Maternal malnutrition is associated with increased risks of infant morbidity and mortality1. Multiple micronutrient deficiencies may develop in pregnant women early during human immunodeficiency virus type 1 (HIV-1) infection2,3. Zinc deficiency, which is common in HIV-infected individuals, has been associated with higher mortality and viral load4,5. Zinc supplementation during pregnancy results in improved neonatal immune status, reduced early neonatal morbidity and fewer infant infections6. Pregnancy increases the risk of vitamin A deficiency in mothers and newborns. Randomized trials of vitamin A supplementation in pregnant women found decreased mortality and a reduced prevalence and duration of infectious episodes in infants7. In a Brazilian study, maternal vitamin A deficiency was strongly associated with infant vitamin A deficiency and low birth weight8. Brazilian HIV-infected pregnant women may have low vitamin A levels even after supplementation9.
HIV infection and pregnancy are accompanied by oxidative stress. Lower levels of vitamin E may play a pathogenic role in the onset and development of acquired immunodeficiency syndrome (AIDS) and other infectious diseases10,11. Several studies have revealed lower vitamin E levels in HIV-infected children in Brazil and elsewhere12–14. Among healthy pregnant women, infant birth weight and length are associated with maternal serum concentrations of antioxidant vitamins15. Anemia is the most frequent hematologic abnormality found in HIV disease16. Standard prenatal care for HIV-infected pregnant women in Latin America includes iron and folate supplements to prevent anemia and neural tube defects.
We hypothesized that nutritional deficiency is frequent among HIV-infected mothers and their infants in Brazil. Our objectives were as follows: 1) to describe the nutritional status of HIV-infected women at delivery and of their infants at sites in Brazil based on anthropometric measurements, including the maternal adjusted for gestational age (adjBMI), and on plasma micronutrient concentrations; 2) to assess the correlation between maternal and infant micronutrient levels at delivery; and 3) to evaluate changes in the micronutrient status of infants from birth to six months of age.
The LILAC study
HIV-infected pregnant women enrolled at three participating Brazilian sites in the Eunice Kennedy ShriverNational Institute of Child Health and Human Development (NICHD), International Site Development Initiative (NISDI) and Longitudinal Study in Latin American Countries (LILAC) dynamic cohort were invited to participate in this nutritional LILAC sub-study. The design and conduct of the LILAC study has been described previously17.
This nutritional sub-study enrolled LILAC participants at three study sites in Brazil. The protocol for this sub-study was approved by the Institutional Review Boards (IRBs) of the participating institutions. This sub-study required the collection of additional blood from mothers (within seven days of delivery) and infants (at birth and then at the 6-12-week and 6-month study visits) for the analysis of micronutrient levels (retinol, α-tocopherol, ferritin, and zinc). The blood samples were stored at -70°C in the dark until processing. Plasma retinol and α-tocopherol concentrations were quantified by high performance liquid chromatography (HPLC) as previously described18. Serum zinc was determined by atomic absorption spectrophotometry using standard procedures19. Maternal C-reactive protein (CRP) levels were quantified to characterize inflammation. CRP and ferritin were measured in plasma samples using immunoassay technology (Immulite 1,000 Immunoassay System – Global Siemens Healthcare Headquarters – Siemens AG; Henkestrasse 127; D-91052 Erlangen – Germany).
