In Vitro Evaluation of the Virulence Attributes of Oropharyngeal Candida Species Isolated from People Living with Human Immunodeficiency Virus with Oropharyngeal Candidiasis on Antiretroviral Therapy.

Background Oropharyngeal Candida species are part commensal microflora in the the oral cavity of health individuals. Commensal Candida species can become opportunist and transition to pathogenic causes of oropharyngeal candidiasis (OPC) in individuals with impaired immunity through ecological cues and expression of virulence factors. Limited studies have evaluated virulence attributes of oropharyngeal Candida species among people living with human immunodeficiency virus (PLHIV) with OPC on antiretroviral therapy (ART) in Uganda. Objective Evaluation of the Virulence Attributes of Oropharyngeal Candida Species Isolated from People Living with Human Immunodeficiency Virus with Oropharyngeal Candidiasis on Antiretroviral Therapy Methods Thirty-five (35) Candida isolates from PLHIV with OPC on ART were retrieved from sample repository and evaluated for phospholipase activity using the egg yolk agar method, proteinase activity using the bovine serum albumin agar method, hemolysin activity using the blood agar plate method, esterase activity using the Tween 80 opacity test medium method, coagulase activity using the classical tube method and biofilm formation using the microtiter plate assay method in vitro . Results Phospholipase and proteinase activities were detected in 33/35 (94.3%) and 31/35 (88.6%) of the strains, respectively. Up to 25/35 (71.4%) of the strains exhibited biofilm formation while esterase activity was demonstrated in 23/35 (65.7%) of the strains. Fewer isolates 21/35 (60%) of the strains produced hemolysin and coagulase production was the least virulence activity detected in 18/35 (51.4%). Conclusion Phospholipase and proteinase activities were the strongest virulence attributes of oropharyngeal Candida species.

5-Flucytosine Longitudinal Antifungal Susceptibility Testing of Cryptococcus neoformans: A Substudy of the EnACT Trial Testing Oral Amphotericin.

Background: The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B in combination with flucytosine for the treatment of cryptococcal meningitis. When flucytosine (5FC) is used as monotherapy in cryptococcosis, 5FC can induce resistant Cryptococcus mutants. Oral amphotericin B uses a novel drug delivery mechanism, and we assessed whether resistance to 5FC develops during oral LNC-amphotericin B therapy. Methods: We enrolled Ugandans with HIV diagnosed with cryptococcal meningitis and who were randomized to receive 5FC and either standard intravenous (IV) amphotericin B or oral LNC-amphotericin B. We used broth microdilution to measure the minimum inhibitory concentration (MIC) of the first and last cryptococcal isolates in each participant. Breakpoints are inferred from 5FC in Candida albicans. We measured cerebral spinal fluid (CSF) 5FC concentrations by liquid chromatography and tandem mass spectrometry. Results: Cryptococcus 5FC MIC50 was 4 µg/mL, and MIC90 was 8 µg/mL. After 2 weeks of therapy, there was no evidence of 5FC resistance developing, defined as a >4-fold change in susceptibility in any Cryptococcus isolate tested. The median CSF 5FC concentration to MIC ratio (interquartile range) was 3.0 (1.7-5.5) µg/mL. There was no association between 5FC/MIC ratio and early fungicidal activity of the quantitative rate of CSF yeast clearance (R2 = 0.004; P = .63). Conclusions: There is no evidence of baseline resistance to 5FC or incident resistance during combination therapy with oral or IV amphotericin B in Uganda. Oral amphotericin B can safely be used in combination with 5FC.

Oral Lipid Nanocrystal Amphotericin B for Cryptococcal Meningitis: A Randomized Clinical Trial.

