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Only a few previous studies have focused on the use or bronchoalveolar lavage (BAL) in patients with community-acquired pneumonia (CAP). Our aim was to evaluate the diagnostic value of BAL in CAP in a routine clinical setting. 71 disease episodes were retrospectively analysed. The patients had undergone BAL for serious or slowly responding pneumonia. All procedures were performed during antimicrobial treatment of the patient. BAL fluid was cultivated for bacteria, fungi, and viruses. In 68 episodes, 1 or several specific polymerase chain reaction tests were performed. Only 1 (1.3%) quantitative bacterial culture was considered diagnostic for CAP, and indicated a change of antimicrobial treatment. The diagnostic yield increased to 9.8% when other methods were used. A respiratory virus was the only aetiology in 3 (6.0%) patients. In slowly responding pneumonia, also hospital-acquired pathogens and malignancies were identified, resulting in a total diagnostic yield of 20.0%. Thus, even when a large array of diagnostic assays was applied, the value of BAL in pretreated patients with CAP was very small, and its therapeutic implications minimal. In a subgroup of slowly responding pneumonia, the procedure was of some usefulness even after commencement of antimicrobial treatment.

To determine the efficiency of reverse-transcriptase polymerase chain reaction (RT-PCR) assays currently used in diagnosing enterovirus and rhinovirus infections, we compared results obtained with RT-PCR methods, which detect both enteroviruses and rhinoviruses simultaneously, with results obtained by conventional virus isolation. Both tests were performed on 591 specimens: 38 samples (6%) had positive results by both RT-PCR and isolation, 90 samples (15%) had positive results by RT-PCR only, and 7 samples (1%) had positive results only by virus isolation. In conclusion, RT-PCR was superior in rapidity and sensitivity to virus isolation and is recommended as the primary diagnostic tool for enterovirus and rhinovirus infections.

Objective: To determine the role of Pogosta virus as a triggering infection in non-specific arthritis., Methods: Serum samples of 142 patients with acute arthritis were screened for the evidence of Pogosta virus infection. Serological tests for Chlamydia trachomatis, salmonella, parvovirus B19, and Borrelia burgdorferi were also carried out. As verified later, 78 of the patients had rheumatoid arthritis and 63 seronegative poly- or oligoarthritis, while one had systemic lupus erythematosus., Results: In the early stage of the joint symptoms 4 patients with rheumatoid arthritis, 1 with seronegative polyarthritis and 1 with systemic lupus erythematosus had recent Pogosta virus infection. Four of them had probably had Pogosta disease at the time of the onset of arthritis. In 11 patients with a diagnosis of seronegative arthritis, serological evidence of preceding infection due to salmonella or Chlamydia trachomatis was found, strongly suggesting classical reactive arthritis in these cases., Conclusions: Our study suggests that also a Sindbis virus infection may be associated both to an acute joint inflammation as a part of Pogosta disease or chronic arthritis. At present, this possibility still needs further research.

To clarify when antibodies against cytomegalovirus (CMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV) develop among young children, 1206 serum samples collected prospectively from 199 children born in 1989 and 1990 were studied. The samples were drawn at the ages of 7 and 13 months, then yearly until the age of 5 y and then at 7 and 8 y. In each age group at least 106 samples were collected. Immunoglobulin G class antibodies to the 3 viruses were measured using an enzyme immunoassay. At the age of 7 months 27% of the children had CMV antibodies, whereas only 3% had antibodies against VZV and 2% against HSV. The prevalence of seropositivity for CMV increased slowly to 41% by the age of 8 y. Seroconversions to VZV antibody positivity occurred frequently after 2 y of age, so that by 8 y 83% of children had VZV antibodies. The proportion of children with HSV antibodies remained low throughout the study, as only 17% of children had HSV antibodies at the age of 8 y. The data show that HSV infection is becoming acquired later in life and the proportion of uninfected children is increasing. The proportion of CMV infections during the perinatal period and early infancy remains high, in one-third of the children, and most children also have VZV infection during the early years of life.

