S1.4d Cryptococcus qPCR assays: the future for routine mycology labs and clinical trials dealing with cryptococcosis

Abstract

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PMBackground: Routine laboratory testing for cryptococcal meningitis currently consists of Cryptococcal antigen (CrAg) testing in blood and cerebrospinal fluid (CSF), CSF India ink, and CSF fungal culture. Quantitative cryptococcal culture (QCC) is labor intensive and not feasible in most settings.ObjectivesWe evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We also investigated the dynamics of fungal DNA and RNA detection during antifungal treatment.MethodsWe developed a qPCR assay that can differentiate serotypes A, D, and B/C of Cryptococcus neoformans and C. gattii based on the amplification of a unique nuclear Quorum sensing protein 1 (QSP1) and a multicopy 28S rRNA gene and evaluated the assays on 205-patient samples from the AMBITION-cm trial in Botswana and Malawi (2018-2021). CSF, plasma, and whole blood samples were stored per patient and were sampled at day 0 (baseline), day 7 and 14 for CSF and at day 1, 3 and 7 for plasma and whole blood post antifungal treatment initiation. A Roche LightCycler480 and Graph pad prism were used for data analysis.ResultsA total of 205/209 stored patient samples (85 from Botswana; 124 from Malawi), were used. For QSP1 qPCR tested in CSF at D0, 138 (81.7%) were serotype A, 28 (16.6%) were serotype B/C and 3 (1.8%) were a mixed infection of serotype A and B/C. There was no amplification with 36 (17.6%) samples. There was no difference in fungal loads at D0, D7, and D14 between serotype A and B/C with the QSP1 qPCR assay, and QCC. QCC showed a good correlation with qPCR quantification with QSP1 qPCR (slope = 0.797, R2 = 0.73) and with 28S rRNA qPCR (Slope = 0.771, R2 = 0.778) assays. The fungal load at D0 was significantly higher in patients who died at week 2 (w2) and at week 10 (w10) as compared with patients who survived post-week 10 (P <.01), with no significant difference in initial fungal load in both treatment regimens (P >.05). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 h of treatment was significantly associated with early mortality at w2 and mortality at w10 (P <.01). QSP1 RT-qPCR showed that detection of DNA was due to viable fungal cells as the quantification of QSP1 whole nucleic acids was systematically higher (X2 to 5) than that of DNA.ConclusionQuantification of C. neoformans and C. gattii load in CSF and plasma at D0 is useful in identifying patients at risk of death and may be a promising tool for monitoring treatment response in the future.