bluuebunny/crossref_metadata_2025 huggingface dataset:
Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model
Authors: Monteiro L , Bonnemaison D , Vekris A , Petry K G , Bonnet J , Vidal R , Cabrita J , Mégraud F
Published: January 2020

A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.
Actinobacillus suis strains isolated from healthy and diseased swine are clonal and carry apxICABDvar. suis and apxIICAvar. suis toxin genes
Authors: Van Ostaaijen J , Frey J , Rosendal S , MacInnes J I
Published: January 2020

Actinobacillus suis isolates recovered from both healthy and diseased pigs were characterized by biochemical testing, serotyping, restriction endonuclease fingerprinting, and apx toxin gene typing. The clinical isolates analyzed were collected over a 10-year period from approximately 40 different locations in southwestern Ontario, Canada. Little variation in the biochemical profiles of these isolates was seen, and all isolates reacted strongly with rabbit antisera prepared against one of the strains. Similarly, by using BamHI and BglII for restriction endonuclease fingerprinting (REF) analysis, all isolates were found to belong to a single REF group. Minor variations could be detected, especially in the BglII fingerprints, but overall the patterns were remarkably similar. Sequences that could be amplified by PCR with primers to the apxICA and apxIICA genes of Actinobacillus pleuropneumoniae were detected in all strains. Although no amplification was obtained with primers to the A. pleuropneumoniae apxIBD genes, sequences with homology to apxIBD were detected by hybridization. There was no evidence of apxIII homologs. Taken together, these data suggest that A. suis isolates are genotypically and phenotypically very similar, regardless of their source, and that they contain genes similar to, but not identical to, the apxICABD and apxIICA genes of A. pleuropneumoniae.
Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus
Authors: Personne P , Bes M , Lina G , Vandenesch F , Brun Y , Etienne J
Published: January 2020

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.
Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995
Authors: Dalsgaard A , Skov M N , Serichantalergs O , Echeverria P , Meza R , Taylor D N
Published: January 2020

Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated.
Evaluation of monoclonal antibodies for subtyping of currently circulating human type A influenza viruses
Authors: Tkácová M , Varecková E , Baker I C , Love J M , Ziegler T
Published: January 2020

The hemagglutinin subtype specificities of six monoclonal antibodies (MAbs) to influenza type A viruses were evaluated in a rapid culture assay by immunoperoxidase staining. Confluent monolayers of MDCK cells in multiwell plates were inoculated with (i) 23 reference viruses, (ii) 200 isolates collected during the influenza season 1995 to 1996, and (iii) 28 clinical specimens previously found to be influenza virus positive. After overnight incubation, the cells were fixed and stained with MAbs IVA1/B10, IIF4/D3, 12L/5, 13L/6, 18L/1, or 18L/4. Type-specific MAbs were included as controls. All antibodies gave intensive cytoplasmic staining with infected cells in the absence of any reaction with uninfected cells. MAbs 12L/5, 13L/6, 18L/1, and 18L/4 exclusively reacted with viruses of the subtype H1, and the antibodies IVA1/B10 and IIF4/D3 exclusively reacted with viruses of the subtype H3. None of these MAbs reacted with viruses of the H2 subtype or with influenza type B viruses. Of the 200 recent isolates, 63 were identified as influenza virus type A, subtype H1, 95 were identified as type A, subtype H3, and 41 were identified as type B. One isolate contained a mixture of a type A (H3) and a type B influenza virus. Of the 28 previously positive clinical specimens, 15 contained an influenza virus A, subtype H3, 1 contained an influenza virus A, subtype H1, and 9 contained an influenza B virus. The subtype of a very weakly positive specimen could not be determined, and two specimens remained negative. The MAbs described here allow for a rapid typing and subtyping of influenza virus isolates and for the type- and subtype-specific detection of influenza viruses in clinical specimens.
Rapid detection of Salmonella enterica with primers specific for iroB
Authors: Bäumler A J , Heffron F , Reissbrodt R
Published: January 2020

