# Human T-Lymphotropic virus type 1 and human immunodeficiency virus co-infection in rural Gabon

Augustin Mouinga-Ondémé, Larson Boundenga, Ingrid Précilya Koumba
Koumba, Antony Idam Mamimandjiami, Abdoulaye Diané, Jéordy Dimitri
Engone-Ondo, Delia Doreen Djuicy, Jeanne Sica, Landry Erik Mombo,
Antoine Gessain, et al.

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Augustin Mouinga-Ondémé, Larson Boundenga, Ingrid Précilya Koumba Koumba, Antony Idam Mamimandjiami, Abdoulaye Diané, et al. Human T-Lymphotropic virus type 1 and human immunodeficiency virus co-infection in rural Gabon. PLoS ONE, 2022, 17 (7), pp.e0271320. 10.1371/journal.pone.0271320. pasteur-04079375

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RESEARCH ARTICLE

# Human T-Lymphotropic virus type 1 and human immunodeficiency virus co-infection in rural Gabon

Augustin Mouinga-Ondémé¹*, Larson Boundenga²,³, Ingrid Précilya Koumba Koumba¹,⁴, Antony Idam Mamimandjiami¹, Abdoulaye Diané¹, Jéordy Dimitri Engone-Ondo¹, Delia Doreen Djuicy¹, Jeanne Sica⁵, Landry Erik Mombo⁴, Antoine Gessain⁶, Avelin Aghokeng Fobang⁷,⁸

1. Unité des Infections Rétrovirales et Pathologies Associées, Centre International de Recherches Médicales de Franceville (CIRMF), Franceville, Gabon, 2. Groupe Evolution et Transmission Inter-espèces des Pathogènes (GETIP), Département de Parasitologie, Centre International de Recherches Médicales de Franceville (CIRMF), Franceville, Gabon, 3. Department of Anthropology, Durham University, Durham, United Kingdom, 4. Laboratoire de Biologie Moléculaire et Cellulaire (LABMC), Université des Sciences et Techniques de Masuku (USTM), Franceville, Gabon, 5. Centre de Traitement Ambulatoire, Franceville, Gabon, 6. Unité d'Epidémiologie et de Physiopathologie des Virus Oncogènes (EPVO), et CNRS UMR3569, Institut Pasteur de Paris, Paris, France, 7. Unité Mixte de Recherche sur le VIH et les Maladies Infectieuses Associées, Centre International de Recherches Médicales de Franceville (CIRMF), Franceville, Gabon, 8. MIVEGEC, Université de Montpellier, CNRS, IRD - Montpellier, Montpellier, France

* ondeme@yahoo.fr

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## OPEN ACCESS

**Citation:** Mouinga-Ondémé A, Boundenga L, Koumba Koumba IP, Idam Mamimandjiami A, Diané A, Engone-Ondo JD, et al. (2022) Human T-Lymphotropic virus type 1 and human immunodeficiency virus co-infection in rural Gabon. PLoS ONE 17(7): e0271320. https://doi.org/10.1371/journal.pone.0271320

**Editor:** Fatah Kashanchi, George Mason University, UNITED STATES

**Received:** June 16, 2021

**Accepted:** June 28, 2022

**Published:** July 22, 2022

**Peer Review History:** PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0271320

**Copyright:** © 2022 Mouinga-Ondémé et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its [Supporting information](#) files.

## Abstract

### Introduction

Human T-cell lymphotropic virus type-1 (HTLV-1) and human immunodeficiency virus (HIV-1) co-infection occur in many populations. People living with HIV-1 and infected with HTLV-1 seem more likely to progress rapidly towards AIDS. Both HTLV-1 and HIV-1 are endemic in Gabon (Central Africa). We investigated HTLV-1 and HIV-1 co-infection in the Haut-Ogooué province, and assessed factors that may favor the rapid evolution and progression to AIDS in co-infected patients.

### Methods

Plasma samples from HTLV-1 patients were tested using ELISA, and positive samples were then tested by western blot assay (WB). We used the polymerase chain reaction to detect HTLV-1 Tax/Rex genes using DNA extracted from the buffy coat of ELISA-positives samples.

### Results

We recruited 299 individuals (mean age 46 years) including 90 (30%) men and 209 (70%) women, all of whom are under treatment at the Ambulatory Treatment Centre of the province. Of these, 45 were ELISA HTLV-1/2 seropositive. According to WB criteria, 21 of 45 were confirmed positive: 20 were HTLV-1 (44%), 1 was HTLV-1/2 (2%), 2 were indeterminate (4%) and 22 were seronegative (49%). PCR results showed that 23 individuals were positive for the Tax/Rex region. Considering both serological and molecular assays, the
**Funding:** The authors received no specific funds for this work.

**Competing interests:** The authors have declared that no competing interests exist.

prevalence of HTLV-1 infection was estimated at 7.7%. Being a woman and increasing age were found to be independent risk factors for co-infection. Mean CD4+ cell counts were higher in HTLV-1/HIV-1 co-infected (578.1 (± 340.8) cells/mm³) than in HIV-1 mono-infected (481.0 (± 299.0) cells/mm³). Individuals. Similarly, the mean HIV-1 viral load was Log 3.0 (± 1.6) copies/ml in mono-infected and Log 2.3 (± 0.7) copies/ml in coinfected individuals.

## Conclusion

We described an overall high prevalence of HTLV-1/HIV-1 co-infection in Gabon. Our findings stress the need of strategies to prevent and manage these co-infections.

