Self-sampling for analysis of respiratory viruses in a large-scale epidemiological study in Sweden.

Viral diagnosis of respiratory tract infections has so far required sampling by health professionals,hampering large-scale epidemiological studies of virus-specific disease outcomes. As part of a population-based, prospective study of work-related risk factors for transmission of viral infections (SWEDE-I), we developed a scheme for self-sampling with nasal swabs. Random selection from the gainfully employed population of a medium-sized town in central Sweden resulted in a study cohort of 2,237 men and women aged 25 to 63 years. From September 2011 through May 2012, the cohort reported all instances of respiratory tract infection or gastroenteritis and participants concomitantly sent self-sampled nasal swabs for analysis using regular mail. Diagnosis of 14 viruses was performed. A total of 1,843 samples were received. The week-wise average delay between disease on set and arrival of the specimens at the laboratory varied between four and six days, and the corresponding median delay was between 3.5 and six days. In line with previous community-based studies, picorna- and coronaviruses dominated in specimens obtained from the self-sampling scheme. The results of self-sampling were contrasted to those from contemporaneous routine clinical sampling, on the same age group, in the adjacent Stockholm county. Although higher proportions of positive samples for respiratory syncytial virus and influenza were observed in the clinical sampling scheme, estimations of seasonality for influenza A and picornaviruses derived from both schemes were similar. Our findings show that nasal self-sampling is feasible in large-scale surveillance of respiratory infections and opens new prospects for population based,virologically verified research on virus spread,burden of disease, and effects of environmental factors or interventions.

Does viral interference affect spread of influenza?

This short communication hypothesises that rhinovirus epidemics occurring after start of school may interfere with the spread of influenza during the period when warm and humid climate decreases the influenza spread by aerosol. Limited laboratory data supporting this hypothesis are included in the article, but the report is written mainly to stimulate interest and research concerning the possibility that viral interaction may affect influenza epidemiology.

Borrelia antibodies in children evaluated for Lyme neuroborreliosis.

BACKGROUND: We wanted to elucidate the value of Borrelia antibodies in serum and cerebrospinal fluid (CSF) for the diagnosis of Lyme neuroborreliosis (LNB). MATERIAL AND METHODS: We analyzed the serological findings, by anti-flagellin assay, in 267 patients with neurological symptoms from the Stockholm area, where Lyme borreliosis is endemic. RESULTS: In the 70 children with LNB, intrathecal Borrelia antibody production was diagnostic and found in 50 (71%). Sixteen (23%) showed an elevated antibody titer in serum only, and 4 (7%) had no serologic findings. Borrelia IgG in serum, with or without concomitant IgM, was a specific (98%), but insensitive (43%) marker of infection. Isolated, false-positive serum IgM titers were common and found in 10 of 67 children (15%) with viral meningitis, as well as in 28 of 111 (25%) with various neurological symptoms and normal CSF. The specificity of an isolated Borrelia IgM titer in serum was 81%, and the positive predictive value for Borrelia infection only 50% in our material. On the other hand, absence of antibodies in blood had a negative predictive value of 94%, which increased to 97% if also CSF findings were included. CONCLUSIONS: Intrathecal antibody production is strongly supportive of an LNB diagnosis. Conversely, isolated, elevated levels of Borrelia IgM in serum occur in up to one-fourth of children with various neurological complaints, and should be interpreted with caution, especially in nonendemic areas.

A case of diffuse leptomeningeal oligodendrogliomatosis associated with HHV-6 variant A.

We describe a rare case of diffuse leptomeningeal oligodendrogliomatosis associated with the human herpes virus 6 variant A (HHV-6 A). A 2-year-old boy presented with progressive neurological symptoms and hydrocephalus. The patient had a VP shunt placement but did not fully recover. HHV-6 A was detected in both CSF and serum by nested PCR. His symptoms improved repeatedly, but temporarily, on antiviral treatment. An open brain biopsy, ten months after presentation, revealed leptomeningeal tumour as well as the presence of viral DNA in the tumour tissue. The role of HHV-6 A could be that of a reactivated opportunist. However, this case also raises the question whether this neurotropic virus, with malignant transforming properties in vitro, may have a role in pathogenesis in some cases of brain malignancy.

Sequence analysis of UL54 and UL97 genes and evaluation of antiviral susceptibility of human cytomegalovirus isolates obtained from kidney allograft recipients before and after treatment.

The frequency of infections caused by drug-resistant cytomegalovirus (CMV) in solid-organ transplant recipients is not known. Only a few resistant strains have been described in transplant recipients. Antiviral susceptibility to ganciclovir (GCV) and foscarnet (PFA) of CMV isolates from 24 renal transplant patients with CMV viremia and CMV disease before and after therapy were investigated by a solid phase ELISA. The CMV DNA polymerase (UL54) and viral phosphotransferase (UL97) genes were also sequenced. Ten patients did not receive antiviral treatment; five and nine patients were treated with PFA and GCV, respectively. No appearance of drug-resistant viruses was observed in the present study, but one isolate showed a reduced sensitivity to PFA after treatment with GCV. This finding could not be explained by the presence or development of mutations that have been associated with drug resistance in UL54. We found no evidence that short-term treatment of CMV with PFA- or GCV-induced resistance.

Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations.

We determined the nucleotide (nt) and amino acid (aa) heterogeneities of three distinct regions of the human cytomegalovirus (CMV) genome for 46 low-passage CMV isolates from four different patient populations (congenitally infected infants, children attending day-care centers, renal transplant recipients, and human immunodeficiency virus-infected individuals) and for two laboratory strains (CMV Ad169 and Towne). The gene regions for the major immediate-early (MIE) exon 4 gene (nt positions 1702 to 1982, aa positions 152 to 244), the DNA polymerase gene (nt positions 2797 to 3046, aa positions 713 to 795), and the glycoprotein B (gB) gene (nt positions 1698 to 1884, aa positions 567 to 628) were sequenced. The sequence information was used to design sets of nested PCR primers directed against the most highly conserved regions identified. MIE was the most variable gene region compared to the variability of the DNA polymerase and gB gene regions. Comparison of the sequences of all 46 isolates with that of Ad169 revealed nt and aa sequence homologies of 87.9 and 87.2%, respectively, within the MIE gene compared to 92.8 and 100% homologies, respectively, within the DNA polymerase gene and 93 and 95.2% homologies, respectively, within the gB gene. Within the MIE gene, compared to the Ad169 nt sequence the homology at the nt level among isolates obtained from children attending day-care centers was high (96.4%), while it was lower (90%) among isolates obtained from the other three patient populations. Preliminary results of a nested PCR with oligonucleotide primers selected from the DNA polymerase gene region with a low level of nt sequence variation indicates that primers selected from this region might be more powerful for use in PCR than primers selected from the MIE gene region.

Detection of respiratory viruses in nasopharyngeal secretions with immunofluorescence technique for multiplex screening-an evaluation of the Chemicon assay.

BACKGROUND: Antigen detection with immunofluorescence is an efficient method for diagnosis of respiratory tract infections, but has previously not allowed for simple screening of many respiratory viruses. Pools of monoclonal antibodies against various respiratory viruses are now available, and are potentially important tools for improvement of antigen detection in nasopharyngeal samples. OBJECTIVE: To evaluate the commercially available Chemicon immunofluorescence assay (IFA; respiratory viruses panel and identification kit), an indirect IFA containing a pool of monoclonal antibodies for screening for influenza A, B, respiratory syncytial virus (RSV), parainfluenza 1, 2, 3 and adenovirus, and the respective individual antibodies. STUDY DESIGN: Ninety-six frozen preparations from nasopharyngeal secretions or bronchoalveolar lavages were retrospectively examined with the assay, and the results compared with other IFAs for antigen detection and cell culture isolation obtained in the everyday routine. Nasopharyngeal preparations from 300 children with lower respiratory tract infections at Beijing Children's Hospital during the 1994-1995 winter season were also examined. RESULTS: The sensitivity of the Chemicon assay compared to the combined results of routine IFA and isolation was 89% and specificity 92%. If five identifications of RSV made with the Chemicon assay alone were regarded to be truly positive, the specificity was 100%. A viral etiology was identified in 105/280 (38%) evaluable samples drawn from the Chinese children (influenza A 20%, RSV 14%, adenovirus 3% and parainfluenza 1, 2 or 3, 7%). CONCLUSION: One problem with the Chemicon assay was that for around 4-13% of samples there was a non-specific staining in the screening assay, necessitating stainings for verification. Despite this, the assay is an excellent tool for identification of viral respiratory tract infections, giving an increased sensitivity compared to direct immunofluorescence assays.