BACKGROUND: Amphotericin B is the gold standard treatment for severe mycoses. A new orally delivered, less-toxic formulation of amphotericin has been developed. METHODS: In our randomized clinical trial, we tested oral lipid nanocrystal (LNC) amphotericin B (MAT2203, Matinas Biopharma) vs intravenous (IV) amphotericin for human immunodeficiency virus-associated cryptococcal meningitis in 4 sequential cohorts. Two pilot cohorts assessed safety and tolerability (n = 10 each), and 2 cohorts assessed efficacy with/without 2 IV loading doses (n = 40 each). The experimental arm received 1.8 g/d oral LNC amphotericin through 2 weeks with 100 mg/kg/d flucytosine, then 1.2 g/d LNC amphotericin through 6 weeks. The randomized control arm (n = 41) received 7 days of IV amphotericin with flucytosine, then 7 days of fluconazole 1200 mg/d. The primary end point was cerebrospinal fluid (CSF) early fungicidal activity (EFA). RESULTS: We randomized 80 participants to oral LNC amphotericin + flucytosine with (n = 40) and without (n = 40) 2 IV loading doses and 41 control participants to IV amphotericin + flucytosine. Mean EFA was 0.40 log10 colony-forming units (CFU)/mL/d for all-oral LNC amphotericin, 0.42 log10  Cryptococcus CFU/mL/d for oral LNC amphotericin with IV loading doses, and 0.46 log10 CFU/mL/d for IV amphotericin controls. LNC amphotericin groups achieved 2-week CSF sterility in 63% (44 of 70) vs 68% (23 of 34) of controls. The 18-week survival was 85% (34 of 40) with all-oral LNC amphotericin, 90% (36 of 40) with oral LNC amphotericin given IV loading doses, and 85% (35 of 41) with IV amphotericin.Grade 3-4 laboratory adverse events occurred less frequently in LNC amphotericin groups (41%) than the IV amphotericin group (61%, P = .05), particularly for anemia (21% vs 44%; P = .01) and potassium (5% vs 17%; P = .04). CONCLUSIONS: This new oral amphotericin B LNC formulation appears promising for cryptococcal meningitis with antifungal activity, similar survival, and less toxicity than IV amphotericin. CLINICAL TRIALS REGISTRATION: NCT04031833.

Sterile Cerebrospinal Fluid Culture at Cryptococcal Meningitis Diagnosis Is Associated with High Mortality.

Cryptococcus is the leading cause of AIDS-related meningitis in sub-Saharan Africa. The clinical implications of a sterile cerebrospinal fluid (CSF) culture among individuals diagnosed with cryptococcal meningitis using CSF cryptococcal antigen (CrAg) are unclear. We prospectively enrolled 765 HIV-positive Ugandans with first-episode cryptococcal meningitis from November 2010 to May 2017. All persons were treated with amphotericin-based induction therapy. We grouped participants by tertile of baseline CSF quantitative Cryptococcus culture burden and compared clinical characteristics, CSF immune profiles, and 18-week mortality. We found 55 (7%) CSF CrAg-positive participants with sterile CSF cultures. Compared to the non-sterile groups, participants with sterile CSF cultures had higher CD4 counts, lower CSF opening pressures, and were more frequently receiving ART. By 18 weeks, 47% [26/55] died in the sterile culture group versus 35% [83/235] in the low culture tertile, 46% [107/234] in the middle tertile, and 56% [135/241] in the high tertile (p < 0.001). The sterile group had higher levels of CSF interferon-gamma (IFN-γ), IFN-α, interleukin (IL)-6, IL-17, G-CSF, GM-CSF, and chemokine CXCL2 compared with non-sterile groups. Despite persons with sterile CSF cultures having higher CD4 counts, lower CSF opening pressures, and CSF cytokine profiles associated with better Cryptococcus control (e.g., IFN-γ predominant), mortality was similar to those with higher fungal burdens. This unexpected finding challenges the traditional paradigm that increasing CSF fungal burdens are associated with increased mortality but is consistent with a damage-response framework model.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50.

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 µg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

Cerebrospinal Fluid Early Fungicidal Activity as a Surrogate Endpoint for Cryptococcal Meningitis Survival in Clinical Trials.

