Despite Excellent Test Characteristics of the cobas 4800 CT/NG Assay, Detection of Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Remains Challenging.

Oropharyngeal Chlamydia trachomatis (CT) infections and, especially, Neisseria gonorrhoeae (NG) infections are common, but few commercial nucleic acid amplification tests (NAATs) specify extragenital samples for intended use. The test characteristics of the cobas 4800 CT/NG assay were evaluated for oropharyngeal swabs. The technical validation included analysis of the specificity, sensitivity, dynamic range, linearity, efficiency, and precision. The probability of detection curve combined with historical data enabled the estimation of potentially missed diagnoses. A clinical evaluation was performed on a subset of 2,798 clinical samples available from routine diagnostics. Results of the cobas 4800 were compared with those from in-house C. trachomatis/N. gonorrhoeae PCR assays. Discrepant samples were tested with resolver assays, and these results were considered decisive. No cross-reactivity was seen in the analytical specificity analysis. High linearity (R2 ≥ 0.983), efficiency (89% to 99%), and precision (cycle threshold [CT ] value of 0.1 to 0.9) were seen for both C. trachomatis and N. gonorrhoeae The limit of detection in oropharyngeal samples was 3.2 × 102 inclusion-forming units (IFU)/ml for C. trachomatis and 6.7 × 102 CFU/ml for N. gonorrhoeae Estimates on potentially missed diagnoses were up to 7.2% for C. trachomatis and up to 24.7% for N. gonorrhoeae Clinical sensitivity and specificity were evaluated with 25 C. trachomatis-positive, 86 N. gonorrhoeae-positive, and 264 negative samples, resulting in 100% and 99.6% for C. trachomatis and 100% and 96.7% for N. gonorrhoeae, respectively. The findings in this study demonstrate the utility of the cobas 4800 CT/NG assay for oropharyngeal samples. Despite its being a highly accurate test, the range of reported CT values, especially for N. gonorrhoeae, suggests relatively low oropharyngeal loads. Hence, consistent detection over the full range of oropharyngeal loads could be impaired.

Multidisciplinary investigation of a multicountry outbreak of Salmonella Stanley infections associated with turkey meat in the European Union, August 2011 to January 2013.

Between August 2011 and January 2013, an outbreak of Salmonella enterica serovar Stanley (S. Stanley) infections affected 10 European Union (EU) countries, with a total of 710 cases recorded. Following an urgent inquiry in the Epidemic Intelligence Information System for food- and waterborne diseases (EPIS-FWD) on 29 June 2012, an international investigation was initiated including EU and national agencies for public health, veterinary health and food safety. Two of three local outbreak investigations undertaken by affected countries in 2012 identified turkey meat as a vehicle of infection. Furthermore, routine EU monitoring of animal sources showed that over 95% (n=298) of the 311 S. Stanley isolates reported from animal sampling in 2011 originated from the turkey food production chain. In 2004­10, none had this origin. Pulsed-field gel electrophoresis (PFGE) profile analysis of outbreak isolates and historical S. Stanley human isolates revealed that the outbreak isolates had a novel PFGE profile that emerged in Europe in 2011. An indistinguishable PFGE profile was identified in 346 of 464 human, food, feed, environmental and animal isolates from 16 EU countries: 102 of 112 non-human isolates tested were from the turkey production chain. On the basis of epidemiological and microbiological evidence, turkey meat was considered the primary source of human infection, following contamination early in the animal production chain.

Analysis of two sequential SARS-CoV-2 outbreaks on a haematology-oncology ward and the role of infection prevention.

Two SARS-CoV-2 nosocomial outbreaks occurred on the haematology ward of our hospital. Patients on the ward were at high risk for severe infection because of their immunocompromised status. Whole Genome Sequencing proved transmission of a particular SARS-CoV-2 variant in each outbreak. The first outbreak (20 patients/31 healthcare workers (HCW)) occurred in November 2020 and was caused by a variant belonging to lineage B.1.221. At that time, there were still uncertainties on mode of transmission of SARS-CoV-2, and vaccines nor therapy were available. Despite HCW wearing II-R masks in all patient contacts and FFP-2 masks during aerosol generating procedures (AGP), the outbreak continued. Therefore, extra measures were introduced. Firstly, regular PCR-screening of asymptomatic patients and HCW; positive patients were isolated and positive HCW were excluded from work as a rule and they were only allowed to resume their work if a follow-up PCR CT-value was ≥30 and were asymptomatic or having only mild symptoms. Secondly, the use of FFP-2 masks was expanded to some long-lasting, close-contact, non-AGPs. After implementing these measures, the incidence of new cases declined gradually. Thirty-seven percent of patients died due to COVID-19. The second outbreak (10 patients/2 HCW) was caused by the highly transmissible omicron BA.1 variant and occurred in February 2022, where transmission occurred on shared rooms despite the extra infection control measures. It was controlled much faster, and the clinical impact was low as the majority of patients was vaccinated; no patients died and symptoms were relatively mild in both patients and HCW.

Controlling a human parainfluenza virus-3 outbreak in a haematology ward in a tertiary hospital: the importance of screening strategy and molecular diagnostics in relation to clinical symptoms.

BACKGROUND: The human parainfluenza virus 3 (HPIV-3) outbreak at the haemato-oncology ward of the Maastricht University Medical Centre in the summer of 2016. AIM: To describe an effective strategy to control the largest reported HPIV-3 outbreak at an adult haematology-oncology ward in the Netherlands by implementing infection control measures and molecular epidemiology investigation. METHODS: Clinical, patient and diagnostic data were both pro- and retrospectively collected. HPIV-3 real-time polymerase chain reaction (HPIV-3 RT-PCR) was validated using oropharyngeal rinse samples. Screening of all new and admitted patients was implemented to identify asymptomatic infection or prolonged shedding of HPIV-3 allowing cohort isolation. FINDINGS: The HPIV-3 outbreak occurred between 9 July and 28 September 2016 and affected 53 patients. HPIV-3 RT-PCR on oropharyngeal rinse samples demonstrated an up to 10-fold higher sensitivity compared with pharyngeal swabs. Monitoring showed that at first positive PCR, 20 patients (38%) were asymptomatic (of which 11 remained asymptomatic) and the average duration of shedding was 14 days (range 1-58). Asymptomatic patients had lower viral load, shorter period of viral shedding (≤14 days) and were mostly immune-competent oncology patients. The outbreak was under control five weeks after implementation of screening of asymptomatic patients. CONCLUSION: Implementation of a sensitive screening method identified both symptomatic and asymptomatic patients which had lower viral loads and allowed early cohort isolation. This is especially important in a ward that combines patients with varying immune status, because both immunocompromised and immune-competent patients are likely to spread the HPIV-3 virus, either through prolonged shedding or through asymptomatic course of disease.

Role of the environment in transmission of Gram-negative bacteria in two consecutive outbreaks in a haematology-oncology department.

In 2019-2020, two subsequent outbreaks caused by phenotypically identical ESBL-producing Enterobacter cloacae and multi-drug-resistant (MDR) Pseudomonas putida were detected in respectively 15 and 9 patients of the haematology-oncology department. Both bacterial species were resistant to piperacillin-tazobactam, used empirically in (neutropenic) sepsis in our hospital, and ciprofloxacin, used prophylactically in selective digestive decontamination for haematology patients. The E. cloacae outbreak was identified in clinical cultures of blood and urine. Despite intensified infection control measures, new cases were found in weekly point-prevalence screening cultures. Environmental samples of sinks and shower drains appeared positive in 18.1%. To diminish the environmental contamination burden, all siphons of sinks were replaced, and disinfection of sinks and shower drains was intensified using chlorine and soda on a daily basis. Replacement of shower drains was not possible. The outbreak of P. putida remained limited to rectal cultures only, and disappeared spontaneously without interventions. During both outbreaks, multiple strains of the incriminated bacterium were found simultaneously (demonstrated by Amplified-Fragment Length Polymorphism and/or Whole-Genome Multi-locus Sequencing Typing) in patients as well as the environment. It was experimentally shown that a biofilm on the toilet edge may act as a source for nosocomial transmission of Gram-negative bacteria. In conclusion, the drainage system of the hospital is an important reservoir of MDR bacteria, threatening the admitted patients. In existing hospitals, biofilms in the drainage systems cannot be removed. Therefore, it is important that in (re)building plans for hospitals a plan for prevention of nosocomial transmission from environment to patients is incorporated.

Social integration of people with intellectual disability: insights from a social psychological research programme.

BACKGROUND: Social integration of people with intellectual disability (ID) moving into regular neighbourhoods tends to be studied and evaluated without detailed knowledge about the social psychological aspects of everyday interaction between neighbours with and without ID. The goal of the present paper is to show how the authors' social psychological research programme may contribute to this field of inquiry. METHODS: The different ways in which societies respond to features and behaviours that may be perceived as deviant are theoretically analysed. Results of empirical studies are reported to clarify how social responses to people with ID are special in terms of perceptions, emotions and interaction desires of people with and without ID during a pre-contact and contact phase. RESULTS: On the basis of the theoretical analysis, it is concluded that regular neighbouring in modern Western society often takes the form of benevolent tolerance, rather than stigmatisation and prejudice. However, empirical studies reveal that, prior to getting people with ID as new neighbours, prospective neighbours without ID experience a specific pattern of emotions that are associated with specific desires (e.g. with respect to information supply or a caring relationship). These anticipatory reactions are dependent on the expected size of the group moving in and on the severity of ID. Furthermore, while actually engaging in neighbouring, neighbours with and without ID appear to have experiences related to behaviour of residents, staff and features of housing facilities that are perceived as (in)congruent with regular neighbouring. CONCLUSIONS: It is concluded that interpersonal relationships between neighbours with and without ID should not be simplified in terms of attitudes that would be primarily prejudiced/stigmatising versus entirely accepting. Rather, our studies paint a more complex picture of sometimes ambivalent thoughts, feelings and interaction needs that all should be taken into account to make social integration a success.

The significance of neighbours: views and experiences of people with intellectual disability on neighbouring.

BACKGROUND: People with intellectual disability (ID) who live in regular neighbourhoods have experiences with their neighbours, which are important to understand when studying social integration. METHOD: This study describes and analyses the opinions on, and experiences with, neighbour relationships of 39 people with ID living in neighbourhood housing facilities. RESULTS: We found that, while the views of people with ID on 'good neighbouring' were consistent with 'neighbouring' described in sociological literature, their experiences may be influenced by an organisational context, the tendency to formalise relationships and apprehension towards meeting unfamiliar people. CONCLUSION: Understanding influential factors to neighbouring for people with ID may shed light on the processes involved in social integration of people with ID at a neighbourhood level. This paper contributes to understanding the opinions of people with ID on satisfactory neighbourhood relationships, and explores opportunities to improve them.

Short-sequence DNA repeats in prokaryotic genomes.

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10(-4) per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.

31P nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. I. Cytoplasmic membrane and total phospholipids.

1. At the growth temperature the total phospholipids isolated from Escherichia coli cells give rise to 31P-NMR spectra which indicate the existence of lamellar, isotropic and hexagonal phases. These phases are also detected by freeze fracture electron microscopy. In particular, the isotropic phase may contain lipidic particles (possibly inverted micelles) associated with the lamellar phase. 2. The cytoplasmic membrane isolated from E. coli cells grown at 37 degrees C is mainly lamellar at 25 degrees C, whereas at 37 and 45 degrees C the presence of some almost isotropic phospholipid motion is indicated. The possible significance of the isotropic phase for the functioning of the cytoplasmic membrane is discussed.

31P nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. II. Lipopolysaccharide and lipopolysaccharide-phospholipid complexes.

1. Freeze-fracture electron microscopy and 31P-NMR spectroscopy on native and electrodialyzed lipopolysaccharide from Escherichia coli K12 cells, both above and below the phase transition temperature, are described. 2. Freeze-fracture electron microscopy of native lipopolysaccharide shows ribbon-like structures below (0 and 22 degrees C) and large vesicles above (37 degrees C) the phase transition temperature. Electrodialyzed lipopolysaccharide (sodium salt) occurs in ribbon-like structures at 0, 22 and 37 degrees C if sodium lipopolysaccharide is hydrated in water. If sodium lipopolysaccharide is hydrated in Tris-HCL/NaCl buffer these ribbon-like structures occur only below the phase transition temperature. Above the phase transition temperature stacked sheets are observed. Moreover, in the latter case, the fracture planes contain particles and pits. Upon etching, sodium lipopolysaccharide when hydrated in water appears to form rods and when hydrated in buffer appears to form mainly stacked lamellae both above (37 degrees C) and below (0 degrees C) the phase transition temperature. 3. High resolution 31P-NMR spectra show that the chemical shifts of the phosphorus atoms in native lipopolysaccharide differ from those in electrodialyzed lipopolysaccharide, probably due to conformational and compositional (the disappearance of ions and (poly)electrolytes) changes. The 31P-NMR spectra of native lipopolysaccharide dispersed in Tris-HCL/NaCl buffer are very broad at 20 and at 40 degrees C indicating little motion. At 22 degrees C electrodialyzed lipopolysaccharide also gives a broad spectrum; at 40 degrees C the spectrum is narrower, indicating more motion, and two peaks are visible. After dispersion in H2o and subsequent addition of buffer, the spectrum of electrodialyzed lipopolysaccharide is narrow both at 20 and 40 degrees C, which can be correlated with the rods observed in freeze etching. After treatment with Ca2+, electrodialyzed lipopolysaccharide shows a very broad spectrum at 40 degrees C probably due to immobilization of the lipopolysaccharide. 4. Freeze-fracture electron microscopy and 31P-NMR spectroscopy of liposomes consisting of native lipopolysaccharide and total phospholipids indicate that the phospholipids and the lipopolysaccharide are mainly organized in bilayers. Lipopolysaccharide in such liposomes undergoes more motion than in the absence of phospholipids. Ca2+ does not influence this behaviour.

31P nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. III. The outer membrane.

1. The outer membrane of a phospholipase A-deficient mutant of Escherichia coli K12, isolated without the use of EDTA and lysozyme, showed the same freeze-fracture morphology as that seen in cells and remained stable for hours as observed by 31P-NMR. 2. 31P-NMR spectroscopy of the isolated outer membranes revealed that the lipopolysaccharide exists in the same physical state as in phospholipid-lipopolysaccharide liposomes and is most probably arranged in a bilayer at 37 degrees C. The outer membrane contains most or all of the phospholipids at 37 degrees C, and all the phospholipids at 20 degrees C, as a bilayer. 3. The 31P-NMR spectroscopy of the outer membranes from a mutant strain lacking the major outer membrane protein b, c and d (60% of the total outer membrane protein) yields virtually the same spectrum as the wild-type outer membranes, although most of the particles and pits which were observed in wild-type outer membranes in freeze-fracture electron microscopy were absent. 4. Whereas treatment of wild-type outer membranes with calcium ions has no effect on the 31P-NMR spectrum, treatment with EDTA results in more motion of the lipopolysaccharide.

Architecture of the outer membrane of Escherichia coli K12. IV. Relationship between outer membrane particles and aqueous pores.

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage lambda receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and equeous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.

Architecture of the outer membrane of Escherichia coli K12. II. Freeze fracture morphology of wild type and mutant strains.

Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.

Architecture of the outer membrane of Escherichia coli K12. I. Action of phospholipases A2 and C on wild type strains and outer membrane mutants.

Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded. It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components. The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants. Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous exogenous phospholipases. Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA. From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to to exogenous phospholipases.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response.

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.

Spectrum of invasive Haemophilus influenzae type b disease in adults.

A prospective nationwide surveillance of invasive Haemophilus influenzae type b disease among adults (greater than or equal to 16 years old) was conducted in Finland during 1985 through 1988. Thirty-one cases were identified (annual incidence, 0.22/100,000). Of these infections, 71% occurred in patients with severe underlying conditions. The overall case fatality rate was 26%. Septicemia (13 patients) and pneumonia (seven patients) were the most common clinical manifestations of H influenzae type b infection; the others were epiglottitis (six patients), meningitis (three patients), and arthritis (two patients). Epiglottitis occurred in significantly younger patients, all of whom were women and four of whom were previously healthy. Subtyping of the H influenzae type b isolates according to the major outer membrane protein subtype, biotype, and lipopolysaccharide serotype showed that patterns that were uncommon (14%) among children were more common (27%) in the adults.

Sequence variation in the hpd gene of nonencapsulated Haemophilus influenzae isolated from patients with chronic bronchitis.

The molecular diversity of protein D of nonencapsulated Haemophilus influenzae strains isolated from persistently infected patients with chronic bronchitis was studied by sequencing the hpd gene of four independently obtained isolates. The nucleotide (nt) sequences of the hpd genes of two strains were identical. The other two hpd sequences showed nt substitutions which were mostly synonymous. As a consequence the deduced amino acid (aa) sequences differed from the consensus sequence only by a few aa. No changes in the hpd genes were observed among the four variants of the four strains persisting in chronic bronchitis patients for 9, 11, 8 and 3 months, respectively, although variation in their major outer membrane proteins P2 and P5 occurred. We conclude that the hpd gene is conserved during chronic infections of nonencapsulated H. influenzae.

An efficient procedure for the generation of human monoclonal antibodies based on activation of human B lymphocytes by a murine thymoma cell line.

A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.