bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal-antibody-based enzyme-linked immunosorbent assay for Trichomonas vaginalis
Authors: Lisi P J , Dondero R S , Kwiatkoski D , Spence M R , Rein M F , Alderete J F
Published: January 2020

Trichomonas vaginalis is estimated to infect 4 million women per year in the United States. The diagnosis of trichomoniasis is predominantly achieved by direct microscopic examination of vaginal exudates. This subjective diagnostic procedure is reported to be 75% sensitive under ideal circumstances. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of T. vaginalis directly from vaginal exudates. The ELISA employs a monoclonal antibody specific for a 65-kilodalton surface polypeptide of T. vaginalis as the capture antibody in a sandwich format. A polyclonal rabbit anti-T. vaginalis antibody labeled with horseradish peroxidase serves as the probe. An evaluation of vaginal specimens from women attending clinics revealed a sensitivity and specificity of the ELISA of 89 and 97%, respectively, versus the culture technique. These results indicate the usefulness of this ELISA as an alternative to microscopic and culture methods for the detection of T. vaginalis in vaginal exudates.
Novel plate culture method to improve the microbiological diagnosis of peritonitis in patients on continuous ambulatory peritoneal dialysis
Authors: Gould I M , Reeves I , Chauhan N
Published: January 2020

A novel economical plate culture technique incorporating Tween 80 was used for the isolation of organisms from dialysis effluent in cases of continuous ambulatory peritoneal dialysis peritonitis. It was found to be convenient for routine laboratory use and increased the yield of positive plate cultures in specimens without antibiotics from 53 to 75% (P less than 0.01) and in specimens containing antibiotics from 24 to 38% (P less than 0.05). It allowed for the identification and susceptibility testing of isolates 24 h earlier than did broth culturing and a more ready appreciation of mixed cultures and contaminants.
Legionella pneumophila serogroup Lansing 3 isolated from a patient with fatal pneumonia, and descriptions of L. pneumophila subsp. pneumophila subsp. nov., L. pneumophila subsp. fraseri subsp. nov., and L. pneumophila subsp. pascullei subsp. nov
Authors: Brenner D J , Steigerwalt A G , Epple P , Bibb W F , McKinney R M , Starnes R W , Colville J M , Selander R K , Edelstein P H , Moss C W
Published: January 2020

Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3.
Restriction endonuclease analysis of Staphylococcus epidermidis DNA may be a useful epidemiological marker
Authors: Renaud F , Freney J , Etienne J , Bes M , Brun Y , Barsotti O , Andre S , Fleurette J
Published: January 2020

We compared the epidemiological markers of 13 Staphylococcus epidermidis strains isolated from an adult inpatient during a febrile episode and 23 S. epidermidis strains isolated during a presumptive outbreak of nosocomial infection in a neonatal ward. The total DNA restriction endonuclease analysis (REA) was processed along with the following conventional markers: biotyping, serotyping, phage typing, antibiotic susceptibility profiles, and plasmid profiles. The REA method was reproducible, giving stable results both in vitro and in vivo. For the hospitalized adult patient, the conventional markers of the 13 strains were concordant and the restriction profiles were identical. Five restriction groups were demonstrated during the course of the outbreak. Within two of the groups, the identities of all of the markers were used to verify whether all of the isolates belonged to the same cell clone. In a third group, combined analysis of the conventional markers and REA had to be used to demonstrate isolate similarity. On the other hand, in another group, none of the markers were similar; interpretation was not easy. An epidemiological study of S. epidermidis infections in hospitals must take into account all of the epidemiological markers: biotypes, serotypes, phage types, antibiograms, plasmid profiles, and REA.
Optimal data processing procedure for automatic bacterial identification by gas-liquid chromatography of cellular fatty acids
Authors: Eerola E , Lehtonen O P
Published: January 2020

Gas-liquid chromatography of cellular fatty acids was used in automatic identification of clinical bacterial isolates. The intraspecies variation in the occurrence of fatty acids and the variation in the relative gas-liquid chromatography peak areas of different fatty acids were evaluated and compared with the relative peak areas of these acids. A new chromatogram comparison method involving the use of an exponential function was developed to adjust to data variation optimally. This method was compared with several previously published methods of correlation analysis with data from representative clinical bacteriological isolates. The efficacies of the methods in separating different bacterial species into distinct clusters were compared. The new exponential function method was superior to the others both in its ability to separate species into different clusters and in giving a greater degree of identity to strains within a proper cluster. The results indicate that the gas-liquid chromatography of bacterial cellular fatty acids can be used effectively in the identification of clinically isolated bacteria. However, the usefulness of the analysis depends on the comparison method used and on its ability to cope with data variations.
Temporal relationship of Vibrio parahaemolyticus in patients and the environment
Authors: Kelly M T , Stroh E M
Published: January 2020

