Evaluation of the Amplex eazyplex® loop-mediated isothermal amplification assay for rapid diagnosis of Pneumocystis jirovecii pneumonia

J Clin Microbiol. 2020 Sep 16;JCM.01739-20. doi: 10.1128/JCM.01739-20. Online ahead of print.

Abstract

Quantitative polymerase chain reaction (qPCR) is the gold standard for diagnosis of Pneumocystis (P.) jirovecii pneumonia (PCP). However, it is laborious and requires skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex®-P. jirovecii assay (eazyplex®-PJA) uses loop-mediated isothermal amplification for detection of P. jirovecii It is performed directly with respiratory specimens without the need for special skills and delivers a result within 3-25 min.The goal of our study was to compare the performance of the eazyplex®-PJA with established P. jirovecii-qPCRs. All archived bronchoalveolar lavage fluids (BALFs) previously tested positive for P. jirovecii by qPCR and 50 control patients (retrospective part) as well as all BALFs received for P. jirovecii analysis over a period of 4 months (prospective part) were tested.49 patients with proven PCP and 126 patients without PCP were included. The sensitivity and specificity of the eazyplex®-PJA (95.7% and 96.5%, respectively) were comparable to three different P. jirovecii-qPCRs. The detection limit of the eazyplex®-PJA was analog to 103 copies of the major surface glycoprotein gene per 25 μl BALF, corresponding to 10-20 P. jirovecii cells. The eazyplex®-PJA reliably discriminated patients with PCP from patients with P. jirovecii colonization. It delivered a positive result within a mean of 9 min 38 sec and required a hands-on-time of 2 min 45 sec.In summary, the eazyplex®-PJA showed an identical performance for the diagnosis of PCP compared to qPCRs. However, in terms of time-to-result, practicability and robustness the eazyplex®-PJA is clearly superior and allows for around-the-clock molecular testing.