Endemic Chromoblastomycosis Caused Predominantly by Fonsecaea nubica, Madagascar1.

Chromoblastomycosis is an implantation fungal infection. Twenty years ago, Madagascar was recognized as the leading focus of this disease. We recruited patients in Madagascar who had chronic subcutaneous lesions suggestive of dermatomycosis during March 2013-June 2017. Chromoblastomycosis was diagnosed in 50 (33.8%) of 148 patients. The highest prevalence was in northeastern (1.47 cases/100,000 persons) and southern (0.8 cases/100,000 persons) Madagascar. Patients with chromoblastomycosis were older (47.9 years) than those without (37.5 years) (p = 0.0005). Chromoblastomycosis was 3 times more likely to consist of leg lesions (p = 0.003). Molecular analysis identified Fonsecaea nubica in 23 cases and Cladophialophora carrionii in 7 cases. Of 27 patients who underwent follow-up testing, none were completely cured. We highlight the persistence of a high level of chromoblastomycosis endemicity, which was even greater at some locations than 20 years ago. We used molecular tools to identify the Fonsecaea sp. strains isolated from patients as F. nubica.

Evaluation of ID Fungi Plates Medium for Identification of Molds by MALDI Biotyper.

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

Sporotrichosis in the Highlands of Madagascar, 2013-20171.

Sporotrichosis is a saprozoonotic fungal infection found mostly in tropical and subtropical areas. Few case reports in Madagascar have been published. To document sporotrichosis epidemiology in Madagascar, we conducted a cross-sectional study. During March 2013-June 2017, we recruited from select hospitals in Madagascar patients with chronic cutaneous lesions suggestive of dermatomycosis. Sporotrichosis was diagnosed for 63 (42.5%) of 148 patients. All but 1 patient came from the central highlands, where the prevalence was 0.21 cases/100,000 inhabitants. Frequency was high (64.7%) among patients <18 years of age. Sporotrichosis was diagnosed for 73.8% of patients with arm lesions, 32.3% with leg lesions, and 15.4% with lesions at other sites. Molecular identification identified 53 Sporothrix schenckii isolates. Among the 32 patients who were followed up, response to itraconazole was complete or major for 15 and minor for 17. Overall, endemicity of sporotrichosis in Madagascar was high, concentrated in the highlands.

New Antifungal Susceptibility Test Based on Chitin Detection by Image Cytometry.

The antifungal susceptibility tests used in clinical laboratories have several limitations. We developed a new test, SensiFONG, based on the detection of chitin levels after exposure to antifungal drugs. The optimal culture conditions were 30°C for 6 h for yeast strains and 26°C for 16 h for molds. The strains were exposed to a range of echinocandin or azole concentrations. Chitin was stained with calcofluor white. The percentage of fungal cells with high chitin levels was determined with an automatic epifluorescence microscope. The SensiFONG results were compared to those with the EUCAST method. Image acquisition and analysis were performed with ScanR software. Fifty-nine strains (28 Candida albicans, 17 Candida glabrata, and 14 Aspergillus fumigatus) were analyzed. Thresholds for the classification of strains as resistant or susceptible were determined for each fungal species. The strains displaying an increase in chitin content of ≥32% for C. albicans, ≥6% for C. glabrata, and ≥17% for A. fumigatus were considered susceptible. The application of these thresholds to all 59 strains resulted in a sensitivity of 0.87, 0.93, and 1.00 and a specificity of 0.93, 0.84, and 0.82 for C. albicans, C. glabrata, and A. fumigatus, respectively. The correlation between the results obtained in the SensiFONG and EUCAST assays was excellent. We developed a new test, SensiFONG, based on a new concept. While current assays assess growth inhibition, our test detects changes in chitin levels after exposure to antifungal drugs. Here, we present preliminary results and we propose a proof of concept of this methodology.

Treatment by Posaconazole Tablets, Compared to Posaconazole Suspension, Does Not Reduce Variability of Posaconazole Trough Concentrations.

The delayed-release tablet formulation of posaconazole (POS-tab) results in higher plasma POS trough concentrations (Cmin) than the oral suspension (POS-susp), which raises the question of the utility of therapeutic drug monitoring (TDM). We aimed to compare the variability of the POS Cmin for the two formulations and identify determinants of the POS-tab Cmin and its variability. Demographic, biological, and clinical data from 77 allogeneic hematopoietic stem cell transplant patients (874 Cmin) treated with POS-tab (n = 41), POS-susp (n = 29), or both (n = 7) from January 2015 to December 2016 were collected retrospectively. Interpatient and within-subject coefficients of variation (CVs) of the Cmin adjusted to dose (D) were calculated for each formulation. Between-group comparisons were performed using a linear mixed effects model. The POS Cmin was higher for the tablet than for the suspension (median [25th-75th percentile]: 1.8 [1.2-2.4] mg/liter versus 1.2 [0.7-1.6] mg/liter, P < 0.0001). Interpatient CVs for the tablet and suspension were 60.8 versus 63.5% (P = 0.7), whereas within-subject CVs were 39.7 and 44.9%, respectively (P = 0.3). Univariate analysis showed that age and treatment by POS-tab were significantly and positively associated with the POS Cmin, whereas diarrhea was associated with a diminished POS Cmin Multivariate analysis identified treatment with POS-tab and diarrhea as independent factors of the POS Cmin, with a trend toward a lower impact of diarrhea during treatment with POS-tab (P = 0.07). Despite increased POS exposure with the tablet formulation, the variability of the POS Cmin was not significantly lower than that of the suspension. This suggests that TDM may still be useful to optimize tablet POS therapy.

Integrating anatomo-physiological changes and pharmacogenomics in anti-infective therapy management: is it a major concern?

Success of anti-infective therapy is a major challenge in some patients given anatomo-physiological changes and genetic variations. In this case anecdote, we report the management strategy of a patient suffering from chronic pulmonary aspergillosis in a context of anorexia nervosa and genetic polymorphism.

Molecular-Type Specific Multiplex PCR produces a distinct VNII PCR pattern among Cryptococcus neoformans species complex.

Prevalence of molecular type VNII among Cryptococcus neoformans species complex is probably underestimated since it can be distinguished from VNI only using molecular typing methods such as URA5-RFLP, AFLP, MLST, or whole genome sequencing. Previously, we described a multiplex polymerase chain reaction (PCR) method able to identify VNI, VNIV, and VNIII hybrids, but, at that time, VNII molecular type was not described yet. In this study, 16 VNII global isolates were analyzed by our multiplex PCR method, and results showed that it was able to produce a specific pattern for all the studied VNII isolates, which was different from those of VNI, VNIV, and VNIII.

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.

The Rim Pathway Mediates Antifungal Tolerance in Candida albicans through Newly Identified Rim101 Transcriptional Targets, Including Hsp90 and Ipt1.

Invasive candidiasis (IC) is a major cause of morbidity and mortality despite antifungal treatment. Azoles and echinocandins are used as first-line therapies for IC. However, their efficacy is limited by yeast tolerance and the emergence of acquired resistance. Tolerance is a reversible stage created due to the yeast's capacity to counter antifungal drug exposure, leading to persistent growth. For Candida albicans, multiple stress signaling pathways have been shown to contribute to this adaptation. Among them, the pH-responsive Rim pathway, through its transcription factor Rim101p, was shown to mediate azole and echinocandin tolerance. The Rim pathway is fungus specific, is conserved among the members of the fungal kingdom, and plays a key role in pathogenesis and virulence. The present study aimed at confirming the role of Rim101p and investigating the implication of the other Rim proteins in antifungal tolerance in C. albicans, as well as the mechanisms underlying it. Time-kill curve experiments and colony formation tests showed that genetic inhibition of all the Rim factors enhances echinocandin and azole antifungal activity. Through RNA sequencing analysis of a rim101-/- mutant, a strain constitutively overexpressing RIM101, and control strains, we discovered novel Rim-dependent genes involved in tolerance, including HSP90, encoding a major molecular chaperone, and IPT1, involved in sphingolipid biosynthesis. Rim mutants were also hypersensitive to pharmacological inhibition of Hsp90. Taken together, these data suggest that Rim101 acts upstream of Hsp90 and that targeting the Rim pathway in combination with existing antifungal drugs may represent a promising antifungal strategy to indirectly but specifically target Hsp90 in yeasts.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Population pharmacokinetics of micafungin in ICU patients with sepsis and mechanical ventilation.

OBJECTIVES: To identify the factors associated with the interindividual pharmacokinetic (PK) variability of micafungin and to evaluate the probability of reaching the previously determined PK/pharmacodynamic efficacy thresholds (AUC/MIC >5000 for non-parapsilosis Candida sp. and ≥285 for Candida parapsilosis) with the recommended 100 mg daily dose in ICU patients with sepsis and mechanical ventilation. METHODS: One hundred patients were included and 436 concentrations were available for PK analysis performed with NONMEM software. PTA was determined by Monte Carlo simulations. RESULTS: Micafungin obeyed a two-compartment model with first-order elimination from the central compartment. Mean parameter estimates (percentage interindividual variability) were 1.34 L/h (34%) for clearance (CL), 11.80 L (38%) and 7.68 L (39%) for central (Vc) and peripheral (Vp) distribution volumes, respectively, and 4.67 L/h (37%) for distribution clearance. CL, Vc and Vp increased by 14% when the albumin level was ≤25 g/L and CL decreased by 25% when SOFA score was ≥10. Body weight was related to CL, Vc and Vp by allometric models. PTA was ≥90% in Candida albicans and Candida glabrata infections, except when the MIC was ≥0.015 mg/L, and ranged between 0% and 40% for C. parapsilosis infections with MIC ≥0.5 mg/L. CONCLUSIONS: A possible increase in the dose should be evaluated for infections due to C. parapsilosis and for infections due to C. albicans and C. glabrata with MICs ≥0.015 mg/L.

Failure of empirical systemic antifungal therapy in mechanically ventilated critically ill patients.

RATIONALE: Systemic antifungal treatments are empirically administered to the sickest critically ill patients, often without documented invasive fungal infection. OBJECTIVES: To estimate the impact of systemic antifungal treatment on 30-day survival of patients suspected to have invasive candidiasis. METHODS: All nonneutropenic, nontransplant recipients managed in five intensive care units intubated for at least 5 days, and free of invasive candidiasis, were included. To account for differences in patients' characteristics recorded daily before study end point, a causal model for longitudinal data was used to assess benefits from antifungal treatment. The composite primary end point was hospital mortality or occurrence of invasive candidiasis. MEASUREMENTS AND MAIN RESULTS: Among 1,491 patients, 100 (6.7%) received antifungal treatment for a suspected infection. Patients treated with antifungals were more severely ill than untreated patients. Within the 30-day follow-up period, 363 (24.3%) patients died, and 22 (1.5%) exhibited documented invasive candidiasis. After adjustment on baseline and time-dependent confounders (underlying illness, severity, invasive procedures, Candida colonization), and using a marginal structural model for longitudinal data, treatment was not associated with a decreased risk of mortality or of occurrence of invasive candidiasis (hazard ratio, 1.05; 95% confidence interval, 0.56-1.96; P = 0.91). CONCLUSIONS: This study failed to show outcome benefits for empirical systemic antifungal therapy in the sickest critically ill, nonneutropenic, nontransplanted patients. The post hoc power did not allow us to conclude to an absence of treatment effect especially for specific subgroups. Studies to refine indications for empirical treatment based on surrogate markers of invasive candidiasis are warranted.

