bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of hepatitis C virus kits
Authors: Chaudhary R K , Frenette S , Mo T
Published: January 2020

Hepatitis C virus (HCV) antibodies were detected in 85.9% of the samples by commercial enzyme immunoassay (EIA) kits. Most EIA-positive samples were reactive (80%) by the RIBA HCV test (Ortho Diagnostics). Samples with optical density values greater than or equal to 2.0 were mostly reactive (87%) by RIBA HCV test, in contrast to those with values less than or equal to 1.0 (6.6%). Samples which were indeterminate by the RIBA HCV test were positive (88.4%) by HCV neutralization EIA (Abbott Laboratories), along with 29.4% of samples which were nonreactive by the RIBA HCV test.
Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium difficile from environmental sites
Authors: Clabots C R , Bettin K M , Peterson L R , Gerding D N
Published: January 2020

Cycloserine-cefoxitin-fructose agar (CCFA) and cycloserine-cefoxitin-fructose broth (CCFB) containing either 500 or 250 micrograms of cycloserine per ml were compared for efficacy in the isolation of Clostridium difficile from hospital ward environmental sites. A RODAC imprint technique was used to inoculate prereduced CCFA. Moistened swabs were used to inoculate prereduced CCFB from environmental sites immediately adjacent to the RODAC sample sites. CCFA (6% positive) was significantly more sensitive than CCFB (3% positive; P less than 0.005), regardless of the cycloserine concentration. When the CCFA cycloserine concentration was decreased from 500 to 250 micrograms/ml, the overall rate of positive cultures rose from 4 to 17%. Medium containing 500 micrograms of cycloserine per ml may be too inhibitory to isolate many moderately sensitive strains of C. difficile from environmental sites. Regardless of the cycloserine concentration, the CCFA RODAC imprint technique is superior to the CCFB method.
Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase
Authors: Lyerly D M , Barroso L A , Wilkins T D
Published: January 2020

Computer analysis showed that the gene encoding the latex test-reactive protein of Clostridium difficile exhibited high levels of homology with glutamate dehydrogenases from various sources. Further analysis demonstrated that the recombinant protein possessed glutamate dehydrogenase activity. Our results show that the protein that reacts in commercial latex tests for C. difficile is a glutamate dehydrogenase.
Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease
Authors: DiPersio J R , Varga F J , Conwell D L , Kraft J A , Kozak K J , Willis D H
Published: January 2020

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.
DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool
Authors: Miranda A G , Singh K V , Murray B E
Published: January 2020

Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.
Evaluation of AN-Ident
Authors: Quentin C , Desailly-Chanson M A , Bebear C
Published: January 2020

AN-Ident (Analytab Products, Inc., Plainview, N.Y.) is a ready-to-use system for anaerobe identification. It is based on the detection of constitutive preformed enzymes, is growth independent, and requires only 4 h of aerobic incubation. This micromethod was evaluated for its ability to identify anaerobic bacteria by using a conventional methodology as a reference. Of 265 clinical isolates, AN-Ident accurately identified 241 (91%) of the isolates to the species level and 259 (98%) of the isolates to the genus level, with limited supplemental testing needed (5%). The AN-Ident system performed well for the most common pathogens but less satisfactorily for infrequently isolated and/or asaccharolytic species; expansion and updating of the data base would be helpful. Although some color reactions were difficult to interpret, the commercial kit was easy to use.
Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies
Authors: Andersen A A
Published: January 2020

Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars.
Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile
Authors: Cundy K V , Willard K E , Valeri L J , Shanholtzer C J , Singh J , Peterson L R
Published: January 2020

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC.
Usefulness of a new serological test (Bio-Rad) to diagnose Helicobacter pylori-associated gastritis
Authors: van den Oever H L , Loffeld R J , Stobberingh E E
Published: January 2020

