Development of a novel mycobiome diagnostic for fungal infection.

BACKGROUND: Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. METHODS: Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. RESULTS: The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. CONCLUSIONS: This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Improving awareness, diagnosis and management of invasive fungal infections in Ghana: establishment of the Ghana Medical Mycology Society.

Invasive fungal infections (IFIs) and medical mycology receive little attention in Ghana. However, the present evolution of biomarker assays for IFIs, offers an opportunity for an increased access to fungal laboratory testing in resource-limited settings, and probably make a case for availability of essential antifungal agents. Using surveys and personal communications, the state of medical mycology and IFI in Ghana were highlighted. Inadequate awareness and insufficient access to fungal diagnostics and therapeutics were identified as the key challenges, the establishment of the Ghana Medical Mycology Society was discussed, and recommendations were made to improve the status quo.

Invasive fungal infections (IFIs) receive little attention in Ghana, despite its growing relevance globally. Using surveys and personal communications, the main challenges were identified, and the formation of the Ghana Medical Mycology Society was discussed as a tool to improve the status quo.

Invasive Aspergillosis Due to Aspergillus Section Usti: A Multicenter Retrospective Study.

BACKGROUND: Aspergillus spp. of section Usti (A. ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. METHODS: Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 countries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. RESULTS: Clinical report forms were obtained for 90 patients, of whom 27 had proven/probable IA. An additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid-organ transplant recipients accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing antimold prophylaxis was present in 47% of cases. Pulmonary IA represented 67% of cases. Primary or secondary extrapulmonary sites of infection were observed in 46% of cases, with skin being affected in 28% of cases. Multiple antifungal drugs were used (consecutively or in combination) in 67% of cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16, and 4 µg/mL for amphotericin B, voriconazole, posaconazole, and isavuconazole, respectively. CONCLUSIONS: Aspergillus ustus IA mainly occurred in nonneutropenic transplant patients and was frequently associated with extrapulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined.

Talaromycosis in a renal transplant recipient returning from South China.

Talaromycosis is a fungal infection endemic in Southeast Asia. We report a case of a renal transplant recipient who developed infection after a trip to South China. She presented with constitutional symptoms and was found to have an FDG-avid lung mass. Histopathology demonstrated small yeast cells and culture grew Talaromyces marneffei. The patient was treated with 2 weeks of liposomal amphotericin B followed by itraconazole. The dose of tacrolimus was significantly reduced because of the interaction with itraconazole. Mycophenolate mofetil was discontinued. After 12 months of treatment, the mass had completely resolved. Talaromycosis has mainly been reported in patients with AIDS and is uncommon among solid organ transplant recipients. The immune response against T. marneffei infection is mediated predominantly by T cells and macrophages. The diagnosis may not be suspected outside of endemic areas. We propose a therapeutic approach in transplant patients by extrapolating the evidence from the HIV literature and following practices applied to other endemic mycoses.

Pulmonary Aspergillosis in Patients with Suspected Ventilator-associated Pneumonia in UK ICUs.

Rationale:Aspergillus infection in patients with suspected ventilator-associated pneumonia remains uncharacterized because of the absence of a disease definition and limited access to sensitive diagnostic tests.Objectives: To estimate the prevalence and outcomes of Aspergillus infection in adults with suspected ventilator-associated pneumonia.Methods: Two prospective UK studies recruited 360 critically ill adults with new or worsening alveolar shadowing on chest X-ray and clinical/hematological parameters supporting suspected ventilator-associated pneumonia. Stored serum and BAL fluid were available from 194 nonneutropenic patients and underwent mycological testing. Patients were categorized as having probable Aspergillus infection using a definition comprising clinical, radiological, and mycological criteria. Mycological criteria included positive histology or microscopy, positive BAL fluid culture, galactomannan optical index of 1 or more in BAL fluid or 0.5 or more in serum.Measurements and Main Results: Of 194 patients evaluated, 24 met the definition of probable Aspergillus infection, giving an estimated prevalence of 12.4% (95% confidence interval, 8.1-17.8). All 24 patients had positive galactomannan in serum (n = 4), BAL fluid (n = 16), or both (n = 4); three patients cultured Aspergillus sp. in BAL fluid. Patients with probable Aspergillus infection had a significantly longer median duration of critical care stay (25.5 vs. 15.5 d, P = 0.02). ICU mortality was numerically higher in this group, although this was not statistically significant (33.3% vs. 22.8%; P = 0.23).Conclusions: The estimated prevalence for probable Aspergillus infection in this geographically dispersed multicenter UK cohort indicates that this condition should be considered when investigating patients with suspected ventilator-associated pneumonia, including patient groups not previously recognized to be at high risk of aspergillosis.

