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.

Diagnostic performance of the IMMY cryptococcal antigen lateral flow assay on serum and cerebrospinal fluid for diagnosis of cryptococcosis in HIV-negative patients: a systematic review.

BACKGROUND: The incidence of cryptococcosis amongst HIV-negative persons is increasing. Whilst the excellent performance of the CrAg testing in people living with HIV is well described, the diagnostic performance of the CrAg LFA has not been systematically evaluated in HIV-negative cohorts on serum or cerebrospinal fluid. METHODS: We performed a systematic review to characterise the diagnostic performance of IMMY CrAg® LFA in HIV-negative populations on serum and cerebrospinal fluid. A systematic electronic search was performed using Medline, Embase, Global Health, CENTRAL, WoS Science Citation Index, SCOPUS, Africa-Wide Information, LILACS and WHO Global Health Library. Studies were screened and data extracted from eligible studies by two independent reviewers. A fixed effect meta-analysis was used to estimate the diagnostic sensitivity and specificity. RESULTS: Of 447 records assessed for eligibility, nine studies met our inclusion criteria, including 528 participants overall. Amongst eight studies that evaluated the diagnostic performance of the IMMY CrAg® LFA on serum, the pooled median sensitivity was 96% (95% Credible Interval (CrI) 68-100%) with a pooled specificity estimate of 96% (95%CrI 84-100%). Amongst six studies which evaluated the diagnostic performance of IMMY CrAg® LFA on CSF, the pooled median sensitivity was 99% (95%CrI 95-100%) with a pooled specificity median of 99% (95%CrI 95-100%). CONCLUSIONS: This review demonstrates a high pooled sensitivity and specificity for the IMMY CrAg® LFA in HIV-negative populations, in keeping with findings in HIV-positive individuals. The review was limited by the small number of studies. Further studies using IMMY CrAg® LFA in HIV-negative populations would help to better determine the diagnostic value of this test.

Population pharmacokinetics and CSF penetration of flucytosine in adults with HIV-associated cryptococcal meningoencephalitis.

BACKGROUND: There are limited data describing clinical flucytosine pharmacokinetics (PK). The variability of flucytosine partitioning into the CNS is not known. We described the interindividual variability in flucytosine PK in patients with HIV-associated cryptococcal meningoencephalitis. In addition, we quantified the extent and variability of CSF partitioning of flucytosine. METHODS: A PK study was conducted in 64 patients with confirmed HIV-associated cryptococcal meningoencephalitis in Blantyre, Malawi. A four-compartment PK model was developed, and Monte Carlo simulations were performed with flucytosine administered at different doses and in different schedules. RESULTS: The estimated mean apparent volume of the central compartment was 17.50 (SD 9.99) L; mean apparent clearance was 5.88 (SD 3.35) L/h; mean apparent volume of the CNS compartment was 41.73 (SD 13.66) L. From the Bayesian posterior estimates, AUC24 values at steady state (144-168 h) with doses of 25 mg/kg q6h were median (IQR) 890.38 (603.81-1213.70) mg.h/L in plasma and 595.66 (425.69-776.64) mg.h/L in CSF. The ratio of CSF:plasma AUC24 was 0.69 (IQR 0.58-0.82). CONCLUSIONS: This study revealed significant interindividual variability in flucytosine PK in plasma and CSF in patients with HIV-associated cryptococcal meningoencephalitis. The population PK model is a first critical step for revised flucytosine regimens that maximize fungal killing and minimize toxicity and the emergence of resistance.

Association of Antibody Immunity With Cryptococcal Antigenemia and Mortality in a South African Cohort With Advanced Human Immunodeficiency Virus Disease.

