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

INTRODUCTION: Human T-cell lymphotrophic 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 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 followed: (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).

Table 1

Patient characteristics associated with HTLV-1/HIV-1 coinfection status according to serology and molecular assays.

Variables	N = 299	HTLV-1/2 Serology		HTLV-1 PCR (Tax/Rex)
		ELISA n [% (95% CI)]	WB n [% (95% CI)]	Positive n [% (95% CI)]
Gender, n (%)
Men	90 (30.1)	13 [14.4(10–19.8)]	3 [3.3 (-0.1–6.7)]	3 [3.3 (-0.1–6.7)]
Women	209 (69.9)	32 [15.3 (10.3–20.3)]	18 [8.6 (4.8–12.4)]	20 [9.6 (5.6–13.6)]
Total	299 (100)	45 [15.1 (10.2–21)]	21 [7.02 (3.3–11.6)]	23 [7.7 (3.9–12.1)]
Age, n (%)
18–30 y	52 (17.4)	7 [13.5 (9–23)]	3 [5.8 (-1-12.6)]	3 [5.8 (-1-12.6)]
31–49 y	129 (43.1)	16 (12.40±5.9)	7 [5.4 (1.7–9.1)]	8 [6.2 (2.1–10.3)]
50–80 y	118 (39.5)	22 [18.8 (11.7–25.9)]	11[9.3 (4–14.6)]	12 [10.2 (4.7–15.7)]
Median (IQR) 46 (34–58)
CD4 cell count, cells/mm3 n (%)
<200	56 (18.7)	6 [10.7 (1.9–19.5)]	3 [5.4 (-0.6–11.4)]	3 [5.4 (-0.6–11.4)]
200–499	108 (36.1)	18 [16.7 (9.6–23.8)]	6 [5.6 (1.2–10)]	6 [5.6 (1.2–10)]
≥500	135 (45.2)	21 [15.6 (9.6–21.6)]	12 [8.9 (5.2–13.6)]	14 [10.4 (5.3–15.5)]
Median (IQR) 468 (256–692)
Virological outcome, n (%)
Viral load undetectable	195(65.2)	32 [16.4 (11.3–21.5)]	18 [09.2 (5.2–13.2)]	19 [09.7(5.6–13.8)]
Viral load ≥300 copies/ml*	7(2.3)	1 [14.3 (5.6–23)]	1 [14.3 (5.6–23)]	1 [14.3 (5.6–23)]
Viral load ≥1000 copies/ml	97(32.5)	12 [12.4 (5.2–19.6)]	2 [2.1 (-0.9–5.1)]	3 [3.1 (-0.2–6)]
Median** (IQR) 2.1 (1.7–3.8)
ART regimen	273/299
AZT + 3TC + EFV, n (%)	107 (39.2)	20 ([18.7 (11.3–26.1)]	6 [5.6 (1.2–10)]	6 [5.6 (1.2–10)]
TDF + 3TC + EFV	46 (16.8)	7 [15.2 (4.725.75)]	4 [8.7 (0.5–16.9)]	4 [8.7 (0.5–16.9)]
AZT + 3TC + NVP	24 (8.8)	4 [16.7 (1.5–31.9)]	2 [8.3 (1.1–15.5)]	2 [8.3 (1.1–15.5)]
TDF + FTC + EFV	79 (28.9)	10 [12.7 (5.3–20.1)]	4 [5.1 (0.2–10)]	5 [6.3 (0.9–11.7)]
Others	17 (6.2)	6 [35.3 (11.9–58.7)]	5 [29.4 (9.1–49.7)]	6 [35.3 (11.9–58.7)]

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

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

3TC: Lamivudine, EFV: Efavirenz, TDF: Tenofovir,

AZT: Zidovudine, FTC: Emtricitabine, NVP: Nevirapines

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. 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).

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.

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)

Click here for additional data file.

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)

Click here for additional data file.

Summary of the studies related to HIV-1 and HTLV-1 survey in Central Africa.

(DOCX)

Click here for additional data file.

25 Aug 2021

PONE-D-21-19756

Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon

PLOS ONE

Dear Dr. Mouinga-Ondemé,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically some technical information (PCR methods, used ELISA, determination of the CD4 count) were missing and deserved to be described in material and method. More importantly, because of the cross-sectional approach a carefull description of the viro-immunological status (point 2 of the reviewer 1) and clinical status of the patient as well as sequence data for the HTLV (point 4 and suggestion of the reviewer 1) deserved to be provided as suggested by the reviewer 1. To note even if not obtained at the time of sampling, clinical data obtained thereafter but for all cases evaluated may provide a tast of longitudinal study.

Please submit your revised manuscript by Oct 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:  http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at  https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“NO”

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in your Competing Interests section:

“NO”

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

This information should be included in your cover letter; we will change the online submission form on your behalf.

4.  In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

5. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ.

Additional Editor Comments (if provided):

Please note that only a rewriting will not be sufficient to push up the article to publication. Additional data have to be included as requested by the two reviewers

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this article by Mouinga-Ondemé et al, the prevalence of HTLV-1 infection was estimated at 7.7% in an HIV-infected population in south-eastern Gabon. Dually infected HIV + HTLV patients have so far been considered at risk of a faster clinical course to AIDS, but data are badly needed to support this hypothesis. Many studies have already been published but Gabon remains one of the right place to carry out such studies due to the superposition of both endemic.

This is of major interest to better understand the factors underlying the progression of HIV in patients infected with HLTV. Unfortunately, the cross sectional aspect of the data collect considerably limits the interest of this paper. The authors proposed that plasma viral load may be slightly lower and CD4 cell count higher in double-infected patients, but these data were not statistically significant.

In addition, before to be considered for publication, various questions remain:

1 / Inclusion criteria for the population infected with HIV in this study. Explain the unbalanced sex ratio. Over representation of the pregnant women ?

2 / EIA + but PCR negative and / or WB not known: additional molecular tests are absolutely necessary in this country with a high STLV / HTLV prevalence. Have you performed additional seological and molecular tests?

3 / The assays used for the RT-PCR viral load and the CD4 count must be specified (manufacturers?)

4 / Could we have more details about HTLV sequencing? Is the population only infected with HTLV-1 B?

5 / Clinical data concerning bi or mono infected patients are essential. HAM / TSP / ATTL reported in dually infected patients? Or more frequent neurological or haematological pathologies in this group?

The study is currently limited by the cross-sectional approach and the scarcity of biological and clinical data.

Suggestion to Authors: Despite the nucleotide diversity between HIV-1 and HTLV-1, their RT enzymes exhibit a fairly similar structure in the palm and fingers. All of these patients are (or have been) treated with 3TC / TFC and 184V and must certainly be present in HIV-RT in the majority of patients. A short sequencing of the HTLV RT pocket could be interesting because the intracellular level of lamivudine will lead to a selection of certain HTLV mutations; V118I and others have been proposed as relevant in explaining the high "resistance" of HTLV to 3TC in the 1980s. But recent interest in resistance to HTLV 3TC at least in vitro (DOI: 10.1086 / 322785) has shown that the question is still open. As the buffy coat was collected, sequencing could be easly performed. The presence of RT 3TC / FTC mutation (s) will confirm a potential replication of HTLV at the cellular level in this doubly infected population and will add original data to this article.

Reviewer #2: 480 / 5000

The article does not have sufficient consistency to support publication in Plos One.

Although the confirmation of HIV/HTLV-1 co-infection in patients from Gabon (Central Africa) is a serious public health problem, from a technical-scientific point of view the objectives are not clearly described, as well as the methodology and, furthermore, the results are insufficient for a publication in Plos One.

Therefore, I recommend that the article be submitted to another journal.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Nov 2021

MOUINGA-ONDÉMÉ Augustin

Unité des Infections Rétrovirales et Pathologies Associées,

Centre International de Recherches Médicales de Franceville (CIRMF),

B.P. 769, Franceville, Gabon. ondeme@yahoo.fr

Dear Editor,

Please find attached a revised version of the manuscript (PONE-D-21-19756) entitled “Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon” by Mouinga-Ondémé and colleagues. We thank you very much for facilitating this set of revisions to our paper.

We appreciate the reviewers’s diligence and we thank them for their constructive and helpful comments on the manuscript. We have modified our manuscript accordingly as detailed below. The main corrections in the new manuscript are highlighted in yellow.

Moreover, the english in the new manuscript was corrected by the native speaker.

We hope that these modifications to the manuscript sufficiently addresses the concerns mentioned by the reviewers and we thank them for their valuable advices.

We hope also that you will now find this manuscript suitable for publication in Plos One.

Yours Sincerely,

Augustin Mouinga Ondémé

Letter

Dear Dr. Mouinga-Ondemé,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically some technical information (PCR methods, used ELISA, determination of the CD4 count) were missing and deserved to be described in material and method.

More importantly, because of the cross-sectional approach a carefull description of the viro-immunological status (point 2 of the reviewer 1) and clinical status of the patient as well as sequence data for the HTLV (point 4 and suggestion of the reviewer 1) deserved to be provided as suggested by the reviewer 1. To note even if not obtained at the time of sampling, clinical data obtained thereafter but for all cases evaluated may provide a tast of longitudinal study.

Please submit your revised manuscript by Oct 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Reviewer 1-

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

_____________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Answer to Reviewer's Questions

Reviewer #1:

In this article by Mouinga-Ondemé et al, the prevalence of HTLV-1 infection was estimated at 7.7% in an HIV-infected population in south-eastern Gabon. Dually infected HIV + HTLV patients have so far been considered at risk of a faster clinical course to AIDS, but data are badly needed to support this hypothesis. Many studies have already been published but Gabon remains one of the right place to carry out such studies due to the superposition of both endemic.

This is of major interest to better understand the factors underlying the progression of HIV in patients infected with HLTV. Unfortunately, the cross sectional aspect of the data collect considerably limits the interest of this paper. The authors proposed that plasma viral load may be slightly lower and CD4 cell count higher in double-infected patients, but these data were not statistically significant.

In addition, before to be considered for publication, various questions remain:

1 / Inclusion criteria for the population infected with HIV in this study. Explain the unbalanced sex ratio. Over representation of the pregnant women ?

In our study, all patients enrolled were HIV-1 infected and receiving antiretroviral therapy (ART) at the only Ambulatory Treatment Centre (ATC) in Haut-Ogooué Province (Franceville is the main city). In other words, our inclusion criterion was receiving treatment at the Centre. We found that the majority of patients followed at the ATC in Franceville were women (209 women and 90 men). This observation is coherent with the study conducted by Liégeois and colleagues in 2012 (http://dx.doi.org/10.7448/IAS.15.2.17985) who showed that the majority of patients followed in the ATC are women. We think this situation may be explained by the fact that men prefer to go to other cities for treatment for fear of being stigmatized. No pregnant women were enrolled in the study. In the discussion section, we raises the concern of vertical transmission of HTLV-1 via breastfeeding in HIV-1/HTLV-1 women, as WHO developed programs to prevent the vertical transmission of HIV from mother to child, especially by using antiretroviral drugs (WHO, 2016).

2 / EIA + but PCR negative and / or WB not known: additional molecular tests are absolutely necessary in this country with a high STLV / HTLV prevalence. Have you performed additional seological and molecular tests?

We thank the reviewer for this question. We used the screening strategy commonly used to detect HTLV-1 and 2 and published in many reports (e.g., Djuicy et al., 2018 https://doi.org/10.1371/journal.pntd.0006832; Ramassamy et al., 2020 doi:10.1111/trf.15838). We acknowledge that other options include additional assays such as the INNO-LIA HTLV I/II Score (Immunogenetics, Gent, Belgium), but we did not do this because we used western blot. In addition, as we discussed in this paper, options are limited for HTLV diagnostic in resource-limited countries. The EIA test used we for serology (HTLV-I/II ELISA 4.0, MP Biomedicals) has high specificity. Because of false EIA+ specimen, we used the WB test for serological confirmation or to discriminate between HTLV-1 and HTLV-2. Without WB results, we consider that PCR results for conserved regions of the genome (tax) and sufficiently different regions of the genome (Env) confirmed or refuted the results of the EIA.

3 / The assays used for the RT-PCR viral load and the CD4 count must be specified (manufacturers?)

As recommended by the reviewer, we added sentences which describe the assays used for the CD4 count and the RT-PCR viral load. Please see Methods section, line 106 to 109 in the new version of manuscript.

4 / Could we have more details about HTLV sequencing? Is the population only infected with HTLV-1 B?

As requested by the reviewers, we provided HTLV sequencing data in the supplementary figure. First, we successfully amplified the PTLV tax region for 23 specimens; however, only 14 specimens had sufficient DNA to amplify a fragment of the Env gene (for more information see the line 135 to 142 of the new version of the manuscript). The phylogenetic tree, in supplementary data, shows that most of the study population is infected with HTLV-1B. Only one patient is infected with HTLV-1D (Gab-2276FCV).

5 / Clinical data concerning bi or mono infected patients are essential. HAM / TSP / ATTL reported in dually infected patients? Or more frequent neurological or haematological pathologies in this group?

