Clinical presentation of invasive disease caused by Neisseria meningitidis serogroup Y in Sweden, 1995 to 2012.

Over the period 1995-2012, the incidence of invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y (NmY) increased significantly in Sweden. This is mainly due to the emergence of a predominant cluster named strain type YI subtype 1, belonging to the ST-23 clonal complex (cc). The aim of this study was to examine the clinical picture of patients with invasive disease caused by NmY and to analyse whether the predominant cluster exhibits certain clinical characteristics that might explain the increased incidence. In this retrospective observational study, the medical records available from patients with IMD caused by Nm serogroup Y in Sweden between 1995 and 2012 were systematically reviewed. Patient characteristics, in-hospital findings and outcome were studied and differences between the dominating cluster and other isolates were analysed. Medical records from 175 of 191 patients were retrieved. The median age was 62 years. The all-cause mortality within 30 days of admission was 9% (15/175) in the whole material; 4% (2/54) in the cohort with strain type YI subtype 1 and 11% (12/121) among patients with other isolates. Thirty-three per cent of the patients were diagnosed with meningitis, 19% with pneumonia, 10% with arthritis and 35% were found to have bacteraemia but no apparent organ manifestation. This survey included cases with an aggressive clinical course as well as cases with a relatively mild clinical presentation. There was a trend towards lower mortality and less-severe disease in the cohort with strain type YI subtype 1 compared with the group with other isolates.

Surveillance of invasive Neisseria meningitidis with a serogroup Y update, Sweden 2010 to 2012.

An increase of invasive meningococcal disease caused by Neisseria meningitidis serogroup Y has been noted in Sweden since 2005, and to a lower extent throughout Europe. The present study describes the epidemiology of invasive N. meningitidis isolates in Sweden in the period between 2010 and 2012, with a focus on serogroup Y. We also aimed to find an optimal molecular typing scheme for both surveillance and outbreak investigations. All invasive N. meningitidis isolates in Sweden during the study period (n=208) were genetically characterised. Serogroup Y predominated with 22/57, 31/61 and 44/90 of all invasive isolates (incidence 0.23, 0.33 and 0.46 per 100,000 population) in 2010, 2011 and 2012 respectively. In each of these years, 15/22, 22/31 and 19/44 of serogroup Y isolates were genetically clonal (Y: P1.5­2,10­1,36­2: F4­1: ST-23(cc23), 'porB allele 3­36, fHbp allele 25 and penA allele 22). Our findings further support those of others that currently recommended FetA typing could be replaced by FHbp. Moreover, in line with a previous study that we conducted, the current results indicate that highly variable multilocus variable-number tandem repeat analysis (HV-MLVA) can be used as a first-hand rapid method for small outbreak investigations.

Evaluation of a commercial multiplex PCR test (SeptiFast) in the etiological diagnosis of community-onset bloodstream infections.

The commercial polymerase chain reaction (PCR) test, SeptiFast, is designed to identify the DNA of individual bacterial and fungal pathogens in whole blood. We aimed to evaluate the usefulness of the test for the detection of community-onset bloodstream infections. We prospectively included adult patients who were subjected to blood culture (BC) at an infectious diseases department. For the evaluation, one BC/PCR set (two BC bottles and one PCR tube) per patient was used. When several sets were obtained and analyzed, the first set with any positive result was evaluated. Among 1,093 consecutively included patients, BC was positive in 138 and PCR was positive in 107. Fifty positive PCR results were supported by BC in the same BC/PCR set, ten were supported by other cultures, and, additionally, ten were supported by the clinical presentation. Compared with BC, PCR showed specificities and negative predictive values of >97% for all detectable pathogens. The following sensitivities and positive predictive values (PPVs) were noted: Staphylococcus aureus, 67% and 43%; Streptococcus pneumoniae, 12% and 67%; other Streptococcus species, 43% and 77%; Escherichia coli, 53% and 56%; and Klebsiella species, 43% and 23%. If support from other cultures and the clinical presentation were included in the reference standard, the PPVs for the detection of these bacteria were 57%, 100%, 92%, 75%, and 69%, respectively. Although the specificities were high, the low sensitivities and suboptimal PPVs noted in the present study discourage routine use of the test in its present form for the detection of community-onset bloodstream infections.

Genetic characterisation of the emerging invasive Neisseria meningitidis serogroup Y in Sweden, 2000 to 2010.

