Many HIV-infected women have detectable nevirapine (NVP) resistance after receiving single-dose NVP (sdNVP) for prevention of mother-to-infant HIV transmission (MTCT) . Emergence of NVP resistance in maternal plasma after sdNVP is associated with high maternal pre-NVP plasma viral load, low maternal pre-NVP CD4 cell count, HIV subtype (C>D>A), and pharmacokinetic factors associated with NVP exposure (e.g., decreased oral clearance) .
NVP is transferred to breast milk after sdNVP exposure [2–4], and transmission of NVP-resistant HIV to infants by breastfeeding has been documented [5,6]. In Zimbabwean women with subtype C HIV infection, NVP resistance was detected in breast milk from 13 (65%) of 20 women 8 weeks after sdNVP administration [7,8]; resistance test results could not be obtained for 12 other women, most of whom had undetectable breast milk HIV RNA. Factors associated with emergence and persistence of NVP resistance in breast milk have not been described.
In Uganda, most HIV infections are caused by subtypes A and D. The risk of developing NVP resistance in plasma HIV after sdNVP is lower in women infected with subtype A and D HIV than in women infected with subtype C HIV . We analyzed NVP resistance in breast milk from Ugandan women following sdNVP administration.
Materials and methods
Source of samples
Breast milk and plasma samples were obtained from HIV-infected women enrolled in an observational study in Kampala, Uganda (‘Pathophysiology of Breast Milk’, 2003–2004). Women received sdNVP according to national treatment guidelines; none of the women received any other antiretroviral drugs, consistent with the standard of care in Uganda at the time of the study. We tested samples from 51 women; this included all available samples from women whose infants were HIV-infected by 6 weeks of age (n = 17) and twice as many samples from women whose infants were HIV-uninfected at 6 weeks of age (n = 34).
Preparation of breast milk supernatants
Breast milk was stored at room temperature and processed within 30 h of collection. Breast milk supernatants were prepared as follows: breast milk was centrifuged at 300g for 15 min, the upper lipid layer was discarded, and the supernatant was removed from the cell pellet and transferred to a separate tube. This process was repeated three to five times to ensure that removal of lipids was complete. Supernatant samples were stored at −70°C.
HIV viral load and CD4 cell count determination
Plasma HIV viral loads were measured using the Roche AMPLICOR Monitor test kit version 1.5 (Roche Molecular Systems Inc., Branchburg, New Jersey, USA, lower limit of detection: 400 copies/ml). HIV RNA was extracted from breast milk supernatant using the Boom method  and was measured using the Roche AMPLICOR Ultrasensitive Monitor test kit (lower limit of detection: 50 copies/ml) . CD4 cell counts were measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, California, USA).
HIV genotyping and subtyping
HIV genotyping was performed using the ViroSeq HIV-1 Genotyping System version 2.7 (Celera, Alameda, California, USA); a nested PCR procedure was used to amplify breast milk samples with less than 500 copies/ml HIV RNA [11,12]. HIV subtypes were determined by phylogenetic analysis of pol region sequences, as described .
Analysis of nevirapine
Plasma and breast milk NVP concentrations were determined using a liquid chromatography tandem mass spectroscopy assay. The lower limit of quantification was 10 ng/ml.
Statistical analyses consisted of computation of medians and proportions. Owing to the small sample size, statistical significance for comparison of proportions and medians was tested using Fisher's exact test and the Wilcoxon test, respectively. All statistical tests were done using the R software  against a two-sided 0.05 α significance level. For statistical analysis, plasma viral loads less than 400 copies/ml were assigned a value of 200 copies/ml, plasma viral loads more than 750 000 copies/ml were assigned a value of 750 000 copies/ml, and breast milk viral loads less than 50 copies/ml were assigned a value of 25 copies/ml.
Written informed consent was obtained from all women for participation in the Pathophysiology of Breast Feeding study. The study was approved by the National AIDS Research Committee and the Uganda National Center for Science and Technology in Uganda, by the Western Institutional Review Board for Johns Hopkins University, and by the US Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia.
GenBank accession numbers
The GenBank accession numbers for 30 breast milk and 30 plasma HIV sequences are GU059279–GU059338.
We analyzed breast milk samples collected from 51 Ugandan women 4 weeks after sdNVP administration. Genotyping results were obtained for all 10 breast milk samples with viral loads more than 500 copies/ml using standard genotyping procedures (median 1208, range 503–8509 copies/ml), and for 21 (51.2%) of 41 samples with viral loads less than 500 copies/ml using a nested PCR procedure (median 25, range 25–200 copies/ml). Overall results were obtained for 31 (60.8%) of the 51 breast milk samples. HIV genotyping results were also obtained for plasma from these 31 women. Results from one woman were excluded because of a possible sample mix-up. The final dataset used for analysis included results from 30 paired maternal plasma and breast milk samples (HIV subtypes: 15A, 1C, 12D, two intersubtype recombinant).
Twelve (40%) of the 30 breast milk samples had at least one NVP resistance mutation; three of those samples had more than one mutation (Table 1). The most common mutations detected were K103N and Y181C. NVP resistance in breast milk was not associated with HIV transmission; NVP resistance was identified in breast milk from four (36.4%) of 11 transmitters vs. eight (42.1%) of 19 nontransmitters (P = 1.0). Among breast milk samples with NVP resistance, the median number of mutations detected was higher for the 11 women who transmitted HIV to their infants (2.0) than for the 19 women whose infants were HIV-uninfected (1.0); however, this difference was not statistically significant (P = 0.18).
Among the 30 women, five had NVP resistance mutations detected in plasma only, four had NVP resistance mutations detected in breast milk only, eight had NVP resistance mutations detected in both samples, and 13 did not have NVP resistance mutations detected in either sample. Five of the eight women who had NVP resistance mutations detected in both plasma and breast milk had different mutations detected in the two samples (Table 1).
We examined persistence of NVP resistance in the 12 women who had NVP resistance detected in breast milk at 4 weeks postpartum (Table 1). Eleven of those women had a 10-week sample available for testing; one sample failed to amplify, leaving 10 follow-up samples with genotyping results. NVP resistance mutations were still detected in four of the 10 women by 10 weeks postpartum. The six women who did not have NVP resistance in breast milk at 10 weeks had only one NVP resistance mutation detected at 4 weeks. In contrast, among the four women who still had NVP resistance mutations detected at 10 weeks, three had multiple NVP resistance mutations detected at the earlier time point. Among the four women who had NVP resistance detected in breast milk at 10 weeks, the number of mutations detected at 10 weeks was lower than the number of mutations detected at 4 weeks; in three of the four women, only K103N was detected in the 10-week sample.
We analyzed the association of breast milk resistance at 4 weeks with clinical and laboratory factors (Table 2). NVP resistance was detected in plasma more frequently among women who had NVP resistance detected in breast milk, but the association was not statistically significant (P = 0.06, Table 2). We found no significant association of breast milk resistance with median maternal pre-NVP viral load, maternal pre-NVP CD4 cell count, median breast milk viral load at 4 weeks, median pre-NVP white blood cell count in breast milk, or breast milk sodium more than 10 mmol/l at 1 or 4 weeks; none of the 30 women had clinical mastitis. Detection of NVP resistance in breast milk was not associated with HIV subtype.
We also measured NVP concentrations in plasma and breast milk. At 1 week postpartum, NVP was detected (>10 ng/ml) in all 29 evaluable plasma samples and in 28 (96.6%) of 29 evaluable breast milk samples. At 2 weeks postpartum, NVP was detected at above 10 ng/ml in 21 (72.4%) of 29 plasma samples and in 16 (69.6%) of 23 breast milk samples. At 2 weeks, detection of NVP in breast milk at above 10 ng/ml was associated with detection of NVP in plasma at above 10 ng/ml (P < 0.001). The median NVP concentrations in plasma and breast milk were similar [at 1 week, for plasma: 168 ng/ml (range 16–744 ng/ml), for breast milk: 117 ng/ml (range <10–576 ng/ml), P = 0.16; at 2 weeks, for plasma: 16 ng/ml (range, <10–83 ng/ml), for breast milk: 15 ng/ml (range <10–64 ng/ml), P = 0.58)]. The concentration of NVP was less than 10 ng/ml in plasma and breast milk from all 30 women by 4 weeks postpartum. There was no association of NVP resistance in breast milk at 4 weeks with either detection of NVP of more than 10 ng/ml in breast milk or plasma, or the level of NVP in either breast milk or plasma, at either study visit (1 or 2 weeks, Table 2).
We detected NVP resistance in 40% of breast milk samples collected 4 weeks after sdNVP exposure; most samples had subtype A or D HIV. Results were not obtained for 20 additional samples with less than 500 copies/ml of HIV RNA. We did not identify any clinical or laboratory factors associated with detection of NVP resistance in breast milk at 4 weeks postpartum, possibly due to small sample size.
In a previous study , NVP resistance was detected in breast milk from 13 (65%) of 20 Zimbabwean women with subtype C HIV at 8 weeks postpartum. It is difficult to compare those results with the results from our study because the two studies analyzed resistance at different times (4 weeks vs. 8 weeks postpartum), used different methods for HIV genotyping, and included samples with different HIV subtypes (predominantly A and D vs. all subtype C).
Administration of sdNVP alone to women in labor continues to be a part of regimens for prevention of MTCT in resource-limited settings. Regimens that combine sdNVP with other antiretroviral drugs (e.g., antenatal zidovudine or lamivudine) are also recommended by the WHO for prevention of MTCT. Studies are needed to test whether women receiving those regimens have a reduced risk for acquiring NVP resistance than women who receive sdNVP alone. Recent studies [15,16] also show that use of extended NVP infant prophylaxis reduces postnatal HIV transmission through breastfeeding. Further studies are needed to evaluate whether emergence or persistence of NVP-resistant HIV in breast milk increases the risk of postnatal transmission in infants receiving NVP-based regimens for prevention of postnatal HIV transmission or is associated with transmission of NVP-resistant strains to infants during breastfeeding.
The authors thank the women in the Pathophysiology of Breast Milk study for their participation in the study and thank the study team for providing the samples and data used in this study. The authors thank the laboratory personnel at Makerere University and Johns Hopkins University for assistance with sample processing and thank Natalia Marlowe (Celera Diagnostics) for providing reagents and protocols for nested PCR.
The Pathophysiology of Breast Milk study was supported by the U.S. CDC. The resistance substudy was supported by the HIV Prevention Trials Network (HPTN) sponsored by the National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Child Health and Human Development (NICHD), National Institute on Drug Abuse, National Institute of Mental Health, and Office of AIDS Research of the National Institutes of Health (NIH) (U01-AI-068613), CDC subcontract to Johns Hopkins University (200-2004-M-09279), and the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (U01-AI-068632, NIAID, NICHD).
The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. CDC. Use of trade names is for identification purposes only and does not constitute endorsement by the U.S. CDC or the Department of Health and Human Services.
None of the authors has financial, consultant, institutional or other relationships that might lead to a bias or conflict of interest.
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