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.

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.

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.

An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens.

BACKGROUND: Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized. METHODS: A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples (30 from malaria-positive and 30 malaria-negative individuals), together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens (AMA1, EBA175, MSP1, MSP2, MSP3, MSP11, Pf41), sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales. RESULTS: Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean-variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation. CONCLUSIONS: For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.

Individuals living in a malaria-endemic area of Cameroon do not have an acquired antibody response to Plasmodium falciparum histidine-rich protein 2.

BACKGROUND: Diagnosis of Plasmodium falciparum is often based on detection of histidine-rich protein 2 (HRP2) in blood. Most HRP2-based assays have high sensitivity and specificity; however, authors have suggested that antibodies (Ab) to HRP2 could reduce assay sensitivity. This study sought to characterize the antibody response to HRP2 with respect to prevalence, class, subclass, affinity, and age distribution in Cameroonian children and adults residing in an area with high P. falciparum transmission. METHODS: Plasma samples from 181 Cameroonian children and adults who had been repeatedly exposed to P. falciparum and 112 samples from American adults who had never been exposed were tested for IgG Ab to HRP2. For comparison, Ab to the merozoite antigens MSP1, MSP2, MSP3 and the pregnancy-associated antigen VAR2CSA were measured using a multiplex bead-based assay. In addition, 81 plasma samples from slide-positive individuals were screened for IgM Ab to HRP2. RESULTS: As expected, children and adults had IgG Ab to MSP1, MSP2 and MSP3, antibody levels increased with age, and only women of child-bearing age had Ab to VAR2CSA; however, no convincing evidence was found that these individuals had an acquired antibody response to HRP2. That is, using two sources of recombinant HRP2, identical results were obtained when plasma from 110 Cameroonian adults and 112 US adults were screened for IgG Ab. Further studies showed that antibody prevalence and levels did not increase with age in Cameroonians between ages 5 and >80 years. Although a few samples from slide-positive Cameroonians had IgM values slightly above the American cut-off, it was unclear if the individuals had a true IgM response to HRP2 or if the values were due to non-specific binding from elevated immunoglobulin levels associated with infection. Data from prediction models showed a paucity of Class II T cell epitopes in HRP2. CONCLUSIONS: These data support the conclusion that most individuals in malaria-endemic areas do not produce an acquired humoral response to HRP2. The absence of Ab helps explain why HRP2-based assays are able to detect nanogram amounts of HRP2 and why HRP2 continues to circulate for a long time after parasite clearance.

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.

Diagnostic accuracy and usefulness of the Genotype MTBDRplus assay in diagnosing multidrug-resistant tuberculosis in Cameroon? a cross-sectional study.

BACKGROUND: Drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB), is a major public health problem. Effective management of MDR-TB relies on accurate and rapid diagnosis. In this study, we assessed the diagnostic accuracy of the Genotype MTBDRplus assay in diagnosing MDR-TB in Cameroon, and then discuss on its utility within the diagnostic algorithm for MDR-TB. METHODS: In this cross-sectional study, 225 isolates of Mycobacterium tuberculosis cultured from sputum samples collected from new and previously treated pulmonary tuberculosis patients in Cameroon were used to determine the accuracy of the Genotype MTBDRplus assay. We compared the results of the Genotype MTBDRplus assay with those from the automated liquid culture BACTEC MGIT 960 SIRE system for sensitivity, specificity, and degree of agreement. The pattern of mutations associated with resistance to RIF and INH were also analyzed. RESULTS: The Genotype MTBDRplus assay correctly identified Rifampicin (RIF) resistance in 48/49 isolates (sensitivity, 98% [CI, 89%-100%]), Isoniazid (INH) resistance in 55/60 isolates (sensitivity 92% [CI, 82%-96%]), and MDR-TB in 46/49 (sensitivity, 94% [CI, 83%-98%]). The specificity for the detection of RIF-resistant and MDR-TB cases was 100% (CI, 98%-100%), while that of INH resistance was 99% (CI, 97%-100%). The agreement between the two tests for the detection of MDR-TB was very good (Kappa = 0.96 [CI, 0.92-1.00]). Among the 3 missed MDR-TB cases, the Genotype MTBDRplus assay classified two samples as RIF-monoresistant and one as INH monoresistant. The most frequent mutations detected by the Genotype MTBDRplus assay was the rpoB S531 L MUT3 41/49 (84%) in RIF-resistant isolates, and the KatG S315 T1 (MUT1) 35/55 (64%) and inhA C15T (MUT1) 20/55 (36%) mutations in INH-resistant isolates. CONCLUSION: The Genotype MTBDRplus assay had good accuracy and could be used for the diagnosis of MDR-TB in Cameroon. For routine MDR-TB diagnosis, this assay could be used for Mycobacterium tuberculosis cultures containing contaminants, to complement culture-based drug susceptibility testing or to determine drug resistant mutations.

