image description  

Detection of Plasmodium species by high resolution melt analysis of DNA from blood smears acquired in Southwestern Uganda

  • 2017/10/18
Type de publication
  • Articles
  • Kassaza
  • K.; Operario
  • D.J.; Nyehangane
  • D.; Coffey
  • K.C.; Namugosa
  • M.; Turkheimer
  • L.; Ojuka
  • P.; Orikiriza
  • P.; Mwanga-Amumpaire
  • J.; Byarugaba
  • F.; Bazira
  • J.; Guler
  • J.L.; Moore
  • C.C.; Boum
  • Y.
  • Paludisme

Background. 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 polymerase chain reaction (PCR) testing to confirm Plasmodium infection or for epidemiologic studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR.Methods. 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 Plasmodium species and compared our results to those of microscopy.Results. We analysed a total of 367 blood smears and corresponding red blood cell pellets that included 185 (50.4%) positive smears by microscopy. Compared to microscopy, PCR-HRM from smear DNA had a sensitivity of 93.0% (Confidence interval [CI]: 88.2-96.2) and a specificity of 96.7% (CI: 93.0-98.8), and PCR-HRM from pellet DNA had a sensitivity of 100.0% (CI: 98.0-100.0) and a specificity of 94.0% (CI: 89.4-96.9%). Speciation of positive PCR-HRM results revealed P. falciparum (92.0%), P. ovale (5.6%), and P. malariae (2.4%).Conclusion 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 Plasmodium species infection and be used for retrospective genetic studies.