Whole genome sequencing analysis demonstrates therapy-induced echinocandin resistance in Candida auris isolates.

Candida auris is an emerging, multidrug-resistant yeast, causing outbreaks in healthcare facilities. Echinocandins are the antifungal drugs of choice to treat candidiasis, as they cause few side effects and resistance is rarely found. Previously, immunocompromised patients from Kuwait with C. auris colonisation or infection were treated with echinocandins, and within days to months, resistance was reported in urine isolates. To determine whether the development of echinocandin resistance was due to independent introductions of resistant strains or resulted from intra-patient resistance development, whole genome sequencing (WGS) single-nucleotide polymorphism (SNP) analysis was performed on susceptible (n = 26) and echinocandin-resistant (n = 6) isolates from seven patients. WGS SNP analysis identified three distinct clusters differing 17-127 SNPs from two patients, and the remaining isolates from five patients, respectively. Sequential isolates within patients had a maximum of 11 SNP differences over a time period of 1-10 months. The majority of isolates with reduced susceptibility displayed unique FKS1 substitutions including a novel FKS1M690V substitution, and nearly all were genetically related, ranging from only three to six SNP differences compared to susceptible isolates from the same patient. Resistant isolates from three patients shared the common FKS1S639F substitution; however, WGS analysis did not suggest a common source. These findings strongly indicate that echinocandin resistance is induced during antifungal treatment. Future studies should determine whether such echinocandin-resistant strains are capable of long-term colonisation, cause subsequent breakthrough candidiasis, have a propensity to cross-infect other patients, or remain viable for longer time periods in the hospital environment.

Axillary Digital Thermometers uplifted a multidrug-susceptible Candida auris outbreak among COVID-19 patients in Brazil.

OBJECTIVES: To describe the first outbreak of Candida auris in Brazil, including epidemiological, clinical and microbiological data. METHODS: After the first Candida auris-colonised patient was diagnosed in a COVID-19 ICU at a hospital in Salvador, Brazil, a multidisciplinary team conducted a local C. auris prevalence investigation. Screening cultures for C. auris were collected from patients, healthcare workers and inanimate surfaces. Risk factors for C. auris colonisation were evaluated, and the fungemia episodes that occurred after the investigation were also analysed and described. Antifungal susceptibility of the C. auris isolates was determined, and they were genotyped with microsatellite analysis. RESULTS: Among body swabs collected from 47 patients, eight (n = 8/47, 17%) samples from the axillae were positive for C. auris. Among samples collected from inanimate surfaces, digital thermometers had the highest rate of positive cultures (n = 8/47, 17%). Antifungal susceptibility testing showed MICs of 0.5 to 1 mg/L for AMB, 0.03 to 0.06 mg/L for voriconazole, 2 to 4 mg/L for fluconazole and 0.03 to 0.06 mg/L for anidulafungin. Microsatellite analysis revealed that all C. auris isolates belong to the South Asian clade (Clade I) and had different genotypes. In multivariate analysis, having a colonised digital thermometer was the only independent risk factor associated with C. auris colonisation. Three episodes of C. auris fungemia occurred after the investigation, with 30-day attributable mortality of 33.3%. CONCLUSIONS: Emergence of C. auris in Salvador, Brazil, may be related to local C. auris clade I closely related genotypes. Contaminated axillary monitoring thermometers may facilitate the dissemination of C. auris reinforcing the concept that these reusable devices should be carefully cleaned with an effective disinfectant or replaced by other temperature monitoring methods.

Miltefosine as an alternative strategy in the treatment of the emerging fungus Candida auris.

OBJECTIVES: Candida auris (C. auris) is an emerging fungal species that is able to develop multidrug resistance and outbreaks of invasive infections worldwide with high mortality rates. To increase the treatment options for C. auris infection this study assessed the efficacy of miltefosine (MFS), that has demonstrated a broad-spectrum antifungal action in vitro. This study aimed to: (i) evaluate the in vitro antifungal activity of MFS against C. auris clinical isolates in the planktonic and biofilm lifestyles; and (ii) compare the activity of MFS in its free form and encapsulated in alginate nanoparticles (MFS-AN) in Galleria mellonella larvae infected by C. auris. METHODS: The antifungal susceptibility test was performed using broth microdilution method and the in vivo treatment in Galleria mellonella larval infection model. RESULTS: MFS exhibited in vitro inhibitory effects at MICs ranging 1-4 µg/mL and fungicidal activity against planktonic cells of C. auris clinical isolates. MFS antibiofilm activity was observed during biofilm formation (0.25-4 µg/mL) and on pre-formed biofilms (16-32 µg/mL). Moreover, the dispersed cells from C. auris biofilms had a similar susceptibility to those obtained for planktonic cells. Treatment with free MFS or MFS-AN resulted in significant improvements in the survival and morbidity rates of Galleria mellonella larvae infected by C. auris. In addition, reduction of fungal burden (0.5-1 log CFU/g) and granuloma formation were observed when compared with the untreated group. CONCLUSIONS: The findings suggest that both the free MFS and MFS-AN have potential for the treatment of fungal infections caused by the emerging C. auris.

