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PURPOSE: Water-bearing instruments and treatments in dental units produce aerosols originating from the dental unit waterlines (DUWLs), which are often microbially contaminated. Particularly, the presence of Legionella mainly realized as aerosols leads to a risk of infection in patients and dental staff. METHODS: Here, we record the general bacteriological status of DUWLs in Germany and investigated the prevalence of Legionella spp., with a focus on identification and occurrence of distinct species considering the various aspects of dental practice such as dental chair equipment, disinfection methods, and temperatures. RESULTS: Out of 3789 water samples of 459 dental practices, collected in the years 2019 and 2020, 36.4% were Legionella positive with predominance of L. anisa (97.89%) identified by MALDI-TOF biotyping. L. pneumophila was detected very rarely. Risk factor analysis revealed that temperatures >20°C are a significant factor for increased Legionella colonization. CONCLUSION: In order to minimize the risk of infection, routine monitoring of the water quality in dental chair units is recommended with regard to general microbiological loads and to the presence of Legionella as opportunistic pathogen as well as the regular application of routine disinfection procedures.
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Legionella , Humanos , Prevalencia , Factores de Riesgo , Alemania/epidemiología , DesinfecciónRESUMEN
BACKGROUND: In addition to the primary health care of refugees, their integration into the regular outpatient care system should be ensured. Initial data suggest that a gap of vaccination among (school) children of refugee families might have emerged in the period between the first general inspection on arrival (the first central health measure) and the transition to the local health care system. OBJECTIVES: The aim of this study was to obtain the opinion of practicing paediatricians regarding the vaccination status of refugee children to examine whether a variance in the measles, mumps, rubella (varicella) vaccination schedule might have emerged between the periods of initial admission and school enrolment examination. Evaluations of both inhibiting and promoting conditions should generate fields of action regarding the systematic integration into the regular health care system. METHOD: Qualitative interviews with experts as well as a quantitative questionnaire survey to measure the opinion of registered paediatricians in Münster were analyzed. RESULTS: The assessments showed that there was no clear vaccination gap among (school) children of refugee families. One challenge was the systematic integration into the local outpatient care system. Critical issues were inadequate vaccination education, language barriers, and frequent changes in location. The vaccination engagement and vaccination behaviour of refugees were assessed as most positive. International standards, in particular the sphere standards, attracted insufficient attention in practical implementation within the refugee relief programs. CONCLUSIONS: Based on the results, it is possible to identify fields of action for the prevention of vaccination gaps among refugees as well as for their systematic integration into the regular outpatient care system. The sphere standards as international standards should be incorporated more consciously.
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Refugiados , Atención Ambulatoria , Niño , Alemania , Humanos , Pediatras , VacunaciónRESUMEN
The worldwide spread of toxin-producing and multi-drug resistant bacteria in water, food and the environment is considered a major threat to human health. Drinking water quality is controlled by inspection of fecal indicators presence whereby viable contaminants will be efficiently reduced by chlorination which is a common process for disinfection. However, the all-out efficiency is arguable, because bacterial regrowth has been documented after disinfection. In this study, we investigated the stability of Shiga toxin producing Escherichia coli (STEC) and ß-lactamase expressing E. coli and Pseudomonas aeruginosa isolates, both equipped with multiple or single ß-lactamase resistance genes. The aim of the study was to analyze the efficiency of chlorine (Cl2) disinfection against shigatoxigenic or ß-lactamase producing bacteria. Cl2 reacts with the bacterial cells after first contact. Counts of antibiotic resistant E. coli were lower after short than upon extended Cl2 treatment. P. aeruginosa counts decreased moderately upon 15-60 min treatment with 1.2 mg Cl2/l, while cells adapted to tap water were not cultivable anymore. We assume that the bacterial physiology changed to a temporary non-cultivatable state at first Cl2 contact followed by resuscitation of some cells at later stages. STEC viability went down continuously at low Cl2 concentrations and these toxigenic E. coli isolates exhibited slightly increased stability to Cl2 treatment compared with non-toxigenic E. coli. Controlling the efficiency of disinfection, realistic counts of cultivatable cells are achieved after extended Cl2 action.
