bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonradioactive colony lift-hybridization assay for detection of Bordetella bronchiseptica infection in swine
Authors: Register K B , Ackermann M R , Dyer D W
Published: January 2020

Current methods for the isolation and identification of Bordetella bronchiseptica from clinical samples are time-consuming and are based, in part, on subjective observations. We describe the use of a Bordetella-specific DNA probe in a nonradioactive colony lift-hybridization assay for the identification of B. bronchiseptica. Eleven of 82 clinical specimens were found to contain B. bronchiseptica by this method, while only 5 of these were reported to contain the organism when the specimens were analyzed by traditional methods. The chromosomal fragment containing a sequence complementary to the probe appeared to be conserved in B. bronchiseptica isolates from swine from a variety of sources. The assay is more rapid than culture and biochemical testing since it can be performed directly on primary culture plates, even when they are heavily contaminated with other bacterial species. Only minimal training is required to accomplish the assay successfully, and the results are easy to interpret.
Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains
Authors: Xu Y , Johnson R C
Published: January 2020

The relationship between plasmid profiles and genospecies of the Lyme disease borreliae was investigated by using 40 strains from diverse biological and geographical sources. The genospecies of the strains were determined by examination of rRNA gene restriction patterns with cDNA probes complementary to the 16S and 23S rRNAs of Escherichia coli. Plasmid profiles were obtained by pulsed-field gel electrophoresis. The number of plasmids per strain and the size of these plasmids ranged from 4 to 10 and from 13.3 to 57.7 kb, respectively. The strains all contained a single large plasmid of 50 to 57.7 kb, with the exception of two Borrelia garinii strains that contained two or three of the large plasmids. The large plasmids of Borrelia burgdorferi sensu stricto strains ranged in size from 51.4 to 52.7 kb and were consistently smaller than the 54.0- to 57.7-kb plasmids present in B. garinii and Borrelia afzelii. The exceptions of this observation were the two B. garinii strains with multiple large plasmids; in this case the large plasmids were 50.6 to 53 kb. Although a large degree of heterogeneity in the sizes and frequencies of occurrence of smaller plasmids was observed, there were some differences among the three genospecies. The differences in plasmids were further studied by using two BamHI DNA fragments from a 28.7-kb plasmid of B. burgdorferi sensu stricto 297 as probes. Both probes hybridized with the 27- to 29-kb plasmids of B. burgdorferi sensu stricto strains. In contrast, two patterns of hybridization were observed with B. garinii and B. afzelii. One pattern was the hybridization of both probes, with each probe hybridizing with a different plasmid. The other pattern was the hybridization of a plasmid of a strain with only one of the two probes. some strains of the three genospecies did not hybridize with either probe. Our results suggest that the plasmid profiles of B. burgdorferi sensu lato have genospecies characteristics and that the hybridization patterns of similar-sized plasmids are different for the three genospecies.
Association between alpha-hemolysin production and HeLa cell-detaching activity in fecal isolates of Escherichia coli
Authors: Marques L R , Abe C M , Griffin P M , Gomes T A
Published: January 2020

Escherichia coli isolates that cause detachment of cell monolayers during in vitro adherence assays (cell-detaching E. coli [CDEC]) were recently reported as a potential new group of enteropathogenic bacteria. In the present study, 269 E. coli isolates from feces of children 1 to 5 years of age were identified as CDEC in a detaching assay developed with HeLa cells. The great majority of these isolates were hemolytic within 3 h of growth on blood agar plates and hybridized with a DNA probe for alpha-hemolysin (93.7%), while most of the non-detaching isolates were hemolytic within 24 h (3.6%) or nonhemolytic (94.8%). E. coli isolates that produced alpha-hemolysin were found in 60 (30%) of 200 children with diarrhea and 47 (24%) of 200 age-matched controls. No statistical significance was found for the differences in alpha-hemolysin production among the matched pairs (P = 0.2). These data suggest that CDEC isolates are not associated with diarrhea in the population studied.
Phenolic acridine orange fluorescent stain for mycobacteria
Authors: Smithwick R W , Bigbie M R , Ferguson R B , Karlix M A , Wallis C K
Published: January 2020

