Different assessment frequencies between study groups (ZDV-BF had monthly hemoglobin measurements) could bias results if treatment was modified in response to anemia (supplementation, change in antiretroviral treatment, or transfusion) and this prevented subsequent detection at the next scheduled 1-month, 3-4 months, or 6-7 months visit. However, we found little evidence of potential bias, as there were no significant differences in the rate of treatment-modifying anemia detected between birth, 1-month, 3-4 months, or 6-7 month visits among the study groups (P = 0.150)-28 in HAART-BF (4.1%), 10 in ZDV-BF (2.0%), and 17 in ZDV-FF (3.4%).
In addition to the effect of exposure group, there were significant positive associations between low maternal income, male sex, and risk of severe infant anemia in univariate analysis. Gestational age at birth was negatively associated with the risk of severe anemia. No significant associations between severe anemia and other maternal or infant characteristics were found, including maternal HIV disease status (Table 3).
In multivariate analysis, antiretroviral and feeding exposure group was identified as the strongest risk factor for severe infant anemia from birth to 6 months of age. Infants in the HAART-BF group had 2.6-fold greater odds of severe anemia as similarly breastfed but HAART-unexposed infants taking 6 months of postnatal ZDV in the ZDV-BF group. HAART-BF infants had 5.8-fold greater odds of developing severe anemia compared with ZDV-FF infants. Decreased gestational age at birth, and male sex were also significantly associated with severe anemia (Table 3).
The majority of the episodes of severe anemia were asymptomatic and resolved with multivitamin and iron supplementation or cessation of ZDV exposure. Median time to improvement to less than grade 3 was 33 days (interquartile range, 16-62 days). Severe anemia persisted for greater than 90 days despite interventions for 18 (17.0%) anemic infants. In addition, 11 infants required transfusion (all in HAART-BF group). None of the transfused infants died before 6 months of age.
Severe anemia may have contributed to the death of 6 infants (0.3%)-1 HAART-BF infant, 2 ZDV-BF infants, and 3 ZDV-FF infants. Severe anemia was reported as the cause of death for 3 of these infants. Alternative causes of death were reported for the remaining 3 infants (2 gastroenteritis, 1 unknown), but unresolved severe anemia may have contributed. Two infants were lost to follow-up before resolution of severe anemia, and their vital status could not be determined.
The MCV was similar between the exposure groups at 1 month of age, however, the change from 1 month to 6 months differed (P < 0.001). Consistent with the observed relationship between ZDV and macrocytosis,37-39 infants exposed to 6 months of postnatal ZDV (ZDV-BF) experienced a mean gain in their standardized MCV (Z score) of +2.25 (P < 0.001). In contrast, infants in the HAART-BF group with more limited ZDV exposure progressed toward microcytosis with mean change in their MCV Z score of −0.60 (P < 0.001). Infants in the ZDV-FF group, receiving iron-fortified infant formula, had a modest increase in their mean MCV Z score of +0.27 from 1 to 7 months of age (P < 0.001).
At the time of severe anemia, microcytosis was more common than macrocytosis, observed in 21.2% and 2.5% of infants, respectively. Similarly hypochromia was present in 29.3% of incident anemias with available MCHC measurement (Fig. 2). At the time of incident anemia, the mean MCV Z score was −0.91 (95% CI: −1.25 to −0.59) and the mean MCHC Z score was −1.31 (95% CI: −1.77 to −0.84).
Estimated hemoglobin iron at birth, the largest iron compartment of total body iron,40 was lower for HAART-BF, than for ZDV-BF, and ZDV-FF infants-133g, 143g, and 142g, respectively (P < 0.001).
In this study of infants born to HIV-infected mothers in Botswana, exposure to maternal HAART was associated with a significantly higher risk of severe anemia during the first 6 months of life compared with exposure to maternal ZDV alone. Among BF infants, assignment to in utero and breast milk HAART exposure (HAART-BF) was associated with an estimated 2.6-fold increased odds of severe anemia compared with infants assigned to in utero and 6 months of postnatal ZDV exposure (ZDV-BF). Formula-fed infants assigned to in utero and 1 month of ZDV (ZDV-FF) experienced the lowest risk of anemia, 5.8-fold and 2.2-fold decreased odds compared with HAART-BF and ZDV-BF, respectively.
The results of this study are in agreement with the findings of observational studies of largely formula-feeding cohorts in the United States and Europe that have demonstrated lower hemoglobin concentrations among infants exposed to in utero HAART compared with infants exposed to monotherapy or no in utero antiretrovirals.22-24,41 Calculating from the incidence of anemia reported for a cohort of 215 German infants,41 HAART-exposed infants had 3.9-fold increased odds of severe anemia compared with infants exposed to mono- or dual-antiretroviral therapy, similar to the findings in our study. Our study, utilizing a controlled design, confirms findings reported in observational studies and extends the findings to African and BF populations.