The maternal body mass index (BMI) at the last study visit during pregnancy was adjusted for gestational age (adjBMI) using a computer algorithm provided by the Ministry of Health of Argentina20 and was used as a criteria for nutritional status classification. Maternal adjBMI values that were <19.8 were considered underweight, ≥26.1 to <29 were considered overweight, and ≥29 to <50 were classified as obese. Maternal and infant micronutrient status was defined at each time point as deficient vs. normal for each micronutrient measured. Deficiencies were defined as follows for each micronutrient: zinc level, <64mg/dL for infants and <50mg/dL for mothers21; retinol level, <0.7mmol/L for both infants and mothers22; α-tocopherol level, <12mmol/L for infants and <7mmol/L for mothers23; and ferritin level, <10ng/mL for mothers, <25ng/mL for infants less than one month of age, <200ng/mL for infants one to two months of age, <50ng/mL for infants aged two months to less than six months, and <7ng/mL for ages six months to 15 years24. The cut-off values for C-reactive protein (CRP) categories in pregnant women were as follows: low, < 1mg/dL; normal, 1-3mg/dL; high, >3mg/dL25. Infant CRP cut-off values were as follows: normal, ≤0.5mg/dL; high, >0.5mg/dL26. HIV disease was classified according to the Centers for Disease Control and Prevention (CDC) definitions27. A low mean corpuscular hemoglobin concentration (MCHC) level was defined as <31g/dL for pregnant women and for infants24. The cut-off values for hemoglobin in pregnant women were as follows: severe deficiency, 6.5-7.4g/dL; low, 7.5-8.4g/dL; mild deficiency, 8.5-10g/dL; and normal, >10g/dL28. The normal hemoglobin range for infants was adjusted for age: one to three days, 14.5-18.5g/dL; two weeks, 13.4-16.6g/dL; one month, 10.7-13.9g/dL; two months, 9.4-11g/dL; and from two to six months, 11.1-12.6g/dL28.
Maternal antiretroviral (ARV) regimens were categorized in order of regimen complexity as follows: 1) one or two nucleoside reverse transcriptase inhibitors (NRTIs), 2) two NRTIs and one non-nucleoside reverse transcriptase inhibitor (NNRTI), 3) two NRTIs and one protease inhibitor (PI), and 4) other. The reason for the use of ARVs during pregnancy was categorized as treatment if ARVs were used before pregnancy and/or continued after the 6-12-week study visit, or as prophylaxis if ARVs were initiated during pregnancy and use was discontinued by the 6-12-week visit.
Associations between maternal adjBMI values and the following characteristics of mothers and infants were examined: maternal age at enrollment, maternal years of formal education, number of persons in the household, mother’s gainful employment outside the home, substance use during pregnancy, reasons for ARV use during pregnancy, maternal ARV regimen, maternal cluster of differentiation 4 (CD4) + T-lymphocyte (CD4) count, plasma Human immunodeficiency virus-ribonucleic acid (HIV-RNA) concentration [viral load(VL)], CDC clinical HIV disease classification27, gestational hypertension, gestational diabetes, preterm birth (<37 weeks of gestational age)1, low infant birth weight (<2,500g)1, infant ARV regimen and CD4+ T-lymphocyte count at birth. Associations between maternal adjBMI and maternal and infant micronutrient levels, which were categorized as deficient vs. normal, were examined. Additionally, associations between micronutrient levels in mothers and infants were examined. The maternal vitamin A, vitamin E, iron, and zinc levels were examined within seven days of delivery, and infant micronutrient status was examined at birth, 6-12 weeks, and six months of age. These associations were examined using Fisher’s exact test or the Fisher-Freemen-Halton extension to Fisher’s exact test. Significant bivariate results were further investigated on the basis of relative risks (RRs) and 95% confidence intervals (95% CIs) were obtained from log-binomial regression modeling. Linear regression and one-way analysis of variance (ANOVA) were used to examine associations between continuous maternal and infant nutritional variables at the study time points. All p-values were two-sided. The p-values<0.05 were considered statistically significant. Analyses were conducted using SAS version 9.3 statistical software (SAS Institute, Cary, NC, USA).
In total, 123 women that were enrolled in the LILAC protocol at the participating sites provided informed consent to participate in this sub-study, and blood samples were available for 97 mother-infant pairs. Thus, the study population was composed of 97 mother-infant pairs. All infants were HIV-uninfected.
