HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin.

OBJECTIVES: Female sex workers (FSWs) play a key role in HIV transmission in West Africa, while they have limited access to antiretroviral therapy (ART). In line with UNAIDS recommendations extending ART to all HIV-infected individuals, we conducted this demonstration project on immediate treatment as prevention (TasP) among FSWs in Cotonou, Benin. We report data on treatment response and its relation to adherence, as well as on ART-resistant genotypes.
METHODS: Complete follow-up varied between 12 and 24 months. At each three-monthly visit, a questionnaire was administered, clinical examinations were carried out and blood samples collected. Adherence to treatment was estimated by self-report. Viral RNA was genotyped at baseline and final visits for drug resistance. Generalized estimating equations for repeated measures with a log-binomial link were used to analyze time trends and the association between adherence and virological response to treatment.
RESULTS: One-hundred-seven HIV-positive and ART-naive FSWs were enrolled; 59.8% remained in the cohort till study completion and 62.6% had a final visit. Viral load<1000 (below quantification limit [<50]) was attained in 73.1% (64.6%) of participants at month-6, 84.8% (71.2%) at month-12, and 80.9% (65.1%) at the final visit. The proportion of women with suppressed (below quantification limit) viral load increased with increasing self-reported adherence (p = 0.06 (0.003), tests for trend). The proportion of participants with CD4≤500 also decreased drastically throughout follow-up (p < .0001). Twelve participants exhibited ART-resistant genotypes at baseline, but only two at their final visit.
CONCLUSION: Our findings indicate that TasP is widely accepted among FSWs in Cotonou and could be implemented with relative success. However, due to mobility in this population, follow-up was sub-optimal, suggesting that large geographical coverage of FSW-friendly clinics is needed for sustained treatment implementation. We also fell short of the UNAIDS objective of 90% viral suppression among treated patients, underlining the need for better adherence support programs.

Introduction

Western and central Africa is the second most affected region by the HIV epidemic with an estimated 5 million people living with HIV (PLHIV) at the end of 2018 (representing 13.2% of all HIV-infected people in the world). Yet, HIV testing and antiretroviral therapy (ART) coverage in this region are among the lowest worldwide [1]. Overall, the UNAIDS 2020 targets remain far from being attained with only 42% of the PLHIV knowing their status, 35% (83% of those knowing their HIV status) on ART, and only 25% (71.4% of those on ART) having suppressed their viral load [2]. Many countries of this region are characterized by an HIV epidemic where specific behaviours relevant to key populations are the main epidemic drivers [3-5]. Female sex workers (FSWs) and their clients, alongside less visible groups involved in part-time transactional sex, including bar workers and mobile fruit sellers [6], play a central role in the HIV transmission dynamics [7, 8]. Several reports in the region suggest that FSWs contributed to between 32–84% of HIV prevalent cases among men [9-11] and to 18% in the general population of women (12), providing evidence that prioritizing this high-risk group with specific preventive interventions could reduce HIV incidence and prevalence in both prioritized groups and the general population at lower risk [12-14]. However, despite such contribution, ART coverage among FSWs remains low in the region [15, 16] For these reasons, we considered FSWs as a priority population for the implementation of new biomedical preventive interventions such as immediate HIV treatment as prevention (TasP) and pre-exposure prophylaxis (PrEP) [17-19]. We conducted a TasP/PrEP demonstration project to assess the acceptability, feasibility, and utility to integrate TasP and PrEP among current prevention methods in FSWs in Cotonou. The present paper reports the main findings of the TasP component of this demonstration project [20] including virological and immunological response to treatment, and resistance to antiretroviral drugs.

Material and methods

Study design

Recruitment was conducted in pre-specified areas of Greater Cotonou (Cotonou and close suburbs) in order to approximately meet target sample sizes of 250 for the PrEP arm, and 100 for the TasP arm. After a run-in phase for community preparedness and the development of a specific education program on adherence, FSWs from the catchment areas were invited to attend the “Dispensaire IST”, a clinic dedicated to FSWs for HIV and sexually transmitted infections (STIs) care in Cotonou, for HIV testing. HIV-positive FSWs not known to be on ART were then invited to participate in the TasP component of the study and received a first-line ART regimen as per the Benin guidelines. Recruitment took from October 2014 to December 2015, followed by an additional one-year follow-up till December 2016, for a total follow-up varying between 12 and 24 months, depending on when a given participant was recruited in the study. A short-term follow-up visit to assess adherence and drug adverse events was scheduled at day 14 with subsequent visits being scheduled quarterly. At baseline and during follow-up visits, clinical examinations were carried out, vaginal and blood samples collected for STIs testing, CD4 count, and viral load quantification. Adherence was assessed during follow-up visits by self-report through a questionnaire. Participants who came back to the clinic for withdrawal were invited to undergo all the procedures of a final visit, while those who stopped participating without informing the clinic, were located by field workers and reasons for dropping out documented.

Study population and eligibility criteria

The study population consisted of professional FSWs (e.g. whose main revenue comes from sex work) 18 years or older, practicing in the pre-defined catchment areas. Those eligible for TasP had to be HIV-1 positive at screening, re-confirmed on a second test at the recruitment visit, and being treatment naïve. The only exclusion criterion was HIV-2 or dual HIV-1/2 infection.

Data collection

Demographic, behavioural and sex work characteristics

Several socio-demographic and sex work characteristics including nationality, age, marital status, education, parity, activities beside sex work, duration in sex work and monthly income were assessed through a questionnaire at baseline. Sexual behaviour including number of clients in the last two weeks, having a regular sexual partner and condom use with both clients and regular partners were assessed through the questionnaire at baseline and during follow-up. In order to assess adherence, at each visit, questions were asked on the number of pills missed in the last month.

Laboratory procedures at the different visits

At screening, all subjects were tested for HIV and syphilis using the SD Bioline HIV/syphilis Duo test (Standard Diagnostics, Yongin, South Korea, which is a treponemal test with confirmation of HIV-positive tests using the Immunoflow HIV-1/2 test (Core Diagnostics, Birmingham, UK). They were also tested for anti-HBc and anti-HBs antibodies and HBs antigen (HBsAg) using the Monolisa-Biorad tests of Biorad (Marnes-la-Coquette, France). FSWs initially testing positive using the treponemal rapid test were then tested with the Rapid Plasma Reagin (RPR) non-treponemal (also provided by Standard Diagnostics). Women testing positive on both the rapid and RPR tests were considered as cases of active syphilis. Subjects negative to hepatitis B antibodies and antigen were vaccinated against this infection. HIV seropositivity was confirmed on a separate blood finger-prick sample at recruitment using the same tests as above. At recruitment and during follow-up visits, vaginal swabs were collected for direct microscopic examination for yeasts and Trichomonas vaginalis; and abnormal vaginal flora using the Nugent score for the diagnosis of bacterial vaginosis. Participants also underwent testing for Neisseria gonorrhoeae and Chlamydia trachomatis on cervical swabs at recruitment and every six months using the BD Probetec NG/CT assay (Becton Dickenson, Cockeysville, MD, USA). Additional tests including glycaemia, blood creatinine serum levels (renal function), and alanine aminotransferase serum levels (liver function), as well as Chest-X ray prior to ART initiation were conducted, and complete blood count carried out using standard assays. Blood samples were collected and stored at screening and final visits for future drug resistance genotyping. In the context of the present study, CD4 count was monitored at screening and every three months using a CyFlow Counter (Partec, Germany), whereas viral load was quantified at the screening visit and then every six months using the NucliSens EasyQ equipment (BioMérieux Laboratories, Marcy l’Etoile, France). The technique allows the quantification of a minimum of 50 copies/ml of HIV-1 RNA in 0.5 ml plasma [21]. For both tests (CD4+ T-cell counting and HIV-1 viral load), positive and negative controls were used in each run. All plasma samples collected at screening and those at final visits with a viral load > 1000 copies per mL were tested for drug resistance. Samples were shipped on dry ice to the Centre Muraz, Bobo Dioulasso, Burkina Faso, where genotyping was carried out using a method developed by the Agence nationale de recherche sur le Sida (ANRS), the French agency for AIDS research described in details on the ANRS website [22]. The interpretation of resistance mutations used the latest updates of the three main algorithms currently available: one from ANRS [23], one from Stanford University [24, 25] and one from the Rega Institute for Medical Research in Leuven, Belgium [26].