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Cloro/farmacología , Infecciones por Escherichia coli/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Escherichia coli Shiga-Toxigénica/efectos de los fármacos , beta-Lactamasas/metabolismo , Animales , Sustancias para la Guerra Química/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Viabilidad Microbiana/efectos de los fármacos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Microbiología del Agua , beta-Lactamasas/genéticaRESUMEN
Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSE's) affecting sheep and goats. Susceptibility of goats to scrapie is influenced by polymorphisms of the prion protein gene (PRNP) of the host. Five polymorphisms are associated with reduced susceptibility to TSE's. In the study presented here caprine samples from a scrapie eradication program on Cyprus were genotyped and further characterized using BioRad TeSeE rapid test, histological, immunohistochemical and biochemical methods. In total 42 goats from 20 flocks were necropsied from which 25 goats showed a positive result in the rapid test, a spongiform encephalopathy and an accumulation of pathological prion protein (PrPSc) in the obex. PrPSc deposits were demonstrated in the placenta, peripheral nervous and lymphoreticular system. Two animals showed PrPSc-accumulations in peripheral tissues only. By discriminatory immunoblots a scrapie infection could be confirmed for all cases. Nevertheless, slight deviations in the glycosylation pattern might indicate the presence of different scrapie strains. Furthermore scrapie samples from goats in the current study demonstrated less long term resistance to proteinase K than ovine or caprine BSE control samples. Reduced scrapie susceptibility according to the PRNP genotype was demonstrated (Fishers Exact test, p < 0.05) for the goats with at least one polymorphism (p = 0.023) at the six codons examined and in particular for those with polymorphisms at codon 146 (p = 0.016). This work characterizes scrapie in goats having implications for breeding and surveillance strategies.
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Enfermedades de las Cabras/genética , Enfermedades por Prión/veterinaria , Animales , Chipre/epidemiología , Femenino , Enfermedades de las Cabras/epidemiología , Enfermedades de las Cabras/patología , Cabras/genética , Enfermedades por Prión/epidemiología , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Proteínas Priónicas/metabolismoRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.
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Células Endoteliales/microbiología , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas Hemolisinas/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Mitocondrias/microbiología , Vesículas Secretoras/metabolismo , Factores de Virulencia/metabolismo , Apoptosis/efectos de los fármacos , Células CACO-2 , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/ultraestructura , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Datos de Secuencia Molecular , Factores de Virulencia/genética , Factores de Virulencia/farmacologíaRESUMEN
Plasma-activated water (PAW) generated from tap water has gained attention as a disinfectant when used directly in its pure form. Little is known about the application of PAW for bacterial inactivation in aqueous environments because its use in fluids results in dilutions. We investigated the effect of PAW in aqueous suspensions simulating such dilutions, and we focused on the minimal addition of PAW volumes to bacterial aqueous suspensions still resulting in high inactivation rates. The antimicrobial effect was highly dependent on the activation of PAW. An increase in activation power from 90 to 100 W resulted in a greater microbial reduction with an identical 10 min activation time. The susceptibility to PAW dilutions was analyzed in detail regarding nine Gram-negative species out of Enterobacterales and other waterborne microorganisms as well as four Gram-positive species present in two different matrices, in saline and in tap water, at high concentrations simulating massive contamination situations. For this purpose, the PAW activation setting of 90 W and 30 min was defined in order to be able to differentiate the limitations of inactivation in individual bacterial species. The Gram-negatives in saline demonstrated susceptibility when one volume unit of PAW was added. However, twice the PAW volume was necessary for inactivation when bacteria were present in tap water. Gram-positive microorganisms were more robust, indicated by prolonged contact times before inactivation. Our results indicate that PAW can be used for bacterial decontamination processes in aqueous environments when added in surplus. Optimized activation settings such as electric power to generate PAW and the contact times to the samples increase the effect of the inactivation a wide range of bacteria, regardless of their resistance profiles.
RESUMEN
Prion diseases entail the conversion of a normal host-encoded prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). Various PrP(C) types differing in banding profiles and detergent solubility are present in different tissues, but only few PrP(Sc) types have been generated although PrP(C) acts as substrate. We hypothesize that distinct PrP(C) subtypes may be converted more efficiently to PrP(Sc) than others. One prerequisite for the analysis is the identification of the PrP(C) subtypes present in the protein complexes. Metal binding to PrP(C) is one of the most prominent features of the protein which induces increased proteolysis resistance and structural changes which might play an important role in the conversion process. Here we analyzed the metal-induced structural PrP(C) transformation of two different Triton X-100 soluble PrP(C) types derived from human platelets and brains by changes in protein solubility. We found that zinc and copper rendered approximately half of total PrP(C) and mainly un- and low-glycosylated PrP(C) to the Triton insoluble fraction. Our results indicate the presence of at least two distinct PrP(C) subtypes by metal interactions. The differentiation of high and low soluble metal bound PrP(C) offers precious information about PrP(C) protein composition and provides approaches for analyzing the transformation efficiency to PrP(Sc).
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Metales/metabolismo , Proteínas PrPSc/química , Enfermedades por Prión/metabolismo , Priones/metabolismo , Cobre/química , Cobre/metabolismo , Glicosilación , Humanos , Metales/química , Octoxinol , Especificidad de Órganos , Proteínas PrPSc/metabolismo , Enfermedades por Prión/patología , Priones/química , Isoformas de Proteínas/metabolismo , Proteolisis , Solubilidad , Zinc/química , Zinc/metabolismoRESUMEN
Water systems in health care facilities can form reservoirs for Gram-negative bacteria. While planning a new neonatal intensive care unit (NICU), we performed a retrospective evaluation of potential risks from water-diverting systems on the existing NICU of our tertiary care University Hospital. During 2017 to 2023, we recorded nine nosocomial cluster events with bacterial pathogens in our NICU. Of these, three clusters of Gram-negative bacteria were potentially related to sink drains: A Klebsiella oxytoca, a Pseudomonas aeruginosa, and an Enterobacter hormaechei cluster were uncovered by clinical routine screening of patients and breastmilk samples. They were confirmed using whole-genome sequencing and a subsequent core genome multilocus sequence typing (cgMLST) algorithm. Our observations highlight that the implementation of sink drains in a NICU may have negative effects on patients' safety. Construction planning should concentrate on the avoidance of washbasins in patient rooms when redesigning sensitive areas such as NICUs.
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Algoritmos , Unidades de Cuidado Intensivo Neonatal , Recién Nacido , Humanos , Estudios Retrospectivos , Instituciones de Salud , Leche HumanaRESUMEN
Bacterial contamination is a problem in dental unit water lines with the consequence of implementing regular disinfection. In this study, the short-term impact of chlorine dioxide (ClO2) treatment was investigated on the microorganisms Legionella pneumophila and L. anisa, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The environmental background was proven as an important factor regarding the tolerance to 0.4 mg/L ClO2 as saline and phosphate-buffered saline resulted in a higher bacterial reduction than tap water. Gram-positive microorganisms demonstrated higher robustness to ClO2 than Gram-negative, and microorganisms adapted to tap water showed increased stability compared to cultured cells. At high densities, substantial numbers of bacteria were able to withstand disinfection, whereby the use of 4.6 mg/L ClO2 increased the inactivation rate. A massive cell decrease occurred within the first 5 minutes with subsequent plateau formation or slowed cell reduction upon further exposure. This biphasic kinetics cannot be explained by a ClO2 depletion effect alone, because the probability of bacterial subpopulations with increased tolerance should be taken into account, too. Our results prove high disinfection efficiency to microorganisms that were rather found in correlation to the level of bacterial contamination and background solutions than the chosen concentration for ClO2 treatment itself.
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The diagnosis of infections and protection against their transmission are aided greatly by determination of indicator proteins. However, protein assays are mostly restricted to single-antigen determinations and are often limited in sensitivity and specificity. Consequently, there is a large demand for high-sensitivity immunoassays for analysis of several antigens in protein suspensions. A novel immuno-polymerase chain reaction (PCR) assay is described for the simultaneous detection of central nervous system (CNS) indicators such as the neuron-specific enolase, the glial fibrillary acid protein, and the cellular prion protein. Coated antigens are immunocomplexed with specific antibodies and a DNA fragment is subsequently amplified by PCR. The PCR product obtained corresponds to the antigen signal. Background signals are a critical factor, primarily when using complex protein suspensions, but we were able to reduce background noise dramatically by including two heating steps, the first for protein denaturation and the second for detachment of immunocomplexed DNA, enabling optimal DNA amplification. Using these methods and depending on the antigen and antibody affinity, a sensitivity enhancement of 2 to 3 orders of magnitude is achieved for CNS indicator detection using the immuno-PCR approach compared with ELISA procedures carried out under identical conditions.
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Sistema Nervioso Central/metabolismo , Proteína Ácida Fibrilar de la Glía/análisis , Fosfopiruvato Hidratasa/análisis , Reacción en Cadena de la Polimerasa , Priones/análisis , Suspensiones/química , Animales , Anticuerpos/química , Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo , Bovinos , Sistema Nervioso Central/enzimología , ADN/química , ADN/metabolismo , Densitometría , Proteína Ácida Fibrilar de la Glía/inmunología , Fosfopiruvato Hidratasa/inmunología , Priones/genética , Priones/inmunología , Desnaturalización Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , OvinosRESUMEN
Prion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) which are essential for developing the disease but are heterogeneously expressed in brains. The disease can be transmitted to humans and animals through blood and blood products, however, little attention has been given to molecular characterization of PrP(C) in blood cells. In this presented study, we characterized phenotypically PrP(C) of platelets (plt) and characterized the proteins regarding their glycobanding profiles by quantitative immunoblotting using a panel of monoclonal antibodies. The glycosylation patterns of plt and brain PrP(C) were compared using the ratios of di-, mono-, and non-glycosylated prions. The detergent solubility of plt and brain PrP(C) was also analyzed. The distinct banding patterns and detergent solubility of plt PrP(C) differed clearly from the glycosylation profiles and solubility characteristics of brain PrP(C). Plt PrP(C) exhibited single or only few prion protein types, whereas brain PrP(C) showed more extensive banding patterns and lower detergent solubility. Plt PrP(C) are post-translational modified differently from PrP(C) in brain. These findings suggest other or less physiological functions of plt PrP(C) than in brain.
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Plaquetas/citología , Encéfalo/citología , Priones/metabolismo , Isoformas de Proteínas/metabolismo , Plaquetas/metabolismo , Encéfalo/metabolismo , Glicosilación , Humanos , Fenotipo , Priones/química , Isoformas de Proteínas/química , Procesamiento Proteico-Postraduccional , SolubilidadRESUMEN
The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.
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Western Blotting/métodos , Proteínas/análisis , Coloración y Etiquetado/métodos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Bovinos , Humanos , Immunoblotting , Ratones , Isoformas de Proteínas/análisis , OvinosRESUMEN
The main feature of prion diseases is the accumulation of infectious proteins (PrP(Sc)). Since PrP(Sc) results from conversion of cellular prion proteins (PrP(C)), differential expressed PrP(C) types may play an important role in the formation and conversion efficiency to specific PrP(Sc) forms. However, little is known about the PrP(C) expression, regulation and differentiation. Here, we demonstrate a new type of differentiation of overlapping PrP(C) isoforms in brain homogenates using differential SDS solubility. Low and highly soluble PrP(C) were detected along with various types of protein which are present in the brain of non-infected humans, sheep and cattle. Our findings provide evidence for the existence of several overlapping PrP(C) proteins exhibiting distinct glycotypes. The selection of defined PrP(C) types offers new possibilities for identifying highly efficient converting proteins and provides the potential for disease control.
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Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Química Encefálica , Bovinos , Detergentes/química , Humanos , Fenotipo , Proteínas PrPC/genética , Proteínas PrPSc/genética , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Isoformas de Proteínas/genética , Ovinos , Dodecil Sulfato de Sodio/química , SolubilidadRESUMEN
Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target's spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.
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Proteínas Bacterianas/análisis , Enterobacteriaceae/enzimología , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/análisis , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana , Enterobacteriaceae/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Meropenem/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
PURPOSE: Homeless persons have a high risk for tuberculosis. The prevalence of latent tuberculosis infection and the risk for a progression to active tuberculosis is higher in the homeless than in the general population. The objective was to assess the prevalence and risk factors of tuberculosis/latent tuberculosis infection in a homeless population in Germany. METHODS: Homeless individuals (n = 150) were enrolled in a cross-sectional study at three shelters in Münster, Germany (October 2017-July 2018). All participants were screened using an ELISPOT interferon-γ release assay (IGRA). Those participants tested positive/borderline by IGRA provided three sputa for microbiological analysis (line probe assay, microscopy, culture) and underwent a chest X-ray to screen for active pulmonary TB. Risk factors for tuberculosis/latent tuberculosis infection were analysed using a standardized questionnaire. RESULTS: Of the 142 evaluable IGRA, 21 (15%) were positive and two (1%) were borderline. No participant with a positive/borderline IGRA had an active tuberculosis as assessed by chest X-ray and microbiology. A negative IGRA was associated with a citizenship of a low-incidence country for tuberculosis (according to WHO, p = 0.01), low-incidence country of birth (p<0.001) or main residence in a low-incidence country in the past five years (p = 0.002). CONCLUSIONS: The prevalence of latent tuberculosis infection (diagnosed by a positive/borderline IGRA) was 16%; no active tuberculosis was detected. The highest risk for latent tuberculosis infection was found in patients from high-incidence countries. This population at risk should be either treated for latent tuberculosis infection or need to be monitored to early detect a progression into active disease.
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Personas con Mala Vivienda/estadística & datos numéricos , Tuberculosis Latente/epidemiología , Adulto , Estudios Transversales , Femenino , Alemania/epidemiología , Humanos , Tuberculosis Latente/terapia , Masculino , Prevalencia , Derivación y Consulta , Factores de RiesgoRESUMEN
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.
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Escherichia coli/metabolismo , Variación Genética , Técnicas para Inmunoenzimas/métodos , Reacción en Cadena de la Polimerasa/métodos , Toxina Shiga II/metabolismo , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Heces/microbiología , Humanos , Sensibilidad y Especificidad , Toxina Shiga II/genéticaRESUMEN
Bacterial intoxications represent a substantial public health concern with enterotoxins produced by Staphylococcus aureus among the most common causes of food poisoning. In addition to their role in the pathogenicity of food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter DNA to secondary detection antibodies. Therefore, the amino-modified reporter DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/microl) of staphylococcal enterotoxin B and staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for staphylococcal enterotoxins. The development of a stable antibody-DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-antigen complexes.
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Antígenos Bacterianos/aislamiento & purificación , Enterotoxinas/aislamiento & purificación , Inmunoconjugados/metabolismo , Técnicas para Inmunoenzimas/métodos , Reacción en Cadena de la Polimerasa/métodos , Staphylococcus aureus/inmunología , Superantígenos/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , Complejo Antígeno-Anticuerpo , Antígenos Bacterianos/inmunología , ADN/metabolismo , Enterotoxinas/inmunología , Enterotoxinas/metabolismo , Reproducibilidad de los Resultados , Superantígenos/metabolismoRESUMEN
Prion diseases are neurodegenerative disorders which cause Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. The infectious agent is a protease resistant isoform (PrP(Sc)) of a host encoded prion protein (PrP(C)). PrP(Sc) proteins are characterized according to size and glycoform pattern. We analyzed the glycoform patterns of PrP(C) obtained from humans, sheep, cattle and mice to find interspecies variability for distinct differentiation among species. To obtain reliable results, the imaging technique was used for measurement of the staining band intensities and reproducible profiles were achieved by many repeated immunoblot analysis. With a set of antibodies, we discovered two distinct patterns which were not species-dependent. One pattern is characterized by high signal intensity for the di-glycosylated isoform using antibodies that bind to the N-terminal region, whereas the other exhibits high intensity for protein bands at the size of the nonglycosylated isoform using antibodies recognizing the C-terminal region. This pattern is the result of an overlap of the nonglycosylated full-length and the glycosylated N-terminal truncated PrP(C) isoforms. Our data demonstrate the importance of antibody selection in characterization of PrP(C).
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Anticuerpos/inmunología , Proteínas PrPC/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Glicosilación , Humanos , Ratones , Datos de Secuencia Molecular , Fenotipo , Proteínas PrPC/química , Homología de Secuencia de Aminoácido , OvinosRESUMEN
Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates.
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Proteínas de Escherichia coli/metabolismo , Linfocinas/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Fenotipo , Estabilidad ProteicaRESUMEN
Contamination of water is a major burden in the public health setting of developing countries. We therefore assessed the quality of water samples in Gabon in 2013. The main findings were a contamination rate with coliforms of 13.5% and the detection of a possible environmental reservoir for extended spectrum beta-lactamase-producing bacteria.