Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM.

Cryptococcosis is a major worldwide disseminated invasive fungal infection. Cryptococcosis, particularly in its most lethal manifestation of cryptococcal meningitis, accounts for substantial mortality and morbidity. The breadth of the clinical cryptococcosis syndromes, the different patient types at-risk and affected, and the vastly disparate resource settings where clinicians practice pose a complex array of challenges. Expert contributors from diverse regions of the world have collated data, reviewed the evidence, and provided insightful guideline recommendations for health practitioners across the globe. This guideline offers updated practical guidance and implementable recommendations on the clinical approaches, screening, diagnosis, management, and follow-up care of a patient with cryptococcosis and serves as a comprehensive synthesis of current evidence on cryptococcosis. This Review seeks to facilitate optimal clinical decision making on cryptococcosis and addresses the myriad of clinical complications by incorporating data from historical and contemporary clinical trials. This guideline is grounded on a set of core management principles, while acknowledging the practical challenges of antifungal access and resource limitations faced by many clinicians and patients. More than 70 societies internationally have endorsed the content, structure, evidence, recommendation, and pragmatic wisdom of this global cryptococcosis guideline to inform clinicians about the past, present, and future of care for a patient with cryptococcosis.

Shared and unique antibody and B cell profiles in HIV-positive and HIV-negative individuals with cryptococcal meningoencephalitis.

Host non-T cell markers to aid in the diagnosis of cryptococcal meningoencephalitis (CM) have not been identified. In this case-control study, we characterized antibody and B cell profiles in HIV-negative and HIV-positive Vietnamese individuals of the Kinh ethnicity recently diagnosed with CM and controls. The study included 60 HIV-negative with no known immunocompromising condition and 60 HIV-positive individuals, with 30 CM cases and 30 controls in each group. Participants were matched by age, sex, HIV serostatus, and CD4 count in the HIV-positive group. Plasma immunoglobulin (Ig) levels, including IgG1, IgG2, IgM, and IgA, Cryptococcus spp. glucuronoxylomannan (GXM)- and laminarin (branched ${\rm{\beta }}$-[1-3]-glucan)-binding IgG, IgM, IgA levels, and peripheral blood B cell subsets were measured. Logistic regression, principal component, and mediation analyses were conducted to assess associations between antibody, B cell levels, and CM. The results showed that GXM-IgG levels were higher and IgG1 and IgG2 were lower in CM cases than controls, regardless of HIV status. In HIV-negative individuals, IgG2 mediated an inverse association between CD19+CD27+CD43+CD5- (B-1b-like) cells and CM. In HIV-positive individuals, lower levels of IgA, laminarin-IgA, and CD19+CD27+IgM+IgD- (IgM+ memory B) cells were each associated with CM. The shared and distinct antibody and B cell profiles identified in HIV-negative and HIV-positive CM cases may inform the identification of non-T-cell markers of CM risk or unsuspected disease, particularly in HIV-negative individuals.

Unlike cryptococcal meningitis (CM) in HIV-positive individuals, there are no known biomarkers of risk in HIV-negative individuals and the diagnosis is often not suspected and delayed. This study identified non-T cells, including antibody and B cell CM-associated profiles that may guide cryptococcal antigen testing in HIV-negative individuals.

Rise in alanine aminotransferase after HCV treatment is a highly sensitive screen for treatment failure.

