Research Article

Prevalence of Herpes simplex Virus in Pregnant Women in Ismailia City

Hanaa H.A. Gomaa1*, Shimaa A.S. Hasan1, Nashwa Harb1, Amal H.A. Gomaa2 and Maha Anani3

1Department of Botany and Microbiology, Faculty of Science, Suez Canal University, Ismailia, Egypt; 2Department of Dermatology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt; 3Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt.

Received | May 30, 2022; Accepted | June 20, 2022; Published | June 28, 2022

*Correspondence | Hanaa H.A. Gomaa, Department of Botany and Microbiology, Faculty of Science, Suez Canal University, Ismailia, Egypt; Email: [email protected]

Citation | Gomaa, H.H.A., S.A.S. Hasan, N. Harb, A.H.A. Gomaa and M. Anani. 2022. Prevalence of Herpes simplex virus in pregnant women in Ismailia city. Journal of Virological Sciences, 10(1): 18-26.

DOI | https://dx.doi.org/10.17582/journal.jvs/2022/10.1.18.26

Keywords | Herpes simplex virus type 1, Herpes simplex virus type 2, Pregnant women, Seroprevalence

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Pregnancy is a unique stage of life that most women go through. One of the most common characteristics of pregnant women is an immune system deficit. As a result, harmful microbes like viruses can overwhelm the host’s defensive system. Human herpes simplex viruses (HSVs) are a well-known microbial pathogen that causes a variety of diseases (Anzivino et al., 2009). They are double-stranded DNA viruses that are, enveloped, and belongs to the Herpesviridae family and subfamily alpha herpesvirinae (Whitley and Baines, 2018). They are transmitted through mucosal membranes and nonintact skin and migrates to nerve tissues, where it remains in latent status. HSV-1 is frequently acquired orally in infancy. It can also be transmitted sexually. Whereas, a person can only get HSV-2 through genital contact (Ayoub et al., 2019). Both viruses have the potential to be transferred vertically during childbirth (Looker et al., 2017). However, the risk is significant when a mother becomes infected with the virus for the first time during late pregnancy (Kimberlin, 2007).

Up to 80% of HSV infections are asymptomatic (Whitley and Baines, 2018) and a large percentage of HSV infections have no or mild manifestations with no detection; hence, pregnant women are the most vulnerable patients to HSV infections. A small number of patients get clinical symptoms such as vaginal lesions and herpetic ulcers (Anzivino et al., 2009; Bochner et al., 2013; Looker et al., 2017).

A first primary infection occurs when a susceptible person (without preexisting HSV-1 and HSV-2 antibodies) is exposed to HSV. A first nonprimary episode happens when a person who has preexisting HSV antibodies (type 1 or 2) has a first episode with the opposing HSV type. Recurrent infection develops when a person has already antibodies against the same HSV type (Gupta et al., 2007). All three kinds of HSV infections are prevalent in pregnant women, which may lead to infection transmission to fetuses and newborns causing eye or skin lesions, meningoencephalitis, fetal abnormalities, and even death (Straface et al., 2012; Stephenson-Famy and Gardella, 2014).

Thus, the present study aimed to assess the seroprevalence of HSV-1 and 2 in pregnant women in Ismailia City and in addition, to assess the impact of the pregnancy duration on the presence of HSV-1 and HSV-2 antibodies. To our knowledge, this is the first report from Ismailia City and gives clues regarding the seroprevalence of the HSVs in Egyptian pregnant women.

Materials and Methods

Study population and data collection

This is a cross-sectional descriptive study conducted in Ismailia city which is located in north-eastern Egypt, near the midpoint of the Suez Canal. Ninety-four pregnant women were randomly chosen from those attending the health care centers in Ismailia city, the sample collection continued from January 2019 to January 2020. After having received written and verbal permission from the participants, they were asked about the following data: name, age and residence, data about the pregnancy semester, and associated complications.

Collection and storage of samples

Each participant’s blood sample (5.0 ml) was taken in a sterile plane tube and centrifuged at 3000 rpm for 5 minutes. Then, the serum obtained from each study subject was divided into three different Eppendorf tubes. One was used for serological tests, the second was used for the real-time PCR test, and the third one was a backup sample. Sera were stored at less than -20˚C until being used.