## Introduction

Human T-Lymphotropic virus type 1 (HTLV-1) was the first HTLV to be discovered and is one of the most prevalent human retroviruses in the world [1, 2]. This oncoretrovirus is highly endemic in some restricted regions of Southwestern Japan, Iran, Australo-Melanesia, sub-Saharan Africa, Caribbean basin and South American [1]. It is estimated that at least 5 to 10 million persons are infected around the world and that 2% to 7% of them develop related pathologies [3, 4]. HTLV-1 is the etiological agent of several pathologies, such as a very severe T-cell lymphoproliferation named Adult T-Cell Leukemia Lymphoma (ATLL) and a progressive disabling neuro-myelopathy, the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM) [2, 5-7]. HTLV-1 can be transmitted through unprotected sexual intercourse, particularly from men to women [8], prolonged breastfeeding from an infected mother to her child [9], and contaminated blood products, during transfusion or intravenous drug injections [10, 11]. HTLV-1 shares transmission routes with Human Immunodeficiency virus (HIV); moreover, the two retroviruses have a common *in vivo* tropism, since CD4+ T-cells are their major targets of infection [12].

At the end 2019, the World Health Organization (WHO) estimated that 38 million people were living with HIV infection in the world [13]. Without treatment, HIV-infected individuals develop Acquired Immunodeficiency Syndrome (AIDS) and may die [14]. Previous studies have reported HTLV-1 and HIV-1 co-infections [15-18] with a prevalence estimated at 0.11% to 10.9% depending on the geographic region and group studied [16, 19-21]. However, there are suggestions that people living with HIV-1 and infected with HTLV-1 are more likely to progress rapidly towards AIDS [22-24]. Moreover, the prevalence of HAM/TSP increases in HIV-1/HTLV-1 coinfected individuals [15, 25].

HTLV-1 and HIV-1 are very prevalent in Gabon. HTLV-1 seroprevalence ranges from 5% to 10.5% in the adult rural population [26, 27] and is 0.7% in blood donors [11]. Similarly, during the last Demographic and Health Survey (DHS), HIV-1 infection was been reported in 4.1% of general population aged 15 years to 49 years [28]. The distribution of HTLV-1 and HIV-1 across the country showed that the Haut-Ogooué province, in the south-east part of the country, is one of the most affected, with 9.5% to 11.6% of adult people living with HTLV-1 [26, 27] and 4.2% with HIV-1 [28]. Despite such very high seroprevalence for both retroviruses, no studies have been published, to our knowledge, on co-infections in populations living in Gabon.

To extend our knowledge of this subject, we investigated HTLV-1 infection in a population of HIV infected patients living in the southern region of Gabon and assessed factors that may favor the rapid evolution and progression to AIDS in such co-infected patients.
# Methods

## 1. Study design and population

We conducted a cross-sectional study from June 2018 to September 2019 in the southern region of the country to investigate HTLV-1 and HIV-1 co-infection in a cohort of patients followed-up and monitored at the major Ambulatory Treatment Centre (CTA) of Franceville (a city of 7,000 inhabitants). The recruitment criteria were as follows: (i) being confirmed HIV-1-positive; (ii) being on antiretroviral therapy (ART), (iii) and provide informed consent. We collected the following information: gender, age, ART regimen, last CD4 counts at ART initiation, and HIV-1 viral load (VL) results obtained as previously described [29].

The study was approved by the National Ethics Committee, registered as PROT N°0011/ 2013/SG/CNE and all participants gave verbal consent to participate in the study.

## 2. HIV viral load and CD4 counts

Blood specimens were collected from each participant using 5 ml EDTA tubes and transferred to a reference national laboratory, the CIRMF Retroviral Infection Laboratory. Samples were processed on arrival at the laboratory and plasma and buffy coat layers were collected and stored at -80°C until testing. CD4 counts at enrolment were determined using flow cytometry (Fascount, Becton Dickinson, San Jose, CA). Plasma HIV-1 VL was determined using the Generic HIV Viral Load test (Biocentric, Bandol, France) performed according to the manufacturer's instructions, using the protocol with a detection limit of 300 copies/mL and 200 ml of plasma.

## 3. HIV diagnosis

HIV-1 RNA was extracted from the plasma using the QIAamp RNA Viral Mini kit (Qiagen, Courtaboeuf, France). Reverse transcriptase HIV fragment was amplified by RT-PCR using SuperScript III One-Step RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA), and the primers pair RT18/RT21, followed by a second-round PCR using Invitrogen Platinum Taq DNA Polymerase (Thermo Fisher Scientific) with RT1/RT4 primers. Genotypic HIV-1 drug resistance testing was performed using the ANRS (French National Agency for Research on AIDS and Viral Hepatitis) version 2015 protocol, targeting the protease (PR) and reverse transcriptase (RT) regions (http://www.hivfrenchresistance.org).

## 4. HTLV diagnosis

To determine HTLV infection, we tested plasma samples using an ELISA assay (HTLV-1/2 ELISA 4.0 MP Biomedicals, Singapore) for the initial screening and detection of HTLV-1/2 antibodies.

The reactions and interpretation of the results were performed according to the manufacturer's instructions. The cut-off value was calculated as (0.25 + non-reactive control mean absorbance).

All ELISA positive samples were tested with a Western Blot (WB) assay (HTLV Blot 2.4 MP Biomedicals, Singapore) to confirm and discriminate between HTLV-1 and HTLV-2 infections.

Briefly, HTLV-1-positive plasma samples were defined as the presence of gag (p19 with/without p24) and two env (GD21 and rgp46-I) bands. Likewise, HTLV-2-positive samples were defined as reactive to gag (p24 with/without p19) and two env (GD21 and rgp46-II) bands. Samples displaying antibodies to both gag (p19 and p24) and env (GD21) bands were defined as HTLV-positive but untypeable. Any other patterns of bands were denoted as
indeterminate. For all samples with a positive ELISA result, we extracted viral DNA from buffy-coat specimens using the Qiagen DNA Blood Mini kit (Qiagen, Courtaboeuf, France). DNA concentration was evaluated with a Nanodrop spectrophotometer (Thermo Fisher Scientific®), and its integrity was verified by amplifying an Albumin gene fragment, as described previously [30]. HTLV DNA amplification was conducted using a generic PCR approach with consensus primers amplifying the PTLVs *tax* region of 220 bp, as previously described [31]. Briefly, for the first round, the *tax* primers used were AV46 and AV45 to amplify the region of 303 bp based on 250 ng of genomic DNA. Then, a 220 bp fragment was amplified during the second round with nested primers AV42 and AV43. All *tax*-positive samples were further analyzed with another semi-nested PCR to amplify a 522 bp fragment of the Env gene with the Env11 and Env22 primers, as previously described [32]. Amplicons of the expected size were sent to Macrogen Europe B.V (Meibergdreef 57, 1105 BA Amsterdam, Pays-Bas) for sequencing.

**5. Phylogenetic analysis**

HTLV-1 genotypes were identified by BLAST searches of the sequences obtained, using the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST). A phylogenetic tree was generated from the alignment of the 522 bp Env gene fragment sequences obtained, by Neighbor-Joining method, with MEGA 7 software.

**6. Statistical analysis**

Statistical analyses were performed using R software. HTLV-1 infection prevalence was compared with gender using a Chi-square (χ²) test. We compared HTLV-1 infection prevalence and age groups using logistic regression. We compared the mean of CD4+ lymphocytes count between mono-infected (HIV-1 only) and co-infected (HIV-1/HTLV-1) individuals using a Chi-square (χ²) test. Finally, we compared the mean of viral load between mono-infected and co-infected individuals using a nonparametric Wilcoxon test. The statistical significance was defined when a *p*-value was <0.05.

**Results**

**Patient population**

Overall, we recruited 299 individuals including 209 (70%) women and 90 (30%) men (Table 1). The median age was 46 years with an interquartile range (IQR) between 34 and 58 years. The median CD4-cell count was 468 (IQR, 256–692) at the time of the inclusion in the study. Viral load was available for all patients and the median VL was Log 2.1 (IQR, 1.7–3.8). All participants were receiving first-line antiretroviral treatment (ART) and the mean time on ART was 17 months. Predominant ART regimens included zidovudine (AZT) plus lamivudine (3TC) plus nevirapine (NVP)/efavirenz (EFV) (48%), and tenofovir (TDF) plus 3TC/emtricitabine (FTC) plus EFV (46%) (Table 1).

**Prevalence of HTLV-1 infection**

Of the 299 plasma samples tested with ELISA, 15% (45/299) were HTLV-1/2 seropositive. Western Blot analyses confirmed HTLV infection for 21 (7%) specimens; HTLV-1 infection was confirmed for 20 participants and one individual was HTLV-1/2 co-infected. Two (4%) samples displayed indeterminate profiles and 22 (49%) specimens which scored positive on ELISA were considered HTLV negative according to the WB results. Molecular investigations were performed on DNA extracted from buffy coat of all 45 ELISA-positive individuals.
<table><caption>Table 1. Patient characteristics associated with HTLV-1/HIV-1 coinfection status according to serology and molecular assays.</caption><thead><tr><th rowspan="2">Variables</th><th rowspan="2">N = 299</th><th colspan="2">HTLV-1/2 Serology</th><th>HTLV-1 PCR (Tax/Rex)</th></tr><tr><th>ELISA n [% (95% CI)]</th><th>WB n [% (95% CI)]</th><th>Positive n [% (95% CI)]</th></tr></thead><tbody><tr><td><strong>Gender, n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Men</td><td>90 (30.1)</td><td>13 [14.4(10–19.8)]</td><td>3 [3.3 (-0.1–6.7)]</td><td>3 [3.3 (-0.1–6.7)]</td></tr><tr><td>Women</td><td>209 (69.9)</td><td>32 [15.3 (10.3–20.3)]</td><td>18 [8.6 (4.8–12.4)]</td><td>20 [9.6 (5.6–13.6)]</td></tr><tr><td>Total</td><td>299 (100)</td><td>45 [15.1 (10.2–21)]</td><td>21 [7.02 (3.3–11.6)]</td><td>23 [7.7 (3.9–12.1)]</td></tr><tr><td><strong>Age, n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>18–30 y</td><td>52 (17.4)</td><td>7 [13.5 (9–23)]</td><td>3 [5.8 (-1-12.6)]</td><td>3 [5.8 (-1-12.6)]</td></tr><tr><td>31–49 y</td><td>129 (43.1)</td><td>16 (12.40±5.9)</td><td>7 [5.4 (1.7–9.1)]</td><td>8 [6.2 (2.1–10.3)]</td></tr><tr><td>50–80 y</td><td>118 (39.5)</td><td>22 [18.8 (11.7–25.9)]</td><td>11[9.3 (4–14.6)]</td><td>12 [10.2 (4.7–15.7)]</td></tr><tr><td><strong>Median (IQR) 46 (34–58)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>CD4 cell count, cells/mm³ n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;200</td><td>56 (18.7)</td><td>6 [10.7 (1.9–19.5)]</td><td>3 [5.4 (-0.6–11.4)]</td><td>3 [5.4 (-0.6–11.4)]</td></tr><tr><td>200–499</td><td>108 (36.1)</td><td>18 [16.7 (9.6–23.8)]</td><td>6 [5.6 (1.2–10)]</td><td>6 [5.6 (1.2–10)]</td></tr><tr><td>≥500</td><td>135 (45.2)</td><td>21 [15.6 (9.6–21.6)]</td><td>12 [8.9 (5.2–13.6)]</td><td>14 [10.4 (5.3–15.5)]</td></tr><tr><td><strong>Median (IQR) 468 (256–692)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Virological outcome, n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Viral load undetectable</td><td>195(65.2)</td><td>32 [16.4 (11.3–21.5)]</td><td>18 [09.2 (5.2–13.2)]</td><td>19 [09.7(5.6–13.8)]</td></tr><tr><td>Viral load ≥300 copies/ml*</td><td>7(2.3)</td><td>1 [14.3 (5.6–23)]</td><td>1 [14.3 (5.6–23)]</td><td>1 [14.3 (5.6–23)]</td></tr><tr><td>Viral load ≥1000 copies/ml</td><td>97(32.5)</td><td>12 [12.4 (5.2–19.6)]</td><td>2 [2.1 (-0.9–5.1)]</td><td>3 [3.1 (-0.2–6)]</td></tr><tr><td><strong>Median** (IQR) 2.1 (1.7–3.8)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>ART regimen</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>AZT + 3TC + EFV, n (%)</td><td>107 (39.2)</td><td>20 ([18.7 (11.3–26.1)]</td><td>6 [5.6 (1.2–10)]</td><td>6 [5.6 (1.2–10)]</td></tr><tr><td>TDF + 3TC + EFV</td><td>46 (16.8)</td><td>7 [15.2 (4.725.75)]</td><td>4 [8.7 (0.5–16.9)]</td><td>4 [8.7 (0.5–16.9)]</td></tr><tr><td>AZT + 3TC + NVP</td><td>24 (8.8)</td><td>4 [16.7 (1.5–31.9)]</td><td>2 [8.3 (1.1–15.5)]</td><td>2 [8.3 (1.1–15.5)]</td></tr><tr><td>TDF + FTC + EFV</td><td>79 (28.9)</td><td>10 [12.7 (5.3–20.1)]</td><td>4 [5.1 (0.2–10)]</td><td>5 [6.3 (0.9–11.7)]</td></tr><tr><td>Others</td><td>17 (6.2)</td><td>6 [35.3 (11.9–58.7)]</td><td>5 [29.4 (9.1–49.7)]</td><td>6 [35.3 (11.9–58.7)]</td></tr></tbody></table>