Treatment regimens

The antiretroviral regimen consisted of Tenolam E® (Hetero Healthcare Ltd., Hyderabad, India) tablets with two nucleotides reverse-transcriptase inhibitors (NRTIs)

(Tenofovir, Lamivudine) and one Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) (Efavirenz) as active ingredients. In case of suspicion of resistance (repeated viral load above 1000 copies/ml; decrease, or lack of increase in CD4 count), a second line treatment was suggested with Lopinavir /Ritonavir (+ lamivudine and tenofovir). For those who desired pregnancy, Tenofovir, Lamivudine and Nevirapine were suggested. Their ART regimen was provided to the participants for a 30-day period. Outside periods of scheduled follow-up visits, they were contacted by field workers every month and, at that time, they were given the choice to come to the clinic just to pick up another monthly supply or to have it delivered at home. All participants diagnosed with STIs were treated immediately according to the Benin national guidelines.

Definition of outcome variables

The primary outcome of interest was the virological response defined as (i) suppressed (<1000 copies/mL) as suggested by the WHO 2013 Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection [27], or (ii) below quantification limit of the assay used during the study. In this study, since we used NucliSENS EasyQ® HIV-1 v2.0 (Semi-Automated), and 0.5ml plasma for viral load quantification, the “quantification limit” was 50 copies/ml. Therefore, all samples in which we could not amplify the viral load were considered as having a viral load below quantification limit (below 50 copies/mL). In relation to this first outcome, we analyzed the evolution of viral load throughout follow-up as well as the association between self-reported adherence and viral load. The second outcome was changes in CD4 count throughout follow-up, considering a CD4 count fall to (or below) the baseline value or persistent CD4 levels below 100 cells/mm3 as indicative of immunologic failure [27]. The last outcome was the development of drug resistance during the study, but we also report on the presence of primary drug resistance at baseline.

Statistical analysis

All data were analyzed using the SAS 9.4 statistical software (SAS Institute, Cary, NC, USA). First, we used descriptive statistics to estimate means and medians for continuous variables, while categorical variables were described using proportions. In particular for immunological and virological parameters, means and medians were estimated using the actual CD4 count and the log of viral load values (the viral load value was set at 50 when below quantification limit) to obtain geometric means for the latter, while proportions were obtained by categorizing the CD4 count into ≤500 cell/μl vs >500 cell/μl; and the viral load into suppressed (<1000 copies/ml) vs unsuppressed, and below quantification limit (<50 copies/ml) vs above quantification limit. We used generalized estimating equations (GEE) models for repeated measures with a log-binomial link and type3 WALD statistical tests to assess trends in CD4 and viral load throughout follow-up, as well as the association between viral load and self-reported adherence while controlling for time duration since ART initiation and other potential confounders, such as age, country of origin and several sexual behaviour variables. Self-reported adherence was categorized into less than 3 pills missed in the last month, corresponding to high adherence (adherence ≥90%), 4–7 pills missed corresponding to medium adherence (adherence between 75–89%), 8–15 pills missed corresponding to low adherence (adherence between 50–74%), and >15 pills missed corresponding to non-adherence (adherence below 50%).

Ethical considerations

After detailed explanation of the study, women provided informed written consent for the screening visit and then again at recruitment for the whole follow-up period. The participants were free to withdraw from the study at any time without any prejudice. The study was approved by the National Committee of Ethics in Health Research of Benin and by the Institutional Review Board of the CHU de Québec–Université Laval, Québec, Canada.

This study is registered with ClinicalTrials.gov, number NCT02237027.

Results

Recruitment and follow-up

Out of the 111 participants tested HIV-positive at screening, 5 declined to participate and one was infected with both HIV-1 and HIV-2, thus excluded from the study and referred for standard treatment. During follow-up, two participants from the PrEP arm seroconverted and were included in the TasP program, summing up to a total of 107 participants recruited for TasP. Participants were then followed for a mean duration of 13.2 ± (Standard Deviation 7.7) months. During follow-up, seven participants withdrew for not being interested to the study anymore (6.5%), 15 went back to their country of origin (14.0%), 19 went to others cities to practice sex work (17.8%), one died and one got married and left sex work; leaving a final number of 64 participants who effectively completed the study. This makes up a strict retention rate of 59.8% and global retention rate of 62.6% since three participants who actively withdrew underwent all the procedures of final visits.

Baseline characteristics of participants

Baseline sociodemographic characteristics of participants are shown in Table 1. Almost half of them were foreigners; median age was 35 years [Interquartile range (IQR): 30–42], and about 40% did not have any formal education. About 70% were either divorced or widowed, and over 80% had at least one child. The median duration of sex work was 2 years (IQR: 0.46–3), with 32.7% being on sex work for less than one year, and 13.0% for more than five years. The median number of sexual clients in the last two weeks was 12 [IQR: 4–30] with 10.3% not having any sexual client during that period; and condom use at last sexual act was reported by 97.1% of participants. The median monthly income was 150,000.00F CFA [IQR: 90,000.00–210,000.00], about USD $270 (IQR: 165–380), with 34.9% earning less than ≤100.000 FCFA per month, about USD $200. Nearly 7.7% of participants were infected by either N. gonorrhoeae or C. trachomatis, 7.2% were positive for vaginal candidiasis, 1% for trichomoniasis; and two thirds had bacterial vaginosis (Nugent Score 7–10). Only 15.5% had WHO clinical stage II with either CD4 count below 200 cell/μl, presence of emaciation, dermatosis, persistent cold, prolonged fever, oral thrush, or cutaneous mucosal lesions. No participant was diagnosed with active syphilis, 13.1% were negative to both anti-HBc and anti-HBs and about 5% had active hepatitis B (see Table 1). All participants started ART with Tenolam-E®.

Table 1

Baseline characteristics of FSWs participating in the TasP demonstration study in Cotonou, Benin.

Variable	Total (n)	Total (%)
Country of origin	107	100%
Benin	56	52.3%
Other countries	51	47.7%
Age (in years)	107	100%
18–24	13	12.1%
25–34	35	32.7%
35–44	40	37.3%
≥45	19	17.7%
Mean age ± SDa	35.5 ±8.8
Median age [IQRb]	35 [30–42]
Marital status	107	100%
Married	2	1.8%
Divorced or Widowed	76	70.9%
Single	29	27.1%
Parity	105	98.1%
0 child	9	8.6%
1 child	27	25.7%
2–3 children	37	35.2%
4–5 children	24	22.8%
>5 children	8	7.6%
Variables related to sex work and sexual behavior
Duration of sex work (years)	105	98.1%
<1	35	33.3%
1–5	56	53.3%
>5	14	13.3%
Median duration of sex work, [IQRb]	2 years [0.46–3.0]
Number of sexual clients during the last 14 days	97	90.6%
0	11	11.3%
1–4	13	13.4%
5–9	15	15.4%
10–19	19	19.6%
20–49	28	28.8%
≥ 50	11	11.3%
Median number of sexual clients, [IQRb]	12 [5–30]
Condom use with last client	105	98.1%
Yes	102	97.1%
Had a regular sexual partner in the last 12 months	105	98.1%
Yes	58	55.24%
Condom use with regular partner at last sex	57	53.2%
No	48	84.2%
Monthly income in FCFAc	103	96.2%
≤100.000	36	34.9%
101.000 à 200.000	41	39.8%
>200.000	26	25.2%
Mean Income ±SDa	170,000 ±100.00
Median Income [IQRb]	150,000 [90,000.00–210,000.00]
Sexually transmitted infections (STIsd)
N. gonorrhoeae	104	97.2%
positive	6	5.7%
C. trachomatis	104	97.2%
positive	3	2.8%
Trichomonas vaginalis	104	97.2%
positive	1	0.9%
Vaginal Candidiasis	104	97.2%
positive	8	7.2%
Bacterial vaginosis	104	97.2%
Normal Flora (Nugent score 0–3)	10	9.6%
Intermediate Flora (Nugent score 4–6)	30	28.8%
Bacterial vaginosis (Nugent score 7–10)	64	61.5%
Serology
Active Syphilis	107	100%
Positive	0.0	0.0%
Anti_HBs	107	100%
Positive	48	46.6%
Anti_HBc	107	100%
Positive	86	83.5%
Ag HBs	107	100%
Positive	5	4.6%

