Comparison of conventional, nested, and real-time PCR assays for rapid and accurate detection of Vibrio vulnificus

We conducted a prospective study to target toxR in the blood of patients with skin and soft tissue infections who were admitted to four tertiary hospitals to assess the clinical usefulness of real-time quantitative PCR (Q-PCR) as a diagnostic technique. We performed conventional PCR (C-PCR), nested PCR (N-PCR), and Q-PCR assays and compared the results to those obtained using the “gold standard” of microbiological culture. The lower detection limit for the Q-PCR assay was 5 × 10⁰ copies/μl. By use of blood samples of patients with skin and soft tissue infections, the sensitivities of the C-PCR and N-PCR assays against the target toxR gene of V. vulnificus as diagnostic tools were determined to be 45% and 86%, respectively. The C-PCR and N-PCR assays had specificities of 100% and 73%, respectively. When we adopted a crossing-point (cp) cutoff value of <38 cp as a positive result, the Q-PCR assay had 100% sensitivity and specificity. Q-PCR to detect V. vulnificus-specific genes is not only the most sensitive and specific of the techniques but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay using blood is useful for the rapid diagnosis and subsequent treatment of V. vulnificus sepsis.     

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples

Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n = 273) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80°C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.     

Associations between the genotypes of Staphylococcus aureus bloodstream isolates and clinical characteristics and outcomes of bacteremic patients

We investigated associations between the genotypic and phenotypic features of Staphylococcus aureus bloodstream isolates and the clinical characteristics of bacteremic patients enrolled in a phase III trial of S. aureus bacteremia and endocarditis. Isolates underwent pulsed-field gel electrophoresis, PCR for 33 putative virulence genes, and screening for heteroresistant glycopeptide intermediate S. aureus (hGISA). A total of 230 isolates (141 methicillin-susceptible S. aureus and 89 methicillin-resistant S. aureus [MRSA]) were analyzed. North American and European S. aureus isolates differed in their genotypic characteristics. Overall, 26% of the MRSA bloodstream isolates were USA 300 strains. Patients with USA 300 MRSA bacteremia were more likely to be injection drug users (61% versus 15%; P < 0.001), to have right-sided endocarditis (39% versus 9%; P = 0.002), and to be cured of right-sided endocarditis (100% versus 33%; P = 0.01) than patients with non-USA 300 MRSA bacteremia. Patients with persistent bacteremia were less likely to be infected with Panton-Valentine leukocidin gene (pvl)-constitutive MRSA (19% versus 56%; P = 0.005). Although 7 of 89 MRSA isolates (8%) exhibited the hGISA phenotype, no association with persistent bacteremia, daptomycin resistance, or bacterial genotype was observed. This study suggests that the virulence gene profiles of S. aureus bloodstream isolates from North America and Europe differ significantly. In this study of bloodstream isolates collected as part of a multinational randomized clinical trial, USA 300 and pvl-constitutive MRSA strains were associated with better clinical outcomes.     

Loop-mediated isothermal amplification method targeting the TTS1 gene cluster for detection of Burkholderia pseudomallei and diagnosis of melioidosis

Melioidosis is a severe infection caused by Burkholderia pseudomallei. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of B. pseudomallei. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and LAMP was positive for 10 clinical B. pseudomallei isolates but negative for 5 B. thailandensis and 5 B. mallei isolates. A clinical evaluation was conducted in northeast Thailand to compare LAMP to an established real-time PCR assay targeting the same TTS1 gene cluster. A total of 846 samples were obtained from 383 patients with suspected melioidosis, 77 of whom were subsequently diagnosed with culture-confirmed melioidosis. Of these 77 patients, a positive result was obtained from one or more specimens by PCR in 26 cases (sensitivity, 34%; 95% confidence interval [CI], 23.4 to 45.4%) and by LAMP in 34 cases (sensitivity, 44%; 95% CI, 32.8 to 55.9%) (P = 0.02). All samples from 306 patients that were culture negative for B. pseudomallei were negative by PCR (specificity, 100%; 95% CI, 98.8 to 100%), but 5 of 306 patients (1.6%) were positive by LAMP (specificity, 98.4%; 95% CI, 96.2 to 99.5%) (P = 0.03). The diagnostic accuracies of PCR and LAMP were 86.7% (95% CI, 82.9 to 89.9%) and 87.5% (95% CI, 83.7 to 90.6%), respectively (P = 0.47). Both assays were very insensitive when applied to blood samples; PCR and LAMP were positive for 0 and 1 of 44 positive blood cultures, respectively. The PCR and LAMP assays evaluated here are not sufficiently sensitive to replace culture in our clinical setting.     

