The HIV pandemic affects over 34 million people worldwide. Southern Africa remains the most affected area, where up to 40% of women live with HIV.1 The number of infants exposed to HIV during pregnancy, delivery, and breast-feeding is thus steadily increasing.2 In addition, the health of exposed noninfected children (ENIC) may also be affected by their mother's HIV status.2
Most studies on clinical outcomes in ENIC have been conducted in industrialized countries, where the disease burden and health systems are not comparable with the African region. In addition, studies conducted in sub-Saharan Africa have often been performed without a control group of HIV-unexposed (UE) children (UEC) or have not been designed to study ENIC.3–6 As a consequence, the impact of maternal HIV infection on their infant's health is not well established. Some studies suggest an increased mortality6–10 and morbidity3,9,11,12 in African ENIC, whereas others have observed similar health indicators compared with UEC.4,13,14 It has also been suggested that adverse outcomes could be because of nonspecific risk factors known to be associated with child morbidity and mortality that are also related to maternal HIV infection, such as maternal disease, type of feeding, child's anemia, low birth weight (LBW), and prematurity.2,10,11,15 However, immunologic alterations have been reported in ENIC as compared with UEC, including an increased level of CD8 T cells, reduced naive CD4 T cells, increased memory T cells, and an increased prevalence of neutropenia16–18 (de Deus N, Moraleda C, Serna-Bolea C, Renom M, Menéndez C, Naniche D, unpublished data, 2012). Although some of these factors specifically associated with HIV exposure, such as decreased CD4 T-cell counts, might have an impact on morbidity and mortality of ENIC,19,20 the overall clinical relevance of these abnormalities is still unclear.
Cotrimoxazol (CTX) prophylaxis (CTXP) has been associated with a decreased morbidity and mortality among HIV-infected children21 and among HIV-negative siblings of HIV patients,22 and it is indicated for ENIC in low resource settings.23 However, this recommendation is being reconsidered because of a lack of information of a protective effect in ENIC, the strengthening and duration of prophylaxis of mother-to-child transmission (PMTCT) programs, and the concern for development of bacterial resistance to CTX.24,25
The objective of this study was to help in guiding targeted clinical and preventive protocols for ENIC by increasing the knowledge of the clinical, immunologic, and hematologic profiles of these children.
Study Setting and Population
The study was conducted at the Manhiça district hospital (MDH) and the Manhiça Health Research Centre, Manhiça District, southern Mozambique. Since 1996, the Manhiça Health Research Centre has run a continuous demographic surveillance system with over 84,700 inhabitants under surveillance in 2009 and a morbidity surveillance, whereby all pediatric hospital admissions and visits to the outpatient department (OPD) at the MDH and other health posts are registered using standardized questionnaires. Malaria transmission is perennial with marked seasonality and mainly because of Plasmodium falciparum.26 At the time of the study, the antenatal clinic prevalence of HIV infection in pregnant women was 29%.27 National guidelines for PMTCT of HIV during the study followed the 2006 World Health Organization recommendation.28 After the first month of the study, the PMTCT national policy for all infants born to HIV mothers changed to 4 weeks of daily zidovudine and a single dose of nevirapine; they also received CTXP from 4 weeks of age until 2 months after weaning, as long as they had a HIV-negative serology test, while the mothers were counseled to exclusively breast-feed until 6 months of age.29 The protocol was approved by the National Committee on Health Bioethics of Mozambique and the Hospital Clinic of Barcelona Ethics Review Committee.
