99
100.0 97.0 91.7 98.0
χ 2 = 1.653491, df = 3, P > 0.05
24 47 68 41
13.3 26.1 37.8 22.8
2 1 1 1
8.3 2.1 1.5 2.4
22 46 67 40
91.7 97.9 98.5 97.6
χ 2 = 3.264087, df = 3, P > 0.05
28 93 59 180
15.6 51.7 32.8 100.0
0 1 4 5
0.0 1.1 6.8 2.8
28 92 55 175
100.0 98.9 93.2 97.2
χ 2 = 5.296946, df = 2, P > 0.05
Sample Collection, Preparation, and Storage Specimen of 5 mL venous blood was aseptically drawn from 180 pregnant women who were HSV-1&2 seropositive into sterile plain tubes. The blood was centrifuged and the serum aspirated into sterile eppendorf tubes. Samples were clearly identified with codes in order to avoid misinterpretation of results. Haemolysed and visibly hyperlipemic samples were discarded as they could generate false results. Samples containing residues of fibrin or heavy particles were also discarded as they could give rise to false results. Sera were stored at +2◦ − 8◦ C for up to five days after collection. For longer storage periods, samples were stored frozen at −20◦ C. Serological Analysis Serum from each samples were assayed for HSV-1&2 IgM using a commercially available ELISA kit (DIA.PRO Diagnostic Bioprobes Srl Via G. Carducci n◦ 27 20099 Sesto San Giovanni (Milano) – Italy) according to the manufacturer’s specifications.
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Principle of the Test Immunoglobulin –M (IgM) antibodies against HSV-1 and HSV-2 were detected by a commercially available enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein M of HSV-1 or HSV-2. The assay was based on the principle of IgM capture where IgM class antibodies in the samples are first captured by the solid phase coated with anti hIgM antibody. After washing out all the other components of the samples and in particular IgG antibodies, the specific IgM captured on the solid phase were detected by the addition of a purified preparation of inactivated HSV-1&2, labeled with a specific antibody conjugated with peroxidase (HRP). After incubation, microwells are washed to remove unbound conjugate and then the chromogen/substrate is added. In the presence of bound conjugate, the colorless substrate is hydrolyzed to a colored end-product, whose optical density may be detected and is proportional to the amount of IgM antibodies to HSV-1&2 present in the sample. Results, Calculation, and Interpretation Results were interpreted according to the manufacturer‘s specifications. The test results were calculated by means of the mean OD450 nm value of the negative control (NC) and a mathematical calculation, in order to define the following cut-off formulation: Cut-Off = NC + 0.250. The values found were used to interpret the results as the ratio of the sample OD450nm and the cut off value (or S/Co) as follows: 1.2 (positive). Data Analysis The data were recorded in and analyzed using Microsoft Excel spreadsheet (Microsoft Corporation). The seroprevalence was calculated as the number of serologically positive samples divided by the total number of samples tested. The Chi-square test was used to determine associations between seropositivity and socio-demographic variables. The level of statistical significance was set at P ≤ 0.05. RESULTS Patient Characteristics The total number of pregnant women included in this study was 180. The socio-demographic data for these samples were stratified and shown in Table 1.
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Prevalence of HSV-1&2 IgM Antibody Of the 180 specimens tested, only 5 (2.8%) were seropositive for HSV-1&2 IgM antibody while most of the specimens 175 (97.2%) were seronegative (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Age Figure 1 shows the rate of seropositivity of HSV-1&2 IgM antibody according to age. In relation to age, only two groups out of the four age groups were reactive. Two (6.7%) in the age group ≤25 years and 3 (4.1%) in the age group 26–30 were found to be seropositive. The other age groups showed no presence of HSV-1&2 IgM antibody. There was no significant relationship between the age groups and prevalence of IgM antibody against HSV-1&2 (χ 2 = 4.359, df = 3, P >0.05) (Table 1).
