# High Resistance Mutation to cART in HIV-1 Exposed Infected Children and Recent Emergence of CRF02_AG Variant in Bouar, A Rural Environment of Central African Republic

Ulrich Vickos, Giovanni Gaiera, Nicola Cotugno, Christelle Luce Bobossi

Gadia, Ornella Anne Sibiro Demi, Angelo Sala, Michela Sampaolo, Alain Le

Faou, Enzo Boeri

► To cite this version:

Ulrich Vickos, Giovanni Gaiera, Nicola Cotugno, Christelle Luce Bobossi Gadia, Ornella Anne Sibiro Demi, et al. High Resistance Mutation to cART in HIV-1 Exposed Infected Children and Recent Emergence of CRF02_AG Variant in Bouar, A Rural Environment of Central African Republic. Journal of Pediatrics, Perinatology and Child Health, 2022, 06 (01), pp.155-176. 10.26502/jp-pch.74050099. hal-03782529

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Research Article

High Resistance Mutation to cART in HIV-1 Exposed Infected
Children and Recent Emergence of CRF02_AG Variant in Bouar, A
Rural Environment of Central African Republic

Ulrich Vickos<fnref n="1" />, <fnref n="2" />*, Giovanni Gaiera<fnref n="3" />, Nicola Cotugno<fnref n="4" />, Christelle Luce Bobossi Gadia<fnref n="5" />,
Ornella Anne Sibiro Demi<fnref n="6" />, Angelo Sala<fnref n="7" />, Michela Sampaolo<fnref n="8" />, Alain Le Faou<fnref n="9" />, Enzo Boeri<fnref n="8" />

<footnote n="1">¹Infectious and tropical diseases Unit, Amitie Hospital, Bangui CAR</footnote>

<footnote n="2">²Laboratory of Immunity, Department of systemics medicine, Faculty of medicine and surgery, Tor Vergata University, Rome, Italy</footnote>

<footnote n="3">³Clinic of Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy</footnote>

<footnote n="4">⁴Academic Department of Pediatrics, Research Unit of Congenital and Perinatal Infection, Children's Hospital Bambino Gesù, Rome, Italy</footnote>

<footnote n="5">⁵Molecular biology Unity, Clinical biology and public health national Laboratory, Bangui, CAR</footnote>

<footnote n="6">⁶Laboratory of viral hepatitis, Institut Pasteur of Bangui, CAR</footnote>

<footnote n="7">⁷Saint Michel Center, Society of Priests of the Sacred Heart of Betharram, Health Pastoral Diocese of Bouar, Bouar, CAR.</footnote>

<footnote n="8">⁸Laboratory of Microbiology, San Raffaele Scientific Institute, Milan, Italy</footnote>

<footnote n="9">⁹Faculty of Pharmacy Brabois Santé, Vandoeuvre-lès-Nancy, France</footnote>

<footnote>*Corresponding Author: Ulrich Vickos, Infectious and tropical diseases Unit, Amitie Hospital, 2, Ouango-Bangui Mango Street, Bangui, 94045, Central African Republic, Email: vickos.ulrich@gmx.com</footnote>

<footnote>Received: 17 February 2022; Accepted: 25 February 2022; Published: 22 March 2022</footnote>

<footnote>**Citation:** Ulrich Vickos, Giovanni Gaiera, Nicola Cotugno, Christelle Luce Bobossi Gadia, Ornella Anne Sibiro Demi, Angelo Sala, Michela Sampaolo, Alain Le Faou, Enzo Boeri. High Resistance Mutation to cART in HIV-1 Exposed Infected Children and Recent Emergence of CRF02_AG Variant in Bouar, A Rural Environment of Central African Republic. Journal of Pediatrics, Perinatology and Child Health 6 (2022): 155-176.</footnote>
**Abstract**

**Introduction:** The emergence of HIV-1 recombinant forms and drug combined antiretroviral therapy (cART) resistance are frequent in the therapeutic course of HIV-infected children in Low and Middle-Income Countries (LMIC) precisely in Central African Republic (CAR) as evidenced by studies carried out in the Bangui capital. Vertical transmission rate including breastfeeding is 12.4. The aim is to analyze retrospectively the molecular characterization of sequencing results and mutation detected in HIV infected children under cART since infancy.

**Methods:** The 2019 retrospective review of the clinical, therapeutic, and immunological-molecular records of six children who were performed the genome sequencing, followed in Bouar, at the St Michel IST/HIV Center, in the north-west of the CAR. These perinatal HIV-infected children were not tested early and started late cART regimens, used for vertical transmission prevention and treatment initiation of HIV infection in CAR.

**Results:** We analyzed results from viral RNA extracted amplification and sequencing of 6 children plasma samples collected under first line ART. Sequencing of viral genomes revealed high level resistance mutations to NRTIs (ABC/FTC/3TC) and to NNRTIs (EFV/NVP used locally and DOR/ETR/ RPV unused) with ambiguous positions in amino-acids comparison and deletion. Three CRF02_AG strains formed a cluster by strongly detaching from other CAR and worldwide strains with robust bootstrap at 91.

**Conclusions:** The genomes sequencing showed that

resistance mutations made the treatment inefficient confirming the clinical, immunological, and virological failure and an emerging CRF02_AG genotype variant, probably of foreign origin. This discovery clearly highlights the importance of ART genetic resistance testing and personalized medicine.

