Ulrike Scharmann1, Lisa Kirchhoff1, Dirk Schmidt1, Jan Buer1, Joerg Steinmann1,2, Peter-Michael Rath1. 1. Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. 2. Institute of Clinical Hygiene, Medical Microbiology and Infectiology, Klinikum Nürnberg, Paracelsus Medical University, Nuremberg, Germany.
Abstract
BACKGROUND: Various tools are obtainable for the detection of Pneumocystis jirovecii, among them qPCR promising highest sensitivity. A novel molecular method is commercially available, the loop-mediated isothermal amplification (LAMP) assay. OBJECTIVES: We compared the performance of the LAMP eazyplex® Pneumocystis jirovecii with the RealStar Pneumocystis jirovecii PCR 1.0 qPCR. MATERIAL/ METHODS: Overall, 162 lower respiratory tract specimens from 146 critically ill patients were investigated. LAMP assay and qPCR were carried out according to the manufacturer's recommendations. Positive results of the LAMP were described as time to positivity (TTP). The limit of detection (LOD) of the LAMP was analysed using 10-fold serial dilutions of a high positive P jirovecii respiratory sample. For each serial dilution, TTP of the LAMP was plotted against cycle threshold (Ct) values of the qPCR. RESULTS: The LOD of the LAMP was determined to be approximately 4 × 103 copies/mL. While the LAMP revealed 28 (17%) positive signals from 20 patients, by using qPCR 41 (25%) positive samples from 28 patients were identified. Overall agreement with qPCR was 92%. Five false-negative, one false-positive and nine invalid results were detected by the LAMP. Positive and negative predictive values were 96% each, and sensitivity and specificity were 84% and 99%, respectively. There was a low correlation between the TTP and the fungal load. CONCLUSION: The LAMP is a time-saving and easy-to-perform method. It can be used as an alternative diagnostic method. However, for quantification purposes the qPCR is still the gold standard.
BACKGROUND: Various tools are obtainable for the detection of Pneumocystis jirovecii, among them qPCR promising highest sensitivity. A novel molecular method is commercially available, the loop-mediated isothermal amplification (LAMP) assay. OBJECTIVES: We compared the performance of the LAMP eazyplex® Pneumocystis jirovecii with the RealStar Pneumocystis jirovecii PCR 1.0 qPCR. MATERIAL/ METHODS: Overall, 162 lower respiratory tract specimens from 146 critically illpatients were investigated. LAMP assay and qPCR were carried out according to the manufacturer's recommendations. Positive results of the LAMP were described as time to positivity (TTP). The limit of detection (LOD) of the LAMP was analysed using 10-fold serial dilutions of a high positive P jirovecii respiratory sample. For each serial dilution, TTP of the LAMP was plotted against cycle threshold (Ct) values of the qPCR. RESULTS: The LOD of the LAMP was determined to be approximately 4 × 103 copies/mL. While the LAMP revealed 28 (17%) positive signals from 20 patients, by using qPCR 41 (25%) positive samples from 28 patients were identified. Overall agreement with qPCR was 92%. Five false-negative, one false-positive and nine invalid results were detected by the LAMP. Positive and negative predictive values were 96% each, and sensitivity and specificity were 84% and 99%, respectively. There was a low correlation between the TTP and the fungal load. CONCLUSION: The LAMP is a time-saving and easy-to-perform method. It can be used as an alternative diagnostic method. However, for quantification purposes the qPCR is still the gold standard.