Seroprevalence and characterisation of herpes simplex virus from human immunodeficiency virus in samples collected from two provinces in South Africa: a retrospective study

Introduction

Herpes simplex virus (HSV) is a prevalent organism that belongs to the sub-family of alpha Herpesviridae (Greninger et al., 2018). The virus is transmitted either through oral or genital route. The routes of transmission of HSV are responsible for its differentiation into two types; herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) (Looker et al., 2008; Schiffer et al., 2014). HSV-1 is transmitted orally and it is the cause of blisters and sores around the mouth, while HSV-2 is sexually transmitted and it is traditionally associated with blisters or ulcers around the genitals (Grinde, 2013; Modi et al., 2008). Conversely, most HSV infections are asymptomatic, a factor that is partly responsible for the high prevalence of HSV infection worldwide. However, some HSV infections show visible signs of infection in the host (Looker et al., 2015), which is often associated with the increase in virus titer that further fuels its transmission (Jaishankar & Shukla, 2016; Mazzarello et al., 2018).

It is important to determine the epidemiology of HSV-1 and HSV-2 because of the disease burden associated with their infection (Chemaitelly et al., 2019). The global prevalence for HSV-1 and HSV-2 is approximately 3.7 billion and 500 million respectively (Feng et al., 2013; WHO, 2015). In 2018, McQuillan and colleagues conducted a survey in the United States and estimated the prevalence of HSV-1 and HSV-2 as 47.8% and 11.9% respectively (McQuillan et al., 2018). However, Africa has a higher rate of HSV-2 prevalence (20–80% and 49.7%) and HSV-1 (10–50% and 50.3%) in women and men, respectively (Looker et al., 2015; WHO, 2015). HSV-2 is the most common virus responsible for genital ulcer diseases (GUD) (Johnston & Corey, 2016); however, research has shown that GUD may also be caused by HSV-1 particularly in the industrialized nations (WHO, 2015). The sudden increase in GUD caused by HSV-1 is attributable to the downward shift in trend of HSV-1 acquisition before sexual relations in this population. In addition, children who do not have HSV-1 antibodies in the early stages of life are vulnerable to genital HSV-1 infection when exposed (Bradley et al., 2014; Forward & Lee, 2003). Daniels et al. (2016), evaluated the incidence of HSV-2 and its risk factors within a cohort of HIV-1 negative women in KwaZulu-Natal, South Africa. They observed that 84% female commercial sex workers in the study population were infected with HSV-2.

The high prevalence of human immunodeficiency virus type 1 (HIV-1) in South Africa (about 7.7 million) has made the Joint United Nations Programme on HIV/AIDS (UNAIDS) to regard the country as the epicenter of HIV-1 in the world (UNAIDS, 2019). Considering the high prevalence of HIV-1 in South Africa and the role that HSV plays in its transmission, the need to determine the prevalence of HSV and HIV-1 co-infection is of great importance. In addition, HSV disrupts the epithelial surface at the infection site, which serves as a port of entry for HIV recruitment that progressively facilitates its transmission by two to three fold. This characteristic makes HSV an important co-factor in HIV acquisition (Looker et al., 2017; Munawwar & Singh, 2016). HSV infection makes the transmission of HIV effortless through a transmission that disrupts the epithelium. Moreover, dual infection with HIV increases the rate of HIV replication, which further suppresses the immune system, thus enhancing disease progression. The effect of HSV in HIV acquisition has been observed in Western Asia, Europe and Africa where the prevalence of HSV-2 in HIV infected populations was 60–90%, 30–70% and 50–90%, respectively, which is three times the rate of infection in normal populations (Anaedobe & Ajani, 2019; Des Jarlais et al., 2014; Mohraz et al., 2018).

It is apparent that a sturdy interaction exists between HSV-2 and HIV-1 infection (Barnabas & Celum, 2012; Kolawole et al., 2016; Todd et al., 2013), although, a contrasting opinions was reported that HSV-2 co-infection with HIV has no role in increasing the transmission of HIV (Mohraz et al., 2018). However, Freeman and colleagues conducted a systematic review on the gender-based effect of HSV-2 in the transmission of HIV infection (Freeman et al., 2006). In their study, they discovered that HSV-2 is a significant facilitator of HIV transmission in both men and women. Another study reported a three-fold risk of HIV-1 acquisition in HSV-2 infected persons in the sub-Saharan Africa (Barnabas & Celum, 2012). Most studies that discovered the association between HSV-2 and HIV-1 were conducted outside of South Africa. Albeit, one study reported an incidence of 41% of the co-infection in South African women (Abbai et al., 2015).