BACKGROUND: In cryptococcal meningitis phase 2 clinical trials, early fungicidal activity (EFA) of Cryptococcus clearance from cerebrospinal fluid (CSF) is used as a surrogate endpoint for all-cause mortality. The Food and Drug Administration allows for using surrogate endpoints for accelerated regulatory approval, but EFA as a surrogate endpoint requires further validation. We examined the relationship between rate of CSF Cryptococcus clearance (EFA) and mortality through 18 weeks. METHODS: We pooled individual-level CSF data from 3 sequential cryptococcal meningitis clinical trials conducted during 2010-2017. All 738 subjects received amphotericin + fluconazole induction therapy and had serial quantitative CSF cultures. The log10-transformed colony-forming units (CFUs) per mL CSF were analyzed by general linear regression versus day of culture over the first 10 days. RESULTS: Mortality through 18 weeks was 37% for EFA > = 0.60 (n = 170), 36% for 0.40-0.59 (n = 182), 39% for 0.30-0.39 (n = 112), 35% for 0.20-0.29 (n = 87), and 50% for those with EFA < 0.20 CFU/mL/day (n = 187). The hazard ratio for 18-week mortality, comparing those with EFA < 0.20 to those with EFA > = 0.20, was 1.60 (95% confidence interval, 1.25, 2.04; P = .002). The lowest EFA group had lower median CD4 T-cell counts (P < .01) and lower proportion of patients with CSF pleocytosis (P < .001). CONCLUSIONS: EFA is associated with all-cause mortality in cryptococcal meningitis. An EFA threshold of > = 0.20 log10 CFU/mL/day was associated with similar 18-week mortality (37%) compared to 50% mortality with EFA < 0.20. This EFA threshold may be considered a target for a surrogate endpoint. This builds upon existing studies to validate EFA as a surrogate endpoint.

Identification of Pathogen Genomic Differences That Impact Human Immune Response and Disease during Cryptococcus neoformans Infection.

Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogen's characteristics. To probe the influence of pathogen genotype on human survival, immune response, and other parameters of disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal Antiretroviral Therapy (ART) Timing (COAT) Trial in Uganda. We measured human participants' survival, meningitis disease parameters, immunologic phenotypes, and pathogen in vitro growth characteristics. We compared those clinical data to whole-genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST), ST93, in our Ugandan participant population and to sequences from an additional 18 strains of 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analysis identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human survival, immunologic response, or clinical parameters. We infected mice with 17 available KN99α gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research identifies important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections.IMPORTANCE Even with the best available care, mortality rates in cryptococcal meningitis range from 20% to 60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine the role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole-genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of some of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

Adjunctive sertraline for HIV-associated cryptococcal meningitis: a randomised, placebo-controlled, double-blind phase 3 trial.

BACKGROUND: Identifying new antifungals for cryptococcal meningitis is a priority given the inadequacy of current therapy. Sertraline has previously shown in vitro and in vivo activity against cryptococcus. We aimed to assess the efficacy and cost-effectiveness of adjunctive sertraline in adults with HIV-associated cryptococcal meningitis compared with placebo. METHODS: In this double-blind, randomised, placebo-controlled trial, we recruited HIV-positive adults with cryptococcal meningitis from two hospitals in Uganda. Participants were randomly assigned (1:1) to receive standard therapy with 7-14 days of intravenous amphotericin B (0·7-1·0 mg/kg per day) and oral fluconazole (starting at 800 mg/day) with either adjunctive sertraline or placebo. Sertraline was administered orally or via nasogastric tube at a dose of 400 mg/day for 2 weeks, followed by 200 mg/day for 12 weeks, then tapered off over 3 weeks. The primary endpoint was 18-week survival, analysed by intention-to-treat. This study is registered with ClinicalTrials.gov, number NCT01802385. FINDINGS: Between March 9, 2015, and May 29, 2017, we screened 842 patients with suspected meningitis and enrolled 460 of a planned 550 participants, at which point the trial was stopped for futility. Three patients in the sertraline group and three patients in the placebo group were lost to follow-up and therefore discontinued before study end. At 18 weeks, 120 (52%) of 229 patients in the sertraline group and 106 (46%) of 231 patients in the placebo group had died (hazard ratio 1·21, 95% CI 0·93-1·57; p=0·15). The fungal clearance rate from cerebrospinal fluid was similar between groups (0·43 -log10 CFU/mL per day [95% CI 0·37-0·50] in the sertraline group vs 0·47 -log10 CFU/mL per day [0·40-0·54] in the placebo group; p=0·59), as was occurrence of grade 4 or 5 adverse events (72 [31%] of 229 vs 75 [32%] of 231; p=0·98), most of which were associated with amphotericin B toxicity. INTERPRETATION: Sertraline did not reduce mortality and should not be used to treat patients with HIV-associated cryptococcal meningitis. The reasons for sertraline inactivity appear to be multifactorial and might be associated with insufficient duration of therapeutic sertraline concentrations. FUNDING: National Institutes of Health and Medical Research Council, Wellcome Trust.

