bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates
Authors: Hirai Y , Shiode J , Masayoshi T , Kanemasa Y
Published: January 2020

We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates. The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons. Cross-reactivity of the antibody with species of mycoplasmas other than M. genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium. Though the absorbed antibody cross-reacted with M. genitalium, the titer was significantly lower than when tested against M. pneumoniae. Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells. Throat smears from 49 patients with serologically confirmed M. pneumoniae infections were examined by our indirect immunofluorescence method. Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection.
Meningitis caused by Psychrobacter immobilis in an infant
Authors: Lloyd-Puryear M , Wallace D , Baldwin T , Hollis D G
Published: January 2020

Psychrobacter immobilis was isolated from the cerebrospinal fluid (CSF) and blood of a 2-day-old infant who appeared well except for a fever and a full anterior fontanelle. The infant was treated with antibiotics intravenously. After 48 h, he became afebrile and CSF and blood cultures were negative; he was then discharged. After 96 h of incubation, CSF and blood cultures yielded a gram-negative organism, P. immobilis. The child was readmitted to the hospital, and the same organism was again isolated from his blood and CSF.
A novel group A rotavirus G serotype: serological and genomic characterization of equine isolate FI23
Authors: Browning G F , Fitzgerald T A , Chalmers R M , Snodgrass D R
Published: January 2020

Equine rotavirus FI23 was shown to be prototypic of a novel G serotype, provisionally G14, by cross-neutralization and VP7 sequence determination. Although distinct, there are as few as six differing amino acid residues (92, 94, 96, 146, 147, and 221) in the VP7 antigenic regions of FI23 and G3 rotaviruses.
Reactivity of monoclonal antibodies to the 41-kilodalton protein of porcine group C rotavirus with homologous and heterologous rotavirus serogroups in immunofluorescence tests
Authors: Ojeh C K , Jiang B M , Tsunemitsu H , Kang S Y , Weilnau P A , Saif L J
Published: January 2020

Three monoclonal antibodies (MAbs) to porcine group C rotavirus immunoprecipitated the major inner capsid protein (41 kDa) but failed to precipitate group A rotavirus proteins. In immunofluorescence tests of rotavirus-infected cell cultures or pig intestines, the MAbs recognized porcine and bovine group C rotaviruses but not group A or B rotaviruses. These MAbs may recognize the group C rotavirus counterpart to VP6 of group A rotaviruses and may be useful as diagnostic reagents.
Evaluation of the E test for quantitative antimicrobial susceptibility testing of Helicobacter pylori
Authors: Glupczynski Y , Labbé M , Hansen W , Crokaert F , Yourassowsky E
Published: January 2020

The Progressive Diagnostics Manufacturers epsilometer test (E test; AB Biodisk, Solna, Sweden), a quantitative variant of the disk diffusion technique, was evaluated comparatively to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. A collection of 79 H. pylori clinical strains, including isolates with known resistance to various antimicrobial agents, was tested against 12 different antimicrobial agents. All strains were tested on Columbia agar supplemented with 10% horse blood. Plates were incubated at 37 degrees C in microaerobic atmosphere (5% O2, 10% CO2), and readings were done after 3 days of incubation. In general, E test MICs were easy to interpret and the correlation between MICs by the agar dilution method and the E test was good, with 86 and 99.5% of results being within, respectively, 1 and 2 log2 dilution steps in a total of 936 tests. All strains of H. pylori with documented resistance to the tested agents were detected by the E test. Thus, the E test appears to be an easy and reliable method for determination of MICs of antibiotics for H. pylori, and it may offer an interesting alternative to MIC determination by the agar dilution technique.
Asymptomatic respiratory tract infection with Chlamydia pneumoniae TWAR
Authors: Hyman C L , Augenbraun M H , Roblin P M , Schachter J , Hammerschlag M R
Published: January 2020

Chlamydia pneumoniae is a newly recognized organism associated with respiratory tract infections. Asymptomatic infection with C. pneumoniae, although it has been suggested to occur, has not been previously documented. We describe two asymptomatic individuals infected with this organism; these infections demonstrate that C. pneumoniae is able to establish a subclinical infection.
Rapid and biologically safe diagnosis of African swine fever virus infection by using polymerase chain reaction
Authors: Steiger Y , Ackermann M , Mettraux C , Kihm U
Published: January 2020

