Abstract
BackgroundThe 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.MethodsPlasmodium 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.ResultsIn 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.ConclusionThe 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.
Highlights
The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas
Improving sensitivity in P. vivax surveillance by us-quantitative PCR (qPCR) is of particular benefit, because the detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission
Plasmodium falciparum‐positivity by qPCR and us‐qPCR By standard Pf_18S rRNA qPCR, positivity was significantly higher in Papua New Guinea (PNG) compared to the other field sites (Chi2–test, p < 0.001)
Summary
The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. The compared assays differed in the copy number of qPCR targets in the parasite genome. Parasite densities in asymptomatic infections are often low, and infections remain undetected by light microscopy (LM) or rapid diagnostic tests (RDT), the standard diagnostics to control malaria in endemic countries. Comparison of the widely used 18S rRNA assays with ultra-sensitive detection methods uncovered a 2–4 times higher parasite prevalence of lowdensity malaria infections at medium and low transmission settings that previously remained undiagnosed by less sensitive detection tools [6, 11, 12]
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