bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a cold-adapted influenza B/Texas/84 reassortant virus (CRB-87) vaccine in young children
Authors: Anderson E L , Newman F K , Maassab H F , Belshe R B
Published: January 2020

A cold-adapted (ca) influenza B reassortant virus vaccine that contained the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus and the neuraminidase and hemagglutinin genes from wild-type influenza B/Texas/1/84 virus was evaluated in children ranging in age from 8 months to 14 years. The children were vaccinated intranasally with doses ranging from 10(3.2) to 10(6.2) 50% tissue culture infective doses (TCID50). Thirty children were seropositive, and 26 were seronegative. Thirty-three children participated as unvaccinated controls. The vaccine was well tolerated by both seronegative and seropositive children. The amount of virus required to infect 50% of seronegative children was approximately 10(4.5) TCID50. Vaccine viruses recovered from airway secretions retained temperature-sensitive and cold-adapted characteristics. The results of this study indicate that the vaccine virus, influenza B/Texas/84 ca reassortant virus, is attenuated, immunogenic, and phenotypically stable when given to young seronegative children.
Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin
Authors: Jayarao B M , Doré J J , Oliver S P
Published: January 2020

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.
The plastic envelope method, a simplified technique for culture diagnosis of trichomoniasis
Authors: Beal C , Goldsmith R , Kotby M , Sherif M , el-Tagi A , Farid A , Zakaria S , Eapen J
Published: January 2020

Although culture of Trichomonas vaginalis is more sensitive than wet mounts in the diagnosis of trichomoniasis, the lack of convenience of culture prevents it from being widely used. To improve the acceptability of diagnosis by culture, a plastic envelope method (PEM) was devised. PEM permits both immediate examination and culture in one self-contained system. The medium consists of dry ingredients that are reconstituted with water before use. The effectiveness of immediate examinations by PEM was compared with that of wet mounts, and the effectiveness of culture by PEM was compared with that of culture in Trichomonas Medium No. 2 (Oxoid). Of 710 vaginal secretion specimens from symptomatic and asymptomatic women that were tested by the four methods, 62 (9%) were positive for T. vaginalis. The sensitivity was 66% by wet mount, 66% by immediate examination by PEM, 89% by cultures in Oxoid medium, and 97% by culture by PEM. The two culture methods had equivalent sensitivities but were significantly (P less than 0.0001) more sensitive than the two immediate methods. The combined immediate examination by PEM plus culture was more convenient to use than wet mounts plus culture in Oxoid medium. The long shelf-life of PEM's dry medium and its anticipated low cost are additional advantages.
Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group
Authors: Scriver S R , Low D E , Simor A E , Toye B , McGeer A , Jaeger R
Published: January 2020

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.
Reverse transcription and polymerase chain reaction amplification of rRNA for detection of Helicobacter species
Authors: Engstrand L , Nguyen A M , Graham D Y , el-Zaatari F A
Published: January 2020

Sequence data on Helicobacter pylori 16S rRNA were used to select two 22-base oligonucleotide primers for use in a polymerase chain reaction (PCR) for detection of H. pylori. H. pylori cells were treated with lysis buffer, boiled, and chloroform extracted. Reverse transcription of rRNA was followed by PCR amplification (RT-PCR) of the synthesized cDNA and 16S rRNA gene. The amplified PCR products were analyzed by agarose gel electrophoresis and Southern blotting. Using ethidium bromide-stained agarose gels, we were able to detect the expected 500-bp DNA fragment from as few as two H. pylori organisms per reaction. The specificity of the RT-PCR assay was tested with 27 clinical isolates and related reference strains; although the number of bacterial cells used per reaction was 10(5)-fold greater than the number of H. pylori organisms used, amplification was detected only with bacteria in the same genus, H. cinaedi and H. mustelae. Ten H. pylori organisms per biopsy specimen were detected on agarose gels when organisms were added to samples prepared from a processed colon biopsy sample. RT-PCR results were consistent with urea breath test and culture results in 14 of 15 gastric biopsy specimens; the specificity was 100%. RT-PCR of rRNA from H. pylori increased the sensitivity of pathogen detection at least 25- to 50-fold compared with that of previous PCR assays. This low level of detection by RT-PCR assay may prove to be well suited for verifying eradication following therapy.
Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay
Authors: Best L M , Veldhuyzen van Zanten S J , Bezanson G S , Haldane D J , Malatjalian D A
Published: January 2020

