CHROMagarTM Candida Research Articles

Differentiation of Candida albicans, Candida glabrata, and Candida krusei by FT-IR and chemometrics by CHROMagar™ Candida

Pathogenic Candida species are detected in clinical infections. CHROMagar™ is a phenotypical method used to identify Candida species, although it has limitations, which indicates the need for more sensitive and specific techniques. Infrared Spectroscopy (FT-IR) is an analytical vibrational technique used to identify patterns of metabolic fingerprint of biological matrixes, particularly whole microbial cell systems as Candida sp. in association of classificatory chemometrics algorithms. On the other hand, Soft Independent Modeling by Class Analogy (SIMCA) is one of the typical algorithms still little employed in microbiological classification. This study demonstrates the applicability of the FT-IR-technique by specular reflectance associated with SIMCA to discriminate Candida species isolated from vaginal discharges and grown on CHROMagar™. The differences in spectra of C. albicans, C. glabrata and C. krusei were suitable for use in the discrimination of these species, which was observed by PCA. Then, a SIMCA model was constructed with standard samples of three species and using the spectral region of 1792–1561cm−1. All samples (n=48) were properly classified based on the chromogenic method using CHROMagar™ Candida. In total, 93.4% (n=45) of the samples were correctly and unambiguously classified (Class I). Two samples of C. albicans were classified correctly, though these could have been C. glabrata (Class II). Also, one C. glabrata sample could have been classified as C. krusei (Class II). Concerning these three samples, one triplicate of each was included in Class II and two in Class I. Therefore, FT-IR associated with SIMCA can be used to identify samples of C. albicans, C. glabrata, and C. krusei grown in CHROMagar™ Candida aiming to improve clinical applications of this technique.

Sensitivity of Candida albicans to essential oils: are they an alternative to antifungal agents?

Candida albicans is an important opportunistic pathogen, responsible for the majority of yeast infections in humans. Essential oils, extracted from aromatic plants, are well-known antimicrobial agents, characterized by a broad spectrum of activities, including antifungal properties. The aim of this work was to assess the sensitivity of 30 different vaginal isolated strains of C.albicans to 12 essential oils, compared to the three main used drugs (clotrimazole, fluconazole and itraconazole). Thirty strains of C.albicans were isolated from vaginal swab on CHROMagar™ Candida. The agar disc diffusion method was employed to determine the sensitivity to the essential oils. The antifungal activity of the essential oils and antifungal drugs (clotrimazole, itraconazole and fluconazole) were investigated using a microdilution method. Transmission and scanning electron microscopy analyses were performed to get a deep inside on cellular damages. Mint, basil, lavender, tea tree oil, winter savory and oregano essential oils inhibited both the growth and the activity of C.albicans more efficiently than clotrimazole. Damages induced by essential oils at the cellular level were stronger than those caused by clotrimazole. Candida albicans is more sensitive to different essential oils compared to the main used drugs. Moreover, the essential oil affected mainly the cell wall and the membranes of the yeast. The results of this work support the research for new alternatives or complementary therapies against vaginal candidiasis.

Candida glabrata species complex prevalence and antifungal susceptibility testing in a culture collection: First description of Candida nivariensis in Argentina.

The presence of the cryptic species belonging to the Candida glabrata complex has not been studied in Argentina. We analyzed a collection of 117 clinical isolates of C. glabrata complex belonging to a National Culture Collection of Instituto Nacional de Microbiología "Dr. Carlos G. Malbrán" from Argentina (40 isolates from blood samples, 18 from other normally sterile sites, 20 from vagina, 14 from urine, 7 from oral cavity, 3 from catheter, 1 from a stool sample and 14 isolates whose clinical origin was not recorded). The aims of this work were to determine the prevalence of the cryptic species Candida nivariensis and Candida bracarensis and to evaluate the susceptibility profile of isolates against nine antifungal drugs. Identification was carried out by using classical phenotypic tests, CHROMagar™ Candida, PCR and MALDI-TOF. The minimal inhibitory concentrations of amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, voriconazole, ketoconazole, posaconazole, caspofungin and anidulafungin were determined according to the EDef 7.3 (EUCAST) reference document. Of the 117 isolates, 114 were identified as C. glabrata and three as C. nivariensis by using PCR and MALDI-TOF. There were no major differences between C. nivariensis and C. glabrata susceptibility profiles. No resistant strains were found to echinocandins. We have found that the percentage of C. nivariensis in our culture collection was 2.56. This is the first description of C. nivariensis in Argentina, and data obtained could contribute to the knowledge of the epidemiology of this cryptic species.