A semiquantitative serological test (G.A.P. test; Bio-Rad) to diagnose Helicobacter (Campylobacter) pylori-associated gastritis has recently become commercially available. This test was evaluated with sera from 72 dyspeptic patients with known H. pylori status. When the instructions provided by the manufacturer were applied strictly, the sensitivity of the test was only 32.7%. With a slight modification of the instructions, the performance characteristics were still unsatisfactory (specificity, 43.5%). When a new cutoff value was determined by a receiver operating curve, the quality of the test (positive predictive value, 95.6%; negative predictive value, 77.8%) was slightly lower than that of a noncommercial enzyme-linked immunosorbent assay (positive predictive value, 95.7%; negative predictive value, 84.0%). It is concluded that the Bio-Rad G.A.P. test has low diagnostic yield, and it is stressed that commercial tests should be evaluated extensively before being applied in daily practice.
Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction
Authors: Aubin J T , Collandre H , Candotti D , Ingrand D , Rouzioux C , Burgard M , Richard S , Huraux J M , Agut H
Published: January 2020

Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.
Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi
Authors: Espinel-Ingroff A , Kerkering T M
Published: January 2020

Homogeneous inoculum suspensions of 29 isolates of clinically important filamentous fungi were adjusted with a spectrophotometer (530 nm) to obtain standardized preparations containing 1 x 10(6) to 5 x 10(6) CFU/ml. Colony counts (CFU per milliliter) of 1 x 10(6) to 5 x 10(6) were achieved on three different days for isolates of Aspergillus spp., Pseudallescheria boydii, and Sporothrix schenckii (80% +/- 2% transmission), and colony counts of 7 x 10(5) to 2.9 x 10(6) (70% +/- 2% transmission) were achieved for Mucor spp. and Rhizopus spp.
Pseudoresistance of Pseudomonas aeruginosa resulting from degradation of imipenem in an automated susceptibility testing system with predried panels
Authors: White R L , Kays M B , Friedrich L V , Brown E W , Koonce J R
Published: January 2020

During the use of a single lot of custom breakpoint panels (Sensititre; Radiometer America Inc., Westlake, Ohio), imipenem susceptibility declined from 70 to 44% for clinical isolates of Pseudomonas aeruginosa. With a new lot, susceptibility increased to 73%. Subsequent evaluations with P. aeruginosa ATCC 27853 revealed a similar susceptibility pattern and an increase in the MIC of imipenem when determined in panels with increasing ages. Imipenem concentrations were determined by high-performance liquid chromatography by using 11 different lots of MIC and breakpoint panels (139 to 893 days of age). The amount of imipenem remaining declined from 94.4 to 13.8% (r = 0.9225) over the age range of the panels. These data suggest that imipenem in Sensititre MIC and breakpoint panels degrades over time and that the decrease in imipenem may be largely responsible for the decline in P. aeruginosa susceptibility.
Survey for Ixodes spp. and Borrelia burgdorferi in southeastern Wisconsin and northeastern Illinois
Authors: Callister S M , Nelson J A , Schell R F , Jobe D A , Bautz R , Agger W A , Coggins J
Published: January 2020

Forested areas adjacent to Milwaukee, Wis., and Chicago, Ill., were investigated for rodents and ticks infected with Borrelia burgdorferi, the causative agent of Lyme disease. White-footed mice (Peromyscus leucopus or Peromyscus maniculatus), meadow voles (Microtus pennsylvanicus), and eastern chipmunks (Tamias striatus) were captured; and specimens from these animals were cultured for B. burgdorferi to define whether the midwestern Lyme disease area currently encompasses these large metropolitan centers. During 1988, B. burgdorferi was successfully cultured from the tissues of two M. pennyslvanicus voles captured from the Chicago area. However, no Ixodes spp. ticks were captured. None of 274 animals captured from sites I3 and 12 additional sites in Wisconsin and Illinois during the summer of 1989 were infected with B. burgdorferi or Ixodes spp. In addition, no ticks were recovered when the underbrush in 11 contiguous areas was flagged. Apparently, B. burgdorferi is rarely found in these areas because of the absence of the appropriate tick vectors. Further studies are needed to monitor the dispersal of B. burgdorferi-infected Ixodes dammini into this heavily populated midwestern region.
Evaluation of five monoclonal antibody-based kits or reagents for the identification and culture confirmation of herpes simplex virus
Authors: Lipson S M , Salo R J , Leonardi G P
Published: January 2020

