Alarming rate of transmitted drug resistance mutations and genetic diversity in newly HIV-1-infected patients in Benin

Drug resistance mutations were detected in (27/248; 10.9%) according to the WHO SDRM 2009 list, with predominance of mutations directed to NNRTIs drugs (24/248; 10%). Phylogenetic and recombination analyses showed a predominance of CRF02_AG strains (165/248; 66.5%) and a high genetic diversity with five other variants and 39 URFs (15.7%) which contained portions of strains that co-circulate in Benin. Eight recent transmission chains revealed active ongoing transmission of HIV-1 strains among ARV naïve patients. Our study complex Regular new


Background
Seventeen years after the start of the IBAARV (Beninese initiative for access to antiretrovirals), transmitted drug resistance mutations in ARV naïve patients and HIV-1 genetic diversity were investigated in Benin.

Conclusions
Our study showed a high primary drug resistance rate and a complex genetic diversity. Regular monitoring of primary drug resistance is required to adapt HIV-1 treatment strategies and adoption of new WHO recommendations in Benin.

Background
In 2019, about 37.9 million people were living with HIV that remains a major global public health problem [1]. Seven hundred seventy thousand people died from AIDS related illnesses and 1.7 million people became newly infected with HIV despite the expansion of antiretroviral treatment (ART) programs in 2019 [1]. An undesired consequence of antiretroviral therapy expansion is the selection of mutations [2,3] allowing viruses to become resistant to treatment. Factors that contribute to this occurrence in Sub-Saharan African are inappropriate use of drugs, changing medication frequently, interruptions in treatment due to financial difficulties. Transmission of these resistant viruses to therapy-naïve individuals could jeopardize the clinical benefits associated with ART [4] and effectiveness of first-line ARV treatment. This leads increasing the need of second-line regimens which are available in many Sub-Saharan Africa countries because the cost of second-line antiretroviral therapy limitation strategies have been reviewed [5]. Several studies reported variable rates of transmitted drug resistance ranging between 2% and 10.8%. In West Africa, rates of 8.3% and 10.8% were reported in Niger [6] and Togo [7] while rates of 4.2% and 8.2% were reported in Morocco [8] and Cameroon [9]. The rate of 3.9% was reported in Benin in 2012 [10]. To maximize the long-term effectiveness of first-line ART and ensure the sustainability of ART programs, it is essential to reduce the further spread of HIV drug resistance mutations. Therefore, WHO recommends that HIV treatment scale-up should always be accompanied by a robust assessment of drug resistance emergence and transmission (https://www.who.int/hiv/topics/drugresistance/protocols/en/). One of the five keys of WHO HIV drug resistance monitoring and surveillance strategy is the Surveillance of Transmitted HIVDR in recently infected populations.
Benin is a small country of 11 340 504 habitants located in West Africa. It is bordered by Nigeria in the East, Togo in the West, Niger in the North, Burkina Faso in North West and the Atlantic Ocean in the south. HIV prevalence was 1.2% in the general population in 2019 and 44231 infected individuals were under antiretroviral (ARV) treatment in 2019. In this study we report 7 years after the first study [10] and after 17 years of ARV circulation in Benin, HIV drug-resistance mutations and analyze whether the pattern of HIV-1 variants that circulate in Benin is stable over time in ARV naive HIV-1 infected individuals.

Study population
A total of 353 samples from antiretroviral-naïve HIV-1 infected individuals were studied. Samples were collected from October 2017 to December 2017 as recommended by WHO on nineteen facilities care covering all the country. The infected individuals were newly diagnosed with HIV-1 infection, and never exposed to antiretroviral therapy.

Blood Sampling and Processing
For all patients, blood samples were collected on EDTA tubes and RNA was extracted from plasma by using the QIAmp Viral RNA kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. The Nested PCR was performed on the entire protease gene and at least the first 240 amino acids encoding the reverse transcriptase (RT) as previously described [8], generating a fragment of 1017 base pairs (bp). After amplification testing, 248 individuals were eligible for HIV-1 drug resistance genotyping.

Phylogenetic Analyses
The newly obtained sequences were aligned with reference sequences representing the overall genetic diversity of HIV-1 in West and Central Africa (available from Los Alamos HIV sequence database: http://hiv-web.lanl.gov/), by using MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/) and G-Blocks to eliminate poorly aligned positions and divergent regions (molevol.cmima.csic.es/castresana/Gblocks_server). Phylogenetic tree reconstruction was done by the maximum likelihood method with the GTR+I+G model as implemented in Seaview v4.4.2 [13]. In addition, bootscanning analysis was done to explore any eventual mosaic structure for each strain with Simplot software and confirmed by phylogenetic analysis of the corresponding sub-segments in case of mosaic viruses [14]. Finally, recent transmission clusters were ascertained using the statistical robustness of ML topologies based on high bootstrap values (98%) with 1000 resamplings and short branch lengths following criteria previously defined [15].
The newly generated sequences were submitted to Genbank database under accession numbers: MT022598 to MT022684, MT022685 to MT022845 and MT022846 to MT023006.

Analysis of drug-resistance mutations
Newly nucleotide sequences of partial pol (protease and/or RT) were translated into amino acids and then inspected to identify mutations associated to drug resistance. Amino acid sequences were analyzed to determine the presence of mutations in protease and rt genes at positions known to be associated with drug resistance by using the latest (2009) version of the WHO list of mutations for surveillance studies [16].

Characteristics of the 248 Study Patients
The median age was 38 years [IQR: 18-82] with 64.1% women and 35.9% men. Nearly 90% of patients were over 25 years of age and nearly 46% were married and living with a partner. Some patients (n=23) were key populations members, 11 of whom were female sex workers (FSW) (blood samples were collected for 4 FSW) and 12 of whom were men who have sex with men (MSM) (blood samples were collected for 8 MSM). Also some patients (n=20) were coming from neighbouring countries such as Togo, Nigeria, Niger, Côte d'Ivoire, Burkina Faso, Ghana, Guinea, Gabon and Cameroon. The median viral load was 5.12log 10 [IQR : 2.20-7log 10 ].
A total of 42 drug resistance-associated mutations have been identified (Table 1)

The distribution of 248 HIV-1 group M variants in Benin
The phylogenetic tree analysis of the 248 pol sequences is presented in figure 1.

Discussion
The study reports the presence of mutations in ARV-naive patients and genetic diversity of HIV-1 variants that circulate in Benin. Globally, the prevalence of transmitted drug resistance was 10,89% after 17 years of ARV circulation in the city, that is consistent with results from other African countries in which prevalence rates are also higher than 10% [7, [17][18][19]. But, studies from some others Sub-Saharan countries have reported rates lower than 10%, by using the same WHO standard protocol and the WHO list of resistance mutations for epidemiological surveys [20,21]. HIV-1 drug resistance mutations were known to be one of the major factors limiting the effectiveness of ARVs. In our study, the mutations encountered were those associated with the ARVs used in first-line treatment since the start of the IBAARV (Beninese initiative for access to antiretroviral) in 2002. This first-line treatment used two NRTIs (AZT/D4T + 3TC) plus a NNRTI (NVP/EFV), NNRTI as well as non-boosted protease inhibitor (indinavir) regimens [22]. At that time, virologic monitoring was not available and patients were followed based on clinical signs and CD4 counts [23]. Moreover those who were in therapeutic failure stayed long periods of time with an ineffective treatment, leading to an accumulation of resistance mutations [24]. This accumulation of resistance mutations may compromise the effectiveness of second line drugs [25] and increases the risk of transmission of drug resistant strains to naïve patients [7]. Among naïve patients in our study, 27 already harboured at least one drug resistance mutations and the NNRTIs represent 10% while the NRTIs and PIs represent 6% and 1% respectively.
NRTIs resistance-associated mutations were present in sixteen patients. M184V was the predominant NRTI encountered which confers resistance to lamivudine (3TC). The K65R confers resistance to abacavir (ABC) and tenofovir (TDF). Thymidine-associated mutations (TAMs) were found but only one patient in our study harboured at least two TAMs conferring intermediate resistance to zidovudine (AZT). TAMs M41L, T215S have been described in Togo [7,19] and also in Burkina Faso with D67N and K219Q [19]. K70R was observed in one case in our study was also found in Guinea-Conakry [21].
For non-adherence reasons to treatment, M184IV is quickly selected in patients under 3TC which explains its presence in high proportion in studies of transmitted resistance [20,21]. Since the study was conducted after the time of TDF use (replacing D4T as recommended by WHO), the presence of K65R in patients could easily be explained.
The major mutations associated to NNRTIs were K103N (33%) encountered in fourteen patients which compromise effectiveness of NNRTIs first generation (NVP and EFV) and G190A (7%) identified in three patients which compromise nevirapine, efavirenz and etravirine [26]. These mutations associated with high-level resistance have been described in one and four patients in Togo and Phylogenetic analysis evidenced eight recent and three probable older transmission chains (6.5-8.9% of the study patients), reflecting active ongoing transmission. Interestingly, 8 patients reported as being MSM and from them, two were involved into the same transmission chain, in which one patient came from Togo and the second one was from Benin. Studies on MSM in Africa also evidenced behavioral links with heterosexual networks, in such a way that the MSM population could serve as a bridge for intermixing of HIV-1 variants between low-risk women and high-risk men. This situation was described in Dakar where the subtype C predominating in the MSM group is increasing in the general population [27,28], confirming the existence of a dual epidemic in the country. In our study we did not find any transmission chain involving both population groups individuals, however the number of MSM patients is too low to conclude. Obviously further studies are needed in key groups in order to assess whether HIV-1 strains from MSM intermix with those from the general population in Benin or with those from other countries.
In our study, the CRF02_AG predominated with 66.5% rate. Overall, the genetic diversity in Benin matches with results found in a neighboring country, Togo