Virological outcome among HIV-1 infected patients on first-line antiretroviral treatment in semi-rural HIV clinics in Togo

Background Access to antiretroviral treatment (ART) in resource-limited countries has increased significantly but scaling-up ART into semi-rural and rural areas is more recent. Information on treatment outcome in such areas is still very limited notably due to additional difficulties to manage ART in these areas. Results 387 HIV-1 infected adults (≥18 years) were consecutively enrolled when attending healthcare services for their routine medical visit at 12 or 24 months on first-line ART in five HIV care centers (four semi-rural and one rural). Among them, 102 patients were on first-line ART for 12 ± 2 months (M12) and 285 for 24 ± 2 months (M24). Virological failure was observed in 70 (18.1 %) patients ranging from 13.9 to 31.6 % at M12 and from 8.1 to 22.4 % at M24 across the different sites. For 67/70 patients, sequencing was successful and drug resistance mutations were observed in 65 (97 %). The global prevalence of drug resistance in the study population was thus at least 16.8 % (65/387). Moreover, 32 (8.3 %) and 27 (6.9 %) patients were either on a completely ineffective ART regime or with only a single drug active. Several patients accumulated high numbers of mutations and developed also cross-resistance to abacavir, didanosine or the new NNRTI drugs like etravirine and rilpivirine. Conclusion The observations on ART treatment outcome from ART clinics in semi-rural areas are close to previous observations in Lomé, the capital city suggesting that national ART-programme management plays a role in treatment outcome.


Background
Scale-up of antiretroviral treatment (ART) programs in resource-limited countries was possible because standardized first and second line antiretroviral (ARV) combinations and clinical and/or immunological criteria to start and monitor ART were used as recommended by WHO [1]. However, heterogeneous treatment outcomes have been observed in the national ART programs from different countries, most likely related to ART-programme management [2]. As such, virological failure can range from less than 3 % to more than 20 % in patients on ART for 12 or 24 months [2][3][4].
In Togo, a country of six million inhabitants in West Africa, scaling-up of ART started in 2007 in Lomé, the capital city, and has expanded to semi-rural areas in 2008. In 2013, almost 50 % of patients who were in need for ART according to WHO guidelines from 2010 (CD4 count <350) were receiving ART [5]. Previous studies in ART clinics in Lomé showed high virological failure related to ARV drug resistance, i.e. in 13 to 25 % of the patients receiving ART for 12 or 24 months [2,6]. Given the additional difficulties to manage ART in these areas

AIDS Research and Therapy
*Correspondence: a.dagnra@yahoo.fr 11 08 B.P. 8742, Lomé 08, Togo Full list of author information is available at the end of the article (distances to clinics, scarce human resources, drug stockouts, etc.) together with high rates of ARV drug resistance in the capital city, it was important to evaluate also virological outcome and emergence of drug resistance in ART clinics located in semi-urban and rural areas in Togo.

Study sites and population
A cross-sectional study was conducted in 2012 between January and July in five HIV care centers that administer ARV drugs and monitor treatment. They were located in four semi-rural cities: Aného (AN), Kpalimé (KP), Atakpamé (AT) and Kara (KA) at respectively 60, 120, 160 and 410 km from Lomé, the capital city, and in one rural city, Kouvé (KO) at 70 km distance from Lomé ( Fig. 1). HIV-1 infected adults (≥18 years) were consecutively enrolled when attending clinics for their routine medical visit at 12 ± 2 months or 24 ± 2 months on first-line ART. This study was approved by the National Ethics Committee (nº751/2014/MS/CAB/ DGS/DPLET/CBRS). Informed consent was obtained for each participating patient. Questionnaires were used to collect epidemiological and demographic information and ART history was obtained from on-site medical records. Whole blood was drawn and plasma was separated by centrifugation. Plasma aliquots were stored at −20 °C for maximum 1 week on site and were subsequently transported by road in a cool box to the Laboratoire de Biologie Moléculaire et d'Immunologie (BIOLIM/FSS-UL) where they were stored at −80 °C until use.

Virological analyses
HIV-1 viral load (VL) was determined with EasyQ HIV assay (Biomerieux, Capronne, France) or RealTime m2000rt (Abbott Pack, IL, USA) in Lomé (BIOLIM/FSS-UL). According to WHO recommendations, genotypic drug resistance testing was done in a WHO accredited laboratory (IRD, Montpellier, France) on patients with VL ≥1000 copies/ml. Protease and partial Reverse Transcriptase (RT) were amplified with the in-house protocol from the Agence Nationale de Recherche sur le Sida et les Hépatites en France (ANRS) [2,7]. Drug resistance mutations (DRM) were identified using the ANRS interpretation algorithm, version 24 [7]. The newly reported sequences are available in GenBank under the following accession numbers: KR047793-KR047859.
As expected, the observed DRM were associated with the drugs used in first-line regimens ( Table 2). M184 V selected by 3TC was the most frequent NRTI mutation, 56/65 (86.2 %). Frequently observed TAMs included M41L, D67 N/D, K70R, K219E/Q and T215Y/F. The K65R mutation was seen in two patients. One-third of the patients had at least 3 or more NRTI mutations and several patients were already predicted to be resistant to ABC (n = 12), DDI (n = 2) or tenofovir (TDF) (n = 3). Among NNRTI mutations, Y181C/Y and K103N were most frequently observed and 15 (23.1 %) patients accumulated at least three NNRTI mutations, with 14 (21.5 %) and 44 (67.7 %) that were predicted to be resistant to second line NNRTIs ETV and RPV, respectively.

Discussion
Overall, we showed that 18 % of patients on ART for 12 or 24 months in semi-rural and rural ART clinics were on virological failure and almost all of them (97 %) were infected with drug resistant HIV-1 strains. These observations are high and close to what has been noticed in previous studies from Lomé, the capital city; for example in a survey conducted about 1 year earlier in 2010/2011 using the same cross-sectional approach, 19 % (124/642) of patients for 12 or 24 months on ART were infected with HIV drug resistant strains [2]. Our study confirms thus a high ART failure in Togo in general and which is higher than observed in other countries, when comparing with studies that used a similar approach [2]. Scaleup of ART started in 2007 in the capital city and was expanded to semi-rural areas in 2008. Between 2006 and 2012, the number of patients on ART increased from 6700 to 31,500 but tools to monitor patients did not follow this scale-up and training of medical personnel was insufficient to allow early detection of side_effects of certain ARVs, evaluate adherence or recognize rapidely decline of clinical status. In addition, the national       AN012  24  K103N  -CRF02_AG  KR047794   AN019  24  Y181C, H221Y  M41L, V75I, M184V, T215F  CRF02_AG  KR047795   AN022  24  Y181C  M184V  URF  KR047797   AN028  24  A98S, Y181C  D67N, K70R, T215F, K219E  CRF02_AG  KR047799   AN032  24  Y181CY, G190AG, H221HY  -URF  KR047800   AN041  24  K103N, Y181C  A62V, K65R, K70T, V75I, F116Y, Q151M, M184V  CRF02_AG  KR047801   AN043  24  K101E, Y181C, G190A  M184V, T215F  CRF02_AG  KR047802   AN048  24  Y181C  D67N, K70R, T215F, K219E  CRF02_AG  KR047803   AN049  24  K103N  M184V  CRF06_cpx  KR047804   AN054  24  K103N  M41L, E44D, L74I, M184V, L210W, T215Y  CRF02_AG  KR047806   AN068  24  Y181C, H221Y  M41LM, M184V, T215Y  CRF02_AG  KR047809   AT400  24  Y181C  K70KR, M184V  CRF02_AG  KR047838   AT403  24  -M184V  A3  KR047839   AT420  24  K103N  M184V  CRF02_AG  KR047842   AT435  24  Y181C, H221Y  M41LM, D67DN, K70KR, T215Y, K219EK  CRF02_AG  KR047845   AT442  24  Y181C  M184V, T215FIST  CRF02_AG  KR047846   AT452  24  Y181C  M41L, M184V, T215Y  CRF02_AG  KR047847   AT456  24  K101E, G190A  M184V, T215F  CRF02_AG  KR047848   AT463  24  --CRF02_AG  KR047849   KA319  24  A98S, K103N,  program encountered problems with stock management resulting in ARV drug substitution with the same molecules, administered separately as individual pills instead as a fixed dosed combination, or even interruption of the treatment. It is known that non-adherence and treatment interruption may favor emergence of drug resistance. Today, only very few studies reported observations on ART outcome from semi-rural or rural areas in resource limited settings, especially from Africa [2,8,9]. In Cameroon, 10 % of patients were infected with drug resistant HIV strains after a median of 12 months on ART in rural district hospitals at 50 to 150 km distance from Yaoundé, the capital city [10], which is close to rates observed in Yaoundé [2]. However, in a rural health center in Kolofata, at the extreme north of Cameroon at 1200 km from the capital city and with difficult connections, almost 30 % of patients on ART (median of 24 months) were resistant to ARV drugs [11]. Another report showed equal proportions of drug resistance in urban and rural areas, between 9.2 to 15.9 % after a median of 36 months on ART in Senegal, Mali and Guinea [12]. In rural and semi-rural settings in Gabon, 21 % of patients were resistant after a median of 33 months on ART [13]. In a rural clinic in Tanzania, rates of drug resistance were low and ranged from 4 to 8 % of patients after 1 or 2 years on ART, respectively [14]. Although, it is important to note that comparing results among the different studies mentioned above has to be taken with caution, because study design can differ as well as viral load capacities and techniques to identify virological failure.
Overall it seems that treatment outcome varies among countries, but within countries treatment outcome in semi-rural settings seem to be similar to those in urban settings except in extreme conditions. These observations are in line with the fact that national ART-programme management plays a role in treatment outcome in resource-limited countries [2]. It is important to note that we provided only information on the proportion of drug resistance in HIV infected patients who are still on ART and have no information on follow-up or mortality rates. Prospective studies where loss of follow-up are considered as treatment failures, would probable yield higher virological failure rates. Previous studies showed a higher mortality rate and loss of follow up in rural areas during the first 3 years [15].
Like in other reports on treatment outcome, several patients in our survey accumulated high numbers of mutations and developed also cross-resistance to potential second and/or third line drugs [16][17][18]. In addition these multi-drug resistant strains can also be transmitted and have a negative impact on future efficiency of first line regimens.

Conclusions
The observations on ART treatment outcome in semirural areas show high failure rate but are close to those in Lomé, the capital city. Lowering the rates of drug resistance represents a challenge for the country. The first goals will be to identify factors associated with drug resistance.