RESUMO
Fungal infections, especially due to Candida species, are on the rise. Multi-drug resistant organisms such as Candida auris are difficult and time consuming to identify accurately. Machine learning is increasingly being used in health care, especially in medical imaging. In this study, we evaluated the effectiveness of six convolutional neural networks (CNNs) to identify four clinically important Candida species. Wet-mounted images were captured using bright field live-cell microscopy followed by separating single-cells, budding-cells, and cell-group images which were then subjected to different machine learning algorithms (custom CNN, VGG16, ResNet50, InceptionV3, EfficientNetB0, and EfficientNetB7) to learn and predict Candida species. Among the six algorithms tested, the InceptionV3 model performed best in predicting Candida species from microscopy images. All models performed poorly on raw images obtained directly from the microscope. The performance of all models increased when trained on single and budding cell images. The InceptionV3 model identified budding cells of C. albicans, C. auris, C. glabrata (Nakaseomyces glabrata), and C. haemulonii in 97.0%, 74.0%, 68.0%, and 66.0% cases, respectively. For single cells of C. albicans, C. auris, C. glabrata, and C. haemulonii InceptionV3 identified 97.0%, 73.0%, 69.0%, and 73.0% cases, respectively. The sensitivity and specificity of InceptionV3 were 77.1% and 92.4%, respectively. Overall, this study provides proof of the concept that microscopy images from wet-mounted slides can be used to identify Candida yeast species using machine learning quickly and accurately.
Fungal infections due to Candida yeasts are increasing worldwide. Existing methods to identify these pathogens are difficult and time consuming. We find that machine learning can identify Candida species from images quickly and accurately, improving the diagnosis of infectious fungal diseases.
Assuntos
Antifúngicos , Candida , Animais , Filogenia , Antifúngicos/uso terapêutico , Candida glabrata , Candida auris , Aprendizado de MáquinaRESUMO
BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.
Assuntos
Antifúngicos , Fungos , Humanos , Antifúngicos/farmacologia , Fungos/genética , Equinocandinas/farmacologia , DNA Ribossômico/genética , Triazóis , Microscopia Eletrônica de Varredura , Esporos FúngicosRESUMO
Echinocandins are frontline antifungal agents in the management of invasive infections due to multidrug resistant Candida auris. The study aimed to evaluate echinocandin resistance in C. auris isolates of multicentric origin, identify the resistance mechanism, and analyze the pharmacodynamic response to caspofungin in a neutropenic mouse model of infection. A total of 199 C. auris isolates originating from 30 centers across India were tested for susceptibility to echinocandins. Isolates with reduced susceptibility were evaluated for FKS1 mutations and in vivo response to caspofungin in a murine model of disseminated candidiasis. In addition, the response to echinocandins was assessed in light of in vitro growth kinetics, chitin content; and transcript levels of chitin synthase and FKS1 genes. We report 10 resistant C. auris isolates with four FKS1 mutations: F635Y (n = 2), F635L (n = 4), S639F (n = 3), and R1354S (n = 1). Of these, F635Y and R1354S exhibited the most profound resistance in mouse model of disseminated infection. S639F and F635L mutations conferred a moderate in vivo resistance, whereas wild-type isolates exhibiting borderline MIC were susceptible in vivo. FKS1 genotype was more accurate predictor of in vivo response than the MIC of the isolates. Isolates with high basal or inducible chitin content exhibited higher in vitro MIC in FKS1 mutant compared to wild type. FKS1 mutations play a major role in clinically relevant echinocandin resistance in C. auris with differential in vivo outcomes. This study could have implications for clinical practice and, therefore, warrants further studies.
Assuntos
Antifúngicos , Candida auris , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica , Equinocandinas , Proteínas Fúngicas , Animais , Antifúngicos/farmacologia , Candida auris/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Genótipo , Camundongos , Testes de Sensibilidade Microbiana , Mutação/genéticaRESUMO
Identification of Candida auris is challenging and requires molecular or protein profiling-based approaches, availability of which is limited in many routine diagnostic laboratories, necessitating the development of a cost-effective, rapid, and reliable method of identification. The objective of this study was to develop a selective medium for C. auris identification. Eighteen C. auris and 30 non-C. auris yeasts were used for the standardization of the selective medium. Sodium chloride (10% to 13% concentration) and ferrous sulfate (8 mM to 15 mM) were added to yeast extract-peptone-dextrose (YPD) agar in various combinations followed by incubation at 37°C, 40°C, or 42°C for 2 to 3 days. For validation, 579 yeast isolates and 40 signal-positive Bactec blood culture (BC) broths were used. YPD agar comprising 12.5% NaCl and 9 mM ferrous sulfate incubated at 42°C for 48 h, named Selective Auris Medium (SAM), allowed selective growth of C. auris A total of 95% (127/133) of C. auris isolates tested grew on the standardized media within 48 h, and the remaining 6 isolates grew after 72 h, whereas the growth of 446 non-C. auris yeast isolates was completely inhibited. The specificity and sensitivity of the test medium were both 100% after 72 h of incubation. The positive and negative predictive values were also noted to be 100% after 72 h of incubation. The formulated selective medium can be used for the detection and identification of C. auris The SAM is inexpensive, can easily be prepared, and can be used as an alternative to molecular diagnostic tools in the clinical microbiology laboratory.
Assuntos
Candida , Candidíase , Ágar , Antifúngicos/uso terapêutico , Candidíase/diagnóstico , Candidíase/tratamento farmacológico , Meios de Cultura , Humanos , LaboratóriosRESUMO
BACKGROUND: Candida auris, an emerging nosocomial pathogen, exhibits phenotypic variation. Non-aggregating C. auris isolates display greater biofilm-forming capacity and virulence than aggregate-forming isolates. Most of the studies till date have focused on clinical isolates. The biofilm-forming capacity of colonising isolates remains uninvestigated. OBJECTIVES: The present study aimed to elucidate the biofilm-forming capacity of the colonising isolates of C. auris, correlate it with their aggregation behaviour and antifungal susceptibility, and compare it with that of the isolates from blood-stream infection. METHODS: Colonising and clinical (candidemia) isolates of C. auris were screened for aggregation behaviour, biofilm-forming capacity and antifungal susceptibility testing. Aggregation behaviour was assessed microscopically. Biofilm-forming capacity was determined on 96-well flat-bottomed microtitre plates. Antifungal susceptibility testing was performed by broth microdilution assay. RESULTS: Aggregative and non-aggregative phenotypes were found to be predominantly associated with colonising and clinical isolates, respectively, with the former ones being stronger biofilm producers in the colonising group. Non-aggregative isolates in the colonising group showed lower susceptibility to amphotericin B and fluconazole than aggregative isolates. In contrast, no association was noted between biofilm formation, aggregation behaviour and antifungal susceptibility amongst the clinical isolates. CONCLUSION: Biofilm formation is a strain-dependent trait in C. auris, strongly associated with the type and phenotypic behaviour of the isolates. Colonising isolates of this fungus were found to be predominantly aggregative in nature, with a higher biofilm-forming capacity than non-aggregative ones.
Assuntos
Antifúngicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida/fisiologia , Candidemia/microbiologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , VirulênciaRESUMO
We report invasive candidiasis caused by Candida viswanathii over 2 time periods during 2013-2015 in a tertiary care hospital in Chandigarh, India. Molecular typing revealed multiple clusters of the isolates. We detected high MICs for fluconazole in the second time period.
Assuntos
Antifúngicos/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Candida/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Farmacorresistência Fúngica , Tipagem Molecular , Antifúngicos/uso terapêutico , Candida/isolamento & purificação , Candidíase/tratamento farmacológico , Infecção Hospitalar , Farmacorresistência Bacteriana , Feminino , Humanos , Índia , Masculino , Testes de Sensibilidade MicrobianaRESUMO
Dermatophytosis, the commonest superficial fungal infection, has gained recent attention due to its change of epidemiology and treatment failures. Despite the availability of several agents effective against dermatophytes, the incidences of chronic infection, reinfection, and treatment failures are on the rise. Trichophyton rubrum and Trichophyton interdigitale are the two species most frequently identified among clinical isolates in India. Consecutive patients (n = 195) with suspected dermatophytosis during the second half of 2014 were included in this study. Patients were categorized into relapse and new cases according to standard definitions. Antifungal susceptibility testing of the isolated Trichophyton species (n = 127) was carried out with 12 antifungal agents: fluconazole, voriconazole, itraconazole, ketoconazole, sertaconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine, griseofulvin, and luliconazole. The squalene epoxidase gene was evaluated for mutation (if any) in 15 T. interdigitale and 5 T. rubrum isolates exhibiting high MICs for terbinafine. A T1189C mutation was observed in four T. interdigitale and two T. rubrum isolates. This transition leads to the change of phenylalanine to leucine in the 397th position of the squalene epoxidase enzyme. In homology modeling the mutant residue was smaller than the wild type and positioned in the dominant site of squalene epoxidase during drug interaction, which may lead to a failure to block the ergosterol biosynthesis pathway by the antifungal drug.
