bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of colorimetric microdilution plate hybridization for rapid genetic identification of 22 Mycobacterium species
Authors: Kusunoki S , Ezaki T , Tamesada M , Hatanaka Y , Asano K , Hashimoto Y , Yabuuchi E
Published: January 2020

Quantitative microdilution plate hybridization was used to identify 22 Mycobacterium species. DNAs of clinical strains were rapidly extracted and labeled with photoreactive biotin. Labeled DNAs were distributed into wells of a microdilution plate in which reference DNAs had been immobilized. After 2 h of hybridization, hybridized DNAs were quantitatively detected with peroxidase-conjugated streptavidin and the substrate, tetramethylbenzidine. This method could differentiate among 20 of the 22 Mycobacterium species tested. The type strains of Mycobacterium tuberculosis and M. bovis were genetically highly related and could not be differentiated by this method. Of 194 biochemically identified human clinical strains, 178 (90%) were genetically identified within 3 h of the small-scale DNA extraction.
Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products
Authors: Shih J W , Cheung L C , Alter H J , Lee L M , Gu J R
Published: January 2020

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.
Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep
Authors: Sugden E A , Brooks B W , Young N M , Watson D C , Nielsen K H , Corner A H , Turcotte C , Michaelides A , Stewart R B
Published: January 2020

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.
Murine intestinal antibody response to heterologous rotavirus infection
Authors: Merchant A A , Groene W S , Cheng E H , Shaw R D
Published: January 2020

Rotavirus is the most important worldwide cause of severe gastroenteritis. Extensive efforts have been devoted to the design of a vaccine that will prevent disease, but development of a more effective vaccine strategy may require progress in the understanding of the mucosal immune response to replicating viral antigens. In this article, we report the characterization of the intestinal antibody response of a murine model to heterologous infection with the rhesus rotavirus vaccine strain. We have adapted the enzyme-linked immunospot assay to measure this response without the difficulties associated with measurement of antibodies in intestinal contents or the artifacts associated with culturing of lymphocytes. The predominant response in terms of antibody-secreting cells (ASC) is seen in the small intestine lamina propria, which can be measured within 4 days of infection, peaks 3 weeks after infection, and remains near that level for longer than 8 weeks. The magnitude of the immunoglobulin A (IgA) cell response is approximately 10 times greater than the intestinal IgG cell response, and IgM cells are rare. Virus-specific ASC constitute approximately 50% of all ASC in the gut at the peak of the virus-specific response. This response is considerably greater than responses to nonreplicating mucosal antigens measured by similar techniques. Enteral infection engenders minimal virus-specific ASC response in the spleen. Rhesus rotavirus-specific enzyme-linked immunosorbent assay and neutralization assays of serum and intestinal contents did not correlate with virus-specific ASC response.
Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid
Authors: Grimprel E , Sanchez P J , Wendel G D , Burstain J M , McCracken G H , Radolf J D , Norgard M V
Published: January 2020

The diagnosis of congenital syphilis continues to pose a difficult clinical challenge. Because the serodiagnosis of congenital syphilis has significant limitations, the direct detection of Treponema pallidum in suspect neonatal tissues or body fluids represents a desirable alternate diagnostic strategy. We developed and applied the polymerase chain reaction (PCR) for the detection of T. pallidum in clinical material relevant to the diagnosis of congenital syphilis but which typically contain factors inhibitory for the PCR. Four methods of specimen processing were examined to circumvent PCR inhibition; clinical materials included amniotic fluids, neonatal sera, and neonatal cerebrospinal fluids. The PCR was 100% specific for T. T. pallidum compared with the sensitive rabbit infectivity test (RIT) for all clinical materials tested. For amniotic fluids, the PCR was 100% sensitive when correlated with the RIT but had a lesser sensitivity when applied to sera or cerebrospinal fluids, which typically contain few treponemes. The combined sensitivity of the PCR for all clinical samples was 78%. Positive PCR results also were obtained among some clinical specimens for which RIT was not performed; these results correlated well with either stigmata or risk factors for congenital syphilis. The combined results suggest that the PCR can be a useful adjunct to the diagnosis and clinical management of congenital syphilis and that it will provide a valuable tool for investigations of the pathogenesis of the disorder.
Variability in commercial histoplasma complement fixation antigens
Authors: Leland D S , Zimmerman S E , Cunningham E B , Barth K A , Smith J W
Published: January 2020

Using Immuno-Mycologics (IMMY; Norman, Okla.) histoplasmal yeast (HY) and mycelial (HM) antibody complement fixation test antigens, we retested 1,386 samples that were initially tested with Meridian Diagnostics, Inc. (Cincinnati, Ohio), antigens. Histoplasma antibody was identified (greater than or equal to 1:16) in 20% of HY and 5% of HM samples reported to have titers of less than 1:8 with Meridian reagents. IMMY titers were at least fourfold higher than Meridian titers in 39% of HY and 54% of HM samples that initially had titers of greater than or equal to 1:8 with Meridian antigens. Because 30 of 58 (52%) samples from confirmed cases of histoplasmosis yielded negative results with Meridian antigens and positive results upon retesting with IMMY antigens, we concluded that the Meridian antigens had less reactivity with human histoplasmal antibody.
Indirect immunofluorescence assay for detection of Helicobacter pylori in human gastric mucosal biopsies
Authors: Rivera E , López-Vidal Y , Luqueño V , Ruiz-Palacios G M
Published: January 2020

To determine sensitivity and specificity of immunofluorescence assay (IFA) for detection of Helicobacter pylori, we studied 151 patients. Biopsies of gastric mucosae were obtained for culture, histological testing, and IFA. H. pylori serum antibodies were tested by enzyme-linked immunosorbent assay. IFA was done on Formalin-preserved, paraffin-embedded biopsies by using rabbit anti-H. pylori and goat anti-rabbit gamma globulin-fluorescein isothiocyanate conjugate. The sensitivity and specificity of IFA compared with culture and Warthin-Starry stain were 93 and 95%, respectively. IFA is an accurate method for the diagnosis of H. pylori infection.
Detection of rubella-specific immunoglobulin G: comparison of the enzyme-linked immunosorbent assay and an automated microparticle enzyme immunoassay (IMx)
Authors: Skurrie I J , Head J L , Garland S M
Published: January 2020

An automated microparticle enzyme immunoassay (IMx Rubella IgG Antibody Assay; Abbott Laboratories, North Chicago, Ill.) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for detection of rubella-specific immunoglobulin G (IgG) in 400 consecutive antenatal patients. There was complete agreement between the two tests in this population, which had a positivity rate of 99% for rubella-specific IgG antibodies. The performance of the IMx was also evaluated at the cutoff zone by assaying 64 selected antenatal serum samples with low or negative rubella antibody titers as determined by ELISA. Overall, the IMx was found to be a specific, sensitive assay for the detection of rubella-specific IgG and is virtually fully automated for easy performance.
Isolation of Mycobacterium shimoidei from a patient with cavitary pulmonary disease
Authors: Tortoli E , Simonetti M T
Published: January 2020

Mycobacterium shimoidei was isolated from the sputum of a man hospitalized for cavitary pulmonary disease. This is the fourth isolation of M. shimoidei to be reported; the organism has also been isolated in Japan, Australia, and Germany.
Effect of agitation of BACTEC 13A blood cultures on recovery of Mycobacterium avium complex
Authors: Jackson K , Sievers A , Dwyer B
Published: January 2020

The effect of agitation of BACTEC 13A bottles (Becton Dickinson) on the recovery of Mycobacterium avium complex (MAC) from blood was compared with that of static incubation. A total of 265 blood specimens was inoculated in duplicate into BACTEC 13A bottles. One specimen was statically incubated at 35 degrees C, and the other was incubated with agitation on a Gyrotory shaker at 35 degrees C for the first 2 weeks and thereafter without shaking for up to 12 weeks. Of the 265 specimens, 77 (29.1%) were positive in either one or both of the paired bottles. The average detection times for the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively. A total of 10.4% of the specimens in the shaken bottles became positive 1 week before those in the nonshaken bottles, and 16.9% of the shaken cultures were positive more than 2 weeks before their counterparts. A further 46.8% of the agitated specimens became positive while the corresponding nonagitated cultures remained negative. When both specimens became positive at the same time, 88% of the shaken cultures had higher growth indices than their nonshaken counterparts. A further 11 paired blood cultures were taken from patients known to be infected with MAC to assess the effect of agitation of bottles on the utility of making twice-weekly readings during the first 2 weeks of incubation. Ten of the 11 sets of specimens in the shaken bottles were positive 1 or more weeks before those in the corresponding nonshaken bottles. In the remaining set, both specimens became positive on the same day; however, the growth index of the agitated culture was higher.(ABSTRACT TRUNCATED AT 250 WORDS)
Rapid purification of hepatitis B virus DNA from serum
Authors: Boom R , Sol C J , Heijtink R , Wertheim-van Dillen P M , van der Noordaa J
Published: January 2020