7
6.7
6
Frequency and Percentage
5 4.1 4 3
3
2
2
1
0
0 ≤25
26–30
0.0
0
31–35
0.0 ≥36
Age (Years) HSV 1&2 IgM +ve frequency
HSV 1&2 IgM +ve2 %
FIGURE 1 The rate of seropositivity of HSV 1&2 IgM antibody according to age.
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Prevalence of HSV-1&2 IgM Antibody in Relation to Marital Status As shown in Figure 2, a higher seroprevalence of HSV-1&2 IgM antibody of 33.1% was observed for the pregnant women that were single which was greater than 2.3% observed for those that were married. Statistically, marital status was significantly associated with prevalence of IgM antibody against HSV-1&2 (χ 2 = 221.5, df = 1, P < 0.05) (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Educational Background While none of the pregnant women with primary education tested positive, 2 (3.0%), 2 (2.0%), and 1 (8.3%) with secondary, tertiary and post-secondary respectively were found to be seropositive, as shown in Figure 3. The level of education of the pregnant women attending antenatal care had no significant relationship with the prevalence of IgM antibody against HSV-1&2 (χ 2 = 1.653491, df = 3, P > 0.05) (Table 1).
35
33.3
30
Frequency and Percentage
25
20
15
10
5
4 2.3 1
0
MARRIED
SINGLE Marital status
HSV 1&2 IgM +ve frequency
HSV 1&2 IgM +ve %
FIGURE 2 The rate of seropositivity of HSV 1&2 IgM antibody according to marital status.
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Frequency and Percentage
7 6 5 4 3.0 3 2
2
2.0
1
1 0
2
0
0.0
PRIMARY
SECONDARY POST SECONDARY Educational status
HSV 1&2 IgM +ve frequency
TERTIARY
HSV 1&2 IgM +ve %
FIGURE 3 The rate of seropositivity of HSV 1&2 IgM antibody according to educational status.
Prevalence of HSV-1&2 IgM Antibody in Relation to Occupational Status Figure 4 shows the prevalence rates of HSV 1&2 IgM antibodies according to occupational status which were found to be 8.3% (2), 2.1% (1), 1.5% (1), and 2.4% (1) for students, unemployed, self-employed, and employed women, respectively. Statistically, there was no significant relationship between occupation and prevalence of IgM antibody against HSV-1&2 (χ 2 = 3.264087, df = 3, P > 0.05) (Table 1). Prevalence of HSV-1&2 IgM Antibody in Relation to Gestation Period Four (6.8%) of the pregnant women that were seropositive for HSV1&2 IgM antibodies were in their third trimester and 1 (1.1%) was in her second trimester, as highlighted in Figure 5. However, no significant association was found between the gestation period and the prevalence of IgM antibody against HSV 1&2 (χ 2 = 5.296946, df = 2, P > 0.05) (Table 1).
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HSV-1 and -2 IgM in Pregnant Women 9 8.3 8
Frequency and Percentage
7 6 5 4 3 2
1.5 1
1 0
2.4
2.1
2
STUDENT
1
UNEMPLOYED SELF EMPLOYED Occupation
HSV 1&2 IgM +ve frequency
1
EMPLOYED
HSV 1&2 IgM +ve %
FIGURE 4 The rate of seropositivity of HSV 1&2 IgM antibody according to occupation.
DISCUSSION The present study was thus undertaken for finding IgM antibodies against HSV-1 and 2 infections among pregnant women and also to evaluate correlation of Serum HSV-1 and 2 IgM in these pregnant women. As HSVs are significant human pathogens, rapid detection and diagnosis of these viruses is imperative.[1] Immunoglobulin–M (IgM) antibodies against HSV-1 and HSV-2 were detected by a commercially available enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein M of HSV-1 or HSV-2.[20] HSV-1 and HSV-2 are highly homologous genetically and antigenically. This results in an antigen-sharing profile for the two serotypes.[20] Seroprevalence estimates of HSV infections allow a better understanding of the burden of infection and how this is evolving with time.[3] The present study showed that of the 180 pregnant women tested, only 5 (2.8%) were seropositive for HSV-1&2 IgM antibody while 175 (97.2%) were seronegative. Detectable IgM antibodies in HSV infection usually reflect primary or recurrent infection. HSV-1&2 IgM, as an indicator of current infection was demonstrated in 2.8% of the pregnant women studied. Like Iraqi women,[21–23] little is known about the seroprevalence of HSV-1&2 in
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7
Frequency and Percentage
6
5 4
4
3
2 1
1
0
0
1.1
0.0
1ST TRIMESTER
2ND TRIMESTER Gestation Period (Months)
HSV 1&2 IgM +ve frequency
3RD TRIMESTER
HSV 1&2 IgM +ve %
FIGURE 5 The rate of seropositivity of HSV 1&2 IgM antibody according to gestation period.