The Mouse Inhalation Model of Cryptococcus neoformans Infection Recapitulates Strain Virulence in Humans and Shows that Closely Related Strains Can Possess Differential Virulence.

Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet the Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with Cryptococcus neoformans strains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.

Evaluation of trypan blue stain in the TC20 automated cell counter as a point-of-care for the enumeration of viable cryptococcal cells in cerebrospinal fluid.

Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log10 transformed cell counter readings with gating of 4-10 µm versus log10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log10 transformed counts were 5.22 (IQR = 4.79-5.44) for the TC20 and 3.99 (IQR = 2.59-5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = -0.0025; P = .92). TC20 automated cell counting with trypan blue staining was poorly predictive of the quantitative CSF culture and could not be used as a substitute for quantitative culture.

Evaluation of a point-of-care immunoassay test kit 'StrongStep' for cryptococcal antigen detection.

BACKGROUND: HIV-associated cryptococcal meningitis is the leading cause of adult meningitis in Sub-Saharan Africa, accounting for 15%-20% of AIDS-attributable mortality. The development of point-of-care assays has greatly improved the screening and diagnosis of cryptococcal disease. We evaluated a point-of-care immunoassay, StrongStep (Liming Bio, Nanjing, Jiangsu, China) lateral flow assay (LFA), for cryptococcal antigen (CrAg) detection in cerebrospinal fluid (CSF) and plasma. METHODS: We retrospectively tested 143 CSF and 77 plasma samples collected from HIV-seropositive individuals with suspected meningitis from 2012-2016 in Uganda. We prospectively tested 90 plasma samples collected from HIV-seropositive individuals with CD4 cell count <100 cells/µL from 2016-2017 as part of a cryptococcal antigenemia screening program. The StrongStep CrAg was tested against a composite reference standard of positive Immy CrAg LFA (Immy, Norman, OK, USA) or CSF culture with statistical comparison by McNemar's test. RESULTS: StrongStep CrAg had a 98% (54/55) sensitivity and 90% (101/112) specificity in plasma (P = 0.009, versus reference standard). In CSF, the StrongStep CrAg had 100% (101/101) sensitivity and 98% (41/42) specificity (P = 0.99). Adjusting for the cryptococcal antigenemia prevalence of 9% in Uganda and average cryptococcal meningitis prevalence of 37% in Sub-Saharan Africa, the positive predictive value of the StrongStep CrAg was 50% in plasma and 96% in CSF. CONCLUSIONS: We found the StrongStep CrAg LFA to be a sensitive assay, which unfortunately lacked specificity in plasma. In lower prevalence settings, a majority of positive results from blood would be expected to be false positives.

Antimicrobial Drug Resistance in Blood Culture Isolates at a Tertiary Hospital, Uganda.

We summarize antimicrobial drug resistance (AMR) patterns from blood cultures at a tertiary hospital in Uganda. High rates of resistance to first-line antibiotic drugs were observed among Staphylococcus aureus and gram-negative organisms. Microbiology services with susceptibility testing should be strengthened to support standardized reporting of AMR data in sub-Saharan Africa.

Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.

BACKGROUND: Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures. RESULTS: Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78-5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59-5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R2 = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R2 = 0.43, P = 0.4). CONCLUSION: Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.

Acridine orange fluorescent microscopy is more sensitive than India ink light microscopy in the rapid detection of cryptococcosis among CrAg positive HIV patients.

BACKGROUND: India ink microscopy on cerebrospinal fluid is still utilized in resource limited settings for the diagnosis of cryptococcal meningitis despite its poor sensitivity. We hypothesized that staining fungal nucleic acids with fluorescent dyes instead of the capsule with India ink might improve sensitivity for the diagnosis of cryptococcal meningitis. METHODS: We enrolled 96 HIV-infected participants with cryptococcal meningitis who provided 194 CSF specimens at serial time points in Kampala, Uganda. Cryptococcosis was diagnosed by cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) test and only positive samples were included. We stained CSF with India ink and acridine orange. We cultured the same samples on standard fungal media. We compared acridine orange to CrAg, India ink and CSF culture. RESULTS: Acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. Acridine orange and India ink had a statistically significant difference (P<0.001) with a 25% correlation for detection of Cryptococcus yeasts. India ink had more negative results (22%) than acridine orange (4%). The sensitivity for India ink increased (86%) while that of acridine orange did not change (97%) when compared to CSF culture. However, both India ink and acridine orange had poor predictive values with reference to culture. CONCLUSION: Acridine orange is a better alternative to India ink in the rapid detection of cryptococcosis among CrAg positive HIV patients.