BACKGROUND: Asymptomatic cryptococcal antigenemia (positive blood cryptococcal antigen [CrAg]) is associated with increased mortality in individuals with human immunodeficiency virus (HIV) even after adjusting for CD4 count and despite receiving antifungal treatment. The association of antibody immunity with mortality in adults with HIV with cryptococcal antigenemia is unknown. METHODS: Cryptococcal capsular glucuronoxylomannan (GXM)- and naturally occurring ß-glucans (laminarin, curdlan)-binding antibodies were measured in blood samples of 197 South Africans with HIV who underwent CrAg screening and were followed up to 6 months. Associations between antibody titers, CrAg status, and all-cause mortality were sought using logistic and Cox regression, respectively. RESULTS: Compared with CrAg-negative individuals (n = 130), CrAg-positive individuals (n = 67) had significantly higher IgG1 (median, 6672; interquartile range [IQR], 4696-10 414 vs 5343, 3808-7722 µg/mL; P = .007), IgG2 (1467, 813-2607 vs 1036, 519-2012 µg/mL; P = .01), and GXM-IgG (1:170, 61-412 vs 1:117, 47-176; P = .0009) and lower curdlan-IgG (1:47, 11-133 vs 1:93, 40-206; P = .01) titers. GXM-IgG was associated directly with cryptococcal antigenemia adjusted for CD4 count and antiretroviral therapy use (odds ratio, 1.64; 95% confidence interval [CI], 1.21 to 2.22). Among CrAg-positive individuals, GXM-IgG was inversely associated with mortality at 6 months adjusted for CD4 count and tuberculosis (hazard ratio, 0.50; 95% CI, .33 to .77). CONCLUSIONS: The inverse association of GXM-IgG with mortality in CrAg-positive individuals suggests that GXM-IgG titer may have prognostic value in those individuals. Prospective longitudinal studies to investigate this hypothesis and identify mechanisms by which antibody may protect against mortality are warranted.

How Applicable Is the Single-Dose AMBITION Regimen for Human Immunodeficiency Virus-Associated Cryptococcal Meningitis to High-Income Settings?

The AmBisome Therapy Induction Optimization (AMBITION-cm) trial, conducted in eastern and southern Africa, showed that a single, high dose (10 mg/kg) of liposomal amphotericin B, given with an oral backbone of fluconazole and flucytosine, was noninferior to the World Health Organization (WHO)-recommended regimen of 7 days of amphotericin B deoxycholate plus flucytosine for treatment of human immunodeficiency virus (HIV)-associated cryptococcal meningitis and has been incorporated into WHO treatment guidelines. We believe that the trial also has important implications for the treatment of HIV-associated cryptococcal meningitis in high-income settings. We advance the arguments, supported by evidence where available, that the AMBITION-cm trial regimen is likely to be as fungicidal as the currently recommended 14-day liposomal amphotericin-based treatments, better tolerated with fewer adverse effects, and confer significant economic and practical benefits and, therefore, should be included as a treatment option in guidance for HIV-associated cryptococcal treatment in high-income settings.

Cryptococcal Antigenemia in Advanced Human Immunodeficiency Virus Disease: Pathophysiology, Epidemiology, and Clinical Implications.

Cryptococcal antigen (CrAg) is detectable in blood prior to the onset of symptomatic cryptococcal meningitis (CM), a leading cause of death among people with advanced human immunodeficiency virus (HIV) disease globally. Highly sensitive assays can detect CrAg in blood, and screening people with HIV with low CD4 counts, followed by preemptive antifungal treatment, is recommended and widely implemented as part of a global strategy to prevent CM and end cryptococcal-related deaths. Cryptococcal antigenemia encompasses a spectrum of conditions from preclinical asymptomatic infection (cerebrospinal fluid [CSF] CrAg-negative) through subclinical (CSF CrAg-positive without overt meningism) to clinical symptomatic cryptococcal disease, usually manifesting as CM. In this review, we summarize current understanding of the pathophysiology, risk factors for, and clinical implications of cryptococcal antigenemia within this spectrum. We also provide an update on global prevalence, recommended screening and treatment strategies, and future considerations for improving outcomes among patients with cryptococcal antigenemia.

High concordance in plasma and CSF HIV-1 drug resistance mutations despite high cases of CSF viral escape in individuals with HIV-associated cryptococcal meningitis in Botswana.

