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

Abstract

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 Plasmodium infections or for epidemiological 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. 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 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 Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (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 Plasmodium species infections and can be used for retrospective genetic studies.

Keywords: PCR; diagnostics; high-resolution melting; malaria.

FIG 1

Comparison of melting profiles for controls and patient samples. (A) Melt peaks of control plasmids used to determine malaria species. (B) Melting curves generated from nucleic acids derived from blood pellets. The data shown were generated by taking the negative of the first derivative of the raw melting data (y axis) derived from each control plasmid and plotting it against temperature (x axis). The fluorescence threshold (solid blue line) was used as a noise filter.

FIG 2

Comparison of melting profiles for controls, patient samples, and cultured parasites. (A) Melting curves for P. malariae and P. falciparum control plasmids and patient samples. Note the double peak shown by the P. falciparum sample. (B) Melting curves for the same P. malariae and P. falciparum control plasmids, compared to the melting curve generated using a laboratory-cultured sample of P. falciparum. The double peak displayed by P. falciparum samples is therefore diagnostic of the species, rather than indicating double infections in patient samples.

FIG 3

Alignment of 18S rRNA sequences for P. falciparum (Pf) and P. malariae (Pm). The PCR products from the four 18S RNA alleles predicted from the P. falciparum genome were aligned with the 18S RNA product from P. malariae obtained from control plasmid sequences, using Geneious 9.1.5 (24). The total product sizes are between 214 and 225 bp. The level of identity between P. falciparum chromosomes 1 and 13 is 97.8% (estimated melting temperature is 71.9°C), that between P. falciparum chromosomes 5 and 7 is 100% (estimated melting temperature is 73.4°C), that between P. falciparum chromosome 1 and 13 and P. malariae is 75.4%, and that between P. falciparum chromosomes 5 and 7 and P. malariae is 72.3%. The estimated P. malariae melting temperature is 71.8°C. Discordant bases are highlighted.