Multiplex Human Malaria Array: Quantifying Antigens for Malaria Rapid Diagnostics.

Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT), which quantifies the antigens commonly used in RDTs-Plasmodium falciparum-specific histidine-rich protein 2 (HRP2), P. falciparum-specific lactate dehydrogenase (Pf LDH), Plasmodium vivax -specific LDH (Pv LDH), and Pan malaria lactate dehydrogenase (Pan LDH), and human C-reactive protein (CRP), a biomarker of severity in malaria. At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.

Simultaneous Quantification of Plasmodium Antigens and Host Factor C-Reactive Protein in Asymptomatic Individuals with Confirmed Malaria by Use of a Novel Multiplex Immunoassay.

Malaria rapid diagnostic tests (RDTs) primarily detect Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, P. vivax LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria. The assay also measures C-reactive protein (CRP) levels as an indicator of inflammation. Validation was conducted with clinical specimens from 397 asymptomatic donors from Myanmar and Uganda, confirmed by PCR for infection, and from participants in induced blood-stage malaria challenge studies. The assay lower limits of detection for HRP2, pan LDH, P. vivax LDH, and CRP were 0.2 pg/ml, 9.3 pg/ml, 1.5 pg/ml, and 26.6 ng/ml, respectively. At thresholds for HRP2, pan LDH, and P. vivax LDH of 2.3 pg/ml, 47.8 pg/ml, and 75.1 pg/ml, respectively, and a specificity ≥98.5%, the sensitivities for ultrasensitive PCR-confirmed infections were 93.4%, 84.9%, and 48.9%, respectively. Plasmodium LDH (pLDH) concentration, in contrast to that of HRP2, correlated closely with parasite density. CRP levels were moderately higher in P. falciparum infections with confirmed antigenemia versus those in clinical specimens with no antigen. The 4-plex array is a sensitive tool for quantifying diagnostic antigens in malaria infections and supporting the evaluation of new ultrasensitive RDTs.

A multiplex immunoassay method for simultaneous quantification of iron, vitamin A and inflammation status markers.

Deficiencies of vitamin A and iron affect a significant portion of the world's population, and efforts to characterize patterns of these deficiencies are hampered by a lack of measurement tools appropriate for large-scale population-based surveys. Vitamin A and iron are not easily measured directly, so reliable proxy markers for deficiency status have been identified and adopted. Measurement of inflammatory markers is necessary to interpret vitamin A and iron status markers, because circulating levels are altered by inflammation. We developed a multiplex immunoassay method for simultaneous measurement of five markers relevant to assessing inflammation, vitamin A and iron status: α-1-acid glycoprotein, C-reactive protein, retinol binding protein 4, ferritin and soluble transferrin receptor. Serum and plasma specimens were used to optimize the assay protocol. To evaluate assay performance, plasma from 72 volunteers was assayed using the multiplex technique and compared to conventional immunoassay methods for each of the five markers. Results of the new and conventional assay methods were highly correlated (Pearson Correlations of 0.606 to 0.991, p<.0001). Inter-assay imprecision for the multiplex panel varied from 1% to 8%, and all samples fell within the limits of quantification for all assays at a single dilution. Absolute values given by the multiplex and conventional assays differed, indicating a need for further work to devise a new standard curve. This multiplexed micronutrient immunoassay technique has excellent potential as a cost effective tool for use in large-scale deficiency assessment efforts.