A simple and efficient DNA extraction from respiratory samples for PCR detection of Pneumocystis jirovecii

Abstract

Background : Pneumonia caused by Pneumocystis jirovecii (PCP) is a leading cause of morbidity and mortality in immunocompromised individuals. Although microscopic detection of stained clinical samples and immunofluorescence assay (IFA) have been widely used for diagnosis of P. jirovecii infections, polymerase chain reaction (PCR)-based detection of P. jirovecii provides a higher diagnostic performance. DNA preparation by simple boiling method could be useful for PCR detection albeit the sensitivity remained remarkably low.

Objective : To refine P. jirovecii DNA preparation by boiling method in order to improve the diagnostic yield of subsequent PCR detection.

Methods : Two induced sputum and 4 bronchoalveolar lavage (BAL) samples from PCP patients were recruited for initial assessment of DNA extraction by boiling for 10, 20, 30, 60 and 90 minutes. Performance of each DNA
preparation was determined by dilution of samples and tested by P. jiroveciispecific nested PCR. Evaluation of the best boiling time for DNA extraction was done with 51 paired sputum and BAL samples from PCP patients using
data from a commercial kit as references.

Results : Boiling of samples for 20 minutes gave the best nested PCR results. Application of DNA extraction of 51 paired sputum and BAL samples by boiling for 20 minutes offered 92.16% and 96.08% sensitivity, respectively;
both yielded 100% specificity.

Conclusion : DNA of P. jirovecii could be efficiently extracted from BAL fluids and sputum samples by boiling for 20 minutes, almost comparable to that obtained from a commercial DNA extraction kit.