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PubMed1992

Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis.

Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment wa specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA< the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.

Base Sequence↗

PubMed1992

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.

Aeromonas↗

PubMed2006

Application of molecular techniques to the study of hospital infection.

Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents up to 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. Increasingly, hospital-acquired infections with multidrug-resistant pathogens represent a major problem in patients. Understanding pathogen relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine whether epidemiologically related isolates are also genetically related. To determine molecular relatedness of isolates for epidemiologic investigation, new technologies based on DNA, or molecular analysis, are methods of choice. These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. The integration of molecular typing with conventional hospital epidemiologic surveillance has been proven to be cost-effective due to the associated reduction in the number of nosocomial infections. Cost-effectiveness is maximized through the collaboration of the laboratory, through epidemiologic typing, and the infection control department during epidemiologic investigations.

Bacteria↗

PubMed1989

Clinical and experimental aspects of viral myocarditis.

Picornaviruses are frequently implicated as the etiological agents of acute myocarditis. This association is based historically on serological evidence of rising antibody titers to specific pathogens and more recently on identification of viral genomic material in endocardial biopsy specimens through in situ hybridization. Only rarely is infectious virus isolated from either the patient or the heart during periods of maximum myocardial inflammation and injury. Thus, despite a probable viral etiology, much interest centers on the role of the immune system in cardiac damage and the likelihood that the infection triggers an autoimmune response to heart-specific antigens. Heart-reactive antibodies and T cells are found in most myocarditis patients, and immunosuppressive therapy has proven beneficial in many, though not all, cases. Furthermore, murine models of coxsackievirus group B type 3-induced myocarditis also demonstrate that virus infection initiates autoimmunity and that these autoimmune effectors are predominately responsible for tissue injury. How virus-host interactions overcome presumed self-tolerance to heart antigens is discussed, and evidence supporting various theories of virus-initiated autoimmunity and disease pathogenesis are delineated.

Animals↗

PubMed2006

Multitarget PCR for diagnosis of pertussis and its clinical implications.

PCR has greatly facilitated pertussis diagnosis due to the speed, sensitivity, and specificity of this assay compared to other detection methods. Various single-target PCR assays are currently utilized, but none is universally considered to be the "gold standard." Our aim was to assess the use of multitarget versus single-target PCR for the diagnosis of pertussis in clinical samples. PCR assays targeting insertion sequence IS481 (IS), pertussis toxin ptxA promoter region (PT), and outer membrane porin (PO), or recA (RA) were evaluated in respiratory specimens collected from 4,442 patients with suspected pertussis. The diagnosis of pertussis was confirmed in 309 (6.96%) patients by the 3-target IS-PT-PO/RA PCR versus 247 (5.56%) by the conventional single-target IS (P=0.007). Compared to single-target IS, the three-target combination increased the proportion of positive specimens by 1.25-fold, and two-target combinations increased the proportion of positive specimens by 1.10- to 1.24-fold. In addition, nine cases of B. parapertussis infection were also confirmed by using the discriminative features of this multitarget PCR. Of the 89 culture-proven pertussis cases, 17 (19.1%) and 5 of the 16 patients (31.3%) admitted to intensive care unit would have been missed had only the single-target IS PCR been applied. Patients with mild disease (P=0.004) and shorter hospitalization (P=0.006) were less likely to have positive cultures. This consensus generating real-time PCR approach permits a sensitive detection, as well as an accurate species identification of the causative Bordetella pathogens for the timely management of patients.

Base Sequence↗

PubMed1985

Demonstration of a common antigen in sonicated cells for identification of Vibrio vulnificus serotypes.

Ouchterlony immunodiffusion of sonicated Vibrio vulnificus cells illustrated a single major precipitation line with antiserum prepared from whole cells of the same species. Antigenic analysis by two-dimensional immunoelectrophoresis verified the presence of a single dominant precipitation line. Tandem two-dimensional immunoelectrophoretic analyses of V. vulnificus antigens from various strains revealed one fused precipitation line of identity. No fused precipitation lines were seen with other Vibrio species tested. This dominant antigen, designated VVA, was not dialyzable, lost antigenicity by heating at 100 degrees C but not at 70 degrees C, and was precipitated by 70%, but not by 50%, saturated ammonium sulfate. VVA was not present in concentrated (20X) spent culture medium. VVA is possibly an intracellular protein specific to V. vulnificus species and may be useful in the serological identification of this important human pathogen.

Antigens, Bacterial↗

PubMed1994

Bacteremia caused by a novel Bordetella species, "B. hinzii".

Bordetella spp. cause respiratory tract diseases in warm-blooded animals. Only Bordetella bronchiseptica has been reported to cause bacteremia in humans, and this rare infection usually occurs with pneumonia in immunocompromised patients. We describe "Bordetella hinzii" bacteremia in an AIDS patient without a respiratory illness. Combining biochemical phenotyping with fatty acid analysis permitted preliminary identification of this previously undescribed pathogen; identity was confirmed by DNA-DNA hybridization. This report extends the spectrum of human infections caused by the bordetellae.

Adult↗

PubMed1995

Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay.

The identification of a new murine pathogen, Helicobacter hepaticus, and its association with chronic active hepatitis and liver tumors prompted an evaluation of the prevalence of H. hepaticus in commercially available mice. Of the 28 different strains or stocks, totaling 160 mice from four major commercial vendors, cultured for H. hepaticus, 100% of mice from two outbred strains from one vendor were infected with H. hepaticus, whereas 9 of 13 inbred mouse strains from another vendor were infected. This high prevalence of H. hepaticus established a need for a rapid and reproducible, noninvasive assay for the screening of colony-maintained mice being used for biomedical research. The culturing of fecal material by using 0.45-microns-pore- size filtration for H. hepaticus consistently yielded reproducible results but required extended periods of time. (1 to 3 weeks) to obtain a definitive answer. Although it is rapid, the use of a direct PCR-based detection assay with fecal specimens is restricted by inhibitory agents. to circumvent these inhibitory agents and to augment our H. hepaticus culture technique, we have developed a novel PCR system in which the bacteria are isolated from fecal material in the presence of polyvinylpyropyrollidone and lysed by treatment with Chelex 100. The PCR is performed with Tth polymerase supplemented with a polymerase enhancer. By this PCR method, 24 H. hepaticus culture-positive and 30 H. hepaticus culture-negative fecal samples were correctly identified. Moreover, two samples which were PCR positive and culture negative initially were positive by both methods upon retesting of fresh material. Southern blot hybridizations and sequencing of PCR products showed them to be H. hepaticus specific. A comparison of results obtained under identical conditions indicated a 100-fold increase in sensitivity with Tth polymerase over Taq polymerase. This PCR method can be used as a noninvasive means of rapidly screening large numbers of colony mice for H. hepaticus.

Animals↗

PubMed1997

Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.

Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities. Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas. The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin. Most results from the two approaches were in agreement, but some discrepancies were discerned. Advantages over previous phenotypic and genetic methods are discussed.

Aeromonas↗

PubMed2000

Accelerated detection and identification of mycobacteria with MGIT 960 and COBAS AMPLICOR systems.

An automated cultivation system for mycobacteria, the MGIT 960 system (MGIT system), was compared in the clinical routine with two variants of Löwenstein-Jensen (L-J) medium. A total of 152 isolates were recovered from 2,015 specimens: 139 (91%) with the MGIT system and 127 (84%) with L-J media (P = 0.05). These included 68 isolates of Mycobacterium tuberculosis, of which 88% grew in the MGIT system and 93% grew in L-J media (P = 0.389), and 84 isolates of mycobacteria other than M. tuberculosis (MOTT), of which 94% grew in the MGIT system and 76% grew in L-J media (P = 0.003). More M. avium complex isolates were detected in the MGIT system (n = 65) than in L-J media (n = 50) (P = 0.001). Growth in the MGIT system was detected in 2 weeks for 78% of the isolates, whereas growth was detected in the two L-J media for 17 and 25% of the isolates, respectively. The mean times to detection of M. tuberculosis were 12 days in the MGIT system and 20 days in L-J media, and for M. avium complex the mean times to detection were 8 and 22 to 25 days, respectively. The contamination rates were similar (8.7 to 8.9%) in all media. A commercial amplification system (COBAS AMPLICOR) was evaluated for its ability to rapidly identify M. tuberculosis, M. avium, and M. intracellulare directly from 393 samples in MGIT system broth. A correct PCR result, as evaluated by culture or clinical data, was obtained for 96% of the samples, with inhibition being detected for 2% of the samples. Of the 89 results positive for M. tuberculosis, 91% were regarded as true positive, 8% were regarded as inconclusive, and 2% were considered false positive. For results positive for M. avium and M. intracellulare, 97 and 79%, respectively, were regarded as true positive. Increased rapidity and enhanced isolation of MOTT were obtained with the MGIT system. COBAS AMPLICOR was suitable for rapid identification of these three common pathogens from MGIT system broth.

Automation↗

PubMed2002

Identification of Listeria species by microarray-based assay.

We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp. demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene. Another virulence factor gene, hly, was used for detection and genotyping all L. monocytogenes, all L. ivanovii, and 8 of 11 L. seeligeri isolates. Other members of the genus Listeria and three L. seeligeri isolates did not contain the hly gene. There was complete agreement between the results of genotyping based on the hly and iap gene sequences. All L. monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species. Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.

Bacterial Proteins↗

PubMed2002

Detection of mycobacterial DNA in Andean mummies.

The identification of genetic material from pathogenic organisms in ancient tissues provides a powerful tool for the study of certain infectious diseases in historic populations. We have obtained tissue samples from the genital areas of 12 mummies in the American Museum of Natural History collection in New York, N.Y. The mummies were excavated in the Andes Mountain region of South America, and radiocarbon dating estimates that the mummies date from A.D. 140 to 1200. DNAs were successfully extracted from all tissues and were suitable for PCR analysis. PCRs were carried out to detect Mycobacterium tuberculosis complex and mycobacteria other than M. tuberculosis (MOTB). M. tuberculosis complex was detected in 2 out of 12 samples, and MOTB were detected in 7 samples. This study confirmed the adequate preservation of genetic material in mummified tissues and the existence of mycobacteria, including M. tuberculosis, in historic populations in South America.

DNA, Bacterial↗

PubMed2003

Fatal case of Salmonella enterica subsp. arizonae gastroenteritis in an infant with microcephaly.

Salmonella enterica subsp. arizonae is a common gut inhabitant of reptiles, with snakes as the most common reservoir. Though human cases due to this organism are exceedingly rare, it may infect young infants and immunocompromised individuals with a history of intimate associations with reptiles. Gastroenteritis is the most common presentation; others include peritonitis, pleuritis, osteomyelitis, meningitis, and bacteremia. We report a fatal case of S. enterica subsp. arizonae gastroenteritis in a 3-month-old child with microcephaly, with a review of earlier cases and problems encountered in identification of this rare human pathogen.

Fatal Outcome↗

PubMed2004

Practical bench comparison of BBL CHROMagar Orientation and standard two-plate media for urine cultures.

A total of 1023 urine samples sent for routine culture were plated onto sheep blood and MacConkey agars and a BBL CHROMagar Orientation (CO; Becton Dickinson, Cockeysville, Md.) plate, and the results were compared. Of these, 250 urine samples (24%) grew >10000 CFU of one or two putative pathogens/ml and 773 showed no growth (NG), mixed growth of <10000 CFU/ml, or three or more strains (mixed). The CO and conventional medium results agreed completely for 595 cultures with NG or <10000 CFU/ml. An additional 178 urine samples yielded clinically insignificant differences. Both medium sets essentially agreed on quantities and identification for 400 single-pathogen cultures and 9 mixed cultures. With the caveat that CO cannot differentiate Klebsiella, Enterobacter, and Serratia spp., enteric pathogens were identified only by morphology and color on CO. Direct visual differentiation of group B streptococci from lactobacilli is not possible, but lactobacillus cells always exhibited easily recognizable morphology on Gram stain. Of 108 paired organism susceptibility results encompassing 2268 drug-pathogen combinations, there were 3% errors and only 1% very major errors. Use of CO allowed a >50% reduction in inoculation time and a >20% reduction in work-up time. For our laboratory, with 50% "no growth" and ca. 25% significant results (50% Escherichia coli), CO allowed time and workup cost savings for a majority of cultures. A cost analysis (time and supplies for our laboratory) showed that if CO is used alone, the break-even level for CO pricing is US dollars 1.78; if CO and blood agar are both used, the break-even pricing for CO is US dollars 1.53.

Agar↗

PubMed2004

Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-linked immunosorbent assay system for direct identification of gram-positive cocci and gram-negative bacilli from positive blood cultures.

We evaluated the Hyplex BloodScreen PCR-enzyme-linked immunosorbent assay (ELISA) system (BAG, Lich, Germany), a new diagnostic test for the direct identification of gram-negative bacilli and gram-positive cocci from positive blood cultures, with 482 positive BACTEC 9240 blood culture bottles. The test involves amplification of the bacterial DNA by multiplex PCR and subsequent hybridization of the PCR product to specific oligonucleotide probes in an ELISA-based format. The available probes allow the separate detection of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella spp., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis/Enterococcus faecium, Streptococcus pyogenes, and Streptococcus pneumoniae and the staphylococcal mecA gene. The Hyplex BloodScreen test showed an overall sensitivity of 100% for the identification of gram-negative bacilli and 96.6 to 100% for the identification of gram-positive cocci (S. aureus, 100%; S. epidermidis, 97.2%; Enterococcus faecalis/Enterococcus faecium, 96.6%; and Streptococcus pneumoniae, 100%). The specificities of the test modules ranged from 92.5 to 100% for gram-negative bacilli and 97.7 to 100% for gram-positive cocci (Escherichia coli, 92.5%; Pseudomonas aeruginosa, 98.5%; Klebsiella spp., 100%; Enterobacter aerogenes, 100%; S. aureus, 100%, S. epidermidis, 97.7%; Enterococcus faecalis/Enterococcus faecium, 99.6%; Streptococcus pyogenes, 100%; and Streptococcus pneumoniae, 99.3%). The result of the mecA gene detection module correlated with the result of the phenotypic oxacillin resistance testing in all 38 isolates of Staphylococcus aureus investigated. In conclusion, the Hyplex BloodScreen PCR-ELISA system is well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of Staphylococcus aureus in positive blood cultures.

Bacteremia↗

PubMed2006

Listeriosis due to infection with a catalase-negative strain of Listeria monocytogenes.

A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection.

Adult↗

PubMed1999

Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events.

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.

Animals↗

PubMed2004

Cough plate versus cough swab in patients with cystic fibrosis; a pilot study.

BACKGROUND: Accurate identification of lower respiratory tract pathogens is important in the management of cystic fibrosis patients. AIM: To evaluate the cough plate as an alternative method of obtaining respiratory samples for microbiological culture. METHODS AND RESULTS: Using sputum culture as the "gold standard", the cough plate method identified significantly more positive cases than either dry or moistened cough swabs. Of 20 patients who had a positive sputum culture, 16 had a positive culture on cough plate compared to seven positive cultures each on moistened swab and on dry swab respectively. CONCLUSIONS: In this study cough plates were more sensitive than cough swabs in isolating respiratory pathogens in sputum producers. As patients prefer it, the cough plate merits further evaluation, particularly in non-sputum producers.

Adolescent↗