Differences in human immunodeficiency virus-1C viral load and drug resistance mutation between plasma and cerebrospinal fluid in patients with human immunodeficiency virus-associated cryptococcal meningitis in Botswana.

To determine effects of cryptococcal meningitis (CM) on human immunodeficiency virus (HIV)-1C cerebrospinal fluid (CSF) viral escape, CSF/plasma viral discordance, and drug resistance mutation (DRM) discordance between CSF and plasma compartments, we compared CSF and plasma viral load (VL) and DRMs in individuals with HIV-associated CM in Botswana.This cross-sectional study utilized 45 paired CSF/plasma samples from participants in a CM treatment trial (2014-2016). HIV-1 VL was determined and HIV-1 protease and reverse transcriptase genotyping performed. DRMs were determined using the Stanford HIV database. CSF viral escape was defined as HIV-1 ribonucleic acid ≥0.5 log10 higher in CSF than plasma and VL discordance as CSF VL > plasma VL.HIV-1 VL was successfully measured in 39/45 pairs, with insufficient sample volume in 6; 34/39 (87.2%) participants had detectable HIV-1 in plasma and CSF, median 5.1 (interquartile range: 4.7-5.7) and 4.6 (interquartile range:3.7-4.9) log10 copies/mL, respectively (P≤.001). CSF viral escape was present in 1/34 (2.9%) and VL discordance in 6/34 (17.6%). Discordance was not associated with CD4 count, antiretroviral status, fungal burden, CSF lymphocyte percentage nor mental status. Twenty-six of 45 (57.8%) CSF/plasma pairs were successfully sequenced. HIV-1 DRM discordance was found in 3/26 (11.5%); 1 had I84IT and another had M46MI in CSF only. The third had K101E in plasma and V106 M in CSF.Our findings suggest that HIV-1 escape and DRM discordance may occur at lower rates in participants with advanced HIV-disease and CM compared to those with HIV associated neurocognitive impairment

HIV-1C env and gag Variation in the Cerebrospinal Fluid and Plasma of Patients with HIV-Associated Cryptococcal Meningitis in Botswana.

HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure

Utility of CD4 count measurement in the era of universal antiretroviral therapy: an analysis of routine laboratory data in Botswana.

OBJECTIVES: National guidelines in Botswana recommend baseline CD4 count measurement and both CD4 and HIV viral load (VL) monitoring post-antiretroviral therapy (ART) initiation. We evaluated the utility of CD4 count measurement in Botswana in the era of universal ART. METHODS: CD4 and VL data were analysed for HIV-infected adults undergoing CD4 count measurement in 2015-2017 at the Botswana Harvard HIV-Reference Laboratory. We determined (1) the proportion of individuals with advanced HIV disease (CD4 count < 200 cells/µL) at initial CD4 assessment, (2) the proportion with an initial CD4 count ≥ 200 cells/µL experiencing a subsequent decline in CD4 count to < 200 cells/µL, and (3) the proportion of these immunologically failing individuals who had virological failure. Logistic regression modelling examined factors associated with advanced HIV disease. CD4 count trajectories were assessed using locally weighted scatterplot smoothing (LOWESS) regression. RESULTS: Twenty-five per cent (3571/14 423) of individuals with an initial CD4 assessment during the study period had advanced HIV disease at baseline. Older age [≥ 35 years; adjusted odds ratio (aOR) 1.9; 95% confidence interval (CI) 1.8-2.1] and male sex were associated with advanced HIV disease. Fifty per cent (7163/14 423) of individuals had at least two CD4 counts during the study period. Of those with an initial CD4 count ≥ 200 cells/µL, 4% (180/5061) experienced a decline in CD4 count to < 200 cells/µL; the majority of CD4 count declines were in virologically suppressed individuals and transient. CONCLUSIONS: One-quarter of HIV-positive individuals in Botswana still present with advanced HIV disease, highlighting the importance of baseline CD4 count measurement to identify this at-risk population. Few with a baseline CD4 count ≥ 200 cells/µL experienced a drop below 200 cells/µL, suggesting limited utility for ongoing CD4 monitoring

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

Diagnostic accuracy of the Biosynex CryptoPS cryptococcal antigen semi-quantitative lateral flow assay in patients with advanced HIV disease.

