Seroprevalence and molecular diversity of Human Herpesvirus 8 among people living with HIV in Brazzaville, Congo

Human herpesvirus 8 (HHV8) is endemic in Africa, although studies of this infection are rare in Congo. We evaluated seroprevalence and HHV-8 diversity among people living with HIV. We included 353 patients receiving highly active antiretroviral therapy. Antibodies against HHV-8 latency-associated nuclear antigen were detected by indirect immunofluorescence. In HHV-8 positive patients, we performed HHV-8 quantification in blood and saliva by real-time PCR and typing by Sanger sequencing of K1 open reading frame. HHV-8 seroprevalence was 19%, being male (odd ratio [OR] = 1.741, [95% Confidence interval {CI}, 0.97–3.07]; p = 0.0581) and having multiple sex partners before HIV diagnosis (OR = 1.682, [CI 95%, 0.97–2.92]; p = 0.0629) tended to be associated with HHV-8 seropositivity. Of the 64 HHV-8 seropositive patients, HHV-8 DNA was detected in 10 (16%) in saliva, 6 (9%) in whole-blood and in 2 (3%) in both whole-blood and saliva. Three out of 6 HHV-8 strains were subtypes A5, 2 subtype B1 and 1 subtype C. HHV-8 seroprevalence was relatively low with more frequent carriage in men, associated with asymptomatic oral excretion and a predominance of subtype A5. These data tend to support the hypothesis of horizontal transmission in people living with HIV in Brazzaville.

The subtypes distribution of HHV-8 varies according to geography and ethnic origin. Molecular epidemiological analysis of the K1 open reading frame (ORF-K1) identified 7 subtypes of HHV-8 (A, B, C, D, E, F and Z). Subtypes A and C are found in Europe, North America, Middle East and North Asia [13][14][15] ; subtypes B and A5 are characteristic of Africa 16 ; subtype D, on Pacific Islands and in Taiwan 17 ; subtype E, in Native Americans and Brazilians 18 ; Subtype F, firstly identified in Uganda 19 and recently described in France 20 , and subtype Z, in a small cohort of Zambian children 21 .
As it stands, no studies have been done on HHV-8/HIV coinfection in Congo-Brazzaville. Although the country is considered an endemic area for these two viruses, the seroprevalence and subtypes of HHV-8 circulating in people living with HIV remains unknown. We conducted this study to evaluated HHV-8 seroprevalence and associated risk factors among people living with HIV in Brazzaville. We also determine the viral load and subtype diversity of HHV-8 circulating in our study population.
HHV-8 seroprevalence. In our study, 12 (3%) patients had equivocal HHV-8 serology results and were not included in our seroprevalence estimation. Characteristic of these patients are described in Table S1.
Overall, the seroprevalence of HHV-8 was 19% (n = 64). The presence of HHV-8 antibodies tended to be higher in males than females (25% versus 16%, p = 0.056) and in patients with history of multiple partners before HIV infection (p = 0.07). However, no difference according to age, CD4 cells count, MSM or IDUs status was found ( Table 1). None of the patients studied reported symptoms associated to HHV-8 related diseases.

HHV-8 viral load and ORF-K1 phylogenetic analysis.
Of the 64 patients with positive antibodies against LANA-1, 10 (16%) had HHV8-DNA oral shedding and 6 (9%) a detectable HHV8-DNA viral load in whole blood. Only 2 (3%) patients had detectable HHV-8 DNA viral load in both saliva and whole blood. The characteristics of the patients in whom the HHV-8 DNA was detectable are described in Table S2. Median HHV8-DNA viral load in oral shedders was 3 [IQR, 2.7 -3.4] log 10 copies/10 6 cells (Table 2). Comparison between HHV8-DNA oral shedders and non-oral shedders showed no statistically significant difference (Table 3). It is noteworthy that all 12 individuals who had HHV-8 equivocal results, did not shed virus either in saliva or in whole blood.
ORF-K1 coding region amplification by nested PCR was successful for 6 of the 12 who had a detectable viral load and a phylogenetic tree based on the same region implemented with reference sequences was constructed (Fig. 1). Subtype A5 was the most common (n = 3; 50%), followed by B1 (n = 2; 33%). Finally, one patient was infected with subtype C (n = 1, 17%).

Risk factors associated with HHV-8 seropositivity.
Risk factors associated with HHV-8 seropositivity were investigated using logistic regression analysis; for a detailed analysis of the association between education level and HHV-8 seropositivity, subjects were divided into 2 categories: secondary/superior and uneducated/ primary. On univariate analysis, being male (odd ratio [ (Table 4). No association was observed with age, education level, sex workers, MSM, blood transfusion, circumcision, history of STIs, unprotected sex, intravenous drug users, history of surgery and CD4 count cells. In multivariate analysis, no particular pattern emerges between HHV-8 status and associated independent factors (Table 4).

Discussion
Congo is a highly informative country to study HHV-8 infection due to a significant proportion of immunosuppressive agents such as HIV, also of its geographic location in an area described as the KS belt 12,22 . This study is first to investigate the seroprevalence and molecular diversity of HHV-8 in the Congolese population by considering their HIV-1 serostatus.
This study reported HHV-8 seroprevalence of 19% among PLWH in Brazzaville, Congo. Our results is in agreement with the low prevalence (12%) found in HIV positive prostitutes in Kenya 23  On the other hand, Gabon, Central African Republic and Democratic Republic of Congo which are neighbouring countries, conducted several studies in non-HIV patients and found that HHV-8 seroprevalence rate was 35%, 94% and 82%, respectively [29][30][31] . The low HHV-8 seroprevalence in our study compared to other countries can be explained by the various degree of immunosuppression of patients between studies, which has an impact on the techniques used to detect HHV-8 antibodies to the point of underestimating the HHV-8 seroprevalence in immunocompromised populations. In other studies conducted in the sub-region, the lytic antigen ELISA assay was the most used which can overestimate HHV-8 seroprevalence, in particular because of EBV-cross-reactive antibodies. Moreover, the low seroprevalence of HHV-8 found in Brazzaville corroborates the prevalence of www.nature.com/scientificreports/    26,33 . The strong representation of women among the participants is explained by the fact that HIV is more prevalent in women than men in Congo, as previously reported in other studies 9,34,35 . However, here men were more frequently HHV-8 infected than women, in line with the sexual route of HHV-8 transmission, according to studies 36,37 . Furthermore in other studies of the sub region on KS, men were more prone to the development of KS than women; almost twice to thrice more affected [38][39][40] . Although MSM population was weakly represented in our study, a trend was observed in patients who had multiple sexual partners before HIV diagnosis, leans in favor of heterosexual transmission in our study group. Ugandan, Kenyan and Iranian studies suggest heterosexual transmission of HHV-8 respectively, in patients who reported having multiple marital unions, sex workers and Iraqi blood donors who reported frequent legal and illegal sex 23,34,36 . Other studies indicate that, in KS belt areas and some countries, the transmission is probably mainly sexual with a risk of infection correlating with the number of sex partners 9,41,42 . We did not identify any signal of parenteral transmission in the present study, as no difference of HHV-8 seroprevalence was evidenced in the two groups for IDU patients, transfusion or circumcision. www.nature.com/scientificreports/ Furthermore, to know HHV-8 subtypes circulating in Brazzaville, we analyzed the sequences of the ORF K1 region of patients with detectable HHV-8 DNA but asymptomatic to HHV-8 associated diseases. Among them, 6 samples were typable, and subtype A5 was the most predominant (50%), followed by B1 variant of subtype B (33%) and a subtype C variant close to C7 (17%). A5 and B subtypes are the most frequent subtypes found in sub-Saharan Africa, with a distribution on the African continent of 42%, 27% and 18% for subtypes B, A5 and C respectively 16,43,44 . Our results are consistent with Betsem et al. which finds subtype A5 and B1 variant of subtype B circulating mainly in the Cameroonian population 33 . Of note, A5 and B subtypes are also actively circulating among KS patients in the Central African Republic 43,45 ; in Zimbabwe, South Africa and Uganda 16,19,46 . In contrast, Lacoste et al. in a sub-regional study including a Congolese patient with MCD, described subtype B as the only one circulating in Congo. Varmazyar et al. reports that subtype A was detected more frequently among HIV-infected patients with or without KS than HIV-negative subjects 15 . Previous studies have demonstrated the clustering patterns of HHV-8 subtypes with geography and ethnicity, and these may have arisen through ancient human migrations 44,47 . In addition, de Oliveira Lopes et al. after analysis of more than a hundred sequences of subtype B from different regions of the world, shows that the HHV-8 subtype B circulating in Congo is one of the oldest; and it is possible that this subtype was brought by African slaves during the colonial period of Brazil 48 . Interestingly, we identified a subtype C variant close to C7 found in Moroccan and Central African populations 43,45,49 , while subtype C occurs predominantly in Europe, USA, the Middle East, the Mediterranean, and Asia 20 . The discovery of subtype C in our study may be explained by human migrations. Our study may have some limitations. The patients in our study were on antiretroviral treatment but did not benefit from regular biological monitoring, which unfortunately did not allow us to get the CD4 cells count and the HIV viral load for each patient. In addition, the sample size studied is limited and probably explain why statistical results did not rich significancy.
In conclusion, we observed a relatively low seroprevalence of HHV-8 in PLWH, with a more frequent carriage in men, asymptomatic oral excretion and a predominance of subtype A5. A larger study, including non-HIV participants is needed to determine global seroprevalence, and to know if other subtypes circulate in Congo, as shown by the presence of a subtype C in Brazzaville.

Materials and methods
Study population. This prospective cross-sectional study was conducted between July and October 2019 in seven health facilities following more than 5890 patients living with HIV in Brazzaville, Congo. In total, 353 patients under highly active antiretroviral therapy (HAART), 18 years of age or older, HIV-1 positive and with signed written consent were included.
Data and sample collection. For each patient, a survey questionnaire was completed with data on sociodemographic characteristics including gender, age, marital status and education level. Information on HIV infection, including clinical stage at inclusion, HAART regimen in accordance with the guidelines of the Congo and date of initiation, CD4 count cells, as well as information's on sexual behaviour (number of different partners, condoms using, homosexuality, history of sexually transmitted infection (STI)) were collected.
For each patient, whole blood, serum and oral dry swab were collected. Within the following hour, the samples were transferred to the Brazzaville University Hospital Laboratory and then stored at -80 °C. All samples were HHV-8 serology. Sera were tested for HHV-8 antibodies directed against LANA-1 IgG (latency associated nuclear antigen) by an indirect immunofluorescence assay using the BC-3 cell line infected with HHV-8 but not with Epstein Barr virus, as described previously 50 . This technique, using unstimulated cells, has a sensitivity between 80 and 85% and a very good specificity (nearly 100%). Note that, the term "equivocal HHV-8 serology results" was used when after several tests in some patients, we had an indeterminate result, i.e. ambiguous, fluorescent slide on reading. Sanger sequencing of ORF-K1 and typing. A 679 base pair (bp) fragment of ORF-K1 including the 2 hypervariable regions, was amplified by nested PCR, as previously described 20 . After multiple alignment of nucleotide ORF-K1 sequences (including reference sequences collected on NCBI database) with Mafft7, phylogenetic analysis was performed with PhyML3.0, GTR model and 1000 bootstraps resampling.

HHV
Statistical analysis. Field and laboratory data were entered in Excel version 2016. Continuous variables were expressed as medians with interquartile ranges [IQR], and discrete variables as numbers and percentages. GraphPad software was used to perform nonparametric tests, Mann-Whitney U tests for quantitative data, Fisher exact t or Chi2 square tests for qualitative data and p < 0.05 was considered significant. Univariable and multivariable (including gender, age, education level, sex workers, men who have sex with men (MSM), blood transfusion, circumcision, history of STIs, multiple sex partners before HIV diagnosis, unprotected sex, intravenous drug users (IDUs), history of surgery and CD4 count cells) logistic regression analyses were performed to identify risk factors associated with HHV-8 infection. Factors with a P value < 0.20 in the univariate logistic regression analyses were included in the multiple logistic regression model.