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

Mbangiwa, Tshepiso, Sturny-Leclère, Aude, Lechiile, Kwana, Kajanga, Cheusisime, Boyer-Chammard, Timothée, Hoving, Jennifer C, Leeme, Tshepo, Moyo, Melanie, Youssouf, Nabila, Lawrence, David S, Mwandumba, Henry ORCID: https://orcid.org/0000-0003-4470-3608, Mosepele, Mosepele, Harrison, Thomas S, Jarvis, Joseph N, Lortholary, Olivier, Alanio, Alexandre, Goodall, J, Mawoko, N, Milburn, J, Mmipi, R, Muthoga, C, Ponatshego, P, Rulaganyang, I, Seatla, K, Tlhako, N, Tsholo, K, April, S, Bekiswa, A, Boloko, L, Bookholane, H, Crede, T, Davids, L, Goliath, R, Hlungulu, S, Hoffman, R, Kyepa, H, Masina, N, Maughan, D, Mnguni, T, Moosa, S, Morar, T, Mpalali, M, Naude, J, Oliphant, I, Sayed, S, Sebesho, L, Shey, M, Swanepoel, L, Chasweka, M, Chimang’anga, W, Chimphambano, T, Dziwani, E, Gondwe, E, Kadzilimbile, A, Kateta, S, Kossam, E, Kukacha, C, Lipenga, B, Ndaferankhande, J, Ndalama, M, Shah, R, Singini, A, Stott, K, Zambasa, A, Banda, T, Chikaonda, T, Chitulo, G, Chiwoko, L, Chome, N, Gwin, M, Kachitosi, T, Kamanga, B, Kazembe, M, Kumwenda, E, Kumwenda, M, Maya, C, Mhango, W, Mphande, C, Msumba, L, Munthali, T, Ngoma, D, Nicholas, S, Simwinga, L, Stambuli, A, Tegha, G, Zambezi, J, Ahimbisibwe, C, Akampurira, A, Alice, A, Cresswell, F, Gakuru, J, Kiiza, D, Kisembo, J, Kwizera, R, Kugonza, F, Laker, E, Luggya, T, Lule, A, Musubire, A, Muyise, R, Namujju, O, Ndyetukira, J, Nsangi, L, Okirwoth, M, Sadiq, A, Tadeo, K, Tukundane, A, Williams, D, Atwine, L, Buzaare, P, Collins, M, Emily, N, Inyakuwa, C, Kariisa, S, Mwesigye, J, Niwamanya, S, Rodgers, A, Rukundo, J, Rwomushana, I, Ssemusu, M, Stead, G, Boyd, K, Gondo, S, Kufa, P, Makaha, E, Moyo, C, Mtisi, T, Mudzingwa, S, Mwarumba, T, Zinyandu, T, Dromer, F, Johnstone, P and Hafeez, S (2024) 'Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study'. The Lancet Microbe, Vol 5, Issue 3, e261-e271.

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Abstract

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 (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).

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.

Item Type:	Article
Subjects:	QU Biochemistry > Genetics > QU 550 Genetic techniques. PCR. Chromosome mapping QW Microbiology and Immunology > Viruses > QW 160 Viruses (General). Virology WC Communicable Diseases > Virus Diseases > Acquired Immunodeficiency Syndrome. HIV Infections > WC 503 Acquired immunodeficiency syndrome. HIV infections
Faculty: Department:	Clinical Sciences & International Health > Clinical Sciences Department Clinical Sciences & International Health > Malawi-Liverpool-Wellcome Programme (MLW)
Digital Object Identifer (DOI):	https://doi.org/10.1016/s2666-5247(23)00362-2
SWORD Depositor:	JISC Pubrouter
Depositing User:	JISC Pubrouter
Date Deposited:	26 Feb 2024 14:24
Last Modified:	19 Mar 2024 16:05
URI:	https://archive.lstmed.ac.uk/id/eprint/24084

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