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Seasonal influenza virus causes significant morbidity and mortality each year. Point-of-care (POC) testing using rapid influenza diagnostic tests (RIDTs), immunoassays that detect viral antigens, are often used for diagnosis by physician offices and urgent care centers. These tests are rapid but lack sensitivity, which is estimated to be 50 to 70%. Testing by PCR is highly sensitive and specific, but historically these assays have been performed in centralized clinical laboratories necessitating specimen transport and increasing the time to result. Recently, Clinical Laboratory Improvement Amendments (CLIA)-waived, POC PCR influenza assays have been developed with >95% sensitivity and specificity compared to centralized PCR assays. To determine the clinical impact of a POC PCR test for influenza, we compared antimicrobial prescribing patterns of one urgent care location using the Cobas LIAT Influenza A/B assay (LIAT assay; Roche Diagnostics, Indianapolis, IN) to other urgent care centers in our health system using traditional RIDT, with negative specimens being reflexed to PCR. Antiviral prescribing was lower in patients with a negative LIAT PCR result (2.3%) than in patients with a negative RIDT result (25.3%; P < 0.005). Antivirals were prescribed more often in patients that tested positive by LIAT PCR (82.4%) than in those testing positive by either RIDT or reflex PCR (69.9%; P < 0.05). Antibacterial prescriptions for patients testing negative by LIAT PCR were higher (44.5%) than for those testing negative by RIDT (37.7%), although the difference was not statistically significant. In conclusion, having results from a PCR POC test during the clinic visit improved antiviral prescribing practices compared to having rapid results from an RIDT.

We compared two real-time PCR assays (both by the use of melting curve analysis) for their ability to identify Staphylococcus species directly from 200 positive blood culture bottles. The PCR assays correctly identified 83% to 94% of the Staphylococcus isolates to species clusters. Molecular testing significantly outperformed commercially available latex tests (sensitivity for both latex tests

Nasal and perianal swab specimens were tested for detection of Staphylococcus aureus and vancomycin-resistant Enterococcus species (VRE) using a laboratory-developed real-time PCR test and microbiological cultures. The real-time PCR and culture results for S. aureus were similar. PCR had adequate sensitivity, but culture was more specific for the detection of VRE.

We evaluated the use of the BD GeneOhm MRSA real-time PCR assay (BD Diagnostics, San Diego, CA) for the detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and an in-house lysis method that was specifically developed to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were 98%, 96%, 77%, and 99.7% with the kit lysate and 98%, 95%, 75%, and 99.7% with the ACP lysate ( P , not significant), respectively. The second evaluation was done after implementation of all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed an NPV of 99.9% and a PPV of 73.5% (prevalence, 6%), consistent with our initial findings. The BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When one is dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.

Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae , the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB . However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB , due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non- N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay.

Real-time PCR testing for bla KPC , bla NDM , bla VIM , bla IMP , and bla CTX-M was performed on rectal swabs obtained from residents of two long-term acute-care facilities. While bla KPC was detected in 69/102 swabs (67.6%), testing for four other targets increased the positivity rate for a broad-spectrum β-lactamase to 73.5% (McNemar's P = 0.03).

Klebsiella pneumoniae carbapenemases (KPCs) have recently been described in Chicago, IL, especially among residents of long-term acute care hospitals (LTACHs). These patients are frequently transferred to local Chicago hospitals for higher acuity of medical care, and rapid detection and isolation of KPC-colonized LTACH residents may interrupt the introduction of KPCs into acute care hospitals. We evaluated the performance of a real-time PCR for bla KPC from enrichment broth versus direct plating of rectal surveillance swabs on two selective culture media, CHROMagar extended-spectrum-β-lactamase (ESBL) and vancomycin, amphotericin B, ceftazidime, and clindamycin (VACC) plates. Rectal surveillance swabs were collected as part of a point prevalence study of KPC carriage rates among 95 residents of two Chicago area LTACHs. Discrepant results between PCR and culture were resolved by subculturing the enrichment broth. Overall, 66 of 95 patients (69.5%) were colonized with KPCs, using the cumulative results of culture as a reference standard. Real-time PCR from enrichment broth was positive in 64 of 66 (97%) colonized patients, including nine surveillance swabs that were missed by both selective culture media. PCR demonstrated higher sensitivity, 97.0%, than culture using either CHROMagar or VACC plates (both with sensitivity of 77.3%). In addition, turnaround time was significantly shorter for the PCR-based method than for culture, with a mean of 24 h versus 64 to 72 h for CHROMagar and VACC plates ( P < 0.0001). Overall, PCR for bla KPC represents the best screening test for KPCs with significantly higher sensitivity and with less hands-on time, resulting in a shorter time to results.

