BackgroundPregnancy poses specific challenges for the diagnosis of Plasmodium falciparum infection due to parasite sequestration in the placenta, which translates in low circulation levels in peripheral blood. The aim of this study is to assess the performance of a new highly sensitive rapid diagnostic test (HS-RDT) for the detection of malaria in peripheral and placental blood samples from pregnant women in Colombia.MethodsThis is a retrospective study using 737 peripheral and placental specimens collected from pregnant women in Colombian malaria-endemic regions. Light microscopy (LM), conventional rapid diagnostic tests (Pf/Pv RDT and Pf RDT), and HS-RDT testing were performed. Diagnostic accuracy endpoints of LM, HS-RDT and RDTs were compared with nested polymerase chain reaction (nPCR) as the reference test.ResultsIn comparison with nPCR, the sensitivity of HS-RDT, Pf RDT, Pf/Pv RDT and LM to detect infection in peripheral samples was 85.7% (95% CI = 70.6–93.7), 82.8% (95% CI = 67.3–91.9), 77.1% (95% CI = 61.0–87.9) and 77.1% (95% CI = 61.0–87.9) respectively. The sensitivity to detect malaria in asymptomatic women, was higher with HS-RDT, where LM and Pf/Pv RDT missed half of infections detected by nPCR, but differences were not significant. Overall, specificity was similar for all tests (>99.0%). In placental blood, the prevalence of infection by P. falciparum by nPCR was 2.8% (8/286), by HS-RDT was 1% and by conventional RDTs (Pf RDT and Pf/Pv RDT) and LM was 0.7%. The HS-RDT detected placental infections in peripheral blood that were negative by LM and Pf/Pv RDT, however the number of positive placentas was low.ConclusionsThe sensitivity of HS-RDT to detect P. falciparum infections in peripheral and placental samples from pregnant women was slightly better compared to routinely used tests during ANC visits and at delivery. Although further studies are needed to guide recommendations on the use of the HS-RDT for malaria case management in pregnancy, this study shows the potential value of this test to diagnose malaria in pregnancy in low-transmission settings.

BackgroundPregnant women frequently show low-density Plasmodium infections that require more sensitive methods for accurate diagnosis and early treatment of malaria. This is particularly relevant in low-malaria transmission areas, where intermittent preventive treatment is not recommended. Molecular methods, such as polymerase chain reaction (PCR) are highly sensitive, but require sophisticated equipment and advanced training. Instead, loop mediated isothermal amplification (LAMP) provides an opportunity for molecular detection of malaria infections in remote endemic areas, outside a reference laboratory. The aim of the study is to evaluate the performance of LAMP for the screening of malaria in pregnant women in Colombia.MethodsThis is a nested prospective study that uses data and samples from a larger cross-sectional project conducted from May 2016 to January 2017 in three Colombian endemic areas (El Bagre, Quibdó, and Tumaco). A total of 531 peripheral and placental samples from pregnant women self-presenting at local hospitals for antenatal care visits, at delivery or seeking medical care for suspected malaria were collected. Samples were analysed for Plasmodium parasites by light microscopy (LM), rapid diagnostic test (RDT) and LAMP. Diagnostic accuracy endpoints (sensitivity, specificity, predictive values, and kappa scores) of LM, RDT and LAMP were compared with nested PCR (nPCR) as the reference standard.ResultsIn peripheral samples, LAMP showed an improved sensitivity (100.0%) when compared with LM 79.5% and RDT 76.9% (p < 0.01), particularly in afebrile women, for which LAMP sensitivity was two-times higher than LM and RDT. Overall agreement among LAMP and nPCR was high (kappa value = 1.0). Specificity was similar in all tests (100%). In placental blood, LAMP evidenced a four-fold improvement in sensitivity (88.9%) when compared with LM and RDT (22.2%), being the only method, together with nPCR, able to detect placental infections in peripheral blood.ConclusionsLAMP is a simple, rapid and accurate molecular tool for detecting gestational and placental malaria, being able to overcome the limited sensitivity of LM and RDT. These findings could guide maternal health programs in low-transmission settings to integrate LAMP in their surveillance systems for the active detection of low-density infections and asymptomatic malaria cases.

