Determination Of Antifungal Susceptibility Research Articles

Photonic Si microwell architectures for rapid antifungal susceptibility determination of Candida auris.

We present the application of a photonic silicon chip-based optical sensor system for expeditious and phenotypic antifungal susceptibility testing. This label-free diagnostic assay optically monitors the growth of Candida auris at varying antifungal concentrations on a microwell-structured silicon chip in real-time, and antifungal susceptibility is detected within 6 h, four times faster than in the current gold standard method.

Trichophyton indotineae, from epidemiology to therapeutic

Trichophyton indotineae is a newly described dermatophyte species. This fungal pathogen has recently emerged in India and is responsible for chronic or recurrent widespread superficial infections. Resistance to terbinafine is frequently associated to this pathogen and is related to point mutations in the gene encoding the squalene epoxidase. T. indotineae infections have been reported outside India, highlighting the risk of worldwide diffusion of this microorganism. Species identification and antifungal susceptibility determination are key points for infection control but still remain challenging. Systemic treatment is usually required and itraconazole is frequently prescribed in case of terbinafine resistance. This review summarizes main features of T. indotineae taxonomy, epidemiology, clinical manifestations, identification, antifungal profile, treatment and prevention.

Antifungal Susceptibility Testing and Drug Discovery in the Dimorphic Fungus Histoplasma Capsulatum.

The thermal dimorphism of the fungal pathogen Histoplasma is linked to its virulence in mammalian hosts. Mammalian body temperature triggers differentiation of the fungus into virulent yeasts which successfully infect host phagocytes. Accurate determination of antifungal susceptibility with relevance to infection requires that the tests be performed specifically using the yeast form, not the filamentous environmental form. However, traditional CLSI methodology for antifungal susceptibility testing of yeasts with Histoplasma is in adequate. We present optimized methodology for performing antifungal susceptibility assays on Histoplasma yeasts with an emphasis on quantitative yeast growth determination. Colorimetric and fluorometric assays for Histoplasma growth overcome challenges associated with quantifying some Histoplasma strains which grow as aggregates of yeasts. We also describe antifungal susceptibility testing of Histoplasma yeasts within macrophages to provide improved accuracy and better physiological relevance of antifungal susceptibility profiles.

P215 Investigation of an outbreak of neonatal Candida emia in the NICU of a 300 -bedded hospital in North India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesNeonatal Candidaemia causes significant morbidity and mortality in very low birth weight neonates. We report the occurrence of an outbreak of neonatal Candidaemia due to Candida krusei in the neonatal intensive care unit (NICU) of a 300-bedded hospital in North India.MethodsA total of 96 blood cultures from 80 neonates admitted in the NICU from October 2020 to April 2021 were received and processed manually in the Microbiology lab. A total of 5 among the 47 yeast isolates were sent to a teaching hospital for identification and antifungal susceptibility testing by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Clinical parameters (very low birth weight, gestational age, birth asphyxia, broad-spectrum antimicrobial therapy, intra-uterine growth restriction, and total parenteral nutrition), lab parameters (CRP, platelet count), and patient outcome were evaluated. A total of 26 environmental samples and hand swabs from six health care workers were collected to trace the source of the outbreak. The samples were inoculated on 5% sheep blood agar and MacConkey agar. Regular rounds of the NICU and training sessions were conducted by the infection control team to intensify the infection control measures.ResultsBlood culture results: In all, 57 neonates (71.25%; 57/80) admitted to the NICU had positive blood cultures. A total of 47 blood cultures yielded non-albicans Candida spp. (82.45%; 47/57). The other microorganisms isolated from blood cultures were Pseudomonas aeruginosa (4), MRSA (3), Klebsiella pneumoniae (4) and Acinetobacter baumanni (1). Polymicrobial bloodstream infections were detected in two neonates. All the yeast isolates subjected to MALDI-TOF were identified as Candida krusei and had similar MICs of 0.25, 4, 16, 1, 0.25 and 16 μg/ml for voriconazole, itraconazole, posaconazole, caspofungin, micafungin and fluconazole respectively.Culture results of environmental sampling: Yeast could not be isolated from any environmental sample and hand swabs of health care workers. However, MRSA was isolated from the hands of one health care worker.Observation during the infection control rounds:Sterile gauze pieces soaked in iodine and kept in plastic bottles were being used for skin antisepsis before collecting samples or before introducing an IV cannula.The nursing staff did not wear sterile gloves while collecting blood samples or introducing IV cannula in case of NICU patients.It was observed that the used bottles of IV fluids (eg. Ringer lactate, normal saline) were not capped and kept at room temperature.Intensive infection control measures and training sessions were effective in controlling the outbreak.ConclusionWe report the occurrence of a massive outbreak of neonatal Candidaemia in the NICU of our hospital with a positivity rate of 58.75% (47/80). Identification of yeast and determination of antifungal susceptibility is important for prompt treatment. Though the source of the outbreak could not be traced but intensifying infection control practices could control the outbreak. This study emphasizes the value of educating healthcare workers and regular monitoring of disinfection practices to prevent health-care-associated infections.