Predominance of methicillin-resistant Staphylococcus aureus among pathogens causing skin and soft tissue infections in a large urban jail: risk factors and recurrence rates

In the 1990s, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains emerged as pathogens outside of the health care environment. Epidemic foci of CA-MRSA infections were reported in jails and prisons, but risk factors for MRSA infection there are not known. All skin and soft tissue infections (SSTIs) cultured in the Cook County Jail in March 2004 to August 2005 were reviewed. Demographic and clinical risk factors were compared among patients with methicillin-susceptible S. aureus (MSSA) SSTIs and those with MRSA SSTIs. Antibiotic susceptibilities were recorded, and we performed multilocus sequence typing on a sample of MRSA isolates. There were 378 SSTIs from different patients requiring culture, of which 240 (63.5%) were of MRSA and 43 (11.4%) were of MSSA; 84.8% of S. aureus isolates were MRSA. MRSA- and MSSA-infected patients were similar with regard to age, gender, ethnicity, previous exposure to the jail, and comorbidities. In the 12 months prior to the index culture, MRSA patients were more likely to have received a β-lactam antibiotic (25% versus 9%; P = 0.02). Among 26 MRSA strains, 24 (92%) had the sequence type 8 (ST8) genotype. Within 6 months, 14% (95% confidence interval, 8.7% to 22.3%) of MRSA SSTI patients in the jail had a recurrent SSTI compared with 8.8% (95% confidence interval, 2.1% to 32.6%) of MSSA SSTI patients (P = 0.004). MRSA is the predominant cause of SSTIs requiring culture in the jail. Few risk factors differentiated MRSA from MSSA SSTIs, and detainee patients with MRSA SSTIs are at high risk for recurrent SSTIs.     

Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice

The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.     

Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections

The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec, agr, and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD (P = 0.01), sea (P < 0.01), and pvl (P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC (P = 0.03), map/eap (P = 0.05), fnbB (P = 0.11), tst (P = 0.02), sea (P = 0.04), sed (P = 0.04), seg (P = 0.11), sej (P = 0.11), agr (P = 0.09), V8 (P = 0.06), sdrD, sdrE, eta, etb, and see (P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.     

