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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.

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates (n = 222) and isolates from a culture collection library (n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.

A quantitative real-time PCR targeting the Pneumolysin (ply) gene of Streptococcus pneumoniae was developed for the LightCycler instrument using Fluorescence Resonance Energy Transfer (FRET) probes. All common S. pneumoniae serotypes were detected while other bacteria and viruses were not. The sensitivity was determined to be between one and ten target copies per reaction. The PCR was applied to six CSF and 16 whole blood specimens from 17 patients with laboratory proven invasive pneumococcal disease. One hundred percent of CSF specimens and 69% of whole blood specimens were PCR positive. The bacterial loads were determined to be 7.6 to 6.01 x 10(5) copies/microL for the six CSF specimens, and 0.08 to 5.4 x 10(2) copies/microL for the 16 whole blood specimens. Ninety-seven percent of 30 culture and Gram's stain negative CSF specimens and 100% of 50 normal whole blood specimens were PCR negative. This highly sensitive and specific PCR assay has the potential to provide sufficiently rapid results to improve antibiotic treatment of S.pneumoniae infections, while bacterial load quantitation has opened up exciting possibilities for patient management.

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

A real-time PCR assay suitable for use on the Roche LightCycler platform was developed to replace an existing gel-based PCR assay for the simultaneous detection of the vanA & vanB genes in enterococcal isolates. Novel Fluorescence Resonance Energy Transfer (FRET) hybridization probes were designed. The multiplex real-time PCR assay and the existing gel-based assay were 100% concordant and both correctly detected the vanA or vanB genes in 4/4 VanA E. faecium and 25/25 VanB E. faecium. Additionally, 1/1 VanC1 E. gallinarum, 1/1 VanC2 E. casseliflavus and 47/47 vancomycin susceptible enterococci were negative for the vanA and vanB genes in both PCR assays. Results were available within 1.5 h for the real-time PCR assay compared to up to 5.5 h for the conventional PCR assay.

The relationship between quantitative PCR (COBAS Amplicor CMV Monitor, Roche Diagnostics) and quantitative antigenemia (Monofluor pp65, Sanofi Diagnostics) was examined for monitoring CMV viraemia. A total of 469 specimens from immunocompromised haematology and solid organ transplant patients were tested by quantitative antigenemia and qualitative PCR. Quantitative PCR (QPCR) was performed on the 245 specimens in which CMV DNA was detected by qualitative PCR. To exclude any effect due to specific anti-CMV treatment, analysis of antigenemia and QPCR results was only performed on the 164 of 245 specimens collected from patients not on ganciclovir or foscarnet treatment. Forty seven specimens had <400 CMV copies/mL and a negative antigen result, four specimens were antigen positive (all between 1 to 10 positive CMV cells/2 x 10(5) leucocytes) and had <400 CMV copies/mL. Fifty-one specimens had a CMV viral load > or = 400 copies/mL and a negative antigen result and 62 specimens had a CMV viral load > or = 400 copies/mL and a positive antigen. The viral load was shown to be as high as 43,000 copies/mL in some patients with a negative antigen and occurred in non-neutropenic patients. The correlation coefficient for antigen and QPCR results for specimens from bone marrow transplant patients, was 0.69 with an average CMV viral load of 3,200 copies/mL (SEM = 800) and an average antigen of nine positive CMV cells/2 x 10(5) leucocytes (SEM = 3). In the corresponding solid organ transplant group, the correlation coefficient for antigen and QPCR results was 0.71 with an average CMV viral load of 9,900 copies/mL (SEM = 2,100) and an average antigen of 26 positive CMV cells/2 x 10(5) leucocytes (SEM = 6). Both the average viral load and the average antigen result in specimens from solid organ transplant patients, were significantly higher than the average viral load and antigen result in the corresponding group of bone marrow transplant patients (Two-Sample-for-Means z-Test, P = 0.001 and P = 0.003, respectively). The differences in the kinetics of the two assays in monitoring CMV and their ability to predict CMV disease was also assessed in a sub-group of patients. In conclusion, the two assays used in this study do not always show parallel changes in CMV viral load, but may be complementary for the diagnosis and management of CMV disease. The observation that non-neutropenic patients can have a high viral load in plasma and a negative antigenemia has implications for laboratories using antigenemia alone to monitor patients for CMV disease., (Copyright 2001 Wiley-Liss, Inc.)

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.

Cell culture has traditionally been considered the most sensitive method for detecting Chlamydia trachomatis from clinical specimens, but depends upon the organisms being viable at the time of cell inoculation. Furthermore, cell culture is slow and labor intensive. Even when a special transport medium is used, there is a progressive loss of viability of C. trachomatis during transport. The detection of C. trachomatis by cell culture is more rapid when immunofluorescence is used to detect early antigen, but requires considerable experience to interpret. The Amplicor C. trachomatis system is a commercial polymerase chain reaction (PCR)-based assay combined with nucleic acid hybridization for the direct detection of C. trachomatis in urine and swabs of appropriate sites, with results available within 6 h. All specimens for C. trachomatis received by the Royal Perth Hospital Department of Microbiology during the period 1 July 1994 to 30 June 1995 that were suitable for culture and Amplicor PCR were tested by both methods (2029 specimens). Discordant results were obtained in nine cases and resolved by additional testing. Seventy-one specimens were confirmed as true positives, of these Amplicor PCR correctly detected 67 (sensitivity 94.4%) and culture correctly detected 62 (sensitivity 87.3%). The Amplicor PCR assay was found to be more sensitive and as specific as culture. It had the added advantages of ease of use, rapid availability of results, standardization and was more suited than culture to processing large number of specimens.