Detection Of Pneumocystis Jirovecii In The Respiratory Samples From Non-pneumocystis Pneumonia Patients

Objective To investigate Pneumocystis jirovecii(Pj) specific DNA fragments from the non-Pneumocystis Pneumonia(non-PCP) patients’ respiratory samples.Methods To extract DNA temples from the definite diagnosed pneumocystis pneumonia patients’ respiratory samples. Pj mitochondrial large subunit r RNA(mt LSUr RNA) gene and major surface glycoprotein(Msg) gene were selected as target genes to design primers. PCR amplification products were cloned and sequenced. After compared with National Center for Biotechnology Information(NCBI) gene bank, mt LSUr RNA and Msg positive standard DNA samples were obtained. By using the conventional PCR, nested PCR(n PCR) and quantitative PCR(q PCR) methods, Pj mt LSUr RNA gene was selected as target gene for conventional PCR(Mt-PCR) and nest PCR(Mt-n PCR). Pj Msg gene was selected as another target gene for conventional PCR(Msg-PCR). Respiratory samples of non-PCP patients were enrolled for investigation. Pharyngolaryngeal aspiration fluid samples and bronchial alveolar lavage fluid(BALF) samples were collected separately. An international PCP diagnostic criteria was selected as diagnostic standard to make sure of the non-PCP patients were enrolled. The non-PCP patients’ respiratory samples screened positive by PCR were further investigated by q PCR targeted mt LSUr RNA and Msg genes.Result The clinic Pj Msg sequences from 57 clones were 55-100% identical to the corresponding region of reference Pj Msg A gene(Gen Bank AAL67137). The clinic Pj mt LSUr RNA sequences were 99% identical to the corresponding region of reference Pj mt LSUr RNA gene(Gen Bank JX855937.1). In the 108 pharyngolaryngeal aspiration fluid samples, the positive rates of Mt-n PCR, Msg-PCR and Mt-PCR were 41.7%(45/108), 15.7%(17/108), and 4.6%(5/108) respectively. Among these, all of the Mt-PCR, Mt-n PCR, and Msg-PCR positive was 1 case(1%), any two of the three tests positive were 11 cases(10%), any one of the three tests positive were 42 cases(39%), and all of the three tests negative were 54 cases(50%). Among the 54 positive cases, 90.7% cases which the copy numbers of mt LSUr RNA gene tested by Mt-q PCR method were from 103 copies/μl to 104 copies/μl. Simultaneously, 90.7% cases which the copy numbers of Msg gene tested by Msg-q PCR method were from 103copies/μl to 106 copies/μl. There were no significant differences in gender comparison(χ2 were 0.017, 0.01 and 2.131, all P>0.05). There were no significant differences in age comparison(χ2 were 0.988, 2.350 and 2.514, all P>0.05). In the 50 BALF samples, the positive rates of Mt-n PCR, Msg-PCR and Mt-PCR were 56%(28/50), 26%(13/50), and 36%(18/50) respectively. Among these, all of the Mt-PCR, Mt-n PCR, and Msg-PCR positive was 5 case(10%), any two of the three tests positive were 13 cases(26%), any one of the three tests positive were 18 cases(36%), and all of the three tests negative were 14 cases(28%). Among the 36 positive cases, 100% cases which the copy numbers of mt LSUr RNA gene tested by Mt-q PCR method were from 103 copies/μl to 104 copies/μl. Simultaneously, 91.7% cases which the copy numbers of Msg gene tested by Msg-q PCR method were from 103copies/μl to 105copies/μl. There were no significant differences in gender comparison(χ2 were 0.253, 0.402 and 0.573, all P>0.05). There were no significant differences in age comparison(χ2 were 0.879, 1.203 and 0.387, all P>0.05).Conclusion In the non-PCP patients, the Pj positive rates were 50% in pharyngolaryngeal aspiration fluid samples, and 72% in the BALF samples. The Mt-q PCR copy numbers were mainly distributed in the level of 103 copies/μl. The Msg-q PCR copy numbers were mainly distributed in the levels of 103, 104, and 105 copies/μl. It must be very careful in interpreting the clinic significance of Pj positive results tested by nucleic acid amplification methods.