Evaluation of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus

Rapid laboratory diagnosis is critical for treating, managing, and preventing methicillin-resistant Staphylococcus aureus (MRSA) infections. We evaluated and compared the potential for MRSA detection of five chromogenic media, Brilliance MRSA agar (Oxoid), ChromID (bioMérieux), MRSASelect (Bio-Rad), CHROMagar (CHROMagar Microbiology), and BBL-CHROMagar (BD Diagnostics). Media were tested with log serial dilutions (10⁰ to 10⁶ CFU) of pure isolates of MRSA (n = 60), non-MRSA (n = 27), and defined mixtures thereof simulating clinical samples (n = 84). Further evaluations were done on pre-enriched nasal and groin screening swabs (n = 213) from 165 hospitalized patients. Randomized samples were spiral plated on each medium and independently scored by five investigators for characteristic colonies at 24 and 48 h of incubation. Confirmatory testing of up to five putative MRSA colonies recovered from each medium was done. The cumulative average sensitivity with isolates, mixtures, and clinical samples was the highest for Brilliance MRSA agar (97%) and similar for the other four media (≥92%). The cumulative average specificity was the highest for BBL-CHROMagar (99%), followed by MRSASelect (98%), CHROMagar (97%), ChromID (89%), and Brilliance MRSA agar (86%). All of the media detected MRSA at 10 and 1 CFU, although at these low loads, few MRSA samples harboring SCCmec type III or IV were misinterpreted as non-MRSA by investigators. False-positive results were mainly due to methicillin-resistant S. epidermidis. For an arbitrary MRSA prevalence of 5% and based on patient sample evaluations, the positive predictive values for BBL-CHROMagar and CHROMagar (∼84%) were the highest. The negative predictive values of all of the media were ≥92% for MRSA prevalences ranging from 5% to 30%. In conclusion, BBL-CHROMagar and CHROMagar gave the best overall results for detection of MRSA, irrespective of the sample concentration, investigator, or incubation period.Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major nosocomial pathogen in the last decade. Patients colonized with MRSA serve as reservoirs of self-infection or dissemination to other patients and to the hospital environment (6, 12, 22). Hence, screening for MRSA carriage and contact isolation of MRSA carriers are crucial for effective hospital infection control (9). Employing rapid and sensitive screening assays for MRSA detection could help to further improve infection control, as well as decrease costs (10, 13).In recent years, the use of chromogenic media has become a key method for the rapid identification of microorganisms in clinical samples (20). These media detect key microbial enzymes as diagnostic markers for pathogens through the use of “chromogenic” substrates incorporated into a solid-agar-based matrix (20). In contrast to conventional culture media, chromogenic media allow direct colony color-based identification of the pathogen from the primary culture. This reduces the need for subculture for further biochemical testing and hence the time until a result is obtained. Currently available chromogenic media for MRSA detection incorporate chromogens to differentiate S. aureus from other pathogens and antibiotics for selective growth of MRSA. These media differ in their chromogenic substrates, antibiotic formulations, and/or concentrations, factors that impact their sensitivity and specificity for MRSA detection (reviewed in reference 13). We compared the potential of five of the most commonly used commercial chromogenic media for MRSA detection using pure MRSA isolates, non-MRSA isolates, and mixtures thereof at defined concentrations simulating clinical samples. Further evaluations of the media were carried out with nasal and groin screening samples from hospitalized patients.(Part of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 28 October 2008.)     

Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Spiked Positive Blood Culture Bottles

To improve the clinical outcome of Staphylococcus aureus septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of S. aureus and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (sa442 and mecA real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) from spiked blood culture bottles (n = 134). In the case of detecting S. aureus (n = 90; for methicillin-susceptible S. aureus, n = 45; for MRSA, n = 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (n = 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of S. aureus and MRSA from blood cultures.Staphylococcus aureus septicemia is associated with high mortality rates, prolonged hospitalization, and increased costs (3, 5). The prevalence of S. aureus septicemia is increasing, primarily due to infections caused by methicillin (meticillin)-resistant S. aureus (MRSA) (20). Several studies have shown that mortality rates among patients suffering from MRSA septicemia is significantly higher than those of patients suffering from infections caused by methicillin-susceptible S. aureus (MSSA) (5, 18, 19).The early selection of an appropriate antibiotic regime for the treatment of MSSA or MRSA is crucial for the patient''s outcome (4, 14, 15). However, bacterial identification and preliminary antibiotic susceptibility testing by standard microbiological procedures still requires 24 to 48 h after growth detection by automated incubation systems. In contrast, new real-time PCR-based methods that use samples directly from positive blood culture bottles allows differentiation of MSSA, MRSA, and coagulase-negative staphylococci (CoNS) within 1.5 to 3 h (7, 12, 13, 16). Such tests promote an early appropriate antibiotic selection, thus avoiding the unnecessary use of vancomycin, and they reduce mortality, the length of hospitalization, and costs associated with bloodstream infections caused by these bacteria (3).We described recently a real-time PCR method for the detection of MSSA, MRSA, and CoNS directly from positive blood cultures; it turned out to have 100% sensitivity and 100% specificity for the detection of MSSA and MRSA (7). In this study, the differentiation between MSSA and MRSA directly from signal-positive blood cultures was achieved by the separate detection of the S. aureus-specific chromosomal fragment sa442 and the mecA gene (encoding methicillin resistance). However, since this test is not a commercialized system and does not run on a common platform like, e.g., the SmartCycler (Cepheid, Sunnyvale, CA), its widespread application is limited. Moreover, in blood cultures containing a mixture of MSSA (sa442⁺ but mecA negative) and methicillin-resistant CoNS (MR-CoNS; sa442 negative but mecA⁺), the test is prone to lead to the incorrect identification of MRSA (sa442⁺ mecA⁺).The BD GeneOhm StaphSR assay (BD Diagnostics GeneOhm, Québec, Canada) provides a rapid, simple, commercially available diagnostic test that runs on the SmartCycler for the detection of S. aureus and MRSA from nasal swabs, wounds, and blood cultures. This multiplex real-time PCR amplifies an S. aureus-specific target sequence and a specific target near the staphylococcal cassette chromosome mec (SCCmec) insertion site and the orfX junction in MRSA, thereby differentiating between MSSA and MRSA (9, 17).Using the herein-described setting, we evaluated the BD GeneOhm StaphSR assay and the PCR that detects sa442 and mecA (designated sa442-mecA-PCR) for the detection of MSSA and MRSA directly from spiked blood cultures.     

Quantitation of Bacteria in Blood of Typhoid Fever Patients and Relationship between Counts and Clinical Features, Transmissibility, and Antibiotic Resistance

Salmonella typhi was isolated from 369 and Salmonella paratyphi A was isolated from 6 of 515 Vietnamese patients with suspected enteric fever. Compared with conventional broth culture of blood, direct plating of the buffy coat had a diagnostic sensitivity of 99.5% (95% confidence interval [CI], 97.1 to 100%). Blood bacterial counts were estimated by the pour plate method. The median S. typhi count in blood was 1 CFU/ml (range, <0.3 to 387 CFU/ml), of which a mean of 63% (95% CI, 58 to 67%) were intracellular. The mean number of bacteria per infected leukocyte was 1.3 (interquartile range [IQR], 0.7 to 2.4) CFU/cell (n = 81). Children (<15 years old; n = 115) had higher median blood bacterial counts than adults (n = 262): 1.5 (range, <0.3 to 387) versus 0.6 (range, <0.3 to 17.7) CFU/ml (P = 0.008), and patients who excreted S. typhi in feces had higher bacteremias than those who did not: a median of 3 (range, <0.3 to 32) versus 1 (range, <0.3 to 68) CFU/ml (P = 0.02). Blood bacterial counts declined with increasing duration of illness (P = 0.002) and were higher in infections caused by multidrug-resistant S. typhi (1.3 [range, <0.3 to 387] CFU/ml; n = 313) than in infections caused by antibiotic-sensitive S. typhi (0.5 [range, <0.3 to 32] CFU/ml; n = 62) (P = 0.006). In a multivariate analysis this proved to be an independent association, suggesting a relationship between antibiotic resistance and virulence in S. typhi.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Specimens from Various Body Sites: Performance Characteristics of the BD GeneOhm MRSA Assay,the Xpert MRSA Assay,and Broth-Enriched Culture in an Area with a Low Prevalence of MRSA Infections

