Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of thick blood films (TBF) as specimens for DNA amplification with the <it>Plasmodium </it>species-specific real-time PCR that was recently validated on whole blood samples.</p> <p>Methods</p> <p>The panel of 135 Giemsa-stained clinical TBFs represented single infections of the four <it>Plasmodium </it>species with varying parasite densities or only gametocytes, mixed infections, and negative samples and was stored for up to 12 years. Half of the Giemsa-stained TBF was scraped off by a sterile scalpel and collected into phosphate buffered saline. DNA was extracted with the Qiagen DNA mini kit with minor modifications. DNA was amplified with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species with four species-specific primers and probes in combination with one genus-specific reverse primer. Results of the PCR on TBF were compared to those of the PCR on whole blood and to microscopy.</p> <p>Results</p> <p>Correct identification for single species infections was obtained for all TBF samples with <it>Plasmodium falciparum </it>(n = 50), <it>Plasmodium vivax </it>(n = 25), <it>Plasmodium ovale </it>(n = 25) and in all but one samples with <it>Plasmodium malariae </it>(n = 10). Compared to whole blood samples, higher Ct-values were observed by PCR on TBF with a mean difference of 5.93. Four out of five mixed infections were correctly identified with PCR on TBF. None of the negative samples (n = 20) gave a PCR signal. PCR on TBF showed a detection limit of 0.2 asexual parasites/μl compared to 0.02/μl for whole blood. Intra-run variation was higher for PCR on TBF (%CV 1.90) compared to PCR on whole blood (%CV 0.54). Compared to microscopy, PCR on TBF generated three more species identifications in samples containing a single species and detected the same four mixed-infections.</p> <p>Conclusions</p> <p>Giemsa-stained TBFs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR analysis, allowing applications in reference and research settings in case whole blood samples are not available.</p