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

2024; Elsevier BV; Volume: 5; Issue: 3 Linguagem: Inglês

10.1016/s2666-5247(23)00362-2

2666-5247

Tshepiso Mbangiwa, Aude Sturny-Leclère, Kwana Lechiile, Cheusisime Kajanga, Timothée Boyer-Chammard, J. Claire Hoving, Tshepo Leeme, Melanie Moyo, Nabila Youssouf, David S. Lawrence, Henry C. Mwandumba, Mosepele Mosepele, Thomas S. Harrison, Joseph N Jarvis, Olivier Lortholary, Alexandre Alanio, Jack Goodall, Norah Mawoko, James Milburn, Refilwe Mmipi, Charles Muthoga, Ponego Ponatshego, Ikanyeng Rulaganyang, Kaelo K. Seatla, Nametso Tlhako, Katlego Tsholo, Samantha April, Abulele Bekiswa, Linda Boloko, Hloni Bookholane, Thomas Crede, Lester M. Davids, René Goliath, Siphokazi Hlungulu, Robert S. Hoffman, Henriette Kyepa, Noma Masina, Deborah Maughan, Trevor Mnguni, Shahida Moosa, Tania Morar, Mkanyiseli Kenneth Mpalali, J. Naudé, Ida Oliphant, Sumaya Sayed, Leago Sebesho, Muki Shey, Loraine Swanepoel, Madalitso Chasweka, Wezi Chimang’anga, Tipatseni Chimphambano, Eltas Dziwani, Ebbie Gondwe, Aubrey Kadzilimbile, Steven Kateta, Evelyn Kossam, Christopher Kukacha, Bright Lipenga, J Ndaferankhande, Maureen Ndalama, Raj C. Shah, Andreas Singini, Katharine E. Stott, Agness Zambasa, T Banda, Tarsizio Chikaonda, Gladys Chitulo, Lorren Chiwoko, Nelecy Chome, Mary Gwin, Timothy Kachitosi, Beauty Kamanga, Mussah Kazembe, Emily Kumwenda, Moses Kumwenda, Camilo Maya, Wilberforce Mhango, Chimwemwe Mphande, Lusungu Msumba, Tapiwa Munthali, D Ngoma, Stephen W. Nicholas, Lusayo Simwinga, Anthony Stambuli, Gerald Tegha, Janet Zambezi, Cynthia Ahimbisibwe, Andrew Akampurira, Anamudde Alice, Fiona V Cresswell, J Gakuru, Daniel Kiiza, John Kisembo, Richard Kwizera, Florence Kugonza, Eva Laker, Tony Luggya, Andrew Lule, Abdu Musubire, Rhona Muyise, O Namujju, Jane Francis Ndyetukira, Laura Nsangi, Michael Okirwoth, A Sadiq, Kizza Kandole Tadeo, Asmus Tukundane, Darlisha A Williams, Lorna Atwine, Peter Buzaare, Matt Collins, Ninsima Emily, Christine Inyakuwa, Samson kariisa, James Mwesigye, S Niwamanya, Anthony Rodgers, Joan Rukundo, Irene Rwomushana, Mike Ssemusu, Gavin Stead, K. Boyd, Secrecy Gondo, Prosper Kufa, Edward Makaha, Crispin Moyo, Takudzwa J. Mtisi, Shepherd Mudzingwa, Taddy Mwarumba, Tawanda Zinyandu, Françoise Dromer, Philippa Johnstone, Shaheryar Hafeez,

Nail Diseases and Treatments

BackgroundHIV-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.MethodsWe 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.FindingsWhen 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 (R2=0·73 and R2=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).InterpretationQSP1 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.FundingEuropean 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.