bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction
Authors: Homberger F R , Smith A L , Barthold S W
Published: January 2020

A polymerase chain reaction (PCR) method was developed for the detection of rodent coronaviruses in biological material by using reverse transcriptase and two primers which flanked an M gene sequence of 375 bp. PCR detected all of 11 different strains of mouse hepatitis virus (MHV) as well as rat sialodacryoadenitis virus but not bovine coronavirus or human coronavirus strains OC43 and 229E. The M gene sequences of bovine coronavirus and human coronavirus OC43 are homologous to that of MHV, but minor differences exist in the primer regions, preventing annealing of the primers. For detecting MHV-Y in tissue samples, PCR was faster than and at least as sensitive as either of the two bioassays (infant mouse bioassay and mouse antibody production test) currently used for MHV diagnostic purposes.
Rickettsiae and Borrelia burgdorferi in ixodid ticks
Authors: Magnarelli L A , Andreadis T G , Stafford K C , Holland C J
Published: January 2020

Nymphs and adults of hard-bodied ticks were collected in Connecticut and tested by direct and indirect immunofluorescence staining methods for rickettsiae and Borrelia burgdorferi. Of the 609 Ixodes dammini ticks examined, 59 (9.7%) harbored rickettsialike microorganisms in hemocytes (blood cells). These bacteria reacted with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of canine ehrlichiosis. Prevalence of infection ranged from 6.8 to 12.7% for males and females, respectively. Although the specific identities of the hemocytic rickettsialike organisms are unknown, they share antigens with ehrlichiae. Electron microscopy revealed rickettsiae in ovarian tissues of I. dammini that also had infected hemocytes. Rickettsialike organisms were also observed in the hemocytes of 5 (6.9%) of 73 Dermacentor variabilis ticks. In analyses for B. burgdorferi, 146 (23.7%) of 617 I. dammini ticks harbored these spirochetes in midguts. Hemocytic rickettsialike microorganisms coexisted with B. burgdorferi in 36 (6.7%) of the 537 nymphs and adults of I. dammini examined. I. dammini, with its broad host range, has the potential to acquire multiple microorganisms.
Evaluation of liquid media for growth of Helicobacter pylori
Authors: Shahamat M , Mai U E , Paszko-Kolva C , Yamamoto H , Colwell R R
Published: January 2020

Helicobacter pylori has routinely been isolated and grown on solid media. Recently, we have succeeded in obtaining growth of this organism in several liquid media in large volumes, including tryptic soy broth, Mueller-Hinton broth, brucella broth, brain heart infusion broth, and Columbia broth. Growth was tested in the media with and without supplementation. Growth was obtained after incubation under microaerobic conditions and with CO2 enrichment. Growth in a stationary system versus that in an agitated system was evaluated. Results from these experiments show that H. pylori can be grown in any of the liquid media tested except buffered yeast extract-alpha-ketoglutarate if serum is added. No growth was observed on buffered yeast extract-alpha-ketoglutarate even with serum and other supplementation. Growth of H. pylori in most of the liquid media with supplements was improved if the culture was incubated in a CO2 atmosphere. The findings reported here may be useful in clinical, industrial, and research laboratories that require harvests of large quantities of H. pylori cells.
Ehrlichia chaffeensis, a new species associated with human ehrlichiosis
Authors: Anderson B E , Dawson J E , Jones D C , Wilson K H
Published: January 2020

The bacterial 16S rRNA genes from blood samples of two patients with human ehrlichiosis and from an isolate recovered from one of the patients were amplified by using the polymerase chain reaction. The amplimers were then cloned and sequenced. The 16S rRNA gene sequence was also determined for Ehrlichia canis (two strains), E. equi, E. phagocytophila (two strains), and E. sennetsu (two strains). These sequences, along with a previously published 16S rRNA gene sequence of E. risticii, were compared. The 16S rRNA gene sequences were identical for all three sources of the human ehrlichiosis agent. The sequence comparisons indicate that the human ehrlichiosis agent is a new species most closely related to E. canis (98.2%) and more distantly related to other Ehrlichia spp. We propose that this species be named Ehrlichia chaffeensis sp. nov., with the Arkansas strain as the type strain.
Aerokey II: a flexible key for identifying clinical Aeromonas species
Authors: Carnahan A M , Behram S , Joseph S W
Published: January 2020

