bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates
Authors: Ksiazek T G , Rollin P E , Jahrling P B , Johnson E , Dalgard D W , Peters C J
Published: January 2020

A sandwich enzyme immunosorbent assay (EIA) using a mixture of mouse monoclonal antibodies for antigen capture and polyclonal hyperimmune rabbit anti-Ebola virus serum for antigen detection was developed and evaluated on the tissues of monkeys naturally or experimentally infected with strains of Ebola viruses. When compared with virus isolation, the antigen detection EIA was both sensitive and specific: 44 of 45 (97.7%) liver homogenates and 38 of 41 (92.7%) spleen homogenates that were culture positive and tested by both techniques were positive for viral antigen, while 85 of 87 (97.7%) culture-negative liver homogenates and 66 of 66 culture-negative spleen homogenates were found to be antigen negative. The assay, initially developed to detect antigens of prototype African strains of Ebola virus, reliably detected related strains of Ebola virus found during two recent outbreaks of Ebola virus infection among imported, quarantined Macaca fascicularis monkeys in the United States. The assay allows economical and rapid testing of large numbers of tissue specimens. Antigen was found in homogenates of spleen and liver and in serum.
Use of a mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest U.S. epidemic of sporotrichosis
Authors: Dixon D M , Duncan R A , Hurd N J
Published: January 2020

Five clinical and 69 environmental isolates from the largest U.S. epidemic of sporotrichosis were evaluated in NYLAR male mice following intravenous injection of 5 x 10(6) to 2 x 10(8) conidia per mouse. The clinical isolates and eight environmental isolates produced 100% mortality in groups of three mice each between 12 and 24 days after injection. These virulent isolates grew at 37 degrees C, were dematiaceous by virtue of melanin (melanized) on permissive media (e.g., potato dextrose agar), produced ovoid conidia borne sympodially on lateral conidiophores and pleurogenously about the main hyphal axis, and were identified as Sporothrix schenckii. Two melanized environmental isolates that grew at 35 degrees C but not at 37 degrees C were not virulent and had subtle morphological differences from S. schenckii. The remaining environmental isolates were not melanized, were not virulent, and were not S. schenckii; five were identified as Ophiostoma stenoceras and the remainder were identified as Sporothrix spp. Quantitative organ cultures revealed that clinical isolates grew exponentially in livers and testes, in contrast to an isolate of O. stenoceras that was eliminated from liver, lung, and spleen but that persisted in the testes throughout the 14-day sample period. This model helped to confirm the identification of S. schenckii isolates obtained from the environment.
Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing
Authors: Brytting M , Wahlberg J , Lundeberg J , Wahren B , Uhlén M , Sundqvist V A
Published: January 2020

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.
Detection of human immunodeficiency virus type 1 (HIV-1) in urine cell pellets from HIV-1-seropositive individuals
Authors: Li J J , Huang Y Q , Poiesz B J , Zaumetzger-Abbot L , Friedman-Kien A E
Published: January 2020

Fresh urine pellets from human immunodeficiency virus type 1 (HIV-1)-seropositive individuals were examined for the presence of the HIV-1 genomic sequence and gene products. By using the polymerase chain reaction technique, HIV-1 DNA proviral sequences were detected in 53 of 80 (66.25%) fresh urine pellets from HIV-1-seropositive individuals, while urine pellets from all 24 healthy heterosexual controls were negative. HIV-1 RNA in urine pellets was detected by reverse transcriptase polymerase chain reaction in 2 of 43 (4.7%) HIV-1-seropositive individuals. In addition, HIV-1 p24 core antigen was demonstrated in 3 of 80 urine pellets from HIV-1-seropositive individuals by enzyme-linked immunosorbent assay. Moreover, HIV-1 p24 core antigen and HIV-1 RNA were shown in the cellular component of urine pellets from HIV-1-seropositive individuals by immunohistochemical staining and in situ hybridization. These results indicate that HIV-1 can be present in urine pellets from HIV-1-infected individuals.
Antigenic characterization of ehrlichiae: protein immunoblotting of Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii
Authors: Brouqui P , Dumler J S , Raoult D , Walker D H
Published: January 2020