Background: Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries. Results: The qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density. Conclusion: This immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

Background: In low transmission settings early diagnosis is the main strategy to reduce adverse outcomes of malaria in pregnancy; however, microscopy and rapid diagnostic tests (RDTs) are inadequate for detecting lowdensity infections. We studied the performance of the highly sensitive-RDT (hsRDT) and the loop mediated isothermal DNA amplification (LAMP) for the detection of P. falciparum in pregnant women. Methods: A cross-sectional study was conducted in two malaria-endemic municipalities in Colombia. We screened pregnant women in the context of an antenatal care program in health facilities and evaluated five tests (microscopy, conventional RDT, hsRDT, LAMP and nested polymerase chain reaction-PCR) for the detection of P. falciparum in peripheral blood, using a quantitative reverse transcription PCR (qRT-PCR) as the reference standard. Diagnostic performance of hsRDT and LAMP were compared with routine testing. Results: The prevalence of P. falciparum was 4.5% by qRT-PCR, half of those infections were subpatent. The sensitivity of the hsRDT (64.1%) was slightly better compared to microscopy and cRDT (59 and 53.8% respectively). LAMP had the highest sensitivity (89.7%) for detecting P. falciparum and the ability to detect very low-density infections (minimum parasite density detected 0.08 p/μL). Conclusions: There is an underestimation of Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits. LAMP methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated.

BackgroundPlasmodium falciparum placental malaria is characterized by the sequestration of infected erythrocytes (IEs) in the placental intervillous space via adherence to chondroitin sulphate A (CSA), production of inflammatory molecules, and leukocytes infiltration. Previous reports suggest that the syncytiotrophoblast (ST) immunologically responds to IEs contact. This study explores the inflammatory response induced in BeWo cells by adherence of IEs and TNFstimulation.MethodsA non-syncitialized BeWo cells (trophoblast model) were used to evaluate its response to CSA-adherents IEs (FCB1csa, FCB2csa, FCR3csa, 3D7csa) and TNF stimulation. Expression of membrane ICAM-1 (mICAM-1) receptor in BeWo cells was quantified by flow cytometry and the IL-8, IL-6 and soluble ICAM-1 (sICAM-1) concentrations were quantified by enzyme-linked immunosorbentassay (ELISA) in BeWo stimulated supernatants.ResultsBeWo cells stimulated with TNF and CSA-adherents IEs of FCB1csa and 3D7csa (strains with higher adhesion) increase the expression of ICAM-1 on the surface of cells and the secretion of immune factors IL-8, IL-6 and sICAM-1. This inflammatory response appears to be related to the level of adherence of IEs because less adherent strains do not induce significant changes.ConclusionsIt was found that BeWo cells responds to CSA-IEs and to TNF favouring a placental pro-inflammatory environment, evidenced by increases in the expression of membrane mICAM-1 and release of soluble ICAM-1, as well as the IL-8 and IL-6 secretion. The expression of ICAM-1 in BeWo cells might be associated to an increase in leukocyte adhesion to the trophoblast barrier, promoting greater inflammation, while the sICAM-1 release could be a protection mechanism activated by trophoblastic cells, in order to regulate the local inflammatory response.

Insect saliva induces significant antibody responses associated with the intensity of exposure to bites and the risk of disease in humans. Several salivary biomarkers have been characterized to determine exposure intensity to Old World Anopheles mosquito species. However, new tools are needed to quantify the intensity of human exposure to Anopheles bites and understand the risk of malaria in low-transmission areas in the Americas. To address this need, we conducted proteomic and bioinformatic analyses of immunogenic candidate proteins present in the saliva of uninfected Anopheles albimanus from two separate colonies—one originating from Central America (STECLA strain) and one originating from South America (Cartagena strain). A ~65 kDa band was identified by IgG antibodies in serum samples from healthy volunteers living in a malaria endemic area in Colombia, and a total of five peptides were designed from the sequences of two immunogenic candidate proteins that were shared by both strains. ELISA-based testing of human IgG antibody levels against the peptides revealed that the transferrin-derived peptides, TRANS-P1, TRANS-P2 and a salivary peroxidase peptide (PEROX-P3) were able to distinguish between malaria-infected and uninfected groups. Interestingly, IgG antibody levels against PEROX-P3 were significantly lower in people that have never experienced malaria, suggesting that it may be a good marker for mosquito bite exposure in naïve populations such as travelers and deployed military personnel. In addition, the strength of the differences in the IgG levels against the peptides varied according to location, suggesting that the peptides may able to detect differences in intensities of bite exposure according to the mosquito population density. Thus, the An. albimanus salivary peptides TRANS-P1, TRANS-P2, and PEROX-P3 are promising biomarkers that could be exploited in a quantitative immunoassay for determination of human-vector contact and calculation of disease risk.

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