Universal surveillance upon patient admission is important in reducing the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).Methicillin-resistant Staphylococcus aureus (MRSA) strains have become a major concern for health care systems. Prevention of the spread of MRSA has, therefore, become a main goal in the past decade, and active screening programs have been established worldwide (4, 27). Compared to infections caused by methicillin-susceptible S. aureus (MSSA), the organism causes severe infections with increased morbidity and mortality and prolonged hospitalization (9, 17). Unlike countries facing a high prevalence of MRSA, such as the United States and Japan, the prevalence in Switzerland has remained low to date (5, 13, 21, 32). In most parts of our country, prevalence rates between 4% and 7% are observed (19). Apart from its spread in the hospital environment, MRSA carriage in our community, as well as in other countries, seems to be more prevalent than previously assumed (31, 32, 37).To facilitate the rapid detection of colonized patients, real-time PCR assays have been developed. The first method to directly detect MRSA from clinical specimens was developed by Huletsky et al. (20). The principle of this method is used in two commercially available tests, the BD GeneOhm MRSA assay (BDGO) (BD, San Diego, CA) and the Xpert MRSA assay (Cepheid, Inc., Sunnyvale, CA).Recent studies have shown that universal admission surveillance for MRSA was associated with a reduction in MRSA disease (18, 28). Likewise, Cunningham et al. have reported a reduction in MRSA transmissions in a critical care unit. The authors attributed these findings, at least partially, to the availability of rapid PCR screening tests, apart from other measures like improved hygiene measures (10). PCR screening methods are cost efficient, especially in an area of low prevalence where high-risk patients are subjected to preemptive contact isolation (6). Our facility is a 1,000-bed tertiary care teaching hospital with a known low prevalence (<5%) of MRSA colonization of patients and follows a surveillance policy similar to that of the University Hospital of Berne, Switzerland (6). As reported in other studies, this means preemptive isolation on admission of all patients who (i) came from or had traveled to countries with known high prevalence rates for MRSA, (ii) were transferred from long-term care facilities, (iii) were transferred from another health care facility, (iv) were hospitalized within the previous 6 months, and/or (v) had a history of MRSA colonization or infection (6, 8, 23). As soon as PCR is negative for MRSA, patient isolation is ended. Under these circumstances, a rapid screening method with a high negative predictive value (NPV) is desirable, because the bulk of costs emerge mainly from noncolonized patients being unnecessarily isolated. In this study, we compared two real-time PCR methods, the BDGO and Xpert MRSA assays, with broth-enriched culture to assess their performance characteristics and rapidity in an area with a low prevalence of MRSA.     

Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from an at-risk community population

We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture with BBL CHROMagar MRSA for nasal surveillance among 602 arrestees from the Baltimore City Jail. The sensitivity and specificity were 88.5% and 91.0%, respectively, and after secondary analysis using enrichment broth, they were 89.0% and 91.7%, respectively. Twenty-three of 42 false-positive PCR lysates contained methicillin-susceptible S. aureus.     

Use of the BacT/Alert Blood Culture System for Culture of Sterile Body Fluids Other than Blood

Studies have demonstrated that large-volume culture methods for sterile body fluids other than blood increase recovery compared to traditional plated-medium methods. BacT/Alert is a fully automated blood culture system for detecting bacteremia and fungemia. In this study, we compared culture in BacT/Alert standard aerobic and anaerobic bottles, BacT/Alert FAN aerobic and FAN anaerobic bottles, and culture on routine media for six specimen types, i.e., continuous ambulatory peritoneal dialysate (CAPD), peritoneal, amniotic, pericardial, synovial, and pleural fluids. Specimen volumes were divided equally among the three arms of the study. A total of 1,157 specimens were tested, with 227 significant isolates recovered from 193 specimens. Recovery by method was as follows: standard bottles, 186 of 227 (82%); FAN bottles, 217 of 227 (96%); and routine culture, 184 of 227 (81%). The FAN bottles recovered significantly more gram-positive cocci (P < 0.001), Staphylococcus aureus (P = 0.003), coagulase-negative staphylococci (P = 0.008), gram-negative bacilli (P < 0.001), Enterobacteriaceae (P = 0.005), and total organisms (P < 0.001) than the routine culture. There were no significant differences in recovery between the standard bottles and the routine culture. The FAN aerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), coagulase-negative staphyococci (P = 0.003), and total organisms (P < 0.001) than the standard aerobic bottle, while the FAN anaerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), Enterobacteriaceae (P = 0.03), and total organisms (P < 0.001) than the standard anaerobic bottle. For specific specimen types, significantly more isolates were recovered from the FAN bottles compared to the routine culture for synovial (P < 0.001) and CAPD (P = 0.004) fluids. Overall, the FAN bottles were superior in performance to both the standard bottles and the routine culture for detection of microorganisms from the types of sterile body fluids included in this study.     

Evaluation of the Murex Hybrid Capture Cytomegalovirus DNA Assay versus Plasma PCR and Shell Vial Assay for Diagnosis of Human Cytomegalovirus Viremia in Immunocompromised Patients

We evaluated a cytomegalovirus (CMV) 24-hour shell vial assay (SVA), the Murex Hybrid Capture CMV DNA assay (HCA), and a CMV plasma PCR for the detection of CMV viremia in renal and bone marrow transplant recipients and human immunodeficiency virus-infected patients. CMV viremia was detected by at least one method in 125 of 317 evaluable samples (39.4%) from 78 patients and was detected in 19.8% of samples by SVA, 26.8% by HCA, and 32.2% by plasma PCR. There was moderate to substantial agreement between the results of the different tests (kappa coefficient = 0.415 to 0.631). However, HCA and plasma PCR were significantly more sensitive than SVA (P = 0.001 and P < 0.0001, respectively; McNemar’s test), and plasma PCR was more sensitive than HCA (P = 0.031; McNemar’s test). HCA and plasma PCR were more consistently positive than SVA during viremic episodes (P = 0.0002 and P < 0.0001, respectively; McNemar’s test). The use of HCA or plasma PCR may therefore improve the diagnosis and management of CMV disease in susceptible patient groups.     

Detection of Penicillin Susceptibility in Streptococcus pneumoniae by pbp2b PCR-Restriction Fragment Length Polymorphism Analysis

A PCR-restriction fragment length polymorphism strategy directed against the pbp2b gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of “not resistant” and “not susceptible,” the sensitivities were 97.5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR.     

Comparison of linear array and line blot assay for detection of human papillomavirus and diagnosis of cervical precancer and cancer in the atypical squamous cell of undetermined significance and low-grade squamous intraepithelial lesion triage study

We evaluated Linear Array (LA), a newly commercialized PGMY09/11 L1 consensus primer PCR test that detects 37 human papillomavirus (HPV) genotypes by reverse line blot hybridization, for the detection of individual HPV genotypes and carcinogenic HPV and its clinical performance for detecting 2-year cumulative cervical precancer and cancer using archived specimens from the Atypical Squamous Cell of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion Triage Study. LA testing was conducted on enrollment specimens from women referred because of an ASCUS Pap test. To gauge the performance of the new test, the results were compared to those of its prototype predecessor assay, Line Blot Assay (LBA), restricted to paired results (n = 3,335). LA testing was done masked to LBA results and clinical outcomes. The results of LA and LBA testing were compared for detection of carcinogenic HPV and clinical outcomes of cervical precancer and cancer. Overall, 50% and 55% of the women tested positive for carcinogenic HPV by LBA and LA, respectively (P < 0.0001). The percent agreement for carcinogenic HPV detection was 88%, percent positive agreement was 80%, and kappa was 0.76 for detection of carcinogenic HPV by the two assays. There was a significant increase in detection by LA for most of the 37 HPV genotypes targeted by both assays, including for 13 of 14 carcinogenic HPV genotypes. LA detected more multiple-genotype infections for all HPV genotypes among HPV-positive women (P < 0.0001) and for carcinogenic HPV genotypes among carcinogenic-HPV-positive women (P < 0.0001). LA was more sensitive (92.3% versus 87.1%; P = 0.003) and less specific (48.2% versus 54.0%; P < 0.0001) than LBA for 2-year cumulative cervical precancer and cancer as diagnosed by the Pathology Quality Control Group. In conclusion, we found LA to be a promising assay for the detection of HPV genotypes and carcinogenic HPV, and it may be clinically useful for the detection of cervical precancer and cancer in women with equivocal cytology.     