A new fluorescence acid-fast staining method with acridine orange as the specific stain is presented. Only two reagents are required: the acridine orange-specific stain and a destaining-counterstaining reagent. Compared with auramine fluorescence acid-fast staining, there was less nonspecific staining of non-acid-fast debris which fluoresced a pale green contrasting color to provide a background in which to search for the red-to-orange fluorescing acid-fast bacilli. The results of the study indicate that the acridine orange method is superior to the auramine method in detecting acid-fast bacilli in specimen smears.
Vancomycin-dependent Enterococcus faecium isolated from stool following oral vancomycin therapy
Authors: Dever L L , Smith S M , Handwerger S , Eng R H
Published: January 2020

The isolation of clinical strains of enterococci requiring vancomycin for growth has only recently been reported. We describe the isolation of Enterococcus faecium requiring vancomycin for growth from the stool of a patient who had completed oral vancomycin therapy. Growth of the vancomycin-dependent E. faecium was supported by ristocetin and D-alanyl-D-alanine but not by daptomycin, teicoplanin, or D,L-alanine. Spontaneous revertants not requiring vancomycin occurred at a rate of 1 in 10(6). Both the vancomycin-dependent E. faecium and the revertant hybridized with a vanB gene probe and had identical contour-clamped homogeneous electrophoresis patterns. The majority of revertant colonies were resistant to teicoplanin, suggesting constitutive production of the vanB ligase. We believe the vancomycin-dependent E. faecium evolved from a vancomycin-resistant, vancomycin-independent E. faecium in the presence of high concentrations of vancomycin in the intestine.
Biochemical fingerprinting compared with ribotyping and pulsed-field gel electrophoresis of DNA for epidemiological typing of enterococci
Authors: Kühn I , Burman L G , Haeggman S , Tullus K , Murray B E
Published: January 2020

The Phene Plate (PhP) biochemical fingerprinting system for bacteria is based on measurements of the kinetics of bacterial biochemical reactions. This system was modified for typing of enterococci and was compared with DNA typing by pulsed-field gel electrophoresis and with ribotyping by using 45 Enterococcus faecalis isolates from international collections. It was also used to study 170 fecal enterococcal isolates from healthy individuals and 28 isolates of E. faecalis from the blood of neonates. The PhP system showed a high degree of discriminatory power for unrelated enterococcal isolates. Among the 170 unrelated fecal isolates, 107 isolates from international collections, PhP typing discriminated 19 types, and ribotyping discriminated 5 types. In most cases, when isolates were of the same DNA type, they were also of the same PhP type, and the level of agreement between these two methods was high (96%). A combination of PhP typing and DNA typing identified 34 different types, but ribotyping did not yield any further discrimination. PhP typing of E. faecalis isolates from healthy individuals (n = 89) and from the blood of neonates with septicemia (n = 28) yielded a diversity of 0.93 for both populations and similar major PhP types in both populations. Thus, the isolates from blood seemed to consist of a normal E. faecalis population, without a dominance of certain strains associated with virulence. We conclude that the PhP system is useful for epidemiological studies of enterococcal isolates, yielding results similar to those obtained with DNA typing by pulsed-field gel electrophoresis. Since PhP typing is a method that is simple and rapid and that is based on automatic evaluation of the data, it is suitable for analyzing large numbers of isolates and can be used alone or in combination with DNA typing or epidemiological and ecological studies of enterococci.
Isolation of Mycoplasma pneumoniae from the human urogenital tract
Authors: Goulet M , Dular R , Tully J G , Billowes G , Kasatiya S
Published: January 2020

Mycoplasma pneumoniae is a common etiologic agent of lower respiratory tract infections in humans. However, it has been reported previously that the organism has occasionally been isolated from sites other than the oropharynx and respiratory tract. We report the isolation of 24 strains of M. pneumoniae from urogenital specimens obtained from 22 female patients. Most isolates were of cervical origin from patients attending several local gynecological clinics over a 2-year period. Strains were also isolated from the urethra of one of three healthy male sexual partners of female patients positive for the organism. Single serum specimens obtained from three female patients and three different male sexual partners showed antibody levels suggestive of either recent respiratory infection or genital tract colonization with M. pneumoniae. Although there is no apparent definitive explanation for the localized outbreak of the organism at these unusual sites, the possible transfer through sexual and/or orogenital contact remains the most likely mode of transmission. The occurrence of an organism with obvious pathogenicity for human epithelial tissue in the urogenital tract suggests such transfer could play a role in genital tract infection.
Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni
Authors: Burnens A , Stucki U , Nicolet J , Frey J
Published: January 2020

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.
Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations
Authors: Pruckler J M , Mermel L A , Benson R F , Giorgio C , Cassiday P K , Breiman R F , Whitney C G , Fields B S
Published: January 2020

Arbitrarily primed PCR (AP-PCR) and pulsed-field gel electrophoresis (PFGE) subtyping were applied to clinical and environmental isolates from seven unrelated outbreaks of Legionnaires' disease. The patterns observed with each method matched patient isolates and the epidemiologically linked source of disease for each of the seven outbreaks. PFGE allowed more discrimination among various isolates, although AP-PCR usually gave comparable results. With both methods, certain patterns appeared to predominate in the comparison of the seven outbreaks. Of five clinical isolates not associated with the outbreaks, three gave profiles distinct from those observed in the outbreaks by both methods. This suggests that there are at least two predominant subtypes of Legionella pneumophila serogroup 1 associated with outbreaks. Investigations of outbreaks of legionellosis should employ either PFGE or AP-PCR in addition to monoclonal antibody analysis.
Use of PCR for prenatal and postnatal diagnosis of congenital rubella
Authors: Bosma T J , Corbett K M , Eckstein M B , O'Shea S , Vijayalakshmi P , Banatvala J E , Morton K , Best J M
Published: January 2020

A reverse transcription-nested PCR assay (RT-PCR) was evaluated for diagnosis of congenitally acquired rubella in utero and during infancy. RT-PCR was compared with virus isolation for retrospective detection of rubella virus in placental and fetal tissues obtained after termination of pregnancy following primary rubella or rubella virus reinfection. Concordant results were obtained for 85% of samples; rubella virus RNA was detected by RT-PCR alone in four samples, and rubella virus was detected by isolation alone in two samples. Samples were also obtained for prenatal diagnosis of congenital infection; rubella virus RNA was detected in three of seven chorionic villus samples and one of three amniotic fluid samples by RT-PCR, while rubella virus was isolated in only one chorionic villus sample. To demonstrate that the RNA extracted from chorionic villus samples contained amplifiable RNA, a nested RT-PCR was used to detect keratin mRNA. Rubella virus was detected in placenta in two cases in which the fetus was uninfected, and there was no evidence of rubella virus in the placenta from one case in which the fetus was infected. Thus, detection of rubella virus in chorionic villus samples by RT-PCR may not always correctly predict fetal rubella virus infection. RT-PCR was successfully used for the diagnosis of congenitally acquired rubella in infancy. Rubella virus RNA was detected in cyropreserved or formalin-fixed lens aspirates obtained from infants in India with serologically confirmed congenital rubella but not in samples from controls with inherited cataract.
Analysis of Enterococcus faecalis isolates from intercontinental sources by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis
Authors: Tomayko J F , Murray B E
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE) were compared in this study of 65 Enterococcus faecalis isolates recovered over a 20-year period from diverse geographic sources. Clonal relationships recognized by PFGE were also recognized by MLEE; however, MLEE recognized as greater number of isolates as belonging to clonal groups than did PFGE. Both techniques were reproducible and discriminatory, but MLEE more readily recognized relationships among large numbers of isolates. MLEE confirmed the previously reported clonal spread of beta-lactamase-producing E. faecalis to six hospitals in five states. MLEE provided a useful population framework of the E. faecalis isolates in this sample, while PFGE was able to differentiate among isolates within some MLEE clonal groups.
Disseminated Encephalitozoon (Septata) intestinalis infection in a patient with AIDS: novel diagnostic approaches and autopsy-confirmed parasitological cure following treatment with albendazole
Authors: Sobottka I , Albrecht H , Schäfer H , Schottelius J , Visvesvara G S , Laufs R , Schwartz D A
Published: January 2020

Encephalitozoon intestinalis is a recently described microsporidian which causes intestinal and disseminated infections in severely immunocompromised patients with AIDS. Preliminary data suggest that albendazole can be an effective therapy for patients with E. intestinalis infection. However, relapses have been reported following treatment in some cases. These results were based upon examination of cytologic, biopsy, or stool samples with an inherent sampling bias. This report documents the first postmortem evaluation of a patient with E. intestinalis infection treated with albendazole. Antemortem microsporidial diagnosis was performed on nasal mucosal smear and duodenal biopsy specimens by electron microscopy and a newly developed indirect fluorescent-antibody method based upon in vitro cultivation of the organism. This case represents the initial report of using nasal cytologic specimens for ultrastructural and antibody-based species-level diagnosis of microsporidiosis. Following successful treatment of this infection with albendazole, the patient died of other causes. A thorough autopsy examination failed to reveal the presence of E. intestinalis in any tissue, providing confirmatory evidence for a complete parasitological cure with albendazole.
Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats
Authors: Gutiérrez M , Samper S , Gavigan J A , García Marín J F , Martín C
Published: January 2020

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.
Genotyping human papillomavirus type 16 isolates from persistently infected promiscuous individuals and cervical neoplasia patients
Authors: van Belkum A , Juffermans L , Schrauwen L , van Doornum G , Burger M , Quint W
Published: January 2020

Nucleotide sequence variation in the noncoding region of the genome of human papillomavirus type 16 (HPV16) was determined by direct sequencing and single-strand conformation polymorphism analysis of DNA fragments amplified by PCR. Individuals of diverse sexual promiscuity and/or cervicopathology were studied. In a group of 14 healthy, monogamous HPV16-positive females, only two HPV16 sequence variants could be documented. Among 17 females and 3 males with multiple sex partners and living in the same geographical region, nine sequence variants were found, whereas among 7 patients with cervical neoplasia from another region, five variants were detected. Although numbers are limited, in the group of individuals at high risk of acquiring a sexually transmitted disease or with cervical neoplasia, a larger number of HPV16 sequence variants was encountered (two types among 14 individuals versus nine types among 20; Fisher's exact test, P = 0.07). Seven of the individuals were sampled repeatedly over time. For these persistently infected women, no differences in HPV16 sequences were detected, irrespective of promiscuity, and persistence of a single viral variant, spread over multiple anatomic sites, for more than 2 years could be demonstrated. This indicates that viral persistence may be a common feature and that successful superinfection with a new variant may be rare, despite a potentially high frequency of viral reinoculation.
Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies
Authors: Dixit V , Quan S , Martin P , Larson D , Brezina M , DiNello R , Sra K , Lau J Y , Chien D , Kolberg J
Published: January 2020

Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.
Use of CHROMagar Candida medium for isolation of yeasts from dental samples
Authors: Beighton D , Ludford R , Clark D T , Brailsford S R , Pankhurst C L , Tinsley G F , Fiske J , Lewis D , Daly B , Khalifa N
Published: January 2020

A new differential medium, CHROMagar Candida, for the isolation of clinically important yeasts was investigated to determine its usefulness in facilitating the study of oral yeasts. The recovery of yeasts on the medium was not significantly different from the recovery on Sabouraud dextrose agar. The identities of 450 green colonies on CHROMagar Candida, presumptively identified as Candida albicans on the basis of the manufacturer's instructions, were confirmed by testing for beta-N-acetylgalactosaminidase. Candida tropicalis also formed distinctive colonies, and other yeasts including Candida (Torulopsis) glabrata, Candida Parapsilosis, Candida Magnoliae, Candida lusitaniae, Candida Famata, Candida kefir, and Saccharomyces cerevisiae were readily distinguished from C. albicans and C. tropicalis isolates. CHROMagar Candida is a very useful medium, and its use will facilitate the study of yeasts associated with dental diseases.
Restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, India, by use of direct-repeat probe
Authors: Sahadevan R , Narayanan S , Paramasivan C N , Prabhakar R , Narayanan P R
Published: January 2020

Large numbers of Mycobacterium tuberculosis isolates that were obtained from patients' sputa on diagnosis and during follow-up after short-course chemotherapy in Madras, India, have either no copy or only a single copy of IS6110. This poses a limitation for DNA fingerprinting with an IS6110-based probe to determine the frequency of exogenous reinfection versus that of endogenous reactivation. In the present study, we overcame this limitation by using an alternate probe, the direct-repeat element. Comparison of pre- and posttreatment isolates by direct-repeat restriction fragment length polymorphism analysis indicated a high degree of endogenous reactivation among patients who have relapses after the successful completion of chemotherapy.
Comparison of MicroScan WalkAway system and Vitek system for identification of gram-negative bacteria
Authors: Rhoads S , Marinelli L , Imperatrice C A , Nachamkin I
Published: January 2020

In a prospective side-by-side comparison conducted from September through November 1994, we compared the MicroScan WalkAway system, a conventional biochemical identification system (Dade MicroScan, Inc., Sacramento, Calif.), with the Vitek system (bioMerieux Vitek, Hazelwood, Mo. [analysis software version AMS-RO8.2] for the identification of gram-negative bacteria. Three-hundred thirty-one nonurine isolates and 493 urine isolates were tested. For nonurinary isolates, there was 91.5% agreement between the two methods. For urinary isolates, there was 97.4% agreement between the two methods. Overall, there was 95% agreement between the two systems. The results suggest that the current version of the MicroScan WalkAway system with conventional panels is essentially comparable to the current Vitek system.
Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera
Authors: Magnarelli L A , Dumler J S , Anderson J F , Johnson R C , Fikrig E
Published: January 2020

Serum specimens from persons with or without Lyme borreliosis were analyzed by indirect fluorescent antibody staining methods for total immunoglobulins to Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). There was serologic evidence of human exposure to multiple tick-borne agents in 15 (6.6%) of 227 serum samples obtained in Connecticut and Minnesota. Of these, 10 serum samples were from Connecticut patients who had erythema migrans and antibodies to Borrelia burgdorferi (range, 1:160 to 1:40, 960). A maximal antibody titer of 1:640 was noted for a B. microti infection, whereas titration end points of 1:640 and 1:1,280 were recorded for E. chaffeensis and E. equi seropositives, respectively. In specificity tests, there was no cross-reactivity among the antisera and antigens tested for the four tick-borne pathogens. On the basis of serologic testing, a small group of persons who had Lyme borreliosis had been exposed to one or more other tick-borne agents, but there was no clinical diagnosis of babesiosis or ehrlichiosis. Therefore, if the clinical picture is unclear or multiple tick-associated illnesses are suspected, more extensive laboratory testing is suggested.
Intestinal cytomegalovirus disease in immunocompromised patients may be ruled out by search for cytomegalovirus DNA in stool samples
Authors: Michel D , Marre E , Hampl W , Roczkos J , Müller S , Hertenstein B , Kern P , Heymer B , Salzberger B , Arasteh K
Published: January 2020

Cytomegalovirus (CMV) PCR from stool specimens was adopted as a diagnostic tool for patients with suspected CMV colitis. After being established, the method was evaluated in 17 AIDS patients and 19 other immunocompromised patients by comparison of PCR results with clinical, histological, and microbiological or virological data. CMV PCR was positive in 4 symptomatic patients with proven CMV colitis and negative in 15 of 16 patients without characteristic histopathology. Neither CMV immunoglobulin G seropositivity nor intestinal symptoms alone were significantly associated with positive PCR results, but severe active systemic CMV infection may lead to a positive PCR. Absence of CMV DNA in stool samples may prove useful in ruling out CMV related colitis.
Use of molecular analysis in pathophysiological investigation of late-onset neonatal Escherichia coli meningitis
Authors: Bingen E , Bedu A , Brahimi N , Aujard Y
Published: January 2020

We describe one case of late-onset neonatal Escherichia coli meningitis associated with urinary tract infection. Two genotypic methods revealed that the E. coli isolates obtained from blood, feces, and cerebrospinal fluid shared the same pattern, which was different from the unique pattern obtained for the isolates recovered from the urine and the tracheal aspirate. Our study supports passage of bacteria through the gastrointestinal mucosa to the bloodstream as the initial event, with subsequent meningeal localization.
Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR
Authors: Flemmig T F , Rüdiger S , Hofmann U , Schmidt H , Plaschke B , Strätz A , Klaiber B , Karch H
Published: January 2020

The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.
Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis
Authors: Sloan L , Schneider S , Rosenblatt J
Published: January 2020