We agree with the reviewer that clinical data concerning the study population are essential. None of the participants included in this study presented signs of TSP / HAM and/or ATLL. Unfortunately, we did not have clinical and epidemiological data for both mono and bi infected groups. Only some bacteriological, virological and parasitological opportunistic diseases have been reported in mono and coinfected patients. There were all correlated with the rate of CD4. We did not find it appropriate to discuss this because the purpose of our study was investigated HTLV-1 and HIV-1 co-infection in Haut-Ogooué Province. We agree that this merits further investigations in the future.

The study is currently limited by the cross-sectional approach and the scarcity of biological and clinical data.

Suggestion to Authors: Despite the nucleotide diversity between HIV-1 and HTLV-1, their RT enzymes exhibit a fairly similar structure in the palm and fingers. All of these patients are (or have been) treated with 3TC / TFC and 184V and must certainly be present in HIV-RT in the majority of patients. A short sequencing of the HTLV RT pocket could be interesting because the intracellular level of lamivudine will lead to a selection of certain HTLV mutations; V118I and others have been proposed as relevant in explaining the high "resistance" of HTLV to 3TC in the 1980s. But recent interest in resistance to HTLV 3TC at least in vitro (DOI: 10.1086 / 322785) has shown that the question is still open. As the buffy coat was collected, sequencing could be easly performed. The presence of RT 3TC / FTC mutation (s) will confirm a potential replication of HTLV at the cellular level in this doubly infected population and will add original data to this article.

The suggestion made by the reviewer is very relevant. Unfortunately, plasma and buffy coat collected from these patients, in 2018 and 2019, was also used for several other studies. After sequencing the HTLV Env gene, no more DNA was available from several patients for further investigations. Finally, we were unable to amplify and sequence HTLV RT. We note this approach for our future studies.

In contrast, we amplified HIV RT to check some mutations in HIV-1/HTLV-1 coinfected patients. The majority of the HIV-1/HTLV-1 co-infected patients had a viral load suppression (VL<2.5 Log); Only one patient had a virological failure (the one co-infected with HTLV-1D). The HIV-1 RT region of 646 bp infected 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). Please see line 209 to 214.

Reviewer #2: 480 / 5000

The article does not have sufficient consistency to support publication in Plos One.

Although the confirmation of HIV/HTLV-1 co-infection in patients from Gabon (Central Africa) is a serious public health problem, from a technical-scientific point of view the objectives are not clearly described, as well as the methodology and, furthermore, the results are insufficient for a publication in Plos One.

Therefore, I recommend that the article be submitted to another journal.

________________________________________

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Mar 2022

6 May 2022

Dr. MOUINGA-ONDÉMÉ Augustin

Unité des Infections Rétrovirales et Pathologies Associées,

Centre International de Recherches Médicales de Franceville (CIRMF),

B.P. 769, Franceville, Gabon. ondeme@yahoo.fr

Dear Editor,

Please find attached a revised version of the manuscript (PONE-D-21-19756R1) entitled “Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon” by Mouinga-Ondémé and colleagues. We thank you very much for facilitating this set of revisions to our paper.

We appreciate the reviewers’s diligence and we thank them for their constructive and helpful comments on the manuscript. We have modified our manuscript accordingly as detailed below.

Corrections in the new manuscript are highlighted in yellow. Concerning the illustrations, we have added supplementary data: the figure (S2 Fig) and the table (S1 table) as suggested by the reviewers.

We have addressed all the points raised by Reviewers #1 and #2 made the required corrections to the manuscript.

Given the new data analysis, we added a new author with approval from all. Delia Doreen DJUICY is now a co-author of this paper, she was added at 7th place.

We hope that these modifications to the manuscript sufficiently addresses the concerns mentioned by the reviewers and we thank them for their valuable advices.

Yours Sincerely,

Dr. Augustin Mouinga Ondémé

Letter

PONE-D-21-19756R1

Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon

PLOS ONE

Dear Dr. Mouinga-Ondemé,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 06 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Fatah Kashanchi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Please address the Reviewers comments

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Answer to Reviewer's Questions

Reviewer #1

Manuscript PONE-D-21-19756R1, Co-infection with human lymphotropic T virus type 1 and human immunodeficiency virus in rural Gabon is a descriptive cross-sectional study. The lack of clarity regarding the objectives and the lack of basic clinical data without any biological and clinical problems are major limitations to its publication in PlosOne

Despite these major limitations, given the importance of HIV / HTLV co-infection in public health and research and the paucity of studies, it is important to report these epidemiological data to the general public of PlosOne.

