Journal of Clinical Microbiology

Quereda, C.; Corral, I.; Laguna, F.; Valencia, M. E.; Tenorio, A.; Echeverria, J. E.; Navas, E.; Martín-Dávila, P.; Moreno, A.; Moreno, V.; Gonzalez-Lahoz, J. M.; Arribas, J. R.; Guerrero, A.

doi: N/Apmid: 10921978

Diagnostic Utility of a Multiplex Herpesvirus PCR Assay Performed with Cerebrospinal Fluid from Human Immunodeficiency Virus-Infected Patients with Neurological Disorders C. Quereda 1 , * , I. Corral 2 , F. Laguna 3 , M. E. Valencia 3 , A. Tenorio 4 , J. E. Echeverria 4 , E. Navas 1 , P. Martín-Dávila 1 , A. Moreno 1 , V. Moreno 3 , J. M. Gonzalez-Lahoz 3 , J. R. Arribas 5 , and A. Guerrero 1 , † Unidad de Enfermedades Infecciosas 1 and Servicio de Neurologı́a, 2 Hospital Ramón y Cajal, 28034-Madrid, Unidad de Enfermedades Infecciosas, Hospital Carlos III, Instituto de Salud Carlos III, 28029-Madrid, 3 Unidad de Virologı́a, Centro Nacional de Microbiologı́a, Instituto de Salud Carlos III, 28220-Majadahonda, 4 and Servicio de Medicina Interna, Hospital “La Paz,” 28046-Madrid, 5 Spain ABSTRACT We used a multiplex nested-PCR assay for the simultaneous detection in cerebrospinal fluid (CSF) of five human herpesviruses (HVs) (cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus (HSV), and human herpesvirus 6 (HHV-6)) in a clinical evaluation of human immunodeficiency virus (HIV)-infected patients with neurological disorders. This method, which has the advantages of being rapid and economical, would be of particular interest for the diagnosis of neurological syndromes caused by more than one HV. We studied 251 CSF samples from 219 patients. HV DNA was demonstrated in 93 (37%) of the CSF samples (34% of the patients). CMV was the HV most frequently detected in our patients (25%), while EBV, VZV, HSV, and HHV-6 DNAs were present in significantly fewer cases (7, 4, 3, and 1%, respectively). When results were compared with the final etiological diagnoses of the patients, the multiplex HV PCR showed high specificity for the diagnosis of CMV and VZV neurological diseases and for cerebral lymphoma (0.95, 0.97, and 0.99, respectively). The sensitivity of the assay was high for CMV disease (0.87), was low for cerebral lymphoma (0.33), and was not evaluable for VZV disease due to the small number of patients with this diagnosis. Nevertheless, detection of VZV DNA had possible diagnostic value in four of the nine cases, and EBV DNA amplification always predicted the diagnosis of cerebral lymphoma in patients with cerebral masses. Detection of HSV DNA was frequently associated with CMV amplification and fatal encephalitis. HHV-6 was not considered to have a pathogenetic role in the three cases in which it was detected. This multiplex HV PCR assay is a specific and clinically useful method for the evaluation of HIV-infected patients with neurological disorders related to HV.

Barenfanger, Joan; Drake, Cheryl; Leon, Nidia; Mueller, Tina; Troutt, Tammy

doi: N/Apmid: 10921934

Clinical and Financial Benefits of Rapid Detection of Respiratory Viruses: an Outcomes Study Joan Barenfanger 1 , * , Cheryl Drake 1 , Nidia Leon 2 , Tina Mueller 1 , and Tammy Troutt 1 Laboratory Medicine, Memorial Medical Center, 1 and Internal Medicine Department, Southern Illinois University School of Medicine, 2 Springfield, Illinois 62781 ABSTRACT To assess the expected benefits of rapid reporting of respiratory viruses, we compared patients whose samples were processed using standard techniques such as enzyme immunoassays, shell vial assays, and culture tube assays (year 1) to patients whose samples were processed with the same standard techniques in addition to immunofluorescent testing (FA) directly on cytocentrifuged samples (year 2). The cytospin FA screened for influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3, and adenovirus (DAKO Diagnostics Ltd.). The specificity of the cytospin FA for all viruses was 100%. The sensitivities for influenza A virus and RSV were 90 and 98%, respectively, but the sensitivities for influenza B virus and adenovirus were unacceptable (14.3 and 0%, respectively). However, since the former viruses account for >85% of our isolates from clinical specimens, the cytospin FA is an excellent screening test since the positive result was available within hours. The mean turnaround time for all positive viruses was 4.5 days in year 1 and 0.9 day in year 2 ( P = 0.001). This rapid reporting resulted in physicians having access to information sooner, enabling more appropriate treatment. The mean length of stay in the hospital for inpatients with respiratory viral isolates was 10.6 days for year 1 versus 5.3 days for year 2. Mean variable costs for these patients was $7,893 in year 1 and $2,177 in year 2. After subtracting reagent costs and technological time, the savings in variable costs was $144,332/year. Summarizing, the cytospin FA markedly decreased turnaround time and was associated with decreased mortality, length of stay, and costs and with better antibiotic stewardship.