We describe two rapid, simple, and reliable procedures for routine purification of hepatitis B virus (HBV) DNA from serum. HBV DNA could be purified from 24 serum samples in 1.5 to 2 h and was recovered in the initial reaction vessel. Both procedures have in common that HBV DNA is complexed with silica particles in the chaotropic agent guanidinium thiocyanate (GuSCN) but differ in lysis conditions and in the conditions used to elute HBV DNA from the silica particles after purification of the silica-DNA complexes. In one procedure (protocol H), serum HBV lysis was mediated by sodium dodecyl sulfate-proteinase treatment and HBV DNA was subsequently complexed with silica particles in the presence of GuSCN. After washing and drying of the silica-DNA complexes, HBV DNA was eluted from the silica particles in a low-salt buffer. In the other procedure (protocol Y*), serum HBV was directly lysed in GuSCN and HBV DNA was simultaneously complexed with silica particles. After washing and drying of the complexes, HBV DNA was eluted by proteinase treatment in low-salt buffer. Omission of proteinase treatment prevented efficient elution, presumably because of copurification of the protein which is covalently bound to the HBV DNA genome. We show, by Southern blot analysis, that HBV DNA could be reproducibly purified from human serum with the same yields by either procedure (30 to 50% relative to a classic procedure) and apparently independent of serum composition. HBV DNA purified by either method was a good substrate in the polymerase chain reaction compared with DNA purified by the classic procedure.(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation of a new tube latex agglutination test for detection of type-specific pneumococcal antigens in urine
Authors: Rosario M , Capeding Z , Nohynek H , Ruutu P , Leinonen M
Published: January 2020

A modified tube agglutination test using type-specific latex reagents for detection of pneumococcal capsular polysaccharide antigens in alkalinized, unconcentrated urine samples was evaluated in reconstituted urine samples and in groups consisting of 26 children with clinical and roentgenographic evidence of acute lower respiratory tract infection, six patients with blood culture-proven infection of nonpneumococcal etiology, and 30 healthy individuals. The sensitivity of the tube latex agglutination method for pneumococcal polysaccharides was 2 to 10 times higher than that of the slide agglutination method. Positive antigen findings were obtained for 42% of urine samples from patients with acute lower respiratory tract infection but in neither patients with nonpneumococcal septicemia nor healthy controls. Fifty-five percent of the antigen-positive patients also showed evidence of pneumococcal involvement by pneumococcal antibody assay or antigen detection in acute-phase serum.
Immunodiagnosis of toxocarosis in humans: evaluation of a new enzyme-linked immunosorbent assay kit
Authors: Jacquier P , Gottstein B , Stingelin Y , Eckert J
Published: January 2020

Excretory/secretory (E/S) antigen derived from second-stage larvae of Toxocara canis maintained in defined medium in vitro has been well established worldwide for the immunodiagnosis of human toxocarosis by enzyme-linked immunosorbent assay. Such an enzyme-linked immunosorbent assay, based on the detection of human anti-T. canis (E/S antigen) serum immunoglobulin G, has recently been commercialized by Biokema-Affinity Products (Crissier-Lausanne, Switzerland). This commercial test kit was evaluated with regard to its application in a routine diagnostic laboratory and the reliability of the results. Of 78 patients with suspected clinical toxocarosis, 71 had anti-T. canis antibodies (positive serological result) corresponding to a diagnostic sensitivity of 91%; 14% of serum samples (n = 199) from patients with protozoan or with helminthic infections also showed positive reactions mainly related to infections with Trichinella, Strongyloides, and Fasciola species. An epidemiological study with 1,000 serum samples from randomly selected healthy blood donors and children in Switzerland demonstrated a seroprevalence of 2.7%. The test kit under evaluation had an overall diagnostic sensitivity of 91% and a relative specificity of 86%, the latter being related to some protozoan and helminthic infections. Because of the scarcity of such infections, potential cross-reactivity does not play a major role under the conditions found in the middle part of Europe. In conclusion, the application of the test kit provided for use in this study can be recommended for routine diagnostic use.
Effect of beta-estradiol on production of the cell-detaching factor of Trichomonas vaginalis
Authors: Garber G E , Lemchuk-Favel L T , Rousseau G
Published: January 2020