We prospectively compared the occurrence of Vibrio parahaemolyticus in patients and the environment in the Pacific Northwest. Inpatient and outpatient stool and wound specimens and water samples from 10 estuarine sites were cultured for V. parahaemolyticus over a period of 3 years. V. parahaemolyticus infections were detected in 13 patients (8 with gastroenteritis; 5 with wound infections), and all of the infections were found in outpatients in physicians' offices. Ten of the infections were locally acquired, and three occurred in patients returning from tropical travel. V. parahaemolyticus was isolated from 11 to 33% of the environmental samples, and each sampling site yielded the organism at some time during the study. V. parahaemolyticus was found in the environment only during the summer months, when water temperatures were greater than or equal to 17 degrees C and salinities were less than or equal to 13% (parts per thousand), and locally acquired infections were detected only when the organism was present in large numbers in the environment. We conclude that V. parahaemolyticus causes locally acquired gastroenteritis and wound infections, as well as traveler's diarrhea, in the Pacific Northwest, that patients with V. parahaemolyticus infections are likely to be seen in physicians' offices rather than hospitals, that locally acquired V. parahaemolyticus infections occur only when the organism is present in the environment, and that the organism is likely to be present during the summer months, when warm, low-salinity water conditions prevail in the coastal marine environment.
Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans
Authors: Aguero-Rosenfeld M E , Edelstein P H
Published: January 2020

We used the Du Pont radioimmunoassay kit for soluble Legionella pneumophila serogroup 1 antigenuria (Du Pont Co., Wilmington, Del.) to test 422 urine samples from patients with and without Legionnaires disease (LD). The urine specimens were collected from 23 patients with culture-proven LD and from 346 patients without LD. L. pneumophila serogroup 1 was isolated from 14 patients with culture-proven LD, and other L. pneumophila serogroups or other Legionella species were isolated from 9 patients; 58 urine specimens were tested from these 23 patients. The non-LD group was composed of 75 bacteremic patients (35 gram-negative and 40 gram-positive bacteremias), 7 patients with candidemia, 48 patients with non-LD pneumonia, 90 patients with gram-negative bacteriuria (greater than 10(5) CFU/ml), 23 patients with gram-positive bacteriuria (greater than 10(5) CFU/ml), 14 patients with candiduria (greater than 10(5) CFU/ml), and 89 outpatients with negative urine cultures. All tests were performed in duplicate, including positive and negative controls. Sample results with values greater than or equal to 3.0 times those of the negative controls were considered positive for L. pneumophila serogroup 1 antigenuria. The average sample-to-negative ratios were 19.1 for the L. pneumophila serogroup 1 specimens, and 1.0 for both the non-serogroup 1 legionella group and the non-LD specimens. All but one of the patients who were culture positive for L. pneumophila serogroup 1 had at least one specimen positive for serogroup 1 antigenuria; none of the non-L. pneumophila serogroup 1 patients had a positive urine test. The test was highly specific (100%) and sensitive (93%) for the detection of L. pneumophila serogroup 1 antigenuria. Concentrations of urine by vacuum evaporation increased test sensitivity without apparently affecting specificity.
Identification of a group-reactive epitope of group B rotaviruses recognized by monoclonal antibody and application to the development of a sensitive immunoassay for viral characterization
Authors: Yolken R , Wee S B , Eiden J , Kinney J , Vonderfecht S
Published: January 2020

Group B rotaviruses (GBRs) are fastidious agents which cause enteric disease in humans and a number of other animal species. Detailed study of the role of GBRs in human disease has been hampered by the lack of immunoreagents suitable for large-scale studies. We developed a monoclonal antibody which recognizes a group-reactive antigen contained in a number of strains of GBRs. When utilized in conjunction with a hyperimmune guinea pig antiserum to GBR, this monoclonal antibody can be used in an enzyme immunoassay system to detect a wide range of GBRs. Alternatively, this monoclonal antibody can be combined with sera obtained from GBR-infected animals to devise assays which are largely specific for the homologous strain of GBR. Reactivity was not noted in either system with strains of group A or group C rotaviruses or with other members of the family Reoviridae. These results indicate that GBRs contain both group-reactive and species-specific antigens which are distinct from those found in group A rotaviruses. The availability of well-defined immunoreagents will facilitate detailed studies of GBR infections in humans and animals.
Newly isolated strains of Rickettsia tsutsugamushi in Japan identified by using monoclonal antibodies to Karp, Gilliam, and Kato strains
Authors: Yamashita T , Kasuya S , Noda S , Nagano I , Ohtsuka S , Ohtomo H
Published: January 2020

Four isolated strains of Rickettsia tsutsugamushi from patients in a new endemic area of Japan were tested for antigenicities by using 12 monoclonal antibodies to Karp, Gilliam, and Kato strains. It was suggested that one isolate was Karp related and that the others were two independent strains.
Skin colonization by Corynebacterium groups D2 and JK in hospitalized patients
Authors: Soriano F , Rodriguez-Tudela J L , Fernández-Roblas R , Aguado J M , Santamaría M
Published: January 2020