Fungicide-driven alterations in azole-resistant Aspergillus fumigatus are related to vegetable crops in Colombia, South America.

Aspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR34/L98H and TR46/Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the ß-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR46/Y121F/T289A (n = 8), TR34/L98H, and TR53. Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.

Breakthrough candidemia after the introduction of broad spectrum antifungal agents: A 5-year retrospective study.

Candidemia is the main invasive fungal disease among hospitalized patients. Several breakthrough candidemia (BrC) cases have been reported, but few studies evaluate the epidemiology, risk factors, molecular characterization, antifungal susceptibility profile and outcome of those patients, especially in developing countries and including patients using broad spectrum antifungals. We conducted a retrospective study from 2011 to 2016, including patients aged 12 years or older with candidemia. Epidemiological characteristics and risk factors for candidemia were evaluated and compared with patients with BrC using univariate and multivariate analysis. Sequential Candida isolates from BrC were identified by internal transcribed spacer sequencing, genotyped with amplified fragment length polymorphism fingerprinting (AFLP), and tested for antifungal susceptibility. From 148 candidemia episodes, 27 breakthrough episodes (18%) were identified, with neutropenia and mucositis being independent risk factors for BrC. Candida non-albicans was more frequent in the BrC group (P < .001). AFLP showed high correlation with conventional methods of identification among breakthrough isolates and a high genetic similarity among isolates from the same patient was observed. C. albicans was the most susceptible species with low MIC values for all antifungal agents tested. In contrast, we found isolates of C. glabrata, C. parapsilosis and C. tropicalis resistant to triazoles and echinocandins. In conclusion, BrC occurred mainly in severely immunosuppressed patients, with neutropenia and mucositis. Mortality did not differ between the groups. Candida non-albicans species were more recovered from BrC, with C. albicans being the most susceptible to antifungals.

Pharmacodynamics of Voriconazole for Invasive Pulmonary Scedosporiosis.

Scedosporium apiospermum is a medically important fungal pathogen that causes a wide range of infections in humans. There are relatively few antifungal agents that are active against Scedosporium spp. Little is known about the pharmacodynamics of voriconazole against Scedosporium Both static and dynamic in vitro models of invasive scedosporiosis were developed. Monoclonal antibodies that target a soluble cell wall antigen secreted by Scedosporium apiospermum were used to describe the pharmacodynamics of voriconazole. Mathematical pharmacokinetic-pharmacodynamic models were fitted to the data to estimate the drug exposure required to suppress the release of fungal antigen. The experimental results were bridged to humans using Monte Carlo simulation. All 3 strains of S. apiospermum tested invaded through the cellular bilayer of the in vitro models and liberated antigen. There was a concentration-dependent decline in the amount of antigen, with near maximal antifungal activity against all 3 strains being achieved with voriconazole at 10 mg/liter. Similarly, there was a drug exposure-dependent decline in the amount of circulating antigen in the dynamic model and complete suppression of antigen, with an area under the concentration-time curve (AUC) of approximately 80 mg · h/liter. A regression of the AUC/MIC versus the area under the antigen-time curve showed that a near maximal effect was obtained with an AUC/MIC of approximately 100. Monte Carlo simulation suggested that only isolates with an MIC of 0.5 mg/liter enabled pharmacodynamic targets to be achieved with a standard regimen of voriconazole. Isolates with higher MICs may need drug exposure targets higher than those currently recommended for other fungi.

Development of Echinocandin Resistance in Candida tropicalis following Short-Term Exposure to Caspofungin for Empiric Therapy.

Isolation of two echinocandin-resistant Candida tropicalis strains from endotracheal secretions of a patient following short-term exposure to caspofungin is described. Both strains exhibited resistance to echinocandins by Etest and reference broth microdilution, showing a homozygous S645P mutation within the hot spot 1 (HS-1) region of FKS1 and belonging to a unique multilocus sequence type. Other C. tropicalis isolates collected from patients in the same intensive care unit within a 60-day period were susceptible to echinocandins and contained wild-type FKS1 sequences.

