Urethritis-associated Pathogens in Urine from Men with Non-gonococcal Urethritis: A Case-control Study.

The aetiology of non-gonococcal urethritis (NGU) remains unexplained in 30-40% of patients. Urine samples from men attending Swedish sexually transmitted disease clinics were examined by species-specific quantitative PCRs for Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, Ureaplasma urealyticum, U. parvum, adenovirus, herpes simplex virus, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. A total of 187 men with acute NGU (symptoms ≤ 30 days) and 24 with chronic NGU (symptoms < 30 days) were cases, and 73 men without NGU were controls. Number of lifetime sexual partners was negatively associated with U. urealyticum bacterial load. C. trachomatis and M. genitalium were associated with NGU, as was U. urealyticum, with bacterial loads ≥ 1.3 × 103 genome equivalents/ml urine. Virus and H. influenzae might explain a few NGU cases, but the aetiology in at least 24% of patients with acute NGU was unexplained. In multivariate analysis, detection of U. urealyticum was significantly more common in acute NGU (20%) compared with controls (11%).

Bacterial vaginosis diagnosed by analysis of first-void-urine specimens.

Bacterial vaginosis (BV) is traditionally diagnosed using vaginal samples. The aim of this study was to investigate whether BV can be diagnosed from first-void urine (FVU). Self-collected vaginal smears, vaginal swabs, and FVU were obtained from 176 women. BV was diagnosed by Nugent's criteria. The FVU and vaginal swabs were analyzed by quantitative PCRs (qPCRs) for selected vaginal bacteria (Atopobium vaginae, Prevotella spp., Gardnerella vaginalis, bacterial vaginosis-associated bacterium 2, Eggerthella-like bacterium, "Leptotrichia amnionii," Megasphaera type 1), and all had an area under the receiver operating characteristic (ROC) curve of >85%, suggesting good prediction of BV according to the Nugent score. All seven bacteria in FVU were significantly associated with BV in univariate analysis. An accurate diagnosis of BV from urine was obtained in this population by a combination of qPCRs for Megasphaera type 1 and Prevotella spp. The same two bacteria remained significantly associated with BV in a multivariate model after adjusting for the other five species. There was no statistically significant difference between the sensitivities and specificities of BV diagnosis by molecular methods performed on swabs and FVU samples. A linear regression analysis showed good agreement between bacterial loads from swabs and FVU, but Prevotella spp. could be detected in high numbers in a few FVU samples without being present in swabs. This method will allow diagnosis of BV in studies where only urine has been collected and where detection of BV is considered relevant.

Vaginal microbiome in women from Greenland assessed by microscopy and quantitative PCR.

BACKGROUND: Bacterial vaginosis (BV) is a common condition, although its aetiology remains unexplained. The aim of this study was to analyse the composition of vaginal microbiota in women from Greenland to provide a quantitative description and improve the understanding of BV. METHODS: Self-collected vaginal smears and swabs were obtained from 177 women. The vaginal smears were graded for BV according to Nugent's criteria. The vaginal swab samples were analysed by 19 quantitative PCRs (qPCRs) for selected vaginal bacteria and by PCR for four sexually transmitted infections (STIs). RESULTS: STIs were common: Mycoplasma genitalium 12%, Chlamydia trachomatis 7%, Neisseria gonorrhoeae 1%, and Trichomonas vaginalis 0.5%. BV was found in 45% of women, but was not associated with individual STIs. Seven of the 19 vaginal bacteria (Atopobium vaginae, Prevotella spp., Gardnerella vaginalis, BVAB2, Eggerthella-like bacterium, Leptotrichia amnionii, and Megasphaera type 1) had areas under the receiver operating characteristic (ROC) curve > 85%, suggesting they are good predictors of BV according to Nugent. Prevotella spp. had the highest odds ratio for BV (OR 437; 95% CI 82-2779) in univariate analysis considering only specimens with a bacterial load above the threshold determined by ROC curve analysis as positive, as well as the highest adjusted odds ratio in multivariate logistic regression analysis (OR 4.4; 95% CI 1.4-13.5). BV could be subdivided into clusters dominated by a single or a few species together. CONCLUSIONS: BV by Nugent score was highly prevalent. Two of seven key species (Prevotella spp. and A. vaginae) remained significantly associated with BV in a multivariate model after adjusting for other bacterial species. G. vaginalis and Prevotella spp. defined the majority of BV clusters.

