Children with multiple viral respiratory infections are older than those with single viruses.

AIM: To study the clinical impact of multiple viral respiratory infections compared to single infections. METHODS: Demographic data from 37 multiple infection periods in children <5 years of age were compared to data from 193 episodes with single infections. Clinical data derived from patient records of the multiple infection episodes were further compared to data from 93 matched control episodes with single infections. RESULTS: The mean age of patients with multiple viral findings was 12.7 months, compared to 5.7 months for those with single findings (p < 0.01). Wheezing was the most common diagnosis in both groups, except among children who were only infected with the coronavirus. No differences were found regarding duration of hospitalisation, oxygen treatment or admittance to the intensive care unit. CONCLUSION: Children with multiple viral findings in their respiratory secretions were older than those with a single detected virus. Otherwise, no major differences in comorbidity, presentation or clinical outcome were observed between the two groups.

Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods.

BACKGROUND: The microbial etiology of community-acquired pneumonia (CAP) is still not well characterized. During the past few years, polymerase chain reaction (PCR)-based methods have been developed for many pathogens causing respiratory tract infections. The aim of this study was to determine the etiology of CAP among adults-especially the occurrence of mixed infections among patients with CAP-by implementing a new diagnostic PCR platform combined with conventional methods. METHODS: Adults admitted to Karolinska University Hospital were studied prospectively during a 12-month period. Microbiological testing methods included culture from blood, sputum, and nasopharyngeal secretion samples; sputum samples analyzed by real-time quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; nasopharyngeal specimens analyzed by use of PCR; serological testing for Mycoplasma pneumoniae, Chlamydophila pneumoniae, and viruses common in the respiratory tract; and urine antigen assays for detection of pneumococcal and Legionella pneumophila antigens. RESULTS: A microbial etiology could be identified for 67% of the patients (n = 124). For patients with complete sampling, a microbiological agent was identified for 89% of the cases. The most frequently detected pathogens were S. pneumoniae (70 patients [38%]) and respiratory virus (53 patients [29%]). Two or more pathogens were present in 43 (35%) of 124 cases with a determined etiology. CONCLUSIONS: By supplementing traditional diagnostic methods with new PCR-based methods, a high microbial yield was achieved. This was especially evident for patients with complete sampling. Mixed infections were frequent (most commonly S. pneumoniae together with a respiratory virus).

Merkel cell polyomavirus in respiratory tract secretions.

Merkel cell polyomavirus (MCPyV), associated with Merkel cell carcinoma, was detected in 27 of 635 nasopharyngeal aspirate samples by real-time PCR. MCPyV was more commonly found in adults than in children. Presence in the upper respiratory tract may be a general property of human PyVs.

A single-tube, real-time PCR assay for detection of the two newly characterized human KI and WU polyomaviruses.

BACKGROUND: Three new human polyomaviruses have been recently described, and investigating their in vivo biology and pathogenicity will require sensitive and rational detection assays. OBJECTIVES: To develop and evaluate a sensitive and rational assay for detection of the newly identified KI and WU polyomaviruses. STUDY DESIGN: A single-tube, dual-probe, real-time PCR assay for simultaneous detection and discrimination of KI and WU polyomaviruses was developed. RESULTS: The assay had near single-molecule sensitivity for both viruses and no cross-reactivity was observed. A panel of 637 nasopharyngeal aspirates was screened, resulting in a frequency of 1.4% for KIPyV and 1.3% for WUPyV. CONCLUSIONS: The dual-probe assay provides a rational approach for further studies of KIPyV and WUPyV.

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses.

Acute respiratory tract infections are caused by a large number of viruses. Diagnostic methods have until recently been available only for a limited number of these viruses. With the objective to achieve sensitive assays for all respiratory viruses, a rational workflow in the laboratory, and a short turn-around time, a real-time PCR diagnostic platform for daily rapid detection of 15 respiratory viruses was developed. The system was evaluated on 585 stored nasopharyngeal aspirates from hospitalized children. Previous analysis by immunofluorescence and virus isolation identified viruses in 37% of the samples while the new PCR diagnostic panel detected 57% virus positive samples. The new platform was introduced in the laboratory in October 2007 and has then fully replaced the standard immunofluorescence assay for rapid detection of viruses and virus isolation.