A small subset (n = 18) of highly discriminatory tests was derived from the feature frequency of 50 tests used in the study of 167 predominantly clinical Aeromonas strains. Seven of these eighteen tests were used to construct a flexible, dichotomous key, Aerokey II, for identifying clinical aerontonads: esculin hydrolysis, gas from glucose, acid from arabinose, indole production, acid from sucrose, Voges-Proskauer reaction, and resistance to cephalothin (30 micrograms). This schema was initially evaluated in a single-blind trial of 60 well-characterized clinical Aeromonas hydrophila (n = 21), A. caviae (n = 19), and A. veronii bv. sobria (n = 20) strains from an independent laboratory. Of the 60 strains tested, 58 (97%) were accurately identified to the species level. Aerokey II was further evaluated with 18 additional American Type Culture Collection and reference strains representing the more recently proposed taxa A. veronii bv. veronii, A. schubertii, A. jandaei, and A. trota and accurately identified all of these strains.
Evaluation of a new method for identification of Cryptococcus neoformans which uses serologic tests aided by selected biological tests
Authors: Kabasawa K , Itagaki H , Ikeda R , Shinoda T , Kagaya K , Fukazawa Y
Published: January 2020

A new method for identifying Cryptococcus neoformans isolates and their serotypes by the slide agglutination test using five kinds of factor sera, with the aid of nitrate reduction, phenol oxidase, and growth at 37 degrees C tests was evaluated by using 36 reference strains and 75 clinical isolates of C. neoformans. The results showed that the reference strains were identified exactly as they were labeled, and clinical isolates were identified as C. neoformans serotypes A, D, and AD. C. neoformans could be distinguished from other Cryptococcus species that cross-reacted with factor sera by their ability to grow at 37 degrees C. These results indicate that the slide agglutination test combined the use of factor sera for isolates which grow at 37 degrees C is a useful method for identification of C. neoformans and their serotypes and that the nitrate reduction test (negative in 100% of the isolates) and the phenol oxidase test (positive in approximately 95% of the isolates) can be used to confirm that the species is C. neoformans.
Disk diffusion susceptibility test interpretive criteria for GR69153, a new catechol-substituted cephalosporin
Authors: Jones R N , Erwin M E
Published: January 2020

GR69153 disk diffusion tests with 10- and 30-micrograms disks were evaluated against 383 rapidly growing aerobic pathogens. Very high absolute agreement (96.1 to 98.1%) was observed between the MICs and disk zone diameters, and no false-susceptible results were encountered with the proposed criteria. The 30-micrograms GR69153 disk with a susceptible-zone diameter of greater than or equal to 17 mm (MIC correlate, less than or equal to 8 micrograms/ml) and a resistant-zone diameter of less than or equal to 13 mm (MIC correlate, greater than 16 micrograms/ml) is recommended. GR69153 had a spectrum of activity slightly wider than that of ceftazidime against the species tested.
Diagnosis of Legionnaires' Disease by Radioimmunoassay of
Legionella
Antigen in Pleural Fluid
Authors: Oliverio Michael J. , Fisher Melanie Ann , Vickers Richard M. , Yu Victor L. , Menon Anil
Published: January 2020

We describe a case of culture-proven Legionnaires' disease (serogroup 1) in which a rapid diagnosis was made by detection of
Legionella
antigen in pleural fluid by use of the Binax radioimmunoassay for urine.
Comparison of commercially available group B streptococcal latex agglutination assays
Authors: Ascher D P , Wilson S , Fischer G W
Published: January 2020

Detection of group B streptococcus (GBS) antigen in urine by latex particle agglutination (LPA) may facilitate the rapid diagnosis of GBS sepsis. We sought to compare three commercial LPA assays with specimens that were spiked with type-specific antigen, group-specific antigen, or type III organisms. There were sensitivity differences between the assays, but the Bactigen assay performed best, detecting as little as 1 ng of GBS group-specific antigen per ml in urine and as few as 10(5) CFU of GBS type III organisms per ml in urine, serum, and cerebrospinal fluid.
Amplification of repetitive DNA for the specific detection of Naegleria fowleri
Authors: McLaughlin G L , Vodkin M H , Huizinga H W
Published: January 2020

