bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intergenic Transcribed Spacer PCR Ribotyping for Differentiation of
Saccharomyces
Species and Interspecific Hybrids
Authors: McCullough Michael J. , Clemons Karl V. , McCusker John H. , Stevens David A. 
Published: December 2019

ABSTRACT

The taxonomy of the genus
Saccharomyces
has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of
Saccharomyces
, allows unknown
Saccharomyces
isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of
Saccharomyces
and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes
Mae
I and
Hae
III could distinguish all species of
Saccharomyces
from each other, as well as from
Candida glabrata
,
Candida albicans
, and
Blastomyces dermatitidis
. The only exception to this was the inability to distinguish between
Saccharomyces bayanus
and
S. pastorianus
(
S. carlsbergensis
). Furthermore, interspecific hybrids resulting from the mating of sibling species of
Saccharomyces
were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.
Nested PCR Assay for Detection of Granulocytic Ehrlichiae
Authors: Massung Robert F. , Slater Kim , Owens Jessica H. , Nicholson William L. , Mather Thomas N. , Solberg Victoria B. , Olson James G. 
Published: December 2019

ABSTRACT

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from
Ixodes scapularis
ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.
Multicenter Evaluation of the COBAS AMPLICOR HCV Assay, an Integrated PCR System for Rapid Detection of Hepatitis C Virus RNA in the Diagnostic Laboratory
Authors: Albadalejo J. , Alonso R. , Antinozzi R. , Bogard M. , Bourgault A.-M. , Colucci G. , Fenner T. , Petersen H. , Sala E. , Vincelette J. , Young C. 
Published: December 2019

ABSTRACT
The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the “gold standard,” indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.
Isolation and Characterization of Verocytotoxin-Producing
Escherichia coli
O157 Strains from Dutch Cattle and Sheep
Authors: Heuvelink A. E. , van den Biggelaar F. L. A. M. , de Boer E. , Herbes R. G. , Melchers W. J. G. , Huis In ’t Veld J. H. J. , Monnens L. A. H. 
Published: December 2019

ABSTRACT

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing
Escherichia coli
(VTEC) of serogroup O157.
E. coli
O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and
E. coli
attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of
E. coli
O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of
E. coli
O157 strains that are potentially pathogenic for humans.
Novel Recombinant-Antigen Enzyme Immunoassay for Serological Diagnosis of Syphilis
Authors: Young H. , Moyes A. , Seagar L. , McMillan A. 
Published: December 2019

ABSTRACTEnzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinantTreponema pallidumantigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the nativeT. pallidumantigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis of syphilis. Specificity was evaluated by screening 1,184 unselected serum specimens in parallel by the ICE Syphilis and SelectSyph-G assays, while sensitivity was tested with a panel of 101 serum specimens containing antitreponemal antibodies (treated and untreated) from patients with various stages of infection. The specificity of the ICE Syphilis EIA (99.8%) on screening was significantly higher (P< 0.02) than that of the SelectSyph-G EIA (99.2%). The sensitivity of the ICE Syphilis EIA was significantly higher (P< 0.01) than that of the SelectSyph-G EIA on both initial (99 versus 91.4%) and repeat (100 versus 92.4%) testing. The ICE Syphilis EIA was also significantly more sensitive (P< 0.01) than the fluorescent treponemal antibody-abs (92.4%) but not theT. pallidumhemagglutination assay (97.1%). Sera containing antitreponemal antibodies gave a much higher antibody index (absorbance of test serum/kit cutoff) by the ICE Syphilis EIA than by the SelectSyph-G EIA. This combined with the overall high sensitivity makes the ICE Syphilis EIA an ideal test for excluding or detecting treponemal infection in human immunodeficiency virus (HIV)-infected patients. The ICE Syphilis EIA was positive with sera from all 15 HIV-infected patients in the study, whereas sera from 3 HIV-infected patients were negative by the SelectSyph-G EIA. We conclude that the high sensitivity and specificity of the ICE Syphilis EIA and its suitability for automation make it an ideal screening test.
Use of Immunoblot Assay To Define Serum Antibody Patterns Associated with
Helicobacter pylori
Infection and with
H. pylori
-Related Ulcers
Authors: Aucher P. , Petit M. L. , Mannant P. R. , Pezennec L. , Babin P. , Fauchere J. L. 
Published: December 2019

