Clinical and Immunological Characteristics of Autoimmune Addison Disease: A Nationwide Swedish Multicenter Study.

Context: Studies of the clinical and immunological features of autoimmune Addison disease (AAD) are needed to understand the disease burden and increased mortality. Objective: To provide upgraded data on autoimmune comorbidities, replacement therapy, autoantibody profiles, and cardiovascular risk factors. Design, Setting, and Participants: A cross-sectional, population-based study that included 660 AAD patients from the Swedish Addison Registry (2008-2014). When analyzing the cardiovascular risk factors, 3594 individuals from the population-based survey in Northern Sweden, MONICA (monitoring of trends and determinants of cardiovascular disease), served as controls. Main Outcome Measures: The endpoints were the prevalence of autoimmune comorbidities and cardiovascular risk factors. Autoantibodies against 13 autoantigens were determined. Results: The proportion of 21-hydroxylase autoantibody-positive patients was 83%, and 62% of patients had ≥1 associated autoimmune diseases, more frequently coexisting in females (P < 0.0001). AAD patients had a lower body mass index (P < 0.0001) and prevalence of hypertension (P = 0.027) compared with controls. Conventional hydrocortisone tablets were used by 89% of the patients, with a mean dose of 28.1 ± 8.5 mg/d. The mean hydrocortisone equivalent dose normalized to the body surface was 14.8 ± 4.4 mg/m2/d. A greater hydrocortisone equivalent dose was associated with a greater incidence of hypertension (P = 0.046). Conclusions: Careful monitoring of AAD patients is warranted to detect associated autoimmune diseases. Contemporary Swedish AAD patients did not have an increased prevalence of overweight, hypertension, type 2 diabetes mellitus, or hyperlipidemia. However, high glucocorticoid replacement doses could be a risk factor for hypertension.

Genome-Based Characterization of Emergent Invasive Neisseria meningitidis Serogroup Y Isolates in Sweden from 1995 to 2012.

Invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y has increased in Europe, especially in Scandinavia. In Sweden, serogroup Y is now the dominating serogroup, and in 2012, the serogroup Y disease incidence was 0.46/100,000 population. We previously showed that a strain type belonging to sequence type 23 was responsible for the increased prevalence of this serogroup in Sweden. The objective of this study was to investigate the serogroup Y emergence by whole-genome sequencing and compare the meningococcal population structure of Swedish invasive serogroup Y strains to those of other countries with different IMD incidence. Whole-genome sequencing was performed on invasive serogroup Y isolates from 1995 to 2012 in Sweden (n = 186). These isolates were compared to a collection of serogroup Y isolates from England, Wales, and Northern Ireland from 2010 to 2012 (n = 143), which had relatively low serogroup Y incidence, and two isolates obtained in 1999 in the United States, where serogroup Y remains one of the major causes of IMD. The meningococcal population structures were similar in the investigated regions; however, different strain types were prevalent in each geographic region. A number of genes known or hypothesized to have an impact on meningococcal virulence were shown to be associated with different strain types and subtypes. The reasons for the IMD increase are multifactorial and are influenced by increased virulence, host adaptive immunity, and transmission. Future genome-wide association studies are needed to reveal additional genes associated with serogroup Y meningococcal disease, and this work would benefit from a complete serogroup Y meningococcal reference genome.

Quantitative data from the SeptiFast real-time PCR is associated with disease severity in patients with sepsis.

BACKGROUND: The commercial test, SeptiFast, is designed to detect DNA from bacterial and fungal pathogens in whole blood. The method has been found to be specific with a high rule-in value for the early detection of septic patients. The software automatically provides information about the identified pathogen, without quantification of the pathogen. However, it is possible to manually derive Crossing point (Cp) values, i.e. the PCR cycle at which DNA is significantly amplified. The aim of this study was to find out whether Cp values correlate to disease severity. METHODS: We used a study cohort of patients with positive results from SeptiFast tests for bacteria from a recent study which included patients with suspected sepsis in the Emergency department. Cp values were compared with disease severity, classified as severe sepsis/septic shock or non-severe sepsis, according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine. RESULTS: Ninety-four patients were included. The prevalence of severe sepsis/septic shock in the study was 29%. SeptiFast positive tests from patients with severe sepsis/septic shock had significantly lower Cp values compared with those from patients with non-severe sepsis, median 16.9 (range: 7.3-24.3) versus 20.9 (range: 8.5-25.0), p < 0.001. Positive predictive values from the SeptiFast test for identifying severe sepsis/septic shock were 34% at Cp cut-off <25.0, 35% at Cp cut-off <22.5, 50% at Cp cut-off <20.0, and 73% at Cp cut-off <17.5. Patients with a positive Septifast test with a Cp value <17.5 had significantly more severe sepsis/septic shock (73% versus 15%, p < 0.001), were more often admitted to the Intensive Care Unit (23% versus 4%, p = 0.016), had positive blood culture (BC) more frequently (100% versus 32%, p < 0.001) and had longer hospital stays (median 19.5 [range: 4-78] days versus 5 [range: 0-75] days, p < 0.001) compared with those with a Cp value >17.5. CONCLUSIONS: Our results suggest that introducing quantitative data to the SeptiFast test could be of value in assessing sepsis severity. Moreover, such data might also be useful in predicting a positive BC result.

Association between population prevalence of smoking and incidence of meningococcal disease in Norway, Sweden, Denmark and the Netherlands between 1975 and 2009: a population-based time series analysis.

OBJECTIVE: To investigate the relationship between the prevalence of smoking in the population and incidence of invasive meningococcal disease (IMD) among children under 5 years of age. DESIGN: Retrospective, longitudinal, observational study. Poisson regression controlled for confounding factors. SETTING: Norway, Sweden, Denmark and the Netherlands between 1975 and 2009. POPULATION: Total population of approximately 35 million people in these four countries. DATA SOURCES: Data were collected from the Ministries of Health, National Statistics Bureaus and other relevant national institutes. RESULTS: In Norway, there was a significant positive relationship between the annual prevalence of daily smokers among individuals aged 25-49 years and the incidence of IMD in children under 5 years of age, unadjusted (RR=1.04-1.06, 95% CI 1.02 to 1.07, p<0.001) and after adjustment for time of year (quarter), incidence of influenza-like illness and household crowding (RR=1.05-1.07, 95% CI 1.03 to 1.09, p<0.001). Depending on age group, the risk of IMD increased by 5.2-6.9% per 1% increase in smoking prevalence among individuals aged 25-49 years in adjusted analyses. Using limited datasets from the three other countries, unadjusted analysis showed positive associations between IMD in children related to older smokers in Sweden and the Netherlands and negative associations related to younger smokers in Sweden. However, there were no demonstrable associations between incidence of IMD and prevalence of smoking, after adjustment for the same confounding variables. CONCLUSIONS: The reduced incidence of IMD in Norway between 1975 and 2009 may partly be explained by the reduced prevalence of smoking during this period. High-quality surveillance data are required to confirm this in other countries. Strong efforts to reduce smoking in the whole population including targeted campaigns to reduce smoking among adults may have a role to play in the prevention of IMD in children.

Comparison of sputum and nasopharyngeal aspirate samples and of the PCR gene targets lytA and Spn9802 for quantitative PCR for rapid detection of pneumococcal pneumonia.

We aimed to compare sputum and nasopharyngeal aspirate (NpA) samples and the PCR gene targets lytA and Spn9802 in quantitative PCR (qPCR) assays for rapid detection of pneumococcal etiology in community-acquired pneumonia (CAP). Seventy-eight adult patients hospitalized for radiologically confirmed CAP had both good-quality sputum and NpA specimens collected at admission. These samples were subjected to lytA qPCR and Spn9802 qPCR assays with analytical times of <3 h. Thirty-two patients had CAP with a pneumococcal etiology, according to conventional diagnostic criteria. The following qPCR positivity rates were noted in CAP cases with and without pneumococcal etiology: 96% and 15% (sputum lytA assay), 96% and 17% (sputum Spn9802 assay), 81% and 11% (NpA lytA assay), and 81% and 20% (NpA Spn9802 assay), respectively. The mean lytA and Spn9802 DNA levels were significantly higher in qPCR-positive sputum samples from cases with pneumococcal etiology than in qPCR-positive sputum samples from CAP cases without pneumococcal etiology or qPCR-positive NpA samples from cases with pneumococcal etiology (P < 0.02 for all comparisons). For detection of pneumococcal etiology, receiver operating characteristic curve analysis showed that sputum specimens were superior to NpA specimens as the sample type (P < 0.02 for both gene targets) and lytA tended to be superior to Spn9802 as the gene target. The best-performing test, the sputum lytA qPCR assay, showed high sensitivity (94%) and specificity (96%) with a cutoff value of 10(5) DNA copies/ml. In CAP patients with good sputum production, this test has great potential to be used for the rapid detection of pneumococcal etiology and to target penicillin therapy.

Genome-wide sequencing of cellular microRNAs identifies a combinatorial expression signature diagnostic of sepsis.

RATIONALE: Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity. OBJECTIVES: We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU). METHODS: Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature. RESULTS: A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation. CONCLUSIONS: Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions.

Gene variability and degree of expression of vaccine candidate factor H binding protein in clinical isolates of Neisseria meningitidis.

The factor H binding protein (fHbp) is currently being evaluated in clinical trials as a vaccine candidate for a meningococcal group B vaccine. We have previously described the prevalence and sequence variation of fHbp (Jacobsson et al., 2009) and here we investigate the expression of the antigen. The present study includes isolates from carriers (n = 62) and patients with invasive Neisseria meningitidis infections (n = 146), of which 62 had a fatal outcome. Among the invasive isolates from patients with fatal and non-fatal infections fHbp allele 1 was most common (42% and 29% respectively), but it was only identified in 3% of the carrier isolates, where allele 16 was most frequent (13%). The Fluorescence-activated cell sorting analysis identified fHbp expression in all except seven isolates and further analysis by Western blot showed that five of these seven samples were indeed negative using a polyclonal anti-fHbp serum. The negative isolates belonged to serogroup B fHbp allele 24, Y allele 104, and W-135 allele 16 (all invasive). Two were non-serogroupable carrier isolates (allele 21 and 101). An interesting finding is that isolates from invasive infections with fatal outcome had lower expression of fHbp or lower affinity for the fHbp antibody compared to isolates from non-fatal invasive infections and carriers.

Evaluation of molecular typing methods for identification of outbreak-associated Neisseria meningitidis isolates.

It is essential in an outbreak investigation that strain characterization of Neisseria meningitidis is performed in a rapid and accurate manner. This study evaluated two new molecular typing methods, multiple-locus variable number tandem repeat analysis (MLVA) and repetitive sequence-based PCR (rep-PCR) (DiversiLab; bioMérieux) and compared them with current recommended methodologies. This retrospective study included 36 invasive N. meningitidis serogroup C isolates collected in Sweden 2001 through 2009 and previously subjected to outbreak investigation. All strains were typed with highly variable-MLVA (HV-MLVA) and rep-PCR. The isolates were further characterized by multilocus sequence typing (MLST) and sequencing of the fetA, fHbp, penA, porA and porB genes. The results showed that HV-MLVA had the highest index of diversity (0.99) and rep-PCR had the highest congruence (40%) with the currently recommended typing methods. The HV-MLVA correlated best to the spatiotemporal connections and had the overall highest Adjusted Wallace coefficients, suggesting that HV-MLVA can predict the results of the other typing methods in the study. We therefore suggest that after initial confirmation of species, serogroup and genosubtype, HV-MLVA should be used as the most discriminatory method for first hand investigation of N. meningitidis serogroup C isolates.

Novel meningococcal 4CMenB vaccine antigens - prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae.

The first cross-protective Neisseria meningitidis vaccine (focus on serogroup B), the protein-based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome-derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0-95.4% and 60.9-94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross-immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined.

Elution of antitransglutaminase antibodies from duodenal biopsies: a novel approach in the diagnosis of celiac disease.

Celiac disease (CeD) is a disease more prevalent and multisymptomatic than was earlier recognized. Whereas prompt initiation of gluten-free diet (GFD) is beneficial in relieving the symptoms, an accurate CeD diagnosis is necessary also to avoid years of restricted diet on uncertain grounds. We propose a new diagnostic method, based on elution of deposited antibodies against transglutaminase (anti-tTG) from duodenal biopsies in patients with symptoms and screening serology analyses suggestive of CeD. The eluates were analyzed in a Phadia 250 fluoroimmunoassay, demonstrating elevated concentrations of anti-tTG in CeD patients, corresponding to serology and histopathology findings. In one case histology was inconclusive, displaying only unspecific inflammation, but eluted anti-tTG was positive. This patient has clinically improved following GFD. We conclude that our novel method represents a new tool in the diagnostic work up in CeD. The detection of deposited anti-tTG at the site of inflammation appears to provide a high sensitivity and specificity using a technique that is quick, simple and reliable. Further studies are needed for optimization and elucidation of suitable applications for this elution method.

Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review.

