From early 2012, all HIV-positive pregnant women started a triple-drug ART regimen of tenofovir disoproxil fumarate (TDF) or zidovudine plus lamivudine plus one of the following: efavirenz or abacavir or lopinavir/ritonavir (LPV/r). The Haiti National HIV guidelines recommend that all HIV-exposed infants receive prophylaxis consisting of zidovudine or nevirapine from birth until either the first PCR test or until six weeks and referring such infants for EID as early as six weeks after birth.¹² The pediatric HIV guidelines recommend initiating ART in all HIV-infected infants. At the time of the study, the first-line regimens were zidovudine or abacavir + lamivudine + nevirapine for children with no or unknown exposure to ART, whereas for children with known exposure to nevirapine or efavirenz, the recommended first-line regimens were zidovudine or abacavir + lamivudine + LVP/r.

Two laboratories provide EID testing services in Haiti: the Laboratoire National de Santé Publique (LNSP) and the Haitian Group for the Study of Kaposi’s Sarcoma and Opportunistic Infections – Institute of Infectious Diseases and Reproductive Health (GHESKIO-IMIS) Rodolphe Mérieux Laboratory.

For EID, whole blood was collected by finger, heel, or toe pricks from HIV-exposed infants. A dried blood spot (DBS) was prepared by spotting a drop of blood onto one of the five circles on a Whatman 903 grade filter paper cards (Whatman Inc., Piscataway, NJ), dried overnight at ambient temperature at the health facility, packaged in humidity-free bags with desiccants, and sent to one of the two EID laboratories in Port-au-Prince for PCR testing. For this study, we included remnant DBS samples collected from children <18 months of age and newly diagnosed with HIV infection by EID at either LNSP or GHESKIO-IMIS between January 1, 2013 and December 31, 2014. The PCR test was performed using the Roche Amplicor HIV DNA test version 1.5 test (Roche Diagnostics, Indianapolis, IN).

HIV genotyping was conducted on the remnant EID DBS specimens at the WHO-designated Specialized Drug Resistance Laboratory at the International Laboratory Branch at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, using a broadly sensitive in-house genotyping assay.^{13, 14} A 1,084 base-pair segment of the 5’ region of the pol gene encompassing the protease and 5’ segment of the reverse transcriptase (RT) region was generated by RT-PCR and nested PCR. The purified PCR products were then sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and analyzed on the ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The customized ReCALL software program was used to edit the raw sequences and generate consensus sequences¹⁵ and sequence quality assurance was performed on each newly obtained sequence using MEGA.¹⁶ HIVDR mutations and drug susceptibility profiles were determined using the HIVdb algorithm (version 8.4) deployed at the Stanford University Drug Resistance Database (http://hivdb.stanford.edu). Drug susceptibility profiles were interpreted such that the presence of any drug resistance mutation that causes low-level, intermediate, or high-level of drug resistance was defined as resistance; those with susceptible or potential low-level of resistance were designated as susceptible. HIV-1 subtypes were determined using the REGA HIV subtyping tool.¹⁷

The data were analyzed using SAS version 9.3 (SAS Institute, Cary, NC) and Epi Info 3.5.4 (CDC, Atlanta, 2013). Frequencies and chi-square tests were used to summarize categorical demographic data and mutation prevalence data while median and interquartile range [IQR] was reported for age. All graphics were produced using Microsoft Excel (Microsoft Corp., Redmond, WA, 2007).

The study protocol was reviewed and approved by the Haiti National Bioethics Committee and the Office of the Associate Director of Science in the Center for Global Health at the Centers for Disease Control and Prevention. The study was determined to be not human subjects research. Upon receiving the HIVDR results, the National HIV Program shared them with clinicians for patient management.