To provide insight into the appearance and longitudinal course of Chlamydia pneumoniae antibodies in childhood, C. pneumoniae immunoglobulin G (IgG), IgA and, in selected children, IgM antibodies were measured annually in 199 healthy children, followed prospectively from age 7 months to age 8 y (number of samples 1225) using a commercial enzyme immunoassay kit. IgG antibodies to C. pneumoniae were common throughout the follow-up, and the values declined rapidly after apparent infections during early childhood. Of the 128 identified seroconversions, 94 were probably primary infections and 34 reinfections. IgM antibodies were detected in 28% of the samples that showed a clear increase in IgG. IgA antibodies were scarce before 2 y of age, but their proportion then increased gradually. At the ages of 7 and 8 y, 10% of the children had clearly positive IgG and IgA antibody values. Increases in IgG were not associated with clinical respiratory symptoms. This study shows that C. pneumoniae infections probably occur commonly already at an early age, and that the infections are often asymptomatic. Consecutive high IgG and IgA antibody concentrations at the ages of 7 and 8 y indicate that persistent seropositivity for both antibodies may already develop in young children.

A randomized, double-blind, placebo-controlled, parallel-group trial performed at 5 residential units of the Finnish Defence Forces was conducted to assess the antiviral activity, efficacy and safety of inhaled zanamivir for the treatment of naturally acquired influenza. Conscripts were recruited within 2 d of onset of typical influenza symptoms and received inhaled zanamivir 10 mg via a Diskhaler twice daily for 5 d or matching placebo. Time to alleviation of clinically significant symptoms of influenza was the primary endpoint. Viral load measurements were made using quantitative real-time polymerase chain reaction assays. 435/588 patients (74%) had laboratory-confirmed influenza infection. The mean area under the curve for viral load during the first 48 h of treatment was 8.48 [95% confidence interval (95% CI) 2.85 to 14.11] log10 vRNA copies/ml x h lower in the zanamivir group compared with placebo (p = 0.003). Zanamivir reduced the time to alleviation of symptoms versus placebo in the influenza-positive group (medians 2.0 vs 2.33 d; 95% CI-0.17 to 1.0 d, p = 0.08). Zanamivir rapidly reduced viral load following the start of therapy compared with placebo and was well tolerated.

Background: Although known as common causes of upper respiratory infections, rhinoviruses, enteroviruses, and corona-viruses are poorly studied as inducers of wheezing in infants, and their possible role in the development of childhood asthma has not been investigated., Objective: The purposes of this study were to assess the occurrence of RV, enterovirus, and coronavirus infections in wheezing infants and to evaluate the association of these viral findings with early school-age asthma., Methods: In 1999, outcome in relation to asthma was studied in 82 of 100 initially recruited children who had been hospitalized for wheezing in infancy during the period 1992-1993. In 2000, etiologic viral studies regarding the index episode of wheezing were supplemented by rhinovirus, enterovirus, and coronavirus detection by RT-PCR from frozen nasopharyngeal aspirates in 81 of the children for whom adequate samples were available. Of these children, 66 had participated in the follow-up in 1999., Results: Rhinoviruses were identified in 27 (33%) of the 81 children, enteroviruses in 10 (12%), and coronaviruses in none. Rhinoviruses were present as single viral findings in 22 (81%) of the 27 rhinovirus-positive cases, and rhinovirus infections were associated with the presence of atopic dermatitis in infancy. Enteroviruses were commonly encountered in mixed infections and had no association with atopy. As single viral findings, rhinoviruses were associated with the development of asthma (P =.047; odds ratio, 4.14; 95% CI, 1.02-16.77 versus rhinovirus-negative cases [by logistic regression adjusted for age, sex, and atopic dermatitis on entry)]., Conclusion: Our results present rhinoviruses as important inducers of wheezing even in infancy. The association with atopy and subsequent asthma calls for reevaluation of the role of rhinoviruses in the development of asthma.

Background: Chlamydia pneumoniae infection has been associated with atherosclerosis by sero-epidemiological, histopathological and interventional studies, and animal experiments. We hypothesized that if chlamydial infection is causative of atherosclerosis, the occurrence of antibodies against C. pneumoniae should be associated with coronary vasomotor dysfunction - an early sign of atherosclerosis., Aim: To study the association between C. pneumoniae infection and coronary vasomotor function in young men without signs of ischemic heart disease., Methods: Serum IgG and IgA antibody concentrations against C. pneumoniae were determined in 125 clinically healthy subjects undergoing positron emission tomography (PET) studies. Myocardial blood flow was measured at rest and during pharmacologically induced hyperemia using [15O]H2O Coronary flow reserve was calculated as the ratio of hyperemic blood flow to resting blood flow., Results: No association was found between serum C. pneumoniae antibody concentrations and myocardial blood flow parameters. In contrast, more conventional risk factors for coronary artery disease, such as total cholesterol and apolipoprotein B, were inversely associated with hyperemic flow and flow reserve., Conclusions: We found no association between C. pneumoniae antibodies and coronary vasomotor function in subjects without ischemic heart disease. Thus, these results do not support the role of C. pneumoniae infection as an early phase risk factor for coronary artery disease.

Epithelial cells of the respiratory tract are the primary targets of measles virus (MV) infection. In this work we have studied the effect of MV infection on the activation of transcription factors nuclear factor (NF)-kappa B and signal transducer and activator of transcription (STAT) and the production of cytokines in the lung epithelial A549 cell line. NF-kappa B and STAT activation were induced by MV in A549 cells as analyzed by electrophoretic mobility shift assay. NF-kappa B activation was rapid and it was not inhibited by the protein synthesis inhibitor cycloheximide, suggesting that MV directly activates NF-kappa B. In contrast, Stat1, Stat3, and interferon-stimulated gene factor 3 (ISGF3) DNA binding was induced by MV infection with delayed kinetics compared to NF-kappa B activation. MV infection also resulted in an efficient interferon (IFN)-alpha/beta and interleukin-6 production. Cycloheximide and neutralizing anti-IFN-alpha/beta antibodies inhibited MV-induced activation of Stat1, Stat3, and ISGF3 DNA binding in A549 cells. In conclusion, the results suggest that MV infection activates transcription factors involved in the initiation of innate immune responses in epithelial cells by two different mechanisms: directly by leading to NF-kappa B activation and indirectly via IFN-alpha/beta leading to STAT activation.

The aim of the study was to evaluate new Mycoplasma pneumoniae IgG, IgA and IgM EIA methods based on the enrichment of P1-protein (ThermoLabsystems, Helsinki, Finland) (L) for the detection of acute infection. This evaluation was performed in two independent routine clinical microbiology laboratories. The first laboratory used samples preselected by IgG and IgM Platelia enzyme immunoassay (P) and the second used samples preseleced by Serion ELISA Classic M. pneumoniae IgG, IgM (V). The L method was also compared to the FDA approved method of ImmunoWell M. pneumoniae IgG and IgM (G). When the agreement of two methods was applied as a serologic criteria for an acute infection, the following ratios of acute to nonacute infection were calculated 32/86 (totally 118) in the first and 20/72 (totally 92) in the second laboratory. In the first laboratory, the corresponding ratios by methods were 35/83 (sensitivity 100%, specificity 96.5%), 31/87 (sensitivity 97%, specificity 100%), and 55/63 (sensitivity 100%, specificity 79%) for the L, P and G methods, respectively. In the second laboratory, the ratios were 21/71 (sensitivity 100%, specificity 99%), 16/76 (sensitivity 83%, specificity 100%), and 53/39 (sensitivity 100, specificity 69%) for the L, V and G methods, respectively. Taking into account that the tested sample material was preselected by the P and V methods, which may have introduced some bias in their favor, the newly developed L method utilizing P1-enriched protein was found reliable for serodiagnosis of acute M. pneumoniae infection. The method G was the least specific in detection of acute infection.

Measles virus (MV)-induced immune suppression is an important reason for MV-associated mortality and morbidity. Despite numerous studies, the mechanisms of immune suppression still remain poorly defined. In the present study we analyzed the effect of MV components on the T-cell recognition of specific non-MV antigens. We demonstrated that even inactivated MV could inhibit the presentation of unprocessed protein antigen to specific T cells, whereas MV did not affect the responses of specific T cells to representative synthetic peptide epitopes derived from complex antigens. The inhibition was induced by MV-infected cell membranes. The kinetics of the MV-dependent inhibition suggested an impaired antigen processing in mononuclear cells as addition of MV-infected cell debris 4 h after the beginning of cell cultures no longer inhibited T-cell responsiveness.

Picornaviruses include several important clinical pathogens which cause diseases varying from common cold to poliomyelitis and hepatitis. Introduction of RT-PCR methods for the detection of these viruses has significantly facilitated the diagnosis of picornavirus infections and elucidated their etiological role in clinical illnesses. Partial sequence analysis of the genomes has been used for typing of the viruses and in studies of molecular epidemiology of picornaviruses. These molecular approaches are likely to become the most predominant techniques for the diagnosis and epidemiological analysis, particularly in the enterovirus infections.

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of Mycoplasma pneumoniae infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for M. pneumoniae-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of M. pneumoniae DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of M. pneumoniae pneumonia in children of any age.

Objective: To determine, by studying patients with early rheumatoid arthritis (RA), whether rubella virus (rubella), mumps virus (mumps), or measles virus (measles) plays a role in the pathogenesis of RA., Methods: Polymerase chain reaction combined with reverse transcription was used to detect viral RNA in peripheral blood mononuclear cells (PBMC) or synovial fluid (SF) cells. The patients with RA had newly diagnosed disease (duration < or = 1 year). The controls were patients with other arthropathies., Results: Rubella genomic RNA was not detected in SF cells from patients with early RA or from controls, or in PBMC from patients with RA. It was found in PBMC of one of 46 patients with other arthropathies. Mumps or measles genomic RNA was detected in PBMC samples from 1.8% (1/54) and 9.3% (5/54) of RA, respectively, and from 4.3% (2/46) and 6.5% (3/46) of control patients. The SF cell samples harbored mumps or measles RNA in 4.8% (2/42) and 7.1% (3/42) of patients with RA, respectively; the corresponding value was 6.5% (2/31) for control patients, for both mumps and measles., Conclusion: Our findings suggest rubella, mumps, or measles do not play a role in the etiopathogenesis of RA.

Although coxsackie B viruses (CBVs) are known to cause viremia during acute infection, the role of the blood cells as a target for virus replication is poorly understood. We have analyzed the susceptibility of human peripheral blood mononuclear cells (PBMCs), granulocytes, bone marrow (BM) cells, and lymphoid cell lines to coxsackievirus B3 infection. Lymphoid cell lines with B- and T-cell characteristics (Raji and Molt-4, respectively) supported virus replication to high titers and virus protein synthesis was detected by metabolic labeling and immunoprecipitation. CBV3 synthesis in the U937 cell line with mononuclear phagocytic characteristics was very limited. The virus was able to infect a small proportion of leukocytes and BM cells, and intracellular virus antigens were detected by immunofluorescent staining. However, only a diminutive amount of infectious virus was produced in isolated PBMCs and granulocytes, and no virus protein synthesis was detected by metabolic labeling and immunoprecipitation in these cells.

Peptides presenting predicted antigenic sites of CBV3 capsid proteins and peptide sequences from conserved regions of the nonstructural proteins were synthesized, and rabbit antipeptide sera were tested for their immunoreactivity. Peptides derived from different capsid regions were able to induce production of neutralizing antibodies in rabbits. As measured by EIA, all peptides representing four different proposed antigenic sites were immunogenic, inducing an antibody response against the homologous peptide and purified CBV3 as measured by EIA. Immunization with inactivated CBV3 induced a secondary response especially in rabbits primed with peptides representing polypeptide VP2. Antisera against the nonstructural protein sequences were highly cross-reactive with other enteroviruses, while the capsid peptide antisera were mainly type-specific when tested by immunoblotting against a panel of enteroviruses. Four of the capsid region peptides also exhibited distinct T-cell reactivity in a mouse T-cell proliferation assay.

We analyzed the effect of exogenous human leukocyte interferon (IFN)-alpha on measles virus (MV) replication in human peripheral blood mononuclear cells (PBMC). The release of infectious virus was progressively reduced by increasing concentrations of IFN-alpha, and blocked with an IFN-alpha concentration of 1000 U/ml. In order to detect a possible target of this inhibitory effect, viral transcription and translation events were analyzed. The synthesis of MV mRNAs was reduced, but not blocked, in the presence of IFN-alpha. However, this effect was not specific on the viral RNAs, but due to a general inhibition of RNA synthesis in IFN-treated PBMC. The expression of viral polypeptides was also inhibited in a dose-dependent manner by exogenous IFN-alpha, but a low level of protein synthesis was detected by both Western blotting and immunofluorescence techniques, even with the maximum amount of IFN-alpha used (1000 U/ml). These findings account for a partial maintenance of the viral replicative cycle, even when the production of infectious virus is blocked. Moreover, the effect of IFN-alpha is not specifically targeted on the virus macromolecular synthesis.

Islet-like cell clusters (ICCs) prepared from human fetal pancreases were infected with mumps or coxsackie B3 virus. Double-labeled antibody technique showed that the viruses infected both insulin-secreting and other pancreatic cells and that secretion of immunoreactive insulin into the culture medium of the mumps virus-infected cells had already ceased on day 7. The mumps virus-infected ICC clusters produced virus for 14 days, and the mumps virus antigen was detected in the ICCs through the whole 22-day observation period. The coxsackie B3 virus-infected ICCs contained cells highly positive for viral antigen during the first 2 days after infection, and the infectious virus was detected in the culture medium for 22 days. This in vitro model indicates that mumps and coxsackie B3 viruses infect human fetal pancreatic endocrine cells and are able to alter beta-cell function. Coxsackie B3 virus infection in ICCs is lytical and seems to lead to rapid cell destruction, but long-lasting, restricted mumps virus infection in human fetal pancreatic ICCs offers an interesting model to study the effects of viral infection in the endocrine pancreas and beta cell.