In order to circumvent the need for infectious virus for the diagnosis of African swine fever (ASF), we established the polymerase chain reaction (PCR) technique for the detection of ASF virus (ASFV) DNA. A 740-bp fragment that originated from the conserved region of the viral genome was partially sequenced. From this sequence, four PCR primers and one oligonucleotide probe were designed and synthesized. A specific 640-bp PCR product was amplified by using oligonucleotides 1 and 5 as primers and extracts of the following samples as templates: organs and plasma obtained from ASFV-infected pigs, ASFV-infected cell cultures, and cloned DNA fragments containing the same conserved genomic region as that in the original 740-bp clone. No specific reaction products were observed in the corresponding controls. The identities of the PCR products were confirmed either by a second amplification with nested primers or by hybridization with a specific, biotinylated oligonucleotide probe. PCR proved to be a quicker and more sensitive method than virus isolation followed by the hemadsorption test when spleen and plasma samples from experimentally ASFV-infected pigs were tested. Furthermore, cloned virus DNA could be used as a positive control in the place of a live virus control. This is advantageous whenever the use of live virus is undesirable.
Reevaluation of the API 20E identification system versus conventional biochemicals for identification of members of the family Enterobacteriaceae: a new look at an old product
Authors: O'Hara C M , Rhoden D L , Miller J M
Published: January 2020

The API 20E bacterial identification system has been used for 19 years, often as the standard with which other identification systems are compared. Because the accuracy of this system compared with conventional biochemical tests has not been determined in many years, we evaluated the API 20E linear strip by using 291 typical and atypical strains of the family Enterobacteriaceae taken from a culture collection. At 24 h, the API 20E correctly identified by genus and species 229 of 291 (78.7%) of the strains, using Salmonella and Shigella serotyping where indicated. At 48 h, 95.2% were correctly identified by using additional biochemical tests as recommended by the manufacturer. The API 20E misidentified eight (2.7%) strains; these strains were not limited to any particular genus. When 81 of these Enterobacteriaceae strains were arranged into a weighted assortment correlating to the frequency with which they might be found in a clinical laboratory, the API 20E correctly identified 71 (87.7%) at 24 h and 78 (96.3%) at 48 h. This evaluation concluded that the accuracy of the identification of Enterobacteriaceae strains at 24 h (78.7%) may be significantly lower than that of earlier evaluations. However, there is no significant difference in the ability of the API 20E to correctly identify "challenge" type organisms (229 of 291) versus routine hospital isolates (71 of 81) (P greater than 0.05), but the system is not as accurate as the conventional biochemical method of identification.
Immunological changes in cats with concurrent Toxoplasma gondii and feline immunodeficiency virus infections
Authors: Lin D S , Bowman D D , Jacobson R H
Published: January 2020

To examine the immunological changes in cats concurrently infected with feline immunodeficiency virus (FIV) and Toxoplasma gondii, kittens (four per group) were inoculated with FIV, T. gondii, both agents, or no pathogens. Blood mononuclear cells and plasma were collected weekly for lymphocyte assays and serology. At week 14, spleen and lymph node cells were used for lymphocyte assays; brains and mesenteric lymph nodes were used for isolation of T. gondii. More T. gondii organisms were present in tissues of the dually infected cats than in tissues of cats with toxoplasmosis alone. Two dually infected cats and one cat infected with T. gondii developed chorioretinitis. Spleen, lymph node, and blood mononuclear cells from dually infected cats had the greatest reduction in mitogenic responses. By week 3, cats infected with FIV underwent a decrease in the number of CD4 cells that was not changed by concurrent T. gondii infection; the number of CD8 cells increased only in cats infected with T. gondii alone. For cats infected with T. gondii, the responses of lymphocytes to T. gondii antigen were not affected by FIV infection; the responses to FIV antigen were negligible in all groups. Overall, this study indicates that FIV infection favors T. gondii proliferation. Also, the establishment of toxoplasmosis may enhance FIV-induced immunodeficiency and is likely to cause a more rapid disease progression than that from infection with FIV alone.
Evaluation of a commercially available second-generation immunoglobulin G enzyme immunoassay for detection of Helicobacter pylori infection
Authors: Goossens H , Glupczynski Y , Burette A , Van den Borre C , Butzler J P
Published: January 2020