Accordingly, the current study aims to establish the prevalence of HSV antibodies and HSV-DNA in HIV-1 sera and further assess the evidence of HSV-2 and HIV-1 co-infection within the study cohort.

Methods

Results

The demographics of the study population showed that majority of the study participants were female (79.5%) with a low percentage of males (20.5%) (Table 1). The mean age and standard deviation of the study population were 33.09 ±11.94 years.

ELISA screening of the samples showed that 36/44 (81.8%) were seropositive for HIV-1 while 35/44 (79.5%) were positive for either HSV-1/2 antibody (Table 1 and Extended data, Figure A). Notably, the study participants within the age group of 21–40 years had the highest HSV and HIV-1 infection rates, as depicted in Table 1.

The PCR amplification of glycoprotein B region of HSV showed that 4/35 (11.4%) of the HSV positive samples were positive for HSV-1 (Table 2 and Extended data, Figure B). Similarly, to differentiate the HSV-1 and HSV-2 positive samples, PCR amplification of the glycoprotein G region of HSV positive samples was performed (Table 2). The data showed that 30/35 (85.7%) were HSV-2 positive [Table 2 and Extended data (Figure C)]. Another finding was that 1/44 (2.3%) males and 2/44 (4.5%) females were HSV-1/HIV-1 co-infected.

Furthermore, a significant proportion of the population, 6/44 (13.6%) males and 24/44 (54.5%) females were HSV-2/HIV-1 co-infected (Figure 1).

Figure 1. Gender categorization of PCR data of the HSV-2/HIV-1 and HSV-1/HIV-1 co-infected sera.

Furthermore, PCR was also used to amplify the integrase gene of HIV-1 genome in the samples and the data showed that 36/44 (81.8%) samples that were positive for HIV-1 p24 using ELISA were also PCR positive.

NGS was performed to confirm the genomes of HSV-2 and HIV-1 viral isolates that were detected using PCR. The samples that were sent for NGS sequencing were those that exhibited high titers with ELISA, that is, HSV-2 sequences 13, 15 and 34 and HIV-1 sequence G20. The sequenced data was compared with reference sequences obtained from NCBI that were selected based on the amplified targeted regions. A maximum likelihood phylogeny method of analysis was used with a bootstrap value of 1,000 replicates to generate evolutionary trees for HSV-2 and HIV-1, respectively. The phylogenetic analysis of HSV-2 sequence data showed that sequences 13 and 15 are more closely related to glycoprotein G2 reference strain than sequence 34, with 98–99% similarity to the G2 strain (Figure 2).

Figure 2. The evolutionary relationship between HSV-2 sequences (13, 15 and 34) and HSV-2 reference genomes was inferred using the maximum likelihood phylogeny method.

The HIV-1 phylogenetic tree in Figure 3 shows that G20 sequence is 100% closely related to subtype A (HM466997) and distantly related to subtype B (AF203332 and LC022388) and subtype C (HM569277 and HM569273) reference genomes.

Figure 3. The evolutionary relationship of the HIV-1 sample (G20) with HIV-1 reference genomes from NCBI database was inferred using the maximum likelihood phylogeny method.

The SPSS v25 statistical software was used to detect whether there is a relationship between age, HSV, and HIV-1. SPSS was also utilized to evaluate the association between HSV and HIV-1 positive samples (Table 3). The two-tailed correlation test exhibited a statistical positive link between age and HSV-1 (0.366**) as shown in Table 3. HSV-2 and HIV-1 samples are also positively correlated (0.690**). In addition, an inverse or negative relationship between HSV-1 and HSV-2 (-0.463**) was detected.

The SPSS chi-square goodness-of-fit test was also used to assess association between HSV and HIV-1 positive samples. The data showed that there was no significant association between HSV-1 and HIV-1 (X² (1) = 0.138, p>0.05). However, there was a strong statistical association between HSV-2 and HIV-1 (X² (1) = 20.952, p< 0.05), as shown in Table 4.