**Keywords:** Children; cART Resistance Mutations; HIV-1 Subtype; Central African Republic

**Abbreviations:** 1st line sub: 1st line substitution; 3TC: Lamivudine; /r: ritonavir; ABC: Abacavir; ARI: Acute respiratory Infections; CAR: Central African Republic, cART: combined ART; CTM: Cotrimoxazole; DRI: Drug Resistance Interpretation; HAART: highly active ART; IR: Intermediate resistance; LLR: Low-level resistance; MoH: Ministry of Health; MS: Mutation Scoring; PI: Protease Inhibitor; NA: Not applicable; NNRTI: Non-nucleoside Reverse Transcriptase Inhibitors; NRTI: Nucleoside Reverse Transcriptase Inhibitors; HLR: High-Level Resistance; PLLR: Potential low-level resistance; PLWH: people living with HIV; S: Sensible/Susceptible; ATV/r: Atazanavir/r; DRV/r: Darunavir/r; LPV/r: Lopinavir/r; AZT: Zidovudine; FTC: Emtricitabine; TDF: Tenofovir; TBC: Tuberculosis; DOR: Doravirine; EFV: Efavirenz; ETR: Etravirine; NVP: Nevirapine; RPV: Rilpivirine

**1. Introduction**

HIV-1 prevalence is lightly decreasing in Central African Republic (CAR) with, in 2019, 4,900 new HIV infections, 3,800 AIDS-related deaths, and 3.5% people living with HIV (PLWH) as compared to 4.9% in 2010 [1, 2]. In 2016, an estimated 100,000 PLWH, among whom only 47% had access to antiretroviral therapy (ART). For the children, about
6,900 were infected and 3,156 were on ART. Treatment or prophylaxis of mother-to-child HIV transmission were available for 81% of pregnant women. Thus, it is estimated that less than 1000 newborns are infected each year [1]. The management of pediatric infections and the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa has dramatically improved. This has been possible by a widespread use of combined ART (cART) and highly active ART (HAART). However, this has been accompanied by the emergence of strains of HIV highly resistant to antiretroviral drugs (ARV). Numerous studies have evaluated the impact of their use on the emergence of drug resistance mutations (DRM) in infected children, mainly infants born to mother presenting either a failure of PMTCT or an absence of prophylaxis [3, 4]. However, less studies have been conducted in Central African rural areas [5-7]. It is easier to carry out studies in major cities given the accessibility and availability of biological and therapeutic means for the monitoring of HIV patients.

In Bangui, this is related to high rates of resistance to nucleotide reverse transcriptase inhibitor (NRTI), non-nucleotide reverse transcriptase inhibitor (NNRTI) DRM (45%) and protease inhibitors (PI) (24%) [8]. Overall, 55% of children receiving first-line treatment are eligible for a second-line regimen of whom 64% need a third-line treatment including an integrase strand transfer inhibitor (INSTI). Most HIV-infected African children fail to respond to the first- or second-line regimens recommended by WHO which is problematic as the available ARV drugs are in limited numbers [9, 10]. For pediatric HIV infections they are: Azidothymidine (AZT), Abacavir (ABC), Lamivudine (3TC), Truvada

(Emtricitabine/Tenofovir (FTC/TDF)), Efavirenz (EFV), Nevirapine (NVP), Duovir-N (Lamivudine/Zidovudine-Nevirapine (3TC/ZDV-NVP)), Atazanavir/Ritonavir (ATV/r) and Kaletra (Lopinavir/Ritonavir (LPV/r) junior). NRTI and NNRTI are favored for first line treatments. Besides no other ARV drug classes are available (Supplementary Table 2).

Two types of resistance to the available drugs are known. First two competitive mechanisms: one by preventing the insertion of the NRTI in the DNA chain, the other specific of thymidine analogues which once incorporated are released by the activity of pyrophosphorylase. The mutations associated with this process are known as thymidine analogue mutations (TAM). Second, non-competitive mechanisms are specific of the NNRTI. These molecules which impair the activity of the transcriptase by fixation in the vicinity of the active site of the enzyme, can no longer adhere to the enzyme [8]. The mutations associated with these resistances are known as primary mutations. Secondary mutations, alone, have in most cases a limited effect, but they increase the resistance level due to primary mutations when both present.

Each mutation is associated with resistance mechanism of its own and the corresponding resistance level varies widely from one to the other [11]. We have investigated the profile of resistance mutations to cARTs in 6 HIV+ children followed in the rural area of Bouar for whom, complete clinical, immunovirological and phylogenetic data have been obtained despite the limited number of patients.
## 2. Methods

### 2.1 Study design

This is a retrospective observational study of 6 HIV-infected children who belong to a cohort of more than 1,639 people living with HIV (PLWH) attending the "Saint Michel HIV/STI Center of Bouar for the Antiretroviral Therapy", a non-profit structure with limited resources. The mean time of ARV therapy initiation for the 6 children was 30 months after birth. These children have been exposed-infected by their HIV-infected mothers. They were prescribed a first-line treatment at inclusion which was not modified thereafter. For the 6 children complete socio-demographic, clinical, immunovirological and therapeutic data were collected from their follow-up medical file, at the time of obtaining blood samples for genotyping analysis. All information were gathered from the patient' files. The Center has only limited possibilities of diagnostic test thus genomic analyses were performed in Italy for the sequencing of integrase, polymerase, and protease genes.

### 2.2 Genomic analysis

Plasma samples from the 6 selected children, sent to the virology laboratory of the San Raffaele Hospital, Milan, Italy in ice pack. GenExpert HIV (Cepheid, Sunnyvale, CA), were used for viral load determination primarily in the Center. The viral integrase (IN), reverse transcriptase (RT) and protease (PR) genes were sequenced in Italy with the ViroSeq HIV-1 genotyping system (Stanford University, Stanford, CA), according to the manufacturer's instructions, as described previously [12].

