Candida albicans and Candida dubliniensis are the only Candida species that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are greater than 30 days old. However, 5-15 day old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were pre-induced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo. Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialised form of growth by C. albicans and C. dubliniensis.
25 Read more
The usefulness of Candida ID 2 (CAID2) reformulated medium (bioMe ´rieux, France) has been com- pared with that of the former Candida ID (CAID; bioMe ´rieux), Albicans ID 2 (ALB2; bioMe ´rieux), and CHROMagar Candida (CAC; Chromagar, France) chromogenic media for the isolation and presumptive identification of clinically relevant yeasts. Three hundred forty-five stock strains from culture collections, and 103 fresh isolates from different clinical specimens were evaluated. CAID2 permitted differentiation based on colony color between Candida albicans (cobalt blue; sensitivity, 91.7%; specificity, 97.2%) and Candida dubliniensis (turquoise blue; sensitivity, 97.9%; specificity, 96.6%). Candida tropicalis gave distin- guishable pink-bluish colonies in 97.4% of the strains in CAID2 (sensitivity, 97.4%; specificity, 100%); the same proportion was reached in CAC, where colonies were blue-gray (sensitivity, 97.4%; specificity, 98.7%). CAC and CAID2 showed 100% sensitivity values for the identification of Candida krusei. However, with CAID2, experience is required to differentiate the downy aspect of the white colonies of C. krusei from other white-colony-forming species. The new CAID2 medium is a good candidate to replace CAID and ALB2, and it compares well to CAC for culture and presumptive identification of clinically relevant Candida species. CAID2 showed better results than CAC in some aspects, such as quicker growth and color development of colonies from clinical specimens, detection of mixed cultures, and presumptive differen- tiation between C. albicans and C. dubliniensis.
Reference strains. Nine C. albicans and four C. glabrata reference strains (including type strains of both species), along with 57 other reference strains representing 31 phylogenetically related or clinically prevalent fungal species and subspecies, were included in the study. Most strains were obtained from the Agricultural Research Service Culture Collection (NRRL), Peoria, IL; the American Type Culture Collection (ATCC), Manassas, VA; and Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Strain identities are given in Table 1. Strains were grown overnight at 37°C on YM agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, 2% agar) (Teknova, Hollister, CA), and a colony was then inoculated into YM broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose) (Teknova) and incu- bated overnight at 37°C. Slides were made the following day as described below. Challenge cultures. Forty-seven blood culture bottles which signaled negative at 7 days were used to challenge the assay. Bottles were selected from those which became available during the study period. Five BacT/ALERT bottle types (10 SA aerobic, 6 PF pediatric, 10 SN anaerobic, 13 FA aerobic, and 8 FN anaerobic) were randomly spiked with representative Candida species, other yeast species, and mixtures of yeasts. All of these strains were clinical isolates whose species identification had been confirmed by sequence analysis of 500 base pairs of the 5 ⬘ end of the nuclear large ribosomal subunit rRNA gene. All seeded bottle assays were performed at Aachen University Hospital using strains from their frozen collection. Strains were thawed and cultured on Sabouraud’s dex- trose agar (BD, Heidelberg, Germany) overnight at 30°C, and then 100 l of a yeast suspension in 0.9% NaCl solution was prepared adjusted to a 0.5 McFarland standard, resulting in an inoculum of 2.5 ⫻ 10 4
allele did not result in increased true hypha formation in C. dubliniensis, suggesting strong repression of the RAS1- cAMP pathway itself or downstream regulators . One of the most important transcriptional regulators involved in the control of morphogenesis in C. albicans is Nrg1, which Staib and Morschh¨auser. showed that it is di ﬀ erentially expressed by C. dubliniensis when grown on media such as Staib agar . In C. albicans this protein targets the negative regulator Tup1 to specific sequences in the promoters of genes involved in hypha formation. NRG1 expression is rapidly downregulated in C. albicans cells incubated under hypha- inducing conditions, including when cells are phagocytosed by murine macrophages, which results in germination and escape from the phagocytes. However, in C. dubliniensis, NRG1 expression remains high under these conditions, preventing hypha formation and causing cells to remain in the yeast phase, which in the murine macrophage model results in failure to escape from the phagocytes and the death of the fungus . Deletion of the NRG1 gene in C. dubliniensis resulted in an increase in the rate of hypha and particularly pseudohypha formation, which in turn led to increased survival when exposed to murine macrophages as well as increased virulence in the reconstituted human epithelial (RHE) cell model of oral candidosis. Surprisingly, the C. dubliniensis Δ nrg1 mutant was no more virulent than its parent strain in the murine systemic infection model and formed mainly pseudohyphae in infected kidneys, suggesting an additional level of repression preventing true hypha formation in vivo .
In East Africa, a study done in Aghakhan hospital- Kenya reported C. albicans as the prominent species with prevalence of 69.3% followed by C. glabrata 12.9% . In Tanzania, non-Candida albicans species were reported to contribute about 37% of Candida vaginitis cases . However, data on azole susceptibility patterns and factors associated with Candida vaginitis are still lim- ited. Here, we report the prevalence and factors associ- ated with laboratory confirmed Candida vaginitis among pregnant women with symptoms of vaginitis attending antenatal clinics in Mwanza, Tanzania. Furthermore, data on the azole susceptibility patterns of these Candida spp. are reported.
Isolates were identified at participant institutions using methods rou- tinely employed at the submitting laboratory, including the use of Vitek, MicroScan, API strips, and AuxaColor systems supplemented by classic methods for yeast and mold identification (29, 30). Isolates were submit- ted to JMI Laboratories (North Liberty, IA), where the identification was confirmed by morphological, biochemical, and molecular methods (31– 34). Yeast isolates were subcultured and screened using CHROMagar Candida (Becton, Dickinson, Sparks, MD) to ensure purity and to differ- entiate Candida albicans/Candida dubliniensis, Candida tropicalis, and Candida krusei. Biochemical tests including Vitek 2 (bioMérieux, Hazel- wood, MO) testing, trehalose assimilation (Candida glabrata), and growth at 45°C (C. albicans/C. dubliniensis) also were used to establish the identification of common Candida species. Molecular methods were used for common species of Candida that could not be definitively identified using phenotypic methods or that presented unusual phenotypic or bio- chemical profiles, as well as all uncommon species of Candida, non-Can- dida yeasts, and all molds. Candida spp. and other yeasts were identified using sequence-based methods for the internal transcribed spacer (ITS) region, 28S ribosomal subunit (D1/D2), and intergenic spacer (IGS) (De- baryomyces spp.) and IGS1 (Trichosporon spp.) (31, 34). All mold isolates were subcultured and analyzed by ITS sequencing followed by specific
Fungal strains. For the testing of primer specificity, a reference strain of Candida albicans (ATCC 10231) was used. For the determination of the ITS2 PCR fragment length, a wide collection of reference strains, American Type Culture Collection or College of American Pathologist specimens were used, as well as patient strains obtained from the Health Sciences Centre Clinical Micro- biology Laboratory stock culture collection. Yeast organisms were grown on Sabouraud dextrose agar plates (BBL, Becton-Dickinson, Cockeysville, Md.) for 24 h at 37°C, and molds were grown on potato dextrose agar (16) for up to 7 days. Species identification was established by using the API 20C kit (BioMerieux, Hazelwood, Mo.) or by conventional morphological analysis (14).