By using hybridization at low C0t values, a genomic library on Naegleria fowleri was screened for clones containing repetitive DNA. Partial sequence information from a repetitive clone, Nf9, showed sequence homologies with the mitochondrial ATPase 6 subunit from yeasts and other organisms. Synthetic DNA primers were selected and tested in amplification reactions. Nonstringent hybridization conditions were defined which allowed amplification of N. fowleri DNA and reduced amplification of DNA from nonpathogenic Naegleria species. Stringent conditions were selected which allowed detection only of N. fowleri. Identity of the amplified DNA was confirmed by using internal restriction sites and an internal primer. In a blind study, tissue from mice experimentally infected with N. fowleri was specifically detected by using stringent hybridization conditions.
Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals
Authors: Simpson W J , Burgdorfer W , Schrumpf M E , Karstens R H , Schwan T G
Published: January 2020

Borrelia burgdorferi expresses a conserved, species-specific 39-kDa protein (P39) that can stimulate antibodies during human infection. To confirm that anti-P39 antibodies are produced consistently in animals exposed to infectious spirochetes, white-footed mice, Peromyscus leucopus, and laboratory white mice, Mus musculus (strain BALB/c), were experimentally inoculated with either infectious or noninfectious B. burgdorferi and the antibody response to P39 was determined by immunoblot at 21 days postinoculation. All mice inoculated with approximately 10(7) infectious B. burgdorferi produced anti-P39 antibodies and were cultured positive for this spirochete. Mice inoculated with similar numbers of inactivated or viable noninfectious B. burgdorferi still producing P39 did not induce anti-P39 antibodies. By contrast, putative antiflagellin antibodies were detected in less than 18% of the infected animals, which supports the notion that antibody reactive with flagellin may not be reliable as a marker for B. burgdorferi exposure as was originally thought. Mice infected with B. burgdorferi following exposure to ticks (Ixodes dammini) produced anti-P39 antibodies no later than 7 days postinfection, indicating that P39 is an effective immunogen in natural infections. Notably, anti-P39 antibodies were the predominant B. burgdorferi reactive antibodies detected early in the infection. Our results indicate that anti-P39 antibodies are produced in response to an active infection and are therefore reliable markers for infection in experimentally and naturally inoculated animals.
Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro
Authors: Tannich E , Burchard G D
Published: January 2020

We previously reported the identification of homologous cDNA clones derived from a pathogenic isolate and a nonpathogenic isolate of Entamoeba histolytica, which had been designated cEh-P1 and cEh-NP1, respectively. Sequence analysis of both clones had revealed 10% nucleic acid substitutions, which were dispersed over the entire sequence. This genetic difference had been found to be conserved between all four pathogenic and all five nonpathogenic laboratory strains of E. histolytica tested. On the basis of nucleic acid substitutions, we have now developed a sensitive assay to distinguish pathogenic from nonpathogenic forms of E. histolytica by using fresh clinical isolates. Comparing the sequence of cEh-P1 and cEh-NP1, we identified a 482-bp segment that contained identical 5' and 3' ends but differed in internal cleavage sites for restriction endonucleases. By using oligonucleotide primers corresponding to the 5' and 3' ends of this segment, the corresponding gene was amplified by the polymerase chain reaction. Endonuclease digestion of the amplified DNA yielded restriction fragments that are characteristic for pathogenic and nonpathogenic forms. This assay allows the detection and classification of fewer than 10 amoebae within a few hours. The differentiation of 48 isolates into pathogenic and nonpathogenic strains by using this method corresponded to the clinical status of the infected individuals and to the classification obtained by isoenzyme determination. The results further support the concept that pathogenic and nonpathogenic strains of E. histolytica constitute distinct subspecies.
Reliability of phenotypic tests for identification of Acinetobacter species
Authors: Gerner-Smidt P , Tjernberg I , Ursing J
Published: January 2020

