Association of Maternal Factors and HIV Infection With Innate Cytokine Responses of Delivering Mothers and Newborns in Mozambique.

Maternal factors and exposure to pathogens have an impact on infant health. For instance, HIV exposed but uninfected infants have higher morbidity and mortality than HIV unexposed infants. Innate responses are the first line of defense and orchestrate the subsequent adaptive immune response and are especially relevant in newborns. To determine the association of maternal HIV infection with maternal and newborn innate immunity we analyzed the cytokine responses upon pattern recognition receptor (PRR) stimulations in the triad of maternal peripheral and placental blood as well as in cord blood in a cohort of mother-infant pairs from southern Mozambique. A total of 48 women (35 HIV-uninfected and 13 HIV-infected) were included. Women and infant innate responses positively correlated with each other. Age, gravidity and sex of the fetus had some associations with spontaneous production of cytokines in the maternal peripheral blood. HIV-infected women not receiving antiretroviral therapy (ART) before pregnancy showed decreased IL-8 and IL-6 PRR responses in peripheral blood compared to those HIV-uninfected, and PRR hyporesponsiveness for IL-8 was also found in the corresponding infant's cord blood. HIV infection had a greater impact on placental blood responses, with significantly increased pro-inflammatory, T H 1 and T H 17 PRR responses in HIV-infected women not receiving ART before pregnancy compared to HIV-uninfected women. In conclusion, innate response of the mother and her newborn was altered by HIV infection in the women who did not receive ART before pregnancy. As these responses could be related to birth outcomes, targeted innate immune modulation could improve maternal and newborn health.

Predictors of personal exposure to black carbon among women in southern semi-rural Mozambique

Sub-Saharan Africa (SSA) has the highest proportion of people using unclean fuels for household energy, which can result in products of incomplete combustion that are damaging for health. Black carbon (BC) is a useful marker of inefficient combustion-related particles; however, ambient air quality data and temporal patterns of personal exposure to BC in SSA are scarce. We measured ambient elemental carbon (EC), comparable to BC, and personal exposure to BC in women of childbearing age from a semi-rural area of southern Mozambique. We measured ambient EC over one year (2014-2015) using a high-volume sampler and an off-line thermo-optical-transmission method. We simultaneously measured 5-min resolved 24-h personal BC using a portable MicroAeth (AE51) in 202 women. We used backwards stepwise linear regression to identify predictors of log-transformed 24-h mean and peak (90th percentile) personal BC exposure. We analyzed data from 187 non-smoking women aged 16-46 years. While daily mean ambient EC reached moderate levels (0.9 µg/m3, Standard Deviation, SD: 0.6 µg/m3), daily mean personal BC reached high levels (15 µg/m3, SD: 19 µg/m3). Daily patterns of personal exposure revealed a peak between 6 and 7 pm (>35 µg/m3), attributable to kerosene-based lighting. Key determinants of mean and peak personal exposure to BC were lighting source, kitchen type, ambient EC levels, and temperature. This study highlights the important contribution of lighting sources to personal exposure to combustion particles in populations that lack access to clean household energy.

Predictors of personal exposure to black carbon among women in southern semi-rural Mozambique.

Sub-Saharan Africa (SSA) has the highest proportion of people using unclean fuels for household energy, which can result in products of incomplete combustion that are damaging for health. Black carbon (BC) is a useful marker of inefficient combustion-related particles; however, ambient air quality data and temporal patterns of personal exposure to BC in SSA are scarce. We measured ambient elemental carbon (EC), comparable to BC, and personal exposure to BC in women of childbearing age from a semi-rural area of southern Mozambique. We measured ambient EC over one year (2014-2015) using a high-volume sampler and an off-line thermo-optical-transmission method. We simultaneously measured 5-min resolved 24-h personal BC using a portable MicroAeth (AE51) in 202 women. We used backwards stepwise linear regression to identify predictors of log-transformed 24-h mean and peak (90th percentile) personal BC exposure. We analyzed data from 187 non-smoking women aged 16-46 years. While daily mean ambient EC reached moderate levels (0.9 µg/m3, Standard Deviation, SD: 0.6 µg/m3), daily mean personal BC reached high levels (15 µg/m3, SD: 19 µg/m3). Daily patterns of personal exposure revealed a peak between 6 and 7 pm (>35 µg/m3), attributable to kerosene-based lighting. Key determinants of mean and peak personal exposure to BC were lighting source, kitchen type, ambient EC levels, and temperature. This study highlights the important contribution of lighting sources to personal exposure to combustion particles in populations that lack access to clean household energy.

