Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study.

Autor:	Mbangiwa T; Botswana-Harvard Health Partnership, Gaborone, Botswana; Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France; Institute of Infectious Disease and Molecular Medicine, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa., Sturny-Leclère A; Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France., Lechiile K; Botswana-Harvard Health Partnership, Gaborone, Botswana., Kajanga C; Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Kamuzu University of Health Science, Blantyre, Malawi., Boyer-Chammard T; Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France; Department of Infectious Diseases and Tropical Medicine, Centre Hospitalier d'Ajaccio, Ajaccio, France., Hoving JC; Institute of Infectious Disease and Molecular Medicine, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; AFRICA CMM Medical Mycology Research Unit, Institute of Infectious Disease and Molecular Medicine (IDM), Cape Town, South Africa., Leeme T; Botswana-Harvard Health Partnership, Gaborone, Botswana., Moyo M; Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Kamuzu University of Health Science, Blantyre, Malawi., Youssouf N; Botswana-Harvard Health Partnership, Gaborone, Botswana; Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK., Lawrence DS; Botswana-Harvard Health Partnership, Gaborone, Botswana; Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK., Mwandumba H; Institute of Infectious Disease and Molecular Medicine, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; Liverpool School of Tropical Medicine, Liverpool, UK., Mosepele M; Botswana-Harvard Health Partnership, Gaborone, Botswana; Department of Internal Medicine, University of Botswana, Gaborone, Botswana., Harrison TS; Centre for Global Health, Institute for Infection and Immunity, St George's University of London, London, UK; Clinical Academic Group in Infection, St George's University Hospitals NHS Foundation Trust, London, UK; MRC Centre for Medical Mycology, University of Exeter, Exeter, UK., Jarvis JN; Botswana-Harvard Health Partnership, Gaborone, Botswana; Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK., Lortholary O; Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France; Necker Pasteur Centre for Infectious Diseases and Tropical Médicine, Hôpital Universitaire Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, Paris, France., Alanio A; Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France; Laboratoire de parasitologie-mycologie, AP-HP, Hôpital Saint-Louis, F-75010, Paris, France. Electronic address: alexandre.alanio@pasteur.fr.
Jazyk:	angličtina
Zdroj:	The Lancet. Microbe [Lancet Microbe] 2024 Mar; Vol. 5 (3), pp. e261-e271. Date of Electronic Publication: 2024 Feb 08.
DOI:	10.1016/S2666-5247(23)00362-2
Abstrakt:	Background: HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25-30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. Methods: We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018-21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. Findings: When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1-99·5) and of the QSP1 assay was 90·4% (85·2-94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R 2 =0·73 and R 2 =0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55-75) and 68% (57-73), respectively, and lower C gattii rates of 21% (14-31) and 8% (4-14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). Interpretation: QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear. Funding: European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust/UK Medical Research Council/UKAID Joint Global Health Trials; and UK National Institute for Health Research. Competing Interests: Declaration of interests AA received honoraria for educational activities and webinars from Gilead Sciences and Pfizer and travel grants from Astellas and Gilead Sciences, outside the submitted work. All other authors declare no competing interests. (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
Databáze:	MEDLINE
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