Innovative quantitative PCR assays for the assessment of HIV-associated cryptococcal meningoencephalitis in Sub-Saharan Africa

BackgroundCryptococcal meningitis (CM) accounts for about 10-20% of AIDS-defining illnesses with a 10-week mortality rate of 25-50%. 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 more rapid and less labor-intensive. MethodsWe developed and validated species-specific qPCR assays based on DNA amplification of a Quorum Sensing Protein 1 (QSP1); QSP1A, QSP1 B/C, and QSP1 D that are specific to C. neoformans, C. deneoformans and C. gattii species, respectively, and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. We tested these assays for species identification (QSP1) and quantification (QSP1 ans 28S) on cerebrospinal fluid (CSF) of 209 CM patients at baseline (Day 0) and during anti-fungal therapy (Day 7 and Day 14), from the AMBITION-cm trial in Botswana and Malawi (2018-2021). FindingsWhen compared to quantitative cryptococcal culture (QCC) as the reference, the sensitivity of the 28S rRNA and QSP1 assays were 98.2% [95% CI: 95.1-99.5] and 90.4% [95% CI: 85.2-94.0] respectively in cerebrospinal fluid (CSF) at Day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with QCC (R2=0.73, R2=0.78, respectively). Both Botswana and Malawi had a predominant C. neoformans prevalence of 67% [95% CI: 55, 75] and 68% [95% CI: 57, 73], respectively and lower C. gattii rates of 21% [95% CI: 14, 31] and 8% [95% CI: 4, 14], respectively. We identified 10 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 correlated well with baseline QCC and showed a similar decline in fungal load during induction therapy. These assays have a quick turnaround time and could be used in place of QCC to determine fungal load clearance. The clinical implications of the detection of possibly viable but non-culturable cells in CSF during induction therapy remain unclear. FundingThe AMBITION-cm clinical trial which was funded by the European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust / Medical Research Council (UK) / UKAID Joint Global Health Trials and National Institute for Health Research (UK).