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. 2019 Sep;5(3):13-18.
doi: 10.18502/cmm.5.3.1741.

Molecular diagnosis and genotyping of Pneumocystis jirovecii in bronchoalveolar lavage samples obtained from patients with pulmonary disorder

Abdolmajid Fata  1   2 , Bahareh Abdollahi  1 , Fariba Rezaeetalab  3   4 , Davood Attaran  3   4 , Mohsen Najjari  1 , Mohammad J Najafzadeh  1   5
Affiliations

Molecular diagnosis and genotyping of Pneumocystis jirovecii in bronchoalveolar lavage samples obtained from patients with pulmonary disorder

Abdolmajid Fata et al. Curr Med Mycol. 2019 Sep.

Abstract

Background and purpose: Pneumocystis pneumonia (PCP) is one of the most common and life-threatening fungal diseases in patients with human immunodeficiency, treated with immunosuppressive medications. Immunocompetent people can also be a spreading agent for PCP. Regarding this, the aim of the present study was to diagnose and identify Pneumocystis jirovecii in bronchoalveolar lavage (BAL) samples obtained from patients with pulmonary disorder using a molecular method.

Materials and methods: For the purpose of the study, BAL samples (n=138) were collected from patients, undergoing bronchoscopy at the different departments of university hospitals affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, during a period of one year (i.e., April 2014 until May 2015). Giemsa staining and molecular identification were carried out for each sample. The samples were also subjected to nested polymerase chain reaction (PCR), sequencing, and genotyping based on mitochondrial ribosomal large subunit (mtLSU rRNA) of P. jirovecii. The phylogenic tree was constructed by MEGA6 software.

Results: The results of direct microscopic examination revealed the presence of P. jirovecii in 3 (2.2%) out of 138 samples; in addition, nested PCR and sequencing led to the detection of species in 17 (12.3%) samples. Out of patients with positive results, 10 (25%) and 7 (7.1%) cases were immunosuppressed and immunocompetent, respectively. The most common clinical symptoms among patients with pneumocystis were fever, dyspnea, and dry cough. In addition, genotypes III and II were the dominant genotypes in our dataset.

Conclusion: Nested PCR and sequencing methods showed higher sensitivity and specificity as compared with a direct staining technique. Genotype III was identified as the most dominant type in patients with pulmonary disorder in Mashhad.

Keywords: Bronchoalveolar Lavage (BAL); Iran; Nested-PCR; Pneumocystis jirovecii; Pneumocystis pneumonia (PCP).

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Conflict of interest statement

The authors declare no conflicts of interest. The authors are responsible for the content and writing of the paper.

Figures

Figure 1
Figure 1
Electrophoresis results of polymerase chain reaction products in agarose gel 1.2% (Fermentas marker 100 bp) (No 1 and 2 are positive and negative controls, respectively, M: Marker, the 260 bp band of the mtLSU rRNA gene was considered positive for P. jirovecii. (No 4 and 5) and no 3, 6, and 7 are negative cases.)
Figure 2
Figure 2
Maximum likelihood tree based on LSU sequence of Pneumocystis jirovecii (Pneumocystis carinii U20169.1 was used as outgroup. Boot strap values above 50% are shown on the branches. Genbank accession numbers are in front of the isolates. Bold isolates were selected from Genbank.)
Figure 3
Figure 3
Aligned Genbank accession number of Pneumocystis jirovecii mtLUS rRNA gene from the current study and Genbank (They have nucleotide variation at only position 85, confirming genotypes III, II, and I. They have a ‘C’ at position 248 and a ‘T’ at position 289 [not shown].)
See this image and copyright information in PMC

References

    1. Chazan R. Contemporary clinical diagnostics of respiratory tract infections. Pol merkur lekarski. 2011;30(179):316–9. - PubMed
    1. Cushion MT, Harmsen A, Matsumoto Y, Stringer JR, Wakefield AE, Yamada M. Recent advances in the biology of Pneumocystis carinii. J Med Vet Myc. 1994;32(suppl1):217–28. - PubMed
    1. Wakefield AE. DNA sequences identical to Pneumocystis carinii f carinii and Pneumocystis carinii f sp hominis in samples of air spora. J Clin Microbiol. 1996;34(7):1754–9. - PMC - PubMed
    1. Respaldiza N, Medrano F, Medrano A, Varela J, De La Horra C, Montes‐Cano M, et al. High seroprevalence of Pneumocystis infection in Spanish children. Clin Microbiol Infect. 2004;10(11):1029–31. - PubMed
    1. Lowe DM, Rangaka MX, Gordon F, James CD, Miller RF. Pneumocystis jirovecii pneumonia in tropical and low and middle income countries: a systematic review and meta-regression. PloS One. 2013;8(8):e69969. - PMC - PubMed

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