This study was a prospective, observational cohort study comparing 2 groups of children: those born to HIV-positive women and those born to HIV-negative women. All women residents in the study area delivering a singleton live born at the MDH were invited to participate. Infants who needed urgent medical care at birth were transferred to the pediatric ward and were not included. After giving informed consent, the mothers were tested for HIV using the Determine HIV-1/2 Rapid Test (Abbott Laboratories, Abbott Park, IL). Positive results were confirmed by the Uni-Gold Rapid Test (Trinity Biotech Co, Wicklow, Ireland). Exclusion criteria included a multiple delivery and/or having an indeterminate HIV test. A 5-mL venous sample was drawn from the HIV-positive mothers for CD4 and viral load determinations. All HIV-positive women were referred for clinical management according to the national guidelines. At the time of delivery, the baby's birth weight was measured on a digital scale and length using a height board. Gestational age was assessed by the Dubowitz's method.30
Study follow-up consisted of passive clinical surveillance of all outpatient visits and hospital admissions included in the morbidity surveillance (as described above) up to 1 year of age; in addition, 4 cross-sectional visits at 1, 3, 9, and 12 months postdelivery were carried out. At each visit, a standardized questionnaire, recording clinical data and feeding habits, was completed. A 1.5-mL venous sample was collected for hematologic and CD4/CD8 determinations, and a filter paper was prepared for HIV DNA polymerase chain reaction (PCR) test. Infants were considered uninfected if the HIV DNA-PCR at 1 month was negative or indeterminate and confirmed negative 2 weeks later. HIV DNA-PCR–negative infants born to HIV-positive mothers were assigned to the ENI group, and infants born to HIV-negative mothers were considered UE. At each subsequent study visit (3, 9, and 12 months), infants were tested for HIV DNA detected by PCR. Those with positive HIV DNA-PCR had a second PCR performed for confirmation, and if it was confirmed, they were referred for clinical management and were withdrawn from further study procedures.
The HIV status of the infants was assessed from dried blood spot on filter paper using the Amplicor HIV-1 DNA-PCR kit (Roche Diagnostics, Branchburg, NJ). Maternal HIV RNA viral load was determined from frozen plasma using the Amplicor HIV-1 Monitor assay version 1.5 (Roche Diagnostics). The assay has a sensitivity of 400 copies per milliliter. For the statistical analyses, plasma HIV-1 RNA concentrations below the limit of detection were assigned the value of 200 copies per milliliter. T-cell counts were determined by flow cytometry in a FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) after whole-blood staining with fluorochrome-labeled antibodies to CD3, CD8, CD4, and CD45 in Truecount tubes. Hematologic determinations were assessed on whole blood using a Coulter counter.
Clinical malaria was defined as either current fever (axillary temperature, ≥37.5°C) or a history of fever in the preceding 24 hours, plus a P. falciparum asexual parasitemia of any density on a blood slide. Diarrhea was defined as ≥3 watery loose stools in the previous 24 hours. Clinical severe pneumonia (CSP) was defined as dyspnea or cough, with increased respiratory rate according to age group and lower chest retractions. Acute lower respiratory infection (ALRI) was defined as fever plus abnormal lung auscultation without criteria for CSP. Acute upper respiratory infection (AURI) was defined as history of cough for less than 15 days without inclusion criteria for CSP or ALRI. Moderate wasting was defined as less than −2 weight-for-age Z score, moderate stunting as less than −2 length-for-age Z score, and moderate underweight as less than −2 weight-for-length Z score. Growth disturbances were considered severe in these 3 anthropometric parameters when Z score was less than −3.31 Exclusive breast-feeding was considered when the infant only received breast milk without any additional solid or liquid foods, including water. Mixed feeding was considered when the infant received breast-feeding and any additional solid or liquid foods and formula feeding when the infant received infant formula with or/without other foods or liquids, without breast milk.32 PMTCT was considered complete for the mother if she had received all treatments according to the guidelines28 and for the newborn if he/she received 1 dose of nevirapine in the first 72 hours after birth plus daily zidovudine for 4 weeks after delivery.28 Anemia was defined as a hematocrit <33%, microcytosis as a mean corpuscular volume <70 fL, and LBW as a birth weight <2.500 grams. An infant was considered premature if the gestational age was <37 weeks and a mother grand multipara if she had ≥5 previous deliveries. High viral load was considered >104 log10 copies per milliliter.33
Microsoft FoxPro version 5.0 (Microsoft Corp, Redmond, WA) was used for data entry, validation, and cleaning of data, and statistical analysis was performed with STATA release 11 (StataCorp, College Station, TX). Comparisons between groups for proportions were assessed using the χ2 test or Fisher exact test where appropriate. The Kruskal–Wallis test was used to compare independent continuous variables between groups. Linear mixed models were used to evaluate differences in hematologic parameters and T-cell counts between the study groups. These models take into account the structure of the data variance with repeated measures for each child and were estimated by maximum likelihood using a random intercept term at the subject level. The model validation is based on assessment of the residuals. Because %CD8 violates the assumption of normality of its residual, it is analyzed in logarithmic scale. According to this, coefficients from the regression models should be read as absolute differences between means for those variables analyzed in linear scale and proportional differences (ratios between geometric means) for those variables analyzed in logarithmic scale.