A numerical approach was used for identification of 198 Acinetobacter strains assigned to DNA groups according to the classification of Tjernberg and Ursing (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989). The matrix used was constructed from data published by Bouvet and Grimont (P.J.M. Bouvet and P.A.D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986) and Bouvet and Jeanjean (P.J.M. Bouvet and S. Jeanjean, Res. Microbiol. 140:291-299, 1989). The tests chosen were those of the simplified identification scheme for Acinetobacter species devised by Bouvet and Grimont (P.J.M. Bouvet and P.A.D. Grimont, Ann. Inst. Pasteur/Microbiol. 138:569-578, 1987), namely, growth at 37, 41, and 44 degrees C, oxidation of glucose, gelatin hydrolysis, and assimilation of 14 carbon sources. Of the strains tested, 181 represented 12 DNA groups in the matrix; at a probability level of greater than or equal to 0.95, 78% of them were correctly identified, 2.2% were misidentified, and 19.8% were not identified. Seventeen strains represented two DNA groups not included in the matrix; nine of them were incorrectly assigned to a DNA group by these phenotypic tests. Because of problems of separating strains belonging to DNA groups 1, 2, 3, and 13 by using the phenotypic tests proposed by Bouvet and Grimont (Ann. Inst. Pasteur/Microbiol.), we suggest that these groups should be referred to as the Acinetobacter calcoaceticus-A. baumannii complex.
Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation
Authors: Nugent R P , Krohn M A , Hillier S L
Published: January 2020

The purpose of the study was to examine intercenter variability in the interpretation of Gram-stained vaginal smears from pregnant women. The intercenter reliability of individual morphotypes identified on the vaginal smear was evaluated by comparing them with those obtained at a standard center. A new scoring system that uses the most reliable morphotypes from the vaginal smear was proposed for diagnosing bacterial vaginosis. This scoring system was compared with the Spiegel criteria for diagnosing bacterial vaginosis. The scoring system (0 to 10) was described as a weighted combination of the following morphotypes: lactobacilli, Gardnerella vaginalis or bacteroides (small gram-variable rods or gram-negative rods), and curved gram-variable rods. By using the Spearman rank correlation to determine intercenter variability, gram-positive cocci had poor agreement (0.23); lactobacilli (0.65), G. vaginalis (0.69), and bacteroides (0.57) had moderate agreement; and small (0.74) and curved (0.85) gram-variable rods had good agreement. The reliability of the 0 to 10 scoring system was maximized by not using gram-positive cocci, combining G. vaginalis and bacteroides morphotypes, and weighting more heavily curved gram-variable rods. For comparison with the Spiegel criteria, a score of 7 or higher was considered indicative of bacterial vaginosis. The standardized score had improved intercenter reliability (r = 0.82) compared with the Spiegel criteria (r = 0.61). The standardized score also facilitates future research concerning bacterial vaginosis because it provides gradations of the disturbance of vaginal flora which may be associated with different levels of risk for pregnancy complications.
Comparison of crossed immunoelectrophoresis, enzyme-linked immunosorbent assays, and tube agglutination for serodiagnosis of Yersinia enterocolitica serotype O:3 infection
Authors: Paerregaard A , Shand G H , Gaarslev K , Espersen F
Published: January 2020

Antibodies against Yersinia enterocolitica serotype O:3 were measured by crossed immunoelectrophoresis (XIE) using whole-cell sonic extract as antigen and by enzyme-linked immunosorbent assays (ELISAs) using either purified lipopolysaccharide or whole formalinized cells expressing virulence plasmid-encoded surface antigens (pYV+ cells). The results were compared with those obtained with the standard tube agglutination method. Sera from three groups of people were examined by using these assays. The first group consisted of healthy blood donors, the second consisted of patients with recent infection due to microorganisms other than Y. enterocolitica O:3, and the third consisted of patients with recent Y. enterocolitica O:3 infection. Sera from the last group were also obtained at regular intervals for 12 months postinfection. Results obtained with XIE and the ELISAs were in good agreement with those obtained with tube agglutination. Variation, diagnostic sensitivity, and diagnostic specificity were satisfactory for all the assays studied. However, the lipopolysaccharide ELISA was less laborious than tube agglutination and XIE and carried a somewhat greater diagnostic specificity than the pYV+ ELISA. XIE and the pYV+ ELISA, on the other hand, also had advantages. XIE enabled simultaneous examination of the individual antibody response against a wide range of chromosome-encoded antigens, and the pYV+ ELISA enabled detection of specific pYV antibodies when sera were adsorbed with formalinized pYV-cured Y. enterocolitica O:3 cells prior to the assay.
Evaluation of an immunofluorescence assay for specific detection of immunoglobulin G antibodies directed against Helicobacter pylori, and antigenic cross-reactivity between H. pylori and Campylobacter jejuni
Authors: Faulde M , Putzker M , Mertes T , Sobe D
Published: January 2020