The serological assessment

Sera from all the participants were subjected to serological studies by indirect ELISA for HSV-1 IgG detection (MyBio Source HSV-1 IgG ELISA kit, Cat no. MBS494292, MyBio Source, Inc., San Diego, California, USA); HSV-1 IgM (MyBioSource HSV-1 IgMELISA kit, Cat no. MBS495633, MyBioSource, Inc., San Diego, California, USA) and HSV-2 IgG/IgM (gG2 purified) (VIRCELL kits for HSV-2 IgG/IgM,Cat.No.G/M1013, VIRCELL, S.L. Pza, Spain). The technique was done according to the manufacturer′s instructions. The absorbance of each well was read at 450nm and determined by ELISA system using ELISA Reader (Hyperion, USA). The concentrations were determined using standard curves. Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph.

The molecular assessment

For confirmation of HSV-1/2 infection, all serum samples positive for IgM were tested to detect the presence of HSV-1/2 DNA using qualitative real-time PCR (RT-PCR) assay.

DNA extraction and qualitative real-time PCR

Since no HSV-1 IgM was detected in any sera, only DNA from all positive HSV-2 IgM serum samples were manually extracted and purified using DNA-sorb-AM nucleic acid extraction kit, Cat no. K1-12-100-CE, supplied by AmpliSens®, Russia.

 

Table 1: Primers and probes used for Taqman detection of the gBregiona.

Primer or probe	Sequence	Label	Tm (°C)	μM
HSV2-F	TGCAGTTTACGTATAACCACATACAGC		59.5	0.9
HSV2-R	AGCTTGCGGGCCTCGTT		60.5	0.9
HSV2-probe	CGCCCCAGCATGTCGTTCACGT	5′FAM or JOE, 3′ TAMRA	70.0	0.2

 

aFAM, 6-carboxyfluorescein; JOE, 6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, TAMRA, 6-carboxytetramethyl-rhodamine. Nucleotides that differ from HSV-1 are underlined.

 

The HSV-2 DNA was detected by using qualitative real-time polymerase chain reaction (RT-PCR) using Ampli Sens® HSV-typing-FRT PCR kit, Cat.No. R-V38-F (RG, iQ)-CE, Supplied by Inter Lab Service Ltd, Russia. The DNA target amplification specific region for HSV-2 was gpB gene by using applied biosystem Step On, USA real-time PCR.

In the real-time PCR, the amplified product was detected with the use of fluorescent dyes. These dyes were linked to oligonucleotide probes, which bind specifically to the amplified product during thermocycling. The real-time monitoring of fluorescence intensities during the real-time PCR allowed the detection of accumulating product without re-opening the reaction tubes after the PCR run.

 

Table 2: Real-time PCR amplification program for amplifying HSV-2 gpB gene.

Step	Temperature °C	Time	Cycles
1	95	15 min	1
2	95	5 s	5
	60	20 s
	72	15 s
3	95	5 s	40
	60	20 s fluorescent signal detection
	72	15s

 

Statistical analysis

Data analyses were performed using SPSS version 23. Quantities data were expressed as means±SD and qualitative data were expressed as numbers and percentages. The Chi-square (χ2) test was used to test the significance of qualitative variables. P < 0.05 was considered to denote statistical significance.

Results and Discussion

Study population

The study population comprised 94 pregnant women; their ages ranged from 16 years up to 42 years (mean±SD= 25.9±5.73 years). Regarding the pregnancyduration at the time of the study, the studied population (n=94) was classified into three groups as follows: only 14.9% were during the 1st trimester; 52.1% were during the 2nd trimester and 33% were during 3rd trimester (Table 3).

 

Table 3: Basic Characteristics of the study population.

Characteristics	(n=94)
Age\ year Mean ± SD Range	25.9 ± 5.73 16–42
Age of pregnancy\ months Mean ± SD Range	5.5±2.13 1–9
Trimester First no. (%) Second no. (%) Third no. (%)	14 (14.9%) 49 (52.1%) 31 (33%)

 

Serological profiles of HSV-1 and 2

Regarding the serological markers for both HSV-1 and 2, the most prevalent immunoglobulins were HSV-1 IgG and HSV-2 IgG which were found to be positive in 74.5% and 98.9% of the studied population, respectively. Whereas, the HSV-2 IgM was detected in only 6.4% of the studied cases who had also HSV-2 IgG, and there was no HSV-1 IgM seropositivity detected at all (Table 4).

Detection of HSV-2 DNA in all positive HSV-2 IgM serum

The qualitative RT-PCR technique confirmed the HSV-2 infection of the six IgM seropositive subjects, while the unknown (equivocal) sample was negative for HSV-2 DNA, and thus it was confirmed to be a negative HSV-2 IgM case.

Interpretation of the serological profile of HSV-1 and 2

The interpretation of the performed serological tests of HSV-1 and 2 revealed that most of the studied subjects had serological evidence of past latent infection with HSV-1 and 2 that representing 74.5% and 92.5%, respectively. HSV-2 recurrent infection was more prevalent (6.4%) in the studied subjects than recurrent HSV-1 which was not detected at all. Otherwise, no primary infections of HSV1 and 2 were detected. Indeed, the results showed a very high prevalence of HSV-1 and 2 infections among the studied pregnant women with percentages of 74.5% and 98.9%, respectively (Table 5).

 

Table 4: Serological results of HSV-1 and 2 among the studied groups.

		Seropositivity in the studied group (n=94)			Titer (U/ml)
			N	%
HSV-1	IgM	Negative	94	100%	Mean±SD	0.39±0.197
		Positive	0	0.0%	Range	0.1–0.9
	IgG	Negative	24	25.5%	Mean±SD	13.26±6.1
		Positive	70	74.5%	Range	3–40
HSV-2	IgM	Negative	87	92.5%	Mean±SD Range	1.41±2.93 0.1–0.9
		Positive	6	6.4%
		Equivocal (unknown)	1	1.1%
	IgG	Negative	1	1.1%	Mean±SD	49.3±31.7
		Positive	93	98.9%	Range	8–200

 

Table 5: Interpretation of the serological results of HSV-1 and 2.

	Interpretation status	N	%
HSV-1	No infection	24	25.5%
	Primary infection	0	0%
	Latent infection	70	74.5%
	Recurrent HSV-1 episode	0	0%
	Total HSV-1 prevalence	70	74.5%
HSV-2	No infection	1	1.1%
	Primary infection	0	0%
	Latent infection	87	92.5%
	Recurrent HSV-2 episode	6	6.4%
	Total HSV-2 prevalence	93	98.9%

 

Relation between pregnancy duration and the HSV-1 and 2 infections among the studied population

Our results showed that regarding the HSV-1 and 2 serological profiles and infection types, there was no significant difference between the studied three groups of pregnant women (p-value > 0.05) (Table 6 and 7). Thus, there was no relationship between the pregnancy duration and the seroprevalences of HSV-1 and 2.

HSV-1 and 2 co-infections among the studied pregnant women

Interestingly, our results showed that all the HSV-1 positive cases (n= 70, 74.5%) had also HSV-2 co-infection. They were classified as follows: 6 (6.4%) had recurrent HSV-2 infection and HSV-1 latent infection, and 64 had evidence of both HSV-1 and 2 past coinfections.

The prevalence of HSV-1 IgM observed in this study was 0%. Similar to the previous study in other different countries such as Saudi Arabia (Al-Hakami et al., 2020), Brazil (Lima et al., 2021) and Turkey (Duran, 2004). On the contrary the high level of HSV-1 IgM, seropositivity was reported also by other countries researchers such as in Serbia 6.25% (Đorđević, 2006), Saudi Arabia two separate studies (5.9%, 4.3%), respectively (Obeid, 2007; Alzahrani et al., 2005), Nigeria 2.8% (Okonko and Cookey, 2015) and India 0.55% (Rajendiran et al., 2021).

The present study found that the prevalence of HSV-1 IgG was 74.5%. This is considerably close to the values obtained in other studies such as in India 69.44% (Rajendiran et al., 2021), Serbia 68.75% (Đorđević, 2006) and Switzerland 79.4% (Kucera et al., 2012). This is also considerably lower than the values obtained in other studies such as in Turkey 98% (Dolar et al., 2006), Saudi Arabia four separate studies (90.9%, 90.5%, 84.1%, 94.7%), respectively (Ghazi et al., 2002; Alzahrani et al., 2005; Obeid, 2007; Al-Hakami et al., 2020), Ghana 99.2% (Agyemang-Yeboah et al., 2019), Yemen 99.4% (Assayaghi et al., 2017), Germany 82% (Sauerbrei et al., 2011) and Brazil 82% (Lima et al., 2021). This is also considerably higher than values obtained in other studies such as in united states two separated studies (59.3%, 63%), respectively (Patton et al., 2018; Xu et al., 2007).

In the current study it was observed that the prevalence of anti-HSV-2 IgM was 6.4%. Different studies had presented different results; in Saudi Arabia 0.5% (Alzahrani et al., 2005), Serbia 6, 25% (Đorđević, 2006), Iraq three separate studies (6.2%, 2.2%, 0.6%), respectively (Mahmood and Najem, 2020; Hasan et al., 2013; Abdulkhaliq et al., 2017), Brazil 1.2% (Lima et al., 2021), India 2.1% (Amar et al., 2015) and Turkey 11.3% (Duran, 2004). In contrast, some studies showed that all the studied cases had no recent HSV- 2 infection with negative IgMtitre such as in Saudi Arabia (Obeid, 2007; Al-Hakami et al., 2020), Turkey (Özdemir et al., 2011), Sudane (El-Amin et al., 2013) and India (Rajendiran et al., 2021).

 

Table 6: The relation between the pregnancy duration and the serological profile of HSV-1 and 2.

			Pregnancy duration			P value
			1st trimester (14)	2ndtrimester (49)	3rdtrimester (31)
HSV-1	IgG	Positive (n=70)	9 (64.3%)	38 (77.6%)	23 (74.2%)	0.664 (NS)
		Negative (n=24)	5 (35.7%)	11 (22.4%)	8 (25.8%)
	IgM	Positive (n=0)	0	0	0	-----
		Negative (n=94)	14 (100%)	49 (100%)	31 (100%)
HSV-2	IgG	Positive (n=93)	13 (92.9%)	49 (100%)	31 (100%)	0.06 (NS)
		Negative (n=1)	1 (7.1%)	0 (0.0%)	0 (0.0%)
	IgM	Positive (n=6)	0 (0.0%)	3 (6.12%)	3 (9.7%)	0.5 (NS)
		Negative (n=87)	14 (100%)	45 (91.84%)	28 (90.3%)
		Equivocal (n=1)	0	1 (2.04%)	0

 

Table 7: The relation between the pregnancy duration and HSV-1 and 2 infection types.

	Interpretation status	Pregnancy duration						P value
		1st trimester (n=14)		2nd trimester (n=49)		3rd trimester (n=31)
HSV-1	No infection	5	35.7%	11	22.4%	8	25.8%	0.5 (NS)
	Primary infection	0	0%	0	0%	0	0%
	Latent infection	9	64.3%	38	77.6%	23	74.2%
	Recurrent HSV-1 episode	0	0%	0	0%	0	0%
	Total HSV-1 prevalence	9	64.3%	38	77.6%	23	74.2%
HSV-2	No infection	1	7.1%	0	0	0	0	0.1 (NS)
	Primary infection	0	0%	0	0	0	0
	Latent infection	13	92.9%	46	93.9%	28	90.3%
	Recurrent HSV-2 episode	0	0%	3	6.1%	3	9.7%
	Total HSV-2 prevalence	13	92.9%	49	100%	31	100%

 

The present study also contrast with other studies such as in Iraq28.9% (Al-Marziqi et al., 2012) and Turkey 28.6% (Sert et al., 2020).

Moreover, some studies found that the state of pregnancy may predispose to the reactivation of latent HSV-2 infection which might result in fetal infection with spontaneous abortion (Sifakis et al., 1998; Goda et al., 2007).

In this study, seroprevalence of anti HSV-2 IgG antibodies among pregnant women was 98.9%. This is considerably close to the values obtained in other studies such as in Nigeria 99.4% (Okonko and Cookey, 2015), Zaria 82.2% (Ameh et al., 2016) and Turkey 73.8% (Sert et al., 2020).

Regarding the low level of HSV-2 IgG, seropositivity was reported also by other countries researchers such as in India five separate studies found that the seropositivity of anti HSV-2 IgG among pregnant women was (64.9% , 16.66% , 8.7% , 7.5% , 6.7%) respectively (Amar et al., 2015; Rajendiran et al., 2021; Biswas et al., 2011; Rathore et al., 2010; Bochner et al., 2013), Nigeria two separate studies (58.9%, 33.3%) respectively (Diamreyan et al., 2021; Anaedobe and Ajani, 2019), Zimbabwe two separate studies (51.1%, 49.1%), respectively (Kurewa et al., 2010; Munjoma et al., 2010), Sudan 32.1% (El-Amin et al., 2013), Ethiopia 32.1% (Anjulo et al., 2016), Turkey two separate studies (63.1%, 4.4%), respectively (Duran, 2004; Özdemir et al., 2011), Saudi Arabia five separate studies (27.1%, 7.6%, 6.8%, 6.5%, 0.5%), respectively (Ghazi et al., 2002; Fazza, 2016; Obeid, 2007; Alzahrani et al., 2005; Al-Hakami et al., 2020), Serbia 12.5% (Đorđević, 2006), Iraq 9% (Mezher et al., 2018), 2.2% (Abdulkhaliq et al., 2017; Hasan et al., 2013), Yemen two separate studies (5.1%, 6%) respectively (Assayaghi et al., 2017; Al-Bana et al., 2021) and Brazil 9.5% (Lima et al., 2021).

