bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme-linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoalveolar fluid
Authors: Jonas D , Rosenbaum A , Weyrich S , Bhakdi S
Published: January 2020

A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5' biotinylated. The amplification product is immobilized on streptavidin-coated microtiter plates. Because of the high binding capacity, no removal of nonincorporated biotin from the PCR product is required. After alkaline denaturation, the single-stranded PCR product is hybridized with a 5' digoxigenin-labeled probing oligomer. The amplification product is then detected by using peroxidase-labeled anti-digoxigenin antibodies in a luminescence or colorimetric reaction. The assay detects as few as 10 legionellae in 1-ml bronchoalveolar lavage fluid specimens. It is specific for medically relevant Legionella species, including Legionella pneumophila, L. bozemanii, and L. longbeachae. Of over 250 clinical specimens examined, 8 were positive for legionellae by both culture and the PCR assay. Six further specimens were culture negative but PCR positive for legionellae; of these, five specimens were from patients receiving high-dose erythromycin therapy for suspected or previously diagnosed legionella pneumonia. None of the remaining 240 specimens that were culture negative for legionellae yielded a positive PCR test, although a total of over 30 different bacterial species were cultured from these specimens. The PCR assay therefore appears to exhibit high sensitivity and specificity and thus could prove suitable for use in the routine microbiological diagnostic laboratory.
Cyclospora species as a gastrointestinal pathogen in immunocompetent hosts
Authors: Ooi W W , Zimmerman S K , Needham C A
Published: January 2020

Previous reports of diarrhea resulting from Cyclospora species have been linked to travelers and immunocompromised patients. We conducted a prospective study of 1,042 formalin-ethyl acetate fecal concentrates collected from patients with diarrhea. Between May and November 1993, we identified three patients for whom studies were positive for nonrefractile spherical organisms measuring 10 microns in diameter and containing a cluster of refractile membrane-bound globules. The cysts exhibited variable acid fastness consistent with Cyclospora species. These three patients had no history of recent travel and presented with relapsing, watery, nonbloody diarrhea that lasted from 12 days to 8 weeks. No other parasitic or bacterial pathogens were identified in their stools. All three instances of diarrhea occurred in May or June. No common source of food or water was identified. None of these patients were immunosuppressed, and their diarrhea resolved spontaneously. We suggest that Cyclospora species should be considered in community-acquired diarrhea.
Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay
Authors: Chapman A , Vallejo V , Mossie K G , Ortiz D , Agabian N , Flisser A
Published: January 2020

Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and treatment of individuals who carry the adult tapeworm. T. solium and Taenia saginata eggs cannot be differentiated on the basis of morphology; thus, in order to improve existing methods for the diagnosis of taeniasis, we have developed highly sensitive, species-specific DNA probes which differentiate T. solium and T. saginata. Recombinant clones containing repetitive DNA sequences which hybridize specifically with genomic DNAs from either species were isolated and characterized. T. solium-specific DNA sequences contained complete and truncated forms of a tandemly repeated 158-bp DNA sequence. An unrelated T. saginata DNA sequence was also characterized and shown to encode a portion of the mitochondrial cytochrome c oxidase I gene. T. solium- and T. saginata-specific DNA probes did not hybridize in dot blot assays either with genomic DNA from the platyhelminths Taenia hydatigena, Taenia pisiformis, Taenia taeniaeformis, Echinococcus granulosus, and Schistosoma mansoni or with genomic DNA from other eukaryotes, including Saccharomyces cerevisiae, Candida albicans, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma gambiense, Trypanosoma brucei, and Giardia lamblia, Caenorhabditis elegans, and human DNA. By using these T. solium and T. saginata DNA probes, a rapid, highly sensitive and specific dot blot assay for the detection of T. solium eggs was developed.
Development of PCR-based hybridization protocol for identification of streptococcal species
Authors: Bentley R W , Leigh J A
Published: January 2020

