bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance of leishmanial phosphatases to inactivation by oxygen metabolites
Authors: Saha A K , Das S , Glew R H , Gottlieb M
Published: January 2020

Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages.
API ZYM and API An-Ident reactions of fastidious oral gram-negative species
Authors: Tanner A C , Strzempko M N , Belsky C A , McKinley G A
Published: January 2020

API ZYM and API An-Ident enzymatic substrate tests were done on six oral species which are difficult to characterize with conventional biochemical tests. "Bacteroides forsythus, the "fusiform" Bacteroides species (A. C. R. Tanner, M. A. Listgarten, M. N. Strzempko, and J. L. Ebersole, manuscript in preparation), is difficult to cultivate in broth media, yet it gave 15 positive tests in these series. The tests were able to separate this new species from species of Capnocytophaga and Fusobacterium. "B. forsythus" reactions were similar but not identical to those of reference Bacteroides species. Positive reactions for alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and alpha-glucuronidase suggest that "B. forsythus" may be saccharolytic. It was the only species tested which was trypsin positive. Wolinella species, Campylobacter concisus, B. gracilis, and Eikenella corrodens are asaccharolytic, and characterization relies heavily on sensitivities to inhibitory agents. These species reacted weakly in the API ZYM and API An-Ident enzymatic substrate tests, and the reactions were not useful for separating these species. The enzyme reactions differentiated Wolinella recta and C. concisus from Selenomonas sputigena, another oral motile but saccharolytic organism.
Plasmid characterization of Salmonella typhimurium transmitted from animals to humans
Authors: Olsvik O , Sørum H , Birkness K , Wachsmuth K , Fjølstad M , Lassen J , Fossum K , Feeley J C
Published: January 2020

The transmission of pathogenic bacteria from animals to humans is widely studied because of its public health importance. In this study, we show the transmission of Salmonella typhimurium from cattle which had received no growth-promoting antibiotics to humans who had direct contact with the ill animals. On one cattle farm, the veterinarian attending the sick animals became ill, and two other individuals living on the farm later developed salmonellosis. The strains isolated from both humans and animals at one farm were identical as to antibiotic susceptibility and phage type, and they were specifically traced by the presence of a common 24-megadalton plasmid. Restriction enzyme digests of this plasmid from both human and animal strains were identical. At another farm, tetracycline-resistant S. typhimurium strains possessing a different profile (eight plasmids) were isolated from both animals and humans. The tetracycline-resistant clone was also isolated from animals at a third farm, but with animals and humans having no known contact with those of the other two farms.
Production of potent Salmonella H antisera by immunization with polymeric flagellins
Authors: Ibrahim G F , Fleet G H , Lyons M J , Walker R A
Published: January 2020

Highly purified polymeric flagellin preparations from 10 different Salmonella serotypes were used to produce specific Salmonella H antisera with high titers by the immunization of rabbits. Antigen emulsions in complete Freund adjuvant were administered at the rate of 50 micrograms per rabbit by multiple intradermal injection. Booster injections were given 110 days after the primary immunization. The immune response was monitored regularly over a period of 200 days. The results showed that the H titers, determined with 125I-labeled antigens, averaged 61,000 +/- 39,000, and the H agglutination titers of 83% of the animals were greater than 40,000. The high titers of the immunized animals persisted for approximately 4 months. The O agglutination titers of the antisera were less than 10 for 9 of the flagellin preparations and ranged from 10 to 320 for the remaining preparation. The antisera obtained were serotype specific after appropriate dilution.
Accuracy and reproducibility of the AutoMicrobic System Gram-Negative General Susceptibility-Plus Card for testing selected challenge organisms
Authors: Nadler H L , Dolan C , Mele L , Kurtz S R
Published: January 2020

A total of 180 selected strains of gram-negative bacilli were tested for susceptibility to nine antibiotics by the prototype General Susceptibility Card and reference broth microdilution MIC method, and 112 of 180 were tested by the modified Gram-Negative General Susceptibility-Plus Card (Vitek Systems, Inc., Hazelwood, Mo.). When category calls--susceptible, intermediate, and resistant for the prototype card and very susceptible, moderately susceptible, and resistant for the modified card--were identical or compatible (very susceptible by one method and moderately susceptible by the other), the methods were considered in qualitative or categorical agreement. The overall categorical agreement improved from 83% for the prototype card to 91.5% for the modified card, and the frequency of major and very major disagreements was reduced from 7.5 to 1%, respectively. Overall agreement between the modified card and reference method for both category calls and MICs (+/- 2 dilutions) was 90%. Of the results, 67% were identical, 22% were more susceptible with the modified card, and 11% were more resistant. Reproducibility of identical or compatible category calls and MICs obtained from three trials was 94.5%. The poorest accuracy and reproducibility were observed when testing Pseudomonas aeruginosa. The study suggests that the modified card can rapidly and efficiently perform susceptibility tests with an acceptable level of accuracy and reproducibility provided that the system is appropriately used for testing strains of the family Enterobacteriaceae with preset criteria for excluding organism-antibiotic combinations. The evaluation also indicates dramatic improvement in the technical performance of the modified card compared with its earlier prototype.
False susceptibility of enterococci to aminoglycosides with blood-enriched Mueller-Hinton agar for disk susceptibility testing
Authors: Jenkins R D , Stevens S L , Craythorn J M , Thomas T W , Guinan M E , Matsen J M
Published: January 2020

Disk diffusion susceptibility tests for enterococci are frequently modified by adding 5% sheep blood (SB) to Mueller-Hinton agar; the performance standards from the National Committee for Clinical Laboratory Standards sanction this addition. Susceptibility testing of aminoglycoside antibiotics is not recommended for enterococci; in actual practice, however, some laboratories do include aminoglycoside antibiotics routinely, and others may test upon request or in selected situations. In examining 50 clinical isolates of enterococci, SB-enriched Mueller-Hinton agar frequently gave enlarged zone sizes that falsely indicated susceptibility (72% for gentamicin and tobramycin), with the average increase in zone size being 6.3 and 7.6 mm, respectively. Comparison agar dilution MICs demonstrated uniform resistance, with or without added SB. The effect was shown to be caused by heme in concentrations as low as 0.03 micrograms/ml, which, when combined with aminoglycoside antibiotics, caused a synergistic growth inhibition of the enterococci, resulting in larger aminoglycoside antibiotic zones. We postulate that the heme effect is related to a catalytic cleavage of intracellular H2O2 and resultant lipid peroxidation. No other organism or antimicrobial agent tested demonstrated a similar effect, although other investigators have shown a similar phenomenon with the broad-spectrum cephalosporins. Because enterococci grow well and give accurate susceptibility results on Mueller-Hinton agar without SB supplementation and because of the spectrum of definable problems with a number of antimicrobial agents, we recommend that enterococci routinely be tested without SB.
Isolation of Capnocytophaga species with a new selective medium
Authors: Rummens J L , Fossepre J M , De Gruyter M , Van de Vyver H , Neyt L , Van Landuyt H W
Published: January 2020

A selective medium (CAP) composed of a GC agar base supplemented with 1% hemoglobin, 1% Polyvitex, and an antibiotic mixture of polymyxin B (15 U/ml), vancomycin (5 micrograms/ml), trimethoprim (2.5 micrograms/ml), and amphotericin B (2.5 micrograms/ml) was compared with another selective medium (TBBP) and two nonselective media--a blood agar and a chocolate agar--to isolate Capnocytophaga species from 725 clinical specimens. These included sputa (467 specimens), throat swabs (116 specimens), oral ulcerations (35 specimens), and periodontal pockets (107 specimens). The recovery rate of Capnocytophaga species was significantly higher with the CAP medium (96%) than with the selective TBBP medium (52.2%), the nonselective blood agar (6.2%), and the chocolate agar (4.6%). Growth of the normal flora was best inhibited on CAP medium. Colony size and yellow-brown pigment formation were maximally expressed on chocolate agar and CAP medium, but gliding motility was mostly absent. We conclude that the CAP medium is an excellent medium for the recovery of Capnocytophaga species from contaminated clinical specimens.
Enzyme-linked immunosorbent assays for detection of thermostable direct hemolysin of Vibrio parahaemolyticus
Authors: Honda T , Yoh M , Kongmuang U , Miwatani T
Published: January 2020

Several systems for enzyme-linked immunosorbent assay (ELISA) of thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus were tested, and single-antibody sandwich ELISA systems gave satisfactory results. ELISA was able to detect as little as several nanograms of purified TDH per milliliter. The method of De Jong (J. Clin. Microbiol. 17:928-930, 1983) and the glutaraldehyde method were successful for preparing conjugates of alkaline phosphatase and anti-TDH antibody. TDH in fluids in intestinal loops of experimental animals challenged with living V. parahaemolyticus was accurately detectable by ELISA.
Evaluation of antibiotic susceptibility testing by agar dilution and the Micro Media system (Fox Panel)
Authors: Welch W D , Layman M A , Chiles L , Ragsdale R , Kutscher E , Southern P M
Published: January 2020

The susceptibilities of 350 gram-positive cocci and 638 gram-negative bacilli to various antimicrobial agents were compared by using the Micro-Media system (MMS) (Fox Panel) (Micro-Media Systems, Inc., Potomac, Md.) and a standard agar dilution procedure. Major discrepancies occurred with enterococci, among which 48 of 53 isolates (91%) were found to be resistant to penicillin G by agar dilution and reported as susceptible by the MMS. Other large discrepancies occurred with Staphylococcus aureus and Acinetobacter calcoaceticus subsp. anitratus, among which more than 40% of the isolates were judged to be resistant to ampicillin by agar dilution and susceptible by the MMS. In terms of overall agreement in interpretation of MICs by the two systems, an agreement of greater than 84% was seen for both gram-positive and gram-negative organisms when ampicillin and cephalothin (68 and 78% agreement for gram-positive cocci, respectively) were excluded. These disagreements in MIC interpretations may result in part from the small number of organisms tested per well (4,000 CFU) in the MMS, as compared with 10,000 CFU per test in the agar dilution method.
New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor
Authors: Iwanaga M , Yamamoto K
Published: January 2020

A new medium that stimulates in vitro production of cholera toxin by Vibrio cholerae O1 El Tor (El Tor vibrios) was developed. The medium contains 0.5% NaCl, 0.3% NaHCO3, 0.4% yeast extract, and 1.5% Bacto-Peptone. El Tor vibrios were cultured in a stationary test tube at 37 degrees C for 20 h. The culture supernatant was assayed for cholera toxin by a reversed passive latex agglutination method. Most vibrios grown in this medium produced 10 to 20 times more toxin than in traditional syncase medium. The number of live vibrios at the end of culture was about 10(8)/ml in the new medium (AKI medium) and about 10(10)/ml in the syncase medium. As a result, each individual organism in the new medium should have produced as much as 1,000 times more toxin than in syncase medium. Sodium bicarbonate was found to be the most important factor in toxin production by El Tor vibrios in the new medium. We recommend this new medium because of its high yield of cholera toxin and its technical simplicity.
Clinical utility of a monoclonal direct fluorescent reagent specific for Legionella pneumophila: comparative study with other reagents
Authors: Edelstein P H , Beer K B , Sturge J C , Watson A J , Goldstein L C
Published: January 2020

Twenty-four lower respiratory tract samples taken from patients with culture-confirmed Legionella pneumophila infection were examined with three different direct immunofluorescent antisera to L. pneumophila, as were 29 samples from similar sources taken from patients without Legionnaires disease. The reagents studied were Genetic Systems Corp. (GS) monoclonal L. pneumophila conjugate, which reacts with all known serogroups of L. pneumophila, BioDx polyvalent L. pneumophila serogroups 1 through 6 conjugate, and Centers for Disease Control polyvalent pool A L. pneumophila serogroups 1 through 4 conjugate. The specimens had been frozen at -70 degrees C for 0.5 to 5 years. Randomization was used in coding the samples, which were stained and read by an independent observer. All three conjugates correctly identified all positive and negative samples. No difference was noted among the conjugates in the absolute numbers of fluorescent L. pneumophila bacteria per sample. The GS conjugate had a much cleaner background than did the other two reagents. Mean staining intensity scores were 3.4, 3.9, and 3.7 for the GS, BioDx, and Centers for Disease Control conjugates, respectively. This study demonstrates that the diagnostic efficiency of all three conjugates is equivalent. Since the GS conjugate is easier to read, does not cross-react with non-L. pneumophila bacteria, and reacts with serogroups 1 through 10 of L. pneumophila, it appears to be preferable for use in diagnostic testing on nonhistopathologically processed specimens.
Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture
Authors: Echevarria J M , de Ory F , Najera R
Published: January 2020

The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella.
Lack of constitutive beta-glucosidase (esculinase) in the genus Fusobacterium
Authors: Edberg S C , Bell S R
Published: January 2020

Esculin has been incorporated into both a medium and test with 20% bile for many years to differentiate Bacteroides from Fusobacterium organisms. After 24 to 48 h, all members of the Bacteroides fragilis group grow in 20% bile and hydrolyze esculin. Fusobacterium mortiferum can both grow in bile and hydrolyze esculin, thus limiting the use of the bile-esculin medium and test. The hypothesis that constitutive esculinase (beta-glucosidase) could differentiate Bacteroides from Fusobacterium organisms was investigated. Clinical isolates and American Type Culture Collection clones of the B. fragilis group and other species of Bacteroides and Fusobacterium were tested. All B. fragilis were positive within 30 min. In no case was a Fusobacterium organism positive for constitutive enzyme in a hydrolyzable substrate-based test. The percentage of positive results for other species of Bacteroides agreed with those published in the literature for the esculin test. The genus Fusobacterium can be separated from Bacteroides organisms based on a lack of constitutive beta-glucosidase in the former in a 30-min one-tube test.
Longitudinal study of immune response in human Chagas' disease
Authors: Mosca W , Plaja J , Hubsch R , Cedillos R
Published: January 2020

Immune response, clinical status, and reactivity to heart tissue were studied longitudinally for 1 year in 42 patients with Chagas' disease (South American trypanosomiasis). The patients were divided into two groups. Group 1 was composed of patients with chagasic infection with no evidence of heart disease. Group 2 patients had chagasic infection and cardiomyopathy. Humoral immune response to Trypanosoma cruzi was measured serologically, and cell-mediated immune responses to T. cruzi and rat heart antigens were evaluated by lymphoblastogenesis. Parasitemia was detected by xenodiagnosis. Serological tests for anti-T. cruzi antibodies were positive in all patients of both groups, and the titers were significantly higher in group 2. A change of titer during the study period was more frequently associated with a positive xenodiagnosis in both groups. Lymphoblastogenesis in response to T. cruzi antigen was positive at least once in all patients of both groups. When rat heart antigen was used, 44.4% of the patients in group 1 and 40.0% of those in group 2 were positive on at least one occasion. Xenodiagnosis revealed that 20% of the patients in group 1 and 50% of those in group 2 (P = 0.01) had detectable circulating parasites during the course of the study. Positive xenodiagnosis was associated with lower lymphoblastogenic responses to T. cruzi in group 1 patients, suggesting the presence of a regulatory or modulatory mechanism which is lost in patients with chagasic cardiomyopathy. No relationship between positive xenodiagnosis and positive lymphoblastogenesis in response to heart antigen could be established. In addition, no correlation was found between clinical heart disease and reactivity to rat heart tissue.
Evaluation of Abbott Quantum II yeast identification system
Authors: Salkin I F , Schadow K H , Bankaitis L A , McGinnis M R , Kemna M E
Published: January 2020

The identity of each of 239 yeasts, encompassing 9 genera and 30 species, was determined with the Quantum II and API 20C identification systems. With API 20C results accepted as being correct, Quantum II proved to be 92% accurate in identification of common isolates, e.g., Candida albicans and Torulopsis glabrata, but only 73% effective with less frequently encountered yeasts, e.g., Trichosporon beigelii and Rhodotorula glutinis. Overall, Quantum II was 86% as accurate as API 20C for the yeast isolates tested. In addition, physical problems were encountered in inoculation of Quantum II cartridges and in the automated reading of some biochemical tests. We conclude that Quantum II is not yet suitable for routine identification of clinical yeast isolates.
Enteritis caused by Pasteurella pneumotropica infection in hamsters
Authors: Lesher R J , Jeszenka E V , Swan M E
Published: January 2020

Pasteurella pneumotropica was isolated in essentially pure cultures from the bowels of hamsters with enteritis 7 days after parturition. Newly received hamsters showed presence of P. pneumotropica in their nasal cavities but not in their uteri, lungs, spleens, or bowels.
Misidentification of mucoid variants of Staphylococcus aureus by standard laboratory techniques
Authors: Notarnicola S M , Zamarchi G R , Onderdonk A B
Published: January 2020

Ten atypical strains of Staphylococcus aureus were examined and compared with previously characterized S. aureus strains. Five of the strains were sent to two hospital microbiology laboratories for identification. All of the mucoid strains were misidentified when tested by standardized coagulase, DNase, and mannitol fermentation criteria, as well as by an automated identification method, but were accurately identified by the API system (Analytab Products, Plainview, N.Y.). These results illustrate the possibility of misidentification when reliance is placed on a few key characteristics rather than a thorough characterization of unusual Staphylococcus isolates.
In vitro susceptibility of Pseudomonas species to carbenicillin and trimethoprim-sulfamethoxazole
Authors: Hill S F , Haldane D J , Ngui-Yen J H , Smith J A
Published: January 2020

We compared susceptibility tests of 47 Pseudomonas aeruginosa isolates and 40 Pseudomonas species to carbenicillin and trimethoprim-sulfamethoxazole by the MS-2 and Sceptor systems and agar dilution. The major and very major errors encountered in these tests in the MS-2 and Sceptor systems raise doubts about the accuracy of these methods for testing P. aeruginosa and confirm that they should not be used for testing the susceptibility of Pseudomonas species to the two drugs tested.
Use of the Autobac system for detection of methicillin-resistant Staphylococcus aureus
Authors: de Rautlin de la Roy Y , Chevalier-Burbaud E , Pannetier S , Souchaud F
Published: January 2020

Using 490 strains of Staphylococcus aureus divided into methicillin-susceptible, -resistant, and -heteroresistant varieties, we compared the results obtained by the agar disk method with those obtained with the automated Autobac system. Susceptible strains exhibited a perfect correlation, whereas there were numerous discrepancies with resistant and still more with heteroresistant varieties. When incubation was increased to 18 h at 37 degrees C (Autobac incubation temperature), 35 degrees C, or 30 degrees C, these differences disappeared, but other problems may arise when incubation is prolonged, especially with erythromycin. We thus recommend carrying out two readings, a normal one after 3 h of incubation and a special reading after 18 h, solely for the detection of heteroresistance to methicillin.
Growth of Aeromonas spp. on cefsulodin-Irgasan-novobiocin agar selective for Yersinia enterocolitica
Authors: Altorfer R , Altwegg M , Zollinger-Iten J , von Graevenitz A
Published: January 2020

Twenty-eight strains of Aeromonas spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan (triclosan; Ciba-Geigy AG, Basel, Switzerland)-novobiocin (CIN) agar containing 15 or 4 mg of cefsulodin per ml and on inositol-bile salts-brilliant green (IBB) agar. Relative to blood agar, 68% of the strains were inhibited by more than 2 logs (i.e., less than 1% growth) at 37 degrees C (39% at 25 degrees C) on CIN I (high cefsulodin concentration), 7% were inhibited at either temperature on CIN II (low cefsulodin concentration), 4% were inhibited on IBB agar at 37 degrees C, and none were inhibited on IBB agar at 25 degrees C. These results reflect the MICs of cefsulodin on CIN Base: the MIC for 50% of the strains was 8 mg/liter at 37 and 25 degrees C, and the MICs for 90% of the strains were 16 mg/liter at 37 degrees C and 64 mg/liter at 25 degrees C. The MICs of Irgasan and novobiocin were far beyond the concentrations used in CIN media. We argue that CIN agar containing 4 mg of cefsulodin per ml (CIN II) can be used for the simultaneous detection of Aeromonas spp. and Yersinia spp.
Second serogroup of Legionella hackeliae isolated from a patient with pneumonia
Authors: Wilkinson H W , Thacker W L , Steigerwalt A G , Brenner D J , Ampel N M , Wing E J
Published: January 2020

A Legionella-like organism (strain 798-PA-H, ATCC 35999) isolated from a lung aspirate was shown by DNA hybridization to belong to the species Legionella hackeliae. Growth, gas-liquid chromatographic, and physiologic characteristics were consistent with those of the type strain of L. hackeliae, Lansing 2 (ATCC 35250). However, 798-PA-H was minimally reactive with L. hackeliae direct immunofluorescence assay conjugate or slide agglutination test antiserum. Cross-absorption studies with 798-PA-H and Lansing 2 antisera supported placing 798-PA-H in a second serogroup of L. hackeliae.
Typing of Pseudomonas cepacia by bacteriocin susceptibility and production
Authors: Govan J R , Harris G
Published: January 2020

The significance of Pseudomonas cepacia as an opportunistic pathogen in immunocompromised patients has become increasingly recognized. Particularly disturbing is its increased incidence, reported by several North American centers, in respiratory tract cultures from patients with cystic fibrosis. Epidemiological studies of P. cepacia have been hampered by a lack of typing methods. In this paper we report the development of a typing scheme based on bacteriocin production and susceptibility. For bacteriocin production, test isolates of P. cepacia were rapidly applied to the surfaces of agar plates with a multiple inoculator. After incubation of these test isolates for 5.5 h and their exposure to chloroform, indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, inhibition zones caused by bacteriocin activity were recognized. A similar procedure was used to examine the bacteriocin susceptibility of the test strain. The bacteriocin type of the test strain was defined based on its bacteriocin production as judged by zones of inhibition against a set of eight indicator strains and by susceptibility or resistance of the test strain to bacteriocin produced by six producer strains. Of 373 strains of P. cepacia, 95.2% were typed into a total of 44 type combinations. Bacteriocin typing provided a suitable procedure for epidemiological studies of colonization or infection by P. cepacia. The technique described in this paper was simple to perform, gave a result within 24 h, provided good strain discrimination, and was suitable for clinical, environmental, and phytopathogenic strains.
Comparison and diagnostic value of the coccidioidin heat-stable (HS and tube precipitin) antigens in immunodiffusion
Authors: Kaufman L , Standard P G , Huppert M , Pappagianis D
Published: January 2020

Coccidioides immitis produces two unrelated heat-stable antigens which are diagnostically useful in immunodiffusion tests. One, the tube precipitin antigen, is valuable for specifically detecting antibody and diagnosing early primary cases of coccidioidomycosis. The other heat-stable antigen, designated HS, is the most useful coccidioidin antigen for specifically immunoidentifying C. immitis cultures. Both of the antigens were compared and evaluated for their usefulness in exoantigen and serologic immunodiffusion tests. Our studies indicated that the detection of tube precipitin antigen is of limited value for immunoidentifying C. immitis isolates because the antigen is common to certain gymnoascaceous saprophytes, such as Arachniotus, Auxarthron, and Malbranchea species, that form alternate arthroconidia. Antibodies to HS antigens are infrequently found in human sera from patients with coccidioidomycosis and are thus of little serodiagnostic value.
Prevalence of antibody to human calicivirus in Japan and Southeast Asia determined by radioimmunoassay
Authors: Nakata S , Chiba S , Terashima H , Nakao T
Published: January 2020

Three hundred ninety single sera from adults in Japan, Indonesia, Singapore, and Papua New Guinea were tested for antibodies to human calicivirus (HCV) by a radioimmunoassay blocking test. A high prevalence of antibodies was observed in samples collected in Japan and Southeast Asia. Of 240 serum specimens collected from five districts in Japan, 209 (87.1%) were positive for HCV antibodies. No striking difference in the prevalence of the antibody was seen among those districts. In sera collected in Southeast Asia, 84.0% (126 of 150) had antibodies to HCV (70% [35 of 50] in Singapore, 88% [44 of 50] in Indonesia, and 94% [47 of 50] in Papua New Guinea). These results indicate that HCV is a common infectious agent in Japan and Southeast Asia.
Production of monoclonal antibodies against parainfluenza 3 virus and their use in diagnosis by immunofluorescence
Authors: Waner J L , Whitehurst N J , Downs T , Graves D G
Published: January 2020

Monoclonal antibodies were produced against parainfluenza virus type 3 (PI-3) and used to identify PI-3 clinical isolates in cell culture and PI-3 antigen in cells obtained from nasopharyngeal (NP) washes of patients. Two (2E9 and 4G5) of the three monoclonal antibodies characterized reacted by immunoblotting with a 67,000-dalton PI-3 protein, and one antibody (4E5) reacted with two viral proteins in the range of 29,000 to 31,000 daltons. The three monoclonal antibodies did not cross-react by indirect immunofluorescence (IFA) with PI-1 or PI-2 and identified by IFA 18 isolates of PI-3 in cell culture. The 2E9 antibody reacted with PI-3 antigen in cells of 8 NP wash specimens that also yielded PI-3 in cell culture. Cells from 12 specimens reactive by IFA for respiratory syncytial virus, 1 specimen yielding adenovirus in cell culture, and 5 specimens yielding influenza virus were not reactive.
Enzyme-linked immunosorbent assay for determining specific immunoglobulin M in infections caused by Leptospira interrogans serovar hardjo
Authors: Milner A R , Jackson K B , Woodruff K , Smart I J
Published: January 2020

An automated enzyme-linked immunosorbent assay detecting specific immunoglobulin M in infections with Leptospira interrogans serovar hardjo was evaluated on 69 patients. The test was sensitive and simple to perform, requiring a single dilution of test serum, with data expressed as units of antibody activity interpolated from a reference serum pool.
Isolation of non-O1 Vibrio cholerae associated with enteric disease of herbivores in western Colorado
Authors: Rhodes J B , Schweitzer D , Ogg J E
Published: January 2020

Non-O1 Vibrio cholerae was isolated from a horse (Equus caballus), a lamb (genus Ovis), and two American buffalo (Bison bison) suffering from enteric disease in the western part of Colorado. In 1981, a foal died of apparent respiratory failure. Necropsy findings included heart failure and gastroenteritis. V. cholerae serovar 347 (Smith) was isolated from the colon of this animal. V. cholerae serovar 27 (Smith) was isolated in 1983 from the intestine of a feedlot lamb suffering from pneumonia and severe watery diarrhea. In 1984, an enteric disease occurred in a herd of American bison. The sick animals were depressed and separated from the herd, dying in about 3 days. Of approximately 100 adult bison, 7 died. Necropsy of one animal revealed that gross lesions were limited to the gastrointestinal tract. V. cholerae serovar 27 (Smith) was isolated from the abomasum, duodenum, and colon of this animal. A swab specimen from the intestine of another dead bison also yielded V. cholerae serovar 27 (Smith).
Neutralization of bacterial lipopolysaccharides by human plasma
Authors: Warren H S , Novitsky T J , Ketchum P A , Roslansky P F , Kania S , Siber G R
Published: January 2020

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli O113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LR50 were prepared. The pool with high LR50 neutralized 166-fold more E. coli 0113 LPS, 190-fold more E. coli 0111B4 LPS, 42-fold more Klebsiella pneumoniae LPS, and 29-fold more Salmonella typhimurium LPS than did the pool with low LR50. Each pool had similar immunoglobulin G (IgG) and IgM antibody levels to homologous LPS, measured by an enzyme-linked immunosorbent assay. Analysis of 212 fresh-frozen plasma units revealed a range of LR50 between 48 and 6,000 ng/ml. Incubation of LPS in a pool of fresh-frozen plasma with high LR50 elicited significantly less fever in the rabbit pyrogen test than did LPS incubated in plasma with low LR50 (fever index, 2.68 +/- 0.61 degrees C X h and 3.52 +/- 0.66 degrees C X h, respectively; P = 0.003). We conclude that there is a 100-fold range in the endotoxin-neutralizing capacity of human plasma and that this variation is not due to LPS-specific IgG or IgM antibodies. Further investigations are needed to determine whether differing susceptibility of patients to the effects of LPS is due to differences in the endotoxin-neutralizing capacity of their plasma and whether plasma screened for high endotoxin-neutralizing capacity may be therapeutically useful in endotoxemia.
Immunoradiometric assay of a recombinant human alpha-2 interferon (SCH 30500)
Authors: Protzman W P , Minnicozzi M , Jacobs S L , Surprenant D I , Schwartz J , Oden E M
Published: January 2020

A sensitive immunoradiometric assay for recombinant alpha-2 interferon (IFN-alpha 2; SCH 30500) was developed by using the monoclonal antibody NK2. A polystyrene bead with polyclonal sheep anti-human IFN-alpha (Hu IFN-alpha) antibody adsorbed to it formed the solid phase of the assay. The monoclonal antibody, labeled with 125I, reacted with the IFN-alpha 2 which bound to the polyclonal antibody on the bead. The assay was sensitive, capable of measuring less than 5 IU/ml, and it was precise. Coefficients of variation were less than 10%, often less than 5%. Interassay coefficients of variation for the same sample were also less than 10%. Specificity of the monoclonal antibody for IFN-alpha 2 was greater than for Hu IFN-alpha produced by leukocytes. In specificity studies it was necessary to add at least 200 IU of Hu IFN-alpha per ml to 25 IU of IFN-alpha 2 per ml before the Hu IFN-alpha affected the accuracy of the assay of IFN-alpha 2. Accuracy as determined by correlation studies with the antiviral cytopathic effect assay was very high. Results of the two assays usually agreed within 15%. This correlation was maintained in studies of IFN-alpha 2 after storage under accelerated degradation conditions, when the IFN was cleaved into smaller sequences of amino acids by cyanogen bromide and when the IFN was carboxymethylated.
Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis
Authors: Le Febvre R B , Foley J W , Thiermann A B
Published: January 2020

We have developed a rapid method for the isolation of leptospiral chromosomal DNA which yields DNA of a purity suitable for restriction endonuclease analysis. A small volume (15 to 20 ml) of an exponentially growing culture of leptospires yielded 2 to 4 micrograms of chromosomal DNA. In a 1-day protocol, the DNA was isolated, restricted with endonucleases, and fractionated on an agarose gel. Chromosomal DNA from dinger zones (visible subsurface zones of leptospiral growth) of first semisolid subcultures of field isolates was also isolated and characterized, thus greatly speeding up the diagnostic process.
Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin
Authors: Karmali M A , Petric M , Lim C , Cheung R , Arbus G S
Published: January 2020

High titers of Verotoxin (VT) were released from cell pellets of VT-producing Escherichia coli (VTEC; corresponding to E. coli strains producing "high" levels of Shiga-like toxin) after incubation in polymyxin B (0.1 mg/ml) for 30 min at 37 degrees C. Maximal titers of polymyxin-releasable VT occurred in cells obtained from 5-h Penassay broth cultures and were up to eightfold higher than the peak culture supernatant VT titers which occurred in 8-h cultures. Polymyxin-releasable cell extracts of 5-h broth cultures inoculated with mixtures of VT-positive (VT+) and VT-negative strains had easily detectable VT titers when the proportion of VT+ cells in the mixture was about 1.0%, but culture supernatants were negative for VT even when this proportion was 20%. The results were the same whether the initial inoculum consisted of broth culture mixtures of VT+ and VT-negative strains or colony sweeps (loopfuls of confluent bacterial growth) taken from solid plate media previously inoculated with the broth mixtures. In a clinical study, 80 stool cultures from patients with hemolytic uremic syndrome and family contacts with diarrhea were tested for free fecal VT, VT in polymyxin extracts of colony sweeps (VT/PECS), and VTEC (examination of 20 separate E. coli colonies from primary media for VT production). Of the 80 samples, 40 were positive for at least one of these three tests; all 40 were positive for free fecal VT, and 20 of these were positive for VT/PECS. VTEC (as few as 1 colony out of 20) were only isolated from 14 of the 20 cultures that were positive for VT/PECS. In six cases, the VT/PECS was positive even when none of 20 colonies tested were VT+, suggesting that the procedure was able to detect a proportion of VTEC that was less than one in 20(5%). We conclude that the VT/PECS method is highly sensitive for detecting low concentrations of VTEC in stools and provides a rapid method for screening out stools that are negative for VTEC. The technique should also be of value in epidemiological studies for detecting low numbers of VTEC in animal feces, foods, and environmental samples.
Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA
Authors: Olsen I , Brondz I
Published: January 2020

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.
Determination of immunoglobulin M antibodies for hepatitis B core antigen with a capture enzyme immunoassay and biotin-labeled core antigen produced in Escherichia coli
Authors: Vilja P , Turunen H J , Leinikki P O
Published: January 2020

A new capture enzyme immunoassay for the determination of immunoglobulin M (IgM) antibodies against hepatitis B core antigen (HBcAg) is described. Core antigen produced in Escherichia coli was labeled with biotin and subsequently detected by an avidin-biotin-peroxidase complex. The biotin-labeled core antigen was effective at concentrations as low as 20 ng/ml. Of 561 serum samples from different groups of patients that were tested, 465 samples were negative for other hepatitis B virus markers and also for anti-HBcAg IgM. Sera from the early stages of hepatitis B infection had high levels of anti-HBcAg IgM, and a clear correlation with the acuteness of the disease was observed in 45 follow-up sera from 23 patients with acute or recent hepatitis B. Sera from 21 patients with past hepatitis B were all negative for anti-HBcAg IgM. Twenty serum samples from chronic carriers of hepatitis B surface antigen showed slightly elevated antibody levels for anti-HBcAg IgM. Ten sera which were positive for anti-HBcAg IgG antibodies and had high levels of rheumatoid factor were negative for anti-HBcAg IgM.
Demonstration of Toxoplasma antigen containing complexes in active toxoplasmosis
Authors: van Knapen F , Panggabean S O , van Leusden J
Published: January 2020

With an enzyme-linked immunosorbent assay antigen, specific circulating immune complexes (CIC) were demonstrated in experimental and human toxoplasmosis. In experimentally infected mice, CIC became demonstrable as soon as antibodies appeared after fatal infection. When a nonvirulent strain of Toxoplasma was used CIC remained detectable for several weeks. This period was characterized by clinically healthy animals with increasing antibody titers and by cysts growing in the brains of the animals, indicating a subacute stage of the toxoplasma infection. In the human sera, a surprisingly high percentage of CIC was demonstrated. Both immunoglobulin G (IgG) and IgM were found in the CIC; however, IgG was seen in the majority. If the humans were grouped according to other serological results, such as a combination with IgM antibodies, circulating antigens, or both, or a positive complement fixation test, increasingly more CIC were observed. When sera were selected from patients with clinical symptoms generally associated with toxoplasmosis, more CIC were also again demonstrated. On the contrary, in healthy individuals (blood donors), CIC were also regularly observed, suggesting that exacerbations of latent infections or reinfections may regularly occur without leading to clinical signs. In conclusion, we propose that the interpretation of a positive CIC test requires great care but may provide useful information about the activity of a toxoplasma infection.
Persistence of Inoue-Melnick virus and antibody in cerebrospinal fluid
Authors: Melnick J L , Seidel E , Wang S S , Muchinik G , Rashti E J , Jacobs L D , Freeman A I , O'Malley J
Published: January 2020

Serial cerebrospinal fluid samples were obtained from 14 multiple sclerosis patients over a period of several months and were tested under code for Inoue-Melnick virus. In five of the positive patients, virus was present in 28 of 32 specimens collected over a period of 2 to 5 months. Four patients, from whom a total of 34 specimens were taken, yielded only a single isolate each. Five patients contributed 28 specimens, all of which were negative. In six patients (three virus positive and three virus negative), neutralizing antibody was detected in serum and in cerebrospinal fluid.
Freezing and rejuvenation of human O erythrocytes for use in the immune adherence hemagglutination test
Authors: Lawrence T G , Wentworth B B
Published: January 2020

A method was established to freeze selected human O erythrocytes and to thaw them as necessary for use in the immune adherence hemagglutination test. This method ensured interrun reproducibility and eliminated the necessity to screen blood donors (fresh cells) for acceptable C3b receptor site sensitivity.
Rapid detection of herpes simplex virus DNA in human brain tissue by in situ hybridization
Authors: Forghani B , Dupuis K W , Schmidt N J
Published: January 2020

A commercial system for detection of herpes simplex virus (HSV) DNA by in situ hybridization gave positive results on 16 of 17 stored human brain specimens that were positive for HSV on initial testing by virus isolation and immunofluorescence staining, and the hybridization system gave negative results on 13 brain specimens that showed no evidence of HSV by isolation or immunofluorescence staining.
Enzyme release of antigen from Streptococcus faecalis and Listeria monocytogenes cross-reactive with Lancefield group G typing reagents
Authors: Hopfer R L , Pinzon R , Wenglar M , Rolston K V
Published: January 2020

Enzyme extraction methods are commonly used to release antigen for Lancefield serogrouping of streptococci. Recently, up to 47% of enterococci have been found to possess antigens cross-reactive with both the group D and the group G grouping reagents. We investigated whether the organisms had recently emerged or whether changes in extraction methods (enzyme extraction) have resulted in our awareness of their existence. We found that approximately 20% of our enterococcal isolates released material cross-reactive with the group G reagents. These organisms have been present for at least 6 years, and probably for many more. In addition, we found that most strains of Listeria monocytogenes also released antigen cross-reactive with the group G reagent. The clinical significance of these findings is also discussed.