Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for differentiation of Pichia kluyveri strains isolated from traditional fermentation processes.

RATIONALE: Non-Saccharomyces yeasts are widespread microorganisms that nowadays have gained importance for their ability to produce volatile compounds which in alcoholic beverages improve aromatic complexity and therefore the overall quality. Their rapid identification and differentiation in fermentation processes is vital for timely decision making. METHODS: A total of 19 strains of Pichia kluyveri isolated from mezcal, tejuino and cacao fermentations were analyzed with rep-PCR fingerprinting using the primer (GTG)5 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT mass spectrometer using Biotyper 3.1 software (Bruker Daltonics). RESULTS: The comparative analysis between MS spectra and rep-PCR patterns obtained from these strains showed a high similarity between both methods. However, minimal differences between the obtained rep-PCR and MALDI-TOF MS clusters could be observed, especially by the presence and/or absence of one or more discriminating peaks even when they have similarities in their main spectra projection, observing that isolates from the same fermentative process were grouped into the same sub-cluster based on their MALDI-TOF MS profiles. CONCLUSIONS: The data shown suggests that MALDI-TOF MS is a promising alternative technique for rapid, reliable and cost-effective differentiation of native yeast strains isolated from different traditional fermented foods and beverages.

Onychomycosis in the 21st Century: An Update on Diagnosis, Epidemiology, and Treatment.

Onychomycosis accounts for 50% of all nail disease cases and is commonly caused by dermatophytes. Diabetes, human immunodeficiency virus, immunosuppression, obesity, smoking, and advancing age are predisposing factors of this fungal infection. Potassium hydroxide and culture are considered the current standard for diagnosing onychomycosis, revealing both fungal viability and species identification. Other diagnostic tests currently available include periodic acid-Schiff staining, polymerase chain reaction techniques, and fluorescent staining. Across 6 recently published epidemiology studies, the global prevalence of onychomycosis was estimated to be 5.5%, falling within the range of previously reported estimates (2%-8%). Newly approved onychomycosis treatments include efinaconazole, tavaborole, and laser therapy with lasers only approved to temporarily increase the amount of clear nail. Additional onychomycosis treatments being investigated include iontophoresis and photodynamic therapy with small open-label studies reported thus far. Preventative strategies, to help decrease recurrence and reinfection rates, include sanitisation of footwear and prophylactic topical antifungal agents.

Simple low cost differentiation of Candida auris from Candida haemulonii complex using CHROMagar Candida medium supplemented with Pal's medium.

BACKGROUND: Candida auris is unique due to its multidrug resistance and misidentification as Candida haemulonii by commercial systems. Its correct identification is important to avoid inappropriate treatments. AIMS: To develop a cheap method for differentiating C. auris from isolates identified as C. haemulonii by VITEK2. METHODS: Fifteen C. auris isolates, six isolates each of C. haemulonii and Candida duobushaemulonii, and one isolate of Candida haemulonii var. vulnera were tested using CHROMagar Candida medium supplemented with Pal's agar for better differentiation. RESULTS: On CHROMagar Candida medium supplemented with Pal's agar all C. auris strains showed confluent growth of white to cream colored smooth colonies at 37°C and 42°C after 24 and 48h incubation and did not produce pseudohyphae. The isolates of the C. haemulonii complex, on the contrary, showed poor growth of smooth, light-pink colonies at 24h while at 48h the growth was semiconfluent with the production of pseudohyphae. C. haemulonii complex failed to grow at 42°C. CONCLUSIONS: We report a rapid and cheap method using CHROMagar Candida medium supplemented with Pal's agar for differentiating C. auris from isolates identified as C. haemulonii by VITEK2.

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents.

Usefulness of MALDI-TOF Mass Spectrometry for Routine Identification of Candida Species in a Resource-Poor Setting.

BACKGROUND: Identification of fungal clinical isolates is essential for therapeutic management. In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. MALDI-TOF has been shown in favored countries to be a reliable and powerful tool for microorganism identification, including yeasts. The aim of this study was to compare MALDI-TOF with routine identification procedures in a resource-poor context. METHODS: A total of 734 clinical specimens (502 vaginal swabs, 147 oral swabs, 61 bronchoalveolar lavage fluids and 24 stool samples) have been tested in the mycology unit of Fann Hospital, Dakar, Senegal. Strains isolated from culture were identified by both conventional phenotypic methods (germ tube formation and biochemical panels) and MALDI-TOF Saramis/VITEK MS, bioMérieux, France. In addition to comparing the final identification, we determined the time of obtaining the results and the cost for both approaches. RESULTS: Overall, 218 (29.7 %) samples were positive for Candida. MALDI-TOF MS enabled the identification of 214 of the 218 strains isolated (98.1 %) at species level. Phenotypic approach yielded identification for 208 strains (95.4 %). Congruence between the tests was observed for 203 isolates. A discrepancy was observed for one isolate identified as Candida krusei with the phenotypic approach and Candida tropicalis with the MALDI-TOF. In addition, ten isolates identified at genus level by phenotypic methods were identified as C. glabrata (n = 8), C. tropicalis (n = 1) and C. parapsilosis (n = 1) by MALDI-TOF. The turnaround time for identification was <1 h using the MALDI-TOF compared to our routine procedures (48 h). The overall cost (reagents + expendables) per isolate was at 1.35 for the MALDI-TOF MS. CONCLUSION: MALDI-TOF clearly outperformed the diagnosis capacities of phenotypic methods by reducing the delay of results and giving accurate identification at species level. Moreover, this approach appears to be cost-effective and should be implemented especially in resource-poor context.

A cost-effective carbohydrate fermentation test for yeast using microtitre plate.

Carbohydrate fermentation test is a well-established technique for speciation of bacteria and fungi. However, long incubation period, cost and procedural complexity may limit its use. Here, we describe a simple modification of conventional carbohydrate fermentation technique using microtitre plate. Thirty-one yeast isolates were identified based on their fermentation property by microtitre plate method and results were compared with conventional method. The modified method had 85.7% sensitivity and 100% specificity. The average time taken to produce positive reaction was 24 hours. When compared with conventional method, modified method reduced turn-around time and cost of processing without significant increase in discordant results.

The economic impact of rapid Candida species identification by T2Candida among high-risk patients.

INTRODUCTION: This study estimates the cost-effectiveness and hospital budget impact of rapid candidemia identification using T2Candida, a novel diagnostic panel with same-day species-specific results. MATERIALS & METHODS: A 1-year decision-tree model estimates hospital costs (2013 US$) and effects (candidemia-related deaths) for faster diagnostics versus blood culture (BC), accounting for disease prevalence, distribution of Candida species, test characteristics (sensitivity/specificity/time to result), antifungal medication and differential length-of-stay and mortality by appropriate treatment timing. RESULTS: The model estimates a hospital with 5100 annual high-risk patients could possibly save $5,858,448 with T2Candida versus BC, a 47.6% decrease in candidemia diagnosis and treatment budget ($1149/patient tested), while averting 60.6% of candidemia-related mortality. CONCLUSION: Hospitals may observe lower candidemia-related inpatient costs and mortality with rapid Candida diagnosis.

Ribosomic DNA intergenic spacer 1 region is useful when identifying Candida parapsilosis spp. complex based on high-resolution melting analysis.

The epidemiology of Candida parapsilosis and the closely related species C. orthopsilosis and C. metapsilosis has changed in recent years, justify the need to identify this complex at the species level. In this study we investigate the intergenic spacer 1 (IGS1) of the ribosomal DNA (rDNA) to evaluate the utility of this gene region as a phylogenetic molecular marker and the suitability of a high-resolution melting (HRM) strategy based on this region for identification of members of the C. parapsilosis spp. complex. We sequenced the IGS1 and the internal transcribed spacer (ITS) regions of the rDNA from 33 C. parapsilosis sensu lato strains. Although both regions are useful in identifying species, comparative sequence analysis showed that the diversity in the IGS1 region was higher than in the ITS sequences. We also developed an HRM analysis that reliably identifies C. parapsilosis spp. complex based on the amplification of 70 bp in the IGS1 region. All isolates were correctly identified with a confidence interval >98%. Our results demonstrate that HRM analysis based on the IGS1 region is a powerful tool for distinguishing C. parapsilosis from cryptic species.

Analysis of strain relatedness using high resolution melting in a case of recurrent candiduria.

