Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype

Clinical Performance of the Mp1p Immunoassay for Rapid Diagnosis of Talaromyces marneffei Infection

P-P9: Cryptococcal Meningitis and other Fungal OIs - Poster abstract no. 747The gold standard to confirm Talaromyces marneffei infection (previously penicilliosis) is culture which can take up to 2 weeks. Diagnostic delay is associated with high mortality. Antigen detecting immunoassays offer rapid, specific and sensitive results, and have transformed management of other mycoses such as cryptococcosis. We describe the performance of the Mp1p immunoassay in a cohort of HIV-infected patients with talaromycosis and matched controls. We evaluated the performance of a novel monoclonal antibody-based immunoassay against Mp1p – an abundant mannoprotein in T. marneffei's cell wall. We used plasma samples from a case-control study: HIV-infected patients with culture-confirmed talaromycosis (N = 284 cases) and HIV-infected patients with other opportunistic infections (N = 332 controls) in Ho Chi Minh City between 2010 and 2015. Controls were matched to cases by age, sex, and CD4 count or WHO disease staging. We describe quantitative antigen dynamics over 90 days for 60 patients on treatment. The median CD4 count was 18 cells/mm3 (IQR: 4-22) in cases and 52 cells/mm3 (IQR: 11-56) in control patients. Amongst cases, blood culture (automated Bactec system) was positive in 200 (70%) patients. The other 84 patients had positive culture from skin lesions or lymph nodes. Common opportunistic infections in control patients included cryptococcal meningitis (N=83), oral or esophageal candidiasis (N=47), tuberculosis (N=46), bacterial pneumonia (N=33), PCP (N=22) toxoplasmosis (N=22), and bacterial sepsis (N=21). Based on an optical density cut-off value generated by a receiver operating characteristic curve (ROC) of 0.208, the sensitivity, specificity, positive predicted value, and negative predicted value of the assay were 89.8%, 92.6%, 91.1%, and 91.5%, respectively. Quantitative antigen testing for 60 patients showed that 90% of patients had cleared antigenemia by 3 months. The rates of antigenemia clearance were similar for patients treated with amphotericin B or itraconazole as induction therapy (P=0.079, Student t-test). The Mp1p immunoassay provides an accurate test for differentiating talaromycosis from other HIV-associated opportunistic infections with sensitivity higher than blood culture, thus allows antifungal therapy to begin sooner, and has the potential to reduce talaromycosis mortality

Do intracerebral cytokine responses explain the harmful effects of dexamethasone in HIV-associated cryptococcal meningitis?

Background The CryptoDex trial showed dexamethasone was associated with poorer clinical outcomes and slower fungal clearance in HIV-associated cryptococcal meningitis. We analysed CSF cytokine concentrations from CrytpoDex participants over the first week of treatment to investigate potential mechanisms of harm and test two hypotheses: dexamethasone reduced pro-inflammatory cytokine concentrations leading to poorer outcomes; and leukotriene A4 hydrolase (LTA4H) genotype (previously associated with dexamethasone responsiveness in tuberculous meningitis) influenced dexamethasone’s clinical impact. Methods We included participants from Vietnam, Thailand, and Uganda. We measured CSF concentrations of IFN-γ, TNF-α, GM-CSF, IL-6, IL-12p70, IL-8, MCP-1, MIP-1α, IL-4, IL-10, and IL-17 from days 1 to 7 of treatment using the Luminex system. We determined LTA4H genotype using a TaqMan genotyping assay of the promoter region SNP rs17525495. We assessed the impact of dexamethasone on cytokine concentration dynamics and the association between cytokine concentration dynamics and fungal clearance with mixed effect models. We measure the influence of LTA4H genotype on outcomes with Cox regression models. Results Dexamethasone increased the rate of TNF-α concentration decline (-0.13 pg/ml/day (95%CI -0.22 to -0.06) p=0.03), which was associated with slower fungal clearance (correlation -0.62 (-0.83 to -0.26)). LTA4H genotype had no statistically significant impact on outcome or response to dexamethasone therapy. Better clinical outcomes were associated with higher baseline concentrations of IFN-γ. Conclusions Dexamethasone may slow fungal clearance and worsen outcomes by increasing the rate of decline of TNF-α concentration

Do intracerebral cytokine responses explain the harmful effects of dexamethasone in HIV-associated cryptococcal meningitis?

