Repetitive-sequence-based PCR using the DiversiLab system for identification of Aspergillus species.

Repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system was investigated for identification of Aspergillus. Ninety-five clinical isolates, identified by conventional methods, and five ATCC strains were tested. Sequencing of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) was performed on 2 isolates with discrepant rep-PCR and conventional results and 15 randomly selected outlier isolates. One isolate not identified by sequencing was excluded from analysis. After resolving discrepancies, all but one A. glaucus strain had >or=85% similarity to one or more strains of the same Aspergillus species in the mold database, thereby providing accurate identification. No isolate was misidentified.

Species identification and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequence-based PCR.

The DiversiLab system, which uses repetitive sequence-based PCR (rep-PCR) to genotype micro-organisms, was evaluated as a molecular typing tool for members of the genus Candida. Initially, 41 clinical Candida spp. (7 Candida krusei, 10 Candida parapsilosis, 7 Candida albicans, 10 Candida tropicalis and 7 Candida glabrata), previously identified at the species level by morphological and biochemical analysis, were analysed with the DiversiLab system. Species identification was confirmed by DNA sequence analysis of the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). On the basis of an 80 % similarity threshold, rep-PCR consistently clustered like species and this set of isolates, along with five ATCC reference strains, was used to create a DNA fingerprint library with the DiversiLab software. Subsequently, an additional set of 115 clinical Candida isolates, identified biochemically as C. albicans (n=94), C. glabrata (n=8), C. parapsilosis (n=5), C. tropicalis (n=3), C. krusei (n=3) and Candida lusitaniae (n=2), isolated at a regional reference laboratory, were typed using DiversiLab. One hundred and six of these isolates clustered with members of the Candida library at >80 % similarity and thus could be assigned species identification, and initial calculations showed that identification via rep-PCR fingerprinting was 95 % concordant (101/106) with the biochemical/morphological identification. However, ITS region sequencing of the five discrepant samples, as well as the nine isolates that were <80 % similar to the database samples, showed that nine were misidentified with traditional biochemical/morphological methods. For the misidentified isolates, the sequence-based identification was in agreement with the DiversiLab clustering, yielding an actual correlation of >99 %. As traditional techniques can take several days to provide information about Candida at the genus/species level, genotyping with the DiversiLab system holds promise for more-rapid speciation of members of this genus. This system may also be useful for epidemiological studies such as source tracking that require Candida subspecies discrimination.

Recovery of live virus after storage at ambient temperature using ViveST™.

BACKGROUND: A major impediment to performing virological field studies in developing nations is the lack of ultra-low freezers as well as the expense and difficulty of shipping frozen samples. A commercially available product, ViveST™, was developed to preserve nucleic acids at ambient temperature for use in specimen storage and transportation. However, its applications as a viral storage, transport and recovery device have not been evaluated. OBJECTIVE: To examine the ability of ViveST to preserve live virus following storage at ambient temperature. STUDY DESIGN: A panel of six viruses was stored at ambient temperature (~22°C) in ViveST with fetal bovine serum (FBS), or ViveST with minimal essential media (MEM) and compared with virus stored in universal transport media (M4RT), MEM, and FBS alone. Stored viruses included: human adenovirus (14p), dengue virus 2 (16608), echovirus 3 (Morrisey), human rhinovirus 15 (1734), Coxsackie virus B5 (Faulkner), and herpes simplex virus 1 (HF). After 7 days storage at ambient temperature, virus recovery was measured via titration using viral plaque assays or focus-forming unit assays. RESULTS: Viral titer studies indicate that ViveST with either FBS or M4RT preserved/recovered 5 different viruses for 1 week at ambient temperature. MEM preserved 4 viruses while FBS and ViveST with MEM preserved 3 viruses each. Statistical analyses indicate that M4RT and ViveST with FBS preserved significantly more virus than the other treatments. CONCLUSIONS: These data suggest that ViveST with either FBS or M4RT may be useful in field specimen collection scenarios where ultra-cold storage is not available.

Repetitive-sequence-PCR-based DNA fingerprinting using the Diversilab system for identification of commonly encountered dermatophytes.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of dermatophytes commonly isolated in a clinical laboratory was assessed by comparing results to those of conventional tests (colony morphology, microscopic examination of slide cultures, and, for suspected Trichophyton species, use of additional media). Sixty-one cultures were tested in phase 1, the feasibility portion of the study; 64 additional cultures were tested in phase 2, the validation portion conducted to assess reproducibility and confirm accuracy. Discrepancies were resolved by repeating rep-PCR and conventional tests and, in phase 2, sequencing the internal transcribed spacers. After initial testing of the cultures in phase 1 (excluding one contaminated culture), agreement between conventional tests and rep-PCR was 90% (54 of 60). Agreement was 98.3% after resolution of discrepancies, and in all but one case the initial rep-PCR result was correct. After initial testing of cultures in phase 2 (excluding one discarded and one contaminated culture), agreement between rep-PCR and conventional testing was 88.7% (55 of 62). After discrepancies were resolved, agreement was 100%. Initial rep-PCR results were correct, except for one Microsporum canis culture containing two colony variants, which could not be initially identified by rep-PCR. The performance of the DiversiLab system for identification of the dermatophytes commonly encountered in a clinical mycology laboratory-Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, and M. canis-was excellent. Moreover, the DiversiLab system is technically simple and provides results in < 24 h once a pure culture is available for testing, which is considerably more rapid than conventional identification tests.