Journal of Clinical Microbiology

Sabato, M. Fernanda; Shiffman, Mitchell L.; Langley, Michael R.; Wilkinson, David S.; Ferreira-Gonzalez, Andrea

doi: 10.1128/JCM.00058-07pmid: 17567786

We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log 10 IU/ml with a linear dynamic range of 1.0 to 7.0 log 10 IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log 10 IU/ml with a linear dynamic range of 2.0 to 7.0 log 10 IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q ( R 2 = 0.84 and R 2 = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation ( R 2 = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log 10 IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0.

Cruz, Andrea T.; Goytia, Veronica K.; Starke, Jeffrey R.

doi: 10.1128/JCM.00359-07pmid: 17537950

Nontuberculous mycobacteria are ubiquitous in the environment but rarely infect immunocompetent patients. We describe a pediatric case of Mycobacterium simiae complex lymphadenitis in an immunocompetent child and review the natural history, clinical manifestations, diagnosis, and current management of the disease.

Sharma, Dharmendra; Sethi, Sunil; Mehta, Swami Das; Sharma, Meera

doi: 10.1128/JCM.00344-07pmid: 17537947

Eno powder (GlaxoSmithKline), an antacid preparation readily available over the counter, was used instead of a CO 2 generator for the growth of 15 strains of Neisseria gonorrhoeae obtained from men with urethritis. Due to its easy accessibility and low cost, Eno powder can be useful in developing countries for transporting clinical specimens from resource-poor peripheral labs to reference laboratories.

Thomson, Kenneth S.; Cornish, Nancy E.; Hong, Seong G.; Hemrick, Kim; Herdt, Christian; Moland, Ellen S.

doi: 10.1128/JCM.00776-07pmid: 17596369

The VITEK 2 and Phoenix extended-spectrum ß-lactamase (ESBL) detection systems, which comprise confirmatory tests and expert systems, were evaluated for their ability to discriminate between 102 well-characterized strains of ESBL-positive or -negative Escherichia coli , Klebsiella pneumoniae , and Klebsiella oxytoca . At least 38 distinct ESBLs were included. The strains were chosen to include some known to cause false-positive and false-negative CLSI ESBL confirmatory test results. Therefore, enzyme characterizations, rather than CLSI tests, were the reference methods for the Phoenix and VITEK 2 evaluations. A third arm of the study was conducted with the Phoenix test using two normally inactive expert rules intended to enhance ESBL detection, in addition to using the currently available software. The Phoenix ESBL confirmatory test and unmodified expert system exhibited 96% sensitivity and 81% specificity for ESBL detection. Activation of the two additional rules increased sensitivity to 99% but reduced the specificity to 58%. The VITEK 2 ESBL confirmatory test exhibited 91% sensitivity, which was reduced to 89% sensitivity by its expert system, while its specificity was 85%. Many of the expert system interpretations of both instruments were helpful, but some were suboptimal. The VITEK 2 expert system was potentially more frustrating because it provided more inconclusive interpretations of the results. Considering the high degree of diagnostic difficulty posed by the strains, both ESBL confirmatory tests were highly sensitive. The expert systems of both instruments require modification to update and enhance their utility.

Bolmstrom, Anne; Karlsson, Asa; Engelhardt, Anette; Ho, Phion; Petersen, Peter J.; Bradford, Patricia A.; Jones, C. Hal

doi: 10.1128/JCM.00089-07pmid: 17522277

A multicenter study was conducted to validate Etest tigecycline compared to the Clinical Laboratory Standards Institute reference broth microdilution and agar dilution methodologies. A large collection of gram-negative ( n = 266) and gram-positive ( n = 162) aerobic bacteria, a collection of anaerobes ( n = 385), and selected collections of nonpneumococcal streptococci ( n = 369), Streptococcus pneumoniae ( n = 372), and Haemophilus influenzae ( n = 372) were tested. Strains with reduced susceptibility to tigecycline were used with all test methods. The Etest showed excellent inter- and intralaboratory reproducibility for all organism groups tested regardless of the test methodology. The essential agreement values with the reference method (±1 dilution) were >99% for the collection of gram-negative and gram-positive aerobes; >98% for the S. pneumoniae , H. influenzae , and anaerobe collections; and 100% for the group of nonpneumococcal streptococci. These results validate the performance accuracy and utility of Etest tigecycline and verify the reproducibility of this convenient predefined gradient methodology for tigecycline susceptibility determination.

