A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

B.C. MILLAR, J. E. MOORE, J. XU, M. J. WALKER, S. HEDDERWICK AND R. MCMULLAN. 2002. Aims: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. Methods: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). Results: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. Impact of the Study: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.

J. E. MOORE, J. XU, B.C. MILLAR, J. COURTNEY AND J. S. ELBORN. 2003. Aims: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). Methods and Results: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30g), bacteriological agar no.1 (10g), yeast extract (5g), crystal violet (2mg), nisin (48mg), novobiocin (5mg), cycloheximide (100mg), amphotericin (2mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1•50×102 CFU ml-1 sputum Pseudomonas aeruginosa, 2•38×102 CFU ml)1 sputum Burkholderia cepacia genomovar IIIb and 6•70×103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. Conclusions: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. Significance and Impact of the Study: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.

This report presents a case of endocarditis due to Haemophilus segnis, which represents a speciation difficulty for the routine laboratory. In this study, a molecular approach provided speciation, which was confirmed phenotypically by a reference laboratory. The use of molecular genotypic analysis is an additional strategy in the investigation of endocarditis. It has applications not only in isolate identification but also in primary detection of infection, particularly in patients whose blood is culture negative by conventional methodologies.