SD: Standard Deviation

IQR: Interquartile range

F CFA: Franc CFA (name of Beninese currency); US$1 is equivalent to about 500 F CFA

STIs: Sexually transmitted Infections

Note: Due to missing values, the total number (N) for each variable may be different from 107. However, the few missing values could not significantly affect the results.

CD4 count

The mean CD4 count increased significantly throughout follow-up, from 526.5 (Standard deviation: 329.2) at baseline to 718.1 (standard deviation: 385.8) at month 24 (p trend < .0001) while the proportion of participants with CD4<500 decreased significantly from 53.2% to 29.4% during the same period (p trend = 0.008) (Fig 1). In addition, among the 64 participants who had regular final visits (either a premature final visit or a final visit at the end of the study), the proportion with CD4 count <500 at that visit was significantly lower (27.9%) compared to baseline (p < .0001) while the mean CD4 count was significantly higher (711.3 vs. 526.5, p < .0001). However, despite such trends in CD4 recovery, three participants experienced immunological failure with persistent CD4 count below 100 cells/μl even after one year of treatment. These participants started treatment with low CD4 counts at baseline, respectively 19, 28, and 52 cells/μl; had adherence level below 50% or sometimes missing; and had all maintained viral load above 1000 copies/ml during follow-up.

Fig 1

Time trends in mean CD4 and proportions of CD4<500 among HIV-infected FSWs on ART, Benin.

N: Number of observations per visit %CD4 count<500 cell/μl: Proportion of participants with CD4<500 cell/μl during follow up Mean CD4 count: Changes in mean CD4 count during follow upFinal visits: Included 63 participants who had final visits from Month 3 onwards and one early final that occurred at Month 3. Subjects with final visits at Day 14 (one at the end of the study for a seroconverting participant from the PrEP component of the study and two early final visits because of active withdrawal) were excluded from this analysis.

Viral load

Out of the 107 samples collected at baseline, 19 (17.8%) had suppressed viral load, and 3 (2.8%) had viral load below quantification limit. At month 6, 73.2% (64.6%) of the 82 participants still followed up, had suppressed (<1000 copies/mL) and below quantification limit (<50 copies/mL) viral load. These percentages increased respectively to 84.8% and 71.2% among the 66 participants remaining in the study after one year (88.2% and 82.3% at month 24). The geometric mean of viral load decreased significantly throughout follow-up, from 12372.0 [95% Confidence Interval (95%CI): 7575.2–202017.4) to 95.1 (95%CI: 31.0–291.0) at month 24 (p trend < .0001), while there was significant increase in the proportions of both suppressed (<1000 copies/mL) (p trend < .0001), and viral load below quantification limit (<50 copies/mL) (p trend < .0001). The geometric mean of viral load at final visits (for both those with a premature final visit and those with a regular final visit) was also significantly lower (144.4; 95%CI: 79.8–261.4) compared to baseline (p < .0001) and over 81.0% (65.1%) of participants with regular final visits had suppressed (below quantification limit) viral load (p < .0001) (Fig 2). However, six participants, who initially suppressed their viral load at month 6, experienced viral rebound at month 12. During follow-up, eight participants experienced virological failure with sustained viral load above 1000 copies/mL for more than two consecutive visits.

Fig 2

Time trends in suppressed and below quantification limit viral load among HIV-infected FSWs on ART, Benin.

N: Number of observations per visit % Suppressed viral load: Proportion of participants with viral load <1000 copies/ml % Below quantification limit: Proportion of participants with viral load <50 copies/ml Final Visits: Included 63 participants with regular final visits from Month 6 onwards; excluded all final visits that occurred prior to M6.

Adherence

Among the 217 follow-up visits for which viral load testing was carried out at month 3 or later, data on self-reported adherence was available for 207 (95.4%). Table 2 shows the association between viral load and self-reported adherence using all observations while controlling for study visit rank (the other potential confounders were dropped from the final model because they did not change by more than 10% the prevalence ratios between the different adherence levels and the prevalence of suppresses or below quantification limit viral load. The proportion of visits where viral load was suppressed or below quantification limit increased with increasing adherence (p = 0.06 and 0.003, respectively) (Table 2). However, this increase was mostly observed when adherence levels went from < 50% to 50–74%, with the prevalence ratios remaining almost identical for the two higher adherence categories.

Table 2

Prevalence ratio of below quantification limit and suppressed viral load according to self-reported adherence levels among FSWs participating in the TasP demonstration study in Benin.

Adherence levels (self-reported)	Prevalences	PRa	95%CIb		p value	p trendc
Suppressed viral load
≥90% (Pill missed ≤3)	129/155 (83.2%)	1.4	1.0	2.0	0.04	0,06
75–89% (Pill missed = 4–7)	16/19 (84.2%)	1.4	0.9	2.2	0.10
50–74% (Pill missed 8–15)	9/11 (81.8%)	1.4	0.9	2.2	0.12
<50% (Pill missed >15)	13/22 (59.1%)	1	-	-	(Reference)
missinge	5/10 (50.0%)	--	--	--
Below quantification limit viral load
≥90% (Pill missed ≤3)	114/155 (73.5%)	3.2	1.5	6.8	0.002	0,003
75–89% (Pill missed = 4–7)	14/19 (73.7%)	3.3	1.5	7.0	0.003
50–74% (Pill missed 8–15)	8/11 (72.7%)	3.3	1.5	7.1	0.003
<50% (Pill missed >15)	5/22 (22.7%)	1	-	-	(Reference)
missinge	4/10 (40.0%)	--	--	--

PR: Prevalence ratio

95%CI: 95% Confidence Interval

cp trend: trend in the prevalence ratio

Missing: No data available for self-reported adherence for these subjects

HIV strains and drug resistance (details in Table 3)

Genotyping for pre-ART drug resistance was attempted on all HIV-positive samples at the screening visit. Out of the 111 samples collected during screening visits and sent for sequencing, 98 (88.3%) were successfully genotyped. Of these, 58.9% were infected with HIV-1 Circulating Recombinant Forms (CRFs) CRF02_AG/CRF02_AG, 7.4% with G/G subtype, 4.2% with CRF01_AE/CRF02_AG subtype, 1.0% with D/D subtype, 4.2% with subtype G/CRF02_AG, and the remaining 24.2% with minor CRFs including CRF02_AG/CRF18_cpx, U/CRF02_AG, CRF06_cpx/CRF02_AG, and CRF11-cpx/CRF02_AG subtypes. Twelve exhibited resistant genotypes making a prevalence of 10.8% (12/111). Among these twelve, four (3.6%) showed resistance to only NRTIs, eleven (10%) to NNRTIs, and one to protease inhibitors (PIs) (Table 3). Mutations associated with resistance to NRTIs included M41L, D67N, T69D, 70R, M184V, T215F, K219Q, T215TS M184I; those associated to NNRTIs included K103N, V179E, Y181C, Y181YF, Y181F, G190A, A98G, Y188L, P225H, Y181S, and those associated to PIs included the major resistance mutation L90M, and other minor resistance mutations L33F, K20I and L10V. All the 12 participants with baseline drug resistance had viral load above 1000 copies/ml, and only one was infected with CRF11-cpx/CRF02_AG strain, the other being infected with either CRF02_AG/CRF02_AG or G/CRF02_AG.