Nucleic acid testing to confirm sustained virological response (SVR) after HCV therapy is technical, often expensive, and frequently unavailable where disease prevalence is highest. Alternative surrogate biomarkers merit evaluation. In a short-treatment trial in Vietnam (SEARCH-1; n = 52) we analysed how changes in alanine transaminase (ΔALT) and aspartate transaminase (ΔAST), from end of treatment (EOT) to EOT + 12 weeks, related to SVR, defined as HCV RNA < lower limit of quantification 12 weeks after EOT. In a separate UK trial (STOPHCV1; n = 202), we then tested the hypothesis that any elevation in ALT or AST between EOT and EOT12 is a sensitive screen for treatment failure. In SEARCH-1, among 48 individuals with data, 13 failed to achieve SVR. Median ΔALT and ΔAST were negative in cured patients but elevated when treatment failed [median ΔALT (IQR): -2 IU/L (-6, +2)] versus +17 IU/L (+7.5, +38) (p< 0.001). Amongst treatment failures, 12/13 had increase in ALT and 13/13 had increase in AST after EOT, compared with 12/35 in those cured. In STOPHCV1, 196/202 patients had evaluable data, of which 57 did not achieve SVR. A rise in ALT after EOT was 100% sensitive (95% C.I. [93.7 - 100%]) and 51% specific (42.4 - 59.7%) for detecting treatment failure. ΔAST >0 IU/L was 98.1% (89.9 - 99.9%) sensitive and 35.8% (27.3 - 45.1%) specific. A rise in ALT or AST after HCV therapy is a highly sensitive screen for treatment failure in mild liver disease. This finding could reduce costs and complexity of managing HCV.

Efficacy of ultra-short, response-guided sofosbuvir and daclatasvir therapy for hepatitis C in a single-arm mechanistic pilot study.

Background: World Health Organization has called for research into predictive factors for selecting persons who could be successfully treated with shorter durations of direct-acting antiviral (DAA) therapy for hepatitis C. We evaluated early virological response as a means of shortening treatment and explored host, viral and pharmacokinetic contributors to treatment outcome. Methods: Duration of sofosbuvir and daclatasvir (SOF/DCV) was determined according to day 2 (D2) virologic response for HCV genotype (gt) 1- or 6-infected adults in Vietnam with mild liver disease. Participants received 4- or 8-week treatment according to whether D2 HCV RNA was above or below 500 IU/ml (standard duration is 12 weeks). Primary endpoint was sustained virological response (SVR12). Those failing therapy were retreated with 12 weeks SOF/DCV. Host IFNL4 genotype and viral sequencing was performed at baseline, with repeat viral sequencing if virological rebound was observed. Levels of SOF, its inactive metabolite GS-331007 and DCV were measured on days 0 and 28. Results: Of 52 adults enrolled, 34 received 4 weeks SOF/DCV, 17 got 8 weeks and 1 withdrew. SVR12 was achieved in 21/34 (62%) treated for 4 weeks, and 17/17 (100%) treated for 8 weeks. Overall, 38/51 (75%) were cured with first-line treatment (mean duration 37 days). Despite a high prevalence of putative NS5A-inhibitor resistance-associated substitutions (RASs), all first-line treatment failures cured after retreatment (13/13). We found no evidence treatment failure was associated with host IFNL4 genotype, viral subtype, baseline RAS, SOF or DCV levels. Conclusions: Shortened SOF/DCV therapy, with retreatment if needed, reduces DAA use in patients with mild liver disease, while maintaining high cure rates. D2 virologic response alone does not adequately predict SVR12 with 4-week treatment. Funding: Funded by the Medical Research Council (Grant MR/P025064/1) and The Global Challenges Research 70 Fund (Wellcome Trust Grant 206/296/Z/17/Z).

