Evaluation of the Performance of a Multiplex Real-Time PCR Assay for the Identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii Simultaneously from Sputum in Multicenter

Abstract

Purpose: To evaluate the performance of a multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii from sputum.

Patients and methods: Sputum samples (n=537) from patients with suspected invasive fungal infection (IFI) were collected from four centers; they were tested by both multiplex real-time PCR assay and DNA sequencing. DNA sequencing was considered as the reference method, and the performance of the multiplex real-time assay was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The interference experiment, repeatability, reproducibility, and stability of the multiplex real-time PCR assay were also evaluated.

Results: The detection performance of the multiplex real-time assay, compared with that of DNA sequencing, for the three pathogens was as follows: sensitivity, specificity, PPV, and NPV for Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were 99.40%, 98.64%, 97.09%, and 99.73%; 100%, 99.59%, 96.36%, and 100.00%; and 99.28%, 98.50%, 95.80%, and 99.75%, respectively. The consistency of the two methods was almost perfect: the kappa value was between 0.97 and 0.98. The minimum detection limit of the multiplex real-time assay for each of the three pathogens was 1250 copies/mL. Interference experiment showed that blood and normally used antifungal drugs had no effect on the results. No cross-reactivity was detected for any bacteria or fungi. In 40 patients, mixed infections by Aspergillus and/or Cryptococcus neoformans and/or Pneumocystis jirovecii were detected by the multiplex real-time assay. Among these patients, those with acquired immune deficiency syndrome (AIDS) ranked first, with Aspergillus and Pneumocystis mixed infection accounting for 75%.

Conclusion: The multiplex real-time PCR assay is fast, sensitive, and specific and has good clinical application prospects.

Keywords: Aspergillus; Cryptococcus neoformans; Pneumocystis jirovecii; invasive fungal infection; multiplex real-time PCR; sequencing.

Figure 1

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Figure 1

PCR amplification curves of simultaneous positive for two or three pathogens. (A) Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were all positive. (B) Aspergillus, and Cryptococcus neoformans were both positive, Pneumocystis jirovecii was negative. (C) Aspergillus and Pneumocystis jirovecii were both positive, Cryptococcus neoformans was negative. (D) Cryptococcus neoformans and Pneumocystis jirovecii were both positive, Aspergillus was negative. PCR channel: Pneumocystis jirovecii: CY5; Aspergillus: FAM; Internal control: ROX; Cryptococcus neoformans: VIC.