Viral etiology of clinically typical parotitis was studied by virus isolation and serology in 100 consecutive patients presented at the Department of Infectious Diseases, University of Turku, during a period of over 2 years. Mumps etiology was confirmed in 95 cases and in one case a probable parainfluenza type 3 infection was diagnosed by IgM serology. In the remaining 4 patients no etiological evidence of any of the viruses studied was obtained. When different methods to diagnose an acute mumps infection were compared, the most sensitive was the assay for specific IgM antibodies.

The virus-specific protein synthesis of the hamster brain cells persistently infected with a wild-type measles virus (M-MB/MVB) was investigated in vivo and in vitro and compared to the protein synthesis of the Vero-cells lytically infected with the wild-type measles virus and the SSPE-measles virus strain LEC. The virus-specific protein synthesis in the M-HB/MVB-cells in vivo was weak. Only two virus-specific polypeptides, corresponding to polypeptide G and nucleocapsid polypeptide NP, were detected. On the other hand, the in vitro translation products, coded by the RNA extracted from the actinomycin D-treated M-HB/MVB-cells, were similar compared to those products coded by the RNAs from the lytically infected Vero-cells. The polypeptides with MWs of 75 000, 61 000, 40 000, 37 000 and 18 000 were coded with all three RNA-extracts. So the similar mRNAs were induced in the actinomycin D-treated M-HB/MVB-cells, but their translation in vivo was limited. In addition to those polypeptides mentioned above the polypeptide with a MW of 42 000 was detected among the in vitro translation products of the M-HB/MVB-cells.

A measles virus (MV) Lec strain conditional-lethal (temperature-sensitive) mutant, designated MV ts38, has been isolated from 5-fluorouracil-mutagenized stock. The mutant has been characterized with regard to growth characteristics at 32 degrees C (permissive temperature) and 39 degrees C (non-permissive temperature). Virus-specific RNA transcription and/or translation appeared to be blocked at the non-permissive temperature as no virus-specific products could be detected by biochemical or immunological procedures. Following initiation of viral replication at 32 degrees C, with subsequent shift-up to 39 degrees C, presynthesized nucleoprotein (NP) was transported to, and accumulated in the cell nucleus whereas other viral proteins could not be detected there. A corresponding accumulation of NP in the nucleus is most often seen in association with MV neurotropic subacute sclerosing panencephalitis isolates in vivo and in vitro.

Coxsackievirus B3 infection in mice was studied histopathologically, by virus isolation and by nucleic acid hybridization after intraperitoneal inoculation of the virus. Extensive viraemia was detected for 1-3 days post-infection. All mice developed necrotizing acute pancreatitis and focal myocarditis. Pancreatitis eventually lead to complete atrophy of the exocrine pancreas. However, the islets of Langerhans and pancreatic ducts remained morphologically intact. Virus could be demonstrated in pancreatic tissue for 1-5 days post-infection by in-situ and spot hybridization as well as by virus isolation. Virus was not detectable on days 7-22 post-infection suggesting an autodigestive aetiology in further destruction of the exocrine pancreas. The mouse model described here permits detailed analysis of viral and host factors in the pathogenesis of enterovirus infections. Since coxsackie B viruses have been proposed to be aetiological agents in human acute pancreatitis, the application of in-situ hybridization allows analysis of enteroviruses directly from pancreatic tissue of clinical routine specimens.

Measles virus persistence in a hamster brain cell line was examined for the appearance of viral antigens and for the synthesis of infectious virus, viral RNA, and virus-specific proteins during 19 serial passages. Cyclical fluctuation was detected at all levels of measles virus replication. After reaching the maximal activity at the passages 9 to 11 viral synthesis diminished rapidly, being lowest after 2 to 3 subsequent passages. No release of infectious virus was detected at the passages 14 and 15 and only 20% of the cells contained measles virus antigens when tested by immunofluorescence. After the nonproductive phase, the cells released virus again and the number of antigen-positive cells increased. The alternation in the amount of the measles virus-specific proteins and viral RNA correlated directly with the parameters mentioned above. No apparent defects in the synthesis of individual viral proteins were observed.