Five immunofluorescence (IF) kits or reagents (Bartels [Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.], Imagen [CellTech Diagnostics, Ltd., distributed by Analytab Products, Plainview, N.Y.], Ortho [Ortho Diagnostics Systems, Inc., Raritan, N.J.], Syva [Syva Co., Palo Alto, Calif.], Whittaker [Whittaker Bioproducts, Walkersville, Md.]) were evaluated for typing and laboratory confirmation of herpes simplex virus (HSV). Of 101 clinical isolates tested by each kit or reagent, results for 97 of them were in agreement. Identification of the four isolates with discordant results was performed by restriction endonuclease analysis of the viral DNA. The sensitivity and specificity of the Imagen and Bartels kits were 100%. For the Ortho, Syva, and Whittaker kits or reagents, the HSV type 1 (HSV-1) and HSV type 2 (HSV-2) sensitivities were 97.4 and 100%, 100 and 100%, and 97.4 and 100%, respectively, and the specificities were 100 and 97.4%, 100 and 92.4%, and 100 and 97.4%, respectively. There was one false-positive HSV-2 isolate identified by each of the Ortho and Whittaker kits or reagents. Three false-positive HSV-2 isolates occurred by staining with Syva, giving the erroneous indication of dual isolates. Several isolates stained with Imagen and Whittaker reagents displayed dull IF patterns. A dull green background occurred in ca. one-third of the HSV-2 isolates tested with the Ortho kit. The intensities of IF staining by the Bartels and Syva kits were satisfactory; however, the latter displayed a specificity of 92.7%. A total of 38 and 63 specimens were finally designated as HSV-1 and HSV-2, respectively. Identification of each isolate with the Bartels kit was consistently interpretable and is recommended as the typing and confirmatory assay of choice.
Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions
Authors: Takimoto S , Grandien M , Ishida M A , Pereira M S , Paiva T M , Ishimaru T , Makita E M , Martinez C H
Published: January 2020

Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P < 0.05), influenza virus type A (K = 0.42; Z = 9.7; P < 0.0001), and adenovirus (K = 0.41; Z = 6.5; P < 0.0001) and of ELISA and IFA for influenza virus type A (K = 0.55; Z = 7.0; P < 0.0001) and adenovirus (K = 0.59; Z = 6.8; P < 0.0001). All of the statistical evaluations demonstrated better agreement between ELISA and VIT for influenza virus type A and adenovirus.
Antibody response to serotype-specific and cross-reactive neutralization epitopes on VP4 and VP7 after rotavirus infection or vaccination
Authors: Taniguchi K , Urasawa T , Kobayashi N , Ahmed M U , Adachi N , Chiba S , Urasawa S
Published: January 2020

By using a competitive solid-phase immunoassay with serotype-specific and cross-reactive neutralizing monoclonal antibodies directed at VP4 and VP7, we tested the antibody responses to some neutralization epitopes on VP4 and VP7 in individuals infected or vaccinated with rotavirus. Antibody responses to VP7 epitopes of the infecting serotype of virus were found at a high frequency in both infants and children. In contrast, antibody responses to VP4 and heterotypic VP7 were observed only when the individuals possessed antibodies to any serotype of rotavirus in their acute-phase or prevaccination sera.
Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum
Authors: Guo K J , Bowden D S
Published: January 2020

