Cryptococcus qPCR assays: the future for routine mycology labs and clinical trials dealing with HIV-associated cryptococcosis

Background: 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. We evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We investigated the dynamics of fungal DNA and RNA detection during antifungal treatment. Methods: We developed a qPCR assay that can differentiate serotypes A, D and B/C of Cryptococcus neoformans and Cryptococcus 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 patients 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. Results: Using the QSP1 qPCR, 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. QCC showed a good correlation 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 10 (w10) as compared to patients who survived post week 10 (p < 0.01). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 hours of treatment was significantly associated with early mortality at w10 (p < 0.01). 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 (2 to 5-fold) than that of DNA. Conclusions: Quantification 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

Chronic and Occult Hepatitis B Virus Infection in Pregnant Women in Botswana

The hepatitis B virus (HBV) is a global problem; however, the burden of HBV infection in pregnant women in Botswana is unknown. We sought to determine the prevalence of chronic and occult HBV infection in human immunodeficiency virus (HIV)-infected and -uninfected pregnant women in Botswana. Samples from 752 pregnant women were tested for hepatitis B surface antigen (HBsAg), and HBsAg-positive samples were tested for hepatitis B e antigen (HBeAg) and HBV DNA load. Samples that were HBsAg negative were screened for occult HBV infection by determining the HBV DNA load. HBV genotypes were determined based on a 415-base-pair fragment of the surface gene. Among the 752 women tested during pregnancy or early postpartum, 16 (2.1%) (95% confidence interval (CI): 2.0–2.2) were HBsAg-positive. The prevalence of chronic HBV infection was higher (3.1%) among HIV-infected (95% CI: 3.0–3.2) compared with HIV-uninfected women (1.1%) (95% CI: 1.07–1.1, p = 0.057). Among the 622 HBsAg-negative women, the prevalence of occult HBV infection was 6.6% (95% CI: 6.5–6.7). Three of thirteen HBsAg-positive participants were HBeAg-positive, and all were HIV-negative. Of the 11 maternal samples successfully genotyped, five (45.5%) were genotype D3, five (45.5%) were genotype A1, and one was genotype E (9%). Low and similar proportions of HIV-infected and -uninfected pregnant women in Botswana had occult or chronic HBV infection. We identified a subset of HIV-negative pregnant women who had high HBV DNA levels and were HBeAg-positive, and thus likely to transmit HBV to their infants

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

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

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

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

The Impact of Human Pegivirus on CD4 Cell Count in HIV-Positive Persons in Botswana

Abstract Background: Human pegiviruses (HPgV)—formerly known as hepatitis G virus or GB virus C (GBV-C)—are common single-stranded RNA viruses that may have a beneficial impact on slowing HIV disease progression. The data on HPgV in resource-limited regions such as Sub-Saharan Africa are scarce. Thus, we conducted the first study of HPgV in Botswana as part of a natural history study of HIV subtype C disease progression. Methods: Plasma samples from 133 HIV-positive adults were evaluated for HPgV RNA, and the 5’UTR was sequenced to determine the HPgV genotype. Results: HPgV RNA was detected in 41 (30.8%) individuals. While the presence of HPgV RNA had no impact on baseline HIV viral load, a significant difference in baseline CD4 cell count was observed. HPgV genotypes were determined for 27 individuals and included 5 individuals (18.5%) with genotype 1 and 22 (81.5%) with genotype 5. Baseline CD4 cell counts were significantly higher for persons infected with HPgV genotype 5 compared with genotype 1. Conclusions: These data suggest that HPgV infection is common among HIV-positive individuals in Botswana and has a significant impact on CD4 cell count. This difference in CD4 cell count based on HPgV genotype suggests that HPgV genotype should be evaluated as a possible predictor of HIV disease progression and highlights the need for additional studies of this virus in resource-limited settings

Hepatitis B virus prevalence and vaccine antibody titers in children HIV exposed but uninfected in Botswana.

BackgroundBotswana introduced the HBV vaccine at birth for all newborns in 2000. To the best of our knowledge, since the introduction of HBV vaccination, there have been limited data for vaccine response to HBV and its impact on early childhood HBV infections among children HIV exposed but uninfected in Botswana.AimsTo determine the prevalence of hepatitis B surface antigen (HBsAg) and HBV vaccine response in 18 months old children HIV exposed but uninfected in Botswana.MethodsStored plasma samples from 304 children at 18 months of age and 287 mothers from delivery were tested for HBsAg. Mothers with positive HBsAg had HBV DNA level tested, and their HBV genotypes were determined by amplifying a 415-base pair (bp) region of the surface gene. Plasma samples from children exposed to HIV were tested for hepatitis B surface antibody (anti-HBs) titers.ResultsNo children (0 of 304) were positive for HBsAg at 18 months while 5 (1.74%) of 287 HIV-positive mothers were HBsAg positive. Four of the HBsAg positive mothers were infected with genotype A1, while 1 was infected with genotype E. The median anti-HBs titer in children was 174 mIU/mL [QR: 70, 457]. Three (1.1%) of 269 children had an inadequate vaccine response (ConclusionNo HBsAg positivity was identified in a cohort of children HIV exposed but uninfected. The absence of HBsAg positives was associated with good HBV vaccine responses and low maternal HBsAg prevalence in Botswana

In Silico Analysis of Hepatitis B Virus Occult Associated Mutations in Botswana Using a Novel Algorithm

Occult hepatitis B infections (OBI) represent a reservoir of undiagnosed and untreated hepatitis B virus (HBV), hence the need to identify mutations that lead to this phenotype. Functionally characterizing these mutations by in vitro studies is time-consuming and expensive. To bridge this gap, in silico approaches, which predict the effect of amino acid (aa) variants on HBV protein function, are necessary. We developed an algorithm for determining the relevance of OBI-associated mutations using in silico approaches. A 3 kb fragment of subgenotypes A1 and D3 from 24 chronic HBV-infected (CHB) and 24 OBI participants was analyzed. To develop and validate the algorithm, the effects of 68 previously characterized occult-associated mutations were determined using three computational tools: PolyPhen2, SNAP2, and PROVEAN. The percentage of deleterious mutations (with impact on protein function) predicted were 52 (76.5%) by PolyPhen2, 55 (80.9%) by SNAP2, and 65 (95.6%) by PROVEAN. At least two tools correctly predicted 59 (86.8%) mutations as deleterious. To identify OBI-associated mutations exclusive to Botswana, study sequences were compared to CHB sequences from GenBank. Of the 43 OBI-associated mutations identified, 26 (60.5%) were predicted by at least two tools to have an impact on protein function. To our knowledge, this is the first study to use in silico approaches to determine the impact of OBI-associated mutations, thereby identifying potential candidates for functional analysis to facilitate mechanistic studies of the OBI phenotype

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

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