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Full text of "Serological and Molecular Detection of Herpes Virus From
Vaginal Samples of Pregnant Women
"
See other formats
CENTRAL ASIAN JOURNAL OF MEDICAL AND NATURAL SCIENCES
—— ee
N\- Volume: 04 Issue: 04 | Jul-Aug 2023 IssN: 2660-4159
http://cajmns.centralasianstudies.org
TTT
ret AL AND
METAL SCIENCES
Serological and Molecular Detection of Herpes Virus From
Vaginal Samples of Pregnant Women
1. Alaa Faroog Rasool
2. Sarah Shareif Akool
3. Hawraa Ali Mahmood
anaes)
Received 2" May 2023,
Accepted 34 Jun 2023,
Online 29" Jul 2023
'23 Central Public Health Laboratory in
Muthanna
Key words: HSV-1, HSV-2, Women,
Pregnant, Molecular, Serological.
Abstract: This investigation was done for serological
and molecular detection of herpes virus from vaginal
samples of pregnant women. This study involved the
enrollment of 210 pregnant women who were admitted
to a private hospital in Baghdad. The data collection
period spanned from 2021 to 2023. Samples of serum
and cervicovaginal lavage were obtained from the
participants and then transferred to the laboratory under
refrigeration using ice. Virus DNA _ from lavage
specimens was extracted by using kit. Two primers were
used in this study for detection of HSV-1 and 2. The
presence of antibodies specific to HSV-1 and HSV-2
was determined in serum samples using a competitive
type-specific ELISA. IgG prevalence was assessed to be
91% (191 out of 210) and IgM prevalence was
determined to be 5.7% (12 out of 210) based on results
from serological tests of total HSV-1l and HSV-2
antibodies. Out of a total of 210 people, 96.7% (203 of
203) were affected. The estimated HSV-1 and HSV-2
frequencies in this research were 5.2% and 8.6%,
respectively, based on molecular studies. The current
study showed that age group 21-30 y were mostly
affected.
Inconclusion, the primary observation derived from this
study is the comparatively reduced prevalence of HSV-1
and HSV-2 among pregnant women, as opposed to the
rates reported by the World Health Organisation
(WHO). However, it is evident that there is a need for
extensive research to be conducted in order to determine
the prevalence of HSV infections among Iraqi women,
particularly in pregnant women, and to analyse the
trends over time. In addition, the utilisation of molecular
and serological techniques can prove to be advantageous
in carrying out such investigations.
588 | Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
Introduction:
The Herpesviridae family is a group of viruses with a reputation for causing long-lasting infections in
humans. Five genera and 45 species make up what is called the subfamily Alphaherpesvirinae. Among
these species, human alphaherpesvirus | and 2 are widely prevalent. They belong to the genus
Simplexvirus and are commonly referred to as herpes simplex type 1 and 2 (HSV-1 and HSV-2)
respectively (1-3).
These viruses have been linked to infections in the orofacial and genital regions, respectively. The
herpes simplex virus (HSV) is widely acknowledged as a highly prevalent cause of sexually
transmitted infections (STIs) on a global scale. It is estimated that between 60% and 95% of the adult
population worldwide either carries HSV viruses or is affected by related infections, which typically
manifest as subclinical or asymptomatic conditions. The severity and frequency of these infections are
contingent upon the individual's immune system and the frequency of viral exposure (4,5).
The transmission of HSV-1 primarily occurs through non-sexual means, specifically mouth-to-mouth
contact, resulting in the development of oral ulcers. On the other hand, HSV-2 is responsible for the
occurrence of genital ulcers, which are transmitted through sexual contact (6, 7). Nevertheless, certain
reports attribute the causation of genital ulcers to HSV-1 (8).
Based on the most recent report released by the World Health Organisation (WHO) in 2017, it was
found that a significant proportion of individuals under the age of 50, specifically 67%, are infected
with HSV-1 globally. Additionally, HSV-2 infection was observed in 13% of individuals aged 15-49
worldwide. According to estimates, the prevalence of HSV-2 infection among women in 2017 was
approximately 313 million (8-9).
Both genital and oral herpes are very infectious during pregnancy, and they may be passed on to the
baby either via the mother's viremia or, more often, through the birth canal. This may cause newborn
herpes, which has been linked to an increased death rate (5, 10). Neonatal herpes is a relatively
infrequent yet highly detrimental complication that arises from infection with the herpes simplex virus
during the gestational period (11).
When compared to pregnant women who have already been exposed to HSV, uninfected women who
come into contact with the virus for the first time in their third trimester have a much greater risk of
transmitting it vertically. It's possible that some mothers get infected for the first time during their
pregnancies (12,13). The acquisition of HSV during gestational status has been associated with various
adverse outcomes, including spontaneous abortion, severe neurologic injuries, prematurity, stillbirth,
preterm birth, as well as congenital and neonatal herpes (14, 15). The implementation of acyclovir
therapy in pregnant women who are infected, along with the consideration of caesarean delivery after
localising genital lesions, are effective strategies that should be considered in order to reduce the risk
of congenital transmission of herpes simplex virus (HSV) (16, 17).
While the prevalence of HSV-1 is increasing worldwide, certain societies have experienced a reversal
in this upward trend due to improvements in their hygienic conditions. Therefore, the implementation
of enhanced sanitation measures would effectively reduce the likelihood of adolescents being exposed
to this virus before reaching puberty, consequently decreasing the risk of post-puberty transmission of
HSV (4, 18). Furthermore, according to Kahlon and Whitley (19), a significant proportion of mothers,
approximately 80%, are not cognizant of their history of HSV infection when their newborns become
infected. Overall, conducting research and monitoring women of reproductive age, particularly prior to
and during pregnancy, is an essential strategy to prevent the occurrence of complications associated
with HSV infection, such as neonatal herpes (20).
This investigation was done for serological and molecular detection of herpes virus from vaginal
samples of pregnant women.
589 | Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
Materials and Methods:
This study involved the enrollment of 210 pregnant women who were admitted to a private hospital in
Baghdad. The data collection period spanned from 2021 to 2023. Serum and cervicovaginal lavage
specimens were collected from the participants and subsequently transported to the laboratory while
being kept at a low temperature using ice. The specimens obtained from cervicovaginal lavage were
subjected to centrifugation at a force of 1000 times the acceleration due to gravity for a duration of 10
minutes. Following centrifugation, the liquid portion above the sediment was removed and discarded.
For either short-term or long-term storage, the cells were resuspended in 1 ml of PBS and frozen at -20
degrees Celsius. After drawing blood from the body's periphery, the serum was separated by
centrifuging the blood at 2000 times the acceleration of gravity for around 10 minutes. The obtained
serum aliquots were subsequently stored at a temperature of -20°C until they were subjected to
serological analyses.
Virus DNA from lavage specimens was extracted by using Genaid kit and according to the
manufacturer instructions.
The forward and reverse primers developed for UL5 and UL27 detection are listed in Table 1 (21).
Denaturation at 95 degrees Celsius for 15 minutes was followed by 35 cycles of denaturation at 94
degrees Celsius for 45 seconds, annealing at 58.5 degrees Celsius for 45 seconds, extension at 72
degrees Celsius for 45 seconds, and a final extension step at 72 degrees Celsius for 7 minutes for the
aforementioned genes.
The PCR products were subjected to analysis using ultraviolet (UV) light following electrophoresis at
a voltage of 100 volts for a duration of 1 hour on a 1.5% agarose gel that had been treated with a
DNA-safe stain.
Table 1. Type specific primers of HSV-1 and HSV-2
Gene F R bp
HSV-1 5'-GACGTCACCGTTTCGCAGGTGT-3' 5'-CGTTGGCCGGTTTCAGCTCCAT-3' | 412
HSV-2 5'-CGCGCCTCCGAAAGATGGTGTT-3' 5'-TCGTCCAGCCCGGCGAAGATAA-3' | 217
The presence of antibodies specific to HSV-1 and HSV-2 was determined in serum samples using a
competitive type-specific ELISA (Mybiosource company). Specimens were coded as seropositive or
seronegative, and the data was entered in Microsoft Excel 2010.
Results:
Based on the results of the serological analysis of total HSV-1 and HSV-2 antibodies, it was found that
the prevalence of IgG was 91% (191 out of 210) and that of IgM was 5.7% (12 out of 210) (Figures 1
and 2). The total prevalence was 96.7% (203 out of 210).
IgG
= Positive m Negative
Figure 1. The prevalence of IgG in the study participants
590 Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
IgM
= Positive = Negative
Figure 2. The prevalence of IgM in the study participants
The estimated HSV-1 and HSV-2 frequencies in this research were 5.2% and 8.6%, respectively,
based on molecular studies (Figures 3, 4, 5, and 6).