A Balanced Proinflammatory and Regulatory Cytokine Signature in Young African Children Is Associated With Lower Risk of Clinical Malaria.

BACKGROUND: The effect of timing of exposure to first Plasmodium falciparum infections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. METHODS: As part of a double-blind, randomized, placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected from participants at age 2.5, 5.5, 10.5, 15, and 24 months were stimulated ex vivo with parasite schizont and erythrocyte lysates. Cytokine messenger RNA expressed in cell pellets and proteins secreted in supernatants were quantified by reverse-transcription quantitative polymerase chain reaction and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. RESULTS: Higher proinflammatory (interleukin [IL] 1, IL-6, tumor necrosis factor) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific T-helper 1 (IL-2, IL-12, interferon-γ) and T-helper 2 (IL-4, IL-5) cytokines by 2 years of age were measured in children undergoing chemoprophylaxis compared to children receiving placebo (P < .03). CONCLUSIONS: Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on T-helper lymphocyte cytokine production >1 year later. Importantly, a balanced proinflammatory and anti-inflammatory cytokine signature, probably by innate cells, around age 2 years was associated with protective clinical immunity during childhood. CLINICAL TRIALS REGISTRATION: NCT00231452.

Impact of age of first exposure to Plasmodium falciparum on antibody responses to malaria in children: a randomized, controlled trial in Mozambique.

BACKGROUND: The impact of the age of first Plasmodium falciparum infection on the rate of acquisition of immunity to malaria and on the immune correlates of protection has proven difficult to elucidate. A randomized, double-blind, placebo-controlled trial using monthly chemoprophylaxis with sulphadoxine-pyrimethamine plus artesunate was conducted to modify the age of first P. falciparum erythrocytic exposure in infancy and assess antibodies and malaria risk over two years. METHODS: Participants (n = 349) were enrolled at birth to one of three groups: late exposure, early exposure and control group, and were followed up for malaria morbidity and immunological analyses at birth, 2.5, 5.5, 10.5, 15 and 24 months of age. Total IgG, IgG subclasses and IgM responses to MSP-1(19), AMA-1, and EBA-175 were measured by ELISA, and IgG against variant antigens on the surface of infected erythrocytes by flow cytometry. Factors affecting antibody responses in relation to chemoprophylaxis and malaria incidence were evaluated. RESULTS: Generally, antibody responses did not vary significantly between exposure groups except for levels of IgM to EBA-175, and seropositivity of IgG1 and IgG3 to MSP-1(19). Previous and current malaria infections were strongly associated with increased IgG against MSP-1(19), EBA-175 and AMA-1 (p < 0.0001). After adjusting for exposure, only higher levels of anti-EBA-175 IgG were significantly associated with reduced clinical malaria incidence (IRR 0.67, p = 0.0178). CONCLUSIONS: Overall, the age of first P. falciparum infection did not influence the magnitude and breadth of IgG responses, but previous exposure was critical for antibody acquisition. IgG responses to EBA-175 were the strongest correlate of protection against clinical malaria. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00231452.

Blood oxidative stress markers and Plasmodium falciparum malaria in non-immune African children.

Converging in vitro evidence and clinical data indicate that oxidative stress may play important roles in Plasmodium falciparum malaria, notably in the pathogenesis of severe anaemia. However, oxidative modifications of the red blood cell (RBC)-membrane by 4-hydroxynonenal (4-HNE) and haemoglobin-binding, previously hypothesized to contribute mechanistically to the pathogenesis of clinical malaria, have not yet been tested for clinical significance. In 349 non-immune Mozambican newborns recruited in a double-blind placebo-controlled chemoprophylaxis trial, oxidative markers including 4-HNE-conjugates and membrane-bound haemoglobin were longitudinally assessed from 2·5 to 24 months of age, at first acute malaria episode and in convalescence. During acute malaria, 4-HNE-conjugates were shown to increase significantly in parasitized and non-parasitized RBCs. In parallel, advanced oxidation protein products (AOPP) rose in plasma. 4-HNE-conjugates correlated with AOPP and established plasma but not with RBC oxidative markers. High individual levels of 4-HNE-conjugates were predictive for increased malaria incidence rates in children until 2 years of life and elevated 4-HNE-conjugates in convalescence accompanied sustained anaemia after a malaria episode, indicating 4-HNE-conjugates as a novel patho-mechanistic factor in malaria. A second oxidative marker, haemoglobin binding to RBC-membranes, hypothesized to induce clearing of RBCs from circulation, was predictive for lower malaria incidence rates. Further studies will show whether or not higher membrane-haemoglobin values at the first malaria episode may provide protection against malaria.