Hepatitis C is a blood-borne virus that causes thousands of deaths from liver cirrhosis and liver cancer each year. Antiviral therapies can cure most cases of infection in 12 weeks. Unfortunately, treatment is expensive, and sticking with the regimen for 12 weeks can be difficult. It may be especially challenging for unhoused people or those who use injection drugs and who have high rates of hepatitis C infection. Shorter durations of therapy may make it more accessible, especially for high-risk populations. But studies of shorter antiviral treatment durations have yet to produce high enough cure rates. Finding ways to identify patients who would benefit from shorter therapy is a key goal of the World Health Organization. Potential characteristics that may predict a faster treatment response include low virus levels before initiating treatment, patient genetics, drug resistance mutations in the virus, and higher drug levels in the patient's blood during treatment. For example, previous research showed that a rapid decrease in virus levels in a patient's blood two days after starting antiviral therapy with three drugs predicted patient cures after three weeks of treatment. To test if high cure rates could be achieved in just four weeks of treatment, Flower et al. enrolled 52 patients with hepatitis C in a study to receive the most widely accessible dual antiviral treatment (sofosbuvir and daclatasvir). Participants received four or eight weeks of treatment, depending on the amount of viral RNA in their blood after two days of treatment. The results indicate that a rapid decrease in virus levels in the blood does not adequately predict cure rates with four weeks of two-drug combination therapy. However, eight weeks may be highly effective, regardless of viral levels early in treatment. Thirty-four individuals with low virus levels on the second day of treatment received four weeks of therapy, which cured 21 or 62% of them. All seventeen individuals with higher viral levels on day two were cured after eight weeks of treatment. Twelve weeks of retreatment was sufficient to cure the 13 individuals who did not achieve cure with four weeks of therapy. Even patients with drug resistance genes after the first round of therapy responded to a longer second round. Flower et al. show that patient genetics, virus subtype, drug levels in the patient's blood, and viral drug resistance genes before therapy, were not associated with patient cures after four weeks of treatment. Given that retreatment is safe and effective, larger studies are now needed to determine whether eight weeks of therapy with sofosbuvir and daclatasvir may be enough to cure patients with mild liver disease. More studies are also necessary to identify patients that may benefit from shorter therapy durations. Finding ways to shorten antiviral therapy for hepatitis C could help make treatment more accessible and reduce therapy costs for both individuals and governments.

Global estimates and determinants of antituberculosis drug pharmacokinetics in children and adolescents: a systematic review and individual patient data meta-analysis.

BACKGROUND: Suboptimal exposure to antituberculosis (anti-TB) drugs has been associated with unfavourable treatment outcomes. We aimed to investigate estimates and determinants of first-line anti-TB drug pharmacokinetics in children and adolescents at a global level. METHODS: We systematically searched MEDLINE, Embase and Web of Science (1990-2021) for pharmacokinetic studies of first-line anti-TB drugs in children and adolescents. Individual patient data were obtained from authors of eligible studies. Summary estimates of total/extrapolated area under the plasma concentration-time curve from 0 to 24 h post-dose (AUC0-24) and peak plasma concentration (C max) were assessed with random-effects models, normalised with current World Health Organization-recommended paediatric doses. Determinants of AUC0-24 and C max were assessed with linear mixed-effects models. RESULTS: Of 55 eligible studies, individual patient data were available for 39 (71%), including 1628 participants from 12 countries. Geometric means of steady-state AUC0-24 were summarised for isoniazid (18.7 (95% CI 15.5-22.6) h·mg·L-1), rifampicin (34.4 (95% CI 29.4-40.3) h·mg·L-1), pyrazinamide (375.0 (95% CI 339.9-413.7) h·mg·L-1) and ethambutol (8.0 (95% CI 6.4-10.0) h·mg·L-1). Our multivariate models indicated that younger age (especially <2 years) and HIV-positive status were associated with lower AUC0-24 for all first-line anti-TB drugs, while severe malnutrition was associated with lower AUC0-24 for isoniazid and pyrazinamide. N-acetyltransferase 2 rapid acetylators had lower isoniazid AUC0-24 and slow acetylators had higher isoniazid AUC0-24 than intermediate acetylators. Determinants of C max were generally similar to those for AUC0-24. CONCLUSIONS: This study provides the most comprehensive estimates of plasma exposures to first-line anti-TB drugs in children and adolescents. Key determinants of drug exposures were identified. These may be relevant for population-specific dose adjustment or individualised therapeutic drug monitoring.

Treatment of Cryptococcal Meningitis: How Have We Got Here and Where are We Going?