Discussion

There are insufficient data to recount the prevalence of HSV genotypes among HIV-1 infected individuals in the Republic of South Africa. This study provides insight into an existing interrelation between HSV and HIV-1 and the potential risk that one of the viruses may have on the other. Our focus was to determine the seroprevalence of HSV among HIV-1 sample cohort and further assess for possible co-infections (HSV-1/HIV-1 or HSV-2/HIV-1). In the study, 81.8% and 79.5% of the samples were positive for HIV-1 and HSV, respectively. Of the positive HSV samples, 85.7% were positive for HSV-2. Conversely, the most prevalent type of HSV (HSV-1) as recorded by previous studies (Debrah et al., 2018; Looker & Garnett, 2005), was not highly prevalent in this study population (11.4%). This may be due to the small sample size as HSV-1 infection is often acquired during childhood and there were no childhood participants in the study (age range 1–10 years). Although, Debrah et al. used a larger sample cohort in their study, there was no record of childhood participants. Most of the high HSV-1 prevalence recorded was for participants aged 25–44 years. This low prevalence was also observed in a study conducted in the USA by Ayoub et al. (2019), where they examined the progression of HSV-1 epidemiology in the country. The study showed that more children will reach the age of sexual debut with no antibody protection against HSV-1. In addition, the low rate is attributable to the change in disease spread in the population since there are reduced viral HSV-1 antibodies at a very young age, a factor influencing HSV-2 acquisition.

Our study also revealed a higher rate of HSV-2/HIV-1 co-infection (13.6% males, 54.5% females) compared with HSV-1/HIV-1 (2.3% males, 4.5% females).Thus, one of the few observations drawn from this study was the relatively high prevalence of HSV-2 compared to HSV-1 in HIV-1 co-infected samples. The validity of the high prevalence of HSV-2/HIV-1 co-infection in this study is supported by previous studies (Looker et al., 2017; Patel et al., 2012). The increase in HSV-2/HIV-1 co-infection is attributed to the route of viral transmission since both viruses share a similar route of transmission. Furthermore, it was also discovered that females were more susceptible to HSV-2 infection 30/35 (85.7%) than their male counterparts 4/35 (11.4%) in this population. This correlates with the findings of Pebody et al. (2004) and Smith & Robinson (2002), who reported that women are more at risk of acquiring HSV-2 infection compared with men. Similar findings were also observed by Celum et al. (2004) that more than half of the female population who are HIV-1 positive suffer from HSV-2 infection. This might be due to their early exposure to sexual relations than their male counterpart (Schiffer et al., 2014). Another probable explanation for the high prevalence of HSV in the female participant could be related to the anatomy of the female reproductive system. That is, the large surface area and the thin lining of the female reproductive system could facilitate HSV-2 entry (Hladik & McElrath, 2008). Furthermore, the hydration and alkalinization of the mucus plug during ovulation in women, hinders the barrier function of the endocervical canal against pathogens hence, facilitating viral entry (Hladik & McElrath, 2008). This is supported by the high number of HSV-2 and HIV-1 prevalence observed in the age group of 21–40 years, with only 3/44 (6.8%) HSV-1/HIV-1 co-infected samples. Of note is increased rate of co-infection with age, which correlates with the steady rise by age for HSV-2/HIV-1 co-infection, as recorded by Beydoun et al. (2010).

PCR was performed to confirm the positivity of the ELISA screened samples and differentiate the HSV samples into types 1 and 2. The HSV-2 results (85.7%) correlate with the high rate of HSV-2 infection in Africa. This was also observed in a similar study by Debrah et al. where they assessed the seroprevalence of HSV-1 and HSV-2 among women in Ghana and discovered that 78.4% of the study population are positive for HSV-2 (Debrah et al., 2018). In addition, the prevalence of HSV-2 in this study is higher than HSV-2 prevalence in other African countries like Zimbabwe (68%) and Uganda (58%) (Kurewa et al., 2010; Nakku-Joloba et al., 2014).

