Detection of Plasmodium Species by High-Resolution Melt Analysis of DNA from Blood Smears Acquired in Southwestern Uganda

• Published in: Journal of clinical microbiology Vol. 56; no. 1
• Main Authors: Kassaza, Kennedy, Operario, Darwin J, Nyehangane, Dan, Coffey, K C, Namugosa, Mary, Turkheimer, Lena, Ojuka, Patrick, Orikiriza, Patrick, Mwanga-Amumpaire, Juliet, Byarugaba, Frederick, Bazira, Joel, Guler, Jennifer L, Moore, Christopher C, Boum, 2nd, Yap
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.01.2018
• Subjects: DNA, Protozoan - genetics; Genetic Techniques; Malaria - blood; Malaria - diagnosis; Malaria - parasitology; Molecular Typing - methods; Nucleic Acid Amplification Techniques; Parasitology; Plasmodium - classification; Plasmodium - genetics; Plasmodium - isolation & purification; Real-Time Polymerase Chain Reaction; Retrospective Studies; RNA, Ribosomal, 18S - genetics; Sensitivity and Specificity; Uganda; Uganda; diagnostics; malaria; high-resolution melting; PCR
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.01060-17

Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed (92.0%), (5.6%), and (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of species infections and can be used for retrospective genetic studies.