Cryptococcal meningitis is a devastating brain infection cause by encapsulated yeasts of the Cryptococcus genus. Exposure, through inhalation, is likely universal by adulthood, but symptomatic infection only occurs in a minority, in most cases, months or years after exposure. Disease has been described in almost all tissues, but it is the organism's tropism for the central nervous system that results in the most devastating illness. While invasive disease can occur in the immunocompetent, the greatest burden by far is in immunocompromised individuals, particularly people living with human immunodeficiency virus (HIV), organ transplant recipients and those on glucocorticoid therapy or other immunosuppressive drugs. Clinical presentation is variable, but diagnosis is usually straightforward, with cerebrospinal fluid microscopy, culture, and antigen testing proving significantly more sensitive than diagnostic tests for other brain infections. Although disease incidence has reduced since the advent of effective HIV therapy, mortality when disease occurs remains extremely high, and has changed little in recent decades. This Therapy in Practice review is an update of a talk first given by JND at the European Congress on Clinical Microbiology and Infectious Diseases in 2019 in the Netherlands. The review contextualizes the most recently published World Health Organization (WHO) guidelines for the treatment of HIV-associated cryptococcal meningitis in terms of the data from large, randomized, controlled trials published between 1997 and 2022. We discuss the rationale for induction and maintenance therapy and the efficacy and undesirable effects of the current therapeutic armamentarium of amphotericin, flucytosine and fluconazole. We address recent research into repurposed drugs such as sertraline and tamoxifen, and potential future treatment options, including the novel antifungals fosmanogepix, efungumab and oteseconazole, and non-pharmaceutical solutions such as neurapheresis cerebrospinal fluid filtration.

Clinical and laboratory factors associated with neonatal sepsis mortality at a major Vietnamese children's hospital.

Sepsis is a major cause of neonatal mortality and children born in low- and middle-income countries (LMICs) are at greater risk of severe neonatal infections than those in higher-income countries. Despite this disparity, there are limited contemporaneous data linking the clinical features of neonatal sepsis with outcome in LMICs. Here, we aimed to identify factors associated with mortality from neonatal sepsis in Vietnam. We conducted a prospective, observational study to describe the clinical features, laboratory characteristics, and mortality rate of neonatal sepsis at a major children's hospital in Ho Chi Minh City. All in-patient neonates clinically diagnosed with probable or culture-confirmed sepsis meeting inclusion criteria from January 2017 to June 2018 were enrolled. We performed univariable analysis and logistic regression to identify factors independently associated with mortality. 524 neonates were recruited. Most cases were defined as late-onset neonatal sepsis and were hospital-acquired (91.4% and 73.3%, respectively). The median (IQR) duration of hospital stay was 23 (13-41) days, 344/524 (65.6%) had a positive blood culture (of which 393 non-contaminant organisms were isolated), and 69/524 (13.2%) patients died. Coagulase-negative staphylococci (232/405; 57.3%), Klebsiella spp. (28/405; 6.9%), and Escherichia coli (27/405; 6.7%) were the most isolated organisms. Sclerema (OR = 11.4), leukopenia <4,000/mm3 (OR = 7.8), thrombocytopenia <100,000/mm3 (OR = 3.7), base excess < -20 mEq/L (OR = 3.6), serum lactate >4 mmol/L (OR = 3.4), extremely low birth weight (OR = 3.2), and hyperglycaemia >180 mg/dL (OR = 2.6) were all significantly (p<0.05) associated with mortality. The identified risk factors can be adopted as prognostic factors for the diagnosis and treatment of neonatal sepsis and enable early risk stratification and interventions appropriate to reduce neonatal sepsis in LMIC settings.

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.

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam, with marked variability by land use type.

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR34 /L98H most frequent (34/38).

Cryptococcal meningoencephalitis: time for action.

