The effect of iron supplementation on maternal iron deficiency anemia does not differ by baseline anemia type among Tanzanian pregnant women without severe iron deficiency anemia.

PURPOSE: Whether anemia type modifies the risk of pregnancy and newborn outcomes and the effectiveness of iron supplementation is unclear. We examined the association of iron deficiency anemia (IDA) and non-iron deficiency anemia (NIDA) on the risks of these outcomes and the extent to which anemia type modifies the impact of prenatal iron supplementation. METHODS: This was a secondary analysis of a placebo-controlled trial of iron supplementation among 1450 HIV-negative women in Tanzania. Eligibility criteria included gestational age < 27 weeks, hemoglobin > 85 g/L, and ferritin > 12 µg/L. Individuals were categorized as non-anemia, IDA or NIDA using hemoglobin, ferritin and CRP. Analyses were conducted using regression models and likelihood ratio tests. RESULTS: Compared to the non-anemia group, delivery hemoglobin was lower by 15 g/L (95% CI 10.9, 19.3) in the baseline IDA group, and 7.3 g/L (95% CI 3.1, 11.5) in the baseline NIDA group. The RRs of anemia severity, iron deficiency, placental malaria, stillbirths, perinatal mortality, birthweight, and preterm birth were not different among women in the baseline NIDA group (vs. non-anemia) compared to the baseline IDA group (vs. non-anemia). The difference in the mean delivery hemoglobin for iron supplementation and placebo arms was 8 g/L (95% CI 6, 11) in the non-anemia group, 7 g/L (95% CI 2, 13) in the NIDA group, and 16 g/L (95% CI 10, 22) in the IDA group. CONCLUSION: Iron supplementation is effective even among pregnant women with NIDA. TRIAL REGISTRATION: NCT01119612 (May 7, 2010).

Iron status among HIV-infected adults during the first year of antiretroviral therapy in Tanzania.

BACKGROUND: The influence of inflammation on iron status among people living with HIV (PLWHIV) has not been well explored. We evaluated the trajectory of iron status among PLWHIV during the first year of highly active antiretroviral therapy (HAART), compared alternative approaches for inflammation correction, and assessed the associations of iron status with HIV-1 viral load and anthropometric outcomes. METHODS: We conducted a secondary analysis of data from a randomized trial among 400 adults initiating HAART in Tanzania. Ferritin and C-reactive protein (CRP) were measured at baseline, 1, 6 or 12 months. Ferritin was considered in four ways: unadjusted, and adjusted for inflammation using higher cut-off (HC), Thurnham-corrected (TC) and regression-corrected (RC) approaches. For unadjusted, TC and RC ferritin, iron deficiency (ID) was defined using ferritin < 15 µg/L and elevated iron status was defined using ferritin > 150 µg/L among females and > 200 µg/L among males. For HC ferritin, elevated iron status was defined based on serum ferritin > 500 µg/L, while ID was defined using ferritin < 70 µg/L in the presence of inflammation and < 15 µg/L in the absence of inflammation. Regression models evaluated the trajectory of ferritin concentration across categories of baseline characteristics, and assessed the association of iron status with viral and anthropometric outcomes. RESULTS: The prevalence of iron deficiency at HAART initiation was 9% for unadjusted, 17% for HC, 12% for TC and 22% for RC ferritin. The prevalence of elevated iron status was 42% for unadjusted, 18% for HC, 31% for TC, and 15% for RC ferritin. The prevalence of iron deficiency for all three methods increased during the first year of HAART, while the prevalence of elevated iron status decreased. Baseline elevated iron status defined using HC ferritin was associated with a greater risk of HIV-1 viral load > 1000 copies/mL [relative risk (RR) = 4.29, 95% CI: 1.38-13.3] and incidence of being underweight [body mass index (BMI) < 18.5 kg/m2 , hazard ratio (HR) = 3.65, 95% confidence interval (CI): 1.38-9.67]. Neither baseline-elevated iron status defined using TC or RC ferritin nor baseline iron deficiency defined using any of the three methods was associated with HIV-1 viral load or anthropometric outcomes. CONCLUSIONS: Whether and how inflammation correction is done influences findings of studies of iron status among PLWHIV.

Factors associated with sub-microscopic placental malaria and its association with adverse pregnancy outcomes among HIV-negative women in Dar es Salaam, Tanzania: a cohort study.

BACKGROUND: Malaria infection during pregnancy has negative health consequences for both mothers and offspring. Sub-microscopic malaria infection during pregnancy is common in most African countries. We sought to identify factors associated with sub-microscopic placental malaria, and its association with adverse pregnancy outcomes among HIV-negative pregnant women in Dar es Salaam, Tanzania. METHODS: We recruited a cohort of pregnant women during their first trimester and assessed for the occurrence of placental malaria and pregnancy outcomes. The follow-up was done monthly from recruitment until delivery. Histopathology placental malaria positive results were defined as the presence of malaria pigment or parasitized erythrocytes on the slide (histology-positive (HP)), and the sub-microscopic placental infection was defined as positive Plasmodium falciparum DNA by polymerase chain reaction (DNA PCR) amplification in a negative histopathology test. Adverse pregnancy outcomes investigated included low birth weight (birth weight below 2.5 kg), prematurity (live birth below 37 weeks), and small-for-gestational-age (SGA) (live born with a birth weight below 10th percentile for gestational age and sex). Weighted baseline category logit, log-binomial, and log-Poisson models were used to assess factors associated with placental malaria, and its association with adverse pregnancy outcomes. RESULTS: Among 1115 women who had histopathology and DNA PCR performed, 93 (8%) had HP placental infection, and 136 (12%) had the sub-microscopic placental infection. The risk of sub-microscopic placental malaria was greater in women who did not use mosquito prevention methods such as bed nets, fumigation, or mosquito coils (odds ratio (OR) = 1.75; 95% confidence interval (CI): 1.05-2.92; P = 0.03) and in women who were anemic (OR = 1.59; 95% CI: 1.20-2.11; P = 0.001). Women who were underweight had reduced odds of sub-microscopic placental malaria infection (OR = 0.33; 95% CI: 0.17-0.62; P = 0.001). Women who were overweight/obese had 1.48 times higher the odds of HP placental malaria compared to normal weight (OR = 1.48; 95% CI: 1.03-2.11; P = 0.03). HP placental malaria infection was associated with an increased risk of SGA births (RR = 1.30, 95% CI: 0.98-1.72, P = 0.07). In contrast, the sub-microscopic infection was associated with a reduced risk of SGA births (RR = 0.61, 95% CI: 0.43-0.88, P = 0.01). Placental malaria was not associated with low birth weight or prematurity. CONCLUSION: Malaria prevention methods and maternal nutrition status during early pregnancy were important predictors of sub-microscopic placental malaria. More research is needed to understand sub-microscopic placental malaria and the possible mechanisms mediating the association between placental malaria and SGA.

Prenatal Zinc and Vitamin A Reduce the Benefit of Iron on Maternal Hematologic and Micronutrient Status at Delivery in Tanzania.

BACKGROUND: Zinc and vitamin A supplementation have both been shown to affect iron status, hemoglobin (Hb) concentration, and anemia in animal and human studies. However, evidence on their combined use in pregnancy, in the context of iron-folic acid (IFA) supplementation, remains limited. OBJECTIVE: This study determined the effects of prenatal zinc, vitamin A, and iron supplementation on maternal hematologic and micronutrient status at delivery in Tanzania. METHODS: We analyzed 2 large randomized controlled trials, using generalized estimating equations, and examined the effect of daily zinc (25 mg) and vitamin A (2500 IU) supplementation starting in the first trimester of pregnancy compared with placebo (n = 2500), and separately evaluated the safety and efficacy of daily iron (60 mg) supplementation among iron-replete pregnant women (n = 1500). Blood samples from baseline and delivery were tested for Hb, serum ferritin, soluble transferrin receptor, plasma zinc, and zinc protoporphyrin. RESULTS: Zinc and vitamin A supplementation were associated with lower Hb concentrations at delivery of  -0.26 g/dL (95% CI: -0.50, -0.02 g/dL) and -0.25 g/dL (95% CI: -0.49, -0.01 g/dL), respectively. Vitamin A increased mean ferritin concentrations at delivery (14.3 µg/L, 95% CI: 1.84, 29.11 µg/L), but was associated with increased risk of severe anemia (RR: 1.41; 95% CI: 1.06, 1.88). Among women who were iron replete at baseline, iron supplementation reduced the risk of iron depletion at delivery by 47% (RR: 0.53; 95% CI: 0.43, 0.65). There was no effect of zinc or iron supplements on plasma zinc concentrations. CONCLUSIONS: Our findings support existing WHO guidelines on prenatal iron, vitamin A, and zinc supplementation among pregnant women. In this setting, scaling uptake of prenatal iron supplements is warranted, but prenatal zinc and vitamin A supplementation did not benefit maternal hematologic status at delivery. In settings where vitamin A deficiency is endemic, the efficacy and safety of the WHO recommended prenatal vitamin A supplementation require further evaluation.

Hemoglobin and hepcidin have good validity and utility for diagnosing iron deficiency anemia among pregnant women.

BACKGROUND/OBJECTIVES: Screening and diagnosis of iron deficiency anemia (IDA) is cumbersome as it may require testing for hemoglobin, ferritin, and an inflammatory biomarker. The aim of this study was to compare the diagnostic capacity of hematologic biomarkers to detect IDA among pregnant women in Tanzania. SUBJECTS/METHODS: We pooled data from an iron supplementation trial of 1500 iron-replete pregnant woman and a prospective cohort of 600 iron-deficient pregnant women. Receiver operating characteristic curves (ROC) for hematologic biomarkers were used to assess the sensitivity, specificity, and area under the curve (AUC) for iron deficiency (ID) and iron deficiency anemia (IDA), crude, or corrected for inflammation. Regression models assessed the relationship of baseline biomarker categories (gestational age <27 weeks) and IDA at delivery. RESULTS: Hemoglobin had the largest AUC for crude ID (0.96), while hepcidin had the largest AUC for corrected ID (0.80). The optimal hepcidin cutoff for the diagnosis of corrected IDA based on maximal sensitivity and specificity was ≤1.6 µg/L. An hepcidin cutoff of <4.3 µg/L had a sensitivity of 95% for regression-corrected ID. Among iron-replete women who did not receive iron, the association of baseline hemoglobin >110 g/L with IDA at delivery (RR = 0.73; 95% CI: 0.47, 1.13) was attenuated. Baseline hepcidin >1.6 µg/L was associated with reduced risk of anemia at delivery by 49% (95% CI: 27%, 45%). CONCLUSIONS: Ascertaining hemoglobin and hepcidin levels may improve the targeting of iron supplementation programs in resource-limited countries, though hepcidin's high costs may limit its use.

Cost-Effectiveness of isoniazid preventive therapy among HIV-infected patients clinicaly screened for latent tuberculosis infection in Dar es Salaam, Tanzania: A prospective Cohort study.

BACKGROUND: One of the reasons why Isoniazid preventive therapy (IPT) for Tuberculosis (TB) is not widely used in low income countries is concerns on cost of excluding active TB. We analyzed the cost-effectiveness of IPT provision in Tanzania having ruled out active TB by a symptom-based screening tool. METHODS: Data on IPT cost-effectiveness was prospectively collected from an observational cohort study of 1283 HIV-infected patients on IPT and 1281 controls; followed up for 24 months. The time horizon for the analysis was 2 years. Number of TB cases prevented and deaths averted were used for effectiveness. A micro costing approach was used from a provider perspective. Cost was estimated on the basis of clinical records, market price or interviews with medical staff. We annualized the cost at a discount of 3%. A univariate sensitivity analysis was done. Results are presented in US$ at an average annual exchange rate for the year 2012 which was Tanzania shillings 1562.4 for 1 US $. RESULTS: The number of TB cases prevented was 420/100,000 persons receiving IPT. The number of deaths averted was 979/100,000 persons receiving IPT. Incremental cost due to IPT provision was US$ 170,490. The incremental cost effective ratio was US $ 405.93 per TB case prevented and US $ 174.15 per death averted. These costs were less than 3 times the 768 US $ Gross Domestic Product (GDP) per capita for Tanzania in the year 2014, making IPT provision after ruling out active TB by the symptom-based screening tool cost-effective. The results were robust to changes in laboratory and radiological tests but not to changes in recurrent, personnel, medication and utility costs. CONCLUSION: IPT should be given to HIV-infected patients who screen negative to symptom-based TB screening questionnaire. Its cost-effectiveness supports government policy to integrate IPT to HIV/AIDS care and treatment in the country, given the availability of budget and the capacity of health facilities.

Strong anti-Epstein Barr virus (EBV) or cytomegalovirus (CMV) cellular immune responses predict survival and a favourable response to anti-tuberculosis therapy.