Empirical Micafungin Treatment and Survival Without Invasive Fungal Infection in Adults With ICU-Acquired Sepsis, Candida Colonization, and Multiple Organ Failure: The EMPIRICUS Randomized Clinical Trial.

Importance: Although frequently used in treating intensive care unit (ICU) patients with sepsis, empirical antifungal therapy, initiated for suspected fungal infection, has not been shown to improve outcome. Objective: To determine whether empirical micafungin reduces invasive fungal infection (IFI)-free survival at day 28. Design, Setting, and Participants: Multicenter double-blind placebo-controlled study of 260 nonneutropenic, nontransplanted, critically ill patients with ICU-acquired sepsis, multiple Candida colonization, multiple organ failure, exposed to broad-spectrum antibacterial agents, and enrolled between July 2012 and February 2015 in 19 French ICUs. Interventions: Empirical treatment with micafungin (100 mg, once daily, for 14 days) (n = 131) vs placebo (n = 129). Main Outcomes and Measures: The primary end point was survival without proven IFI 28 days after randomization. Key secondary end points included new proven fungal infections, survival at day 28 and day 90, organ failure, serum (1-3)-ß-D-glucan level evolution, and incidence of ventilator-associated bacterial pneumonia. Results: Among 260 patients (mean age 63 years; 91 [35%] women), 251 (128, micafungin group; 123, placebo group) were included in the modified intent-to-treat analysis. Median values were 8 for Sequential Organ Failure Assessment (SOFA) score, 3 for number of Candida-colonized sites, and 99 pg/mL for level of (1-3)-ß-D-glucan. On day 28, there were 82 (68%) patients in the micafungin group vs 79 (60.2%) in the placebo group who were alive and IFI free (hazard ratio [HR], 1.35 [95% CI, 0.87-2.08]). Results were similar among patients with a (1-3)-ß-D-glucan level of greater than 80 pg/mL (n = 175; HR, 1.41 [95% CI, 0.85-2.33]). Day-28 IFI-free survival in patients with a high SOFA score (>8) was not significantly different when compared between the micafungin vs placebo groups (HR, 1.69 [95% CI, 0.96-2.94]). Use of empirical micafungin decreased the rate of new invasive fungal infection in 4 of 128 patients (3%) in the micafungin group vs placebo (15/123 patients [12%]) (P = .008). Conclusions and Relevance: Among nonneutropenic critically ill patients with ICU-acquired sepsis, Candida species colonization at multiple sites, and multiple organ failure, empirical treatment with micafungin, compared with placebo, did not increase fungal infection-free survival at day 28. Trial Registration: clinicaltrials.gov Idenitfier: NCT01773876.

An in-house assay is superior to Sepsityper for direct matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification of yeast species in blood cultures.

We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification).

Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles.

OBJECTIVES: MDR Candida strains are emerging. Next-generation sequencing (NGS), which enables extensive and deep genome analysis, was used to investigate echinocandin and azole resistance in clinical Candida isolates. METHODS: Six genes commonly involved in antifungal resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1 and FKS2) were analysed using NGS in 40 Candida isolates (18 Candida albicans, 15 Candida glabrata and 7 Candida parapsilosis). The strategy was validated using strains with known sequences. Then, 8 clinical strains displaying antifungal resistance and 23 sequential isolates collected from 10 patients receiving antifungal therapy were analysed. RESULTS: A total of 391 SNPs were detected, among which 6 coding SNPs were reported for the first time. Novel genetic alterations were detected in both azole and echinocandin resistance genes. A C. glabrata strain, which was resistant to echinocandins but highly susceptible to azoles, harboured an FKS2 S663P mutation plus a novel presumed loss-of-function CgPDR1 mutation. This isolate was from a patient with deep-seated and urinary candidiasis. Another C. glabrata isolate, with an MDR phenotype, carried a new FKS2 S663A mutation and a new putative gain-of-function CgPDR1 mutation (T370I); this isolate showed mutated (80%) and WT (20%) populations and was collected after 75 days of exposure to caspofungin from a patient who underwent complicated abdominal surgery. CONCLUSIONS: This study shows that NGS can be used for extensive assessment of genetic mutations involved in antifungal resistance. This type of wide genome approach will become very valuable for detecting mechanisms of resistance in clinical strains subjected to multidrug pressure.