Nigerian pregnant women. These three studies reported a wide range of HSV-2 seropositivity ranging from 8.1–73.9% for HSV IgM in Iraq,[24] which are higher than the 2.8% reported in our present study. This low seropositivity rate (2.8%) recorded in this study is in agreement with the low rates reported by previous authors.[1, 24–26] Alzahrani et al.[27] reported 0.5% for HSV-2 IgM in Arab countries. Ozdemir et al.[25] reported a seropositivity of 0.0% and 1.4% for HSV type 1 and 2 respectively in Turkey. Sadik et al.[26] reported 1.69% seropositivity in their study. Peña et al.[1] reported a 0.15% seropositivity for both HSV-1 and HSV-2 in women. Zainab et al.[24] demonstrated HSV-2 IgM seropositivity 3.2% of pregnant women. Our 2.8% HSV IgM seropositivity reported in these pregnant women in this study was higher than that reported the 0.0% reported by Ozdemir et al.[28] in Turkey; the 0.5% reported by Alzahrani et al.[25] in Saudi Arabia; the 1.2% reported by Vilibik-Cavlek et al.[29] in Croatia and the 1.8% reported by Nabi et al.[30] in Bangladesh. All these figures correlated favourably with our present findings. Thus, 2.8% current infection with HSV-1&2 reported in this study was lower to that reported in other studies elsewhere. It is lower than the 5.8% HSV IgM seroprevalence reported by Zainab et al.[24] in pregnant women
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with bad obstetrics history (BOH). It was lower than the seropositivity rates reported for India,[31–34] Baghdad, Iraq,[21] Waset, Iraq,[23] and Mosul, Iraq.[22] The overall Serum HSV-1 and 2 IgM seroprevalence was 15.66% in the study by Tada and and Khandelwal.[35] Al-Marzoqi et al.[36] reported seroprevalence of 28.9% HSV-2 IgM in Iraq pregnant women. Other studies in Arab countries reported a range of 0.5–7.6% for HSV2 IgM[24,37] and 6.5% to 27.1%[24, 27, 38] for HSV-2 IgG in pregnant women. Our 2.8% rate is also lower than the 6.25% reported by Dordevic;[39] the 28.9% reported by Al-Marzoqi et al.[36] in Babylon, Iraq; the 13.8% reported by Duran et al.[40] in Turkey and the 7.6% reported by Abu-Madi et al.[37] in Qatar. It is also lower than the 14.0% reported by Peña et al.[1] for either HSV-1 alone, HSV-2 alone, or both HSV-1 and HSV-2. It is also lower than the 48.0% seropositivity reported for HSV-1 alone, 7.0% for HSV-2 alone and 12.0% for co-infections with HSV-1 and HSV-2.[41] In a previous small-cohort analysis by Adelson et al.,[42] an 11.0% HSV positivity rates for HSV-1 and/or HSV-2 was reported. This 2.8% HSV-1&2 seropositivity value is much lower than 28.0% HSV-1 positivity rate found in a previous study reported by Pena et al. in 2005. However, our present study therefore suggests a very low acute HSV infection in the population of study. Now a days, serum HSV-1&2 IgM positive in patients of non herpetic clinical manifestations is quite common. This has also been reported by many authors outside Nigeria.[35, 43–45] At first glance, our 2.8% seropositivity value from this first analysis in Port Harcourt, Nigeria seems comparable to the results of a focused study which reported a 16.0% positivity rate.[1, 46] However, contrary to the study by Peña et al.,[1] our study was specifically derived from specimens from pregnant women who were not known to have genital herpes or any clinical lesions. Our study population focused exclusively on pregnant women who were not known to have genital HSV disease. This shows that these pregnant women may have either simultaneous co-infection with HSV-1 and/or 2, although the virus is not clinically manifesting the herpetic symptoms and patients show asymptomatic seroconversion or HSV-1 and/or -2 IgM reactivation.[35,47] Seropositivity of HSV-1&2 IgM antibody and its association with sociodemographic variables showed that marital status was the main correlate of HSV-1&2 IgM seropositivity. HSV IgM seropositivity was significantly higher in singles compared to married women. Whereas age, level of education, occupation, and gestational period were not consistently associated with HSV-1/HSV-2 infection and co-infection. This association to marital status can be as a result of the married women being infected by their husbands, thereby leading to an acute HSV infection. This differs from the findings of Beydoun et al.[41] which reported that age, sex, race, and level of education independently predicted all three outcomes, lifetime sexual activity, as well as use of tobacco products and recreational drugs mainly correlated