We suggest:

Improve the quality of the prevalence of coinfection data by matching the HIV HTLV population with the general population of the same place in the country in order to compare the prevalence of these two retroviruses in these different populations. This could at least lead to showing that the prevalence of HTLV, as expected, is higher in the HIV-infected population.

We agree with the Reviewer for improving the quality of the prevalence. Concerning general population of the same place of the study, prevalence was obtained from data used for our previous study published by Djuicy et al. (doi.org/10.1371/journal.pntd.0006832). A paragraph was added in the discussion section from the lines 281 to 285. Also, a supporting information file, S2 Fig. B, about the HTLV-1 prevalence in the general population, was provided and mentionned at the line 284.

And to make this article more attractive, we suggest that the discussion include a review of the literature on this double infection. A summary in the form of a table of epidemiological studies on HIV / HTLV co-infections which comment on the specific situation in Central Africa would be very necessary.

We thank the Reviewer for this suggestion. At the line 299 to 300, we mentionned that « 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.” on the specific situation in Central Africa, we do not find published studies about HIV-1/HTLV-1 co-infection. We found only studies based on serological survey or seroprevalence evaluation of HTLV-1, HIV-1 and others pathogens in different populations; and some of these studies are summarized in the S1 table. Lines 307 to 311.

Reviewer #2

In this new version the manuscript presents sufficient consistency to support publication in the Plos One. However some modifications are necessary.

The title of the article sounds appropriate. However the data presented do not support the hypothesis the factors evaluated may favor the rapid evolution and progression to AIDS in HTLV-1/ HIV co-infected patients. This hypothesis must be reviewed since the corresponding author informed to reviewer #1 it is not possible to include new evaluations using the plasma and buffy coat.

Points to improve:

- Line 100: Please define ART.

As recommended by the Reviewer, ART was defined as « antiretroviral therapy » (line 101 in the new manuscript version).

- Line 114: Western Blot: please write Western with initial capital letter.

As recommended by the Reviewer, « Western » was written with initial capital letter (line 132).

- Line 130: Please define ANRS.

We agree with the Reviewer, and ANRS was defined as « French National Agency for Research on AIDS and Viral Hepatitis. » (lines 122 to 123).

METHODS

The methodology should be described more organized and coherent. A good way is to insert subtitles:

1.Study design an population

2.HIV Viral Load and CD4 counts

3.HTLV diagnosis (include serological and molecular diagnosis. e.g.: serological diagnosis was performed..../ Molecular diagnosis was performed for all samples with a positive ELISA result. We extracted viral DNA from buffy-coat. )

4.HIV diagnosis

We agree with the Reviewer. The METHODS section was edited and organized as recommended :

1. Study design and population (Line 96)

2. HIV Viral Load and CD4 counts (Line 107)

3. HIV diagnosis (Line 116)

4. HTLV diagnosis (Line 127)

5. Phylogenetic analysis (Line154)

6. Statistical analysis (Line 160)

- Line 150: The study was approved ...(It would be better moving this topic to the end of subtitle 1.: “Study design an population”

As recommended by the Reviewer, this topic was moved to the end of « Study design and population » section (lines 104 to 105)

RESULTS

- Patient Population: Please, describe the results in the order they appear in the table 1. This will make it easier for the reader to follow the data.

We agree with the Reviewer. Results was organized by following the order they appear in the table 1 (lines 173 to 175)

I suggest adding a bar graph figure of HTLV-1 prevalence PCR among men, women and total, according to the age groups evaluated.

As suggested by the Reviewer, a bar graph has been added as a supplementary data (S2 Fig. A), and this reference was inserted in the main text (line 218).

DISCUSSION

It is important to review some points of the discussion after reviewing of the hypothesis that factors evaluated may favor the rapid evolution and progression to AIDS in HTLV-1/ HIV co-infected patients.

We agree with the Reviewer. In the discussion section, a paragraph was added at the lines 270 to 277

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Jun 2022

Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon

PONE-D-21-19756R2

Dear Dr. Mouinga-Ondemé,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Fatah Kashanchi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

13 Jul 2022

PONE-D-21-19756R2

Human T-Lymphotropic virus type 1 and Human Immunodeficiency Virus co-infection in rural Gabon

Dear Dr. Mouinga-Ondémé:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Fatah Kashanchi

Academic Editor

PLOS ONE