cdubliniensis

cdubliniensis Identification of Candida dubliniensis using a rapid PCR test
   Candida dubliniensis was first described by Sullivan et al. in 1995 , it is closely related phylogenetically to Candida albicans and is commonly associated with oral candidiasis in Human Immunodeficiency Virus (HIV) patients. C. dubliniensis is a yeast species. C. dubliniensis and C. albicans differ significantly at the genetic level . The appearance of colonies on a chromogenic medium is a dark green colonies after incubation at 37?C for 48 hours in contrast to the light green colonies seen with C. albicans .
   Phenotypically, C. dubliniensis and C. albicans have many similarities, including their microscopic morphology and ability to form germ tubes in serum. Both species produce blastoconidia with pseudohyphae, true hyphae, and chlamydospores. Some investigators have noted more abundant production and different arrangements (pairs, triplets, or clusters) of chlamydospores with C. dubliniensis, but this can be subjective for clear separation of the two species .
   C. dubliniensis can be readily differentiated from C. albicans on the basis of its inability to grow at 45?C. Although easy to perform, this test requires that isolates be incubated for 24 to 48 hours before the isolates can be discriminated .
   Phylogenetic analysis of large and small subunit rRNA gene sequences provided the basis for the designation of C. dubliniensis as a separate species. The first isolates identified as C. dubliniensis were recovered from the oral cavities of Irish HIV infected individuals. Since the discovery there have been increasing numbers of reports of the recovery of C. dubliniensis isolates by laboratories throughout the world, including Europe, North and South America, and Australia. The majority of these isolates have been associated with oral candidosis in HIV infected and AIDS patients. And also been recovered from the oral cavities, gastrointestinal tracts and vaginas of HIV-negative individuals and also there have been isolates associated with systemic disease .
   A variety of tests have been developed to discriminate between C. dubliniensis and C. albicans based on their phenotypic characteristics. These include carbohydrate assimilation profiles and colonial coloration on differential media such as CHROMagar Candida and methyl blue-Sabouraud agar. However, some of these assays have been shown to be unreliable in some instances and should only be used for the presumptive identification of C. dubliniensis from clinical specimens. The accuracy of C. dubliniensis isolate identification based on carbohydrate assimilation profiles has been improved by the recent inclusion of the assimilation profiles of some C. dubliniensis strains in the databases of commercially available yeast identification systems, including the API ID 32C and 20C AUX systems. The most reliable tests available to differentiate between these species are based on molecular techniques such as DNA fingerprinting with repetitive-sequence-containing probes, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis, but these are not suitable for the analysis of large sample numbers in routine diagnostic laboratories. However, since the differences between C. dubliniensis and C. albicans are most pronounced at the genetic level such differences should provide the basis for a specific and rapid identification test. One molecular technique with the required degree of specificity and ease of use is the polymerase chain reaction (PCR) .
   The time to identify C. dubliniensis was reduced from 3.5 days to 7 hours by the PCR method .

Materials and Methods
Microorganisms
Cultures obtained from the American Type Culture Collection (ATCC). Isolates of Candida spp., Cryptococcus humicolus, Stefanoascus ciferrii, and Trichosporon cutaneum were grown in 50mL Erlenmeyer flasks by seeding one 10-?L loopful of growth from an agar slant into 10mL of YPD broth (1% yeast extract, 2% Bacto Peptone, 2% dextrose). Cryptococcus neoformans serotypes A, B, C, and D were grown similarly; however, YPD broth was supplemented with 2.9% NaCl to reduce capsule formation. All broth cultures were grown at 35?C for 18 hours in a rotary shaker set at 150 rpm prior to DNA extraction for prototype testing.

DNA isolation
DNA was extracted from all yeast species by using the Pure-gene DNA Isolation Kit. This kit facilitates the rapid recovery of sufficient DNA for PCR amplification and allows multiple samples to be extracted in parallel. For example, multiple yeast isolates could be extracted at the same time so that a large number of samples could be processed quickly and efficiently on a given day. DNAs from filamentous and dimorphic fungi were obtained. Quantification of DNA was performed by using a fluorometer and Hoechst 33258 Dye. DNA was diluted in TE buffer (10mM Tris, 1mM EDTA{pH 8.0}) so that a total of 1ng of template DNA was added to each PCR vial.

Oligonucleotide synthesis of primers and probes
Oligodeoxyribonucleotide primers and probes were synthesized. Universal fungal primers ITS3 and ITS4 were used to amplify the ITS2 region. Oligonucleotide probes were designed from sequence data for the ITS2 region of the Candida sp. rDNA.

PCR amplification
The reaction mixture (100 ?L) contained 10 ?L of 10x PCR buffer (100 mM Tris-HCl, 500 mM KCl {pH 8.3}), 6 ?L of 25 mM MgCl2, 8 ?L of a deoxynucleotide triphosphate mixture (1.25 mM each dATP, dCTP, dGTP, and dTTP), 1 ?L of each primer (20 ?M), 2.5 U of Taq DNA polymerase, 2 ?L of template DNA (0.5 ng/?L), and sterile distilled water to bring the total volume to 100 ?L. Vials were placed in heating block of a model 9600 thermal cycler equilibrated at 95?C, followed by 30 cycles of 95?C for 30 s, 58?C for 30 s, and 72?C for 1 min. A final extension step of 72?C for 5 min was then conducted. Appropriate positive and negative controls were included.

Agarose gel electrophoresis
Electrophoresis was conducted in TBE (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA {pH 8.4}) buffer at 76 V for approximately 1 hour in gels composed of 1% (wt/vol) agarose and 1% (wt/vol) NuSieve. Gels were stained with 0.5 ?g ethidium bromide per mL of deionized water for 30 min, followed by a 30 min wash in deionized water. DNA bands confirming a positive PCR were visualized with a UV transilluminator and photographed.

PCR-EIA (Enzyme Immunoassay)
   PCR-amplified DNA was hybridized to species-specific digoxigenin-labeled probes and to a generic biotinylated probe, and then the complex was added to streptavidin-coated microtitration plates and captured. A colorimetric EIA was then conducted to detect captured DNA by using horseradish peroxidase-conjugated anti-digoxigenin antibodies. All probes were tested in a matrix format against DNA from other Candida species, as well as against DNAs from other fungi. All probes were tested against all of the target DNAs so that fungi could be identified by a discrete pattern of reactivity. When a probe cross-reacted with heterologous DNA, probes specific to the heterologous DNA were designed. Therefore, use of both probes as part of the matrix allows species-specific identification by a process of elimination and does not require additional steps or retesting of samples because all probes and all targets are included in the complete matrix from the beginning .
   The probes in the method above discriminated C. albicans from C. dubliniensis. The CA probe which detected C. albicans DNA did not react with DNA from any C. dubliniensis strain tested, and the DB probe for C. dubliniensis identification did not hybridize with DNA from any C. albicans strain tested. The CA probe also detected both C. stellatoidea type I and II DNAs and differentiated C. stellatoidea DNA from C. dubliniensis DNA .
    Previous research in the same laboratory where the above method was carried out demonstrated that five Candida species-specific probes could be designed and adapted to a simple PCR-EIA format to detect Candida species DNA. The above method extends the range of probes to include a test matrix of 18 Candida species that is capable of complementing species identification by the API 20C Carbohydrate assimilation system. Sixteen of the probes were totally specific and can be used to identify their respective Candida species, including C. dubliniensis.
    The currently available commercial tests for species identification, such as the API 20C, RapID, etc. require subculturing from clinical specimens to obtain pure cultures before inoculation of the test panels. Therefore, even if an overnight culture were required prior to PCR-EIA testing, the time to species identification after obtaining a pure culture is still reduced to 7 hours from 3.5 days by conventional phenotypic identification methods.
    C. dubliniensis was only recently identified as a separate species, and since it is difficult to distinguish it from C. albicans in clinical samples there is still very little information available concerning its epidemiology and clinical significance. It has been shown that C. dubliniensis is able to readily develop fluconazole resistance under selective pressure. To determine whether this or other factors are implicated in the emergence of C. dubliniensis as a pathogen, and to measure what the true incidence of this species is in humans, novel methods for discriminating between C. dubliniensis and C. albicans are required.

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