P102 Nuclear magnetic resonance -based identification of metabolites in dermatophytes

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesNuclear magnetic resonance (NMR) spectroscopy provides a holistic snapshot of the metabolome of an organism. There is a dearth of studies till date that had exploited NMR metabolomic platform to study dermatophytes, despite its potential for rapid identification and subsequent application of the knowledge in performing faster antifungal susceptibility of dermatophytes. Here we attempted to study the frequency of various species of dermatophytes in clinically suspected cases of dermatophytosis and perform NMR-based identification of metabolites in the culture suspensions/cell extracts of T. mentagrophyte and T. rubrum.MethodsThis was a hospital-based prospective study conducted in the isolates obtained from clinically suspected cases of Dermatophytosis in the patients. Skin, nails, and hair samples of patients suspected with superficial fungal infections were processed for dermatophytes using conventional microbiological methods. NMR-based identification of metabolites was carried out in cell extracts prepared from the culture suspensions of T. mentagrophytes and T. rubrum obtained during the study from a subset of the clinical isolates from the samples.ResultsDermatophytes were isolated in 85.88% (219/255) cases, with T. mentagrophyte being isolated in 65% (143/219) of isolates, followed by T. rubrum in 31.5% (69/219) isolates. In NMR study was done in the standard ATCC strains (T. mentagrophyte ATCC9533 and T. rubrum ATCC28188) and representative clinical isolates of both the species. Overall, 24 metabolites were identified in T. rubrum and 23 metabolites in T. mentagrophyte amongst which 22 metabolites were common to both fungus, however, ‘4-hydroxyproline’ and ‘acetate’ was found specific to T. rubrum, and ‘allantoin’ was found specific to T. mentagrophyte.Conclusion T. mentagrophyte was the predmominant dermatophyte species in the study. Amongst the number of metabolites detected in T. rubrum and T. mentagrophyte, ‘4-hydroxyproline’ and ‘acetate’ was found specific to T. rubrum, and ‘allantoin’ was found specific to T. mentagrophyte. These specific metabolites could be useful for as early identification of these dermatophytes as well early determination of antifungal susceptibility by using metabolic endpoints, further large-scale study will be helpful in this regard.

P071 Isolate profiling and antifungal susceptibility determination for terbinafine and itraconazole among dermatophytes in a tertiary care hospital of western rajasthan

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectiveTo determine the species distribution of causative agents causing dermatophytosis, and their antifungal susceptibility pattern for terbinafine and itraconazole in trichophyton spp among samples collected in patients with dermatophytosis clinical suspicion during the period of December 1, 2020 to January 31, 2022.Materials and MethodsThis is a prospective study conducted in the Department of Microbiology of a tertiary care super specialty and referral Centre of western Rajasthan from December 1, 2020 to January 31, 2022.Skin scraping, nail clipping, and hair pluckings were collected in mycology lab from clinically suspected cases of dermatophytosis presenting to the department of dermatology for conventional identification, and antifungal susceptibility testing.The specimens were subjected to direct KOH and calcofluor white microscopy and conventional fungal culture on SDA at 25 ͦ C and 37 ͦ C.The cultures positive for dermatophytes were speciated by microslide culture lactophenol cotton blue mount, hair perforation test, and urease test.The isolates identified as Trichophyton spp were taken up for antifungal susceptibility testing against terbinafine and itraconazole by microbroth dilution according to CLSI-M38 A2 guidelines. Further terbinafine resistance gene evaluation for detection of C1191A and T1189C single nucleotide polymorphism in Squalene epoxide by Amplified Refractory Mutation System-Polymerase chain Reaction (ARMS-PCR) is undergoing for trichophyton spp.ResultsOver the 14-month study period, the laboratory processed total of 174 specimens: 134 skin scraping, 36 nail clipping, and 4 hair pluckings. Of them, 106 (61.62%) specimens were microscopy positive and 111 (63.79%) were culture positive. Out of the 111 culture-positive agents isolated, 94 (84.68%) were found to be dermatophytes. On isolate profiling of 94 dermatophytes T. mentagrophyte was found to be most common 45 (48%) followed by T. rubrum 27 (29%), T. tonsurans 20 (21%), T. verrucosum 1 (1.1%), and Microsporum spp 1(1.1%). Antifungal susceptibility of 93 Trichophyton spp against terbinafine showed resistance among 58.06% isolates with 83.33% isolates among terbinafine resistant cases showing ≥4 μg/ml minimum inhibitory concentration. There was no resistance detected for itraconazole with microbroth dilution.ConclusionA total of (54.02%) skin, hair, nail infections were found to be caused by dermatophytes.On isolate profiling, T. mentagrophyte, T. rubrum, and T. tonsurans were found to be predominant species among our isolates showing altered trend of local isolates from T. tonsurans being second most common spp isolated in past.On antifungal susceptibility >55% isolates showed resistance for Terbinafine with >80% having higher MIC of ≥4 μg/ml on the contrary there was no observed resistance for itraconazole.There is a need for encouraging dermatologists for prescribing routine fungal microscopy, culture, and AFST for dermatophytes in Western Rajasthan, to reduce the indiscriminate use of antifungals.