Due to unacceptably high interlaboratory variation in caspofungin MIC values, we evaluated the use of micafungin as a surro- gate marker to predict the susceptibility of Candida spp. to caspofungin using reference methods and species-specific interpre- tive criteria. The MIC results for 3,764 strains of Candida (eight species), including 73 strains with fks mutations, were used. Caspofungin MIC values and species-specific interpretive criteria were compared with those of micafungin to determine the per- cent categorical agreement (%CA) and very major error (VME), major error (ME), and minor error rates as well as their ability to detect fks mutant strains of Candida albicans (11 mutants), Candida tropicalis (4 mutants), Candida krusei (3 mutants), and Candida glabrata (55 mutants). Overall, the %CA was 98.8% (0.2% VMEs and MEs, 0.8% minor errors) using micafungin as the surrogate marker. Among the 60 isolates of C. albicans (9 isolates), C. tropicalis (5 isolates), C. krusei (2 isolates), and C. glabrata (44 isolates) that were nonsusceptible (either intermediate or resistant) to both caspofungin and micafungin, 54 (90.0%) contained a mutation in fks1 or fks2. An additional 10 C. glabrata mutants, two C. albicans mutants, and one mutant each of C. tropicalis and C. krusei were classified as susceptible to both antifungal agents. Using the epidemiological cutoff val- ues (ECVs) of 0.12 g/ml for caspofungin and 0.03 g/ml for micafungin to differentiate wild-type (WT) from non-WT strains of C. glabrata, 80% of the C. glabrata mutants were non-WT for both agents (96% concordance). Micafungin may serve as an acceptable surrogate marker for the prediction of susceptibility and resistance of Candida to caspofungin.
oratory at Royal North Shore Hospital, Sydney, Australia, and the Molecular Mycology Research Laboratory at Westmead Hospital, Westmead, Australia. Clinical isolates were obtained from the Mycology Laboratory at Westmead Hospital. Isolates were identified using standard colonial and microscopic char- acteristics (for molds) (20, 37) and the VITEK I (bioMerieux Vitek, Hazelwood, MO) and/or ID 32C (bioMerieux, Marcy-l’Etoile, France) commercial systems (for yeasts). Canavanine-glycine bromothymol blue (CGB) agar was used to differentiate between Cryptococcus neoformans (Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. grubii) and Cryptococcus gattii (19). A total of 159 (32 reference and 127 clinical) isolates belonging to 22 fungal species were studied; all species were represented by species-specific probes in the RLB assay (Table 1). Isolates comprised 16 Candida species (101 strains; Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida haemulonii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida norvegica, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Can- dida tropicalis, Candida utilis, Candida viswanathii, Candida zeylanoides), C. neo- formans complex (five strains of C. neoformans var. grubii, four strains of C. neoformans var. neoformans, and eight strains of C. gattii), and five Aspergillus species (40 strains; Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans).
Pathogenic fungi such as Candida, Cryptococcus, and Aspergil- lus spp. and species of other genera commonly cause life-threat- ening infections of immunocompromised hosts, including patients receiving immunosuppressive therapy. In the United States, Can- dida species are the fourth leading cause of nosocomial blood- stream infections. With an estimated total of 72.8 million oppor- tunistic Candida infections per year worldwide, the case/fatality rate due to Candida species is in the range of 33.9% (32). Al- though Candida albicans remains the leading and most important pathogenic yeast species, the incidence rates of non-C. albicans yeast infections have been increasing in recent years. Moreover, outbreaks in perinatal intensive care and oncohematological units have been described (23). Recently, infections caused by less common yeasts and yeast-like species such as Pichia, Rhodotorula, Saccharomyces, and Trichosporon species and other rarely en- countered species have been reported (17).
FIG. 1. (A) Typical design of a circularizable padlock probe as exemplified by the Candida albicans-specific (CAL) probe. The probe comprises (i) a 5 ⬘ -phosphorylated end, (ii) a “backbone” containing binding sites for the RCA primers (RCA primers 1 and 2, respectively; designated by bold uppercase letters) as well as the nonspecific linker regions (designated by bold lowercase letters), and (iii) a 3 ⬘ end. The 5 ⬘ and 3 ⬘ ends of the probe are complementary to the 5 ⬘ and 3 ⬘ termini of the target sequence in reverse, in this example to the C. albicans sequence (GenBank accession no. AF455531). Abbreviations: 5 ⬘ -P, 5 ⬘ -phosphorylated binding arm; 3 ⬘ , 3 ⬘ binding arm. (B) Pictorial representation of the RCA method. Step 1, hybridization. Hybridization of padlock probe, containing target-complementary segments, to a target DNA sequence. Step 2, ligation. The probe is circularized by DNA ligase. Step 3, RCA and primer extension I. Ligated probe and binding of RCA primer 1 for RCA. Tandem repeat sequences complementary to the circular probe are generated by RCA. The reverse primer (RCA primer 2) binds to each tandem repeat generated by the rolling circle. Step 4, RCA and primer extension II. As the original RCA strand elongates, further priming events are initiated by primer 2, generating displaced DNA strands. As a result, new priming sites for the first primer (primer 1) are generated. The two primers thus function to generate a self-propagating pattern of DNA fragment release events (20). Step 5, detection of amplified product. RCA may be monitored using real-time PCR or agarose gel electrophoresis. ssDNA, single-stranded DNA.