Table 3

Summary of pre-ART drug resistance mutations among FSWs participating in the TasP demonstration study in Benin.

Baseline			Mutations			Resistance levels			HIV Subtypes
Sample ID	CD4 count (cell/μl)	Viral load(copies/ml)	NRTIa	NNRTIb	PIsc	NRTI	NNRTI	PIs	RT / PR regionsd
CD009	321	11220	M41L, D67N, T69D, 70R, M184V, T215F, K219Q	K103N, V179E		Intermediate to High	High		G/CRF02_AG
CD011	700	6309		K103N, Y181C			Intermediate to High		CRF02_AG/CRF02_AG
CD068	305	2800	T215TS	Y181YF, G190A		low	Low to High		G/CRF02_AG
CD083	506	2786	T215S	A98G V179E Y188L		Low	High		CRF02_AG/CRF02_AG
CD140	395	140000		Y181YF			Intermediate to High		CRF02_AG/CRF02_AG
CD191	90	36 000		Y181YF			Low to High		CRF02_AG/CRF02_AG
CD244	720	1900		K103N, P225H			High		CRF02_AG/CRF02_AG
CD330	78	100968		Y181S			Low to High		CRF02_AG/CRF02_AG
CD333	686	3466	M184I	Y181F		Low to high	Low to High		CRF11-cpx/CRF02_AG
CD343	448	2044		K103N			High		CRF02_AG/CRF02_AG
CD371	1365	1000		Y181YF			Low to High		CRF02_AG/CRF02_AG
CD314	590	12657			L90M, K20I, L33F			High	CRF02_AG/CRF02_AG

NRTI: Nucleoside Reverse Transcriptase Inhibitors

NNTI: NoNucleoside Reverse Transcriptase Inhibitors

PIs: Protease Inhibitors

RT / PR regions: Sequencing was conducted at the Protease (PR) and the reverse transcriptase (RT) regions

At the end of the study, 12 samples with viral load above 1000 copies/ml were sent for genotyping. Out of them, 10 were successfully genotyped while two were not, despite many attempts. Among those successfully genotyped, two (20.0%) exhibited major mutations associated with resistance to NNRTI or NRTI. Mutations associated with resistance to NRTIs included K70E, M184V, and D67G; those associated to NNRTIs included K103N, V179E, Y188L, V106M, G190A; V82L, and G73GA. These two participants experienced treatment failure and had self-reported adherence levels either missing or very low, varying between 50 and <90%. Among the 12 participants with resistance mutations at baseline, the four who had final visits had all suppressed their viral load.

Discussion

Our study, one of the first in the West and Central Africa region, revealed wide acceptance of early HIV treatment as prevention (TasP) among FSWs in Cotonou, Benin. This strategy, coupled with adherence to treatment, resulted in restoration of CD4 count, suppression at below quantification limit of viral load, and low emergence of drug resistance. Of note, the World Health Organisation (WHO), in the 2016 WHO Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection, defines a viral load <1000 copies/mL as treatment success [28]. The term “undetectable” viral load was considered as a viral load below the quantification limit of the assay used in the study. FSWs in this region are disproportionally affected by the HIV infection, play a key role in its spread to the general population [29] but have limited access to antiretroviral therapy [15, 16]. It is in this context that, in line with the recent UNAIDS recommendations extending antiretroviral therapy to all HIV-infected individuals regardless their CD4 count levels [30], we conducted the present demonstration project among FSWs in Cotonou Benin. Globally, if TasP was accepted by the majority of HIV-infected FSWs, its feasibility was highly affected by mobility in this group of population. For instance, despite 96.4% enrolled (107/111) at the beginning of the follow-up, about 40.2% dropped out between recruitment and the end of the study. Reasons for dropping out were not necessarily related to TasP intervention itself, rather to seeking clients in other cities or countries. Considering the diversity of origin of FSWs included in this study, this could be predictable since almost half of them were not Beninese nationals. Furthermore, even when they were Beninese, they had frequent trips to other cities within the country (or even moved from Cotonou to such cities) to practice sex work, trips during which their access to treatment may be limited by the absence of se-worker dedicated clinics.

Other similar studies have also shown that loss to follow up is very common in FSWs and a recent TasP demonstration project carried out in South Africa among FSWs also found similar results with one third of the TasP participants not completing their follow-up visits [31]. Indeed, mobility in FSWs is mostly associated with work location (number of clients), being HIV-positive, earning less per client, and experiencing violence among other reasons [32-34], which are not necessarily associated with treatment program. Therefore, the rate of dropout observed in this study should not be interpreted as lack of acceptability or feasibility itself since more than 90% of FSWs who were offered TasP accepted it, and reasons for dropping out were related to mobility, not to drug uptake. However, this suggests that future studies or interventions should consider mobility as a serious barrier to the intervention. Indeed, only few cities in West Africa have sex-worker dedicated clinics that provide ART services, such as Dispensaire IST in Cotonou. In other clinical settings, FSWs could face individual, social, or structural barriers, including anxiety, stigmatization, lack of social support, violence, or discrimination even from medical staff, that could prevent them from being fully adherent [35, 36]. For the participants who were relatively stable in Cotonou during the study period, such barriers seemed to be unlikely at Dispensaire IST as most of our participants achieved relatively good virological response with about 4 in 5 participants having suppressed, and 3 in 4 having below quantification limit viral load at month 12. Similar studies investigating combination HIV prevention and care package along with systematic reviews have also shown that the fraction of FSWs on ART who reached a below quantification limit viral load ranged between 40%–82% across varying time periods and definitions of viral suppression (≤50 copies/ml, ≤100 copies/ml, ≤180 copies/ml, ≤300 copies/ml)[37-39]. A strong association and a positive trend were found between self-reported adherence and viral suppression. However, this association was weaker for suppressed compared to below quantification limit viral load. This could be explained by the fact that viral suppression is a transition step, while below quantification limit is permanent stage when patients keep taking their drugs. Following ART initiation, most of patients suppress their viral load, and when the medication is taken on a regular basis, the viral load reaches below quantification limit levels where it is maintained as long as patients keep taking their medication. Viral suppression is mainly associated with control of transmission [40, 41], while below quantification limit levels, in addition to reducing transmission, are associated with well-being, meaning better health [42] and a stronger immune system [43]. On the other hand, viral suppression, coupled with both reduced opportunistic infections and good recovery of CD4 levels, could provide a sensation of absolute well-being and health trust that in return pushes such participants to be less adherent. Although modest, our findings of less than 20% of participants having their CD4 count below 500 at the final visit are encouraging, and in line with previous findings in this population. In their cohort study, Diabaté et al. (2011), when comparing the immunologic response to ART between FSWs and the general population at a time when, in Benin, ART was only initiated in patients with CD4 < 200 cells/μl, observed a median increase in CD4 count of 103 cells/μl from a baseline of 134 cells/μl among FSWs over a one-year period [15]. This increase however, was somewhat lower compared to that of the general population (103 cells/μl versus 129 cells /μl; p = 0.085).

