Environmentally Resilient Microfluidic Point-of-Care Immunoassay Enables Rapid Diagnosis of Talaromycosis.

Point-of-care tests (POCTs) are increasingly being used in field settings, particularly outdoors. The performance of current POCTs─most commonly the lateral flow immunoassay─can be adversely affected by ambient temperature and humidity. We developed a self-contained immunoassay platform─the D4 POCT─that can be conducted at the POC by integrating all reagents in a capillary-driven passive microfluidic cassette that minimizes user intervention. The assay can be imaged and analyzed on a portable fluorescence reader─the D4Scope─and provide quantitative outputs. Here, we systematically investigated the resilience of our D4 POCT to varied temperature and humidity and to physiologically diverse human whole blood samples that span a wide range of physiological hematocrit (30-65%). For all conditions, we showed that the platform maintained high sensitivity (0.05-0.41 ng/mL limits of detection). The platform also demonstrated good accuracy in reporting true analyte concentration across environmental extremes when compared to the manually operated format of the same test to detect a model analyte─ovalbumin. Additionally, we engineered an improved version of the microfluidic cassette that improved the ease-of-use of the device and shortened the time-to-result. We implemented this new cassette to create a rapid diagnostic test to detect talaromycosis infection in patients with advanced HIV disease at the POC, demonstrating comparable sensitivity and specificity to the laboratory test for the disease.

Impact of pathogen genetics on clinical phenotypes in a population of Talaromyces marneffei from Vietnam.

Talaromycosis, a severe and invasive fungal infection caused by Talaromyces marneffei, is difficult to treat and impacts those living in endemic regions of Southeast Asia, India, and China. While 30% of infections result in mortality, our understanding of the genetic basis of pathogenesis for this fungus is limited. To address this, we apply population genomics and genome-wide association study approaches to a cohort of 336 T. marneffei isolates collected from patients who enrolled in the Itraconazole vs Amphotericin B for Talaromycosis trial in Vietnam. We find that isolates from northern and southern Vietnam form two distinct geographical clades, with isolates from southern Vietnam associated with increased disease severity. Leveraging longitudinal isolates, we identify multiple instances of disease relapse linked to unrelated strains, highlighting the potential for multistrain infections. In more frequent cases of persistent talaromycosis caused by the same strain, we identify variants arising over the course of patient infections that impact genes predicted to function in the regulation of gene expression and secondary metabolite production. By combining genetic variant data with patient metadata for all 336 isolates, we identify pathogen variants significantly associated with multiple clinical phenotypes. In addition, we identify genes and genomic regions under selection across both clades, highlighting loci undergoing rapid evolution, potentially in response to external pressures. With this combination of approaches, we identify links between pathogen genetics and patient outcomes and identify genomic regions that are altered during T. marneffei infection, providing an initial view of how pathogen genetics affects disease outcomes.

Impact of pathogen genetics on clinical phenotypes in a population of Talaromyces marneffei from Vietnam.

Talaromycosis, a severe and invasive fungal infection caused by Talaromyces marneffei , is difficult to treat and impacts those living in endemic regions of southeast Asia, India, and China. While 30% of infections result in mortality, our understanding of the genetic basis of pathogenesis for this fungus is limited. To address this, we apply population genomics and genome wide association study approaches to a cohort of 336 T. marneffei isolates collected from patients who enrolled in the Itraconazole versus Amphotericin B for Talaromycosis (IVAP) trial in Vietnam. We find that isolates from northern and southern Vietnam form two distinct geographical clades, with isolates from southern Vietnam associated with increased disease severity. Leveraging longitudinal isolates, we identify multiple instances of disease relapse linked to unrelated strains, highlighting the potential for multi-strain infections. In more frequent cases of persistent talaromycosis caused by the same strain, we identify variants arising over the course of patient infections that impact genes predicted to function in the regulation of gene expression and secondary metabolite production. By combining genetic variant data with patient metadata for all 336 isolates, we identify pathogen variants significantly associated with multiple clinical phenotypes. In addition, we identify genes and genomic regions under selection across both clades, highlighting loci undergoing rapid evolution, potentially in response to external pressures. With this combination of approaches, we identify links between pathogen genetics and patient outcomes and identify genomic regions that are altered during T. marneffei infection, providing an initial view of how pathogen genetics affects disease outcomes.

A global call for talaromycosis to be recognised as a neglected tropical disease.

Talaromycosis (penicilliosis) is an invasive mycosis that is endemic in tropical and subtropical Asia. Talaromycosis primarily affects individuals with advanced HIV disease and other immunosuppressive conditions, and the disease disproportionally affects people in low-income and middle-income countries, particularly agricultural workers in rural areas during their most economically productive years. Approximately 17 300 talaromycosis cases and 4900 associated deaths occur annually. Talaromycosis is highly associated with the tropical monsoon season, when flooding and cyclones can exacerbate the poverty-inducing potential of the disease. Talaromycosis can present as localised or disseminated disease, the latter causing cutaneous lesions that are disfiguring and stigmatising. Despite up to a third of diagnosed cases resulting in death, talaromycosis has received little attention and investment from regional and global funders, policy makers, researchers, and industry. Diagnostic and treatment modalities remain extremely insufficient, however control of talaromycosis is feasible with known public health strategies. This Viewpoint is a global call for talaromycosis to be recognised as a neglected tropical disease to alleviate its impact on susceptible populations.

