Methods for the relative quantitation of human plasma lipidome using liquid chromatography coupled with mass spectrometry using minimal sample manipulation.

Extraction protocols and liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) methods for the measurement of four lipid categories, namely glycerophospholipids, glycerolipids, sphingolipids and sterol lipids in human plasma, are described here. Step-by-step instructions are provided for the liquid-liquid extraction methods, including solution preparation and the non-endogenous lipid internal standards used. All instrumental conditions, chromatography columns and solutions required for the LC-MS and LC-MS/MS methods are also provided in detail.

Are high avidity antibodies to Plasmodium falciparum antigens preferentially transferred across the placenta of premature and term babies?

INTRODUCTION: Transplacental transport of maternal IgG via the neonatal Fc receptor (FcRn) provides babies with passive immunity. Several factors are reported to influence transport, including the avidity of antibodies (Abs) for their cognate antigens. Unfortunately, information on the role of antibody (Ab) avidity is limited. This study investigated if i) antibodies (Abs) with high avidity for 6 Plasmodium falciparum antigens and tetanus toxoid (TTx) were preferentially transferred to premature and term Cameroonian babies and ii) if Ab avidity was increased in babies whose mothers had placental malaria (PM), implicating the involvement of immune complexes. METHODS: Total IgG (mg/ml) and Abs to malarial antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3, DBL5 of VAR2CSA) and TTx were measured in paired mother-cord samples obtained from premature and term deliveries in Cameroon. Half the women had PM at delivery. Avidity Indices (AIs) were determined by treating antigen-bound-Abs with different molar concentrations of NH4SCN and calculating 50% endpoints. RESULTS: Total IgG and antigen-specific Abs increased in cord blood with gestational age; however, AIs did not. AIs in paired maternal-cord blood samples were strongly associated for all antigens (r = 0.77-0.96). However, no significant different in AIs was found between paired mother-cord blood samples for any of the antigens (p values > 0.05). Similarly, Ab avidity was not increased in cord blood of babies whose mothers had PM or hypergammaglobulinemia. DISCUSSION: Overall, there was no evidence that higher avidity Abs to any of the malarial antigens or TTx were preferentially transferred to Cameroonian babies.

Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry.

RATIONALE: The present work shows comprehensive chromatographic methods and MS conditions that have been developed based on the chemical properties of each lipid subclass to detect low-abundance molecular species. This study shows that the developed methods can detect low- and/or very-low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipid (GP) method; dihydroceramide (dhCer) and dihydrosphingosine/sphinganine (dhSPB) in the sphingolipid (SP) method; and lysophosphatidic acid (LPA), LPI, LPG and sphingosine-1-phosphate (SPBP) in the lysolipid method. METHODS: An optimised method for the extraction of lysolipids in plasma is used in addition to Folch extraction. Then, four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches are described here. Three of the methods use a tertiary pumping system to enable the inclusion of a gradient for analyte separation (pumps A and B) and an isocratic wash (pump C). This wash solution elutes interfering compounds that could cause background signal in the subsequent injections, reducing column lifetime. RESULTS: Semi-quantitative values for 37 lipid subclasses are reported for a plasma sample (NIST SRM 1950). Furthermore, the methods presented here enabled the identification of 338 different lipid molecular species for GPs (mono- and diacyl-phospholipds), SPs, sterols and glycerolipids. The methods have been validated, and the reproducibility is presented here. CONCLUSIONS: The comprehensive analysis of the lipidome addressed here of glycerolipids, GPs, sterols and SPs is in good agreement with previously reported results, in the NIST SRM 1950 sample, by other laboratories. Ten lipid subclasses LPS, LPI, alkyl-lysophosphatidic acid/alkenyl-lysophosphatidic acid, alkyl-lysophosphatidylethanolamine/alkenyl-lysophosphatidylethanolamine, dhCer (d18:0), SPB (d18:1), dhSPB (d18:0) and SPBP (d18:2) have been detected using this comprehensive method and are uniquely reported here.

Characterization of the primary antibody response to Plasmodium falciparum antigens in infants living in a malaria-endemic area.

BACKGROUND: The primary antibody (Ab) response to Plasmodium falciparum is a critical step in developing immunity to malaria. Information on the initial Ab responses of babies in malaria-endemic areas is incomplete, in part, because babies receive maternal IgG via transplacental-transfer and usually become infected before maternal IgG wanes. The study aimed to identify the primary IgM and IgG Ab responses to malarial antigens in Cameroonian babies. METHODS: Infants (n = 70) living in a high malaria transmission area were followed from birth throughout the first year of life (mean 341 ± 42 days, an average of 8.5 time points per infant). Malaria infection was assessed by microscopy and PCR, and IgM and IgG antibodies (Abs) were measured using a multiplex immunoassay to AMA1, EBA-175, MSP1-42, MSP2, MSP3, RESA, LSA1, and CSP. RESULTS: The half-life of maternal IgG varied among the antigens, ranging from 0.7 to 2.5 months. The first infection of 41% of the babies was sub-microscopic and only 11 to 44% of the babies produced IgM to the above antigens; however, when the first infection was detected by microscopy, 59-82% of the infants made IgM Abs to the antigens. Infants were able to produce IgM even when maternal IgG was present, suggesting maternal Abs did not suppress the baby's initial Ab response. Using longitudinal regression models that incorporated time-varying covariates, infants were found to produce IgG Ab to only AMA-1 when the first infection was sub-microscopic, but they produced IgG Abs to MSP1-42 (3D7, FVO), AMA1 (3D7, FVO) MSP2-FC27, MSP3, RESA, and LSA1, but not MSP 2-3D7, EBA-175, and CSP during their first slide-positive infection. Notably, the primary and secondary IgG responses were short-lived with little evidence of boosting. CONCLUSIONS: The primary Ab response of babies who had maternal IgG was similar to that reported for primary infections in malaria-naïve adults.

Transplacental transfer of total immunoglobulin G and antibodies to Plasmodium falciparum antigens between the 24th week of gestation and term.

Full-term newborns have antibody (Ab) repertoires and levels similar to their mothers to help protect them from environmental pathogens. Unfortunately, preterm babies, especially those born < 34 weeks, have reduced levels of protective antibodies. In Africa, antibodies to Plasmodium falciparum are important in protection from malaria. This study investigated the transfer of total IgG and antibodies to 9 P. falciparum antigens and tetanus toxoid between 24 weeks and term. Paired maternal and cord samples from 166 preterm (24-37 weeks) and 154 term deliveries were used. Transfer efficiency was expressed as the ratio of Ab levels in cord to maternal plasma (CMR). At 24-25 weeks, CMR ranged from 0.31 to 0.94 for the different antigens; the rate of transfer was similar for all antigens between 24 and 40 weeks; resulting in median CMR of 0.49-0.95 at term. Babies of mothers with hypergammaglobulinemia and normal IgG levels had similar amounts of IgG, supporting data that saturation of the neonatal Fc-receptor occurs at ~ 16 mg IgG/ml. Thus, babies born prior to 34-35 weeks in Africa are likely to have reduced Ab levels to some, but not all antigens. Since IgG transfer is Fc-mediated, why differences exist in CMR among the antigens warrants further investigation.

Does Antibody Avidity to Plasmodium falciparum Merozoite Antigens Increase with Age in Individuals Living in Malaria-Endemic Areas?

High-avidity antibodies (Abs) are acquired after a few Plasmodium falciparum infections in low transmission areas, but it remains unclear if Ab avidity to different merozoite antigens increases with age in individuals with persistent antigenemia and, if so, when a fully mature Ab response occurs. The study used plasma samples collected between 1996 and 1998 from 566 individuals aged 4 to 84 years in Simbok, Cameroon, where residents received an estimated 1.6 infectious mosquito bites/person/night. Plasma samples were examined for Ab levels (median fluorescence intensity [MFI]) and Ab avidity index (AI) (where AI = [MFI after treatment with 2 M NH4SCN/MFI without salt] × 100) using a bead-based multiplex immunoassay for recombinant AMA1, EBA-175, MSP1-42 (3D7, FVO), MSP2 (3D7, Fc27), and MSP3. Blood-smear positivity for P. falciparum declined with age from 54.3% at 4 to 5 years to 18% at 16 to 40 years and <11% at >40 years of age, although most individuals had submicroscopic parasitemia. Ab affinity maturation, based on age-related patterns of median AI, percentage of individuals with AI of ≥50, and strength of association between MFI and AI, occurred at different rates among the antigens; they developed rapidly before age 4 years for AMA1, increased gradually with age for EBA-175 and MSP1 until ∼16 to 25 years, but occurred negligibly for MSP2 and MSP3. In a hyperendemic area with perennial transmission, affinity maturation resulting in an increase in the proportion of high-avidity Abs occurred for some merozoite antigens, in parallel with a decline in malaria slide passivity, but not for others.

The Development, Fine Specificity, and Importance of High-Avidity Antibodies to VAR2CSA in Pregnant Cameroonian Women Living in Yaoundé, an Urban City.

Pregnant women infected with Plasmodium falciparum often produce antibodies (Abs) to VAR2CSA, a ligand that binds to placental chondroitin sulfate A causing placental malaria (PM). Antibodies to VAR2CSA are associated with improved pregnancy outcomes. Antibody avidity is a surrogate marker for the extent of maturation of the humoral immune response. Little is known about high avidity Abs to VAR2CSA for women living in urban African cities. Therefore, this study sought to determine: i) if high avidity Abs to full-length VAR2CSA (FV2) increase with gravidity in women in Yaoundé, Cameroon exposed to ~ 0.3-1.1 infectious mosquito bites per month, ii) if high avidity Abs to FV2 are directed against a specific region of VAR2CSA, and iii) if having high avidity Abs to FV2 improve pregnancy outcomes. Plasma samples collected at delivery from 695 women who had Abs to FV2 were evaluated. Ab levels and the Avidity Index (AI), defined as the percent Abs remaining bound to FV2 after incubation with 3M NH4SCN, were determined. Similar Ab levels to FV2 were present in women of all gravidities (G1 through 6+; p=0.80), except significantly lower levels were detected in PM-negative (PM-) primigravidae (p <0.001). Median Ab avidities increased between gravidity 1 and 2 (p<0.001) and remained stable thereafter (G3-G6+: p=0.51). These results suggest that B cell clonal expansion began during the first pregnancy, with clonal selection primarily occurring during the second. However, the majority of women (84%) had AI <35, a level of high avidity Abs previously reported to be associated with improved pregnancy outcomes. When plasma from 107 Cameroonian women was tested against 8 different regions of FV2, high avidity Abs were predominately restricted to DBL5 with median AI of 50 compared to AI <25 for the other domains. The only significance influence of high avidity Abs on pregnancy outcome was that babies born to mothers with AI above the median were 104 g heavier than babies born to women with AI below the median (p=0.045). These results suggest that a vaccine that boosts maturation of the immune response to VAR2CSA may be beneficial for women residing in urban areas.

The immunoglobulin G antibody response to malaria merozoite antigens in asymptomatic children co-infected with malaria and intestinal parasites.