We evaluated a commercially available second-generation anti-H. pylori immunoglobulin G enzyme immunoassay (EIA) (Cobas Core Anti-Helicobacter pylori EIA; Roche S. A., Basel, Switzerland) for serodiagnosis of H. pylori infection. The results of the assay were assessed in relation to the results of bacterial culture, urease testing, and histological Giemsa stain of gastric biopsy specimens from 1,134 patients with a variety of symptoms relating to the upper gastrointestinal tract. H. pylori was detected in biopsy specimens from 660 (58.2%) patients: 6 had a normal mucosa, 123 had chronic gastritis only, and 531 were found to have chronic active gastritis by histology; endoscopy showed duodenal and gastric ulcers in 137 and 64 patients of the last two groups, respectively. The test was evaluated with different age and ethnic groups. The prevalence, sensitivity, specificity, and positive and negative predictive values were, respectively, (i) for Belgian patients between 18 and 40 years old, 34, 93, 95, 91, and 96%; (ii) for Belgian patients more than 40 years old, 53, 96, 91, 93, and 95%; and (iii) the Mediterranean patients more than 17 years old, 87, 94, 70, 95, and 64%. All sera showing discordant immunoassay results compared with the results of histology and culture of biopsy specimens, as well as those with borderline immunoassay results, were tested further by immunoblotting. Among the EIA results considered false negative, we demonstrated an absence of seroconversion in 14 of 19 patients tested by immunoblotting. Among the EIA results considered false positive, immunoblotting showed the presence of specific antibodies in 28 of 37 patients tested. Among the borderline results obtained in the first assay with 22 patients' sera, a second assay showed positive results in 10 patients (8 were positive by immunoblotting) and negative reactions in 10 patients (9 were negative by immunoblotting), whereas 2 remained borderline. These data indicate that sera showing borderline immunoassay results must be tested again. In conclusion, this commercially available second-generation EIA, which is easy and quick to perform, was found highly reliable for the serodiagnosis of H. pylori infection.
Serotype distribution of Campylobacter jejuni and Campylobacter coli isolated from hospitalized patients with diarrhea in central Australia
Authors: Albert M J , Leach A , Asche V , Hennessy J , Penner J L
Published: January 2020

Campylobacter jejuni and/or Campylobacter coli was cultured from 218 of 1,078 patients of all age groups admitted to Alice Springs Hospital, Alice Springs, central Australia, between July 1988 and June 1989 for treatment of diarrhea. One hundred sixty-six Campylobacter colonies from 127 patients were subjected to O serotyping by using the Penner typing scheme. All except 29 colonies could be serotyped. A total of 46 serotypes were identified, and the predominant serotypes were O:8, 17, O:22, O:1,44, and O:19. A large proportion of colonies reacted with more than one antiserum, and nine serotypes had antigenic compositions not observed previously. Several patients had multiple infections with more than one serotype, and some patients were shown for the first time to be infected with up to three different serotypes. Repeated reinfections with different serotypes were seen in some patients. In some patients, provided it was not due to reinfection with the same serotype, long-term excretion of the same serotype was seen, and for the first time, one patient showed evidence of excretion of the same serotype for up to 73 days.
Capnocytophaga canimorsus septicemia caused by a dog bite in a hairy cell leukemia patient
Authors: Ndon J A
Published: January 2020

Fatal septicemia developed in a splenectomized patient with hairy cell leukemia following a dog bite. Capnocytophaga canimorsus, a slowly growing gram-negative bacillus, was isolated from the patient's blood. Although a rare complication of dog bites in the normal population, this bacterium should be suspected and promptly treated in immunologically compromised dog bite victims. Furthermore, immunocompromised patients should be made aware of the dangers of dog ownership.
Detection of human immunodeficiency virus type 1 antibody by using commercially available whole-cell viral lysate, synthetic peptide, and recombinant protein enzyme immunoassay systems
Authors: Johnson J E
Published: January 2020

A total of 575 serum specimens received for human immunodeficiency virus type 1 (HIV-1) antibody determination were tested prospectively by enzyme immunoassay with a whole-cell viral lysate (VL) (Genetic Systems Corp.), a synthetic peptide (SP) (United Biomedical, Inc.), and a recombinant protein (RCP) (Syva Co.). Concordance of all three antigens was noted for 559 of 575 (97.2%) specimens tested. Of the specimens tested, 90 (15.7%) were positive and 469 (84.3%) were negative. Retrospective testing by SP and RCP of 86 specimens indeterminate for HIV-1 antibody by VL, Western blot (immunoblot), and immunofluorescence was also performed. The results of both phases of this study indicate that the specificity of the three antigens is RCP greater than SP greater than VL. As noted from the prospective phase, the sensitivities of the antigens appear to be equivalent, indicating that the RCP and SP systems could be used in place of, or to confirm, a VL enzyme immunoassay screening test.
Rapid diagnosis of Brucella melitensis in blood: some operational characteristics of the BACT/ALERT
Authors: Solomon H M , Jackson D
Published: January 2020

A clinical isolate of Brucella melitensis was detected in a blood culture with the BACT/ALERT after an incubation period of 2.8 days. Dilution studies revealed an inverse linear relationship between the log of the initial concentration of the organism and the time to detection of a positive result. Reproducibility studies demonstrated a mean detection time of 48 +/- 1 h in 8 of 10 replicates seeded with a stock containing 10(2) CFU/ml.
Recovery of Histoplasma capsulatum from blood in a commercial radiometric Mycobacterium medium
Authors: Merz W G , Kodsy S , Merz C S
Published: January 2020

We report the recovery of Histoplasma capsulatum from blood specimens cultured for Mycobacterium sp. in BACTEC 13A radiometric medium. H. capsulatum was recovered from six of eight blood specimens submitted for mycobacterial cultures from five human immunodeficiency virus-positive individuals. Initial positive metabolic signals occurred at a mean of 11 days, but no organisms were detected with acid-fast stains. The bottles remained positive, and after an additional incubation (mean, 8 days), yeast cells morphologically compatible with H. capsulatum were detected when aliquots were stained with acridine orange. Therefore, when radiometric mycobacterial blood cultures with persistent positive metabolic signals and negative acid-fast stains are encountered, acridine orange staining and subculturing for a variety of microorganisms, including fungi, e.g., H. capsulatum, should be considered.
Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide sequence within the insertion sequence-like element
Authors: Wang R Y , Hu W S , Dawson M S , Shih J W , Lo S C
Published: January 2020

A new assay using the polymerase chain reaction to amplify a 206-nucleotide specific gene sequence within the insertion sequence-like element of Mycoplasma fermentans has been developed. The unique insertion sequence-like element exists in multiple copies in the M. fermentans genome. The assay selectively amplifies DNA from all strains of M. fermentans tested. In contrast, DNA from other species of human and nonhuman mycoplasmas, common tissue culture-contaminating mycoplasmas, and bacteria, as well as human, monkey, and mouse tissues do not produce the amplified DNA products specific for M. fermentans.
Comparison of six different culture media for isolation of Treponema hyodysenteriae
Authors: Achacha M , Messier S
Published: January 2020

Fecal material from pigs experimentally infected with Treponema hyodysenteriae was collected and inoculated on six different selective media to compare their abilities for recovery of T. hyodysenteriae. Additionally, various types of samples were compared to find the most appropriate for submitting material to laboratories. Isolation rates for T. hyodysenteriae were 89.7% on BJ medium, 88.3% on TSA-BJ medium, 76.6% on SVC medium, 76.6% on BA2-BJ medium, 75.2% on BA2-SVC medium, and 52.4% on TSA-S400 medium. BJ medium was the most useful, yielding good growth of T. hyodysenteriae and inhibition of the normal fecal flora. Also, undiluted fecal material kept at refrigeration temperature allowed better recovery of T. hyodysenteriae during storage than swabs.
Detection of enteroviruses in cell cultures by using in situ transcription
Authors: Carstens J M , Tracy S , Chapman N M , Gauntt C J
Published: January 2020

In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells. A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays. Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time. Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio. Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity. Exogenous RNase H added to the transcription reaction ablated the signal. Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes. The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes.
Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in U.S. hospitals: epidemiological significance
Authors: Zierdt C H , Hosein I K , Shively R , MacLowry J D
Published: January 2020

For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, greater than 4 micrograms/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7/47/53/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 micrograms/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-beta-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-beta-lactamase producing S. aureus are distinct epidemic strains.
Use of polymerase chain reaction for rapid diagnosis of tuberculosis
Authors: Cousins D V , Wilton S D , Francis B R , Gow B L
Published: January 2020

A DNA amplification assay using the polymerase chain reaction technique designed for the rapid identification of Mycobacterium bovis organisms was used to test 211 human mycobacterial isolates and 177 clinical specimens previously submitted for routine mycobacterial culture. The procedures described could be used by routine or specialist laboratories for identification of M. tuberculosis complex organisms in 4 h and/or as a rapid screening method for the direct detection of M. tuberculosis complex organisms in specimens.
Detection of human immunodeficiency virus type 1 infection in young pediatric patients by using polymerase chain reaction and biotinylated probes
Authors: Brandt C D , Rakusan T A , Sison A V , Josephs S H , Saxena E S , Herzog K D , Parrott R H , Sever J L
Published: January 2020

