Todd M. Pryce

Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.

Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients

We report the development and evaluation of a real-time PCR assay using the LightCycler instrument for the detection of C. albicans and A. fumigatus DNA in whole blood. Recently published consensus criteria for the diagnosis of invasive fungal infection (IFI) were used for all patient samples. Unique and published primer pairs were developed and assessed for sensitivity, specificity, and reproducibility to detect C. albicans and A. fumigatus DNA in samples spiked with purified DNA, and whole blood samples from 8 high-risk patients and 45 negative controls. The real-time assay demonstrated an analytical sensitivity of 10 fg of purified C. albicans and A. fumigatus DNA and was found to be specific for each species. The standardized approach was highly reproducible and detected C. albicans and A. fumigatus DNA in two patients with proven IFI and in one patient with a possible IFI. In addition, we report for the first time the use of recently published international consensus criteria for the diagnosis of IFI in the evaluation of a mildly invasive fungal diagnostic assay. Standardized clinical criteria and a more standardized approach to detect fungal DNA in less invasive patient samples, may permit a more reliable comparison of future studies. A rapid real-time detection of fungal DNA in whole blood, combined with standard clinical markers of response, may be more useful for monitoring patients at risk of developing IFI than other diagnostic methods currently available.

Feasibility of real-time polymerase chain reaction in whole blood to identify Streptococcus pneumoniae in patients with community-acquired pneumonia.

We assessed a real-time quantitative polymerase chain reaction (PCR) assay targeting the lytA and ply gene of Streptococcus pneumoniae. Both assays were applied to whole blood samples from 28 adult patients with community-acquired pneumonia. Our findings suggest the lytA PCR is more sensitive, and the quantitative aspect of the assay shows promise as an aid to clinical judgment.

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.

Cutaneous mucormycosis and motor vehicle accidents: Findings from an Australian case series

Cutaneous disease is the third most frequent manifestation of mucormycosis. The clinical manifestations of and subsequent mortality due to cutaneous mucormycosis are dependent on the mode of acquisition and the host immune status. Here, we describe the epidemiology, clinical presentation, microbiology, and outcomes of 16 cutaneous mucormycosis infections managed in an Australian tertiary hospital over a 15-year period. The proportion with localized (56%), deep (38%), and disseminated (6%) cutaneous disease as well as the overall mortality (25%) were consistent with findings reported in the published literature. Two novel forms of hospital-acquired infection were reported following a sacral pressure sore and insertion of a foreign body during a bone graft procedure. The majority of patients were immunocompetent (75%) and/or suffered trauma (56%) with associated environmental contamination. A novel finding was that motor vehicle accidents (MVAs) accounted for 78% of all trauma-related cases, suggesting MVAs should receive greater recognition as a potential precipitant of cutaneous mucormycosis. Aggressive decontamination and debridement of devitalized tissue following trauma is therefore likely to play an important role in the prevention of this rare but potentially devastating infection.

Low vancomycin MICs and fecal densities reduce the sensitivity of screening methods for vancomycin resistance in Enterococci.

ABSTRACT Active surveillance is part of a multifaceted approach used to prevent the spread of vancomycin-resistant enterococci (VRE). The impact of fecal density, the vancomycin MIC of the isolate, and the vancomycin concentration in liquid medium on test performance are uncertain. Using fecal specimens spiked with a collection of 18 VRE (predominantly vanB) with a wide vancomycin MIC range, we compared the performances of commercial chromogenic agars (CHROMagar VRE, chromID VRE, Brilliance VRE, and VRE Select) and 1 liquid medium (Enterococcosel enrichment broth) for VRE detection. The specificity of solid media was excellent; however, the sensitivity at 48 h varied from 78 to 94%. Screening using liquid medium was less sensitive than screening with solid media, particularly as the vancomycin content increased. Sensitivity declined (i) as the fecal VRE density decreased, (ii) when the media were assessed at 24 h (versus 48 h), and (iii) for isolates with a low vancomycin MIC (sensitivity, 25 to 75% versus 100% for isolates with vancomycin MIC of <16 mg/liter versus >32 mg/liter on solid medium using 106 CFU/ml of feces). Depending on local epidemiology and in particular VRE vancomycin MICs, the sensitivity of culture-based methods for VRE screening of stool or rectal specimens may be suboptimal, potentially facilitating secondary transmission.

Quantitative rt‐PCR holds promise as a screening tool for patients with severe sepsis

Objective: The aim of the present study was to determine if the quantification of bacterial 16S rDNA could be clinically useful in predicting patients at increased risk of developing septic shock.

Universal Detection and Identification of Fungi by PCR and DNA Sequencing

This assay was developed to rapidly (within 24 h) detect and identify fungi from positive blood cultures and from clinical samples where initial laboratory evidence of fungal infection is suspected or the clinical pre-test probability of invasive fungal disease is high. Two PCR-DNA sequence based methods (PCRS) are described. The first method (PCRS-B) is used for the identification of fungi from culture plates and positive blood cultures. The second method (PCRS-D) is used for the detection and identification of fungi from microscopy-positive clinical samples such as fresh tissue or fluid. Requests for fungal PCR directly from clinical specimens are screened by a Clinical Microbiologist to determine whether it is worth testing based on laboratory and clinical evidence of invasive fungal disease.