OBJECTIVES: We compared the patterns of HIV-1 drug resistance mutations between the CSF and plasma of individuals with HIV-associated cryptococcal meningitis. METHODS: This is a cross-sectional study of archived CSF and plasma samples collected from ART-exposed participants recruited in the Phase 3 AmBisome Therapy Induction Optimisation randomized controlled trial (ISRCTN72509687) conducted in Botswana between 2018 and 2021. HIV-1 RT and protease genes were genotyped using next-generation sequencing and HIV-1 drug resistance mutations were compared between the CSF and plasma compartments stratified by thresholds of ≥20% and <20%. RESULTS: Overall, 66.7% (16/24) of participants had at least one HIV-1 drug resistance mutation in the CSF and/or plasma. A total of 15/22 (68.2%) participants had HIV-1 drug resistance mutations at ≥20% threshold in the plasma and of those, 11 (73.3%) had been on ART longer than 6 months. HIV-1 drug resistance mutations were highly concordant between the CSF and plasma at ≥20% threshold despite a substantial number of individuals experiencing CSF viral escape and with only 54.5% with CSF WBC count ≥20 cells/mm3. Minority HIV-1 drug resistance mutations were detected in 20.8% (5/24) of participants. There were no mutations in the CSF that were not detected in the plasma. CONCLUSIONS: There was high concordance in HIV-1 drug resistance mutations in the CSF and plasma, suggesting intercompartmental mixing and possibly a lack of compartmentalization. Some individuals harboured minority HIV-1 drug resistance mutations, demonstrating the need to employ more sensitive genotyping methods such as next-generation sequencing for the detection of low-abundance mutations.

Cost-effectiveness of single, high-dose, liposomal amphotericin regimen for HIV-associated cryptococcal meningitis in five countries in sub-Saharan Africa: an economic analysis of the AMBITION-cm trial.

BACKGROUND: HIV-associated cryptococcal meningitis is a leading cause of AIDS-related mortality. The AMBITION-cm trial showed that a regimen based on a single high dose of liposomal amphotericin B deoxycholate (AmBisome group) was non-inferior to the WHO-recommended treatment of seven daily doses of amphotericin B deoxycholate (control group) and was associated with fewer adverse events. We present a five-country cost-effectiveness analysis. METHODS: The AMBITION-cm trial enrolled patients with HIV-associated cryptococcal meningitis from eight hospitals in Botswana, Malawi, South Africa, Uganda, and Zimbabwe. Taking a health service perspective, we collected country-specific unit costs and individual resource-use data per participant over the 10-week trial period, calculating mean cost per participant by group, mean cost-difference between groups, and incremental cost-effectiveness ratio per life-year saved. Non-parametric bootstrapping and scenarios analyses were performed including hypothetical real-world resource use. The trial registration number is ISRCTN72509687, and the trial has been completed. FINDINGS: The AMBITION-cm trial enrolled 844 participants, and 814 were included in the intention-to-treat analysis (327 from Uganda, 225 from Malawi, 107 from South Africa, 84 from Botswana, and 71 from Zimbabwe) with 407 in each group, between Jan 31, 2018, and Feb 17, 2021. Using Malawi as a representative example, mean total costs per participant were US$1369 (95% CI 1314-1424) in the AmBisome group and $1237 (1181-1293) in the control group. The incremental cost-effectiveness ratio was $128 (59-257) per life-year saved. Excluding study protocol-driven cost, using a real-world toxicity monitoring schedule, the cost per life-year saved reduced to $80 (15-275). Changes in the duration of the hospital stay and antifungal medication cost showed the greatest effect in sensitivity analyses. Results were similar across countries, with the cost per life-year saved in the real-world scenario ranging from $71 in Botswana to $121 in Uganda. INTERPRETATION: The AmBisome regimen was cost-effective at a low incremental cost-effectiveness ratio. The regimen might be even less costly and potentially cost-saving in real-world implementation given the lower drug-related toxicity and the potential for shorter hospital stays. FUNDING: European Developing Countries Clinical Trials Partnership, Swedish International Development Cooperation Agency, Wellcome Trust and Medical Research Council, UKAID Joint Global Health Trials, and the National Institute for Health Research. TRANSLATIONS: For the Chichewa, Isixhosa, Luganda, Setswana and Shona translations of the abstract see Supplementary Materials section.