Diagnostic markers and risk factors of cytomegalovirus infection and disease in renal allograft recipients.

In a prospective study, the incidences of CMV infection and disease were 56 and 23%, respectively, during the first 6 months following kidney transplantation. Viremia was found in all patients with CMV disease and arthralgia was present in 71% prior to the development of CMV disease. The positive predictive value for CMV disease reached up to 90% for viremia and arthralgia in combination. Viruria was poorly correlated to viremia and hence CMV disease. The majority of patients (93%) who developed CMV disease had a seropositive donor, and viremia was significantly more common in patients who received CMV-seropositive kidneys. CMV disease was more common in CMV-seronegative recipients than in seropositive recipients. The 1-year graft survival rate was 75% in the entire study group. In patients with CMV viremia and disease, the rates were 78 and 73%, respectively. Antiviral treatment was initiated within 3 weeks of viremia detection in the 6 patients with CMV disease who survived. We found that the combination of arthralgia and viremia was a useful predictor of CMV disease and that recipients of CMV-seropositive allografts were at a greater risk of developing CMV disease. To obtain an early diagnosis and commence an early treatment of CMV disease, patients prone to develop CMV disease should be identified and clinical examination and viremia surveillance should be performed regularly.

Diagnosis of cytomegalovirus in bronchoalveolar lavage by polymerase chain reaction, in comparison with virus isolation and detection of viral antigen.

Bronchoalveolar lavage (BAL) products from 52 immunocompromised patients with symptoms of pulmonary infection was examined for cytomegalovirus (CMV) by virus isolation, polymerase chain reaction (PCR) and detection of CMV antigen by immunofluorescence or immunoperoxidase staining after short-term incubation in tissue culture and directly in BAL cells. We found that PCR detected all cases positive by virus isolation (15/52 samples) and the result was obtained within 5 h. PCR detected more cases of CMV than did virus isolation (22/52 samples). Positive PCR and negative virus isolation were consistent with probable CMV infection in 3/7 patients when other clinical and laboratory parameters of CMV infection were considered. The negative predictive value of PCR was high; none of 30 patients negative by PCR developed CMV pneumonia within the subsequent 2 months. Detection of CMV antigen after short-term incubation was rapid enough to be used in clinical practice, specific (100%) and with a sensitivity of 60%. Demonstration of CMV antigen in alveolar cells was highly specific (100%) but had too low a sensitivity (26.7%) to be used as the only rapid method. Our conclusion is that a combination of PCR and detection of CMV antigen after short-term incubation and directly in alveolar cells is optimal for rapid identification of CMV.

Early detection of cytomegalovirus in cell culture by a new monoclonal antibody, CCH2.

A CMV monoclonal antibody, CCH2, produced in this laboratory was evaluated for rapid detection of CMV. Two staining procedures, immunofluorescence and an immunoenzymatic technique using biotin-streptavidin peroxidase, were compared. The CCH2 monoclonal antibody was used to demonstrate early CMV antigen in cell culture 24 h after inoculation of 598 urine samples from kidney transplanted patients by indirect immunofluorescence in comparison with virus isolation. One hundred and sixty of the specimens were stained additionally by an immunoenzymatic technique and the results were compared. CMV was isolated from 170 out of 598 specimens within 6 weeks. Early CMV antigen was demonstrated in 114 of these specimens by immunofluorescence giving a sensitivity of 67% and a specificity of 95%. In the comparison with the immunoenzymatic staining procedure the results for all three tests agreed for 81% (130/160) of the specimens. After resolving discordant results into true positives and true negatives, the sensitivity was 87, 85 and 70%, respectively for virus isolation, immunoenzymatic staining and immunofluorescence and the specificity 100, 96 and 99%. The CCH2 monoclonal antibody proved to be useful for rapid detection of CMV in urine specimens and using immunoenzymatic staining with biotin-streptavidin a sensitivity comparable to that of virus isolation was found.