An immunofluorescence assay (IFA) for the detection of immunoglobulin G antibodies directed against Helicobacter pylori was evaluated by comparing 20 serum specimens from patients with a positive urease test on biopsy material and 20 serum specimens from patients with a negative test and with defined clinical symptoms. The resulting anti-H. pylori titers were classified as follows: negative, less than or equal to 64; borderline, 128; and positive, greater than or equal to 256. By using these criteria, the IFA was subsequently tested, using 100 serum specimens from patients with gastric complaints. Overall, the titers were 71% positive, 10% borderline, and 19% negative. Depending on the patients' biopsy urease test results, the sensitivity and specificity of the assay were calculated to be 96%. Furthermore, these sera were classified into three subgroups on the basis of clinical manifestations: gastritis with 74% positive and 10% borderline titers, duodenal ulcer with 84% positive and 4% borderline titers, and gastric ulcer with 52% positive and 16% borderline titers. A serologic follow-up study was carried out with three patients with gastric ulcers who had been treated with colloidal bismuth subcitrate for 4 weeks and erythromycin for the final 2 weeks. The results indicate that a significant decrease in titer could be expected within 9 to 12 months after successful therapy, as determined by repeated negative CLO tests. Absorption experiments demonstrated that possible cross-reactivity between H. pylori and C. jejuni did not influence serodiagnosis.
Effect of hydrogen peroxide on growth of Candida, Cryptococcus, and other yeasts in simulated blood culture bottles
Authors: Huahua T , Rudy J , Kunin C M
Published: January 2020

The addition of hydrogen peroxide to blood contained in liquid culture medium increased the dissolved-O2 partial pressure in direct proportion to the volume injected. The effect of hydrogen peroxide on the growth of subcultured clinical isolates of Candida albicans, Cryptococcus neoformans, Torulopsis glabrata, and other yeasts and on the growth of blood culture isolates of representative pathogenic bacteria was compared with its effect on their growth in vented and unvented stationary bottles. C. albicans and C. neoformans grew significantly better in bottles to which hydrogen peroxide had been added than in vented or unvented bottles. The advantage of hydrogen peroxide over venting was most marked with several slowly growing strains. Similar results were obtained in shaker cultures with strains of C. albicans which were inoculated directly from positive blood cultures. The effect of hydrogen peroxide tended to diminish during serial passage. T. glabrata grew less well when hydrogen peroxide was added, perhaps because of the absence of oxidase. The growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis was not significantly inhibited or augmented by the addition of hydrogen peroxide. The growth of Escherichia coli was inhibited slightly. The value of the addition of hydrogen peroxide to blood cultures to improve the isolation of yeasts needs to be established by a clinical trial which would compare this method with established methods.
Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay
Authors: Furuya Y , Yamamoto S , Otu M , Yoshida Y , Ohashi N , Murata M , Kawabata N , Tamura A , Kawamura A
Published: January 2020

Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody-positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test.
Phage typing of Staphylococcus intermedius
Authors: Overturf G D , Talan D A , Singer K , Anderson N , Miller J I , Greene R T , Froman S
Published: January 2020

Staphylococcus intermedius, a coagulase-positive staphylococcal species, is a common canine pathogen and a rare human wound pathogen. A total of 145 strains of S. intermedius (ATCC 29663, 4 reference strains, 4 human isolates, 44 canine infection isolates, and 92 isolates from canine gingiva) were screened for lysogenic phage by a modified Fisk method. Nineteen phage preparations were prepared for preliminary typing experiments. Lytic activity was observed on 93 of 145 (64.1%) isolates, yielding 44 lytic patterns with individual strains susceptible to one or more phages. Five phages lysed only a single strain, but lytic patterns varied from 1 to 11 lytic phages per isolate. A distinct lytic pattern did not separate canine or human wound isolates from canine gingival isolates. All human wound isolates fell into the two most common canine gingival or wound patterns; the single human nasopharyngeal isolate was not lysed by any phage. Twenty-two of 44 (55%) canine wound isolates and 65 of 92 (71%) gingival isolates yielded lytic patterns. Lysogenic phages are common in S. intermedius. This preliminary study suggests that phage typing may be a useful tool in distinguishing epidemiologically related strains.
Identification of coagulase-negative staphylococci by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and rRNA restriction patterns
Authors: Pennington T H , Harker C , Thomson-Carter F
Published: January 2020