ABSTRACT

Serology has been used worldwide to detect
Helicobacter pylori
infection. Using an immunoblot assay with an antigen from strain ATCC 43579, we sought to determine the antibodies which were good markers of colonization and the antibody patterns associated with ulcers or atrophy. Out of 98 dyspeptic patients, 41 were colonized by
H. pylori
, based on a positive culture or on positive results of both a urease test and direct examination. These 41 patients were seropositive by an enzyme immunoassay, and 12 of them had ulcers and 29 had evidence of atrophy. Fifty-seven of the 98 patients were noncolonized. Twenty-five of the 57 had evidence of gastric atrophy, and 10 were seropositive; 5 of these 10 had ulcers. By Western blot analysis, 12 antibodies were significantly more frequent in sera from colonized patients, and they produced immunoreactive bands at 125, 87, 74, 66, 54, 48, 46, 42, 35, 30, 16 and 14 kDa. The presence of at least one band at 54, 35, or 42 kDa was the best marker of infection (sensitivity, 95%; specificity, 82%). In the group of colonized patients, none of the antibody patterns were correlated to gastric atrophy. Conversely, the presence of a band at 125, 87, or 35 kDa was statistically associated with the presence of an ulcer. The simultaneous presence of bands at 87 and 35 kDa predicted the risk of ulcers with 83% sensitivity and 69% specificity. By using CagA-positive and VacA-positive strains and CagA-negative and VacA-negative isogenic mutants, the antigens corresponding to the bands at 125 and 87 kDa were shown to be CagA and VacA, respectively. On the other hand, the 35-kDa antigen is a novel uncharacterized component of
H. pylori
. These results may help to optimize the composition of antigenic preparations for serologic detection of
H. pylori
colonization. Immunoblot assay would be useful for screening patients at high risk of ulcers.
Comparison of PCR and Microscopy for Detection of
Cryptosporidium parvum
in Human Fecal Specimens: Clinical Trial
Authors: Morgan U. M. , Pallant L. , Dwyer B. W. , Forbes D. A. , Rich G. , Thompson R. C. A. 
Published: December 2019

ABSTRACT

PCR technology offers alternatives to conventional diagnosis of
Cryptosporidium
for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect
Cryptosporidium
but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different
Cryptosporidium
genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of
Cryptosporidium
infections.
Evaluation of BACTEC MYCO/F Lytic Medium for Recovery of Mycobacteria and Fungi from Blood
Authors: Waite Rebecca T. , Woods Gail L. 
Published: December 2019

ABSTRACT

The reliability of MYCO/F Lytic medium in the BACTEC 9240 blood culture system was evaluated by comparing its performance to that of the Isolator system for the recovery of fungi and to that of the ESP II system for the recovery of mycobacteria. Of 717 specimens of blood cultured for fungi, 24 were positive; 12 samples were positive with both systems, 7 samples were positive with the Isolator system only, and 5 samples were positive with MYCO/F Lytic medium only. Fourteen samples grew
Histoplasma capsulatum
; both systems detected
H. capsulatum
in seven samples but the Isolator system alone detected
H. capsulatum
in seven samples. The mean times to the detection of
H. capsulatum
were 8 days (range, 4 to 13 days) for MYCO/F Lytic medium and 9 days (range, 6 to 18 days) for the Isolator system; the mean times to identification were 20 days (range, 15 to 24 days) for isolates recovered with MYCO/F Lytic medium and 11 days (range, 6 to 18 days) for those recovered with the Isolator system (
P
< 0.05).
Cryptococcus neoformans
was isolated from 10 fungal cultures; five isolates grew in both systems, and five isolates grew in MYCO/F Lytic medium only. The mean times to detection of
C. neoformans
were 4 days (range, 2 to 6 days) for MYCO/F Lytic medium and 7 days (range, 5 to 7 days) for the Isolator system (
P
< 0.05); the mean times to identification were 15 days (range, 7 to 27 days) for isolates recovered with MYCO/F Lytic medium and 8 days (range, 7 to 11 days) for those recovered with the Isolator system. Of the 687 samples of blood cultured for mycobacteria, 64 blood samples from 42 patients grew mycobacteria (58 grew
Mycobacterium avium
complex, 4 grew
Mycobacterium kansasii
, and 2 grew
Mycobacterium tuberculosis
); 42 isolates were recovered with both systems, 18 were isolated with MYCO/F medium only, and 4 were isolated with the ESP II system only alone (
P
< 0.05). The mean time to detection of mycobacteria with MYCO/F Lytic medium was 14 days, whereas it was 17 days with the ESP II system (
P
< 0.05). In summary, the combination of MYCO/F Lytic medium and the BACTEC 9240 instrument is an excellent blood culture system for the growth and detection of mycobacteria. A valid assessment of MYCO/F Lytic medium with regard to fungal isolation, however, was not possible due to the small number of isolates recovered.
Insertion Element IS
3
-Based PCR Method for Subtyping
Escherichia coli
O157:H7
Authors: Thompson Curt J. , Daly Claire , Barrett Timothy J. , Getchell Jane P. , Gilchrist Mary J. R. , Loeffelholz Mike J. 
Published: December 2019

ABSTRACT

An
Escherichia coli
O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS
3
was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS
3
PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS
3
PCR subtyping method was applied to 35 epidemiologically related and unrelated
E. coli
O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS
3
PCR subtypes were identified. While not as sensitive as PFGE, IS
3
PCR subtyping grouped all outbreak-related isolates. IS
3
PCR banding patterns were reproducible between amplifications and between subcultures. IS
3
PCR could serve as a simple, rapid screening method for the identification of unrelated
E. coli
O157:H7 isolates.
Temporal Correlations between Tick Abundance and Prevalence of Ticks Infected withBorrelia burgdorferiand Increasing Incidence of Lyme Disease
Authors: Stafford Kirby C. , Cartter Matthew L. , Magnarelli Louis A. , Ertel Starr-Hope , Mshar Patricia A. 
Published: December 2019

ABSTRACTThe abundance of host-seekingIxodes scapularisnymphs, the principal vector for the Lyme disease agent,Borrelia burgdorferi, in Old Lyme, Lyme, and East Haddam, Connecticut, was compared with the incidence of reported human Lyme disease in the 12-town area around the Connecticut River and the State of Connecticut for the period 1989 to 1996. Ticks were sampled from lawns and woodlands by dragging flannel over the vegetation and examined for the presence ofB. burgdorferiby indirect fluorescent antibody staining. The infection rate of the nymphal ticks byB. burgdorferiduring the 9-year period was 14.3% (of 3,866), ranging from 8.6% (1993) to 24.4% (1996). The incidence of Lyme disease was positively correlated with tick abundance in the 12 town area (r= 0.828) and the State of Connecticut (r= 0.741). An entomological risk index based upon the number ofI. scapularisticks infected byB. burgdorferiwas highest in 1992, 1994, and 1996 and was highly correlated with the incidence of Lyme disease in Connecticut (r= 0.944). The number of Lyme disease cases has been influenced, in part, by annual changes in population densities ofI. scapularisand, presumably, a corresponding change in the risk of contact with infected ticks. Based upon tick activity and spirochetal infection rates, epidemiologically based Lyme disease case reports on a regional scale appear to reflect real trends in disease.
Detection of Bovine Torovirus in Fecal Specimens of Calves with Diarrhea from Ontario Farms
Authors: Duckmanton Lynn , Carman Susy , Nagy Éva , Petric Martin 
Published: December 2019

ABSTRACTBreda virus (BRV), a member of the genusTorovirus, is an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in both Iowa and Ohio and in several areas of Europe. However, this virus has been reported only once in Canada. Therefore, a study was performed to determine the extent to which bovine torovirus is present in calves with diarrhea from farms in southern Ontario. A total of 118 fecal samples from symptomatic calves and 43 control specimens from asymptomatic calves were examined by electron microscopy (EM) and reverse transcription-PCR (RT-PCR) for the presence of torovirus. Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%) by RT-PCR with primers designed in the conserved 3′ end of the torovirus genome. By EM, torovirus particles were observed in 37 of the 118 specimens (31.4%). All but one of these samples were also positive by RT-PCR. The incidence of torovirus in the asymptomatic control specimens by RT-PCR was only 11.6%. To establish the identity of the particles observed in the diarrheic specimens, five of the amplicons from samples positive by both RT-PCR and EM were cloned and sequenced. Nucleotide sequence analysis revealed that the bovine torovirus found in southern Ontario manifests between 96 and 97% sequence identity to the BRV type 1 strain found in Iowa. This study shows that bovine torovirus is a common virus in the fecal specimens of calves with diarrhea from farms in southern Ontario and thus may be an important pathogen of cattle.
Rapid Diagnosis of Extrapulmonary Tuberculosis by Ligase Chain Reaction Amplification
Authors: Gamboa Fredy , Dominguez José , Padilla Eduardo , Manterola José M. , Gazapo Elena , Lonca Joan , Matas Lurdes , Hernandez Agueda , Cardona Pere Joan , Ausina Vicente 
Published: December 2019

ABSTRACT

A rapid amplification-based test for the diagnosis of extrapulmonary tuberculosis, the LCx
Mycobacterium tuberculosis
Assay from Abbott Laboratories, was evaluated. Results from the LCx
M. tuberculosis
Assay were compared with those from culture and the final clinical diagnosis for each patient. A total of 526 nonrespiratory specimens from 492 patients were tested. The specimens included urine; feces; lymph node exudates; pleural, cerebrospinal, articular, and ascitic fluids; tissue biopsies; gastric aspirates; purulent exudates; blood; and bone marrow aspirates. After combination of the culture results and the patient’s clinical data, a total of 135 specimens were collected from 122 patients with a diagnosis of extrapulmonary tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx
M. tuberculosis
Assay were 77.7, 98.7, 95.2, and 93.1%, respectively; these values rose in resolved cases of TB to 78.5, 100, 100, and 93.1%, respectively. For 37 (27.4%) specimens from patients smear positive for the disease and 98 (72.6%) specimens from patients smear negative for the disease, the sensitivities of the LCx
M. tuberculosis
Assay were 100 and 71.1%, respectively. Statistically significant differences (
P
< 0.01) in sensitivities were found between culture and the LCx
M. tuberculosis
Assay. These differences were even greater among smear-negative specimens. The results demonstrate that the LCx
M. tuberculosis
Assay will provide rapid and valuable information for the diagnosis of extrapulmonary tuberculosis.