Isavuconazole Therapeutic Drug Monitoring during Long-Term Treatment for Chronic Pulmonary Aspergillosis.

Isavuconazole is the newest triazole antifungal, and it displays a favorable pharmacokinetic and safety profile. Less is known about its long-term use in immunocompetent hosts. We performed a retrospective service evaluation of isavuconazole therapeutic drug monitoring in patients with chronic pulmonary aspergillosis. Adverse events (AEs) and dose adjustments made during routine clinical practice were recorded, and AEs were classified based on Common Terminology Criteria for Adverse Events v5.0. Forty-five patients (mean age, 64 years) had 285 isavuconazole blood drug levels measured (mean level, 4.1 mg/liter). A total of 117 measurements (41%) were performed on patients on a 100-mg daily dose instead of 200 mg, and all had blood levels of >1 mg/liter. Age (P = 0.012) and a daily dose of 200 mg versus 100 mg (P = 0.02) were independent predictors of levels of >6 mg/liter. AEs were recorded for 25 patients (56%). The mean drug level at the first measurement was 5.5 ± 2 mg/liter for patients reporting AEs, compared with 4.2 ± 1.7 mg/liter for those not reporting AEs (P = 0.032). The cutoff threshold best predictive of an AE was 4.6 mg/liter (area under the concentration-time curve, 0.710). Sixteen patients (36%) discontinued isavuconazole therapy due to AEs. Twenty-six patients (58%) continued on isavuconazole beyond 6 months. Asthma (P = 0.022) and a daily dose of 200 mg versus 100 mg (P = 0.048) were associated with AEs of grade 2 or higher. A reduced daily dose (100 mg versus 200 mg) of isavuconazole resulted in satisfactory drug levels in a substantial number of patients; it was better tolerated and enabled continuation of therapy for prolonged periods.

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

BACKGROUND: Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. OBJECTIVES: To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. METHODS: Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017-March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. RESULTS: The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. CONCLUSIONS: The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Positive Aspergillus PCR as a marker of azole resistance or sub-therapeutic antifungal therapy in patients with chronic pulmonary aspergillosis.

BACKGROUND: Chronic pulmonary aspergillosis (CPA) is a progressive respiratory disease, caused most commonly by A fumigatus, with significant morbidity and mortality. Azole resistance in A fumigatus is a growing concern worldwide, with resistance to itraconazole reported in up to 50% of patients. AIM: The aim of this study was to determine whether a positive Aspergillus PCR (polymerase chain reaction) is a marker of resistance in CPA patients on azole therapy. METHODS: Patients were selected via a consecutive database search for the first 50 CPA patients with a positive Aspergillus PCR from January to September 2016. Data were collected regarding concurrent and subsequent culture results, current therapy and serum antifungal levels. PCR-positive patients not on therapy were included as the control group. RESULTS: Twenty-three patients were on therapy (15 itraconazole, 4 voriconazole and 4 posaconazole). Cycle threshold (Ct) values ranged from 20.8 to 37.9; no significant difference was found between each treatment and the control group (P = .47). In treated patients, concurrent azole-resistant A fumigatus was found in 75% of A fumigatus-positive cultures (6/8). All of the resistant isolates in the itraconazole group showed therapy resistance. Twenty per cent of all itraconazole levels were sub-therapeutic. No significant difference was found in serum itraconazole levels for patients on itraconazole with a positive PCR versus negative PCR (P = .44). CONCLUSION: Positive sputum, Aspergillus-specific PCR can be associated with azole resistance in CPA patients on therapy.

European confederation of medical mycology expert consult-An ECMM excellence center initiative.