We evaluated a commercially available enzyme-linked immunoassay (ELISA) from LMD Laboratories, Inc., Carlsbad, Calif., for the detection of antibodies in serum to the cysticercus of Taenia solium. The ELISA was performed on 308 serum samples; 198 from a pool of healthy individuals, 42 from patients who had antibodies against a variety of parasites other than T. solium, and 68 from patients suspected of having cysticercosis. All of these 68 specimens were tested both by the ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot assay [EITB]) developed at the Parasitic Serology Laboratory of the Centers for Disease Control and Prevention. Twenty-seven of the 68 serum samples from patients suspected of having cysticercosis were positive by both EITB and ELISA, while 31 were negative by both assays. ELISA results for three and two samples were considered false positive and false negative, respectively, when compared with the results of EITB. Results for an additional five samples were considered equivocal but were technically positive because their optical density readings were slightly above the cutoff value. Three of the 198 serum samples from the bank of serum samples from healthy individuals were also false positive by ELISA (the EITB result for the samples was negative). Six other serum samples from healthy individuals which had equivocal results and the five serum samples from patients with equivocal results were EITB negative. Serum samples containing antibodies against Echinococcus spp. frequently cross-reacted with the cysticercus ELISA antigen (13 of 16 specimens), but serum samples with antibodies against other parasites did not (2 of 26 specimens); all of these serum samples were EITB negative. The commercially available ELISA that we describe is a simple and rapid test. Considering all 308 specimens, the ELISA had a specificity of 93% (when samples with equivocal results were considered negative) or 89% (when samples with equivocal results were considered positive); the sensitivity was 93%.
Comparison of different PCR approaches for characterization of Burkholderia (Pseudomonas) cepacia isolates
Authors: Liu P Y , Shi Z Y , Lau Y J , Hu B S , Shyr J M , Tsai W S , Lin Y H , Tseng C Y
Published: January 2020

In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.
Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCR products
Authors: Locher F , Suryanarayana V V , Tratschin J D
Published: January 2020

Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 degrees C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.
Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus
Authors: Bannerman T L , Hancock G A , Tenover F C , Miller J M
Published: January 2020

Bacteriophage typing (BT) (World Health Organization method) has been used at the Centers for Disease Control and Prevention for over 30 years to type isolates of Staphylococcus aureus. Since studies have shown that BT patterns have poor reproducibility and because BT fails to type a high percentage (15 to 20%) of isolates, the Centers for Disease Control and Prevention has converted from using BT to using pulsed-field gel electrophoresis (PFGE) for strain typing S. aureus. We compared the results of BT with results of PFGE for typing 300 isolates of S. aureus, including strains from several well-characterized outbreaks. Ninety-six isolates were BT group I, 19 were group II, 82 were group III, 7 were group V, and 96 were nontypeable. PFGE identified subgroups within each phage group and thus was more discriminating than BT, which identified no subgroups. PFGE was able to type all isolates and distinguish related from unrelated strains of S. aureus. Our modified, standardized PFGE methodology should enable typing laboratories to obtain rapid, reliable results in 3 to 4 days when starting with an isolated colony on agar media.
Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis
Authors: van Deventer A J , Goessens W H , van Belkum A , van Vliet H J , van Etten E W , Verbrugh H A
Published: January 2020

A PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C. albicans-specific probe, +/- 10 to 15 C. albicans cells are detected in 100 microliters of whole blood by Southern analysis. A DNase pretreatment was critical in the purification process of yeast DNA from whole blood. Omission of the DNase pretreatment decreased assay sensitivity 10-fold. PCR analysis of blood specimens collected from mice with invasive candidiasis is more sensitive than blood culture (100 versus 67%, respectively) at 72 h after intravenous (i.v.) inoculation with C. albicans. Furthermore, the intensity of the hybridization signals increased with the progression of infection. In contrast, multiple blood samples from gastrointestinally colonized mice were all negative by PCR, indicating that the PCR assay is also specific and may, therefore, make a positive contribution to the detection and follow-up of invasive candidiasis.
Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media
Authors: Coudron P E , Stratton C W
Published: January 2020

In order to increase the database for in vitro growth and/or susceptibility testing in liquid media, we evaluated the growth of Helicobacter pylori in broth media containing 5% sheep blood. We also compared the effect of bismuth on the growth of H. pylori in broth media containing 10% fetal calf serum with the effect on growth in media containing 0.5% starch. In contrast to the result seen with agar, we found that sheep blood, whether whole or laked, inhibited the growth of H. pylori in broth media. In addition, we found that bismuth inhibited growth in media with starch but that this inhibition was negated in media with serum.
Differentiation of Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil by plasmid content, serotyping, and pulsed-field gel electrophoresis
Authors: Ng L K , Carballo M , Dillon J A
Published: January 2020

A combination of DNA macrorestriction analysis using pulsed-field gel electrophoresis and a serotyping method using three panels of monoclonal antibody was used to discriminate 43 epidemiologically unrelated Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil (PCU-) into 35 groups. This indicates that PCU- isolates of N. gonorrhoeae are not clonal.
Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting
Authors: Taylor N S , Fox J G , Akopyants N S , Berg D E , Thompson N , Shames B , Yan L , Fontham E , Janney F , Hunter F M
Published: January 2020