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

Emergence of the new variant of Chlamydia trachomatis in a defined area of Sweden before 2002?

OBJECTIVES: To compare the proportions of Chlamydia trachomatis-positive specimens detected by Cobas Amplicor CT/NG (CA PCR) with C trachomatis positives in cell culture from 1999 to 2006 in order to estimate when the new variant of C trachomatis (nvCT) with a deletion in the cryptic plasmid (in the target region for CA PCR that resulted in false-negative results) emerged in Örebro County, Sweden. METHODS: The annual number of specimens analysed using CA PCR in 1999-2006 ranged from 5077 to 11,622 and using cell culture (McCoy cells) from 5201 to 7425. Logistic regression was applied to evaluate the change in the proportion of C trachomatis-positive tests over the years between the two methods. The statistical interaction effect of year and method was estimated using both unadjusted and adjusted (age, gender and clinic) models. RESULTS: From 2002, the proportion of C trachomatis-positive specimens identified using CA PCR decreased annually, whereas the proportion of culture-positive specimens increased annually. Logistic regression showed a statistically significant interaction effect between periods (1999-2006) and groups of specimens analysed using CA PCR or cell culture. A statistically significant association between the interaction of CA PCR/cell culture and period was observed in the unadjusted and adjusted models. CONCLUSION: This study indicates that in Orebro County, Sweden, nvCT was already present before 2002, that is, when the difference between the proportions of C trachomatis-positive specimens identified by CA PCR compared with cell culture-positive specimens began to show a statistically significant decline.

Multicenter study for defining the breakpoint for rifampin resistance in Neisseria meningitidis by rpoB sequencing.

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of > or = 0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of < 0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of > 1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of < or = 1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of < or = 1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

Definite, probable, and possible bacterial aetiologies of community-acquired pneumonia at different CRB-65 scores.

According to the recommendations of the Swedish Community-Acquired Pneumonia (CAP) guidelines, the selection of empirical antibiotic therapy should be based on the CRB-65 rule. The guidelines recommend empirical therapy directed predominantly against Streptococcus pneumoniae for patients with low CRB-65 scores and broad-spectrum therapy for patients with high CRB-65 scores. In order to study the utility of the recommendations, we analyzed the data from an aetiological study previously performed on 235 hospitalized adult CAP patients at our medical centre. A definite, probable, or possible bacterial aetiology was noted in 194 cases (83%), including 112 cases (48%) with S. pneumoniae aetiology. The following frequencies of definite-probable aetiologies were noted in the patients with CRB-65 score 0-1 (n=155) and CRB-65 score 2-4 (n=80): S. pneumoniae 30% and 35%, Haemophilus influenzae 6.5% and 14% (p=0.063), Mycoplasma pneumoniae 15% and 5.0% (p=0.019), Chlamydophila species 2.6% and 1.2%, Legionella pneumophila 1.9% and 0%, and Staphylococcus aureus 1.3% and 1.2%, respectively. The high frequency of S. pneumoniae in the study supports the recommendations to predominantly cover this bacterium in the empirical therapy of patients with low CRB-65 scores. In the case of treatment failure in these patients, the study indicates that coverage against M. pneumoniae and H. influenzae should be considered.

The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization.

Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms - the genes for central metabolism, development cycle and virulence are conserved - or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population.

Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden--the meningococcal population remains susceptible.

The susceptibility to 7 antibiotics was determined for all Swedish invasive Neisseria meningitidis isolates from 1995 to 2008 (N = 717). In general, these remain highly susceptible to the antibiotics recommended for use. Accordingly, penicillin G remains effective for the treatment of invasive meningococcal disease and ciprofloxacin appropriate for prophylaxis.

Isolation and characterization of Neisseria meningitidis in the vaccine era. Who needs what and when?

Diagnosing invasive meningococcal disease can require a number of different approaches. The logic behind and the practical steps for diagnosis are described and discussed. The characterization of Neisseria meningitidis can be performed with a number of methods, depending on the problem/question that is formulated. Increased availability and use of meningococcal vaccines with new immunizing proteins requires diagnostic and surveillance systems of high quality. It is foreseen that a combination of genetic characterization and proof of corresponding production/exposure of the protein by a phenotypic method will be an optimal strategy for dealing with candidate vaccine antigens that have genetic and phenotypic variability. Such an approach will make use of the best qualities of both systems. Who needs what and when concerning the characterization is outlined, including future situations when new meningococcal vaccines are available.