In recent years a febrile illness apparently associated with tick bite in patients in the United States has been attributed to infection by an Ehrlichia species. This implication is based on serologic responses to E. canis, morphologic demonstration of ehrlichiae in clinical materials, and a single isolate distinct from E. canis which was obtained from a human patient by the Centers for Disease Control. Little is known about the antigens of the ehrlichiae. This report expands the breadth of available knowledge concerning the antigenic components and serologic responses to component antigens of E. canis, E. sennetsu, and E. risticii. Protein immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using density gradient-purified ehrlichiae and homologous antisera demonstrated reproducible and characteristic antigens within each species (for E. sennetsu, 91, 64, 54, 44, 36, 34, 28, 25, and 24 kDa; for E. risticii, 70, 52, 48, 44, 35, 28, 24, 23, and 20 kDa; for E. canis, 110, 64, 52, 42, 33, 28, 24, 23, and 20 kDa). When antisera were reacted with heterologous antigens, cross-reactivity among these species was virtually restricted to the 70-kDa antigen. Furthermore, when serum samples obtained from 10 patients who were convalescing from ehrlichiosis were tested against each antigen, only three serum samples had any reactivities, and these serum samples reacted with only a few of the antigenic bands. These results documented the molecular sizes of electrophoretically separated antigens of the three Ehrlichia species, confirm their serologic relationships, and support the novel nature of the agent(s) of human ehrlichiosis in the United States.
Differentiation of Corynebacterium spp., Listeria spp., and related organisms by using fluorogenic substrates
Authors: Kämpfer P
Published: January 2020

A total of 228 strains of Arcanobacterium haemolyticum, Corynebacterium spp., Erysipelothix rhusiopathiae, and Listeria spp. were investigated for their abilities to hydrolyze 60 different fluorogenic 4-methylumbelliferyl-linked and beta-naphthylamide-linked substrates within 6 and 24 h of incubation. The hydrolysis of a group of 16 fluorogenic substrates, and in particular, the glycosidase tests, in most cases showed high separation values at the genus level. When used in combination with other biochemical tests, these tests improved the differentiation of coryneform bacteria and phenotypically similar organisms.
Extensive allelic variation in Cryptococcus neoformans
Authors: Casadevall A , Freundlich L F , Marsh L , Scharff M D
Published: January 2020

The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains.
Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii
Authors: Parmley S F , Sgarlato G D , Mark J , Prince J B , Remington J S
Published: January 2020

The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.
Performance of Haemophilus Test Media prepared with 12 different lots of Mueller-Hinton agar from four manufacturers
Authors: Barry A L , Packer R R
Published: January 2020

Haemophilus Test Media (HTM) were prepared from 12 different lots of Mueller-Hinton agar. When tested with Haemophilus influenzae ATCC 49247, most lots were initially rejected because of small zones of inhibition for cefaclor, cefuroxime, and cefamandole disks, whereas five other drugs performed satisfactorily on the 11 lots that supported growth of the control strain. At the same time, tests of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 documented the acceptability of these agar media, with or without HTM supplements. The current control limits for cefaclor, cefuroxime, and cefamandole appear to be unrealistic. Because the beta-lactamase-negative, ampicillin-resistant control strain of H. influenzae (ATCC 49247) has altered penicillin-binding proteins, it is resistant to ampicillin and is probably also resistant to cefaclor, cefuroxime, and cefamandole. Consequently, media that produce no or very small zones of inhibition with those three drugs might be clinically correct and should not be rejected. One manufacturer provided three lots of Mueller-Hinton agar that gave unusually large zones of inhibition with all beta-lactams. The three other manufacturers provided eight Mueller-Hinton agars that were satisfactory for the preparation of HTM agar, provided that small zones of inhibition with cefaclor, cefuroxime, and cefamandole disks are accepted as the preferred result.
Enhanced shedding of cytomegalovirus in semen of human immunodeficiency virus-seropositive homosexual men
Authors: Rinaldo C R , Kingsley L A , Ho M , Armstrong J A , Zhou S Y
Published: January 2020

Site-specific shedding of cytomegalovirus (CMV) was assessed in a longitudinal study of homosexual and bisexual men. At initial testing, CMV was cultured from the semen of 33% (19 of 58) of asymptomatic and mildly symptomatic men who were seropositive for human immunodeficiency virus (HIV) at the time of entry into the study, whereas it was cultured from the semen of 17% (10 of 58) of the men who were HIV seronegative. CMV was isolated much more frequently from semen than from urine or throat washing specimens, and it was rarely recovered from stool or blood, regardless of the subject's HIV serostatus. CMV was cultured from the semen of 31% (16 of 52) of the men relatively early after seroconversion to HIV (mean, 12.8 months). CMV was persistently isolated from the semen of a greater proportion of the HIV-seropositive men than from the semen of the HIV-seronegative men during a 4.5-year follow-up period (52 of 110 - [47%] and 15 of 58 [26%] men, respectively). There was an increased relative risk for shedding of CMV in semen in association with decreased CD4+ cell numbers and increased levels of serum immunoglobulin A. However, there was no association of CMV shedding with an increased risk for the development of AIDS.
Microbial flora in eyes of current and former contact lens wearers
Authors: Fleiszig S M , Efron N
Published: January 2020