OBJECTIVES: Difficult-to-treat invasive fungal infections require infectious diseases expert consultation to improve treatment outcome and increase survival rates. METHODS: The European Confederation of Medical Mycology (ECMM) intends to provide expert help free of charge by a newly founded ECMM Expert Consultation Service for medical centres around the globe seeking advice when there is no fungal infection consultant available. The expert consult will provide recommendations and broad expertise on difficult-to-treat invasive fungal infections (eg azole-resistant Aspergillus species, Candida auris, mucormycosis) to improve diagnostic and therapeutic management and outcome. RESULTS: The initiative plans global outreach through video conferencing between ECMM Excellence Centers and treating physicians. FungiScope® registries will be used to structure case information and to evaluate the impact of the collegial advice system at regular intervals. Advice will follow recent guidelines, and EQUAL Scores will be used to measure guideline adherence. CONCLUSIONS: Infectious diseases expert consultation should be an integral component of care for patients with difficult-to-treat invasive fungal infections. The ECMM Expert Consult will attend to this matter on a global scale.

Chronic pulmonary aspergillosis commonly complicates treated pulmonary tuberculosis with residual cavitation.

Chronic pulmonary aspergillosis (CPA) complicates treated pulmonary tuberculosis (TB), with high 5-year mortality. We measured CPA prevalence in this group.398 Ugandans with treated pulmonary TB underwent clinical assessment, chest radiography and Aspergillus-specific IgG measurement. 285 were resurveyed 2 years later, including computed tomography of the thorax in 73 with suspected CPA. CPA was diagnosed in patients without active TB who had raised Aspergillus-specific IgG, radiological features of CPA and chronic cough or haemoptysis.Author-defined CPA was present in 14 (4.9%, 95% CI 2.8-7.9%) resurvey patients. CPA was significantly more common in those with chest radiography cavitation (26% versus 0.8%; p<0.001), but possibly less frequent in HIV co-infected patients (3% versus 6.7%; p=0.177) The annual rate of new CPA development between surveys was 6.5% in those with chest radiography cavitation and 0.2% in those without (p<0.001). Absence of cavitation and pleural thickening on chest radiography had 100% negative predictive value for CPA. The combination of raised Aspergillus-specific IgG, chronic cough or haemoptysis and chest radiography cavitation had 85.7% sensitivity and 99.6% specificity for CPA diagnosis.CPA commonly complicates treated pulmonary TB with residual chest radiography cavitation. Chest radiography alone can exclude CPA. Addition of serology can diagnose CPA with reasonable accuracy.

Evaluation of LDBio Aspergillus ICT Lateral Flow Assay for IgG and IgM Antibody Detection in Chronic Pulmonary Aspergillosis.

Detecting Aspergillus-specific IgG is critical to diagnosing chronic pulmonary aspergillosis (CPA). Existing assays are often cost- and resource-intensive and not compatible with resource-constrained laboratory settings. LDBio Diagnostics has recently commercialized a lateral flow assay based on immunochromatographic technology (ICT) that detects Aspergillus antibodies (IgG and IgM) in less than 30 min, requiring minimal laboratory equipment. A total of 154 CPA patient sera collected at the National Aspergillosis Centre (Manchester, United Kingdom) and control patient sera from the Peninsula Research Bank (Exeter, United Kingdom) were evaluated. Samples were applied to the LDBio Aspergillus ICT lateral flow assay, and results were read both visually and digitally. Results were compared with Aspergillus IgG titers in CPA patients, measured by ImmunoCAP-specific IgG assays. For proven CPA patients versus controls, sensitivity and specificity for the LDBio Aspergillus ICT were 91.6% and 98.0%, respectively. In contrast, the routinely used ImmunoCAP assay exhibited 80.5% sensitivity for the same cohort (cutoff value, 40 mg of antigen-specific antibodies [mgA]/liter). The assay is easy to perform but challenging to read when only a very faint band is present (5/154 samples tested). The ImmunoCAP Aspergillus IgG titer was also compared with the Aspergillus ICT test line intensity or rate of development, with weak to moderate correlations. The Aspergillus ICT lateral flow assay exhibits excellent sensitivity for serological diagnosis of CPA. Quantifying IgG from test line intensity measurements is not reliable. Given the short run time, simplicity, and limited resources needed, the LDBio Aspergillus ICT is a suitable diagnostic tool for CPA in resource-constrained settings.

An introduction to current standards of CARE in invasive fungal disease.

