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PubMed2006

Rapid detection and identification of bacterial pathogens using novel molecular technologies: infection control and beyond.

The rapid detection and reporting of antimicrobial-resistant pathogens, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and multidrug-resistant Mycobacterium tuberculosis, is a challenge for the clinical microbiology laboratory. Molecular-based diagnostic tests can provide data on the presence of methicillin-resistant S. aureus in the nares in approximately 1 h, whereas testing for the vanA and vanB resistance genes in enterococci isolated from perirectal samples can be completed in approximately 4 h. Novel pyrosequencing assays can provide data regarding the presence of multidrug-resistant M. tuberculosis directly from positive mycobacterial broth cultures in <1 day. These data can assist physicians in both therapeutic and infection control decisions.

Bacteria↗

PubMedDate not supplied

[Etiologic profile of acute diarrhea in children in São Paulo]

OBJECTIVE: To evaluate the etiologic profile of acute diarrhea in socioeconomically deprived children assisted at a regional pediatric emergency care service. METHODS: During two years all children with acute diarrhea assisted at a previously established day and week time schedule were included in the study. Other selective criteria were: a) age less than 5 years; b) nonuse of antibiotics in the previous month; and c) no travel outside the city in the previous month. Stool examination was used for the detection of the following microorganisms: a) rotavirus (immunofluorescence and counterimmunoelectrophoresis); b) bacteria - culture in MacConkey agar, SS agar, Columbia agar, bright green, serotyping, detection of toxins - INV, LT,ST,SLT I, SLT II, Sereny test, detection of virulence factors- EAF, eae, BFP; and c) protozoa (Hoffman and Faust). In the same period, a control group without diarrhea was also evaluated for the same fecal pathogens. RESULTS: Between March 1994 and June 1996, 154 children with acute diarrhea (AD) and 42 control children (WAD), that is, without acute diarrhea, were selected. In the AD group, intestinal pathogens were detected in 112 (72.8%) cases, and in 9 (21.5%) cases in the WAD group. The association of two or more intestinal pathogens occurred in 47 (30.5%) cases in the AD group, and in 3 (7.1%) cases in the WAD group. The pathogens identified in the AD cases were: Rotavirus: 32 (20.8%), bacteria: 53 (34.4%), both: 25 (16.2%), and 2 (1.4%) with Giardia lamblia (in one case associated with Rotavirus and in another one associated with bacteria). In the WAD group, only bacteria were detected in 8 (19.1%) cases, and bacteria associated with Giardia lamblia in 1 (2.4%) case. Altogether, there were 105 bacteria isolated in the AD group: 90 were Escherichia coli (EPEC 27, DAEC 24, ETEC 21, EAEC 18), 12 were Shigella sp, 2 were Salmonella sp, and one was Yersinia sp. Children with mixed infections (viral and bacterial) had increased incidence of severe vomiting, dehydration and hospitalization. CONCLUSIONS: Bacteria were the most frequent pathogens detected in acute diarrhea cases, among which Escherichia coli was highly predominant. The majority of Escherichia coli strains belong to non-EPEC varieties, strains that are not routinely evaluated in clinical laboratories of pathology. Rotavirus was found in a great number of diarrhea cases, often associated with bacteria. Protozoa showed reduced importance.

Journal Article↗

PubMed2002

Beta-lactamase detection in nosocomial bacterial pathogens in Lagos, Nigeria.

Beta-lactamase production was investigated in 1033 bacterial pathogens--262 gram-positive bacteria, 482 Enterobacteriaceae, 289 non-fermenting bacilli. These were isolated from nosocomial infections in paediatric patients. Beta-lactamase was detected using chromogenic cephalosporin and starch paper methods. More than 80% of the isolates produced the enzyme, which was not detected in enterococci and streptococci. There was no appreciable difference ([symbol: see text] 2 = 5.31, P > 0.05) in the number of Beta-lactamase producers obtained using the two methods. Great caution must be exercised in the therapeutic use of Beta-lactam drugs for nosocomial bacterial infections.

