Diagnostic Performance of Conventional and Ultrasensitive Rapid Diagnostic Tests for Malaria in Febrile Outpatients in Tanzania

Abstract

Background: A novel ultrasensitive malaria rapid diagnostic test (us-RDT) has been developed for improved active Plasmodium falciparum infection detection. The usefulness of this us-RDT in clinical diagnosis and fever management has not been evaluated.

Methods: Diagnostic performance of us-RDT was compared retrospectively to that of conventional RDT (co-RDT) in 3000 children and 515 adults presenting with fever to Tanzanian outpatient clinics. The parasite density was measured by an ultrasensitive qPCR (us-qPCR), and the HRP2 concentration was measured by an enzyme-linked immunosorbent assay.

Results: us-RDT identified few additional P. falciparum-positive patients as compared to co-RDT (276 vs 265 parasite-positive patients detected), with only a marginally greater sensitivity (75% vs 73%), using us-qPCR as the gold standard (357 parasite-positive patients detected). The specificity of both RDTs was >99%. Five of 11 additional patients testing positive by us-RDT had negative results by us-qPCR. The HRP2 concentration was above the limit of detection for co-RDT (>3653 pg of HRP2 per mL of blood) in almost all infections (99% [236 of 239]) with a parasite density >100 parasites per µL of blood. At parasite densities <100 parasites/µL, the HRP2 concentration was above the limits of detection of us-RDT (>793 pg/mL) and co-RDT in 29 (25%) and 24 (20%) of 118 patients, respectively.

Conclusion: There is neither an advantage nor a risk of using us-RDT, rather than co-RDT, for clinical malaria diagnosis. In febrile patients, only a small proportion of infections are characterized by a parasite density or an HRP2 concentration in the range where use of us-RDT would confer a meaningful advantage over co-RDT.

Keywords: HRP2; Malaria; PCR; RDT; Tanzania; diagnosis; fever; quantitative; ultrasensitive.

Figure 1.

Venn diagram of Plasmodium falciparum positivity by different diagnostic methods. A, Positivity by a conventional rapid diagnostic test (co-RDT), an ultrasensitive RDT (us-RDT), and an ultrasensitive quantitative polymerase chain reaction (us-qPCR) in 3515 febrile children and adults attending outpatient clinics in Dar es Salaam, Tanzania. B, Positivity by RDT, us-qPCR, and an HRP2 enzyme-specific immunosorbent assay (ELISA) in the subset of 566 children and adults who were tested by HRP2 ELISA. For RDT positivity, the larger, continuous circle delineates positivity by us-RDT, and the smaller, dashed circle delineates positivity by co-RDT.

Figure 2.

Limit of detection and diagnostic sensitivity of a conventional rapid diagnostic test (co-RDT) and an ultrasensitive RDT (us-RDT) in relation to parasite density and HRP2 concentration. A and B, Relationship between the probability of testing positive for Plasmodium falciparum by co-RDT and us-RDT and parasite density (A) or HRP2 concentration (B). Shaded areas represent 95% confidence intervals (CIs) of the logistic regression model. C and D, Diagnostic sensitivity of co-RDT and us-RDT in relation to parasite density (C) and HRP2 concentration (D). Diagnostic sensitivities were calculated as a rolling means of 18 observations, using us-qPCR as a gold standard, and are shown with 95% CIs (shaded areas). Curves were smoothed using the lowess function (span, 0.05).

Figure 3.

Distributions of parasite density and HRP2 concentration in febrile children and adults attending outpatient clinics in Dar es Salaam, Tanzania. A and B, Distribution of parasite densities in 309 of 3000 children (A) and 48 of 515 adults (B) with positive results of an ultrasensitive quantitative polymerase chain reaction (us-qPCR). C and D, Distribution of HRP2 concentrations in 270 of 3000 children (C) and 49 of 515 adults (D) with positive us-qPCR, conventional rapid diagnostic test (co-RDT), or ultrasensitive RDT (us-RDT) results who had a positive HRP2 enzyme-linked immunosorbent assay (ELISA) result. In addition, of 200 randomly selected samples with negative us-qPCR, co-RDT, and us-RDT results that were tested by HRP2 ELISA, 11 HRP2-positive samples are shown. −, negative; +, positive.

Figure 4.

Correlation of parasite density and HRP2 concentration in febrile children (A) and febrile adults (B) attending outpatient clinics in Dar es Salaam, Tanzania. Data points are color-coded according to the sample’s positivity by a conventional rapid diagnostic test (co-RDT) and an ultrasensitive RDT (us-RDT). ELISA, enzyme-linked immunosorbent assay; neg, negative; us-qPCR, ultrasensitive quantitative polymerase chain reaction; −, negative; +, positive.