Comparison of Clinical Laboratory Standards Institute (CLSI) Microdilution Method and VITEK 2 Automated Antifungal Susceptibility System for the Determination of Antifungal Susceptibility of Candida Species.

IntroductionChanges in the epidemiology of Candida infections, increasing resistance, and advances in treatment have increased the need to perform antifungal susceptibility testing in clinical laboratories. Standardized reference, the microbroth dilution method, and various commercial antifungal susceptibility test systems are used to determine antifungal susceptibility. This study aims to determine and compare the antifungal susceptibility of various Candida species isolated from blood cultures in our laboratory with the CLSI M27 microdilution reference method and VITEK 2 automated system (bioMérieux, Marcy-l'Étoile, France).MethodsThe antifungal susceptibility of a total of 140 Candida strains to fluconazole, voriconazole, and amphotericin B, and a total of 92 strains to anidulafungin was tested with the CLSI M27 method and the VITEK 2 automated system. For fluconazole, voriconazole, and amphotericin B, essential and categorical agreement percentages were calculated between the two methods. Because there is no anidulafungin in the VITEK 2 system, anidulafungin results obtained with CLSI were compared with micafungin only in terms of categorical agreement. In the category comparison, CLSI clinical breakpoints were used; the epidemiological cut-off values were used when they were not available. Very major error, major error, and minor error rates were calculated.ResultsIn general, the minimum inhibitory concentration (MIC) values obtained with VITEK 2 for azole group drugs were found to be one-fold higher than the CLSI MICs read at the 24th hour. While the essential agreement between the two methods was >90% for amphotericin B and voriconazole, it remained at 85% for fluconazole. Overall, the best categorical agreement was obtained with amphotericin B (99.3%), and the least categorical agreement was obtained with voriconazole (85.7%). A very major error was seen with amphotericin B (0.7%) and fluconazole (0.7%) in one C. parapsilosis strain each. No resistance was detected with VITEK 2 in one C. glabrata strain found to be resistant to fluconazole by the reference method. Major and minor error rates were higher for azole drugs than amphotericin B and anidulafungin/micafungin.ConclusionThe VITEK 2 system is a fast and highly applicable system, and with these features, it is advantageous for routine laboratories. In this study, although the error rate was not very high, one fluconazole-resistant C. parapsilosis and C. glabrata strain could not be detected with VITEK 2. The increase in data on the antifungal performance of the VITEK 2 system, which is available in many routine laboratories due to its ability to be used for bacteria identification and sensitivity, will contribute to the usability of the system for this purpose. In this study, data that will support the literature information in terms of the antifungal performance of the VITEK 2 system are presented.