Incidence of outpatient visits and admissions was calculated using the observation period for the infants from the day of birth until the study participant reached any of the following: (1) withdrawal or lost of follow-up of the study, (2) death, (3) became HIV positive, or (4) reached 12 months of age. The date when an infant was considered HIV positive was the midpoint between the visit in which the result was positive and the previous visit. Children were excluded from the morbidity analysis for the periods spent outside the study area. An arbitrary lag of 15 days (28 for malaria outcomes) was applied after each registered episode during which children did not contribute to the time at risk or to the cases. Negative binomial regression models were estimated to compare incidence rates (IR), and P values were calculated using the likelihood ratio test.
A logistic regression model was created to test the association between mortality and the HIV status group [results reported as odds ratio and 95% confidence interval (CI)]. The probability of mother-to-child transmission during the first year of life was calculated as 1 − (negative proportion at 1 month visit × negative proportion at 3 months visit × negative proportion at 9 months visit × negative proportion at 12 months visit) × 100.
Between August 2008 and June 2009,158 HIV-positive mothers and 160 HIV-negative mothers and their children were enrolled. Characteristics of mother–infant pairs at delivery are shown in Table 1. Birth outcomes were similar in both groups. Among HIV-positive mothers, 5% (7/151) had CD4 below 200 counts per microliter at delivery (Table 2) and 13% (21/158) had received highly active antiretroviral therapy before delivery.
Mother-to-Child Transmission of HIV and Feeding Habits
Eighty-two percent (129/158) of the mothers and 70% (110/158) of their children had received complete PMTCT of HIV. At 1 month of age, HIV transmission rate was 9% [10/109 (95% CI: 3.7 to 14.7)]. Feeding methods were similar in the 2 groups until 6 months of age. Thereafter, the percentage of ENIC who were weaned increased progressively reaching 18% [10/55 (95% CI: 9.1 to 31.0)] at 9 months of age and 47% [20/42 (95% CI: 32.0 to 63.6)] at 12 months of age, whereas at both ages, 100% of UEC were still breast-feeding (P = 0.005 and P < 0.001 for 9 and 12 months, respectively). Mixed feeding in ENIC was 10% (95% CI: 4.9 to 17.6) at 1 month, 43% (95% CI: 33.7 to 58.1) at 3 months, 75% (95% CI: 61.0 to 85.2) at 9 months, and 52% (95% CI: 36.4 to 68.0) at 12 months of age. The rate of HIV transmission through breast-feeding assessed at 3, 9, and 12 months was 8% [6/76 (95% CI: 1.7 to 14.1)], 8% [5/59 (95% CI: 1.2 to 15.8)], and 5% [2/44 (95% CI: 1.9 to 10.9)], respectively. The estimated rate of mother-to-child transmission of HIV in the first year of life was 27%.
Hematologic Parameters During Infancy
The hematologic parameters of ENIC and UEC were compared at 1, 3, 9, and 12 months of age and assessed longitudinally. Median values of hematocrit were significantly lower in ENIC compared with UEC at 1, 3, and 9 months of age (P = 0.024, 0.025, and 0.012, respectively) (Fig. 1A). Multivariate longitudinal analysis showed that hematocrit remained lower in ENIC compared with UEC after adjusting for age (difference between means: −2.09; 95% CI: −3.34 to −0.84; P = 0.001). A similar but nonsignificant trend for lower hemoglobin in ENIC vs. UEC was also observed (Fig. 1B). The prevalence of anemia was consistently higher in ENIC vs. UEC but only reached statistical significance at 9 months of age. [64% (49/77) vs. 52% (30/58) at 1 month, P = 0.164; 86% (37/43) vs. 77% (24/31) at 3 months, P = 0.336; 94% (50/53) vs. 78% (25/32) at 9 months, P = 0.025; and 92% (35/38) vs. 91% (29/32) at 12 months, P = 0.826]. Overall, the mean corpuscular volume values (Fig. 1C) and the prevalence of microcytosis were similar between the 2 groups [0% vs. 0% at 1 month; 9% (4/43) vs. 6% (2/31) at 3 months, P = 0.658; 66% (35/53) vs. 59% (19/32) at 9 months, P = 0.536; and 71% (27/38) vs. 82% (27/33) at 12 months, P = 0.289]. Total leukocyte (Fig. 1D), platelet, neutrophil, and lymphocyte counts did not differ between groups nor over time (data not shown).
