Comparison of microscopy, nested-PCR, and Real-Time-PCR assays using high-throughput screening of pooled samples for diagnosis of malaria in asymptomatic carriers from areas of endemicity in Myanmar

Abstract

Asymptomatic infection is an important obstacle for controlling disease in countries where malaria is endemic. Because asymptomatic carriers do not seek treatment for their infections, they can have high levels of gametocytes and constitute a reservoir available for new infection. We employed a sample pooling/PCR-based molecular detection strategy for screening malaria infection in residents from areas of Myanmar where malaria is endemic. Blood samples (n = 1,552) were collected from residents in three areas of malaria endemicity (Kayin State, Bago, and Tanintharyi regions) of Myanmar. Two nested PCR and real-time PCR assays showed that asymptomatic infection was detected in about 1.0% to 9.4% of residents from the surveyed areas. The sensitivities of the two nested PCR and real-time PCR techniques were higher than that of microscopy examination (sensitivity, 100% versus 26.4%; kappa values, 0.2 to 0.5). Among the three regions, parasite-positive samples were highly detected in subjects from the Bago and Tanintharyi regions. Active surveillance of residents from regions of intense malaria transmission would reduce the risk of morbidity and mitigate transmission to the population in these areas of endemicity. Our data demonstrate that PCR-based molecular techniques are more efficient than microscopy for nationwide surveillance of malaria in countries where malaria is endemic.

FIG 1

Map of three surveyed areas in Myanmar.

FIG 2

Sensitivities of Plasmodium genus- and species-specific nested PCR and real-time PCR assays performed with serial dilutions of Plasmodium genomic DNA extracted from field samples. (A and B) Amplification with a Plasmodium genus-specific nested PCR and real-time PCR assays. (C to F) Amplification plots showing the P. falciparum, P. vivax, P. malariae, and P. ovale nested PCR-1 assays. (G to J) Amplification plots showing the P. falciparum, P. vivax, P. malariae, and P. ovale nested PCR-2 assays. (C, D, G, and H) Lanes 1, 1,000 parasites/μl; lanes 2, 500 parasites/μl; lanes 3, 100 parasites/μl; lanes 4, 50 parasites/μl; lanes 5, 10 parasites/μl; lanes 6, 5 parasites/μl; lanes 7, 1 parasite/μl; lanes 8, 0.1 parasite/μl; and lanes 9, 0 parasites/μl. (E, F, I, and J) Lanes 1, 200 parasites/μl; lanes 2, 100 parasites/μl; lanes 3, 50 parasites/μl; lanes 4, 25 parasites/μl; lanes 5, 10 parasites/μl; lanes 6, 5 parasites/μl; lanes 7, 1 parasite/μl; lanes 8, 0.1 parasite/μl; and lanes 9, 0 parasites/μl. (K to N) Amplification plots showing the P. falciparum, P. vivax, P. malariae, and P. ovale real-time PCR assays.