bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of [3H]thymidine uptake method for studying growth of spiroplasmas under various conditions
Authors: Bastian F O , Baliga B S , Pollock H M
Published: January 2020

[3H]thymidine uptake and colony counts are quantitative and inexpensive methods for studying Spiroplasma growth. Using these techniques, we demonstrated subtle effects on the growth of suckling mouse cataract agent of medium alterations, inoculum size, and freezing of cultures. In addition, suckling mouse cataract agent multiplied more actively under aerobic than under anaerobic conditions. These techniques have wide application for the study of Spiroplasma growth and will be useful for the development of a defined medium.
Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay
Authors: Perera L P , Marques L R , O'Brien A D
Published: January 2020

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five lines had kappa light chains. These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies. The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value.
Detection of antibodies to Ureaplasma urealyticum in pregnant women by enzyme-linked immunosorbent assay using membrane antigen and investigation of the significance of the antibodies
Authors: Liepmann M F , Wattre P , Dewilde A , Papierok G , Delecour M
Published: January 2020

Optimal conditions of a microenzyme-linked immunosorbent assay using a group-specific membrane antigen of Ureaplasma urealyticum serotype 7 were established with rabbit antisera and applied for the evaluation of immunoglobulin M (IgM) and IgG antibodies in 139 serum specimens from pregnant women between 26 and 38 weeks of gestation, and the assay was compared with microorganism culture and investigated to determine the role of U. urealyticum in perinatal morbidity and mortality. U. urealyticum was isolated from 75 (54%) of 139 patients; 40 had a colonization greater than or equal to 10(6) cells per ml of swab (29%); 64 (85%) of 75 culture-positive patients had IgG antibodies (absorbance mean, 0.650), versus 4 (6%) of 64 culture-negative patients (absorbance mean, 0.103) (P less than 0.001). There was no cross-reactivity with Chlamydia trachomatis infection from patients from whom no mycoplasmas were isolated, but this cross-reactivity occurred in 24% of patients with other mycoplasma infections. There was a good correlation between quantitative evaluation of U. urealyticum colonization and antibody level (P less than 0.05). However, IgM antibody was found in 30% of culture-positive patients but also in 25% of the culture-negative group. Frequency of U. urealyticum colonization was greater in unmarried young women (less than 25 years old) with a history of genital infection, and a significantly greater frequency was detected in patients who smoked (P less than 0.01) and had a lower socioeconomic status (P less than 0.001). A lower infant birth weight was more associated with U. urealyticum colonization greater than or equal to 10(5) cells per ml. The enzyme-linked immunosorbent assay provided an additional means to diagnose and evaluate U. urealyticum infection in pregnant women.
Isolation of Campylobacter jejuni from the bile of a cholecystic patient
Authors: Drion S , Wahlen C , Taziaux P
Published: January 2020

Described is the isolation of Campylobacter jejuni from the bile of a cholecystic patient admitted for abdominal pain to an intensive care unit. A cholecystectomy was performed, and the patient responded to erythromycin therapy. Details of this uncommon isolation are given.
Modified spin-amplified adsorption procedure with conventional tissue culture tubes for rapid detection and increased recovery of herpes simplex virus from clinical specimens
Authors: Oefinger P E , Loo S H , Gander R M
Published: January 2020

Conventional culture tubes were used in a modification of the spin-amplified adsorption procedure for recovery of herpes simplex virus (HSV) from clinical specimens. The sensitivity of isolation of HSV from 864 specimens adsorbed by the spin-amplified method was 100% (127 of 127), compared with 88.2% (112 of 127) for stationary-phase-adsorbed specimens. Cytopathic effect developed more rapidly in 32.1% (36 of 112) of isolates adsorbed by spin amplification than in those adsorbed by stationary means. In a separate quantitative study, cultures of HSV type 1 adsorbed by spin amplification yielded higher antigen levels and greater cytopathic effect than stationary-phase-adsorbed cultures. Cells grown in conventional tissue culture tubes may be used in a spin-amplified adsorption for rapid detection and increased sensitivity of HSV isolation.
Enzyme profiles of oral spirochetes in RapID-ANA system
Authors: Syed S A , Salvador S L , Loesche W J
Published: January 2020

