Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma.

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.

Performance of a highly sensitive rapid diagnostic test (HS-RDT) for detecting malaria in peripheral and placental blood samples from pregnant women in Colombia.

BACKGROUND: Pregnancy 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. METHODS: This 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. RESULTS: In 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. CONCLUSIONS: The 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.

Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for screening malaria in peripheral and placental blood samples from pregnant women in Colombia.

BACKGROUND: Pregnant 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. METHODS: This 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. RESULTS: In 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. CONCLUSIONS: LAMP 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.