Cox proportional-hazards models showed CPT exposure was associated with an increased hazard of severe neutropenia (hazard ratio 1.97, 95% CI 1.01–3.86) (Table 2). Unlike severe anemia, most initial episodes of severe neutropenia during follow-up occurred by 18 weeks of age (Fig. 1b), regardless of CPT exposure. Antiretroviral treatment arm was not significantly associated with severe neutropenia (Table 2, Fig. 1d).
Neither CPT exposure nor antiretroviral treatment arm assignment was associated with severe elevated ALT (Table 2). Models were also run with interaction terms between CPT exposure and weeks of age, and between CPT exposure and antiretroviral treatment arm for all of the outcomes studied, but none of the interactions tested showed statistical significance (data not shown).
Linear mixed models, used to analyze the longitudinal effects of CPT exposure on continuous hematologic outcomes, showed that CPT exposure was associated with increases in mean concentration of hemoglobin compared to infants not exposed to CPT, and that this association was modified by both antiretroviral treatment and infant age. The significant interactions in this model mean the beta coefficients presented for this model in Table 3 represent changes in mean hemoglobin concentration compared to CPT-unexposed infants in the control arm at 12 weeks of age. They indicate that the observed association of CPT with mean hemoglobin was greater for infants in the control arm and for older infants. Using the mixed-model coefficients, mean hemoglobin concentration was 0.18 g/dl (95% CI 0.03, 0.33) higher for those exposed to CPT in the control group at 12 weeks of age (Table 3). Mean hemoglobin concentration was higher in the maternal antiretroviral and infant nevirapine arms compared to the control arm when not exposed to CPT (increases of 0.25 and 0.18 g/dl, respectively, at 12 weeks of age), but these effects of antiretroviral treatment were diminished when exposed to CPT (decreases of 0.22 and 0.24 g/dl for maternal antiretroviral and infant nevirapine arms, respectively) due to the significant interaction terms.
Cotrimoxazole preventive therapy exposure was associated with a 191.8 cells/μl decrease in mean neutrophil count (95% CI −303.1, −80.6) in the control arm at 12 weeks of age; however, this decrease was reduced by 7.8 cells/μl for each additional week of age due to a significant interaction term. There was no significant interaction between CPT and study arm in the model with neutrophil count as the dependent variable.
Cotrimoxazole preventive therapy exposure was not associated with ALT levels (increase of 0.53 IU/l; 95% CI −0.68, 1.74) (Table 3).
Results of the sensitivity analysis where follow-up was cut off at the discontinuation of the control group yielded similar results to the main analysis (data not shown).
In this cohort of breastfed HEU infants followed until 48 weeks of life, severe anemia was relatively common, whereas severe neutropenia was rare. There was a significant decrease in severe anemia and a significant increase in severe neutropenia was associated with CPT. Longitudinal mixed models showed corresponding increases in hemoglobin levels and decreases in neutrophil counts associated with CPT exposure. Associations between CPT and hemoglobin levels increased with age, but were reduced by exposure to antiretroviral (either maternal antiretroviral or infant nevirapine). The reduction in neutrophil count associated with CPT exposure lessened with age.
A previous study on the effects of CPT on hematologic outcomes in HEU infants in Botswana showed that severe anemia and neutropenia were rare among HEU infants, and there was no association between CPT and either outcome . Our research confirms the low frequency of severe neutropenia in HEU infants, whereas the difference in frequency of severe anemia may be attributable to the different populations studied. The BAN study took place in a region where malaria, a cause of anemia , is prevalent and all infants are breastfed, whereas the previous study took place in a nonmalarial region of Botswana and very few infants receiving CPT were breastfed. Nearly 50% of HEU infants in another study in Uganda and Zimbabwe (HPTN-046) who were taking CPT developed severe anemia and/or neutropenia . However, the analysis focused on the effects of antiretrovirals in the studied population, and did not present results for severe anemia and severe neutropenia separately; also, because all participants were also exposed to CPT, the effects of each on the outcomes could not be determined.
Although there is evidence from studies in adults that the combination of CPT and zidovudine-containing antiretroviral treatment may cause hematologic toxicities , our analysis shows that for HEU infants in areas of high malarial prevalence, it is possible that CPT may reduce the occurrence of severe anemia. In a separate analysis of data from the BAN trial, it was shown that CPT was associated with a reduction in subclinical malaria, although it had no effect on clinical malaria . Thus, CPT may cause a reduction in malaria-related anemia separate from the drug's direct effects on blood hemoglobin. Severe anemia in sub-Saharan Africa is associated with adverse clinical outcomes, including death [19,34,35], and the results of this analysis suggest that in populations with a high malaria prevalence, CPT has the potential to reduce the frequency of these outcomes among HIV-exposed infants.