### 2.3 Drug resistance associated mutations and genomic analysis

Mutations of RT and PR genes associated with

resistance to NRTIs, NNRTIs and PIs were identified and their consequences on resistance to ARV drugs interpreted according to the mutations scoring of the Stanford University genotypic resistance interpretation algorithm and the HIV Drug Resistance Database (https://hivdb.stanford.edu/page/release-notes/). The scores are the sum of each mutation penalty score for a given drug. Scores less than 10 indicate susceptibility; between 10 and 14 a potential low-level resistance; between 15 and 29 low-level resistance; between 30 and 59 intermediate resistance. Scores of 60 or greater indicate high-level resistance.

For genomic analysis, first, HIV-1 subtypes were determined using the IN gene sequences according to the genotyping tool of the NIH available on line (https://www.ncbi.nlm.nih.gov/projects/genotyping/fordmpage.cgi). From INSDC database, 1,227,800 coding sequences (CDS) belonging to taxonomic IDs 11,676 (HIV-1 M), 11,709 (HIV-2) and 12,721 (others HIV-1 O et N) were retrieved. CDS were dereplicated (884,488 unique CDS) and clustered (636 groups) with Biomanda Data to group similar CDS based on the sequence similarity and the gene annotation. CDS cluster composed of 578 229 CDS for 454,944 unique ones was selected.

Second, for strains comparisons, after the raw sequences from the 6 patients have been curated to remove sequencing errors, the nucleotide positions 2 096 to 2 551 of the genome of the reference HIV strain AF033819 was retained. It corresponds to the 195 first amino acids of the A chain of the reverse transcriptase. Eighty-four unique sequences of the target region and from the gag-pol gene CDS cluster were collected from GenBank. They correspond to
the most frequent sequences available from African
Countries and other main countries (USA, France,
Canada, United Kingdom, China, etc.). Moreover,
300 sequences from CAR were included. CDS were
aligned to the reference genome sequence using
Clustal Omega algorithm. A phylogenetic reconstr-
uction by maximum likelihood from PhyML
algorithm (Model GTR) with a bootstrap analyzes of
1 000 replicates were performed on nucleic acid
sequences. Phylogenetic tree was annotated with the
available metadata on sequences. HIV-1 IN, RT and
PR sequences were deposited at the GenBank
Nucleotide Archive database with the accession
numbers MW373071 to MW373076.

## 2.4 Statistical analyses

The studied children characteristics and the analyzes
results were entered into a Microsoft Excel data
sheet.

## 3. Results

The available and complete 6 patients' data are
displayed in Table 1. The mutation scorings and drug
used are detailed (Supplementary Table 1 and Figure
1a/1b).

* Patient 1: In the PR gene an accessory mutation of resistance to PI (V32S) was found. Several major mutations of the RT gene and were associated with resistance to NRTI (L74LI, M184V) and NNRTI (K103N, E138Q, Y188F). The mutation scoring is from -10 to 130. The patient was an immunological and virological partial responder.

* Patient 2: The PR gene sequence was not obtained. The resistance mutations to NRTI (L74V, Y115F, M184V) and NNRTI (Y181C) give a mutation scoring varying from -10 to 120, high-level resistance for ABC (Supplementary Table 1). A high viral load was associated with a low CD4 count.

* Patient 3: Accessory resistance mutations in PR gene was found (G73A along with several others) and comprising the highly uncommon L76 deletion. In the RT gene, resistance mutations to NRTIs (L174V, Y115F) and NNRTIs (K103N, Y181C) were noted. The mutations scoring was from -10 to 120 (Supplementary Table 1), high-level resistance for TDF, ABC and NVP. Despite a high CD4 count, the viral load was elevated. Patient 2 and patient 3 were from the same mother.

* Patient 4: No major resistance mutations were observed in the PI gene. The presence in the RT gene of low-level resistance mutations to NRTI (M184I) and intermediate one to NNRTI (Y181C) were found. The mutation scoring varied from -10 to 60 (Supplementary Table 1). This child has been lost to follow-up for 2 years. The last analyzes were made 1 year and a half after his return to follow-up.

* Patient 5: No major resistance mutation for PIs has been noted. However, resistance mutations were found solely to NNRTI (K103N, V106I, H221Y, F227L, Y181C) and none to NRTI. In this patient, mutation scoring varied from 25 to 115 (Supplementary Table 1), resistance exclusively to NNRTI. The viral load was remarkably high and the CD4 count low.

* Patient 6: The sequencing of the PI gene was unsuccessful. Resistance mutations to NRTI
(M184V) and to NNRTI (K103N, V108I) were observed with a scoring of -10 to 75. The viral load was moderately elevated while the CD4 count result was satisfactory.

The 6 patients' strains harbored the M184V mutation (corresponding to a hyper susceptibility to Zidovudine/Azidothymidine (ZDV or AZT). This mutation has been shown to make HIV susceptible to AZT(ZDV) by reduction of HIV-1 replication (13,14) which is the case of our patients (Figure 1a/b). Two accessory RT resistance mutations are also present in

the 6 strains (Q174E/K, V35I/K/T). The phylogenetic comparison of RT sequences shows that HIV-1 A (patient 1), G-J (patient 5) and 1 of the CRF02_AG strains (patient 4) were found into their respective genotype. On the contrary, 3 CRF02_AG strains (patients 2, 3 and 6), are isolated in the corresponding genotype with a branch longer than the others and a bootstrap at 90, testifying the robustness of the relationship between these three strains when compared to Central African strains (Figure 2). The results were identical when compared to strains of different origin (Supplementary Figure 1).
<table><thead><tr><th>Characteristic collected</th><th>Patient 2</th><th>Patient 3</th><th>Patient 4</th><th>Patient 5</th><th>Patient 6</th></tr></thead><tbody><tr><td>Age, years (y) months (m)</td><td>7 y</td><td>5 y</td><td>3 y</td><td>11 y</td><td>19 m</td><td>16 y</td></tr><tr><td>Gender, Male and Female</td><td>Male</td><td>Female</td><td>Male</td><td>Female</td><td>Female</td><td>Female</td></tr><tr><td colspan="7"><strong>Clinical</strong></td></tr><tr><td>CTM prophylaxis (Systematic)</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Immunization history (MoH)</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td colspan="7"><strong>Infectious antecedents</strong></td></tr><tr><td>Malaria (number of episodes)</td><td>10</td><td>8</td><td>8</td><td>6</td><td>8</td><td>8</td></tr><tr><td>ARI (number of episodes)</td><td>8</td><td>7</td><td>6</td><td>3</td><td>4</td><td>7</td></tr><tr><td>Others</td><td>HCV+</td><td>Scabiosis</td><td>Otitis</td><td>Herpes</td><td>Herpes</td><td>Herpes</td></tr><tr><td></td><td>Taeniasis</td><td>Candidosis</td><td>Mycosis</td><td>TBC, Zona</td><td>Candidosis</td><td>TBC, Zona</td></tr><tr><td></td><td></td><td></td><td>Enteritis</td><td>Scabiosis, Polyparasitosis</td><td></td><td>Dental infection</td></tr><tr><td>WHO Aids clinical stage</td><td>2</td><td>2</td><td>2</td><td>3</td><td>2</td><td>4</td></tr><tr><td colspan="7"><strong>Immunovirological data</strong></td></tr><tr><td>CD4 T cell count/µl (%)</td><td>487 (12.9)</td><td>146 (14.5)</td><td>1482 (44.4)</td><td>197 (8.2)</td><td>192 (3.1)</td><td>646 (24.8)</td></tr><tr><td>CD4 Nadir</td><td>219</td><td>146</td><td>1137</td><td>101</td><td>192</td><td>371</td></tr><tr><td>VL copies/ml (log)</td><td>1 360 (3.13)</td><td>1 930 000 (6.29)</td><td>1 690 000 (6.23)</td><td>101 000 (5.00)</td><td>3 220 000 (6.51)</td><td>5 200 (3.72)</td></tr><tr><td>Genotype/Subtype</td><td>A</td><td>CRF02_AG</td><td>CRF02_AG</td><td>CRF02_AG</td><td>G-J</td><td>CRF02_AG</td></tr><tr><td>Therapeutic line (1st, 2nd)</td><td>1<sup>st</sup>-line<br>ABC/3TC/NVP</td><td>1<sup>st</sup>-line<br>ABC/3TC/NVP</td><td>1<sup>st</sup>-line<br>ABC/3TC/NVP</td><td>1<sup>st</sup>-line sub:<br>EFV/FTC/TDF</td><td>1<sup>st</sup>-line<br>ABC/3TC/NVP</td><td>1<sup>st</sup>-line sub:<br>EFV/FTC/TDF</td></tr><tr><td>Duration of treatment</td><td>5 years</td><td>4 years</td><td>2 years</td><td>2 years</td><td>1 year</td><td>4 years</td></tr><tr><td>Adherence</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td></tr><tr><td>Status</td><td>Alive</td><td>Alive</td><td>Alive</td><td>Alive</td><td>Death</td><td>Alive</td></tr><tr><td colspan="7"><strong>Resistance to ARV drugs</strong></td></tr></tbody></table>
<table><thead><tr><th>Genotyping resistance mutation (N)</th><th>33</th><th>23</th><th>35</th><th>30</th><th>27</th><th>17</th></tr></thead><tbody><tr><td>PI</td><td>V32S (1)</td><td>NA</td><td>G73A (1)</td><td>None</td><td>None</td><td>NA</td></tr><tr><td>Accessory PI</td><td>L10I, I13V, E35D, M36I, R41K, L63ST, H69K, I72IT, L89M (9)</td><td>NA</td><td>I13V, K14R, L19I, K20I, E35D, M36I, R41K, L63P, H69K, T74G, V75I, L76Deletion, L89M, I93IF (14)</td><td>K14E, G16E, K20I, M36I, R41K, K43R, H69K, T74A, V75G, L76I, L89M (11)</td><td>I13V, G17D, K20M, E34K, M36I, Q61N, I62M, L63S, C67E, H69K, V75I, V77I (12)</td><td>NA</td></tr><tr><td>NRTI</td><td>L74LI, M184V (2)</td><td>L74V, Y115F, M184V (3)</td><td>L74V, Y115F, M184V (3)</td><td>M184I (1)</td><td>None</td><td>M184V (1)</td></tr><tr><td>NNRTI</td><td>K103N, E138Q, Y188F (3)</td><td>Y181C (1)</td><td>K103N, Y181C (2)</td><td>Y181C (1)</td><td>K103N, V106VI, H221Y, F227FL (4)</td><td>K103N, V108I (2)</td></tr><tr><td>Accessory RT</td><td>K22R, V35K, T39D, W88C, V90I, D121H, K122E, D123S, I135V, S162A, K173S, Q174K, G196E, T200K, K201M, Q207S, R211S, V245KLMQ (18)</td><td>E6EK, K11KQ, V35T, T39TA, K49R, V60I, T69N, K101R, K122E, D123S, I135T, Q145M, S162A, K173T, Q174K, D177EK, I178M, G196E, T200A (19)</td><td>K20R, V35T, K49R, V60I, K122E, D123N, I135IT, Q145V, S162A, K173T, Q174K, D177E, I178M, G196E, T200A (15)</td><td>I31L, V35T, E36A, T39ILM, V60I, V90I, K122P, I142V, V148VG, L149LF, S162A, K166KR, K173KR, Q174EK, D177E, V179I, T200E (19)</td><td>V35I, I142T, K173A, Q174EK, T200E, I202IM, Q207EK, R211K, F214L, V245Q, E248D (11)</td><td>K20R, V35T, K49R, V60I, K122E, D123N, I135T, I142IV, Q145M, S162AD, K173AT, Q174K, D177EG, I178IM (14)</td></tr></tbody></table>