Population Pharmacokinetics and Pharmacodynamics of Itraconazole for Disseminated Infection Caused by Talaromyces marneffei.

First-line treatment of talaromycosis with amphotericin B deoxycholate (DAmB) is labor-intensive and toxic. Itraconazole is an appealing alternative antifungal agent. Pharmacokinetic data were obtained from 76 patients who were randomized to itraconazole in the Itraconazole versus Amphotericin B for Talaromycosis (IVAP) trial. Plasma levels of itraconazole and its active metabolite, hydroxyitraconazole, were analyzed alongside longitudinal fungal CFU counts in a population model. Itraconazole and hydroxyitraconazole pharmacokinetic variability was considerable, with areas under the concentration-time curve over 24 h (AUC24) of 3.34 ± 4.31 mg·h/liter and 3.57 ± 4.46 mg·h/liter (mean ± standard deviation), respectively. Levels of both analytes were low; itraconazole minimum concentration (Cmin) was 0.11 ± 0.16 mg/liter, and hydroxyitraconazole Cmin was 0.13 ± 0.17 mg/liter. The mean maximal rates of drug-induced killing were 0.206 and 0.208 log10 CFU/ml/h, respectively. There were no associations between itraconazole Cmin/MIC and time to sterilization of the bloodstream (hazard ratio [HR], 1.01; 95% confidence interval [CI], 0.99 to 1.03; P = 0.43), time to death (HR, 0.99; 95% CI, 0.96 to 1.02; P = 0.77), or early fungicidal activity (EFA) (coefficient, -0.004; 95% CI, -0.010 to 0.002; P = 0.18). Similarly, there was no relationship between AUC/MIC and time to sterilization of the bloodstream (HR, 1.00; 95% CI, 0.99 to 1.00; P = 0.50), time to death (HR, 1.00; 95% CI, 0.99 to 1.00; P = 0.91), or EFA (coefficient, -0.0001; 95% CI, -0.0003 to 0.0001; P = 0.19). This study raises the possibility that the failure of itraconazole to satisfy noninferiority criteria against DAmB for talaromycosis in the IVAP trial was a pharmacokinetic and pharmacodynamic failure.

Cost-Effectiveness of Amphotericin B Deoxycholate Versus Itraconazole for Induction Therapy of Talaromycosis in Human Immunodeficiency Virus-Infected Adults in Vietnam.

BACKGROUND: Talaromycosis (penicilliosis) is an invasive fungal infection and a major cause of human immunodeficiency virus (HIV)-related deaths in Southeast Asia. Guidelines recommend induction therapy with amphotericin B deoxycholate; however, treatment with itraconazole has fewer toxic effects, is easier to administer, and is less expensive. Our recent randomized controlled trial in Vietnam found that amphotericin B was superior to itraconazole with respect to 6-month mortality. We undertook an economic evaluation alongside this trial to determine whether the more effective treatment is cost-effective. METHODS: Resource use, direct and indirect costs, and health and quality-of-life outcomes (measured using quality-adjusted life-years [QALYs]) were evaluated for 405 trial participants from 2012 to 2016. Both a Vietnamese health service and a broader societal costing perspective were considered. Mean costs and QALYs were combined to calculate the within-trial cost-effectiveness of amphotericin vs itraconazole from both perspectives. RESULTS: From a Vietnamese health service perspective, amphotericin increases costs but improves health outcomes compared to itraconazole, at a cost of $3013/QALY gained. The probability that amphotericin is cost-effective at a conventional (World Health Organization CHOICE) threshold of value for money is 46%. From a societal perspective, amphotericin is cost-reducing and improves outcomes compared to itraconazole, and is likely to be a cost-effective strategy at any value for money threshold greater than $0. CONCLUSIONS: Our analysis indicates that induction therapy with amphotericin is a cost-effective treatment strategy for HIV-infected adults diagnosed with talaromycosis in Vietnam. These results provide the evidence base for health care providers and policy makers to improve access to and use of amphotericin.

Neurocognitive Trajectories After 72 Weeks of First-Line Anti-retroviral Therapy in Vietnamese Adults With HIV-HCV Co-infection.