The analysis did show an increase in severe neutropenia and a corresponding reduction in neutrophil counts associated with CPT. However, the effect on neutrophil count lessened with age, and the frequency of severe neutropenia was low even for those exposed to CPT. We did not find any association between CPT exposure and elevated ALT.
There was no overall difference in severe anemia or severe neutropenia according to antiretroviral treatment arm, although those infants receiving daily nevirapine did have a lower estimated hazard of severe anemia compared with the control arm. Likewise, in longitudinal mixed models, both maternal antiretroviral and infant nevirapine were associated with higher levels of hemoglobin in the absence of cotrimoxazole .
The study instructed mothers to wean rapidly after 28 weeks, and self-reported adherence to the breastfeeding schedule was high. Median breastfeeding duration was 169 days, and did not vary by study arm. Median breastfeeding duration was 172 days for infants born before CPT implementation and 169 days for those born after (Kruskal–Wallis test, P < 0.001). Current guidelines for HIV-infected women on antiretroviral therapy (ART) recommend breastfeeding until 12–24 months . HIV-uninfected mothers in Malawi generally continue breastfeeding until about 24 months, which means that the infants will continue to be exposed to antiretrovirals and CPT. The reassuring findings of this study related to drug toxicities, combined with results from BAN and other studies showing increased morbidity and mortality in HIV-exposed infants after early weaning suggest that the benefits of continued breastfeeding should outweigh concerns related to these exposures.
Whereas a randomized controlled trial would be ideal to evaluate the hematologic effects of CPT in HEU infants in areas of high malaria prevalence, this is not possible or ethical following the release and implementation of CPT guidelines from the WHO and Malawi Ministry of Health. Thus, our analysis took advantage of the unplanned experiment caused by the BAN study's implementation of CPT in June 2006 by treating CPT as a time-varying exposure. To account for secular trends in participants’ environments and health that may have confounded the association between CPT and the studied outcomes, longitudinal mixed models included factors indicative of maternal and infant health before entry into the analysis cohort at 6 weeks of age, along with variables to account for seasonal changes. Despite our ability to control for these factors, there may have been changes over time in participants’ health unrelated to CPT that we could not account for.
Changes in the study protocol over time may have affected results. An increase in the minimum CD4+ cell count for enrollment eligibility, from less than 200 to less than 250, 6 months after CPT implementation and changes to the maternal antiretroviral regimen over time , may have led to the inclusion of healthier individuals later in the study, and changes in study population health unrelated to CPT. These changes should be at least partially addressed by the factors adjusted for in longitudinal mixed models, which included maternal CD4+ cell count. If changes to the maternal antiretroviral regimen improved outcomes at around the same time as CPT implementation and inflated the observed positive associations between CPT and hemoglobin levels, this effect should show up in the maternal antiretroviral arm. Instead, in linear mixed models, the positive association between CPT and hemoglobin levels was reduced in the maternal antiretroviral arm compared to the control arm, and associations between CPT and other outcomes did not differ according to study arm (Table 3). Likewise, the similarity of the results of the sensitivity analysis to the main analysis indicates that the observed effect of CPT was not due to the shift, later in the study, of participants to the two intervention arms. Rates of analysis-specific exclusions were nondifferential with respect to CPT implementation, so we do not have reason to expect bias to results. Most of the 146 HIV-related exclusions were due to in-utero infection (n = 119); postnatal before 6-week visit infections (n = 27) were more likely in the control group. If these excluded infants were more likely to suffer adverse study outcomes due to poor health, this could bias results related to the two antiretroviral arms towards significant associations, but the number involved is small and any effect is unlikely to be large. Exclusions due to loss to follow-up (LTFU) and infant deaths before the 6-week visit were nondifferential by study arm. The results of this analysis may only apply to populations similar to the one studied.
The findings of this analysis further support the use of daily CPT among HEU infants, especially in areas of high malaria prevalence. There has been concern about the potential for the combined use of CPT and antiretroviral treatment to cause hematologic toxicities. Whereas our data suggest that CPT may increase the hazard of severe neutropenia, the overall prevalence of this outcome remained low. The reduction in severe anemia associated with CPT is an additional benefit for HEU infants at increased risk for adverse health outcomes and mortality.
Author contributions: A.P.K., C.S.C., C.vd.H., and D.J.J. designed the trial. C.S.C., D.K., G.T., M.C.H., and M.G.H. collected data. A.C.E., A.F., and C.C.K. analyzed data. A.C.E., A.P.K., C.C.K., J.B.W., C.vd.H., and D.J.J. interpreted data. A.C.E. wrote the manuscript. All authors reviewed versions of the report and contributed to the intellectual content of the article.
We are grateful to the BAN Study Team at University of North Carolina Chapel Hill, Centers for Disease Control and Prevention, Atlanta, and UNC Project in Lilongwe: Linda Adair, Yusuf Ahmed, Mounir Ait-Khaled, Sandra Albrecht, Shrikant Bangdiwala, Ronald Bayer, Margaret Bentley, Brian Bramson, Emily Bobrow, Nicola Boyle, Sal Butera, Charles Chasela, Charity Chavula, Joseph Chimerang’ambe, Maggie Chigwenembe, Maria Chikasema, Norah Chikhungu, David Chilongozi, Grace Chiudzu, Lenesi Chome, Anne Cole, Amanda Corbett, Amy Corneli, Nicole Davis, Anna Dow, Ann Duerr, Henry Eliya, Sascha Ellington, Joseph Eron, Sherry Farr, Yvonne Owens Ferguson, Susan Fiscus, Valerie Flax, Ali Fokar, Shannon Galvin, Laura Guay, Chad Heilig, Irving Hoffman, Elizabeth Hooten, Mina Hosseinipour, Michael Hudgens, Stacy Hurst, Lisa Hyde, Denise Jamieson, George Joaki (deceased), David Jones, Elizabeth Jordan-Bell, Zebrone Kacheche, Esmie Kamanga, Gift Kamanga, Coxcilly Kampani, Portia Kamthunzi, Deborah Kamwendo, Cecilia Kanyama, Angela Kashuba, Damson Kathyola, Dumbani Kayira, Peter Kazembe, Caroline C. King, Rodney Knight, Athena P. Kourtis, Robert Krysiak, Jacob Kumwenda, Hana Lee, Edde Loeliger, Dustin Long, Misheck Luhanga, Victor Madhlopa, Maganizo Majawa, Alice Maida, Cheryl Marcus, Francis Martinson, Navdeep Thoofer, Chrissie Matiki (deceased), Douglas Mayers, Isabel Mayuni, Marita McDonough, Joyce Meme, Ceppie Merry, Khama Mita, Chimwemwe Mkomawanthu, Gertrude Mndala, Ibrahim Mndala, Agnes Moses, Albans Msika, Wezi Msungama, Beatrice Mtimuni, Jane Muita, Noel Mumba, Bonface Musis, Charles Mwansambo, Gerald Mwapasa, Jacqueline Nkhoma, Megan Parker, Richard Pendame, Ellen Piwoz, Byron Raines, Zane Ramdas, John Rublein, Mairin Ryan, Ian Sanne, Christopher Sellers, Diane Shugars, Dorothy Sichali, Wendy Snowden, Alice Soko, Allison Spensley, Jean-Marc Steens, Gerald Tegha, Martin Tembo, Roshan Thomas, Hsiao-Chuan Tien, Beth Tohill, Charles van der Horst, Esther Waalberg, Elizabeth Widen, Jeffrey Wiener, Cathy Wilfert, Patricia Wiyo, Innocent Zgambo, Chifundo Zimba. Finally and most especially, all the women and infants that have agreed to participate in the study.
The Breastfeeding, Antiretrovirals, and Nutrition Study was supported by grants from the Prevention Research Centers Special Interest Project of the Centers for Disease Control and Prevention (SIP 13–01 U48-CCU409660-09, SIP 26-04 U48-DP000059-01, and SIP 22-09 U48-DP001944-01); the National Institute of Allergy and Infectious Diseases, the University of North Carolina Center for AIDS Research (P30-AI50410); the Carolina Population Center (R24 HD050924); the National Institutes of Health Fogarty International Programs [AIDS International Training and Research Program (D43 TW001039) and Scholars and Fellows Program (R24 TW007988); the American Recovery and Reinvestment Act]; and the Bill and Melinda Gates Foundation (Grant # OPP53107).
The antiretrovirals used in the BAN study were donated by Abbott Laboratories, GlaxoSmithKline, Boehringer Ingelheim, Roche Pharmaceuticals, and Bristol-Myers Squibb. The Call to Action PMTCT program was supported by the Elizabeth Glaser Pediatric AIDS Foundation, the United Nations Children's Fund, the World Food Program, the Malawi Ministry of Health and Population, Johnson & Johnson, and the U.S. Agency for International Development.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2017 Wolters Kluwer Health, Inc.
anemia; antiretrovirals; BAN; breastfeeding; cotrimoxazole; hematologic toxicities; HIV-exposed uninfected; infants; malaria; neutropenia