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.
Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school
Authors: Vandamme P , Pugina P , Benzi G , Van Etterijck R , Vlaes L , Kersters K , Butzler J P , Lior H , Lauwers S
Published: January 2020

In the autumn of 1983, an outbreak of recurrent abdominal cramps occurred in a nursery and primary school in the Rovigo area in Italy. None of the 10 affected children had diarrhea. An atypical Campylobacter-like organism was isolated from feces in all cases. Conventional enteropathogens were searched for but not detected. The Campylobacter-like organism was identified as Arcobacter butzleri by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and cellular fatty acid analysis. Its identity was confirmed by DNA-DNA hybridizations versus Arcobacter reference strains. All of the preserved outbreak strains have identical protein profiles and phenotypic characteristics and belong to serogroup 1 of the Lior serotyping scheme on the basis of slide agglutination of crude and absorbed antisera of A. butzleri reference strains versus heat-labile antigens of live bacteria. These data point to an epidemiological relationship. The successive timing of the cases suggests person-to-person transmission.
Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies
Authors: Mourton C , Romestand B , de Kinkelin P , Jeffroy J , Le Gouvello R , Pau B
Published: January 2020

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA. The efficiency of the anti-N system in detecting purified and crude viruses as well as the virus in infected-organ extracts and infected blood was compared with that of a recently described antigen capture ELISA based on the detection of viral envelope glycoprotein Gp (anti-G system). For the detection of purified virus, the anti-N system was found to be as sensitive as the anti-G system (detection limit, 1 ng of total viral protein per ml), but the anti-N system was much more sensitive than the anti-G system for the detection of crude VHSV I (detection limits, 1 x 10(4) PFU/ml versus 5 x 10(5) PFU/ml). In organ extracts, VHSV I could be detected by both systems 3 days postinfection. The signal for the assay of VHSV I in blood 24 h postinfection was higher with the anti-N system than the anti-G system. Furthermore, VHSV I could be detected in 80% of the brain samples of surviving trout by the anti-N system and also by the anti-G system, but with a lower signal. In conclusion, we have developed a highly sensitive immunoassay for VHSV I that is more rapid and easier to perform than the currently used plaque assay.
Usefulness of the ID32 staph system and a method based on rRNA gene restriction site polymorphism analysis for species and subspecies identification of staphylococcal clinical isolates
Authors: Chesneau O , Aubert S , Morvan A , Guesdon J L , el Solh N
Published: January 2020

The usefulness of the ID32 Staph System and a method based on rRNA gene restriction site polymorphism was evaluated by the study of 42 staphylococcal clinical isolates phenotypically difficult to identify. The ID32 Staph micromethod and the genomic method are adapted for recognition of 27 and 31 staphylococcal taxa, respectively. The genomic method is based on a Dice analysis of the hybridization patterns obtained by cutting the cellular DNA either with EcoRI or with HindIII and by probing with pBA2, containing the Bacillus subtilis gene encoding 16S rRNA, labeled either with [alpha-32P]dCTP or with acetylaminofluorene. This study showed that the nonradioactive labeling provided a better resolution of the hybridizing bands than radioactive labeling. Of the 42 isolates selected, only 22 could be assigned to a staphylococcal species by the ID32 Staph System, whereas 35 could be identified by the genomic method. This latter method also enabled the screening of three unclassified isolates having hybridization patterns more closely related to each other than to any of the 31 staphylococcal taxa investigated. These three isolates could belong to a staphylococcal taxon not yet described.
Restriction endonuclease analysis and ribotyping differentiate genital and nongenital strains of Bacteroides ureolyticus
Authors: Akhtar N , Eley A
Published: January 2020

Thirty-three clinical isolates from male nongonococcal urethritis and 28 isolates from soft tissue infections and ulcers were identified as Bacteroides ureolyticus by conventional bacteriological tests and were compared with five reference strains of the species. Whole-cell proteins from these clinical isolates and the reference strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The majority of the strains from the two sources could be divided into five different groups, named phenons I to V; phenons I to IV have been described previously by others, while phenon V has been described recently by us. Digestion of chromosomal DNA from 16 of the clinical isolates (including strains representative of each of the five SDS-PAGE phenons) and the five reference strains was attempted with restriction endonucleases EcoRI, PstI, SmaI, and HindIII. After electrophoresis in agarose gels, good digestion was observed with HindIII only, and 12 different banding patterns (restriction endonuclease analysis [REA] profiles) were obtained for the 19 strains digested; one nongonococcal urethritis isolate and one reference strain did not show any digestion. From the agarose gels, HindIII-digested fragments of DNA were transferred to nylon membranes by use of vacuum blotting and subjected to hybridization with 32P-labelled 16S-23S rRNA from Escherichia coli. The resultant pattern of bands (ribotypes), which depends on the restriction fragment length polymorphisms in the rRNA genes, was used as a measure of genomic variation within the species. In total, 13 different ribotypes were obtained for the 19 strains. For some strains, good correlation was achieved among the SDS-PAGE phenons, REA profiles, and ribotypes. However, for others, REA analysis and ribotyping were able to discriminate between strains which shared the same SDS-PAGE phenon. Interestingly, these two techniques of DNA characterization were able to differentiate between isolates from the genital tract and those associated with soft tissue infections and ulcers.
Antigenic and molecular characterization of bat rabies virus in Europe
Authors: Bourhy H , Kissi B , Lafon M , Sacramento D , Tordo N
Published: January 2020

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.
Serological studies of antigenic similarity between Japanese spotted fever rickettsiae and Weil-Felix test antigens
Authors: Amano K , Hatakeyama H , Okuta M , Suto T , Mahara F
Published: January 2020

Acute and convalescent-phase sera obtained from 10 patients infected with a Japanese strain of spotted fever group (SFG) rickettsia were tested by the indirect immunoperoxidase test, the Weil-Felix test, an enzyme-linked immunosorbent assay (ELISA), and immunoblotting. By the Weil-Felix test, the reactivity of these sera to the OX2 antigen was higher than those to the OX19 antigen, as is the case with sera from persons infected with other SFG rickettsiae. By ELISA, the titers of immunoglobulin M (IgM) antibodies against OX2 corresponded to the Weil-Felix test titers of these sera against OX2 but not to the titers obtained with IgG antibodies. The reactivity of the patient sera with the OX2 antigen in the Weil-Felix test was probably due to IgM antibodies against antigens which OX2 and SFG rickettsiae have in common. By immunoblotting tests, both IgG and IgM antibodies from the patient sera reacted with lipopolysaccharides from SFG rickettsiae and Proteus strain OX2. These results may show that these lipopolysaccharides contain similar epitopes.
Bordetella bronchiseptica pneumonia and bacteremia following bone marrow transplantation
Authors: Bauwens J E , Spach D H , Schacker T W , Mustafa M M , Bowden R A
Published: January 2020

Bordetella bronchiseptica is a frequent cause of respiratory infections in animals but rarely causes disease in humans. We describe a patient with B. bronchiseptica pneumonia and bacteremia that developed following bone marrow transplantation. B. bronchiseptica infection persisted despite antimicrobial therapy and may have progressed because of the combined effects of the patient's underlying immunosuppression and the antimicrobial antagonism between doxycycline and ciprofloxacin.
Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method
Authors: Nakajima H , Inoue M , Mori T , Itoh K , Arakawa E , Watanabe H
Published: January 2020

We developed a polymerase chain reaction method in order to detect and identify both Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica. Polymerase chain reaction was performed by using a mixture of primers against the inv gene from Y. pseudotuberculosis and the ail gene from pathogenic Y. enterocolitica. Further addition of primers against the plasmid-coded virF gene from Y. enterocolitica made it possible to detect a virulence-associated gene of both species at the same time. This method was proved to be an adequate and convenient procedure for routine detection and identification of these bacilli.
Spectrum of monoclonal antibodies to coxsackievirus B-3 includes type- and group-specific antibodies
Authors: Yagi S , Schnurr D , Lin J
Published: January 2020

Fifteen monoclonal antibodies (MAbs) made to coxsackievirus B-3 were tested against a panel of enteroviruses by indirect immunofluorescence. The MAbs included seven which reacted with coxsackievirus B-3 only, five which reacted with more than one enterovirus included in the panel, one which reacted with broad enteroviral specificity and did not react with any heterologous virus (group specific), and two which reacted with all enteroviruses tested and at least one heterologous virus. The group-specific MAb identified 44 of 45 clinical isolates as enteroviruses, while the two broadly reactive MAbs reacted with all 45 of the clinical isolates. These MAbs are potentially important diagnostic reagents for grouping and typing enteroviruses by indirect immunofluorescence.
Salmonella choleraesuis subsp. indica serovar bornheim causing urinary tract infection
Authors: Snehalatha S , Mathai E , Jayasheela M , Chandy M , Lalitha M K , John T J
Published: January 2020