Microbial flora from the right eye conjunctival sac of 84 consecutively presenting contact lens patients were compared with cultures from both surfaces of their lens after aseptic removal and with the flora of their storage cases. Similar results were obtained from contact lens and conjunctival cultures of each individual; however, there was no correlation between storage case isolates and lens or conjunctival flora, suggesting that in uncomplicated lens wear, the eye is highly efficient in eradicating microorganisms introduced via handling. Conjunctival flora during daily contact lens wear was similar to the conjunctival flora of a matched control group of non-lens wearers. However, bacteria that are considered to be part of the normal ocular flora were isolated significantly more often from former contact lens wearers. The data also indicated that the use of nonperoxide chemical lens disinfection was associated with a higher proportion of positive cultures for pathogenic microorganisms than the use of other forms of disinfection, for both current and former contact lens wearers. The isolation of potential pathogens was particularly common among elderly subjects using thick contact lenses for extended wear. These changes to conjunctival flora may contribute to the increased risk of ocular infection associated with contact lens wear.
Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for optimal quantitation of human cytomegalovirus antigenemia
Authors: Gerna G , Revello M G , Percivalle E , Morini F
Published: January 2020

The main parameters of immunostaining techniques, i.e., the type of fixative, immunocytochemical reaction, and quality of monoclonal antibodies (MAbs), for quantitation of human cytomegalovirus (HCMV) antigenemia in peripheral blood polymorphonuclear leukocytes (currently performed by the indirect immunofluorescence or immunoperoxidase reaction by using MAbs to HCMV pp65) were investigated in order to optimize procedural steps and reagents. Significantly better results (in terms of the number of positive cells) were obtained on multiple cytospin preparations from heart transplant recipients with HCMV viremia when we used (i) formalin instead of methanol-acetone fixation and (ii) the indirect immunofluorescence reaction instead of the immunoperoxidase reaction, the avidin-biotin complex method, or the alkaline phosphatase antialkaline phosphatase procedure. In addition, comparison of the staining capabilities of three MAbs to pp65, which were developed in the laboratory and which were reactive to different epitopes of the protein, with a commercially available MAb (Clonab CMV) for determination of HCMV antigenemia showed that, while individual MAbs did not provide better results, the pool of MAbs detected a significantly higher number of positive peripheral blood polymorphonuclear leukocytes than Clonab CMV did. In addition, the sensitivity of the pool in detecting patients with low levels of viremia (less than 5/2 x 10(5) cells inoculated) as antigenemia positive was 100%, whereas the sensitivity of Clonab CMV was 47%. No differences in the specificities between the two MAb preparations were observed.
Measurement of fecal lactoferrin as a marker of fecal leukocytes
Authors: Guerrant R L , Araujo V , Soares E , Kotloff K , Lima A A , Cooper W H , Lee A G
Published: January 2020

While diarrheal illnesses are extremely common in communities and hospitals throughout the world, an etiologic diagnosis may be expensive and cost-ineffective. Although the presence of fecal leukocytes are helpful in the diagnosis and specific therapy of inflammatory diarrheas, this requires prompt microscopic examination of fecal specimens (preferably obtained in a cup rather than a swab or diaper) by a trained observer. We developed a simple, sensitive test for the detection of leukocytes in fecal specimens using antilactoferrin antibody. Whereas radial immunodiffusion detected 0.02 micrograms of lactoferrin (LF) per microliter or greater than or equal to 2,000 leukocytes per microliter, latex agglutination (LA) readily detected greater than or equal to 0.001 micrograms of LF per microliter or greater than or equal to 200 leukocytes per microliter added to stool specimens. Despite the destruction or loss of morphologic leukocytes on storage for 1 to 7 days at 4 degrees C or placement of specimens on swabs, measurable LF remained stable. Initial studies of stool specimens from six patients with Salmonella or Clostridium difficile enteritis were positive and those from three controls were negative for LF by LA. Of 17 children in Brazil with inflammatory diarrhea (greater than or equal to 1 leukocyte per high-power field), 16 (94%) had LF titers of greater than 1:50 by LA, whereas only 3 of 12 fecal specimens with less than 1 leukocyte per high-power field on methylene blue examination and none of 7 normal control specimens had an LF titer of greater than 1:50 by LA. Of 16 fecal specimens from patients with C. difficile diarrhea (cytotoxin titers, >/= 1:1,000), 95% (n = 15) had detectable LF by LA (in titers of 1:100 to 1: 800). Finally, of 48 fecal specimens from healthy adult U.S. volunteers before and after experimental shigellosis and of 29 fecal specimens from children with documented shigellosis and hospitalized controls in northeastern Brazil, fecal LF titers ranged from 1:200 to >/= 1:5,000 in 96% (25 of 26) samples from patients with shigellosis (and reported positive for fecal leukocytes), while 51 controls consistently had fecal LF titers of </= 1:200. We conclude that fecal LF is a useful marker for fecal leukocytes, even when they are morphologically lost swab specimens or when they are destroyed on transport or storage or by cytotoxic fecal specimens.
Seroepidemiological studies of Borrelia burgdorferi infection in sheep in Norway
Authors: Fridriksdóttir V , Nesse L L , Gudding R
Published: January 2020

An enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi in sheep was established. The assay was used in a seroepidemiological survey in which serum samples from 327 Norwegian rams were screened for antibodies to B. burgdorferi. These rams were randomly chosen, clinically healthy animals from all parts of Norway. They came both from areas where Ixodes ricinus is common and from areas where the tick has not been found. The age of the animals varied from 0.5 to 7.5 years. Sera from 10% of the animals tested were seropositive by the enzyme-linked immunosorbent assay, with the percentage of positive animals varying from 0 to 20% between counties. The distribution of seropositive animals was generally in good agreement with the known distribution of I. ricinus, with the highest proportion of seropositive animals being in southern coastal areas of Norway. There were some exceptions, however, because seropositive animals were also found in areas where the tick has not been recorded. The majority of animals appeared to become infected during the first 2 years of life, with 12% of animals that were 1.5 years old being seropositive. The animals were all healthy at the time of serum sampling, and the clinical significance of B. burgdorferi in sheep is still uncertain.
High prevalence of Kingella kingae in joint fluid from children with septic arthritis revealed by the BACTEC blood culture system
Authors: Yagupsky P , Dagan R , Howard C W , Einhorn M , Kassis I , Simu A
Published: January 2020

In an effort to improve detection of fastidious organisms, joint fluid aspirates of pediatric patients were inoculated into BACTEC 460 aerobic blood culture bottles, in addition to cultures on solid media. Culture records for the 1988 to 1991 period were reviewed to compare the performance of both methods for the recovery of pathogens. Overall, 216 children underwent a diagnostic joint tap, and 63 specimens grew significant organisms, including Kingella kingae in 14. While both methods were comparable for recovery of usual pathogens, with a single exception, K. kingae isolates were detected by the BACTEC system only. K. kingae appears to be a more common cause of septic arthritis in children than has been previously recognized. The BACTEC blood culture system enhances the recovery of K. kingae from joint fluid and improves bacteriologic diagnosis of pediatric septic arthritis.
Application of quantitative immunofluorescence to clinical serology: antibody levels of Treponema pallidum
Authors: Picciolo G L , Kaplan D S
Published: January 2020

A previously reported method of quantitative immunofluorescence, employing a calibrated photometric system and chemically stabilized fluorescence intensity, was used to replace the subjective, visual method of endpoint determination with a quantitative, calibrated measurement of antibodies to Treponema pallidum in serum. The results of the quantitative immunofluorescence method showed a 90% correlation with the subjective determinations of the visual method.
Preliminary disk diffusion susceptibility testing criteria for cefdaloxime (RU29246, HR-916 metabolite), a new orally administered cephalosporin
Authors: Jones R N , Erwin M E
Published: January 2020

Cefdaloxime (formerly RU29246; Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.) a new active component of the HR-916 ester, was tested by dilution and two disk (10- and 30-micrograms) diffusion susceptibility tests against 391 clinical isolates. Interpretive criteria were proposed for three potential MIC breakpoints of less than or equal to 1, less than or equal to 2, and less than or equal to 4 micrograms/ml. Analyses by regression line and error rate bounding methods minimized false-susceptible (very major) errors and produced a greater than or equal to 90% absolute interpretive agreement between susceptibility test methods. The less than or equal to 2-micrograms/ml breakpoint seemed optimal when 10-micrograms disks and the available human pharmacokinetics were used. The following inhibition zone diameter criteria were proposed: susceptible, greater than or equal to 19 mm; resistant, less than or equal to 15 mm. These recommendations for clinical trials should remain tentative until additional information about cefdaloxime formulations, pharmacokinetics, and patient outcomes can be correlated with in vitro susceptibility test results.
Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia
Authors: Rice E W , Sowers E G , Johnson C H , Dunnigan M E , Strockbine N A , Edberg S C
Published: January 2020

The antigenic relatedness of Escherichia coli O157 and four sorbitol-negative species of the genus Escherichia was examined. Isolates of Escherichia hermannii, E. fergusonii, E. vulneris, and E. blattae were tested in the tube agglutination assay by using polyclonal antisera and in the slide agglutination assay by using latex reagents. Only four isolates (17%) of E. hermannii exhibited serological cross-reactivity.
Defined medium simulating genital tract secretions for growth of vaginal microflora
Authors: Geshnizgani A M , Onderdonk A B
Published: January 2020

A chemically defined medium that simulates female genital tract secretions was developed for the growth of the vaginal microflora. Qualitative and quantitative studies of the growth of predominant components of the vaginal microflora indicated that all vaginal isolates tested were able to grow in this defined medium.
Characterization of Aeromonas caviae antigens which cross-react with Shigella boydii 5
Authors: Albert M J , Qadri F , Ansaruzzaman M , Kibriya A K , Haider K , Neogi P K , Alam K , Alam A N
Published: January 2020

Live and boiled cells of 16 strains of Aeromonas caviae, isolated from patients with diarrhea, agglutinated with Shigella boydii 5 antiserum in a slide test. Further studies with seven selected strains showed agglutination with boiled cells in a tube test. Lipopolysaccharide antigen extracted from one of these strains cross-reacted with S. boydii 5 in enzyme-linked immunosorbent assay and immunoblot studies. Either all or the majority of the seven strains possessed properties deemed to be diarrheagenic.
Campylobacter upsaliensis isolated from a breast abscess
Authors: Gaudreau C , Lamothe F
Published: January 2020

Campylobacter upsaliensis is a rare human pathogen recovered so far only from stools or blood from patients with enterocolitis or bacteremia. We report the isolation of C. upsaliensis from a breast abscess.
Comparison of the Bactec Peds Plus pediatric blood culture vial with Roche pediatric Septi-Chek for blood cultures from pediatric patients
Authors: Welby P L , Zusag T M , Storch G A
Published: January 2020

Equal volumes of blood from 4,112 blood cultures were inoculated into one Bactec Peds Plus bottle and one Roche Septi-Chek bottle. There were no significant differences in the recoveries of bloodstream pathogens. Initial detection occurred earlier with Bactec Peds Plus, while growth on solid media occurred earlier with Roche Septi-Chek.
Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA
Authors: Fahrimal Y , Goff W L , Jasmer D P
Published: January 2020

Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites.
Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis
Authors: Lariviere S , Leblanc L , Mittal K R , Martineau G P
Published: January 2020

A total of 137 strains of Pasteurella multocida isolated from the nasal tracts of pigs with and without clinical atrophic rhinitis (AR) were studied for their biochemical, antigenic, and toxigenic characteristics. There were no major biochemical differences among the P. multocida isolates. Capsular antigen types A and D were both present in the nasal cavities of the pigs with or without clinical AR. However, the prevalence of type D was higher on farms with pigs with AR. Types A and D with different somatic antigens could both be present in the same pig. There was no correlation between somatic types and/or capsular types with the clinical AR status of the pigs on the farm. Toxigenic isolates were found only in pigs which had a problem of clinical AR, and a great majority of these isolates belonged to type D. Since there was a high level of heterogeneity of the strains in the P. multocida population on a farm, several strains should be characterized before the diagnosis of AR could be excluded on the basis of the absence of isolation of rhinopathogenic P. multocida strains.
Molecular epidemiology of trimethoprim-resistant Shigella boydii serotype 2 strains from Bulgaria
Authors: Bratoeva M P , John J F , Barg N L
Published: January 2020

In 1990 an increased number of strains of Shigella boydii serotype 2 were isolated from different regions of Bulgaria. Strains were reported as sporadic, although they showed identical phenotypic characteristics, including resistance to ampicillin, carbenicillin, streptomycin, sulfonamide, tetracycline, ticarcillin, and trimethoprim. The objective of this study was to determine the genetic relatedness of the strains and the mechanism of their antimicrobial resistance. Plasmid fingerprinting showed an identical pattern for 23 of 25 of the selected strains. All 25 strains tested transferred their resistances en bloc to an Escherichia coli recipient. Transconjugants contained a 112-kb R plasmid which carried all the resistance genes, including that conferring type I dihydrofolate reductase-mediated trimethoprim resistance (MIC greater than 2,000 micrograms/ml). Riboprobe analysis showed identical restriction length fragment polymorphisms, suggesting a highly conserved genome. All findings indicate that strains of S. boydii serotype 2 isolated in 1990 from different regions of Bulgaria were highly related genetically and can be considered representatives of a single bacterial clone. The presence of an R plasmid and selection pressure because of the usage of antimicrobial agents, particularly trimethoprim, have likely facilitated the spread of the clone throughout the country.
Use of enzyme-linked immunosorbent assays with chimeric fusion proteins to titrate antibodies against Epstein-Barr virus nuclear antigen 1
Authors: Inoue N , Kuranari J , Harada S , Nakajima H , Ohbayashi M , Nakamura Y , Miyasaka N , Ezawa K , Ban F , Yanagi K
Published: January 2020