Generic PCR results showed that a PTLVs tax region was successfully amplified for 23 specimens (8%) the 20 HTLV-1 positive individuals in WB, the HTLV-1/2 co-infected individual and the two indeterminate individuals (WB reactivity was shown only for GD21 and P19). Thus, the combination of serological and molecular testing led to an estimated overall HTLV-1 prevalence of 7.7%. All data are summarized in Table 1.

## Phylogenetic analysis

Blast comparative and phylogenetic analyses of the 522bp fragment in the env gene showed that 13 of the new 14 characterized strains belonged to the Central African HTLV-1b genotype. Only one strain belonged to the HTLV-1d genotype. Within the HTLV-1b subclade, new strains grouped into two groups (S1 Fig).

<footnote>*300 copies/ml is the assay lower detection limit.</footnote>

<footnote>**The median viral load level was calculated for results ≥300 copies/ml</footnote>

<footnote>3TC: Lamivudine, EFV: Efavirenz, TDF: Tenofovir,</footnote>

<footnote>AZT: Zidovudine, FTC: Emtricitabine, NVP: Nevirapines</footnote>
## Risk factors for HTLV-1 infection in the study population

Women were more likely to be HTLV-1 infected than men: 20 of 209 women (9.6%) and 3 of 90 men (3.3%) were HTLV-1 infected, but this difference was not statistically significant (*p* > 0.05). Seroprevalence gradually increases with age in this study population (*p* < 0.01). Adult individuals of the 50–80 y (10%) and the 31–49 y (6.2%) age groups are at increased risk of being HTLV-1 seropositive compared to younger people of the 18–31 y age group (5.8%) (Table 1, S2A Fig).

Clinical parameters showed higher levels of CD4+ T cells in HTLV-1/HIV-1 co-infected individuals than in HIV-1 mono-infected: respectively 578.1 (273.3–918.3) cells/mm³ and 481.0 (182–780) cells/mm³. However, this difference was not statistically significant (*p* = 0.28). The mean viral loads were Log 3.0 (Log1.4-Log4.6) copies/ml and Log 2.3 (Log1.6-Log3.0) copies/ml in mono-infected and co-infected individuals, respectively, a statistically significant difference (*p* = 0.02) (Table 1).

Thirteen of 14 HIV-1/HTLV-1 co-infected patients had a viral load suppression (VL<2.5 Log); Only one patient had a virological failure (Gab-2276FCV, co-infected with the HTLV-1D genotype). The HIV-1 RT region of 646 bp infecting this patient was successfully sequenced. This patient was receiving ABC+3TC+EFV first-line antiretroviral therapy. Several HIV drug resistance mutations (DRMs) were identified: L74I, V75T, Y115F, M184V (NRTI), K103N, Y181C (NNRTI).

## Discussion

Previous studies of HTLV-1 conducted in Gabon focused on infection in different types of population including pregnant women, blood donors, pygmy people and rural Bantou communities [11, 26, 27, 33]. We report here HTLV-1/2 infection among people living with HIV/AIDS (PLWHA) in the southeast of the country. Most of the study participants were women. This was expected as most HIV-1 infected patients receiving ART in the Ambulatory Treatment Centre (CTA) in Gabon are women [29, 34]. We found that HTLV-1 is highly endemic among PLWHA. Women were at higher risk of HTLV/HIV co-infection compared to men. The infection increases with age but no significant difference was found in the CD4+ T-cell count between HTLV-1/HIV-1 co-infected individuals and those mono-infected with HIV-1. The mean viral load was significantly higher in HIV-1 mono-infected than in HTLV-1/HIV-1 co-infected individuals, although one should interpret this with caution, since patients were all on ART. Most HTLV-1 strains from HIV-1/HTLV-1 co-infected patients belonged to the central African HTLV-1b genotype. This genotype is the most frequent in Gabon [11]. The unique HTLV-1d case found confirms that this rare genotype is occasionally reported in this country [27].

The prevalence of 7.7% for HTLV-1/HIV-1 co-infection, found in this study, confirms the high rate of HTLV-1 infection in Gabon and particularly in the rural region of Franceville and around. This prevalence is in the range of that reported in rural adults at the national level in Gabon, estimated from 7.3% to 8.7%, including recent data reported in the Haut-Ogooué province ranging from 9.5% to 11.6% [26, 27].

This high rate of HTLV-1/HIV-1 co-infection is an important public health concern in a country like Gabon where both infections are endemic. Infection with HTLV-1 can stimulate an abnormal increase of CD4+ T-cells which may bias ART strategies as CD4+ is a major immunological marker in HIV/AIDS monitoring and lead to a delay in ART initiation in regions where the WHO “test and treat” approach for HIV-1 infection is still to be implemented [15, 20]. Moreover, the consequence of the virological failure is the emergence of mutations that could induce ART drug resistance to the virus. The description of the M184V
mutation in a HTLV-1/HIV-1 co-infected patient (Gab-2276FCV) treated with 3TC confirms the replication of HIV-1 and potentially the HTLV-1 one [35, 36]. HTLV-1/HIV-1 co-infected individuals are predisposed to develop neurological diseases, which are hardly investigated in resource-limited settings, but which may represent a significant public health concern [16]. Moreover, a study of 29 coinfected patients from Mozambique (representing 4.5%) of a series of 704 HIV/AIDS patients showed that co-infected patients exhibited higher levels of CD4+ T cells expressing activation markers and a massive loss of naïve cells, suggesting that co-infected patients may progress faster to AIDS [24].

Several factors are involved in HTLV-1 transmission and probably to the evolution to related diseases in mono-infected and HIV-1/HTLV-1 co-infected individuals. Clinical aspects and outcomes in persons co-infected with both retroviruses were recently summarized in the review [37]. And as previously shown, co-infection with both retroviruses may lead to rapid evolution to AIDS and development of opportunistic infections in the absence of ART [38, 39]. Similarly, higher rates of the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (HAM/TSP) and peripheral neuropathy were observed in contexts of HIV-1/HTLV-1 co-infections [16], and higher mortality rates were also reported [40-42].

We observed, in our study, a significant increase of HTLV-1 infection with age and with a high prevalence of HTLV-1 in women. Similar findings have been reported for a rural area in Gabon [26, 27]; furthermore, most of the co-infected patients in our study were aged from 50 to 80 years. More importantly, in the same place, at Franceville, HTLV-1 prevalence among HIV-1 infected women was higher (9.6%) than among those from the general population (4.4%). In contrast, this prevalence was lower among HIV-1 infected men (3.3%) comparatively to men in general population (4.7%) (S2B Fig). Data from the general population was extracted from the paper by Djuicy et al [26]. A similar result was found in Brazil [21]. This observation of the increased rate of infection with age especially in women is considered as a specificity of HTLV-1 virus [26]. These findings have been attributed to sexual transmission of HTLV-1, especially from males to females [43, 44]. The high rate of HIV-1/HTLV-1 co-infection in women also highlights the risks of vertical transmission of these viruses. Efficient programs have been developed over the past 4 decades to prevent the vertical transmission of HIV from mother to child, especially by using antiretroviral drugs [45]. However, although mother to child transmission is currently recognized as a major transmission route for HTLV-1, there is still no program developed to address this issue in most African countries. Our results indicate that such programs should be urgently developed and implemented. Also, none of the study participants knew they were infected with HTLV before we conducted this study. In a country like Gabon, where both viruses are known to circulate at high prevalence, routine testing for both infections should be considered to help develop prevention and care strategies.

Although the prevalence of these two retroviruses is very high in many African countries, limited number of studies have been carried on the co-infection. Studies of different types of population in African countries estimate HTLV-1 and HIV-1 co-infection prevalences from 1.55% to 3.9%, highlighting the importance of the HTLV-1 sexual and vertical transmission, but also blood transfusion. For example, a co-infection rate of 3.45% was reported in inmate females from Mozambique [46]; similarly, the prevalence of HIV-1/HTLV-1 was 2.8% among women attending two sexual health clinics in Guinea Bissau [47]. HTLV-1 was also described among HIV-seropositive children (3.9%), suggesting that HTLV-1 is mostly acquired early in life [48]. In contrast, no data on HIV-1/HTLV-1 co-infection was found in the specific situation of Central Africa. However, the only studies available were based either serological survey or seroprevalence evaluation of HTLV-1, HIV-1 and others pathogens in different populations (S1 Table) [49-53].
In summary, we described a high prevalence of HTLV-1 infection among HIV-1 infected patients in rural Gabon. Our study highlights the needs for prevention and care programs for HTLV infection in this region and specific attention to populations that may be co-infected with both retroviruses. Implementing larger investigations and targeting both the general and specific populations will help develop these public health strategies.

