Evaluation of Real-Time Polymerase Chain Reaction in Pneumocystis jirovecii Laboratory Diagnosis


MIKROBIYOLOJI BULTENI, vol.54, no.3, pp.418-428, 2020 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 54 Issue: 3
  • Publication Date: 2020
  • Doi Number: 10.5578/mb.69711
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, EMBASE, MEDLINE, TR DİZİN (ULAKBİM)
  • Page Numbers: pp.418-428
  • Keywords: Pneumocystis jirovecii, real-time polymerase chain reaction, nested-PCR, microscopy, PCR ASSAY, QUANTITATIVE PCR, PNEUMONIA, COLONIZATION, INFECTION, PREVALENCE, MANAGEMENT, LAVAGE, ERA
  • Dokuz Eylül University Affiliated: Yes


Pneumocystis jirovecii is a human-specific species and causes fatal infections like P. jirovecii pneumonia (PCP) in immunocompromised persons. Although direct microscopy is the gold standard in the diagnosis of the microorganism, molecular methods such as polymerase chain reaction (PCR) are needed in non-human immune deficiency virus (HIV) immunosuppresive patients with low P.jirovecii burden. In this study, we aimed to evaluate the value of real-time PCR (Rt-PCR) in the laboratory diagnosis of P. jirovecii. Bronchoalveolar lavage (BAL) specimens of 658 patients sent to Dokuz Eylul University Hospital Central Medical Parasitology Laboratory on suspicion of PCP were included in the study. BAL fluids were evaluated for identification of P. jirovecii mitochondria! gene coding ribosomal large subunit (mtLSUrRNA) using Rt-PCR. In addition, Giemsa and Gomori's methenamine silver (GMG) staining assays were applied to all samples and nested PCR (n-PCR) assay was applied to positive samples detected by real time PCR. Ninety-two (14.3%) of these samples were positive by Rt-PCR. Of these 92 patients, 85 (92.4%) were positive with n-PCR. Only seven of the specimens had P. jirovecii cysts and trophozoites with microscopic examination. The mean cycle threshold (C-T) value of Rt-PCR positive patients was 29.7 (18.17 <= C-T <= 37.96). P.jirovecii load in these patients was calculated as 2.6 x 10(1)-6.15 x 10(7) copies/ml. The difference between the mean C-T values of n-PCR positive and negative results was statistically significant (p< 0.01). The C-T values of Rt-PCR of the samples with positive microscopy were; 18.2, 20.9, 22.2, 24.3, 24.7, 26.5, 29.7. The difference between the C-T means of the samples with positive and negative microscopy was statistically significant (p< 0.05). When positive patients were grouped according to their diagnosis; the lowest mean C-T value (C-Tmean = 24.8) was found in HIV-positive patients. On the other hand, C-r values were found to be significantly lower in the organ transplantation patients (C-Tmean = 26.15) and in the collagen-vascular-inflammatory patient group (C-Tmean = 27.8). This study demonstrated that Rt-PCR was the effective method in the diagnosis of P.jirovecii in the laboratory. Conventional n-PCR method was found to be more unsuccessful than Rt-PCR in the presence of very low density organism; direct microscopy is generally found to be positive in samples with a higher burden of P.jirovecii.