16S rRNA of Streptococcus agalactiae, S. uberis, and S. parauberis was bound to streptavidin-coated magnetic beads by using a biotinylated oligonucleotide probe complementary to a highly conserved region of the molecule. In-solution hybridization of radiolabelled oligonucleotide probes to immobilized 16S rRNA allowed the specific identification of S. agalactiae and S. parauberis but not S. uberis. PCR was used to amplify a species-specific region of the 16S rRNA gene from these species. One of the PCR primers was biotinylated at the 5' end to allow purification of the amplified product on streptavidin-coated magnetic beads and subsequent denaturation to yield immobilized single-stranded DNA. Radiolabelled oligonucleotide probes were hybridized in solution to the single-stranded target molecule and enabled species-specific identification of the target organism. This protocol overcame problems associated with hybridization of the S. uberis-specific probe to 16S rRNA in solution. A similar procedure may enable the specific detection of other streptococci which exhibit a species-specific sequence in this region of the gene.
Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method
Authors: Gehrt A , Peter J , Pizzo P A , Walsh T J
Published: January 2020

Inoculum size is a critical variable in development of methods for antifungal susceptibility testing for filamentous fungi. In order to investigate the influence of different inoculum sizes on MICs of amphotericin B, 5-fluorocytosine, itraconazole, and miconazole, 32 clinical isolates (8 Aspergillus fumigatus, 8 Aspergillus flavus, 5 Rhizopus arrhizus, 8 Pseudallescheria boydii, and 3 Fusarium solani isolates) were studied by the broth microdilution method. Four inoculum sizes were studied: 1 x 10(2) to 5 x 10(2), 1 x 10(3) to 5 x 10(3), 1 x 10(4) to 5 x 10(4), and 1 x 10(5) to 5 x 10(5) CFU/ml. The National Committee for Clinical Laboratory Standards reference method for antifungal susceptibility testing in yeasts was modified and applied to filamentous fungi. The inoculum was spectrophotometrically adjusted, and all tests were performed in buffered medium (RPMI 1640) at pH 7.0 with incubation at 35 degrees C for 72 h. MICs were read at 24, 48, and 72 h. Amphotericin B showed a minimum effect of inoculum size on MICs for all species with the exception of P. boydii (P < 0.05). A significant effect of inoculum size on MICs was observed with 5-fluorocytosine, for which there was an increase of more than 10-fold in MICs against all Aspergillus spp. between inoculum concentrations of 10(2) and 10(4) CFU/ml (P < 0.001). For itraconazole, the results showed a more species-dependent increase of MICs, most strikingly for R. arrhizus and P. boydii. Miconazole, which was tested only with P. boydii, did not demonstrate a significant effect of inoculum size on MICs. In summary, the effect of inoculum size on MICs for filamentous fungi was dependent upon the organism and antifungal compound tested. Thus, among antifungal compounds, itraconazole and 5-fluorocytosine demonstrated significant inoculum effects, while amphotericin B and miconazole showed comparatively minimum inoculum effects against pathogenic filamentous fungi. Moreover, among filamentous fungi, P. boydii and R. arrhizus exhibited the greatest inoculum effect.
Escherichia coli adherence to HEp-2 cells with prefixed cells
Authors: Zepeda-Lopez H M , Gonzalez-Lugo G M
Published: January 2020

We describe a new method which uses cold absolute methanol-prefixed cells for adherence of enteropathogenic Escherichia coli to HEp-2 cells. We found that a method using bacteria grown in Penassay broth to 10(6) to 10(7) CFU/ml and incubated with prefixed cells for 3 h at 37 degrees C, showed 100% sensitivity and specificity against a method using live cells.
Diagnosis of congenital toxoplasmosis by immunoblotting and relationship with other methods
Authors: Chumpitazi B F , Boussaid A , Pelloux H , Racinet C , Bost M , Goullier-Fleuret A
Published: January 2020

Immunoblot has been evaluated as a diagnostic method for congenital toxoplasmosis. Like enzyme-linked immunofiltration assay (ELIFA), immunoblot can be used to compare antibody patterns and to determine if the antibodies are transmitted by the mother or synthesized by the fetus or infant. Among the 48 infants tested, 27 had congenital toxoplasmosis and 21 were suspected but had none. Reproducibility, sensitivity, specificity, and positive predictive values in immunoblot for immunoglobulins (Igs) G+M+A and/or G+M were 90, 92.6, 89.1, and 92.4%, respectively. G+M immunoblot and G+M ELIFA have better sensitivities than the conventional IgM immunosorbent agglutination assay, IgM enzyme-linked immunosorbent assay (ELISA), IgM immunofluorescence antibody test, in vitro culture, and mouse inoculation. The novel antibodies, i.e., those synthesized by infants against Toxoplasma gondii, were of the IgG class in most cases, although a confident diagnosis could be related to the number of observed Ig classes (G+M, G+A, and G+M+A). Immunoblot has a better resolution than ELIFA. In prenatal diagnosis, immunoblot could be complementary to in vitro culture and mouse inoculation. In the other cases, early detection by immunoblot appears to give the best results when compared with the other serological methods.
Geographic distribution of human rotavirus VP4 genotypes and VP7 serotypes in five South African regions
Authors: Steele A D , van Niekerk M C , Mphahlele M J
Published: January 2020

The rotavirus outer capsid proteins elicit the production of neutralizing antibodies and are known to play a role in inducing resistance to disease. In this study, cDNA probes directed at the six most common human rotavirus VP7 serotypes (G1 to G4, G8, and G9) and five human rotavirus VP4 genotypes (P4, P6, P8, P9, and P10) were utilized. Hybridization analysis of 572 human rotavirus strains collected from five regions in South Africa was performed to determine the distribution of the VP7 serotypes and VP4 genotypes in nature. VP7 serotype G1 was identified most frequently, occurring in 51% of the rotavirus strains tested. VP7 serotypes G2 and G4 occurred in similar numbers, although their distribution varied regionally. Few serotype G3 strains and no G8 or G9 strains were identified. The P8 VP4 genotype occurred most frequently overall (66%), and the P4 genotype was detected next most frequently. The P6 genotype was identified in 28 symptomatically infected neonates and in 8 symptomatic infants. Few P9 strains were identified. The potential for reassortment events was demonstrated by dual infections with different viruses.
Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains
Authors: van Belkum A , Kluytmans J , van Leeuwen W , Bax R , Quint W , Peters E , Fluit A , Vandenbroucke-Grauls C , van den Brule A , Koeleman H
Published: January 2020

Fifty-nine isolates of Staphylococcus aureus and a single strain of Staphylococcus intermedius were typed by arbitrarily primed PCR (AP-PCR). To study reproducibility and discriminatory abilities, AP-PCR was carried out in seven laboratories with a standardized amplification protocol, template DNA isolated in a single institution, and a common set of three primers with different resolving powers. The 60 strains could be divided into 16 to 30 different genetic types, depending on the laboratory. This difference in resolution was due to differences in technical procedures (as shown by the deliberate introduction of experimental variables) and/or the interpretation of the DNA fingerprints. However, this did not hamper the epidemiologically correct clustering of related strains. The average number of different genotypes identified exceeded those of the more traditional typing strategies (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hebert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407-415, 1994). Comparison of AP-PCR with pulsed-field gel electrophoresis (PFGE) indicated the existence of strains with constant PFGE types but variable AP-PCR types. The reverse (constant AP-PCR and variable PFGE patterns) was also observed. This indicates additional resolution for combined analyses. It is concluded that AP-PCR is well suited for genetic analysis and monitoring of nosocomial spreading of staphylococci. The interlaboratory reproducibility of DNA-banding patterns and the intralaboratory standardization need improvement.
Comparison of BioStar Strep A OIA optical immune assay, Abbott TestPack Plus Strep A, and culture with selective media for diagnosis of group A streptococcal pharyngitis
Authors: Roe M , Kishiyama C , Davidson K , Schaefer L , Todd J
Published: January 2020

We directly compared three techniques for the diagnosis of group A streptococcal pharyngitis in 500 symptomatic children seen in the Emergency Department or Child Care Clinic of The Children's Hospital of Denver. Throats were vigorously swabbed with two rayon swabs, which were transported immediately to the Microbiology Laboratory. Each swab was cultured aerobically on Strep A Isolation Agar (Remel) and then tested for antigen-one swab by the Strep A OIA optical immune assay (BioStar) and the other by the TestPack Plus Strep A (Abbott) technique. Each test was performed blind to the others. The refrigerated pledget was cultured in Todd-Hewitt broth if an antigen test was positive and both direct plate cultures were negative (the "gold standard" was any culture positive). All isolates were serologically grouped. Of 500 complete patient cultures, 151 (30%) were positive for group A streptococcal growth. The two antigen tests gave comparable results with an average sensitivity of 83%. Each was significantly (P < 0.02) less sensitive than its corresponding culture. The BioStar Strep A OIA optical immune assay produced significantly (P < 0.003) more false-positive results than did the Abbott test. Rapid antigen testing is not sensitive enough to eliminate the need for backup cultures.
Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus
Authors: Lin D , Lehmann P F , Hamory B H , Padhye A A , Durry E , Pinner R W , Lasker B A
Published: January 2020

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.
Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains
Authors: Hermans P W , Sluijter M , Hoogenboezem T , Heersma H , van Belkum A , de Groot R
Published: January 2020

The aim of this study was to identify the strengths and weaknesses of five DNA fingerprint methods for epidemiological typing of Streptococcus pneumoniae. We investigated the usefulness of (i) ribotyping, (ii) BOX fingerprinting with the BOX repetitive sequence of S. pneumoniae as a DNA probe, (iii) PCR fingerprinting with a primer homologous to the enterobacterial repetitive intergenic consensus sequence, (iv) pulsed-field gel electrophoresis of large DNA fragments, and (v) restriction fragment end labeling to detect restriction fragment length polymorphism of small DNA fragments. Twenty-eight S. pneumoniae strains isolated from the blood and/or cerebrospinal fluid of 21 patients were analyzed. Genetic clustering among the 28 strains was independent of the DNA fingerprint technique used. However, the discriminatory power and the similarity values differed significantly among the individual techniques. BOX fingerprinting, pulsed-field gel electrophoresis, and restriction fragment end labeling provided the highest degree of discriminatory power. Furthermore, the ease with which computerized fingerprint analysis could be conducted also varied significantly among the techniques. Ribotyping, BOX fingerprinting, and restriction fragment end labeling were very suitable techniques for accurate computerized data analysis. Because of their high discriminatory potential and ease of accurate analysis, we conclude that BOX fingerprinting and restriction fragment end labeling are the most suitable techniques to type pneumococcal strains.
Investigation of the reformulated Remel Synergy Quad plate for detection of high-level aminoglycoside and vancomycin resistance among enterococci
Authors: Free L , Sahm D F
Published: January 2020

We investigated the accuracy of the recently released Remel Synergy Quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the National Committee for Clinical Laboratory Standards recommended guidelines (National Committee for Clinical Laboratory Standards, M7-A3, 1993). The Synergy Quad correctly determined the gentamicin and streptomycin resistance status for > or = 97% of 147 Enterococcus faecalis and Enterococcus faecium isolates tested. Detection of vancomycin resistance also was reliable, as no false susceptibility occurred with 36 vancomycin-resistant E. faecalis and E. faecium strains and false resistance occurred only once with the 47 susceptible strains tested. One strain each of Enterococcus gallinarum and Enterococcus casseliflavus failed to grow on the screen, but because the true nature and significance of resistance in such isolates is unknown the implication of their screen negativity is uncertain. In summary, the Remel Synergy Quad provides a highly accurate and convenient method for susceptibility testing of enterococci against gentamicin, streptomycin, and vancomycin.
Quantitative competitive PCR with bronchoalveolar lavage fluid for diagnosis of toxoplasmosis in AIDS patients
Authors: Bretagne S , Costa J M , Fleury-Feith J , Poron F , Dubreuil-Lemaire M L , Vidaud M
Published: January 2020

Of 121 AIDS patients, 12 (10%) had Toxoplasma gondii DNA detected by competitive PCR in their bronchoalveolar lavage samples. Quantitation of the PCR results showed a correlation between the parasite burden and the serum lactic dehydrogenase titer. Quantitative competitive PCR could be useful to assess the diagnosis and the severity of pulmonary toxoplasmosis.
False-positive cryptococcal antigen latex agglutination caused by disinfectants and soaps
Authors: Blevins L B , Fenn J , Segal H , Newcomb-Gayman P , Carroll K C
Published: January 2020

Five disinfectants or soaps were tested to determine if any could be responsible for false-positive results obtained with the Latex-Crypto Antigen Detection System kit (Immuno-Mycologics, Inc., Norman, Okla.). Three disinfectants or soaps (Derma soap, 7X, and Bacdown) produced false-positive agglutination after repeated washing of ring slides during testing of a known negative cerebrospinal fluid specimen.
Identification of microsporidia in stool specimens by using PCR and restriction endonucleases
Authors: Fedorko D P , Nelson N A , Cartwright C P
Published: January 2020

We report the development of a PCR-based assay for the detection of microsporidia in clinical specimens. A single primer pair complementary to conserved sequences of the small-subunit rRNA enabled amplification of DNA from the four major microsporidian pathogens of humans: Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, and Septata intestinalis. The extraction method allowed PCR amplification of E. bieneusi and S. intestinalis DNA from sodium hypochlorite-treated stool specimens. Differentiation of the microsporidian gastrointestinal pathogens E. bieneusi and S. intestinalis could be accomplished by restriction endonuclease digestion of PCR products using PstI and HaeIII.
Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization
Authors: Ylitalo N , Bergström T , Gyllensten U
Published: January 2020

Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associated with infection by certain types of human papillomavirus (HPV). To facilitate the detection and molecular typing of HPV in clinical samples, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing temperatures. This method eliminates cross-contamination between samples during transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the E1 system and used to distinguish 19 genital HPV types. The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M.M. Manos, Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky, Cancer Cells 7:209-214, 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears. The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitable for research as well as clinical analyses.
Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus
Authors: Fingerle V , Hauser U , Liegl G , Petko B , Preac-Mursic V , Wilske B
Published: January 2020

A total of 472 field-collected Ixodes ricinus ticks from southern Germany were investigated by immunofluorescence for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and with monoclonal antibodies specific for outer surface proteins A and C (OspA and OspC, respectively). Borreliae were detected in 90 ticks with the polyvalent immunofluorescence assay. Infection rates in adults (females, 20.2%; males, 25.2%) were significantly higher than in nymphs (12.1%). OspA was detected in 77 ticks and OspC was detected in only 1 tick with the respective monoclonal antibodies. We therefore conclude that B. burgdorferi in unfed I. ricinus ticks usually expresses OspA and very rarely OspC.
Characterization of Finnish Borrelia burgdorferi sensu lato isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with monoclonal antibodies
Authors: Tuomi J , Rantamäki L K , Tanskanen R , Junttila J
Published: January 2020

Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis.
Human Bordetella bronchiseptica infection related to contact with infected animals: persistence of bacteria in host
Authors: Gueirard P , Weber C , Le Coustumier A , Guiso N
Published: January 2020

Within a period of 2 1/2 years, Bordetella bronchiseptica was isolated four times from a 79-year-old woman with bronchopneumonia. We have demonstrated by pulsed-field gel electrophoresis that this infection was related to contact with infected rabbits. The initial human B. bronchiseptica isolate had a phenotype characteristic of usual B. bronchiseptica clinical isolates; it produced toxin and adhesins, such as adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin, and was able to induce lethality in a murine respiratory model. By contrast, although the three successive human isolates produced adhesins, they did not express adenylate cyclase-hemolysin and were unable to induce lethality. This implies that adenylate cyclase-hemolysin is required to induce lethality. We suggest that B. bronchiseptica may persist in the host, with expression of adenylate cyclase-hemolysin being essential for the initiation of infection and expression of adhesins being essential for persistence.
Molecular epidemiology of tuberculosis in Denmark in 1992
Authors: Yang Z H , de Haas P E , Wachmann C H , van Soolingen D , van Embden J D , Andersen A B
Published: January 2020

The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark.
Utility of recombinant flagellar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection
Authors: Godsel L M , Tibbetts R S , Olson C L , Chaudoir B M , Engman D M
Published: January 2020

The protozoan Trypanosoma cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America and of growing concern in the United States as the number of infected immigrants increases. There is currently no testing of U.S. blood products for T. cruzi infection, and the best tests available, although highly sensitive, are not of high enough specificity to be useful for widespread screening of the blood supply in this country. Among the parasite antigens detected by sera of infected humans and mice, those in the range of 24 to 26 kDa are particularly reactive. With an aim of developing a sensitive, specific, recombinant antigen-based serologic test for T. cruzi infection, we used two antibody reagents specific for these 24- to 26-kDa antigens to isolate cDNA clones from a T. cruzi expression library. One clone was found to encode a previously characterized T. cruzi antigen, a 24-kDa flagellar calcium-binding protein (FCaBP). Recombinant FCaBP was found to be a sensitive, specific reagent for distinguishing T. cruzi-infected individuals from uninfected persons, and it therefore could potentially be used for screening purposes, especially if combined with other recombinant T. cruzi antigens that have similarly high degrees of diagnostic sensitivity and specificity.
Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare
Authors: Nishimori K , Eguchi M , Nakaoka Y , Onodera Y , Ito T , Tanaka K
Published: January 2020

The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR-amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex.
Rapid detection of methicillin resistance in coagulase-negative staphylococci by commercially available fluorescence test
Authors: Ieven M , Jansens H , Ursi D , Verhoeven J , Goossens H
Published: January 2020

A commercially available fluorescence test (Crystal MRSA ID System; BBL) for the rapid detection of methicillin resistance in Staphylococcus aureus was evaluated for the detection of methicillin resistance in coagulase-negative staphylococci. The assay was compared with the agar dilution method and mecA detection by PCR. Provided that one uses an inoculum equivalent to a no. 2 McFarland standard instead of a no. 0.5 McFarland standard, which is used for S. aureus, the Crystal MRSA ID System detects methicillin resistance among coagulase-negative staphylococci with sensitivity, specificity, and positive and negative predictive values of 98.5, 95.4, 96.3, and 98.1%, respectively.
Detection of anti-toxoplasma immunoglobulin A antibodies by Platelia-Toxo IgA directed against P30 and by IMx Toxo IgA for diagnosis of acquired and congenital toxoplasmosis
Authors: Decoster A , Gontier P , Dehecq E , Demory J L , Duhamel M
Published: January 2020

Platelia-Toxo IgA and IMx Toxo IgA assays were used with 260 serum samples, of which 93 were from seroconverted patients, 58 were from 21 congenitally infected children, and 109 were from uninfected patients, to detect anti-P30 immunoglobulin A antibodies. Because of its enhanced sensitivity, Platelia-Toxo IgA is more efficient in diagnosing acute or congenital toxoplasmosis. IMx Toxo IgA must not be used to diagnose congenital toxoplasmosis.
Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis
Authors: Duhamel G E , Muniappa N , Mathiesen M R , Johnson J L , Toth J , Elder R O , Doster A R
Published: January 2020

Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.
Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients
Authors: Kersulyte D , Struelens M J , Deplano A , Berg D E
Published: January 2020

Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency.
Comparison of in-house and commercially prepared haemophilus test media for disk diffusion testing of ampicillin against Haemophilus species
Authors: Laue R R , Kohner P C , Cockerill F R
Published: January 2020

The performance characteristics of two lots of Haemophilus Test Medium (HTM) prepared in-house at Mayo Clinic, one lot obtained from BBL (Cockeysville, Md.) and one lot obtained from Remel (Lenexa, Kans.), for disk diffusion susceptibility testing of ampicillin against 81 Haemophilus strains were assessed. Insufficient growth occurred in 2.5 to 13.6% of strains, depending on the HTM used, and was most frequent for beta-lactamase-negative, ampicillin-resistant strains when they were tested with one lot of HTM prepared in-house. Results for all 14 beta-lactamase-positive Haemophilus strains were in complete agreement for all lots of HTM when either 1990 or 1993 National Committee for Clinical Laboratory Standards (NCCLS) interpretive guidelines were followed. However, these beta-lactamase-positive Haemophilus strains could be as precisely, but more rapidly, identified by the cefinase disk method. Results for 56 beta-lactamase-negative, ampicillin-susceptible strains and 11 beta-lactamase-negative, ampicillin-resistant strains varied significantly among all HTM lots tested and as to whether 1990 or 1993 NCCLS interpretive guidelines were followed. We conclude that in our hands disk diffusion testing with HTM prepared in-house or obtained from two commercial sources and following either 1990 or 1993 NCCLS guidelines is an unacceptable method for determining the susceptibilities of Haemophilus strains to ampicillin.
Characterization of a 17-kilodalton antigen of Bartonella henselae reactive with sera from patients with cat scratch disease
Authors: Anderson B , Lu E , Jones D , Regnery R
Published: January 2020

A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay. Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision. All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen. The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes. Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD. The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.
Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae
Authors: Hookey J V , Birtles R J , Saunders N A
Published: January 2020

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species within the family Legionellaceae. The sizes of the PCR products varied from 1,353 to 350 bp. Strains of Legionella pneumophila were characterized as having products of approximately 900 and 530 bp, and L. birminghamensis had products of 1,390, 960, and 380 bp. Of the 38 species of legionellae examined, only 7 were indistinguishable (L. erythra from L. rubrilucens, L. anisa or L. cherrii from L. tucsonensis, and L. quateirensis from L. shakespearei). Two environmental isolates were identified as L. pneumophila. Strain LLAP-3, which was a symbiont of amoebae, could not be associated with any Legionella sp. studied.
Geographic spread of epidemic multiresistant Staphylococcus aureus clone in Brazil
Authors: Teixeira L A , Resende C A , Ormonde L R , Rosenbaum R , Figueiredo A M , de Lencastre H , Tomasz A
Published: January 2020

Staphylococcus aureus isolates from five large teaching hospitals and one medium-size community hospital located in geographically distant parts of Brazil, in the south and southeast (Rio de Janeiro, Niteroi, Sao Paulo, Porto Alegre) and in the north (Manaus), were tested for their antibiotic resistance patterns and genetic backgrounds. Eighty-five of the 152 isolates were identified as methicillin-resistant S. aureus (MRSA) by using a combination of an agar dilution screen and a mecA gene-specific DNA probe. All MRSA isolates were resistant to penicillin, erythromycin, gentamicin, oxacillin, and cephalothin, and the majority of isolates (74%) were also resistant to chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, and clindamycin as well and were susceptible only to vancomycin. Isolates obtained from hospitals in Sao Paulo, Rio de Janeiro, Niteroi, and Porto Alegre (1,600 km from one another) and Manaus (3,700 km from Rio de Janeiro) were examined by a variety of molecular fingerprinting techniques: the nature of the mecA polymorph and Tn554 attachment sites and restriction fragment length polymorphism of genomic DNAs after SmaI restriction and separation of the digested DNA by pulsed-field gel electrophoresis. The overwhelming majority of the isolates shared a common pulsed-field gel electrophoresis pattern and carried mecA polymorph III in combination with Tn554 pattern B, indicating the presence of a single, epidemic MRSA clone spread over large geographic distances of Brazil.
Evaluation of an enterovirus group-specific anti-VP1 monoclonal antibody, 5-D8/1, in comparison with neutralization and PCR for rapid identification of enteroviruses in cell culture
Authors: Trabelsi A , Grattard F , Nejmeddine M , Aouni M , Bourlet T , Pozzetto B
Published: January 2020

We evaluated the usefulness of a commercially available monoclonal antibody (MAb) directed against a group-specific epitope of the capsid protein VP1 of enteroviruses for the rapid identification of these viruses in cell culture. The MAb was assayed in an indirect immunofluorescence test with cultured cells infected by various serotypes of enterovirus; all 39 serotypes tested, including echoviruses 22 and 23, which are considered atypical enteroviruses, were reactive. The MAb was also tested with 61 strains recovered from clinical specimens inoculated into cell cultures in comparison with seroneutralization with intersecting pools of hyperimmune sera and PCR with primers from the 5' untranslated region of enteroviruses. There was total agreement between the results obtained with the MAb and those obtained by PCR, even for those strains of enteroviruses which were found to be untypeable with polyclonal antisera. These data demonstrate the usefulness of the MAb for rapid identification of enteroviruses in cell culture.
Peritonitis involving a Capnocytophaga sp. in a patient undergoing continuous ambulatory peritoneal dialysis
Authors: Esteban J , Albalate M , Caramelo C , Reyero A , Carriazo M A , Hernandez J , Ortiz A , Soriano F
Published: January 2020

Capnocytophaga is a genus of gram-negative rods involved mainly in bacteremia in immunocompromised hosts. Here we report a case of Capnocytophaga peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis. The disease appeared as a superinfection during antimicrobial therapy of a previous episode of peritonitis.
Screening urine samples by leukocyte esterase test and ligase chain reaction for chlamydial infections among asymptomatic men
Authors: Anestad G , Berdal B P , Scheel O , Mundal R , Odinsen O , Skaug K , Khalil O S , Plier P , Lee H
Published: January 2020

Urine samples from 358 asymptomatic males were screened for urethral inflammation by the leukocyte esterase (LE) test and for Chlamydia trachomatis by the ligase chain reaction (LCR). LE and LCR positivity rates were 7.5% (27 of 358 samples) and 2.8% (10 of 358 samples), respectively. Eight of the 10 LCR-positive samples were detected by the LE screening test. The urine LE prescreening test in combination with the LCR assay may be a reasonable approach for genitourinary chlamydial disease control.
Survival of anaerobic bacteria in various thioglycolate and chopped meat broth formulations
Authors: Claros M C , Citron D M , Goldstein E J
Published: January 2020

Three commercially available formulations of thioglycolate broth and of chopped meat broth were evaluated for their abilities to maintain the viabilities of 32 strains of anaerobic bacteria during a period of 8 weeks. While thioglycolate broth supported the initial (48-h) growth of all strains tested, approximately half of the strains died off within 4 weeks. Chopped meat broths maintained the viabilities of almost all cultures during the test period.
Novel method for rapid identification of Nocardia species by detection of preformed enzymes
Authors: Biehle J R , Cavalieri S J , Felland T , Zimmer B L
Published: January 2020

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.
Identification of a 25-kilodalton protein of Mycobacterium bovis BCG to distinguish BCG strains from Mycobacterium tuberculosis
Authors: Kumar D , Srivastava B S , Singh N B , Srivastava R
Published: January 2020

Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M. tuberculosis.