High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a

Introduction
HIV is the etiological agent of Acquired Immunodeficiency Syndrome (AIDS), a slowly progressive disease characterized by chronic immune activation resulting from the loss of cell-mediated immune function (Costin, 2007;Perkins et al., 2015). Host genetic factors (e.g., HLA genotypes) and host immune response (e.g., release of proinflammatory cytokines and chemokines), as well as the presence of co-infections, can directly influence the rate of disease progression (Claiborne et al., 2015;Kouri et al., 2015;Perkins et al., 2015).
Neglected Tropical Diseases (NTDs) are endemic among the most impoverished populations in Africa, Asia and Latin America. Among these NTDs, visceral leishmaniasis (VL), caused by infection with Leishmania infantum, is associated with increased morbidity and mortality in people with HIV-1 (PWH) but has not been recognized as a diagnostic criterion for AIDS (van Griensven et al., 2014). The World Health Organization (WHO) identified the geographical overlap of both infections as an emerging challenge in successfully controlling the HIV-1 infection in countries considered endemic for leishmaniasis, such as Brazil (World Health Organization, 2015).
In addition, the increase of geographical overlap between HIV-1 and Leishmania infection in Bahia (Secretaria de Saúde do Estado da Bahia, 2022a, 2022b) has raised public health concerns. However, no epidemiological studies have attempted to investigate the prevalence of co-infection to date. Therefore, we investigated the seroprevalence of L. infantum in PWH in the state of Bahia, Brazil and attempted to identify virological and immunological factors associated with co-infection, supported by publicly available transcriptomic data.

Study population and design
A two-stage cross-sectional cohort (CSC) study (CSC-I, 1998-2013and CSC-II, 2014 was designed to determine the seroprevalence of L. infantum in PWH and to evaluate the association with demographic, clinical, virological, and immunological parameters ( Figure 1). From 1998 to 2015, individuals with a new confirmed diagnosis of HIV-1 at the Specialized Center for Diagnosis, Care and Research (CEDAP), a state government public health reference service located in the city of Salvador, Bahia-Brazil, were included in this study. Their remaining blood sample was utilized for the outlined assays herein.
First, serological reactivity to L. infantum was assessed in plasma or serum samples from the retrospective cross-sectional cohort (n = 5,346) collected between 1998 and 2013. In the second stage, serological reactivity to L. infantum was assessed in a prospective cross-sectional cohort of 317 PWH (sampled between July 2014 and December 2015), with demographic and clinical data (sex, age, HIV-1 viral load, CD4 + , CD8 + , CD4 + /CD8 + ratio and CD45 + T cell counts) prospectively obtained from the clinical records. Additionally, we performed a subpopulation analysis on a subset of PWH identified as either double-positive or double-negative using two independent anti-Leishmania immunoassays. Based on these stringently defined categories, we compared virological and immunological factors influenced by L. infantum seropositivity. The study's recruitment did not interfere with standard medical care, and patients were treated according to the general Brazilian/WHO guidelines. This study was conducted following the Declaration of Helsinki and was approved by the Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ) (protocol number 1.764.505).

Detection of anti-Leishmania and anti-Trypanosoma antibodies
All samples underwent a previously described and validated in-house enzyme-linked immunosorbent assay (ELISA) for the detection of IgG against Soluble Leishmania infantum Antigen (SLA), which we previously demonstrated as a sensitive biomarker (90% sensitivity and 95% specificity) of current or past infection with the parasite (Charlab et al., 2000). SLA preparation and ELISA procedures were performed as previously described (Souza et al., 2013). For the prospective cohort of 317 samples from PWH with digitalized clinical records, results of the SLA immunoassay were compared with a second, previously described ELISA employing recombinant L. infantum heat shock protein 70 (HSP70), for which we once found a sensitivity of 74% and a specificity of 73% (Soto et al., 2015). To avoid false-positive results for L. infantum due to cross-reactivity among Trypanosomatidae parasites, an ELISA for anti-Trypanosoma cruzi IgG (Euroimmun) was employed in a subset of 42 samples with dual-positive (DP) (n = 15) and double-negative (n = 27) serology for anti-SLA and anti-HSP70.

Control samples and ELISA cutoff value
Positive (n = 6) Leishmania-infected patient samples and negative (n = 47) control samples previously identified by the intradermal Leishmanin (also known as Montenegro) Delayed-type  Figure S1).

