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

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.

Funding: 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.

Figure 1

Schematic representation of the assays used in this study C neoformans=Cryptococcus neoformans. CSF=cerebrospinal fluid. qPCR=quantitative PCR. RT-qPCR=reverse transcriptase quantitative PCR. WNA=whole nucleic acid.

Figure 2

Comparison of QSP1 and 28S rRNA assays to QCC and description of the proportion of positive quantitative tests in the cohort (A) Correlation between QSP1 quantification (log₁₀ Cn cells per mL) and QCC (log₁₀ Cn cells per mL), between 28S rRNA quantification (log₁₀ Cn cells per mL) and QCC, and between 28S rRNA and QSP1 using CSF pellet results at baseline (before starting antifungal treatment). (B) Proportion of CSF pellet and supernatant sample fractions amplified by QSP1A, QSP1B/C (combined in the figure as QSP1), and 28S rRNA assays at day 0 (baseline), day 7, and day 14 of antifungal treatment. (C) Fungal loads in CSF at day 0 with QSP1 and 28S rRNA assays in C neoformans and C gattii. The boxes show median and IQR, and the whiskers show the range. Cn=Cryptococcus neoformans or Cryptococcus gattii. Cq=quantification cycles. CSF=cerebrospinal fluid. NS=not significant. QCC=quantitative cryptococcal culture. WNA=whole nucleic acid.

Figure 3

Cryptococcal fungal load quantification Cryptococcal fungal load was quantified at day 0 (baseline), day 7, and day 14 in the cerebrospinal fluid of participants by QCC, QSP1 assay, and 28S rRNA assay in total populations (A–C) and in the control or single-dose regimens (D–F). Serotype-specific fungal load was quantified at day 0 by QCC, QSP1 assay, and 28S rRNA assay (G–I). The boxes show median and IQR, and the whiskers show the range. Cn=Cryptococcus neoformans or Cryptococcus gattii. NS=not significant. QCC=quantitative cryptococcal culture.

Figure 4

Quantification of the fungal load in the cerebrospinal fluid of participants with a positive T1 and T2 band, a positive T1 band, or who were negative in CryptoPS testing in plasma at baseline (day 0) Quantification is shown for serotype A and B/C with QCC (A), QSP1 assay (B), and 28S rRNA assay (C). The boxes show median and IQR, and the whiskers show the range. Cn=Cryptococcus neoformans or Cryptococcus gattii. QCC=quantitative cryptococcal culture.

Figure 5

QSP1 DNA and WNA quantification in CSF pellet and comparison between QCC and QSP1 WNA load QSP1 DNA (qPCR) and WNA (RT-qPCR) were quantified in CSF pellet at baseline (day 0; A), day 7 (B), and day 14 (C) using QSP1 assays. The boxes show median and IQR, and the whiskers show the range. QSP1 WNA load was compared with QCC at baseline (D), day 7 (E), and day 14 (F). Red circles are participants with negative QCC and positive QSP1 WNA detection. Cn=Cryptococcus neoformans or Cryptococcus gattii. CSF=cerebrospinal fluid. QCC=quantitative cryptococcal culture. qPCR=quantitative PCR. RT-qPCR=reverse transcriptase quantitative PCR. WNA=whole nucleic acid.