BACKGROUND: Several genotyping protocols have been described to study Candida albicans strains with different sensitivity values. In this study we have analyzed the genetic relatedness and the antifungal susceptibility of several Candida albicans strains isolated from a patient who from suffered recurrent candiduria for a period of five years. Strains were genotyped using Microsatellite Length Polymorphism (MLP) with three microsatellite markers (HIS 3, EF 3 and CDC 3), and a new method based on high resolution melting (HRM) was developed to analyze the microsatellite region. This method was compared with the conventional technique that uses capillary electrophoresis. RESULTS: MICs of the isolates showed the existence of fluconazole susceptible and resistant strains. An inter-colony test using single concentration (8 and 16 mg/l) of fluconazole revealed the coexistence of both fluconazole susceptible and resistant strains. Both genotyping analysis methods showed that all the patient's isolates had a clonal origin. HRM analysis method developed was able to accurately establish strain relatedness and presented a discriminatory power of 0.77. CONCLUSIONS: Although HRM analysis method presented a lower discriminatory power compared to methods based on capillary electrophoresis, it provided a more cost-effective and suitable alternative for genotyping C. albicans in a clinical laboratory.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals.

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

Rapid, simple, and low-cost identification of Candida species using high-resolution melting analysis.

The feasibility of using high-resolution melting analysis (HRMA) was examined for its rapid and simple detection of 5 clinically relevant Candida species (C. albicans, C. glabrata, C. kefyr, C. parapsilosis, and C. guilliermondii). HRMA was able to differentiate clinical Candida species and resulted in being sensitive, reproducible, and inexpensive.

'Spiking' as a rapid method for differentiation of Candida albicans from other yeast species.

This paper describes a simple and rapid method for the differentiation of Candida albicans from other yeast species in primary cultures based on colonial morphology following incubation in carbon dioxide. The technique has superior sensitivity to the traditional germ-tube method and requires no additional laboratory tests. In a busy laboratory, this can result in significant savings in cost and time, as well as improvements in patient care.

Use of CHROMagar medium in the differentiation of Candida species: is it cost-effective in developing countries?

The opportunistic infection of humans with Candida is becoming more common. As several species of Candida are relatively insusceptible to the commonly used antifungal drugs, rapid identification of the species involved can facilitate effective treatment. CHROMagar Candida medium (CCM) is a commercial product designed to allow the rapid identification of Candida to species level. To explore the potential usefulness of CCM in a developing country, attempts were made to identify the Candida species in 107 Indian isolates (obtained, consecutively, from 90 clinical specimens, over a year-long period, in a tertiary-level teaching hospital), using CCM and more conventional methods in parallel. The most common species appeared to be C. tropicalis (representing 40% of the isolates), followed by C. albicans (28%) and C. glabrata (23%). Although use of CCM allowed the isolates from 84 (93.3%) of the clinical specimens to identified to species level within 48 h, it took at least 7 days to identify the yeasts in 90% of the specimens using the more conventional procedures. With the results of the conventional methods set as the 'gold standard', the use of CCM appeared to allow all of the C. albicans, C. tropicalis and C. krusei isolates and most (92%) of the C. glabrata to be correctly identified. The costs/isolate identified with the CCM were no more than those of the conventional methods. As many (67%) of the isolates examined were of potentially drug-resistant yeasts (C. tropicalis, C. glabrata or C. krusei), there is clearly a need to identify local isolates quickly, to prevent treatment with ineffective drugs. In terms of both performance and cost, CCM appears to be a good method to use.

[Comparison of different methods for the identification of Candida species isolated from clinical specimens]. / Klinik örneklerden izole edilen Candida türlerinin tanimlanmasinda farkli yöntemlerin karsilastirilmasi.

