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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.

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