Cryptococcal meningoencephalitis was first described over a century ago. This fungal infection is preventable and treatable yet continues to be associated with excessive morbidity and mortality. The largest burden of disease resides in people living with HIV in low-income and middle-income countries. In this group, mortality with the best antifungal induction regimen (7 days of amphotericin B deoxycholate [1·0 mg/kg per day] and flucytosine [100·0 mg/kg per day]) in a clinical trial setting was 24% at 10 weeks. The world is now at an inflection point in terms of recognition, research, and action to address the burden of morbidity and mortality from cryptococcal meningoencephalitis. However, the scope of interventional programmes needs to increase, with particular attention to implementation science that is specific to individual countries. This Review summarises causes of excessive mortality, interventions with proven survival benefit, and gaps in knowledge and practice that contribute to the ongoing high death toll from cryptococcal meningoencephalitis. TRANSLATIONS: For the Vietnamese and Chichewa translations of the abstract see Supplementary Materials section.

Superiority of a Novel Mp1p Antigen Detection Enzyme Immunoassay Compared to Standard BACTEC Blood Culture in the Diagnosis of Talaromycosis.

BACKGROUND: Talaromycosis is an invasive mycosis endemic in Southeast Asia and causes substantial morbidity and mortality in individuals with advanced human immunodeficiency virus (HIV) disease. Current diagnosis relies on isolating Talaromyces marneffei in cultures, which takes up to 14 days and is detectable only during late-stage infection, leading to high mortality. METHODS: In this retrospective case-control study, we assessed the accuracy of a novel Mp1p antigen-detecting enzyme immunoassay (EIA) in stored plasma samples of 372 patients who had culture-proven talaromycosis from blood or sterile body fluids (reference standard) and 517 individuals without talaromycosis (338 healthy volunteers; 179 with other infections). All participants were recruited between 2011 and 2017 in Vietnam. RESULTS: Of cases and controls, 66.1% and 75.4%, respectively, were male; the median age was 33 and 37, respectively. All cases were HIV infected; median CD4 count was 10 cells/µL. At an optical density cutoff of 0.5, the specificity was 98.1% (95% CI, 96.3%-99.0%); the sensitivity was superior to blood culture (86.3% [95% CI, 82.3%-89.5%] vs 72.8% [95% CI, 68.0%-77.2%]) (P < .001, McNemar test). The time to diagnosis was 6 hours vs 6.6 ± 3.0 days for blood culture. Paired plasma and urine testing in the same patients (n = 269) significantly increased sensitivity compared to testing plasma alone or testing urine alone (P < .001 and P = .02, respectively, McNemar test). CONCLUSIONS: The Mp1p EIA is highly specific and is superior in sensitivity and time to diagnosis compared to blood culture for the diagnosis of talaromycosis. Paired plasma and urine testing further increases sensitivity, introducing a new tool for rapid diagnosis, enabling early treatment and potentially reducing mortality.

Antifungal Susceptibility Does Not Correlate With Fungal Clearance or Survival in AIDS-Associated Cryptococcal Meningitis.

We investigated the value of susceptibility testing in predicting response in AIDS-associated cryptococcal meningitis using clinical isolates from a randomized controlled trial of antifungal treatment (amphotericin monotherapy, amphotericin with flucytosine, or amphotericin with fluconazole). We found no correlation between antifungal susceptibility and either early or late survival, or fungal clearance.

Occult Talaromyces marneffei Infection Unveiled by the Novel Mp1p Antigen Detection Assay.

Talaromyces marneffei causes fatal invasive mycosis in Southeast Asia. Diagnosis by culture has limited sensitivity and can result in treatment delay. We describe the use of a novel Mp1p enzyme immunoassay (EIA) to identify blood culture-negative talaromycosis, subsequently confirmed by bone marrow cultures. This EIA has the potential to speed diagnosis, enabling early therapy initiation.

Drug-Resistant Aspergillus flavus Is Highly Prevalent in the Environment of Vietnam: A New Challenge for the Management of Aspergillosis?

The burden of aspergillosis, especially Chronic Pulmonary Aspergillosis, is increasingly recognized, and the increasing presence of azole-resistant environmental Aspergillus fumigatus has been highlighted as a health risk. However, a sizable minority of aspergillosis is caused by Aspergillus flavus, which is assumed to be sensitive to azoles but is infrequently included in surveillance. We conducted environmental sampling at 150 locations in a rural province of southern Vietnam. A. flavus isolates were identified morphologically, their identity was confirmed by sequencing of the beta-tubulin gene, and then they were tested for susceptibility to azoles and amphotericin B according to EUCAST methodologies. We found that over 85% of A. flavus isolates were resistant to at least one azole, and half of them were resistant to itraconazole. This unexpectedly high prevalence of resistance demands further investigation to determine whether it is linked to agricultural azole use, as has been described for A. fumigatus. Clinical correlation is required, so that guidelines can be adjusted to take this information into account.

Population Pharmacokinetic Properties of Antituberculosis Drugs in Vietnamese Children with Tuberculous Meningitis.

Optimal dosing of children with tuberculous meningitis (TBM) remains uncertain and is currently based on the treatment of pulmonary tuberculosis in adults. This study aimed to investigate the population pharmacokinetics of isoniazid, rifampin, pyrazinamide, and ethambutol in Vietnamese children with TBM, to propose optimal dosing in these patients, and to determine the relationship between drug exposure and treatment outcome. A total of 100 Vietnamese children with TBM were treated with an 8-month antituberculosis regimen. Nonlinear mixed-effects modeling was used to evaluate the pharmacokinetic properties of the four drugs and to simulate different dosing strategies. The pharmacokinetic properties of rifampin and pyrazinamide in plasma were described successfully by one-compartment disposition models, while those of isoniazid and ethambutol in plasma were described by two-compartment disposition models. All drug models included allometric scaling of body weight and enzyme maturation during the first years of life. Cerebrospinal fluid (CSF) penetration of rifampin was relatively poor and increased with increasing protein levels in CSF, a marker of CSF inflammation. Isoniazid and pyrazinamide showed good CSF penetration. Currently recommended doses of isoniazid and pyrazinamide, but not ethambutol and rifampin, were sufficient to achieve target exposures. The ethambutol dose cannot be increased because of ocular toxicity. Simulation results suggested that rifampin dosing at 50 mg/kg of body weight/day would be required to achieve the target exposure. Moreover, low rifampin plasma exposure was associated with an increased risk of neurological disability. Therefore, higher doses of rifampin could be considered, but further studies are needed to establish the safety and efficacy of increased dosing.

A multi centre randomized open label trial of chloroquine for the treatment of adults with SARS-CoV-2 infection in Vietnam.

Background: COVID-19 is a respiratory disease caused by a novel coronavirus (SARS-CoV-2) and causes substantial morbidity and mortality. There is currently no vaccine to prevent COVID-19 or therapeutic agent to treat COVID-19. This clinical trial is designed to evaluate chloroquine as a potential therapeutic for the treatment of hospitalised people with COVID-19. We hypothesise that chloroquine slows viral replication in patients with COVID-19, attenuating the infection, and resulting in more rapid decline of viral load in throat/nose swabs. This viral attenuation should be associated with improved patient outcomes. Method: The study will start with a 10-patient prospective observational pilot study following the same entry and exclusion criteria as for the randomized trial and undergoing the same procedures. The main study is an open label, randomised, controlled trial with two parallel arms of standard of care (control arm) versus standard of care with 10 days of chloroquine (intervention arm) with a loading dose over the first 24 hours, followed by 300mg base orally once daily for nine days. The study will recruit patients in three sites in Ho Chi Minh City, Vietnam: the Hospital for Tropical Diseases, the Cu Chi Field Hospital, and the Can Gio COVID hospital. The primary endpoint is the time to viral clearance from throat/nose swab, defined as the time following randomization until the midpoint between the last positive and the first of the negative throat/nose swabs. Viral presence will be determined using RT-PCR to detect SARS-CoV-2 RNA. Discussion: The results of the study will add to the evidence-based guidelines for management of COVID-19. Given the enormous experience of its use in malaria chemoprophylaxis, excellent safety and tolerability profile, and its very low cost, if proved effective then chloroquine would be a readily deployable and affordable treatment for patients with COVID-19. Trial registration: Clinicaltrials.gov NCT04328493 31/03/2020.