Trichosporon asahii as an emerging panresistant agent of urinary tract infection: A case report

Objectives: Identification and determination of antifungal susceptibility of this yeast. Case Report: A 65 yrs. male patient k/c/o Diabetes and Hypertension since 5years was admitted with the complaints of decreased urine output and burning micturition since 15 days. After the relevant investigations he was clinically diagnosed with AKI on CKD. Materials and Methods: Urine sample was examined microscopically and Inoculated onto Blood agar, MacConkey agar and SDA and incubated overnight at 37?C. Growth was subjected to gram stain and further processed as per standard protocols conventionally. Isolate was identified by conventional and automation methods. Antifungal susceptibility was performed by Vitek2 system. Sample was collected for two more times and processed similarly to confirm pathogenicity. Results: Wet mount of urine showed many pus cells, yeast cells and fungal hyphae. On SDA cream colored, smooth colonies this later became dry folded. Gram stain of the isolate showed gram positive with lateral budding yeast cells, septate hyphae and rectangular arthrospores. Isolate identified as Trichosporon species conventionally and confirmed as T.asahii by MALDITOF. Antifungal susceptibility revealed multiresistance with preserved susceptibility to Voriconazole. All the three samples showed similar results. Patient was started on Voriconazole and showed improvement clinically. Discussion: T.asahii causes white piedra and onychomycosis in immunocompetent host and disseminated infection in immunocompromised host. Isolation of same yeast in three consecutive urine samples establishes T. asahii as an etiological agent of the infection in our patient. Conclusion: We need to have an increased awareness of disseminated infections due to Trichosporon species which is inherently resistant to AmphotericinB and Caspofungin. Such infections require early diagnosis and treatment for fast recovery of the patient. Keywords: Trichosporon, Asahii, Panresistant, Ur

E-test Determination of Antifungal Susceptibility of Candida Species Isolated from Turkeys.

IntroductionCandida species are a natural component of the intestinal tract microflora, but in favourable conditions they can cause superficial, mucosal, or even systemic candidiasis. Poultry production might be a source of human drug-resistant yeast infections, including Candida spp. The limited data concerning the antifungal susceptibility of poultry Candida isolates prompted us to carry out research to determine the susceptibility of isolates from turkey intestinal tracts.Method and MaterialsThe beak cavity, crop and cloaca were swabbed of 580 turkeys from 58 flocks in western Poland. The susceptibility tests were conducted using the E-test method with amphotericin B, fluconazole, itraconazole, and voriconazole on 52 isolates of C. albicans, C. catenulata, C. glabrata, C. palmioleophila, C. rugosa, C. krusei and C. lusitaniae.ResultsAll isolates were susceptible to voriconazole. According to the MIC values obtained for amphotericin B and fluconazole, all Candida spp. isolates were classified as susceptible according to the described breakpoints except for C. krusei, which was the only isolate that was amphotericin B-, fluconazole- and itraconazole-resistant. The susceptibility to itraconazole varied: 11 of the Candida isolates were susceptible (21.1%), 29 were dose-dependently susceptible (55.8%), and 12 isolates were resistant (23.1%).ConclusionThere are few resistant strains of Candida in turkeys, and the drug resistance varies. When Candida passes from turkeys to humans, there is a wide range of antifungal treatment options.

Necessity to identify candida species accurately with minimum inhibitory concentration determination in each case of bloodstream infections

BackgroundBloodstream Candida infection is a life-threatening event among ICU admitted patients. This infection is caused by a diverse range of Candida species having varied minimum inhibitory concentrations. ObjectivesTo identify Candida species causing bloodstream infections with their antifungal susceptibility determination. MethodsCandida species isolated from the blood of ICU admitted patients were identified by phenotypic as well as by molecular methods including PCR-RFLP using MspI restriction enzyme and MALDI TOF MS. The minimum inhibitory concentration of fluconazole, voriconazole, amphotericin B and caspofungin was determined against isolated Candida species by CLSI M27A3 guidelines. ResultsA total of 119 Candida species were isolated. Among them, C. tropicalis(n=29) was the predominant isolate followed by C. parapsilosis(n=18), C. glabrata (n=12), C. krusei (n=11) and C. albicans(n=11). Uncommon Candida species isolated were; Wickerhamomyces anomalus(n=15), Kodaemia ohmeri(n=8), C. lusitaniae (n=5) and C. auris (n=2). A varied antifungal MIC values were observed. Caspofungin had the lowest MIC among the tested antifungals. Increased fluconazole MIC was observed against the isolated Candida species including C. tropicalis. All the isolated C. lusitaniae and C. auris strains have ≥1mcg/ml amphotericin B MIC. In comparison to fluconazole, voriconazole was more effective when tested in vitro. ConclusionEmergence of uncommon Candida species having varied antifungal MIC warns the physicians to have a prompt, accurate identification with antifungal MIC determination in each case of bloodstream Candida infections.

Identification of Drug Resistant Candida auris.

Candida auris is a multidrug resistant yeast, recognized as a cause of invasive infections and health care associated outbreaks around the world. C. auris is of great public health concern, due to its propensity for drug resistance, mode and pace of its transmission, and the possibility that biologic and epidemiologic factors could exacerbate worldwide emergence of C. auris infections. Currently, outbreak response is complicated by limited treatment options and inadequate disinfection strategies, as well as by issues (misidentification, long turnaround time) associated with application of commonly used diagnostic tools. Misdiagnosis of C. auris is common since many diagnostic platforms available in clinical and public health laboratories depend on reference databases that have not fully incorporated C. auris. Moreover, the correlation between minimal inhibitory concentration values (MICs) and clinical outcomes is poorly understood resulting in the absence of C. auris-specific breakpoints. New, accurate and fast diagnostic methods have emerged to facilitate effective patient management and improve infection control measures, ultimately reducing the potential for C. auris transmission. This review provides an overview of available C. auris detection/identification and antifungal susceptibility determination methods and discusses their advantages and limitations. A special emphasis has been placed on culture-independent methods that have recently been developed and offer faster turnaround times.

Aspergillosis in domestic and wild birds from Argentina

Aspergillus species are widely distributed throughout the world and can develop parasitic and saprophytic ways of life, allowing Aspergillus to infect living hosts, including plants, insects, birds and mammals. The most common form of aspergillosis in poultry and other birds is respiratory infection. Clinical manifestations depend on the infective dose, pre-existing diseases, and the immune response of the host. The aim of the present research was to study aspergillosis in domestic and wild birds from Argentina. We carried out morphological and molecular identification, and determination of antifungal susceptibility against seven antifungal drugs. Six birds from different cities of Buenos Aires Province of Argentina were studied. Three of the samples belonged to broiler chicks, while the other three belonged to an eagle, a pheasant, and a kelp gull. Two isolates were identified as Aspergillus fumigatus by morphological characteristics and growth at 50 °C. Morphology and BenA sequencing enabled us to identify three isolates as Aspergillus flavus, and one as Aspergillus sydowii. All antifungal drugs tested showed low MIC values, ranging from 0.008 to 1 mg/L. Aspergillosis in birds causes high economic losses and could be controlled by sanitation, avoidance of moldy food, nest and litter and reducing stress factors.

Reidentification and antifungal susceptibility profile of Candida guilliermondii and Candida famata clinical isolates from a culture collection in Argentina.

The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.

Identification of Candida strains with nested PCR in bovinemastitis and determination of antifungal susceptibilities

Mastitis is one of the biggest problems of dairy cattle industries. In recent years, fungal agents have been frequently reported among the causative agents for mastitis. Among the fungal agents, Candida species are the most common. This study's aims were the isolation, identification, and determination of antifungal susceptibility of Candida species causing mycotic mastitis in cattle. A total of 260 mastitic milk samples were collected from different farms. Identification was conducted by rapid diagnostic tests and nested PCR method; 6 different antifungal agents were examined. Candida sp. was detected in 46 (17.7%) of the 260 mastitic milk samples. Based on API 20 C AUX and nested PCR test results, 6 different species of Candida were identified. C. tropicalis was the predominant one (26.1%), followed by C. parasilosis (21.7%), C. kefyr and C. krusei (17.4% each), C. rugosa (13%), and C. glabrata (4.4%). A total of 46 strains were confirmed by PCR. According to antifungal test results, the isolates were found to be susceptible to ketoconazole (78.3%) and resistant to flucytosine (91.3%), amphotericin (82.6%), miconazole and nystatin (73.9%), and fluconazole (69.5%).

Evaluation of MALDI-TOF-MS for the Identification of Yeast Isolates Causing Bloodstream Infection.

Infections due to Candida species are major causes of morbidity and mortality in humans, causing a diverse spectrum of clinical disease ranging from superficial and mucosal infections to invasive disease. Several authors have demonstrated that mortality is closely linked to both timing of therapy and/or source control. The rapid identification of pathogenic species is helpful to start timely and effective antifungal therapy. The aim of this study was to assess the performance of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the correct and rapid identification of yeast isolates causing bloodstream infection. Between January 2014 and January 2015, a total of 117 yeast like organisms isolated from blood culture samples of 117 episodes from 102 patients who had blood stream infections were included in the study. The isolates were identified by MALDI-TOF MS. The results were compared with those obtained by the standard mycological methods and/or sequence analysis. One hundred and seventeen yeast isolates including 115 Candida spp and two non-Candida yeasts were analysed. The Biotyper correctly identified 115 (98.3%) isolates to the genus level and 102 (87.2%) isolates to the species level using the manufacturer's recommended cutoff scores. The Bruker Biotyper is a rapid, easy, inexpensive, and highly reliable system for the identification of yeast isolates. Early identification with MALDI-TOF MS would save time for determination of antifungal susceptibility and proper treatment strategy. The expansion of the database of the library by addition of less common species will improve the performance of the system.

Antifungal Breakpoints of Non-albicans Candida Clinical Isolates in Vitek-2 Compact.

Sir, The incidence and diversity of infections caused by non-albicans Candida species (NAC) have shown a substantial increase in recent years. Given the growing number of susceptible patients with compromised immunity as a consequence of HIV infection, cancer chemotherapy and transplantation associated immunosuppression, NAC has emerged as an eminent fungal pathogen worldwide. Pertaining to the diverse mechanisms of resistance to various antifungals agents among NAC, it has become imperative to identify the effective antifungals. A prospective study was conducted in Department of Microbiology in our tertiary care hospital from May to August, 2014. A total of 36 candida isolates (16 C. albicans, 11 C. tropicalis, 5 C. krusei, 2 C. glabrata and 2 C. parapsilosis) recovered from blood and urine were identified as per standard procedures. The NAC isolates (n=20) were selected for antifungal susceptibility testing (AFST) in Vitek-2 Compact using AST-YS07 card. MIC for fluconazole, voriconazole, caspofungin, micafungin and flucytosine were interpreted [Table/Fig-1] as per CLSI guideline [1]. Among the various AFST methods currently in use for yeasts, macrobroth dilution, microbroth dilution and disc diffusion tests are recommended by CLSI [2]. However, the utility of these tests are limited by the technically challenging procedure, difficulty of inoculums standardization and subjective variation in interpretation. Unlike bacterial pathogens, the slow growth rate of candida also precludes a short turnaround time. Early initiation of appropriate therapy is a critical factor determining the clinical outcome. The introduction of automated AFST has ensured prompt results in a standardized platform. However, there is a lack of published data of fungal resistance pattern determined by automated system in India. In this study, the mean analysis time for determining antifungal susceptibility by Vitek-2 Compact was 14.15 ± 2.49 hour. Vitek-2 utilizes a self-calibrating optic system comprising of light emitting diodes and photo detectors to continually monitor the growth of organisms inside the wells of the test cards, reducing the analysis time [3]. All five C. krusei isolates had fluconazole and flucytosine resistance with MIC > 8μg/dl. Two C. tropicalis strains showed fluconazole resistance and one had intermediate susceptibility to flucytosine. The median MICs for fluconazole, voriconazole, caspofungin, micafungin and flucytosine among C. tropicalis were 1, 0.12, 0.25, 0.06 and 18μg/dl respectively. In C. krusei the median MICs for these drugs were 16, 0.12, 0.25, 0.12 and 8μg/dl respectively. Our findings are in accordance with other studies. Tellapragada et al., found all NAC isolates except C. krusei were uniformly sensitive to fluconazole, while one C. parapsilosis and one C. tropicalis strain displayed voriconazole and caspofungin resistance respectively [4]. Echinocandins are novel synthetic lipopeptide antifungals which non-competitively inhibit the fungal β [1,3]-D-glucan synthase enzyme [5]. Echinocandin resistance among NAC species have been mainly reported in C. parapsilosis with FKS1 mutation [5]. In our study, caspofungin and micafungin were sensitive in all isolates, indicating their critical role in treatment of infections by azole resistant strains. However, further study with a larger sample size comparing Vitek-2 Compact results with other methods of MIC testing is required to confirm our findings. In conclusion, Vitek-2 Compact is essential in rapid determination of antifungal susceptibility of candida. However, the cost constraints may deter its incorporation in routine diagnostic protocol in resource poor settings. [Table/Fig-1]: Resistance pattern of non-albicans candida isolates All isolates were uniformly sensitive to voriconazole, caspofungin and micafungin

Épico project. Development of educational recommendations using the DELPHI technique on invasive candidiasis in non-neutropenic critically ill adult patients