The aim of this study was to compare the different methods for the identification of Candida strains isolated from clinical specimens. The methods of germ tube examination, chlamydospore examination formed on the rice Tween-80 (RT-80) agar and evaluation of colony morphologies on the two chromogenic agars (CHROMagar Candida, Albicans ID), were compared with a reference API 20C AUX (bioMerieux, France) automated system based on the carbohydrate assimilation, for the identification of a total 255 Candida isolates. Of them, 173 (67.8%) were identified as C. albicans, 37 (14.5%) were C. glabrata, 23 (9%) were C. krusei, 9 (3.5%) were C. tropicalis, 9 (3.5%) were C. kefyr, 2 (0.8%) were C. guillermondii and 2 (0.8%) were C. parapsilosis, by API 20C AUX system. In the view of these results, 146 (84.4%) of C. albicans strains were identified by germ tube examination, 161 (93.1%) of C. albicans strains and 208 (81.5%) of total strains were identified by chlamydospore examination. 169 (97.7%) of C. albicans strains and 231 (90.6%) of total strains were identified by CHROMagar Candida method, and 168 (97.1%) of C. albicans strains were identified by Albicans ID method, correctly. In the CHROMagar Candida medium, 169 C. albicans isolates have produced bright green colored colonies, whereas 33 (89.2%) isolates which produced dark pink/purple colored colonies were identified as C. glabrata, 7 (77.8%) isolates which produced metalical blue colored colonies were identified as C. tropicalis and 22 (95.6%) isolates which produced pale pink colored colonies were identified as C. krusei. In the Albicans ID medium, four of the 172 isolates which were evaluated as C. albicans initially by producing blue colored colonies, have been identified as C. tropicalis by API 20C AUX system. The sensitivities and specificities of germ tube examination, RT-80, CHROMagar Candida and Albicans ID methods were found as follows, respectively; 84.4% and 100%, 93.1% and 100%, 97.7% and 100%, 99.4% and 95.3 percent. In conclusion, CHROMagar Candida medium seems the most favorable rapid and practical method with high sensitivity and specificity for the identification of Candida species, but its cost-effectiveness should be kept in view.

Novel microtiter plate format for testing germ tube formation and proposal of a cost-effective scheme for yeast identification in a clinical laboratory.

The germ tube test is most widely used for presumptive identification of Candida albicans. Conventional testing is relatively time-consuming due to the hands-on time involved in preparing and viewing each isolate. In order to reduce work-load and costs we have developed a novel microtiter plate test format that offers several advantages: (i) use of removable strips of microtiter wells placed in lockwell frames, (ii) only one pipetting step for each isolate, (iii) direct micromorphological evaluation using an inverse microscope, (iv) use of a novel synthetic germ tube test medium, (v) reduction of the inoculum, permitting testing of minute colonies, (vi) thus, testing of different colonies in potentially mixed primary cultures of clinical specimens is encouraged and facilitated. Implementing this microtiter based germ tube test with simultaneous trehalase test for presumptive identification of Torulopsis (Candida) glabrata, we propose an identification scheme including this test format. This has been implemented in our routine laboratory permitting cost-effective presumptive identification of almost all clinically relevant yeast species.

Diagnosing candidiasis. A new, cost-effective technique.

OBJECTIVE: To demonstrate the efficacy of normal saline as a culture medium for the rapid growth and detection of Candida. STUDY DESIGN: During a six-month period in 1995, the authors examined 302 patients with vulvovaginal complaints. A wet smear diagnosis was accomplished in 271 patients; 31 had symptoms suggestive of a Candida infection, which was not confirmed by microscopy. Two patients were excluded, leaving 29 in the study group. Two samples of the vaginal discharge were collected from the vaginal fornices, with one sample placed in a tube of liquid Sabouraud medium and the second placed in a sterile, red-topped tube containing 5 mL of normal saline. Each saline culture was placed in a test tube rack and left to incubate at room temperature. The samples containing the Sabouraud medium were incubated in the hospital microbiology laboratory. Both samples were evaluated microscopically within 24-72 hours to detect the presence or absence of Candida organisms. RESULTS: Of the 29 patients who had symptoms suggestive of Candida, 16 had a confirmed diagnosis of Candida employing both the saline method and Sabouraud medium. Eleven patients were negative for Candida with both the saline and Sabouraud; there was one false positive and one false negative in each group. The positive predictive value of the normal saline culture technique was 94.1%, with a negative predictive value of 91.7%. Sensitivity and specificity were 94.1% and 91.7%, respectively. CONCLUSION: Using normal saline instead of Sabouraud's medium to culture Candida proved inexpensive, cost-effective and highly accurate for rapid diagnosis.