Distribution of HIV-1 and HSV-2 epidemics in Chad revealing HSV-2 hot-spot in regions of high-risk HIV spread

Introduction: Herpes Simplex Virus-2 (HSV-2) is known to be a potent co-factor of Human Immunodeficiency type 1 virus (HIV-1) heterosexual transmission. We were interested in assessing the distribution of HIV-1 and HSV-2 epidemics at the national level in Chad. Methodology: In 2007, a population-based anonymous serosurvey for HIV-1 and HSV-2 infections, using dried blood spots, was conducted. The study included 548 adults living in 15 regions of Chad. After specimen elution, serological testing for HIV and HSV-2 infections was performed. Results: Countrywide, the HIV-1 and HSV-2 seroprevalences were 11.1% and 15.7%, respectively. A positive correlation was observed with the highest HIV-1 prevalence seen in regions of the highest HSV-2 prevalence, especially in two conflict-affected eastern provinces of Darfur. Conclusion: Urgent public health interventions are needed in regions of Chad where high HSV-2 prevalence may be increasing the risk of HIV propagation.


Introduction
Chad is a west central African country with a population of ~9.8 million people and recently marred by conflict and humanitarian crisis.The HIV-1 prevalence in Chad among adults in 2008 was estimated to be ~3.3%[1].A previous molecular epidemiology survey pointed to the high genetic diversity of HIV-1 variants in Chad, indicating the existence of a relatively older epidemic [2].The HIV epidemic in Chad is more concentrated in urban areas (7% prevalence) as compared to rural areas (~2.3%) and women are much more affected than men (4% vs. 2.6%) within the same age group [1].Of note, the HIV prevalence among refugees and displaced persons is not known, but given current overcrowding conditions in camps, with rape, prostitution, and spread of other sexually transmitted infections (STIs), this population is particularly vulnerable to infection with HIV.
Among various factors possibly involved in the heterogeneous distribution of HIV-1 in Chad is the variable distribution of infection by herpes simplex virus 2 (HSV-2), as recently shown in Senegal [3].HSV-2 is considered a potent co-factor of HIV-1 heterosexual bi-directional transmission in sub-Saharan Africa, according to both biological plausibility and epidemiological evidence [4,5].A higher risk of HIV transmission may therefore be expected in HSV-2 hotspot regions in Chad.In the present study, the distribution of HIV-1 and HSV-2 epidemics in Chad was estimated at the national level to identify regions of possible increased HIV spread in at-risk populations.

Methodology
A population-based national unlinked anonymous testing sero-survey was conducted by the National AIDS program of Chad in 2007, according to the World Health Organization guidelines [6].The survey received ethical approval from the Chad Ministry of Health.In brief, several counting areas were defined, and the number of subjects in each of them was decided according to the results of the prior population census.In this way, samples accurately representing the global population were obtained in rural as well as urban areas.All participants provided written informed consent.The cluster sampling method was employed, resulting in a random choice of unit samples, which are themselves subunits of the total population.A total of 10,587 adults, living in 15 of 18 different regions of Chad, were included.Three regions of Chad could not be reached because of logistical difficulties for accessibility.
The Chi-square test and Spearman test were used for statistical analyses.

Results
A median of 28 individuals (interquartile range = 10-53) were included by region.Figure 1 depicts the map of HIV-1 and HSV-2 seroprevalence in 548 randomly selected adults from the 15 study regions of Chad.The sex ratio of selected populations was close to one in each region.
The prevalence of both HIV and HSV-2 varied significantly by region (P < 0.001).The highest HIV-1 prevalence was observed in regions exhibiting the highest HSV-2 prevalence.Finally, HSV-2 and HIV-1 prevalence within the 15 study regions were positively correlated (r = 0.59; P = 0.02).

Discussion
The utility of dried blood spots for HIV-1 surveillance in remote developing world settings was previously shown [9].In the present serosurvey, this blood sample collection procedure was further optimized to perform both HIV-1, as well as the HSV-2-specific, serology.The results allowed estimation of the HIV-1 and HSV-2 prevalence in randomly selected populations from 15 Chad regions, representing the Chad adult population.
As a result, the HIV-1 epidemic in Chad appeared generalized throughout the country, without significant differences between southern and northern regions.Unexpectedly, high levels of the HSV-2 epidemic could be observed in Chad, with higher prevalence in the southern regions, in contrast to the northern regions or desert areas.Interestingly, two eastern provinces of Chad (Ouaddaï and Wadi-Fira), harboured very high prevalences of both HIV-1 and HSV-2 infections; these latter regions from Darfur are characterized by conflict-affected and displaced people and refugees, constituting vulnerable populations generally assumed to be at high risk for HIV infection and STIs [10].
Our findings showed the countrywide ecological distribution of HIV-1 and HSV-2 epidemics in the general adult population living in 15 distinct regions of Chad, emphasizing and anticipating the role of HSV-2 infection as a possible biological co-factor of HIV-1 acquisition and transmission in Chad.Although the sample size of included volunteers in each region of Chad was limited, our observations demonstrating ecological association between HIV-1 and HSV-2 in Chad are consistent with the dynamics of HIV-1 and HSV-2 epidemics in sub-Saharan Africa [5].These findings mirror the association of HIV-1 and HSV-2 infections in Senegal where the HIV epidemic is concentrated, and where the seroprevalence of HIV-1 and HSV-2 infections were found to be both very high and associated with female sex workers [3].Female sex workers constitute the major high-risk group for HIV dissemination in the general Senegalese adult population [3].
Of note, suppressive HSV-2 therapy however did not reduce the incidence in a Tanzanian cohort of women [11].Nonetheless, these data do not argue against the role of HSV-2 as a potent cofactor of heterosexual transmission of HIV, but rather suggests a more complex relationship in the replication of both viruses at the genital level [12].
In conclusion, further study on the impact of HSV-2 upon HIV-1 transmission in African populations is needed.Moreover, urgent public health interventions are critically important in regions of Chad revealed as HSV-2 hotspots of high-risk HIV propagation.

Figure 1 .
Figure 1.Seroprevalences of HIV-1 (striped box) and HSV-2 (black box) infections among 548 adults randomly selected living in 15 distinct regions of Chad.Three regions (in gray) could not be investigated (Guera, Salamat, Borkou-Ennedi-Tibesti).The distribution of HIV and HSV-2 seroprevalences and their correlation curve are shown in the insert.