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

Humans Meningitis, Cryptococcal / diagnosis Longitudinal Studies RNA, Ribosomal, 28S Cryptococcus neoformans / genetics Cryptococcosis Malawi HIV Infections / complications Polymerase Chain Reaction

Journal

The Lancet. Microbe
ISSN: 2666-5247
Titre abrégé: Lancet Microbe
Pays: England
ID NLM: 101769019

Informations de publication

Date de publication:
Mar 2024
Historique:
received: 26 01 2023
revised: 27 09 2023
accepted: 26 10 2023
medline: 11 3 2024
pubmed: 12 2 2024
entrez: 11 2 2024
Statut: ppublish

Résumé

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. 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. 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 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. 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.

Sections du résumé

BACKGROUND 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 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 RESULTS
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
INTERPRETATION CONCLUSIONS
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 BACKGROUND
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.

Identifiants

DOI: 10.1016/S2666-5247(23)00362-2 PMID: 38342110 PMC: PMC10914677
pubmed: 38342110
pii: S2666-5247(23)00362-2
doi: 10.1016/S2666-5247(23)00362-2
pmc: PMC10914677
pii:
doi:

Substances chimiques

RNA, Ribosomal, 28S 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

e261-e271

Subventions

Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/V033417/1
Pays : United Kingdom

Investigateurs

J Goodall (J)
N Mawoko (N)
J Milburn (J)
R Mmipi (R)
C Muthoga (C)
P Ponatshego (P)
I Rulaganyang (I)
K Seatla (K)
N Tlhako (N)
K Tsholo (K)
S April (S)
A Bekiswa (A)
L Boloko (L)
H Bookholane (H)
T Crede (T)
L Davids (L)
R Goliath (R)
S Hlungulu (S)
R Hoffman (R)
H Kyepa (H)
N Masina (N)
D Maughan (D)
T Mnguni (T)
S Moosa (S)
T Morar (T)
M Mpalali (M)
J Naude (J)
I Oliphant (I)
S Sayed (S)
L Sebesho (L)
M Shey (M)
L Swanepoel (L)
M Chasweka (M)
W Chimang'anga (W)
T Chimphambano (T)
E Dziwani (E)
E Gondwe (E)
A Kadzilimbile (A)
S Kateta (S)
E Kossam (E)
C Kukacha (C)
B Lipenga (B)
J Ndaferankhande (J)
M Ndalama (M)
R Shah (R)
A Singini (A)
K Stott (K)
A Zambasa (A)
T Banda (T)
T Chikaonda (T)
G Chitulo (G)
L Chiwoko (L)
N Chome (N)
M Gwin (M)
T Kachitosi (T)
B Kamanga (B)
M Kazembe (M)
E Kumwenda (E)
M Kumwenda (M)
C Maya (C)
W Mhango (W)
C Mphande (C)
L Msumba (L)
T Munthali (T)
D Ngoma (D)
S Nicholas (S)
L Simwinga (L)
A Stambuli (A)
G Tegha (G)
J Zambezi (J)
C Ahimbisibwe (C)
A Akampurira (A)
A Alice (A)
F Cresswell (F)
J Gakuru (J)
D Kiiza (D)
J Kisembo (J)
R Kwizera (R)
F Kugonza (F)
E Laker (E)
T Luggya (T)
A Lule (A)
A Musubire (A)
R Muyise (R)
O Namujju (O)
J Ndyetukira (J)
L Nsangi (L)
M Okirwoth (M)
A Sadiq (A)
K Tadeo (K)
A Tukundane (A)
D Williams (D)
L Atwine (L)
P Buzaare (P)
M Collins (M)
N Emily (N)
C Inyakuwa (C)
S Kariisa (S)
J Mwesigye (J)
S Niwamanya (S)
A Rodgers (A)
J Rukundo (J)
I Rwomushana (I)
M Ssemusu (M)
G Stead (G)
K Boyd (K)
S Gondo (S)
P Kufa (P)
E Makaha (E)
C Moyo (C)
T Mtisi (T)
S Mudzingwa (S)
T Mwarumba (T)
T Zinyandu (T)
F Dromer (F)
P Johnstone (P)
S Hafeez (S)

Informations de copyright

Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Déclaration de conflit d'intérêts

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.

Auteurs

Tshepiso 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.

Aude Sturny-Leclère (A)

Institut Pasteur, Université Paris Cité, Translational Mycology Group, Centre National de Référence Mycoses Invasives et Antifongiques, Department of Mycology, Paris, France.

Kwana Lechiile (K)

Botswana-Harvard Health Partnership, Gaborone, Botswana.

Cheusisime Kajanga (C)

Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Kamuzu University of Health Science, Blantyre, Malawi.

Timothée 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.

Jennifer C 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.

Tshepo Leeme (T)

Botswana-Harvard Health Partnership, Gaborone, Botswana.

Melanie Moyo (M)

Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Kamuzu University of Health Science, Blantyre, Malawi.

Nabila 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.

David S 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.

Henry 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 Mosepele (M)

Botswana-Harvard Health Partnership, Gaborone, Botswana; Department of Internal Medicine, University of Botswana, Gaborone, Botswana.

Thomas S 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.

Joseph N 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.

Olivier 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.

Alexandre 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.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Development and validation of quantitative PCR...
questionsmedicales.fr › Maladies du système nerveux › Méningite › Méningite fongique › Méningite cryptococcique › Development and validation of quantitative PCR...
questionsmedicales.fr › Environnement et santé publique › Santé publique › Méthodes épidémiologiques › Caractéristiques des études épidémiologiques › Études épidémiologiques › Études de cohortes › Études longitudinales › Development and validation of quantitative PCR...
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › ARN ribosomique › ARN ribosomique 28S › Development and validation of quantitative PCR...
questionsmedicales.fr › Eucaryotes › Champignons › Levures › Cryptococcus › Cryptococcus neoformans › Development and validation of quantitative PCR...
questionsmedicales.fr › Infection › Infections bactériennes et mycoses › Mycoses › Cryptococcose › Development and validation of quantitative PCR...
questionsmedicales.fr › Régions géographiques › Afrique › Afrique subsaharienne › Afrique australe › Malawi › Development and validation of quantitative PCR...
questionsmedicales.fr › Maladies du système immunitaire › Déficits immunitaires › Infections à VIH › Development and validation of quantitative PCR...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Techniques d'amplification d'acides nucléiques › Réaction de polymérisation en chaîne › Development and validation of quantitative PCR...