IgG1 anti-cell wall and IgG2 anti-phosphopeptidomannan antibodies in the diagnosis of invasive candidiasis and heavy Candida colonization.

We conducted a retrospective study to evaluate the usefulness of immunoglobulin G (IgG) subclasses against Candida cell wall fragments (CW) and phosphopeptidomannan (PPM) for the diagnosis of invasive candidiasis (IC). We analyzed 54 patients with IC (n = 19), Candida heavy colonization (HC; n = 16), and controls (no IC or HC, n = 19).In nonneutropenic patients (n = 47), the sensitivity and specificity values of IgG1 anti-CW and IgG2 anti-PPM in IC were 88%, 59%, and 88%, 94%, respectively. The areas under the receiver operating characteristic curves were 0.69 (0.51-0.88) and 0.901 (0.78-1.02), respectively. IgG1 mean values (arbitrary units) and 95% confidence interval were 46 (20-71), 42 (-0.38 to 84) and 20 (8.3-32) in IC, HC, and in controls, respectively, and discriminated IC but not HC from controls (P = .032, and P = .77, respectively). IgG2 mean values were 26 (9.2-42), 19 (4.4-33), and 3.2 (0.28-6.6) in IC, HC, and in controls, respectively, and discriminated both IC and HC from controls (P < .0001 and P = .035, respectively) but did not separate IC from HC (P = .2). IgG2 showed positivity as early as one day after the IC diagnosis. Antibodies were detected in only two out of a total of seven neutropenic patients.For both IC and HC patients, the diagnostic performance of IgG2 anti-PPM was better than the one of IgG1 anti-CW. In nonneutropenic patients, IgG2 anti-PPM accurately identified not only IC patients but also HC patients at high risk for IC. This marker may help clinicians in the initiation of early preemptive therapy.

Contribution of a Simple Bioassay in Effective Therapeutic Drug Monitoring of Posaconazole and Voriconazole.

BACKGROUND: With the constantly growing incidence of invasive fungal infections, any failure of antifungal treatment is worrying. Azole antifungals present high variability of their plasma trough concentrations (Cmin), justifying their therapeutic drug monitoring (TDM). The authors aimed to develop a simple bioassay to determine the in vitro growth inhibition diameter (ID) and to correlate this ID with Cmin in patients treated with voriconazole or posaconazole. METHODS: The bioassay determined the ID for Candida parapsilosis using a disk diffusion method. Calibration curves were built for posaconazole and voriconazole in water and in 45% plasma. ID was determined in plasma from patients currently undergoing TDM for posaconazole (n = 73) or voriconazole (n = 90). RESULTS: In water or plasma spiked with antifungals and patient samples, cubic regression between ID and Cmin gave coefficient of determination values of 0.997, 0.999, and 0.819, respectively, for posaconazole and 0.996, 0.990 and 0.925, respectively, for voriconazole (P < 0.001 for each curve). Calibration curves with or without plasma did not differ. For voriconazole, Cmin of 1 and 4.7 mg/L corresponded to 54% and 90% of maximal ID, respectively. For posaconazole, Cmin of 0.5, 0.7, and 1 mg/L corresponded to 26%, 40%, and 53% of maximal ID, respectively. CONCLUSIONS: Bioassay could be useful to better characterize the antifungal therapeutic range and brings additional information to the interpretation of TDM in patients for whom Cmin alone is insufficient to adjust the antifungal dosage.

pH signaling in human fungal pathogens: a new target for antifungal strategies.