Using the ANRS-AC 11- HIV genotypic drug resistance interpretation’s algorithms, [23] a prevalence of 12.2% for primary drug resistance prior to ART initiation was quite high compared to results from a previous study conducted in the same population [44]. In that study, Chamberland et al. found a prevalence rate of 3.9% for resistance mutations, with no major resistance to NRTIs, two resistances (K103N, G190A) to NNRTIs, and only one (F53Y) to protease inhibitors. Compared to our findings, in addition to K103N, and G190A, new resistant mutants included V179E, Y181C, Y181YF, Y181F, A98G, Y188L, P225H, and Y181S for NNRTIs, while those associated with resistance to NRTIs included M41L, D67N, T69D, 70R, M184V, T215F, K219Q, T215TS, and M184I. These findings point out both emergences of new circulating mutants, and/or arrival of new resistant mutants from surrounding countries where other ARV regimens including PIs are already in use. However, this high prevalence should be interpreted with caution since most participants with primary resistance were Beninese (only three Togolese). These could have initiated ART prior to inclusion in our study but hid the information during enrollment, as the services offered in our study could have been better than those provided in standard care. Luckily, this rate of primary drug resistance observed at baseline in our study did not seem to be much alarming since none of participants diagnosed with resistance mutations, developed clinical resistance during the study, and even better, those who completed their final visit had all suppressed viral load and CD4 count above 500 cell/μl at their final visit. Two participants who developed drug resistance during follow-up had treatment failure (repeated viral load >1000).

Study limitations

Two major caveats could have affected our results during this demonstration study project. The first is related to the high number of dropouts observed from the beginning to the end of the study reaching up to 40%. These dropouts were mainly due to the mobility of sex workers who either returned to their countries of origin or traveled to other cities to change work settings. In addition, we were not able to have access to data related to adherence, CD4 count, and viral load for those participants who went back to their countries of origin where they could also have accessed ART program and continued treatment. These losses to follow-up could have reduced the power of the study and have impact on our results since drop out subjects could be differentially related to treatment adherence and treatment response, causing either an overestimation or an underestimation of the association.

The second is related to the method of assessing adherence which used self-report. Indeed, self-reported adherence is subject to social desirability bias but also recall bias. These biases could overestimate adherence levels and affect the association between viral load and adherence to treatment. However, although not showing a clear trend of increasing viral load suppression with increasing adherence, especially in the upper adherence categories, the association we found between self-reported adherence and below quantification limit viral load is somewhat reassuring about the validity of self-reported adherence.

Conclusion

Findings from this TasP demonstration project on FSWs indicate that immediate HIV treatment initiation following diagnosis is widely accepted, while feasibility could be affected by FSWs mobility and treatment adherence. Considering the sub-optimal follow up observed in this highly mobile population, a regional collaboration between FSW-friendly clinics is needed for sustained treatment implementation. We fell short of the UNAIDS objective of 90% viral suppression among those treated, underlining the need for better programs for enhancing treatment adherence, including structural interventions for reducing stigma and discrimination towards female sex workers and HIV-infected people.

Key messages

Our results show that immediate treatment as prevention is quite accepted among female sex workers in Cotonou, Benin, and that suppressed viral load can be achieved in over 80% of female sex workers after one year, with good recovery in CD4 count and low emergence of drug resistance.

The study also highlights the need to consider the mobility of sex workers, their access to ARVs in their workplaces. It thus underlines the necessity of a regional collaboration between FSWs dedicated STI-clinics for effective implementation and effectiveness of TasP in FSWs in the sub-region.

Excel data to reproduce our results.

(XLSX)

Click here for additional data file.

28 Jul 2019

PONE-D-19-15725

HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin

PLOS ONE

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Additional Editor Comments (if provided):

The authors report on the results of a TasP demonstration study among a small cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin. In general the study is clear and well written and interesting. However, the number of tables versus size of study is uncomparebly high. Table 1 could be removed and explained in the text; A graph of vl and cd4 versus visits could replace tables 3 and 4. Table 5 is unclearly presented. On the other hand, a table summarizing the prevlence and type of baseline DRM in the different class of drugs versus subtypes is missing. The outline of Table 6 is also unclear and if a new summary table is provided- than table 6 can be in appendix. Please also check if all references are correctly written and according to PLOS policy.

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #2: No

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Reviewer #1: General comments:

The authors report on the results of a Treatment as Prevention demonstration study among a cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin.

Their findings on acceptability, retention and viral load provide important and relevant information which should be taken into account when implementing TasP among key populations in Africa.

The general methods seem adequate. Statistical methods and analysis however should be verified. Some of the statistical tests used are not mentioned. The authors use generalized estimating equations to analyse time trends and the association between adherence and virological response however the results of the model are not clear and complete. It is also a pity that no attempt for identifying or adjusting for confounders have been made.

Specific comments:

Introduction

- P5, line 59: “an estimated 6.1 million people living with HIV at the end of 2016”

Please update the numbers and add a reference.

- P5, line 73: “(12,13).(14)”

Please correct this typo.

Methods

- P.6, line 90: “FSW not known to be HIV-positive on ART”

This expression is not clear, why the specification “on ART”?. Do you mean “FSW not known to be HIV-positive” ?

- P. 8, lines 130-132. Syphilis tests.

There may be some confusion when using the terms “rapid test” and “RPR”. Can you make it clear that the rapid is a treponemal, and the RPR is a nontreponemal test. One could also argue that one positive RPR test is not sufficient to detect active syphilis. According international guidelines there is a need of a fourfold change in titer, equivalent to a change of two dilutions to demonstrate an active infection.

Results

- P. 13, line 237: “13.2 ± 7.7”

Please specify that 7.7 is a standard deviation (I guess)

- P. 13, line 241: “the main reasons for not completing final visits”

The reason for not completing final visits is not the main issue I guess, but the reason for not being retained on ART, whether they come for a final visit or not.

- P. 15, line 277: “were infected or had a history of NG/CT”

Was this a history of an infection “ever”? It would make more sense to separate the biological results (STI at baseline) and the interview data “did you ever had an STI”, because they are two separate things.

- P .20, table 3 “Mean”

Please add “Mean CD4” for clarity in the title and the column head

- P. 20, table 3: p-values

The statistical methods (tests) for this table are not shown, please indicate which tests were used to obtain both type of p-values (p-trend and p-value), and explain in the methods.

- P. 23, table 4: p-values

Idem as previous comment.

- P. 24, lines 363-367: “the proportion … increased with increasing adherence (p=0.06 and 0.003)”

The results in the table do not confirm this statement: the proportion of visits where viral load was suppressed was 83.2% for the group with 90% adherence and 84.2% for the group 75-89% adherent.

- P. 24, table 5: results GEE

The table is quite difficult to read. The column with the head “Ratio” seems to present prevalences, not prevalence ratios. A prevalence is also a ratio in theory, but in this situation the term “ratio” adds to confusion.

- P. 24, table 5, p for trend (p=0.06 and 0.003)

Which test has led to these p’s? It is curious to see a p for trend of 0.06, when the PR are exactly the same in all adherence categories (with the exception of the reference category). Idem for the p for trend 0.03 for the outcome “undetectable”, where PR go from 3.2 for the group with the best adherence, vs 3.3 for the group with adherence 75% and 50%.

- P. 24, table 5: results GEE

The table only show the PR of the virological response per adherence level, the time factor is not presented. Have the authors considered to check potential confounders including age, behaviour factors such as condom use ? This may provide interesting alternative explanations for the relation adherence/virological outcome.

- P. 28-30, table 6

Please revise lay-out of the table, as the text in the lines is not positioned in a standardized way, which make the table difficult to read.

Discussion

- P. 31, line 440-441

Revise structure. The logical flow can be improved by replacing: “since half of them are not Beninese, and even when they were …”

- P. 32, line 453: “mobility”

Mobility is indeed be a serious barrier. The authors may discuss here the access of the FSW to ART services if they move to another place, another country.

- P. 32, line 466

Correct typo: “fact”

- P. 34, lines 509-512: “The results revealed … interventions”

This paragraph is repeated later in the discussion. It is a general conclusion and should be moved to the end of the paper.

- P. 35, lines 527-529

The authors should be cautious in their conclusions about the association between self-reported adherence and viral load, taking into account the results (see higher)

Reviewer #2: General comments

1. Although the study describes a TasP/PrEP demonstration project, limited data are provided about PrEP

2. The accession numbers of the nucleotide sequences generated in this project are not provided

3. The number of Tables should be reduced

Page 26, HIV strains and drug resistance (details in Table 6)

1 Authors provide subtype classification twice (i.e. CRF02_AG/CRF02_AG). This should be explained

2 The prevalence of resistance mutations should be reported

**********

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Reviewer #2: No

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27 Sep 2019

Dr Orna Mor, Ph.D.

Academic Editor

PLoS ONE

RE: Response to reviewers - Manuscript ID: PONE-D-19-15725

Dear Editor,

We would like to sincerely thank the editor and the reviewers for the comprehensive evaluation of our manuscript entitled “HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin” that has been submitted for publication in PLOS ONE.

We are excited the reviewers found our manuscript to be of significance to the field. Their pertinent comments and suggestions have definitely contributed to strengthen our paper, and as such, we have done our best to address all the editor’s and reviewers’ concerns. These changes are highlighted in the manuscript with the Word “Track changes” function. Since major revisions were brought and as requested in the letter from the academic editor, a “clean” version of the manuscript is also provided. As the revised manuscript also includes two figures, we have uploaded the figure files separately and have written the figure title and legends following the text that explains about the figure.

Please find below the comments and questions (numbered and italicized) followed by our answers and details about the corrections that we performed on the original manuscript. Please note that when we refer to line numbers, those refer to line numbers as read in the clean version of our revised paper without tracked changes.

The revised manuscript is now ready for further consideration by PLoS ONE. We sincerely hope that it will now be suitable for publication in your very interesting journal.

Best Regards,

Mamadou Diallo and Michel Alary (on the behalf of all coauthors)

Note from the Editor:

1) We note that you have indicated that data from this study are available upon request.

Response: Indeed, as we mentioned while submitting the manuscript, data are available and can be made accessible. We have now provided a minimal anonymized data set necessary to replicate our study findings. This file uploaded as a supporting Information file can be accessed in the PloS ONE website.

Additional Editor Comments (if provided):

The authors report on the results of a TasP demonstration study among a small cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin. In general, the study is clear and well written and interesting. However, the number of tables versus size of study is uncomparebly high.

2) Table 1 could be removed and explained in the text

Response: We have removed the table 1 and explained the corresponding data in the text (page 12-13: line 230-233).

3) A graph of cd4 and vl versus visits could replace tables 3 and 4.

Response: We have replaced tables 3 and 4 by Figures 1 and 2 (2 figures needed to provide enough details) that we have uploaded as separate files. Figure titles and legends are provided in the manuscript itself (see page 19, lines 282-289 for changes in CD4, and page20-21: lines 313-318 for viral response). We have also slightly modified the text describing these 2 figures to provide some of the actual numbers that were in the table and that are somewhat less clear in a figure format (see page 18-19, lines 269-276 for changes in CD4 and page 20, lines 300-308 for viral load).

4) Table 5 is unclearly presented

Response: We have entirely reformatted table 5, which is now table 2 (see page 21).

5) On the other hand, a table summarizing the prevalence and type of baseline DRM in the different class of drugs versus subtypes is missing.

Response: The information on baseline DRM was already present in former Table 6; indeed, we agree it was badly formatted. We have also entirely reformatted this table that is now Table 3 (see page 24-25). In addition, we now provide data on baseline prevalence of DRM in the text (page 23, lines 350-367).

6) The outline of Table 6 is also unclear and if a new summary table is provided- then table 6 can be in appendix.

Response: See our response to the previous comment.

7) Please also check if all references are correctly written and according to PLOS policy.

Response: We have checked the references and adjusted them to PLOS policy where necessary.

Reviewer #1: General comments:

8) The authors report on the results of a Treatment as Prevention demonstration study among a cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin. Their findings on acceptability, retention and viral load provide important and relevant information which should be taken into account when implementing TasP among key populations in Africa. The general methods seem adequate. Statistical methods and analysis however should be verified. Some of the statistical tests used are not mentioned. The authors use generalized estimating equations to analyse time trends and the association between adherence and virological response however the results of the model are not clear and complete. It is also a pity that no attempt for identifying or adjusting for confounders have been made.

Response: We thank the reviewer for addressing this aspect of the methodology of data analysis, and his willing to have more information in the methodology. We hereby provide detail information on the methodology and reasons we used for the analysis. Indeed, since we had repeated data, the use of generalized estimating equations with the log –link and the type3 test of Wald seemed to be more appropriate for the analysis of time trend changes as well as the association between viral load and self-reported adherence. While analyzing the association between viral load and self-reported adherence, the final model controlled for time duration since ART initiation. We also initially included potential confounders such as age, country of origin, duration in sex work, having a regular partner (or not), and number of clients (but not reported condom use as it was reported to be extremely high: see Table) in the multivariate model, however, all these variables did not confound the association between self-reported adherence and viral load. Therefore, we did not keep them in the final model in order to maximize statistical power. We have made some changes in the section on statistical methodology to better describe our approach (see page 11, lines 197-202) as well as in the results section when reporting the associations between self-reported adherence and viral load (see page 21, lines 324-332).

Specific comments:

Introduction

9) - P5, line 59: “an estimated 6.1 million people living with HIV at the end of 2016”

Please update the numbers and add a reference.

Response: We have updated both the numbers and the reference. (Page 5: line 58-60).

10) - P5, line 73: “(12,13).(14)”

Please correct this typo.

Response: Corrected (page 5: line 73).

11) Methods

- P.4, line 93: “FSW not known to be HIV-positive on ART”

This expression is not clear, why the specification “on ART”? Do you mean “FSW not known to be HIV-positive”?

Response: We thank the reviewer for pointing out this ambiguity. Indeed, this expression was misplaced. It’s only after the confirmation of the screening test at the Dispensaire IST, that we restrained recruitment to HIV-positive FSWs who were not known to be on ART. Being ART naïve were confirmed following responses to questions, and confirmation the clinic records as the vast majority of HIV-positive FSWs in Cotonou who are on ART receive their treatment at Dispensaire IST). In the manuscript, the deletion at the inappropriate location of the manuscript is on page 6: line 90-91 and the addition is on page 6, lines 93-95.

IST (in order to confirm that participants were ART-naïve)

12) - P. 8, lines 130-132. Syphilis tests.

There may be some confusion when using the terms “rapid test” and “RPR”. Can you make it clear that the rapid is a treponemal, and the RPR is a nontreponemal test. One could also argue that one positive RPR test is not sufficient to detect active syphilis. According international guidelines there is a need of a fourfold change in titer, equivalent to a change of two dilutions to demonstrate an active infection.

Response: We have now specified that the rapid test was treponemal and that the RPR was non-treponemal (page 8: Line 130-133). Note also than in cross-sectional studies (and all data presented in table 1 are from baseline and thus cross-sectional), it is usual to apply the definition we used for active syphilis. We don not report follow-up data on this issue, but please note that, in addition to not having a single case of active syphilis at baseline, there was no new case of syphilis during follow-up.

Results

13) - P. 13, line 237: “13.2 ± 7.7”

Please specify that 7.7 is a standard deviation (I guess)

Response: This is now specified (page 12: line 230).

14) - P. 13, line 241: “the main reasons for not completing final visits”

The reason for not completing final visits is not the main issue I guess, but the reason for not being retained on ART, whether they come for a final visit or not.