BackgroundAlthough there has been an improved management of invasive candidiasis in the last decade, controversial issues still remain, especially in the diagnostic and therapeutic approaches. AimsWe sought to identify the core clinical knowledge and to achieve high level agreement recommendations required to care for critically ill adult patients with invasive candidiasis. MethodsA prospective Spanish survey reaching consensus by the DELPHI technique was made. It was anonymously conducted by electronic mail in a first term to 25 national multidisciplinary experts in invasive fungal infections from five national scientific societies, including intensivists, anesthesiologists, microbiologists, pharmacologists and infectious diseases specialists, who answered to 47 questions prepared by a coordination group after a strict review of the literature in the last five years. The educational objectives spanned five categories, including epidemiology, diagnostic tools, prediction rules, and treatment and de-escalation approaches. The level of agreement achieved among the panel experts in each item should exceed 75% to be selected. In a second term, after extracting recommendations from the selected items, a face to face meeting was performed where more than 80 specialists in a second round were invited to validate the preselected recommendations. ResultsIn the first term, 20 recommendations were preselected (Epidemiology 4, Scores 3, Diagnostic tools 4, Treatment 6 and De-escalation approaches 3). After the second round, the following 12 were validated: (1) Epidemiology (2 recommendations): think about candidiasis in your Intensive Care Unit (ICU) and do not forget that non-Candida albicans–Candida species also exist. (2) Diagnostic tools (4 recommendations): blood cultures should be performed under suspicion every 2–3 days and, if positive, every 3 days until obtaining the first negative result. Obtain sterile fluid and tissue, if possible (direct examination of the sample is important). Use non-culture based methods as microbiological tools, whenever possible. Determination of antifungal susceptibility is mandatory. (3) Scores (1 recommendation): as screening tool, use the Candida Score and determine multicolonization in high risk patients. (4) Treatment (4 recommendations): start early. Choose echinocandins. Withdraw any central venous catheter. Fundoscopy is needed. (5) De-escalation (1 recommendation): only applied when knowing susceptibility determinations and after 3 days of clinical stability. The higher rate of agreement was achieved in the optimization of microbiological tools and the withdrawal of the catheter, whereas the lower rate corresponded to de-escalation therapy and the use of scores. ConclusionsThe management of invasive candidiasis in ICU patients requires the application of a broad range of knowledge and skills that we summarize in our recommendations. These recommendations may help to identify the potential patients, standardize their global management and improve their outcomes, based on the DELPHI methodology.

Proyecto Épico: formulación de unas recomendaciones educativas con metodología DELPHI para pacientes adultos críticos no neutropénicos y con candidiasis invasiva

BackgroundAlthough there has been an improved management of invasive candidiasis in the last decade, controversial issues still remain, especially in the diagnostic and therapeutic approaches. AimsWe sought to identify the core clinical knowledge and to achieve high level agreement recommendations required to care for critically ill adult patients with invasive candidiasis. MethodsA prospective Spanish survey reaching consensus by the DELPHI technique was made. It was anonymously conducted by electronic mail in a first term to 25 national multidisciplinary experts in invasive fungal infections from five national scientific societies, including intensivists, anesthesiologists, microbiologists, pharmacologists and infectious diseases specialists, who answered to 47 questions prepared by a coordination group after a strict review of the literature in the last five years. The educational objectives spanned five categories, including epidemiology, diagnostic tools, prediction rules, and treatment and de-escalation approaches. The level of agreement achieved among the panel experts in each item should exceed 75% to be selected. In a second term, after extracting recommendations from the selected items, a face to face meeting was performed where more than 80 specialists in a second round were invited to validate the preselected recommendations. ResultsIn the first term, 20 recommendations were preselected (Epidemiology 4, Scores 3, Diagnostic tools 4, Treatment 6 and De-escalation approaches 3). After the second round, the following 12 were validated: (1) Epidemiology (2 recommendations): think about candidiasis in your Intensive Care Unit (ICU) and do not forget that non-Candida albicans–Candida species also exist. (2) Diagnostic tools (4 recommendations): blood cultures should be performed under suspicion every 2–3 days and, if positive, every 3 days until obtaining the first negative result. Obtain sterile fluid and tissue, if possible (direct examination of the sample is important). Use non-culture based methods as microbiological tools, whenever possible. Determination of antifungal susceptibility is mandatory. (3) Scores (1 recommendation): as screening tool, use the Candida Score and determine multicolonization in high risk patients. (4) Treatment (4 recommendations): start early. Choose echinocandins. Withdraw any central venous catheter. Fundoscopy is needed. (5) De-escalation (1 recommendation): only applied when knowing susceptibility determinations and after 3 days of clinical stability. The higher rate of agreement was achieved in the optimization of microbiological tools and the withdrawal of the catheter, whereas the lower rate corresponded to de-escalation therapy and the use of scores. ConclusionsThe management of invasive candidiasis in ICU patients requires the application of a broad range of knowledge and skills that we summarize in our recommendations. These recommendations may help to identify the potential patients, standardize their global management and improve their outcomes, based on the DELPHI methodology.This article is also published in English in this issue. It can be found inhttp://dx.doi.org/10.1016/j.riam.2013.05.006.