BACKGROUND: Co-infection with malaria and intestinal parasites is common in children in Africa and may affect their immune response to a malaria parasite infection. Prior studies suggest that co-infections may lead to increased susceptibility to malaria infection and disease severity; however, other studies have shown the reverse. Knowledge on how co-morbidities specifically affect the immune response to malaria antigens is limited. Therefore, this study sought to determine the prevalence of co-infection of malaria and intestinal parasites and its association with antibody levels to malaria merozoite antigens. METHODS: A cross sectional study was carried out in two villages with high transmission of malaria in Cameroon (Ngali II and Mfou) where mass drug administration (MDA) had been administered at ~6-month intervals (generally with albendazole or mebendazole). Children aged 1-15 years were enrolled after obtaining parental consent. A malaria rapid diagnostic test was used on site. Four (4) ml of peripheral blood was collected from each participant to determine Plasmodium falciparum infections by microscopy, haemoglobin levels and serology. Fresh stool samples were collected and examined by wet mount, Kato-Katz method and modified Ritchie concentration techniques. A Multiplex Analyte Platform assay was used to measure antibody levels. RESULTS: A total of 320 children were enrolled. The prevalence of malaria by blood smear was 76.3% (244/320) and prevalence of malaria and intestinal parasites was 16.9% (54/320). Malaria prevalence was highest in young children; whereas, intestinal parasites (IP+) were not present until after 3 years of age. All children positive for malaria had antibodies to MSP142, MSP2, MSP3 and EBA175. No difference in antibody levels in children with malaria-co infections compared to malaria alone were found, except for antibody levels to EBA-175 were higher in children co-infected with intestinal protozoa (p = 0.018), especially those with Entamoeba histolytica infections (p = 0.0026). CONCLUSION: Antibody levels to EBA175 were significantly higher in children co-infected with malaria and E. histolytica compared to children infected with malaria alone. It is important to further investigate why and how the presence of these protozoans might modulate the immune response to malaria antigens.

Antibodies to full-length and the DBL5 domain of VAR2CSA in pregnant women after long-term implementation of intermittent preventive treatment in Etoudi, Cameroon.

In high malaria transmission settings, the use of sulfadoxine-pyrimethamine-based intermittent preventive treatment during pregnancy (IPTp-SP) has resulted in decreased antibody (Ab) levels to VAR2CSA. However, information of Ab levels in areas of low or intermediate malaria transmission after long-term implementation of IPTp-SP is still lacking. The present study sought to evaluate antibody prevalence and levels in women at delivery in Etoudi, a peri-urban area in the capital of Yaoundé, Cameroon, that is a relatively low-malaria transmission area. Peripheral plasma samples from 130 pregnant women were collected at delivery and tested for IgG to the full-length recombinant VAR2CSA (FV2) and its most immunogenic subdomain, DBL5. The study was conducted between 2013 and 2015, approximately ten years after implementation of IPTp-SP in Cameroon. About 8.6% of the women attending the clinic had placental malaria (PM). One, two or 3 doses of SP did not impact significantly on either the percentage of women with Ab to FV2 and DBL5 or Ab levels in Ab-positive women compared to women not taking SP. The prevalence of Ab to FV2 and DBL5 was only 36.9% and 36.1%, respectively. Surprisingly, among women who had PM at delivery, only 61.5% and 57.7% had Ab to FV2 and DBL5, respectively, with only 52.9% and 47.1% in PM-positive paucigravidae and 77.7% of multigravidae having Ab to both antigens. These results suggest that long-term implementation of IPTp-SP in a low-malaria transmission area results in few women having Ab to VAR2CSA.

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

BACKGROUND: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration. METHODS: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability. RESULTS: Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2 M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8 M ± 0.23 M (infants) released 50% of bound Ab from the merozoite antigens. CONCLUSIONS: An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Impact of HIV-1 infection on the IGF-1 axis and angiogenic factors in pregnant Cameroonian women receiving antiretroviral therapy.