Background The CryptoDex trial showed dexamethasone was associated with poorer clinical outcomes and slower fungal clearance in HIV-associated cryptococcal meningitis. We analysed CSF cytokine concentrations from CrytpoDex participants over the first week of treatment to investigate potential mechanisms of harm and test two hypotheses: dexamethasone reduced pro-inflammatory cytokine concentrations leading to poorer outcomes; and leukotriene A4 hydrolase (LTA4H) genotype (previously associated with dexamethasone responsiveness in tuberculous meningitis) influenced dexamethasone’s clinical impact. Methods We included participants from Vietnam, Thailand, and Uganda. We measured CSF concentrations of IFN-γ, TNF-α, GM-CSF, IL-6, IL-12p70, IL-8, MCP-1, MIP-1α, IL-4, IL-10, and IL-17 from days 1 to 7 of treatment using the Luminex system. We determined LTA4H genotype using a TaqMan genotyping assay of the promoter region SNP rs17525495. We assessed the impact of dexamethasone on cytokine concentration dynamics and the association between cytokine concentration dynamics and fungal clearance with mixed effect models. We measure the influence of LTA4H genotype on outcomes with Cox regression models. Results Dexamethasone increased the rate of TNF-α concentration decline (-0.13 pg/ml/day (95%CI -0.22 to -0.06) p=0.03), which was associated with slower fungal clearance (correlation -0.62 (-0.83 to -0.26)). LTA4H genotype had no statistically significant impact on outcome or response to dexamethasone therapy. Better clinical outcomes were associated with higher baseline concentrations of IFN-γ. Conclusions Dexamethasone may slow fungal clearance and worsen outcomes by increasing the rate of decline of TNF-α concentration

A randomized open label trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis is a leading cause of death in HIV-infected patients. International treatment guidelines recommend induction therapy with amphotericin B and flucytosine. This antifungal combination is most effective, but unfortunately flucytosine is expensive and unavailable where the burden of disease is greatest. Where unavailable, guidelines recommend treatment with amphotericin and fluconazole, but this is less effective, with mortality rates of 40-50%. Faster rates of clearance of yeast from cerebrospinal fluid (CSF) are associated with better outcomes - improving the potency of antifungal therapy is likely to be an effective strategy to improve survival. Tamoxifen, a selective estrogen receptor modulator used to treat breast cancer, has anti-cryptococcal activity, appearing synergistic when combined in vitro with amphotericin, and fungicidal when combined with fluconazole. It is concentrated in the brain and macrophages, off-patent, cheap and widely available. We designed a randomized trial to deliver initial efficacy and safety data for tamoxifen combined with amphotericin and fluconazole.Method: A phase II, open-label, randomized (1:1) controlled trial of tamoxifen (300mg/day) combined with amphotericin (1mg/kg/day) and fluconazole (800mg/day) for the first 2 weeks therapy for HIV infected or uninfected adults with cryptococcal meningitis. The study recruits at Cho Ray Hospital and the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. The primary end point is Early Fungicidal Activity (EFA-the rate of yeast clearance from CSF), over the first two weeks of treatment. 50 patients will be recruited providing ≈80% and 90% power to detect a difference in the EFA of -0.11 or -0.13 log10CFU/ml/day, respectively.Discussion: The results of the study will inform the decision to proceed to a larger trial powered to mortality. The size of effect detectable has previously been associated with reduced mortality from this devastating disease. Particular side effects of interest include QT prolongation

A randomized open label trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis is a leading cause of death in HIV-infected patients. International treatment guidelines recommend induction therapy with amphotericin B and flucytosine. This antifungal combination is most effective, but unfortunately flucytosine is expensive and unavailable where the burden of disease is greatest. Where unavailable, guidelines recommend treatment with amphotericin and fluconazole, but this is less effective, with mortality rates of 40-50%. Faster rates of clearance of yeast from cerebrospinal fluid (CSF) are associated with better outcomes - improving the potency of antifungal therapy is likely to be an effective strategy to improve survival. Tamoxifen, a selective estrogen receptor modulator used to treat breast cancer, has anti-cryptococcal activity, appearing synergistic when combined in vitro with amphotericin, and fungicidal when combined with fluconazole. It is concentrated in the brain and macrophages, off-patent, cheap and widely available. We designed a randomized trial to deliver initial efficacy and safety data for tamoxifen combined with amphotericin and fluconazole.Method: A phase II, open-label, randomized (1:1) controlled trial of tamoxifen (300mg/day) combined with amphotericin (1mg/kg/day) and fluconazole (800mg/day) for the first 2 weeks therapy for HIV infected or uninfected adults with cryptococcal meningitis. The study recruits at Cho Ray Hospital and the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. The primary end point is Early Fungicidal Activity (EFA-the rate of yeast clearance from CSF), over the first two weeks of treatment. 50 patients will be recruited providing ≈80% and 90% power to detect a difference in the EFA of -0.11 or -0.13 log10CFU/ml/day, respectively.Discussion: The results of the study will inform the decision to proceed to a larger trial powered to mortality. The size of effect detectable has previously been associated with reduced mortality from this devastating disease. Particular side effects of interest include QT prolongation.</br

An open label randomized controlled trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anticancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods: Open label randomized controlled trial. Participants received standard care – amphotericin combined with fluconazole for the first 2 weeks – or standard care plus tamoxifen 300 mg/day. The primary end point was Early Fungicidal Activity (EFA) – the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031. Results: Fifty patients were enrolled (median age 34 years, 35 male). Tamoxifen had no effect on EFA (−0.48log10 colony-forming units/mL/CSF control arm versus −0.49 tamoxifen arm, difference −0.005log10CFU/ml/day, 95% CI: −0.16, 0.15, p=0.95). Tamoxifen caused QTc prolongation. Conclusions: High-dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding: The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA