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Background: Polymerase chain reaction (PCR) has high sensitivity and specificity for detection of group A streptococcus (GAS) in throat swabs and is routinely used for GAS pharyngitis diagnosis at our institution. Herein we defined the natural history of throat swab GAS PCR and culture positivity during and following treatment of GAS pharyngitis., Methods: Fifty children with a PCR positive GAS throat swab were recruited for participation. Four additional throat swabs were collected over 2 weeks following the initial positive PCR result (during and following a standard course of antibiotic therapy) and tested for GAS using rapid real-time PCR and culture., Results: After the initial positive swab, 45% had a positive PCR 2-4 days, 20% 5-7 days, 18% 8-10 days, 25% 11-13days, and 20% 14-18days later. The median time to a negative PCR was 4 days with the nadir in positive PCR results approximating the end of a typical 10-day treatment interval. Seven subjects remained persistently PCR positive. Culture results remained positive at a stable rate for each time interval, ranging from 5-10%., Conclusions: If a patient presents with symptoms of GAS pharyngitis after previous positive GAS PCR testing and treatment with appropriate antibiotics, it is reasonable to use PCR testing for GAS pharyngitis testing beginning one week after initial testing. Further studies are warranted to determine if this time frame can be applied to PCR testing used to detect other infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We report Mycobacterium lepromatosis infection in a US-born person with an extensive international travel history. Clinical symptoms, histopathology, and management are similar to those of infections caused by M. leprae. Clinicians should consider this pathogen in the diagnosis of patients with symptoms of leprosy who have traveled to endemic areas.

Staphylococcus aureus is a leading cause of bacteremia in hospitalized patients. Whether or not S. aureus bacteremia (SAB) is associated with clonality, implicating potential nosocomial transmission, has not, however, been investigated. Herein, we examined the epidemiology of SAB using whole genome sequencing (WGS). 152 SAB isolates collected over the course of 2015 at a single large Minnesota medical center were studied. Staphylococcus protein A (spa) typing was performed by PCR/Sanger sequencing; multilocus sequence typing (MLST) and core genome MLST (cgMLST) were determined by WGS. Forty-eight isolates (32%) were methicillin-resistant S. aureus (MRSA). The isolates encompassed 66 spa types, clustered into 11 spa clonal complexes (CCs) and 10 singleton types. 88% of 48 MRSA isolates belonged to spa CC-002 or -008. Methicillin-susceptible S. aureus (MSSA) isolates were more genotypically diverse, with 61% distributed across four spa CCs (CC-002, CC-012, CC-008 and CC-084). By MLST, there was 31 sequence types (STs), including 18 divided into 6 CCs and 13 singleton STs. Amongst MSSA isolates, the common MLST clones were CC5 (23%), CC30 (19%), CC8 (15%) and CC15 (11%). Common MRSA clones were CC5 (67%) and CC8 (25%); there were no MRSA isolates in CC45 or CC30. By cgMLST analysis, there were 9 allelic differences between two isolates, with the remaining 150 isolates differing from each other by over 40 alleles. The two isolates were retroactively epidemiologically linked by medical record review. Overall, cgMLST analysis resulted in higher resolution epidemiological typing than did multilocus sequence or spa typing.

Background.  Emerging data implicate long-term care facilities (LTCFs) as reservoirs of fluoroquinolone-resistant (FQ-R) Escherichia coli of sequence type 131 (ST131). We screened for ST131 among LTCF residents, characterized isolates molecularly, and identified risk factors for colonization. Methods.  We conducted a cross-sectional study using a single perianal swab or stool sample per resident in 2 LTCFs in Olmsted County, Minnesota, from April to July 2013. Confirmed FQ-R E. coli isolates underwent polymerase chain reaction-based phylotyping, detection of ST131 and its H30 and H30-Rx subclones, extended virulence genotyping, and pulsed-field gel electrophoresis (PFGE) analysis. Epidemiological data were collected from medical records. Results.  Of 133 fecal samples, 33 (25%) yielded FQ-R E. coli, 32 (97%) of which were ST131. The overall proportion with ST131 intestinal colonization was 32 of 133 (24%), which differed by facility: 17 of 41 (42%) in facility 1 vs 15 of 92 (16%) in facility 2 (P = .002). All ST131 isolates represented the H30 subclone, with virulence gene and PFGE profiles resembling those of previously described ST131 clinical isolates. By PFGE, certain isolates clustered both within and across LTCFs. Multivariable predictors of ST131 colonization included inability to sign consent (odds ratio [OR], 4.16 [P = .005]), decubitus ulcer (OR, 4.87 [ P = .04]), and fecal incontinence (OR, 2.59 [P = .06]). Conclusions.  Approximately one fourth of LTCF residents carried FQ-R ST131 E. coli resembling ST131 clinical isolates. Pulsed-field gel electrophoresis suggested intra- and interfacility transmission. The identified risk factors suggest that LTCF residents who require increased nursing care are at greatest risk for ST131 colonization, possibly due to healthcare-associated transmission.