Changes in the vaginal microbiota following antibiotic treatment for Mycoplasma genitalium, Chlamydia trachomatis and bacterial vaginosis.

The human vagina harbor a rich microbiota. The optimal state is dominated by lactobacilli that help to maintain health and prevent various diseases. However, the microbiota may rapidly change to a polymicrobial state that has been linked to a number of diseases. In the present study, the temporal changes of the vaginal microbiota in patients treated for sexually transmitted diseases or bacterial vaginosis (BV) and in untreated controls were studied for 26 days. The patients included 52 women treated with azithromycin, tetracyclines or moxifloxacin for present or suspected infection with Chlamydia trachomatis or Mycoplasma genitalium. Women with concurrent BV were also treated with metronidazole. The controls were 10 healthy women of matching age. The microbiota was analyzed by 16S rRNA gene deep sequencing, specific qPCRs and microscopy. There was generally good correlation between Nugent score and community state type (CST) and qPCR confirmed the sequencing results. By sequencing, more than 600 different taxa were found, but only 33 constituted more than 1 ‰ of the sequences. In both patients and controls the microbiota could be divided into three different community state types, CST-I, CST-III and CST-IV. Without metronidazole, the microbiota remained relatively stable regarding CST although changes were seen during menstrual periods. Administration of metronidazole changed the microbiota from CST-IV to CST-III in approximately 50% of the treated patients. In contrast, the CST was generally unaffected by azithromycin or tetracyclines. In 30% of the BV patients, Gardnerella vaginalis was not eradicated by metronidazole. The majority of women colonized with Ureaplasma parvum remained positive after azithromycin while U. urealyticum was eradicated.

Season of Birth Impacts the Neonatal Nasopharyngeal Microbiota.

Objective: Pathogenic airway bacteria colonizing the neonatal airway increase the risk of childhood asthma, but little is known about the determinants of the establishment and dynamics of the airway microbiota in early life. We studied associations between perinatal risk factors and bacterial richness of the commensal milieu in the neonatal respiratory tract. Methods: Three hundred and twenty-eight children from the Copenhagen Prospective Studies on Asthma in the Childhood2000 (COPSAC2000) at-risk birth cohort were included in this study. The bacterial richness in each of the nasopharynxes of the 1-month old, asymptomatic neonates was analyzed by use of a culture-independent technique (T-RFLP). Information on perinatal risk factors included predisposition to asthma, allergy and eczema; social status of family; maternal exposures during pregnancy; mode of delivery; and postnatal exposures. The risk factor analysis was done by conventional statistics and partial least square discriminant analysis (PLSDA). Results: The nasopharyngeal bacterial community at 1-month displayed an average of 35 (IQR: 14-55, range 1-161) phylogenetically different bacteria groups. Season of birth was associated with nasopharyngeal bacterial richness at 1-month of age with a higher bacterial richness (p = 0.003) and more abundant specific bacterial profiles representing Gram-negative alpha-proteobacteria and Gram-positive Bacilli in the nasopharynx of summer-born children. Conclusion: Early postnatal bacterial colonization of the upper airways is significantly affected by birth season, emphasizing a future focus on the seasonality aspect in modelling the impact of early dynamic changes in airway bacterial communities in relation to later disease development.

Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates.

Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR-32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate.

Detection of ureaplasmas and bacterial vaginosis associated bacteria and their association with non-gonococcal urethritis in men.

No aetiology is found in up to 40% of men with symptomatic urethritis. Male partners of women with bacterial vaginosis (BV) may be at higher risk of non-gonococcal urethritis (NGU). The aim of this study was to examine the role of BV associated bacteria in first-void urine (FVU) in 97 asymptomatic men without urethritis (controls) and 44 men (cases) with NGU including 20 men with idiopathic urethritis (IU) attending a Swedish STD-clinic between January and October 2010. BV-associated bacteria and ureaplasmas were detected by quantitative PCR assays. All BV associated bacteria, except Megasphaera-like type 1, were strongly positively correlated with U. urealyticum p<0.005 and even stronger with the combined U. urealyticum and U. parvum load (p<0.0005) suggesting that ureaplasma induced elevated pH may stimulate the growth of BV associated bacteria. No statistically significant differences were found between IU cases and controls in the prevalence or load of BV associated bacteria or ureaplasmas. In multiple logistic regression, Megasphaera-like type 1 was associated with IU (p = 0.03), but most positive FVU samples contained very few bacteria and the finding may not be clinically relevant.

Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a recessive genetic disorder that is characterized by sinopulmonary disease and reflects abnormal ciliary structure and function. Situs inversus totalis occurs in approximately 50% of PCD patients (Kartagener's syndrome in PCD), and there are a few reports of PCD with heterotaxy (situs ambiguus), such as cardiovascular anomalies. Advances in diagnosis of PCD, such as genetic testing, allow the systematic investigation of this association. METHODS AND RESULTS: The prevalence of heterotaxic defects was determined in 337 PCD patients by retrospective review of radiographic and ultrasound data. Situs solitus (normal situs) and situs inversus totalis were identified in 46.0% and 47.7% of patients, respectively, and 6.3% (21 patients) had heterotaxy. As compared with patients with situs solitus, those with situs abnormalities had more ciliary outer dynein arm defects, fewer inner dynein arm and central apparatus defects (P<0.001), and more mutations in ciliary outer dynein arm genes (DNAI1 and DNAH5; P=0.022). Seven of 12 patients with heterotaxy who were genotyped had mutations in DNAI1 or DNAH5. Twelve patients with heterotaxy had cardiac and/or vascular abnormalities, and most (8 of 12 patients) had complex congenital heart disease. CONCLUSIONS: At least 6.3% of patients with PCD have heterotaxy, and most of those have cardiovascular abnormalities. The prevalence of congenital heart disease with heterotaxy is 200-fold higher in PCD than in the general population (1:50 versus 1:10 000); thus, patients with PCD should have cardiac evaluation. Conversely, mutations in genes that adversely affect both respiratory and embryological nodal cilia are a significant cause of heterotaxy and congenital heart disease, and screening for PCD is indicated in those patients.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12.

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides (cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.

The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12.

The aim of the present study was to investigate the organisation of the genes (cps) involved in biosynthesis the capsular polysaccharide (CPS) of Actinobacillus pleuropneumoniae serotypes 6, 7, and 12 and to compare these to the corresponding genes previously described in other A. pleuropneumoniae serotypes. In serotypes 6 and 7 the sequenced DNA regions comprised five and four open reading frames, respectively, designated cps6ABCDE and cps7ABCD, whereas the sequenced DNA region in serotype 12 comprised only two open reading frames designated cps12AB. At the amino acid level, CpsA, CpsB, and CpsC of A. pleuropneumoniae serotypes 2, 6, 7, and 8 contained a high degree of homology. At the amino acid level Cps6D revealed a high degree of homology to Cps8D, whereas Cps7D contained a high degree of homology to the Cps2D. The deduced gene product of the partially sequenced cps6E gene showed no homology to any deduced gene products of any cps genes of A. pleuropneumoniae investigated so far. None of the deduced gene products of the cps genes involved in encapsulation of A. pleuropneumoniae serotypes 2, 6, 7, and 8 revealed homology to the deduced gene products of the cps genes of serotypes 1, 5A, and 12. For some genes, a local homology was found to genes probably involved in teichoic acid synthesis in other bacteria. The results obtained revealed a high degree of homology among the genes involved in CPS biosynthesis for serotypes 2, 6, 7, and 8 and a different group of homologous cps genes for serotypes 1 and 12. In some serotype 7 strains, including the serotype 7 reference strain, WF83, the cps genes were not located adjacent to the genes responsible for CPS export (cpx), probably due to genetic rearrangements.

The bacterial microbiota in first-void urine from men with and without idiopathic urethritis.

BACKGROUND: Non-gonococcal urethritis (NGU) is a common syndrome in men. NGU may have several causes, but many cases are caused by sexually transmitted infections that may also cause complications in their female partners. Chlamydia trachomatis and Mycoplasma genitalium are the most common causes of NGU, but in up to 35% of the cases, none of the known viral or bacterial causes are found. Traditionally, pathogens have been detected using various culture techniques that may not identify all species present in the urethra. To address this, we used culture-independent methods for analysis of the male urethral microbiota. METHODS: This case-control study analysed first void urine samples, collected at STD clinics in Stockholm, Sweden from men with idiopathic urethritis (IU), i.e. negative for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis, adenovirus, and herpes simplex virus type 1 and -2 together with samples from men without urethritis. Forty-six controls and 39 idiopathic urethritis patients were analysed. RESULTS: The microbiota was highly diverse: None of the 302 operational taxonomic units (OTUs) found in negative controls and IU patients were found in all of the samples or even in all of the samples in one group. More than 50% of the OTUs were only found in one or two of the total of 85 samples. Still the most dominant 1/6 of the genera constituted 79% of the sequences. Hierarchical clustering in a heatmap showed no specific clustering of patients or controls. A number of IU patient samples were dominated by a single genus previously related to urethritis (Gardnerella, Haemophilus, Ureaplasma). CONCLUSION: The male urethra contain a very diverse composition of bacteria, even in healthy controls. NGU may be caused by a number of different bacteria but more studies including a higher number of samples are needed for elucidation of the role of each species.

Development of an improved species specific PCR test for detection of Haemophilus parasuis.

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.

Favourable effect of TNF-alpha inhibitor (infliximab) on Blau syndrome in monozygotic twins with a de novo CARD15 mutation.

Blau syndrome is a hereditary granulomatous disease caused by mutations in the CARD15 gene that is diagnosed in children of young age with exanthema/erythema, arthritis/periarthritis and/or uveitis. We report two cases of Blau syndrome in Danish Caucasian monozygotic male twins, exhibiting a heterozygous de novo R334W mutation in codon 334 of CARD15. The patients were initially diagnosed as having sarcoidosis. In both twins, symptoms (exanthema, arthritis/periarthritis) started at 1 year of age, and were followed by uveitis at 7-10 years of age. There was no involvement of the lungs or other organs. An initial course of standard antituberculous treatment had no effect on the symptoms. Hydroxychloroquine and cyclosporine A were also ineffective, and the latter caused impaired renal function. Partial symptomatic relief was obtained with prednisolone and increased benefit was observed in combination with methotrexate. Subsequent introduction of the TNF-alpha inhibitor eternacept did not discernibly benefit the clinical condition, but was associated with recurrent infections. In contrast, a trial of infliximab therapy demonstrated clinical efficacy and eliminated all symptoms, restoring a high quality of life. At follow up at 20 years of age (after 2-5 years of infliximab treatment) the twins had an almost normal physical appearance and a normal psychomotoric development, indicating a favourable short-term prognosis of the disease. Blau syndrome has pathologic, clinical and therapeutic features in common with sarcoidosis, but rarely involves the lungs or other parenchymatous organs. In children, discrimination between early onset sarcoidosis and Blau syndrome should include a CARD15 mutation analysis.

