Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola

Monitoring genetic diversity and drug resistance mutations (DRMs) is critical for understanding HIV epidemiology. Here, we report HIV-1 genetic diversity and DRMs in blood samples from 42 HIV-positive pregnant women naive to antiretroviral therapy (ART), in Luanda. The samples were subjected to nested-PCR, followed by sequencing of HIV-1 pol gene, targeting the protease and reverse transcriptase fragments. HIV-1 diversity was analyzed using the REGA HIV-1 subtyping tool and DRMs were identified using the Calibrated Population Resistance tool. A total of 34 sequences were obtained. The data revealed wide HIV-1 subtypes heterogeneity, with subtype C (38%, 13/34) the most frequent, followed by the subtypes F1 (18%, 6/34), A1 (9%, 3/34), G (9%, 3/34), D (6%, 2/34) and H (3%, 1/34). In addition, recombinants strains were detected, with CRF02_AG (6%, 2/34) the most frequent, followed by CRF37_cpx, F1/C, A1/G and H/G, all with 3% (1/34). A total of 6/34 (18%) of the sequences presented DRMs. The non-nucleoside reverse transcriptase inhibitors presented 15% (5/34) of resistance. Moreover, 1/34 (3%) sequence presented resistance against both non-nucleoside reverse transcriptase inhibitors and nucleoside reverse transcriptase inhibitors, simultaneously. Despite the small sample size, our results suggest the need to update currently used ART regimens. Surveillance of HIV-1 subtypes and DRMs are necessary to understand HIV epidemiology and to guide modification of ART guidelines in Angola.

The emergence of HIV-1 subtypes with drug resistance mutations (DRMs) during pregnancy represents a challenge for the efficacy of ART, especially in low-and middle-income countries [15]. There is a lack of recent data on HIV-1 genetic diversity and prevalence of DRMs in Angola [15,16]. In this study, we investigated the genetic diversity and DRM prevalence in blood samples from HIV-positive pregnant women naive to ART in Luanda, to better understand HIV epidemiology and to allow a timely modification of ART guidelines in Angola.

Study design and sample collection
A cross-sectional study was carried out at the Lucrecia Paim Maternity clinic, located in Luanda, capital city of Angola, during the months of April to June of 2018. The study involved 1612 pregnant women who were screened for HIV infection using the rapid antibody detection test Determine HIV1/2™ (Alere, Japan) and the Unigold™ HIV (Trinity Biotech, Ireland) during prenatal care. Sociodemographic characteristics and blood samples were collected from HIV-positive pregnant women. The main criterion for inclusion of HIV-positive pregnant women was that they had not been previously exposed to any ART. The blood samples were collected in a tube with EDTA, centrifuged and the plasma was aliquoted and stored at -80˚C. The blood samples preparation was performed at the Molecular Biology Laboratory, of the National Institute for Health Research of Angola (INIS). Following the recommendations of the National Institute of Fighting against AIDS (INLS), the HIV-positive women, were prescribed ART with TDF, 3TC and EFV, and were medicated with AZT until child birth [13,14].

RNA extraction, cDNA synthesis, PCR and sequencing
Total viral RNA was extracted from 140μL of plasma using QIAamp Viral RNA kit (QIAGEN, Germany) following the manufacturer instructions. The cDNA synthesis was carried out using 10μL of the RNA in a final reaction volume of 20μL. The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer (5'-TTTTACATCATTAGTGTGGG-3'), and 200U of M-MLV enzyme (Life Technologies, USA) [17].
The obtained cDNA was subjected to a nested-PCR, targeting the protease (PR) and reverse transcriptase (RT) fragments of the HIV-1 pol gene, with an expected size of 1302 bp, using the protocol previously described [17]. Successful amplification was checked using a 1% agarose gel. The amplicons were purified using the NZYGelpure Kit (Nzytech, Portugal), and sequenced using the ABI BigDye Terminator v3.1 reaction kit (Applied Biosystems, USA). For each sample, eight primers were used for the complete sequencing of the PR (nucleotide range: 2253-2549) and the first 335 codons of RT (nucleotide range: 2550-3554), considering the genome of the HXB2 strain (nucleotide range: 2252-3554) [17]. Sequencing was performed on an ABI 3500 sequencer (Applied Biosystems, USA) at the Molecular Biology Laboratory of the INIS, in Luanda.

Statistical analysis
Chi-square (X 2 ) tests were performed to evaluate the association between sociodemographic characteristics and HIV prevalence at 5% statistical significance in the SPSS v25 statistical program (IBM SPSS Statistics, USA).

Ethical considerations
The pregnant women were informed of the study and consented (oral and written) to participation and follow-up until delivery. Consent from parents or guardians of the minors (under 15 years) was also obtained. All pregnant women underwent pre-and post-test counseling individually. The HIV study results were provided to the clinical staff to ensure appropriate patient clinical management. The study protocol was reviewed and approved by the National Ethics Committee of Angola (nr. 13

Sociodemographic characteristics
From the 1612 pregnant women tested for HIV, 42 (2.6%) tested positive and not been exposed to any ART previously. All pregnant women were from Luanda province. A total of 25/42 HIV-positive pregnant women were in the age group of 25-34 years, 18/42 had basic and secondary education, respectively, and 19/42 were unemployed. Moreover, a total of 12/42 pregnant women were in the second trimester of gestation, followed by 11/42 in the third trimester and 10/42 pregnant women diagnosed and the sample was obtained just before labor (Table 1).