EQUAL Candida Score: An ECMM score derived from current guidelines to measure QUAlity of Clinical Candidaemia Management.

Candida species frequently cause blood stream infections and are reported to be the third to tenth most commonly isolated pathogens. Guidelines and standardised treatment algorithms provided by professional organisations aim to facilitate decision-making regarding diagnosis, management and treatment of candidaemia. In routine clinical practise, however, it may be challenging to comply with these guidelines. The reasons include lack of familiarity or feasibility to adherence, but also their length and complexity. There is no tool to measure guideline adherence currently. To provide such a tool, we reviewed the current guidelines provided by the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and by the Infectious Diseases Society of America (IDSA), and selected the strongest recommendations for management quality as the bases for our scoring tool. Factors incorporated were diagnostic (blood cultures, echocardiography, ophthalmoscopy, species identification) and follow-up procedures (repeat blood cultures until negative result) as well as key treatment parameters (echinocandin treatment, step down to fluconazole depending on susceptibility result, CVC removal). The EQUAL Candida Score weighs and aggregates factors recommended for the ideal management of candidaemia and provides a tool for antifungal stewardship as well as for measuring guideline adherence.

Current antifungal treatment of fusariosis.

Fungi of the genus Fusarium are well known as major plant pathogens and soil inhabitants, but also cause a broad spectrum of human infections. Fusariosis is the second most common mould infection after aspergillosis, and keratitis is the most encountered implantation infection in immunocompetent individuals. Natamycin is active against Fusarium species both in vitro and in vivo, and is used along with voriconazole as the mainstay of treatment for Fusarium keratitis. Onychomycosis is treated with terbinafine, voriconazole and sometimes itraconazole. Cure is possible despite high in vitro minimum inhibitory concentrations (MICs). Recently, disseminated infections have increased dramatically, mainly affecting severely immunocompromised patients. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in this patient population. Recovery of neutropenia is essential for patient survival and treatment should include voriconazole or amphotericin B as first-line and posaconazole as salvage therapy.

Triazole Resistance Is Still Not Emerging in Aspergillus fumigatus Isolates Causing Invasive Aspergillosis in Brazilian Patients.

Aspergillus fumigatus azole resistance has emerged as a global health problem. We evaluated the in vitro antifungal susceptibility of 221 clinical A. fumigatus isolates according to CLSI guidelines. Sixty-one isolates exhibiting MICs at the epidemiological cutoff value (ECV) for itraconazole or above the ECV for any triazole were checked for CYP51A mutations. No mutations were documented, even for the isolates (1.8%) with high voriconazole MICs, indicating that triazoles may be used safely to treat aspergillosis in Brazil.

Azole-Resistant Aspergillosis: Epidemiology, Molecular Mechanisms, and Treatment.

Aspergillus fumigatus remains the most common species in all pulmonary syndromes, followed by Aspergillus flavus which is a common cause of allergic rhinosinusitis, postoperative aspergillosis and fungal keratitis. The manifestations of Aspergillus infections include invasive aspergillosis, chronic pulmonary aspergillosis and bronchitis. Allergic manifestations of inhaled Aspergillus include allergic bronchopulmonary aspergillosis and severe asthma with fungal sensitization. Triazoles are the mainstay of therapy against Aspergillus infections for treatment and prophylaxis. Lately, increased azole resistance in A. fumigatus has become a significant challenge in effective management of aspergillosis. Earlier studies have brought to light the contribution of non-cyp51 mutations along with alterations in cyp51A gene resulting in azole-resistant phenotypes of A. fumigatus. This review highlights the magnitude of azole-resistant aspergillosis and resistance mechanisms implicated in the development of azole-resistant A. fumigatus and address the therapeutic options available.

Pharmacodynamics of Voriconazole against Wild-Type and Azole-Resistant Aspergillus flavus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis.

Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice.

Home Environment as a Source of Life-Threatening Azole-Resistant Aspergillus fumigatus in Immunocompromised Patients.

A case of fatal aspergillosis due to a TR46/Y121F/T289A azole-resistant Aspergillus fumigatus is reported. Environmental investigations at the patient's residence led to the recovery of TR46/Y121F/T289A isolates, genotypically indistinguishable from the clinical isolate, supporting for the first time the direct role of household as potential source of azole-resistant invasive aspergillosis.

