Variability in patterns of BK viral load after renal transplantation.

BK virus (BKV) may cause nephropathy in renal transplant patients, resulting in graft dysfunction and possible graft loss. We used a sensitive quantitative BKV assay to monitor plasma BK viral loads in 11 renal transplant patients for periods ranging from 37 to 189 weeks posttransplant. Five patients remained negative for BKV, and 6 developed viremia, including 1 patient with a transient viremia. Of the viremic patients, 2 were diagnosed with BKV nephropathy after increasing serial BK viral loads, prompting a renal biopsy that established the diagnosis. A 3rd patient had high initial BK viral load and biopsy-proven disease that resolved with reduced immunosuppression. Two patients did not develop nephropathy despite persistent viral loads of 10(4) copies/mL. Five of 6 patients experienced viral clearance from the plasma (BK viral load <500 copies/mL), which was associated with their renal function becoming stabilized, and the remaining patient experienced a downward trend in viral load and stable renal function. Thus, the BKV quantitative assay was useful in aiding the diagnosis of BKV nephropathy, monitoring the response to reductions in immunosuppression and identified that some patients can have persistent viremia and still develop stable renal function without specific antiviral therapy.

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.

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

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.

Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction.

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.

Eradication of a large outbreak of a single strain of vanB vancomycin-resistant Enterococcus faecium at a major Australian teaching hospital.

OBJECTIVE: To demonstrate that nosocomial transmission of vancomycin-resistant enterococci (VRE) can be terminated and endemicity prevented despite widespread dissemination of an epidemic strain in a large tertiary-care referral hospital. INTERVENTIONS: Two months after the index case was detected in the intensive care unit, 68 patients became either infected or colonized with an epidemic strain of vanB vancomycin-resistant Enterococcus faecium despite standard infection control procedures. The following additional interventions were then introduced to control the outbreak: (1) formation of a VRE executive group; (2) rapid laboratory identification (30 to 48 hours) using culture and polymerase chain reaction detection of vanA and vanB resistance genes; (3) mass screening of all hospitalized patients with isolation of carriers and cohorting of contacts; (4) environmental screening and increased cleaning; (5) electronic flagging of medical records of contacts; and (6) antibiotic restrictions (third-generation cephalosporins and vancomycin). RESULTS: A total of 19,658 patient and 24,396 environmental swabs were processed between July and December 2001. One hundred sixty-nine patients in 23 wards were colonized with a single strain of vanB vancomycin-resistant E. faecium. Introducing additional control measures rapidly brought the outbreak under control. Hospital-wide screening found 39 previously unidentified colonized patients, with only 7 more nonsegregated patients being detected in the next 2 months. The outbreak was terminated within 3 months at a cost of dollar 2.7 million (Australian dollars). CONCLUSION: Despite widespread dissemination of VRE in a large acute care facility, eradication was achievable by a well-resourced, coordinated, multifaceted approach and was in accordance with good clinical governance.

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.

A quantitative LightCycler PCR to detect Streptococcus pneumoniae in blood and CSF.

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.

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay.

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.

Real-time PCR for the rapid detection of vanA and vanB genes.

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.