The higher prevalence in our study might be due to various factors. First, the prevalence of HSV might be high among general population especially in men. Financial constraints might be the second factor as it was difficult to cover screening cost in our country and simultaneously creating awareness. Educational status might be the other factor. Lack of health information might play important role to increase the prevalence.

In this study, real-time PCR confirmed the positivity of the HSV-2 IgM cases (n=6), while it cleared that the equivocal sample was HSV-2 IgM negative. This proved the importance of the DNA assays in cases of doubtful serological findings, due to their sensitivity and specificity.

Our study showed high HSV PCR positivity compared with the previously published studies such as in Iraq 1.2% (Ali et al., 2019), Brazil; 0% (Lima et al., 2021), 1.49% (Lima et al., 2018).

In contrast, another study performed by (Finger-Jardim et al., 2017) in Brazil reported about 28% and 12.6% positivity results of HSV-1 DNA and HSV-2 DNA, respectively.

Miranda et al. (2014) reported that about (16% and 12.3%) of pregnant women in Brazil had positive result to HSV-1 DNA and HSV-2 DNA, respectively.

In this study we found that all IgM positive cases with recent HSV-2 infection had old HSV-1 and HSV-2 infection too with positive IgG test result and about 75.3% of cases with old HSV-2 infection gave positive results for old HSV-1 infection. Also, one case showed to be negative for old infection of both types.

In contrast to this, (Duran, 2004) found that about 44.4% of cases with recent herpes type 2 infection had old infection too with positive IgG test results. Another study by (Abdulkhaliq et al., 2017) reported that only one case with recent herpes type 2 infection had also old infection too with positive IgG test result.

Also, other studies showing that none of the cases that had old infection with herpes type 2 had also recent infection with negative IgM test results such as (Duran, 2004; Obeid, 2007; Özdemir et al., 2011; El-Amin et al., 2013; Al-Hakami et al., 2020; Rajendiran et al., 2021).

The present study found that none of the demographic factors age and duration of pregnancy has significant influence on the HSV positivity rate, these results are in agreement with (Al-Bana et al., 2021; Hasan et al., 2013) and are inconsistent with certain studies such as (Lima et al., 2021; Rathore et al., 2010; Biswas et al., 2011).

Our results clearly suggested that the prevalence of HSV were high and can contribute to various congenital infections and other complications. Periodic screening for antenatal mothers thus becomes necessary to avoid HSV complications, which results in pregnancy related and neonatal morbidity.

Conclusions and Recommendations

The current study results showed that there was a high seroprevalence of both HSV-1 and HSV-2 among pregnant women in Ismailia city. Thus, pregnancy screening for HSVs is recommended to manage the complications caused by these viruses. HSVs can be a significant problem in pregnancy since they can infect newborns vertically and produce difficulties that can affect mother health and even cause maternal mortality. Furthermore, we discovered that detecting DNA in maternal serum using PCR improves the diagnostic value of serological testing.

Novelty Statement

The study presents the results of original research and reported have not been published elsewhere.

Author’s Contribution

Conceptualization: Hanaa H.A. Gomaa, Maha Anani and Amal H.A. Gomaa; Methodology: Hanaa H.A. Gomaa, Maha Anani and Amal H.A. Gomaa; Formal analysis and investigation: Shimaa A.S. Hasan and Maha Anani; Writing-original draft preparation: Shimaa A.S. Hasan; Writing-review and editing: Hanaa H.A. Gomaa, Maha Anani, Amal H.A. Gomaa and Nashwa Harb; Supervision: Hanaa H.A. Gomaa and Maha Anani

Conflict of interest

The authors have declared no conflict of interest.

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