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Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed.
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Doença de Crohn , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Humanos , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Ruminantes , Meios de Cultura , Fezes/microbiologiaRESUMO
Autoimmune diseases are thought to develop as a consequence of various environmental and genetic factors, each of which contributes to dysfunctional immune responses and/or a breakdown in immunological tolerance towards native structures. Molecular mimicry by microbial components is among the environmental factors thought to promote a breakdown in immune tolerance, particularly through the presence of cross-reactive epitopes shared with the human host. While resident members of the microbiota are essential promoters of human health through immunomodulation, defence against pathogenic colonisation and conversion of dietary fibre into nutritional resources for host tissues, there may be an underappreciated role of these microbes in the aetiology and/or progression of autoimmune disease. An increasing number of molecular mimics are being identified amongst the anaerobic microbiota which structurally resemble endogenous components and, in some cases, for example the human ubiquitin mimic of Bacteroides fragilis and DNA methyltransferase of Roseburia intestinalis, have been associated with promoting antibody profiles characteristic of autoimmune diseases. The persistent exposure of molecular mimics from the microbiota to the human immune system is likely to be involved in autoantibody production that contributes to the pathologies associated with immune-mediated inflammatory disorders. Here-in, examples of molecular mimics that have been identified among resident members of the human microbiota and their ability to induce autoimmune disease through cross-reactive autoantibody production are discussed. Improved awareness of the molecular mimics that exist among human colonisers will help elucidate the mechanisms involved in the breakdown of immune tolerance that ultimately lead to chronic inflammation and downstream disease.
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Doenças Autoimunes , Microbiota , Humanos , Mimetismo Molecular , Anaerobiose , Doenças Autoimunes/etiologia , AutoanticorposRESUMO
AIM: To develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP). METHODS AND RESULTS: Seven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM). CONCLUSIONS: MAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 µg ml-1 Mycobactin J. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.
Assuntos
Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Ágar , Animais , Técnicas Bacteriológicas/métodos , Meios de Cultura , Fezes/microbiologia , Indicadores e Reagentes , Paratuberculose/microbiologia , PolissorbatosRESUMO
A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M.caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture.
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Cromatografia de Afinidade/métodos , Mycobacterium bovis/isolamento & purificação , Tuberculose Bovina/diagnóstico , Medicina Veterinária/métodos , Animais , Bovinos , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e Especificidade , Espanha , Reino UnidoRESUMO
BACKGROUND: The European badger is an important wildlife reservoir of Mycobacterium bovis implicated in the spread of bovine tuberculosis in the United Kingdom and Ireland. Infected badgers are known to shed M. bovis in their urine and faeces, which may contaminate the environment. To aid bovine tuberculosis control efforts novel diagnostic tests for detecting infected and shedding badgers are needed. We proposed development of a novel, rapid immunochromatographic lateral flow device (LFD) as a non-invasive test to detect M. bovis cells in badger faeces. Its application in combination with immunomagnetic separation (IMS) to detect Mycobacterium bovis cells in badger faeces is reported here. RESULTS: A novel prototype LFD for M. bovis cells was successfully developed, with unique specificity for M. bovis and a limit of detection 50% (LOD50%) of 1.7 × 104 M. bovis cells/ml. When IMS was employed to selectively capture and concentrate M. bovis cells from badger faeces prior to LFD testing, the LOD50% of the IMS-LFD assay was 2.8 × 105 M. bovis cells/ml faecal homogenate. Faeces samples collected from latrines at badger setts in a region of endemic bovine tuberculosis infection were tested; 78 (18%) of 441 samples tested IMS-LFD assay positive, whereas 140 (32%) tested IMS-qPCR positive (Kappa agreement -0.009 ± 0.044, p = 0.838). Subsequently, when 130 faeces samples from live captured, or captive, badgers of known infection status (on the basis of StatPak, interferon-γ and/or culture results) were tested, the IMS-LFD assay had higher relative diagnostic specificity (Sp 0.926), but poorer relative diagnostic sensitivity (Se 0.081), than IMS-qPCR (Sp 0.706, Se 0.581) and IMS-culture (Sp 0.794, Se 0.436). CONCLUSIONS: The novel IMS-LFD assay, although very specific for M. bovis, has low analytical sensitivity (indicated by the LOD50%) and would only detect badgers shedding high numbers of M. bovis (>104-5 cells/g) in their faeces. The novel LFD would, therefore, have limited value as a non-invasive test for badger TB surveillance purposes but it may have value for alternative veterinary diagnostic applications.
