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Resistance mutation patterns in plasma and breast milk of HIV-infected women receiving highly-active antiretroviral therapy for mother-to-child transmission prevention

2007; Lippincott Williams & Wilkins; Volume: 21; Issue: 17 Linguagem: Inglês

10.1097/qad.0b013e3282f190a6

1473-5571

Mauro Andreotti, Giovanni Guidotti, Clementina Maria Galluzzo, Sandro Mancinelli, Paola Germano, Maria Franca Pirillo, Maria Cristina Marazzi, Stefano Vella, Leonardo Palombi, Marina Giuliano,

HIV Research and Treatment

Mother-to-child transmission via breastfeeding accounts for a relevant proportion of pediatric HIV infections in resource-limited countries [1–2]. Highly-active antiretroviral therapy (HAART) prophylaxis administered to breastfeeding mothers has been proposed as a possible strategy to prevent postnatal transmission [3] and, indeed, the recently reported results of two studies assessing this approach are encouraging [4,5]. One of the possible obstacles to the use of this preventive strategy is that the different penetration of antiretroviral drugs into the breast milk could favour the emergence of resistance and be associated to the transmission of resistant strains. In this study, we analyzed the resistance mutation patterns of plasma and breast milk viral populations in women receiving HAART for mother-to-child transmission prevention in order to assess the frequency of mutations in these women and to evaluate if different patterns arise between plasma and breast milk. We studied 26 pregnant women attending the AnteNatal Clinic in Matola, Maputo, Mozambique [part of the Drug Resource Enhancement against AIDS and Malnutrition (DREAM) program, designed and managed by the Community of S. Egidio] who had received zidovudine (or stavudine if Hb < 8 g/dl), lamivudine and nevirapine from 28 weeks of gestation until 1 month postpartum [6]. Within 1 week after delivery, 10 ml of blood and 10 ml of breast milk were collected from all women. Plasma and 1 ml of unprocessed breast milk were stored at −80°C. The remaining 9 ml of breast milk were centrifuged at 1000 g × 10 min. Breast milk cells were washed once in phosphate-buffered saline and stored at −80°C as dry pellets. HIV-RNA in plasma and whole breast milk and proviral DNA in breast milk cells were quantified as previously described [6]. Concentrations of nevirapine, lamivudine and zidovudine were determined by the high-performance liquid chromatography method with ultraviolet detection [6]. Sequence analysis of viruses in plasma, breast milk and in breast milk cells was performed with the TruGene HIV-1 Assay (Bayer Diagnostics, Milan, Italy). Only resistance mutations included in the IAS-USA 2006 classification were considered as significant. The hivdb6 internet-accessible database (http://hivdb6.stanford.edu) was used for subtype assignment. Women had received a median of 77 days of prophylaxis (range 27–137 days), the median CD4+ cell count at delivery was 620/mm3 (range 183–1275/mm3), the median HIV-RNA level was 2.6 log10 copies/ml (range 1.7–4.7 log10 copies/ml) in plasma, and 2.7 log10 copies/ml (range 1.7–4.7 log10 copies/ml) in breast milk. Median HIV-DNA content was 10 copies/106 breast milk cells (range 10–667 copies/106 breast milk cells). Sequences were obtained from plasma and from breast milk in 23 cases and from breast milk cells in 18 women (Table 1). All strains belonged to subtype C with the exception of one subtype A. Major resistance mutations (K103N + M184V, K103N, V108I, M184I) were detected in the plasma viruses of four women; two of them had the same pattern in breast milk (although in one woman only in the cell-free virus) whereas the other two had no mutation in breast milk. In two cases, viral strains present in breast milk harboured major resistance mutations (M184I + M46I in the cell-associated virus in one patient and V106A in the cell-free virus in the second patient) not present in the plasma virus. Although the patterns of resistance were different, the prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI)-associated mutations was 13% (3/23) both in plasma and in breast milk and that of lamivudine-associated mutations was 8.7% (2/23) in both plasma and breast milk (considering either cell-free or cell-associated viruses). Several patients had differences in the number and type of minor protease mutations in the different viral populations.Table 1: Drug-resistance mutations in plasma, breast milk and breast milk cells.Patients with or without mutations did not have significantly different plasma or breast milk drug concentrations that could possibly explain the differences in resistance mutations, although it must be emphasized that the number of our patients was small and the statistical power to detect these differences was limited. The frequency of major resistance mutations in plasma in our women (4/23, 17.4%) is comparable to that reported in previous studies performed in women receiving HAART [7,8] whereas the only available study assessing the rate of mutations in breast milk reported a considerably higher frequency with the use of single-dose nevirapine (NNRTI resistance was present in breast milk in 65% of the women tested) [9]. In conclusion, in our study, the same proportion of women receving HAART prophylaxis had resistance-associated mutations in plasma and in breast milk. However, since there were differences in the mutational patterns, our data indicate that postnatal transmission may occur with viral variants that cannot predicted by those present in plasma. Acknowledgements The authors wish to thank Roberta Amici, Maria Grazia Mancini, Patrizia Cocco, Daniela Diamanti and Fernando Costa for technical support and Alessandra Mattei for secretarial assistance.