In-host adaptation and acquired triazole resistance in Aspergillus fumigatus: a dilemma for clinical management.

Aspergillus fumigatus causes a range of diseases in human beings, some of which are characterised by fungal persistence. A fumigatus can persist by adapting to the human lung environment through physiological and genomic changes. The physiological changes are based on the large biochemical versatility of the fungus, and the genomic changes are based on the capacity of the fungus to generate genetic diversity by spontaneous mutations or recombination and subsequent selection of the genotypes that are most adapted to the new environment. In this Review, we explore the adaptation strategies of A fumigatus in relation to azole resistance selection and the clinical implications thereof for management of diseases caused by Aspergillus spp. We hypothesise that the current diagnostic tools and treatment strategies do not take into account the biology of the fungus and might result in an increased likelihood of fungal persistence in patients. Stress factors, such as triazole exposure, cause mutations that render resistance. The process of reproduction-ie, sexual, parasexual, or asexual-is probably crucial for the adaptive potential of Aspergillus spp. As any change in the environment can provoke adaptation, switching between triazoles in patients with chronic pulmonary aspergillosis might result in a high-level pan-triazole-resistant phenotype through the accumulation of resistance mutations. Alternatively, when triazole therapy is stopped, an azole-free environment is created that could prompt selection for compensatory mutations that overcome any fitness costs that are expected to accompany resistance development. As a consequence, starting, switching, and stopping azole therapy has the risk of selecting for highly resistant strains with wildtype fitness. A similar adaptation is expected to occur in response to other stress factors, such as endogenous antimicrobial peptides; over time the fungus will become increasingly adapted to the lung environment, thereby limiting the probability of eradication. Our hypothesis challenges current management strategies, and future research should investigate the genomic dynamics during infection to understand the key factors facilitating adaptation of Aspergillus spp.

Fatal disseminated infection with Fusarium petroliphilum.

Members of the Fusarium solani species complex (FSSC) are causing the majority of the fusariosis in humans. Disseminated fusariosis has a high mortality and is predominantly observed in patients with leukemia. Here, we present the case of a fatal infection by a Fusarium strain with a degenerated phenotype, in a patient with acute lymphatic leukemia. Multiple nasal and skin biopsies as well as blood cultures yielded fungal growth, while in direct and histopathological examination of biopsy material septate hyphae were visible. Initial colonies were white with slimy masses with microconidia reminiscent of Fusarium/Acremonium, but with conidiospore production directly on the hyphae. Multi-locus sequence typing discerned a pionnotal-morphologically degenerated-colony of the recently recognized F. petroliphilum as etiological agent. The culture returned to a typical F. solani species complex morphology only after several weeks of growth in culture. Antifungal susceptibility tests indicate amphotericin B as best drug for this FSSC member rather than any of the azoles or echinocandins.

Keratitis by Fusarium temperatum, a novel opportunist.

BACKGROUND: Fusarium species are among the most common fungi present in the environment and some species have emerged as major opportunistic fungal infection in human. However, in immunocompromised hosts they can be virulent pathogens and can cause death. The pathogenesis of this infection relies on three factors: colonization, tissue damage, and immunosuppression. A novel Fusarium species is reported for the first time from keratitis in an agriculture worker who acquired the infection from plant material of maize. Maize plants are the natural host of this fungus where it causes stalk rot and seeding malformation under temperate and humid climatic conditions. The clinical manifestation, microbiological morphology, physiological features and molecular data are described. METHODS: Diagnosis was established by using polymerase chain reaction of fungal DNA followed by sequencing portions of translation elongation factor 1 alpha (TEF1 α) and beta-tubulin (BT2) genes. Susceptibility profiles of this fungus were evaluated using CLSI broth microdilution method. RESULTS: The analyses of these two genes sequences support a novel opportunist with the designation Fusarium temperatum. Phylogenetic analyses showed that the reported clinical isolate was nested within the Fusarium fujikuroi species complex. Antifungal susceptibility testing demonstrated that the fungus had low MICs of micafungin (0.031 µg/ml), posaconazole (0.25 µg/ml) and amphotericin B (0.5 µg/ml). CONCLUSION: The present case extends the significance of the genus Fusarium as agents of keratitis and underscores the utility of molecular verification of these emerging fungi in the human host.

Molecular characterization and in vitro antifungal susceptibility of 80 clinical isolates of mucormycetes in Delhi, India.

