Identification Of Clinical Yeast Isolates Research Articles

P402 Comparison of PCR-RFLP with 21-plex PCR and rDNA: sequencing for identification of clinical yeast isolates

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesNon-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which is time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR.MethodsA total of 173 yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method.ResultsThe results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR.ConclusionThe application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

Molecular identification and antifungal susceptibility pattern of Candida species isolated from HIV infected Patients with candisiasis.

Background and Purpose:Opportunistic fungal infections have been on a growing trend since the last two decades. Among the opportunistic fungal agents, Candida species, Cryptococcus neoformans, and Aspergillus fumigatus account for most of the life-threatening infections in immunocompromised individuals. Regarding this, the present study aimed to investigate the molecular identification and antifungal susceptibility pattern of Candida species isolated from HIV-infected patients.Materials and Methods:This study was conducted on 80 clinical samples collected from HIV-infected patients with suspected candidiasis referring to Tagore Medical College and Hospital, Rathinamangalam and Government Hospital of Thoracic Medicine, in Chennai, India, for 18 months (i.e., May 2016-December 2017). Phenotypic and molecular identification was accomplished using internal transcribed spacer region 1 (ITS1) and ITS4 primers. The antifungal susceptibility pattern of the isolates against four antifungal agents was also determined by both disk diffusion and broth dilution methods.Results:In the present study, the prevalence of candidiasis was obtained as 75% (n=60). Candida tropicalis was the predominant identified species. All the emerging species (i.e., Kodamaea ohmeri, Hanseniaspora opuntiae, and C. orthopsilosis) were identified through molecular identification since the phenotypic identification was inconclusive. In terms of the susceptibility pattern, 63.3% and 18.3% of the isolates were resistant to fluconazole and voriconazole, respectively. Candida albicans was also found to be resistant to amphotericin B.Conclusion:Molecular assay led to the identification of K. ohmeri, H. opuntiae, and C. orthopsilosis, which were multidrug-resistant. This study highlighted the need for the prompt and timely identification of clinical yeast isolates given the emergence of many rare species and their capability of causing life-threatening infections and outbreaks. In the laboratories where molecular diagnostic methods are not available, alternative services of reference laboratories can be utilized as cost-effective measures. With regard to the growing prevalence of antifungal drug resistance, antifungal susceptibility testing should be made mandatory for effective patient management.

Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (T_m) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, T_m values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

Microdilution in vitro Antifungal Susceptibility Patterns of Candida Species, From Mild Cutaneous to Bloodstream Infections.

BackgroundCandida species, as opportunistic organisms, can cause various clinical manifestations, ranging from mild cutaneous infections to systemic candidiasis in otherwise healthy individuals. Remarkably, the incidence and mortality rates of candidemia have significantly increased worldwide, even after advances in medical interventions and the development of novel antifungal drugs.ObjectivesGiven the possible resistance to antifungal agents, susceptibility testing can be useful in defining the activity spectrum of antifungals and determining the appropriate treatment regime.Materials and MethodsThe in vitro susceptibilities of molecularly identified Candida strains (n = 150) belonging to seven species recovered from clinical specimens, including vaginal, cutaneous, sputum, bronchoalveolar lavage (BAL), and blood samples, were determined for six antifungal drugs (amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin), based on the clinical and laboratory standards institute’s M27-A3 and M27-S4 documents.ResultsCandida albicans was the most frequently isolated species (44.66%), followed by non-albicans Candida, including C. glabrata (20%), C. parapsilosis (13.33%), C. krusei (8%), C. tropicalis (7.3%), C. dubliniensis (4%), and C. africana (3.33%). Posaconazole had the lowest geometric mean minimum inhibitory concentration (MIC) (0.0122 µg/ml), followed by amphotericin B (0.0217 µg/mL), voriconazole (0.1022 µg/mL), itraconazole (0.1612 µg/mL), caspofungin (0.2525 µg/mL), and fluconazole (0.4874 µg/mL) against all isolated Candida species. Candida africana and C. parapsilosis were significantly more susceptible to fluconazole, compared to C. albicans and other Candida species (P < 0.001). However, their clinical effectiveness in the treatment of Candida infections remains to be determined.ConclusionsThese findings highlight the importance of precise and correct species identification of clinical yeast isolates via molecular approaches, and of monitoring the antifungal susceptibility of Candida species recovered from clinical sources. Laboratories should consider routine MIC testing of C. glabrata isolates collected from sterile sites. Surveillance studies of Candida species and new analyses of antifungal treatment outcomes will allow more informed determinations of the value of these drugs in the antifungal armamentarium.