Enzyme profiles of oral Treponema species were determined by using RapID-ANA (Innovative Diagnostic System, Atlanta, Ga.), a 4-h test system which detects 18 enzymatic reactions, including aminopeptidases and glycosidases. Seventy-two clinical isolates of Treponema denticola, four reference strains of T. denticola (ATCC 35404, ATCC 35405, ATCC 35520, and ATCC 33521), one strain of T. vincentii (ATCC 35580), and two strains of T. socranskii subspecies (T. socranskii subsp. buccale ATCC 35534 and T. socranskii subsp. socranskii ATCC 35536) were used in this study. All T. denticola strains produced indole and a variety of aminopeptidases and glycosidases. These organisms could be differentiated into two groups on the basis of tetrazolium reductase and serine, phenylalanine, and glycine aminopeptidase activities. T. vincentii produced N-acetylglucosaminidase and arginine aminopeptidase, which facilitated the differentiation of this organism from T. socranskii subspecies and the T. denticola group. T. socranskii subspecies gave positive reactions for alkaline phosphatase only. These findings suggest that the RapID-ANA system is useful for enzymatic characterization and differentiation of oral spirochetes.
Microparticle enzyme immunoassay for determination of immunoglobulin G antibodies to human cytomegalovirus
Authors: Duverlie G , Roussel C , Driencourt M , Orfila J
Published: January 2020

A microparticle enzyme immunoassay (LEIA) that uses passive latex agglutination was used to detect cytomegalovirus immunoglobulin G antibodies in 495 serum samples. LEIA was in excellent concordance with latex agglutination (97%) and an enzyme-linked immunosorbent assay (98.3%). The sensitivity and specificity of LEIA compared with those of the enzyme-linked immunosorbent assay were 100 and 92.5%, respectively.
Propagation of the virus of porcine epidemic diarrhea in cell culture
Authors: Hofmann M , Wyler R
Published: January 2020

Porcine epidemic diarrhea virus (PEDV) was adapted to serial propagation in Vero cell cultures by adding trypsin to the medium. PEDV-infected cells showed a distinct cytoplasmic fluorescence when examined by a fluorescent-antibody-staining technique. Cytopathic effects, such as vacuolation, formation of syncytia, and fusion of cells, were detected even at passage 1 of the PEDV in Vero cells. Once adapted, the virus induced numerous syncytia containing over 100 nuclei. From virus passage 5 on, all cells forming the monolayer were fused and totally destroyed within 24 h after inoculation. Cell culture-grown PEDV had typical coronavirus morphology when viewed by electron microscopy. Attempts to propagate PEDV in several primary and secondary fetal porcine cell cultures in the presence or absence of trypsin were unsuccessful.
Comparison of broth enrichment and direct plating for the isolation of Campylobacter jejuni from dogs
Authors: Monfort J D , Stills H F , Bech-Nielsen S
Published: January 2020

Two techniques for the isolation of Campylobacter jejuni from feces, direct plating and thioglycolate broth enrichment, were compared. A total of 272 rectal swab cultures were performed on 156 laboratory dogs. Campylobacter blood agar plates and Campylobacter thioglycolate broth were inoculated immediately upon sampling of the dogs. After incubation at 4 degrees C for 12 to 16 h, material from the Campylobacter thioglycolate medium was inoculated onto Campylobacter blood agar plates. A total of 157 samples were positive for C. jejuni; 154 were positive by the direct method and 112 were positive by the enrichment technique. Forty-five samples which were negative by the enrichment were positive by the direct method, and three samples which were negative by the direct method were positive for C. jejuni by the enrichment method. The use of the enrichment step resulted in an increase in the isolation rate from 56.6 to 57.7%.
Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract
Authors: Baseman J B , Dallo S F , Tully J G , Rose D L
Published: January 2020

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.
Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli
Authors: Sommerfelt H , Kalland K H , Raj P , Moseley S L , Bhan M K , Bjorvatn B
Published: January 2020

Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with 32P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.
Heterogeneity in immunoblot patterns obtained by using four strains of Borrelia burgdorferi and sera from naturally exposed dogs
Authors: Greene R T , Walker R L , Burgess E C , Levine J F
Published: January 2020

This study evaluated the differences in immunoblot patterns when serum specimens from seropositive dogs were reacted against four strains of Borrelia burgdorferi. Intense bands were consistently detected for the 83-, 66-, 61- to 60-, 41-, and 31-kilodalton regions for all four strains. Most variations were observed in the regions of 45 to 34 and 26 to 15 kilodaltons. Adsorption studies suggested that one reason for the observed variability was a difference in proteins among the organisms, rather than a difference in migration of proteins. Therefore, knowledge and consistency of the test antigen are essential when evaluating and comparing canine immunoblot patterns to B. burgdorferi, but for diagnostic purposes all of the serum samples would have been considered positive regardless of the strain used.
Collaborative evaluation in seven laboratories of a standardized micromethod for yeast susceptibility testing
Authors: Guinet R , Nerson D , de Closets F , Dupouy-Camet J , Kures L , Marjollet M , Poirot J L , Ros A , Texier-Maugein J , Volle P J
Published: January 2020

The new micromethod for yeast susceptibility testing, MYCOTOTAL, was evaluated with 10 reference strains in seven laboratories. Ready-to-use microtitration plates and the same synthetic medium were used with two dilutions of imidazoles, flucytosine, and amphotericin B, permitting the categorization of each strain as susceptible, intermediate, or resistant. The results were compared with the MIC for each reference strain, and the repeatability and reproducibility were evaluated. The yeasts tested presenting different patterns of susceptibilities in reference MICs included six strains of Candida albicans, two strains of Candida tropicalis, one strain of Candida parapsilosis, and one strain of Torulopsis glabrata. For 4,200 antifungal agent-yeast results, the repeatability was 99.3% and the reproducibility was 96.3%. The correlation between the reference MICs and the category results was 91.5% for seven laboratories (and 92.7% for six laboratories excluding the laboratory which did not follow exactly the same protocol). We observed only 7.9% minor discrepancies, 0.5% (0.29% for six laboratories) major discrepancies, and 0.1% uninterpretable results. The percentages of concording results were similar for each strain and each antifungal agent tested. The overall results indicated that MYCOTOTAL was a reliable and reproducible method, well correlated with reference MICs. This ready-to-use micromethod with the same medium for all antifungal agents would be an important step in the necessary standardization of yeast susceptibility testing.
Susceptibilities of 14 cell lines to bluetongue virus infection
Authors: Wechsler S J , McHolland L E
Published: January 2020

The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations.
Evaluation of the modified Bac-T-Screen and FiltraCheck-UTI urine screening systems for detection of clinically significant bacteriuria
Authors: Murray P R , Niles A C , Heeren R L , Pikul F
Published: January 2020

Previous evaluations of the Bac-T-Screen system (Vitek Systems, Inc., Hazelwood, Mo.) demonstrated excellent sensitivity with specimens from patients with clinically significant bacteriuria (including infections with small numbers of uropathogens) but poor specificity with specimens from noninfected patients. In the study reported here, the sensitivity and specificity of the Bac-T-Screen system with a modified decolorizing reagent were evaluated. A manual filtration system, FiltraCheck-UTI (Vitek Systems), for screening urine specimens, Gram stains of mixed urine specimens, and quantitative cultures were also evaluated. The test sensitivity for clinically significant bacteriuria was greater than 96% with the original Bac-T-Screen system as well as the modified system and the manual system. In comparison, the sensitivities of the Gram stains and quantitative cultures (greater than or equal to 10(5) CFU/ml) were 82 and 77%, respectively. Of the 375 patients classified as noninfected by clinical parameters, 34% had positive screening tests with the original Bac-T-Screen system, as compared with 13 and 11% with the modified Bac-T-Screen and FiltraCheck-UTI systems, respectively. Thus, the modified Bac-T-Screen system and the manual FiltraCheck-UTI system have sensitives comparable to that of the original Bac-T-Screen system and markedly improved test specificities.
Improved selective medium for the isolation of Treponema hyodysenteriae
Authors: Kunkle R A , Kinyon J M
Published: January 2020

An agar medium with improved selection for Treponema hyodysenteriae was developed. Cultures of T. hyodysenteriae and T. innocens, feces from 11 clinically normal pigs, and colonic contents from 6 pigs with gross lesions consistent with swine dysentery were diluted in phosphate-buffered saline and plated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% citrated bovine blood (TSA), TSA with 400 micrograms of spectinomycin per ml (TSA-S400), TSA-S400 with 25 micrograms each of colistin and vancomycin per ml, and TSA with 5% pig feces extract and five antimicrobial agents (spiramycin, rifampin, vancomycin, colistin, and spectinomycin) (BJ). Viable numbers of T. hydodysenteriae grown on BJ were virtually identical to those for TSA, TSA-S400, and TSA-S400 with colistin and vancomycin. Pure cultures of four isolates of T. hyodysenteriae and three isolates of T. innocens were sustained through six subcultures on BJ. Fecal floras were completely inhibited on BJ for 14 of 17 fecal samples from both groups of pigs. A total of 461 colonic specimens from naturally occurring cases of porcine intestinal disease were plated on TSA-S400 and BJ. T. hyodysenteriae was isolated on both TSA-S400 and BJ for 69 specimens and on BJ alone for an additional 19 specimens.
Visual and clinical analysis of Bac-T-Screen urine screen results
Authors: Baron E J , Tyburski M B , Almon R , Berman M
Published: January 2020

