Operational Performance of a Plasmodium falciparum Ultrasensitive Rapid Diagnostic Test for Detection of Asymptomatic Infections in Eastern Myanmar

Abstract

In the Greater Mekong Subregion in Southeast Asia, malaria elimination strategies need to target all Plasmodium falciparum parasites, including those carried asymptomatically. More than 70% of asymptomatic carriers are not detected by current rapid diagnostic tests (RDTs) or microscopy. An HRP2-based ultrasensitive RDT (uRDT) developed to improve the detection of low-density infections was evaluated during prevalence surveys within a malaria elimination program in a low-transmission area of eastern Myanmar. Surveys were conducted to identify high-prevalence villages. Two-milliliter venous blood samples were collected from asymptomatic adult volunteers and transported to the laboratory. Plasmodium parasites were detected by RDT, uRDT, microscopy, ultrasensitive qPCR (uPCR), and multiplex enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, and predictive positive and negative values of RDT and uRDT were calculated compared to uPCR and ELISA. Parasite and antigen concentrations detected by each test were defined using uPCR and ELISA, respectively. A total of 1,509 samples, including 208 P. falciparum-positive samples were analyzed with all tests. The sensitivity of the uRDT was twofold higher than that of RDT, 51.4% versus 25.2%, with minor specificity loss, 99.5% versus 99.9%, against the combined reference (uPCR plus ELISA). The geometric mean parasitemia detected by uRDT in P. falciparum monospecific infections was 3,019 parasites per ml (95% confidence interval [95% CI], 1,790 to 5,094; n = 79) compared to 11,352 parasites per ml (95% CI, 5,643 to 22,837; n = 38) by RDT. The sensitivities of uRDT and RDT dropped to 34.6% and 15.1%, respectively, for the matched tests performed in the field. The uRDT performed consistently better than RDT and microscopy at low parasitemias. It shows promising characteristics for the identification of high-prevalence communities and warrants further evaluation in mass screening and treatment interventions.

Keywords: Plasmodium falciparum; asymptomatic infection; low density parasitemia; low transmission setting; malaria; rapid tests; ultrasensitive RDT.

FIG 1

Flowchart of study participants and results. Species results obtained by the combination of Plasmodium DNA detection by uPCR and Plasmodium antigens by Quansys ELISA (reference C) are displayed.

FIG 2

Increased range of PfHRP2 detection in uRDT compared to RDT and corresponding increase in detection of lower parasitemias for rapid tests performed in the laboratory (A) or in the field (B). Parasitemia measured by uPCR and the corresponding PfHRP2 concentration by Quansys ELISA are presented for samples positive for P. falciparum only by uPCR (monospecific infection) and by type of detection method. Vertical lines indicate PfHRP2 concentrations of 100 and 2,000 pg/ml, while horizontal lines correspond to 1,000 and 100,000 parasites/ml (see the text). Mixed P. falciparum plus P. vivax uPCR-positive samples and Plasmodium DNA-positive samples with PfHRP2 are presented in Fig. S1 in the supplemental material.

FIG 3

Probability of a P. falciparum-positive test for uRDT compared to RDT according to the parasitemia in uPCR-positive single P. falciparum infections. (A) Lab test results (solid line) and 95% CI (shaded area); field test results (dashed line). (B) Field test results (solid line) and 95% CI (shaded area); lab test results (dashed line).

FIG 4

Readability of the uRDT in the laboratory. Agreement between readers, semiquantitative assessment of signal intensity, corresponding results according to reference C, and matching results of field uRDT testing.

FIG 5

Theoretical distribution of P. falciparum parasitemia among carriers in the Greater Mekong Subregion (reference data from Imwong et al. [3] shown by the gray bars) and the corresponding fraction of each class that would be detected by uRDT (Table 4, laboratory uRDT results shown by the black bars). If used in optimal conditions, uRDT is predicted to detect up to 56% of the carriers in the GMS setting.