Investigation of Pneumocystis jirovecii colonization in patients with chronic pulmonary diseases in the People's Republic of China

Abstract

Background: The detection of Pneumocystis jirovecii DNA in respiratory specimen from individuals who do not have signs or symptoms of pneumonia has been defined as colonization. The role of P. jirovecii colonization in the development or progression of various lung diseases has been reported, but little information about P. jirovecii colonization in patients is available in the People's Republic of China.

Objective: To determine the prevalence of P. jirovecii colonization in patients with various pulmonary diseases, including the acute and stable stage of COPD, interstitial lung diseases, cystic fibrosis, and chronic bronchiectasis.

Materials and methods: A loop-mediated isothermal amplification (LAMP) and a conventional polymerase chain reaction (PCR) method for detecting P. jirovecii were developed. Ninety-eight HIV-negative patients who were followed-up and who had undergone bronchoscopy for diagnosis of various underlying respiratory diseases were included in the study. Sputa of these patients were analyzed with LAMP amplification of P. jirovecii gene. In addition, conventional PCR, Giemsa and Gomori's methenamine silver nitrate staining assays were applied to all specimens.

Results: The sensitivity and specificity test showed that there was no cross-reaction with other fungi or bacteria in detecting the specific gene of P. jirovecii by LAMP, and the minimum detection limits by LAMP was 50 copies/mL. P. jirovecii DNA was detected in 62 of 98 (63.3%) sputa specimens by LAMP assay and 22.45% (22/98) by conventional PCR. However, no P. jirovecii cysts were found by Giemsa and Gomori's methenamine silver nitrate in all of gene-positive specimens.

Conclusion: The results of our study showed that prevalence of P. jirovecii colonization is particularly high in patients with chronic pulmonary diseases in the People's Republic of China, and the LAMP method is better for evaluation of the colonization of P. jirovecii in sputum specimen than conventional PCR.

Keywords: Pneumocystis jirovecii; chronic pulmonary diseases; colonization; loop-mediated isothermal amplification.

Figure 1

Sensitivity of LAMP method compared with PCR. Notes: Detection limit of LAMP or PCR assays were performed using serial tenfold dilutions of the Pneumocystis plasmid. (A) LAMP method: line 1, ×10⁹ copies/mL; line 2, ×10⁸ copies/mL; line 3, ×10⁷ copies/mL; line 4, ×10⁶ copies/mL; line 5, ×10⁵ copies/mL; line 6, ×10⁴ copies/mL; line 7, ×10³ copies/mL; line 8, ×10² copies/mL; line 9, 50 copies/mL; line 10, 10 copies/mL. (B) PCR method: lane M, 100 bp DNA marker; lane 1, ×10⁹ copies/mL; lane 2, ×10⁸ copies/mL; lane 3, ×10⁷ copies/mL; lane 4, ×10⁶ copies/mL; lane 5, ×10⁵ copies/mL; lane 6, ×10⁴ copies/mL; lane 7, ×10³ copies/mL; lane 8, ×10² copies/mL; lane 9, 50 copies/mL; lane 10, 10 copies/mL. Abbreviations: LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction.

Figure 2

Examination of the products of LAMP for detecting the Pneumocystis jirovecii gene. Notes: The products of LAMP were examined by three methods. (A) By real-time turbidity; (B) by either the naked eye or ultraviolet light after adding SYBR Green I; (C) by gel electrophoresis. (A and B) 1, lung of PCP positive rat; 2, sputum specimen of patient; 3, lung of PCP negative rat. (C) Lane M, 2,000 bp DNA marker; lane 1, lung of PCP positive rat; lane 2 and lane 3, sputum specimen of patients; lane 4, lung of PCP negative rat. Abbreviations: LAMP, loop-mediated isothermal amplification; PCP, Pneumocystis pneumonia.