Population pharmacokinetics of liposomal amphotericin B in adults with HIV-associated cryptococcal meningoencephalitis.

BACKGROUND: Single, high-dose liposomal amphotericin B (LAmB; AmBisome, Gilead Sciences) has demonstrated non-inferiority to amphotericin B deoxycholate in combination with other antifungals for averting all-cause mortality from HIV-associated cryptococcal meningitis. There are limited data on the pharmacokinetics (PK) of AmBisome. The aim of this study was to describe population PK of AmBisome and conduct a meta-analysis of the available studies to suggest the optimal dosing for cryptococcal meningoencephalitis. METHODS: Data from a Phase II and Phase III trial of high-dose, short-course AmBisome for cryptococcal meningoencephalitis were combined to develop a population PK model. A search was conducted for trials of AmBisome monotherapy and meta-analysis of clinical outcome data was performed. RESULTS: A two-compartment model with first-order clearance of drug from the central compartment fitted the data best and enabled the extent of inter-individual variability in PK to be quantified. Mean (SD) population PK parameter estimates were: clearance 0.416 (0.363)  L/h; volume of distribution 4.566 (4.518) L; first-order transfer of drug from central to peripheral compartments 2.222 (3.351)  h-1, and from peripheral to central compartment 2.951 (4.070)  h-1. Data for the meta-analysis were insufficient to suggest optimal dosing of AmBisome for cryptococcal meningoencephalitis. CONCLUSIONS: This study provides novel insight into the PK of AmBisome at the population level and the variability therein. Our analysis also serves to highlight the paucity of data available on the pharmacodynamics (PD) of AmBisome and underscores the importance of thorough and detailed PK/PD analysis in the development of novel antifungals, by demonstrating the challenges associated with post hoc PK/PD analysis.

The acceptability of the AMBITION-cm treatment regimen for HIV-associated cryptococcal meningitis: Findings from a qualitative methods study of participants and researchers in Botswana and Uganda.

BACKGROUND: The AMBITION-cm trial for HIV-associated cryptococcal meningitis demonstrated that a single, high-dose of liposomal amphotericin (AmBisome) plus 14-days of oral flucytosine and fluconazole was non-inferior in terms of all-cause mortality to 7-days of amphotericin B deoxycholate and flucytosine followed by 7-days of fluconazole (Control). The AmBisome regimen was associated with fewer adverse events. We explored the acceptability of the AmBisome regimen from the perspective of participants and providers. METHODS: We embedded a qualitative methods study within the AMBITION-cm sites in Botswana and Uganda. We conducted in-depth interviews with trial participants, surrogate decision makers, and researchers and combined these with direct observations. Interviews were transcribed, translated, and analysed thematically. RESULTS: We interviewed 38 trial participants, 20 surrogate decision makers, and 31 researchers. Participant understanding of the trial was limited; however, there was a preference for the AmBisome regimen due to the single intravenous dose and fewer side effects. More time was required to prepare the single AmBisome dose but this was felt to be acceptable given subsequent reductions in workload. The AmBisome regimen was reported to be associated with fewer episodes of rigors and thrombophlebitis and a reduction in the number of intravenous cannulae required. Less intensive monitoring and management was required for participants in the AmBisome arm. CONCLUSIONS: The AmBisome regimen was highly acceptable, being simpler to administer despite the initial time investment required. The regimen was well tolerated and associated with less toxicity and resultant management. Widespread implementation would reduce the clinical workload of healthcare workers caring for patients with HIV-associated cryptococcal meningitis.

Single-Dose Liposomal Amphotericin B Treatment for Cryptococcal Meningitis.

BACKGROUND: Cryptococcal meningitis is a leading cause of human immunodeficiency virus (HIV)-related death in sub-Saharan Africa. Whether a treatment regimen that includes a single high dose of liposomal amphotericin B would be efficacious is not known. METHODS: In this phase 3 randomized, controlled, noninferiority trial conducted in five African countries, we assigned HIV-positive adults with cryptococcal meningitis in a 1:1 ratio to receive either a single high dose of liposomal amphotericin B (10 mg per kilogram of body weight) on day 1 plus 14 days of flucytosine (100 mg per kilogram per day) and fluconazole (1200 mg per day) or the current World Health Organization-recommended treatment, which includes amphotericin B deoxycholate (1 mg per kilogram per day) plus flucytosine (100 mg per kilogram per day) for 7 days, followed by fluconazole (1200 mg per day) for 7 days (control). The primary end point was death from any cause at 10 weeks; the trial was powered to show noninferiority at a 10-percentage-point margin. RESULTS: A total of 844 participants underwent randomization; 814 were included in the intention-to-treat population. At 10 weeks, deaths were reported in 101 participants (24.8%; 95% confidence interval [CI], 20.7 to 29.3) in the liposomal amphotericin B group and 117 (28.7%; 95% CI, 24.4 to 33.4) in the control group (difference, -3.9 percentage points); the upper boundary of the one-sided 95% confidence interval was 1.2 percentage points (within the noninferiority margin; P<0.001 for noninferiority). Fungal clearance from cerebrospinal fluid was -0.40 log10 colony-forming units (CFU) per milliliter per day in the liposomal amphotericin B group and -0.42 log10 CFU per milliliter per day in the control group. Fewer participants had grade 3 or 4 adverse events in the liposomal amphotericin B group than in the control group (50.0% vs. 62.3%). CONCLUSIONS: Single-dose liposomal amphotericin B combined with flucytosine and fluconazole was noninferior to the WHO-recommended treatment for HIV-associated cryptococcal meningitis and was associated with fewer adverse events. (Funded by the European and Developing Countries Clinical Trials Partnership and others; Ambition ISRCTN number, ISRCTN72509687.).

Equity in clinical trials for HIV-associated cryptococcal meningitis: A systematic review of global representation and inclusion of patients and researchers.

BACKGROUND: It is essential that clinical trial participants are representative of the population under investigation. Using HIV-associated cryptococcal meningitis (CM) as a case study, we conducted a systematic review of clinical trials to determine how inclusive and representative they were both in terms of the affected population and the involvement of local investigators. METHODS: We searched Medline, EMBASE, Cochrane, Africa-Wide, CINAHL Plus, and Web of Science. Data were extracted for 5 domains: study location and design, screening, participants, researchers, and funders. Data were summarised and compared over 3 time periods: pre-antiretroviral therapy (ART) (pre-2000), early ART (2000 to 2009), and established ART (post-2010) using chi-squared and chi-squared for trend. Comparisons were made with global disease burden estimates and a composite reference derived from observational studies. RESULTS: Thirty-nine trials published between 1990 and 2019 were included. Earlier studies were predominantly conducted in high-income countries (HICs) and recent studies in low- and middle-income countries (LMICs). Most recent studies occurred in high CM incidence countries, but some highly affected countries have not hosted trials. The sex and ART status of participants matched those of the general CM population. Patients with reduced consciousness and those suffering a CM relapse were underrepresented. Authorship had poor representation of women (29% of all authors), particularly as first and final authors. Compared to trials conducted in HICs, trials conducted in LMICs were more likely to include female authors (32% versus 20% p = 0.014) but less likely to have authors resident in (75% versus 100%, p < 0.001) or nationals (61% versus 93%, p < 0.001) of the trial location. CONCLUSIONS: There has been a marked shift in CM trials over the course of the HIV epidemic. Trials are primarily performed in locations and populations that reflect the burden of disease, but severe and relapse cases are underrepresented. Most CM trials now take place in LMICs, but the research is primarily funded and led by individuals and institutions from HICs.

The Lived Experience Of Participants in an African RandomiseD trial (LEOPARD): protocol for an in-depth qualitative study within a multisite randomised controlled trial for HIV-associated cryptococcal meningitis.