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Cromatografia de Afinidade/veterinária , Fezes/microbiologia , Separação Imunomagnética/veterinária , Mustelidae/microbiologia , Mycobacterium bovis/isolamento & purificação , Animais , Anticorpos Antibacterianos/análise , Separação Imunomagnética/métodos , Sensibilidade e EspecificidadeRESUMO
Introduction. Bacteroides fragilis is a Gram-negative anaerobe that is a member of the human gastrointestinal microbiota and is frequently found as an extra-intestinal opportunistic pathogen. B. fragilis comprises two distinct groups - divisions I and II - characterized by the presence/absence of genes [cepA and ccrA (cfiA), respectively] that confer resistance to ß-lactam antibiotics by either serine or metallo-ß-lactamase production. No large-scale analyses of publicly available B. fragilis sequence data have been undertaken, and the resistome of the species remains poorly defined.Hypothesis/Gap Statement. Reclassification of divisions I and II B. fragilis as two distinct species has been proposed but additional evidence is required.Aims. To investigate the genomic diversity of GenBank B. fragilis genomes and establish the prevalence of division I and II strains among publicly available B. fragilis genomes, and to generate further evidence to demonstrate that B. fragilis division I and II strains represent distinct genomospecies.Methodology. High-quality (n=377) genomes listed as B. fragilis in GenBank were included in pangenome and functional analyses. Genome data were also subject to resistome profiling using The Comprehensive Antibiotic Resistance Database.Results. Average nucleotide identity and phylogenetic analyses showed B. fragilis divisions I and II represent distinct species: B. fragilis sensu stricto (n=275 genomes) and B. fragilis A (n=102 genomes; Genome Taxonomy Database designation), respectively. Exploration of the pangenome of B. fragilis sensu stricto and B. fragilis A revealed separation of the two species at the core and accessory gene levels.Conclusion. The findings indicate that B. fragilis A, previously referred to as division II B. fragilis, is an individual species and distinct from B. fragilis sensu stricto. The B. fragilis pangenome analysis supported previous genomic, phylogenetic and resistome screening analyses collectively reinforcing that divisions I and II are two separate species. In addition, it was confirmed that differences in the accessory genes of B. fragilis divisions I and II are primarily associated with carbohydrate metabolism and suggest that differences other than antimicrobial resistance could also be used to distinguish between these two species.
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Infecções Bacterianas , Bacteroides fragilis , Humanos , Bacteroides fragilis/genética , Filogenia , Genômica , Bases de Dados FactuaisRESUMO
Footbaths containing disinfectants are used on dairy farms to reduce the spread of digital dermatitis; however, they commonly become contaminated with manure. This trial investigated the physical properties and microbial composition of dairy cow manure from two production systems and examined whether the source of manure impacted the efficacy of footbathing disinfectants. Manure was collected from eighteen dairy cows, nine housed and fed grass silage (HOUSED) and nine at pasture (PASTURE). The pH and dry matter content was determined, total DNA was extracted and the region v3-v4 of the 16s rRNA gene sequenced. The efficacy of formalin and two trial products (TP1: peracetic acid and hydrogen peroxide; TP2: chlorocresol and triamine) was evaluated when mixed with manure from the two production systems. Production system differences were found in manure dry matter content, bacterial microbiome and the efficacy of both trial footbathing products but not formalin. The properties of manure affected the results of laboratory testing and therefore have the potential to influence footbathing disinfectant efficacy when footbaths are contaminated with manure. Further research into the impact of organic contaminants on the efficacy of disinfectants could facilitate the development of improved testing programmes and disinfectant products.
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Bacterial pathogens represent a safety concern in the food industry, and this is amplified by the lack of sensing devices that can be applied on-site by non-trained personnel. In this study, peroxidase-mimicking activity of gold nanostars was exploited to develop a user-friendly colourimetric sensor. A smartphone was exploited as an image reader and analyser, empowered with a novel App developed in-house. The mobile App was evaluated and compared with a commercial smartphone App for its capability to quantify generated colourimetric signals. A major obstacle found with sensors relying on gold nanozymes is the fact that modification of the surface of gold nanoparticles with biorecognition elements generally lead to a suppression of their nanozyme activity. This drawback was overcome by introducing an autocatalytic growth step, which successfully restored the peroxidase-mimicking activity through generation of new gold nanoseeds acting as catalytic centres. A proof-of-concept using this sensing mechanism was developed targeting Mycobacterium bovis, a zoonotic pathogen primarily found in cattle but that can be transmitted to humans by consumption of contaminated food and cause tuberculosis disease. The resulting smartphone-based immunological sensor has shown promising results with a linear response between 104 - 106 CFU/mL, enabling detection of M. bovis at concentrations as low as 7.2·103 CFU/mL in buffer conditions. It is anticipated that the concept of the developed approach will have applicability in many fields relying on smartphone-based biosensing.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Mycobacterium bovis , Técnicas Biossensoriais/métodos , Ouro , Ligantes , Mycobacterium bovis/isolamento & purificação , Peroxidases , SmartphoneRESUMO
This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.
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Anticorpos Antibacterianos , Técnicas Bacteriológicas/métodos , Separação Imunomagnética/métodos , Mycobacterium bovis/isolamento & purificação , Peptídeos , Tuberculose Bovina/diagnóstico , Animais , Anticorpos Monoclonais , Bovinos , Linfonodos/microbiologia , Camundongos , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Sensibilidade e Especificidade , Tuberculose Bovina/microbiologiaRESUMO
In the present study, a facile, eco-friendly, and controlled synthesis of gold nanoparticles (Au NPs) using Prunus nepalensis fruit extract is reported. The biogenically synthesized Au NPs possess ultra-active intrinsic peroxidase-like activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Chemical analysis of the fruit extract demonstrated the presence of various bioactive molecules such as amino acids (l-alanine and aspartic acids), organic acids (benzoic acid and citric acid), sugars (arabinose and glucose), phenolic acid, and bioflavonoids (niacin and myo-inositol), which likely attributed to the formation of stable biogenic Au NPs with excellent peroxidase-mimicking activity. In comparison with the natural horseradish peroxidase (HRP) enzyme, the biogenic Au NPs displayed a 9.64 times higher activity with regard to the reaction velocity at 6% (v/v) H2O2, presenting a higher affinity toward the TMB substrate. The Michaelis-Menten constant (KM) values for the biogenic Au NPs and HRP were found to be 6.9 × 10-2 and 7.9 × 10-2 mM, respectively, at the same concentration of 100 pM. To investigate its applicability for biosensing, a monoclonal antibody specific for Mycobacterium bovis (QUBMA-Bov) was directly conjugated to the surface of the biogenic Au NPs. The obtained results indicate that the biogenic Au NPs-QUBMA-Bov conjugates are capable of detecting M. bovis based on a colorimetric immunosensing method within a lower range of 100 to 102 cfu mL-1 with limits of detection of â¼53 and â¼71 cfu mL-1 in an artificial buffer solution and in a soft cheese spiked sample, respectively. This strategy demonstrates decent specificity in comparison with those of other bacterial and mycobacterial species. Considering these findings together, this study indicates the potential for the development of a cost-effective biosensing platform with high sensitivity and specificity for the detection of M. bovis using antibody-conjugated Au nanozymes.
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Nanopartículas Metálicas , Mycobacterium bovis , Prunus , Frutas/química , Ouro/química , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/análise , Nanopartículas Metálicas/química , Mycobacterium bovis/metabolismo , Prunus/metabolismoRESUMO
The process of risk assessment which should inform and help identify clinical needs is often seen as a tick box and task-focussed approach. While on the surface this provides a sense of security that forms have been completed, we often fail to communicate in a meaningful manner about the clinical needs identified, which would assist in supporting the care planning delivery processes. A clinical practice improvement (CPI) project implemented a care zoning framework as an evidenced-based process that provides pragmatic support to nurses who are required to continually assess, implement, and evaluate plans to address clinical need across three acute mental health inpatient settings. Risk descriptors informed by the New South Wales (NSW) Mental Health Assessment & Outcome Tools (MHAOT) criteria were developed and described in behavioural contexts in order to improve the project's reliability and translation. A pragmatic traffic light tool was used to share clinical information across three agreed care zones, red (high clinical need), amber (medium clinical need), and green (low clinical need). Additionally nurses were asked to utilise a shift review form in the context of supporting the recording of care zoning and promoting action-orientated note writing. The introduction of care zoning has enthused the nursing teams and the mental health service to adopt care zoning as a supervisory framework that increases their capacity to communicate clinical needs, share information, and gain invaluable support from one another in addressing clinical needs. This includes increased opportunities for staff to feel supported in asking for assistance in understanding and addressing complex clinical presentations.
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Hospitalização , Serviços de Saúde Mental/organização & administração , Necessidades e Demandas de Serviços de Saúde , Humanos , Avaliação de Processos em Cuidados de Saúde , Medição de RiscoRESUMO
A ferromagnetic gold nanoparticle based immune detection assay, exploiting the enhanced signal amplification of inorganic nanozymes, was developed and evaluated for its potential application in the detection of Mycobacterium tuberculosis complex (MTBC) organisms, and simultaneous identification of Mycobacterium bovis. Ferromagnetic gold nanoparticles (Au-Fe3O4 NPs) were prepared and their intrinsic peroxidase-like activity exploited to catalyse 3,3',5',5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). When the Au-Fe3O4 NPs were functionalised by direct coupling with MTBC-selective antibodies, a nanoparticle based immune detection assay (NPIDA) was developed which could detect Mycobacterium tuberculosis (MTB) and differentiate M. bovis. In the assay, the intrinsic magnetic capability of the functionalised Au-Fe3O4 NPs was used in sample preparation to capture target bacterial cells. These were incorporated into a novel immunoassay which used species selective monoclonal antibodies (mAb) to detect bound target. The formation of a blue TMB oxidation product, with a peak absorbance of 370 nm, indicated successful capture and identification of the target. The detection limit of the NPIDA for both MTB and M. bovis was determined to be comparable to conventional ELISA using the same antibodies. Although limited matrix effects were observed in either assay, the NPIDA offers a reduced time to confirmatory identification. This novel NPIDA was capable of simultaneous sample concentration, purification, immunological detection and speciation. To our knowledge, it represents the first immune-based diagnostic test capable of identifying MTBC organisms and simultaneously differentiating M. bovis.
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Nanopartículas Metálicas , Mycobacterium bovis , Mycobacterium tuberculosis , Catálise , Diferenciação Celular , Testes Diagnósticos de Rotina , Ouro , Peróxido de Hidrogênio , ImunoensaioRESUMO
A novel electrochemical immunosensor for the detection of the important marine biotoxins domoic acid (DA) and okadaic acid (OA) was developed. The sensors used carbon black modified screen-printed electrodes (CB-SPE) obtained using a high-throughput method. The electrochemical performance and stability of CB modified SPEs and bare carbon SPEs (c-SPEs) were compared using cyclic voltammetry and electrochemical impedance spectroscopy. CB-SPEs showed improved long-term (at least six months) stability and electro-catalytic properties compared with c-SPEs. The CB-SPEs were bio-functionalized with DA or OA protein-conjugates and used to develop two indirect competitive immunosensors using differential pulse voltammetry (DPV). The DPV signals obtained for the OA and DA immunosensors fitted well to four-parameter dose-response curves (R2 > 0.98) and showed excellent LODs (LOD = 1.7 ng mL-1 for DA in buffer; LOD = 1.9 ng mL-1 for DA in mussel extract; LOD = 0.15 ng mL-1 for OA in buffer; LOD = 0.18 ng mL-1 for OA in mussel extract). No significant interference of the naturally co-occurring marine toxins saxitoxin, tetrodotoxin and OA was detected for the DA immunosensor. Similarly, for the OA immunosensor saxitoxin, tetrodotoxin and DA did not cross-react and very limited interference was observed for the dinophysis toxins DTX-1, DTX-2 and DTX-3 (OA congeners). Moreover, both immunosensors remained stable after at least 25 days of storage at 4 °C. This work demonstrates the potential of affordable, mass-produced nanomaterial-modified SPEs for marine toxin detection in shellfish.
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Técnicas Biossensoriais , Animais , Técnicas Eletroquímicas , Eletrodos , Imunoensaio , Ácido Caínico/análogos & derivados , Toxinas Marinhas , Ácido Okadáico/análise , FuligemRESUMO
A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 microg/kg, a limit of detection of 31 microg/kg, and a working range of 31-174 microg/kg. The midpoint on the standard matrix calibration curve is 80 microg/kg, half the current regulatory limit. Inter- and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were obtained using a certified reference material (R(2) = 0.99), laboratory reference material, and naturally contaminated mussel samples (R(2) = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins.