The characteristics of the 97 mother-infant pairs overall and according to maternal adjBMI at the last study visit during pregnancy are shown in Table 1. Based on the adjBMI measurements, 26 (26.8%) of the mothers were classified as overweight/obese, 52 (53.6%) were normal weight, and 19 (19.6%) were underweight. The reason for using ARVs during pregnancy was significantly associated with adjBMI (p-value=0.03); a higher proportion of overweight/obese women received ARVs for prophylaxis (as opposed to treatment) during pregnancy compared with normal weight or underweight women. No associations were observed between maternal adjBMI and years of education (p-value=0.45), the number of persons in the household (p-value=0.41), gainful employment outside the home (p-value=0.54), tobacco use during pregnancy (p-value=0.74), alcohol use during pregnancy (p-value=0.71), cocaine use during pregnancy (p-value=0.42) or marijuana use during pregnancy (p-value=0.42). Additionally, maternal adjBMI was not associated with preterm birth (p-value=0.77), low birth weight (p-value=0.28) or infant CD4 count at birth (p-value=0.59). The median gestational age at the time of adjBMI assessment was 33 weeks (minimum, 25 weeks; maximum, 39 weeks). Underweight women were significantly more likely to have low MCHC after delivery than their normal and overweight counterparts (p-value<0.01; data not shown).
|Age at enrollment (years)||0.17|
|Reason for use of ARVs during pregnancy||0.03|
|Most complex ARV regimen used ≥28 days of pregnancy||0.37|
|2 NRTIs and 1 PI||19||76.0||39||79.6||16||88.9||74||80.4|
|2 NRTIs and 1 NNRTI||3||12.0||8||16.3||1||5.6||12||13.0|
|CD4 count (cells/mm3) at enrollment||0.09|
|Viral load (copies/mL) at enrollment||0.09|
|CDC disease stage at enrollment||0.53|
adjBMI: body mass index adjusted for gestational age; ARV: antiretroviral; NRTI: nucleoside reverse transcriptase inhibitors; PI: protease inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor; CD4: cluster of differentiation 4; CDC: Centers for Disease Control and Prevention.
Nutritional and inflammatory marker data for the mothers and infants are summarized [mean, standard deviation (SD)], median, and range) in Table 2. The test results are categorized as normal versus deficient in Table 3. The most frequent maternal deficiencies were zinc (41.1%), followed by retinol (12.5%), ferritin (6.5%) and α-tocopherol (1%) (Table 3). Additionally, 46.4% of the women had low hemoglobin levels, and the MCHC was <31g/dL in 5.2% of the women. Approximately 54% of the women had high levels of CRP in their blood at hospital discharge following delivery. At birth, 81.1% of the infants were deficient in α-tocopherol, with the proportion decreasing with age to only 18.5% by six months of age. High proportions of neonates also had deficient levels of zinc (36.8%) and retinol (29.5%) at birth, and these numbers fell to only 17.5% and 18.5%, respectively, by six months of age. The proportion of infants with low levels of retinol increased to 41.1% by 6-12 weeks of age before decreasing by six months of age. Although none of the infants had low ferritin levels at birth, a sharp increase was observed in the proportion of infants with low ferritin levels at 6-12 weeks of age (58.7%) and at 6 months of age (25.8%). Additionally, low hemoglobin levels were observed in 43.8% and 7.5% of the infants at 6-12 weeks of age and at 6 months of age, respectively. All infants were formula-fed after birth.