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with HSV-2 infection and HSV-1/HSV-2 co-infection, whereas lifetime use of alcohol was not consistently associated with HSV-1/HSV-2 infection and co-infection.[41] HSV-1&2 IgM was not detected in women with age of greater than 30 years, however, the higher seropositivity rate (6.7%) was observed in pregnant women with age of less than 25 years and 4.1% in ages 26–30 years. This deviates from what was reported by Pena et al.[1] Pena et al.[1] reported a zero seropositivity of HSV-2 IgM in women with ages less than 20 years and higher seroprevalence rate (5.5%) in women with age of 20–29 years. Peña et al.[41] also reported that percentages of genital HSV-1 positivity vary with age. In their study, samples which were positive for both HSV-1 and HSV2 were detected in all age groups.[41] In variance with our present finding, Zainab et al.[24] found both HSV-2 IgG and IgM to significantly vary with age groups, with trends of increasing with older ages. In this study, level of education was not consistently associated with HSV infection. HSV-1&2 IgM was not detected in women with primary education, however, the higher seropositivity rate (8.3%) was observed in pregnant women with post-secondary education, 3.0% in secondary education, and 2.0% in women with tertiary education. In disagreement with our finding, significant association was found between HSV-2 seroprevalence and education levels.[24] In this study, occupation was not consistently associated with HSV infection. HSV-1&2 IgM was lower in women who are self-employed (1.5%), unemployed (2.1%) and employed (2.4%) however, higher seropositivity rate (8.3%) was observed in pregnant women who were students. This also deviated from the previous reports. Zainab et al.[24] suggested occupation as a correlate of HSV seropositivity. Their study found HSV-2 infection to be statistically significantly higher in working women as compared to those who were housewives.[24] Of importance to note is the higher seropositivity rate of HSV-1&2 IgM observed in pregnant women that were in their third trimester (6.8%) than those in their second trimester (1.1%), however, HSV-1&2 IgM was not detected in pregnant women in their first trimester. This was not statistically associated with HSV infection. It suggests that these women in their third trimester are undergoing a primary or recurrent infection and are therefore at a higher risk of neonatal transmission. This is because when primary HSV infection occurs during late pregnancy, there is no adequate time to develop antibodies needed to suppress viral replication before labor. Previous studies have also shown that genital HSV infection acquired during pregnancy is associated with preterm labor, intrauterine growth retardation, and spontaneous abortion.[18,19] The risk of neonatal infection varies from 30–50% for HSV infections that onset in late pregnancy (third trimester).[12] Even though genital herpes infection in
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pregnant women is common and rarely serious, the risk of vertical transmission to the infant when the mother develops a primary infection during the third trimester is high and this risk increases the closer to the time of delivery.[10] We observed a high overall anti-HSV-1&2 IgM seronegativity of 97.2% among these pregnant women. Group-specific seronegativity was also high ranging from 93.3–100%. Though the age-groups (