A process employing patient- or parent-collected pharyngeal swabs for group A Streptococcus (GAS) testing would expedite diagnosis and treatment, reduce patient exposure to the health care setting, and decrease health care costs. Our aim was to determine the concordance between patient- or parent-collected (self-collected) and health care worker (HCW)-collected pharyngeal swabs for detection of GAS by PCR. From 9 October 2012 to 21 March 2013, patients presenting with a sore throat meeting criteria for GAS testing and not meeting criteria for severe disease were offered the opportunity to collect their own pharyngeal swab. The HCW also collected a swab. Paired swabs were tested by GAS real-time PCR, allowing semiquantitative comparisons between positive results. Of the 402 participants, 206 had a swab collected by the patient and 196 a swab collected by the parent. The percent positivity results were 33.3% for HCW-collected swabs and 34.3% for self-collected swabs (P = 0.41). The overall concordance between the two collection strategies was 94.0% (95% confidence interval [CI], 91.3 to 96.0). Twenty-four of the paired swabs had discordant results, with 10 and 14 positives detected only with the HCW- and self-collected swabs, respectively (P = 0.41). The person collecting the swab in the self-collected arm, the order of collection, and prior swab collection training did not influence results. Among the 124 specimens that were positive by both collection methods, the amount of GAS DNA was higher in the self-collected versus the HCW-collected swabs (P = 0.008). Self-collected pharyngeal swabs provide a reliable alternative to HCW collection for detection of GAS and offer a strategy for improved health care delivery., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

PCR coupled with electrospray ionization mass spectrometry applied to synovial fluid specimens had an 81% sensitivity and a 95% specificity for the diagnosis of prosthetic joint infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We compared PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) to culture using sonicate fluid from 431 subjects with explanted knee (n = 270) or hip (n = 161) prostheses. Of these, 152 and 279 subjects had prosthetic joint infection (PJI) and aseptic failure, respectively. The sensitivities for detecting PJI were 77.6% for PCR-ESI/MS and 69.7% for culture (P = 0.0105). The specificities were 93.5 and 99.3%, respectively (P = 0.0002).

PCR coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) is a novel technology that has recently been used to identify pathogens from clinical specimens or after culture within about 6 h. We evaluated the MDR-TB (multidrug-resistant tuberculosis) assay, which uses PCR-ESI-MS for detection and identification of Mycobacterium spp. and Mycobacterium tuberculosis complex (MTBC) resistance determinants from solid and broth Middlebrook culture media. The performance of the MDR-TB assay was compared to identification using nucleic acid hybridization probes and 16S rRNA gene sequencing for 68 MTBC and 97 nontuberculous mycobacterial (NTM) isolates grown on agar and 107 cultures grown in Bactec MGIT broth. MTBC resistance profiles from the MDR-TB assay were compared to results with the agar proportion method. The PCR-ESI-MS system correctly identified all MTBC isolates and 97.9% and 95.8% of the NTM isolates from characterized agar cultures and MGIT broth cultures to the species level, respectively. In comparison to the agar proportion method, the sensitivity and specificity for the detection of drug resistance using the MDR-TB assay were 100% and 92.3% for rifampin, 100% and 93.8% for isoniazid, 91.6% and 94.4% for ethambutol, and 100% and 100% for fluoroquinolones, respectively. The MDR-TB assay appears to be a rapid and accurate method for the simultaneous detection and identification of mycobacterial species and resistance determinants of MTBC from culture.

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.