Genetic diversity analysis
From the 42 plasma samples subjected to nested-PCR, a total of 34 amplicons and respective sequences were obtained. It was not possible to obtain amplicons from the remaining 8 samples, even after repeated PCR attempts using different primers. The genotyping analysis revealed that 28/34 sequences were HIV-1 pure subtypes and 6/34 recombinants strains. From the detected pure subtypes, subtype C (38%, 13/34) was the most frequent HIV-1 subtype, followed by the subtypes F1 (18%, 6/34), A1 (9%, 3/34), G (9%, 3/34), D (6%, 2/34) and H (3%, 1/ 34). From the recombinants strains detected, CRF02_AG (6%, 2/34), was marginally more frequent, followed by CRF37_cpx, F1/C, A1/G and H/G, all with 3% (1/34) (Fig 1).  Genetic distance analysis showed genetic similarity (more than 70%) of HIV-1 subtype C isolates with isolated from Botswana, Mozambique, Tanzania, South Africa and India. The subtype F1 was more similar to isolates from Brazil and Spain. The subtype A1 was most similar to isolates from Cameroon, South Africa, and Pakistan. The subtype G was most similar to isolates from Portugal, and subtype D, subtype H, and recombinant strain H/G to isolates from Democratic Republic of Congo. The recombinants strains A1/G, CRF02_AG, and CRF37_cpx were similar to isolates from Cameroon and Ivory Coast (Fig 2).

Discussion
This study presents an important update on molecular epidemiology of circulating HIV-1 strains in Luanda. The HIV prevalence was 2.6% (42/1612). A significant difference in HIV positivity was observed between pregnant women of different age groups and pregnancy stage (P<0.05). On the other hand, residence, level of education and occupation did not show significant differences (P>0.05) ( Table 1).
Genetic analysis of 34 isolates revealed a wide diversity of HIV-1 strains (Fig 1), similar to that observed in previous studies performed in Angola [5][6][7][8][9][10][11]. Though it is hard to prove statistical significance with our small sample size, our study indicates an increase in HIV-1 subtype C, but a slight decrease in subtype F1 in Luanda [5,8]. The genetic similarity of HIV-1 subtype C with isolates from Botswana, Mozambique, Tanzania, South Africa, India and the subtype F1 with isolates from Brazil and Spain (Fig 2), may be attributed to high mobility between countries or to the fact that, after colonial war, and the end of civil war in 2002, thousands of refugees returned to Angola [27].
The RT inhibitors are important components of ART regimen [12][13][14]. The identification of pre-treatment drug resistance in pregnant women naïve to ART (Table 2), may threaten ART based strategy to HIV control in Luanda [12][13][14]. The K103N and G190A mutations are associated with EFV and NVP resistance [28]. The Y181I and P225H mutations are often associated with second generation RPV and ETR resistance [29]. The thymidine analogue associated mutations (TAMs) at positions M41L and D67N have the greatest impact on susceptibility of AZT and Stavudine (d4T) [28]. The T69D mutation when present with T215S mutation, is associated with broad resistance to NRTIs [28]. The non-polymorphic PI-selected mutation at position I85V has minimal effects on PIs susceptibility [30,31].
The identification of K103N and G190A mutations, may be attributed to the long use of NNRTIs as part of the first-line ART regimens in Angola [13,14]. Displacement of people to countries where ART is available for a longer period also may help to explain the origin of the HIV-1 subtypes with DRMs in Luanda [27,32].
The reasons for PCR failure of 8/42 samples were not identified. It may be that the high HIV-1 genetic diversity compromised the binding of primers, even though they were targeted at highly conserved regions of the HIV-1. Other studies with more representative sampling  P10  A1/G  -G190A  -ETR, RPV  EFV  NVP   P12  F1  -K103N, P225H  -ETR, RPV  -EFV, NVP   P26  H  -G190A  -ETR, RPV  EFV  NVP   P27  G  M41L, D67N, T69D, T215S  Y181I  -ABC, TDF  AZT, EFV  ETR, RPV, NVP   P31  G  --I85V  ---P41  C  -K103N  -- monitoring of the HIV-1 subtypes and DRMs are necessary to guide a timely modification of ART guidelines in Angola. Despite the small sample size, our findings suggest that the Angolan Ministry of Health should prompt consideration of moving to generic integrase strand transfer inhibitors (INSTIs) in the ART regimen in Angola [33].

Conclusions
Our results show a wide HIV-1 subtypes heterogeneity, with subtype C the most frequent. A total of 6/34 (18%) of the sequences presented DRMs. Of these, 15% (5/34) were associated with resistance against NNRTIs. Moreover, 1/34 (3%) sequence presented resistance against both NNRTIs and NRTIs, simultaneously. Our findings suggest the need to update currently used ART regimens. Better understanding is needed of emergence of HIV-1 subtypes and DRMs, to allow a timely modification of ART guidelines in Angola.