|Parameter||Mothers||Infants (at birth)||Infants (at 6-12 weeks)||Infants (at 6 months)|
|mean (SD)||median||range||mean (SD)||median||range||mean (SD)||median||range||mean (SD)||median||range|
|Retinol (µmol/l)||1.1 (0.4)||1.1||0.2-2.2||0.9 (0.2)||0.8||0.3-1.6||0.8 (0.4)||0.8||0.2-2.0||1.0 (0.4)||0.9||0.4-2.5|
|α-Tocopherol (µmol/L)||17.1 (4.9)||16.3||6.3-35.4||8.5 (3.4)||7.7||3.2-20.2||17.2 (7.9)||15.7||3.7-36.4||18.0 (6.5)||17.0||7.1-44.1|
|Hemoglobin (g/dL)||10.2 (1.8)||10.3||4.3-15.0||14.9 (2.1)||15.2||8.5-19.0||9.9 (1.5)||9.7||7.5-19.8||11.6 (1.3)||11.7||8.8-18.9|
|MCHC (g/dL)||34.0 (2.0)||34.0||31.0-47.0||33.5 (1.4)||34.0||27-37||33.5 (1.2)||33.0||31-37||33.9 (2.8)||34.0||31-58|
|Ferritin (ng/mL)||61.1 (55.1)||44.5||4.0-305.0||268.5 (207.5)||217.0||26.6-1447.0||181.7 (115.5)||158.5||7.3-793.0||43.0 (39.6)||38.0||2.4-251.0|
|Zinc (mg/dL)||56.1 (31.1)||56.0||15.7-314.5||74.9 (31.4)||72.5||28.5-276.0||102.8 (153.5)||69.5||39.9-995.0||83.6 (21.6)||80.5||44.8-137.0|
|C-reactive protein (mg/dL)||5.2 (5.7)||3.3||0.0-29.3||0.4 (1.0)||0.1||0.0-7.8||0.3 (1.2)||0.0||0.0-10.0||0.2 (0.6)||0.0||0.0-4.2|
|Mothers||at birth||6-12 weeks||6 months|
|Age-adjusted hemoglobin (g/dL)||normal||52||53.6||77||79.4||54||56.3||86||92.5|
|C-reactive protein (mg/dL)||low||18||18.6||0||0.0||0||0.0||0||0.0|
When we compared the underweight adjBMI category with the normal adjBMI category, no association was found between maternal underweight adjBMI during pregnancy and maternal zinc (RR=1.2, 95% CI: 0.7, 2.3), retinol (RR=0.7, 95% CI: 0.2, 2.9), α-tocopherol (RR not calculable), or ferritin (RR=1.4, 95% CI: 0.5, 4.4) deficiency at delivery, nor was any observed association found between maternal underweight adjBMI during pregnancy and infant zinc (RR=0.9, 95% CI: 0.4, 1.9), retinol (RR=0.6, 95% CI: 0.2, 1.7), α-tocopherol (RR=0.9, 95% CI: 0.6, 1.2), ferritin (RR=0.7, 95% CI: 0.4, 1.3), or MCHC at birth (RR=2.7, 95% CI: 0.2, 41.6; Table 4). In addition, maternal zinc, α-tocopherol, ferritin, and retinol deficiencies were not associated with age, years of education, the number of persons in the household, gainful employment outside the home, tobacco use during pregnancy, alcohol use during pregnancy, cocaine use during pregnancy, marijuana use during pregnancy, preterm birth, low birth weight or infant CD4 count at birth (data not shown). Women with acquired immunodeficiency syndrome (AIDS) were less likely to have normal retinol levels than women with asymptomatic or mildly symptomatic HIV disease (RR=0.2, 95% CI: 0.1, 0.6; data not shown). Low maternal hemoglobin was associated only with low maternal CD4+ T-lymphocyte counts at enrollment. Women who had CD4 cell counts that were <200 cells/mm3 were less likely to have normal hemoglobin levels than were women with CD4+ T-lymphocyte counts that were ≥ 500 cells/mm3 (RR=0.4, 95% CI: 0.3, 0.7; data not shown). Low maternal zinc, ferritin, and hemoglobin were not associated with elevated maternal CRP (p-value>0.8). No observed associations were found between low maternal levels of zinc (RR=1.0, 95%CI: 0.6, 1.7), retinol (RR=1.5, 95%CI: 0.7, 3.2), α-tocopherol (RR not calculable), or ferritin (RR=0.9, 95%CI: 0.5, 1.6) after delivery and corresponding low levels of these micronutrients in the infants at birth (p-value>0.3; Table 5). Additionally, linear regression analysis did not reveal any significant associations between maternal and infant zinc, retinol, α-tocopherol, or ferritin levels at birth when the levels were examined on a continuous scale (p-value>0.15).