Efficacy of adjunctive sertraline for the treatment of HIV-associated cryptococcal meningitis: an open-label dose-ranging study.

BACKGROUND: Cryptococcus is the most common cause of adult meningitis in Africa. We assessed the safety and microbiological efficacy of adjunctive sertraline, previously shown to have in-vitro and in-vivo activity against cryptococcus. METHODS: In this open-label dose-finding study, we recruited HIV-infected individuals with cryptococcal meningitis who presented to Mulago Hospital in Kampala, Uganda between Aug 14, 2013, and Aug 30, 2014. To assess safety and tolerability, the first 60 participants were given sertraline at escalating doses of 100 mg/day, 200 mg/day, 300 mg/day, or 400 mg/day as induction therapy for 2 weeks, followed by consolidation therapy with 200 mg/day for an additional 8 weeks. From Nov 29, 2013, participants were randomly assigned (1:1) to receive open-label sertraline at predetermined doses of 200 mg/day, 300 mg/day, or 400 mg/day as induction therapy for 2 weeks, followed by consolidation therapy with 200 mg/day for 8 weeks. Dose assignment was made via computer-generated, permuted block randomisation stratified by antiretroviral therapy (ART) status for people with a first episode of meningitis. The primary outcome was 2-week cerebrospinal fluid (CSF) clearance rate of cryptococcus, termed early fungicidal activity, measured in patients with a first episode of culture-positive meningitis and two or more CSF cultures. This study is registered with ClinicalTrials.gov, number NCT01802385. FINDINGS: Of the 330 individuals assessed, 172 HIV-infected adults with cryptococcal meningitis were enrolled. We gave 100 mg/day sertraline to 17 patients, 200 mg/day to 12 patients, 300 mg/day to 14 patients, and 400 mg/day to 17 patients. 112 participants were randomly assigned to receive sertraline at 200 mg (n=48), 300 mg (n=36), or 400 mg (n=28) daily for the first 2 weeks, and 200 mg/day thereafter. The final population consisted of 17 participants in the 100 mg group, 60 in the 200 mg group, 50 in the 300 mg group, and 45 in the 400 mg in group. Participants receiving any sertraline dose averaged a CSF clearance rate of -0·37 colony forming units per mL per day (95% CI -0·41 to -0·33). Incidence of paradoxical immune reconstitution inflammatory syndrome was 5% (two of 43 newly starting ART) and no cases of relapse occurred over the 12-week study period. 38 (22%) of 172 participants had died at 2 weeks, and 69 (40%) had died at 12 weeks. Six grade 4 adverse events occurred in 17 participants receiving 100 mg, 14 events in 60 participants receiving 200 mg, 19 events in 50 participants receiving 300 mg, and eight events in 45 participants receiving 400 mg. Grade 4 or 5 adverse event risk did not differ between current US Food and Drug Administration-approved dosing of 100-200 mg/day and higher doses of 300-400 mg/day (hazard ratio 1·27, 95% CI 0·69-2·32; p=0·45). INTERPRETATION: Participants receiving sertraline had faster cryptococcal CSF clearance and a lower incidence of immune reconstitution inflammatory syndrome and relapse than that reported in the past. This inexpensive and off-patent oral medication is a promising adjunctive antifungal therapy. FUNDING: National Institutes of Health, Grand Challenges Canada.

Human Immune Response Varies by the Degree of Relative Cryptococcal Antigen Shedding.