Manzur, Adriana; Tubau, Fe; Pujol, Miquel; Calatayud, Laura; Dominguez, Maria Angeles; Pena, Carmen; Sora, Mercedes; Gudiol, Francesc; Ariza, Javier

doi: 10.1128/JCM.02546-06pmid: 17581932

Enterobacter cloacae has been associated with several outbreaks, usually involving strains that overproduce chromosomal ß-lactamase or, uncommonly, strains expressing extended-spectrum ß-lactamases (ESBL). Only sporadic cases of ESBL-producing E. cloacae have been identified in our hospital in recent years. We describe the epidemiology and clinical and microbiological characteristics of an outbreak caused by ESBL-producing E. cloacae in a cardiothoracic intensive care unit (CT-ICU). Prospective surveillance of patients with infection or colonization by ESBL-producing E. cloacae among patients admitted to the CT-ICU was performed during the outbreak. Production of ESBL was determined by decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk test result. Clone relatedness was determined by pulsed-field gel electrophoresis (PFGE). From July to September 2005, seven patients in the CT-ICU with ESBL-producing E. cloacae were identified (four males; median age, 73 years; range, 45 to 76 years); six patients had cardiac surgery. Four patients developed infections; three had primary bacteremia, one had ventilator-associated pneumonia, and one had tracheobronchitis. ESBL-producing E. cloacae showed resistance to quinolones and aminoglycosides. PFGE revealed two patterns. Five isolates belonged to clone A; two carried a single ESBL (pI 8.2 and a positive PCR result for the SHV type), and three carried two ESBLs (pIs 8.1 and 8.2 and positive PCR results for the SHV and CTX-M-9 types). Isolates belonging to clone B carried a single ESBL (pI 5.4 and a positive PCR result for the TEM type). Review of antibiotic consumption showed increased use of cefepime and quinolones during June and July 2005. The outbreak was stopped by the implementation of barrier measures and cephalosporin restriction. ESBL production could be increasingly common in nosocomial pathogens other than Escherichia coli or Klebsiella pneumoniae .

Musher, Daniel M.; Manhas, Atisha; Jain, Pranav; Nuila, Franziska; Waqar, Amna; Logan, Nancy; Marino, Bernard; Graviss, Edward A.

doi: 10.1128/JCM.00686-07pmid: 17567791

Several kinds of laboratory techniques are available to detect Clostridium difficile toxin in fecal samples. Because questions have been raised about the reliability of immunoassays compared to the accepted standard, cytotoxicity assay, we studied three enzyme immunoassays (EIAs) and one rapid EIA, which demonstrated relatively good sensitivities and specificities compared to cytotoxicity assay.

Nakano, Kazuhiko; Lapirattanakul, Jinthana; Nomura, Ryota; Nemoto, Hirotoshi; Alaluusua, Satu; Gronroos, Lisa; Vaara, Martti; Hamada, Shigeyuki; Ooshima, Takashi; Nakagawa, Ichiro

doi: 10.1128/JCM.02343-06pmid: 17567784

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes ( c , e , f , and k ). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans , in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans . Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10 10 sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e , f , and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c . No geographic specificity was identified. However, the distribution of the collagen-binding protein gene ( cnm ) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.

Gotsch, Antje; Schubert, Andreas; Bombis, Armin; Wiedmann, Michael; Zauke, Michael; Schorling, Stefan

doi: 10.1128/JCM.00647-07pmid: 17553976

Molecular diagnostic tests based on the PCR or alternative nucleic acid amplification technologies are commonly used for pathogen screening at blood drawing centers. Contrived process surveillance using test-specific external and internal controls is critical for the efficient leverage of PCR power. We describe here novel control constructs for use in nucleic acid amplification assays for pathogens with a single-stranded DNA genome, e.g., parvovirus B19. These controls are derived from a deletion mutant of the filamentous phage fd-tet, fKN16, and consist of single-stranded DNA packaged in a protein coat. They are essentially noninfectious to Escherichia coli and highly resistant to nuclease degradation. fKN16 based controls can be readily manufactured and highly purified. Despite their confirmed filamentous morphology, they can be precisely and accurately diluted over a wide range. Stability studies reveal that the novel control constructs are highly resistant to temperature stress, regardless of whether they are tested as concentrated stocks in storage buffer or diluted in buffer or human plasma. Real-time amplification curves derived from recombinant control constructs containing a parvovirus B19 specific sequence fragment match those derived from native virus. In summary, our data demonstrate the feasibility of novel nuclease-resistant single-stranded DNA controls as surrogates for parvovirus B19 and their applicability in routine molecular diagnostics.

Makristathis, Athanasios; Hirschl, Alexander M.; Russmann, Holger; Koletzko, Sibylle

doi: 10.1128/JCM.00846-07pmid: 17675456