|Micronutrient||Maternal adjusted BMI||p-value**||RR (95% CI)* for maternal adjBMI|
|underweight vs normal||overweight/obese vs normal|
|Maternal micronutrient levels|
|deficient||11 (42.3%)||19 (38.0%)||9 (47.4%)||39 (41.1%)||1.2 (0.7, 2.3)||1.1 (0.6, 2.0)|
|normal||15 (57.7%)||31 (62.0%)||10 (52.6%)||56 (58.9%)||1.0|
|deficient||2 (7.7%)||8 (15.7%)||2 (10.5%)||12 (12.5%)||0.7 (0.2, 2.9)||0.5 (0.1, 2.1)|
|normal||24 (92.3%)||43 (84.3%)||17 (89.5%)||84 (87.5%)||1.0|
|deficient||1 (3.8%)||0 (0.0%)||0 (0.0%)||1 (1.0%)||NC||NC|
|normal||25 (96.2%)||51 (100.0%)||19 (100.0%)||95 (99.0%)|
|deficient||4 (16.0%)||7 (14.6%)||4 (21.1%)||15 (16.3%)||1.4 (0.5, 4.4)||1.1 (0.4, 3.4)|
|normal||21 (84.0%)||41 (85.4%)||15 (78.9%)||77 (83.7%)||1.0|
|Infant micronutrient levels at birth|
|deficient||7 (28.0%)||19 (41.3%)||6 (37.5%)||32 (36.8%)||0.9 (0.4, 1.9)||0.7 (0.3, 1.4)|
|normal||18 (72.0%)||27 (58.7%)||10 (62.5%)||55 (63.2%)||1.0|
|deficient||10 (38.5%)||15 (29.4%)||3 (16.7%)||28 (29.5%)||0.6 (0.2, 1.7)||1.3 (0.7, 2.5)|
|normal||16 (61.5%)||36 (70.6%)||15 (83.3%)||67 (70.5%)||1.0|
|deficient||21 (80.8%)||43 (84.3%)||13 (72.2%)||77 (81.1%)||0.9 (0.6, 1.2)||1.0 (0.8, 1.2)|
|normal||5 (19.2%)||8 (15.7%)||5 (27.8%)||18 (18.9%)||1.0|
|deficient||9 (42.9%)||30 (63.8%)||8 (47.1%)||47 (55.3%)||0.7 (0.4, 1.3)||0.7 (0.4, 1.2)|
|Normal||12 (57.1%)||17 (36.2%)||9 (52.9%)||38 (44.7%)||1.0|
|deficient||0 (0.0%)||1 (1.9%)||1 (5.3%)||2 (2.1%)||2.7 (0.2, 41.6)||NC|
|normal||26 (100.0%)||51 (98.1%)||18 (94.7%)||95 (97.9%)||1.0|
*The relative risks (RRs) and 95% confidence intervals (CIs) for predicting maternal and infant micronutrient deficiencies as a function of maternal adjBMI were obtained using log binomial regression models.
|Infant micronutrient||Maternal micronutrient level||p-value*||RR (95% CI)|
|Zinc||1.00||1.0 (0.6, 1.7)|
|deficient||22 (62.7%)||32 (62.7%)||54 (62.8%)|
|normal||13 (37.1%)||19 (37.2%)||32 (37.2%)|
|Retinol||0.33||1.5 (0.7, 3.2)|
|deficient||7 (58.3%)||59 (72.0%)||66 (70.2%)|
|normal||5 (41.7%)||23 (28.0%)||28 (29.8%)|
|deficient||0 (0.0%)||18 (19.3%)||18 (19.1%)|
|normal||1 (100.0%)||75 (80.7%)||76 (80.9%)|
|Ferritin||0.76||0.9 (0.5, 1.6)|
|deficient||6 (50.0%)||30 (44.1%)||36 (45.0%)|
|normal||6 (50.0%)||38 (55.9%)||44 (55.0%)|
No observed associations were found between low infant zinc, retinol or α-tocopherol levels at birth and low levels of the same micronutrients at 6-12 weeks or at six months of age (p-value>0.1). Low infant ferritin levels at birth were associated with normal infant ferritin levels at 6-12 weeks of age (p-value=0.04). This association persisted but was only marginally associated with levels at six months of age (p-value=0.07). Overall, a slightly lower proportion of infants had micronutrient deficiencies at the six-month visit than at birth (data not shown).
Micronutrient deficiencies are common in HIV-infected pregnant women and their infants in Brazil. The prevalences of most infant micronutrient deficiencies were lower at six months of age than at birth. Being underweight at the end of pregnancy was not associated with micronutrient deficiency in HIV-infected women or their infants at birth.
