Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities

Abstract

Background: The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome.

Methods: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities.

Results: In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG.

Conclusion: The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Keywords: Low-density; Molecular diagnostics; Ultra-sensitive; mtCOX1; qPCR; varATS.

Fig. 1

Proportion of P. falciparum and P. vivax infections detected by qPCR versus us-qPCR by country. Proportion of P. falciparum (a) and P. vivax infections (b) positive by species-specific 18S rRNA qPCR among all samples positive by any qPCR assay (standard qPCR or us-qPCR) and parasite densities of P. falciparum (c) and P. vivax infections (d) detected by 18S rRNA qPCR versus only by us-qPCR. White circles: infection detected by standard qPCR; black circles: infection detected only by us-qPCR. ¹Owing to exhaustion of DNA, the parasite densities of 5 Brazilian samples (white circles) were quantified by 18S rRNA qPCR

Fig. 2

Age trends in P. vivax positivity and parasite density in Thailand, Brazil and PNG. P. vivax infections detected by Pv_18S rRNA qPCR and Pv_mtCOX1 us-qPCR (white circles) and P. vivax infections only detected by Pv_mtCOX1 us-qPCR (black circles). a–c P. vivax positivity by age group with 95% confidence intervals (black vertical lines). d–f Log₁₀ transformed P. vivax parasite density by Pv_mtCOX1 us-qPCR per age group with median Pv_mtCOX1 copy numbers (horizontal lines)