The PLEX-ID system is a novel technology that couples PCR amplification and electrospray ionization-mass spectrometry to identify pathogens directly in clinical specimens. The analytical performance of the PLEX-ID Broad Fungal assay was compared with that of traditional culture identification by using 91 characterized fungal culture isolates (64 manufacturer-claimed and 27 nonclaimed organisms) and directly by using 395 respiratory specimens. Discordant results were resolved by D2 large-subunit ribosomal DNA fungal sequencing. Environmental studies were performed to monitor for potential contamination. The PLEX-ID Broad Fungal assay correctly identified 95.6% (87/91) and 81.3% (74/91) of the culture isolates to the genus and species levels, respectively. Of the manufacturer-claimed organisms, 100% (64/64) and 92.2% (59/64) were correctly identified to the genus and species levels, respectively. Direct analysis of respiratory specimens resulted in 67.6% (267/395) and 66.6% (263/395) agreement with culture results to the genus and species levels, respectively, with 16.2% (64/395) of the results discordant with culture and 16.2% (64/395) not detected by the system. The majority (>95%) of the isolates not detected directly by the PLEX-ID system ultimately grew in low quantities in culture (≤ 20 colonies). In 20.3% (35/172) of the respiratory specimens where no growth was observed in culture, the PLEX-ID system identified a fungus, suggesting a potential increase in sensitivity over culture in some instances. The PLEX-ID system provides a rapid method for the detection of a broad array of fungi directly in respiratory specimens and has the potential of impacting turnaround times and patient care by reducing the need to wait for the growth of an organism in culture.

Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis.

A total of 120 pleural fluid specimens from 113 pediatric patients were tested using two rapid antigen detection assays for Streptococcus pyogenes. Results were compared to culture, Gram stain, and PCR results. Each rapid antigen assay detected 9 out of 10 (90%) PCR-positive samples, with 100% specificity. These antigen detection assays are useful to provide microbiological diagnosis of empyema caused by S. pyogenes.

Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2.

Pneumocystis jiroveci is an important cause of pneumonia in immunocompromised individuals. This organism cannot be cultured, and therefore, diagnosis relies on microscopic identification of the organism using stains or antibodies. Although simple, these tests are insensitive and require expertise for accurate interpretation. We developed a real-time polymerase chain reaction (PCR) assay that provides sensitive and objective detection of Pneumocystis from bronchoalveolar lavage fluid. Primers and fluorescence resonance energy transfer probes were developed that target the cdc2 gene of P. jiroveci. Assay sensitivity is 6 copies of target per microliter of sample. No cross-reactivity occurs with other pathogens, and the PCR assay has a 21% increase in clinical sensitivity as compared with Calcofluor white staining. The real-time PCR assay provides a sensitive, rapid, and objective method for the detection of Pneumocystis from bronchoalveolar lavage fluid.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

The results for a real-time PCR assay, using the LightCycler Strep B analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis, Ind.), were compared to a direct plate method combined with a broth enrichment culture method for detection of group B streptococcus colonization in pregnant women. Two separate evaluations were conducted using two different automated nucleic extraction instruments, the MagNA Pure LC instrument (Roche Diagnostics Corporation) and the lower-capacity MagNA Pure Compact instrument (Roche Diagnostics Corporation). The sensitivities, specificities, and positive and negative predictive values for the different evaluation methods were as follow: for the LightCycler Strep B assay with MagNA Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively. The LightCycler Strep B assay combined with either MagNA Pure LC or MagNA Pure Compact extraction is a suitable method for detecting group B streptococcus colonization in pregnant women. An advantage of the LightCycler assay over culture is the considerably reduced turnaround time for results.

DNA from 101 specimens containing herpes simplex virus (HSV) produced atypical intermediate melting curves compared with those expected for HSV type 1 or HSV type 2 subsequent to real-time PCR. Nucleic acid sequence analysis of amplified target DNA revealed 1- or 3-bp polymorphisms in the probe region which allowed designation of these viruses as HSV genotype 1 or HSV genotype 2. These two subpopulations of HSV were also identified as HSV genotype 1 or HSV genotype 2 using another commercially available PCR method. Amplified HSV target DNA producing intermediate melting curves could be designated as HSV genotype 1 or HSV genotype 2 without performing sequencing or another PCR method with 96/101 (95%) specimens by adding known intermediate HSV DNA characteristic for the two subpopulations as controls.

A total of 563 specimens (234 dermal and 329 genital swabs) from patients suspected of having herpes simplex virus (HSV) infections were processed using two different extraction methods (the MagNA Pure LC system and the swab extraction tube system [SETS]); HSV DNA was amplified by LightCycler PCR. HSV DNA was detected in 157 of 563 specimens (27.9%) processed by the MagNA Pure LC system and in 179 of 563 specimens (31.8%) processed by SETS (P < 0.0001). There was no specimen processed by the MagNA Pure LC extraction method that was positive only for HSV DNA. Of 157 specimens positive by both methods, HSV DNA copy levels were higher (using cycle crossover points [cycle threshold {C(T)}]) with SETS (mean C(T), 25.9 cycles) than with the MagNA Pure LC system (mean C(T), 32.0 cycles) (P < 0.0001). The time to process 32 samples was longer with the MagNA Pure LC extraction system (90 min) than with SETS (35 min). HSV DNA extraction using SETS is faster, less expensive, and more sensitive than the MagNA Pure LC system and could replace the latter for the laboratory diagnosis of HSV infections using LightCycler PCR.

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (n = 58) than either culture (n = 55) or the Directigen immunoassay (n = 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted "gold standard," bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (n = 7) or the Directigen method (n = 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (P = 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs; P < 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs; P < 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time ( approximately 1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per microl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

Objective: To determine whether autoclaving suspensions of vaccinia virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Bacillus anthracis inactivate infectivity of these agents but allow detection of target DNA by LightCycler polymerase chain reaction (PCR)., Material and Methods: Swabs were inserted into tubes containing serial 10-fold dilutions (10(-1) to 10(-5); 500 microL; 6 samples per dilution) of vaccinia virus, HSV, VZV, or a single suspension of 10(8) colony-forming units of B anthracis (2 samples). One half of the samples were autoclaved, and the remainder were not. An aliquot of each not autoclaved sample served as a control for infectivity., Results: Autoclaving swabs saturated with suspensions of vaccinia virus, HSV, or VZV eliminated the infectivity of these agents; however, DNA was detectable in most autoclaved samples in dilutions of 10(-1) to 10(-4) by LightCycler PCR. All not autoclaved specimens were detected by culture (infectivity) except for VZV and, in most dilutions of 10(-1) to 10(-3), by assay of target DNA by LightCycler PCR. Similarly positive results were obtained for PCR assessment of sporulated B anthracis., Conclusions: Standard autoclaving procedures eliminated the infectivity of viruses (and B anthracis), but target DNA was often retained for detection by LightCycler PCR. Current recommendations indicate that the laboratory diagnosis of smallpox virus infection be performed only within Biosafety Level 4 facilities. We suggest that, in addition to the requirement for immediate coordination with public health officials, the federal government consider expanding the existing guidelines for processing these specimens to encourage immediate collection, autoclaving, and testing by LightCycler PCR to differentiate smallpox virus from other dermal pathogens such as HSV and VZV by specific qualified laboratories.

Rapid-cycle real-time polymerase chain reaction has immediate and important implications for diagnostic testing in the clinical microbiology laboratory. In our experience this novel testing method has outstanding performance characteristics. The sensitivities for detecting microorganisms frequently exceed standard culture-based assays, and the time required to complete the assays is considerably shorter than that required for culture-based assays. We describe the principle of real-time polymerase chain reaction and present clinical applications, including the detection of Bacillus anthracis, the causative agent of anthrax. This latter test is commercially available as the result of a collaborative venture between Mayo Clinic and Roche Applied Science, hence the designation The Mayo-Roche Rapid Anthrax Test.

Escherichia coli serotype O157:H7 is a leading cause of diarrhea and hemolytic uremic syndrome (HUS). Because of the limitations of current diagnostic techniques, the prevalence of non-O157:H7 Shiga toxin-producing E coli strains is not known. We describe two patients with HUS in whom no E coli O157:H7 was demonstrable in stool cultures. On culture of the urine, the first patient was found to have E coli O113:H21 strain, and the second patient had E coli O6:H1 serotype. Shiga toxin production (stx2) by the O113:H21 isolate was confirmed. The first patient required 15 days of peritoneal dialysis and subsequently recovered renal function. At last follow-up, serum creatinine was 0.9 mg/dL. The second patient had preservation of renal function throughout the acute illness with serum creatinine of 0.5 mg/dL. The clinical presentation, bacteriology, course, and outcome as well as epidemiologic implications of the increasing number of patients with E coli urinary tract infections associated with HUS are discussed. These cases illustrate the need to investigate patients with nondiarrheal HUS for infection with Shiga toxin-producing E coli of the non-O157 strain variety.

Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Resistance to the glycopeptide antibiotic vancomycin in enterococci, is phenotypically and genotypically heterogeneous. Three glycopeptide resistance phenotypes, VanA, VanB, and VanC, account for most glycopeptide resistance in enterococci; they can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin.

Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of Staphylococcus lugdunensis among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as S. lugdunensis.

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (n = 197; 23.1%) than cell cultures (n = 150) for the routine laboratory detection of herpes simplex virus infections.

Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the "gold standard" for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.