Two new enzyme-linked immunosorbent assays (ELISAs) with chimeric fusion polypeptides for the detection of human antibodies specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1) are described. One is an indirect ELISA with affinity-purified beta-galactosidase-EBNA-1 fusion protein as the antigen. The other is a "sandwich" assay based on the use of anti-beta-galactosidase antibody to capture beta-galactosidase-EBNA-1 fusion proteins in bacterial extracts. A good correlation was shown between antibody titers determined by the ELISA with the EBNA-1 fusion proteins and those determined by a conventional anticomplement immunofluorescence test which is being widely performed with Raji cells for the purpose of research and clinical diagnosis. The advantage of the ELISAs for seroepidemiologic studies on Epstein-Barr virus was demonstrated by sensitive detection of marginal immunoglobulin G antibody to the EBNA-1 domain in serum samples from patients with infectious mononucleosis.
Critical assessment of blood culture techniques: analysis of recovery of obligate and facultative anaerobes, strict aerobic bacteria, and fungi in aerobic and anaerobic blood culture bottles
Authors: Murray P R , Traynor P , Hopson D
Published: January 2020

Recent reports have documented a decrease in anaerobic bacteremias and have questioned the need for routine anaerobic blood cultures. At the same time, we and others have noted an increase in fungal bloodstream infections. In this two-part study, we first compared recoveries of obligate anaerobic bacteria with those of fungi over a 13-year period and then examined the recoveries of all bacteria and fungi in aerobic and anaerobic blood culture bottles during a 12-month period. During the 13-year period, the number of patients with anaerobic bacteremia remained relatively constant (average, 39 patients per year), while the incidence of fungemia steadily increased, from 12 patients in 1978 to 117 patients in 1990. Of the 1,090 anaerobic isolates, 55.1 and 90.2% were recovered in aerobic and anaerobic bottles, respectively, compared with 98.6 and 37.0% of the 2,582 fungi. During the 12-month period of evaluation, 2,980 bacteria and fungi were recovered in cultures collected from 1,555 patients. Overall, 21.1% more organisms were recovered in aerobic bottles than in anaerobic bottles, including significantly more Staphylococcus species; gram-positive aerobic bacilli; Escherichia, Enterobacter, Pseudomonas, Xanthomonas, and Acinetobacter species; miscellaneous gram-negative bacilli; and yeasts. Only anaerobic gram-negative bacilli and non-spore-forming gram-positive bacilli were isolated more commonly in anaerobic bottles. These data support the concepts that bacteremia caused by obligate anaerobic bacteria is decreasing relative to sepsis caused by other bacteria and fungi and that the routine use of unvented anaerobic blood culture bottles reduces the recovery of common aerobic bloodstream pathogens.
Tracking laboratory contamination by using a Bacillus cereus pseudoepidemic as an example
Authors: Morrell R M , Wasilauskas B L
Published: January 2020

From 1 March to 31 May 1990, Bacillus cereus was recovered from 24 of 5,534 (0.49%) blood cultures and 22 of 1,088 (2.02%) other body fluid cultures. The rarity of this organism as a pathogen and comparison with previous baseline rates led to the conclusion that it was a pseudoepidemic involving some form of culture contamination. Generalized precautions taken without specific knowledge of the contaminant source reduced the recovery rate of the organism. Recovery rates for the organism returned to normal baseline prevalence after environmental cultures and epidemiological analysis led to the sterilization of a contaminated water bath used for boiling thioglycollate media. The problems encountered in this investigation are examined, and a systematic approach to clinical laboratory epidemiology is outlined.
Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection
Authors: Meurman O , Waris M , Hedman K
Published: January 2020

The titer and avidity of respiratory syncytial virus-specific antibodies were measured in 196 serum specimens from 93 children with an acute, laboratory-confirmed respiratory syncytial virus infection. An enzyme immunoassay method based on the ability of urea to dissociate the bound antibodies with low avidity from the antigen was used. Three patterns of immune responses were observed. Children less than 6 months of age usually had low titers of antibodies with high avidity in their acute-phase serum samples. These antibodies were concluded to be of maternal origin, since their reaction pattern was similar to that of healthy adults. During the next few weeks, a slight increase in titers with a concurrent decrease in antibody avidity was observed. All children 6 to 24 months of age had low-avidity antibodies in their acute-phase serum samples, which matured to high avidity during the follow-up. On the contrary, about half of the children greater than 24 months of age had high-avidity antibodies already in the acute-phase serum samples. We conclude that the former children were experiencing primary infections with respiratory syncytial virus and the latter were experiencing reinfections. All adults with remote immunity had antibodies with high avidity.
Rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool
Authors: Rabalais G P , Stout G G , Ladd K L , Cost K M
Published: January 2020