Neisseria meningitidis serogroups B and C have been responsible for the majority of invasive meningococcal disease in Europe. Recently, an increase of N. meningitidis disease due to serogroup Y has been noted in Sweden (in 2010, the proportion was 39%, with an incidence of 0.23 per 100,000 population), as well as in other northern European countries. We aimed to investigate the clonal pattern of the emerging serogroup Y in Sweden during 2000 to 2010. The serogroup Y isolates identified during this time (n=85) were characterised by multilocus sequence typing and sequencing of the fetA, fHbp, penA, porA and porB genes. The most frequent clone (comprising 28 isolates) with identical allele combinations of the investigated genes, was partly responsible for the observed increased number of N. meningitidis serogroup Y isolates. It was sulfadiazine resistant, with genosubtype P1.5-2,10-1,36-2, sequence type 23, clonal complex 23, porB allele 3-36, fetA allele F4-1, fHbp allele 25 and penA allele 22. The first case with disease due to this clone was identified in 2002: there was a further case in 2004, six during 2006 to 2007, eight during 2008 to 2009, with a peak of 12 cases in 2010. An unusual increase of invasive disease in young adults (aged 20­29 years) caused by this clone was shown, but no increase in mortality rate was observed.

Experiences with the new genetic variant of Chlamydia trachomatis in Orebro county, Sweden - proportion, characteristics and effective diagnostic solution in an emergent situation.

A Chlamydia trachomatis variant that contains a 377 bp deletion in the cryptic plasmid was recently reported in Sweden. This deletion includes the targets for Cobas Amplicor, Cobas TaqMan48, and Abbott m2000. We examined the proportion and characteristics of this variant in Orebro county, Sweden and developed an effective diagnostic solution. In total, 2,401 consecutive C. trachomatis culture samples and 536 PCR samples from symptomatic and asymptomatic patients and screened females were included. Culture, Cobas Amplicor, and LightMix 480HT were used for diagnosis. A mutant-specific PCR, plasmid sequencing, omp1 sequencing and multilocus sequence typing (MLST) were used to identify and characterise mutants. In total, 162 (6.7%) of the cultured samples were positive for C. trachomatis. However, 61 (38%) of those were negative when using Cobas Amplicor, and 60 of these were subsequently confirmed as the new variant. 13 of these mutant isolates were further characterised genetically, and all were of identical genotype E and the unique MLST sequence type: 21, 19, 1, 2, 1. Of all culture-positive samples, 161 of 162 were positive in the LightMix 480HT assay. The single negative sample was only weakly positive in culture, and negative in all PCRs. Of the 536 PCR samples, 37 were positive in both Cobas Amplicor and LightMix 480HT, 13 were only positive in LightMix 480HT (mutants), and two were only positive in Cobas Amplicor. Mutated C. trachomatis were prevalent in Orebro county in the period from October 2006 to February 2007, and it appeared to be a single clone. LightMix 480HT seemed sensitive, specific, and enabled high throughput diagnostics. However, rare low positive samples may be false-negative. Frequent surveillance and evaluations of diagnostic methods worldwide are crucial.

Increased prevalence of anti-gliadin IgA-antibodies with aberrant duodenal histopathological findings in patients with IgA-nephropathy and related disorders.

BACKGROUND: Antibodies present in coeliac disease may occur in IgA-nephropathy. This raises the question of food intolerance in the disease. Evidence for a true correlation between the two disorders has however been scarce. DESIGN: Sera from 89 patients with IgA-nephropathy and 13 other patients with IgA deposits in the glomeruli of kidney biopsies were analysed for IgA-antibodies to gliadin, endomysium and tissue transglutaminase (92/102 patients). RESULTS: Eleven out of 89 (12.4%) of the patients with IgA-nephropathy and five of the 13 others (38%) had elevated titres of IgA-antibodies to gliadin but, in all cases but one, normal IgA-antibodies to endomysium. Patients with IgA-nephropathy and elevated IgA-antibodies to gliadin had elevated total serum IgA more frequently than patients who had not (p<0.01). Two patients with IgA-nephropathy and one with Hennoch Schönlein's purpura had elevated IgA-antibodies to tissue transglutaminase. Small bowel biopsy in 7 out of 11 IgA-antibodies to gliadin positive patients with IgA-nephropathy was pathologic in three cases (two with Marsh I) . One patient with chronic glomerulnephritis also had Marsh I. CONCLUSIONS: We found no increased frequency of verified coeliac disease in 89 patients with IgA-nephropathy. Two patients with IgA-nephropathy and one patient with chronic glomerulonephritis with IgA deposits in the kidney biopsy had a Marsh I histopathology. The findings suggest a possible link of celiac disease to IgA-nephropathy and a role for antibodies to food antigens in this disorder.

Pharyngeal carriage of serogroup W135 Neisseria meningitidis in Hajjees and their family contacts in Morocco, Oman and Sudan.

In 2000 the global outbreak that began in Saudi Arabia was caused by a W135:2a:P1.5,2 strain of Neisseria meningitidis belonging to the ET-37 complex and to ST-11. There was concern that introduction of this epidemic clone (EC) might lead to a wave of outbreaks in the African meningitis belt. The WHO therefore initiated studies of meningococcal carriage among pilgrims and their family contacts in Morocco, Oman and Sudan, 3 to 12 months after the Hajj 2000. In Morocco, 1186 persons were swabbed 3 times. Ninety-five meningococcal strains were isolated from 2.7% of the specimens. Pulsed-field gel electrophoresis showed that 32 (33.6%) were identical with the EC. In Sudan, 5 strains identical with the EC were obtained after sampling 285 persons. In Oman, among 18 meningococcal strains isolated from 399 subjects, 11 (61.1%) belonged to the EC. The important pharyngeal carriage of W135 (EC) and its role in the 2001-2002 outbreaks in Burkina Faso argues for the necessity of reinforcing surveillance, and adapting and planning responses in Africa and the Middle East using the most appropriate vaccine.

A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by Neisseria meningitidis.

Luminol-enhanced chemiluminescence (CL) was used to study the ability of various strains of Neisseria meningitidis (MC) to induce oxidative metabolism of polymorphonuclear leukocytes (PMNL); an indirect measure of phagocytic activity. To circumvent variations related to different PMNL donors, a MC serogroup X strain was used as a control for indexing the CL responses induced by other MC strains. This procedure, with pooled serum from healthy blood donors to standardize opsonising conditions, gave reproducible and comparable results, irrespective of PMNL donors. Under these conditions, there was a highly significant difference between pathogenic and non-pathogenic MC strains as regards their ability to induce CL responses (p less than 0.001). The results indicated that the differences were due partly to opsonizing antibodies, partly to other differences related to pathogenicity of tested MC strains. These differences in leukocyte/MC interaction were also confirmed by phagocytic-killing experiments. The index procedure of CL measurements may be a suitable method to study the appearance of natural immunity to MC disease, as well as the pathogenicity of particular MC strains.

Genosubtyping by sequencing group A, B and C meningococci; a tool for epidemiological studies of epidemics, clusters and sporadic cases.

Genosubtyping, by sequencing variable regions (VRs) 1, 2 and 3 of the porA gene, was evaluated as a tool to detect clonality of isolates in meningococcal epidemics in Africa and clusters of disease in Sweden. All 63 examined meningococcal isolates were successfully genosubtyped. The isolates belonging to group A type 4 with genosubtype P1.20,9,35a showed little heterogeneity in African epidemics in 1988 and onwards. In Sweden, two meningococcal clones of group B type 15, with genosubtypes P1.7,16,35 and P1.7,16f,35, dominated during two clusters of meningococcal disease in 1995-96 and in sporadic cases thereafter. The characterisation of group C meningococci isolated during 1992 in Sweden indicated a cluster (type 2a with genosubtype P1.5a,10d,36b) connected with a discotheque visit. Two variants of VR2 (10p and 25b), not previously described, were found among the examined isolates. Nucleotide sequence analysis of VRs in the porA gene proved a valuable epidemiological tool since almost all isolates could be genosubtyped, in contrast to the phenotypic methods presently used.

Early development in healthy children of serum opsonins against nonpathogenic Neisseria meningitidis.

In an earlier study, with the use of chemiluminescence (CL) and phagocytic killing, we could show that in the presence of serum from healthy adults polymorphonuclear leukocytes (PMNL) efficiently handle nonpathogenic Neisseria meningitidis strains, in sharp contrast to those associated with clinical disease. The major part of this difference was dependent on serum factors. In the present study 84 serum samples from children 1-3, 4-6, 7-9, and 10-14 years old were studied by the CL technique according to their ability to opsonize meningococci. There was a highly significant difference (p less than 0.001) in all four age groups when the CL indexes obtained with the pathogenic meningococci of the serogroups A, B and C were compared with those of the nonpathogenic menigococci: serogroup 29E and nongroupable meningococci. These findings imply that the ability to opsonize so-called nonpathogenic meningococci is developed early in life and may explain why they are only occasionally able to cause disease.