**Supporting information**

**S1 Fig. Phylogenetic tree generated by the Neighbor-Joining method with a 522bp fragment of the env gene.** Phylogenetic comparisons were performed with the 522-nucleotide *env gp21* gene fragment obtained from 43 HTLV-1 isolates, including the 14 sequences from coinfected HIV-1/HTLV-1 patients (in red) and 29 previously published sequences. The Genbank accession numbers of the new sequences from the coinfected HIV-1/HTLV-1 patients are OL546372- OL546385. The phylogeny was derived by the Neighbor-Joining method with the GTR model. Horizontal branch lengths are drawn to scale, with the bar indicating 0.005 nucleotide replacements per site.
(TIF)

**S2 Fig. HTLV-1 and HIV/HTLV-1 prevalence distribution by sex and age class.** The figure shows the overall prevalence of infection by age group and the prevalence of infection by sex: A) In the HIV-1/HTLV-1 coinfected population; B) In the general population at the same place. Red color indicates global prevalence, yellow and green ones represent prevalence in men and women, respectively.
(TIF)

**S1 Table. Summary of the studies related to HIV-1 and HTLV-1 survey in Central Africa.**
(DOCX)

**Acknowledgments**

We thank all patients enrolled in this study, the Ambulatory Treatment Centre (CTA) in Franceville, and the medical unit from the Centre International de Recherches Médicales de Franceville (CIRMF).

**Author Contributions**

**Conceptualization:** Augustin Mouinga-Ondémé.

**Data curation:** Ingrid Précilya Koumba Koumba, Antony Idam Mamimandjiami, Abdoulaye Diané, Jéordy Dimitri Engone-Ondo, Landry Erik Mombo.

**Formal analysis:** Larson Boundenga, Jéordy Dimitri Engone-Ondo, Delia Doreen Djuicy.

**Funding acquisition:** Antoine Gessain, Avelin Aghokeng Fobang.

**Investigation:** Larson Boundenga, Ingrid Précilya Koumba Koumba, Antony Idam Mamimandjiami, Abdoulaye Diané.

**Methodology:** Augustin Mouinga-Ondémé.

**Project administration:** Augustin Mouinga-Ondémé.

**Resources:** Jeanne Sica.

**Supervision:** Augustin Mouinga-Ondémé.

**Validation:** Avelin Aghokeng Fobang.
**Writing – original draft:** Augustin Mouinga-Ondémé.

**Writing – review & editing:** Larson Boundenga, Landry Erik Mombo, Antoine Gessain, Avelin Aghokeng Fobang.

## References

1. Afonso P.V., Cassar O., and Gessain A., Molecular epidemiology, genetic variability and evolution of HTLV-1 with special emphasis on African genotypes. Retrovirology, 2019. 16(1): p. 39. https://doi.org/10.1186/s12977-019-0504-z PMID: 31842895

2. Poiesz B.J., et al., Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A, 1980. 77(12): p. 7415-9. https://doi.org/10.1073/pnas.77.12.7415 PMID: 6261256

3. Gessain A. and Cassar O., Epidemiological Aspects and World Distribution of HTLV-1 Infection. Front Microbiol, 2012. 3: p. 388. https://doi.org/10.3389/fmicb.2012.00388 PMID: 23162541

4. Proietti F.A., et al., Global epidemiology of HTLV-I infection and associated diseases. Oncogene, 2005. 24(39): p. 6058-68. https://doi.org/10.1038/sj.onc.1208968 PMID: 16155612

5. Gessain A., et al., Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet, 1985. 2(8452): p. 407-10. https://doi.org/10.1016/s0140-6736(85)92734-5 PMID: 2863442

6. Osame M., et al., HTLV-I associated myelopathy, a new clinical entity. Lancet, 1986. 1(8488): p. 1031-2. https://doi.org/10.1016/s0140-6736(86)91298-5 PMID: 2871307

7. Yoshida M., et al., Monoclonal integration of human T-cell leukemia provirus in all primary tumors of adult T-cell leukemia suggests causative role of human T-cell leukemia virus in the disease. Proc Natl Acad Sci U S A, 1984. 81(8): p. 2534-7. https://doi.org/10.1073/pnas.81.8.2534 PMID: 6326131

8. Kaplan J.E., et al., Male-to-female transmission of human T-cell lymphotropic virus types I and II: association with viral load. The Retrovirus Epidemiology Donor Study Group. J Acquir Immune Defic Syndr Hum Retrovirol, 1996. 12(2): p. 193-201. https://doi.org/10.1097/00042560-199606010-00014 PMID: 8680892

9. Hino S., Establishment of the milk-borne transmission as a key factor for the peculiar endemicity of human T-lymphotropic virus type 1 (HTLV-1): the ATL Prevention Program Nagasaki. Proc Jpn Acad Ser B Phys Biol Sci, 2011. 87(4): p. 152-66. https://doi.org/10.2183/pjab.87.152 PMID: 21558754

10. Okochi K., Sato H., and Hinuma Y., A retrospective study on transmission of adult T cell leukemia virus by blood transfusion: seroconversion in recipients. Vox Sang, 1984. 46(5): p. 245-53. https://doi.org/10.1111/j.1423-0410.1984.tb00083.x PMID: 6328765

11. Ramassamy J.L., et al., High prevalence of human T-cell leukemia virus type-1b genotype among blood donors in Gabon, Central Africa. Transfusion, 2020. 60(7): p. 1483-1491. https://doi.org/10.1111/trf.15838 PMID: 32415686

12. Brites C., Sampalo J., and Oliveira A., HIV/human T-cell lymphotropic virus coinfection revisited: impact on AIDS progression. AIDS Rev, 2009. 11(1): p. 8-16. PMID: 19290030