NA: not applicable; Immunization/Vaccination program: BCG, VPO, DTC-HepB-Hib1, VPO/VPI1, PCV13, Rota, VAM/MM, VAA; 1st line sub: 1st line substitution (cf.

supplementary Table 2), CTM: Cotrimoxazole; ARI: Acute respiratory Infections; TBC: Tuberculosis; MoH: Ministry of Health.

<caption>**Table 1:** Demographic, clinical, immunovirological, and Drug cART characteristics of HIV-1 Exposed Infected (HEI) children.</caption>
<figure><img src="image_3.png" /><figcaption>**Figure 1a:** Heatmap of mutations analysis to pharmacoresistance sequencing. PI: Protease Inhibitor, NRTI: Nucleoside Reverse Transcriptase Inhibitors, NNRTI: Non-nucleoside Reverse Transcriptase Inhibitors, ritonavir: /r, Atazanavir/r (ATV/r), Darunavir/r (DRV/r), Lopinavir/r (LPV/r), Abacavir (ABC), Zidovudine (AZT), Emtricitabine (FTC), Lamivudine (3TC), Tenofovir (TDF), Doravirine (DOR), Efavirenz (EFV), Etravirine (ETR), Nevirapine (NVP), Rilpivirine (RPV).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 1b:** Crystal structure (cartoon style 3D representation) of the HIV reverse transcriptase (PDB id: 3LAK) for the 6 patients. A (Patient 1), B (Patient 5), C (Patient 2), D (Patient 3), E (Patient 4) and F (Patient 6). Chain A and B are colored in magenta and blue, respectively. Only NRTI resistance and NNRTI resistance mutations are shown, in yellow and green, respectively. Here we show the location of the main resistance mutations for each patient on their HIV-1 RT 3D representation.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 2:** Phylogenetic tree of HIV-1 RT pol gene of our 6 HIV-1 isolates of present study in 300 Central African HIV-1 reference strains of clade A, B, C, D and CRF submitting in GenBank representing the various genetic</figcaption></figure>
subtypes. The viral strains from our study are named in Red Color. 585 conserved sites were used to perform the phylogenetic reconstruction by maximum likelihood and with 1000 bootstrap replicates. For each leaf, accession number of the sequence, number of replicons and collection date were indicated. Bootstrap score over 70% are shown in red with the characteristic amino acid mutations in blue. Six clusters are highlighted in color: in blue for the cluster I, in green for the cluster II, in yellow for the cluster III, in orange for the cluster IV, in pink for the cluster V and in purple for cluster VI. Patients are highlighted in red. Tree is rooted with the reference HIV sequence AF033819's cluster.

<figure><img src="image_6.png" /><figcaption>**Figure 3a/b:** 3a) Mutation comparison of 6 patients with the reference genome of HIV1 for the pol gene. 3b) Mutation comparison of 6 patients with the reference genome of HIV1 for the protease gene. Points indicate same amino acids with the reference, hyphen a gap and X an ambiguous position.</figcaption></figure>

## 4. Discussion

This is the first study performed in a rural area of CAR. Its interest resides in the fact that it concerns people suffering hardship in their everyday life

because of the recurrent troubles which plague the country. No children were administered PI and two of them (patients 4 and 5) had no major PI resistance mutations while strains of patients 1 and 3 harbors
resistance mutation: V32S is highly unusual while G73S/T/C/A are non-polymorphic accessory PI-selected mutations. Both are associated primarily with reduced susceptibility to ATV [11, 15]. L76V, a non-polymorphic mutation selected by LPV and DRV, reduces susceptibility to the three PIs. The highly unusual L76 deletion was found in patient 3 but his strain remains susceptible to LPV [16]. However, the strains of the two former patients remained susceptible or hypersusceptible to ATV, and LPV (Figure 1a). Similar results have been reported in CAR [17-20] and other African countries such as Cameroon, Democratic Republic of Congo (DRC) and Congo Republic [21, 22]. The mutations described in these countries are identical to those found in Bouar and 96% of these strains were susceptible to PI [21].