Although mother-to-child transmission of HIV has dramatically declined, the number of in utero HIV-exposed, uninfected infants is on the increase. HIV-exposed infants are at an increased risk of mortality, morbidity and slower early growth than their non-HIV exposed counterparts. Maternal HIV increases the risk of having preterm deliveries, intrauterine growth restriction and low birth weight babies. However, the mechanism underlying dysregulation of fetal growth in HIV-infected pregnant women is unknown. We sought to determine whether maternal HIV is associated with dysregulation of the insulin-like growth factor (IGF) axis, some angiogenic factors or other related biomarkers that regulate fetal growth. A total of 102 normotensive pregnant women were enrolled in a small cross-sectional study. Amongst these were thirty-one HIV-1 positive women receiving combination antiretroviral therapy (cART) (Mean age: 30.0 ± 5.1 years; % on ART: 83.9%; median plasma viral load: 683 copies/ml; median CD4 count: 350 cells/ul) and 71 HIV uninfected women (mean age: 27.3 ± 5.8) recruited at delivery. A panel of biomarkers including IGF1 and IGF binding proteins (IGFBP1, IGFBP3), angiopoietins (ANG) 1 and 2, matrix metalloproteinases (MMP) 2 and 9, and galectin 13, was measured in plasma collected from the placental intervillous space. The levels of IGF1, IGFBP1, ANG1, ANG2, MMP2, MMP9 and Gal-13 were not affected by maternal HIV, even when adjusted for maternal factors in linear regression models (all p>0.05). It was observed that HIV-infection in pregnancy did not significantly affect key markers of the IGF axis and angiogenic factors. If anything, it did not affect women. These findings highlight the importance of the use of ART during pregnancy, which maintains factors necessary for fetal development closer to those of healthy women. However, decrease in IGF1 levels might be exacerbated in women con-infected with HIV and malaria.

A comparison of thick-film microscopy, rapid diagnostic test, and polymerase chain reaction for accurate diagnosis of Plasmodium falciparum malaria.

BACKGROUND: Accurate diagnosis of malaria is important for effective disease management and control. In Cameroon, presumptive clinical diagnosis, thick-film microscopy (TFM), and rapid diagnostic tests (RDT) are commonly used to diagnose cases of Plasmodium falciparum malaria. However, these methods lack sensitivity to detect low parasitaemia. Polymerase chain reaction (PCR), on the other hand, enhances the detection of sub-microscopic parasitaemia making it a much-needed tool for epidemiological surveys, mass screening, and the assessment of interventions for malaria elimination. Therefore, this study sought to determine the frequency of cases missed by traditional methods that are detected by PCR. METHODS: Blood samples, collected from 551 febrile Cameroonian patients between February 2014 and February 2015, were tested for P. falciparum by microscopy, RDT and PCR. The hospital records of participants were reviewed to obtain data on the clinical diagnosis made by the health care worker. RESULTS: The prevalence of malaria by microscopy, RDT and PCR was 31%, 45%, and 54%, respectively. However, of the 92% of participants diagnosed as having clinical cases of malaria by the health care worker, 38% were malaria-negative by PCR. PCR detected 23% and 12% more malaria infections than microscopy and RDT, respectively. A total of 128 (23%) individuals had sub-microscopic infections in the study population. The sensitivity of microscopy, RDT, and clinical diagnosis was 57%, 78% and 100%; the specificity was 99%, 94%, and 17%; the positive predictive values were 99%, 94%, and 59%; the negative predictive values were 66%, 78%, and 100%, respectively. Thus, 41% of the participants clinically diagnosed as having malaria had fever caused by other pathogens. CONCLUSIONS: Malaria diagnostic methods, such as TFM and RDT missed 12-23% of malaria cases detected by PCR. Therefore, traditional diagnostic approaches (TFM, RDT and clinical diagnosis) are not adequate when accurate epidemiological data are needed for monitoring malaria control and elimination interventions.

PCR-based detection of Plasmodium falciparum in saliva using mitochondrial cox3 and varATS primers.