A total of 1,417 staphylococcal and micrococcal strains were collected from the beards and scalps of 10 subjects over a period of 8 months. Sixteen strains identified as Staphylococcus epidermidis with an API system had distinctive yellow colonies on nutrient agar plates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell polypeptide profiles similar to those of Staphylococcus capitis; this identification was confirmed by analysis of rRNA gene restriction patterns.
Comparison of isolates of Helicobacter pylori and Helicobacter mustelae
Authors: Morgan D R , Fox J G , Leunk R D
Published: January 2020

On the basis of analysis of protein profiles, isolates of Helicobacter pylori and Helicobacter mustelae were less than 40% similar. Cytotoxin produced by H. pylori was not detected in isolates of H. mustelae. Both bacterial species agglutinated human erythrocytes. These results substantiate a taxonomic difference between H. pylori and H. mustelae.
Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection
Authors: Herchline T E , Ayers L W
Published: January 2020

Consecutive record review over a 63-month period revealed 229 Staphylococcus lugdunensis isolates, or 10.1% of the staphylococcal species that were not Staphylococcus aureus or Staphylococcus epidermidis. A total of 155 S. lugdunensis specimens were isolated from sites over the entire bodies of the 143 patients studied. The most common clinical diagnoses were skin and skin structure infections (55.4%) and blood and vascular catheter infections (17.4%). For 40% of the reviewed specimens, S. lugdunensis was the sole agent isolated, and for 60% of specimens, S. lugdunensis was isolated as part of mixed flora. In only 15.4% of clinically reviewed specimens was S. lugdunensis clearly a culture contaminant or colonizing organism. The pattern of human infection identified in this study emphasizes the predominance of skin and soft tissue S. lugdunensis infections over deep serious infections such as endocarditis, peritonitis, infected hip prosthesis, and osteomyelitis and vascular-associated infections. S. lugdunensis should be included along with S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus as a coagulase-negative species of Staphylococcus pathogenic for humans.
Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction
Authors: Johnson W M , Tyler S D , Ewan E P , Ashton F E , Pollard D R , Rozee K R
Published: January 2020

Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.
New oligopeptide immunoglobulin G test for human parvovirus B19 antibodies
Authors: Schwarz T F , Modrow S , Hottenträger B , Höflacher B , Jäger G , Scharti W , Sumazakl R , Wolf H , Middeldorp J , Roggendorf M
Published: January 2020

A new, highly sensitive and specific enzyme immunoassay using oligopeptides as antigen (enzyme-linked immunosorbent assay [ELISA] B19-OP) for detecting parvovirus B19-specific immunoglobulin G (IgG) was established. As antigens, B19-specific oligopeptides of 24 and 30 kDa derived from a 196-kDa fusion protein of beta-galactosidase and viral capsid protein (VPI) of B19 after CNBr cleavage and separation by high-pressure liquid chromatography were used. Of 139 serum specimens tested in parallel for anti-B19 IgG by standard ELISA using B19 particles as antigen and by ELISA B19-OP, 73 (52.5%) were positive and 63 (45.3%) were negative in both tests, and 3 (2.2%) were negative by standard ELISA but positive by ELISA B19-OP and by immunoblot. By using ELISA B19-OP, it was possible to detect anti-B19 IgG in an asymptomatic blood donor 4 weeks after acute infection, and anti-B19 IgG titers of 10(-5) could be measured in convalescent-phase sera.
Comparison of two enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen
Authors: Gonik B , Seibel M , Berkowitz A , Woodin M B , Mills K
Published: January 2020

Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.
Specific immunofluorescence staining of Treponema pallidum in smears and tissues
Authors: Ito F , Hunter E F , George R W , Swisher B L , Larsen S A
Published: January 2020

To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent-antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the indirect IFA technique after either trypsin or NH4OH pretreatment.
Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus
Authors: Waner J L , Todd S J , Shalaby H , Murphy P , Wall L V
Published: January 2020