The treatment combining NRTI and NNRTI failed to render undetectable the viral genome but ensured a satisfactory CD4 count in patients 1, 3 and 6. Patient 3 despite a high CD4 count, had absolutely no control of viral replication. The three remaining patients have both high viral load and low CD4 counts. Considering the NRTI, five patients harbored the M184V/I resistance mutation of the RT gene (Figure 3a/b). The 19 months infant (patient 5) alone had no major resistance to NRTI which may be due to his young age and a treatment for only one year. However, the virus remained resistant to NNRTI and the administration of two NRTI (ABC/3TC) did not prevent an extremely high viral load. This may be related to an observance problem which favored resistance outcome. The mutations L74V/I (patients 1, 2 and 3) and Y115F (patients 2 and 3) were associated with NRTI resistance. The patient 5 was susceptible to the 6 NRTI (Figure 1a). All the six

viral strains have retained susceptibility to TDF which may explain the partial efficacy of the treatments of patient 4 and 6. HIV of five patients have developed high level resistance to ABC, FTC, 3TC varying from 60 to 130 on the scoring mutation. The Y181C mutation causes intermediate resistance to ABC and low-level resistance to TDF. The number of resistance mutations to NNRTIs is elevated. Accessory mutations are mainly compensatory which have alone little effect on drug resistance. However, their accumulation can increase the level of phenotypic resistance [23]. Major mutations associated with resistance are K103N (patients 1, 3, 5, 6) and Y181C (patients 2, 3, 4) the latter being the only one present in patients 2 and 4. Patient 6 have the K103N, associated to the V106I. Y181C is a non-polymorphic mutation associated with a reduced response to an EFV-containing regimen in NNRTI-experienced patients [15, 24]. K103N is a non-polymorphic mutation that causes high-level resistance to NVP and EFV. Mutation V106I (patient 5) occurs in 1% to 2% of viruses from untreated persons [25], (https://hi vdb.stanford.edu/dr-summary/resistance-notes/ NNRTI/). It is associated with a reduced NNRTI susceptibility in association with other mutations. H221Y is a non-polymorphic accessory mutation selected primarily by NVP. It frequently occurs in combination with Y181C (but it is found isolated in patients 5). In the same patient, F227L is a non-polymorphic mutation which in most cases is associated to V106A. The former is selected in vivo and in vitro by NVP and is associated with high-level resistance to NVP and intermediate activity of EFV. The major V106I mutation was present in patient 5 and 6. NVP has been and remains the frequent first molecule administered even if it is no longer used for prophylaxis of vertical maternal-fetal transmission. It
is commonly part of first line treatment and as in
patients 1, 2, 3 and 5. Resistance to this drug was
observed in patient 6 and an intermediate activity in
patient 4. There is structural difference between EFV
(given to patients 4 and 6) and the other NNRTIs
which explains the differences of susceptibility
between this drug and the other NNRTI. The
previous studies on HIV and resistance to ARVs in
children which were held in CAR were limited to
Bangui. They confirm the resistance to the different
classes of ARVs which has grown over time since in
different social groups [18, 26-28]. HIV-1 infected
children born to HIV-infected mothers living in rural
areas have an underestimated prevalence of clinical,
virologic and immunologic failures. It is interesting
to note that some mutations correspond to resistance
to drugs unavailable in CAR. The IP Darunavir
(DRV) remains susceptible which is not surprising as
IP are not prescribed to the 6 children. On the
contrary, intermediate resistances are present for
Doravirine (DOR) in patients 1 and 5, Rilpivirine
(RPV) in patients 1 to 5) and Etravirine (ETR) in
patients 2 and 4, three NNRTI not available yet in the
country [29]. For example, the rare mutation F227I/V
which is said to be selected in vitro by DOR is
present in patient 5 with a corresponding low-level
resistance (figure 1a). The presence of numerous
resistance mutations of the PI (mean 8) et RT genes
(mean of 2 for NRTI, and NNRTI but 16 for
accessory mutations) is surprising considering the
young age of patients and sometimes the short time
since they were treated. Some of these mutations are
likely due to the transmission of virus by mother at
birth but for some an occurrence by chance is also a
possibility. In the absence of maternal results, one
cannot conclude in favor of one or the other
hypothesis. For example, patients 2 and 3 are from

the same mother and were administered the same
cART. They both harboured the L74F, Y115F,
M184V, Y181C mutations which may come from
their mother. However, the patient 3 has an additional
one (K103N) who may have been selected during his
treatment despite his younger age. Nonetheless, these
two children respond differently to treatment. While
the viral load is high for both, patient 3 have a far
higher CD4 count maybe, as being younger, its
treatment duration was shorter, and he has retained a
good immunity. Considering the patient 5, the viral
strain was susceptible to all NRTI. Because he is still
an infant, the mutations responsible for resistance to
NNRTI are likely of maternal origin. The lowest
number of mutations of the RT in this patient may be
related to his young age. Considering that an
undetectability of the viral load was never achieved,
all these patients are prone to develop mutations of
the RT genes (15 to 23 were observed). Studies
carried out in Bangui in HIV+ children at the Bangui
Pediatric University Hospital Center found 60% of
non-responders among the cohort of pediatric
patients. The main mutations of the RT found were
M184V and K103N [20, 30, 31]. The 6 patients have
presented opportunistic infections which is related to
the absence of control of the viral replication (Table
1/Figure 3a/b). Besides the maternal transmission,
and a limited efficacy of the administered ART, local
factors may contribute to the poor evolution of the
virological and immunological results of these young
patients. The prescription was adapted to age, weight,
and body surface. Parents were given treatment for
one month with the recommendations for their
administration. However, the drug supply,
particularly of pediatric formulations may be
interrupted and solely a limited number of ART is
available. Moreover, they are not always available,
the limited quantity of syrup formulation renders the administration of ART difficult in young infant. The viral load is not systematically performed at each visit because of the limited resources. The follow-up consists of quarterly visits with a clinical examination, hematological, chemical, and immunological tests and finally the distribution of ARTs. Once the treatment is initiated and if the evolution is satisfactory, visits are programmed every three months. The households are visited weekly by community staff. Self-report is not a good predictor of adherence, and the actual level of non-adherence is certainly higher than the one reported. Adherence to therapy plays a central role in the development of resistance mutations. Compliance must be greater than 95% to obtain an adequate virological response [21, 32]. Moreover, in places lacking electricity, a good conservation of the drug is not ascertained and in a hot climate they may lose their efficacy with time [32, 33]. The recurring politico-military crises which have repercussions on the supply of ART. Nonetheless the Center can continuously deliver these molecules to its planned reserve stock if deliveries are insured. The insecurity may prevent the attendance to visits, with, consequently, an interruption of treatment. All these factors are in favor of a discontinuity of drug administration and thus, of the emergence of resistances. The frequency of drug administration may also be a factor in the development of resistance mutations [34]. Some studies have shown the development of mutations at different times, without correlation with frequency 60 to 80%. Additionally, the health center cannot afford the sequencing of the viral genome. Thus, the actual resistance of the virus to ART drugs is not determined and the treatment cannot be adapted adequately, and a mere switch of cART does not