Association of Diarrheagenic Escherichia coli Pathotypes with Infection and Diarrhea among Mexican Children and Association of Atypical Enteropathogenic E. coli with Acute Diarrhea

Seventy-six children ≤2 years old were prospectively followed for 1 year in a peri-urban community of Mexico City to determine asymptomatic infection and acute diarrhea associated with diarrheagenic Escherichia coli pathotypes (DEPs). By use of a pathogen-specific multiplex PCR, DEPs were sought in 795 stool samples, of which 125 (16%) were positive for DEP; of these, 4 represented shedding episodes and 4 parasite coinfections. Most single-DEP infections (85/117) were asymptomatic (P < 0.001), and of the 32 DEP diarrhea episodes, 41% were associated with atypical enteropathogenic E. coli (aEPEC), 37.5% with enterotoxigenic E. coli, 9% with typical EPEC, 9% with enteroinvasive E. coli, and 3% with Shiga toxin-producing E. coli strains. Among the 76 children, 54 had at least one stool positive for DEP, of which 23 experienced a DEP-associated diarrhea episode. In the last group of children, DEP infection was significantly associated with a diarrhea episode (relative risk [RR] = 2.5; 95% confidence interval [CI], 1.79 to 3.57; P < 0.001), with ETEC (RR = 2.30; 95% CI, 1.49 to 3.54; P = 0.003) and aEPEC (RR = 1.92; 95% CI, 1.23 to 3.0; P = 0.019) being the pathotypes associated with diarrhea. aEPEC-associated diarrhea episodes were frequently in the <12-month age group (RR = 2.57; 95% CI, 1.05 to 6.27; P = 0.04). aEPEC infections were distributed all year round, but associated diarrheal episodes were identified from April to October, with a May-June peak (rainy season). Most ETEC infections and diarrhea episodes characteristically occurred during the summer (rainy season), with a diarrhea peak in August. Of all DEPs, only aEPEC was associated with acute diarrhea episodes lasting 7 to 12 days (P = 0.019). DEPs are important causes of community-acquired enteric infection and diarrhea in Mexican children.     

Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia

In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by Streptococcus pneumoniae. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy.     

Disease severity associated with presence in subgingival plaque of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, singly or in combination, as detected by nested multiplex PCR

This study used a nested multiplex PCR method to detect three periodontal pathogens in subgingival plaque collected before treatment and at 2 and 6 months posttreatment from 107 patients with severe, generalized periodontitis. The proportions of the patients who harbored these bacteria before periodontal treatment were as follows: Tannerella forsythia, 81%; Porphyromonas gingivalis, 78%; and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, 47%. At 2 months posttreatment there was a significant reduction in the numbers of patients harboring P. gingivalis (46%; P < 0.001) or T. forsythia (63%; P = 0.043) but not A. actinomycetemcomitans (50%) compared to pretreatment data. At 6 months posttreatment, significantly fewer patients harbored P. gingivalis (43%; P < 0.001); A. actinomycetemcomitans, (31%; P = 0.025), or T. forsythia (63%; P = 0.030). Interestingly, at baseline and at 2 months posttherapy, subjects who harbored only a single pathogen had a greater level of periodontal disease than subjects who harbored two, or all three, of these periodontal pathogens. These data suggest that a reduction in the number of species present may be associated with an increase in the severity of periodontal diseases.