BACKGROUND: Sampling of saliva for diagnosing Plasmodium falciparum infections is a safe, non-invasive alternative to sampling of blood. However, the use of saliva presents a challenge because lower concentrations of parasite DNA are present in saliva compared to peripheral blood. Therefore, a sensitive method is needed for detection of parasite DNA in saliva. This study utilized two recently reported "ultra-sensitive" PCR assays based on detection of the P. falciparum mitochondrial cox3 gene and the multi-copy nuclear varATS gene. The ultra-sensitive assays have an advantage over standard 18S rRNA gene-based PCR assay as they target genes with higher copy numbers per parasite genome. Stored saliva DNA samples from 60 Cameroonian individuals with infections previously confirmed by 18S rRNA gene PCR in peripheral blood were tested with assays targeting the cox3 and varATS genes. RESULTS: Overall, the standard 18S rRNA gene-based PCR assay detected P. falciparum DNA in 62% of the stored saliva DNA samples, whereas 77 and 68% of the samples were positive with assays that target the cox3 and varATS genes, respectively. Interestingly, the ultra-sensitive assays detected more P. falciparum infections in stored saliva samples than were originally detected by thick-film microscopy (41/60 = 68%). When stratified by number of parasites in the blood, the cox3 assay successfully detected more than 90% of infections using saliva when individuals had > 1000 parasites/µl of peripheral blood, but sensitivity was reduced at submicroscopic parasitemia levels. Bands on electrophoresis gels were distinct for the cox3 assay, whereas faint or non-specific bands were sometimes observed for varATS and 18S rRNA that made interpretation of results difficult. Assays could be completed in 3.5 and 3 h for the cox3 and varATS assays, respectively, whereas the 18S rRNA gene assays required at least 7 h. CONCLUSIONS: This study demonstrates that a PCR assay targeting the cox3 gene detected P. falciparum DNA in more saliva samples than primers for the 18S rRNA gene. Non-invasive collection of saliva in combination with the proposed cox3 primer-based PCR assay could potentially enhance routine testing of P. falciparum during disease surveillance, monitoring, and evaluation of interventions for malaria elimination.

Association of Antibodies to VAR2CSA and Merozoite Antigens with Pregnancy Outcomes in Women Living in Yaoundé, Cameroon.

Plasmodium falciparum infections are serious in pregnant women, because VAR2CSA allows parasitized erythrocytes to sequester in the placenta, causing placental malaria (PM). In areas of endemicity, women have substantial malarial immunity prior to pregnancy, including antibodies to merozoite antigens, but produce antibodies to VAR2CSA only during pregnancy. The current study sought to determine the importance of antibodies to VAR2CSA and merozoite antigens in pregnant women in Yaoundé, Cameroon, where malaria transmission was relatively low. A total of 1,377 archival plasma samples collected at delivery were selected (at a 1:3 ratio of PM-positive [PM+] to PM-negative [PM-] women) and screened for antibodies to full-length VAR2CSA and 7 merozoite antigens. Results showed that many PM+ women and most PM- women lacked antibodies to VAR2CSA at delivery. Among PM+ women, antibodies to VAR2CSA were associated with a reduced risk of having high placental parasitemia (odds ratio [OR], 0.432; confidence interval [CI], 0.272, 0.687; P = 0.0004) and low-birth-weight (LBW) babies (OR = 0.444; CI, 0.247, 0.799; P = 0.0068), even during first pregnancies. Among antibodies to the 7 merozoite antigens, i.e., AMA1, EBA-175, MSP142, MSP2, MSP3, MSP11, and Pf41, only antibodies to MSP3, EBA-175, and Pf41 were associated with reduced risk for high placental parasitemias (P = 0.0389, 0.0291, and 0.0211, respectively) and antibodies to EBA-175 were associated with reduced risk of premature deliveries (P = 0.0211). However, after adjusting for multiple comparisons significance declined. Thus, in PM+ women, antibodies to VAR2CSA were associated with lower placental parasitemias and reduced prevalence of LBW babies in this low-transmission setting.

Increased Susceptibility to Plasmodium falciparum in Infants is associated with Low, not High, Placental Malaria Parasitemia.