Épico project. Development of educational recommendations using the DELPHI technique on invasive candidiasis in non-neutropenic critically ill adult patients

BackgroundAlthough there has been an improved management of invasive candidiasis in the last decade, controversial issues still remain, especially in the diagnostic and therapeutic approaches. AimsWe sought to identify the core clinical knowledge and to achieve high level agreement recommendations required to care for critically ill adult patients with invasive candidiasis. MethodsA prospective Spanish survey reaching consensus by the DELPHI technique was made. It was anonymously conducted by electronic mail in a first term to 25 national multidisciplinary experts in invasive fungal infections from five national scientific societies, including intensivists, anesthesiologists, microbiologists, pharmacologists and infectious diseases specialists, who answered to 47 questions prepared by a coordination group after a strict review of the literature in the last five years. The educational objectives spanned five categories, including epidemiology, diagnostic tools, prediction rules, and treatment and de-escalation approaches. The level of agreement achieved among the panel experts in each item should exceed 75% to be selected. In a second term, after extracting recommendations from the selected items, a face to face meeting was performed where more than 80 specialists in a second round were invited to validate the preselected recommendations. ResultsIn the first term, 20 recommendations were preselected (Epidemiology 4, Scores 3, Diagnostic tools 4, Treatment 6 and De-escalation approaches 3). After the second round, the following 12 were validated: (1) Epidemiology (2 recommendations): think about candidiasis in your Intensive Care Unit (ICU) and do not forget that non-Candida albicans–Candida species also exist. (2) Diagnostic tools (4 recommendations): blood cultures should be performed under suspicion every 2–3 days and, if positive, every 3 days until obtaining the first negative result. Obtain sterile fluid and tissue, if possible (direct examination of the sample is important). Use non-culture based methods as microbiological tools, whenever possible. Determination of antifungal susceptibility is mandatory. (3) Scores (1 recommendation): as screening tool, use the Candida Score and determine multicolonization in high risk patients. (4) Treatment (4 recommendations): start early. Choose echinocandins. Withdraw any central venous catheter. Fundoscopy is needed. (5) De-escalation (1 recommendation): only applied when knowing susceptibility determinations and after 3 days of clinical stability. The higher rate of agreement was achieved in the optimization of microbiological tools and the withdrawal of the catheter, whereas the lower rate corresponded to de-escalation therapy and the use of scores. ConclusionsThe management of invasive candidiasis in ICU patients requires the application of a broad range of knowledge and skills that we summarize in our recommendations. These recommendations may help to identify the potential patients, standardize their global management and improve their outcomes, based on the DELPHI methodology.This article is also published in Spanish in this issue. It can be found in 10.1016/j.riam.2013.05.005

In-vitro Activity of 10 Antifungal Agents against 320 Dermatophyte Strains Using Microdilution Method in Tehran.

Dermatophyte fungi are the etiologic agents of skin infections commonly referred to as ringworm. These infections are not dangerous but as a chronic cutaneous infections they may be difficult to treat and can also cause physical discomfort for patients. They are considered important as a public health problem as well. No information is available regarding the efficacy of antifungal agents against dermatophytes in Tehran. Therefore, in this study we evaluated the efficacy of 10 systemic and topical antifungal medications using CLSI broth microdilution method (M38-A). The antifungal agents used included griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole, voriconazole, clotrimazole, ciclopirox olamine, amorolfine and naftifine.Fifteen different species of dermatophytes which were mostly clinical isolates were used as follows; T. mentagrophytes, T. rubrum, E. floccosum, M. canis, T. verrucosum, T. tonsurans, M. gypseum, T. violaceum, M. ferruginum, M. fulvum, T. schoenleinii, M. racemosum, T. erinacei, T. eriotrephon and Arthroderma benhamiae. The mean number of fungi particles (conidia) inoculated was 1.25 ×10⁴ CFU/mL. Results were read after 7 days of incubation at 28 °C. According to the obtained results,itraconazole and terbinafine showed the lowest and fluconazole had the greatest MIC values for the most fungi tested. Based on the results, it is necessary to do more research and design a reliable standard method for determination of antifungal susceptibility to choose proper antibiotics with fewer side effects and decrease antifungal resistance and risk of treatment failure.