Of 337 urine specimens evaluated, 75 of the 113 that showed positive readings on the Bac-T-Screen urine-screening instrument were found by subsequent semiquantitative culture to yield either less than 10,000 CFU of mixed bacteria per ml or no growth (less than 100 CFU/ml by our criteria). We tried to determine what factors contributed to the positive Bac-T-Screen results by examining the 75 Bac-T-Screen-positive urine specimens with three visual methods: Gram staining, hemacytometer chamber counting, and filtering through a 5.0-microns-pore-size nitrocellulose filter with subsequent microscopic examination of the stained filter. Somatic cells and other particles present in those urine specimens that yielded positive readings by the Bac-T-Screen included epithelial cells in 43%, crystals and amorphous material in 33%, and leukocytes in 17% of the specimens. There was no relationship between the numbers of particles seen in urine and the magnitude of the relative absorbance reading obtained with the Bac-T-Screen. A retrospective chart review was conducted for patients with positive Bac-T-Screen results and negative cultures. Of the 75 patients, 6 were thought to have urinary tract infections on the basis of clinical criteria; the majority of the remaining 69 patients had clinical histories revealing systemic or urogenital conditions consistent with shedding of particles in the urine. A positive reading by the Bac-T-Screen system seemed to be related to the presence of somatic cells and other particles in urine; bacteriuria was not always detectable in these cases.
Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype O8:KX105 and O8:K"2829" strains isolated from piglets with diarrhea
Authors: Broes A , Fairbrother J M , Mainil J , Harel J , Lariviere S
Published: January 2020

Twelve pathogenic and seven nonpathogenic enterotoxigenic Escherichia coli strains which were previously identified as belonging to serogroup O8:KX105 (A. Broes, J. M. Fairbrother, S. Larivière, M. Jacques, and W. M. Johnson, Infect. Immun. 56:241-246, 1988) were further examined for their phenotypic and genotypic properties. Only the 12 pathogenic strains were confirmed to possess the capsular antigen KX105. The seven nonpathogenic strains did not possess this antigen and thus were incorrectly assigned to have capsular antigen KX105. All seven nonpathogenic strains apparently possessed a previously unrecognized capsular antigen which has been designated K"2829". Studies with antisera prepared against F1 (type 1) fimbriae from three E. coli strains suggested that at least three antigenic subtypes of F1 fimbriae were represented among the O8:KX105 strains examined. By using serotyping, biotyping, and outer membrane protein profile analyses, the O8:KX105 strains were divided into at least two distinct clusters, whereas the O8:K"2829" strains were grouped into a single unique cluster. Most of the strains of the same cluster were further differentiated by testing for antibiotic resistance and colicin production and resistance and by analysis of plasmid content. With the exception of one strain which lost its enterotoxicity during storage, all of the O8:KX105 strains hybridized with the gene probes for the heat-labile (LT) and heat-stable (STb) enterotoxins. For each O8:KX105 strain, a single plasmid ranging in size from 61 to 77 megadaltons carried the LT and STb genes.All of the enterotoxigenic O8: KX105 strains fermented sorbose, whereas the nonenterotoxigenic strain did not. All of the O8:K "2829" strains hybridized with the STb probe only. For each O8:K "2829" strain, the STb genes were located on a single plasmid of 61 or 22 megadaltons. None of the strains demonstrated homology with the genes encoding the F4 (K88), F5 (K99), F6 (987P), and F41 fimbrial antigens and STaP and STaH.
Rotavirus epidemiology in Vellore, south India: group, subgroup, serotype, and electrophoretype
Authors: Brown D W , Mathan M M , Mathew M , Martin R , Beards G M , Mathan V I
Published: January 2020

Rotaviruses were detected in 163 of 916 (17.8%) specimens collected from children under 3 years of age with gastroenteritis in Vellore, South India, between August 1983 and July 1985. Rotaviruses were detected throughout the study period, with a peak prevalence in December to February (winter) and June to August (southwest monsoon season). A total of 117 rotavirus strains were tested for subgroup, serotype, and rotavirus double-stranded RNA electrophoretic migration pattern; 24.8% of the strains were subgroup I, 69.2% were subgroup II, and 6.0% were neither subgroup I nor subgroup II. Subgroup I and II strains were circulating concurrently throughout the study. Of the 117 rotavirus strains, 32 (27.4%) were serotyped; 15 were serotype 1, 3 were serotype 2, 2 were serotype 3, and 12 were serotype 4. Three serotypes were circulating concurrently during the periods of peak rotavirus prevalence. In 100 of the 117 strains (85.4%) an RNA pattern was detected. One unusual subgroup I group A rotavirus with a long migration pattern and four atypical rotaviruses serologically related to group C were also detected.
Evaluation of a commercial latex test for Clostridium difficile for reactivity with C. difficile and cross-reactions with other bacteria
Authors: Miles B L , Siders J A , Allen S D
Published: January 2020

Seventy-eight species of bacteria (739 isolates) were tested for reactivity with a commercial latex test for Clostridium difficile. All noncytotoxic as well as cytotoxic strains of C. difficile reacted positively. Immuno-specific cross-reactions were found only with C. sporogenes, proteolytic C. botulinum, and Peptostreptococcus anaerobius.
Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity
Authors: Hunter P R , Gaston M A
Published: January 2020

An index of discrimination for typing methods is described, based on the probability of two unrelated strains being characterized as the same type. This index may be used to compare typing methods and select the most discriminatory system.
Campylobacter cryaerophila isolated from a human
Authors: Tee W , Baird R , Dyall-Smith M , Dwyer B
Published: January 2020

Campylobacter cryaerophila was recovered from a single stool specimen of a 35-year-old homosexual man who presented with intermittent diarrhea for 4 to 6 months. The isolate was identified as C. cryaerophila by using biochemical reactions and confirmed by DNA-DNA hybridization and gas-liquid chromatograph profiles. The base composition of DNA of the isolate was found to be 31.1 +/- 1 mol% (G+C). C. cryaerophila was previously reported to be associated with bovine and porcine abortions in animals. We believe this is the first report of recovery of C. cryaerophila from human stool.
Species-specific oligonucleotide probes for rRNA of Clostridium difficile and related species
Authors: Wilson K H , Blitchington R , Hindenach B , Greene R C
Published: January 2020

The large copy number of rRNA makes it an appealing target for oligonucleotide probes designed to identify microorganisms. Given that nucleotide sequences in rRNA are known to reflect phylogeny, species-specific rRNA probes should be feasible if the sequences found in closely related species are different. We sequenced portions of the 16S rRNA of three closely related clostridia found in the human colonic microflora: Clostridium bifermentans, C. sordellii, and C. difficile. The rRNAs of these three species showed 97 to 98% sequence similarity. Five oligonucleotide probes complementary to unique segments of the sequences were end labeled with 32P and hybridized on a nylon filter to the immobilized rRNA of each clostridium. Each probe efficiently hybridized only to the rRNA of the species to which it was directed. Complementary probes emitted a signal that exceeded by a factor of 100 to 1,000 the signal of probes that mismatched the target rRNA by 2 to 5 bases. Even a 1-base difference in rRNA sequence allowed a clear distinction between species. A systematic approach can efficiently yield taxon-specific oligonucleotide probes directed at rRNA.
Enhanced detection of cytomegalovirus in confluent MRC-5 cells treated with dexamethasone and dimethyl sulfoxide
Authors: West P G , Aldrich B , Hartwig R , Haller G J
Published: January 2020

Optimum growth conditions for human cytomegalovirus (HCMV) include the use of subconfluent, actively growing cultures of human embryonic fibroblasts. Many clinical virology laboratories, however, use tissue culture cells from commercial sources. These cells are usually confluent, static cultures that tend to be less sensitive to viral infection. To determine whether dimethyl sulfoxide (DMSO) or dexamethasone (DEX), which are known enhancers of HCMV, facilitates the detection of the virus in confluent cells, we tested both HCMV AD169 and a number of clinical specimens suspected to contain HCMV on MRC-5 cells in both shell vials and conventional tube cultures. We found that, in the shell vial test, treatment of the cultures with either DMSO or DEX before and after inoculation increased the number of cells staining positive by three- to sixfold compared with untreated controls. Best results were obtained by pretreating the cultures with DEX alone and by treating the cultures with a combination of DEX and 1% DMSO postinfection. In the conventional MRC-5 culture tubes, treatment with the reagents resulted in the more rapid appearance of cytopathic effect and a more extensive infection of the cell sheet. The experimental findings indicate that the enhancing effect of DEX is attributable mainly to the increased production of a cellular mRNA during the period preceding viral infection.
Comparison between viremia and antigenemia for detection of cytomegalovirus in blood
Authors: van der Bij W , Schirm J , Torensma R , van Son W J , Tegzess A M , The T H
Published: January 2020

In a prospective study, 139 serial blood samples from 15 transplant recipients were assessed for the presence of cytomegalovirus (CMV) by virus isolation (CMV viremia) and by direct staining of CMV antigens (CMV Ag) in blood leukocytes (CMV antigenemia). CMV was isolated from 23 samples, whereas CMV Ag was detected in 44 specimens. All positive samples were from a total of nine patients who were diagnosed as having active CMV infections. In seven patients, active CMV infections were diagnosed by virus isolation from blood and urine and by a significant rise of CMV-specific antibodies. In these patients, 21 of the 23 blood samples which were positive for CMV by cell culture were also positive by direct CMV Ag detection. Moreover, CMV Ag were detected in 23 of the 116 culture-negative samples. Twenty of these samples were from the acute phase of infection in the same seven patients. The remaining three CMV Ag-positive specimens were from the other two patients, from whom CMV was not isolated but who had serological evidence of concomitant active CMV infections. These results suggest that direct detection of CMV Ag in peripheral blood leukocytes is as specific as and more sensitive than current isolation techniques. Furthermore, by its sensitivity and inherent rapidity the antigen detection test proved to be the earliest diagnostic marker of active CMV infection in eight of the nine patients. Finally, it was shown that monoclonal antibodies to CMV immediate early antigens are a prerequisite for demonstration of CMV antigenemia.
Comparison of cell cultures for rapid isolation of enteroviruses
Authors: Chonmaitree T , Ford C , Sanders C , Lucia H L
Published: January 2020

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.
Production and characterization of monoclonal antibodies to Mobiluncus species
Authors: Fohn M J , Lukehart S A , Hillier S L
Published: January 2020

Members of the genus Mobiluncus are anaerobic motile curved rods which are associated with bacterial vaginosis (BV). Murine monoclonal antibodies (MAbs) to the ATCC type strains of M. curtisii subsp. curtisii, M. curtisii subsp. holmesii, and M. mulieris were produced and characterized by enzyme-linked immunosorbent assay and indirect immunofluorescence assay. Four MAbs were subspecies specific and reacted with M. curtisii subsp. curtisii but not with M. curtisii subsp. holmesii; four were specific for M. mulieris. The remaining antibodies demonstrated some cross-reactivity: three were species specific and reacted with both subspecies of M. curtisii, and one defined an epitope shared by M. curtisii subsp. holmesii and M. mulieris but not by M. curtisii subsp. curtisii. None of the MAbs reacted with a panel of other bacteria commonly present in the vaginas of normal women or women with BV. Examination of the molecular specificities of the antibodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed four antibodies which were specific for an 82,000-dalton molecule of M. curtisii subsp. curtisii and five antibodies which bound a major band of M. mulieris at 93,000 daltons. Selected MAbs reacted in the indirect immunofluorescence assay with 24 of 25 Mobiluncus spp. clinical isolates from local women with BV and could be used for direct detection of Mobiluncus spp. in vaginal fluid from a patient with BV.
Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity
Authors: Namdari H , Bottone E J
Published: January 2020

Certain strains of mesophilic aeromonads (Aeromonas hydrophila, A. sorbria, and A. caviae), when grown in broth containing 0.5% glucose, undergo growth inhibition concomitant with acetate accumulation. Because these strains are nonviable after 24 h, this phenomenon is termed suicide. We investigated suicidal strains of Aeromonas species as a means of understanding animal virulence and enteropathogenicity. To assess virulence, batches of five white mice were inoculated intraperitoneally with 10(7) cells (washed) of suicidal and nonsuicidal strains of A. hydrophila and A. sobria and suicidal strains of A. caviae. The three nonsuicidal strains of A. sobria tested showed lethality as early as 12 h and were uniformly fatal within 36 h postinoculation. After 36 h, the three suicidal strains killed only 1 of 15 mice inoculated. Four A. hydrophila strains tested which showed the suicide phenomenon at 37 degrees C were variably lethal (40 to 100%). None of three suicidal strains of A. caviae were lethal. Enteropathogenicity was studied by orally inoculating three white mice each with the same Aeromonas strains (10(8) cells, in skim milk) and assessing diarrhea and intestinal fluid accumulation. Diarrhea and fluid accumulation were present in all mice inoculated with two nonsuicidal strains of A. sobria and in 4 of 12 mice given four suicidal strains of A. hydrophila. Two suicidal strains each of A. sorbria and A. caviae failed to elicit any gastrointestinal disturbances. These data suggest that the suicide phenomenon may explain strain-specific (A. sobria and A. hydrophila) and species-specific (A. caviae) virulence and enteropathogenicity.
Effects of blood and different media on the production of toxic shock syndrome toxin 1 by Staphylococcus aureus in the tampon sac method
Authors: Reiser R F , Denzin L K , Bergdoll M S
Published: January 2020

The use of three different agar concentrations in the tampon sac method resulted in slightly higher fluid uptake by the tampons when a 0.5% agar concentration was used. However, there was essentially no difference in the total amount of toxin produced. The largest amount of toxic shock syndrome toxin 1 was produced with brain heart infusion agar, followed closely by 3% NZ-amine NAK-1% yeast extract medium. The addition of plasma and serum to the inoculum resulted in increases (62 and 73%, respectively) in toxin production. The addition of whole blood to the inoculum had a variable effect on toxin production, with an increase in the amount of toxin produced with some tampons and not with others. Over fivefold differences in the amount of toxin produced were obtained when duplicate experiments were done on successive days, whereas the differences were less than twofold for experiments done on the same day. This was related to the effect of small changes in the parameters on toxic shock syndrome toxin 1 production.
Pseudoappendicitis caused by Plesiomonas shigelloides
Authors: Fischer K , Chakraborty T , Hof H , Kirchner T , Wamsler O
Published: January 2020

A 20-year-old patient was hospitalized with clinical signs of acute appendicitis. After surgery, the histological findings in the appendix and a lymphatic node suggested the diagnosis of pseudoappendicitis caused by Plesiomonas shigelloides, which was isolated in pure culture from the lymphatic node. The strain of P. shigelloides was found to elaborate a heat-stable toxin and harbored two plasmids of 280 and 4 kilobases. A large plasmid has previously been implicated as a virulence marker in P. shigelloides infections.
Increased detection rate for varicella-zoster virus with combination of two techniques
Authors: West P G , Aldrich B , Hartwig R , Haller G J
Published: January 2020

Standard cell culture and centrifugation culture were compared for the isolation of varicella-zoster virus from 337 clinical specimens. Of a total of 85 (25%) positive specimens, 67 (78.8%) were positive by centrifugation culture alone and 18 (21.2%) were positive by both methods. When epithelial cells from the specimen were included in the vial inoculum, these cells stained positive and increased the varicella-zoster virus detection rate by 30%.
Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus
Authors: Pacini D L , Brady M T , Budde C T , Connell M J , Hamparian V V , Hughes J H
Published: January 2020

Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE were subjected to blocking EIA with rabbit or goat anti-SA11. Rotavirus was detected by PAGE and Pathfinder in 62 stools, but only 47 of these were positive by Rotazyme II. Blocking assays of EIA-positive, PAGE-negative samples suggested the presence of rotavirus in four additional stools. Sensitivity and specificity measured against PAGE and blocking assays were: Pathfinder, 0.985 and 0.934; and Rotazyme II, 0.731 and 0.927, respectively. False-positive rates were 0.134 for Pathfinder and 0.149 for Rotazyme II. The specificity and rate of false-positive results of Pathfinder were improved by using an adjusted optical density cutoff 4.36 times greater than that recommended by the manufacturer (specificity, 0.993; sensitivity, 0.985; false-positive rate, 0.015).
Severe progressive subcutaneous abscesses and necrotizing tenosynovitis caused by Rhodococcus aurantiacus
Authors: Tsukamura M , Hikosaka K , Nishimura K , Hara S
Published: January 2020

A case of severe progressive subcutaneous abscesses and necrotizing tenosynovitis of the right arm of a 30-year-old woman caused by Rhodococcus aurantiacus is reported.
Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures
Authors: Flynn W T , Saif L J
Published: January 2020

A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures. Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful. A low-pH medium (pH 6.8) was also ineffective in virus propagation. Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium. The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation. Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation. Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16. Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13. The infected cells became separated, rounded, and detached, forming holes in the cell monolayer. Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy. Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.
Kinetics of intestinal replication of group B rotavirus and relevance to diagnostic methods
Authors: Vonderfecht S L , Eiden J J , Miskuff R L , Yolken R H
Published: January 2020

Non-group-A rotaviruses have been implicated with increasing frequency as causes of acute gastroenteritis in humans and other animals. However, the incidence and significance of infection with these agents, as well as appropriate diagnostic strategies for making these determinations, are largely unknown. Studies to make these determinations could be more accurately conducted if the relationship between the viral replication kinetics and the particular diagnostic method used is understood. We thus utilized the murine model of group B rotavirus infection to establish the viral replication kinetics by a variety of commonly used diagnostic methods. Enzyme immunoassay, routine negative-stain electron microscopy, solid-phase immunosorbent electron microscopy, polyacrylamide gel electrophoresis, and a dot hybridization assay were used in these studies. By enzyme immunoassay, 100% of experimentally infected suckling rats tested positive for group B rotaviral antigens at 1, 4, and 5 days postinoculation. However, only 70 and 20% of infected animals tested positive at days 2 and 3 postinoculation, respectively. Dot hybridization with a complementary DNA probe also suggested a biphasic pattern of viral antigen excretion. Evidence of the virus causing infectious diarrhea in infant rats was found only on day 1 postinoculation in samples examined by routine negative-stain electron microscopy and by polyacrylamide gel electrophoresis. Rotaviruslike particles were observed by solid-phase immunosorbent electron microscopy on days 1, 2, and 4 after viral inoculation suckling rats but were clearly the most numerous on day 1. Additionally, the enzyme immunoassay was used to quantitate the kinetics of group B rotaviral replication in the intestines of the experimentally infected animals. Levels of murine group B rotaviral antigens in intestinal samples peaked on days 1 and 4 postinoculation; however, only peak 1 represented actual intraepithelial replication of the virus. These studies thus indicate that early sample collection and selection of the appropriate diagnostic method are critical if the incidence and significance of group B and possibly other non-group-A rotaviral infections are to be accurately assessed.
Clinical evaluation of the Vitek ANI card for identification of anaerobic bacteria
Authors: Schreckenberger P C , Celig D M , Janda W M
Published: January 2020

An evaluation of the Vitek Anaerobe Identification (ANI) card was performed with 341 bacterial isolates, including 313 clinical isolates and 28 stock strains of anaerobic microorganisms. Identifications obtained with the ANI card were compared with those determined by conventional methods. The card identified 73.2% of 149 anaerobic gram-negative bacilli, 63.6% of 44 Clostridium spp., 65.8% of 38 anaerobic nonsporeforming gram-positive bacilli, and 69.1% of 110 anaerobic cocci, with no further testing required. When genus-level identifications were included, 83.9% of the anaerobic gram-negative bacilli, 70.5% of Clostridium spp., 73.7% of the anaerobic nonsporeforming gram-positive bacilli, and 73.6% of the anaerobic cocci were identified. Nineteen isolates (5.6%) produced identifications of good confidence but marginal separation or questionable biotype, in which the correct identification was listed with one or two other possible choices and extra tests were required and suggested. A total of 28 (8.2%) were not identified and 29 isolates (8.5%) were misidentified by the ANI card. Among the commonly isolated clinically significant anaerobes, the ANI card identified 100% of 55 Bacteroides fragilis and 100% of 8 Clostridium perfringens. Use of supplemental tests and expansion of the data base to include additional strains of organisms that are difficult to separate even with conventional methods may improve the accuracy of the ANI card as a method for identification of anaerobic bacteria in the clinical laboratory.
Optimization of a rapid test by using fluorescein-conjugated monoclonal antibodies for detection of Chlamydia trachomatis in clinical specimens
Authors: Pouletty P , Martin J , Catalan F , Garcia-Gonzalez M , Morellet I , Bettinger S , Kadouche J
Published: January 2020

A mixture of two fluorescein isothiocyanate-conjugated monoclonal antibodies (MAbs) was used to optimize a direct specimen test (Chlamydia Direct Specimen Test IF; Clonatec, Paris, France) for detection of chlamydial elementary bodies in clinical specimens. One MAb reacted with a subspecies-specific epitope of the major outer membrane protein (molecular weight 43,000) of Chlamydia trachomatis, whereas the other reacted with the periodate-sensitive genus-specific antigen (molecular weight 11,000) of Chlamydia spp. Nonfat dry milk was the most efficient additive at suppressing the fluorescent background and was included in the antibody preparation. Fc-dependent binding of fluorescein-conjugated MAbs to protein A-containing Staphylococcus aureus was inhibited by addition of purified rabbit immunoglobulin. The Chlamydia Direct Specimen Test IF was compared with tissue culture isolation by using 309 genital specimens. The sensitivity and specificity were 77.4 and 98%, respectively.