BACKGROUND: Intact immune responses to cytomegalovirus (CMV) and Epstein-Barr virus (EBV) represent a biologically and clinically relevant correlate of 'immunological fitness' in humans. However, there is a lack of knowledge concerning anti-EBV or anti-CMV responses in patients with pulmonary tuberculosis (TB), in whom aberrant immune responses may promote progression of clinical disease. METHODS: Venous blood samples were obtained at the time of (sputum smear positive) pulmonary TB diagnosis. A whole blood assay was performed by exposing PBMCs (peripheral blood mononuclear cells) to a panel of infectious antigens, including CMV, EBV and mycobacterial proteins. Cell culture supernatants were collected after seven days and interferon gamma (IFN-γ) was measured using a sandwich ELISA. Patients received standard first line anti-tuberculosis rifampicin (R)/isoniazid (H)/ethambutol (E)/pyrazinamide (Z) for two months followed by RH for four months. RESULTS: PBMCs from cured patients (after treatment completion) exhibited significantly stronger IFN-γ responses to CMV (p=0.035), EBV (p=0.006) or Mycobacterium tuberculosis ESAT-6 (p=0.043) at the time of diagnosis as compared to patients who succumbed to TB during treatment. IFN-γ responses to other viral (H5N1, HSV-1) as well as other mycobacterial (Ag85A, Rv2958c, Rv0447c) antigens were not found to be significantly different among patients who were cured or those who succumbed to TB. CONCLUSIONS: Increased cellular immune responses to CMV and EBV antigens at the time of diagnosis of pulmonary tuberculosis are associated with increased survival after a standard six months anti-TB therapy. CVM and EBV antigens may represent "intrinsic markers for immune fitness" and guide improved TB therapies including host-directed therapies.

Performance of FACSPresto Point-of-Care Instrument for CD4-T Cell Enumeration in Human Immunodeficiency Virus (HIV)-Infected Patients Attending Care and Treatment Clinics in Belgium and Tanzania.

BACKGROUND: CD4 T-cell counts are widely used to assess treatment eligibility and to follow-up HIV-infected patients. The World Health Organization prequalification of in vitro diagnostics program conducted a performance evaluation of the FACSPresto (BD Biosciences), a new point-of-care instrument to measure absolute CD4-T cell (CD4) counts and percentages in venous and capillary blood samples from HIV-infected patients. METHODS: Patients were recruited in Belgium (200 patients) and in Tanzania (247 patients). Venous blood samples were analyzed in two nearby reference laboratories. In addition, nurses/technicians collected a capillary blood sample by finger prick directly into a FACSPresto CD4 cartridge. Assay precision was assessed on fresh blood and on external quality control samples. Trueness (bias) was assessed by comparing results from FACSPresto with the reference (single-platform FACSCalibur). Clinical misclassification was measured at 200, 350 and 500 cells/µL thresholds. RESULTS: Intra-assay precision was < 6%, and inter-assay < 8%. CD4 results from FACSPresto and reference method resulted in regression slopes of 0.99-1.11 using either venous or capillary blood. Correlation was better for venous than for capillary blood (minimum 0.97 vs 0.93 respectively). Capillary blood resulted in a larger bias than venous blood, with 24 and 83 cells/µL for absolute CD4 counts on capillary blood in Antwerp and Dar es Salaam respectively, vs 12 and 41 cells/µL on venous blood. Bias on CD4% was < 1% on both venous and capillary blood, and was proportionally better than for absolute CD4 counts. Clinical misclassification was in line with the average overestimation, showing a very good specificity, but sensitivity around 70-90%. The rejection rate was 11% on first reading, leading to 6% of all samples without final result after a second reading. CONCLUSIONS: The FACSPresto performed very well on venous blood samples, and well on capillary blood samples.

Iron Supplementation Affects Hematologic Biomarker Concentrations and Pregnancy Outcomes among Iron-Deficient Tanzanian Women.