Background: High cryptococcal antigen (CrAg) titers in blood are associated with subclinical meningitis and mortality in CrAg-positive individuals with advanced HIV-disease (AHD). We evaluated a novel semi-quantitative lateral flow assay (LFA), CryptoPS, that may be able to identify individuals with high CrAg titers in a cohort of AHD patients undergoing CrAg screening.Methods: In a prospective cohort of patients with AHD (CD4 ≤200 cells/μL) receiving CD4 count testing, CryptoPS and IMMY LFA CrAg testing were performed on whole blood by two operators blinded to results of the other assay. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CryptoPS were assessed against IMMY LFA as a reference. CryptoPS low-titer (T1 band) and high-titer (T2 band) results were compared against IMMY LFA titers obtained through serial dilution.Results: 916 specimens were tested. Sensitivity of the CryptoPS assay was 61.0% (25/41, 95% confidence interval [95%CI]: 44.5-75.8), specificity 96.6% (845/875, 95%CI: 95.1-97.7), PPV 45.5% (95%CI: 32.0-59.4), and NPV 98.1% (95%CI: 97.0-98.9). All (16/16) CryptoPS false-negatives were samples with IMMY titers ≤1:160. Of 29 patients (30 specimens) who tested positive on CryptoPS but negative on IMMY LFA, none developed cryptococcal meningitis over 3-months follow-up without fluconazole. Median CrAg titers were 1:20 (interquartile range [IQR] 0-1:160) in CryptoPS T1-positive samples and 1:2560 (IQR 1:1280-1:10240) in T2-positives.Conclusions: Diagnostic accuracy of the CryptoPS assay was sub-optimal in the context of CrAg screening, with poor sensitivity at low CrAg titers. However, the CryptoPS assay reliably detected individuals with high titers associated with poor outcomes

Prevalence and Sequelae of Cryptococcal Antigenemia in Antiretroviral Therapy-Experienced Populations: An Evaluation of Reflex Cryptococcal Antigen Screening in Botswana.

BACKGROUND: Evidence to inform cryptococcal antigen (CrAg)-screening guidelines among ART-experienced populations is lacking. We performed a study evaluating the utility of reflex CrAg screening in Gaborone, Botswana. METHODS: CD4 count data were collected from the HIV reference laboratory from 2014-2016. CrAg screening was performed on samples with CD4 ?100 cells/µL beginning January 2015. The proportion of CD4 counts ?100 cells/µL was determined and the frequency of repeat CrAg testing described. Analyses ascertained the impact of ART status on CrAg prevalence and outcomes, and whether CrAg titers could be used for risk stratification. RESULTS: Overall, 5.6% (3335/59 300) of individuals tested had CD4 ?100 cells/?L; 2108 samples with CD4 ?100 cells/?L from 1645 unique patients were CrAg tested. Over half of samples were from ART-experienced individuals: 40.9% (863) on ART and 12.1% (255) defaulters; 22% (463) of CrAg tests were on repeat samples. CrAg prevalence was 4.8% (72/1494; 95% CI, 3.8-6.0%) among outpatients and 21.9% (32/151; 95% CI, 15.3-28.5%) among inpatients. CrAg prevalence rates did not differ by ART status, but 6-month mortality was significantly lower in CrAg-positive individuals on ART at screening. Ten CrAg positives were identified through repeat testing. A CrAg titer cutoff ?1:80 provided the best discrimination for 6-month survival. CONCLUSIONS: CrAg-positivity rates in an ART-experienced population were comparable to those seen in ART-naive populations. Repeat screening identified individuals who seroconverted to CrAg positivity and were at risk of cryptococcal disease. CrAg titers ?1:80 can help identify the individuals at highest risk of death for more intensive management

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

Reversal of CSF HIV-1 Escape during Treatment of HIV-Associated Cryptococcal Meningitis in Botswana.

Cerebrospinal fluid (CSF) viral escape has been poorly described among people with HIV-associated cryptococcal meningitis. We determined the prevalence of CSF viral escape and HIV-1 viral load (VL) trajectories in individuals treated for HIV-associated cryptococcal meningitis. A retrospective longitudinal study was performed using paired CSF and plasma collected prior to and during the antifungal treatment of 83 participants recruited at the Botswana site of the phase-3 AMBITION-cm trial (2018-2021). HIV-1 RNA levels were quantified then CSF viral escape (CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma) and HIV-1 VL trajectories were assessed. CSF viral escape occurred in 20/62 (32.3%; 95% confidence interval [CI]: 21.9-44.6%), 13/52 (25.0%; 95% CI: 15.2-38.2%) and 1/33 (3.0%; 95% CI: 0.16-15.3%) participants at days 1, 7 and 14 respectively. CSF viral escape was significantly lower on day 14 compared to days 1 and 7, p = 0.003 and p = 0.02, respectively. HIV-1 VL decreased significantly from day 1 to day 14 post antifungal therapy in the CSF but not in the plasma (β = -0.47; 95% CI: -0.69 to -0.25; p &lt; 0.001). CSF viral escape is high among individuals presenting with HIV-associated cryptococcal meningitis; however, antifungal therapy may reverse this, highlighting the importance of rapid initiation of antifungal therapy in these patients