Assessing the virulence of Cryptococcus neoformans causing meningitis in HIV infected and uninfected patients in Vietnam.

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in individuals who are uninfected with human immunodeficiency virus (HIV). This disease was associated with a single genotype of Cryptococcus neoformans (sequence type [ST]5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans, we selected 30 representative Vietnamese isolates and compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37°C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (P < .001). Counterintuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF-α concentrations (P < .001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

The combination of tamoxifen with amphotericin B, but not with fluconazole, has synergistic activity against the majority of clinical isolates of Cryptococcus neoformans.

BACKGROUND: Cryptococcal meningitis has fatality rates of 40%-70%, resulting in 200 000 deaths each year. The best outcomes are achieved with amphotericin combined with flucytosine but flucytosine is expensive and unavailable where most disease occurs. More effective and affordable treatments are needed. Tamoxifen, a selective oestrogen receptor modulator frequently indicated for breast cancer, has been found to have synergistic activity against the Cryptococcus neoformans type strain when combined with amphotericin or fluconazole. It is cheap, off-licence, widely available and well-tolerated, and thus a pragmatic potential treatment for cryptococcal disease. OBJECTIVES: We wanted to determine the susceptibility of clinical isolates of C. neoformans to tamoxifen alone and in combination with other antifungals, to determine whether there is sufficient evidence of activity to justify a clinical trial. METHODS: We used the CLSI broth microdilution protocol to test the susceptibility of 30 randomly selected clinical isolates of C. neoformans to tamoxifen, in dual combination with amphotericin, fluconazole or flucytosine, and in triple combination with amphotericin and fluconazole. Evidence of drug interactions was assessed using the fractional inhibitory concentration index. RESULTS: The MIC50 and MIC90 of tamoxifen were 4 and 16 mg/L, respectively. The combination of tamoxifen and amphotericin suggested a synergistic interaction in 20 of 30 (67%) isolates. There was no interaction between tamoxifen and either fluconazole or flucytosine. Synergy was maintained in 3-Dimensional chequerboard testing. There was no evidence of antagonism. CONCLUSIONS: Tamoxifen may be a useful addition to treatment with amphotericin and fluconazole for cryptococcal meningitis; a trial is justified.

Multilocus sequence typing of Cryptococcus neoformans var. grubii from Laos in a regional and global context.

Cryptococcosis causes approximately 180 000 deaths each year in patients with human immunodeficiency virus (HIV). Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii, responsible for the majority of cases, is distributed globally. We used the consensus ISHAM Multilocus sequence typing (MLST) scheme to define the population structure of clinical C. neoformans var. grubii isolates from Laos (n = 81), which we placed into the global context using published MLST data from other countries (total N = 1047), including a reanalysis of 136 Vietnamese isolates previously reported. We observed a phylogeographical relationship in which the Laotian population was similar to its neighbor Thailand, being dominated (83%) by Sequence Types (ST) 4 and 6. This phylogeographical structure changed moving eastwards, with Vietnam's population consisting of an admixture of isolates dominated by the ST4/ST6 (35%) and ST5 (48%) lineages. The ST5 lineage is the predominant ST reported from China and East Asia, where it accounts for >90% of isolates. Analysis of genetic distance (Fst) between different populations of C. neoformans var. grubii supports this intermediate structure of the Vietnamese population. The pathogen and host diversity reported from Vietnam provide the strongest epidemiological evidence of the association between ST5 and HIV-uninfected patients. Regional anthropological genetic distances suggest diversity in the C. neoformans var. grubii population across Southeast Asia is driven by ecological rather than human host factors. Where the ST5 lineage is present, disease in HIV-uninfected patients is to be expected.