The emergence of a novel pandemic human strain of influenza A (H1N1/09) virus in April 2009 has demonstrated the need for well-validated diagnostic tests that are broadly applicable, rapid, sensitive, and specific. The analytical performance and clinical validity of results generated with the novel Roche RealTime Ready Influenza A/H1N1 Detection Set using the LightCycler 2.0 instrument were characterized. Analytical performance was assessed by processing respiratory samples spiked with H1N1/09 and seasonal influenza A virus, a set of seasonal influenza A virus subtypes, and samples containing common viral and bacterial respiratory pathogens. The clinical validity of results was assessed in comparison to other assays by analyzing 359 specimens at three clinical sites and one reference laboratory. Direct sequencing was used to resolve samples with discrepant results. The assay detected virus concentrations down to

The 2009 H1N1 influenza virus has rapidly spread all over the world. At this time, regions in the northern hemisphere are at the beginning of the typical annual respiratory virus season, although the 2009 H1N1 virus has been highly prevalent for the last several months. This year, it is likely that

A real-time PCR assay was developed to identify common staphylococcal species. A single set of consensus primers was designed to amplify a portion of the 16S rRNA gene, and a pair of fluorescence resonance energy transfer probes was used to identify species based on the unique melt properties of the probes resulting from sequence variations in the amplicons from each species. Nine common staphylococcal strains ( S. aureus , S. capitis , S. epidermidis , S. haemolyticus , S. hominis , S. lugdunensis , S. schleiferi , S. simulans , and S. warneri ) were used for assay development. The species-specific melting profiles were validated by correctly identifying 36 of 37 coagulase-negative staphylococcal (CoNS) isolates identified by ribotyping. In a study of clinical isolates, the PCR/melt curve approach correctly identified 56/56 S. aureus isolates identified by coagulase/protein A latex agglutination. Fifty-four CoNS clinical isolates characterized using the API Staph assay were studied, with the PCR/melt curve approach yielding matching identifications for 32/54 (59%). The API Staph assay was unable to identify 18 CoNS isolates, and differing results were obtained for 4 isolates. Sequencing of the 22 discrepant or unidentified CoNS samples revealed that the PCR/melt curve results were correct for all but one isolate. Thus, PCR/melt curve analysis achieved a nearly 100% accuracy and performed better than biochemical testing. Performance of the PCR/melt curve approach requires less than 2 h after colony selection. This method thus provides a rapid and accurate approach to the identification of staphylococcal species in the clinical laboratory.

PCR has been used successfully for the direct detection of Mycobacterium tuberculosis in uncultured patient samples. Its potential is hindered by the risk of false-positive results as a result of either amplicon carryover of cross-contamination between patient samples. In the present study, we investigated whether residual amplifiable human or M. tuberculosis DNA could remain in sterile bronchoscopes and potentially be a cause of false-positive PCR results in subsequent patient samples. Sterilized bronchoscopes were flushed with sterile saline, and the collected eluate was submitted for PCR amplification of IS6110 sequences and exon 8 of the human p53 gene. Of a total of 55 washes of sterile bronchoscopes from two institutions, 2 (3.6%) contained amplifiable M. tuberculosis DNA and 11 (20%) contained residual human DNA. These findings indicate that residual DNA can remain in sterilized bronchoscopes and can be a source of false-positive PCR results.

A real-time PCR assay was developed targeting the bla KPC responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.