A nonradioactive hybridization assay for the detection of hepatitis B virus (HBV) DNA in serum with a digoxigenin-labeled probe is described. The probe was sensitive, being able to detect 0.25 pg of homologous HBV DNA, equivalent to 7 x 10(4) genome copies. After extraction of DNA from clinical samples, the probe detected HBV DNA in 11 of 12 hepatitis B e antigen-positive sera and did not react with 6 hepatitis B surface antigen-negative sera. This result was comparable to that obtained with a radiolabeled probe. When serum samples were treated by the alkaline denaturation method, some false-positive reactions were apparent with the digoxigenin-labeled probe, although their frequency could be reduced to around 8% by modifying the sample treatment with a centrifugation step. Overall, the sensitivity and specificity of the digoxigenin-labeled probe indicate that it is a viable alternative to the radiolabeled probe for the detection of HBV DNA in serum. The lack of radioactive reagents in the digoxigenin labeling and detection system and its long shelf-life make this system suitable for routine use in laboratories.
Aeromonas jandaei and Aeromonas veronii dual infection of a human wound following aquatic exposure
Authors: Joseph S W , Carnahan A M , Brayton P R , Fanning G R , Almazan R , Drabick C , Trudo E W , Colwell R R
Published: January 2020

Exudate removed from an infection that developed below the left eye of a 10-year-old male following a previously inflicted wound after aquatic exposure was cultured and revealed two different Aeromonas spp. Further characterization showed that one strain was phenotypically identical to Aeromonas veronii, while the other strain was confirmed by DNA hybridization analysis to be Aeromonas jandaei sp. nov. This is the first report of these more recently described aeromonads, thus far rarely reported from clinical disease, occurring simultaneously in a human infection.
Comparison of the affinities to bovine and human prothrombin of the staphylocoagulases from Staphylococcus intermedius and Staphylococcus aureus of animal origin
Authors: Raus J , Love D N
Published: January 2020

Staphylocoagulases of Staphylococcus aureus (40 strains originally isolated from horses, dogs, cats, cows, sheep, opossums, pigs, humans, and a goat) and Staphylococcus intermedius (19 isolates from dogs and 1 pigeon strain) were tested for their affinity to prothrombins of either bovine or human origin. The tests used were the coagulase tube test (using human, bovine, or equine fibrinogen with either bovine or human prothrombin as the source of coagulase-reacting factor) and a chromogenic assay which enabled quantification of the amidolytic activity of the staphylocoagulase-prothrombin complex. S. intermedius showed weak specificity for human prothrombin, with 15% of the coagulases clotting human fibrinogen, 25% clotting equine fibrinogen, and 40% clotting bovine fibrinogen. However, 65% of coagulases clotted equine fibrinogen, 75% of coagulases clotted human fibrinogen, and 100% of coagulases clotted bovine fibrinogen when bovine prothrombin was used. The animal isolates of S. aureus displayed more diverse specificity toward prothrombin than S. intermedius strains. While 85% of coagulase preparations clotted human fibrinogen when human prothrombin was used, only 45% of preparations clotted bovine fibrinogen when bovine prothrombin was used. However, 62.5% of coagulases clotted human fibrinogen when bovine prothrombin was used and 85% of coagulases clotted bovine fibrinogen when human prothrombin was used. This may be a reflection of the diversity of the animal origins of S. aureus isolates.
DNA fingerprinting by pulsed field gel electrophoresis and ribotyping to distinguish Pseudomonas cepacia isolates from a nosocomial outbreak
Authors: Anderson D J , Kuhns J S , Vasil M L , Gerding D N , Janoff E N
Published: January 2020

We typed 40 isolates of Pseudomonas cepacia obtained from patients involved in a single outbreak using pulsed field gel electrophoresis and ribotyping. All isolates from the majority of the patients, 16 of 18 (89%), were included in a single group. These typing methods should aid in the clarification of the epidemiology of infection with P. cepacia.
Prevalence of respiratory syncytial virus subgroups A and B in France from 1982 to 1990
Authors: Freymuth F , Petitjean J , Pothier P , Brouard J , Norrby E
Published: January 2020

A fluorescence antibody test with monoclonal antibodies was used to determine the subgroup (A or B) of respiratory syncytial virus from infants hospitalized in Caen, France, over eight consecutive epidemics from 1982 to 1990. From 1982 to 1985, 27 (30%) out of 90 frozen nasal slides were classified as subgroup A strains and 63 (70%) were classified as subgroup B. B strains predominated over A in 1983-1984 and 1984-1985. From 1985 to 1990, 284 respiratory syncytial virus field strains were reisolated from frozen materials; 115 (40.5%) were typed as subgroup A and 169 (59.5%) were typed as subgroup B. In 1985-1986, 1986-1987, and 1988-1989, both subgroups were present in almost equal numbers; subgroup A (88.3%) predominated in 1987-1988, and subgroup B (84.5%) predominated in 1989-1990. In conclusion, both subgroups occur together each year, and one subgroup rarely predominates, e.g., subgroup A in 1987-1988 and subgroup B in 1983-1984 and 1989-1990. Therefore, there is a gradual change of the predominant subgroup into another over a period of about 5 years; the relative frequency of subgroup A strains increased from 1983 to 1988, whereas the percentage of subgroup B decreased during the same period.
Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea
Authors: Lortie L A , Dubreuil J D , Harel J
Published: January 2020

Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay. The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I). Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains. Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E. coli strains. The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.
Inhibition of human immunodeficiency virus gene amplification by heparin
Authors: Holodniy M , Kim S , Katzenstein D , Konrad M , Groves E , Merigan T C
Published: January 2020

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.
Evaluation of 10 methods to distinguish epidemic-associated Campylobacter strains
Authors: Patton C M , Wachsmuth I K , Evins G M , Kiehlbauch J A , Plikaytis B D , Troup N , Tompkins L , Lior H
Published: January 2020

We compared four phenotypic and six genotypic methods for distinguishing Campylobacter jejuni strains from animals and humans involved in four epidemics. Based on a comparison with epidemiologic data, the methods that correctly identified all strains in three milkborne outbreaks and one waterborne outbreak were heat-stable and heat-labile serotyping; multilocus enzyme electrophoresis (MEE); DNA restriction endonuclease analysis with BglII, XhoI, PvuII, or PstI; and Southern blot and hybridization of PvuII- and PstI-digested DNA with Escherichia coli 16S and 23S rRNA (ribotyping). Biotyping, phage typing, plasmid analysis, and probing of BglII and XhoI DNA digests with C. jejuni 16S rRNA genes failed to correctly separate one or more strains. MEE, restriction endonuclease analysis, and ribotyping were the most sensitive methods and identified nine types among the 22 strains. These methods were also capable of further distinguishing strains within the same serotype. Data from MEE were also analyzed to calculate genetic relatedness among strains. Serotyping was the most discriminating phenotypic method, with eight and seven types distinguished by the heat-stable and heat-labile methods, respectively. MEE and ribotyping had several advantages over the other methods because they measure relatively stable and significant chromosomal differences and are applicable to other species and genera. These methods, however, are complex and not easily quantified; they are currently limited to specialized laboratories. When antisera are available, serotyping appears to be an effective and more practical approach to the identification of epidemic-related strains.
Serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the microimmunofluorescence test
Authors: Andersen A A
Published: January 2020

A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chlamydia psittaci was prepared. The panel included one monoclonal antibody to each of the 10 serovars. Monoclonal antibodies were selected for their specificity in the indirect microimmunofluorescence test. Each of the monoclonal antibodies had a titer of 1:1,280 or higher to the homologous strain, with only two showing any cross-reactivity at a dilution of 1:10. Chlamydial antigen derived from organisms growing in tissue culture of one well of a 96-well multiwell dish was usually sufficient for the serotyping of an isolate. Infected yolk sac preparations were also suitable for serotyping. The panel of monoclonal antibodies was used to serotype 55 mammalian and avian strains. All except five of the strains were successfully serotyped; these five strains are presumed to represent at least two additional serovars. The use of a panel of monoclonal antibodies in the indirect microimmunofluorescence test provides a rapid and reliable method for serotyping new isolates. Monoclonal antibodies to new serovars can easily be added to the panel.
Blocking enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serotype 2
Authors: Nielsen R , Plambeck T , Foged N T
Published: January 2020

A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes of A. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work.
Reliability of two new test kits for rapid diagnosis of respiratory syncytial virus infection
Authors: Rothbarth P H , Hermus M C , Schrijnemakers P
Published: January 2020

Two new rapid enzyme immunoassays (EIAs) for detecting respiratory syncytial virus (RSV), Directigen (Becton Dickinson Microbiology Systems) and TestPack (Abbott Diagnostics) were compared with virus isolation and direct immunofluorescence by using fresh specimens. The sensitivities of both EIAs were low (72 to 73%), but when initial specimens were used, TestPack had a high sensitivity (92%) in contrast to that of Directigen (76%). Because of its high sensitivity and specificity, TestPack can be used for diagnosis of RSV in acute disease.
Acridine Orange Stain for Determining Intracellular Enteropathogens in HeLa Cells
Authors: Miliotis Marianne D.
Published: January 2020

Green-fluorescent intracellular enteropathogenic bacteria were observed after infected HeLa cell monolayers were stained with acridine orange and counterstained with crystal violet at least 3 h after infection.
Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila
Authors: Sampson J S , Plikaytis B B , Aloisio C H , Carlone G M , Pau C P , Stinson A R
Published: January 2020

Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays.
Restriction fragment length polymorphism analysis of Vibrio cholerae strains associated with a cholera outbreak in Hong Kong
Authors: Yam W C , Lung M L , Ng M H
Published: January 2020

We studied Vibrio cholerae El Tor isolates associated with an outbreak of cholera among Vietnamese refugees interned in Hong Kong. The restriction fragment length polymorphism of the enterotoxin gene was used as an epidemiological marker. All outbreak strains were indistinguishable. They were distinct from strains isolated in Hong Kong prior to the outbreak.
Identification of the innate human immune response to surface-exposed proteins of coagulase-negative staphylococci
Authors: Plaunt M R , Patrick C C
Published: January 2020

The presumed host defense against coagulase-negative staphylococci (ConS), recognized pathogens in hosts with compromised immunity or indwelling medical devices, is opsonophagocytosis. Targets for opsonization remain unclear. Using radiolabeling techniques, we identified the surface-exposed proteins of ConS and determined the innate humoral immune responses to them among healthy adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface proteins extrinsically labeled with 125I demonstrated 20 to 30 proteins with molecular weights of 15,000 to greater than 130,000. Five to ten of these proteins were immunogenic and recognized by normal human sera, including predominant 18-, 41-, 48-, and 51-kDa proteins. We also evaluated the humoral response of cancer patients with ConS bacteremia. Patients' sera obtained before bacteremic episodes demonstrated a pattern of reactivity similar to that of normal human sera. When patients' sera obtained after bacteremic episodes were used to determine whether an expanded immune response followed infection, only one of seven showed reactivity with more proteins than seen with the innate response. Western blot (immunoblot) analysis and whole-cell enzyme-linked immunosorbent assays were also evaluated. This study identifies (i) the surface-exposed proteins available for host interaction, (ii) the innate human antibody response to these proteins, and (iii) the immune response of cancer patients with ConS bacteremia.
Persistent cardiac and urinary tract infections with Borrelia burgdorferi in experimentally infected Syrian hamsters
Authors: Goodman J L , Jurkovich P , Kodner C , Johnson R C
Published: January 2020

The heart can be severely affected in humans with Lyme disease, causing conduction defects and, rarely, heart failure. Although immunodeficient and young mice may develop cardiac lesions, cultivation of Borrelia burgdorferi from cardiac tissues of experimentally infected animals has not been reported previously. We infected Syrian hamsters with B. burgdorferi 297 and found a marked tropism of the spirochete for myocardial and urinary tract tissues. Fifty-six of 57 hearts (98%) and 52 of 58 bladders (90%) were culture positive. The cardiac infection was persistent and could be documented in 21 of 22 hearts (96%) cultured from days 28 to 84 postinfection. The urinary tract was also a site of persistent infection in most animals, with 18 of 23 bladders (78%) being culture positive from days 28 to 84. The persistence of spirochetes was specific for the heart and bladder, as indicated by negative cultures of specimens from the liver and spleen, in which only 1 of 23 cultures was positive from days 28 to 84. Because of the high isolation rates, tropism, and persistence that we found for B. burgdorferi in the hamster heart and bladder, these sites will be useful and important for the cultivation of spirochetes in experimental studies that evaluate the efficacies both of candidate vaccines in preventing infection and of antibiotics in eradicating organisms from privileged sites. In addition, the clear demonstration of persistent cardiac infection with B. burgdorferi may provide a useful model for studying the pathogenesis of cardiac Lyme disease.
Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe
Authors: Todd D , Creelan J L , McNulty M S
Published: January 2020

A dot blot hybridization assay capable of detecting chicken anemia agent (CAA)-specific DNA in tissues from infected birds has been developed. The assay uses a 32P-labeled DNA probe prepared from cloned CAA-specific fragments representing the entire virus genome and has a sensitivity limit of between and 1 and 10 pg. DNAs from CAA isolates originating in the Federal Republic of Germany, Japan, the United States, the United Kingdom, and Australia were detected. Investigation of specimens from experimentally infected chicks indicated that virus-specific DNA was detected in the tissues of birds from 5 through 42 days after infection and that greater amounts were usually detected in the thymus than in the spleen, liver, feces, or blood. Tissues from specific-pathogen-free and broiler chicks which had become infected at an older age through contact with experimentally infected anemic chicks also contained CAA-specific DNA detectable by the assay. Thymuses from 1- to 2-week-old chicks from eight commercial broiler flocks which had been showing clinical signs characteristic of anemia-dermatitis syndrome were found positive by the hybridization technique, but thymuses from chicks obtained from broiler flocks which did not show such signs were found negative. Of the 35 positive samples (from 46 samples tested), 19 (54%) contained virus-specific DNA in sufficiently great amounts to permit 4-h autoradiography exposures and sample throughput times of 2 days. When compared with virus isolation, the CAA dot blot hybridization assay is time- and labor-saving.
Effects of FP2 and a mercury resistance plasmid from Pseudomonas aeruginosa PA103 on exoenzyme production
Authors: Johnson J , Warren R L , Branstrom A A
Published: January 2020

Plasmids encoding mercury resistance carried by Pseudomonas aeruginosa PAO1161 and PA103 were found to be involved in regulating the secretion of protease, phospholipase C, and alkaline phosphatase. Previously, mutations in Pseudomonas strains that caused pleiotropic effects on the production of extracellular enzymes were mapped to the bacterial chromosome. We show that pleiotropic changes in extracellular enzyme production can also be regulated by plasmids. In this study, the effects on secretion of exoenzymes by two mercury resistance plasmids, FP2 from PAO1161 and pRLW103 from PA103, were assayed in P. aeruginosa PAO1 and PAO18. The introduction of either plasmid into PAO1 resulted in a significant decrease in exoprotease production. Additionally, pRLW103 significantly increased the production of alkaline phosphatase by both strains. Phospholipase C was produced only in strain PAO18 containing the pRLW103 plasmid. FP2 had no effect on alkaline phosphatase or phospholipase C production in either strain and was found to decrease exoprotease secretion only in strain PAO1. The results indicate the P. aeruginosa mercury resistance plasmids vary in their ability to modify exoenzyme expression, and this ability is influenced by the host strain.
Repeated DNA in Pneumocystis carinii
Authors: Stringer S L , Hong S T , Giuntoli D , Stringer J R
Published: January 2020

A 16-kb DNA fragment designated Rp3-1 and cloned from the genome of rat-derived Pneumocystis carinii was found to contain sequences that were repeated approximately 70 times per genome. The repeated sequences in Rp3-1 spanned at least 10.4 kb. Sequences in Rp3-1 were present on each of the 16 P. carinii chromosomes resolved by field inversion gel electrophoresis. Most of the P. carinii genomic sequences homologous to those in the Rp3-1 clone appeared to be as long as those in the Rp3-1 clone but were highly polymorphic with respect to restriction enzyme cleavage sites. The Rp3-1 DNA fragment appears to be a member of a family of large, degenerate, dispersed repeats.
Molecular characterization and proposal of a neotype strain for Bartonella bacilliformis
Authors: Brenner D J , O'Connor S P , Hollis D G , Weaver R E , Steigerwalt A G
Published: January 2020

Bartonella bacilliformis, the etiologic agent of bartonellosis, was characterized biochemically and by DNA hybridization, guanine-plus-cytosine content, genome size, and 16S rRNA sequencing. DNAs from the two strains in our collection exhibited 97% relatedness in hydroxyapatite reactions done at 55 degrees C (optimal reassociation criterion) and 100% relatedness in reactions done at 70 degrees C (stringent reassociation criterion). There was no evidence of divergence within the related sequences. B. bacilliformis DNA showed no relatedness to the cat scratch disease bacillus or to a strain of a second species in the same genus as the cat scratch disease bacillus in hybridization reactions done at 65 degrees C. The guanine-plus-cytosine contents of DNAs from the two B. bacilliformis strains were 39 and 40 mol%. Time course reassociation, done by determining spectrophotometrically the time required for one-half of the denatured DNA to form duplexes, indicated that B. bacilliformis has a genome size of approximately 4 x 10(8). The 16S rRNA sequence analysis indicated that B. bacilliformis is in the alpha-2 subgroup of the purple bacteria, class Proteobacteria, and that its closest relatives are Rochalimaea quintana and Brucella abortus. Strain KC583 (= Herrer 020/F12,63 = ATCC 35685) is proposed as the type strain of B. bacilliformis.
Antigenic variation among strains of Chlamydia pneumoniae
Authors: Black C M , Johnson J E , Farshy C E , Brown T M , Berdal B P
Published: January 2020

The antigenic profiles of six strains of Chlamydia pneumoniae were analyzed by the microimmunofluorescence test (MIF) and immunoblotting with human serum and murine monoclonal antibody. MIF-derived antibody titers in serum samples from culture-positive patients were four- to eightfold higher against autologous isolate antigen than they were against the prototype antigen strain TW-183. Sera of patients with respiratory illness that were culture negative and complement fixation positive for Chlamydia spp. produced higher titers by MIF against a strain of C. pneumoniae isolated in the area than they did against TW-183. For two of five cases, the criteria for establishing the diagnosis of acute infection were met only with use of the antigen from the local strain; TW-183 was inadequate for this purpose. Immunoblot profiles revealed antigenic differences between strains that varied with the human serologic response; i.e., unique antigens were recognized by the sera of some individuals and not by the sera of others. Using the reactivity of a genus-specific monoclonal antibody against a major outer membrane protein, we found that strain CWL-011, isolated in Atlanta, Ga., may possess a major outer membrane protein with a molecular mass between those of C. trachomatis L2 and other C. pneumoniae strains. These data provide evidence of several new and unique serotypes of C. pneumoniae and suggest that the serologic diagnosis of C. pneumoniae infection may require the use of antigens from more than one strain of this species.
Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotypes 4 and 7
Authors: Mittal K R , Bourdon S
Published: January 2020

Actinobacillus pleuropneumoniae strains of serotypes 4 and 7 were studied for their antigenic properties by means of agglutination, coagglutination, indirect hemagglutination, immunodiffusion, and counterimmunoelectrophoresis tests. Strains of serotype 4 showed cross-reactivity with those of serotype 7 in various serological tests. Serotype 7 strains were antigenically heterogeneous and shared common antigens with several other serotypes. By using boiled whole-cell saline extract as the antigen in the immunodiffusion test, serotype 7 strains could be divided into four subgroups. Subgroup I strains did not have antigens in common with other serotypes, whereas subgroup II strains had antigens in common with serotype 4; subgroup III strains had antigens in common with serotype 10, and subgroup IV had antigens in common with serotypes 1, 9, and 11. The indirect hemagglutination test using unheated whole-cell saline extract as the antigen detected serotype-specific activity. Quantification of serotype-specific and group-specific antigens by coagglutination and immunodiffusion tests was found useful for identifying strains that belonged to serotype 4 or 7.