Evaluación de la espectrometría de masas: MALDI-TOF MS para la identificación rápida y confiable de levaduras

The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification.

Epidemiological study of a large cluster of fungaemia cases due to Kodamaea ohmeri in an Indian tertiary care centre

While performing molecular confirmation of phenotypically identified Candida tropicalis isolates, we re-identified a few isolates as Kodamaea ohmeri. This led us to the present epidemiological investigation of K. ohmeri fungaemia cases. All phenotypically identified C. tropicalis blood isolates during October 2008 through to December 2009 at our advanced paediatric centre were included for molecular identification by sequencing of the internal transcribed spacer and D1/D2 regions of rDNA. After identifying a large cluster K. ohmeri fungaemia cases, a case–control study was carried out retrospectively to analyse potential risk factors for K. ohmeri fungaemia. Molecular typing of the isolates was performed using a fluorescent amplified fragment length polymorphism (FAFLP) technique. The antifungal susceptibility testing was performed as per the M27-A3 protocol of CLSI. Thirty-eight (25.7%) of 148 phenotypically identified C. tropicalis isolates were confirmed as K. ohmeri by sequencing and FAFLP. By case–control analysis, piperacillin-tazobactam was significantly associated with the K. ohmeri fungaemia. The FAFLP analysis showed that all K. ohmeri isolates had >92% similarity. The azoles and echinocandins had good in vitro activity against K. ohmeri, though 86.8% of the isolates had MIC of 1 mg/L for amphotericin B. The response to antifungal therapy could be evaluated in 27 patients and 70.4% of patients recovered after antifungal therapy. The present study reports the largest cluster of K. ohmeri fungaemia from a single centre. The study also stresses the need for accurate identification of clinical yeast isolates.

Evaluation of a Short, On-Plate Formic Acid Extraction Method for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Identification of Clinically Relevant Yeast Isolates

This report describes a short, on-plate formic acid (FA) extraction method for the identification of clinical yeast isolates using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 41.1% (78/190) and 63.7% (121/190) of yeasts were identified using species log score thresholds of >2.0 and >1.9, respectively. Overall, 97.4% (185/190) of yeasts were identified in combination with conventional FA extraction.

Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis

BackgroundCandida albicans is the most pathogenic Candida species but shares many phenotypic features with Candida dubliniensis and may, therefore, be misidentified in clinical microbiology laboratories. Candidemia cases due to C. dubliniensis are increasingly being reported in recent years. Accurate identification is warranted since mortality rates are highest for C. albicans infections, however, C. dubliniensis has the propensity to develop resistance against azoles more easily. We developed a duplex PCR assay for rapid detection and differentiation of C. albicans from C. dubliniensis for resource-poor settings equipped with basic PCR technology and compared its performance with three phenotypic methods.MethodsDuplex PCR was performed on 122 germ tube positive and 12 germ tube negative isolates of Candida species previously identified by assimilation profiles on Vitek 2 ID-YST system. Typical morphologic characteristics on simplified sunflower seed agar (SSA), and reaction with a commercial (Bichro-Dubli) latex agglutination test were also performed. The assay was further applied on 239 clinical yeast and yeast-like fungi and results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA.ResultsThe results of duplex PCR assay for 122 germ tube positive and 12 germ tube negative isolates of Candida species were comparable to their identification by Vitek 2 ID-YST system, colony characteristics on SSA and latex agglutination test. Application of duplex PCR also correctly identified all 148 C. albicans and 50 C. dubliniensis strains among 239 yeast-like fungi.ConclusionsThe data show that both, duplex PCR and Bichro-Dubli are reliable tests for rapid (within few hours) identification of clinical yeast isolates as C. dubliniensis or C. albicans. However, duplex PCR may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or latex Candida agglutination.

Performance of matrix‐assisted laser desorption‐time of flight mass spectrometry for identification of clinical yeast isolates

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.

Optimization of the Preanalytical Steps of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Identification Provides a Flexible and Efficient Tool for Identification of Clinical Yeast Isolates in Medical Laboratories

We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers, appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed, when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since 95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation, respectively.

Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)

Optimising antifungal treatment requires the fast and species-specific identification of yeast isolates. We evaluated a modified protocol for the rapid identification of clinical yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) technology. First, we evaluated a simplified extraction procedure using 54 clinical yeast isolates. Second, we validated a new protocol with this simplified extraction procedure and lower identification threshold by analysing 167 isolates with either MALDI-TOF or conventional identification techniques. MALDI-TOF analysis with both the standard and short extraction procedure yielded identical identification results, although the log-scores were lower with the latter. With the modified protocol, 163/167 (97.6%) isolates showed a correct identification as compared to conventional identification techniques. A total of 135 out of the 163 (82.8%) correct identifications showed log-scores above 1.7, which we considered as the minimum log-score for secure species identification. The rapid identification of clinical yeast isolates is crucial in patient management. The MALDI-TOF technique using a short extraction procedure can be an alternative for the labourious standard procedure, although the log-scores will be lower. The identification of clinical yeast isolates with the modified protocol is a practical and accurate alternative for conventional identification techniques. If the isolate shows a log-score below 1.7, the standard extraction procedure should be used.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Fast and Reliable Identification of Clinical Yeast Isolates

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of Candida (n = 250), Cryptococcus, Saccharomyces, Trichosporon, Geotrichum, Pichia, and Blastoschizomyces spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.

Candidaspp.in vitrosusceptibility profile to four antifungal agents. Resistance surveillance study in Venezuelan strains

The aim of this study was to determine in vitro susceptibility profiles of Venezuelan strains of Candida spp. to four antifungal agents. One hundred and forty five (145) isolates were recovered during a 1-year period (June 2006 to June 2007) from clinical specimens of patients with severe Candida spp. infections in 15 hospitals. In vitro susceptibilities to amphotericin B, fluconazole, itraconazole and voriconazole were determined by modified Etest. Non Candida albicans Candida spp. were the most frequently isolated yeasts (72.4%) in comparison with C. albicans (27.6%). Candida spp. strains showed MIC ranges between <0.002 and 0.5 mug/ml to amphotericin B. While none were found to be resistant to voriconazole, 5.5% and 27.6% of the test strains were resistant to fluconazole and itraconazole, respectively. C. albicans remains the most susceptible of the yeasts studied to fluconazole and itraconazole (P<0.05) when compared with non C. albicans Candida spp. C. krusei showed the greater cross-resistance to azoles, followed by C. glabrata, C. tropicalis and C. parapsilosis, while C. albicans isolates did not demonstrate this characteristic. It is very important to carry out the correct species identification of clinical yeast isolates because they show up variations in both distribution and susceptibility profiles according to the hospital, patient's underlying disease, clinical specimen analyzed, and the geographical region in which the studies were conducted. The Mycology Department of the INHRR is the national reference center responsible for antifungal resistance surveillance, performing the susceptibility tests with isolates recovered from hospitalized patients in public health centres which do not have mycological diagnosis laboratories.

Rapid and Accurate Identification of Candida albicans Isolates by Use of PNA FISH Flow

We developed the simple, rapid (1 h), and accurate PNA FISH(Flow) method for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy.

Rapid identification of clinical yeast isolates using the colorimetric AUXACOLOR system☆

The AUXACOLOR colorimetric system (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) for the identification of clinical yeast isolates, was compared in its identification of 100 yeast strains to conventional identification methods. Of the 94 correctly identified isolates, 47% ( n = 44) were identified by 24 h, and 100% ( n = 94) were identified by 48 h. AUXACOLOR is a simple, rapid and accurate method for the identification of yeast pathogens.

Clinical Evaluation of the Uni-Yeast-Tek System for Rapid Presumptive Identification of Medically Important Yeasts

The results of over 400 tests for identification of clinical yeast isolates as to species using the Uni-Yeast-Tek (UYT) system in comparison with a more conventional system are reported. The conventional system utilized a total of 23 individual tests, including both fermentation and assimilation tests, whereas the UYT system included only 11 separate tests. In the initial phase of the study, coded unknown isolates were evaluated by each of two technologists using both methods independently. After this initial evaluation, the two methods were used in parallel for routine testing of yeast isolates as they were obtained from clinical specimens. A further evaluation of the UYT system was carried out by retrospectively analyzing the species reported from a clinical mycology laboratory during two separate time periods in which different approaches to yeast identification were employed. A total of 92% of the isolates tested with the UYT system were correctly reported within 72 h, 96% were correctly named after 1 week of incubation, and 97% were correctly reported after 2 weeks of incubation of UYT plates at 30 degrees C when results of the two phases of the study were analyzed together. With the conventional system, 88% of the isolates were correctly reported at 72 h, 96% at 1 week, and 98% after 2 weeks of incubation of biochemical tests. Retrospective analysis of laboratory records revealed no major changes in species reported after adoption of the UYT system for routine testing of clinical isolates. The data presented in this report suggest that the UYT system can be expected to yield rapid presumptive identification of clinical yeast isolates with reasonable confidence when certain minor limitations that are discussed in the text are taken into account.