Lo Presti, Francois; Riffard, Serge; Jarraud, Sophie; Le Gallou, Florence; Richet, Herve; Vandenesch, Francois; Etienne, Jerome

doi: N/Apmid: 10921997

Two cases of Legionnaire's disease caused by Legionella oakridgensis were diagnosed at the university hospital in Nantes, France. The two patients' isolates were identified by means of phenotyping and genotyping methods. Epidemiological investigations concluded that the first case was hospital acquired while the second case was considered community acquired.

Beggs, Marjorie L.; Eisenach, Kathleen D.; Cave, M. Donald

doi: N/Apmid: 10921952

Mapping of IS 6110 Insertion Sites in Two Epidemic Strains of Mycobacterium tuberculosis Marjorie L. Beggs 1 , * , Kathleen D. Eisenach 1 , 2 , and M. Donald Cave 3 Departments of Pathology, 1 Anatomy, 3 and Microbiology and Immunology, 2 University of Arkansas for Medical Sciences and J. L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas ABSTRACT A widely distributed strain designated 210 was identified in a study of the diversity of Mycobacterium tuberculosis DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS 6110 , and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS 6110 insertions in strain 210 and compare these to IS 6110 insertion sites in strain W. Our hypothesis is that an IS 6110 insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the M. tuberculosis H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS 6110 insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS 6110 insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS 6110 has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS 6110 was found to reside 55 bp in the ctpD gene. This gene is expressed, indicating that IS 6110 can provide a promoter sequence for the transcription of genes.

Echavarria, Marcela; Kolavic, Shellie A.; Cersovsky, Steven; Mitchell, Felicia; Sanchez, Jose L.; Polyak, Christina; Innis, Bruce L.; Binn, Leonard N.

doi: N/Apmid: 10921963

Since 1954, adenoviruses (AdV) have been recognized as an important cause of acute respiratory disease (ARD) among U.S. military recruits. Until recently, routine oral vaccination for AdV serotypes 4 and 7 eliminated epidemic AdV-associated ARD in this population. Now that the manufacturer has ceased production, vaccination has ended and AdV epidemics have reappeared. As part of a prospective epidemiological study during the high-risk ARD season, serial samples were obtained from ventilation system filters and tested for AdV by culture and PCR. An outbreak occurred during this surveillance. Of 59 air filters, 26 (44%) were AdV positive only by PCR. Sequence analysis confirmed the presence of AdV serotype 4, the implicated outbreak serotype. The number of AdV-related hospitalizations was directly correlated with the proportion of filters containing AdV; correlation coefficients were 0.86 (Pearson) and 0.90 (Spearman's rho). This is the first report describing a PCR method to detect airborne AdV during an ARD outbreak. It suggests that this technique can detect and quantify AdV-associated ARD exposure and may enable further definition of environmental effects on AdV-associated ARD spread.

von Eiff, Christof; Reinert, Ralf Rene; Kresken, Michael; Brauers, Johannes; Hafner, Dieter