The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin. We conclude that H. pylori genotypes, as measured by several sensitive DNA fingerprinting methods, can remain stable for years in vivo, despite the acquisition or loss of drug resistance, circulating antibody, or exposure to antibiotics or sucralfate.
Production of elastase, exotoxin A, and alkaline protease in sputa during pulmonary exacerbation of cystic fibrosis in patients chronically infected by Pseudomonas aeruginosa
Authors: Jaffar-Bandjee M C , Lazdunski A , Bally M , Carrère J , Chazalette J P , Galabert C
Published: January 2020

Secretion of Pseudomonas aeruginosa elastase, exotoxin A, and alkaline protease in sputum during bronchopulmonary exacerbations was examined in 18 cystic fibrosis patients chronically infected with this microorganism. The patients were studied during one or several exacerbation periods necessitating hospitalizations of 12 to 20 days. In all cases, P. aeruginosa was present in bronchial secretions at admission and was not eradicated after treatment. The P. aeruginosa density decreased significantly after antibiotic therapy but remained greater than 10(6) CFU/g of sputum in most cases. Significant amounts of P. aeruginosa exoproteins were measured in total homogenized bronchial secretions by immunoenzymatic assays. The detection of higher levels of exoproteins at admission, the significant decrease after treatment, and the absence of exoproteins during intercrisis phases constituted arguments for a renewal of virulence of P. aeruginosa during exacerbations. Nevertheless, the concomitant changes in bacteria load and the triggering of the inflammatory process and immune complex formation could also contribute to pulmonary exacerbations.
Evidence for nosocomial transmission of Candida albicans obtained by Ca3 fingerprinting
Authors: Schmid J , Tay Y P , Wan L , Carr M , Parr D , McKinney W
Published: January 2020

The moderately repetitive sequence Ca3 was used to fingerprint Candida albicans isolates from 32 patients hospitalized for more than 3 days, 17 recent admissions or outpatients, and 8 recently readmitted patients and 10 commensal isolates from the community in Wellington, New Zealand, plus isolates from 21 hospitalized patients, 26 outpatients or recent admissions, 4 recently readmitted patients, and 10 healthy individuals in the community in Auckland, New Zealand. In Wellington, isolates from patients hospitalized in Wellington Hospital for more than 3 days were genetically significantly less diverse than were isolates from outpatients or recent admissions or isolates from healthy individuals in the community. In addition, two clusters of genetically similar strains were isolated from hospitalized patients significantly more often than from other individuals. These observations provide evidence (albeit indirectly) for nosocomial transmission of hospital-specific C. albicans strains. In contrast, no indication of hospital-specific transmission of C. albicans was found in Auckland Hospital. Since these results were obtained under conditions in which no candidiasis outbreak occurred in either hospital, they also suggest that Ca3 fingerprinting may be a useful tool in preventive nosocomial infection control programs, allowing assessment of the extent of C. albicans transmission occurring in a hospital.
Reappraisal with meta-analysis of bacteremia, endotoxemia, and mortality in gram-negative sepsis
Authors: Hurley J C
Published: January 2020

Among patients with suspected sepsis, endotoxemia is variably present in association with gram-negative bacteremia. A total of 738 patients with suspected sepsis from 11 studies could be classified into four groups: 131 (18%) patients had both endotoxemia and gram-negative bacteremia (group 1), 87 (12%) had only gram-negative bacteremia (group 2), and 143 (19%) had only endotoxemia (group 3); in 377 (51%) patients neither could be detected (group 4). By the statistical techniques of meta-analysis, the fatality risk for patients with either endotoxemia or gram-negative bacteremia or both was estimated with group-specific case fatality rates from these studies and expressed as an odds ratio (OR; 95% confidence interval [95% CI]) versus patients with these factors absent. This risk was increased marginally when endotoxemia was detected without gram-negative bacteremia (OR, 2.3; 95% CI, 1.3 to 4.0) or the converse (OR, 2.0; 95% CI, 1.0 to 3.8), in contrast to the striking increase when both endotoxemia and gram-negative bacteremia were detected (OR, 6.3; 95% CI, 4.0 to 10.0). Alone, neither endotoxemia nor gram-negative bacteremia is a strong predictor of outcome in patients with suspected sepsis. However, in combination, the two identify a subpopulation with a substantially increased risk of mortality.