We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.

Nested polymerase chain reaction (nPCR) demonstrated the presence of Chlamydia pneumoniaespecific DNA in peripheral blood mononuclear cells (PBMC). PBMC samples were obtained from 103 consecutive patients (62 male, 41 female) aged 22 -85 years (mean, 64) admitted for coronary angiography because of suspected coronary heart disease and from 52 blood donors (43 male, 9 female) aged 40 -64 years (mean, 49). Of the 101 evaluable patients, 60 (59%) were identified by nPCR assay as C. pneumoniae DNA carriers; C. pneumoniae -specific microimmunofluorescence (MIF) serology confirmed exposure to the bacterium in 57 (95%) of the 60 nPCR-positive patients. Among the 52 blood donors, the nPCR assay identified 24 (46%) C. pneumoniae DNA carriers, all of whom were positive by C. pneumoniae-specific serology. Thirty-two patients (32%) and 23 blood donors (44%) were MIF antibody -positive but repeatedly nPCR-negative; Bartonella henselae -or Bartonella quintana -specific antibodies were not detected among any of these subjects. In this study, C. pneumoniae DNA was common in PBMC of patients with coronary heart disease and in middle-aged blood donors.Chlamydia pneumoniae has been established as a common polymerase chain reaction (PCR) detection is a technically feasible approach that is becoming an important tool in the diagnoand important pathogen causing upper and lower respiratory tract infections in humans [1]. Also, several recent studies have sis of pulmonary tuberculosis and for monitoring the efficacy of antituberculosis therapy. suggested that C. pneumoniae may be implicated in atherosclerotic disease [2 -5], and treatment with antichlamydial antibiot-The advent of PCR technology has resulted in major advantages in the diagnosis of chlamydial infections, and we have ics may reduce the risk of recurrent ''cardiac events'' in patients with established atherosclerotic coronary heart disease recently shown that nested PCR (nPCR) is a sensitive and specific method for diagnosing patients with acute respiratory [6, 7].A properly performed serology may be of value in diagnos-C. pneumoniae infections [8]. The purpose of the present study was to investigate prospectively whether blood-based nPCR is ing an acute C. pneumoniae infection [8]. In the case of persistent C. pneumoniae infection, however, serology is less useful useful in identifying individuals carrying circulating C. pneumoniae DNA. [9]. Therefore, there is an obvious need for methods that could identify individuals persistently infected by C. pneumoniae.C. pneumoniae may be harbored in human monocytes [10], Methods and monocytes are therefore a potential reservoir and source Samples. Venous whole blood (10 mL) was collected in for diagnostic efforts. Mycobacterium tuberculosis is a case in EDTA-treated tubes from 103 consecutive patients (62 male, 41 point. In a study of patients with active M. tuberculosis infecfemale) aged 22-85 years (mean, 64) who were admitted to Umeå tion, Condos et al. [11] showed that peripheral blood-based University Hospital for coronary ...

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This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of "clean" mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.