Polymorphisms of haemostasis genes as risk factors for preterm delivery.

Clinical trials evaluating the potential benefit of anticoagulant treatment in pregnant women with inherited thrombophilia are based on the observation that a genetic predisposition to thrombosis is associated with frequent abortions and preterm birth. It was the aim of our study to delineate the impact of genetic polymorphisms with prothrombotic and antithrombotic effects on the occurrence of preterm birth in a large cohort of very-low-birth-weight (VLBW)-infants and their mothers. We examined the factor V Leiden and the prothrombin G20210A mutation, the factor VII 121del/ins and the factor XIII Val34Leu polymorphism in preterm very-low-birth-weight (VLBW, n=593) and term-born-infants (n=278) and their mothers (n=785). The primary outcome was preterm vs.term birth. From all polymorphisms tested, the maternal factor VII-121del/ins polymorphism (26.2 vs. 17.6 %; p=0.009) and the infant's factor VII-121del/ins polymorphism (29.0 vs. 20.0 %; p=0.009) were more frequent in singleton VLBW and their mothers compared to term infants and their mothers. Furthermore, the frequency of the factor XIII-Val34Leu polymorphism was significantly lower in singleton VLBW than in term infant controls (5.1 vs. 9.6%, p=0.025). In a multivariate regression analysis, previous preterm delivery (OR=3.8, 95% CI: 1.7-8.4), the maternal carrier status of the factor-VII-121del/ins polymorphism (OR=1.7, 95% CI: 1.12-2.5, p=0.007) and the lower frequency of infant's factor-XIII-Val34Leu polymorphism (OR=0.53; 95% CI: 0.29-0.96; p=0.038) were found to be independently associated with preterm delivery. InVLBW mothers with pathological CTG as cause of preterm delivery, the frequency of factor V Leiden mutation was significantly increased compared to VLBW mothers without pathological CTG (14.1 vs. 6.1%, p=0.01). The investigated haemostasis gene polymorphisms have a much lower impact on subsequent preterm delivery than known risk factors such as previous preterm birth. The reported association of the factor-VII-121del/ins polymorphism on preterm delivery and its clinical relevance needs to be further elucidated.

Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes.

Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S. chromogenes producing exfoliative toxin type B, ExhB, was identified by the use of a multiplex PCR specific for the exfoliative toxins from S. hyicus. The exfoliative toxin from S. chromogenes reacted in immunoblot analysis with polyclonal and monoclonal antibodies specific to ExhB from S. hyicus and had an apparent molecular weight of 30 kDa. Sequencing the gene encoding the exfoliative toxin from S. chromogenes revealed that the molecular weight of the toxin with the signal peptide and the mature toxin was 30,553 and 26,694 Da, respectively. Comparison of the exhB genes from S. chromogenes strain VA654 and S. hyicus strain 1289D-88 showed differences in seven base pairs of the DNA sequences and in two amino acid residues in the deduced amino acid sequences. Pigs were experimentally inoculated with S. chromogenes strain VA654. By clinical observations and histopathological evaluation of the skin alterations, all pigs revealed development of generalized exudative epidermitis. No toxin producing S. hyicus was isolated from the pigs and all ExhB-positive bacterial isolates were identified as S. chromogenes. This confirmed that the disease-causing agent was the inoculated S. chromogenes strain VA654. The results of this study show that S. chromogenes may cause exudative epidermitis in pigs.

Parallel phylogenetic analyses using the N, G or Nv gene from a fixed group of VHSV isolates reveal the same overall genetic typing.

Different genetic regions representing the viral phospho- (P), nucleocapsid- (N) or glyco-protein (G) gene have been used for phylogenetic studies of viral haemorrhagic septicaemia virus (VHSV). Since these analyses were performed on different virus isolates using various genomic regions, it has been difficult to evaluate how the choice of target region affects the output of the analyses. To address this, we sequenced and performed parallel phylogenetic analysis of an N gene fragment, the entire Nv (non-structural protein) and G genes, and 4 different fragments of the G gene from a fixed virus panel. The overall genotyping of the selected isolates was identical for the 7 target regions, but separation of Genotype I sub-lineages was best when the analysis was performed on the full length G gene (1524 nucleotides, nt). Good resolution was furthermore obtained using smaller sequencing windows represented by a G gene fragment (nt 360 to 720) or the Nv gene (366 nt), although these regions had different characteristics with respect to resolution of Genotype I sublineages and resolution within Sub-lineage Ia. Phylogenetic analysis based on the deduced amino acid sequences was also performed. The phylogenetic relationship between the nucleotide and amino acid sequences of the isolates corresponded best in the case of the N gene/protein. For the 6 other genomic regions, genetically distant isolates occasionally grouped together when compared at protein levels. No clear relationship between the G gene genotyping and serotyping with neutralising (G protein specific) antibodies was observed, stressing that epidemiological analysis based on phenotypic characteristics such as serotype could be misleading.

Improved asthma control in patients with severe, persistent allergic asthma after 12 months of nightly temperature-controlled laminar airflow: an observational study with retrospective comparisons.

INTRODUCTION: Continuous or episodic allergen exposure is a major risk factor of frequent symptoms and exacerbations for patients with allergic asthma. It has been shown that temperature-controlled laminar airflow (TLA) significantly reduced allergen exposure and airway inflammation and improved quality of life of patients with poorly controlled allergic asthma. OBJECTIVE: The objective was to evaluate the effects of nighttime TLA when used during real-life conditions for 12 consecutive months in addition to the patients' regular medication. METHODS: This multicenter, pre- and postretrospective observational study included patients with inadequately controlled moderate-to-severe allergic asthma who received add-on treatment with TLA for 12 consecutive months. Data on medication use, asthma control, asthma symptoms, lung function, use of hospital resources, and exacerbations were collected after 4 and 12 months and compared with corresponding data collected retrospectively from medical records during the year prior to inclusion in the study. RESULTS: Data from 30 patients (mean age 28; range 8-70) completing 4 months and 27 patients completing 12 months of TLA use are presented. The mean number of exacerbations was reduced from 3.6 to 1.3 (p<0.0001), and the ratio of asthma-related emergency room visits or hospitalizations diminished from 72.4 to 23.3% (p=0.001) or from 44.8 to 20.0% (p<0.05), respectively, after 12 months of TLA use. The Asthma Control Test index increased from 14.1 to 18.5 (p<0.0001). After 4 months of TLA use, clear improvements can be shown for most variables in line with the data collected after 12 months. CONCLUSIONS: The addition of TLA to the patients' regular medication significantly reduced exacerbations, asthma symptoms, and the utilization of hospital resources. The data support that TLA may be an important new non-pharmacological approach in the management of poorly controlled allergic asthma.

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis.

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.

Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers.

The pathogenesis of campylobacter infection in man is largely unknown, although cytolethal distending toxin (CDT) has been incriminated as a virulence factor. However, little is known about the cdt genes in Campylobacter spp. isolated from broiler chickens. A total of 350 cloacal swabs was collected and tested by conventional culture and PCR. Of the 114 Campylobacter isolates obtained, 101 (88.6%) were identified as C. jejuni and 13 (11.4%) as C. coli by conventional methods. cdt genes were detected by PCR in all the isolates except one C. jejuni isolate. Cytotoxic effects were produced in a Vero cell line, by 100 of the C. jejuni isolates. In contrast, 10 C. coli isolates produced much lower levels of toxin and 3 produced no detectable toxin. These results confirm the common occurrence of campylobacter infection in chickens and indicate that cdt genes are commonly present in both C. jejuni and C. coli isolates from broilers, but that there are distinct differences in CDT production in these two closely related species.