Between January 1, 2013 and December 31, 2014, DBS samples collected from 3,555 HIV-exposed children from all 10 of Haiti’s geographic departments were submitted to the LNSP for EID by PCR (Figure 1). Of these, 360 (10.1%) were PCR-positive. Among the 360 HIV-positive DBS specimens, 355 had sufficient residual DBS sample for inclusion in the study. Of the specimens submitted for genotyping, 304 (85.6%) were successfully genotyped, including 139 DBS samples collected in 2013 and 165 collected in 2014 (Figure 1). The mean age of the children tested in 2013 was 6.8 months (standard deviation, SD 5.3 months), whereas the mean age of the children tested in 2014 was 6.2 months (S.D. 5.1 months); 243 (79.9%) of the children were under 6 months of age.

Among the 304 children for whom genotyping results were obtained, 217 (71.4%) had at least one DR mutation (Table 1), with 123 (40.5%) children having at least one DR mutation conferring resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and 210 (69.1%) having at least one DR mutation conferring resistance to non-NRTIs (NNRTIs). Moreover, 121 (39.8%) children harbored viruses with DR mutations conferring resistance to both NRTIs and NNRTIs, and 122 (40.1%) had two or more NNRTI mutations. Twenty-nine (9.5%) of the children had additional NNRTI mutations (A98G, E138A/G/K/Q, H221Y, and M230L) that confer resistance to second generation NNRTI drugs etravirine and rilpivirine. Forty-four (14.5%) of the children had one thymidine analogue mutation (TAM) and 28 (9.2%) had two or more TAMs. The most frequent mutations observed were K103N/S (48.0%), M184V (37.5%), G190A/S/E/Q/R (15.1%), and Y181C/G/V (13.8%) (Table 1).

The frequency of HIVDR mutations increased from 44.6% (62/139) in 2013 to 93.9% (155/165) in 2014 (P <0.0001). Similarly, the frequency of mutations conferring resistance to NRTIs increased from 8.6% (12/139) in 2013 to 67.3% (111/165) in 2014 (P <0.0001), whereas the frequency of mutations conferring resistance to NNRTIs increased from 41.0% in 2013 to 92.7% in 2014 (P <0.0001).

Ten (3.3%) of the children had at least one mutation associated with protease inhibitor (PI) resistance. One child had a major M46I PI mutation, which causes low level of resistance to all PIs except darunavir and tipranavir, and another had the I50L major PI mutation, which causes high level resistance to atazanavir (Table 1). The other eight children had accessory PI mutations (L10F, L33F, K43T, and Q58E).

The numbers of children with low-, intermediate-, and high-level drug resistance mutations are shown in Figure 2. Among the 119 children with resistance mutations to abacavir, 25 (21.0%) had mutations conferring high-level resistance, 10 (8.4%) had mutations conferring intermediate resistance, and 84 (70.6%) had mutations resulting in low-level resistance. Forty-two children had resistance mutations to zidovudine: 16 (38.1%) had high-level resistance, 19 (45.2%) had intermediate-level resistance, and 9 (16.7%) low-level resistance. Twenty-seven children had resistance to tenofovir: 5 had high-level resistance (18.5%), 9 had intermediate-level resistance (33.3%), and 13 had low-level resistance (48.1%). All of the 209 children with resistance to efavirenz/nevirapine harbored virus with intermediate- to high-level mutations. As for newer generation NNRTIs, 71 children had resistance mutations to etravirine: 10 (14.1%) high-level, 57 (80.3%) intermediate, and 4 (5.6%) low-level and 110 had resistance mutations to rilpivirine: 48 high-level (43.6%), 28 intermediate (25.5%), and 34 (30.9%) low-level resistance.

Predicted resistance to abacavir, zidovudine, and lamivudine were 39.1%, 14.1%, and 37.8%, respectively, among the children whereas predicted resistance to tenofovir was detected in 9.2% of the children (Figure 2). Predicted resistance was detected to nevirapine and efavirenz in 68.8% of children, whereas predicted resistance to the second generation of NNRTIs etravirine and rilpivirine were detected in 23.4% and 36.2%, respectively.

The main HIV-1 subtype circulating in this population was B or B-like in 267 children (87.8%). Other variants included recombinant forms of B/D (22; 7.2%), B/F1 (3; 1.0%) and others (9; 4.0%).