Directigen FLU-A, an enzyme immunoassay membrane test, was compared prospectively to isolation in cell culture and direct immunofluorescence (IF) for the detection of influenza A virus. One hundred ninety specimens were evaluated by Directigen FLU-A and cell culture; 184 of these specimens were also tested by direct IF. The sensitivity of Directigen FLU-A compared to isolation in cell culture and direct IF was 100%. The specificities of Directigen FLU-A compared to isolation and direct IF were identical, 91.6%. Fourteen specimens that were positive by Directigen FLU-A did not yield virus in culture; two of the specimens, however, were positive by direct IF, and four other specimens were not specimens of choice for the test. A positive Directigen result had positive predictive values of 62.6 and 75.0% compared to isolation and direct IF, respectively; a positive Directigen result with an intensity reading of 2+ or greater, however, had positive predictive values of 85 and 100% compared to isolation and direct IF, respectively. In all comparisons, the negative predictive value was 100%. There was no evidence that cross-reactivity occurred with non-influenza A antigens. Directigen FLU-A should serve as a convenient screening test for influenza A and as a rapid test supported by isolation in cell culture during an influenza outbreak.
Detection of group B and C rotaviruses by polymerase chain reaction
Authors: Gouvea V , Allen J R , Glass R I , Fang Z Y , Bremont M , Cohen J , McCrae M A , Saif L J , Sinarachatanant P , Caul E O
Published: January 2020

We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.
Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction
Authors: Eiden J J , Wilde J , Firoozmand F , Yolken R
Published: January 2020

A combined reverse transcriptase reaction-polymerase chain reaction (RT-PCR) was developed to achieve the sensitive detection of group B rotaviruses (GBR). Sequences derived from genomic segment 3 of the IDIR (intestinal disease of infant rats) strain of GBR permitted the detection of greater than or equal to 0.08 pg of purified IDIR genomic RNA (4,000 genome copies). Primers complementary to the terminal sequences of gene 11 of GBR strain ADRV (adult diarrhea rotavirus) allowed for the detection of as little as 0.008 pg of purified ADRV genomic RNA. Detection of heterologous strains of GBR was also observed with these primer pairs. IDIR gene 3 primers recognized greater than or equal to 8 pg of RNA from bovine GBR obtained from a variety of geographic locations. RNA from IDIR, but not bovine GBR, strains was detected by means of RT-PCR with ADRV gene 11 primers. Neither set of GBR primers was reactive in RT-PCR with fecal specimens containing group A rotaviruses or fecal specimens from uninfected controls. This RT-PCR assay permits the sensitive and specific detection of a variety of GBR in fecal specimens.
Detection of antibodies to hepatitis C virus in U.S. blood donors
Authors: Dawson G J , Lesniewski R R , Stewart J L , Boardway K M , Gutierrez R A , Pendy L , Johnson R G , Alcalde X , Rote K V , Devare S G
Published: January 2020

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.
Antigenic characterization of respiratory syncytial virus group A and B isolates in Rio de Janeiro, Brazil
Authors: Siqueira M M , Nascimento J P , Anderson L J
Published: January 2020

The antigenic characteristics of 87 strains of respiratory syncytial virus isolated in Rio de Janeiro, Brazil, from 1982 to 1988 were determined with a panel of monoclonal antibodies (MAbs) in an enzyme immunoassay. Four of these MAbs immunoprecipitated the fusion protein, and five immunoprecipitated the large glycoprotein. On the basis of the patterns of reaction of these MAbs to respiratory syncytial virus isolates in an enzyme immunoassay, we were able to separate isolates into the two major groups, A and B, and also to identify four variants within group A and three within group B. Strains from groups A and B were isolated each year, and the prevalence of the two groups varied over the seven study years.
Rapid detection of bovine viral diarrhea virus by polymerase chain reaction
Authors: Lopez O J , Osorio F A , Donis R O
Published: January 2020

The polymerase chain reaction was used to detect genomic sequences of the positive-stranded RNA of bovine viral diarrhea virus (BVDV), a member of the family Togaviridae. Using a set of 20-bp primers located within the conserved 3' region of the BVDV genome, we were able to consistently amplify a 205-bp target sequence from BVDV cDNA. BVDV RNAs from cell culture-propagated BVDV reference strains, diverse unrelated cytopathic and noncytopathic field isolates, and clinical serum samples were transcribed to cDNA by using avian myeloblastosis virus reverse transcriptase and further specifically amplified by using the polymerase chain reaction assay. The amplification assay was sensitive enough to detect one molecule of cloned BVDV cDNA. Reconstitution experiments conducted by adding decreasing amounts of BVDV (NADL strain) to BVDV-free serum indicated that the threshold of sensitivity of the assay was less than or equal to 1 50% tissue culture infective dose. These results show that the polymerase chain reaction may be used for the rapid detection of diverse strains of BVDV in cell cultures, biological products, and clinical specimens from cattle.
Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis
Authors: Kutner S , Pellerin P , Breniere S F , Desjeux P , Dedet J P
Published: January 2020