Statistical analysis
Since data were not normally distributed, as identified by the Shapiro-Wilk and D' Agostino-Pearson tests (GraphPad Prism v7.0), results were analyzed using non-parametric tests: Mann-Whitney test and Spearman's correlation, as well as Fisher's exact test, with p < 0.05 considered statistically significant.

Detection of IgG antibodies against
Leishmania infantum antigens (anti-SLA and anti-HSP70) in people living with HIV-1 To determine the seroprevalence of L. infantum among PWHs, 5,346 plasma or serum samples from treatment-näive HIV-1-infected individuals diagnosed between 1998 and 2013 were tested for the presence of IgG antibodies against soluble L. infantum antigen (anti-SLA) ( Figure 1).
Positivity for anti-SLA serology was detected in 870/5,346 samples, resulting in a prevalence rate of 16.27% (Figure 2). We included additional positive control samples from patients with a clinically confirmed diagnosis of visceral leishmaniasis (VL) and laboratory-confirmed L. infantum infection in the absence (n = 16) or presence of HIV-1 co-infection (n = 10) (Zacarias et al., 2017). The detection rate of anti-SLA antibodies of the positive control samples was 81.25% for HIV-1 − VL + patients and 100% for HIV-1 + VL + patients ( Figure 2). Besides, we found humoral immune response against SLA in VL patients quantitatively similar regardless of the presence of HIV-1 co-infection (Mann Whitney p = 0.71, HIV-negative vs. HIV-positive). Thus, we could confirm the high specificity of the L. infantum anti-SLA IgG serology protocol employed herein.
Based on these findings, DP (n = 15) was adopted as the most stringent criterium for L. infantum infection. Assessment of standard diagnosis of previous or ongoing visceral leishmaniasis (VL) documented in the clinical records showed that one of the 15 DP subjects had VL reported >20 years ago (anti-SLA index = 4.71; anti-HSP70 index = 2.67).
After two decades of clinical diagnosis, this pronounced humoral response most likely reflects chronic antigen exposure, supporting the concept that Leishmania infection is lifelong in most patients as observed in murine models of disease (Bogdan et al., 2000;Seyed et al., 2018;Paduch et al., 2019). These results, taken together, demonstrated that a significant number of PWH in the state of Bahia are unaware of their exposure to and/or infection by L. infantum and are at risk of developing new or relapsing visceral leishmaniasis.
Comparative analyses between the DP (n = 15) and total doublenegative (DN total ) (n = 257) groups indicated that individuals with antibodies against both SLA and HSP70 L. infantum were older (p = 0.0430), presented lower baseline CD4 + T cell counts (p = 0.0208) and lower CD4 + /CD8 + T cell ratios (p = 0.0282), as well as a tendency towards higher HIV-1 viral load levels (p = 0.1268) suggestive of an advanced stage of HIV-1 infection than individuals without L. infantum antibodies (Table 1). Indeed, a large proportion of DP individuals (10/15, 66.67%) had <200 CD4 + T cells/μL at the time of HIV-1 diagnosis, which is considered AIDS-defining.

Subpopulation analysis reveals virological and immunological markers associated with dual-positive L. infantum serology in PWH
To ensure specificity for L. infantum in the DP serological samples and to exclude the possibility of cross-reactivity due to Clinical control Cross sectional cohort Detection of anti-L. infantum IgG antibodies in plasma samples collected from PWH between 2014 and 2015. Violin plot with a dotplot overlay of OD index values representative of L. infantum anti-SLA and anti-HSP70 serology in the PWH prospective cohort (n = 317). The red symbols represent the DP individual diagnosed with visceral leishmaniasis >20 years ago.
Frontiers in Microbiology 06 frontiersin.org polyclonal activation and hypergammaglobulinemia, anti-Trypanosoma cruzi IgG serology was performed. Samples demonstrating positivity for anti-T. cruzi IgG (n = 2) were excluded from subsequent analyses (Supplementary Figure S2). Next, we performed a subpopulation analysis by matching DN individuals to the DP group by gender, age, viral load and CD4 + T cell count. This strategy resulted in the establishment of a third group (DN paired ) ( Figure 1), thereby mitigating the possibility of bias arising from polyclonal activation, hypergammaglobulinemia, high viral load or low CD4 + T cell levels. Controlling for these parameters limited the potential for variations in the HIV-1 transcriptomic profile and the state of immune activation. No statistical differences were observed between the DP and DN paired groups regarding sociodemographic and clinical parameters, which underscores the pairing strategy's equity (Table 2).

Subpopulation analysis reveals virological and immunological markers associated with dual-positive L. infantum serology in PWH
To investigate possible associations between transmission clusters of a more-aggressive HIV subtype and worsen HIV clinical status, HIV-1 subtyping was performed in the DP and DN paired subset to infer. After excluding samples with a viral load <1,000 copies/mL, both groups were pooled together (n = 19) and submitted to phylogenetic analysis using the jpHMM-HIV model (Supplementary Figure S3). Concerning subtype distribution, subtype B (63.16%, 12/19) was the most prevalent, followed by subtypes C (15.79%, 3/19), recombinant BF (15.79%, 3/19), and D (5.26%, 1/19). No statistical differences in subtype were observed between the DP and DN paired groups (p = 0.67) (Supplementary Table S2). No transmission cluster was identified. HIV-1 subtyping showed no impact on L. infantum serology titers, viral load, T cell count, or any other host immunological marker evaluated, indicating that HIV-1 subtypes B, C, D, F1, and BF exhibit similar virological and immunological outcomes in HIV/L. infantum co-infection.
Next, we quantified circulating cytokines and chemokines that might reflect an increase in systemic immune activation and have been previously associated with infection to L. infantum (Caldas et al., 2005;Peruhype-Magalhães et al., 2006;Frade et al., 2011;Carneiro et al., 2016). The systemic levels of cytokines tested (IL-1β, IL-6, IL-10, IL-12p70, and TNF) were below the detection limits in all samples (DP and DN paired groups), concordant with our previous findings in a large Cuban cohort of PWH (Kouri et al., 2015). Conversely, most chemokines (except IL-8) were readily detected in most PWH, demonstrating a significant increase in CXCL10/ IP-10 (p = 0.0076) and a tendency towards higher CXCL9/MIG levels (p = 0.061) (Figure 4) in the DP group compared to DN paired , suggesting increased immune activation in the group with DP serology for L. infantum.  This finding, together with the decreased CD4 + T cell counts found in our previous analysis (Table 2), corroborates the hypothesis that therapy-naïve HIV-1-infected subjects previously or currently infected with L. infantum exhibit a more intensified state of immune activation than HIV-1 positive, L. infantum seronegative subjects. During clinical follow-up, the introduction of antiretroviral therapy restored CD4 + T cell counts (median values of CD4 + T cell counts after 4-5 years of follow-up: DP = 620 cells/μL and DN paired = 870 cells/μL) and reduced viral load levels to undetectable limits similarly in each group ( Figure 5), with no statistically significant differences between groups (AUC comparison, Mann-Whitney test: VL: p = 0.6773; CD4 + : p = 0.5239; CD8 + : p = 0.6986; Ratio CD4 + /CD8 + : p = 0.9044; CD45 + : p = 0.7540).
3.5. CXCL10 is shared between the HIV and visceral leishmaniasis gene signatures, while CXCL10, CXCL9, and CCL2 positively correlate with HIV viral load in untreated PWH Given the small number of immune activation markers and modest cohort size for the paired DP and DN groups, we aimed to validate and extend our findings by cross-examining independent cohorts of HIV-1 (Swiss HIV Cohort, n = 137) or Leishmania-infected individuals with and without active disease (Piaui Northeast Brazil, n = 30) with publicly available transcriptome-wide data (Rotger et al., 2010;Gardinassi et al., 2016). As shown in Figures 6A,B Long-term follow-up of CD4 + , CD8 + , CD4/CD8 ratio, CD45 + , and viral load levels after therapy introduction in DP and DN paired groups. Frontiers in Microbiology 09 frontiersin.org overlap (41 genes, enrichment p < 0.0001) was found between the HIV and visceral leishmaniasis gene signatures when comparing the top 500 transcripts significantly upregulated during active disease in both untreated HIV patients and untreated visceral leishmaniasis patients, as compared to their paired post-treatment groups. A systems biology analysis of the 41 genes shared by HIV and leishmaniasis active disease signatures revealed several significantly enriched biological processes, encompassing immune activation broadly and, more specifically, type I IFN signaling and antiviral response. The overlapping genes included CXCL10, which we found to be overexpressed in the DP group of our prospective cohort, as well as STAT1, the major transcription factor activated by IFN signaling. As detailed in Figure 6A and Supplementary Tables S3-S5, (Caldas et al., 2005;Peruhype-Magalhães et al., 2006).
To define a specific 'Leishmania infection' gene signature, we used the top 500 significantly upregulated transcripts from Leishmania-infected individuals who did not develop disease, for which Leishmania infection was documented by a positive skin DTH reaction, as compared to matched DTH-negative individuals from the same endemic area (Gardinassi et al., 2016). Surprisingly, the overlap between the 'untreated HIV' and 'Leishmaniaseropositive (DP)' gene signatures was even more pronounced than the overlap between the 'untreated HIV' and 'untreated visceral leishmaniasis' gene signatures, both by quantitative (65 shared genes, Figure 6B) and qualitative measures (median gene set overlap 9.4% vs. 3.2%, Mann-Whitney p < 0.0001, Supplementary Table S3), indicating that even asymptomatic Leishmania infection has a profound effect on the systemic immune response. Again, CXCL10 and STAT1 were shared among the two disease signatures ( Figure 6B), and enriched gene sets included type I IFN signaling (genes upregulated by IFN-beta treatment of multiple sclerosis patients in vivo and by IFN-alpha treatment of fibroblasts in vitro) and immune activation (inflammatory response to LPS, TNF signaling).
Validation of our DP cohort results revealed that CXCL10, but not CCL2, CCL5 or CXCL8 transcripts, were significantly upregulated by Leishmania infantum infection with or without clinically definite visceral leishmaniasis ( Figure 6C upper  CXCL10 is shared between the HIV and visceral leishmaniasis disease signatures while CXCL10, CXCL9, and CCL2 transcripts positively correlate with HIV viral load in untreated PWH. Venn diagrams show overlap between (A) active disease gene signatures, i.e., genes upregulated in untreated PWH (Swiss cohorts) and visceral leishmaniasis patients (as compared to post-treatment) and (C) genes upregulated in untreated PWH and asymptomatic Leishmania-infected individuals (who did not develop disease). Systems biology analysis shows a significant enrichment of gene sets comprising immune activation and type I IFN signaling (right-hand panels). Frontiers in Microbiology 10 frontiersin.org lower panel). In the Swiss HIV Cohort, we found that CXCL10, CCL2 and, to a lesser extent, CXCL9 transcripts were significantly upregulated during untreated HIV-1 infection. In contrast, no significant effect was observed for CXCL8 in either asymptomatic or symptomatic Leishmania infantum infection, supporting our negative findings for CXCL8 genetic variation in visceral leishmaniasis patients from the same endemic region (Frade et al., 2011). However, CXCL8 transcripts were significantly downregulated during untreated HIV-1 infection ( Figure 6D upper panel). Finally, as shown in Figure 6D (lower panel), CCL2, CXCL9, and CXCL10 transcripts were significantly correlated (even after stringent correction for genome-wide testing) with set point viral load in untreated PWH from the Swiss HIV Cohort (n = 137), confirming the results from our prospective crosssectional cohort ( Figure 4F).

Discussion
The geographic overlap of HIV-1 and L. infantum infections raises the possibility of co-infection, which several groups have shown to increase morbidity and mortality (Coura-Vital et al., 2014;Lima et al., 2017Lima et al., , 2018Viana et al., 2017;Diniz et al., 2018;Fontoura et al., 2018;Luz et al., 2018;Rezaei et al., 2018;Sousa et al., 2018;Botana et al., 2019;Diro et al., 2019;Horrillo et al., 2019;Maia-Elkhoury et al., 2019;Prestes-Carneiro et al., 2019;Santos et al., 2019;Silva de Lima et al., 2019;van Griensven et al., 2019). Several groups have studied the extent of this overlap in several Brazilian regions. However, no studies have attempted to determine the seroprevalence of L. infantum among PWH in the state of Bahia, despite the significant prevalence of both infections (Secretaria de Saúde do Estado da Bahia, 2022a, 2022b). We used a two-stage cross-sectional cohort approach (CSC-I, n = 5,346 and CSC-II, n = 317) and identified positive serology for L. infantum ranging from 4.73% (DP for anti-SLA and anti-HSP70) to 18.93% in the CSC-II (union of all positive samples for anti-SLA and anti-HSP70), with an intermediate seroprevalence for only anti-SLA of 16.27% in the CSC-I and 15.46% in the CSC-II.
The observed discrepancy between the results obtained with the two different immunoassays might be explained by the rigor employed to determine cutoffs for each serological test and the imbalance in protein fractions between these two antigen-based diagnostic methods. SLA is a heterogeneous mix of antigens and has a lower concentration of HSP70 than parasite structural proteins (actin, tubulin, etc.). Even though it will pick up a broader set of samples, some samples with low antibody levels will fail. On the other hand, the recombinant HSP70 antigen is homogenous and highly concentrated and has a better detection limit with better sensitivity than other recombinant proteins (rH2A, rH2B, rH3, rH4, and rKMP11) and high specificity when evaluated against sera from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus (Souza et al., 2013). We considered only those samples demonstrating dual positivity to increase confidence regarding past or present L. infantum infection. This strategy was underscored by the identification of one individual with a previous history of VL in the DP group and no cases with an earlier history of VL in the DN paired group. Furthermore, we confirmed that no statistical differences regarding epidemiological, virological, and immunological characteristics were observed comparing DP, SLA, HSP70 and HSP70 + SLA groups.
Unfortunately, only serum or plasma samples were available for HIV-1 RNA extraction, thus precluding DNA analysis to detect L. infantum by qPCR, which we acknowledge as one of the limitations of our study. However, Fukutani et al. (2014) demonstrated a concordance of 68% between anti-SLA serology and PCR, and found a 5.40% rate of ongoing asymptomatic L. infantum infection among blood donors in Salvador, Bahia. Of note, this concordance between PCR and serology could be compromised by the reduced serology sensitivity (66%) to identify true positive ongoing Leishmaniainfected cases among PWH. In addition, we also highlight as a limitation the absence of Leishmanin skin test in our study (DTH) to access cellular immune response present during Leishmania infection, which is more sensitive and specific to determine ongoing asymptomatic infection than serological assays (Botana et al., 2019). Nevertheless, positive IgG serology for L. infantum indicates immunological memory of at least a previous Leishmania infection before HIV diagnosis. Strikingly, we identified a DP individual with a pronounced humoral response two decades after a VL clinical diagnosis. This ongoing humoral response most likely reflects chronic antigen exposure, emphasizing the concept that Leishmania infection is lifelong in most patients, as well as murine models of disease (Bogdan et al., 2000;Seyed et al., 2018;Paduch et al., 2019). A wealth of studies worldwide (Oryan and Akbari, 2016;Grande et al., 2017;Zacarias et al., 2017;Bruhn et al., 2018;Guedes et al., 2018;Luz et al., 2018) and a recent systematic review (Fontoura et al., 2018) have demonstrated a high frequency of relapse of VL in PWH, as well as increased mortality. Relapse of VL has been reported after anti-leishmaniasis treatment, even years after the patient was considered clinically cured, especially in PWH and/or immunosuppressive conditions (Horrillo et al., 2019;Silva de Lima et al., 2019).
Studies conducted in populations of newly diagnosed HIV-1infected individuals from regions endemic for leishmaniasis in Brazil reported a prevalence of HIV-1/L. infantum co-infection ranging from 4.20 to 20.20%. In these studies, L. infantum infection was determined by parasitological, serological and/or molecular testing (Soares et al.,  -Tamayo et al., 2009;Orsini et al., 2012;Carvalho et al., 2013). However, in co-infection studies focused on VL patients, which were diagnosed by parasitological, molecular or immunofluorescence methods, the prevalence of HIV-1/L. infantum coinfection ranged from 36.60 to 55.60% (Souza et al., 2012;Cota et al., 2014;Druzian et al., 2015). The present study also investigated possible differences in the prevalence of HIV-1 variants associated with L. infantum infection in PWH. With these subtyping results, we would like to investigate if a possible worsen disease progression in PWH with positive serology for L. infantum cases was related to cluster of transmission of a moreaggressive HIV subtype (e. g., HIV Subtype D) (Khouri and Vandamme, 2015). However, no differences were found concerning HIV-1 isolates between matched samples of patients infected (DP) or not (DN paired ) to L. infantum. Within these isolates, subtype B (63.16%) was found to be the most prevalent, followed by C (15.79%), recombinant BF (15.79%) and D (5.26%). Recent studies in Bahia that evaluated HIV-1 variant distribution have described the wide distribution of subtype B, followed by a smaller proportion of recombinant BF units (Araujo et al., 2010;Monteiro-Cunha et al., 2011;Santos et al., 2011). Of note, the higher prevalence of subtype C (15.79%) found herein stands in contrast to other studies also conducted in this region, which reported a very low prevalence ranging from 1.70 to 2.50% (Chavez et al., 2007;Santos et al., 2009;Monteiro-Cunha et al., 2011).