Invasive fungal infections are life-threatening conditions that require rapid diagnosis and optimal management to alleviate their high morbidity and mortality. They are also associated with a high economic burden, owing to prolonged hospitalization, the need for intensive supportive care, and the consumption of costly new antifungal agents. Many standards of care and guidelines have been published by national and international medical societies and organizations over the past 20 years that have embraced new diagnostic technologies and strategies, and new antifungal drugs. Recognizing the ongoing need and debate on the topic of standards for optimal diagnostics and patient care, the Scientific Committee of the Continuing Antifungal Research and Education (CARE) programme devised the scientific agenda for the 10th CARE (CARE X) meeting to review current practice and recommendations. Specialists in haematology, infectious diseases, medical microbiology and medical mycology met in Barcelona in November 2017. The meeting was organized and funded by Gilead Sciences Europe Ltd.

Therapeutic drug monitoring and adverse events of delayed-release posaconazole tablets in patients with chronic pulmonary aspergillosis.

BACKGROUND: Posaconazole delayed-release tablets offer better bioavailability than the liquid suspension, but no post-marketing data are available in immunocompetent hosts such as those with chronic pulmonary aspergillosis (CPA). OBJECTIVES: To explore the pharmacokinetics and adverse event (AE) profile of posaconazole tablets in patients with CPA. METHODS: Patients started on posaconazole tablets at the National Aspergillosis Centre (NAC), Manchester, UK between February 2014 and October 2015 were identified from the NAC database and analysed retrospectively. The medical records were reviewed for factors that could affect posaconazole serum levels and the development of AEs. RESULTS: Seventy-two patients were included; 50 (69%) were male and the mean age was 48.5 ±âŸ12 years. Therapeutic levels (≥1 mg/L) were achieved in 90% of cases on 200 mg versus 90% of cases on 300 mg daily (P = not significant). Based on multivariate analysis, female sex (P = 0.041), a 100 mg daily dose (P < 0.001), asthma (P = 0.01) and bronchiectasis (P = 0.001) were associated with subtherapeutic levels. Forty-nine (68%) patients developed AEs, mainly fatigue (37%), dyspnoea (18%) and nausea (12%). AEs were present on 115/196 (59%) occasions on 300 mg/day and on 45/115 (39%) occasions on 200 mg/day (P < 0.01). The mean level was 1.81 ±âŸ0.96 mg/L for patients reporting no AEs and 1.90 ±âŸ1.11 mg/L for those reporting AEs (P = not significant). Factors associated with AEs of grade ≥2 were a daily dose of 300 versus 200 mg (P = 0.001) and asthma (P = 0.008). CONCLUSIONS: A lower-than-recommended posaconazole tablet dose achieved therapeutic levels in most patients and was better tolerated. Males were more likely to achieve a therapeutic level. Underlying conditions affected the degree and frequency of AEs.

Diagnosis and treatment of invasive fungal infections: looking ahead.

Improved standards of care depend on the development of new laboratory diagnostic and imaging procedures and the development of new antifungal compounds. Immunochromatography technologies have led to the development of lateral flow devices for the diagnosis of cryptococcal meningitis and invasive aspergillosis (IA). Similar devices are being developed for the detection of histoplasmosis that meet the requirements for speed (∼15 min assay time) and ease of use for point-of-care diagnostics. The evolution of molecular tools for the detection of fungal pathogens has been slow but the introduction of new nucleic acid amplification techniques appears to be helpful, for example T2Candida. An Aspergillus proximity ligation assay has been developed for a rapid near-patient bedside diagnosis of IA. CT remains the cornerstone for radiological diagnosis of invasive pulmonary fungal infections. MRI of the lungs may be performed to avoid radiation exposure. MRI with T2-weighted turbo-spin-echo sequences exhibits sensitivity and specificity approaching that of CT for the diagnosis of invasive pulmonary aspergillosis. The final part of this review looks at new approaches to drug discovery that have yielded new classes with novel mechanisms of action. There are currently two new classes of antifungal drugs in Phase 2 study for systemic invasive fungal disease and one in Phase 1. These new antifungal drugs show promise in meeting unmet needs with oral and intravenous formulations available and some with decreased potential for drug-drug interactions. Novel mechanisms of action mean these agents are not susceptible to the common resistance mechanisms seen in Candida or Aspergillus. Modification of existing antifungal susceptibility testing techniques may be required to incorporate these new compounds.