High diagnostic yield by CSF-PCR for entero- and herpes simplex viruses and TBEV serology in adults with acute aseptic meningitis in Stockholm.

Acute aseptic meningitis (AAM) affects 10-20/100,000 inhabitants per years in Sweden. Up to the beginning of the 1980s the diagnoses were made by virus isolation and/or determination of viral antibodies in serum. The development of PCR for detection of viruses in CSF samples has increased the sensitivity and diagnostic efficiency considerably. We investigated the aetiology of AAM and the diagnostic efficiency in an adult population in Stockholm, using a limited first-line combination of microbiological assays. CSF and serum samples, consecutively collected in 419 patients with clinical symptoms of AAM in northern Stockholm during 1999-2004, were included. PCR assays for herpes simplex virus (HSV) DNA and enterovirus (EV) RNA in the CSF as well as ELISA for IgM in serum to tick-borne encephalitis virus (TBEV) were performed routinely. A viral diagnosis was obtained in 255 of the 419 cases (62%) with these routinely performed assays. Clinical findings in combination with additional diagnostic tests resulted in an overall aetiological yield of 72%. EV was the major causative agent (27%) followed by TBEV (21%) and HSV-2 (19%). We conclude that consistent use of CSF-PCR for EV and HSV and TBEV serology established a diagnosis in the majority of AAM patients.

Norovirus strains belonging to the GII.4 genotype dominate as a cause of nosocomial outbreaks of viral gastroenteritis in Sweden 1997--2005. Arrival of new variants is associated with large nation-wide epidemics.

BACKGROUND: In recent years an increase of the incidence of nosocomial outbreaks caused by noroviruses has been observed throughout Sweden, with high peaks noted in the winter seasons 2002/2003 and 2004/2005, respectively. OBJECTIVES: To phylogenetically characterize norovirus strains causing nosocomial outbreaks from 1997 to 2005 and estimate the impact of norovirus-like disease on the Swedish health care system during the peak season 2002/2003 when a new variant of norovirus occurred. STUDY DESIGN: Stool samples from 115 randomly selected nosocomial outbreaks occurring during 1997--2005 throughout Sweden were studied by RT-PCR and sequencing. In addition, to investigate the impact on the health-care system, a questionnaire was distributed to infection control units (n=90) serving all Swedish hospitals, nursing homes and other health-care institutions during the largest epidemic of nosocomial outbreaks. RESULTS: Sequencing of 279 nucleotides of the norovirus RNA polymerase gene in stools containing norovirus RNA showed that strains belonging to the GII.4 genotype dominated. Each of the two large epidemics was due to a new variant within this cluster. The questionnaire revealed that 30,000-35,000 episodes of nosocomial norovirus-like infections occurred in 80 of 82 major Swedish hospitals affected in 2002/2003. CONCLUSION: New norovirus variants within the cluster GGII.4 may have a major impact on the health-care system.

High occurrence of a new variant of Chlamydia trachomatis escaping diagnostic tests among STI clinic patients in Stockholm, Sweden.

BACKGROUND: In 2006, a genetic variant of Chlamydia trachomatis not detectable with the most commonly used diagnostic tests was identified. Initial reports suggested that as many as 10% to 13% of all chlamydia cases would have remained undiagnosed. The aim of the study was to find the occurrence and clinical findings of this genetic variant among a high-risk population in Stockholm, Sweden. METHODS: Samples were analyzed using the Cobas TaqMan CT test (Roche Diagnostics). To detect the new variant, an additional PCR-analysis, artus C. trachomatis LC MOMP PCR Kit (Qiagen) was performed on all negative samples. Positive results in the artus test were confirmed by a mutant specific PCR. Clinical data were retrospectively collected from medical records. RESULTS: Among 1009 samples analyzed, 115 were positive for C. trachomatis and among those, 27 were found to belong to the genetic altered strain. This variant constituted 23% of all chlamydia cases diagnosed, and 29% were found in the age group 20 to 29 years. Women with the new variant were younger and had more often performed another chlamydia test within the previous 6 months compared with those infected with the wild type. CONCLUSION: These results indicate that a large number of sexually active individuals might be infected despite a negative chlamydia test, thus facilitating a rapid transmission of the new variant. Accordingly, it is of great importance to be aware of limitations of the diagnostic methods used.