Anti-Bacterial Agents↗

PubMed1999

Blood cultures have limited value in predicting severity of illness and as a diagnostic tool in ventilator-associated pneumonia.

STUDY OBJECTIVES: To define the usefulness of blood cultures for confirming the pathogenic microorganism and severity of illness in patients with ventilator-associated pneumonia (VAP). DESIGN: Prospective observational study using BAL and blood cultures collected within 24 h of establishing a clinical diagnosis of VAP. SETTING: A 15-bed medical and surgical ICU. PATIENTS: One hundred and sixty-two patients receiving mechanical ventilation hospitalized for > 72 h who had new or progressive lung infiltrate plus at least two of three clinical criteria for VAP. INTERVENTIONS: BAL and blood culture performed within 24 h of establishing a clinical diagnosis of VAP. MEASUREMENTS AND RESULTS: Ninety patients were BAL positive (BAL+), satisfying a microbiological definition of VAP (>/= 10(4) cfu/mL), 72 patients were BAL negative (BAL-). Bacteremia was diagnosed when at least two sets of blood cultures yielded a microorganism or when only one set was positive, but the same bacteria was present at a concentration >/= 10(4) cfu/mL in the BAL fluid. Bacteremia was significantly more frequent in the BAL+ than in the BAL- group (22/90 patients vs 5/72 patients; p = 0.006). In 6 of 22 BAL+ patients with bacteremia, an extrapulmonary site of infection was the source of bacteremia. Sensitivity of blood culture for disclosing the pathogenic microorganism in BAL+ patients was 26%, and the positive predictive value to detect the pathogen was 73%. Factors associated with mortality were age > 50 years, simplified acute physiology score > 14, prior inadequate antibiotic therapy, PaO(2)/fraction of inspired oxygen < 205, and use of H(2) blockers. By multivariate analysis, only the use of prior inadequate antimicrobial therapy (odds ratio [OR], 6.47) and age > 50 years (OR, 5.12) were independently associated with higher mortality. The rate of complications was not different in patients with bacteremia. CONCLUSIONS: Blood cultures have a low sensitivity for detecting the same pathogenic microorganism as BAL culture in patients with VAP. The presence of bacteremia does not predict complications, it is not related to the length of stay, and it does not identify patients with more severe illness. Inadequacy of prior antimicrobial therapy and age > 50 years were the only factors associated with mortality in a multivariate analysis. Blood cultures in patients with VAP are clearly useful if there is suspicion of another probable infectious condition, but the isolation of a microorganism in the blood does not confirm that microorganism as the pathogen causing VAP.

Adult↗

PubMed2004

A review of conventional detection and enumeration methods for pathogenic bacteria in food.

With continued development of novel molecular-based technologies for rapid, high-throughput detection of foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique approaches and variations on existing techniques are currently on the market or are being implemented that offer ease of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a food microbiology setting.

Bacteria↗

PubMed2005

A novel semiquantitative fluorescence-based multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood.

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.

Animals↗

PubMed1999

Development of a polymerase chain reaction diagnostic test for the detection of the biotrophic pathogen Plasmopara halstedii in sunflower.

The obligate parasitic fungus-like organism Plasmopara halstedii (Farl.) Berl. et De Toni, is the causal agent of downy mildew disease in sunflower (Helianthus annuus). New races of this economically important parasite are regularly detected throughout the world. In addition, fungicide-resistant isolates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by infected seeds, means that it is necessary to guarantee the absence of Plasmopara halstedii in seed shipments. We report here the development of a rapid assay that can be used to detect infection by Plasmopara halstedii in plant tissues. Based on the nucleotide sequence information obtained from one cloned random amplified polymorphic DNA fragment, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by polymerase chain reaction. An amplification product was detected on agarose gel stained with ethidium bromide when DNA from various Plasmopara halstedii races was tested, whereas no amplified DNA was detected when DNA from other origins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Plasmopara halstedii in infected sunflower plants prior to sporulation.

