Selection of resistance mutations in pregnant women receiving zidovudine and lamivudine to prevent HIV perinatal transmission

Two zidovudine/lamivudine regimens for the prevention of HIV perinatal transmission were studied for the selection of resistance mutations. The M184V mutation was detected one week after delivery in six out of fifty women (12%) who received the regimen prepartum, intrapartum and postpartum, and was no longer present 3 months later. No nucleoside reverse transcriptase inhibitor resistance-associated mutations were detected in 50 women who received zidovudine/lamivudine intrapartum and postpartum only. No association with the risk of perinatal transmission was found. One of the potential obstacles to the continuing effectiveness of drug interventions for the prevention of perinatal transmission is the development of antiretroviral drug resistance. Such resistance could not only decrease the effectiveness of the intervention but also affect future responses to antiretroviral therapy and prophylactic regimens for other pregnancies. The results of the HIVNET 012 study carried out in Uganda using nevirapine for the prevention of vertical transmission [1,2] showed that a single dose administered to women during labour and to infants within 72 h of birth was able to select resistance mutations in 19% of women and 46% of children, tested 6–8 weeks after delivery. The PETRA study [3] is a multicentre, double-blind, placebo-controlled study carried out in five hospitals in sub-Saharan Africa. Women were randomly selected to receive zidovudine/lamivudine 300/150 mg twice a day, according to one of four regimens: arm A, pre-partum (from week 36), intrapartum and postpartum (one week mother and child); arm B, intrapartum and postpartum; arm C, intrapartum only; arm P, placebo. A significant decrease in the transmission rate at week 6 was seen for arms A and B (5.7 and 8.9%, respectively, versus 15.3% in the placebo group, corresponding to a 63 and 42% reduction). The aim of the present study was to determine if the selection of resistance mutations occurs in mother–baby pairs enrolled in arms A and B of the PETRA study. Week 1 postpartum (time of discontinuation of drugs for arms A and B) was selected as the timepoint to evaluate the presence of mutations in women. When resistance-associated mutations were found, samples collected at enrolment (before the administration of study drugs) and 3 months after delivery were also analysed. For HIV-infected children either week 1 or month 3 samples were analysed. The TruGene HIV-1 genotyping kit and OpenGene DNA sequencing system (Visible Genetics, Toronto, Canada) were used for amplifying, sequencing and analysing the HIV pol gene (codons 4–99 of the protease gene and codons 37–248 of the reverse transcriptase gene). Two sets of reverse transcriptase–polymerase chain reaction (RT–PCR) primers were utilized in the study for the amplification step: the standard primers included in the current version 1.0 of the TruGene kit, and a set of novel RT–PCR primers (prototype version 1.5) supplied by Visible Genetics for research use only. These alternative primers were designed to improve the sequencing rate in non-B clades. The subtype was determined using software developed at Frontier Science Technology Research Foundation (Amherst, NY, USA), which compares the aligned sequences with a library of wild-type sequences from 14 HIV-1 clades. A total of 124 samples were processed. Eighty-two were analysed using the standard primers on the first line, and were retested with the alternative primers when amplification was unsuccessful (16 cases). The remaining 42 samples were analysed straight with the alternative primers; amplification was successful in 34 out of 42 whereas eight were sequenced with the standard primers. Subtyping was performed in a total of 93 samples, resulting in 45.2% subtype A, 7.5% subtype C, and 47.3% subtype D. No differences in primer performance were seen according to the HIV-1 subtype. In six out of 50 samples (12%) belonging to women enrolled in arm A the lamivudine-associated mutation M184V, was detected one week after delivery. In four of these mothers enrolment samples could be tested; in five of them samples collected 3 months after delivery could also be tested. None of these samples was positive for M184V. Eleven of the 50 women had an HIV-infected child. The M184I mutation was present in one out of 11 transmitting mothers (9.1%), and five out of 39 non-transmitting mothers (12.8%) had the M184V mutation (not significant). No sample of the HIV-infected child born to the M184V-positive mother was available. The M41L mutation was detected in one sample at week 1 (arm A). This mutation was already present in the enrolment sample and was still present 3 months after delivery. No nucleoside reverse transcriptase inhibitor resistance-associated mutations were detected in 50 samples from women in arm B. Table 1 summarizes the information on mutations in the RT gene in the women of our study.Table 1: Specific reverse transcriptase mutations of HIV-1 in women at different study timepoints, transmission status and HIV-1 subtype.Twelve samples from HIV-infected children (five born to mothers in arm A and seven born to mothers in arm B) were sequenced: the twins of one mother harbouring the V118I and V179E mutations showed the same pattern. No resistance mutations were found in the other samples. A total of 95 out of 119 sequenced isolates (79.8%) carried minor mutations in the protease gene. The M46I mutation was detected in one woman, who also carried the K20R and M36I mutations. Her HIV-infected child shared the same pattern. In our samples of non-B viral subtypes, the performance of the TruGene HIV-1 Genotyping System was optimized by using an alternative prototype primer mix (v1.5 primers). These findings are similar to those obtained in patients with clade G infection [4,5], and underline the need for developing and validating reagents suitable for the highly prevalent non-B subtypes. In our study, the lamivudine resistance-associated mutation M184V was selected in 12% of women receiving zidovudine/lamivudine prepartum, intrapartum and one week postpartum, whereas it was not found either in women receiving drugs only intrapartum and one week postpartum or in HIV-infected children (who had received one week's prophylaxis after birth). In the absence of pharmacological pressure, the mutation seems to be fading rapidly, as expected as a result of its impact on viral fitness. No apparent relationship was seen between positivity for M184V and the risk of vertical transmission or viral subtype. Our data confirm previous findings [6], which showed that the duration of prophylaxis is critical for the selection of resistant variants, and suggest that the administration of lamivudine for short periods for the prevention of mother-to-child transmission may have a low impact on the selection of resistance. Further research is needed to confirm the limited impact of M184V on HIV transmission, and to clarify whether it may persist in minority species, potentially affecting the outcome of prophylaxis in future pregnancies.

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