To determine the prevalence of Corynebacterium group D2 and JK organisms on the skin of different types of patients, 200 hospitalized subjects, half of them admitted to a university hospital and the others in a chronic care institution, were surveyed. Samples were taken from the axilla, groin, and abdominal wall. Corynebacterium group D2 and JK organisms were isolated from at least one of the three skin sites in both groups of patients. Only five patients harbored groups D2 and JK at the same time but a different skin sites. The rate of colonization by group D2 organisms was higher in females (43.3%) than in males (17.7%); on the contrary, group JK organisms were isolated more frequently from males (32.1%) than from females (13.5%). All these differences were statistically significant. Corynebacterium group D2 and JK organisms are widely distributed on the skin of hospitalized patients, and the prevalence is sex related.
Evaluation of BACTEC system for urine culture screening
Authors: Colombrita D , Ravizzola G , Pirali F , Manni M , Manca N , Savoldi E , Turano A
Published: January 2020

We compared urine culturing performed by the calibrated loop method with a screening system (BACTEC). A total of 852 urine specimens were examined by both the conventional loop method and the BACTEC system. With the loop method, 193 (22.6%) urine samples were positive, whereas with the BACTEC system, 185 (21.7%) were positive (sensitivity, 96.01%). At a breakpoint of 10(4) CFU/ml, eight false-negatives were detected (sensitivity, 87.09%), and at a breakpoint of 10(5) CFU/ml, four false-negatives were observed (sensitivity, 97.6%). The specificity of the BACTEC system was 100%. We propose the BACTEC method as an effective alternative to the other growth-dependent screening tests for bacteriuria.
Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp
Authors: Kuijper E J , Steigerwalt A G , Schoenmakers B S , Peeters M F , Zanen H C , Brenner D J
Published: January 2020

Phenotypic characteristics were used to identify 189 Aeromonas strains isolated from human feces. One hundred forty-two of these strains were placed in 11 DNA hybridization groups, and the genetic and phenotypic data were compared. According to the criteria of Popoff, 66% of the strains were identified as Aeromonas caviae, 18% were identified as A. sobria, and 16% were identified as A. hydrophila. Some biochemical characteristics differed from the criteria of Popoff; 19 of 40 (48%) of tested strains were encapsulated, 42 of 124 (34%) of A. caviae strains were nonmotile, and all A. sobria strains were resistant to KCN. Gas production from D-glucose was temperature dependent; 11 of 64 (17%) A. hydrophila and A. sobria strains produced gas only at 22 degrees C. Of 142 Aeromonas strains, 57% belonged to hybridization group 4, 25% belonged to group 8, 11% belonged to group 1, 4% belonged to group 5A, 2% belonged to group 3, and 1% belonged to group 2. Of 26 strains phenotypically identified as A. hydrophila, 8 (31%) were in hybridization group 8, which contains strains of the new species A. veronii. It therefore appears that our ability to identify Aeromonas strains phenotypically is not sufficiently specific. Either additional definitive biochemical markers must be found or phenotypic identification, at least for some Aeromonas groups, must be regarded as only presumptive.
Sensitivity and specificity of an enzyme-linked immunosorbent assay using the recombinant DNA-derived Treponema pallidum protein TmpA for serodiagnosis of syphilis and the potential use of TmpA for assessing the effect of antibiotic therapy
Authors: Ijsselmuiden O E , Schouls L M , Stolz E , Aelbers G N , Agterberg C M , Top J , van Embden J D
Published: January 2020

The recombinant DNA-derived Treponema pallidum membrane protein TmpA, purified from Escherichia coli K-12, was used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its suitability in a screening test for syphilis and to monitor the effect of antibiotic treatment. The sensitivity of the TmpA ELISA was 76% for primary syphilis, 100% for secondary syphilis, and 98% for early latent syphilis. All except 1 of 15 serum samples positive for yaws were positive in this test. A specificity of 99.6% was found by testing more than 938 donor samples. The sensitivity and specificity of the TmpA ELISA are comparable to that of the T. pallidum hemagglutination assay, and therefore the test may be useful for the diagnosis of untreated syphilis. After antibiotic treatment, the level of anti-TmpA antibodies in sera of syphilis patients dropped sharply within 1 year. Thus, TmpA might be a useful antigen for monitoring successful treatment of syphilis.
Detection of human papillomaviruses in exfoliated cervicovaginal cells by in situ DNA hybridization analysis
Authors: Pao C C , Lai C H , Wu S Y , Young K C , Chang P L , Soong Y K
Published: January 2020