Background. Cerebrospinal fluid (CSF) cryptococcal glucuronoxylomannan antigen (CrAg) titers generally correlate with quantitative fungal culture burden; however, correlation is not precise. Some patients have higher CrAg titers with lower fungal burdens and vice versa. We hypothesized that the relative discordancy between CrAg titer and quantitative culture burden reflects the relative degree of CrAg shedding by Cryptococcus neoformans and is associated with human immune responses. Methods. One hundred ninety human immunodeficiency virus-infected individuals with cryptococcal meningitis were enrolled in Uganda and South Africa. We compared initial CSF CrAg titers relative to their CSF quantitative cultures to determine low (n = 58), intermediate (n = 68), or high (n = 64) CrAg shedders. We compared cytokines measured by Luminex multiplex assay on cryopreserved CSF and 10-week mortality across shedding groups using linear and logistic regression and distribution of genotypes by multilocus sequence typing. Results. The relative degree of CrAg shedding was positively associated with increasing CSF levels of the following: interleukin (IL)-6, IL-7, IL-8, and tumor necrosis factor-α (each P < 0.01), which are all secreted by antigen-presenting cells and negatively associated with vascular endothelial growth factor (P = .01). In addition, IL-5, IL-13, granulocyte colony-stimulating factor, and macrophage chemotactic protein were decreased in low-CrAg shedders compared with intermediate shedders (each P ≤ .01). Type 1 T-helper cells (Th1) cytokine responses and 10-week mortality did not differ between the shedding groups. Cryptococcal genotypes were equally distributed across shedding groups. Conclusions. Discordancy between CrAg shedding and expected shedding based on quantitative fungal burden is associated with detectable immunologic differences in CSF, primarily among secreted cytokines and chemokines produced by antigen-presenting cells and Th2.

Reproducibility of CSF quantitative culture methods for estimating rate of clearance in cryptococcal meningitis.

Quantitative cerebrospinal fluid (CSF) cultures provide a measure of disease severity in cryptococcal meningitis. The fungal clearance rate by quantitative cultures has become a primary endpoint for phase II clinical trials. This study determined the inter-assay accuracy of three different quantitative culture methodologies. Among 91 participants with meningitis symptoms in Kampala, Uganda, during August-November 2013, 305 CSF samples were prospectively collected from patients at multiple time points during treatment. Samples were simultaneously cultured by three methods: (1) St. George's 100 mcl input volume of CSF with five 1:10 serial dilutions, (2) AIDS Clinical Trials Group (ACTG) method using 1000, 100, 10 mcl input volumes, and two 1:100 dilutions with 100 and 10 mcl input volume per dilution on seven agar plates; and (3) 10 mcl calibrated loop of undiluted and 1:100 diluted CSF (loop). Quantitative culture values did not statistically differ between St. George-ACTG methods (P= .09) but did for St. George-10 mcl loop (P< .001). Repeated measures pairwise correlation between any of the methods was high (r≥0.88). For detecting sterility, the ACTG-method had the highest negative predictive value of 97% (91% St. George, 60% loop), but the ACTG-method had occasional (∼10%) difficulties in quantification due to colony clumping. For CSF clearance rate, St. George-ACTG methods did not differ overall (mean -0.05 ± 0.07 log10CFU/ml/day;P= .14) on a group level; however, individual-level clearance varied. The St. George and ACTG quantitative CSF culture methods produced comparable but not identical results. Quantitative cultures can inform treatment management strategies.

Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda.

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.

Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda.

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/µl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 µg/ml and an MIC90 of 8 µg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 µg/ml and an MIC90 value of 32 µg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 µg/ml. We observed an amphotericin B MIC50 of 0.5 µg/ml and an MIC90 of 1 µg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 µg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).

Epidemiology of meningitis in an HIV-infected Ugandan cohort.

There is limited understanding of the epidemiology of meningitis among human immunodeficiency virus (HIV)-infected populations in sub-Saharan Africa. We conducted a prospective cohort study of HIV-infected adults with suspected meningitis in Uganda, to comprehensively evaluate the etiologies of meningitis. Intensive cerebrospiral fluid (CSF) testing was performed to evaluate for bacterial, viral, fungal, and mycobacterial etiologies, including neurosyphilis,16s ribosomal DNA (rDNA) polymerase chain reaction (PCR) for bacteria, Plex-ID broad viral assay, quantitative-PCR for HSV-1/2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Toxoplasma gondii; reverse transcription-PCR (RT-PCR) for Enteroviruses and arboviruses, and Xpert MTB/RIF assay. Cryptococcal meningitis accounted for 60% (188 of 314) of all causes of meningitis. Of 117 samples sent for viral PCR, 36% were EBV positive. Among cryptococcal antigen negative patients, the yield of Xpert MTB/RIF assay was 22% (8 of 36). After exclusion of cryptococcosis and bacterial meningitis, 61% (43 of 71) with an abnormal CSF profile had no definitive diagnosis. Exploration of new TB diagnostics and diagnostic algorithms for evaluation of meningitis in resource-limited settings remains needed, and implementation of cryptococcal diagnostics is critical.