The mechanism for the observed increase in severe anemia due to maternal HAART exposure is uncertain. Infants in the HAART-BF and ZDV-BF groups seemed to experience a deeper and more prolonged physiologic nadir in hemoglobin. Infants in the HAART-BF group were exposed to in utero ZDV for a median of 11.5 weeks compared with 5.6 weeks in the ZDV-FF and ZDV-BF groups, but direct infant ZDV receipt occurred for a longer period in the ZDV-BF group. If ZDV were the principal cause of severe anemia, we would have expected the greatest incidence of severe anemia in the ZDV-BF group exposed to 6 months of postnatal ZDV, particularly given minimal exposure to ZDV via breast milk in the HAART-BF group.45 The fact that the HAART-BF group had more severe anemias suggests that either the timing of exposure or the combination of ZDV with other antiretrovirals may be important considerations. In a randomized trial in Malawi of postnatal maternal HAART, rates of severe anemia were similar between infants with exposure to HAART via breast milk and those without exposure to antiretrovirals in breast milk,15 suggesting that it is in utero exposure that increases risk for anemia. The addition of lamivudine may also promote the hematologic toxicity of ZDV as suggested by several case reports46-48 and 1 controlled trial49 in adults. However, other larger controlled trials have not reported this effect.50,51
The type of infant anemias that we observed is noteworthy. Despite universal exposure to ZDV, which promotes macrocytosis, microcytic (and hypochromic) anemia were common, particularly among HAART-exposed infants, suggesting a role of iron deficiency. Mothers in both the Mashi and Mma Bana studies were provided iron supplementation as part of antenatal care. It is possible that HAART exposure could increase vulnerability of hematopoiesis to nutrient deficiencies, infection, or other insults. Additionally, HAART could impair the transfer of iron from mother to fetus as suggested by decreased total hemoglobin iron estimated in HAART-BF infants. The observed morphology of infant red blood cells and the decreased hemoglobin iron suggest that infant iron supplementation should be considered.
This study is subject to several limitations. The assigned exposures of the Mashi and Mma Bana trials preclude separation of antiretroviral and feeding effects because almost all HAART-exposed infants were breastfed. A comparator group of HIV-exposed infants without exposure to antiretroviral treatment would be informative but unethical. Our analysis cannot separate contributions of antenatal and postnatal antiretroviral exposure toward anemia. Another potential limitation of our analysis was the difference in study timing-HAART-BF infants were from the Mma Bana study, whereas the ZDV-BF and ZDV-FF groups were from the earlier Mashi study. However, mothers assigned to HAART were more likely to have higher incomes, more education, and live in urban areas, reflecting the economic development in the years between Mashi and Mma Bana. Consequently, the effect of measured or unmeasured socioeconomic and nutritional factors would be expected to be favorable to HAART-BF infants, decreasing their incidence of severe anemia. Additionally, measured differences in maternal or infant characteristics between comparator groups were controlled for in multivariate analyses, and indeed some of these factors (such as small for gestational age and prematurity) may be associated with in utero HAART exposure.52-54 For these reasons, our findings may underestimate the impact of maternal HAART on the incidence of severe early infant anemia. Nevertheless, our study design cannot conclusively establish causality between in utero HAART exposure and infant anemia, and further study of this important question is warranted.
The clinical implications of our findings also require further study. The majority of infants with severe anemia in the Mashi and Mma Bana trials did not have symptoms reported. Observational studies in Africa suggest that severe anemia is associated with increased mortality, however, these studies have typically used a much lower hemoglobin threshold (<5.0 g/dL) to define severe anemia28 than was used in this study. Our analysis used the DAIDS toxicity table definitions for severe anemia-less than 10 g/dL at birth, 8 g/dL at 1 month, and 9 g/dL at 3 and 6 months.33 Data to describe the impact of less severe anemia are scant. In Tanzania, using a similar hemoglobin threshold of < 8.5 g/dL, HIV-exposed infants with iron deficiency anemia were approximately twice as likely to die before 2 years of age.55 Anemia may have contributed to 6 infant deaths in our study, but only one of these infants was HAART-exposed in utero. It is possible that routine screening and access to transfusion prevented additional deaths, particularly among the HAART-exposed infants.
Although our analysis focused on severe anemia, HAART-BF infants also had lower hemoglobin measurements at 3 and 6 months and were more likely to have any grade of anemia. Mild or moderate anemia due to iron deficiency is of significant public health importance due to its association with impaired child development.56 It remains uncertain whether anemia in the absence of iron deficiency impairs child development,57 however, the possibility of an adverse effect of maternal HAART, mediated via anemia, on subsequent infant development should be explored.
In summary, we have shown that maternal HAART started in pregnancy and continued during BF was associated with increased severe infant anemia. Confirmation of this finding and the etiology of anemia in HAART-exposed infants deserve further study. Given the established benefits of HAART for the prevention of MTCT11-14,58 and promotion of maternal health,59 the clinical implications of laboratory-detected toxicity should be considered carefully. Mitigating strategies such as iron supplementation to HIV-exposed breastfed infants or alternative antiretrovirals should be evaluated to maximize the benefits of maternal HAART although minimizing potential risks.
We are indebted to the participating patients and staff of the Mashi and Mma Bana studies, to the support of the Botswana Ministry of Health, and to our collaborating partners in the clinics and hospitals of Molepolole, Lobatse, Gaborone, and Mochudi. In addition, we thank the comments and suggestions offered by Drs Parth Mehta, Kenneth McIntosh, and Heather Ribaudo and the programing assistance of Jean Leidner.
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