A total of 851 specimens of exfoliated cervicovaginal cells and 27 specimens of male urethral smears obtained from 706 individuals with various clinical findings were examined for the presence of human papillomavirus (HPV) types 6, 11, 16, 18, 31, and 33 by in situ DNA hybridization analysis. The nonradioactive DNA in situ hybridization method used in this study showed no detectable cross-hybridization either among different types of HPV (except between types 6 and 11) or between HPV DNA and human cellular DNA. Furthermore, this system was found to be more sensitive than the Southern blotting method in detecting HPV. HPV was found in 233 of 276 (84.4%) and in 34 of 47 (72.3%) samples of cervicovaginal cells from patients with urogenital condylomata and cervical dysplasia, respectively. HPV was also detected in 6 of 39 (15.4%) women with normal cytological findings who were also symptom-free. Young women who were at low risk but were infected with HPV showed significantly reduced ratios of helper-inducer T lymphocytes to suppressor-cytotoxic T lymphocytes compared with those of uninfected normal controls (1.28 +/- 0.31 versus 2.47 +/- 0.64; P less than 0.001). This in situ DNA hybridization method can have broad application to the screening of HPV in early lesions and in normal-looking tissues and may be used to identify patients at risk of more serious or possibly malignant progression.
Evaluation of four methods for cytomegalovirus antibody detection for use by a bone marrow transplantation service
Authors: Leland D S , Barth K A , Cunningham E B , Jansen J , Tricot G J , French M L
Published: January 2020

Four methods, latex agglutination, indirect fluorescent antibody, enzyme immunoassay, and complement fixation, were compared for cytomegalovirus antibody screening and for pre- and posttransplant determinations on bone marrow transplant recipients. Latex agglutination was most sensitive (98%) and specific (97%) for screening and pretransplant determinations and was quickest and easiest to perform. In posttransplant sera from allogeneic bone marrow transplant recipients, all methods except complement fixation detected cytomegalovirus antibody from therapeutically administered globulin preparations; this made it difficult to determine the significance of changes in cytomegalovirus antibody titer.
Detection of sialidase (neuraminidase) activity in Actinomyces species by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid in a filter paper spot test
Authors: Moncla B J , Braham P
Published: January 2020

A rapid method for the detection of acetylneuraminyl hydrolase, EC 3.2.1.18 (sialidase or neuraminidase), was developed by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid as substrate in a filter paper spot test. The method was compared to conventional assays that use 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid and bovine submaxillary mucin and was found to be in excellent agreement. Organisms with greater than 10 U of enzyme activity (in nanomoles per minute per milligram of cell protein) gave positive reactions, while those with 2.7 to 9.0 U gave only weak reactions. Isolates with less than 2.7 U of activity were detected upon prolonged incubation. Sialidase activity was detected in 79% of 71 clinical isolates representing five species of Actinomyces. The percentage of sialidase-producing isolates of each species varied considerably: Actinomyces israelii, 63%; A. meyeri, 73%; A. naeslundii, 85%; A. odontolyticus, 73%; and A. viscosus, 100%.
Comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses
Authors: de Souza M , Smith A L
Published: January 2020

The sensitivity of the mouse antibody production test with intraperitoneal or intrasplenic inoculation of mice with reovirus type 3, minute virus of mice, lymphocytic choriomeningitis virus, or mouse hepatitis virus was compared with that of direct isolation in cultured cells. The mouse antibody production test for detection of mouse hepatitis virus was significantly more sensitive than virus isolation in permissive cells, but differences in sensitivity were less marked for the other three viruses. The intrasplenic route of inoculation did not yield seroconversion that occurred earlier or more consistently than that detected after intraperitoneal inoculation.
Quality control guidelines for testing cefotetan in the reference agar dilution procedure for susceptibility testing of anaerobic bacteria
Authors: Zabransky R J , Bobey D G , Barry A L , Allen S D , Fuchs P C , Gerlach E H , Thornsberry C , Sheikh W , Jones R N
Published: January 2020

Reference values for quality control of in vitro susceptibility tests with cefotetan against anaerobic bacteria were determined in two independent multilaboratory studies with the approved National Committee for Clinical Laboratory Standards agar dilution method and three control strains (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium perfringens ATCC 13124). The results of the two studies were in agreement. The recommended MIC control limits for B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 are 4.0 to 16 micrograms/ml and 32 to 128 micrograms/ml, respectively. MICs for C. perfringens ATCC 13124 were too variable to be useful for controlling tests with cefotetan.
Quality control limits for the standard anaerobic reference agar dilution susceptibility test procedure of the National Committee for Clinical Laboratory Standards
Authors: Barry A L , Fuchs P C , Allen S D , Thornsberry C , Gerlach E H , Jones R N
Published: January 2020

Multilaboratory studies were performed to develop MIC quality control limits for the National Committee for Clinical Laboratory Standards reference agar dilution method for anaerobic susceptibility tests. Acceptable MICs were defined as those which include greater than 95% of all 100 MICs generated by the study. Most MIC control limits included either 2- or 3-dilution intervals rather than the more traditional 3-dilution intervals that are described as the mode +/- 1 doubling dilution.
Rapid fluorogenic assay for differentiation of the Candida parapsilosis group from other Candida spp
Authors: Smitka C M , Jackson S G
Published: January 2020

