Evaluation of the Amplex eazyplex Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Pneumocystis jirovecii Pneumonia

Quantitative PCR (qPCR) assays are the gold standard for diagnosis of pneumonia (PCP). However, they are laborious and require skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex assay (PJA) uses loop-mediated isothermal amplification for detection of It is performed directly with respiratory specimens, without the need for special skills, and delivers a result within 3 to 25 min. The goal of our study was to compare the performance of the eazyplex PJA with that of established qPCR assays. All archived bronchoalveolar lavage fluid (BALF) samples that had previously tested positive for by qPCR assay and 50 control samples (retrospective part), as well as all BALF samples received for analysis over a period of 4 months (prospective part), were tested. Forty-nine 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 those for three different qPCR assays. The detection limit of the eazyplex PJA was analogous to 10 copies of the major surface glycoprotein gene per 25 μl of BALF, corresponding to 10 to 20 cells. The eazyplex PJA reliably discriminated patients with PCP from patients with colonization. It delivered a positive result within a mean of 9 min 38 s and required a hands-on time of 2 min 45 s. In summary, the eazyplex PJA showed identical performance for the diagnosis of PCP, compared to qPCR assays. However, in terms of time to result, practicability, and robustness, the eazyplex PJA is clearly superior and allows for around-the-clock molecular testing.