We found that 46.4% of HIV-infected mothers were anemic and that 6.5% had low levels of ferritin immediately postpartum. Few studies have reported iron deficiency in HIV-infected pregnant women in Latin America; however, some studies have reported an extremely high prevalence of anemia in HIV-infected pregnant women in other countries28; 78% of HIV-infected pregnant women in Burkina Faso were reported to be anemic29, and 83% of HIV-infected pregnant women in Cote d’Ivoire were reported to be anemic30.
Factors such as poor nutritional status, HIV infection31, ARV therapy32, and even vitamin D deficiency33could explain the high prevalence of anemia found in the present sub-study. However, the acute phase response34 was not associated with anemia. Few studies have examined lipid-soluble vitamins in HIV-infected pregnant women35–37. In Tanzania38, approximately 35 and 51% of HIV-infected pregnant women had low levels of vitamins A and E, respectively. In contrast, only 12.5 and 1% of women in our sub-study had vitamin A and E deficiencies, respectively, although our cut-off for vitamin E deficiency was lower than that used in Tanzania38 (vitamin E level<9.7 µmol/L). Zinc deficiency has been reported in children39 and in HIV-infected individuals5,40 and is thought to be widely prevalent in pregnant women due to hemodilution, decreased albumin levels and insufficient intake41–43.
Infants in our sub-study had lower rates of micronutrient deficiencies and higher mean levels of vitamin A and vitamin E than those found by Monteiro et al. in a group of HIV-infected Latin American children14. Nevertheless, 81% of infants at birth were deficient in α-tocopherol, which could partially be explained by increased oxidative stress during delivery. Increased levels of oxidative stress and lipid peroxidation induced by reactive oxygen species (ROS) production play a critical role in the stimulation of HIV replication and immunodeficiency and can be attenuated by the presence of vitamin E44. However, vitamin E may facilitate HIV entry into cells, and higher plasma vitamin E levels have been associated with adverse outcomes in HIV-infected individuals45. Thus, lower vitamin E levels may constitute a physiological defense mechanism against HIV in infants during delivery.
A high proportion of infants (41%) had vitamin A deficiency at 6 to 12 weeks, with mean and SD values of 0.8 and 0.4µmol/L, respectively. Chatterjee A et al. also found vitamin A deficiency at six weeks of age; The percentages of infants with plasma vitamin A concentrations less than 0.70mmol/L or 0.35mmol/L were 89% and 22%, respectively, in this sub-study46. These authors found that higher vitamin A concentrations in plasma at six weeks of age were protective against mortality in children born to HIV-infected women.
Our sub-study had several limitations. Vitamin A deficiency in this sub-study was defined based on WHO recommendations, which are widely accepted and used28; however, no consensus regarding a definition of vitamin E deficiency exists. Maternal adjBMI during pregnancy, which is used as a measure of maternal nutritional status, may not actually be a good surrogate for nutritional assessment. Additionally, maternal specimens collected up to seven days postpartum may not accurately represent nutritional deficiencies during pregnancy. We did not measure serum lipoprotein and cholesterol, which may be important because serum α-tocopherol (vitamin E) concentrations are highly dependent on serum lipid concentrations2,3. Likewise, this sub-study did not account for the dietary intake of vitamin A, vitamin E, iron or zinc. All infants were formula-fed and thus received at least some supplementation after birth, although the exact amounts of supplementation are not known. Moreover, the interpretation of serum micronutrient levels is not straightforward due to the wide range of unknown nutrient-nutrient and gene-nutrient interactions and physiological and environmental processes involved.
The present sub-study suggests that low serum micronutrient levels are frequently found in HIV-infected pregnant women and their infants and that neither underweight women nor their infants at birth were at increased risk for low serum micronutrient levels. Future studies in different countries that have appropriate designs and that examine genetic and proteomic profiles should be pursued to gain a greater understanding of the interactions among genes, nutrients, and the environment and of micronutrient requirements in HIV-infected and HIV-exposed individuals47–50.
CONFLICT OF INTEREST
The authors have no competing interests to declare. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the US National Institutes of Health or the US Department of Health and Human Services.