Organisms. A total of 2,656 clinical isolates obtained from 60 different medical centers internationally in 2004 and 2005 were tested. The collection included 1,476 strains of Candida albicans, 383 of Candida parapsilosis, 356 of Candida glabrata, 269 of Candida tropicalis, 63 of Candida krusei, 45 of Candida guillier- mondii, 24 of Candida lusitaniae, 17 of Candida kefyr, 10 of Candida famata, 4 of Candida dubliniensis, 4 of Candida lipolytica, 3 of Candida pelliculosa, and 1 each of Candida rugosa and Candida zeylanoides. All isolates were obtained from blood or other normally sterile sites and represented individual infectious epi- sodes. The isolates were collected at the individual study sites and were sent to the University of Iowa (Iowa City) for identification and susceptibility testing as described previously (6, 13–16). The isolates were identified by standard methods (3) and stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice onto potato dextrose agar (Remel) and CHROMagar Candida (Hardy Diagnostics, Santa Maria, Calif.) to ensure purity and viability.
Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilli- ermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. nor- vegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.
Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropi- calis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of sus- ceptibility to fluconazole of < 8 g/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25- g fluconazole disk was calculated to be > 18 mm for C. albicans and > 22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5- g disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 g/ml, whereas a 2.5- g disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two mea- surements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.
A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Cryp- tococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.
The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans, C. tropicalis, and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.
A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Further- more, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.
Identification of isolated fungal pathogens from clinical samples. Eighty-six fungal pathogens isolated from clinical samples were processed and hybridized as described above and were then identified according to their specific hybridization maps. All 86 strains were distinguished by their hybridization maps on the microarray. The comprehensive identification re- sults by classical methods are regarded as the final standards. All the hybridization assay results were consistent with those of the conventional methods; the consistency was 100% (86/86). Testing of spiked blood samples. To examine the specificity of the designed probes and to assess their potential applicabil- ity in clinical testing, the performance of the array was vali- dated by blind testing of blood samples from 16 patients at the FHRCMM. Additionally, the identification of all isolates was confirmed by conventional morphological and physiological methods. Comparison of the array results with those of con- ventional methods showed that the array was able to unequiv- ocally identify the contents of all 16 samples. Ten samples were identified as C. albicans, three as Candida dubliniensis, and three as A. fumigatus. All the hybridization assay results were consistent with those of the conventional methods.
Table 3 shows the comparative study of 10 homologues. The results are in tabular form [Table 3].Compound 1, 4, 8, 12 and 13 proved active against S. aureus 209p. Compound 11 have shown its activity against bacterial strain S. aureus 209pand fungal speciesAspergillusfumigatus, Candida albicans. This indicates the structure activity relationship (SAR) that when halogen occupies the meta position of phenyl ring of coumarinchalcone; a broad spectrum antimicrobial activity is favoured. Compound 18 have shown activity against Candida albicansATCC10231. Compound 2, 7 and 9 exhibited no promising activity. The anti S. aureus 209p activity was found tobe in the order of substituents :
The emerging phenomenon of antifungal resistance is primarily a concern for invasive candidiasis. The knowledge about the antifungal resistance is less when compared to that of the antibiotic resistant bacterial infections, as they are the threat to public health. Thus, importance for understanding the emergence of antifungal resistance, awareness among medical and public health communities about these infections, and methods used to prevent and control them should be highlighted. The changing epidemiology of Candida infection has been partly attributed to the selection of less sensitive Candida strains by the widespread use of the azole fluconazole as a prophylactic and therapeutic agent.
163 Read more