Multicentric prospective evaluation of <i>Pneumocystis jirovecii</i> fungal load in bronchoalveolar lavage fluid fractions using qPCR

Bronchoalveolar lavage fluid (BALF) is the key sample type for diagnosing Pneumocystis jirovecii pneumonia, with quantitative PCR (qPCR) providing high sensitivity and specificity. However, sample processing varies considerably between laboratories, and optimal nucleic acid extraction method for BALF remains undetermined. This retrospective multicenter study, conducted in 12 centers as part of the fungal PCR initiative, assessed the efficacy of P. jirovecii detection by qPCR in different BALF fractions, including whole (WHO), pellet (PEL), and supernatant (SUP). Samples that were P. jirovecii-qPCR-positive during routine testing were divided into the three predefined fractions prior to nucleic acid extraction and qPCR, comparing detection rates and quantification cycle (Cq) values. Out of 113 P. jirovecii-qPCR-positive BALF samples, 339 qPCR measurements were analyzed. The PEL fraction demonstrated a similar detection rate to the WHO fraction, with positivity rates of 92.9% and 88.5%, respectively. The SUP fraction showed a lower positivity rate of 71.7%, dropping to 47% for high Cq values (Cq > 35). Quantitative analysis showed that the SUP fraction consistently yielded higher Cq values, trailing by 3.05 cycles compared to WHO, while PEL showed a smaller deviation (0.49 cycles), confirming its efficiency in retaining P. jirovecii genetic material for qPCR detection. The study concludes that the SUP fraction is suboptimal for P. jirovecii detection due to higher Cq values, suggesting lower fungal loads. The PEL and WHO fractions are comparable, suggesting that the PEL is a viable alternative, permitting the concentration of larger BALF volumes to levels that can be extracted across a range of platforms.

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Publication Details

Journal
Medical Mycology
Published
2025-02-28
DOI
https://doi.org/10.1093/mmy/myaf023
Citations
3
Primary Topic
Pneumocystis jirovecii pneumonia detection and treatment
Type
article
Field-Weighted Citation Impact
4.06
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article

Multicentric prospective evaluation of Pneumocystis jirovecii fungal load in bronchoalveolar lavage fluid fractions using qPCR

Anne Totet, Catriona L Halliday, Rosemary A. Barnes, P. Lewis White et al.
3 citations
Medical Mycology
Pneumocystis jirovecii pneumonia detection and treatment
4.06
article

Multicentric prospective evaluation of Pneumocystis jirovecii fungal load in bronchoalveolar lavage fluid fractions using qPCR

Anne Totet, Catriona L Halliday, Rosemary A. Barnes, P. Lewis White, Joerg Steinmann, Guillaume Désoubeaux, Stefan Fuchs, Mario Cruciani, Lize Cuypers, Théo Ghelfenstein-Ferreira, Solène Le Gal, Alexandre Alanio, J. Peter Donnelly, Sean X. Zhang, Anaïs Pinto, Vicente Friaza, Ulrike Scharmann, Juergen Loeffler
article en
3 citations

Abstract

Bronchoalveolar lavage fluid (BALF) is the key sample type for diagnosing Pneumocystis jirovecii pneumonia, with quantitative PCR (qPCR) providing high sensitivity and specificity. However, sample processing varies considerably between laboratories, and optimal nucleic acid extraction method for BALF remains undetermined. This retrospective multicenter study, conducted in 12 centers as part of the fungal PCR initiative, assessed the efficacy of P. jirovecii detection by qPCR in different BALF fractions, including whole (WHO), pellet (PEL), and supernatant (SUP). Samples that were P. jirovecii-qPCR-positive during routine testing were divided into the three predefined fractions prior to nucleic acid extraction and qPCR, comparing detection rates and quantification cycle (Cq) values. Out of 113 P. jirovecii-qPCR-positive BALF samples, 339 qPCR measurements were analyzed. The PEL fraction demonstrated a similar detection rate to the WHO fraction, with positivity rates of 92.9% and 88.5%, respectively. The SUP fraction showed a lower positivity rate of 71.7%, dropping to 47% for high Cq values (Cq > 35). Quantitative analysis showed that the SUP fraction consistently yielded higher Cq values, trailing by 3.05 cycles compared to WHO, while PEL showed a smaller deviation (0.49 cycles), confirming its efficiency in retaining P. jirovecii genetic material for qPCR detection. The study concludes that the SUP fraction is suboptimal for P. jirovecii detection due to higher Cq values, suggesting lower fungal loads. The PEL and WHO fractions are comparable, suggesting that the PEL is a viable alternative, permitting the concentration of larger BALF volumes to levels that can be extracted across a range of platforms.

Medical MycologyVol. 63(3)
Rockefeller Foundation (US), Innsbruck Medical University (AT), Johns Hopkins University (US), Inserm (FR), Institut Pasteur (FR), Université de Bretagne Occidentale (FR), Universität Innsbruck (AT), Université Paris Cité (FR), Rega Institute for Medical Research (BE), Paracelsus Medical University (AT), Johns Hopkins Medicine (US), Fairfield Infectious Diseases Hospital (AU), University Hospital of Wales (GB), Westmead Hospital (AU), Public Health Wales (GB), Centre Hospitalier Universitaire Amiens-Picardie (FR), Assistance Publique – Hôpitaux de Paris (FR), Hôpital Bretonneau (FR), Nuremberg Hospital (DE), Institut de Myologie (FR), Paracelsus Medizinische Privatuniversität (DE), Universitätsklinikum Würzburg (DE), Centre Hospitalier Régional Universitaire de Brest (FR), Instituto de Biomedicina de Sevilla (ES), Hôpital Saint-Louis (FR), Westmead Institute for Medical Research (AU), Hospital Universitario Virgen del Rocío (ES), Centre d'Étude des Pathologies Respiratoires (FR), Agents infectieux, résistance et chimiothérapie (FR), Université de Picardie Jules Verne (FR), University of Duisburg-Essen (DE), Universidad de Sevilla (ES), Cardiff University (GB), KU Leuven (BE)
Openalex Percentile: Top 7%
Pneumocystis jirovecii pneumonia detection and treatment
4.06
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