Polymerase chain reaction (PCR) testing using up to four primer pairs and biotinylated probes was 97.9% sensitive (188 of 192 specimens positive) and 100% specific (267 of 267 specimens negative) for detecting the presence or absence of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells from pediatric patients whose HIV status has been confirmed. SK38/39 and SK145/150 were the most sensitive primer pairs, respectively detecting HIV DNA in 95.6 and 95.9% of peripheral blood mononuclear cell specimens from HIV-infected children and collectively detecting all adequately tested PCR-positive specimens. Primer pairs SK29/30 and SK68/69 respectively detected HIV DNA in only 76.4 and 76.6% of HIV-positive specimens. Among infants born to HIV-seropositive mothers, 30 who subsequently were confirmed to be infected were sampled when they were less than or equal to 6 months of age; in all but one infant, HIV DNA was found in the first specimen collected. Among the nine youngest infected infants tested, all were PCR positive by 38 days of age. PCR methods thus have reliably detected vertically transmitted HIV infection early in life.
Susceptibility testing of clinical isolates of Enterococcus faecium and Enterococcus faecalis
Authors: Louie M , Simor A E , Szeto S , Patel M , Kreiswirth B , Low D E
Published: January 2020

We collected 103 clinical Enterococcus faecium isolates from across Canada, performed standard broth microdilution susceptibility testing, and compared these results with results from the MicroScan Pos MIC Type 6 panel (Baxter Health Care Corp., West Sacramento, Calif.) and the AMS-Vitek Gram-Positive Susceptibility card (Vitek Inc., St. Louis, Mo.). High-level aminoglycoside resistance to gentamicin and streptomycin was detected by a single-concentration agar method with 1,000 micrograms of each aminoglycoside per ml. In addition, we tested the effect of the lower calcium content in broth media as recommended in National Committee for Clinical Laboratory Standards (NCCLS) guideline M7-A2 on the activity of the highly calcium-dependent agent daptomycin. Of the 103 E. faecium isolates, there were 4 and 30 isolates with high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR), respectively. An additional 39 (37 with HLGR and 36 with HLSR) E. faecium isolates were tested by both the MicroScan and the AMS-Vitek systems. The AMS-Vitek card demonstrated sensitivities of 95 and 82% for detecting HLGR strains and HLSR strains, respectively. The MicroScan panel demonstrated improved sensitivities for detecting HLGR (42 to 97%) and HLSR (64 to 84%) when readings were performed manually instead of being generated automatically. Ampicillin resistance (MIC, greater than or equal to 16 micrograms/ml) was detected in 23 of the 103 E. faecium isolates. Only 14 and 20 of these were detected by the MicroScan panels and AMS-Vitek cards, respectively. beta-Lactamase activity was not detected in any isolates. The lower calcium content in broth media recommended by NCCLS guideline M7-A2 markedly reduced the in vitro activity of daptomycin against Enterococcus spp.
Isolation of an avianlike group A rotavirus from a calf with diarrhea
Authors: Brüssow H , Nakagomi O , Gerna G , Eichhorn W
Published: January 2020

An atypical group A rotavirus (993/83) was isolated from a 3-day-old German calf with diarrhea. It differed from 35 conventional German bovine rotavirus isolates analyzed previously with respect to subgroup (strain 993/83 was non-subgroup I and non-subgroup II), serotype (strain 993/83 showed a two-way cross-reaction with serotype 7 and a one-way cross-reaction with serotype 3), and electropherotype (strain 993/83 showed comigrating gene segments 10 and 11). Isolate 993/83 reacted with only one of four monoclonal antibodies that recognized a common VP6 epitope(s). In addition, VP6 and VP2 of isolate 993/83 showed one-dimensional peptide maps that differed substantially from the peptide maps of VP6 and VP2 from all bovine rotavirus isolates. By RNA-RNA hybridization, the 993/83 probe failed to react with a panel of mammalian rotavirus strains, including bovine rotaviruses. It hybridized, however, to genomic RNA of an avian rotavirus strain. Isolate 993/83 could thus represent a candidate for a natural interspecies transmission of rotavirus between different classes of vertebrates.
Isolation and characterization of two distinct human rotavirus strains with G6 specificity
Authors: Gerna G , Sarasini A , Parea M , Arista S , Miranda P , Brüssow H , Hoshino Y , Flores J
Published: January 2020