Development and validation of the European QUALity (EQUAL) score for mucormycosis management in haematology.

BACKGROUND: Mucormycosis is a life-threatening infection in immunocompromised patients and in haematological malignancy patients in particular. OBJECTIVES: Our aim was to develop and evaluate a scoring tool to measure adherence to current guidelines for mucormycosis. METHODS: Current guidelines of scientific societies on mucormycosis management were reviewed. We assembled diagnostic, treatment and follow-up milestones and designed the EQUAL Mucormycosis Score. The EQUAL Mucormycosis Score was evaluated in the ECMM Excellence Centres. RESULTS: An 18-item tool with one to three points per item resulted in a maximum achievable score depending on disease complexity and ranging from 25 to 32 points. Given variable patient disease course, the diagnostic score is higher in patients with positive fungal culture and biopsy, thus reflecting more decision points and higher management complexity. Eleven patients from two centres were included during the study period. A total of 200 EQUAL Mucormycosis Score points were achieved, which is 62.7% of the maximum EQUAL Mucormycosis Score of 319 points achievable in that cohort (median 18 points, range 7-27). The total score accomplished for diagnostic procedures was 112 of 165 points (67.9%), for first-line treatment 54 of 88 (61.4%) and for follow-up management 34 of 66 points (51.5%). CONCLUSIONS: The EQUAL Mucormycosis Score quantitates adherence to current guideline recommendations for mucormycosis management. With 62.7% of maximum achievable score points, a first result is obtained that may serve as a reference for future evaluations. It remains to be shown whether guideline adherence and mortality rates correlate.

The human lung and Aspergillus: You are what you breathe in?

The diversity of fungal species comprising the lung mycobiome is a reflection of exposure to environmental and endogenous filamentous fungi and yeasts. Most lung mycobiome studies have been culture-based. A few have utilized next generation sequencing (NGS). Despite the low number of published NGS studies, several themes emerge from the literature: (1) moulds and yeasts are present in the human respiratory tract, even during health; (2) the fungi present in the respiratory tract are highly variable between individuals; and (3) many diseases are accompanied by decreased diversity of fungi in the lungs. Even in patients with the same disease, different patients have been shown to harbor distinct fungal communities. Those fungal species present in any one individual may represent a patient's unique environmental exposure(s), either to species restricted to the indoor environment, for example, Penicillium, or species found in the outdoor environment such as Aspergillus, wood and vegetation colonizing fungi and plant pathogens. In addition to causing clinical fungal infections, the lung mycobiome may have inflammatory effects that can cause or worsen lung disease. Most respiratory diseases that have been studied, have been associated with decreases in fungal diversity. However, none of these diversity studies distinguish between accidental, transient fungal colonizers and true residents of the respiratory tract. Where does Aspergillus feature in the mycobiomes of the respiratory tract? Do these mycobiomes reflect the diversity of fungi in outdoor and internal environments? These intriguing questions are explored here.

Siemens Immulite Aspergillus-specific IgG assay for chronic pulmonary aspergillosis diagnosis.

Chronic pulmonary aspergillosis (CPA) complicates underlying lung disease, including treated tuberculosis. Measurement of Aspergillus-specific immunoglobulin G (IgG) is a key diagnostic step. Cutoffs have been proposed based on receiver operating characteristic (ROC) curve analyses comparing CPA cases to healthy controls, but performance in at-risk populations with underlying lung disease is unclear. We evaluated optimal cutoffs for the Siemens Immulite Aspergillus-specific IgG assay for CPA diagnosis in relation to large groups of healthy and diseased controls with treated pulmonary tuberculosis. Sera from 241 patients with CPA attending the UK National Aspergillosis Centre, 299 Ugandan blood donors (healthy controls), and 398 Ugandans with treated pulmonary tuberculosis (diseased controls) were tested. Radiological screening removed potential CPA cases from diseased controls (234 screened diseased controls). ROC curve analyses were performed and optimal cutoffs identified by Youden J statistic. CPA versus control ROC area under curve (AUC) results were: healthy controls 0.984 (95% confidence interval 0.972-0.997), diseased controls 0.972 (0.959-0.985), screened diseased controls 0.979 (0.967-0.992). Optimal cutoffs were: healthy controls 15 mg/l (94.6% sensitivity, 98% specificity), unscreened diseased controls 15 mg/l (94.6% sensitivity, 94.5% specificity), screened diseased controls 25 mg/l (92.9% sensitivity, 98.7% specificity). Results were similar in healthy and diseased controls. We advocate a cutoff of 20 mg/l as this is the midpoint of the range of optimal cutoffs. Cutoffs calculated in relation to healthy controls for other assays are likely to remain valid for use in a treated tuberculosis population.