doi: N/Apmid: 10921933

Antibiotic-resistant gram-positive bacteria have become an increasing problem in the last two decades. In order to evaluate the prevalence of antibiotic resistance in staphylococcal bloodstream isolates in Germany, 2,042 staphylococci collected in 21 tertiary-care hospitals were investigated during a 3-year period (March 1996 to March 1999). Altogether, 1,448 S. aureus isolates and 594 coagulase-negative staphylococci (CoNS) that comprised 13 different species were included. Furthermore, the antistaphylococcal activities of quinupristin-dalfopristin were compared with those of eight other compounds by the broth microdilution method. The rates of oxacillin resistance in Staphylococcus aureus , S. epidermidis , S. haemolyticus , and other CoNS were 13.5, 69, 90, and 34%, respectively. In oxacillin-resistant strains high rates of resistance (up to 100%) to erythromycin, clindamycin, ciprofloxacin, and gentamicin were also observed. However, no strain appeared to be resistant to vancomycin or quinupristin-dalfopristin. The streptogramin combination exhibited excellent in vitro activity against all staphylococcal species tested, regardless of the patterns of resistance to other drug classes. In terms of MICs at which 90% of the isolates are inhibited, quinupristin-dalfopristin was 2 times more active against S. aureus isolates, 4 to 16 times more active against S. haemolyticus , and 8 to 32 times more active against S. epidermidis than vancomycin or teicoplanin.

Meis, Jacques F. G. M.; Verduyn Lunel, Frans M.; Verweij, Paul E.; Voss;, Andreas; Coleman, D.

doi: N/Apmid: 11001667

Coetsier, Christophe; Vannuffel, Pascal; Blondeel, Nathalie; Denef, Jean-Francois; Cocito, Carlo; Gala, Jean-Luc

doi: N/Apmid: 10921976

Duplex PCR for Differential Identification of Mycobacterium bovis , M. avium , and M. avium subsp. paratuberculosis in Formalin- Fixed Paraffin-Embedded Tissues from Cattle Christophe Coetsier 1 , Pascal Vannuffel 2 , Nathalie Blondeel 1 , Jean-Francois Denef 1 , Carlo Cocito 1 , and Jean-Luc Gala 2 , 3 , * Histology Unit 1 and Laboratory of Applied Molecular Technology, 2 Medical Faculty, Universitécatholique de Louvain, and Section MSW, Operational Epidemiology and Infectious Diseases, Queen Astrid Military Hospital, 3 Brussels, Belgium ABSTRACT We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis , namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis . Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli ( M. tuberculosis , M. bovis , and M. africanum ), for both nontuberculous mycobacteria M. avium and M. intracellulare , or for M. avium subsp. paratuberculosis . The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

Whyte, Tom; Hanahoe, Belinda; Collins, Tom; Corbett-Feeney, Geraldine; Cormican;, Martin; Alcaide, Fernando; Benitez, Miguel Angel; Martin, Rogelio; Escriba, Josep M.

doi: N/Apmid: 11001663

Yu, Ming-Lung; Chuang, Wan-Long; Dai, Chia-Yen; Chen, Shinn-Cherng; Lin, Zu-Yau; Hsieh, Ming-Yuh; Wang, Liang-Yen; Chang, Wen-Yu

doi: N/Apmid: 10921954

A second-generation hepatitis C virus (HCV) quantitative assay (COBAS AMPLICOR HCV MONITOR Test, version 2.0; COBAS HCM-2) has been developed, with the intention of achieving equivalent quantification of all HCV genotypes and improving assay performance. To evaluate the clinical performance of COBAS HCM-2 and its utility in predicting the response to alpha interferon treatment, sera from 215 chronic hepatitis C patients were analyzed and the results were compared with those obtained by the Quantiplex bDNA HCV RNA, version 2.0, assay (bDNA-2). The COBAS HCM-2 had significantly greater sensitivity than bDNA-2 (94.9 versus 88.4%; P < 0.001) when performed with sera from chronic hepatitis C patients who were viremic by a qualitative PCR test. The standard deviations for the within-run and between-run reproducibilities of COBAS HCM-2 were <0.1 and <0.2, respectively, and it showed an improved linear range between genotypes with the threefold serial dilutions tested ( r 2 = 0.986 to 0.995). The COBAS HCM-2 results were positively correlated with the bDNA-2 results, but the values for COBAS HCM-2 were on average 0.96 log lower than the values for bDNA-2. The mean difference in quantification values between these two assays did not differ among samples with different genotypes (0.70 to 1.00 log). No genotype-dependent difference in viral load was observed. The pretreatment viral load was significantly lower in complete responders. By using multivariate analysis, the viral load 2 weeks after the initiation of alpha interferon treatment was the strongest predictor of a complete response. In conclusion, COBAS HCM-2 demonstrated good sensitivity, linearity, and reproducibility and efficiency equal to that of bDNA-2 for the quantification of HCV genotypes 1 and 2. Hence, this assay provides a rapid and reliable method for the quantification of HCV RNA in serum and is useful for the planning of interferon treatment.