warrant an efficacy. The insecurities also favor the migration of populations seeking stability on the other side of the borders. This may explain the presence in three patients of the CRF02_AG genotype, constituting a single cluster, totally separate from all the CRF02_AG strains in CAR and the world and probably of foreign origin or the emergence of sub-CRFs with specific mutations. In Bangui, strains consist mainly of CRF (especially CRF11) and BA and rarely CRF02_AG [26, 28].

## 5. Conclusions

Children in Bouar, a rural area, show high level resistance to RT and susceptibility to PI and emergence of CRF_AG variant. It is striking that all these young patients were in treatment failure although at different levels. In this difficult environment, the success of cART is not warranted. More complete survey must be done to evaluate the rate of success of ART administration in this population to make a good comparison with what occurs in Bangui urban environment. Thus, some of the difficulties and low success of cART described in Bouar must exist in Bangui [28]. Making the accessibility to viral load determination for all patients would ameliorate their follow-up with better budget. The viral RNA sequencing would permit a better prescription of ARV. Additionally, the introduction of more recent ART would permit to overcome the observed dramatic emergence of resistances. The more recent PI, NRTI and NNRTI may have already a reduced activity (e.g. DOR, ETR, RPV), due to the accumulation of mutations, thus drugs of other classes (e.g. integrase inhibitors) would be helpful for controlling the infection.
## Ethics Considerations and Patient Consent Statement

The expert committee of the HIV/AIDS national control program of the CAR Ministry of Health of the approved this program of resistance surveillance to cART (Arrêté n°0277/MSPP/CAB/DGSPP/DMPM/ SMEE du 05 août 2002) as part of the surveillance of communicable diseases requiring compulsory coverage. Our retrospective study does not require patient consent to participate.

## Consent for Publication

Not applicable

## Availability of Data and Materials

The data and materials used are available on request.
The sequences of the strains are already in GenBank.

## Competing Interests

Any conflicts of interests for all authors should be declared

## Funding

This work was supported by personal authors
contributions.
No funding.
Not applicable.

## Authors' Contributions

UV acquired the data and wrote the draft paper. GG, EB AS and UV conceived and designed the experiments. SM and EB performed the experiments and analyzed the data with AS, GG and UV. NC helped to draft. GG, EB, ALF, NC and UV reviewed the paper. All authors have read and approved the final manuscript.

## Acknowledgments

The authors would like to express their sincere thanks to all the Participants; Healthcare workers of HIV/STI Centre St Michel of Bouar (CAR) who helped of data collection with patient recruitment and performing health assessments. The authors thank Julien Gardès (Biomanda, Nice in France) for the expert biotechnical assistance in phylogeny.

## Author Information

¹Infectious and tropical diseases Unit, Amitie Hospital, Bangui CAR.

²Laboratory of Immunity, Department of systemics medicine, Faculty of medicine and surgery, Tor Vergata University, Rome, Italy.

³Clinic of Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

⁴Academic Department of Pediatrics, Research Unit of Congenital and Perinatal Infection, Children's Hospital Bambino Gesù, Rome, Italy.

⁵Molecular biology Unity, Clinical biology and Public health national Laboratory, Bangui, CAR,

⁶Laboratory of viral hepatitis, Institut Pasteur of Bangui, CAR.

⁷Saint Michel Center, Society of Priests of the Sacred Heart of Betharram, Health Pastoral Diocese of Bouar, Bouar, CAR.

⁸Laboratory of Microbiology, San Raffaele Scientific Institute, Milan, Italy.

⁹EA 3452 CITHEFOR, Faculty of Pharmacy Brabois Santé, Vandoeuvre-lès-Nancy, France.

ALF is Emeritus Professor in faculty of pharmacy Brabois Santé and faculty of maieutical medicine and health professions of Nancy university in France.