Response: In fact, we cannot know if the women who did not complete follow-up in the study were retained on ART or not. It depends if they accessed ARV clinics after leaving the study (most of them were not in Cotonou anymore and many were in other countries). Therefore, the reasons given in the manuscript are for withdrawing from the study. The wording of this section has also been changed because of the deletion of original table 1 and the addition of new text following a request from the editor (see page 9: 230-236).

15) - P. 15, line 277: “were infected or had a history of NG/CT”

Was this a history of an infection “ever”? It would make more sense to separate the biological results (STI at baseline) and the interview data “did you ever had an STI”, because they are two separate things.

Response: We thank the reviewer for pointing out this mistake. The prevalence we provided was in fact for the actual biological results of the nucleic acid amplification tests for NG and CT and did not include past history collected by questionnaire. This is now corrected (see page 13, lines 249-252).

16) - P .20, table 3 “Mean”

Please add “Mean CD4” for clarity in the title and the column head

Response: As per the editor’s request, we have replaced table 3 by figure 1, but mean CD4 count is now specified in this figure’s title and legend (page 19: Line 282-292).

17) - P. 20, table 3: p-values

The statistical methods (tests) for this table are not shown, please indicate which tests were used to obtain both type of p-values (p-trend and p-value), and explain in the methods.

Response: We described the analysis methodology in the method section. For all the analysis, we used generalized estimating equations with the log–link and the type3 test of Wald (see page 11: Lines 197-202), (see also our response to comment 8 above). Note that table 3 has now been replaced by figure 1.

18) - P. 23, table 4: p-values

Idem as previous comment.

Response: Same response as to the previous comment. Note also that table 4 has now been replaced by figure 2. (see page 11: Lines 197-202).

19) - P. 24, lines 363-367: “the proportion … increased with increasing adherence (p=0.06 and 0.003)”. The results in the table do not confirm this statement: the proportion of visits where viral load was suppressed was 83.2% for the group with 90% adherence and 84.2% for the group 75-89% adherent.

Response: We used 4 adherence categories in this analysis and evaluated a test for trend over the 4 categories and these are the p-values we obtained (see our response to comment 8 above). This can happen even if the various categories yield similar prevalence ratios when compared to the reference category as long as the association is strong enough. However, we agree with the reviewer in the fact that it would be important to qualify this statement by evoking the stability of the prevalence ratios as long as the adherence level is >50%. We have thus added a sentence in this regard in the result section (see page 21, lines 330-332).

20) - P. 24, table 5: results GEE

The table is quite difficult to read. The column with the head “Ratio” seems to present prevalences, not prevalence ratios. A prevalence is also a ratio in theory, but in this situation the term “ratio” adds to confusion.

Response: As per the editor’s request, we have completely reformatted this table, that is now table 2, and we have changed the column header as suggested by the reviewer. (see page 21-22: line 334-340).

21) - P. 24, table 5, p for trend (p=0.06 and 0.003)

Which test has led to these p’s? It is curious to see a p for trend of 0.06, when the PR are exactly the same in all adherence categories (with the exception of the reference category). Idem for the p for trend 0.03 for the outcome “undetectable”, where PR go from 3.2 for the group with the best adherence, vs 3.3 for the group with adherence 75% and 50% (page 15: line 334).

Response: See our response to comments 8, 17 and 19 above.

22) - P. 24, table 5: results GEE

The table only show the PR of the virological response per adherence level, the time factor is not presented. Have the authors considered to check potential confounders including age, behaviour factors such as condom use? This may provide interesting alternative explanations for the relation adherence/virological outcome.

Response: As time since treatment initiation was treated as a confounding factor for the association between adherence levels and viral load and that we were mainly interested by this latter association, we do not present prevalence ratios for this time variable to keep the table as simple as possible for the readers. Concerning the other potential confounders, see our response to comment 8 above.

23) - P. 28-30, table 6

Please revise lay-out of the table, as the text in the lines is not positioned in a standardized way, which make the table difficult to read.

Response: This table has now been entirely reformatted and its number as changed to table 3 (see page 24-26).

Discussion

24) - P. 31, line 440-441

Revise structure. The logical flow can be improved by replacing: “since half of them are not Beninese, and even when they were …”

Response: We have reviewed and rephrased the sentence as suggested (page28: line 418-423).

25) - P. 32, line 453: “mobility”

Mobility is indeed be a serious barrier. The authors may discuss here the access of the FSW to ART services if they move to another place, another country.

Response: We added some discussion on this issue (see page 29, lines 433– 443).

26) - P. 32, line 466

Correct typo: “fact”

Response: Corrected (page 29: line 433)

27) - P. 34, lines 509-512: “The results revealed … interventions”

This paragraph is repeated later in the discussion. It is a general conclusion and should be moved to the end of the paper.

Response: We removed this paragraph as suggested by the reviewer (see page 32, lines 499-502). At the same time, since this sub-section now only present limitations, we changed the name of the sub-section “Study strengths and caveats” to “Study limitations”.

28) - P. 35, lines 527-529

The authors should be cautious in their conclusions about the association between self-reported adherence and viral load, taking into account the results (see higher)

Response: We have made some changes in order to be more cautious with this conclusion (see page 32, line 506-510).

Reviewer #2: General comments

29) Although the study describes a TasP/PrEP demonstration project, limited data are provided about PrEP

Response: We thank the reviewer for his interest in the PrEP data. Although our project focused on TasP and PrEP, we only report data on TasP in this paper. Indeed, we have already published a paper on the overall resust of our TasP/PrEP study in the Journal of the International AIDS Society (JIAS) (see 20 of the present study or DOI: 10.1002/jia2.25208). The title of this paper is “Early antiretroviral therapy and daily pre-exposure prophylaxis for HIV prevention among female sex workers in Cotonou, Benin: a prospective observational demonstration study. However, the results presented in the present paper include some additional TasP subjects and present analyses that are not included in our JIAS paper.

30) The accession numbers of the nucleotide sequences generated in this project are not provided

Response: We perfectly agree with the reviewer that the nucleotide sequences generated in this project should be provided. Unfortunately, we did not think about recording these sequences in Genbank. However, if requested, we can provide s the sequences for verification.

31) The number of Tables should be reduced

Response: This has been done: we went from 6 tables to 3 tables and 2 figures following the editor’s comments.

32) Page 26, HIV strains and drug resistance (details in Table 6)

Authors provide subtype classification twice (i.e. CRF02_AG/CRF02_AG). This should be explained

Response: As to our understanding of the question, the reviewer asks us to explain why we reported the same subtype twice (i.e. CRF02_AG/CRF02_AG). This is a standard terminology for presenting the results of genotyping for drug resistance. Indeed, sequencing the HIV-1 pol and env genes is routinely used to identify mutations associated with resistance to reverse transcriptase (RT) or protease (PR) inhibitors in HIV-1 circulating recombinant forms (CRFs) including CFR02_AG and CRF01_AE. Samples were genotyped at both env and pol regions. When the results yielded identical sequences in the two regions, we reported the same sequence twice (ex.: CRF02_AG/CRF02_AG), otherwise we reported differently (i.e. CRF11-cpx/CRF02_AG). This appears in the revised table 3 (i.e. G/CRF02_AG and CRF11-cpx/CRF02_AG) and shows the different strains in our study.

The prevalence of resistance mutations should be reported

Response: We now report the prevalence of resistance mutations in the text of the manuscript (see page 22-23, lines 345-353).

Submitted filename: Response to reviewers.docx

Click here for additional data file.

25 Oct 2019

PONE-D-19-15725R1

HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin

PLOS ONE

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Additional Editor Comments (if provided):

Thanks you for submitting the revised manuscript. The previous corrections requested by the reviewers were met. However, there are still few minor remarks on the revised version. I do not find that the correction requested by reviewer 1 is necessary as indeed as stated and discussed t is possible that some of the FSW were on ART previous the study. Therefore I suggest to leave the sentence "HIV positive FSW not known to be on ART"

Regarding the remark of reviewer 2- I agree, if it is possible.