Moreover, our confirmation of the circulation of subtype D in the state is following work by Monteiro et al. (2009), who previously reported the presence of subtype D in individuals from Feira de Santana (0.60% of pol F/ env D subtype), a city located ~100 km from Salvador. It is worth noting that the subtyping analysis performed herein involved three different methodologies (REGA HIV-1 Subtyping Tool, phylogeny and jpHMM-HIV probabilistic modeling), which lends support to the presently reported distribution. Furthermore, our subtype identification was performed using HIV-1 pol sequences, inferring subtype frequencies can vary when complete genome sequencing is performed.
Prior research into host immune responses has demonstrated that L. infantum infection induces a higher level of immune activation in co-infected PWH, which could lead to a more rapid progression to AIDS (Ipp et al., 2014;Claiborne et al., 2015;Hoffmann et al., 2016;Utay and Hunt, 2016). Our study found a roughly five-fold increase in circulating CXCL10/IP-10 and CXCL9/MIG chemokines in PWH exposed to L. infantum, supporting a link between positive serology for L. infantum and immune activation, which we validated at the transcript level for CXCL10 in independent HIV-1 and Leishmaniainfected cohorts. These chemokines are strongly induced by both type I (IFN-α/β) and type II IFN (IFN-γ) (Kedzierska and Crowe, 2001;Mehla, 2013;Kenway-Lynch et al., 2014;Utay and Douek, 2016). It has been previously reported that Th1 subpopulations of CD4 + CD95 + T cells in HIV-1 infected individuals are highly susceptible to HIV-1 envelope gp120-induced apoptosis (Accornero et al., 1997;Cummins and Badley, 2010), which may corroborate the deleterious role of intensified immune activation (Costa et al., 2012;Sun et al., 2015;Rodrigues et al., 2016). In a submitted manuscript (Khouri et al.), we demonstrated an increase in both systemic soluble Fas as well as membrane-bound Fas/CD95 + expression in CD8 + cells in HIV/VL co-infection, as compared to HIV-1 or VL mono-infected groups, in a large cohort of individuals recruited during an outbreak of VL in Piaui, another state in the same northeast region of Brazil. Since we previously demonstrated STAT1-mediated IFN signaling as a preferential inducer of Fas/CD95 + at both the protein and transcript level (Farre et al., 2008;Khouri et al., 2018), this study as well as Khouri et al. (submitted) underscore the pivotal role of IFN-driven immune activation in HIV-1 and Leishmania infantum co-infection and its deleterious effect on both AIDS progression and visceral leishmaniasis.
The present study aimed to describe the prevalence and consequences of L. infantum infection in a population of antiretroviral treatment-naïve PWH at diagnosis. We found a high seroprevalence of L. infantum in treatment-naïve PWH in Bahia (Brazil) associated with a more intensified state of immune activation as compared to HIV-1 mono-infected subjects, possibly exacerbating progression to AIDS, which was observed in 66.67% in co-infected individuals at diagnosis. This immune activation and disease progression in PWH is reversed by immediate ART implementation (Buzón et al., 2010). Brazil adopted therapy as prevention (TasP) for HIV in 2013, which through our National Health System (SUS) offers and treats every HIV diagnosed patient in the country, independent of the CD4 + T cell level. However, in Brazil, Visceral Leishmaniasis cases are investigated only when severe VL-like symptoms are already present, precluding ongoing asymptomatic or mild visceral leishmaniasis diagnosis. Thus, Leishmania-infected and ART-naïve PWH with low CD4 + T cells (<200 cell/μL) and high viral load are at high risk of developing severe visceral leishmaniasis (Viana et al., 2017). These findings underscore the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

Ethics statement
The studies involving human participants were reviewed and approved by Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ) (protocol number 1.764.505). The patients/participants provided their written informed consent to participate in this study. None of the funding organizations had any role in the study design, data collection, data interpretation or writing of this report.