Increased detection rate in diagnosis of herpes simplex virus type 2 meningitis by real-time PCR using cerebrospinal fluid samples.

Efficient and sensitive diagnostic methods are needed in the management of virus infections in the central nervous system. There is a demand for an evaluation of the sensitivity of PCR methods for early diagnosis of meningitis due to herpes simplex type 2 (HSV-2) and varicella-zoster virus (VZV). The objective of this study was to evaluate real-time PCR in the detection of HSV-2 and VZV DNA from cerebrospinal fluid (CSF) for etiological diagnoses in clinically well-characterized cases of primary and recurrent aseptic meningitis. Samples from 110 patients, 65 of whom were diagnosed with or were strongly suspected of having HSV-2 meningitis and 45 with acute aseptic meningitis of unknown causes, were analyzed. Results were compared with the outcome of nested PCR for HSV-2 infection. Clinical parameters were analyzed in relation to CSF viral load. With real-time PCR, HSV-2 DNA was found in CSF from 80% (52/65) of patients with clinical HSV-2 meningitis compared to 72% (47/65) found by nested PCR. The sensitivity of real-time HSV-2 PCR was found to be 87% (33/38) in primary and 70% (19/27) in recurrent meningitis. The HSV-2 viral load was significantly higher in primary than in recurrent meningitis and correlated with the degree of inflammation. VZV DNA was detected in 2 of 45 samples (4.4%). Real-time PCR for the diagnosis of HSV-2 meningitis was evaluated in a large, clinically well-characterized sample of material and found to identify more cases than nested PCR in the group of patients with recurrent meningitis. Quantification of DNA enables further research of disease prognosis and treatment.

Human bocavirus and acute wheezing in children.

BACKGROUND: Human bocavirus is a newly discovered parvovirus. It has been detected primarily in children with acute lower respiratory tract infection, but its occurrence, clinical profile, and role as a causative agent of respiratory tract disease are not clear. METHODS: We investigated the presence of human bocavirus by quantitative polymerase chain reaction of nasopharyngeal aspirate specimens and selected serum samples obtained from 259 children (median age, 1.6 years) who had been hospitalized for acute expiratory wheezing. The samples were analyzed for 16 respiratory viruses by polymerase chain reaction, virus culture, antigen detection, and serological assays. RESULTS: At least 1 potential etiologic agent was detected in 95% of children, and >1 agent was detected in 34% of children. Human bocavirus was detected in 49 children (19%). A large proportion of the cases were mixed infections with other viruses, but human bocavirus was the only virus detected in 12 children (5%). High viral loads of human bocavirus were noted mainly in the absence of other viral agents, suggesting a causative role for acute wheezing. In addition, infections that had uncertain clinical relevance and low viral loads were prevalent. Human bocavirus DNA was frequently detected in serum specimens obtained from patients with acute wheezing, suggesting systemic infection. CONCLUSIONS: Human bocavirus is prevalent among children with acute wheezing and can cause systemic infection. Results suggest a model for bocavirus infection in which high viral loads are potentially associated with respiratory symptoms and low viral loads indicate asymptomatic shedding. Therefore, quantitative polymerase chain reaction analysis may be important for additional studies of human bocavirus.

Cloning of a human parvovirus by molecular screening of respiratory tract samples.

The identification of new virus species is a key issue for the study of infectious disease but is technically very difficult. We developed a system for large-scale molecular virus screening of clinical samples based on host DNA depletion, random PCR amplification, large-scale sequencing, and bioinformatics. The technology was applied to pooled human respiratory tract samples. The first experiments detected seven human virus species without the use of any specific reagent. Among the detected viruses were one coronavirus and one parvovirus, both of which were at that time uncharacterized. The parvovirus, provisionally named human bocavirus, was in a retrospective clinical study detected in 17 additional patients and associated with lower respiratory tract infections in children. The molecular virus screening procedure provides a general culture-independent solution to the problem of detecting unknown virus species in single or pooled samples. We suggest that a systematic exploration of the viruses that infect humans, "the human virome," can be initiated.