Evaluation of DNA extraction by boiling method for PCRbased detection of Pneumocystis jirovecii in sputum samples from immunocompromised patients

Abstract

Problem/background : Pneumocystis jirovecii can be detected by staining methods but the sensitivity is low. Polymerase chain reaction (PCR) technique is more sensitive and accurate but it requires high quality of DNA template. Although DNA prepared by boiling method may not generate high quality of DNA, it has been used for extraction of DNA from several organisms such as malaria, several bacteria and fungi for PCR detection with favorable results. Because boiling method is not time-consuming and inexpensive, we evaluated whether or not DNA of P. jirovecii could be prepared from sputum samples and amplifiable by diagnostic PCR. Results were also compared with those obtained from 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, Chulalongkorn University

Materials and Methods : An appropriate boiling time was determined for DNA preparation from 28 sputum samples from immunocompromised patients collected from April to October 2006 at King Chulalongkorn Memorial Hospital. DNA of these samples was also prepared using a commercial kit. All samples were stained with Giemsa for comparison.

Results : Boiling of sputum samples for 60 minutes gave positive PCR results in 67.86% (n = 19) of samples (n = 28) whereas boiling for either 30 or 90 minutes yielded positive results in 39.29% (n = 11). On the other hand, positive PCR results were obtained from all 28 samples (100%) that were prepared by using a commercial kit. Although DNA prepared with a commercial kit was superior to boiling method, it is more time-consuming and more 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 is far superior to Giemsa stain. The lower sensitivity of PCR using DNA prepared by boiling method than that by a commercial kit indicates that further improvement of the former method will be required before it can be used for general diagnostic laboratory.