Assuntos
Alilamina/farmacologia , Antifúngicos/farmacologia , Esqualeno Mono-Oxigenase/metabolismo , Trichophyton/efeitos dos fármacos , Trichophyton/enzimologia , Alilamina/análogos & derivados , Arthrodermataceae/efeitos dos fármacos , Ciclopirox/farmacologia , Farmacorresistência Fúngica/genética , Fluconazol/farmacologia , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana , Morfolinas/farmacologia , Esqualeno Mono-Oxigenase/genética , Terbinafina/farmacologia , Trichophyton/genética , Voriconazol/farmacologiaRESUMO
Antimicrobial resistance is a global health crisis to which pathogenic fungi make a substantial contribution. The human fungal pathogen C. auris is of particular concern due to its rapid spread across the world and its evolution of multidrug resistance. Fluconazole failure in C. auris has been recently attributed to antifungal "tolerance". Tolerance is a phenomenon whereby a slow-growing subpopulation of tolerant cells, which are genetically identical to susceptible cells, emerges during drug treatment. We use microbroth dilution and disk diffusion assays, together with image analysis, to investigate antifungal tolerance in C. auris to all three classes of antifungal drugs used to treat invasive candidiasis. We find that (1) C. auris is tolerant to several common fungistatic and fungicidal drugs, which in some cases can be detected after 24 h, as well as after 48 h, of antifungal drug exposure; (2) the tolerant phenotype reverts to the susceptible phenotype in C. auris; and (3) combining azole, polyene, and echinocandin antifungal drugs with the adjuvant chloroquine in some cases reduces or eliminates tolerance and resistance in patient-derived C. auris isolates. These results suggest that tolerance contributes to treatment failure in C. auris infections for a broad range of antifungal drugs, and that antifungal adjuvants may improve treatment outcomes for patients infected with antifungal-tolerant or antifungal-resistant fungal pathogens.
RESUMO
INTRODUCTION: Cladophialophora bantiana, a neurotropic phaeoid fungus, is the primary agent of cerebral phaeohyphomycosis. The disease more commonly affects immunocompetent males and is associated with a high mortality rate. CASE REPORT: We report a case of brain abscess caused by Cladophialophora bantiana in a 50-year-old immunocompetent male who presented with headache for two months, weakness of both lower limbs for 15 days, and altered sensorium and aphasia for one day. Contrast-enhanced MRI of the brain showed multiple coalescent abscesses in the right basal ganglia and corpus callosum. Based on clinical and radiological suspicion of tuberculoma, treatment with antitubercular drugs was initiated. A month after discharge, the patient was re-admitted with history of loss of consciousness, altered sensorium, respiratory distress and aphasia. Brain CECT revealed multiple ring-enhancing lesions in the right basal ganglia with mass effect and a leftward midline shift. The patient underwent craniotomy and evacuation of abscess. Direct microscopy of pus aspirated from the lesions showed pigmented septate fungal hyphae, which was identified as C. bantiana in fungal culture. The patient was administered intravenous liposomal amphotericin B and voriconazole. However, he died due to multiple organ failure on day 19 after surgery. CONCLUSIONS: Fungal etiology should be considered in the differential diagnosis of intracranial space occupying lesions, regardless of the host immune status. An early diagnosis, together with aggressive medical and neurosurgical interventions are imperative for improving the survival in such patients.
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Sporotrichosis is one of the neglected tropical diseases causing subcutaneous chronic granulomatous lesion by thermally dimorphic fungi belonging to Sporothrix species. Sporothrix brasiliensis, Sporothrix mexicana and Sporothrix globosa are the common pathogenic species. In Asian countries, S. globosa constitutes nearly 99.3% of all Sporothrix species. We studied 63 cases of sporotrichosis of geographically diverse origin from India and Sporothrix isolates were characterised for its growth in different media, temperatures, ability to assimilate sugars and antifungal susceptibility profile. Molecular characterization was performed by sequencing of the calmodulin (CAL), beta tubulin (BT) and translational elongation factor 1-alpha (TEF-1α) and typing by fluorescent amplified fragment length polymorphism (FAFLP). In patients who presented with fixed (49.2%), lymphocutaneous lesions (23.8%), in 26.9% the details were not known, none had systemic dissemination. All the isolates tested were Sporothrix globosa and that could grow up to 35 °C and unable to grow at and beyond 37 °C. The assimilation of sucrose, ribitol and raffinose helps in identifying S. globosa. Sequences of CAL or BT or TEF-1α can differentiate S. globosa from other species in the complex. FAFLP results exhibited low genetic diversity. No correlation was noted between genotypes and clinical presentation, or geographic distribution. Itraconazole, terbinafine and posaconazole showed good in vitro antifungal activity against S. globosa whereas fluconazole and micafungin had no activity. S. globosa of Indian origin is relatively less pathogenic than other pathogenic Sporothrix species as it does not cause systemic dissemination and in the diagnostic laboratory, incubation of the cultures below 37 °C is essential for effective isolation.
Assuntos
Sporothrix/genética , Sporothrix/isolamento & purificação , Esporotricose/microbiologia , Adulto , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antifúngicos/farmacologia , Feminino , Proteínas Fúngicas/genética , Genótipo , Humanos , Índia , Itraconazol/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Filogenia , Sporothrix/classificação , Sporothrix/efeitos dos fármacosRESUMO
Dermatophytosis has gained interest in India due to rise in terbinafine resistance and difficulty in management of recalcitrant disease. The terbinafine resistance in dermatophytes is attributed to single nucleotide polymorphisms (SNPs) in squalene epoxidase (SE) gene. We evaluated the utility of amplified refractory mutation system polymerase chain reaction (ARMS PCR) for detection of previously reported point mutations, including a mutation C1191A in the SE gene in Trichophyton species. ARMS PCR was standardized using nine non-wild type isolates and two wild type isolates of Trichophyton species. Study included 214 patients with dermatophyte infection from March through December 2017. Antifungal susceptibility testing of isolated dermatophytes was performed according to CLSI-M38A2 guidelines. Among dermatophytes isolated in 68.2% (146/214) patients, Trichophyton species were predominant (66.4%). High (>2 mg/L, cut off) minimum inhibitory concentrations to terbinafine were noted in 15 (15.4%) Trichophyton mentagrophytes complex isolates. A complete agreement was noted between ARMS PCR assay and DNA sequencing. C to A transversion was responsible for amino acid substitution in 397th position of SE gene in terbinafine resistant isolates. Thus, the ARMS PCR assay is a simple and reliable method to detect terbinafine-resistant Trichophyton isolates.
Assuntos
Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Mutação Puntual , Reação em Cadeia da Polimerase , Esqualeno Mono-Oxigenase/genética , Terbinafina , Trichophyton/genética , Proteínas Fúngicas/metabolismo , Humanos , Esqualeno Mono-Oxigenase/metabolismo , Tinha/tratamento farmacológico , Tinha/enzimologia , Tinha/genética , Trichophyton/enzimologiaRESUMO
PURPOSE: To determine the prevalence of Candida auris candidaemia in our ICU patients and its molecular epidemiology. METHODS: A prospective observational study was conducted on candidaemia in our ICU patients over 18 months during 2016-2017. Demographics, underlying disease, risk factors, antifungal therapy and outcome were studied. Risk factors of C. auris and non-auris candidaemia were compared. RESULTS: During the study period, among 108 candidaemia cases recorded, the incidence was 6.75/1000 ICU bed days. C. auris topped the list (n = 42, 39.9%), followed by C. tropicalis (34.3%), and C. parapsilosis (15.7%). On bivariate analysis prior antibiotic therapy, long central line days, mechanical ventilation and length of ICU stay were significant risk factors for C. auris candidaemia compared to non-auris candidaemia. Multivariate analysis showed underlying respiratory and neurological diseases as significantly associated with risk of C. auris candidaemia. Fluconazole, amphotericin B, and caspofungin resistance were noted in 97.0%, 93.7% and 3% of C. auris isolates respectively. CONCLUSION: Longer duration of central line days, prior antibiotic use, mechanical ventilation and prolonged ICU stay were important risk factors associated with C. auris candidaemia along with underlying respiratory or neurological disease. The isolates are non-clonal in origin, but they belong to a single clade.