13. UNAIDS, GLOBAL AIDS MONITORING. https://www.unaids.org/en/resources/documents/2020/unaids-data 2020.

14. Morlat P., et al., Causes of death among HIV-infected patients in France in 2010 (national survey): trends since 2000. Aids, 2014. 28(8): p. 1181-91. https://doi.org/10.1097/QAD.0000000000000222 PMID: 24901259

15. Beilke M.A., et al., Tropical spastic paraparesis/human T leukemia virus type 1-associated myelopathy in HIV type 1-coinfected patients. Clin Infect Dis, 2005. 41(6): p. e57-63. https://doi.org/10.1086/432890 PMID: 16107970

16. Araujo A.Q., Neurological Aspects of HIV-1/HTLV-1 and HIV-1/HTLV-2 Coinfection. Pathogens, 2020. 9(4). https://doi.org/10.3390/pathogens9040250 PMID: 32231144

17. Beilke M.A., Retroviral coinfections: HIV and HTLV: taking stock of more than a quarter century of research. AIDS Res Hum Retroviruses, 2012. 28(2): p. 139-47. https://doi.org/10.1089/AID.2011.0342 PMID: 22171689

18. Dhasmana D. and Taylor G.P., Human T-lymphotropic virus/HIV co-infection: a clinical review. Curr Opin Infect Dis, 2014. 27(1): p. 16-28. https://doi.org/10.1097/QCO.0000000000000027 PMID: 24305042

19. Silva M.T., et al., Neurological manifestations of coinfection with HIV and human T-lymphotropic virus type 1. Aids, 2012. 26(4): p. 521-3. https://doi.org/10.1097/QAD.0b013e32834c4a3e PMID: 21881482
20. Ribeiro M.L., et al., HTLV 1/2 Prevalence and risk factors in individuals with HIV/AIDS in Pernambuco, Brazil. Rev Soc Bras Med Trop, 2019. 52: p. e20180244. https://doi.org/10.1590/0037-8682-0244-2018 PMID: 31340354

21. Marcon C.E.M., et al., The first survey of human T-cell lymphotropic viruses (HTLV) in HIV/AIDS patients in Santa Catarina State, Brazil. Rev Inst Med Trop Sao Paulo, 2019. 61: p. e53. https://doi.org/10.1590/S1678-9946201961053 PMID: 31618373

22. Bahia F., et al., The impact of human T-cell lymphotropic virus I infection on clinical and immunologic outcomes in patients coinfected with HIV and hepatitis C virus. J Acquir Immune Defic Syndr, 2011. 57 Suppl 3(03): p. S202-7. https://doi.org/10.1097/QAI.0b013e31821e9a1e PMID: 21857319

23. Casoli C., Pilotti E., and Bertazzoni U., Molecular and cellular interactions of HIV-1/HTLV coinfection and impact on AIDS progression. AIDS Rev, 2007. 9(3): p. 140-9. PMID: 17982939

24. Gudo E.S., et al., Co-infection by human immunodeficiency virus type 1 (HIV-1) and human T cell leukaemia virus type 1 (HTLV-1): does immune activation lead to a faster progression to AIDS? BMC Infect Dis, 2009. 9: p. 211. https://doi.org/10.1186/1471-2334-9-211 PMID: 20028500

25. Berger J.R., et al., The role of HTLV in HIV-1 neurologic disease. Neurology, 1991. 41(2 (Pt 1)): p. 197-202. https://doi.org/10.1212/wnl.41.2_part_1.197 PMID: 1671529

26. Djuicy D.D., et al., Risk factors for HTLV-1 infection in Central Africa: A rural population-based survey in Gabon. PLoS Negl Trop Dis, 2018. 12(10): p. e0006832. https://doi.org/10.1371/journal.pntd.0006832 PMID: 30312301

27. Caron M., et al., Revisiting human T-cell lymphotropic virus types 1 and 2 infections among rural population in Gabon, central Africa thirty years after the first analysis. PLoS Negl Trop Dis, 2018. 12(10): p. e0006833. https://doi.org/10.1371/journal.pntd.0006833 PMID: 30359373

28. EDSG, Enquête Démographique et de Santé du Gabon 2012: Rapport de synthèse 2012, DGS et ICF International Calverton, MD.

29. Liégeois F., et al., Virological failure rates and HIV-1 drug resistance patterns in patients on first-line antiretroviral treatment in semirural and rural Gabon. J Int AIDS Soc, 2012. 15(2): p. 17985. https://doi.org/10.7448/IAS.15.2.17985 PMID: 23199801

30. Laurendeau I., et al., TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency. Clin Chem, 1999. 45(7): p. 982-6. PMID: 10388473

31. Vandamme A.M., et al., Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I, II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology. J Med Virol, 1997. 52(1): p. 1-7. PMID: 9131450

32. Vermeulen M., et al., The prevalence of human T-lymphotropic virus type 1 & 2 (HTLV-1/2) in South African blood donors. Vox Sang, 2019. 114(5): p. 451-458. https://doi.org/10.1111/vox.12778 PMID: 30950074

33. Etenna S.L., et al., New insights into prevalence, genetic diversity, and proviral load of human T-cell leukemia virus types 1 and 2 in pregnant women in Gabon in equatorial central Africa. J Clin Microbiol, 2008. 46(11): p. 3607-14. https://doi.org/10.1128/JCM.01249-08 PMID: 18845819

34. Mongo-Delis A., et al., Factors associated with adherence to ARV treatment in people living with HIV/AIDS in a rural area (Koula-Moutou) in East Gabon. Afr J AIDS Res, 2019. 18(1): p. 51-57. https://doi.org/10.2989/16085906.2018.1552878 PMID: 30880584