Cytokine and antibody responses to Plasmodium falciparum in naïve individuals during a first malaria episode: effect of age and malaria exposure.

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n=48), European adults (naïve travelers, n=22; expatriates with few prior malaria exposures, n=15) and Mozambican adults with long-life malaria exposure (n=99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-1(42) (P=0.030) and a P. falciparum isolate (P=0.002), as well as higher IL-12 (P=0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P ≤ 0.0090), IL-2 (P ≤ 0.0379) and IL-8 (P ≤ 0.0233). Children also had higher IL-12 (P=0.0001), IL-4 (P=0.003), IL-1ß (P=0.024) and TNF (P=0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A T(H)1/pro-inflammatory response was the hallmark of non-immune subjects.

Population characteristics of young African women influencing prenatal exposure to DDT (Manhiça, Mozambique).

The concentrations of dichlorodiphenyltrichloroethane (DDT) compounds in cord blood of 214 children born between 2003 and 2006 in Manhiça (Mozambique) have been determined. In this time interval, corresponding to the period before DDT reintroduction for indoor residual spraying, the observed values averaged 0.8 and 0.4 ng/ml for 4,4'-dichlorodiphenyldichloroethylene (4,4'-DDE) and 4,4'-DDT, respectively, and were similar to those found in western countries. However, the 4,4'-DDT/4,4'-DDE ratio was high indicating that the inputs of these compounds arriving to children in utero originated from recent uses of the insecticide. The strongest factor affecting DDT concentration was parity. A well-defined decreasing concentration trend was observed for the cord blood concentrations in the period of study. The trend was also observed for multiparae and primiparae mothers independently. Children from multiparae women showed much lower concentrations than primiparae women. Children from mothers with secondary school level exhibited lower concentrations of these pesticides than mothers with lower degree of education.

A direct comparison of real time PCR on plasma and blood to detect Plasmodium falciparum infection in children.

BACKGROUND: Estimation of Plasmodium falciparum parasitaemia can vary with the method used and time of sampling. Quantitative real time PCR (qPCR) on whole blood or plasma samples has previously been shown to be more sensitive than thick film microscopy. However the efficiencies of each method have not been compared using samples obtained from infants less than one year old. METHODS: A multiple of statistical approaches were used to compare the performance of qPCR on whole blood or plasma to detect the 18 S ribosomal gene of P. falciparum in 548 samples from children aged 2.5 or 24 months. Parasite prevalence in matched samples was compared using Mcnemar's test and agreement of positive results quantified as Kappa scores. Parasite prevalences between different age groups were compared by Fisher's test. Results from analyses by thick film microscopy were also available from children at 24 months and their correlation to each qPCR method examined by the Spearman's test. Finally the association of P. falciparum infection with the incidence of multiple malaria episodes from contact to 24 months of age was evaluated using negative binomial regression. RESULTS: These analyses showed that qPCR from whole blood detected approximately 3-fold more cases of infection than plasma qPCR. Both qPCR methods agreed well with each other although qPCR from plasma had a greater agreement with microscopy (96.85%) than did qPCR from blood (69.7%). At 24 months the prevalence of infection detected by all methods was associated with anaemia (p<0.05). CONCLUSIONS: The data presented here demonstrates that low levels of parasitaemia are better detected by qPCR using parasite DNA from whole blood than from plasma. However plasma samples provide a viable substitute when parasite smears are unavailable.

Pyrethroid use-malaria control and individual applications by households for other pests and home garden use.

Presence of pyrethroid insecticides in human breast milk and in thatch wall material of dwellings from Southern Africa subtropical area (Manhiça, Mozambique) was investigated to assess potential pyrethroid route of human exposure. Human breast milk samples were collected during 2002 when pyrethroids were widely used as insecticides for mosquito bed nets in Mozambique for malaria control. The median concentration value of total pyrethroids ranged between 87 and 1200ng/glw, with λ-cyhalothrin being the most predominant pyrethroid in human breast milk contributing for 35% of the total amount. Moreover, and for the first time, an isomer-specific enrichment was found in human breast milk, showing a selective isomeric accumulation or metabolism in the human body. Based on the calculated pyrethroid concentrations in human breast milk, the daily ingestion rate of pyrethroid was estimated. The nursing infant dietary intake ranged from 0.67 to 9.0µg(kg of body weight)(-1)day(-1). In addition, thatch materials collected after the reintegration of dichlorodiphenyltrichloroethene (DDT) as insecticide residual spraying (IRS) in Mozambique, showed the presence of pyrethroids with concentration values ranging between 6.9 and 700ng/gdw. In thatch material as well as in human breast milk, pyrethroid contamination was mainly attributed to the agriculture usage of this insecticide knowing that agriculture represent the 80% of the economy in Mozambique. However, a possible usage of this insecticide as IRS in Mozambique cannot be excluded despite their low efficiency for malaria control. The continued use of these compounds (both for agriculture and malaria prevention) and the ingestion rates calculated from the breast milk concentrations indicate that these insecticides cannot be overlooked for the assessment of the lactation risks of breastfeeding infants from the Manhiça region.

Assessment of exposure to DDT and metabolites after indoor residual spraying through the analysis of thatch material from rural African dwellings.

INTRODUCTION: We report on the analysis of 4,4'-dichlorodiphenyltrichloroethane (4,4'-DDT) and its metabolites in thatch and branch samples constituting the wall materials of dwellings from South African subtropical areas. This approach was used to assess the exposure to DDT in the residents of the dwellings after indoor residual spraying (IRS) following recommended sanitation practices against malaria vectors. DISCUSSION: Examination of the distributions of DDT compounds (2,4'-DDT, 4,4'-DDT and its metabolites) in 43 dwellings from the area of Manhiça (Mozambique) has shown median concentrations of 19, 130, and 23 ng/g for 2,4'-DDT, 4,4'-DDT, and 4,4'-DDE, respectively, in 2007 when IRS implementation was extensive. The concentrations of these compounds at the onset of the IRS campaign (n = 48) were 5.5, 47, and 2.2 ng/g, respectively. The differences were statistically significant and showed an increase in the concentration of this insecticide and its metabolites. Calculation of 4,4'-DDT in the indoor air resulting from the observed concentrations in the wall materials led to the characteristic values of environments polluted with this insecticide.

Concentration of DDT compounds in breast milk from African women (Manhiça, Mozambique) at the early stages of domestic indoor spraying with this insecticide.

Breast milk concentrations of 4,4'-DDT and its related compounds were studied in samples collected in 2002 and 2006 from two populations of mothers in Manhiça, Mozambique. The 2006 samples were obtained several months after implementation of indoor residual spraying (IRS) with DDT for malaria vector control in dwellings and those from 2002 were taken as reference prior to DDT use. A significant increase in 4,4'-DDT and its main metabolite, 4,4'-DDE, was observed between the 2002 (median values 2.4 and 0.9 ng/ml, respectively) and the 2006 samples (7.3 and 2.6 ng/ml, respectively, p<0.001 and 0.019, respectively). This observation identifies higher body burden intakes of these compounds in pregnant women already in these initial stages of the IRS program. The increase in both 4,4'-DDT and 4,4'-DDE suggest a rapid transformation of DDT into DDE after incorporation of the insecticide residues. The median baseline concentrations in breast milk in 2002 were low, and the median concentrations in 2006 (280 ng/g lipid) were still lower than in other world populations. However, the observed increases were not uniform and in some individuals high values (5100 ng/g lipid) were determined. Significant differences were found between the concentrations of DDT and related compounds in breast milk according to parity, with higher concentrations in primiparae than multiparae women. These differences overcome the age effect in DDT accumulation between the two groups and evidence that women transfer a significant proportion of their body burden of DDT and its metabolites to their infants.