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products.

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.

Epiglottitis in Sweden before and after introduction of vaccination against Haemophilus influenzae type b.

BACKGROUND: Acute epiglottitis is an important manifestation of invasive Haemophilus influenzae type b (Hib) infection. In 1992 and 1993 Hib vaccination was introduced in the general childhood vaccination program in Sweden. The aim of the present investigation was to study the impact of Hib vaccination on the diagnosis of epiglottitis in Sweden in children as well as adults. METHODS: A retrospective national population-based study on the incidence of epiglottitis in Sweden was performed for the 10-year period 1987 to 1996. The incidence calculations were based on figures from the national register of all patients treated at Swedish hospitals. The incidence (cases/100,000/year) for the prevaccination period 1987 to 1991 was compared with the incidence after Hib vaccination was introduced. RESULTS: In children a substantial decrease was found after introduction of large scale vaccination against Hib. Below 5 years of age the annual incidence decreased from 20.9 in 1987 to 0.9 in 1996. In adults a tendency toward a decrease in incidence was evident. CONCLUSIONS: Introduction of Hib vaccination in a general childhood program was followed not only by a >90% reduction in the incidence in the youngest age group but also by a reduction in the incidence in the older age groups and among adults.

Protective effect of breastfeeding: an ecologic study of Haemophilus influenzae meningitis and breastfeeding in a Swedish population.

BACKGROUND: In Orebro County, Sweden, a 2.5-fold increase in the incidence of Haemophilus influenzae (HI) meningitis was found between 1970 and 1980. In a case-control study of possible risk factors for invasive HI infection conducted in the same area, 1987-1992, breastfeeding was found to be a strong protective factor. MATERIAL AND METHODS: In order to study the relation between incidence rates of HI meningitis between 1956-1992 and breastfeeding rates in the population an ecologic study was performed. RESULTS: A strong (negative) correlation between breastfeeding and incidence of HI infection 5 to 10 years later (rho(xy) (s) approximately -0.6) was seen, whereas no relation seems to exist for the time lag 15 years and beyond. The correlation for contemporary data was intermediate. There were similar results for the breastfeeding proportions at 2, 4 as well as 6 months of age. DISCUSSION: Our ecologic data are consistent with results from our case-control study. The time-lag for the delayed effect on the population level could be estimated although sparse data make the estimates vulnerable to sampling fluctuations. Limitations with ecologic studies are discussed. CONCLUSION: There seems to be an association between high breastfeeding rate in the population and a reduced incidence of HI meningitis 5 to 10 years later. These results do have implications on strategies for breastfeeding promotion, especially in countries where Hib vaccination is too costly and not yet implemented.

Protective effect of breastfeeding on invasive Haemophilus influenzae infection: a case-control study in Swedish preschool children.

BACKGROUND: In Orebro County a 2.5-fold increase in the incidence of Haemophilus influenzae (HI) meningitis was found between 1970 and 1980, an observation that initiated the present study. MATERIALS AND METHODS: In order to search for associations between morbidity in invasive HI infection and possible risk factors, a case-control study was conducted over a 6-year period from 1987 to 1992, before general Hib vaccination was introduced in Sweden. Fifty-four cases with invasive HI infection 139 matched controls were studied for possible risk factors such as day-care outside the home, short duration of breastfeeding, passive smoking, low socioeconomic level of the household, many siblings in the family, allergy, frequent, infections, repeated antibiotic treatments and immunoglobulin deficiency. RESULTS: Multivariate analysis showed a significant association between invasive HI infection and two independent factors, i.e. short duration (< 13 weeks) of exclusive breastfeeding, odds ratio (OR) 3.79 (95% confidence interval [CI] 1.6-8.8) and history of frequent infections, OR 4.49 (95% CI : 1.0-21.0). For the age at onset 12 months or older, the associations were stronger, OR 7.79 (95% CI : 2.4-26.6) and 5.86 (95% CI : 1.1-30.6), respectively. When breastfeeding duration in weeks was analysed as a continuous variable the OR was 0.95 (95% CI : 0.92-0.99), indicating a decreased risk with each additional week. Increased OR were observed for other risk factors as well but not of the magnitude found for short duration of breastfeeding. DISCUSSION: The association of decreased risk for invasive HI infection and long duration of breastfeeding was persisting beyond the period of breastfeeding itself. This finding supports the hypothesis of a long-lasting protective effect of breastfeeding on the risk for invasive HI infection. CONCLUSION: A decreased risk for invasive HI infection with long duration of breastfeeding was found. Our results do have implications for strategies in breastfeeding promotion, especially in countries where Hib vaccination is too costly and not yet implemented.

Glycosphingolipid binding specificities of Neisseria meningitidis and Haemophilus influenzae: detection, isolation, and characterization of a binding-active glycosphingolipid from human oropharyngeal epithelium.

The glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.

Rapid serotyping of groups A, B, and C meningococci by rocket-line immunoelectrophoresis and co-agglutination.

Rocket-line immunoelectrophoresis (R-LIE) with antigen containing intermediate gel, and co-agglutination utilising protein A-containing staphylococci coated with specific antibodies, were adapted for serotyping the prototypes of group B meningococci. Both were found to have the same specificity as agar gel double diffusion (AGDD) but they were more sensitive and more rapid than AGDD. R-LIE required, like AGDD, the extraction of relatively large quantities of bacteria, while the co-agglutination method, performed as a slide agglutination tests with results within a few minutes and no need of special equipment, required only a small amount of heated whole meningococcal cells. Meningococcal strains of serogroups B, C, and A from patients with carriers were serotyped and the results with all three methods were in agreement.

Culture diagnosis of meningococcal carriers: yield from different sites and influence of storage in transport medium.

Different specimens and techniques have been used in the diagnosis of carriers of Neisseria meningitidis, reflecting the uncertainty about the optimal diagnostic procedure. In the present investigation the culture yield of meningococci from throat specimens was compared to that from nasopharyngeal speimens in 178 persons: 44 carriers were diagnosed. All of them were detected by culture of throat specimens while 34% of them would have remained undiagnosed if only nasopharyngeal specimens had been examined. Storage of throat specimens in a transport medium for 24 hours before culture gave a negative culture for meningococci in 41% of the carriers. This loss was surprisingly high, the reasons for which are discussed.

Antibody response in Staphylococcus aureus septicaemia--a prospective study.

Formation of serum antibodies against alpha-toxin, teichoic acid and lipase was followed in 63 patients with Staphylococcus aureus septicaemia in 240 consecutive serum samples. Control subjects comprised 23 patients with septicaemia due to other causes and 21 febrile patients without septicaemia. An antibody response against alpha-toxin, measured by ELISA, was most common (40%) in the initial serum, but antibody to teichoic acid was present in the highest number of positive patients (60%) when samples were drawn between 0 and 30 days: 74% of the patients showed a positive antibody response to at least one of the three antigens. When complicated versus uncomplicated septicaemia was compared (samples taken 8-14 days), 14 (45%) of 31 patients had a positive response against alpha-toxin versus 12 (75%) of 16, against teichoic acid 16 (51%) of 31 versus 12 (75%) of 16 and against lipase 15 (48%) of 31 versus 8 (50%) of 16. Patients with low initial antibody levels displayed a poorer antibody response than those with higher initial antibody levels. This phenomenon was observed with all three antigens, but was most pronounced with alpha-toxin. The initial antibody levels may predict the antibody response during the course of the disease. ELISA titres against alpha-toxin correlated (r=0.87) with biological neutralising activity of the antisera. The results may indicate a biological role of serum antibodies in staphylococcal septicaemia.

PCR assay or culture for diagnosis of Bordetella pertussis in the routine diagnostic laboratory?

A nested PCR method was compared with culture for the detection of Bordetella pertussis in a routine clinical diagnostic laboratory. A total of 241 clinical nasopharyngeal aspirates were examined in parallel in the laboratory. Both methods were positive for 75 samples (31%), eight samples were positive by nested PCR only (3.3%), and one sample was positive by culture only (0.4%). The mean time actually required in the clinical laboratory (not operating with pertussis diagnosis during weekends) from the day of arrival to the diagnosis of a positive or negative sample by the nested PCR assay was 1.8 +/- 1.3 days (mean +/- SD), for positive culture 4.5 +/- 1.4 days and for negative culture 10.5 +/- 1.0 days. The hands-on time in the laboratory to perform the nested PCR was 2 h, for a positive culture 25 min, and for a negative culture 15 min. The cost analysis of the methods, when running one sample at a time, showed that the laboratory cost for PCR was six times higher than culture. When running four samples together the cost for PCR was three times higher than culture. In conclusion, the nested PCR is the more rapid and sensitive method compared to culture. With the present design, the PCR-protocol involves higher material expenditure and claims more hands-on time.