Purpose: Preretinal neovascularization has been previously observed in neonatal rats with spontaneously occurring diarrhea. This neovascularization appears analogous to retinopathy of prematurity (ROP), which occurs in human neonates. A new enterococcus species, designated Enterococcus rattus, has been isolated from the duodenum of these rats. In the present controlled study, the effect of the enteropathy induced by this organism on the retinal vasculature in the neonatal rat was further investigated., Methods: One hundred fifty newborn Sprague-Dawley rats were randomly assigned to 6 expanded litters (n = 25). On the second day of life, animals were gavaged with either 100 microl of E. rattus suspension (1.0 X 10(7) colony forming units, inoculated group, n = 100 rats) or 100 microl saline (control group, n = 50 rats). All rats were raised in room air and were killed on day 13 of life. Duodenal and blood samples were cultured. The retinal vasculature was assessed using fluorescent microscopy and ADPase staining in a masked manner. Two additional inoculated litters and one control litter were studied for evaluation of arterial blood gases and validation of the grading method for preretinal neovascularization., Results: One hundred percent of rats in the inoculated group developed severe diarrhea and had duodenal cultures positive for E. rattus compared with 0% in the control group. Preretinal neovascularization similar to ROP occurred in 55% of rats in the inoculated group compared with 2% in the control group (P = 0.001). Retinal vascular areas were reduced in the inoculated group (mean +/- SD, 89% +/- 5% versus 96% +/- 2%; P < 0.001). Rats in the inoculated group demonstrated severe growth retardation (final weight, 9.7 +/- 2.2 versus 16.7 +/- 2.7 g, P < 0.001). Inoculated animals also experienced acidosis (pH 7.31 +/- 0.06 versus 7.39 +/- 0.06 control, P = 0.04)., Conclusions: A previously undescribed enterococcal enteropathy was associated with preretinal neovascularization similar to ROP in the neonatal rat. This supports an independent role for factors other than inspired oxygen in the development of ROP.

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.

The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

During surveillance for various tickborne pathogens in the upper Midwest during the summer and early fall of 1995, a Bartonella-like agent was detected in the blood of mice that were concurrently infected with Borrelia burgdorferi or Babesia microti (or both). The organism was isolated in pure culture after inoculation of blood from wild-caught mice into C.B-17 scid/scid mice. Phylogenetic analysis of the 16S rRNA and the citrate synthase genes showed that the novel Bartonella species and a Bartonella isolate from a mouse captured on Martha's Vineyard, Massachusetts, were closely related to each other and secondarily related to Bartonella grahamii and Bartonella vinsonii. Further analysis of Peromyscus leucopus blood and tissue samples demonstrated that the novel Bartonella species was exclusively found in conjunction with B. burgdorferi and B. microti. Patent coinfection with these agents may be relatively frequent in naturally infected mice.

Aims: Recent studies suggest that Helicobacter pylori is an invasive enteropathogen. However, the efficiency with which this pathogen invades mammalian cells remains unknown. Therefore, this study was designed to investigate the invasion frequencies of HEp-2 cells by clinical strains of H pylori., Methods: An acridine orange assay and cultured HEp-2 cell monolayers were used to determine the HEp-2 cell penetration frequencies of 17 clinical isolates and one American Type Culture Collection (ATCC) strain of H pylori, and single clinical strains of Yersinia enterocolitica, Shigella flexneri, and a non-invasive ATCC Escherichia coli strain., Results: The acridine orange assay demonstrated that invasion frequencies of HEp-2 cells by all H pylori isolates were significant and, in most instances, exceeded those for the S flexneri strain and equalled those for the Y enterocolitica strain. The assay also showed that internalised H pylori organisms remained viable for at least six hours, the maximum time that bacteria and HEp-2 cells were co-incubated., Conclusions: These results may have important implications for treatment and prevention strategies for this gastric pathogen. Furthermore, the acridine orange assay may be useful for assessing, in vitro, the ability of conventional and newer antibiotics, alone or in combination, to kill intracellular H pylori organisms.

We studied the DNA sequence variation of van genes of 34 isolates of Enterococcus spp. The isolates containing the vanB gene exhibited between 0 and 41 base pair changes per 801 bp studied when the vanB sequences were compared to that of the reference strain Enterococcus faecalis V583. The isolates carrying the vanC-2 gene exhibited between 0 and 23 base pair changes per 346 bp studied when the vanC-2 sequences were compared to that of the reference strain E. casseliflavus ATCC 25788. Little variation was noted in the vanA and vanC-1 genes.