We examined the expression and the antigenicity of the major surface polypeptides of Leishmania braziliensis braziliensis and Leishmania donovani chagasi, parasites which commonly coexist in the same endemic areas of Bolivia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles from surface-iodinated promastigotes showed the presence of a unique iodinatable polypeptide of 72 kDa on the L. b. braziliensis surface and of two major components of 65 and 50 kDa exposed at the surface of L. d. chagasi. Comparison of the peptide digestion profiles of the major iodinated polypeptides of both strains showed no similarity between the maps of the 72- and the 65-kDa polypeptides of L. b. braziliensis and L. d. chagasi, respectively. Immunoprecipitation of surface-labeled L. b. braziliensis Nonidet P-40 extracts with 35 serum specimens obtained from Bolivian patients with cutaneous and mucocutaneous leishmaniasis showed that all serum specimens recognized predominantly the 72-kDa antigen and high-molecular-mass proteins in some cases. The recognition patterns were independent of the geographical origin of the patient, the type of lesion, and the serum antibody titer. Serum specimens from children with visceral leishmaniasis did not precipitate the L. b. braziliensis 72-kDa antigen. Hamster hyperimmune serum against L. b. braziliensis also recognized the 72-kDa surface antigen. However, this recognition was inhibited in the presence of the homologous nonlabeled antigen but not in the presence of heterologous (L. d. chagasi and Trypanosoma cruzi) antigens. The specific recognition of 72-kDa surface antigen in both natural and experimental L. b. braziliensis infections suggests that this antigen could be a good candidate for use in the differential immunodiagnosis and prognosis of the disease.
Characterization of a reduction-sensitive factor from human plasma responsible for apparent false activity in competitive assays for antibody to hepatitis B core antigen
Authors: Robertson E F , Weare J A , Randell R , Holland P V , Madsen G , Decker R H
Published: January 2020

Addition of reducing agents to competitive assays for antibody to hepatitis B core antigen (anti-HBc) eliminates apparent false reactivity of specimens obtained from individuals with no prior history of hepatitis B virus (HBV) infection and without other serological markers of HBV infection. We have purified and characterized a reduction-sensitive factor (RSF) isolated from the plasma of several volunteer blood donors. Column fractions were assayed fro anti-HBc by using a highly sensitive chemiluminescence assay with a detection of 0.15 Paul Ehrlich Institut units per ml at 50% inhibition. Gel filtration on Sephacryl S-300 indicated that reductant-sensitive samples possessed anti-HBc activity that was associated with immunoglobulin M (IgM), whereas reductant-stable activity was associated with IgG. Gel filtration followed by metal chelate affinity chromatography resulted in a 55-fold purification and demonstrated that RSF activity copurifies with IgM. RSF was recovered from a recombinant hepatitis B core antigen matrix and shown to be an IgM species by immunoblot. In addition, RSF activity coeluted with IgM protein from anti-mu-chain Sepharose. Discrepancies between enzyme immunoassay and radioimmunoassay procedures for anti-HBc (Corzyme and Corab, respectively: Abbott Laboratories, North Chicago, Ill.) appear to be due to the relative sensitivity of the enzyme immunoassay for IgM anti-HBc (sevenfold greater than the radioimmunoassay using a specific panel). The biological basis for the occurrence of low levels of nonspecific IgM anti-HBc reactivity in individuals not previously exposed to HBV remains to be elucidated.
Seroprevalence of helicobacter pylori infection in couples
Authors: Perez-Perez G I , Witkin S S , Decker M D , Blaser M J
Published: January 2020

We investigated the prevalence of Helicobacter pylori in 277 couples attending an infertility clinic. In total, 96 (17.3%) of the 554 persons were positive; in only 18 (6.6%) of the couples were both persons seropositive. Age was an important predictor for H. pylori infection. For 177 couples, information regarding birthplace, duration of cohabitation, history of ulcer or gastritis, and use of antacid or bismuth compounds was available. None of these variables correlated with H. pylori infection except place of birth; 69.1% of 55 persons born outside the United States were seropositive compared with 8.7% of persons born within the United States (P less than 0.0001). Being a partner of an H. pylori-infected person increased the risk of being infected; however, by multiple logistic regression analysis this effect was entirely explained by age and national origin. These data suggest that in young sexually active adults, person-to-person transmission of H. pylori does not occur or at most occurs infrequently.