At 1 month of age, ENIC had significantly lower median %CD4 T cells [44% interquartile range (IQR) (39–50) vs. 47% IQR (43–53), P = 0.034] and higher %CD8 T cells [19% IQR (15–25) vs. 15% IQR (13–21), P = 0.0057] than UEC (Figs. 1E, F). Multivariate longitudinal analysis showed that after adjusting for age, the %CD4 T cells was lower in ENIC compared with UEC (difference between means: −3.01; 95% CI: −5.15 to −0.86; P = 0.006). Similarly, after adjusting for age, ENIC had higher %CD8 T cells (ratio of geometric means: 1.5; 95% CI: 1.06 to 1.25; P = 0.001) compared with UEC.
Morbidity During Infancy
One hundred fifty-three study children with PCR results at 1 month were included in the morbidity analysis. Among these infants under follow-up, 540 OPD visits and 34 admissions were registered during passive clinical surveillance. IR of overall outpatient visits at 1 year of age were lower in ENIC than in UEC (P = 0.042). During the first month of life, no differences in IR were observed (Table 3). ENIC had significantly fewer OPD visits with the diagnoses of diarrhea and AURI than did UEC (P = 0.001 and 0.042, respectively; Table 3). The IR of OPD visits with ALRI (0.77, 95% CI: 0.53 to 1.11), conjunctivitis (0.65, 95% CI: 0.29 to 1.45), and skin lesions (0.76, 95% CI: 0.46 to 1.26) also tended to be lower among ENIC compared with UEC (Table 3). There were no differences in the incidences of hospital admissions between the 2 groups of children neither during the first month of life (P = 0.175) nor during the next 12 months of follow-up (P = 0.282). Hospital admissions for CSP only occurred in 7 children, but its IR ratio in ENIC vs. UEC was 5.6 (95% CI: 0.67 to 46.55; P = 0.057) (Table 4). During the study follow-up, 4 children died among ENIC and 1 child among UEC (odds ratio: 3.74; 95% CI: 0.41 to 34.26; P = 0.195).
Multivariate longitudinal analysis showed that after adjusting for age, the weight-for-age Z score was lower in ENIC than in UEC (difference between means: −0.27; 95% CI: −0.53 to 0.00; P = 0.049). The proportions of moderate and severe wasting, stunting, and underweight were similar among ENIC and UEC (data not shown).
The findings of this study show that compared with UE infants, HIV ENI infants had an increased frequency of anemia, poorer nutritional status, and alterations in some immunologic profiles. In addition, although the risk of outpatient attendances was lower in ENIC, the risk of severe pneumonia was increased in these children compared with UE infants.