Two new human rotavirus (HRV) strains, PA151 and PA169, with subgroup I specificity and a long RNA pattern, yet with a serotype G (VP7) specificity different from those of any of the six well-established HRV serotypes (G1 to G4, G8, and G9), were isolated 3 months apart from two children with acute gastroenteritis in Sicily, southern Italy, in the winter season of 1987 and 1988. The HRV isolates were adapted to growth in cell cultures and were then characterized by neutralization and RNA-RNA (Northern blot) hybridization. Cross-neutralization studies with type-specific immune sera to RV serotypes 1 to 10 showed the antigenic relatedness of the two strains with serotype 6 bovine strains UK and NCDV. Monoclonal antibodies to VP7 of UK were able to recognize UK and NCDV strains as well as both HRV isolates. Cross-hybridization studies showed a genetic relatedness of PA151 and PA169 to bovine strains for all genes except gene 4. Gene 4 of PA151 appeared to be genetically related to that of AU228 (a human strain of subgroup I and with serotype G3 specificity that belongs to a feline genogroup), whereas gene 4 of PA169 appeared to be unique, yet it was related to gene 4 of two recently reported subgroup I HRV strains, one (PA710) with serotype G3 specificity and the other (HAL1271) with serotype G8 specificity. The new HRV strains must be taken into consideration when deciding strategies for the development of an effective RV vaccine.
Detection of Norwalk virus in stool by polymerase chain reaction
Authors: Jiang X , Wang J , Graham D Y , Estes M K
Published: January 2020

A method of reverse transcription (RT) and polymerase chain reaction (PCR) for the detection of Norwalk virus in human stools was developed. A cationic detergent, cetyltrimethylammonium bromide, was found to effectively remove from stool extracts factors that inhibit the RT-PCR assay. The specificities of the tests were shown by hybridization of the amplified DNA with Norwalk virus-specific cDNA probes and a consistent correlation between virus detection in stools and infection of volunteers. RT-PCR detected virus in stool samples diluted 10(-4) and was about 100 times more sensitive than dot blot hybridization. In serial stool samples collected before and at different times after inoculation of 10 volunteers with Norwalk virus, 37 of 55 were positive by RT-PCR, but only 27 were positive by dot blot hybridization (chi 2 = 22.96; P less than 0.001). Further application of this method should allow detection of Norwalk virus in food or environmental samples such as shellfish and shellfish waters.
Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system
Authors: Kolk A H , Schuitema A R , Kuijper S , van Leeuwen J , Hermans P W , van Embden J D , Hartskeerl R A
Published: January 2020

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.
Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification
Authors: Figueroa J V , Chieves L P , Johnson G S , Buening G M
Published: January 2020

A SpeI-AvaI fragment (0.3 kbp) from pBbi16 (a pBR322 derivative containing a 6.3-kbp Babesia bigemina DNA insert) was subcloned into the pBluescript phagemid vector and was sequenced by the dideoxy-mediated chain termination method. Two sets of primers were designed for the polymerase chain reaction (PCR) assay. Primer set IA-IB was used to amplify a 278-bp DNA fragment, and primer set IAN-IBN was used to prepare a probe directed to a site within the PCR-amplified target DNA. Digoxigenin-dUTP was incorporated into the probe during the amplification reaction. PCR amplification of target DNA obtained from in vitro-cultured B. bigemina and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 278-bp fragment could be detected when as little as 100 fg of parasite genomic DNA was used in the assay. A fragment of similar size was amplified from genomic DNAs from several B. bigemina isolates but not from DNAs from Babesia bovis, Anaplasma marginale, or six species of bacteria or bovine leukocytes. Similarly, the PCR product could be detected in DNA samples purified from 200 microliters of blood with a parasitemia of as low as 1 in 10(8) cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 microliters of packed erythrocytes with a calculated parasitemia of 1 in 10(9) cells. With the analytical sensitivity level of the PCR-DNA probe assay, six cattle with inapparent, 11-month chronic B. bigemina infection were found to be positive. No PCR product was observed in bovine blood samples collected from a splenectomized, A. marginale-infected bovine, a 4-year chronic B. bovis-infected animal, or 20 uninfected cattle from Missouri which were subjected to amplification. The PCR-DNA probe assay was shown to be sensitive in detecting latently infected cattle. The specificity and high analytical sensitivity of the test provide valuable tools for performing large-scale epidemiological studies.
Genotyping Naegleria spp. and Naegleria fowleri isolates by interrepeat polymerase chain reaction
Authors: van Belkum A , De Jonckheere J , Quint W G
Published: January 2020

All six Naegleria species recognized to date were studied by interrepeat polymerase chain reaction (PCR). Priming at repeat sequences, which are known to be variable among eukaryotes, yielded electrophoretic DNA banding patterns that were specific for any single species. With a single PCR and simple gel electrophoresis, species determination could be performed in less than 1 day. Unambiguous discrimination between the pathogen N. fowleri and nonpathogenic Naegleria species appeared to be possible. Analysis of DNAs obtained from 20 separate isolates of N. fowleri revealed that geographic variation of the genetic fingerprints rarely occurs. All but 3 of 20 isolates of N. fowleri which were investigated showed identical banding patterns; for two isolates from New Zealand and one from Australia, a limited number of additional bands was detected, independent of the PCR primers used. These data corroborate previous findings on the genetic stability of pathogenic N. fowleri.
Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris
Authors: Kappos T , John M A , Hussain Z , Valvano M A
Published: January 2020

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.
Detection of Mycoplasma pneumoniae in clinical samples from pediatric patients by polymerase chain reaction
Authors: Skakni L , Sardet A , Just J , Landman-Parker J , Costil J , Moniot-Ville N , Bricout F , Garbarg-Chenon A
Published: January 2020

The polymerase chain reaction (PCR) technique was used to detect Mycoplasma pneumoniae DNA in clinical samples (nasopharyngeal aspirations or bronchoalveolar lavages) obtained from 100 children, 1 month to 16 years old. PCR allowed the detection of M. pneumoniae DNA from 20 out of the 100 patients studied. In 16 cases, PCR positivity was associated with acute respiratory symptomatology. For five PCR-positive patients, a positive culture or a serological response evidenced acute M. pneumoniae infections. A lack of antibody response was observed particularly with immunocompromised children and infants less than 12 months old. The amount of M. pneumoniae DNA in the PCR was estimated in a semiquantitative way by comparison of its hybridization signal with those obtained for 100, 10, and 1 color-changing unit (CCU) of the M. pneumoniae FH strain. Small amounts (less than or equal to 10(2) CCU/ml) of M. pneumoniae were found in samples from asymptomatic patients, while larger amounts (greater than or equal to 10(2) to greater than or equal to 10(4) CCU/ml) were found for 8 out of 10 patients with acute pneumonia.
Characterization of full-length and polymerase chain reaction-derived partial-length Gottfried and OSU gene 4 probes for serotypic differentiation of porcine rotaviruses
Authors: Rosen B I , Parwani A V , Gorziglia M , Larralde G , Saif L J
Published: January 2020

To determine the VP4 (P type) specificity of porcine rotaviruses, full- and partial-length gene 4 probes were produced from cloned Gottfried and OSU porcine rotavirus genomic segment 4 cDNAs. The gene 4 segments from the prototype Gottfried (VP7 serotype 4) and OSU (VP7 serotype 5) porcine rotavirus strains were selected for study because of their distinct P types and the occurrence of rotaviruses with similar serotypes among swine. Partial-length gene 4 cDNAs were produced and amplified by the polymerase chain reaction (PCR) and encompassed portions of the variable region (nucleotides 211 to 612) of VP8 encoded by genomic segment 4. The hybridization stringency conditions necessary for optimal probe specificity and sensitivity were determined by dot or Northern (RNA) blot hybridizations against a diverse group of human and animal rotaviruses of heterologous group A serotypes and against representative group B and C porcine rotaviruses. The PCR-derived gene 4 probes were more specific than the full-length gene 4 probes but demonstrated equivalent sensitivity. The Gottfried PCR-derived probe hybridized with Gottfried, SB2, SB3, and SB5 G serotype 4 porcine rotaviruses. The OSU PCR-derived probe hybridized with OSU, EE, A580, and SB-1A porcine rotaviruses and equine H1 rotavirus. Results of the hybridization reactions of the PCR-derived gene 4 probes with selected porcine rotavirus strains agreed with previous serological or genetic analyses, indicating their suitability as diagnostic reagents.
Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen
Authors: Zygmunt M S , Gilbert F B , Dubray G
Published: January 2020

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis.
Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex
Authors: Gerner-Smidt P
Published: January 2020

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex consists of four genotypically distinct but phenotypically very similar bacterial species or DNA groups: A. calcoaceticus (DNA group 1), A. baumannii (DNA group 2), unnamed DNA group 3 (P. J. M. Bouvet and P. A. D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986), and unnamed DNA group 13 (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989). Because strains in this complex cause nosocomial outbreaks, it is important to be able to identify them as completely as possible. Ribotyping could provide such identification. Therefore, ribotyping was done on 70 strains in the A. calcoaceticus-A. baumannii complex with known DNA group affiliations by use of restriction enzymes EcoRI, ClaI, and SalI. A nonradioactive digoxigenin-11-dUTP-labeled Escherichia coli rRNA-derived probe was used. With any of the three restriction enzymes, banding patterns that were specific for each DNA group were seen. All 70 strains showed banding patterns that could identify them to the correct DNA group by use of any two of the three enzymes. In addition, banding patterns that could separate strains within any one DNA group were present. The discriminatory index of P. Hunter and M. Gaston (J. Clin. Microbiol. 26:2465-2466, 1988), applied to all strains with the combined results obtained with all three enzymes, revealed a value of 0.99. For strains in each DNA group, the value varied from 0.93 to 0.98. These results indicate the high discriminatory power of the system when used for epidemiological typing.
Purification of listeriolysin O and development of an immunoassay for diagnosis of listeric infections in sheep
Authors: Low J C , Davies R C , Donachie W
Published: January 2020

A protein of 58,000-Da molecular mass was purified from the supernatant fluid of a dialysis sac culture of Listeria monocytogenes by cation-exchange chromatography. The purified protein, homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and possessing the characteristics of listeriolysin O (LLO), was used to develop an indirect enzyme-linked immunosorbent assay. Anti-LLO antibodies were shown to be consistently produced in sheep after experimental challenge with L. monocytogenes serovar 4b. The assay also successfully detected and measured specific anti-LLO antibodies in the sera of silage-fed sheep among which listeric enteritis and abortions had occurred.
Development of cDNA probes for typing group A bovine rotaviruses on the basis of VP4 specificity
Authors: Parwani A V , Rosen B I , McCrae M A , Saif L J
Published: January 2020

Dot and Northern (RNA) blot hybridization assays were developed for the P typing of group A bovine rotaviruses (BRV) by using cDNA probes prepared from gene segment 4. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions (nucleotides 211 to 686) of BRV strain UK, IND, NCDV, and Cr VP4 cDNA by using specific oligonucleotide primers. The probes were P type specific (VP4) and exhibited little or no cross-reactivity with double-stranded RNA from heterologous rotavirus P types. Our studies indicate that at least three P types, as defined by polymerase chain reaction-derived VP4 gene probes from the UK, NCDV, and Cr strains, exist among the seven BRV isolates tested.
Identification of Streptococcus pneumoniae with a DNA probe
Authors: Denys G A , Carey R B
Published: January 2020

The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae.
Septicemia with Ewingella americana
Authors: Devreese K , Claeys G , Verschraegen G
Published: January 2020

Ewingella americana was isolated from two blood cultures from a 75-year-old male after cholecystectomy. The characteristics of this strain are compared with the reported biochemical characteristics of 44 American strains. Previously described infections and pseudoinfections with E. americana are reviewed.
Evaluation of three immunoassays for detection of Chlamydia trachomatis in urine specimens from asymptomatic males
Authors: Leonardi G P , Seitz M , Edstrom R , Cruz J , Costello P , Szabo K
Published: January 2020

The performances of three commercially available immunoassays (Chlamydiazyme/Antibody Blocking Assay [Abbott Diagnostics, Abbott Park, Ill.], IDEIA [Analytab Products, Plainview, N.Y.], and Microtrak EIA [Syva Co. Palo Alto, Calif.]) were evaluated for the detection of Chlamydia trachomatis in urine specimens from asymptomatic males. Assay results were compared with direct specimen immunofluorescence (DFA) analysis of urine sediment (Syva Microtrak; Syva Co.), which was chosen as the study confirmation assay. An overall Chlamydia prevalence of 7% (24 of 340) was found in our study population, with peak incidences occurring in the adolescent (8 of 93 specimens) and young adult (11 of 146 specimens) age groups. Sensitivity and specificity data among the Chlamydiazyme, IDEIA, and Microtrak enzyme immunoassays (EIAs) were determined to be 79.1 and 99%, 91.7 and 98%, and 95.8 and 99%, respectively. The Microtrak EIA and IDEIA products demonstrated sensitivities and specificities equal to or greater than those claimed for urine specimens. The diagnostic accuracies of these assays on asymptomatic subjects, along with the ease of this collection method, suggest a role for these products as screening tools. The sensitivity of the Chlamydiazyme assay was lower than that claimed previously in symptomatic patients, with 5 of 24 positive specimens demonstrating false-negative results. In those cases, centrifugation of the original immunoassay aliquot material and then DFA examination confirmed specimen positivity. Urine immunoassay screening in combination with DFA confirmation (which was chosen because it has antibody epitopic specificity different from that of the primary assay) provides a high degree of diagnostic precision. The use of noninvasive collection methods could result in greater testing compliance among asymptomatic males and, subsequently, could reduce the incidences of both symptomatic and silent chlamydial infections.