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Supplementary

<table><thead><tr><th colspan="2">Drugs Resistance</th><th colspan="6">HIV-Exposed Infected Children ID (DRI, MS)</th></tr><tr><th>Class</th><th>Generic name</th><th>Patient 1</th><th>Patient 2</th><th>Patient 3</th><th>Patient 4</th><th>Patient 5</th><th>Patient 6</th></tr></thead><tbody><tr><td rowspan="3">PI</td><td>Atazanavir/r (ATV/r)</td><td>S, 0</td><td>NA</td><td>PLLR, 10</td><td>S, 0</td><td>S, 0</td><td>NA</td></tr><tr><td>Darunavir/r (DRV/r)</td><td>S, 0</td><td>NA</td><td>S, 0</td><td>S, 0</td><td>S, 0</td><td>NA</td></tr><tr><td>Lopinavir/r (LPV/r)</td><td>S, 0</td><td>NA</td><td>S, 5</td><td>S, 0</td><td>S, 0</td><td>NA</td></tr><tr><td rowspan="5">NRTI</td><td>Abacavir (ABC)</td><td>HLR, 60</td><td>HLR, 120</td><td>HLR, 120</td><td>LLR, 15</td><td>S, 0</td><td>LLR, 15</td></tr><tr><td>Zidovudine (AZT)</td><td>S, -10</td><td>S, -10</td><td>S, -10</td><td>S, -10</td><td>S, 0</td><td>S, -10</td></tr><tr><td>Emtricitabine (FTC)</td><td>HLR, 60</td><td>HLR, 60</td><td>HLR, 60</td><td>HLR, 60</td><td>S, 0</td><td>HLR, 60</td></tr><tr><td>Lamivudine (3TC)</td><td>HLR, 60</td><td>HLR, 60</td><td>HLR, 60</td><td>HLR, 60</td><td>S, 0</td><td>HLR, 60</td></tr><tr><td>Tenofovir (TDF)</td><td>S, 5</td><td>S, 5</td><td>S, 5</td><td>S, -10</td><td>S, 0</td><td>S, -10</td></tr><tr><td rowspan="5">NNRTI</td><td>Doravirine (DOR)</td><td>IR, 30</td><td>PLLR, 10</td><td>LLR, 20</td><td>PLLR, 10</td><td>IR, 55</td><td>LLR, 15</td></tr><tr><td>Efavirenz (EFV)</td><td>HLR, 130</td><td>IR, 30</td><td>HLR, 90</td><td>IR, 30</td><td>HLR, 85</td><td>HLR, 70</td></tr><tr><td>Etravirine (ETR)</td><td>PLLR, 10</td><td>IR, 30</td><td>IR, 30</td><td>IR, 30</td><td>LLR, 20</td><td>S, 0</td></tr><tr><td>Nevirapine (NVP)</td><td>HLR, 130</td><td>HLR, 60</td><td>HLR, 120</td><td>HLR, 60</td><td>HLR, 115</td><td>HLR, 75</td></tr><tr><td>Rilpivirine (RPV)</td><td>IR, 45</td><td>IR, 45</td><td>IR, 45</td><td>IR, 45</td><td>LLR, 25</td><td>S, 0</td></tr></tbody></table>

DRI: Drug Resistance Interpretation, MS: Mutation Scoring, PI: Protease Inhibitor, NRTI: Nucleoside Reverse Transcriptase
Inhibitors, NNRTI: Non-nucleoside Reverse Transcriptase Inhibitors. NA: Not applicable, HLR: High-Level Resistance, PLLR: Potential low-level resistance, LLR: Low-level resistance, IR: Intermediate resistance, S: Susceptible

<table><caption>**Supplementary Table 1:** Analysis of correlation of mutations to pharmacoresistance sequencing.</caption><thead><tr><th colspan="2">The ARV available for pediatric HIV in CAR are ABC, 3TC, Duovir-N (3TC/ZDV)-NVP), Kaletra (LPV/r) junior, EFV, NVP, ATV, Truvada (Emtricitabine/Tenofovir = FTC/TDF) and Ritonavir (r)</th></tr></thead><tbody><tr><td rowspan="4">- 1<sup>st</sup> line:</td><td>Atripla (TDF + FTC + EFV)</td></tr><tr><td>Duovir (AZT + 3TC) + EFV or NVP</td></tr><tr><td>Abacavir + 3TC + EFV or NVP</td></tr><tr><td>• Substitution 1<sup>st</sup> line 1 or Substitution 1<sup>st</sup> line 2</td></tr><tr><td rowspan="4">- 2<sup>nd</sup> line:</td><td>Kaletra (LPV/r) + Abacavir (ABC) + 3TC (Lamivudine)</td></tr><tr><td>Kaletra (LPV/r) + Truvada (TDF+FTC)</td></tr><tr><td>Kaletra (LPV/r) + Duovir (AZT+3TC)</td></tr><tr><td>Kaletra (LPV/r) + Atazanavir (ATV)</td></tr><tr><td rowspan="2">- 3<sup>rd</sup> line:</td><td>• Substitution 2<sup>nd</sup> line 1 or Substitution 2<sup>nd</sup> line 2:</td></tr><tr><td>Introduct this year: Dolutegravir + Tenofovir + Lamivudine</td></tr></tbody></table>

<caption>**Supplementary Table 2:** ART used in CAR National Program Against Aids.</caption>
<figure><img src="image_7.png" /><figcaption>**Supplementary Figure 1:** Phylogenetic tree of HIV-1 RT pol gene of our 6 HIV-1 isolates of present study in 84 worldwide HIV-1 reference strains of clade A, B, C, D and CRF representing the various genetic subtypes.</figcaption></figure>
The viral strains from our study are in Red Colour. 585 conserved sites were used to perform the phylogenetic reconstruction by maximum likelihood and with 1 000 bootstrap replicates. For each leaf, accession number of the sequence, number of replicated sequences, the serotype and the country location were indicated. Bootstrap score over 70% are shown in red with the characteristic amino acid mutations in blue. Five robust clusters are highlighted in color: in red for the cluster I: B, D and C, in blue for the cluster II: HIV-1 C, in pink for the cluster III: CRF11 and G-J, in green for the cluster IV: CRF02_AG and in orange for the cluster V: HIV-1 A.

<figure><img src="image_8.png" /></figure>

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