HSV-1
= Positive # Negative
Figure 3. Molecular prevalence of HSV-1
HSV-2
= Positive m= Negative
Figure 4. Molecular prevalence of HSV-2
591 | Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
The current study showed that age group 21-30 y were mostly affected (Table 2).
Table 2. Prevalence according to age
Age IgG IgM HSV-1 HSV-2
groups(y)
15-20 (52) 46 (88.5%) 3 (5.8%) 2 (3.8%) 4 (7.7%)
21-30 (113) 109 (96.5%) 7 (6.2%) 8 (7.1%) 13 (11.5%)
31-42 (45) 36 (80.0%) 2 (4.4%) 1 (2.2%) 1 (2.2%)
Total (210) 191 (96.7%) 12 (5.7%) 11(5.2%) 18 (8.6%)
Figure 6. Gel electrophoresis for HSV-2 gene, showed positive results at 217 bp
Discussions:
According to a study conducted by Monavari et al., the prevalence rates of HSV1 and HSV-2 were
reported to be 22.9% and 14.3% respectively (23). The findings of the present study align with a
previous investigation conducted in Sudan, which examined a cohort of 100 pregnant women. In that
study, the prevalence of HSV-1 and HSV-2 was reported to be 3% and 2% respectively (24), mirroring
the results obtained in the current research. Within the scope of our investigation, it was determined
that 5.2% (11 out of 210) and 8.6% (18 out of 210) of the clinical specimens analysed exhibited
positive results for the presence of the HSV-1 and HSV-2 genomes, respectively. The aforementioned
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
variables may account for the extensive variability observed. Molecular typing results from this
research show that HSV-2 was the most common type, with an 8.6% prevalence rate.
Furthermore, the enzyme-linked immunosorbent assay (ELISA) was employed to identify antibodies
(specifically IgM, IgG, and total) targeting HSV-1 and HSV-2 in serum samples. A total of 800
women were surveyed for a research by Rezaei-Chaparpordi et al. (25) in Iran's northwestern
provinces. The research found that the prevalence of HSV-1 was 58.4 percent, whereas that of HSV-2
was 3.5 percent. According to their findings, it has been reported that the prevalence of HSV-1 is
higher than that of HSV-2. They also observed an age-related rise in HSV-1 sero-prevalence (25).
However, when it comes to HSV-1 and HSV-2 infections, the ELISA results showed that the
frequency of IgM and IgG antibodies were 5.7% and 91%, respectively.
Serological survey results showed a significant increase in the sero-prevalence of HSV infections
among the pregnant women surveyed in this research (96.7%). One notable constraint of the current
study was the absence of examination into the specific antibodies targeting HSV-1 and HSV-2 using
the enzyme-linked immunosorbent assay (ELISA) method. However, prior studies have shown that
knowing the sero-prevalence of both herpes kinds may aid in epidemiological studies and the creation
of healthcare policy. The high sero-prevalence of HSV infections found in our research might be due
to a number of causes, including widespread virus spread, low female education about the issue, and
environmental influences (22).
According to the data collected on HSV-positive patients, the largest incidence of infection was seen
in women between the ages of 24 and 33. Results from a study in South Africa were consistent with
ours (26, 27). Consistent with earlier studies, this one found a favourable connection between HSV
infectivity and younger ages. This association could potentially be attributed to heightened sexual
activity among individuals within this age group. Furthermore, it was observed in the previous study
(28) that the age group of 20-29 years was the most prevalent among the female participants.
Conclusion:
The primary observation derived from this study is the comparatively reduced prevalence of HSV-1
and HSV-2 among pregnant women, as opposed to the rates reported by the World Health
Organisation (WHO). However, it is evident that there is a need for extensive research to be conducted
in order to determine the prevalence of HSV infections among Iraqi women, particularly in pregnant
women, and to analyse the trends over time. In addition, the utilisation of molecular and serological
techniques can prove to be advantageous in carrying out such investigations.
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Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
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CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
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594 | Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
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CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
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595 | Published by “ CENTRAL ASIAN STUDIES" http://www.centralasianstudies.org
Copyright (c) 2023 Author (s). This is an open-access article distributed under the terms of Creative Commons
Attribution License (CC BY).To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/