BACKGROUND: Iron deficiency is a highly prevalent micronutrient abnormality and the most common cause of anemia globally, worsening the burden of adverse pregnancy and child outcomes. OBJECTIVE: We sought to evaluate the response of hematologic biomarkers to iron supplementation and to examine the predictors of the response to iron supplementation among iron-deficient pregnant women. METHODS: We identified 600 iron-deficient (serum ferritin ≤12 µg/L) pregnant women, aged 18-45 y, presenting to 2 antenatal clinics in Dar es Salaam, Tanzania using rapid ferritin screening tests, and prospectively followed them through delivery and postpartum. All women received 60 mg Fe and 0.25 mg folate daily from enrollment until delivery. Proportions meeting the thresholds representing deficient hematologic status including hemoglobin <110 g/L, ferritin ≤12 µg/L, serum soluble transferrin receptor (sTfR) >4.4 mg/L, zinc protoporphyrin (ZPP) >70 mmol/L, or hepcidin ≤13.3 µg/L at baseline and delivery were assessed. The prospective change in biomarker concentration and the influence of baseline hematologic status on the change in biomarker concentrations were assessed. Regression models were estimated to assess the relation of change in biomarker concentrations and pregnancy outcomes. RESULTS: There was significant improvement in maternal biomarker concentrations between baseline and delivery, with increases in the concentrations of hemoglobin (mean difference: 15.2 g/L; 95% CI: 13.2, 17.2 g/L), serum ferritin (51.6 µg/L; 95% CI: 49.5, 58.8 µg/L), and serum hepcidin (14.0 µg/L; 95% CI: 12.4, 15.6 µg/L) and decreases in sTfR (-1.7 mg/L; 95% CI: -2.0, -1.3 mg/L) and ZPP (-17.8 mmol/L; 95% CI: -32.1, 3.5 mmol/L). The proportions of participants with low hemoglobin, ferritin, and hepcidin were 73%, 93%, and 99%, respectively, at baseline and 34%, 12%, and 46%, respectively, at delivery. The improvements in biomarker concentrations were significantly greater among participants with poor hematologic status at baseline - up to 12.1 g/L and 14.5 µg/L for hemoglobin and ferritin concentrations, respectively. For every 10-g/L increase in hemoglobin concentration, there was a 24% reduced risk of perinatal mortality (RR = 0.76; 95% CI: 0.59, 0.99) and a 23% reduced risk of early infant mortality (RR = 0.77; 95% CI: 0.60, 0.99). The risk of anemia at delivery despite supplementation was predicted by baseline anemia (RR = 2.11; 95% CI: 1.39, 3.18) and improvements in ferritin concentration were more likely to be observed in participants who took iron supplements for up to 90 d (RR = 1.41; 95% CI: 1.13, 1.76). CONCLUSION: Iron supplementation decreases the risk of maternal anemia and increases the likelihood of infant survival among iron-deficient Tanzanian pregnant women. Interventions to promote increased duration and adherence to iron supplements may also provide greater health benefits.

Inflammatory and Angiogenic Factors at Mid-Pregnancy Are Associated with Spontaneous Preterm Birth in a Cohort of Tanzanian Women.

RESEARCH QUESTION: Preterm birth (PTB) is the leading cause of perinatal mortality worldwide, with the greatest burden occurring in resource-constrained settings. Based on the hypothesis that altered placental angiogenesis and inflammation early in pregnancy lead to PTB, we examined whether levels of inflammatory and angiogenic mediators, measured early in pregnancy, were predictive of spontaneous PTB (sPTB). STUDY DESIGN: Plasma samples were collected from a prospective cohort of primigravid Tanzanian women between 12-27 weeks gestation. A panel of 18 markers was screened on a training cohort of 426 women. Markers associated with sPTB in the training cohort were repeated in a test cohort of 628 women. All markers were measured by ELISA. FINDINGS: In both the training and test cohorts plasma levels of IL-18BP, sICAM-1, sEndoglin and CHI3L1 were elevated and Leptin was lower at enrollment in women who subsequently experienced sPTB. In multivariate analysis women with plasma levels of CHI3L1, C5a, sICAM-1, AngptL3, sEndgolin, sFlt-1 and IL-18BP in the highest quartile had an increased risk of sPTB compared with those in the lowest quartile. Women with Leptin and Ang2 in the highest quartile had a reduced risk of sPTB compared with women in the lowest quartile. IMPLICATIONS: Levels of angiogenic and inflammatory mediators measured at mid-pregnancy were associated with subsequent sPTB. These findings provide insight into mechanisms underlying sPTB and suggest biomarkers that may have clinical utility in risk-stratifying pregnancies.

Iron Supplementation in Iron-Replete and Nonanemic Pregnant Women in Tanzania: A Randomized Clinical Trial.

IMPORTANCE: Anemia is common in pregnancy and increases the risk of adverse outcomes. Iron deficiency is a leading cause of anemia in sub-Saharan Africa, and iron supplementation is the standard of care during pregnancy; however, recent trials among children have raised concerns regarding the safety of iron supplementation in malaria-endemic regions. There is limited evidence on the safety of iron supplementation during pregnancy in these areas. OBJECTIVE: To evaluate the safety and efficacy of iron supplementation during pregnancy in a malaria-endemic region. DESIGN, SETTING, AND PARTICIPANTS: We conducted a randomized, double-blind, placebo-controlled clinical trial among pregnant women presenting for antenatal care in Dar es Salaam, Tanzania, from September 28, 2010, through October 4, 2012. Iron-replete, nonanemic women were eligible if they were uninfected with human immunodeficiency virus, primigravidae or secundigravidae, and at or before 27 weeks of gestation. Screening of 21,316 women continued until the target enrollment of 1500 was reached. Analyses followed the intent-to-treat principle and included all randomized participants. INTERVENTIONS: Participants were randomized to receive 60 mg of iron or placebo, returning every 4 weeks for standard prenatal care, including malaria screening, prophylaxis with the combination of sulfadoxine and pyrimethamine, and treatment, as needed. MAIN OUTCOMES AND MEASURES: The primary outcomes were placental malaria, maternal hemoglobin level at delivery, and birth weight. RESULTS: Among 1500 study participants (750 randomized for each group), 731 in iron group and 738 in placebo group had known birth outcomes and 493 in iron group and 510 in placebo group had placental samples included in the analysis. Maternal characteristics were similar at baseline in the iron and placebo groups, and 1354 (91.7%) used malaria control measures. The risk of placental malaria was not increased by maternal iron supplementation (relative risk [RR], 1.03; 95% CI, 0.65-1.65), and iron supplementation did not significantly affect birth weight (3155 vs 3137 g, P = .89). Compared with placebo, iron supplementation significantly improved the mean increase from baseline to delivery for hemoglobin (0.1 vs -0.7 g/dL, P < .001) and serum ferritin (41.3 vs 11.3 µg/L, P < .001). Iron supplementation significantly decreased the risk of anemia at delivery by 40% (RR, 0.60; 95% CI, 0.51-0.71) but not severe anemia (RR, 0.68; 95% CI, 0.41-1.14). Iron supplementation significantly reduced the risk of maternal iron deficiency at delivery by 52% (RR, 0.48; 95% CI, 0.32-0.70) and the risk of iron deficiency anemia by 66% (RR, 0.34; 95% CI, 0.19-0.62). CONCLUSIONS AND RELEVANCE: Prenatal iron supplementation among iron-replete, nonanemic women was not associated with an increased risk of placental malaria or other adverse events in the context of good malaria control. Participants receiving supplementation had improved hematologic and iron status at delivery compared with the placebo group. These findings provide support for continued administration of iron during pregnancy in malaria-endemic regions. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01119612.

Angiogenic and inflammatory biomarkers in midpregnancy and small-for-gestational-age outcomes in Tanzania.

OBJECTIVE: We sought to investigate the relationship between a panel of angiogenic and inflammatory biomarkers measured in midpregnancy and small-for-gestational-age (SGA) outcomes in sub-Saharan Africa. STUDY DESIGN: Concentrations of 18 angiogenic and inflammatory biomarkers were determined in 432 pregnant women in Dar es Salaam, Tanzania, who participated in a trial examining the effect of multivitamins on pregnancy outcomes. Infants falling below the 10th percentile of birthweight for gestational age relative to the applied growth standards were considered SGA. Multivariate binomial regression models with the log link function were used to determine the relative risk of SGA associated with increasing quartiles of each biomarker. Restricted cubic splines were used to test for nonlinearity of these associations. RESULTS: A total of 60 participants (13.9%) gave birth to SGA infants. Compared to those in the first quartile, the risk of SGA was reduced among those in the fourth quartiles of vascular endothelial growth factor-A (adjusted risk ratio [RR], 0.38; 95% confidence interval [CI], 0.19-0.74), placental growth factor (adjusted RR, 0.28; 95% CI, 0.12-0.61), soluble fms-like tyrosine kinase-1 (adjusted RR, 0.48; 95% CI, 0.23-1.01), monocyte chemoattractant protein-1 (adjusted RR, 0.48; 95% CI, 0.25-0.92), and leptin (adjusted RR, 0.46; 95% CI, 0.22-0.96). CONCLUSION: Our findings provide evidence of altered angiogenic and inflammatory mediators, at midpregnancy, in women who went on to deliver SGA infants.

Iron status predicts treatment failure and mortality in tuberculosis patients: a prospective cohort study from Dar es Salaam, Tanzania.

