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Production, characterisation and potential application of a novel monoclonal antibody for rapid identification of virulent Listeria monocytogenes.

A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) 'mAb2B3' was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non-Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1x10(7) cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody 'mAb2B3', described herein.

Animals↗

Bovine tuberculosis in India: potential basis for zoonosis.

Our laboratory has designed a specific nested-PCR (N-PCR) assay, based on the hupB gene of Mycobacterium tuberculosis (Rv2986c) and Mycobacterium bovis (Mb3010c) as a method to differentiate these closely related species. The present paper deciphers the utility of this assay for identification of pathogenic Mycobacteria in clinical samples. Extra-pulmonary clinical samples obtained from cattle and humans were investigated. Pre-dominance of M. tuberculosis (15.7%) and M. bovis (26.8%) was seen in humans and cattle, respectively. However, more importantly, both mycobacterial pathogens (mixed infection) were identified in a number of samples. In humans 8.7% of the samples and 35.7% in cattle were classified as mixed infection. The detection of mixed infection with the mycobacterial pathogenic duo in humans and bovines denotes the prospect of potential transmission of these pathogens from humans to cattle (zoonosis) and vice versa (reverse zoonosis).

Animals↗

An amplification-free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up-converting phosphor technology.

OBJECTIVES: Development of a generally applicable sensitive hybridization-based assay devoid of any target amplification for the detection and identification of (pathogenic) bacterial and viral species. DESIGN AND METHODS: Using a sandwich hybridization format, the presence of a species-specific nucleic acid sequence is detected by means of Lateral Flow (LF) and Up-converting Phosphor Technology (UPT, a luminescent tracer). As a model, detection of the pathogen Streptococcus pneumoniae was investigated using a probe against the single-copy lytA gene. RESULTS: Detection of S. pneumoniae (in particular a 1200 p lytA sequences) required less than 1 ng of genomic DNA (approximate size 2.2 Mb). Hybridization and detection were performed in a complex background containing 10 microg fish sperm DNA. CONCLUSIONS: The results indicate the possibility to detect nucleic acid targets in nonamplified DNA samples using easy, inexpensive, amplification-free hybridization-based assays and the ultra sensitive UPT reporters. Employment of UPT allows to by-pass target amplification and therefore brings genetic-based testing a step closer to the point-of-care environment. Detection of S. pneumoniae with only 1 ng of DNA indicates a potential for applications in the field of infectious diseases.

DNA, Bacterial↗

Applications of high-performance liquid chromatography in bacteriology. I. Determination of metabolites.

The chemotaxonomic approach to the identification of pathogenic bacteria for clinical purposes is surveyed. Primary interest is focused on the applications of HPLC to the determination of metabolic products from anaerobic bacteria. The use of HPLC is attractive as different classes of short-chain acids can be determined in a single analysis. Chromatographic conditions are extensively described, emphasizing the effects of changing variables on the HPLC profiles of analytes. The application of labelling procedures to bacterial metabolites can markedly increase the sensitivity of the analysis of pathological fluids. HPLC appears to be potentially useful in clinical bacteriology for the diagnosis of anaerobic infections.

Bacteria, Anaerobic↗

What diagnostic tests are needed for community-acquired pneumonia?

Diagnostic tests play an important part in the evaluation and management of patients with CAP. Tests have key roles in diagnosing the presence of CAP and in assessing severity. An ideal test for microbiologic diagnosis in CAP is not yet available, and initial antimicrobial therapy usually is empiric. Nonetheless, when appropriately applied and correctly performed, tests for the identification of pathogens in CAP are useful and cost-effective.

Biopsy, Needle↗

Intravenous immunoglobulin treatment of immunodeficiency disorders.

ADDs can occur as primary genetic disorders or may develop secondary to various other conditions, including infections, trauma, malnutrition, and protein-losing states. Although antibiotics are the first-line therapy for acute infection, using them prophylactically can select for resistant organisms. IM ISG and fresh frozen plasma were the principal agents for antibody-replacement therapy until the advent of IVIG 2 decades ago. IVIG is now the definitive product for antibody-replacement therapy. Although IVIG has a long history of safety regarding the infectious pathogens, the identification of more than 100 patients with non-A, non-B hepatitis apparently acquired from a single product prompted additional modifications, improving the safety profile of IVIG. Despite the excellent safety record of IVIG, the unexpected occurrence of hepatitis in some recipients served as a reminder that IVIG is a biologic product derived from human plasma. Newer products are being developed that may supplement polyvalent IVIG including humanized MAbs and hyperimmune IVIG preparations to address specific clinical requirements.

Child↗

Antimicrobial therapy of acute otitis media: review of treatment recommendations.

OBJECTIVE: This paper reviews 3 previously published articles that provided recommendations for antimicrobial therapy of acute otitis media (AOM) and combines them to provide revised recommendations. BACKGROUND: AOM is one of the most common pediatric infections requiring a prescription for an antimicrobial agent. The optimal approach to treatment of AOM requires early, efficacious, and practical therapy. Several experts and organizations have developed recommendations for the management of AOM, but the number of these may overwhelm the busy primary care practitioner. A MEDLINE search of the pediatric and infectious disease literature on AOM treatment recommendations was used to select 3 representative, previously published articles for this review. When selecting an agent, physicians should consider in vitro activity, particularly against drug-resistant Streptococcus pneumoniae; pharmacokinetics; adverse events; palatability of the suspension; and cost. In addition, physicians' clinical experience is an important determinant. CONCLUSIONS: Amoxicillin is recommended as the first-line agent to treat uncomplicated AOM. For clinical treatment failures after 3 days of amoxicillin, recommended antimicrobial agents include oral amoxicillin/clavulanate, cefuroxime axetil, cefprozil, cefpodoxime proxetil, and intramuscular (i.m.) ceftriaxone. I.m. ceftriaxone should be reserved for severe cases or patients in whom noncompliance is expected. Tympanocentesis for identification of pathogens and susceptibility to antimicrobial agents is recommended for selection of third-line agents.

Anti-Infective Agents↗

Identification of a novel pathogen-induced gene encoding a leucine-rich repeat protein expressed in phloem cells of Capsicum annuum.

The CALRR1 gene, expressed in pepper leaves following infection by Xanthomonas campestris pv. vesicatoria, encodes a secreted leucine-rich repeat (LRR) with five tandem repeats of a 24-amino-acid LRR motif. Northern blot analyses revealed that CALRR1 is not constitutively expressed in pepper plants, but is strongly induced upon the infection by X. campestris pv. vesicatoria, Phytophthora capsici, Colletotrichum coccodes and Colletotrichum gloeosporioides on leaves. CALRR1 was not systemically induced in upper leaves by bacterial infection. The inoculation of bacterial live cells, and treatment with dead cells and culture filtrates of pathogenic or nonpathogenic bacteria triggered the accumulation of CALRR1 transcripts. Treatment with signaling molecules, including salicylic acid (SA), ethylene (ET), methyl jasmonate (MeJA), dl-beta-amino-n-butyric acid (BABA) and benzothiadiazole (BTH), did not activate the transcription of the CALRR1 gene, indicating that CALRR1 expression is not regulated by defense signaling pathways activated by these molecules. CALRR1 was induced by treatment with high salinity, abscisic acid (ABA) and wounding, but not by drought and cold stress. An in situ hybridization study showed that CALRR1 mRNA was localized in phloem tissues of leaves, stems, and green fruits of pepper plants during the pathogen infection and ABA exposure. The location characteristics and the spatio-temporal expression pattern of CALRR1 suggest that it may play a role in protecting phloem cells against biotic and abiotic stresses affecting phloem function.

Amino Acid Sequence↗

Evaluation of a wound scoring method for patients undergoing cardiac surgery.

The main endpoint of studies evaluating antimicrobial prophylaxis in surgical patients is wound morbidity. This should consist of more than the solitary declaration of the rate of wound infection. In this study, a modified version of ASEPSIS (a wound scoring system) has been prospectively evaluated in 1000 patients undergoing cardiac surgery. Twenty-one patients (2.1%) had an infected sternal wound. The components of ASEPSIS most significantly associated with a sternal wound infection were: the presence of a purulent exudate, a postoperative stay > 14 days, the identification of pathogenic organisms, and the use of antibiotics (P < 0.0001). Although ASEPSIS is a useful mechanism for conveying information about wounds, it does ignore bleeding into wounds and the extent that troublesome wounds need to be dressed after surgery.

Aged↗

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.

Base Sequence↗

Is multiple chemical sensitivity a clinically defined entity?

In 1996 a WHO/IPCS Workshop has suggested to use as an appropriate descriptor of MCS the broader term "Idiopathic Environmental Intolerances (IEI)", in order to incorporate "a number of disorders sharing similar symptomatologies". Research was strongly encouraged. The following points have been put forward as a precondition to define MCS as a clinical entity: (a) establishment of diagnostic criteria, (b) identification of pathogenic mechanisms, together with, (c) an explanation of relationship between exposures and symptoms. Against this background, progress made in the fields of sensory physiology and neurobehaviour research must be debated. In particular, recent results on processing of cognitive stimuli have to be considered. IEI/MCS patients exhibited differences vs. controls in their reactions to intranasal challenge, consistent with changes in cognitive processing of suprathreshold chemosensory information. Trait anxiety and focus of attention have clearly been identified as major components in eliciting neurobehavioural MCS symptoms. Hence, the question as to whether MCS should be regarded as a clinically defined entity remains controversial, but important progress can be noticed in elucidating and defining the nature of this phenomenon, by a combined effort of several disciplines (toxicology and behavioural toxicology, psychology and psychophysiology, and clinical medicine). The new situation will call for a re-evaluation of traditional positions.

Cognition↗

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins.

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

Adhesins, Escherichia coli↗

Signal transduction in plant immunity.

Significant recent advances in the understanding of plant defense mechanisms include the isolation and characterization of resistance genes against bacterial, fungal and viral pathogens, the identification of genes involved in cell death, and the demonstration of the involvement of reactive oxygen species and salicylic acid in the signal-transduction pathways for expression of induced resistance.

Cell Death↗

Cytotoxic T cell responses to intracellular pathogens.

Host defense against intracellular pathogens is thought to require cytotoxic T cells. Recent studies have investigated the impact of host cell lysis and cytokine production by cytotoxic T lymphocytes on the fate of intracellular pathogens. The identification of two mechanisms of lysis induced by cytotoxic T lymphocytes--the granule exocytosis pathway and the Fas-FasL interaction--have provided new insight into the role of cytotoxic T lymphocyters in immunity to infection.

Animals↗

Respiratory chain defects in hereditary spastic paraplegias.

Hereditary Spastic Paraplegias (HSPs) are heterogeneous neurodegenerative disorders whose etiopathogenesis is still unclear. The identification of pathogenic mutations in a gene (SPG7) encoding a mitochondrial metalloprotease suggested that oxidative phosphorylation (OXPHOS) alterations might underlie HSP in a subgroup of patients. We performed clinical, morphological, biochemical, and molecular genetic studies in six HSP patients and in six sporadic patients to investigate OXPHOS in muscle biopsies. Complicated and pure forms were included in our study. Morphological alterations of the type seen in OXPHOS-related disorders were found in three patients. Five patients showed an isolated defect of complex I activity. No mutations in the SPG7 gene were detected. Our results suggest that OXPHOS defects in HSP patients are more common than previously believed.

Adolescent↗

Fungal infection in lung transplantation.

Fungal infection remains a significant cause of postoperative morbidity and mortality in lung transplant recipients. The lung recipient remains the only solid-organ allograft continuously open to the environment and to the myriad of fungal spores and pathogens. Many factors may predispose to fungal infection in these patients, including: preoperative chronic lung diseases and inherent palliative immunosuppression, intraoperative complications such as abnormalities in the bronchial anastomosis or lung injury, and postoperative complications such as enhanced immunosuppression for early rejection, graft dysfunction, concurrent viral and bacterial infections, and bronchiolitis obliterans syndrome. The risk factors and time course for fungal infection in lung transplant recipients parallel the observations in other solid-organ transplant recipients. Early fungal infections are related to surgical complications, while the period of 1-6 months reflect opportunistic, relapsed, or residual infections; fungal infections greater than 6 months and thereafter are usually associated with treatments for chronic rejection or bronchial airway mechanical abnormalities. The majority of fungal infections in lung transplant recipients involve Aspergillus species, followed by Candida, Pneumocystis, Cryptococcus, geographically-restricted agents, and newly emerging fungal pathogens. The identification of at-risk patients (preoperatively and postoperatively) is essential in implementing prophylaxis or preemptive management. Some anti-fungal strategies and future options for clinical research are discussed.

Antibiotic Prophylaxis↗

Analysis of structure and conservation for supporting functional evaluation of PMS2 missense variants.

Germline defects in mismatch repair (MMR) genes are known to significantly increase the risk of developing certain types of cancers, notably colorectal and endometrial cancers. These conditions are characterized under Lynch syndrome. Accurate diagnosis of this predisposition, along with meaningful predictive testing for family members, necessitates the identification of pathogenic variants. However, classifying small coding genetic variants identified in cancer patients is very challenging, specifically in the case of PMS2 variants, since PMS2 pathogenic variants display a lower penetrance and less severe phenotype and therefore a lower tumor burden in affected families. We have assembled clinical data on four PMS2 missense variants of uncertain significance (VUS) identified in 23 patients (p.(Asp286Gly), p.(Asn335Ser), p.(Ile679Thr) and p.(Arg799Trp)). For these variants, functional testing was performed (RNA splicing, protein stability and catalytic activity). Since many protein ortholog sequences and accurate predictive models from AlphaFold2 are available, we also included a systematic analysis of residue conservation and structural role (ConStruct assessment). Overall, our findings indicate that p.(Asp286Gly) and p.(Arg799Trp) behave similarly to wild-type PMS2 and are thus probably neutral. In contrast, p.(Asn335Ser) and p.(Ile679Thr) conferred defects in protein expression or MMR activity. These could be explained by the relevant roles of these amino acids in MLH1-PMS2-N-terminal dimerization (p.Asn335) and C-terminal dimerization (p.Ile679). Our data thus suggest that p.(Asp286Gly) and p.(Arg799Trp) are benign, while the tumor risk in the other two variants remains to be established. Taken together, we suggest roadmaps for the individualized evaluation of difficult uncertain variants by comprising information from all available sources.

Humans↗

Clinical and morphological correlations for transglutaminase 1 gene mutations in autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited disorders of cornification in which progress has recently been made in the identification of pathogenic mechanisms causing the disease. Transglutaminase 1 (TGM1) has been found as a defective gene in a large fraction of patients with lamellar ichthyosis (LI), a severe inherited scaling disorder of the skin. We have previously performed molecular genetic studies of 38Finnish ARCI families and found six different mutations in 13 families of 38 (34%). In this study we compared the molecular genetic alterations with clinical and electron microscopic findings of these patients. Families were classified by electron microscopy in ichthyosis congenita (IC) types I, II, III, IV and a non-defined group. TGM 1 gene mutation was found in all of the IC type II and 1/3 of the IC type 1 families. Although electron microscopy is not always used to classify ARCI patients, it can distinguish groups which are parallel with molecular genetic findings. This finding might be useful in the classification of ARCI patients for further linkage studies. Clinically typical phenotype of the TGM1 mutation carrier includes large, thick, brownish scales, but ichthyosis of some of these patients tends to be milder.

Adolescent↗