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Abstract 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.

Abstract The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/thaw impacts the profiles of these analytes in plasma was not thoroughly understood. We utilized flow cytometric analysis to examine the effect of differential centrifugation and a freeze/thaw cycle on EV profiles. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/thaw processing removes and retains EV subpopulations. Additionally, cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Our findings suggest the importance of minimizing confounding variation attributed to plasma processing and platelet contamination.

Abstract 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.

Abstract Background Adequate knowledge of malaria prevention and control can help in reducing the growing burden of malaria among vulnerable groups, particularly pregnant women and children aged under 5 years living in malaria endemic settings. Similar studies have been conducted but with less focus on these vulnerable groups. This study assessed knowledge of malaria prevention and control among the pregnant women and non-pregnant mothers of children aged under 5 years in Ibadan, Oyo State, South West Nigeria. Methods In this cross sectional study, data on socio-demographic, clinical and knowledge on malaria prevention was collected using interviewer administered questionnaires from consenting study participants attending Adeoyo maternity hospital between May and November 2016. Data was described using percentages and compared across the two maternal groups in the study population. Knowledge scoring from collected data was computed using the variables on causes, symptoms and prevention of malaria and thereafter dichotomised. Multivariate analyses were used to assess the interactive effect of socio demographic and clinical characteristics with malaria knowledge. Level of statistical significance was set at p

Cancer metastasis is a dynamic process during which cancer cells separate from a primary tumor, migrate through the vessel wall into the bloodstream, and extravasate at distant sites to form secondary colonies. During this process, circulating tumor cells are subjected to shear stress forces from blood flow, and in contact with plasma proteins and blood cells of the immune and hemostatic system, including platelets. Many studies have shown an association between high platelet count and cancer metastasis, suggesting that platelets may play an occult role in tumorigenesis. This mini-review summarizes recent and emerging discoveries of mechanisms by which cancer cells activate platelets and the role of activated platelets in promoting tumor growth and metastasis. Moreover, the review discusses how aspirin has the potential for being clinically used as an adjuvant in cancer therapy.

Abstract 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.

Abstract 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®•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®•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®•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.

In this review, we discuss the interaction between cancer and markers of inflammation (such as levels of inflammatory cells and proteins) in the circulation, and the potential benefits of routinely monitoring these markers in peripheral blood measurement assays. Next, we discuss the prognostic value and limitations of using inflammatory markers such as neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios and C-reactive protein measurements. Furthermore, the review discusses the benefits of combining multiple types of measurements and longitudinal tracking to improve staging and prognosis prediction of patients with cancer, and the ability of novel in silico frameworks to leverage this high-dimensional data.

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.