Download Clinical Evaluation of RIDA®GENE Pneumocystis jirovecii

Clinical Evaluation of RIDA®GENE Pneumocystis jirovecii for the qualitative detection of Pneumocystis jirovecii
from human bronchoalveolar lavage fluid
R. Wurlitzer1, K. Beyser1, A. Lindauer2, L. Kastl3, A. Simons3
synlab Medizinisches Versorgungszentrum Weiden GmbH, Weiden, Germany
Labor Team w AG, Goldach, Switzerland
3
R-Biopharm AG, Darmstadt, Germany
1
2
Introduction:
Pneumocystis pneumonia (PCP), is an important opportunistic infection in immunocompromised patients like HIV/AIDS patients, chemotherapy-treated patients and patients receiving an organ transplant. According to the Centers
For Disease Control and Prevention (CDC), an infection with Pneumocystis jirovecii causes 100 % mortality in patients without treatment and the mortality rate in immunocompromised patients is between 5 – 40 % in treated patients.1 The mortality rate due to Pneumocystis jirovecii in HIV-uninfected patients
can be as high as 40 %.2 Hence, fast and sensitive detection of Pneumocystis jirovecii in patients is required. Detection by quantitative real-time PCR
using multi-copy targets enables a fast diagnosis and implementation of appropriate treatment measures. In this study, the RIDA®GENE Pneumocystis jirovecii real-time PCR assay for the qualitative, direct detection of Pneumocystis jirovecii from human bronchoalveolar lavage fluid (BAL) was evaluated.
Methods:
RIDA®GENE Pneumocystis jirovecii is a qualitative and quantitative assay targeting the mitochondrial large subunit (LSU) of Pneumocystis jirovecii with
fluorogenic target-specific hydrolysis probes. An included Internal Control
DNA (ICD) detects PCR inhibition, monitors reagent integrity and confirms
that nucleic acid extraction was sufficient and hence ensures reliable results.
Evaluation of the RIDA®GENE Pneumocystis jirovecii assay was performed with
203 prospectively collected BAL samples. DNA extraction was either performed
on the Chemagic Magnetic Separation Modul I (Chemagen) with the Chemagic DNA/RNA Kit or on King-Fisher Flex (Thermo Scientific) with the Innu-PrepBlood Kit DNA Kfflx (Figure 2). Extracted BAL specimens were analyzed on the
LightCycler® 2.0 (Roche) with the RIDA®GENE Pneumocystis jirovecii real-time
PCR assay and clinical performance was compared to a routine in-house realtime PCR assay which targets another multi-copy target of Pneumocystis jirovecii.
BAL specimen
DNA extraction (Chemagic Magnetic Separation Modul I or King-Fisher Flex)
Real-time PCR (LightCycler® 2.0)
In-house real-time PCR
RIDA®GENE Pneumocystis jirovecii
Figure 2: Study design
Figure 1: RIDA®GENE Pneumocystis jirovecii real-time PCR
Results:
Overall, 198 tested samples (97.5 %) showed concordant results with both
real-time PCR methods, where 28 samples were found to be positive and
170 samples were found to be negative (Figure 3). In addition, three samples showed a positive result with the in-house real-time PCR method but
were negative with the RIDA®GENE Pneumocystis jirovecii real-time PCR
assay. All three samples had a Ct value > 35 with the in-house real-time PCR
which is at the limit of detection (LoD) of the RIDA®GENE Pneumocystis jirovecii
assay (Figure 3). Conversely, also two samples were negatively identified by
the in-house real-time PCR but showed a positive result with the RIDA®GENE
Pneumocystis jirovecii assay (Figure 3). Compared to the in-house real-time
PCR, positive agreement and negative agreement of the RIDA®GENE Pneumocystis jirovecii assay was 91,8 % and 98,6 %, respectively (Table 1).
Figure 3: In-house real-time PCR vs. RIDA®GENE Pneumocystis jirovecii
Table 1: Clinical performance of the RIDA®GENE Pneumocystis jirovecii
real-time PCR assay
Positive agreement:
91,8 %
Negative agreement:
98,6 %
Overall Agreement:
97.5 %
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Figure 4: Example of Pneumocystis jirovecii run on the LightCycler® LC2.0
Conclusion:
The RIDA®GENE Pneumocystis jirovecii real-time PCR assay shows good correlation with an established in-house real-time PCR method. Three discrepant
samples were near the limit of detection (LoD) of the RIDA®GENE Pneumocystis
jirovecii real-time PCR, however samples above the LoD are of most clinical relevance. The RIDA®GENE Pneumocystis jirovecii assay proved to be a sensitive
and specific real-time PCR assay for the diagnosis of pneumocystosis. Results
are available in less than 2 hours enabling implementation of timely treatment
measures. The RIDA®GENE Pneumocystis jirovecii assay also contains three
DNA standards that allow direct quantification of the amount of Pneumocystis
jirovecii present in a positive sample as well as monitoring treatment success.
References:
1
Centers for Disease Control and Prevention. Pneumocystis pneumonia
Statistics 2012.
2
Krajicek, B.J., Thomas, C.F. Jr., Limper, A.H. (2009) Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin
Chest Med 30:265-278.