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Técnicas Biossensoriais , Imunoensaio , Toxinas Marinhas/química , Ácido Okadáico/análise , Animais , Cromatografia Líquida , Piranos/análise , Frutos do Mar , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The American College of Surgeons (ACS) and the Accreditation Council for Graduate Medical Education (ACGME) are committed to promoting patient safety through education. In view of the critical role of residents in the delivery of safe patient care, the ACS and ACGME sponsored jointly a national consensus conference to initiate the development of a curriculum on patient safety that may be used across all surgical residency programs. CONCLUSIONS: National leaders in surgery with expertise in surgical care and surgical education, patient safety experts, medical educators, key stakeholders from national organizations, and surgical residents were invited to participate in the conference. Attendees considered patient safety issues within the context of the 6 core competencies defined by the ACGME and American Board of Medical Specialties (ABMS). Discussions resulted in the development of a curriculum matrix that includes listings of patient safety topics, teaching and learning strategies, and assessment methods. Guidelines for implementation and dissemination are also provided. The curriculum content underscores the need to create an organizational culture of safety and focuses on both individuals and systems. Individual residency programs may prioritize the curriculum content based on their specific needs. The ACS and ACGME will pursue development of educational modules to address the curriculum content, disseminate helpful information, and assist in implementation of new educational interventions. This effort has the potential to positively impact residency education in surgery, help surgical program directors address the core competencies, and enhance patient safety.
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Currículo/normas , Internato e Residência/normas , Assistência Perioperatória/normas , Segurança/normas , HumanosRESUMO
The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.
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Anticorpos Imobilizados/química , Anticorpos Monoclonais/química , Separação Imunomagnética/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Peptídeos/química , Animais , Técnicas Bacteriológicas/métodos , Sítios de Ligação , Biotinilação , Técnicas de Visualização da Superfície Celular , Feminino , Camundongos Endogâmicos BALB C , Leite/microbiologia , Paratuberculose/diagnóstico , Ruminantes/microbiologiaRESUMO
Immunomagnetic separation (IMS) represents a simple but effective method of selectively capturing and concentrating Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), from tissue samples. It is a physical cell separation technique that does not impact cell viability, unlike traditional chemical decontamination prior to culture. IMS is performed with paramagnetic beads coated with M. bovis-specific antibody and peptide binders. Once captured by IMS, M. bovis cells can be detected by either PCR or cultural detection methods. Increased detection rates of M. bovis, particularly from non-visibly lesioned lymph node tissues from bTB reactor animals, have recently been reported when IMS-based methods were employed.
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Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Separação Imunomagnética/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mycobacterium bovis/genética , Mycobacterium bovis/isolamento & purificação , Tuberculose/veterinária , Animais , BovinosRESUMO
BACKGROUND: Enteroviral meningitis has traditionally been diagnosed by cell culture. More recently, molecular techniques and shell vials with a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (BGMK cells) (Super E-Mix) have been described. OBJECTIVE: We compared the results of this new cell culture technique with two reverse transcriptase polymerase chain reaction (RT-PCR) techniques and conventional cell culture to assess the accuracy of these various methods. STUDY DESIGN: 2 ml of cerebrospinal fluid (CSF) was obtained from 72 patients with suspected viral meningitis. 600 microl was used for conventional cell culture and 200 microl was inoculated to each of two Super E-Mix vials. One vial was incubated for 24 h, then stained with pan-enterovirus antibody, and the other was examined for CPE daily for 5 days and stained when positive or at the end of incubation. The final aliquot (500 microl) was centrifuged at 25,000g, and the pellet in 140 microl of supernatant was used for ribonucleic acid (RNA) extraction and tested by commercial single-step (Chemicon, Temecula, CA) and two-step (Argene, Varilhes, France) RT-PCR methods. RESULTS: Conventional culture was 51% sensitive and 100% specific. The Super E-Mix was slightly higher (sensitivity 76%, specificity 100%). The Chemicon and Argene PCR methods had a sensitivity of 93% and 88% and specificity of 90% and 97%, respectively. CONCLUSION: The Super E-Mix procedure had greater sensitivity than conventional cell culture, but RT-PCR was the most sensitive technique with this type of specimen. The RT-PCR methods performed equally.
Assuntos
Técnicas de Cocultura/métodos , Infecções por Enterovirus/diagnóstico , Meningite Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adolescente , Adulto , Animais , Células CACO-2 , Líquido Cefalorraquidiano/virologia , Criança , Pré-Escolar , Chlorocebus aethiops , Enterovirus/genética , Enterovirus/crescimento & desenvolvimento , Enterovirus/isolamento & purificação , Infecções por Enterovirus/líquido cefalorraquidiano , Infecções por Enterovirus/virologia , Feminino , Engenharia Genética , Humanos , Lactente , Masculino , Meningite Viral/líquido cefalorraquidiano , Meningite Viral/virologia , RNA Viral/análise , Cultura de VírusRESUMO
Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 191 (68.2%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 79 M. bovis positive lymph node samples (27 (13.1%) VL and 52 (70.3%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 70.3% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.