A 30-min fluorogenic test was developed for differentiation of members of the Candida parapsilosis group from other Candida species commonly encountered in clinical material. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucoside, was utilized to assay beta-glucosidase activity. A total of 50 C. parapsilosis isolates and 135 isolates of four other Candida species were tested. Assay sensitivity and specificity were 100 and 99.3%, respectively. The procedure was adapted for use with a spectrofluorometer.
Ovine-associated Staphylococcus aureus protein with immunochemical similarity to toxic shock syndrome toxin 1
Authors: Ho G , Campbell W H , Carlson E
Published: January 2020

A toxic shock syndrome toxin 1 (TSST-1) antibody-binding protein produced by an ovine-associated strain of Staphylococcus aureus was examined. The protein showed total identity to TSST-1 by immunodiffusion analysis. Western blots (immunoblots) of proteins separated by isoelectric focusing revealed that the TSST-1 antibody-binding protein had a pI of 8.6 rather than 7.0, the pI of standard TSST-1.
Detection of pathogenic Yersinia enterocolitica by using congo red-magnesium oxalate agar medium
Authors: Riley G , Toma S
Published: January 2020

A Congo red-magnesium oxalate agar medium was developed to detect expression of virulence-associated calcium dependency and Congo red absorption in Yersinia enterocolitica. Of the 157 pathogenic serotypes tested, 119 (75.8%) were positive; 98% of nonpathogenic serotypes and strains of three other Yersinia species were negative.
Endocarditis caused by relatively penicillin-resistant Stomatococcus mucilaginosus
Authors: Pinsky R L , Piscitelli V , Patterson J E
Published: January 2020

We report a case of Stomatococcus mucilaginosus endocarditis in which the isolate was relatively resistant to penicillins and cephalothin. The patient was treated successfully with vancomycin and valve replacement.
Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only
Authors: Figura N , Guglielmetti P , Rossolini A , Barberi A , Cusi G , Musmanno R A , Russi M , Quaranta S
Published: January 2020

A total of 66.6% of Campylobacter pylori strains isolated from patients with peptic ulcers produced a cytotoxin active against mammalian cells in vitro, versus 30.1% of strains isolated from patients with chronic gastritis of various degrees of severity only. This difference was statistically significant and suggests that the toxic substance could be involved in the development of peptic ulcers.
Dot hybridization assay for distinction of rotavirus serotypes
Authors: Flores J , Green K Y , Garcia D , Sears J , Perez-Schael I , Avendaño L F , Rodriguez W B , Taniguchi K , Urasawa S , Kapikian A Z
Published: January 2020

We have developed a hybridization assay that permits distinction of rotavirus serotypes 1, 2, 3, and 4. The serotype of rotaviruses from stool samples or tissue culture was recognized by hybridization of specific probes to (i) blots of viral double-stranded RNAs electrophoresed in agarose gels (Northern blots) or (ii) heat-denatured double-stranded RNAs directly dotted on nylon membranes. The probes consisted of 32P-labeled cDNA synthesized by reverse transcription of in vitro derived rotavirus mRNA from rotavirus serotypes 1 to 4. To prepare these probes, mRNAs were primed with a 17-mer nucleotide common to all four serotypes whose sequence is complementary to bases 375 to 391 of the rotavirus gene encoding the VP7 glycoprotein (gene 8 or 9 depending on the rotavirus strain). The resulting downstream transcripts encompassed areas of major sequence divergence among the four serotypes. Hybridization at high stringency (50 degrees C, 50% formamide, 4 x SSC [1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) was performed for 16 to 48 h. Autoradiograms of the washed membranes allowed recognition of the rotavirus serotype present in the blotted or dotted specimens since each of them hybridized preferentially to one of the four probes. Twenty-four laboratory specimens and 103 clinical specimens from Washington, D.C., Venezuela, and Chile were "serotyped" with this assay. The results were similar to those obtained with a monoclonal antibody serotyping assay.
Oropharyngeal and fecal carriage of Pseudomonas aeruginosa in hospital patients
Authors: Murthy S K , Baltch A L , Smith R P , Desjardin E K , Hammer M C , Conroy J V , Michelsen P B
Published: January 2020

This prospective study was designed to determine the incidence of rectal and/or oropharyngeal colonization rates of patients with Pseudomonas aeruginosa upon admission to a general hospital and the risk of becoming colonized while hospitalized. Consecutive 186 admissions (180 patients) to one medical ward, one surgical ward, and the intensive care unit were studied over a period of 5 months. Rectal and oropharyngeal swabs for P. aeruginosa were obtained on admission, weekly thereafter, and/or upon discharge. Forty-two patients (22.6%) were colonized on admission, 20 patients (10.8%) acquired P. aeruginosa during hospitalization. Colonization on admission was observed twice as frequently on the surgical ward and in the intensive care unit as on the medical ward. Positive rectal cultures were more frequent than oropharyngeal cultures throughout the study (P less than 0.01). For patients admitted culture positive or culture negative, the probabilities of remaining culture positive or culture negative, respectively, remained at 44 and 72% after 35 days of hospitalization. The most common P. aeruginosa serotypes were 1, 6, and 10, and pyocin types 1, 3, and 10 were predominant. There was no statistical difference in the serotypes or pyocin types detected on admission or acquired during hospitalization. Except for two hospital-acquired first isolates which were resistant to moxalactam, all first isolates were susceptible to the four antibiotics tested. During the study, one isolate became resistant to azlocillin, gentamicin, and tobramycin, while two isolates became resistant to moxalactam. A statistical analysis was performed for 13 risk factors for all colonized and noncolonized patients. Colonization detected at the time admission was positively associated with age ( > 65 years), previous surgery of the gastrointestinal tract for neoplasm, and anemia ( P< 0.05). In contrast, for patients who entered the study culture negative, none of the analyzed 13 risk factors was associated with an increased probability for colonization. This observation included the administration of antimicrobial agents singly or in combination or both.
Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus
Authors: Rappocciolo G , Toso J F , Torpey D J , Gupta P , Rinaldo C R
Published: January 2020

Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities.
Monoclonal antibody assay for detection of double-stranded RNA and application for detection of group A and non-group A rotaviruses
Authors: Kinney J S , Viscidi R P , Vonderfecht S L , Eiden J J , Yolken R H
Published: January 2020

Fastidious viruses are generally detected in human body fluids by means of immunoassay or nucleic acid hybridization systems. These approaches can be difficult to apply to the detection of viruses which display variations in antigenic or genetic composition. Rotaviruses are examples of viruses which can display such variations. Recently identified antigenic variants, designated as non-group A rotaviruses, cannot be detected by immunoassays or nucleic acid hybridization assays which utilize reagents directed at group A rotavirus strains. The incomplete understanding of the extent of antigenic and genetic variation has inhibited the development of assay systems for all of the non-group A rotaviruses and has limited the study of their role in human disease. While rotaviruses display genetic variation, they all contain a genome which consists of double-stranded RNA. We utilized a monoclonal antibody to devise a sensitive assay for the measurement of double-stranded RNA and applied it to the detection of a wide range of rotaviruses. We found that the assay could detect double-stranded RNA from as few as 10 PFU of standard strains of group A rotaviruses. The assay system was also capable of detecting double-stranded RNA from several strains of group B rotaviruses isolated from calves, rats, and pigs at levels below those at which viral RNA could be visualized by means of polyacrylamide gel electrophoresis. When applied to the detection of double-stranded RNA in serial stools shed by rotavirus-infected children, the assay system was capable of detecting double-stranded RNA in samples in which antigen could not be detected by immunoassay. The specific nature of the double-stranded RNA detected by this assay system could be determined by the elution of the nucleic acids from the monoclonal antibody and the reaction of the RNA with specific nucleotide probes. The measurement of double-stranded RNA offers a potential method for the sensitive detection of a wide range of rotaviruses and other members of the family Reoviridae.
Evaluation of performance parameters of a membrane-based dot immunoassay for meningococcal polysaccharide
Authors: Oprandy J J , Sippel J E
Published: January 2020

Increasingly, membrane-based enzyme immunoassays are being developed as the preferred solid-phase enzyme immunoassay format. We describe the rate kinetics of a polyvinylidene difluoride membrane-based dot immunoassay for meningococcal group A polysaccharide. Antigen detection sensitivity decreased logarithmically with linear decreases in incubation time. The sensitivity of a 30-min assay (5-min incubation steps) was increased to nearly the level of the standard assay (1-h incubation steps) by increasing the concentration of assay reagents fourfold. These results support the idea that existing microtiter plate assays can be transferred to rapid dot immunoassay formats with little or no loss of sensitivity.
Improved method for bacteriological diagnosis of spontaneous bacterial peritonitis
Authors: Bobadilla M , Sifuentes J , Garcia-Tsao G
Published: January 2020

The definitive diagnosis of spontaneous bacterial peritonitis is made by a positive ascitic fluid culture. Causative organisms cannot be isolated in up to 65% of patients with well-defined spontaneous bacterial peritonitis, probably due to inadequate ascites culture techniques. We prospectively compared two ascites culture methods: conventional (on chocolate agar and thioglycolate broth) and modified (inoculation of 10 ml of ascites in a tryptic soy broth blood culture bottle at the patient's bedside). In a 10-month period, 31 cirrhotic patients met our diagnostic criteria for spontaneous bacterial peritonitis; both culture methods were performed on their ascitic fluid. The conventional method grew bacteria in only 16 of the 31 episodes (52%), whereas the modified method grew bacteria in 25 (81%), a significantly higher sensitivity (P less than 0.05). The modified method also shortened significantly the time for detection of bacterial growth. We conclude that ascites inoculation into a blood culture bottle at the patient's bedside should be the routine method for ascites culture.
Electrophoretic karyotypes of Torulopsis glabrata
Authors: Kaufmann C S , Merz W G
Published: January 2020