INTRODUCTION: Individuals recruited into clinical trials for life-threatening illnesses are particularly vulnerable. This is especially true in low-income settings. The decision to enrol may be influenced by existing inequalities, poor healthcare infrastructure and fear of death. Where patients are confused or unconscious the responsibility for this decision falls to relatives. This qualitative study is nested in the ongoing AMBIsome Therapy Induction OptimisatioN (AMBITION) Trial. AMBITION is recruiting participants from five countries in sub-Saharan Africa and is trialling a novel treatment approach for HIV-associated cryptococcal meningitis, an infection known to affect brain function. We aim to learn from the experiences of participants, relatives and researchers involved in AMBITION. METHODS AND ANALYSIS: We will collect data through in-depth interviews with trial participants and the next of kin of participants who were confused at enrolment and therefore provided surrogate consent. Data will be collected in Gaborone, Botswana; Kampala, Uganda and Harare, Zimbabwe. Interviews will follow a narrative approach including participatory drawing of participation timelines. This will be supplemented by direct observation of the research process at each of the three recruiting hospitals. Interviews will also take place with researchers from the African and European institutions that form the partnership through which the trial is administered. Interviews will be transcribed verbatim, translated (if necessary) and organised thematically for narrative analysis. ETHICS AND DISSEMINATION: This study has been approved by the Health Research Development Committee, Gaborone (Reference: HPDME:13/18/1); Makerere School of Health Sciences Institutional Review Board, Kampala (Reference: 2019-061); University of Zimbabwe Joint Research Ethics Committee, Harare (Reference: 219/19), and the London School of Hygiene and Tropical Medicine (Reference: 17957). Study findings will be shared with research participants from the sites, key stakeholders at each research institution and ministries of health to help inform the development and implementation of future trials. The findings of this study will be published in journals and presented at academic meetings. TRIAL REGISTRATION: Registered at www.clinicaltrials.gov:NCT04296292.

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/µL; 2108 samples with CD4 ≤100 cells/µL 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.

Outcomes of Reflex Cryptococcal Antigen (CrAg) Screening in Human Immunodeficiency Virus (HIV)-Positive Patients With CD4 Counts of 100-200 Cells/µL in Botswana.

Increasing the CD4-count threshold for cryptococcal antigen (CrAg) screening from ≤100 to ≤200 cells/µL resulted in a 3-fold increase in numbers screened. CrAg-prevalence was 3.5% at CD4 101-200 and 6.2% ≤100 cells/µL. Six-month mortality was 21.4% (9/42) in CrAg-positive CD4 ≤100 cells/µL and 3.2% (1/31) in CrAg-positive CD4 101-200 cells/µL.

Cryptococcal meningitis: a review of cryptococcal antigen screening programs in Africa.

INTRODUCTION: Cryptococcal meningitis remains a significant contributor to AIDS-related mortality despite widened access to antiretroviral therapy. Cryptococcal antigen (CrAg) can be detected in the blood prior to development of meningitis. Development of highly sensitive and specific rapid diagnostic CrAg tests has helped facilitate the adoption of CrAg screening programs in 19 African countries. AREAS COVERED: The biological rationale for CrAg screening and the programmatic strategies for its implementation are reviewed. We describe the approach to the investigation of patients with cryptococcal antigenemia and the importance of lumbar puncture to identify individuals who may have cryptococcal meningitis in the absence of symptoms. The limitations of current treatment recommendations and the potential role of newly defined combination antifungal therapies are discussed. A literature review was conducted using a broad database search for cryptococcal antigen screening and related terms in published journal articles dating up to December 2019. Conference abstracts, publicly available guidelines, and project descriptions were also incorporated. EXPERT OPINION: As we learn more about the risks of cryptococcal antigenemia, it has become clear that the current management paradigm is inadequate. More intensive investigation and management are required to prevent the development of cryptococcal meningitis and reduce mortality associated with cryptococcal antigenemia.

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.