Phylogenetic analysis revealed that HSV-2 samples 13 and 15 from this study do not share the same ancestral lineage with a more virulent clinical isolate SD90e (accession number KF781518) from South Africa (Newman et al., 2015) and the HSV-2 laboratory strain 333 (M15118). However, sample 34 is closely related to SD90e. Thus, the distant relation between SD90e reference genome and samples 13 and 15 may suggest geographical diversity in viral transmission within South Africa. This was also discovered in a study conducted by Newman et al. (2015), where they explored the geographical diversity between HSV-2 sequences and observed that HSV-2 sequences from Uganda are strongly related to the HSV-2 sequence from USA. Furthermore, a close relation of the two samples (G13 and G15) was observed with the less virulent glycoprotein G-2 strain originating from Scotland, which might be attributed to international migration. This was also observed by Szpara et al. (2014) where African strains isolated in Kenya were shown to cluster closely with Europe, North America, and Asian strains.

The HIV-1 phylogenetic analysis confirmed that the G20 sequence is an HIV-1 sequence however, it did not cluster with any of the subtype A, B or C reference sequences that were used. The G20 distant clustering may reflect another instance of international migration. For example, Lurie et al. (2003) demonstrated that migrants have a two-fold odd of contracting HIV than non-immigrants in South Africa. Similarly, migration from different geographical locations could influence viral transmission of different HIV-1 subtypes. This was demonstrated by von Wyl et al. (2011), where they revealed that 80% of non-B HIV-1 subtypes recorded in their study population originated outside the study region, Switzerland.

A previous study on the relationship between age and HSV-1 prevalence by Smith & Robinson (2002) reported that global HSV-1 prevalence increases with age. The statistical relationship between age, HSV and HIV-1 infection in our study revealed a similar significant relationship between age and HSV-1 (p = 0.366*). Similarly, we discovered a discreet relationship between age, HSV-2 and HIV-1. However, HSV-1 showed an inverse correlation with HSV-2 (p = -0.463^**). This may suggest that an increase in HSV-2 prevalence in the population will result in a decline in HSV-1 as supported by Ayoub et al. (2019). It was also discovered that a robust significant relationship exists between HSV-2 and HIV-1 (p =0.690^**) suggesting that a steady rise in HSV-2 contributes to an increase in HIV-1 infection in the population. A probable reason for this relationship is that the viruses share a similar route of entry and the impact of one is significant on the other as seen in the micro-ulceration of the genitalia in HSV-2 patients, which provides a port of entry for HIV-1 (Sheth et al., 2008).

The association between HIV-1 and HSV was analysed using chi-square goodness-of-fit test and it was discovered that no significant association exist between HSV-1 and HIV-1 (X² (1) = 0.138, p >0.05) but a strong statistical association was found between HSV-2 and HIV-1 (X² (1) = 20.952, p <0.05). Although, there has been contrasting opinions on the association of HSV-2 and HIV-1, the current study is consistent with Sudenga et al. (2012) and Omori & Abu-Raddad (2017) who suggested that the infection of one virus may fuel the transmission of the other. In their studies, Sudenga et al. (2012) observed that HIV-1 positive individuals with higher CD4+ counts at baseline and those with lower viral load were associated with HSV-2 acquisition, while Omori & Abu-Raddad (2017) used the sexual network determinants as components to determine the prevalence of HIV-1/HSV-2. They deduced that HIV is an agent of HSV-2 transmission in the population. However, another study led by Kouyoumjian et al. (2018) discovered a robust association between HSV-2 and HIV-1, with HSV-2 prevalence being consistently higher than HIV-1 in the global population. The data from this study suggests that contracting one virus (either HSV-2 or HIV-1) will influence the acquisition of the other. This highlights the importance for data collation on HSV-2 and HIV-1 infected persons in South Africa.

Conclusion

This study revealed a clustering variation of HSV-2 sequences. The clustering differentiated the sequences from the prominent sequence (SD90e) found in South Africa, which suggests diversity in the transmission of the virus. Similarly, a different HIV-1 sub-type was isolated from the study population, different from other HIV-1 sub-type C isolates in South Africa. Despite the small sample size, a high prevalence of HSV-2/HIV-1 co-infection (68.2%) was recorded which shows that a positive association exists between HSV-2 and HIV-1, suggesting that an increase in one of the viruses may influence the spread of the other. Thus, acknowledging that a relationship exists between these two viruses, and to identify how the transmission of one could affect the other, requires a larger cohort that is well described with longitudinal measurements of HSV-1, HSV-2, and HIV-1 as well as measurements of potential confounders such as condom use, partner change and other sexually transmitted diseases.

Data availability