Abstract: First-line antiretroviral treatment regimens in resource-limited settings used in breastfeeding mothers often include stavudine (d4T). Limited data describing d4T concentrations in breast milk are available. We analyzed d4T concentrations in 52 mother–infant pairs using ultra-performance liquid chromatography–tandem mass spectrometry (lower limit of quantification: 5 ng/mL in plasma, 20 ng/mL in breast milk). Median (interquartile range) d4T concentrations were 86 (36–191) ng/mL in maternal plasma, 151 (48–259) ng/mL in whole milk, 190 (58–296) ng/mL in skim milk, and <5 (<5 to <5) ng/mL in infant plasma. Although d4T is concentrated in breast milk relative to maternal plasma, the infant d4T dose received from breast milk is very small and not clinically significant.
*Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD
†Department of Epidemiology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD
‡Department of Statistics and Biostatistics and Institute for Health, Health Care Policy and Aging Research, Rutgers University, Piscataway, NJ
§Department of Medicine (Clinical Pharmacology), Johns Hopkins University School of Medicine, Baltimore, MD
‖Department of Medicine, College of Medicine, University of Malawi, Blantyre, Malawi
¶Pediatric, Adolescent, and Maternal AIDS Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Rockville, MD
#Department of Pediatrics, Boston University School of Medicine, Boston, MA.
Correspondence to: Jessica M. Fogel, MSc, Department of Pathology, The Johns Hopkins Medical Institutions, Ross Building, Room 646, 720 Rutland Avenue, Baltimore, MD 21205 (e-mail: email@example.com).
Supported by (1) the National Institute of Allergy and Infectious Diseases (NIAID/NIH) R01-AI087139 (to S.H.E); (2) the International Maternal Pediatric and Adolescent AIDS Clinical Trials (IMPAACT) Network (UM1-AI068632); and (3) the HIV Prevention Trials Network (HPTN) sponsored by NIAID, the National Institute on Drug Abuse, the National Institute of Mental Health, and the Office of AIDS Research, of the NIH, Department of Health and Human Services (UM1-AI068613).
All authors contributed to preparation of the article. In addition, individual authors had the following contributions to the study: J.M.F. Designed the study, prepared samples for ARV testing, performed resistance testing, analyzed data, wrote the article; T.E.T U.S. PI for the PEPI-Malawi trial; J.S. Data analyst for this study and the PEPI-Malawi trial; D.R.H. Statistician for the PEPI-Malawi trial; T.L.P. Performed ARV concentration assays; J.J.K. Clinician in charge of ARV treatment and clinical care for participants in the PEPI-Malawi trial; L.M.M. NICHD Medical Officer for the PEPI-Malawi trial; M.G.F. Former CDC Medical Officer for the PEPI-Malawi trial; helped with the design and conduct of the trial; C.W.H. assisted with study design and data analysis, direction of ARV laboratry, and article writing; N.I.K. Malawi PI for the PEPI-Malawi trial; S.H.E. Designed the study, analyzed the data, wrote the article; M.M. assisted with data analysis, wrote the article.
The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the National Institutes of Health. Use of trade names is for identification purposes only and does not constitute endorsement by the National Institutes of Health or the Department of Health and Human Services.
The authors have no conflicts of interest to disclose.
Received April 3, 2012
Accepted May 4, 2012
Access to antiretroviral (ARV) drugs is increasing in many resource-limited settings. Breastfeeding HIV-infected mothers may receive ARV regimens for HIV treatment or for prevention of mother-to-child HIV transmission. Nursing infants whose mothers take ARV drugs may receive subtherapeutic doses of maternally administered ARV drugs via breast milk.1–4 This may lead to development of ARV drug resistance in infants who are HIV infected.5–7 Data describing breast milk transfer exist for some ARV drugs, including nevirapine (NVP), lamivudine (3TC), zidovudine (ZDV), efavirenz, nelfinavir, and indinavir, but not for stavudine (d4T).1–4,8 While the World Health Organization (WHO) recently recommended against including d4T in ARV treatment regimens because of side-effects, such as lactic acidosis and mitochondrial toxicity, d4T-based treatment regimens are still widely used in many resource-limited settings.9 In Malawi, d4T-based ARV regimens are recommended for both infants and adults, including lactating women.10
In the PEPI-Malawi trial, HIV-infected women with CD4 cell counts <250 cells per microliter who were eligible for antiretroviral therapy (ART) according to WHO guidelines at the time initiated therapy postpartum while nursing.11,12 Most of those women received NVP, 3TC, and d4T, which is the first-line ART regimen in Malawi. We analyzed d4T concentrations in maternal plasma and breast milk from PEPI-Malawi trial participants who initiated ART by 6 months postpartum and in plasma from their breastfeeding infants.
Samples Used for Analysis
ARV drug concentrations were measured in maternal plasma, breast milk, and infant plasma collected in the PEPI-Malawi trial. The main objective of the PEPI-Malawi trial (2004–2009) was to compare three ARV regimens to prevent postnatal HIV transmission: (1) single-dose nevirapine (sdNVP) plus 1 week of daily ZDV (control); (2) control plus daily NVP from day 8 to 14 weeks of age (extended NVP); and (3) control plus daily NVP and ZDV from day 8 to 14 weeks of age (extended NVP/ZDV).11 Women whose infants were HIV–uninfected were counseled to exclusively breastfeed for 6 months, and women whose infants became HIV–infected were counseled to breastfeed as long as possible, based on WHO recommendations at the time. Study visits took place at birth, 3, 6, 9, and 14 weeks and then every 3 months up to 18–24 months postpartum to monitor safety and to test infants for HIV infection. Women who met WHO criteria for ART initiation during the postpartum period received NVP (200 mg), 3TC (150 mg), and d4T (30 mg if <60kg, 40 mg if >60kg) twice daily. This treatment was administered outside of the PEPI-Malawi trial. Maternal ARV drug use was recorded on a structured questionnaire. However, the exact date of ART initiation was not consistently recorded. Postpartum visits usually occurred in the morning; the exact time of maternal dosing, and sample collection were not recorded. In this report, HIV-infected infants who also received ART were excluded from the analysis after ART initiation.
Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was used to measure ARV concentrations and was performed at the Clinical Pharmacology Analytical Laboratory at Johns Hopkins University School of Medicine. The instruments used were AB-Sciex API4000 triple quadrupole mass spectrometer (Foster City, CA) interfaced with a Waters Acquity UPLC (Milford, MA). Maternal plasma samples (50 μL) were assayed for d4T and NVP concentrations and infant plasma samples (50 μL) were assayed for d4T concentrations. Breast milk (both whole milk and breast milk supernatant/skim milk) were also assayed for d4T. Skim milk was prepared by centrifuging whole milk at 3000 rpm for 15 minutes at room temperature; the upper lipid layer was discarded and the supernatant was transferred to a clean tube. The process was repeated 2–4 times to ensure complete removal of lipids. The lower limit of detection [below quantification limit (BQL)] was 5 ng/ml in plasma and 20 ng/mL in breast milk. Samples with NVP concentration below the limit of assay quantification are referred to as undetectable. Precision within the assay for plasma was <7%, and accuracy was <7%. Precision within the assay for whole and skim breast milk was <11% and accuracy was <6%. Deuterated d4T was added to all samples as an internal standard followed by protein precipitation with acetonitrile. Samples were vortexed and centrifuged at 3000g for 15 minutes. The organic layer was dried and reconstituted with mobile phase before injection. All drugs and internal standards were separated on an acquity UPLC HSS C18 column (2.1 × 50mm, 1.8 μm) using methanol and water containing 0.1% acetic acid as the mobile phases with a gradient elution. Detection was through electrospray ionization in the positive multiple reaction monitoring mode.
HIV drug resistance was assessed by analyzing infant plasma (100 μL) using the ViroSeq HIV Genotyping System (ViroSeq, Celera, Alameda, CA) and was performed at the HIV Genotyping Laboratory at Johns Hopkins University School of Medicine.
Detection of NVP in maternal plasma was used as a surrogate measure of maternal drug adherence because of the long half-life of NVP in plasma which, if not detected, provides evidence that NVP doses had not been taken within 2 weeks. Women with no NVP detected in plasma were presumed to be poorly adherent, and they and their infants were excluded from the analysis. Concentrations of d4T that were BQL were assigned values of 2.5 ng/mL for plasma and 10 ng/mL for breast milk. Fisher exact test compared proportions and Wilcoxon rank sum test compared medians between 2 variables. Data were analyzed using SAS 9.3 (SAS Institute, Inc, Cary, NC).
Written informed consent was obtained from all women for participation in the PEPI-Malawi trial. The trial was approved by Institutional Review Boards in Malawi and the United States, including the US Centers for Disease Control and Prevention.
GenBank accession numbers
Sequences analyzed in this report have the following GenBank Acession numbers: HM635507, HM635530, HM635536, HM635636, HM635500, HM635630, HM635521, HM635599, HM635489, HM635481, HM635559, and HM635558.
Overall, 246 women who were enrolled in the PEPI-Malawi trial initiated ART by 6 months postpartum; 221 (89.8%) were nursing at the time of ART initiation. Eighty-three mother–infant pairs had samples available from all 4 sample types (maternal plasma, whole milk, skim milk, and infant plasma) from the same visit. Thirty-one women were excluded from analysis; 10 women did not have sufficient plasma volumes available for testing and 21 women had undetectable NVP concentrations in their plasma. Fifty-two mother–infant pairs had d4T results obtained from all 4 sample types. We compared clinical and laboratory characteristics of the 52 mother/infant pairs with d4T results with the 169 nursing mother/infant pairs not included in the study (Table 1). There was no significant difference between the groups in terms of maternal viral load or CD4 cell count at enrollment, maternal sdNVP administration, maternal report of ART use by 3 months postpartum, infant HIV infection before maternal ART initiation, or infant study regimen (Table 1).
Detection and quantification of d4T in study samples
Concentrations of d4T in maternal plasma, whole milk, skim milk, and infant plasma samples were measured using the first available set of samples collected after the mother reported that she had started ART (median: 1.5 months after the mother reported ART initiation, range: 0–7.5 months). For the 52 mother–infant pairs with results from all 4 sample types, d4T was detected in 46 (88.5%) of the maternal plasma samples, 44 (84.6%) of the whole milk samples, and 45 (86.5%) of the skim milk samples. In contrast, d4T was only detected in 7 (13.5%) of the infant plasma samples. The median concentrations of d4T were 86 ng/mL in maternal plasma [interquartile range (IQR): 36–191 ng/mL], 151 ng/mL in whole milk (IQR: 48–259 ng/mL), 190 ng/mL in skim milk (range: 58–296 ng/mL), and BQL (<5 ng/mL) in infant plasma (IQR: BQL–BQL ng/mL, Fig. 1). The median ratio of d4T concentration in skim milk and whole milk was 1.12 (range 0.90–2.41). The median ratio of d4T concentration in whole milk, and maternal plasma was 1.45 (range 0.17–5.44). The median ratio of maternal plasma and infant plasma of the seven infants with detectable d4T concentrations was 0.05 (0.01–0.15). The estimated median daily infant d4T dose from breast milk (whole milk) was 22.7 μg/kg, assuming a daily breast milk intake of 150 mL/kg/day.
ARV Drug Resistance
Fifteen (28.8%) of the 52 infants analyzed were HIV infected; 12 (80.0%) of those infants were HIV infected before the mother reported ART initiation. HIV genotyping results were obtained from 12 (80.0%) of 15 infants. HIV from all 12 infants had mutations associated with resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs). In addition, HIV from 5 (41.7%) of the 12 infants had mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs, 1 infant had M184V, 3 infants had K65R, and 1 infant had T69N and L74V). No d4T-specific thymidine analog mutations were detected.
This is the largest study to date reporting concentrations of d4T in plasma and breast milk from lactating women and in plasma from their breastfeeding infants. The majority of maternal plasma and breast milk samples had detectable concentrations of d4T [only 11%–15% of plasma and breast milk samples had d4T concentrations below the limit of quantification (<5 ng/mL)]. D4T is concentrated in breast milk, with whole milk d4T concentrations on average 45% greater than in plasma. The estimated infant median daily exposure to d4T from breast milk was 22.7 μg/kg, which corresponds to approximately 1% of the recommended pediatric treatment dose of 2 mg/kg/day.13 Only 7 (13.5%) of 52 infants studied had detectable d4T concentrations; all 7 of those infants had d4T concentrations of <10 ng/mL, which falls in the lower limit of the range of the 50% inhibitory concentration (IC50) for d4T for wild-type HIV (2–897 ng/mL).14 The low concentrations of d4T detected in the infants reflects the small dose of d4T from breast milk and the short plasma d4T half-life, which averages 2.2 hours in 1-week-old infants and 1.5 hours in 6-week-old infants.15 One limitation of the study is that data on the time of dosing were not available, which precluded more detailed analysis of the kinetics of d4T transfer from plasma to breast milk and the resulting infant d4T exposure. We are only aware of one prior study that described d4T concentrations in maternal and infant plasma.16 In that study, d4T concentrations were undetectable (<5 ng/mL) in 8 serum samples collected from 4 infants who were exposed to maternally administered d4T.16 In another study, d4T was detected in breast milk from 2 women in a method validation study.17
All HIV-infected infants in this study were analyzed for drug resistance mutations. All infants had NNRTI resistance mutations detected. The infants had several sources of NNRTI exposure, including maternal and infant sdNVP, infant NVP prophylaxis regimen (up to 14 weeks), and maternal NVP-containing ART. Any of these exposures could have contributed to the acquisition of NNRTI resistance mutations. In contrast, none of the HIV-infected infants had d4T-specific thymidine analog mutations. Other NRTI mutations were observed, such as M184V and K65R. M184V increases susceptibility to d4T and is often selected after 3TC exposure. The K65R mutation is selected by several NRTIs; however, in this study, it was likely selected by 3TC in the maternal ART regimens. Selection of K65R by d4T was less likely because of the low concentrations of d4T detected in infant plasma. In a previous study of ARV drug resistance in HIV-infected infants from the PEPI-Malawi trial who were exposed to maternal ART, we detected only 1 of 37 infants with a mutation that may have been selected by exposure to maternally administered d4T (D67N); however, that mutation is not sufficient to cause resistance to d4T.5 The absence of d4T mutations in infants is supported by the limited transfer of d4T from breast milk and the little antiviral effect it has on infants as the majority of infants have d4T concentrations below the in vitro IC50 of d4T.
In conclusion, our data demonstrate that although breast milk d4T concentrations generally exceed those in maternal plasma, the d4T dose provided via breast milk is very small and does not result in clinically significant d4T exposure in breastfeeding infants whose mothers are receiving d4T.
The authors thank the women and infants who participated in the PEPI-Malawi trial and the PEPI-Malawi study team in Malawi. The authors also thank the laboratory staff at the College of Medicine, University of Malawi-Johns Hopkins University Research Project in Blantyre, Malawi for their assistance with sample processing and shipping, and thank Matthew Cousins for assisting with creating the Figure.
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