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PubMed2004

Emerging gram-negative enteric infections.

This is an overview describing the microbiology, epidemiology, clinical presentations, laboratory diagnosis, and treatment of emerging gram-negative enteric infections. This review illustrates the shifts in epidemiology of infections caused by new and reemerging bacteria and highlights the role of the clinical microbiology laboratory in detecting these pathogens. It concludes with suggestions for the implementation of molecular techniques to ensure rapid surveillance and enhanced patient care.

Communicable Diseases, Emerging↗
PubMed2019

Development of a multiplex real-time RT-PCR assay for simultaneous detection and differentiation of influenza A, B, C, and D viruses.

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

Animals↗
PubMed2005

Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads.

Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1,000-fold improvements in the PCR detection limit, from 10(6) Cfu/g (no sample processing) to 10(3) Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10(1)-10(6) Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10(5) Cfu/g for chicken salad, 10(4) Cfu/g for macaroni salad, and 10(3) Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.

Blotting, Southern↗
PubMed2006

Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1.

The efficacy of two selective chromogenic culture media, Agar Listeria Ottaviani and Agosti (ALOA) and RAPID' L. mono for the detection of Listeria monocytogenes in food, was compared with that of an official culture method according to the EN/DIN 11290-01 and -02 protocols [corresponding to the section 35 LMBG (German Food Act) method]. A total of 310 pre-packed ready-to-eat food samples (100 of graved and cold smoked salmon, 130 of different raw and cooked sausages and 80 of delicatessen and mixed salads) were examined. L. monocytogenes was identified in 52 investigated salmon samples. Using two chromogenic media, 50 samples were found positive for L. monocytogenes. Compared to the reference method there were no false-positive results. By the EN/DIN 11290-01 culture procedure after the selective enrichment in Fraser broth 12 out of 130 samples of sausages were positive for L. monocytogenes. These 12 samples were also positive for L. monocytogenes with the chromogenic medium RAPID' L. mono. One sample was false negative with ALOA. Three additional samples were found positive with ALOA and four with RAPID' L. mono. The standard method was inadequate to confirm these samples as positive. Listeria spp. were isolated from 7 samples of mixed salads with both methods. One, 3 and 3 samples were found to contain L. monocytogenes, L. innocua and L. seeligeri, respectively. Both chromogenic media enabled a rapid and specific detection of L. monocytogenes within 24h after enrichment. Visual detection of pathogenic L. monocytogenes and other Listeria spp. was easier on chromogenic media.

Agar↗
PubMed2005

Regulation of Toxoplasma gondii multiplication in BeWo trophoblast cells: cross-regulation of nitric oxide production and polyamine biosynthesis.

Materno-foetal transmission causes one of the most severe forms of infection with the protozoan parasite Toxoplasma gondii. Several studies have shown T. gondii in placental trophoblast cells, which form the barrier between maternal blood circulation and foetal tissue. Parasite multiplication in trophoblast cells is thus a critical step leading to infection of the foetus. Here, we show that multiplication of T. gondii tachyzoites was slow in BeWo trophoblast cells, compared with MRC-5 fibroblast cells. However, unlike MRC-5 cells, even combined stimulation with interferon-gamma and tumor necrosis factor-alpha did not reduce T. gondii replication in BeWo cells. This was associated with a lack of indoleamine-2,3-dioxygenase induction by these cytokines. Neither low availability of iron salts, nor an immunosuppressive action of cyclooxygenase-2 could be attributed to the low T. gondii multiplication rate in BeWo cells. However, treatment with the nitric oxide synthesis inhibitor N(G)-methyl-l-arginine and addition of ornithine enhanced the proliferation rate of the intracellular pathogen. Despite detection of inducible nitric oxide synthase-II mRNA in BeWo cells, nitric oxide production could not be detected during cell culture. Thus, inhibition of arginase activity by nitric oxide synthesis may be partially responsible for the lower multiplication rate in BeWo cells.

Animals↗
PubMed2006

Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids.

