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Evaluation of analyte-specific reagents for the direct detection of Pneumocystis jirovecii
Samantha R Giffen1, Elizabeth Stoeppler1, Avian Elliott1
1McLendon Clinical Laboratories, University of North Carolina Medical Center, Chapel Hill, North Carolina, USA.
Abstract:
Pneumocystis jirovecii pneumonia (PJP) is a serious and sometimes fatal infection occurring in immunocompromised individuals. High-risk patients include those with low CD4 counts due to human immunodeficiency virus infection and transplant recipients. The incidence of PJP is increasing, and rapid detection of PJP is needed to effectively target treatment and improve patient outcomes. A common method used is an immunofluorescent assay (IFA), which has limitations, including labor costs, low sensitivity, and requirement for expert interpretation. This study evaluates the performance of the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) in a laboratory-developed test (LDT) for the direct detection of P. jirovecii DNA without prior nucleic acid extraction. Respiratory samples (n = 135) previously tested by IFA from 111 patients were included. Using a composite standard of in-house IFA and reference lab PJP PCR, the percent positive agreement for the LDT using the DiaSorin ASR was 97.8% (90/92). The negative percent agreement was 97.7% (42/43). The lower limit of detection of the assay was determined to be 1,200 copies/mL in bronchoalveolar lavage fluid. Analytical specificity was assessed using cultures of oropharyngeal flora and common respiratory bacterial and fungal pathogens. No cross-reactivity was observed. Our study suggests that the DiaSorin Pneumocystis ASR accurately detects P. jirovecii DNA and demonstrates improved sensitivity compared to the IFA method.
Importance:
Our study is unique compared to other previously published studies on the DiaSorin analyte-specific reagent (ASR) because we focused on microbiological diagnostic methods commonly used (immunofluorescent assay) as opposed to pathology findings or reference PCR. In addition, in our materials and methods, we describe the protocol for the use of the DiaSorin ASR as a singleplex assay, which will allow other users to evaluate the ASR for clinical use in their lab.
Insights
This study shows the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) accurately detects P. jirovecii DNA. This laboratory-developed test (LDT) offers improved sensitivity over traditional immunofluorescent assays for diagnosing Pneumocystis jirovecii pneumonia (PJP).
Area of Science:
- Clinical Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Pneumocystis jirovecii pneumonia (PJP) is a severe infection in immunocompromised individuals, with increasing incidence.
- Current detection methods like immunofluorescent assay (IFA) have limitations including low sensitivity and high labor costs.
- Rapid and accurate PJP diagnosis is crucial for timely treatment and improved patient outcomes.
Purpose of the Study:
- To evaluate the performance of the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) as a laboratory-developed test (LDT).
- To assess the direct detection of P. jirovecii DNA without prior nucleic acid extraction.
- To compare the diagnostic accuracy and sensitivity of the DiaSorin ASR LDT against established methods like IFA and PCR.
Main Methods:
- Respiratory samples (n=135) from 111 patients, previously tested by IFA, were analyzed using the DiaSorin ASR in an LDT.
- A composite standard including in-house IFA and reference laboratory PJP PCR was used for performance evaluation.
- The assay's lower limit of detection (LLOD) and analytical specificity against common respiratory pathogens were determined.
Main Results:
- The DiaSorin ASR LDT demonstrated high diagnostic agreement: 97.8% positive agreement and 97.7% negative agreement.
- The lower limit of detection was established at 1,200 copies/mL in bronchoalveolar lavage fluid.
- No cross-reactivity was observed when tested against common respiratory flora and pathogens, indicating strong analytical specificity.
Conclusions:
- The DiaSorin Molecular P. jirovecii ASR, when used in an LDT, accurately detects P. jirovecii DNA directly from respiratory samples.
- The assay shows improved sensitivity compared to the traditional immunofluorescent assay (IFA) method.
- This LDT offers a valuable tool for rapid and sensitive PJP diagnosis, potentially improving patient management.
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