Detection of HSV and CMV in Pregnant and Miscarriage women by ELISA and real time PCR Assay

 

Mariam Kareem Ali¹, Jaafar Sataar Shia², Huda Dhahar Al- marsome³

¹Department of Microbiology, College of Medicine – University of Baghdad, Iraq

²Ministry of Health, Baghdad, Iraq

³Department of Microbiology, College of Medicine Al-Nahrain University

 

ABSTRACT:

Herpes Simplex Viruses (HSV) and Human Cytomegaloviruses (HCMV) are the major cause of serious viral complications in pregnant women. Screening methods that is ELISA for detecting Herpes Simplex virus(HSV) and Human Cytomegalovirus(HCMV) tend to be slow and insensitive. Therefore in this work, a rapid Real Time PCR (qT-PCR) and ELISA based assay was designed to detect HSV and CMV among pregnant women in Iraq from serum samples. Our study revealed that among the ELISA screened 420 sera samples, 81 (19.3%) of CMV and 17(4.05 %) of HSV were positive, while 64 (12.9%) had positive results for CMV DNA and 5(1.2%) had HSV DNA through real-time PCR. Real time PCR was more sensitive and reliable method in diagnosis of CMV and HSV infections in pregnant women in this comparative study.

 

 

INTRODUCTION:

Viral infections accounts for major part of maternal infections which was responsible of the unfavorable outcome of pregnancy, mainly rubella, cytomegalovirus (CMV) and Herpes Simplex Virus (HSV) infections. Herpes Simplex Virus (HSV) and Cytomegalovirus (CMV) most common sexually transmitted diseases [1, 2], and these species of viruses that belong to Herpesviridae or Herpesviruses family. These viruses are among the most ubiquitous viruses found in the adult population. This family has a characteristic of lifetime latency after primary infection and the latent virus can reactivate in infected individuals at any time [3]. CMV and HSV play important role in causing maternal infections and these are known to have an intrauterine route of transmission with significant mortality and morbidity [4,5].

 

 

 

 

In developing countries, CMV is the most common cause of congenital deformity which usually occurred during viral intrauterine infection [6]. Due to virus reactivation during the child bearing age, Cytomegalovirus infection during pregnancy is more complex than other infections during pregnancy and can be transmitted to the fetus despite maternal immunity [7, 8]. On the other hand, HSV infection of the newborn can be acquired in ex utero intrapartum therapy and after child0 birth [8]. These infections are usually symptomatic and asymptomatic, and they are to diagnose clinically [9,10].   

 

Laboratory confirmation can be achieved using serological and molecular techniques. Conventional methods for detection of antibodies to HSV and CMV include various assays like Immunofluorescence assay (IFA), Enzyme-linked fluorescent assay (ELFA), Enzyme immunoassay (EIA), and Enzyme-linked immunosorbent assay (ELISA). These techniques have been used widely for both diagnostic and screening protocols for CMV, HSV and other viral infections [11]. Recently Real time PCR due to its high specificity and sensitivity emerged as a novel approach in detection of molecular response to various infectious diseases [12]. Real time PCR allows simultaneous detection and identification of multiple samples [13-15].

In Iraq, and especially in Baghdad rural and peripheral areas, along with other diseases, infectious diseases are becoming more day by day, due to no awareness, low health care facilities and low literacy rate. Due to lack of a national screening program, there is no baseline serological data regarding the seroprevalence of such infections in patients. Therefore, this comparative study was undertaken for the assessment of ELISA and PCR accuracy in detection of CMV and HSV exposure to pregnant women at high risk for miscarriages and other pregnancy related complications in Baghdad.

 

MATERIALS AND METHODS:

Blood samples collection and handling:

The study population was 420 females having obstetrical problems Causes Habitual Abortion like TORCH infection.

 

Thyroid dysfunction and Anti cardiolipin antibody (ACA) – IgG/IgM etc and 15 females having no obstetrical problem as a control group. Blood specimen were collected from pregnant women during period of January 2017 to March 2017 from different hospitals in Baghdad. Inclusion criteria for all of these was; complications found during pregnancy or some other chronic diseases like hypertension, diabetes associated with pregnancy. All those female patients having other TORCH infections were excluded from this study. The blood samples were collected aseptically by using venipuncture techniques and centrifuged at 3000 rpm for 5 min. The sera collected were refrigerated (2-8°C) upon collection or frozen (-20°C) if the test could not be performed within 7 days. For detection of CMV and HSV antibodies, [NOVALisa™, Germany test], ELISA kit instructions were followed.

 

Enzyme-linked Immunosorbent Assay (ELISA tests):

Detection of Herpes Simplex virus IgM and Cytomegalovirus IgM by (ELISA; (NovaLisa™, Germany).

 

These tests were done using human diagnostic ELISA kits, Germany, for the qualitative determination of human antibodies of the IgM against herpes simplex virus andCytomegalovirus IgM in serum or plasma.

 

Molecular Study:

Singleiplex RT-PCR kit (Sacace ™ Biotecnologies) for the direct, qualitative detection of herpes simplex virus and Cytomegalovirus. The principle of (DNA extraction Nano drop, Agarose electrophoresis, and RT-PCR) preparation sample, and interpretation of the results were as same as those in HSV and CMV determination method.

 

Real Time PCR Assay:

PCR–mix-1-FL HSV/CMV, PCR-mix-2-FRT and polymerase (TaqF) was done for few seconds. For amplification of DNA from the test and control samples, required number of the tubes were prepared.

 

For N reactions (including 2 controls of amplification) mixed in a new tube:

10*(N+1) ul of PCR-mix-1-FLHSV/CMV, 5.0* (N+1) ul of PCR-mix-2-FRT and 0.5* (N+1) ul of polymerase (Taq F).

 

The prepared mixture was stirred and then centrifuged for about 1-2 s in order to remove all drops from the walls of tubes. About 15ul of the prepared mix was transferred to each tube and 10ul of extracted DNA obtained from the extraction test was added into the prepared tubes. Centrifugation was again done.

 

Amplification of DNA:

The tubes were inserted into the device reaction module cells, According to Manufacturer’s manual. The amplification program was set with fluorescence detection, Results were analyzed after completion of the amplification program. In the end, fluorescent signal was detected in FAM, JOE and ROX channels.

 

HSV DNA was detected in the JOE Channel, CMV DNA was detected in the FAM Channel, and in the ROX fluorescence channel internal control DNA was detected [Figure 1]

 

Nano drop:

Extracted DNA was measured by Nano drop instrument were aspirated using special tips and inserted in specified socket in the machine, DNA was quantified by the refractive index using the wave length260nm, 280nm. DNA concentration was calculated with the OD260nm. The purity was estimated with the OD260nm/OD280nm ratio, a ratio of 1.8-2 was generally accepted as pure for DNA.

 

Agarose electrophoresis:

DNA extraction was successfully observed from samples by agarose electrophoresis (1.5%). This DNA was used as a template for RT- PCR assay (Figure 1).

 

 

Figure 1: Agarose electrophoresis of total DNA extraction

Table 1.qT-PCR and ELISA results for CMV and HSV infection

 

Table 2. Performance of different diagnostic tests in detection of CMV infections in women grouped

*Pearson Chi-square analysis

 

Table 3. Performance of different diagnostic tests in detection of HSV infections in women grouped.

*Pearson Chi-square analysis

 

Statistical analysis Statistics Analysis:

Statistical analysis was performed by using SPSS computing program for the analysis of the results.

 

Ethics approval and consent to participate:

Research Ethical approval was obtained from the research ethics committee of Baghdad University, college of medicine, and permission letters was obtained from the hospital management committee. Before commencement of data collection, the purpose of the study was explained to the participants and all of them provided written informed consent. CMV and HSV screening was performed free of charge, and those found to have infection were managed by physicians.

 

RESULTS:

All blood samples from 420 patients in this study were tested using ELISA and qT-PCR. The results obtained from the serological test for 420 samples, the seropositive of CMV IgM was 81(19.3%), and ratio for IgM positive samples among HSV positive sera samples 17(4.05%). The results for PCR showed that 64 (12.9%) out of 420 patients were found to be CMV positive, While5 (1.2%) were found to be HSV positive, The control group did not detect any viral DNA presence as shown in table (1), figure (2).

 

Table (2, 3) shows that types of vaginal infection according to method of diagnostics test, in ElISA test shows 57(20.6%) of patients with CMV infection were pregnant women group, and only 24(16.8%)were aborted women group, In qT-PCR show48(17%) were complaining from pregnant women group, and only 16(11.2%)were aborted women group, for HSV infection in ElISA test shows 9(3.2%) of patients with HSV infection were pregnant women group, and only 8(5.6%)were aborted women group, in qT-PCR show2(0.7%) were complaining from pregnant women group, and only 3(2.1%)were aborted women group. On application of Chi square test, highly significant difference between typesvaginal infection in relation to infections in women grouped

 

 

Fig. 2: Amplification plots of Real time PCR

 

DISCUSSION:

The prevalence of HSV &CMV infection is increasing in our country, and most individuals are unaware of their infection. Most cases of genital HSV infection in women occur without signs or symptoms of disease and are associated with cervical viral shedding. Almost none of this viral shedding is accompanied by clinically detectable genital lesions [16-18].

 

Festary et al. (2015) [19] conducted a study in which they found that 89.5% pregnant women tested positive for CMV and 83.2% for HSV. Our study showed high rate to CMV IgM (17%) antibodies. In 2015, it was also reported that miscarriage women had highest percentage of seropositive to HCMV for IgG (40%) and (25%) for IgM out of 40 samples [201,21].

 

Primary CMV infection in pregnancy has a higher incidence of symptomatic congenital infection and fetal loss. This infection, being asymtomatic in adults, is difficult to diagnose clinically. Demonstration of IgM antibodies is indicative of primary infection. The need of serological evaluation of CMV specific IgM during pregnancy has been supported by various investigators [21-23].

 

There was also low prevalence rate of HSV among pregnant women as indicated by Hasanet al. (2013) [24] in their seroprevalence study of HSV through ELISA. According to Rozimanet al. in 2007 [25], most people acquire HSV in childhood therefore HSV IgM is rarely found in adults and not all such patients have elevated HSV IgM. Study correlates with our findings in which HSV IgM antibodies had lower percentage of IgM antibodies in pregnant women (Rozimanet al., 2007) [25]. However, the role of HSV in causing infections among pregnant women could not be denied as the incidence and prevalence of HSV infections are increasing rapidly globally [26].

 

In this study, real time PCR was run on all positive samples of extracted DNA obtained from pregnant women that have no history of hypertension, diabetes and other complications. The results for the RT-PCR showed that; 17 out of 420 pregnant women having miscarriages were found to be HCMV DNA Positive while 403 out of 420 pregnant women were found HCMV DNA Negative. 15 out of 420 pregnant women having miscarriages were found to be HSV DNA Positive while 415 out of 420 pregnant women were found HSV DNA Negative. Festary et al. in 2015 [19] detected CMV DNA in vaginal swab samples of pregnant women i.e. 9(9.5%) and HSV DNA in 1(1. 1%) among 95 sera samples. This finding is quite like our study; however, we found CMV and HSV DNA in sera in another study conducted to find out CMV DNA, it was found out that 10 out of 57 samples had positive CMV DNA which showed similarity with our work [27-29]. HSV genome confirmation by simple PCR was in aborted material which gave positive DNA confirmation in 3 samples same as in our case we had only 7 HSV DNA in 175 pregnant women sera samples. These findings showed that most of the ELISA results were confirmed by PCR which means that seropositive results by ELISA were not specific or less significant due to probability of false positive results as a result of other microbial infection [30,31]. The seroprevalence of HCMV IgM and HSV IgM of our study agreed with (Saeed Noor al huda, et al 2016 [33]) and disagreed with (Noor Al- Huda et al., 2016 [23]) the best method to detect CMV and HSV was RT-PCR as Real time PCR was considered to be active, rapid and useful technique for diagnosis of active disease and monitoring response to therapy [34,35].

 

CONCLUSION:

Severe life-threatening complications of CMV and HSV in pregnant women may not be as rare as previously considered therefore proper diagnosis must be done before pregnancy in order to reduce miscarriage rate and different congenital infant infections. The accurate diagnosis of Cytomegalovirus and Herpes Simplex virus must be done by sensitive molecular methods such as Real Time PCR while ELISA should be used as screening method as Real Time PCR is the best technique and has more sensitive and specific effect than conventional PCR and ELISA.

 

COMPETING INTERESTS:

The authors declare that they have no competing interests.

 

AUTHORS' CONTRIBUTIONS:

Dr. Mariam Kareem Ali, Dr. Jaafar Sataar Shia, Dr. Huda Daher Al-Marsome conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

 

FUNDING:

Self –funding

 

ACKNOWLEDGEMENT:

Authors would like to gratefully acknowledge staff in lab for their timely help and critical suggestion during work.

 

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Accepted on 14.06.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4090-4094.