Background: Long-term neurocognitive outcomes following first-line suppressive anti-retroviral therapy (ART) remain uncertain for individuals with HIV and hepatitis C (HCV) co-infection. The study examined neurocognitive performance before and after 72 weeks of ART using repeated multivariate analyses and latent trajectory models. Methods: One hundred and sixty adults with chronic, untreated HIV infection (n = 80 with HCV co-infection and n = 80 HIV mono-infected) and 80 demographically similar healthy controls were recruited from the Hospital for Tropical Diseases in Ho Chi Minh City and the surrounding community, respectively. Neurocognitive measures (adapted for use in Vietnam) and liver enzyme tests were compared across groups at baseline. Repeated multivariate and group-based trajectory analyses (GBTA) examined neurocognitive subgroup profiles of the co-infected individuals after 72 weeks of de novo efavirenz- (n = 41) or raltegravir-based (n = 39) ART. Results: Baseline analyses revealed worse motor function in HIV-HCV co-infected individuals compared to both comparison groups. Longitudinal analyses revealed improved neurocognitive performance by week 48 for most participants regardless of treatment arm. GBTA identified a subgroup (35% of HIV-HCV sample) with persistent motor impairment despite otherwise successful ART. Higher HIV viral load and lower CD4+ T cell count at baseline predicted persistent motor dysfunction. Liver indices and ART regimen did not predict neurocognitive outcomes in HIV-HCV co-infected individuals. Conclusions: Most HIV-HCV co-infected individuals achieve normative neurocognitive performance after 48 weeks of de novo suppressive ART. However, individuals with more severe HIV disease prior to ART exhibited motor impairment at baseline and 72 weeks after otherwise successful treatment. Interventions aimed at improving motor symptoms at the time of HIV treatment onset may improve long-term clinical outcomes in HIV-HCV co-infected adults.

Prognosis and treatment effects of HIV-associated talaromycosis in a real-world patient cohort.

Talaromycosis is a leading cause of AIDS-associated opportunistic infections and death in Southeast Asia. We have recently shown in the Itraconazole versus Amphotericin for Talaromycosis (IVAP) trial that induction therapy with amphotericin B reduced mortality over 24 weeks, but not during the first 2 weeks. Antifungal treatment effects in real-world settings have not been rigorously evaluated. Using data obtained from patient records at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam from 2004 to 2009, we first developed a prognostic model using Bayesian logistic regression to identify predictors of death. Second, we developed a causal model using propensity score matching to assess the treatment effects of amphotericin B and itraconazole. Our prognostic model identified intravenous drug use (odds ratio [OR] = 2.01), higher respiratory rate (OR = 1.12), higher absolute lymphocyte count (OR = 1.62), a concurrent respiratory infection (OR = 1.67) or central nervous system infection (OR = 2.66) as independent predictors of death. Fever (OR = 0.56) was a protective factor. Our prognostic model exhibits good in-sample performance and out-of-sample validation, with a discrimination power of 0.85 and 0.91, respectively. Our causal model showed no significant difference in treatment outcomes between amphotericin B and itraconazole over the first 2 weeks (95% credible interval: 0.62, 2.50). Our prognostic model provides a simple tool based on routinely collected clinical data to predict individual patient outcome. Our causal model shows similar results to the IVAP trial at 2 weeks, demonstrating an agreement between real-world data and clinical trial data.

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.

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.

Population Pharmacodynamics of Amphotericin B Deoxycholate for Disseminated Infection Caused by Talaromyces marneffei.

Amphotericin B deoxycholate (DAmB) is a first-line agent for the initial treatment of talaromycosis. However, little is known about the population pharmacokinetics and pharmacodynamics of DAmB for talaromycosis. Pharmacokinetic data were obtained from 78 patients; among them, 55 patients had serial fungal CFU counts in blood also available for analysis. A population pharmacokinetic-pharmacodynamic model was fitted to the data. The relationships between the area under the concentration-time curve (AUC)/MIC and the time to blood culture sterilization and the time to death were investigated. There was only modest pharmacokinetic variability in the average AUC, with a mean ± standard deviation of 11.51 ± 3.39 mg·h/liter. The maximal rate of drug-induced kill was 0.133 log10 CFU/ml/h, and the plasma concentration of the DAmB that induced the half-maximal rate of kill was 0.02 mg/liter. Fifty percent of patients sterilized their bloodstreams by 83.16 h (range, 13 to 264 h). A higher initial fungal burden was associated with a longer time to sterilization (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.36 to 0.70; P < 0.001). There was a weak relationship between AUC/MIC and the time to sterilization, although this did not reach statistical significance (HR, 1.03; 95% CI, 1.00 to 1.06, P = 0.091). Furthermore, there was no relationship between the AUC/MIC and time to death (HR, 0.97; 95% CI, 0.88 to 1.08; P = 0.607) or early fungicidal activity {slope = log[(0.500 - 0.003·(AUC/MIC)]; P = 0.319} adjusted for the initial fungal burden. The population pharmacokinetics of DAmB are surprisingly consistent. The time to sterilization of the bloodstream may be a useful pharmacodynamic endpoint for future studies. (This study has been registered at the ISRCTN registry under no. ISRCTN59144167.).