Risk of malaria in infants can be influenced by prenatal factors. In this study, the potential for placental parasitemia at delivery in predicting susceptibility of infants to Plasmodium falciparum (Pf) infections was evaluated. Seventy-two newborns of mothers who were placental malaria negative (PM-) and of mothers who were PM+ with below (PM+ Lo) and above (PM + Hi) median placental parasitemia, were actively monitored during their first year of life. Median time to first PCR-detected Pf infection was shorter in PM + Lo infants (2.8 months) than in both PM- infants (4.0 months, p = 0.002) and PM + Hi infants (4.1 months, p = 0.01). Total number of new infections was also highest in the PM + Lo group. Only 24% of infants experienced clinical malaria episodes but these episodes occurred earlier in PM + Lo infants than in PM + Hi infants (p = 0.05). The adjusted hazard ratio (95% CI) of having Pf infection was 3.9 (1.8-8.4) and 1.5 (0.7-3.4) for infants in the PM + Lo and PM + Hi groups, respectively. Collectively, low placental parasitemia was associated with increased susceptibility to malaria during infancy. Therefore, malaria in pregnancy preventive regimens, such as sulfadoxine-pyremethamine, that reduce but do not eliminate placental Pf in areas of drug resistance may increase the risk of malaria in infants.

Atmospheric modeling to assess wind dependence in tracer dilution method measurements of landfill methane emissions.

The short-term temporal variability of landfill methane emissions is not well understood due to uncertainty in measurement methods. Significant variability is seen over short-term measurement campaigns with the tracer dilution method (TDM), but this variability may be due in part to measurement error rather than fluctuations in the actual landfill emissions. In this study, landfill methane emissions and TDM-measured emissions are simulated over a real landfill in Delaware, USA using the Weather Research and Forecasting model (WRF) for two emissions scenarios. In the steady emissions scenario, a constant landfill emissions rate is prescribed at each model grid point on the surface of the landfill. In the unsteady emissions scenario, emissions are calculated at each time step as a function of the local surface wind speed, resulting in variable emissions over each 1.5-h measurement period. The simulation output is used to assess the standard deviation and percent error of the TDM-measured emissions. Eight measurement periods are simulated over two different days to look at different conditions. Results show that standard deviation of the TDM- measured emissions does not increase significantly from the steady emissions simulations to the unsteady emissions scenarios, indicating that the TDM may have inherent errors in its prediction of emissions fluctuations. Results also show that TDM error does not increase significantly from the steady to the unsteady emissions simulations. This indicates that introducing variability to the landfill emissions does not increase errors in the TDM at this site. Across all simulations, TDM errors range from -15% to 43%, consistent with the range of errors seen in previous TDM studies. Simulations indicate diurnal variations of methane emissions when wind effects are significant, which may be important when developing daily and annual emissions estimates from limited field data.

A versatile, high through-put, bead-based phagocytosis assay for Plasmodium falciparum.

Antibody-mediated phagocytosis is an important immune effector mechanism against Plasmodium falciparum-infected erythrocytes (IE); however, current phagocytosis assays use IE collected from infected individuals or from in vitro cultures of P. falciparum, making them prone to high variation. A simple, high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The assay is highly repeatable, provides both the overall percent phagocytosis and semi-quantitates the number of antigen-coupled beads internalized.

Detection of Plasmodium falciparum DNA in saliva samples stored at room temperature: potential for a non-invasive saliva-based diagnostic test for malaria.

BACKGROUND: Current malaria diagnostic methods require blood collection, that may be associated with pain and the risk of transmitting blood-borne pathogens, and often create poor compliance when repeated sampling is needed. On the other hand, the collection of saliva is minimally invasive; but saliva has not been widely used for the diagnosis of malaria. The aim of this study was to evaluate the diagnostic performance of saliva collected and stored at room temperature using the OMNIgene®â€¢ORAL kit for diagnosing Plasmodium falciparum malaria. METHODS: Paired blood and saliva samples were collected from 222 febrile patients in Cameroon. Saliva samples were collected using the OMNIgene®â€¢ORAL (OM-501) kit and stored at room temperature for up to 13 months. Thick blood film microscopy (TFM) was used to detect P. falciparum blood-stage parasites in blood. Detection of P. falciparum DNA in blood and saliva was based on amplification of the multi-copy 18 s rRNA gene using the nested-polymerase chain reaction (nPCR). RESULTS: Prevalence of malaria detected by TFM, nPCR-saliva and nPCR-blood was 22, 29, and 35%, respectively. Using TFM as the gold standard, the sensitivity of nPCR-saliva and nPCR-blood in detecting P. falciparum was 95 and 100%, respectively; with corresponding specificities of 93 and 87%. When nPCR-blood was used as gold standard, the sensitivity of nPCR-saliva and microscopy was 82 and 68%, respectively; whereas, the specificity was 99 and 100%, respectively. Nested PCR-saliva had a very good agreement with both TFM (kappa value 0.8) and blood PCR (kappa value 0.8). At parasitaemia > 10,000 parasites/µl of blood, the sensitivity of nPCR-saliva was 100%. Nested PCR-saliva detected 16 sub-microscopic malaria infections. One year after sample collection, P. falciparum DNA was detected in 80% of saliva samples stored at room temperature. CONCLUSIONS: Saliva can potentially be used as an alternative non-invasive sample for the diagnosis of malaria and the OMNIgene®â€¢ORAL kit is effective at transporting and preserving malaria parasite DNA in saliva at room temperature. The technology described in this study for diagnosis of malaria in resource-limited countries adds on to the armamentarium needed for elimination of malaria.

Statistical prediction of immunity to placental malaria based on multi-assay antibody data for malarial antigens.

BACKGROUND: Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman's risk of anemia, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. METHODS: Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL 1-6 of the FCR3, 3D7 and 7G8 lines, ID1-ID2a (FCR3 and 3D7) and 11 antigens that have been reported to be associated with immunity to P. falciparum (AMA-1, CSP, EBA-175, LSA1, MSP1, MSP2, MSP3, MSP11, Pf41, Pf70 and RESA)). Ab levels along with clinical variables (age, gravidity) were used in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. RESULTS: The best and simplest model proved to be the logistic regression reduced model. AMA-1, MSP2, EBA-175, Pf41, and MSP11 were found to be the top five most important predictors for the PM status based on overall prediction performance. CONCLUSIONS: Not surprising, significant differences were observed between PM positive (PM+) and PM negative (PM-) groups for Ab levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among seven different classifiers considered, the reduced logistic regression model produces the best overall predictive performance.

Timing of the human prenatal antibody response to Plasmodium falciparum antigens.

Plasmodium falciparum (Pf)-specific T- and B-cell responses may be present at birth; however, when during fetal development antibodies are produced is unknown. Accordingly, cord blood samples from 232 preterm (20-37 weeks of gestation) and 450 term (≥37 weeks) babies were screened for IgM to Pf blood-stage antigens MSP1, MSP2, AMA1, EBA175 and RESA. Overall, 25% [95% CI = 22-28%] of the 682 newborns were positive for IgM to ≥1 Pf antigens with the earliest response occurring at 22 weeks. Interestingly, the odds of being positive for cord blood Pf IgM decreased with gestational age (adjusted OR [95% CI] at 20-31 weeks = 2.55 [1.14-5.85] and at 32-36 weeks = 1.97 [0.92-4.29], with ≥37 weeks as reference); however, preterm and term newborns had similar levels of Pf IgM and recognized a comparable breadth of antigens. Having cord blood Pf IgM was associated with placental malaria (adjusted OR [95% CI] = 2.37 [1.25-4.54]). To determine if in utero exposure occurred via transplacental transfer of Pf-IgG immune complexes (IC), IC containing MSP1 and MSP2 were measured in plasma of 242 mother-newborn pairs. Among newborns of IC-positive mothers (77/242), the proportion of cord samples with Pf IC increased with gestational age but was not associated with Pf IgM, suggesting that fetal B cells early in gestation had not been primed by IC. Finally, when cord mononuclear cells from 64 term newborns were cultured in vitro, only 11% (7/64) of supernatants had Pf IgM; whereas, 95% (61/64) contained secreted Pf IgG. These data suggest fetal B cells are capable of making Pf-specific IgM from early in the second trimester and undergo isotype switching to IgG towards term.