Chulalongkorn Medical Journal


Background : Pneumonia caused by Pneumocystis jirovecii (PCP) is a leading cause ofmorbidity and mortality in immunocompromised individuals. Althoughmicroscopic detection of stained clinical samples and immunofluorescenceassay (IFA) have been widely used for diagnosis of P. jirovecii infections,polymerase chain reaction (PCR)-based detection of P. jirovecii provides ahigher diagnostic performance. DNA preparation by simple boiling methodcould be useful for PCR detection albeit the sensitivity remained remarkablylow.Objective : To refine P. jirovecii DNA preparation by boiling method in order to improvethe diagnostic yield of subsequent PCR detection.Methods : Two induced sputum and 4 bronchoalveolar lavage (BAL) samples fromPCP patients were recruited for initial assessment of DNA extraction byboiling for 10, 20, 30, 60 and 90 minutes. Performance of each DNApreparation was determined by dilution of samples and tested by P. jiroveciispecificnested PCR. Evaluation of the best boiling time for DNA extractionwas done with 51 paired sputum and BAL samples from PCP patients usingdata 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 byboiling 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 andsputum samples by boiling for 20 minutes, almost comparable to thatobtained from a commercial DNA extraction kit.



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