DNA, Fungal↗

PubMed2002

Detection of the plant pathogenic bacterium Xanthomonas campestris pv. Campestris in seed extracts of Brassica sp. Applying fluorescent antibodies and flow cytometry.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers. Seed lots and plants are screened for contamination with this pathogen using plating or serological assays. These methods, however, are time consuming and not very sensitive, respectively. Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X. campestris isolate (Xc), and in crude seed extracts. METHODS: The mAb 18G12, conjugated with FITC, was tested at dilutions of 1:50, 1:100, 1:200, and 1:400. For mixed suspensions of Xcc and Psf, mAb 18G12 was used at a dilution of 1:100. The combination of mAbs 18G12, 2F4, and 20H6, all conjugated with FITC, was used at a dilution of 1:100 for the detection and quantification of Xcc cells in mixed suspensions containing Xcc and Xc and in crude seed extracts. The analyses were performed with a Coulter EPICS XL-MCL flow cytometer, at low flow rate during 2 min. RESULTS: Using FCM, Xcc cells labeled with FITC-conjugated mAbs (18G12, 2F4, and 20H6) were detected and quantified rapidly at low numbers, i.e., 10(3) colony-forming units per milliliter in pure and in mixed cultures with Psf. The presence of the nonpathogenic Xc in the seed extracts did not interfere with the FCM results. Xcc cells were distinguished from the cells of other organisms and from small particles present in the seed extract based on the high-intensity fluorescence of the labeled cells. CONCLUSION: The application of FCM in combination with FITC-conjugated mAbs appears to be a promising technique for the detection and quantification of Xcc cells in seed extracts of crucifers.

Antibodies, Monoclonal↗

PubMed2002

Comparison of 16S rDNA-based PCR and checkerboard DNA-DNA hybridisation for detection of selected endodontic pathogens.

Molecular methods have been used recently to investigate the bacteria encountered in human endodontic infections. The aim of the present study was to compare the ability of a 16S rDNA-based PCR assay and checkerboard DNA-DNA hybridisation in detecting Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Peptostreptococcus micros, Porphyromonas endodontalis, Por. gingivalis and Treponema denticola directly from clinical samples. Specimens were obtained from 50 cases of endodontic infections and the presence of the target species was investigated by whole genomic DNA probes and checkerboard DNA-DNA hybridisation or taxon-specific oligonucleotides with PCR assay. Prevalence of the target species was based on data obtained by each method. The sensitivity and specificity of each molecular method was compared with the data generated by the other method as the reference--a value of 1.0 representing total agreement with the chosen standard. The methods were also compared with regard to the prevalence values for each target species. Regardless of the detection method used, T. denticola, Por. gingivalis, Por. endodontalis and B. forsythus were the most prevalent species. If the checkerboard data for these four species were used as the reference, PCR detection sensitivities ranged from 0.53 to 1.0, and specificities from 0.5 to 0.88, depending on the target bacterial species. When PCR data for the same species were used as the reference, the detection sensitivities for the checkerboard method ranged from 0.17 to 0.73, and specificities from 0.75 to 1.0. Accuracy values ranged from 0.6 to 0.74. On the whole, matching results between the two molecular methods ranged from 60% to 97.5%, depending on the target species. The major discrepancies between the methods comprised a number of PCR-positive but checkerboard-negative results. Significantly higher prevalence figures for Por. endodontalis and T. denticola were observed after PCR assessment. There was no further significant difference between the methods with regard to detection of the other target species.

Aggregatibacter actinomycetemcomitans↗

PubMedDate not supplied

[Detection of exotoxins of the pathogens of anaerobic gas infection using enzyme immunoassay].

