Abstract
To determine HIV-1 subtypes and transmitted HIV-1 drug-resistant mutations among HIV-1-positive children born to HIV-positive mothers in Busia County, blood samples were collected from 53 children aged between 6 weeks and 5 years in 2011. Their mothers were HIV-1 positive and on antiretroviral therapy at the time the children were born. The samples were analyzed for HIV-1 drug resistance and subtypes through sequencing of portions of the HIV-1 pol gene. The generated sequences were analyzed for subtype diversity using the REGA and BLAST subtyping tools. HIV-1 drug resistance was determined using the Stanford University HIV database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C. The prevalence of HIV-1 drug resistance mutations of any kind was 22.6% (12/53).
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Reduction in maternal viral load in breast milk and indirect infant prophylaxis by ingestion of antiretrovirals in breast milk are some of the mechanisms used to protect infants from becoming infected with HIV from their mothers. Studies have shown that antiretroviral drugs taken by nursing women are present in breast milk. 5,6 Between 25% and 40% of mothers who are not on medication can transmit HIV to their children while those on short-course antiretrovirals during the peripartum period have transmission rates of between 8% and 10%. 7 For mothers on triple therapy throughout pregnancy, transmission is less than 5%. 8 However, for infants who become infected before or during breastfeeding, the presence of antiretroviral (ARV) drugs in breast milk may induce the development of drug resistance due to viral replication in the presence of low drug concentrations. 7
Mutations that occur in the HIV genome and lead to resistance can be classified into primary and secondary mutations. Primary mutations lead to a several-fold decrease in sensitivity to one or more drugs. Secondary mutations may not result in a significant decrease in drug sensitivity but are associated with increases in viral fitness in the presence of existing drug resistance mutations. 9 Transmission of resistant HIV variants has serious implications among infected infants since it might reduce the choices of active ARV drugs.
This study was carried out to determine the common transmitted HIV-1 drug resistance mutations in children who were born to HIV-infected mothers in Busia District. This was a retrospective study conducted in Busia County in 2011. After obtaining informed consent from the parents of the children, 5 ml of blood was collected in EDTA tubes and plasma was separated and stored for future analysis. The blood samples were collected from children aged between 1 and 5 years. Scientific and ethical approval was sought from the KEMRI National Ethical Review Committee (ERC).
RNA was extracted from 140 μl of plasma samples using the Qiagen RNA extraction kit according to the manufacturer's instructions. One-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR were performed using in-house primers targeting the protease and reverse transcriptase enzymes in the HIV-1 pol gene as previously described. 10 Both the reverse transcriptase and protease genes were amplified and sequenced.
The generated sequences were analyzed for subtype diversity using the REGA subtyping tool 11 and BLAST tool. 12 Drug resistance was determined using the International Aids Society (IAS) algorithm and the Stanford University HIV database. Of the 53 samples that were successfully amplified and sequenced, 69.8% (37/53) were determined to be HIV-1 subtype A, 22.6% (12/53) were subtype D, 5.6% (3/53) were subtype C, and 1.8% (1/53) were subtype A1C.
The generated sequences were also analyzed for HIV-1 drug resistance mutations. The prevalence of HIV-1 drug resistance mutations of any kind in these children was found to be 22.6% (12/53). Further analysis showed that none of the children had any major protease inhibitor (PI)-related mutations. The only minor mutation that was seen was L10I/V, which was present in 41.5% (22/53) of the children. This is a polymorphic PI-selected accessory mutation that reduces PI susceptibility and may increase the replication of viruses with other PI resistance mutations. Analysis of the reverse transciptase region showed that there were major nucleoside RT inhibitor (NRTI) and nonnucleoside RT inhibitor (NNRTI) resistance mutations present in the children. These are as indicated in Table 1. The results showed that 15% of the children (8/53) had viruses with mutations against the common NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Twelve (22.6%) of the children had resistance mutations toward NNRTI drugs such as efavirenz and nevirapine. Seven of the children (13%) had mutations against both NRTI and NNRTI drugs as indicated in Table 1.
NRTI, nucleotide reverse transcriptase inhibitator; NNRTI, nonnucleotide reverse transcriptase inhibitor.
The findings of this study indicate that HIV-1 subtype A was the most prevalent subtype circulating in the study population with a percentage of 69.8%, with 22.6% being subtype D, 5.6% (3/53) being subtype C, and 1.8% (1/53) being subtype A1C. This mirrors the general picture of HIV-1 subtypes in Kenya where HIV-1 subtype A is predominant. Unlike other areas of Kenya in which HIV-1 subtype A is very dominant, in this population a significant percentage of the viruses was subtype D (22.6%), which is much higher than what has been reported in other parts of Kenya. 13 This could be a result of the fact that Busia borders Uganda where there is a significant percentage of HIV-1 subtype D in circulation.
The study showed that a significant number of the children (15%) had HIV-1 drug-resistant mutations to NRTI drugs such as lamivudine, abacavir, tenofovir, or zidovudine. Additionally, 22.6% of the children had resistance mutations toward NNRTI drugs, while 13% of the children had mutations conferring resistance to both NRTI and NNRTI drugs. The resistance could have developed in the children during breastfeeding or after they were started on lifesaving ARV therapy. 14 It is apparent that some of the mothers were on single-dose nevirapine resulting in incomplete suppression of viral replication. In Kenya, single dose nevirapine has long been used as the main option for preventing MTCT of HIV. The use of triple-drug ART prophylaxis for pregnant and breastfeeding women has been suggested to be a more efficacious means of preventing MTCT of HIV. However, this has resulted in debates on issues involving treatment interruption, risk of maternal drug toxicities, fetal exposure to multiple drugs, and cost implications of the same. 15 All these issues require due consideration and need to be looked at in light of the larger picture of stemming new infections and preventing the development of drug resistance.
The greatest limitation of the study resulted from the fact that the samples were collected from children whose mothers did not have a definitive history of the medication that they were taking. As a result we were not able to provide a link between the development of resistance and the type of medication the mother was taking. However, the study was able to show that development of resistance in children born to ART-experienced mothers can be high, especially in resource-limited settings.
The 53 sequences generated from this study were submitted to GenBank under accession numbers KM016171–KM016223.
Footnotes
Acknowledgments
We thank all the study participants for consenting to be involved in this study. The study was carried out in Kenya Medical Research Institute, Centre for Virus Research. This work was published with permission from the Director, KEMRI.
Author Disclosure Statement
No competing financial interests exist.