Chromosome-sized DNA molecules of clinical isolates of Torulopsis glabrata were resolved by a pulsed-field electrophoretic method, contour-clamped homogeneous electric fields. With the conditions established in this study, 8 to 12 bands (ranging from 445 to 3,000 kilobases) were observed. There were differences in the intensities and migrations of bands, consistent with T. glabrata being either haploid or diploid. A total of 22 distinctive electrophoretic patterns were noted among single isolates of T. glabrata recovered from 33 patients. When strains were delineated by an electrophoretic pattern, individuals usually harbored only one strain.
Turnover of nontypable Haemophilus influenzae in the nasopharynges of healthy children
Authors: Trottier S , Stenberg K , Svanborg-Edén C
Published: January 2020

The nasopharyngeal Haemophilus influenzae flora of healthy children in a day care center was analyzed by repeated sampling during 4 winter months. The average carrier rate was 39%, but 74% of the children became colonized at some time during the study. The H. influenzae isolates were identified by capsular type, biotype, and multilocus enzyme electrophoresis. The turnover of the flora in individual children and in the day-care group was characterized. Four patterns of colonization were defined among the 38 children and 49 H. influenzae strains. Depending on the persistence in the group, the strains were designated as endemic, i.e., shared between several children on several occasions, or as epidemic, i.e., occurring only once but in several children. The individual hosts had two patterns; resident strains persisted for 2 weeks or more, and transient strains showed no evidence of persistence. The results suggest that there is considerable sharing of certain nontypable H. influenzae among healthy children in day care, whereas other strains remain restricted to a single host. The properties of host and bacteria determining these patterns remain to be defined.
Enzyme-linked immunosorbent assay and immunoblot analysis of the immunoglobulin G response to whole-cell and lipooligosaccharide antigens of Pasteurella pneumotropica in laboratory mice with latent pasteurellosis
Authors: Manning P J , Gaibor J , Delong D , Gunther R
Published: January 2020

The immunoglobulin G (IgG) response to whole-cell and lipooligosaccharide (LOS) antigens of Pasteurella pneumotropica was evaluated in mice with latent pasteurellosis by enzyme-linked immunosorbent assay (ELISA) and immunoblots. Antibodies to cell wall proteins of P. pneumotropica also reacted with several protein antigens from isolates of Actinobacillus spp. and other pasteurellae. Conversely, antibodies to LOS antigens of P. pneumotropica demonstrated no cross-reactivity with LOSs of other Pasteurella or Actinobacillus species. IgG to cell wall proteins was detected initially by ELISA 4 weeks after experimental oronasal inoculation of specific-pathogen-free mice; antibody to LOSs was first detected 7 weeks after infection and at that time exceeded titers to other cell wall antigens. Naturally infected conventional mice from a colony with endemic latent pasteurellosis had high IgG titers to P. pneumotropica antigens at 8 to 10 weeks of age, and, as in the experimentally infected mice, antibody to LOSs predominated. Thus, LOSs of P. pneumotropica can be used as an ELISA or immunoblot antigen to detect serospecific antibodies in laboratory mice with latent pasteurellosis.
Identification of pseudorabies virus-exposed swine with a gI glycoprotein enzyme-linked immunosorbent assay
Authors: Mellencamp M W , Pfeiffer N E , Suiter B T , Harness J R , Beckenhauer W H
Published: January 2020

A monoclonal antibody specific for the gI glycoprotein of virulent pseudorabies virus was produced and used to affinity purify gI glycoprotein. The purified gI was used in an enzyme-linked immunosorbent assay (ELISA) that identified and differentiated field virus-exposed animals from animals vaccinated with gI-deleted virus. The gI ELISA was evaluated by comparing it with the virus neutralization test and with a standard ELISA which does not distinguish between vaccinated and naturally infected animals. Pigs vaccinated with a gI-deleted vaccine were seropositive by the virus neutralization or standard ELISA but were seronegative in the gI ELISA. Nonvaccinated and vaccinated animals were detected as seropositive in the gI ELISA only after exposure to gI-containing field virus. Exposed animals were detected as early as day 7 and for as long as 141 days after field virus exposure. As little as 10(2.7) PFU of field virus was sufficient to seroconvert negative animals in the gI ELISA. Pseudorabies virus-seronegative animals which received multiple doses of gI-deleted vaccine remained seronegative in the gI ELISA. The use of this test to monitor swine for pseudorabies virus infection would offer significant benefits towards eradication of the disease.
Development of an enzyme immunoassay using recombinant expressed antigen to detect hepatitis delta virus antibodies
Authors: Puig J , Fields H A
Published: January 2020