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.

Elevated levels of soluble TNF receptors 1 and 2 correlate with Plasmodium falciparum parasitemia in pregnant women: potential markers for malaria-associated inflammation.

Plasmodium falciparum-infected erythrocytes (IEs) sequester in the intervillous space (IVS) of the placenta causing placental malaria (PM), a condition that increases a woman's chances of having a low-birth-weight baby. Because IEs sequester, they frequently are not observed in peripheral blood smears, resulting in women with PM being misdiagnosed and thus not treated. Because sequestered IEs induce inflammation in the IVS, detection of inflammatory mediators in the peripheral blood may provide an approach for diagnosing PM. Two counterregulatory molecules, TNF-αR (TNFR) 1 and TNFR2, modulate the pathological effects of TNF-α. Levels of these soluble TNFRs (sTNFRs) are reported to be elevated in children with severe malaria, but it is unclear if they are increased in the peripheral blood of PM-positive women with asymptomatic infections. In this study, sTNFR levels were measured throughout the course of pregnancy, as well as at delivery, in women with asymptomatic infections and those who remained uninfected. Results showed that both sTNFRs were significantly increased in the peripheral blood of women with asymptomatic malaria (p < 0.0001) and were positively correlated with parasitemia (p < 0.0001 for sTNFR1 and p = 0.0046 for sTNFR2). Importantly, levels of sTNFR2 were elevated in the peripheral blood of women who were PM-positive but peripheral blood-smear negative (p = 0.0017). Additionally, sTNFR2 levels were elevated in the blood of malaria-positive women who delivered low-birth-weight babies. In vitro studies demonstrated that syncytiotrophoblasts were not a major source of sTNFR. These data suggest that sTNFR2 may be a valuable biomarker for detection of malaria-associated inflammation.

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.

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.

Genetic polymorphisms of mannose-binding lectin do not influence placental malaria but are associated with preterm deliveries.

During pregnancy, Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta where they induce pathology and increase the risk of low-birth-weight (LBW) babies. The innate immune mediator, mannose-binding lectin (MBL), enhances phagocytosis of pathogens. Since MBL is reported to bind to IE, we hypothesized that it might aid in clearance of IE from the placenta, thereby reducing the risk of LBW babies. To test this hypothesis, molecular genotyping was used to detect polymorphisms at codon 57 (A/C) in exon 1 of MBL2 in 401 pregnant Cameroonian women, with or without placental malaria, who had LBW and normal-weight babies. Polymorphisms in the promoter region at positions -550 (H/L), -221 (X/Y), and +4 (P/Q) were also determined, and plasma MBL levels were measured during pregnancy and at delivery. The expected correlation between genotype and plasma MBL levels was confirmed. However, asymptomatic infections were not associated with an increase in MBL levels in the peripheral blood, and MBL levels were similar in the placental and cord blood of women with or without placental malaria at delivery. There was no evidence that MBL levels at delivery were associated with malaria-related poor pregnancy outcomes. Women with the LXPA haplotype, however, were more likely to have LBW babies, but the risk was not related to malaria. These results do not support the hypothesis that MBL aids in the clearance of parasites from the placenta but suggest that Cameroonian women with LXPA are at risk of having LBW babies due to other causes.

Influence of human chorionic gonadotropin (hCG) on in vitro growth of Plasmodium falciparum.

BACKGROUND: During pregnancy, women are more susceptible to Plasmodium falciparum infections and frequently have a higher parasitaemia than non-pregnant women. Several mechanisms are responsible for their increased susceptibility, including down-modulation of immune responses that aid in parasite clearance and sequestration of infected erythrocytes in the placenta. Early in pregnancy, a third mechanism may contribute to higher parasitaemia, since it has been reported that addition of human chorionic gonadotropin (hCG) to in vitro cultures of the NF54-strain of P. falciparum results in increased parasite growth rates. The goal of this study was to further examine the effect of hCG on P. falciparum growth. METHODS: The NF54-3D7, FVO and 7G8 strains of P. falciparum were cultured in vitro with various physiological concentrations of hCG purchased from three sources. Infected erythrocytes were also co-cultured with a human cell line that naturally secretes hCG. RESULTS: Results from 14 experiments using different combinations of parasite strains and concentrations of hCG from different sources, as well as the co-culture studies, failed to provide convincing evidence that hCG enhances parasite growth in vitro. CONCLUSION: Based on these data, it seems unlikely that hCG has a direct effect on the rate of parasite growth early in pregnancy.