The followings are few remarks that if corrected, could better clear this paper:

Line 139: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT). In Table 1 : N. gonorrhoeae or C. trachomatis. Please choose either NG/CT or N. gonorrhoeae - C. trachomatis throughout the text.

Page 9 148-151: nuclisens can officially assess HIV-1 only (not HIV-2). Only one publication (ref 21) assessed its performance on HIV-2 subtype A samples only and claims detection limit of 200 c/ml. This is not what is stated here. I suggest to remove HIV-2 from this sentence.

Also the limit of quantitation for HIV -1 has changed during the years. Please provide the version number of the VL kit used in this study and give the updated LOQ for the version used herein (as far as I recall it was never 40c/ml).

LINE 151: Replace HIV by HIV-1 (to exclude HIV-2)

Line 179: vl <40 cannot considered undetectable. If 40 is the quantification limit It could be considered as below quantification limit. If <40 is the LOQ for the assay used herein, please amend the term “undetectable” ( in cases of VL <40 C/ML) to “below quantification limit” all through this paper.

Line 282: Fi 1 change to Figure 1

Line 296: term “suppressed viral load” and “undetectable viral load” should be explained in the text and not only in the figure legend, especially as <1000 is not regularly considered “suppressed…”. Please discuss to these numbers in the discussion section as today VL >200 (SOMETIMES >50) is considered virological failure.

Table 1 : need restructuring. Numbers are not in lanes and some do not fit the space

Table 3: needed reformatting and detailing. What does NA mean? Why some spaces are empty? The list of ART for each sample is the drugs which could be taken and are no affected by the identified DRM? Why to include drugs that are not in use any more? The subtype is given for both RT and PR? Please provide detailed information so that the reader will easily understand.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Just one small correction to make: revised clean text, line 93 "HIV positive FSW not known to be on ART" is confusing, should be "HIV positive FSW not on ART"

Reviewer #3: This is a strong paper that I enjoyed reading. The one remaining issue that I would strongly recommend the authors to consider is to present a comparison of baseline characteristics of participants who did and not complete the follow-up to give readers a better sense of the types of participants who dropped out of the study.

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Reviewer #1: No

Reviewer #3: Yes: Timothy P Johnson

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9 Dec 2019

Note from the Editor:

1) Page 8 line 139: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT). In Table 1: N. gonorrhoeae or C. trachomatis. Please choose either NG/CT or N. gonorrhoeae - C. trachomatis throughout the text.

Response: We have chosen to use N. gonorrhoeae and C. trachomatis throughout the text (page 8, line 140-141; page 14 line 257; table 1); except for the name of the Probetec assay used to test for these infections, as NG/CT is part of the brand name of the assay.

2) Page 9 148-151: nuclisens can officially assess HIV-1 only (not HIV-2). Only one publication (ref 21) assessed its performance on HIV-2 subtype A samples only and claims detection limit of 200 c/ml. This is not what is stated here. I suggest to remove HIV-2 from this sentence.

Response: Indeed, Biomerieux-diagnostics mentioned that NucliSens® HIV-1 QT assay is generally insensitive for detection of HIV-2 RNA. In addition, samples from individuals infected with HIV-2 may exhibit cross-reactivity in this assay. We therefore, have removed HIV-2 from this sentence, as suggested (page 9: line 151). Please also note that, as already stated in the manuscript, subjects with evidence of HIV-2 infection were excluded from the study.

3) Also the limit of quantitation for HIV-1 has changed during the years. Please provide the version number of the VL kit used in this study and give the updated LOQ for the version used herein (as far as I recall it was never 40c/ml).

Response: We used the NucliSens EasyQ® HIV-1 v2.0 kit. 48 tests - Ref: 28 5033. The kit is of high-level sensitivity with validated input volumes plasma, can detect the majority of HIV-1 subtypes including: A, B, C, D, F, G, H, J, CRF01_AE and CRF02_AG with a viral load measuring range between: 10-10 000 000 copies/ml.

This kit provides detection limits according to volume of plasma used:

Flexible Input Limit of Detection (LoD)

1 ml plasma 25 cps/ml

0.5ml plasma 50 cps/ml

0.1 ml plasma 292 cps/ml

Dry Blood Spot (0.1 ml) 802 cps/ml

We used 0.5 ml plasma, therefore the lowest viral load we could amplify was 50 copies/ml (page 9: line 151).

4) Page 9 line 151: Replace HIV by HIV-1 (to exclude HIV-2)

Response: We have replaced HIV by HIV-1 (page 9, line 152).

5) Page 10 line 179: vl <40 cannot considered undetectable. If 40 is the quantification limit it could be considered as below quantification limit. If <40 is the LOQ for the assay used herein, please amend the term “undetectable” (in cases of VL <40 C/ML) to “below quantification limit” all through this paper.

Response: Thank you very much for underlining this ambiguous misuse of “undetectable” instead of “below quantification limit”, “undetectable” being a term too often used in viral quantification. Since we used the NucliSens EasyQ® HIV-1 v2.0 kit. 48 tests - Ref: 28 5033 and 0.5 ml plasma, the lowest viral load we could amplify was 50 copies/ml. We therefore have amended the term “undetectable” (in all cases of VL <40 C/mL) to “below quantification limit” all through the manuscript, and the figure 2. Please, see (page 3: line 43-45; page 9: line 151; page 10 line 178-185; page 11 line 201-202; page 18 line 304-307; page 19 line 313-327; page 20 line 338-340, table 2; page 26 line 397).

6) Page 18 line 282: Fi 1 change to Figure 1

Response: We have changed Fi 1 to Figure 1 (page 18: line 290).

7) Page 19 line 296: term “suppressed viral load” and “undetectable viral load” should be explained in the text and not only in the figure legend, especially as <1000 is not regularly considered “suppressed…” Please discuss to these numbers in the discussion section as today VL >200 (SOMETIMES >50) is considered virological failure.

Response: The term viral suppression or “suppressed viral load”, when viral load is <1000 copies/mL was used to evaluate treatment success by the World Health Organisation (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS). According to the 2016 WHO Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection, a viral load <1000 copies/mL defines treatment success. The term “undetectable” viral load was defined as viral load below detection limits of the assay used for viral load quantification in the study (page 10 line 180-184, page 26 line 400-402). For the latter, we however now use “below quantification limit” instead of “undetectable” throughout the manuscript.

8) Table 1: Needs restructuring. Numbers are not in lanes and some do not fit the space

Response: We have restructured Table 1 so that numbers are in lanes and fit the space (page 14-17).

9) Table 3: needed reformatting and detailing. What does NA mean? Why some spaces are empty? The list of ART for each sample is the drugs which could be taken and are no affected by the identified DRM? Why to include drugs that are not in use any more? The subtype is given for both RT and PR? Please provide detailed information so that the reader will easily understand.

Response table 3: NA meant not applicable as there was no resistance mutation to this class of ARVs; some spaces were empty for the same reason. We have now decided to leave all these spaces empty when no resistance mutation was present to a given ARV class. The list of specific drugs is quite useless at the end and we have suppressed this column entirely in order to make the table easier to read. We have provided the sequences corresponding to the protease and the reverse transcriptase regions (PR/RT), and this is now better explained (table page 23-24: table 3).

Submitted filename: Response to reviewers_ii.docx

Click here for additional data file.

16 Dec 2019

HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin

PONE-D-19-15725R2

Dear Dr. Alary,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing all issue and correcting the paper. I have enjoyed reading it and look forward to seeing it on pubmed.

I still find Table 3 not lined up- but I guess you can improve it when the paper is processed for publication.

Reviewers' comments:

8 Jan 2020

PONE-D-19-15725R2

HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin

Dear Dr. Alary:

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