35. Gagliardini R., et al., Impact of the M184V Resistance Mutation on Virological Efficacy and Durability of Lamivudine-Based Dual Antiretroviral Regimens as Maintenance Therapy in Individuals With Suppressed HIV-1 RNA: A Cohort Study. Open Forum Infect Dis, 2018. 5(6): p. ofy113. https://doi.org/10.1093/ofid/ofy113 PMID: 29977967

36. García-Lerma J.G., Nidtha S., and Heneine W., Susceptibility of human T cell leukemia virus type 1 to reverse-transcriptase inhibitors: evidence for resistance to lamivudine. J Infect Dis, 2001. 184(4): p. 507-10. https://doi.org/10.1086/322785 PMID: 11471110

37. Montaño-Castellón I., et al., Clinical and Laboratory Outcomes in HIV-1 and HTLV-1/2 Coinfection: A Systematic Review. Front Public Health, 2022. 10: p. 820727. https://doi.org/10.3389/fpubh.2022.820727 PMID: 35359787

38. de Mendoza C., et al., HIV co-infection in HTLV-1 carriers in Spain. Virus Res, 2019. 266: p. 48-51. https://doi.org/10.1016/j.virusres.2019.04.004 PMID: 30998953

39. Schechter M., et al., Coinfection with human T-cell lymphotropic virus type I and HIV in Brazil. Impact on markers of HIV disease progression. Jama, 1994. 271(5): p. 353-7. PMID: 7904317

40. Page J.B., et al., HTLV-I/II seropositivity and death from AIDS among HIV-1 seropositive intravenous drug users. Lancet, 1990. 335(8703): p. 1439-41. https://doi.org/10.1016/0140-6736(90)91456-k PMID: 1972217
41. Brites C., et al., Severe and Norwegian scabies are strongly associated with retroviral (HIV-1/HTLV-1) infection in Bahia, Brazil. Aids, 2002. 16(9): p. 1292–3. https://doi.org/10.1097/00002030-200206140-00015 PMID: 12045498

42. Brites C., et al., Early and Successful Combination Antiretroviral Therapy Normalizes Survival Time in Patients Coinfected With Human Immunodeficiency Virus and Human T-cell Lymphotropic Virus Type 1. Clin Infect Dis, 2020. 71(1): p. 196–200. https://doi.org/10.1093/cid/ciz756 PMID: 31406994

43. Mueller N., et al., Findings from the Miyazaki Cohort Study. J Acquir Immune Defic Syndr Hum Retrovirol, 1996. 13 Suppl 1: p. S2–7. https://doi.org/10.1097/00042560-199600001-00002 PMID: 8797696

44. Caterino-de-Araujo A., et al., Short Communication: Current Prevalence and Risk Factors Associated with Human T Lymphotropic Virus Type 1 and Human T Lymphotropic Virus Type 2 Infections Among HIV/AIDS Patients in São Paulo, Brazil. AIDS Res Hum Retroviruses, 2015. 31(5): p. 543–9. https://doi.org/10.1089/AID.2014.0287 PMID: 25464979

45. WHO, WHO Guidelines Approved by the Guidelines Review Committee, in Guideline: Updates on HIV and Infant Feeding: The Duration of Breastfeeding, and Support from Health Services to Improve Feeding Practices Among Mothers Living with HIV. 2016, World Health Organization. Copyright © World Health Organization 2016.. Geneva.

46. Augusto Â., et al., First description of HTLV-1/2 seroprevalence in HIV-infected inmates in Mozambique. J Med Virol, 2017. 89(8): p. 1498–1502. https://doi.org/10.1002/jmv.24801 PMID: 28240367

47. Månsson F., et al., High prevalence of HIV-1, HIV-2 and other sexually transmitted infections among women attending two sexual health clinics in Bissau, Guinea-Bissau, West Africa. Int J STD AIDS, 2010. 21(9): p. 631–5. https://doi.org/10.1258/ijsa.2010.009584 PMID: 21097735

48. Manhiça I., et al., First Report of Prevalence of HTLV-1 Among HIV-1/2-Infected Children in Mozambique. J Acquir Immune Defic Syndr, 2017. 74(2): p. e54–e56. https://doi.org/10.1097/QAI.0000000000001194 PMID: 27753683

49. Geffray L., et al., [African histoplasmosis: clinical and therapeutic aspects, relation to AIDS. Apropos of 4 cases, including a case with HIV-1-HTLV-1 co-infection]. Ann Med Interne (Paris), 1994. 145(6): p. 424–8. PMID: 7864504

50. Mauclere P., et al., A new HTLV type II subtype A isolate in an HIV type 1-infected prostitute from Cameroon, Central Africa. AIDS Res Hum Retroviruses, 1995. 11(8): p. 989–93. https://doi.org/10.1089/aid.1995.11.989 PMID: 7492447

51. Pegha Moukandja I., et al., Non-malarial infectious diseases of antenatal care in pregnant women in Franceville, Gabon. BMC Pregnancy Childbirth, 2017. 17(1): p. 185. https://doi.org/10.1186/s12884-017-1362-0 PMID: 28606185

52. Schrijvers D., et al., Seroprevalence of retroviral infection in women with different fertility statuses in Gabon, western equatorial Africa. J Acquir Immune Defic Syndr (1988), 1991. 4(5): p. 468–70. PMID: 2016684

53. Getchell J.P., et al., Detection of human T cell leukemia virus type I and human immunodeficiency virus in cultured lymphocytes of a Zairian man with AIDS. J Infect Dis, 1987. 155(4): p. 612–6. https://doi.org/10.1093/infdis/155.4.612 PMID: 3029238