Template preparation is important in reverse-transcription polymerase chain reaction (RT-PCR)-based detection methods. A TissueLyser with tungsten carbide beads was used for simultaneous processing of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for RT reaction. A sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. It was also demonstrated that the combination use of random primers and oligo dT primer in an RT reaction was efficient for simultaneous cDNA synthesis of viral and viroid RNAs in plant extracts. These template preparation methods were used for the amplification of Grapevine leafroll-associated virus-1,-2, and -3; Grapevine virus A and B; Grapevine rupestris stem pitting-associated virus; Grapevine fleck virus; and Grapevine fanleaf virus. All these viruses tested in this study were reliably detected up to a 10(3)-fold or higher dilution of the original extract. Besides, Hop stunt viroid and Grapevine yellow speckle viroid 1 were well amplified in the same manner as the template preparation and following PCR for virus detection. These methods would contribute to cost-effective testing of a large number of samples through the year and help to detect viral pathogens in grapevine.

DNA Primers↗
PubMed2026

Identification of Two Novel Compound Heterozygous ADAMTS17 Variants Associated With Weill-Marchesani Syndrome 4.

PURPOSE: Weill-Marchesani syndrome 4 (WMS4) is frequently underdiagnosed when standard exome sequencing fails to detect noncoding pathogenic variants. We aimed to identify the genetic cause in a patient with suspected WMS4 and to characterize the splicing-altering mechanism of a deep intronic variant in ADAMTS17. MATERIALS AND METHODS: Whole-exome sequencing combined with whole-genome sequencing was conducted in the proband, who presented with ocular and skeletal manifestations of WMS4. A minigene splicing assay was applied to verify the splicing abnormality caused by the deep intronic variant. RESULTS: The patient showed high myopia, brachydactyly, accelerated growth velocity, and advanced bone age. Two novel compound heterozygous variants in ADAMTS17, c.1655G>A and c.450+38C>A, were identified. The deep intronic variant c.450+38C>A was confirmed by minigene assay to disrupt normal pre-messenger RNA splicing by inducing retention of a 35-base pair intronic segment, leading to a frameshift and premature termination (p.G152Lfs*23). CONCLUSIONS: This study broadens the mutational spectrum of ADAMTS17. Whole-genome sequencing combined with functional splicing validation is essential for resolving molecularly undiagnosed cases of WMS4, particularly when deep intronic variants are suspected, and supports clinical genetic testing and genetic counseling of hereditary connective tissue disorders.

ADAMTS17↗
PubMed2004

Sensitive and specific detection of Xanthomonas campestris pv campestris by PCR using species-specific primers based on hrpF gene sequences.

A sensitive and specific assay was developed to detect bacterial black rot of crucifers caused by Xanthomonas campestris pv. campestris (X. c. pv. campestris), in cabbage seed and plant. Primers XCF and XCR from hrpF homologous to nolX, host recognition protein, were used to amplify a 525 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally infected seed and plant of cabbage. The PCR product was only produced from X. c. pv. campestris among 40 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference bacteria.

Bacterial Proteins↗
PubMed2005

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences.

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.

Citrus↗
PubMed2004

Nasal colonization with Staphylococcus aureus is not associated with the severity of symptoms or the extent of the disease in chronic rhinosinusitis.

OBJECTIVES: There is an increasing knowledge that the severity of perennial allergic rhinitis is associated with nasal carriage of Staphylococcus aureus (S. aureus). The aim of this study was to evaluate the rate of bacterial colonization with S. aureus in the nose of subjects with and without chronic rhinosinusitis (CRS) and to correlate these findings with the severity of symptoms and the extent of the disease. STUDY DESIGN AND SETTING: Open, prospective controlled trial. METHODS: 190 subjects with CRS and 42 subjects with septal deviation without sinusitis (control subjects) were included in this study. Swabs were taken endoscopically from the middle meatus and bacteria were cultured and identified. Airway symptoms were assessed by subjects in standardized questionnaires and frequencies of respiratory tract infections were noted. The rhinosinusitis extent was graded by CT scan assessment. Analysis of variance, chi-square test, and Pearson's correlation test were applied for statistical analyses. RESULTS: The S. aureus carriage rate was 25.5% in CRS and 31.4% in control subjects. Further facultative pathogens were cultured in 20.6% of subjects with CRS and in 8.5% of controls. 73.8% of S. aureus were ampicillin-resistant, multiresistant strains were cultured in 5.8%. Most airway symptoms and the frequencies of respiratory tract infections were significantly higher in the CRS group compared with control subjects. In post hoc comparison between the subgroups with and without S. aureus colonization, no significant differences were found between the extent of rhinosinusitis and the severity of airway symptoms. CONCLUSIONS: Facultative pathogens were detected in more than 40% of CRS patients without acute respiratory tract infections. In contrast to the findings in atopic dermatitis and perennial allergic rhinitis, the colonization rate with S. aureus is not increased and S. aureus carriage is not a risk factor for the severity or the extent of rhinosinusitis.

Adult↗
PubMed2004

Expressing an antibacterial protein in bacteria for raising antibodies.

Magainins are small peptides with broad-spectrum activity against a range of plant and animal microbial pathogens. To detect magainin peptides in applications such as Western blot analysis and enzyme-linked immunosorbent assays, specific antibodies that recognize magainin peptides are required. The production of antibodies against small peptides injected into host animals poses problems with respect to eliciting an adequate immunogenic response due to the small size of the molecules. To increase the immunogenicity of a target peptide, it may be expressed as part of a larger fusion protein. However, expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases. To overcome these potential problems, we fused the DNA coding sequence of a magainin gene analogue within the sequence of a bacterial thioredoxin gene. The subsequent gene fusion comprising a bacterial thioredoxin gene with a magainin coding sequence ligated at the active site of thioredoxin was successfully translated in a bacterial expression system. The fusion protein was non-toxic to the host bacteria. This represents a novel strategy to express antimicrobial peptides in a bacterial expression system. The fusion protein, purified by molecular size separation, was recovered in a soluble form following electroelution from polyacrylamide gels. Sufficient fusion protein was obtained for injection into rabbits and antibodies were obtained from rabbit sera that selectively recognized magainin peptides in Western blot analysis.

Amino Acid Sequence↗
PubMed2007

Infectious canine hepatitis: an "old" disease reemerging in Italy.

Four outbreaks of infectious canine hepatitis (ICH) occurring in Italy between 2001 and 2006 are reported. Three outbreaks were observed in animal shelters of southern Italy, whereas a fourth outbreak involved two purebred pups imported from Hungary few days before the onset of clinical symptoms. In all outbreaks canine adenovirus type 1 (CAV-1) was identified by virus isolation and PCR. In three outbreaks, other canine viral pathogens were detected, including canine distemper virus, canine parvovirus or canine coronavirus. The present study shows that CAV-1 is currently circulating in the Italian dog population and that vaccination is still required.

Adenoviruses, Canine↗
PubMed2006

Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials.

In recent years it has been recognized that bacteriophages have several potential applications in the modern biotechnology industry: they have been proposed as delivery vehicles for protein and DNA vaccines; as gene therapy delivery vehicles; as alternatives to antibiotics; for the detection of pathogenic bacteria; and as tools for screening libraries of proteins, peptides or antibodies. This diversity, and the ease of their manipulation and production, means that they have potential uses in research, therapeutics and manufacturing in both the biotechnology and medical fields. It is hoped that the wide range of scientists, clinicians and biotechnologists currently researching or putting phages to practical use are able to pool their knowledge and expertise and thereby accelerate progress towards further development in this exciting field of biotechnology.

Anti-Bacterial Agents↗
PubMed2003

Molecular interactions between DNA and an aminated glass substrate.

With the development of DNA arrays, the immobilization of DNA strands onto solid substrates remains an essential research topic. DNA arrays have potential applications in DNA sequencing, mutation detection, and pathogen identification. DNA bound to solid substrates must still be accessible and retain the ability to hybridize with its complementary strands. One technology to produce these arrays involves linking DNA molecule probes to a silanized substrate in microspot patterns and exposing them to a solution of fluorescently labeled samples of DNA targets. The behavior of both the target and probe DNA and their interactions with each other at the substrate surface, particularly with respect to molecular interactions, are poorly understood at the present time. The objective of this work is to model simply the interface interactions between DNA and glass slides modified with an aminosilane (gamma-aminopropyltriethoxysilane, APTS). In aqueous solutions, DNA behaves as a polyacid over a wide range of pH. A glass substrate treated with APTS is positively or negatively charged, depending on the pH. A model of the surface charge of APTS-treated glass has been developed from results of wetting experiments performed at various pH. It has been demonstrated that the surface charge of APTS-treated glass is well described by a model of constant capacitance of the electrical double layer. A good correlation between experimental data on DNA retention at various pH's and the variation of the surface charge of the APTS-treated glass is obtained. This provides an indication of the role of ionic interactions in the adsorption of DNA molecules onto aminated glass slides.

Adsorption↗
PubMed1999

Retracted: Rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment.

The classic India ink test is positive in only half of cryptococcal meningitis cases, and reliable, rapid cryptococcal antigen (CRAG) testing requires technical expertise and facilities not always available. We therefore examined cerebrospinal fluid (CSF) sediment using May-Giemsa, periodic acid-Schiff, and Gram stains in 16 patients with cryptococcal meningitis. The India ink test was positive in seven patients (44%), while microscopic examination of sediment revealed cryptococci in 13 (81%); in six of these 13 the India ink test was negative. Both methods failed to detect the pathogen in the remaining three patients. CRAG testing in CSF was negative in two patients (one with acquired immunodeficiency syndrome, one with diabetes mellitus) whose India ink test also was negative while cryptococci were identified in their CSF sediment. No false positives occurred with CSF May-Giemsa staining in 27 cases of aseptic meningitis with negative cultures for Cryptococcus. In all, microscopic examination of centrifuged and stained CSF sediment proved more sensitive for rapid diagnosis of cryptococcal meningitis than the India ink method, and in two of our patients cryptococci were seen in centrifuged CSF sediment despite negative CRAG and India ink tests.

Adolescent↗
PubMed2002

[Community-acquired pneumonia associated with influenza virus].

BACKGROUND: Pneumonia is one of the most common complications of influenza infection. However, its characteristics are not well-known. Our aim was to assess the frequency of community-acquired pneumonia (CAP) associated with influenza virus and to determine its clinical and epidemiological characteristics. PATIENTS AND METHOD: A total of 240 patients with CAP were evaluated and cases with serological evidence of influenza virus infection were identified. Patients in whom there was no evidence of infection by other microbial agents were selected and defined as influenza virus infection-associated CAP (CAP-i). Clinical and epidemiological features of CAP-i patients were compared with the rest of patients with CAP. RESULTS: A microbial diagnosis was achieved in 143 (59.6%) of 240 patients with CAP. In 12 cases (5%) there was serological evidence of infection by influenza A virus. In 8 patients no other respiratory pathogens were detected (CAP-i). In a multivariate analysis, the presence of arthromyalgias (odds ratio [OR] = 30, 68; 95% confidence interval [CI], 1-946), plasmatic sodium < 133 mEq/l (OR = 16.92; 95% CI, 1.48-193), bilateral infiltrates in chest X-ray (OR = 8.83; 95% CI, 1.35-57.6), diagnosis of CAP in January or February (OR = 5.60; 95% CI, 0.87-35.8) and blood neutrophil cell count >= 14,000/l, (OR = 5.23; 95% CI, 0.85-32) were all independently associated with CAP-i. The simultaneous presence of all these characteristics had a positive and negative predictive value of 95% and 99%, respectively, to differentiate CAP-i from other CAP. CONCLUSIONS: Influenza virus infection-associated CAP is uncommon. Some clinical and radiographic variables could differentiate CAP-i from other CAP. These parameters could also help identify patients who are more likely to benefit from specific antiviral therapy.

Adolescent↗
PubMedDate not supplied

Applications of the polymerase chain reaction to diagnosis of ophthalmic disease.

The polymerase chain reaction (PCR) is a powerful molecular biologic technique for the analysis of very small amounts of DNA. This technique has found increasing use in the past 10 years for the detection of pathogenic organisms associated with many forms of ocular inflammatory and infectious disease. PCR has shown utility in the diagnosis of viral uveitis, infectious endophthalmitis, and parasitic eye disease. The strengths and weaknesses of this diagnostic technique are discussed. Additionally, uses of PCR in linking known pathogens to disease, and to discovering novel pathogens, are addressed.

Eye Infections↗