BACKGROUND: Experimental data suggest a role for iron in the course of tuberculosis (TB) infection, but there is limited evidence on the potential effects of iron deficiency or iron overload on the progression of TB disease in humans. The aim of the present analysis was to examine the association of iron status with the risk of TB progression and death. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed plasma samples and data collected as part a randomized micronutrient supplementation trial (not including iron) among HIV-infected and HIV-uninfected TB patients in Dar es Salaam, Tanzania. We prospectively related baseline plasma ferritin concentrations from 705 subjects (362 HIV-infected and 343 HIV-uninfected) to the risk of treatment failure at one month after initiation, TB recurrence and death using binomial and Cox regression analyses. Overall, low (plasma ferritin<30 µg/L) and high (plasma ferritin>150 µg/L for women and>200 µg/L for men) iron status were seen in 9% and 48% of patients, respectively. Compared with normal levels, low plasma ferritin predicted an independent increased risk of treatment failure overall (adjusted RR = 1.95, 95% CI: 1.07 to 3.52) and of TB recurrence among HIV-infected patients (adjusted RR = 4.21, 95% CI: 1.22 to 14.55). High plasma ferritin, independent of C-reactive protein concentrations, was associated with an increased risk of overall mortality (adjusted RR = 3.02, 95% CI: 1.95 to 4.67). CONCLUSIONS/SIGNIFICANCE: Both iron deficiency and overload exist in TB patients and may contribute to disease progression and poor clinical outcomes. Strategies to maintain normal iron status in TB patients could be helpful to reduce TB morbidity and mortality.

Post-natal anaemia and iron deficiency in HIV-infected women and the health and survival of their children.

Prenatal iron supplementation may improve pregnancy outcomes and decrease the risk of child mortality. However, little is known about the importance of post-natal maternal iron status for child health and survival, particularly in the context of HIV infection. We examined the association of maternal anaemia and hypochromic microcytosis, an erythrocyte morphology consistent with iron deficiency, with child health and survival in the first two to five years of life. Repeated measures of maternal anaemia and hypochromic microcytosis from 840 HIV-positive women enrolled in a clinical trial of vitamin supplementation were prospectively related to child mortality, HIV infection and CD4 T-cell count. Median duration of follow-up for the endpoints of child mortality, HIV infection and CD4 cell count was 58, 17 and 23 months, respectively. Maternal anaemia and hypochromic microcytosis were associated with greater risk of child mortality [hazard ratio (HR) for severe anaemia = 2.58, 95% confidence interval (CI): 1.66-4.01, P trend < 0.0001; HR for severe hypochromic microcytosis = 2.36, 95% CI: 1.27-4.38, P trend = 0.001]. Maternal anaemia was not significantly associated with greater risk of child HIV infection (HR for severe anaemia = 1.46, 95% CI: 0.91, 2.33, P trend = 0.08) but predicted lower CD4 T-cell counts among HIV-uninfected children (difference in CD4 T-cell count/µL for severe anaemia: -93, 95% CI: -204-17, P trend = 0.02). The potential child health risks associated with maternal anaemia and iron deficiency may not be limited to the prenatal period. Efforts to reduce maternal anaemia and iron deficiency during pregnancy may need to be expanded to include the post-partum period.

Predictors of anaemia and iron deficiency in HIV-infected pregnant women in Tanzania: a potential role for vitamin D and parasitic infections.

OBJECTIVE: Anaemia is common during pregnancy, and prenatal Fe supplementation is the standard of care. However, the persistence of anaemia despite Fe supplementation, particularly in HIV infection, suggests that its aetiology may be more complex and warrants further investigation. The present study was conducted to examine predictors of incident haematological outcomes in HIV-infected pregnant women in Tanzania. DESIGN: Prospective cohort study. Cox proportional hazards and binomial regression models were used to identify predictors of incident haematological outcomes: anaemia (Hb < 110 g/l), severe anaemia (Hb < 85 g/l) and hypochromic microcytosis, during the follow-up period. SETTING: Antenatal clinics in Dar es Salaam, Tanzania. SUBJECTS: Participants were 904 HIV-infected pregnant women enrolled in a randomized trial of vitamins (1995-1997). RESULTS: Malaria, pathogenic protozoan and hookworm infections at baseline were associated with a two-fold increase in the risk of anaemia and hypochromic microcytosis during follow-up. Higher baseline erythrocyte sedimentation rate and CD8 T-cell concentrations, and lower Hb concentrations and CD4 T-cell counts, were independent predictors of incident anaemia and Fe deficiency. Low baseline vitamin D (<32 ng/ml) concentrations predicted a 1.4 and 2.3 times greater risk of severe anaemia and hypochromic microcytosis, respectively, during the follow-up period. CONCLUSIONS: Parasitic infections, vitamin D insufficiency, low CD4 T-cell count and high erythrocyte sedimentation rate were the main predictors of anaemia and Fe deficiency in pregnancy and the postpartum period in this population. A comprehensive approach to prevent and manage anaemia, including micronutrient supplementation and infectious disease control, is warranted in HIV-infected women in resource-limited settings - particularly during the pre- and postpartum periods.

Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania.

BACKGROUND: We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. METHODS: Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1mg intradermally (id), n=20, or 3.8mg intramuscularly (im), n=20, or placebo, n=20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (10(8)pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. RESULTS: The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8(+) and CD4(+) T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01_AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. CONCLUSIONS: This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher and broader cell-mediated immune responses to Env after HIV-MVA boost compared to a higher HIV-DNA priming dose given im. Three HIV-DNA priming immunizations followed by two HIV-MVA boosts efficiently induced Env-antibody responses.

Effect of vitamin supplements on HIV shedding in breast milk.

BACKGROUND: Supplementation in lactating HIV-1-infected women with preformed vitamin A and ß-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. OBJECTIVE: The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. DESIGN: We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected ≤15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1-infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 × 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. RESULTS: The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent ≥6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin had no effects. In observational analyses, ß-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. CONCLUSIONS: VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. ß-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756.

Strong HIV-specific CD4+ and CD8+ T-lymphocyte proliferative responses in healthy individuals immunized with an HIV-1 DNA vaccine and boosted with recombinant modified vaccinia virus ankara expressing HIV-1 genes.

We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag, env, rev, and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag, env, and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [(3)H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed (n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [(3)H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4(+) T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8(+) T-cell responses. There was strong correlation between the proliferative responses measured by the [(3)H]thymidine uptake assay and CD4(+) T-cell FASCIA-WB (r = 0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4(+) T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8(+) T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [(3)H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [(3)H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania.

BACKGROUND: Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. METHODS: Five rapid HIV assays: Determine HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold HIV-1/2 (Trinity Biotech) were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). RESULTS: Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9), respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold, Determine and SD Bioline assays. CONCLUSION: An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive samples on the Determine or SD Bioline gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold as tiebreaker for discordant results.

Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara.

BACKGROUND: A human immunodeficiency virus (HIV) vaccine that limits disease and transmission is urgently needed. This clinical trial evaluated the safety and immunogenicity of an HIV vaccine that combines a plasmid-DNA priming vaccine and a modified vaccinia virus Ankara (MVA) boosting vaccine. METHODS: Forty healthy volunteers were injected with DNA plasmids containing gp160 of HIV-1 subtypes A, B, and C; rev B; p17/p24 gag A and B, and RTmut B by use of a needle-free injection system. The vaccine was administered intradermally or intramuscularly, with or without recombinant granulocyte macrophage colony-stimulating factor, and boosted with a heterologous MVA containing env, gag, and pol of CRF01A_E. Immune responses were monitored with HIV-specific interferon (IFN)-gamma and interleukin (IL)-2 ELISpot and lymphoproliferative assays (LPAs). RESULTS: Vaccine-related adverse events were mild and tolerable. After receipt of the DNA priming vaccine, 11 (30%) of 37 vaccinees had HIV-specific IFN-gamma responses. After receipt of the MVA boosting vaccine, ELISpot assays showed that 34 (92%) of 37 vaccinees had HIV-specific IFN-gamma responses, 32 (86%) to Gag and 24 (65%) to Env. IFN-gamma production was detected in both the CD8(+) T cell compartment (5 of 9 selected vaccinees) and the CD4(+) T cell compartment (9 of 9). ELISpot results showed that 25 (68%) of 37 vaccinees had a positive IL-2 response and 35 (92%) of 38 had a positive LPA response. Of 38 subjects, a total of 37 (97%) were responders. One milligram of HIV-1 DNA administered intradermally was as effective as 4 mg administered intramuscularly in priming for the MVA boosting vaccine. CONCLUSION: This HIV-DNA priming-MVA boosting approach is safe and highly immunogenic. TRIALS REGISTRATION: International Standard Randomised Controlled Trial number: ISRCTN32604572 .