To detect the exotoxins of the causative agents of the main anaerobic gas infection (Clostridium perfringens, C.oedematiens, C.histolyticum, C.septicum) a rapid and easily reproducible variant of the enzyme-linked immunosorbent assay (ELISA), based on the use of the sandwich modification activated due to avidin-biotin interaction, was proposed. The possibility of using the avidin-biotin variant of ELISA for the detection of C.perfringens, C.oedematiens, C.histolyticum and C.septicum toxins in experimental gas gangrene in guinea pigs was shown. The method made it possible to reproducibly detect 0.02-0.2 ng of antigens (gangrene toxins-toxoids) with the immunoassay being highly specific.

Anaerobiosis↗

PubMed2000

Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia.

The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7 % of the 'lightly' infected oysters, and 66.7 % of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia.

Animals↗

PubMedDate not supplied

[The screening detection of the antigens of pathogenic enterobacteria in the biological fluids of patients with chronic intestinal diseases].

Screening detection of the antigens of some pathogenic enterobacteria in the coagglutination test with the use of Shigella (S. sonnei, S. flexneri 2a, 1b, 6), Salmonella (groups A, B, C, D, E) and Yersinia (Y. pseudotuberculosis, serovars I and III, Y. enterocolitica 03 and 09) diagnostica in patients with chronic enteric diseases, hospitalized in a noninfectious ward, has demonstrated a high incidence of the above antigens. S. flexneri antigens have shown the highest detection rate. The results thus obtained indicate that these infective agents probably take part in the development of exacerbations of chronic enteric diseases and, therefore, a comprehensive examination of this group of patients is necessary with a view to the detection of bacterial enteric infection.

Adolescent↗

PubMed2002

Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum.

AIMS: This study developed a new diagnostic method for the bacterium Flavobacterium psychrophilum based on a TaqMan polymerase chain reaction (PCR) assay. METHODS AND RESULTS: Based on reported and newly designed PCR probes, a rapid procedure, that requires no post-PCR processing, was developed for the detection of F. psychrophilum by measuring the fluorescence produced during PCR amplification. Primers were designed to amplify a 971-bp fragment of the 16S rRNA as the target. When different F. psychrophilum strains and other bacterial species, that are taxonomically and ecologically related, were assayed the fluorogenic test was 100% specific in identifying all of the F. psychrophilum strains. The sensitivity of the assay was found to be 1.1 pg DNA and the assay was linear over a range of 0.1 pg-11.2 ng. With pure cultures of F. psychrophilum, the assay was linear over the range 0.4-4.7 x 104 cfu and was able to detect 4.7 cfu per reaction. The analysis was reproducible using either extracted DNA or pure culture. Results using artificially infected fish and diseased fry from natural fish farm outbreaks showed that the assay was useful for diagnosis. CONCLUSIONS: The data showed that the assay was as specific, sensitive, reproducible and rapid but less toxic than the PCR assays described and so very useful for the diagnosis of these micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach permits a rapid, easy and safe routine laboratory diagnosis of F. psychrophilum.

Animals↗

PubMed2026

First Report of Enterocytozoon bieneusi, Encephalitozoon spp. and Blastocystis spp. in Hair Goats in Türkiye.

INTRODUCTION: Blastocystis spp., Enterocytozoon bieneusi and Encephalitozoon spp. are prevalent zoonotic gastrointestinal parasites that cause severe diarrhoea and enteric diseases in humans and animals worldwide. This study investigated the molecular occurrence, genetic diversity and zoonotic potential of E. bieneusi, Blastocystis spp. and Encephalitozoon spp. in domestic hair goats in Central Anatolia. METHODS: A total of 300 faecal samples were collected from hair goats in Kayseri, Aksaray and Sivas Provinces and genomic DNA was extracted from these samples. The presence of the three pathogens was detected using PCR and nested PCR targeting the SSU rRNA for Blastocystis spp., the internal transcribed spacer (ITS) for E. bieneusi and Encephalitozoon spp., respectively. Positive PCR products were sequenced to determine the species, subtypes and genotypes of the pathogens. RESULTS: The overall prevalence rates of E. bieneusi and Blastocystis spp. were 5.3% (16/300) and 6.7% (20/300), respectively. None of the faecal samples tested was positive for Encephalitozoon spp. and no co-infections among these three pathogens were detected. The SSU rRNA sequence analysis revealed Blastocystis spp. ST10, a subtype predominantly associated with animals. Additionally, one known E. bieneusi genotype (BEB6) was identified. The BEB6 genotype falls into zoonotic Group 2 of E. bieneusi in the phylogenetic tree. CONCLUSION: Our study is the first report of Blastocystis spp. and E. bieneusi infection in hair goats in Türkiye, highlighting their potential role in zoonotic transmission within a One Health framework. Our results provide scientific data for the prevention and control of these two intestinal pathogens. Further investigations are necessary to better understand their genetic characteristics and zoonotic potential in Türkiye.

Enterocytozoon bieneusi↗

PubMed2002

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR.

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.

Cucumis sativus↗

PubMed1979

[Idiopathic haemochromatosis. Pathogenic and genetic aspects. Detection and prevention (author's transl)].

The basic disorder of iron metabolism in idiopathic haemochromatosis finds expression on at least two levels: the intestinal mucosa (increased iron absorption) and the liver. Its exact nature, however, remains obscure. The role of iron overload in the pathogenesis of the disorder seems clear. Lysosome disruption has recently been proposed as a possible pathogenic factor. Phenotypic family studies have lent considerable weight to the hypothesis of a recessive transmission of idiopathic haemochromatosis. Demonstration of a close link between the disease and the HLA antigen A3 and haplotype A3, B14 has made it possible: to remove all doubt as to the hereditary nature of the disease; identify the underlying gene as located on chromosome 6 near the A locus of the HLA system; demonstrate a recessive mode of transmission; and achieve the early detection of individuals at risk in the family of a patient with the disease. Thanks to this possibility of early detection, the feasability of preventive measures is greatly enhanced.

Alleles↗

PubMed2006

Screening for melanocortin-4 receptor mutations in a cohort of Belgian morbidly obese adults and children.

OBJECTIVE: To investigate whether pathogenic melanocortin-4 receptor (MC4R) mutations are a common cause of obesity in Belgium. DESIGN: Cross-sectional mutation analysis. SUBJECTS: In total, 95 morbidly obese adults (mean age 44.02+/-11.35 years; mean BMI 47.87+/-4.17 kg/m2 and 123 obese children and adolescents were screened for mutations in MC4R (mean age 16.56+/-2.58 years; BMI>95th percentile for age and sex; mean % overweight 170.86 +/- 23.63). MEASUREMENTS: A series of anthropometric (e.g. weight, height, waist, hip), biochemical and clinical measurements were performed on all subjects. The entire coding region of MC4R was screened using DHPLC, a highly sensitive and specific method for mutation analysis. Direct sequencing was performed when the chromatogram deviated from the WT pattern. RESULTS: Mutation screening of a cohort of Belgian obese adults and children did not detect any pathogenic mutations as only the previously described polymorphisms Val103Ile, Thr112Met and Ile251Leu were detected. CONCLUSION: Pathogenic mutations in MC4R are not a common cause of obesity in a Belgian population of obese adults, children and adolescents.

Adolescent↗

PubMed2004

Applications of single-molecule detection to the analysis of pathogenic DNA.

We have devised a new technique based on single fluorescent molecule detection for the analysis of specific sequences of DNA. The method consists of synthesizing a fluorescent reporter molecule using a polymerase extension reaction and labeled nucleotides. The fluorescent reporter products are analyzed in a laser-based single-molecule detection system. We have applied this method to the detection of pUC19 and Bacillus anthracis DNA targets. We expect that this method will have applications in rapid detection and identification of DNA from pathogens as well as other sources, and that it will be used for processing of large number of samples in a short period of time.

Bacillus anthracis↗