Comparison of two malaria multiplex immunoassays that enable quantification of malaria antigens.

BACKGROUND: Immunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. This study presents the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species. Moreover, correlation between the malaria antigen measurements performed at these platforms is assessed after calibrating with either assay standards or international standards and the cross-reactivity among Plasmodium species is examined. METHODS: A 77-member panel of specimens composed of the World Health Organization (WHO) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens. Assays for HRP2, P. falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex and the xMAP platforms were evaluated with these panels. RESULTS: The xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH (Pearson r = 0.9926) and PvLDH (r = 0. 9792), moderate positive correlation for HRP2 (r = 0.7432), and poor correlation for PanLDH (r = 0.6139). In Pearson correlation analysis between the two platforms on the DBS, the same assays were r = 0.9828, r = 0.7679, r = 0.6432, and r = 0.8957, respectively. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS. CONCLUSIONS: Q-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform.

Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi.

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

Assessment of Plasmodium antigens and CRP in dried blood spots with multiplex malaria array.

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12639-020-01325-2) contains supplementary material, which is available to authorized users.

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 new highly sensitive enzyme-linked immunosorbent assay for the detection of Plasmodium falciparum histidine-rich protein 2 in whole blood.

BACKGROUND: The detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda. RESULTS: The HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z'-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4-100) diagnostic sensitivity and 97.9% (95% CI 94.8-99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4-100) and 88.9% (95% CI 70.8-97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96-100%) and 97.3% (CI 95% 94-99%). CONCLUSIONS: The HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.