Abstract
Problem/background : Pneumocystis jirovecii can be detected by staining methodsbut the sensitivity is low. Polymerase chain reaction (PCR)technique is more sensitive and accurate but it requires highquality of DNA template. Although DNA prepared by boilingmethod may not generate high quality of DNA, it has been usedfor extraction of DNA from several organisms such as malaria,several bacteria and fungi for PCR detection with favorableresults. Because boiling method is not time-consuming andinexpensive, we evaluated whether or not DNA of P. jiroveciicould be prepared from sputum samples and amplifiable bydiagnostic PCR. Results were also compared with those obtainedfrom a commercial kit and Giemsa staining method.Objective : To develop a rapid boiling method for detection of P. jirovecii.Design : A descriptive study.Setting : Department of Parasitology, Faculty of Medicine, ChulalongkornUniversityMaterials and Methods : An appropriate boiling time was determined for DNA preparationfrom 28 sputum samples from immunocompromised patientscollected from April to October 2006 at King ChulalongkornMemorial Hospital. DNA of these samples was also preparedusing a commercial kit. All samples were stained with Giemsafor comparison.Results : Boiling of sputum samples for 60 minutes gave positive PCRresults in 67.86% (n = 19) of samples (n = 28) whereas boilingfor either 30 or 90 minutes yielded positive results in 39.29%(n = 11). On the other hand, positive PCR results were obtainedfrom all 28 samples (100%) that were prepared by using acommercial kit. Although DNA prepared with a commercial kitwas superior to boiling method, it is more time-consuming andmore expensive. Meanwhile, Giemsa stain could detected P.jirovecii in sputum of 5 patients (18%).Conclusion : The sensitivity of PCR using DNA isolation by boiling method isfar superior to Giemsa stain. The lower sensitivity of PCR usingDNA prepared by boiling method than that by a commercial kitindicates that further improvement of the former method will berequired before it can be used for general diagnostic laboratory.
Publisher
Faculty of Medicine, Chulalongkorn University
First Page
459
Last Page
470
Recommended Citation
Tia, T; Pattanawong, U; Kongpolprom, N; and Putaporntip, C.
(2012)
"Evaluation of DNA extraction by boiling method for PCRbaseddetection of Pneumocystis jirovecii in sputumsamples from immunocompromised patients,"
Chulalongkorn Medical Journal: Vol. 56:
Iss.
4, Article 7.
Available at:
https://digital.car.chula.ac.th/clmjournal/vol56/iss4/7