We compared the detection of seven respiratory viruses by using a commercially available monoclonal antibody pool in a 2-day shell vial assay with that by using standard cell culture with respiratory syncytial virus (RSV) enzyme-linked immunosorbent assay (ELISA)-negative nasal secretions from hospitalized children. We found 179 respiratory virus isolates by either method in 675 specimens. Overall, the shell vial assay detected 147 of 179 (79%) of the positives after 2 days; cell culture detected 148 of 179 (80%) after a mean incubation period of 7.6 days (range, 1 to 14 days). The sensitivity of the shell vial assay was 78% for RSV, 94% for influenza B virus, 83% for adenovirus, and 80% for parainfluenza viruses. The sensitivity of the cell culture was 70% for RSV, 79% for influenza B virus, 90% for adenovirus, and 89% for parainfluenza viruses. The 2-day shell vial assay allowed the detection of respiratory viruses in a clinically relevant time frame and rapidly detected RSV in specimens lacking RSV antigen by ELISA.
Detection of Mycoplasma hyopneumoniae by using rRNA-oligodeoxynucleotide hybridization
Authors: Futo S , Seto Y , Mitsuse S , Mori Y
Published: January 2020

A system that uses rRNA-oligodeoxynucleotide hybridization was developed for the detection of Mycoplasma hyopneumoniae. Synthetic oligonucleotide MHP1 was hybridized specifically with M. hyopneumoniae. Furthermore, the detection of M. hyopneumoniae in clinical samples, such as bronchoalveolar lavage fluid and lung lesions from experimentally infected pigs, was evaluated by this assay. The evidence obtained from the assay indicated that the system can be used to efficiently diagnose mycoplasmal pneumonia of swine. Additionally, a nonradioisotopic system with chemiluminescence detection was tested. This system was 10-fold less sensitive than a test that used radioisotopes.
Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction
Authors: Victor T , du Toit R , van Helden P D
Published: January 2020

A method is described for the routine preparation of sputum samples for detection of Mycobacterium tuberculosis by using polymerase chain reaction amplification. Liquefaction of sputum samples with NaOH and subsequent removal of inhibitors of the polymerase reaction with a 50% sucrose centrifugation step (5 min) in a desktop centrifuge allow direct amplification of a 123-bp repetitive region of the M. tuberculosis genome. We have evaluated the sensitivity and specificity of the sucrose method with 155 sputum specimens from patients suspected of having tuberculosis and from normal healthy volunteers. This method, in our opinion, is reproducible, sensitive, and reliable. Multiple samples can be handled simultaneously, and results can be obtained in one working day.
Evaluation of the performance of commercial test kits for detection of Helicobacter pylori antibodies in serum
Authors: Hoek F J , Noach L A , Rauws E A , Tytgat G N
Published: January 2020

We have compared the sensitivities, specificities, and predictive values of three commercial serological assays for the diagnosis of Helicobacter pylori infection. A qualitative latex method (Pyloriset; Orion Diagnostics), a semiquantitative enzyme-linked immunosorbent assay (ELISA) (GAP test IgG; Bio-Rad), and a quantiative ELISA (Helico-G; Porton Cambridge) were used in 109 untreated dyspeptic patients. The presence of H. pylori was established when the results of culture and/or histology of the gastric biopsies taken were positive. The prevalence of H. pylori infection was 62% (52% in 42 patients younger than 45 years of age and 69% in 67 patients older than 45 years of age). Sensitivities and specificities were 68 and 76% for Pyloriset, 89 and 77% for GAP test IgG, and 82 and 83% for Helico-G. The positive predictive values for all three tests were between 85 and 90%. The predictive values for the absence of disease with a negative result were 62, 82, and 74% for Pyloriset, the GAP test, and Helico-G, respectively. With Helico-G in the younger group (less than 45 years), sensitivity significantly lower (71 versus 87%) and a positive predictive value lower than those for the older group (greater than 45 years) were found. Either the sensitivities and specificities of commercial methods for the measurement of antibodies to H. pylori in serum must be improved or the relationship between the presence of antibodies and the presence of bacteria in the stomach at the time of investigation is too weak to allow the use of serological techniques instead of culture and histological investigation of gastric biopsy material.
National hospital survey of anaerobic culture and susceptibility testing methods: results and recommendations for improvement
Authors: Goldstein E J , Citron D M , Goldman R J
Published: January 2020