Fungi are exposed to broadly fluctuating environmental conditions, to which adaptation is crucial for their survival. An ability to respond to a wide pH range, in particular, allows them to cope with rapid changes in their extracellular settings. PacC/Rim signaling elicits the primary pH response in both model and pathogenic fungi and has been studied in multiple fungal species. In the predominant human pathogenic fungi, namely, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, this pathway is required for many functions associated with pathogenesis and virulence. Aspects of this pathway are fungus specific and do not exist in mammalian cells. In this review, we highlight recent advances in our understanding of PacC/Rim-mediated functions and discuss the growing interest in this cascade and its factors as potential drug targets for antifungal strategies. We focus on both conserved and distinctive features in model and pathogenic fungi, highlighting the specificities of PacC/Rim signaling in C. albicans, A. fumigatus, and C. neoformans. We consider the role of this pathway in fungal virulence, including modulation of the host immune response. Finally, as now recognized for other signaling cascades, we highlight the role of pH in adaptation to antifungal drug pressure. By acting on the PacC/Rim pathway, it may therefore be possible (i) to ensure fungal specificity and to limit the side effects of drugs, (ii) to ensure broad-spectrum efficacy, (iii) to attenuate fungal virulence, (iv) to obtain additive or synergistic effects with existing antifungal drugs through tolerance inhibition, and (v) to slow the emergence of resistant mutants.

The strategy for the diagnosis of invasive pulmonary aspergillosis should depend on both the underlying condition and the leukocyte count of patients with hematologic malignancies.

The identification of the causative organism in invasive pulmonary aspergillosis (IPA) is recommended. We investigated whether a mycologic diagnostic strategy could be optimized based on patient characteristics. Fifty-five patients were enrolled in a prospective study. The presence of Aspergillus in respiratory samples occurred more frequently in non-acute leukemia (AL) patients than in AL patients (P = .0003), and in patients with leukocyte counts more than 100/mm(3) (P = .002). In a logistic regression model, these 2 factors appeared to be independent, with an adjusted odds ratio of 7.14 (95% confidence interval, 1.40-36.5) for non-AL patients and an adjusted odds ratio of 6.97 (95% confidence interval, 1.33-36.5) for patients with leukocyte counts more than 100/mm(3). A positive mycologic result was also more frequent among patients with lung CT scan signs of airway-invasive disease than among other patients (P = .043). Airway-invasive signs were more frequent among non-AL patients (P = .049), whereas angioinvasive disease was more frequent among both AL patients (P = .01) and patients with leukocyte counts less than 100/mm(3) (P = .001). A concomitant pulmonary infection was identified more frequently among non-AL patients (P = .005 vs allogeneic hematopoietic stem cell transplant and P = .048 vs others). Our results suggest that different strategies for diagnosing IPA should be considered based on the underlying condition.

Easy-to-use imaging-cytometry assay to analyze chitin patterns in yeasts.

BACKGROUND & AIM: Pathogenic fungi are a major threat to public health, and fungal infections are becoming increasingly common and treatment resistant. Chitin, a component of the fungal cell wall, modifies host immunity and contributes to antifungal resistance. Moreover, chitin content is regulated by chitin synthases and chitinases. However, the specific roles and mechanisms remain unclear. In this study, we developed a cytometric imaging assay to quantify chitin content and identify the distribution of chitin in the yeast cell wall. METHODS: The Candida albicans SC5314 and Nakaseomyces glabratus (ex. C. glabrata) ATCC2001 reference strains, as well as 106 clinical isolates, were used. Chitin content, distribution, and morphological parameters were analysed in 12 yeast species. Moreover, machine learning statistical software was used to evaluate the ability of the cytometric imaging assay to predict yeast species using the values obtained for these parameters. RESULTS: Our imaging-cytometry assay was repeatable, reproducible, and sensitive to variations in chitin content in C. albicans mutants or after antifungal stimulation. The evaluated parameters classified the yeast species into the correct clade with an accuracy of 85 %. CONCLUSION: Our findings demonstrate that this easy-to-use assay is an effective tool for the exploration of chitin content in yeast species.