Background: The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established.Objectives: To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease.Study Design: Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated.Results: Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged 15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively.Conclusions: This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC. (C) 2008 Elsevier B.V. All rights reserved.

Se han descrito 6 genotipos y hasta 80 subtipos del virus de la hepatitis C (VHC). El conocimiento del genotipo infectante es crucial para tratar adecuadamente al paciente. El método de referencia para la tipificación molecular del VHC es la secuenciación completa del genoma viral y su posterior análisis filogenético. Sin embargo, esta posibilidad no está al alcance de la mayoría de los laboratorios hospitalarios. Existen varios métodos alternativos comercializados; los de uso más extendido se basan en el análisis de la región 5’UTR mediante amplificación y posterior secuenciación directa (TRUGENE 5’NC Genotyping Kit; Visible Genetics, Toronto, Ontario, Canadá) o hibridación inversa de los amplicones con sondas dispuestas en línea sobre membrana-LiPA- (INNO-LiPA HCV I/II/ VERSANT HCV Genotype Assay 1.0; Innogenetics, N.V., Zwijnaarde, Bayer Healthcare, Bélgica). Los resultados obtenidos con estos ensayos son generalmente superponibles; identifican correctamente los genotipos en un 95-100% (clasifican algunos genotipos 4 y 6 como genotipos 1), pero su índice de acierto es menor (70-85%) con los subtipos, particularmente 1a,1b, 2a/2c y 4a/4c. Existe una nueva generación de pruebas comercializadas con mejores prestaciones: GEN-ETI-K DEIA kit (Sorin, Saluggia, Italy) que analiza la región core; TRUGENE NS5B Genotyping Assay (Bayer Healhcare, Bélgica), la región NS5B; Real Time HCV Genotyping Test (Abbott, MS) las regiones 5’UTR y la NS5B y VERSANT 2.0 (Bayer), las regiones 5’UTR y core; estos ensayos son muy certeros en la determinación del genotipo y de la mayoría de subtipos conflictivos, particularmente 1a y 1b.Six genotypes and up to 80 subtypes of hepatitis C virus (HCV) have been described. Knowledge of the infecting genotype is crucial for appropriate therapeutic management of HCV infection. Whole genome sequencing and subsequent phylogenetic analysis is the gold standard for HCV molecular typing; however, this procedure cannot be implemented in routine clinical laboratories. A number of alternative methods have been commercialized, the most widely used being those that target the 5’UTR region. In these assays, amplicons are first generated and then analyzed by direct sequencing (TRUGENE 5’NC genotyping kit; Visible Genetics, Toronto, Notario, Canada) or by reverse hybridization of amplicons with membrane immobilized line probes -LiPA- (INNO-LiPA HCV I/II/ VERSANT HCV genotype assay 1.0; Innogenetics, N.V., Zwijnaarde, Belgium/Bayer Healthcare). Both assays produce comparable results: the overall accuracy for genotype determination is 95-100% (a number of genotypes 4 and 6 are misclassified as genotype 1), but only 70-85% for subtyping (with frequent mistyping of subtypes 1a, 1b, 2a/2c, 4a/4c). A new generation of commercialized assays with better performance is currently available: GEN-ETI-K DEIA kit (Sorin, Saluggia, Italy), which analyzes the core region, TRUGENE NS5B genotyping assay (Bayer Healhcare) for the NS5B region, RealTime HCV Genotyping Test (Abbott, MS) for both 5’UTR and NS5B regions and VERSANT 2.0 (Bayer), both for the 5’UTR and core regions; these assays are highly accurate for genotype determination and typing of most difficult subtypes, particularly 1a and 1b.

ABSTRACT A single-tube 5′ nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport ( ctrA ) , capsulation ( bexA ), and pneumolysin ( ply ) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number ( n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive ( P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.