The methods for performing anaerobic bacterial isolation and identification continue to change and improve. Anaerobic susceptibility testing has become controversial, and method-dependent variability has been noted. To assess the status of clinical anaerobic bacteriology in the United States, we surveyed, by means of a questionnaire, 120 hospitals, selected at random, with bed capacities of 200 to 1,000, and we received responses from 88 (73%). All hospitals performed cultures for anaerobes. The media and methods used for transport, initial processing, incubation, and identification varies between the different regions in the United States. Thirty percent of laboratories did not perform susceptibility studies, 16% used a reference laboratory, and 54% performed them in house. For half the laboratories, susceptibility testing was performed on isolates depending on the source; in this case, blood cultures were tested by 97% of the laboratories, serious infections were tested by 60%, sterile body sites were tested by 73%, pure cultures were tested by 47%, and tests were done by physician request by 39%. For laboratories doing testing, the broth disk method, no longer sanctioned by the National Committee for Clinical Laboratory Standards, was used most often (56%), followed by microdilution (33%), beta-lactamase testing (25%), macrotube dilution (2%), and agar dilution (2%). The antimicrobial agents tested were as follows: penicillin-ampicillin, 94%; clindamycin, 94%, metronidazole, 90%; chloramphenicol, 80%; cefoxitin, 76%; tetracyclines, 51%; and erythromycin, 45%. All other agents were tested by less than or equal to 25% of laboratories; the methods used could be improved to make the results more timely and consequently more clinically relevant.
Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays
Authors: Müller F , Stiegler C , Hadding U
Published: January 2020

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.
Ribotyping of Helicobacter pylori from clinical specimens
Authors: Tee W , Lambert J , Smallwood R , Schembri M , Ross B C , Dwyer B
Published: January 2020

Ribotyping is a method used to type strains of bacteria by analyzing the restriction enzyme digestion patterns of the rRNA genes. This method was applied to 126 strains of Helicobacter pylori from 100 unrelated symptomatic patients who had endoscopies done and to 15 strains from 15 infected subjects from seven families. Analysis of the rRNA gene patterns revealed 77 distinct ribotypes from the 100 patients. From 15 of these subjects, isolates were recovered from antral mucosal biopsies at follow-up endoscopy. All follow-up isolates from the same patient, with one exception, yielded identical digest patterns. This patient had strains with two distinct digest patterns obtained from a set of three isolates cultured from biopsy specimens taken at different times. Five patients who had isolates recovered from different sites in the stomach (antrum, gastric body, duodenum, and pyloric channel) showed ribotyping patterns which were identical for each patient yet distinct between patients. In seven family groups studied, identical digest patterns were detected in members of two families, with variability in strains detected among members of the remaining families. This study demonstrates that ribotyping provides a useful, reliable, reproducible, and highly discriminatory typing scheme for the study of H. pylori infection.
Involvement of Gardnerella vaginalis in urinary tract infections in men
Authors: Smith S M , Ogbara T , Eng R H
Published: January 2020

Fifteen male patients from whose urine samples Gardnerella vaginalis was isolated (clinical incidence of 0.1%) were evaluated for clinical signs and symptoms of urinary tract infection and modality of acquisition of the organism. Ten of 15 (67%) patients were symptomatic or had signs of inflammation as manifested by an increased number of urinary neutrophils. One patient had two bouts of infection caused by this organism which required two courses of antibiotic therapy. Colonies of diphtheroidlike organisms found in urine cultures should not be ignored as insignificant but should be further investigated to determine whether G. vaginalis is present.
Pseudomonas mendocina, an environmental bacterium isolated from a patient with human infective endocarditis
Authors: Aragone M R , Maurizi D M , Clara L O , Navarro Estrada J L , Ascione A
Published: January 2020

Pseudomonas mendocina has been isolated from soil and water samples. Although it has been recovered from some human clinical samples, its pathogenic role has not yet been documented. We report the first known case of endocarditis in humans due to P. mendocina.
Isolation of vibriostatic agent O/129-resistant Vibrio cholerae non-O1 from a patient with gastroenteritis
Authors: Abbott S L , Cheung W K , Portoni B A , Janda J M
Published: January 2020

A strain of Vibrio cholerae non-O1 was isolated from a 48-year-old male in California who presented with a chief complaint of watery diarrhea. Laboratory investigations of this strain revealed the bacterium to be resistant to trimethoprim-sulfamethoxazole and to the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine).