New DNA Extraction Method for the Detection of Pneumocystis in Lung Tissue Samples of Colonized Individuals

Background: Pneumocystis jirovecii colonization in patients is associated with a low organism burden, which supports the need to use highly sensitive molecular techniques, such as nested-PCR to determine the presence of the organism. However, few studies have considered the effect of nucleic acid extraction methods on the detection of P. jirovecii. Here, we evaluate how pre-treatment affects microbial detection. Methods: Lung tissue samples from fifteen autopsied infants were processed using two different DNA extraction protocols: 1) the QIAamp DNA Mini kit (Qiagen) (method A) and the QIAamp DNA Mini kit (Qiagen) with additional steps including a pre-treatment, bead-beating and phenol-chloroform steps (method B). Detection of P. jirovecii was performed using the conventional nested-PCR in lung tissue samples. In addition, we estimated the bacterial, fungal and human DNA using qPCR method. Results: Higher DNA yields was obtained with DNA extraction method B compared to method A. P. jirovecii was detected in 10 out of 15 lung tissue samples for both methods at the first or second round of PCR. However, P. jirovecii was detected in 9 out of 10 at the first round with the DNA extraction method B, rather than those processed with DNA extraction method A that were detected in 5 out of 10. Moreover, the faint PCR band intensity observed in 2 out of 6 samples processed with DNA extraction method A was increased with method B. We also observed that DNA extraction method B increased significantly the bacterial and fungal DNA, but not the human DNA, compared to method A. Conclusions: Our findings show that the additional steps (i.e. pre-treatment, bead-beating and Phenol: Chloroform: Isoamyl alcohol steps) supplemented to the QIAamp DNA Mini kit (Qiagen) improves the detection of P. jirovecii in lung tissue samples.