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.

Soluble Tumor Necrosis Factor-α Receptor 2 in Urine Is a Potential Biomarker for Noninvasive Diagnosis of Malaria During Pregnancy.

Background. During pregnancy, the placenta is inaccessible for diagnosis of placental malaria (PM), but soluble tumor necrosis factor-α receptors (sTNFR) are elevated in the plasma of women with PM. Methods. In this study, sTNFR-1 and sTNFR-2 were quantified in urine of pregnant and nonpregnant Cameroonian women who were positive or negative for malaria by blood-smear microscopy. Results. We found that levels of both sTNFR in urine were higher in pregnant compared with nonpregnant women, but malaria-positive pregnant women excreted substantially more sTNFR-1 (P = .005) and sTNFR-2 (P < .001) than malaria-negative pregnant women. The amount of sTNFR-1(rs = 0.784, P < .001) and sTNFR-2 (rs = 0.816, P < .001) in urine correlated with parasitemia, even in afebrile pregnant women. Urine sTNFR-2 predicted maternal malaria with an area under curve of 0.892 (95% confidence interval, .787-.898). At cutoff concentrations of 9.8 ng and 13.6 ng of sTNFR-2 per mL urine, the sensitivity/specificity were 82.6%/87.0% and 78.3%/95.7%, respectively. Conclusions. The sTNFR-2 in noninvasive urine samples may be useful for diagnosis of malaria during pregnancy.

Influence of Intermittent Preventive Treatment on Antibodies to VAR2CSA in Pregnant Cameroonian Women.

Intermittent preventive treatment (IPT) and insecticide-treated bed nets are the standard of care for preventing malaria in pregnant women. Since these preventive measures reduce exposure to malaria, their influence on the antibody (Ab) response to the parasite antigen VAR2CSA was evaluated in pregnant Cameroonian women exposed to holoendemic malaria. Ab levels to full-length VAR2CSA (FV2), variants of the six Duffy binding like (DBL) domains, and proportion of high avidity Ab to FV2 were measured longitudinally in 92 women before and 147 women after IPT. As predicted, reduced exposure interfered with acquisition of Ab in primigravidae, with 71% primigravidae being seronegative to FV2 at delivery. Use of IPT for > 13 weeks by multigravidae resulted in 26% of women being seronegative at delivery and a significant reduction in Ab levels to FV2, DBL5, DBL6, proportion of high avidity Ab to FV2, and number of variants recognized. Thus, in women using IPT important immune responses were not acquired by primigravidae and reduced in a portion of multigravidae, especially women with one to two previous pregnancies. Longitudinal data from individual multigravidae on IPT suggest that lower Ab levels most likely resulted from lack of boosting of the VAR2CSA response and not from a short-lived Ab response.

Maternal Human Immunodeficiency Virus-Associated Hypergammaglobulinemia Reduces Transplacental Transfer of Immunoglobulin G to Plasmodium falciparum Antigens in Cameroonian Neonates.

Background. Human immunodeficiency virus (HIV) infection reduces placental transfer of antibodies from mother to the fetus for many antigens; however, conflicting data exist for transfer of immunoglobulin G (IgG) to malarial antigens. The mechanism(s) underlying reduced placental transfer is unknown. Methods. Levels of maternal and cord total IgG, IgG subclasses, and cord-to-mother ratios (CMRs) were measured in 107 mother-cord pairs to 3 malarial antigens: circumsporozoite protein (CSP), apical membrane antigen 1 (AMA-1), merozoite surface protein 1 (MSP-1), and tetanus toxoid C-fragment (TTc). Results. Immunoglobulin G levels to CSP and TTc were lower in HIV+ mothers, and cord IgG to CSP, MSP-1, and TTc were significantly lower in neonates born to HIV+ mothers (all P values <.05). The prevalence of mothers with hypergammaglobulinemia was significantly higher among HIV+ women (68%) compared with HIV- mothers (8%) (P < .0001). Maternal hypergammaglobulinemia was associated with reduction in transplacental transfer of antibodies to CSP (P = .03), MSP-1 (P = .004), and TTc (P = .012), and CMRs <1 were found for MSP-1 (odds ratio [OR] = 6.5), TTc (OR = 4.95), and IgG1 to CSP (OR = 3.75, P = .025) in statistical models adjusted for maternal IgG. Conclusions. Data confirmed that HIV infections are associated with lower cord antibody levels to malarial antigens and that hypergammaglobulinemia may contribute to reduced antibody transfer.