Mucormycosis is a highly aggressive disease which is usually fatal in immunocompromised patients. The species of mucormycetes show significant differences in susceptibility to amphotericin B, azoles and terbinafine. The precise species level identification for this fungal group could be achieved by internal transcribed-spacer (ITS) region sequencing. Herein, we present the largest series of antifungal susceptibility data of molecularly characterised isolates of mucormycetes reported so far from India. Eighty isolates originating from 71 patients comprised 50 (62.5%) from pulmonary cases, 15 (19%) from rhino-orbital-cerebral, 13 (16.2%) from cutaneous and 2 (2.5%) from disseminated mucormycosis. ITS and D1/D2 regions sequencing of the isolates identified, Rhizopus arrhizus var. delemar (n = 25), R. arrhizus var. arrhizus (n = 15), R. microsporus (n = 17), R. stolonifer (n = 3), Syncephalastrum racemosum (n = 11), Apophysomyces elegans (n = 2), A. variabilis (n = 2), Lichtheimia ramosa (n = 3) and Mucor circinelloides f. lusitanicus (n = 2). Amplified fragment length polymorphism analysis was done to genotype Rhizopus isolates and revealed 5 clusters of R. arrhizus, which were well separated from R. microsporus. Amphotericin B was the most potent antifungal followed by posaconazole, itraconazole and isavuconazole. Etest and CLSI MICs of amphotericin B showed 87% agreement. Overall, the commonest underlying condition was uncontrolled diabetes mellitus. Records of 54 patients revealed fatalities in 28 cases.

Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms.

Aspergillus fumigatus, a ubiquitously distributed opportunistic pathogen, is the global leading cause of aspergillosis. Azole antifungals play an important role in the management of aspergillosis. However, over a decade, azole resistance in A. fumigatus isolates has been increasingly reported with variable prevalence worldwide and it is challenging the effective management of aspergillosis. The high mortality rates observed in patients with invasive aspergillosis caused by azole-resistant A. fumigatus (ARAF) isolates pose serious challenges to the clinical microbiologist for timely identification of resistance and appropriate therapeutic interventions. The majority of ARAF isolates contain alterations in the cyp51A gene; however, there have been increasing reports on non-cyp51A mutations contributing to azole resistant phenotypes. This review highlights the emergence and various mechanisms implicated in the development of azole resistance in A. fumigatus. We further present recent developments related to the environmental route in the emergence of ARAF isolates and discuss the therapeutic options available.

Effect of antibiotic streamlining on patient outcome in pneumococcal bacteraemia.

OBJECTIVES: In blood culture-proven pneumococcal infections, streamlining empirical therapy to monotherapy with a penicillin is preferred in order to reduce the use of broad-spectrum antibiotics. However, adherence to this international recommendation is poor, and curiously it is unclear whether antibiotic streamlining may be harmful to individual patients. We investigated whether streamlining in bacteraemic pneumococcal infections is associated with mortality. METHODS: Adults admitted to two Dutch hospitals between 2001 and 2011 with bacteraemic pneumococcal infections were retrospectively included. Detailed clinical data on patient characteristics, comorbidities and severity and outcome of disease were obtained in addition to data on antibiotic treatment. Those eligible for streamlining were selected for further analyses. RESULTS: In the 45.8% of cases (126 of 275) where antibiotic treatment was streamlined, a lower mortality rate was observed (6.3% versus 15.4%, P = 0.021). The decision to streamline was only marginally explained by the 38 determinants accounted for. After correction for potential confounders, the OR for death while streamlining was 0.45 (95% CI: 0.18-1.11, P = 0.082) in all cases and 0.35 (95% CI: 0.12-0.99, P = 0.048) specifically in pneumonia cases. CONCLUSIONS: Our results suggest that streamlining in eligible pneumococcal bacteraemia cases is safe, irrespective of patient characteristics, severity of disease or empirical treatment regimen.

Subcutaneous phaeohyphomycosis caused by Exophiala equina, with susceptibility to eight antifungal drugs.

A case of subcutaneous phaeohyphomycosis caused by Exophiala equina is reported in a 75-year-old female, who showed subcutaneous abscesses on both forearms for 8 months. A lesion was initiated by inoculation with a spine from a tree. Histopathologically, suppurative granulomatous inflammation was present and short hyphal elements were observed. Upon culture greyish-black, velvety colonies of a black yeast were obtained after 3 weeks. The strain grew well at 25 °C, but poorly at 37 °C. After sequencing the internal transcribed spacer domain and the partial ß-tubulin gene, the fungus was identified as E. equina. The patient was successfully treated with fluconazole for 3 months.