Reduced hematocrit and hemoglobin and increased anemia have previously been described in ENIC.34 It has been suggested that these changes might be associated with exposure to antiretrovirals for PMTCT35 or because of a direct impact of the virus on fetal erythropoiesis.36 This type of anemia is mainly macrocytic and usually resolves within the first 3 months of life.35,37–39 In this study, ENIC were more frequently anemic than UEC at all times, especially at 9 months of age, and in many, the anemia was microcytic, thus arguing against zidovudine as the main cause of anemia. It could be speculated that the anemia in these children may be associated with inadequate nutrition in ENIC, as suggested by the early weaning in the HIV-exposed group or to increased exposure of ENIC to infectious diseases.2 CTXP exposure may also result in anemia as a secondary effect of this drug.40 However, in this study, the lower level of hematocrit was already present at 1 month, before initiation of CTXP. Additionally, CTXP-associated anemia is characteristically megaloblastic,41 which was not the case in this study. In addition, it has been observed that HIV-infected children taking CTXP have significantly higher hemoglobin levels than children with placebo.42 Although it is known that anemia in children is associated with increased morbidity and mortality,43 the clinical impact of reduced hematocrit levels in ENIC is still unclear. As previously reported, we found an imbalance in T-cell populations with higher CD8 levels and lower CD4 levels in ENIC compared with UEC.16,18,44 The clinical implications of these findings are not well understood.
Children born to HIV-positive mothers with CD4 <200 cells per microliter are more likely to have a poor birth outcome and increased infant morbidity and mortality.7,11,15,45,46 In this study, the prevalence of LBW and prematurity in ENIC was not significantly higher than in UEC. This is probably because of the few HIV-positive women with CD4 counts below 200 cells per microliter in this study.
Interestingly, ENIC had a significantly lower risk of OPD visits than did UEC, mainly because of a decreased risk of diarrhea and AURI episodes. This lower OPD attendance is probably related to the antibacterial effect of CTXP that ENIC routinely receive through a special clinic at the MDH. Alternatively, this additional clinical follow-up might have affected the health-seeking behavior of mothers of ENIC reducing their attendance to the OPD. It is important to note that previous studies documenting an increased morbidity in ENIC were performed in contexts where CTXP was not available.3,9,11 Contrary to the other reports suggesting a nonsignificant increased risk of diarrhea with CTXP,47–49 the findings of this study showed a significant effect of CTXP in decreasing the risk of AURI (by 30%) and diarrhea (by 50%). This was observed even in the presence of high levels of bacterial resistance to CTX in this setting.50 The contribution of CTXP to these findings is also supported by the lack of difference in the incidence of OPD visits between the 2 groups of infants in the first month of life before the initiation of CTXP. Discrepancies between this and other reports regarding the effect of CTXP on morbidity in ENIC might be explained by the differences in age, feeding modes, duration of CTXP, or follow-up. It has been shown that CTXP confers protection against malaria.49,51 However, we were unable to assess the effect of CTXP on malaria incidence because of the small number of malaria episodes registered during the follow-up. We did, however, observe a borderline significant increased incidence of hospital admissions for CSP in ENIC. This could be explained by a misdiagnosis of tuberculosis.12 Tuberculosis is frequent among infants born to HIV-positive mothers,2 but it is difficult to diagnose in small children in poor resource settings,52 and it is not preventable by CTXP. Alternatively, it may be possible that CTXP is less effective in preventing CSP than mild respiratory infections53 or that those ENIC with CSP were poor compliers for CTX. Larger studies will be necessary to elucidate CSP risk in ENIC.
CTXP for ENIC is being reconsidered because of the concerns regarding the development of bacterial resistance.24,25 In the current study, despite an established PMTCT program, the rate of vertical transmission at 12 months of age was 27%, which illustrates the shortcomings of the health-care system. It might be premature to discontinue the CTXP in ENIC in the current context of continued breast-feeding up to 12 months of age and poor uptake of antiretrovirals.1 More information is needed on the impact of CTXP on infant health and uptake of PMTCT interventions before changing the current policy.
In conclusion, the findings of this study have shown that ENIC have more anemia, are nutritionally disadvantaged, and have immunologic alterations, which may all have clinical consequences that need to be studied in larger prospective studies. We have also shown that CTXP may have a role in reducing morbidity in ENIC. This information is crucial to assist in tailoring public health policies for the management of this group of vulnerable infants in sub-Saharan Africa.
The authors are grateful to all the mothers and their infants who participated in the study, also to the dedicated staff of the Manhiça District Hospital, and to the field, clinic, and data management staff at the Manhiça Health Research Centre, Mozambique.
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Keywords:© 2014 by Lippincott Williams & Wilkins
HIV-exposed noninfected infants; morbidity; anemia; sub-Saharan Africa; cotrimoxazol