Despite over 40 years of study, the pathogenetic mechanisms of Trichomonas vaginalis are just starting to be elucidated. We have recently reported that T. vaginalis produces a virulence factor, cell-detaching factor (CDF), that likely causes the cell sloughing seen in clinical disease. This 200-kDa glycoprotein is acid and heat labile and correlates with clinical symptoms. We applied a McCoy cell culture system to study the effects of various concentrations of beta-estradiol (10(-6) to 10(-10) M) on T. vaginalis growth and CDF production. T. vaginalis growth was unaffected by the different concentrations of beta-estradiol studied, in comparison with the growth of control cultures without beta-estradiol. However, beta-estradiol significantly diminished the activity of CDF at all concentrations and did so most profoundly at 10(-7) and 10(-8) M (P less than 0.0001). This suggests that the symptoms of T. vaginalis infection may be influenced by the vaginal concentration of estrogens, and further studies of the interactions between T. vaginalis and estrogens are warranted.
Enzyme immunoassays for invasive Candida infections: reactivity of somatic antigens of Candida albicans
Authors: Zöller L , Krämer I , Kappe R , Sonntag H G
Published: January 2020

The main problem encountered with serodiagnostic tests for Candida infections is their failure to differentiate between invasive and superficial candidosis. Recent immunoblotting studies suggested that the use of selective somatic proteins of Candida albicans as antigens might be a promising approach toward developing a new generation of serodiagnostic assays. In this study major cytoplasmic protein antigens with molecular weights of 47,000 (47K), 46,000 (46K), 45,000 (45K), and 29,000 (29K) were identified as potential marker antigens for antibody detection in invasive candidosis. Continuous-flow isoelectric focusing was employed to enrich the proteins in two fractions, one of them containing the 47K and 29K proteins and the other one containing predominantly the 47K and 45K major proteins. These antigens and a whole somatic antigen extract were used to establish enzyme immunoassays (EIAs) for antibody detection. Whereas all tests were able to discriminate between patients with invasive candidosis (n = 27) and normal healthy volunteers (n = 167), as proved by graphic marker analysis, the selective antigen EIAs were highly superior to the whole somatic antigen EIA and two serological standard assays (indirect immunofluorescence assay and indirect hemagglutination assay) when a panel of sera from patients with superficial candidosis (n = 34) was used as a negative control group. The use of the 47K-29K antigen fraction allowed the best differentiation between invasive and noninvasive candidosis. The corresponding immunoglobulin G class-specific EIA had a sensitivity of 81.5% and a specificity of 97% for both negative control groups as well.
Human cytomegalovirus structural proteins: immune reaction against pp150 synthetic peptides
Authors: Landini M P , Ripalti A , Sra K , Pouletty P
Published: January 2020

In the present study, several peptides of the major structural antigen (pp150) of human cytomegalovirus (CMV) have been chemically synthesized and tested by a modified slot blotting procedure for their ability to bind CMV-specific immunoglobulin G (IgG) and IgM present in human sera. The sequences of the peptides were deduced on the basis of either (i) their presence in a fusion protein already known to be frequently recognized by human antibody or (ii) their high content of hydrophilic amino acids as deduced from the published nucleotide sequence. An important IgM-binding epitope was found to be located in the last 38 amino acids at the carboxy terminus of the molecule. This region reacts with anti-CMV IgM present in the great majority (83.3%) of IgM-positive human sera, and adsorption experiments have shown that IgM titers to the entire pp150 decrease 25 to 50% in most sera previously absorbed with this region. The overall results obtained endorse the continued synthesis of other sequences in order to define a group of peptides sensitive and specific enough to replace the virus and infected cells as an antigenic substrate in the serological evaluation of anti-CMV antibody.
Evaluation of commercial enzyme immunoassays for detection of hepatitis delta antigen and anti-hepatitis delta virus (HDV) and immunoglobulin M anti-HDV antibodies
Authors: Shattock A G , Morris M C
Published: January 2020

Panels of hepatitis B virus surface antigen-positive sera from drug abusers were used to evaluate 14 commercial enzyme immunoassays from six companies for detecting hepatitis delta virus (HDV) markers. For detecting hepatitis delta virus antigen (HDAg), the Wellcome, Pasteur and Noctech assays had 100% sensitivity for all 42 HDAg-positive serum specimens that were confirmed in-house; the Organon reagents gave 59.5% sensitivity without detergent and 64.3% sensitivity with detergent, but there were 14 discrepant results with and without detergent. The Sorin assay detected HDAg in only 10 of the positive samples (23.8% sensitivity). For the detection of antibody to HDV (anti-HDV) all six commercial enzyme immunoassays were reactive with all 36 anti-HDV-positive specimens that were confirmed in-house. There were no false-positive results with the Wellcome, Noctech, or Sorin assay, but one specimen was false positive by the Organon assay. One HDAg-positive specimen gave a false anti-HDV-positive result in the Abbott assay and an equivocal result in the Pasteur assay (97.8% specificity). For the detection of immunoglobulin M anti-HDV, the Wellcome, Noctech, and Sorin assays agreed for the 38 positives confirmed in-house, except for one false negative with the Sorin test. We conclude that there has been a substantial improvement over previously evaluated assays in sensitivity and specificity of commercial assays for anti-HDV detection, and the sensitivities of immunoglobulin M anti-HDV assays are also comparable. However, there are still major differences in sensitivity among some assays for HDAg detection.
Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina
Authors: Binsztein N , Jouve M J , Viboud G I , López Moral L , Rivas M , Orskov I , Ahrén C , Svennerholm A M
Published: January 2020

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.
Maternal immunity and antibody response of neonatal mice to pneumococcal type 19F polysaccharide
Authors: Lee C J , Ching E D , Vickers J H
Published: January 2020

The effect of immunization of mothers on the antibody response of their young to pneumococcal type 19F polysaccharide was studied. When 2-week-old BALB/c mice from mothers immunized with 23-valent pneumococcal vaccine during gestation were given an additional dose of the same vaccine, mouse pneumococcal antiserum, or both, they produced higher titers of antibodies to the 19F polysaccharide (1.87 to 4.66 micrograms of 19F immunoglobulin M [IgM] antibody per ml of serum; 0.45 to 0.81 micrograms of IgG antibody per ml of serum) than the control group that did not receive any treatment after birth (0.69 micrograms of 19F IgM antibody per ml; 0.28 micrograms of 19F IgG antibody per ml) (P less than 0.01). Furthermore, all 11- to 12-week-old monkeys that received an additional dose of 23-valent vaccine, pneumococcal immunoglobulin, or both produced statistically higher titers of IgG antibody to the 19F polysaccharide than did controls at various ages. The titers (micrograms of IgG antibody per milliliter of serum) were as follows: vaccine group, 7.12 +/- 0.96; control group at 4 months of age, 3.82 +/- 0.74 (P less than 0.01); immunoglobulin-treated group, 6.85 +/- 0.76; vaccinated and immunoglobulin-treated group, 7.80 +/- 1.40; control group at 3 months of age, 3.01 +/- 0.61 (P less than 0.01). These results suggest that immunization of mothers under certain conditions, such as with an optimum dose of antigen at a critical period of gestation or postnatal development, could provide young infants with an enhanced antibody response to pneumococcal polysaccharide immunogens.
Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization
Authors: Hurst J W , Forghani B , Chan C S , Oshiro L , Darai G
Published: January 2020

A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy.
Detection of Pneumocystis carinii sequences by polymerase chain reaction: animal models and clinical application to noninvasive specimens
Authors: Kitada K , Oka S , Kimura S , Shimada K , Serikawa T , Yamada J , Tsunoo H , Egawa K , Nakamura Y
Published: January 2020

Pneumocystis carinii is a eukaryotic microbe which causes fatal pneumonia in patients with AIDS. Oligonucleotide primers were used to amplify the 5S rDNA sequence of P. carinii by the polymerase chain reaction (PCR) in various clinical and animal samples. Of 35 independent lung specimens tested, PCR detected the P. carinii sequence in all 23 cases which were known to be P. carinii infected, i.e., 15 from mice, 1 from rat, 3 from human autopsy, and 4 from biopsy of AIDS patients by needle aspiration. The results were consistent with clinical and microscopic diagnosis. The detection was highly sensitive and specific. Direct sequencing of these amplified DNAs revealed homogeneity of 5S rDNA sequences of independent isolates from mice, rats, and humans. Preliminary trials manifested efficacy of the PCR method to detect P. carinii sequences in induced sputum or blood from AIDS patients, the latter case suggesting that P. carinii might enter peripheral blood via phagocytosis or direct intrusion. Development of less-invasive or noninvasive PCR diagnostic techniques to detect P. carinii infection would greatly facilitate therapeutic and prophylactic management of P. carinii pneumonia.
Pathogenic properties of Edwardsiella species
Authors: Janda J M , Abbott S L , Kroske-Bystrom S , Cheung W K , Powers C , Kokka R P , Tamura K
Published: January 2020

The pathogenic characteristics of 35 Edwardsiella strains from clinical and environmental sources were investigated. Overall, most Edwardsiella tarda strains were invasive in HEp-2 cell monolayers, produced a cell-associated hemolysin and siderophores, and bound Congo red; many strains also expressed mannose-resistant hemagglutination against guinea pig erythrocytes. Edwardsiella hoshinae strains bound Congo red and were variable in their invasive and hemolytic capabilities while Edwardsiella ictaluri strains did not produce either factor; neither E. hoshinae nor E. ictaluri expressed mannose-resistant hemagglutination nor elaborated siderophores under the tested conditions. Selected strains of each species tested for mouse lethality indicated strain variability in pathogenic potential, with E. tarda strains being the most virulent; 50% lethal doses in individual strains did not correlate with plasmid content, chemotactic motility, serum resistance, or expression of selected enzyme activities. The results suggest some potential important differences in pathogenic properties that may help explain their environmental distribution and ability to cause disease in humans.
Cross-reacting antigens between Neorickettsia helminthoeca and Ehrlichia species, shown by immunofluorescence and Western immunoblotting
Authors: Rikihisa Y
Published: January 2020

Dogs orally infected with Neorickettsia helminthoeca developed immunoglobulin G titers against Erlichia risticii, Erlichia sennetsu, and Erlichia canis similar to those against N. helminthoeca antigen, as determined by immunofluorescence. Western immunoblotting showed that the major common antigens shared among the microorganisms were 80- or 78-kDa and 64-kDa polypeptides. In contrast, horse anti-E. risticii and anti-E. sennetsu and dog anti-E. canis sera reacted more weakly to N. helminthoeca antigen than to homologous antigens in both immunofluorescence and Western immunoblotting. Antisera raised in other species of animals, i.e., mouse anti-E. canis and rabbit anti-E. risticii and anti-E. sennetsu sera, however, all reacted with the 64-kDa antigen of N. helminthoeca. This strong antigenic cross-reactivity and similarity in Western immunoblotting reaction profiles indicate that N. helminthoeca is antigenically closely related to E. risticii and E. sennetsu and less so to E. canis. In both immunofluorescence and Western immunoblotting, E. canis shared fewer common antigens with E. risticii and E. sennetsu than N. helminthoeca did. It is reasonable to conclude that these results may have both diagnostic and taxonomic significance.
Assay for detection of Trypanosoma cruzi antibodies in human sera based on reaction with synthetic peptides
Authors: Vergara U , Lorca M , Veloso C , Gonzalez A , Engstrom A , Aslund L , Pettersson U , Frasch A C
Published: January 2020

Synthetic peptides modelled according to the amino acid sequences derived from the repeated domains of five Trypanosoma cruzi antigens were used in an immunoradiometric assay to detect antibodies appearing after natural human infections. An enzyme-linked immunosorbent assay and an indirect immunofluorescence assay performed with a complex antigenic mixture from parasites were used as controls. The results indicate that the synthetic peptides were recognized by a large proportion of serum samples collected from 34 patients with Chagas' disease in Chile and point to their possible use in diagnosis.
Monoclonal antibodies to the fusion protein of bovine respiratory syncytial virus
Authors: Mulkey K M , Anderson G A
Published: January 2020

Five monoclonal antibodies specific for bovine respiratory syncytial virus were characterized by Western immunoblotting, radioimmunoprecipitation, and epitope mapping assays. The monoclonal antibodies were found to be specific for the fusion protein, and there were at least two antigen binding sites, one of which was neutralizing.
Rabbit Syncytium Virus Is a Kemerovo Serogroup Orbivirus
Authors: Theil Kenneth W. , McCloskey Christine M.
Published: January 2020

Polyacrylamide gel genome electropherotyping and negative-stain electron microscopic studies, along with immunofluorescent staining and immune electron microscopy reactions, indicate that rabbit syncytium virus has the morphologic, genomic, and antigenic attributes of a Kemerovo serogroup orbivirus.
Abscess caused by vancomycin-resistant Lactobacillus confusus
Authors: Bantar C E , Relloso S , Castell F R , Smayevsky J , Bianchini H M
Published: January 2020

Several isolates of vancomycin-resistant Lactobacillus confusus from human sources have been described, but to our knowledge, no well-documented infection attributable to this organism has been published. A thumb abscess caused by this bacterium in a healthy 49-year-old male is reported here. He was successfully treated by surgical drainage and cephalothin.
Comparison of antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in sera from HIV-1-infected individuals from Tanzania and from the United States
Authors: Warren R Q , Nkya W M , Shao J F , Anderson S A , Wolf H , Hendrix C W , Kanda P , Wabuke M , Boswell R N , Redfield R R
Published: January 2020

In this study, we compared sera from 159 human immunodeficiency virus type 1 (HIV-1)-infected individuals from Tanzania and 103 infected individuals from the United States for antibodies reactive with 10 HIV-1 gp160 epitopes defined by synthetic peptides. Our data indicate that the anti-gp160 antibody fine specificity differs between infected individuals from these two geographically diverse populations. For example, 50% of the Tanzanian sera contained antibodies reactive with an immunodominant HIV-1 gp41 epitope defined by peptide 600-611, whereas 91% of the sera from the United States were reactive. Differences in serologic reactivity between HIV-1-infected individuals from Tanzania and the United States were also observed with gp160 epitopes defined by peptides 503-528 and 846-860. Included among the peptides examined were four which corresponded to the V3 region of gp120. The majority of sera from either country contained antibodies reactive with peptide RP142, whose V3 sequence is based upon that of HIV-1 isolate MN. Further characterization of serologic reactivity suggested that sera from Tanzania were more likely to neutralize HIV-1 isolate IIIB or MN in vitro than were sera from the United States. These differences in antibody fine specificity between HIV-1-infected individuals from Tanzania and the United States suggest that regional isolates of HIV-1 may exist.
Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection
Authors: Rikihisa Y , Ewing S A , Fox J C , Siregar A G , Pasaribu F H , Malole M B
Published: January 2020

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.
Serotype prevalence of Candida albicans from blood culture isolates
Authors: Brawner D L , Anderson G L , Yuen K Y
Published: January 2020

Blood culture isolates of Candida albicans were collected from 102 patients in Seattle, Wash., hospitals (n = 77) and Hong Kong (n = 25). The patients were classified by immune status into two groups. Group I patients were severely immunosppression, and group II patients had underlying risk factors for candidemia but no underlying immunosuppression. Serotyping by Hasenclever tube agglutination was done. In the Seattle area, the odds of fungemia with type B C. albicans were 3.62 times greater than the odds of type B fungemia in group II patients. Although the odds ratio could not be computed for Hong Kong patients, the direction of the relationship in this population was consistent with the data on Seattle patients. Despite the magnitude of the odds ratios, the relative prevalence of type B over type A in group I compared with group II was not significant when analyzed separately by region, probably because of relatively low numbers of isolates in group II. Accepting that the effect of immune status on serotype is equivalent across regions but presupposing that a regional effect on type B prevalence exists, the pooled odds for fungemia with serotype B in group I patients are increased 5.4-fold over those of group II patients. Logistic regression analysis controlling for region gave similar results.
General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections
Authors: Zoll G J , Melchers W J , Kopecka H , Jambroes G , van der Poel H J , Galama J M
Published: January 2020

The aim of this study was to determine the applicability of the polymerase chain reaction (PCR) for routine diagnostic use and for the detection of persistent enteroviral infections. To this end, general primers were selected in the highly conserved part of the 5'-noncoding region of the enteroviral genome. They were tested on 66 different enterovirus serotypes. A specific fragment was amplified from 60 of 66 serotypes. An amplification product was not observed from coxsackievirus types A11, A17, and A24 and echovirus types 16, 22, and 23. Enteroviral RNA was detected by the PCR in routinely collected throat swabs and stool specimens that were found to be positive for enterovirus by isolation in tissue culture. Enteroviral RNA was detected in one of five myocardial biopsy specimens from patients with dilated cardiomyopathy, implicating virus persistence. No amplification product was obtained from eight control samples. Our results demonstrate the significance of the PCR for the detection of enteroviral RNA and, in particular, for the demonstration of persistent enteroviral infections.
Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows
Authors: Sweeney R W , Whitlock R H , Rosenberger A E
Published: January 2020

Milk and supramammary lymph node samples were obtained from asymptomatic cows infected with Mycobacterium paratuberculosis at the time of slaughter. Of 81 supramammary lymph node samples, 22 (27%) were culture positive for M. paratuberculosis. Of 77 milk samples, 9 (11.6%) were culture positive. The prevalence of supramammary lymph node or milk infection was highest with heavy fecal shedding of M. paratuberculosis and lowest with light shedding. The serologic status of the cow was not useful for predicting the risk of supramammary lymph node or milk infection. Shedding of M. paratuberculosis occurs in the milk of asymptomatic infected cows but, apparently, less frequently than previously reported for symptomatic cows.
Comparison of the E test and microdilution for detection of beta-lactam-resistant mutants that are stably derepressed for type I beta-lactamase
Authors: Knapp C C , Washington J A
Published: January 2020

The activities of cefotaxime, ceftazidime, piperacillin, and aztreonam were compared in the E test and broth microdilution test against 30 gram-negative bacterial mutants derepressed for type I beta-lactamases. The results demonstrated complete agreement between 24-h MICs of 80 to 83% and essential agreement between 24-h MICs of 90 to 97%. When sufficient growth was present for the E test to be read at 6 h, the essential agreement between 6- and 24-h E-test MICs was 100% for ceftazidime, piperacillin, and aztreonam and 85% for cefotaxime.
Occurrence of resistance to vibriostatic compound 0/129 in Vibrio cholerae 01 isolated from clinical and environmental samples in Bangladesh
Authors: Huq A , Alam M , Parveen S , Colwell R R
Published: January 2020

Fifty-one Vibrio cholerae 01 strains isolated from 734 natural water and plankton samples and 31 rectal swabs were examined. Of these strains, 32 (62.7%) were found to be resistant to vibriostatic compound 0/129. When antibiograms using the antibiotics ampicillin, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, furoxan, and gentamicin were done, it was observed that there was a correlation of sensitivity to 0/129 with selected antibiotics. Only the Ogawa E1 Tor (72% of strains resistant) and Inaba classical (28% of strains resistant) biotypes of V. cholerae 01 showed resistance to 0/129. On the other hand, all Inaba E1 Tor and Ogawa classical strains were susceptible to 0/129. The 32 0/129-resistant and 19 0/129-sensitive isolates of V. cholerae 01 were tested for the presence of plasmid DNA. Only two strains isolated from the environment were found to carry a plasmid, and they were also found to be resistant to 0/129 and gentamicin. Thus, 0/129 resistance, although more common than previously suspected, is concluded not to be plasmid mediated in the strains tested in this study.
Clinical demonstration of isolation of Nocardia asteroides on buffered charcoal-yeast extract media
Authors: Vickers R M , Rihs J D , Yu V L
Published: January 2020

Nocardia asteroides was isolated only from sputum samples, obtained from three patients with pulmonary nocardiosis, that had been cultured onto buffered charcoal-yeast extract (BCYE) and selective BCYE media as part of laboratory workups for Legionella species. A decontamination procedure with low-pH pretreatment (KCl-HCl solution) had been performed on the sputa prior to culture onto the BCYE media because direct cultures on the media were overgrown with commensal microflora. Chalky white colonies, 0.5 to 1.0 mm in diameter, that were subsequently identified as N. asteroides grew well on the BCYE media. Thus, the techniques and the selective media used for Legionella species were useful for isolating Nocardia species from sputum.
Ovalbumin blocking improves sensitivity and specificity of immunoglobulin M immunoblotting for serodiagnosis of patients with erythema migrans
Authors: Lange R , Bocklage H , Schneider T , Kölmel H W , Heesemann J , Karch H
Published: January 2020

To improve the serodiagnosis of erythema migrans, we evaluated how sensitivity and specificity of immunoblotting are influenced by antigen concentration and blocking conditions. We found that an antigen concentration of 0.5 micrograms per lane in concert with ovalbumin blocking of the nitrocellulose provided the best results. In this case, 81% of the erythema migrans had positive immunoglobulin M tests, whereas only 33% were positive in a flagellum enzyme-linked immunosorbent assay (ELISA) and 28% were positive in a sonicate ELISA.
Amplification of human papillomavirus type 16 transforming genes from cervical cancer biopsies and lymph nodes of Hungarian patients
Authors: Czeglédy J , Póka R , Veress G , Gergely L
Published: January 2020

We have used a polymerase chain reaction (PCR) to examine cervical cancer biopsy specimens and pelvic lymph nodes for the presence of human papillomavirus type 16 (HPV 16) DNA. Of the 75 cervical specimens tested, 36 (48%) were positive for HPV 16 in the PCR. A total of 65 pelvic lymph nodes removed during radical surgery on 35 women were also analyzed. Lymph nodes originating from 19 patients whose cervical biopsy specimens were negative for HPV 16 seemed to lack HPV 16 sequences. For 16 women with positive PCR results for cervical biopsy specimens, 9 of 10 lymph node metastases were positive in the PCR, while 11 of their 36 histologically negative lymph nodes were also shown to contain HPV 16 DNA.
Isolation of Yersinia enterocolitica serovar O8 from free-living small rodents in Japan
Authors: Iinuma Y , Hayashidani H , Kaneko K , Ogawa M , Hamasaki S
Published: January 2020

Yersinia species were isolated from 65 of 223 free-living small mammals trapped in 10 regions on Honshu Island in Japan. Of the 65 strains isolated, 1 was Yersinia enterocolitica serovar O3, 8 were Y. enterocolitica O5, 6 were Y. enterocolitica O8, 3 were Y. enterocolitica O9, and 1 was Yersinia pseudotuberculosis 4b. Of the six Y. enterocolitica O8 strains, five were positive for autoagglutination, Ca2+ dependence, and the 45-MDa virulence plasmid and showed high pathogenicity for mice.