An unusual Salmonella species, S. choleraesuis subsp. indica serovar bornheim, was isolated from the urine of a patient with aplastic anemia, diabetes mellitus, and a healed urethral injury. An immune response to this isolate was demonstrated by whole-bacterial-cell agglutination.
Osteomyelitis and intervertebral discitis caused by Pseudomonas pickettii
Authors: Wertheim W A , Markovitz D M
Published: January 2020

Pseudomonas pickettii, a nonfermenting, gram-negative rod, is rarely pathogenic. Previous reports of infection with P. pickettii have largely involved direct contamination of supplies presumed to be sterile. We describe a case of vertebral osteomyelitis and intervertebral discitis caused by P. pickettii in a debilitated patient. The aggressive nature of this infection demonstrates that P. pickettii may be a more invasive organism than previously noted, particularly in hosts with weakened immunity secondary to underlying disease.
Evaluation of an indirect immunofluorescence assay for confirmation of human immunodeficiency virus type 1 antibody in U.S. blood donor sera
Authors: Sullivan M T , Mucke H , Kadey S D , Fang C T , Williams A E
Published: January 2020

An indirect immunofluorescence assay (IFA) was evaluated as a confirmatory test for antibody to human immunodeficiency virus type 1 in U.S. blood donor sera previously found to be repeatedly reactive by enzyme immunoassay. IFA results were 100% concordant with a licensed Western blot (immunoblot) for 53 negative and 49 positive samples. Four samples which exhibited antibody to viral proteins from more than one gene, yet were indeterminate by Western blot by the manufacturer's criteria, were also reactive by IFA, whereas 49 additional indeterminate samples were IFA negative.
Identification of some uncommon monounsaturated fatty acids of bacteria
Authors: Moss C W , Daneshvar M I
Published: January 2020

Location of the double-bond position of monounsaturated fatty acids of various bacteria was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Twenty-one relatively novel monoenoic fatty acids were identified among the bacteria studied. All Flavobacterium species contained i17:1 omega 8c, Bacillus alvei contained i16:1 omega 11c and i17:1 omega 12c, and Psychrobacter immobilis contained 12:1 omega 9c. Resolution of cis and trans isomers with capillary gas chromatography and subsequent mass spectrometry permitted positive identification of 16:1 omega 7c and 16:1 omega 7t in Arcobacter (Campylobacter) cryaerophila and 16:1 omega 9c and 16:1 omega 9t in Aerococcus viridans.
Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine
Authors: Chaicumpa W , Ruangkunaporn Y , Burr D , Chongsa-Nguan M , Echeverria P
Published: January 2020

A monoclonal antibody specific for group D Salmonella antigen 9 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the antigen in urine specimens collected from patients with clinical typhoid fever in Jakarta, Indonesia. The ELISA had a sensitivity of 95% in identifying patients in whom Salmonella typhi was isolated from hemocultures, 73% in patients in whom S. typhi was isolated from stool specimens, and 40% in patients in whom the organism was isolated from bone marrow cultures. Among patients in whom S. typhi was isolated from blood cultures, the ELISA had a sensitivity of 65% when a single urine specimen was examined and 95% when serially collected urine specimens were examined. A dot blot immunoassay performed on a nitrocellulose filter in parallel had a sensitivity of 85%, versus 83% for the plate ELISA in which S. typhi was isolated from blood, bone marrow, and/or stool specimens. Since S. typhi antigen is intermittently excreted in the urine of patients with typhoid fever, serially collected urine from patients with typhoid should be tested for antigen 9.
Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies
Authors: Coulson B S
Published: January 2020

Two different neutralization specificities exist on the outer capsid of group A rotaviruses. At least seven VP7 (G) antigenic types are distinguishable among human rotaviruses. Four distinct antigenic (P) types of human rotavirus VP4 corresponding to separate rotavirus gene 4 groups have been described. The aim of this study was to identify P types in clinical specimens by developing an enzyme immunoassay, using P-type-specific neutralizing monoclonal antibodies (N-MAbs). Three N-MAbs primarily or solely recognizing each of P types 4, 6, and 8 and binding to VP4 or its subunit VP5* were derived. These N-MAbs served as detector antibodies in an enzyme immunoassay P-typing system similar to that in use for G typing. P-type specificity was highest when the G-type specificity of the capture antiserum was matched to the G type of the rotavirus in the test sample. The method correctly identified the P types of 13 well-characterized, cell culture-adapted human rotaviruses and was used to classify a further six strains. P typing of 118 rotavirus-positive stools gave results consistent with the P type inferred from the G type for 98 (83%) samples. Twelve (10%) of the stools showed no reaction with any N-MAb and eight (7%) samples were untypeable because of cross-reactivity between N-MAbs or high background readings. This P-typing enzyme immunoassay system is economical and amenable to large-scale use in epidemiological studies. Its use will facilitate assessment of the distribution of P types worldwide and of the role of VP4 in eliciting protective immune responses.
Comparison of the fluorescent treponemal antibody absorption (FTA-ABS) test with the FTA-ABS double staining test for detection of antitreponemal immunoglobulin M in the 19S fraction of human serum
Authors: Ozanne G , d'Halewyn M A , Larsen S A
Published: January 2020

A widely used immunoglobulin M (IgM) detection assay for the diagnosis of neonatal congenital syphilis is the fluorescent treponemal antibody absorption test used with fractionated serum (FTA-ABS 19S IgM test). Reading the results of the FTA-ABS test is more cumbersome than reading those of the FTA-ABS double staining (FTA-ABS-DS) test, a confirmatory test for specific IgG. To verify that the FTA-ABS-DS test used with an anti-human IgM conjugate could detect specific IgM in fractionated serum samples (FTA-ABS-DS 19S IgM test), 164 fractionated (QUIK-SEP IgM Isolation System; ISOLAB, Inc., Akron, Ohio) serum specimens from infected neonates or adults or from IgG-seronegative subjects were tested by both techniques. The sensitivity limits of the two tests were assessed with reactive serum samples diluted to an endpoint titer. Samples nonreactive by the FTA-ABS 19S IgM test (n = 74) were either nonreactive (n = 65), minimally reactive (n = 5), or reactive (n = 4) by the FTA-ABS-DS 19S IgM test. Samples minimally reactive by the FTA-ABS 19S IgM test (n = 32) were minimally reactive (n = 1) or reactive (n = 31) by the double staining test. All samples reactive by the FTA-ABS 19S IgM test (n = 58) were also reactive by the FTA-ABS-DS 19S IgM test. There was a directly proportional linear relationship (r = 0.9794) between titers obtained by both tests. FTA-ABS-DS 19S IgM titers were constantly equal to or higher than FTA-ABS 19S IgM titers. Fluorescence intensity reading repeatability was 91.4% for the FTA-ABS-DS 19S IgM test and 81.7% for the FTA-ABS 19S IgM test (P = 0.015). Because the more easily read FTA-ABS-DS 19S IgM test is at least as sensitive as, if not more sensitive than, the FTA-ABS 19S IgM test, it is a good alternative to the latter test for the detection of specific IgM in human fractionated sera for those using fluorescence microscopes with incident light.
Serotyping of European isolates of Chlamydia psittaci from poultry and other birds
Authors: Vanrompay D , Andersen A A , Ducatelle R , Haesebrouck F
Published: January 2020

A panel of five serovar-specific monoclonal antibodies which distinguish the five known avian serovars of Chlamydia psittaci was used to serotype 45 European avian Chlamydia psittaci isolates. Chlamydial antigen was grown in Buffalo green monkey (BGM) cells or in embryonated chicken eggs and was then inoculated into BGM cells. Serotyping was performed in an indirect immunofluorescence test. The 45 European isolates included 22 isolates from the order Psittaciformes, 9 isolates from the order Columbiformes, 6 isolates from the order Galliformes, 5 isolates from the order Passeriformes, and 3 isolates from the order Anseriformes. All of these were successfully serotyped. No additional serovars were found. One isolate from a duck and two isolates from psittacine birds gave positive immunofluorescences with two monoclonal antibodies considered to be specific for two different serovars. These three isolates were cloned by an agar overlay method. Serotyping of the clones demonstrated that the duck and one psittacine bird each were infected with two different serovars. After cloning, one isolate from a psittacine bird reacted only with serovar A. From these results it was concluded that this serotyping system allows the classification of all isolates tested so far. The results show that similar serovars are prevalent in avian species in Europe and the United States. The results also indicate that birds from a certain order are more susceptible to a distinct serovar. The use of a panel of serovar-specific monoclonal antibodies in the immunofluorescence test provides a reliable method for serotyping avian isolates. Monoclonal antibodies to new avian isolate serovars can easily be added to the panel, which makes the system useful for epidemiological studies.
Enzyme-linked immunosorbent assay for detection of human immunoglobulin G to lipopolysaccharide of spotted fever group rickettsiae
Authors: Jones D , Anderson B , Olson J , Greene C
Published: January 2020

An enzyme-linked immunosorbent assay for detecting human immunoglobulin G to spotted fever group rickettsiae was developed and tested. The assay uses proteinase K-resistant material, characteristic of the rickettsial lipopolysaccharides shown to be group specific by immunoblots, as the antigen. The results indicate that the assay provides a sensitive, yet specific, alternative method for diagnosing rickettsial diseases.
Use of electrophoretic polymorphisms of esterases for differentiation of Clostridium argentinense strains
Authors: Bories P N , Antoniotti C , Pons J L , Leluan G
Published: January 2020

Esterase electrophoresis was used to study 10 strains of Clostridium argentinense, including 7 toxigenic and 3 nontoxigenic strains. On the basis of the electrophoretic mobilities and hydrolytic specificities toward five synthetic substrates, different esterase profiles could be defined for almost all strains, revealing the heterogeneity of bacterial clones. Therefore, electrophoretic polymorphism of esterases can be used for differentiation of C. argentinense in population genetic or epidemiological studies.
Investigation of sera reactive to hepatitis C virus by second-generation enzyme immunoassay
Authors: Frost E H
Published: January 2020

Hepatitis C virus antibodies were detected by a second-generation enzyme immunoassay and investigated with a second-generation recombinant immunoblot assay. Most sera with optical density values of > or = 2.0, particularly those with optical densities of > or = 2.0 after a further 10-fold dilution, were positive in the second-generation recombinant immunoblot assay. Repeat testing of hemodialysis patients revealed excellent reproducibility and increased sensitivity of the second-generation enzyme immunoassay.
Serotypic and genotypic characterization of human serotype 10 rotaviruses from asymptomatic neonates
Authors: Dunn S J , Greenberg H B , Ward R L , Nakagomi O , Burns J W , Vo P T , Pax K A , Das M , Gowda K , Rao C D
Published: January 2020

Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.
Rapid immunodot technique for identifying Bordetella pertussis
Authors: Sanden G N , Cassiday P K , Barbaree J M
Published: January 2020

We developed and evaluated a rapid test with monoclonal antibodies to identify cultures of Bordetella pertussis. Samples of 5 microliters of cells suspended in formalin-saline were dried onto a nitrocellulose disk. The disk was placed in a filtration device, and 5-microliters volumes of murine monoclonal antibody directed against B. pertussis lipooligosaccharide and peroxidase conjugate were added consecutively, with washing after each addition. The disk was removed and immersed in peroxidase substrate solution. All of 66 B. pertussis isolates confirmed by direct fluorescent-antibody assay were correctly identified by using four different monoclonal antibodies. One of the monoclonal antibodies did not react with over 20 bacterial species tested, including other Bordetella, Acinetobacter, Haemophilus, Moraxella, Mycobacterium, Neisseria, and Staphylococcus spp. This technique detected > or = 2 micrograms of lipooligosaccharide per ml or > or = 5 x 10(8) B. pertussis cells per ml. This rapid procedure used small amounts of reagents, needed less equipment, and was less subjective and more specific than the direct fluorescent-antibody assay.
Biochemical properties and fatty acid composition of Mycobacterium haemophilum: study of 16 isolates from Australian patients
Authors: Portaels F , Dawson D J , Larsson L , Rigouts L
Published: January 2020

The biochemical properties and fatty acid compositions of 16 strains of Mycobacterium haemophilum from Australian patients were studied. The strains proved to be indistinguishable from each other but could readily be differentiated from other slowly growing mycobacteria with similar cultural features. Mycolic acid analyses revealed the presence of alpha-, methoxy-, and ketomycolates. The fatty acid composition supports the validity of the fact that M. haemophilum is a distinct species. The fatty acid composition was consistent among the 16 strains, but it was unusual in that there was some resemblance to the fatty acid composition of M. leprae. The wide range of pHs (5.4 to 7.4) that supported growth of M. haemophilum on artificial medium is in keeping with suggestions that M. haemophilum exists in an environmental habitat.
Do temperature-sensitive auxotrophs of Escherichia coli have special virulence?
Authors: Kunin C M , Hua T H , Krishnan C , White L V
Published: January 2020

To determine whether temperature (42 degrees C)-sensitive auxotrophs of Escherichia coli have special virulence properties (W. D. Welch, D. Kitts, H. S. Moyed, and L. D. Thrupp, J. Clin. Microbiol. 13:606-608, 1981), we examined 301 strains isolated from patients with bacteremia or acute cystitis and from the stools of healthy subjects. Of these strains, 49.5% grew at 42 degrees C without supplements, 39.2% required a nutritional supplement, and 11.3% failed to grow even with selected nutrients. Nicotinamide restored growth for 35.2% of strains at either 37 or 42 degrees C. Some of strains required methionine, glutamic, aspartic, and amino acid mixtures or NaCl for growth at 42 degrees C. Temperature-sensitive strains were significantly more abundant in isolates from blood and urine than in stool, but temperature-sensitive auxotrophs were isolated at about the same frequency from each site. There were no discernible clonal patterns, by serotype, among of the nicotinamide-requiring temperature-sensitive auxotrophs. Resistance to trimethoprim-sulfamethoxazole was associated with ability to grow at 42 degrees C. This was not observed with any other antimicrobial drug. Temperature-sensitive strains are a heterogenous group. The relationship of temperature-sensitive auxotrophy to virulence is uncertain.
Detection of Epstein-Barr virus and human papillomavirus in head and neck tumors
Authors: Tyan Y S , Liu S T , Ong W R , Chen M L , Shu C H , Chang Y S
Published: January 2020

The presence of Epstein-Barr virus (EBV) DNA and human papillomavirus (HPV) DNA in 74 head and neck tumor tissues was examined by the polymerase chain reaction and DNA sequencing analysis. EBV DNA sequence was detected in all 30 nasopharyngeal-carcinoma tissue samples and in 30 of 44 other head and neck tumor samples. HPV DNA sequence was detected in 14 of 30 nasopharyngeal-carcinoma tissue samples and in 11 of 44 other tumor samples. Coinfection of both viruses was observed in 14 nasopharyngeal-carcinoma tissue samples but only in 5 other head and neck tumor samples including 3 hypopharyngeal-carcinoma tissue samples. Our data indicate that EBV is closely associated with nasopharyngeal- carcinoma and may also be related to hypopharyngeal-carcinoma. In addition, a relatively high percentage of EBV-positive nasopharyngeal- and hypopharyngeal-carcinoma tissue specimens contained HPV sequence. The significance of the coexistence of EBV and HPV in these tumor tissues requires further study.
Development and use of species-specific oligonucleotide probes for differentiation of Streptococcus uberis and Streptococcus parauberis
Authors: Bentley R W , Leigh J A , Collins M D
Published: January 2020

Oligonucleotide probes specific for 16S rRNA and capable of differentiating Streptococcus uberis and S. parauberis from each other and other esculin-hydrolyzing streptococci were developed. Use of a mini-RNA extraction technique for gram-positive cocci associated with bovine mastitis has allowed the probes to be used for identification of esculin-hydrolyzing streptococci from two dairy herds at the Institute for Animal Health, Compton, United Kingdom. One hundred seventy-nine of 206 isolates were identified as S. uberis, 3 were identified as S. parauberis, and 24 were not identified. Isolates not identified by the probes were tested biochemically and found to be mainly Enterococcus faecium, E. faecalis, or S. bovis.
Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods
Authors: Shawar R M , el-Zaatari F A , Nataraj A , Clarridge J E
Published: January 2020

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.
Characterization of ehrlichial organisms isolated from a wild mouse
Authors: Kawahara M , Suto C , Rikihisa Y , Yamamoto S , Tsuboi Y
Published: January 2020

An infectious agent was isolated from the enlarged spleen of a wild mouse, Eothenomys kageus, by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed splenomegaly, and the agent was maintained by serial passage of spleen homogenates in laboratory mice. The agent in the spleen homogenate was inactivated after incubation at 37 or 50 degrees C. Tetracyclines were effective in preventing infection of mice with this agent, but penicillin and sulfonamides were ineffective. Cytoplasmic inclusion bodies were observed in the peritoneal macrophages of infected mice. Electron microscopy revealed numerous small pleomorphic cocci within membrane-lined vacuoles in the cytoplasm of splenic macrophages. Morphologically similar to the ehrlichial organisms, each organism was surrounded by a distinct plasma membrane and rippled outer cell membrane without a distinct peptidoglycan layer. The agent did not grow in chicken embryos, and the Weil-Felix test result was negative. In the indirect fluorescent-antibody test, the agent reciprocally cross-reacted with Ehrlichia canis and cross-reacted somewhat with Ehrlichia sennetsu but did not cross-react with Ehrlichia risticii, Neorickettsia helminthoeca, Rickettsia tsutsugamushi, or Chlamydia spp. The mouse antiserum against this agent reacted with 64-, 47-, 46-, 44-, and 40-kDa proteins of E. canis by Western blotting (immunoblotting). Since E. canis and closely related Ehrlichia chaffeensis and Ehrlichia ewingii are not known to proliferate or cause splenomegaly in mice, these results suggest that the agent is a new species within the tribe Ehrlichieae of the family Rickettsiaceae. The finding suggests that wild rodents may serve as reservoirs for pathogenic ehrlichiae.
Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli
Authors: van Gelder P , Bosman F , de Meuter F , van Heuverswyn H , Hérion P
Published: January 2020

A 330-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii 54-kDa rhoptry protein (ROP2) was expressed in Escherichia coli as a fusion polypeptide containing a 48-amino-acid sequence derived from phage lambda protein Cro and E. coli protein LacI followed by six consecutive histidyl residues. Metal chelate affinity chromatography provided an easy way to isolate the recombinant product in a highly purified form (> 95%). When this material was used to develop an immunoglobulin G (IgG) enzyme-linked immunosorbent assay for diagnosis of toxoplasmosis, the test reached a sensitivity of 89%. The sensitivity of the assay was similar whether the sera contained T. gondii-specific IgM or were devoid of such IgM. It was also found that ROP2 is a conserved antigen since antibodies against the recombinant antigen could be detected in mice experimentally infected with 11 independent T. gondii isolates originating from infected human tissues tested. Thus, the 54-kDa rhoptry antigen could advantageously complement other previously described T. gondii antigens for the development of more-sensitive and more-informative recombinant antigen-based tests for toxoplasmosis diagnosis.
Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital
Authors: Bingen E H , Desjardins P , Arlet G , Bourgeois F , Mariani-Kurkdjian P , Lambert-Zechovsky N Y , Denamur E , Philippon A , Elion J
Published: January 2020

Over a 12-month period, 43 children in eight different wards of our hospital (Hôpital Robert Debré) were infected or colonized with Klebsiella pneumoniae strains producing extended broad-spectrum beta-lactamases. The epidemiology of the outbreak was studied by a molecular approach including the determination of the beta-lactamase physicochemical parameters and plasmid profiles, as well as analysis of the restriction fragment length polymorphisms of the rDNA regions (ribotyping). The last approach produced 12 and 5 different patterns with EcoRI and HindIII, respectively, thus identifying 15 different ribotypes among the 43 clinical K. pneumoniae strains. However, 60% of the strains in six wards belonged to only two ribotypes, whereas nine ribotypes were observed only once. Twelve isolates from different wards that were representative of the eight most common ribotypes showed four different beta-lactamase isoelectric focusing patterns and seven different plasmid profiles by direct analysis or after EcoRI digestion. Thus, at least two genetically unrelated strains in the same ward were found to have the same plasmid content. Our results show the complexity of the outbreak, which was associated with patient-to-patient cross-contamination with several epidemic strains with different plasmid contents, interspersed sporadic cases with nonepidemic strains, and the possible spread of a plasmid. The combination of plasmid profile analysis and ribotyping therefore seems to be powerful at deciphering the details of such outbreaks.
Identification of clinical yeast isolates by using the Microring YT
Authors: McGowan K L , Mortensen J E
Published: January 2020

The Microring YT (MYT; Medical Wire & Equipment Co., Victory Gardens, N.J.) is a system for the rapid (24 to 48 h) identification of yeasts. The MYT system was evaluated and compared with the API20C (Analytab Products, Plainview, N.Y.) system for its ability to identify 677 clinical yeast isolates. Only 458 isolates (68%) were correctly identified by the MYT system, and the accuracy of the system varied considerably (0 to 96%), depending on the species. While MYT was less expensive and convenient to use and results were available 24 h sooner, it is inadequate for identification of many commonly isolated yeasts and is not designed for the identification of Cryptococcus species.