The iroB gene of Salmonella enterica is absent from the chromosome of the related organism Escherichia coli. We determined the distribution of this gene among 150 bacterial isolates, representing 51 serotypes of different Salmonella species and subspecies and 8 other bacterial species which are frequent contaminants during routine enrichment procedures by Southern hybridization. An iroB-specific DNA probe detected homologous sequences in all strains of S. enterica, including serotypes of S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), and houtenae (IV). No hybridization signal was obtained with strains of Salmonella bongori or other bacterial species. In contrast, hybridization with a DNA probe specific for purD, a purine biosynthesis gene, detected homologs in all bacterial species tested. Primers specific for iroB were used to amplify this gene from 197 bacterial isolates by PCR. The iroB gene could be PCR amplified from S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), houtenae (IV), arizonae (IIIa), and indica (VI), but not from S. bongori or other bacterial species. Thus, PCR amplification of iroB can be used to distinguish between S. enterica and other bacterial species, including S. bongori. A combination of preenrichment in buffered peptone water supplemented with ferrioxamine E and amplification of iroB by magnetic immuno-PCR allowed detection of S. enterica in albumen within 24 h. In conclusion, PCR amplification of iroB is a new sensitive and selective method which has the potential to rapidly detect S. enterica serotypes.
Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme-linked immunosorbent assay
Authors: Livingston R S , Riley L K , Steffen E K , Besch-Williford C L , Hook R R , Franklin C L
Published: January 2020

Helicobacter hepaticus is a newly recognized bacterium associated with chronic active hepatitis, hepatic carcinoma, and inflammatory bowel disease in mice. Currently, fecal or tissue PCR, fecal culture, or histologic examination of silver-stained liver sections is used to diagnose H. hepaticus infection. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of H. hepaticus infection in mice with a membrane digest preparation of H. hepaticus as the antigen. Sera from mice positive for H. hepaticus by PCR or histologic examination (n = 88), positive for Helicobacter bilis by PCR (n = 13), positive for other helicobacters (not identifiable to species level) by PCR (n = 25), or negative for all Helicobacter species by PCR (n = 162) were used to evaluate the ELISA. Results indicated that ELISA provided 93.2% sensitivity, 94% specificity, 87.2% positive predictive value, and 96.9% negative predictive value. Cross-reactive antibodies were detected in some mice infected with helicobacters not identifiable to species level. To further define ELISA sensitivity and specificity, groups of 10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter muridarum, or H. bilis. Sera were collected and examined by the ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and maintained ELISA reactivity throughout the 18-week study, while mice infected with H. muridarum and H. bilis were negative by ELISA. These results indicate that this reported ELISA is highly sensitive and specific for the serodiagnosis of H. hepaticus infection in mice.
Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification
Authors: Badak F Z , Kiska D L , O'Connell M , Nycz C M , Hartley C , Setterquist S , Hopfer R L
Published: January 2020

Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures.
PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems
Authors: Qu W , Jiang G , Cruz Y , Chang C J , Ho G Y , Klein R S , Burk R D
Published: January 2020

Human papillomavirus (HPV) is an etiologic agent of cervical cancer and is the most common sexually transmitted disease in women. PCR amplification of HPV genomes is the most sensitive method for the detection of cervicovaginal HPV. We have compared the two most commonly used PCR primer sets, MY09/MY11 (MY-PCR) and GP5+/GP6+ (GP+-PCR), for the detection of HPV DNA in cervicovaginal lavage samples from 208 women. Oligonucleotide probes for 39 different HPV types were used. Both primer sets amplified a wide spectrum of HPV genotypes and detected similar overall prevalences of 45% (94 of 208) and 43% (89 of 208), respectively. The MY-PCR system detected 27 of 30 (90%) samples with multiple HPV types, whereas the GP+-PCR system detected 14 of 30 (47%) samples with multiple HPV types. Differences in the detection of HPV types 35, 53, and 61 were noted between the two primer systems. Serial dilution of plasmid templates indicated a 3-log decrease in the amplification of HPV type 35 by MY-PCR and HPV types 53 and 61 by GP+-PCR. These results indicate that although the MY-PCR and GP+-PCR identified nearly equivalent prevalences of HPV in a set of clinical samples, differences in the detection of specific types and infections with multiple types were found. Differences in the sensitivities and characteristics of the PCR systems for the detection of HPV within clinical samples should be considered when comparing data between studies and/or in designing new studies or clinical trials.
Molecular epidemiology of antibiotic resistance of Salmonella enteritidis during a 7-year period in Greece
Authors: Tassios P T , Markogiannakis A , Vatopoulos A C , Katsanikou E , Velonakis E N , Kourea-Kremastinou J , Legakis N J
Published: January 2020

A significant increase in the frequency of isolation of Salmonella enteritidis has been observed during recent years in Greece, parallelled by an increasing rate of resistance of this organism to antibiotics. A substantial proportion of ampicillin- and doxycycline-resistant isolates exhibited cross-resistance to drugs of other classes, such as sulfonamides and streptomycin. Isolates of human origin were overall less resistant than those of animal or food-feed origin. Indeed, strains associated with animal infections were characterized by the highest rates of resistance to several antibiotics. These phenotypic data were correlated with genotypic information concerning two distinct populations: isolates from all sources that were resistant only to ampicillin, the drug toward which resistance rates were highest, and a control group of sensitive isolates. Ampicillin resistance was due to a 34-MDa conjugative plasmid. DNA fingerprinting by macrorestriction of genomic DNA revealed two types, A and B, common to both ampicillin-resistant and -sensitive strains, with 80 to 90% of strains being of type A. However, a third type, C, was specific for the sensitive population, representing 17% of those strains. Therefore, although the majority of resistant isolates were genetically related to sensitive ones, there existed a susceptible clone which had not acquired any resistance traits.
Etiological agents of infectious diarrhea: implications for requests for microbial culture
Authors: Rohner P , Pittet D , Pepey B , Nije-Kinge T , Auckenthaler R
Published: January 2020

Gastrointestinal infections remain a frequent disease worldwide. In order to increase our knowledge of the epidemiology for our patient population, we retrospectively analyzed the results obtained for stool samples received at the clinical microbiology laboratory of the University Hospital of Geneva during a 4-year period. A total of 13,965 specimens from 7,124 patients (1.96 specimens per patient) were cultured, yielding 369 (2.6%) Salmonella spp., 408 (2.9%) Campylobacter spp., and 79 (0.6%) Shigella spp. The cumulative positivity rate of 6.1% decreased to 2.7% when patients received antimicrobial agents (P < 0.001). The positivity rate for 5,912 specimens obtained from patients hospitalized for < or = 3 days was 12.6%, whereas it dropped to 1.4% for patients hospitalized for > 3 days (P < 0.001). Of 3,837 stool samples originating from pediatric patients, 8.8% were positive, and 5.1% of 10,128 samples from adults were positive (P < 0.001). The cytotoxin of Clostridium difficile was detected in 379 of 3,723 samples analyzed (10.2%), and rotaviruses were detected in 190 of 1,601 samples (11.9%). We recommend that the use of cultures for enteric bacterial pathogens be restricted to patients hospitalized for < or = 3 days, with the exceptions of follow-up samples, specimens from immunocompromised patients, and patients whose first sample was culture negative or in the rare event of nosocomial food-borne outbreaks. For patients under antimicrobial therapy, testing for cytotoxin of C. difficile should primarily be requested; this analysis should also be accepted for samples from patients not receiving antimicrobial agents at the time of specimen collection. By applying these restrictions, we could have saved at least $5,000 annually.
Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups
Authors: Weig M , Klinker H , Bögner B H , Meier A , Gross U
Published: January 2020

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.
Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay
Authors: Smith J H , Buxton D , Cahill P , Fiandaca M , Goldston L , Marselle L , Rigby S , Olive D M , Hendricks A , Shimei T , Klinger J D , Lane D J , Mahan D E
Published: January 2020

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.
Molecular characterization of two Bordetella bronchiseptica strains isolated from children with coughs
Authors: Stefanelli P , Mastrantonio P , Hausman S Z , Giuliano M , Burns D L
Published: January 2020

During a surveillance program associated with the Italian clinical trial for the evaluation of new acellular pertussis vaccines, two bacterial isolates were obtained in cultures of samples from immunocompetent infants who had episodes of cough. Both clinical isolates were identified as Bordetella bronchiseptica by biochemical criteria, although both strains agglutinated with antisera specific for Bordetella parapertussis, suggesting that the strains exhibited some characteristics of both B. bronchiseptica and B. parapertussis. Both children from whom these strains were isolated exhibited an increase in serum antibody titer to pertussis toxin (PT), a protein that is produced by Bordetella pertussis but that is not thought to be produced by B. bronchiseptica. We therefore examined whether the clinical isolates were capable of producing PT. Neither strain produced PT under laboratory conditions, although both strains appeared to contain a portion of the ptx region that encodes the structural subunits of PT. In order to determine whether the ptx genes may encode functional proteins, we inserted an active promoter directly upstream of the ptx region of one of these strains. Biologically active PT was produced, suggesting that this strain contains the genetic information necessary to encode an active PT molecule. Sequence analysis of the ptx promoter region of both strains indicated that, while they shared homology with the B. bronchiseptica ATCC 4617 sequence, they contained certain sequence motifs that are characteristic of B. parapertussis and certain motifs that are characteristic of B. pertussis. Taken together, these findings suggest that variant strains of B. bronchiseptica exist and might be capable of causing significant illness in humans.
Rapid discrimination of Mycobacterium avium strains from AIDS patients by randomly amplified polymorphic DNA analysis
Authors: Matsiota-Bernard P , Waser S , Tassios P T , Kyriakopoulos A , Legakis N J
Published: January 2020

A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform.
Use of heteroduplex analysis to classify legionellae on the basis of 5S rRNA gene sequences
Authors: Pinar A , Ahkee S , Miller R D , Ramirez J A , Summersgill J T
Published: January 2020

Seventeen different species of Legionella, 12 serogroups of Legionella pneumophila, and 2 Legionella-like amoebal pathogens (LLAP1 and Sarcobium lyticum) were examined by heteroduplex analysis of PCR products of the 5S rRNA gene. Eight different banding patterns were identified, indicating that heteroduplex analysis of this gene can be used to classify these bacteria according to base substitutions between species. This classification may have future applications in clinical and epidemiological studies.
A novel method for isolation of Chlamydia pneumoniae by treatment with trypsin or EDTA
Authors: Kazuyama Y , Lee S M , Amamiya K , Taguchi F
Published: January 2020

To establish a novel method for the efficient isolation of Chlamydia pneumoniae, experiments were performed to determine the effects of EDTA or trypsin treatment of C. pneumoniae on its adsorption and inclusion body formation. Treatment of C. pneumoniae with 0.1% trypsin or 1 mM EDTA significantly increased inclusion body-forming activity from 8,000- to 10,000-fold higher than that of the control. C. pneumoniae was successfully isolated in cultured cells which were inoculated with clinical specimens after treatment with 0.1% trypsin.
Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heat-stable enterotoxin gene (yst)
Authors: Ibrahim A , Liesack W , Griffiths M W , Robins-Browne R M
Published: January 2020

The chromosomal gene yst, which encodes a heat-stable enterotoxin of Yersinia enterocolitica, is a useful diagnostic marker because it occurs only in invasive strains of this species. A homologous gene also occurs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' ends of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 codons were found in the yst of Y. kristensenii. These findings were used to develop a PCR-based assay for yst of Y. enterocolitica which yielded a detectable product in as little as 50 min. The assay was 100% specific in terms of its ability to identify potentially pathogenic strains of Y. enterocolitica regardless of biotype or serotype. The PCR yielded an amplicon that was visible on agarose gel electrophoresis from as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that corresponded to an internal sequence of yst. These results establish the value of the yst gene as a target for the identification of pathogenic bioserotypes of Y. enterocolitica and the usefulness of PCR for this purpose.
Coli surface antigens associated with enterotoxigenic Escherichia coli strains isolated from persons with traveler's diarrhea in Asia
Authors: Serichantalergs O , Nirdnoy W , Cravioto A , LeBron C , Wolf M , Svennerholm A M , Shlim D , Hoge C W , Echeverria P
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) strains were isolated from travelers or military personnel who developed diarrhea after visiting Nepal or who were deployed to Thailand, Indonesia, or the Philippines. ETEC isolates were examined for colonization factor antigen (CFA). CFAs were identified on 59% (40 of 68) of the isolates examined. The lack of a detectable CFA on 41% (28 of 68) of the isolates is of concern for the development of an effective ETEC vaccine.
Prevalence of the virulence-associated gene of Rhodococcus equi in isolates from infected foals
Authors: Haites R E , Muscatello G , Begg A P , Browning G F
Published: January 2020

The prevalence of the plasmid-encoded virulence-associated gene (vapA) of Rhodococcus equi, as determined by PCR, was found to be 98% in isolates from 154 foals with pneumonia, confirming the strong association of vapA with virulence. The vapA genes from 60 representative isolates were compared by digestion with the restriction endonuclease HinfI, and no evidence of sequence variation was detected.
Production of monoclonal antibodies against Rickettsia massiliae and their use in antigenic and epidemiological studies
Authors: Xu W , Raoult D
Published: January 2020

Rickettsiae are gram-negative, obligate intracellular bacteria which have historically been divided into three groups: the typhus group, the scrub typhus group, and the spotted fever group (SFG). Recently, several new SFG rickettsiae have been characterized, and most of these species are associated with ticks and have, as yet, no known pathogenicity toward humans. Rickettsia massiliae, which is widely distributed in Europe and Africa, is one such rickettsia. In order to investigate the antigenic relationships between R. massiliae and other rickettsial species and to develop a more convenient methodology for identifying R. massiliae, we produced monoclonal antibodies against the type strain (Mtu1T) of R. massiliae by fusing immunized splenocytes with SP2/0-Ag14 myeloma cells. A panel of 16 representatives were selected from the 163 positive hybridomas identified on initial screening, and their secreted monoclonal antibodies were further characterized. The reactivities of these 16 monoclonal antibodies with a large panel of rickettsial species were assessed by the microimmunofluorescence assay. All species of the SFG rickettsiae reacted with the monoclonal antibodies directed against epitopes on lipopolysaccharide, which is the common antigen among the SFG rickettsiae. Some closely related species of the SFG, such as Bar29, "R. aeschlimanni," and R. rhipicephali, showed strong cross-reactivities with the monoclonal antibodies directed against epitopes on the two major high-molecular-mass heat-labile proteins (106 and 120 kDa). In addition, species-specific monoclonal antibodies demonstrated that R. massiliae is antigenically different from other rickettsial species. Moreover, these species-specific monoclonal antibodies were successfully used for identifying R. massiliae in the ticks collected from southern France, and are therefore potentially useful tools in the identification and investigation of R. massiliae in ticks in large-scale field work.
Use of cloned excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological assay
Authors: Poot J , Kooyman F N , Dop P Y , Schallig H D , Eysker M , Cornelissen A W
Published: January 2020

The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with exposure levels based on cumulative egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitive tool for estimating exposure levels in cattle.
Genotypic distribution of hepatitis C virus in different regions of Thailand
Authors: Kanistanon D , Neelamek M , Dharakul T , Songsivilai S
Published: January 2020

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.
Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory
Authors: Brumback B G , Bolejack S N , Morris M V , Mohla C , Shutzbank T E
Published: January 2020

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.
Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline
Authors: Wen B , Rikihisa Y , Mott J M , Greene R , Kim H Y , Zhi N , Couto G C , Unver A , Bartsch R
Published: January 2020

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.
Microscopic observation of progressive immobilization of leishmania promastigotes in acridine orange stain
Authors: Barreca G S , Berlinghieri M C , Foti F , Matera G , Foca A
Published: January 2020

To rapidly isolate Leishmania donovani promastigotes in samples from Novy-MacNeal-Nicolle (NNN) cultures, a method of staining with acridine orange was developed. Such vital staining combines the advantages of direct microscopic examination (e.g., observation of motility) with more accurate cytological and structural imaging of the stained parasites (usually obtained by Giemsa staining). Progressive immobilization of Leishmania promastigotes associated with a change in fluorescence color was also studied. Our findings may be useful for the early confirmation of a positive culture inoculated with a clinical sample.
Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus
Authors: Green K Y , Kapikian A Z , Valdesuso J , Sosnovtsev S , Treanor J J , Lew J F
Published: January 2020

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.
Evaluation of transport media for Pasteurella multocida isolates from rabbit nasal specimens
Authors: Kawamoto E , Sawada T , Maruyama T
Published: January 2020

A suitable medium for the transport of Pasteurella multocida in nasal specimens from rabbits was investigated by using pure cultures of the organism and nasal swabs from infected rabbits. First, the ability of eight transport media to preserve the viabilities of P. multocida strains isolated from rabbits was studied. Cary-Blair medium and Leibovitz medium no. 15 (L-15) were found to be superior to the other six media tested, enabling survival of the organism for more than 14 days at room temperature. Second, the survival of P. multocida in nasal specimens was evaluated on both Cary-Blair medium and L-15. The recovery rate of the organism from these two media was more than 80 to 90% during 4 days of storage and decreased gradually with increasing preservation time. There were no significant differences (P > 0.05) in recovery rates of the organism between Cary-Blair medium and L-15. On the basis of these results, we recommend the use of Cary-Blair medium for the transport of P. multocida in rabbit nasal specimens because of the ease of transport of nasal swabs by mail.
Microbiologic and clinical value of primary broth cultures of wound specimens collected with swabs
Authors: Silletti R P , Ailey E , Sun S , Tang D
Published: January 2020

In order to assess the microbiologic and clinical value of primary broth culture of wound specimens collected with swabs and submitted to the laboratory in transport medium, we compared the results of primary agar culture with the results of a corresponding primary broth culture for 344 aerobic specimens and 176 anaerobic specimens. While 8.7% (45 of 520) of the specimens yielded organisms from the primary broth culture that were not recovered from the corresponding primary agar culture, only 5.0% (26 of 520) of the specimens yielded organisms from the primary broth culture other than Staphylococcus epidermidis, viridans group streptococci, and Corynebacterium spp. Moreover, the primary broth culture of only 0.6% (3 of 520) of the specimens yielded organisms not recovered from the primary agar culture that caused a change in the therapy of the patient. Our conclusion is that primary broth cultures are unnecessary for the processing of wound specimens properly collected with swabs.
Controlled comparison of bioMérieux VITAL and BACTEC NR-660 systems for detection of bacteremia and fungemia in pediatric patients
Authors: Zaidi A K , Mirrett S , McDonald J C , Rubin E E , McDonald L C , Weinstein M P , Gupta M , Reller L B
Published: January 2020

The bioMérieux VITAL automated blood culture system measures a decrease in fluorescence to detect the presence of microorganisms in blood. To assess the performance of VITAL with AER aerobic medium versus that of the nonradiometric BACTEC NR-660 PEDS PLUS medium for the detection of sepsis in children, a total of 12,146 blood specimens were collected at three university medical centers and inoculated into AER and PEDS PLUS bottles that were weighed before and after filling. The sample volumes were considered adequate in 6,276 bottle pairs. The total yield of isolates was 629, of which 489 (78%) were judged to be the cause of true infections. Staphylococci (P < 0.001) and yeasts (P < 0.05) were detected more often in PEDS PLUS bottles, as were all microorganisms combined (P < 0.001). The improved detection in the PEDS PLUS medium was most marked for patients on antimicrobial therapy (P < 0.001), but remained statistically significant even for patients not on therapy (P < 0.025). There were 431 episodes of sepsis, including 407 considered adequate for analysis. Of the 363 unimicrobial episodes, 278 were detected by both bottles, 64 were detected by PEDS PLUS bottles only, and 21 were detected by AER bottles only (P < 0.01). No false-negative cultures were detected by terminal subculture of the PEDS PLUS bottles when the companion AER bottle was positive. However, there were 14 false-negative cultures (7 yeasts, 5 staphylococci, 1 Enterococcus faecalis, and 1 Enterobacter sp.) on terminal subculture of the AER bottles when the companion PEDS PLUS bottle was positive. When both systems were positive, the VITAL system detected bacteria earlier than did the BACTEC system by a mean of 1.6 h. Also, false-positive signals were less common with the VITAL system. We conclude that the VITAL system with AER medium must be modified to improve the detection of clinically important staphylococci and yeasts if it is to perform comparably to the BACTEC NR-660 nonradiometric system with PEDS PLUS medium for a pediatric population.
Pretreatment of clinical specimens with sodium dodecyl (lauryl) sulfate is not suitable for the mycobacteria growth indicator tube cultivation method
Authors: Pfyffer G E , Welscher H M , Kissling P
Published: January 2020

When using the Mycobacteria Growth Indicator Tube (MGIT), pretreatment of clinical specimens with N-acetyl-L-cysteine-NaOH is recommended by the manufacturer. Processing of clinical specimens (n = 1,000) with sodium dodecyl (lauryl) sulfate-NaOH resulted in both poor recovery and delayed mean time to detection of acid-fast bacilli. Values were comparable to those obtained on solid media.
Indwelling device-related bacteremia caused by serum-susceptible Campylobacter coli
Authors: Hsueh P R , Teng L J , Yang P C , Ho S W , Luh K T
Published: January 2020

Two isolates of serum-susceptible Campylobacter coli were recovered in a 7-day interval from blood from a patient with hepatocellular carcinoma and liver cirrhosis whose peritoneal-caval (Denver's) shunt malfunctioned. Identical random amplified polymorphic DNA fingerprints, cellular fatty acid chromatograms, and antibiograms of the two isolates indicate that C. coli has the ability to cause catheter-related bacteremia following its colonization of the catheter.
Prevalence of Bartonella species in domestic cats in The Netherlands
Authors: Bergmans A M , de Jong C M , van Amerongen G , Schot C S , Schouls L M
Published: January 2020

Cats have been shown to provide the only known reservoir of Bartonella henselae, the causative agent of cat scratch disease. To determine the prevalence of Bartonella bacteremia and antibodies in Dutch cats, blood samples from 113 cats from shelters (sheltered cats), 50 pet cats, and 25 specific-pathogen-free (SPF) cats were analyzed. Culture and subsequent PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA intergenic region and 16S rRNA gene PCR-hybridization assays revealed a prevalence of Bartonella bacteremia in 22% of the sheltered cats and showed no bacteremia in the SPF cats. Three spacer RFLP types were found: types A, B, and G, with type B being predominant over types A and G. An important finding was the existence of mixtures of different Bartonella species. Bartonella DNA was detected in 7 of 27 DNA extracts from fleas combed from the sheltered cats (26%). Seropositivity was 50% for sheltered cats and 56% for pet cats, as determined by a B. henselae enzyme-linked immunoassay.
Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis
Authors: San Millán R , Quindós G , Garaizar J , Salesa R , Guarro J , Pontón J
Published: January 2020

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.