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Objective.  To evaluate molecular tools to detect low-level parasitemia and the five species of  Plasmodium  that infect humans for use in control and elimination programs, and in reference laboratories.    Methods.  We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar ®  Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar ®  Malaria Screen &amp; Type PCR Kit (alt-S&amp;T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear.    Results.  The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&amp;T had a sensitivity of 100%, except for identifying  P. falciparum , for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for  P. vivax, P. malariae  and  P. falciparum . For all  Plasmodium  species, the correlation between cycle threshold values of alt-S&amp;T and alt-Gen compared with qPCR was significant ( P  &lt; 0.0001, Spearman’s test), with  r  = 0.8621 for alt-S&amp;T and  r  = 0.9371 for alt-Gen. When all  Plasmodium  species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values ( P  &lt; 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&amp;T.    Conclusions.  The alt-Gen and alt-S&amp;T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories