bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
Cryptosporidium
“Mouse” Genotype Is Conserved across Geographic Areas
Authors: Morgan Una M. , Sturdee Anthony P. , Singleton Grant , Gomez M. Soledad , Gracenea Mercedes , Torres Jordi , Hamilton Steven G. , Woodside D. P. , Thompson R. C. Andrew 
Published: December 2019

ABSTRACT

A 298-bp region of the
Cryptosporidium parvum
18S rRNA gene and a 390-bp region of the acetyl coenzyme A synthetase gene were sequenced for a range of
Cryptosporidium
isolates from wild house mice (
Mus domesticus
), a bat (
Myotus adversus
), and cattle from different geographical areas. Previous research has identified a distinct genotype, referred to as the “mouse”-derived
Cryptosporidium
genotype, common to isolates from Australian mice. Comparison of a wider range of Australian mouse isolates with United Kingdom and Spanish isolates from mice and cattle and also an Australian bat-derived
Cryptosporidium
isolate revealed that the “mouse” genotype is conserved across geographic areas. Mice are also susceptible to infection with the “cattle”
Cryptosporidium
genotype, which has important implications for their role as reservoirs of infection for humans and domestic animals.
Comparison of Direct Plating and Broth Enrichment Culture for the Detection of Intestinal Colonization by Glycopeptide-Resistant Enterococci among Hospitalized Patients
Authors: Ieven M. , Vercauteren E. , Descheemaeker P. , van Laer F. , Goossens H. 
Published: December 2019

ABSTRACT

The results of prevalence studies on glycopeptide-resistant enterococci (GRE) in the intestine may be influenced by the detection methods applied. In most studies different media, different concentrations of antibiotics, and different methods are used, and these differences result in differences in recovery rates. In this cross-sectional study on the carrier state of GRE among patients at the University Hospital Antwerp, Antwerp, Belgium, performed on 21 May 1996, direct plating and broth enrichment were compared by using the same media. Stool samples (
n
= 213) or rectal swabs (
n
= 122) were plated directly on Enterococcosel agar (bioMérieux) and after enrichment in Enterococcosel broth. The prevalence of GRE was 12.8%. Direct plating recovered 53.4% of the GRE isolates, and broth enrichment recovered an additional 46.5% of them; in the latter test the isolates were thus present at less than 10
3
CFU per g of feces. The prevalence of GRE among dialysis patients was higher than among the other patients, but the difference was not significant (
P
= 0.06), possibly as a result of the small numbers of dialysis patients examined. The GRE species isolated included 19
E. gallinarum
(44.2%), 13
E. faecium
(30.2%), 6
E. faecalis
(13.9%), and 5
E. casseliflavus
(11.6%) isolates. All
E. faecalis
and
E. faecium
strains isolated carried the
vanA
gene, and
E. gallinarum
and
E. casseliflavus
carried the
vanC1
and
vanC2
gene, respectively. The majority of isolates were polyclonal. Our data indicate that the rate of detection of GRE from both stool samples and rectal swabs is significantly increased with enrichment cultures.
Mycobacterium malmoense
-Specific Nested PCR Based on a Conserved Sequence Detected in Random Amplified Polymorphic DNA Fingerprints
Authors: Kauppinen Juha , Mäntyjärvi Rauno , Katila Marja-Leena 
Published: December 2019

ABSTRACT

Mycobacterium malmoense
is an opportunistic human pathogen of increasing clinical importance. Since it is difficult to detect and identify the organism by conventional techniques, it was decided to seek a nucleic acid amplification method specific for
M. malmoense
. The method was based on detection of a conserved band in random amplified polymorphic DNA (RAPD) fingerprints of 45
M. malmoense
strains. This band was a 1,046-bp product which was proven to be
M. malmoense
specific in dot blot hybridization analysis with a panel of mycobacterial strains belonging to 39 other species. The fragment was sequenced, and oligonucleotide primers were synthesized to evaluate the specificity of the PCR. Two primer pairs were found to be specific and sensitive in the nested PCR that was developed. All 49
M. malmoense
strains analyzed produced a PCR product of the expected size. In contrast, no strains belonging to the other mycobacterial species tested produced amplicons with these primers under specified reaction conditions. The results of the electrophoresis were confirmed by the hybridization with the
M. malmoense
-specific oligonucleotide probe. This method could be applied to the analysis of clinical or environmental samples, permitting the rapid detection of
M. malmoense
.
Pulsed-Field Gel Electrophoresis Used To Investigate Genetic Diversity ofHaemophilus influenzaeType b Isolates in Australia Shows Differences between Aboriginal and Non-Aboriginal Isolates
Authors: Moor Patricia Ezekiel , Collignon Peter C. , Gilbert Gwendolyn L. 
Published: December 2019

ABSTRACTWe used pulsed-field gel electrophoresis to study the epidemiology and population structure ofHaemophilus influenzaetype b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n= 76), non-Aboriginal children (n= 106), and non-Aboriginal adults (n= 5) in urban and rural regions across Australia were digested with theSmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments (size range, ∼8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Two types predominated. One type (n= 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) of the isolates from non-Aboriginals, and the other type (n= 41) accounted for 2 (3%) of the isolates from Aboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a genetic distance of ∼50% similarity in a tree-like dendrogram. They included two highly divergent groups representing 50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinct group representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The results showed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of the sample. There was no association between RFLP type and the diagnosis of meningitis or epiglottitis, age, sex, date of collection, or geographic location, but there was a strong association between the origin of isolates from Aboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP type A8b. The methodology discriminated well among the isolates (D= 0.91) and will be useful for the monitoring of postvaccine isolates ofH. influenzaetype b.
Second Generation of the Automated Cobas Amplicor HCV Assay Improves Sensitivity of Hepatitis C Virus RNA Detection and Yields Results That Are More Clinically Relevant
Authors: Doglio Alain , Laffont Catherine , Caroli-Bosc François X. , Rochet Pascale , Lefebvre J.-Claude 
Published: December 2019

ABSTRACT
The first and second generations of the Cobas Amplicor HCV assay were compared among patients at risk of hepatitis C virus (HCV) infection. The second-generation test was found to be of greater sensitivity and of good specificity among clinical specimens containing HCV RNA of different genotypes. Finally, this new test is shown to predict the outcome of interferon therapy better.
Molecular Cloning and Sequencing of thearoAGene fromActinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification
Authors: Moral Carmen Hernanz , Soriano Alberto Cascón , Salazar María Sánchez , Marcos Javier Yugueros , Ramos Susana Suárez , Carrasco German Naharro 
Published: December 2019

ABSTRACTThe gene (aroA) ofActinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of thearoAmutation inEscherichia coliK-12 strain AB2829, and the nucleotide sequence was determined. A pair of primers from the 5′ and 3′ termini were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,025 bp was amplified from lysedA. pleuropneumoniaeserotypes 1 to 12 of biovar 1 or from isolated DNA. No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified whenActinobacillus equulichromosomal DNA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was very sensitive, with lower detection limits of 12 CFU withA. pleuropneumoniaecells and 0.8 pg with extracted DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis ofA. pleuropneumoniaeinfections.
False-Positive Results Obtained with the Alexon ProSpecT
Cryptosporidium
Enzyme Immunoassay
Authors: Doing Kirk M. , Hamm Jill L. , Jellison Jo Ann , Marquis Jessica A. , Kingsbury Cindy 
Published: December 2019

ABSTRACT

Cryptosporidium
is known to cause diarrhea in immunocompromised patients and is also associated with outbreaks of disease due to food-borne and waterborne parasites. Traditional procedures, involving iodine staining of wet mounts of stool sediments and trichrome staining, lack the sensitivity to detect
Cryptosporidium
. Special staining procedures, such as the modified acid-fast and safranin stains, are generally employed. Less labor-intensive antigen detection assays have simplified detection; however, careful attention to local epidemiology is important because false-positive tests occur. Here, we report two incidents involving 62 false-positive results obtained with the Alexon ProSpecT
Cryptosporidium
enzyme immunoassay, which were deemed false-positive based on negative results obtained from extensive microscopic examinations.
Multisite Reproducibility of Results Obtained by the Broth Microdilution Method for Susceptibility Testing of
Mycobacterium abscessus
,
Mycobacterium chelonae
, and
Mycobacterium fortuitum
Authors: Woods Gail L. , Bergmann John S. , Witebsky Frank G. , Fahle Gary A. , Wanger Audrey , Boulet Betty , Plaunt Marianne , Brown Barbara A. , Wallace Richard J. 
Published: December 2019

ABSTRACT

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four
Mycobacterium fortuitum
group, three
Mycobacterium abscessus
, and three
Mycobacterium chelonae
isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin (
M. chelonae
only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode ± 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of
M. fortuitum
against clarithromycin;
M. abscessus
and
M. chelonae
against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.
Effect of Multiple Freeze-Thaw Cycles on Hepatitis B Virus DNA and Hepatitis C Virus RNA Quantification as Measured with Branched-DNA Technology
Authors: Krajden Mel , Minor James M. , Rifkin Oretta , Comanor Lorraine 
Published: December 2019

ABSTRACT

Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (
P
< 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable.
VP4 and VP7 Genotyping of Rotavirus Samples Recovered from Infected Children in Ireland over a 3-Year Period
Authors: O’Mahony Jim , Foley Barbara , Morgan Sheila , Morgan John G. , Hill Colin 
Published: December 2019

ABSTRACT
Between September 1995 and August 1998, the incidence and diversity of the main human rotavirus genotypes (G1, G2, G3, and G4 and P[8], P[4], P[6], and P[9]) among Irish children were determined by using established and adapted reverse transcriptase PCR-based genotyping methods. From a total of 193 rotavirus-positive specimens collected from nine hospitals we successfully identified the P type in 182 (94%) of the samples and the G type in 165 (85.5%) of the samples. Only four samples could not be assigned a G or P type. Two P types existed in Ireland, P[8] (78%) and P[4] (16%), and their relative incidence varied over the 3 years of this study. No P[6] or P[9] types were detected. G1 was the most predominant G type (55%), and the incidences of G2, G3, and G4 isolates were 15.5, 1, and 11%, respectively. Three percent of the samples tested had a mixed G type. A P and G type was assigned to 158 (81.8%) of samples. Of the typeable samples, G1 P[8] was the most prevalent (65%), whereas G2 P[4] (17%), G3 P[8] (1%), G4 P[8] (12%), and mixed types (all G1/ G4 P[8]) (4%) were detected less frequently. In the third year a significant genotypic shift from G1 P[8] to G2 P[4] and G4 P[8] was observed. During the study, we noticed that the inclusion of random primers during cDNA synthesis greatly increased the specificity of the PCR typing assays. No correlation was seen between the contributing hospitals and a specific genotype. In conclusion, the coverage of infection given by the recently licensed tetravalent vaccine would be significantly high in Ireland, although future monitoring of genotypic changes among Irish isolates should be encouraged.
Multidrug-Resistant
Streptococcus pneumoniae
in Poland: Identification of Emerging Clones
Authors: Overweg Karin , Hermans Peter W. M. , Trzcinski Krzysztof , Sluijter Marcel , de Groot Ronald , Hryniewicz Waleria 
Published: December 2019

ABSTRACT

Penicillin resistance among
Streptococcus pneumoniae
isolates has rapidly emerged in Poland during the last decade and has reached prevalence levels of up to 14.4% in 1997. In order to investigate the nature of this increase, a molecular epidemiological analysis of non-penicillin-susceptible multidrug-resistant pneumococci isolated in 1995 and 1996 was conducted. Thirty-seven patients who suffered mainly from upper respiratory tract infections and pneumococcal pneumonia were enrolled in this study. The medical centers to which the patients were admitted were located in 16 Polish towns across the country. Eight distinct BOX PCR types were observed, representing 14 subtypes. Restriction fragment end labeling (RFEL) analysis divided the pneumococcal strains into 16 distinct types. By combining the BOX PCR and RFEL data, four genetically distinct clusters of strains were identified. Two clusters represented the genetic clones 23F and 9V, which have recently emerged all over the world. The two other genetic clusters, which represented serotypes 23F and 6B, clearly predominated in the analyzed collection of Polish non-penicillin-susceptible pneumococcal strains. Since the latter clusters did not match any of the 133 RFEL types of non-penicillin-susceptible pneumococci collected in 15 other countries, their Polish clonal origin is most likely.
Molecular Epidemiologic Evaluation of Transmissibility and Virulence of
Mycobacterium tuberculosis
Authors: Rhee Jeanne T. , Piatek Amy S. , Small Peter M. , Harris Lisa M. , Chaparro Sandra V. , Kramer Fred Russell , Alland David 
Published: December 2019

ABSTRACT

Discovery of genotypic markers associated with increased transmissibility in
Mycobacterium tuberculosis
would represent an important step in advancing mycobacterial virulence studies.
M. tuberculosis
strains may be classified into one of three genotypes on the basis of the presence of specific nucleotide substitutions in codon 463 of the
katG
gene (
katG
-463) and codon 95 of the
gyrA
gene (
gyrA
-95). It has previously been reported that two of these three genotypes are associated with increased IS
6110
-based clustering, a potential proxy of virulence. We designed a case-control analysis of U.S.-born patients with tuberculosis in San Francisco, Calif., between 1991 and 1997 to investigate associations between
katG
-463 and
gyrA
-95 genotypes and epidemiologically determined measures of strain-specific infectivity and pathogenicity and IS
6110
-based clustering status. We used a new class of molecular probes called molecular beacons to genotype the isolates rapidly. Infectivity was defined as the propensity of isolates to cause tuberculin skin test conversions among named contacts, and pathogenicity was defined as their propensity to cause active disease among named contacts. The molecular beacon assay was a simple and reproducible method for the detection of known single nucleotide polymorphisms in large numbers of clinical
M. tuberculosis
isolates. The results showed that no genotype of the
katG
-463- and
gyrA
-95-based classification system was associated with increased infectivity and pathogenicity or with increased IS
6110
-based clustering in San Francisco during the study period. We speculate that molecular epidemiologic studies investigating clinically relevant outcomes may contribute to the knowledge of the significance of laboratory-derived virulence factors in the propagation of tuberculosis in human communities.
Simultaneous Detection of Bovine
Theileria
and
Babesia
Species by Reverse Line Blot Hybridization
Authors: Gubbels J. M. , de Vos A. P. , van der Weide M. , Viseras J. , Schouls L. M. , de Vries E. , Jongejan F. 
Published: December 2019

ABSTRACT

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of
Theileria
and
Babesia
simultaneously. We included
Theileria annulata
,
T. parva
,
T. mutans
,
T. taurotragi
, and
T. velifera
in the assay, as well as parasites belonging to the
T. sergenti-T. buffeli-T. orientalis
group. The
Babesia
species included were
Babesia bovis
,
B. bigemina
, and
B. divergens
. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all
Theileria
and
Babesia
species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from
Bos taurus
or other hemoparasites (
Trypanosoma
species,
Cowdria ruminantium
,
Anaplasma marginale
, and
Ehrlichia
species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain.
T. annulata
,
T. orientalis
, and
B. bigemina
were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.
Development of a Simple and Highly Sensitive Enzyme Immunoassay for Hepatitis C Virus Core Antigen
Authors: Aoyagi Katsumi , Ohue Chiharu , Iida Kumiko , Kimura Tatsuji , Tanaka Eiji , Kiyosawa Kendo , Yagi Shintaro 
Published: December 2019

ABSTRACT

A highly sensitive enzyme immunoassay (EIA) for the hepatitis C virus (HCV) core antigen (HCVcAg) was developed, and its performance was compared with that of the AMPLICOR HCV test (Roche Molecular Systems). The developed one-step pretreatment method, 30-min incubation of the specimen with a solution containing three different types of detergents (Triton X-100, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], and sodium dodecyl sulfate), does not require any special device. Because the interfering anti-core antibody in the sample was sufficiently inactivated by the pretreatment, HCVcAg in the sample could be detected. The immunoreactivity on gel filtration was shifted from void fractions to those corresponding to the molecular mass range from 20 to 25 kDa, which is equal to the estimated molecular mass of HCVcAg, after the pretreatment. By the recovery test with HCVcAg-positive serum, the recovery rate was 93.5 to 106.5%. There was no interference with the EIA by anticoagulants or blood components in the serum. When the cutoff value was tentatively set at 0.5 mU/ml based on the distribution of healthy subjects’ sera, the sera of all healthy subjects (
n
= 125) and patients with hepatitis B (
n
= 50) were negative. HCVcAg was detected in sera from 57 of 73 individuals (78.1%) with anti-HCV antibody. Similarly, HCV RNA was detected in sera from 59 individuals (80.8%) with the AMPLICOR HCV as the qualitative test (AMPLICOR HCV test) and in sera from 54 individuals (74.0%) by the AMPLICOR HCV Monitor as the quantitative test (AMPLICOR Monitor test). Concentrations of HCVcAg and HCV RNA (measured by the AMPLICOR Monitor test) correlated significantly (
r
= 0.8,
P
< 0.001). On seroconversion panels, HCVcAg was detected during the early stage of infection, when anti-HCV antibodies had not been produced. This assay for HCVcAg is simpler than assays for HCV RNA based on gene technology and shows specificity and sensitivity equivalent to those of the AMPLICOR HCV test.