The role of occupational Aspergillus exposure in the development of diseases.

Aspergillus spp. have a high nutritional versatility and good growth on a large variety of construction materials. They also colonize soil or food, but decaying vegetation is their primary ecological niche. Therefore, exposure to fungi may occur at home, during hospitalization, during specific leisure activities, or at the workplace. The development of Aspergillus infections depends on the interplay between host susceptibility and the organism. Environments with high counts of fungal elements (conidia, hyphal fragments and others), high levels of bioarerosols, and elevated concentrations of mycotoxins or other volatile organic compounds should be considered as potential hazards, since they may present a risk to the exposed person. Rural tasks as well as work related to wood and food industries, poultries, swineries, waste handling plants, and other occupational environments involving contaminated organic material are among the ones posing higher respiratory risks to the workers. This paper presents a review of several studies related to occupational and indoor exposure to Aspergillus, potential health effects related to that exposure, and associated exposure assessment procedures.

Interlaboratory Analysis of Isavuconazole Plasma Concentration Assays Among European Laboratories.

BACKGROUND: Under certain circumstances, clinicians treating patients with isavuconazole for invasive aspergillosis or mucormycosis may use therapeutic drug monitoring. However, the accuracy and reproducibility of the various assays used by different laboratories for the quantification of isavuconazole plasma concentrations have yet to be determined. METHODS: Human plasma samples spiked with known concentrations of isavuconazole were provided to 27 European laboratories that took part in a "round-robin" test (an interlaboratory test performed independently at least 2 times; 2 rounds performed in the current study). Assay methods included liquid chromatography-tandem mass spectrometry (LC-MS/MS), LC with ultraviolet detection (LC-UV), LC with fluorescence detection (LC-FL), and bioassay. The accuracy and reproducibility compared with the known concentrations for each sample in each round were compared overall, between assays, and between laboratories. RESULTS: Twenty-seven laboratories participated in the study (LC-MS/MS, n = 15; LC-UV; n = 9; LC-FL, n = 1; bioassay, n = 2). In round 1, for nominal concentrations of 1000, 1700, 2500, and 4000 ng/mL, the mean (SD) determined concentrations were 1007 (183), 1710 (323), 2528 (540), and 3898 (842) ng/mL, respectively. In round 2, for nominal concentrations of 1200, 1800, 2400, and 4000 ng/mL, the mean (SD) determined concentrations were 1411 (303), 2111 (409), 2789 (511), and 4723 (798) ng/mL, respectively. Over both rounds, determined concentrations were consistently within 15% of the nominal concentrations for 10 laboratories (LC-MS/MS, n = 4; LC-UV, n = 5; bioassay, n = 1) and consistently exceeded the upper 15% margin for 7 laboratories (LC-MS/MS and LC-UV, n = 3 each; LC-FL, n = 1). CONCLUSIONS: Alignment of methodologies among laboratories may be warranted to improve the accuracy and reproducibility of therapeutic drug measurements.

Case Definition of Chronic Pulmonary Aspergillosis in Resource-Constrained Settings.

Chronic pulmonary aspergillosis (CPA) is a recognized complication of pulmonary tuberculosis (TB). In 2015, the World Health Organization reported 2.2 million new cases of nonbacteriologically confirmed pulmonary TB; some of these patients probably had undiagnosed CPA. In October 2016, the Global Action Fund for Fungal Infections convened an international expert panel to develop a case definition of CPA for resource-constrained settings. This panel defined CPA as illness for >3 months and all of the following: 1) weight loss, persistent cough, and/or hemoptysis; 2) chest images showing progressive cavitary infiltrates and/or a fungal ball and/or pericavitary fibrosis or infiltrates or pleural thickening; and 3) a positive Aspergillus IgG assay result or other evidence of Aspergillus infection. The proposed definition will facilitate advancements in research, practice, and policy in lower- and middle-income countries as well as in resource-constrained settings.