Two generic enzyme immunoassays (EIAs) were developed for detection of anti-hepatitis delta virus antibodies (anti-HD) and compared with a commercially available radioimmunoassay. Both generic assays were configured as blocking assays and used hepatitis delta antigen (HDAg) derived from infected chimpanzee liver (EIA-1) or from Escherichia coli transformed with a plasmid containing an insert from within an open reading frame encoding HDAg (EIA-2). Absolute sensitivity was ascertained by endpoint titration, which demonstrated essentially identical endpoints for EIA-1 and EIA-2. The absolute sensitivities of the EIAs were approximately four times greater than that of the radioimmunoassay. Specificity and sensitivity were ascertained by testing a panel of 176 serum specimens by each assay. The specimens were selected to represent a panel composed of sera from individuals with or without markers of viral hepatitis as follows: (i) serologically confirmed by exclusion as posttransfusion non-A, non-B hepatitis; (ii) acute or chronic hepatitis B virus infection, positive for hepatitis B surface antigen; (iii) resolved hepatitis B virus infection, positive for anti-hepatitis B surface antigen; (iv) acute hepatitis A virus infection, positive for anti-hepatitis A virus immunoglobulin M; and (v) normal human sera. All three assays for anti-HD gave similar specificity and sensitivity values. In conclusion, the recombinant expressed HDAg can replace antigen derived from infected liver tissue as a diagnostic reagent used to configure an EIA for detection of anti-HD. Furthermore, the results suggest that the expressed antigen contains the important immunodominant epitope(s).
Diagnostic considerations and interpretation of microbiological findings for evaluation of chronic prostatitis
Authors: Krieger J N , McGonagle L A
Published: January 2020

Seventy-five patients attending a clinic for chronic prostatitis were evaluated by use of lower urinary tract localization cultures. Coagulase-negative staphylococci, alpha-hemolytic streptococci, and diphtheroids were the most common isolates, but none of these organisms were pathogens, based on the absence of bacteriuria or evidence of an inflammatory response in prostatic secretions. Recognized uropathogens were isolated in 12 (16%) of the 75 cases and included Escherichia coli in 6 cases, Enterococcus spp. in 2 cases, and Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus saprophyticus in 1 case each. Laboratory evaluation of men with chronic prostatitis should concentrate on the isolation and antimicrobial susceptibility testing of bacteria that have an established pathogenic potential in the genitourinary tract.
Methods for identification of flavobacteria
Authors: Pickett M J
Published: January 2020

Published reports disagree on the best features for detecting and distinguishing between Flavobacterium meningosepticum (biovar IIa) and Flavobacterium species CDC group IIb (biovar IIb; Flavobacterium indologenes). This report discloses that at least some of these disagreements may reflect the methods used. To detect production of indole, a modified Kovács reagent (not Ehrlich) and a buffered tryptophan medium were optimal, but not all strains of these two biovars produced indole. To distinguish the two biovars, hydrolysis of corn starch was preferable to that of soluble potato starch. Both biovars may hydrolyze DNA; the differentiation achieved varied with the methods used. Both biovars presented pigmented growth; only IIb, however, was obviously pigmented on a 2-day blood agar plate. Acidification of D-arabinose definitively distinguished these two biovars; several additional features were useful but not definitive.
Antibody response to recombinant lambda gt11 fusion proteins in cytomegalovirus infection
Authors: Landini M P , Lazzarotto T , Ripalti A , Guan M X , La Placa M
Published: January 2020

Significant epitopes of two of the major cytomegalovirus antigens, a nonstructural DNA-binding protein of 52 kilodaltons (kDa) and a structural phosphoprotein of 150 kDa, expressed as fusion proteins with the beta-galactosidase, were induced in Escherichia coli after infection with recombinant lambda gt11 clones. The epitopes were then used in immunoblotting to assay specific immunoglobulin G (IgG) and IgM in several groups of sera from long-term seropositive subjects and from patients undergoing primary or secondary virus infection. The data obtained showed that IgM reacting with the 52-kDa nonstructural antigen are linked to primary virus infection and can therefore be considered a serological marker of this infection.
Application of different chromosomal DNA restriction digest fingerprints to specific and subspecific identification of Campylobacter isolates
Authors: Owen R J , Costas M , Dawson C
Published: January 2020

Eleven strains comprising representatives of different subspecies, biotypes, and serotypes of Campylobacter jejuni and reference strains of C. coli, C. fetus subsp. fetus, C. hyointestinalis, and C. sputorum subsp. sputorum were studied to assess the utility of different DNA profiles for measuring fine differences between allied bacteria. Strains were compared by analyses of HaeIII and XhoI digest patterns of chromosomal DNA and Southern blot hybridization patterns of XhoI digests obtained with an Escherichia coli 16S + 23S rRNA gene probe. Visual comparisons and numerical analyses of the HaeIII and XhoI digest patterns both revealed clear differences between the five Campylobacter species and between representatives of C. jejuni subspecies and biotypes. Only strains with the same Penner serotype gave identical total digest polymorphisms. The advantages of XhoI total digests and Southern blot hybridization patterns were that they were less complex than the HaeIII patterns and easier to compare visually. However, numerical analysis of XhoI data resulted in reduced discrimination. We conclude that DNA fingerprinting using either HaeIII or XhoI fragment polymorphisms has considerable potential as a generally applicable method for identification of Campylobacter isolates, especially at the infrasubspecific level.