Comparison Between Phenotypic Characterization and Semi-Nested PCR for the Identification of Candida Species in Clinical Isolates

Background: Nowadays, due to the increasing number of immunocompromised patients, mycosis infections caused by Candida species are on the rise, especially among hospitalized patients. Objectives: In the current study, the chromogenic medium CHROMagar™ Candida and semi-nested polymerase chain reaction (snPCR) were compared concerning their ability to detect the species of Candida in 65 clinical isolates. Materials and Methods: We used snPCR with universal and species-specific primers to detect Candida species in the culture of clinical isolates. By using universal primers, we carried out the amplification of the 3/end of 5.8S ribosomal DNA (rDNA) and the 5/end of 28S rDNA, including the internal transcribed spacer 2 (ITS2) and production of 350 to 410-bp fragments from 4 Candida species, vis. Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results: The phenotypic identification system identified 60 (92.3%) yeasts isolates, including C. albicans (n = 33), C. glabrata (n = 14), C. tropicalis (n = 11), and C. parapsilosis (n = 2), and 5 isolates were mixed culture. By snPCR, 63 (96.6%) isolates were identified, including C. albicans (n = 37), C. glabrata (n = 11), C. tropicalis (n = 14), and C. parapsilosis (n = 1), and the species of 2 isolates could not be identified. Additionally, snPCR for the specific identification of the Candida species of the 65 clinical Candida isolates revealed 70.3% results agreement with the chromogenic medium CHROMagar™ Candida. Conclusions: In this study, snPCR was specific and more sensitive than the chromogenic medium CHROMagar™ Candida for the detection of Candida spp. insofar as it failed to identify only a few isolates.

P248 Sensitivity of the amsel’s criteria compared to the nugent score in absence and in presence oftrichomonas vaginalis(TV) and/orcandidaSPP among women with symptomatic vaginitis/vaginosis

Background/introduction In a multicenter clinical trial funded by BD, we observed less accurate clinician diagnosis of bacterial vaginosis (BV) based on clinical observations when Trichomonas vaginalis (TV) and/or Candida spp. were also detected by the trial Reference Methods than when only BV was detected. Aim(s)/objectives To determine the sensitivity of each criterion and of the overall Amsel’s criteria (3/4 criteria met), the results of the Amsel’s corresponding to the sub-population of specimens that gave a Nugent score of 7–10 were analysed. Methods Following informed consent , women with symptoms of vaginitis/vaginosis were included in the trial. The four Amsel’s criteria and the Nugent score were performed. Evaluation for trichomoniasis by wet mount and culture (InPouch™ TV, Biomed) were performed. Candida colonies were isolated (BBL™ Sabouraud Dextrose Agar, Emmons and BBL™ CHROMAgar™ Candida plate, BD) and identified by ITS-2 bi-directional sequencing (Accugenix ® ). Results In total, 269/497 (54.1%) specimens gave a Nugent score of 7–10. Amongst them, TV and/or Candida spp. were found in 100 specimens (37.2%). The sensitivity of clue cells, amine test, vaginal pH, BV vaginal discharge, and overall Amsel’s criteria in absence of TV and/or Candida spp. was 86.3%, 82.7%, 91.1%, 71.0%, and 84.6% respectively. In presence of TV and/or Candida spp., the sensitivity was 63.6%, 64.0%, 75.0%, 42.0%, and 60.0% respectively (p values ≤ 0.0009 for all comparisons). Discussion/conclusion The sensitivity of the Amsel’s criteria in women with BV decreases when TV and/or Candida spp. are present. The BV vaginal discharge is the least sensitive criterion.

Prevalence of Yeast Other than Candida albicans in Denture Wearers

The isolation of yeast species other than Candida albicans from the oral mucosa has been increasing in frequency, suggesting that those may constitute emerging potential oral colonizers. The purpose of this work was to determine whether yeast species other than C. albicans are associated with factors related to wearing of dental prostheses. tRNA-PCR fingerprinting and sequencing of the 26S rDNA D1/D2 domain were used to identify all yeasts isolated from CHROMagar™ Candida cultures of oral swabs collected from 178 patients. Besides C. albicans, 13 other species were identified, corresponding to 34% of the yeast isolates. The majority of the non-C. albicans species were not detected as single colonizers but rather in co-colonization with one or two other yeasts, often with C. albicans. No significant associations were found with non-C. albicans species. On the contrary, the best-fitted logistic regression model predicts that either wearing a denture (adjusted odds = 4.6) or insufficient oral hygiene (adjusted odds = 2.3) are risks for colonization by yeast, in general. The colonization with non-C. albicans species and co-colonization were not independently associated with any of the analyzed host-related factors. In particular, neither wearing a removable denture nor being elderly were significant predictors.

Candida colonisation in asymptomatic HIV patients attending a tertiary hospital in Benin City, Nigeria

Background: Candidiasis is the commonest opportunistic fungal infection in patients infected with human immunodeficiency virus (HIV). CD4+ lymphocyte counts have been found to be a marker of HIV disease progression.Aim: This study focused on determining the spectrum of Candida isolates in urine, stool, and oral specimens among HIV patients in a tertiary hospital.Methods: A total of 300 subjects comprising of 200 HIV patients and 100 non-HIV subjects were used for this study. Three samples (urine, stool, and oral swab) were collected from each subject. Each specimen was processed using standard microbiological techniques and emergent Candida isolates were identified with CHROMagar™ Candida and sugar fermentation tests.Results: The overall prevalence of Candida colonisation among HIV patients was 52.5%. HAART-naive patients had a significantly higher prevalence (OR = 3.65; 95% CI = 2.03–6.56; p<0.0001) than their counterpart on highly active antiretroviral therapy (HAART) (OR = 1.99; 95% CI = 1.13–3.50; p=0.0232). Female gender was a significant risk factor for acquiring Candida infection (OR = 3.40; 95% CI = 1.14–10.13; p=0.0289). The effect of age on prevalence of candidiasis was observed among HIV patients on HAART (p=0.0161). A CD4+ count <200 cells/µl was a significant risk factor for acquiring candidal infection only among HAART-naive patients (OR = 4.37; 95% CI = 1.60–11.95; p=0.0042). The five species of Candida recovered from this study were C. albicans, C. krusei, C. parapsilosis, C. tropicalis, and C. glabrata.Conclusion: There is a significant relationship between antiretroviral therapy, CD4+ counts, and the prevalence of candidiasis.

Complete denture biofilm after brushing with specific denture paste, neutral soap and artificial saliva

This study compared the levels of biofilm in maxillary and mandibular complete dentures and evaluated the number of colony-forming units (cfu) of yeasts, after using auxiliary brushing agents and artificial saliva. Twenty-three denture wearers with hyposalivation and xerostomia were instructed to brush the dentures 3 times a day during 3 weeks with the following products: Corega Brite denture dentifrice, neutral liquid soap, Corega Brite combined with Oral Balance (artificial saliva) or tap water. For biofilm quantification, the internal surfaces of the dentures were disclosed, photographed and measured using a software. For microbiological analysis, the biofilm was scrapped off, and the harvested material was diluted, sown in CHROMagar™ Candida and incubated at 37°C for 48 h. Data were analyzed statistically by two-way ANOVA and Tukey's test (α=0.05). Mandibular dentures presented a mean biofilm percentage (µ=26.90 ± 21.10) significantly greater than the maxillary ones (µ=18.0 ± 15.0) (p<0.05). Brushing using Corega Brite combined with Oral Balance (µ=15.87 ± 18.47) was more effective (p<0.05) than using the denture dentifrice (µ=19.47 ± 17.24), neutral soap (µ=23.90 ± 18.63) or tap water (control; µ=32.50 ± 20.68). For the microbiological analysis, the chi-square test did not indicate significant difference between the hygiene products for either type of denture. The more frequently isolated species of yeasts were C. albicans, C. tropicalis and C. glabrata. In conclusion, mandibular dentures had more biofilm formation than maxillary ones. Denture brushing with Corega Brite dentifrice combined with the use of Oral Balance was the most effective method for reduction of biofilm levels, but the use of products did not show difference in yeast cfu counts.

Candida nivariensis isolated from a renal transplant patient with persistent candiduria—Molecular identification using ITS PCR and MALDI-TOF

We report on the isolation of Candida nivariensis from a renal transplant patient with persistent candiduria. Biochemical profiling misidentified isolates as Candida glabrata (3/5) and Candida inconspicua (2/5). All isolates produced white colonies on CHROMagar™ Candida medium. Internal transcribed spacer (ITS) ribosomal gene sequence analysis and MALDI-TOF-MS analysis (Bruker Biotyper™ 2.0) identified all isolates as C. nivariensis, demonstrating the utility of MALDI-TOF as a rapid, accurate approach for the identification of cryptic Candida species.

Strengths and Limitations of different Chromogenic Media for the Identification of &lt;i&gt;Candida&lt;/i&gt; Species

The treat ment of invasive candidiasis and other Candida infections with the appropriate antifungal agent is assisted by the identification of Candida isolates to the species level. Rapid and accurate methods of differentiation are therefore imperat ive if t reat ment is to be effective, particularly in HIV-positive patients and in pregnant mothers where intervention may be necessary to reduce the risk for preterm delivery. The time used for isolation, identificat ion and detection of mixed cultures may be reduced with the help of available chro mogenic med ia. In this study, five commercial chro mogenic med ia were evaluated for the differentiation of Candida species. Six type-strains of Candida species were streaked onto each of five different chro mogenic media and incubated for up to 4 days at the different temperatures recommended by the manufacturers. This co mparative evaluation demonstrated the strengths and weaknesses of each mediu m employed and found CHROMagar™ Candida and Chro mogenic Candida Agar to be the most effective for distinguishing between different Candida species.

Identification of Candida spp. by phenotypic tests and PCR

The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans.