bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryptosporidium parvum
and
Giardia lamblia
Recovered from Flies on a Cattle Farm and in a Landfill
Authors: Szostakowska Beata , Kruminis-Lozowska Wieslawa , Racewicz Maria , Knight Ronald , Tamang Leena , Myjak Przemyslaw , Graczyk Thaddeus K.
Published: June 2004

Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for
Cryptosporidium parvum
and
Giardia lamblia
on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of
C. parvum
and 84% of
G. lamblia
pathogens were presumptively viable.
Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India
Authors: Parashar Deepti , Chauhan D. S. , Sharma V. D. , Chauhan Aradhana , Chauhan S. V. S. , Katoch V. M.
Published: June 2004

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.
Diverse Tetracycline Resistance Genotypes of
Megasphaera elsdenii
Strains Selectively Cultured from Swine Feces
Authors: Stanton Thaddeus B. , McDowall Jennifer S. , Rasmussen Mark A.
Published: June 2004

A
total of 30
Megasphaera elsdenii
strains, selectively isolated
from the feces of organically raised swine by using Me109 M medium, and
one bovine strain were analyzed for tetracycline resistance genotypic
and phenotypic traits. Tetracycline-resistant strains carried
tet
(O),
tet
(W), or a
tet
gene mosaic of
tet
(O) and
tet
(W).
M. elsdenii
strains
carrying
tet
(OWO) genes exhibited the highest tetracycline
MICs (128 to >256 μg/ml), suggesting that
tet
(O)-
tet
(W) mosaic genes provide the selective
advantage of greater tetracycline resistance for this species. Seven
tet
genotypes are now known for
M. elsdenii
, an
archetype commensal anaerobe and model for
tet
gene evolution
in the mammalian intestinal
tract.
Molecular and Biological Characterization of a
Cryptosporidium molnari
-Like Isolate from a Guppy (
Poecilia reticulata
)
Authors: Ryan Una , O'Hara Amanda , Xiao Lihua
Published: June 2004

Histological, morphological, genetic, and phylogenetic analyses of a
Cryptosporidium molnari
-like isolate from a guppy (
Poecilia reticulata
) identified stages consistent with those of
C. molnari
and revealed that
C. molnari
is genetically very distinct from all other species of
Cryptosporidium
. This study represents the first genetic characterization of
C. molnari
.
Immobilization of
Saccharomyces cerevisiae
Cystathionine γ-Lyase and Application of the Product to Cystathionine Synthesis
Authors: Yamagata Shuzo , Akamatsu Tsuyoshi , Iwama Tomonori
Published: June 2004

Cystathionine γ-lyase of
Saccharomyces cerevisiae
was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5′-phosphate and was characterized with respect to its cystathionine γ-synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.
Novel Epibiotic
Thiothrix
Bacterium on a Marine Amphipod
Authors: Gillan David C. , Dubilier Nicole
Published: June 2004

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean
Urothoe poseidonis
. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus
Thiothrix
. FISH and denaturing gradient gel electrophoresis revealed that the
Thiothrix
filaments are present on the majority of the amphipods examined.
Distribution of
Giardia duodenalis
Genotypes and Subgenotypes in Raw Urban Wastewater in Milwaukee, Wisconsin
Authors: Sulaiman Irshad M. , Jiang Jianlin , Singh Ajaib , Xiao Lihua
Published: June 2004

Giardia
cysts in 131 raw wastewater samples from Milwaukee, Wis., were genotyped by sequence analysis of the triosephosphate isomerase gene which showed the presence of two distinct genotypes (assemblages A and B) of
Giardia duodenalis
. Of the 131 samples, 111 belonged to assemblage A, and the remaining samples belonged to assemblage B. A high degree of genetic polymorphism was evident within the assemblage B cluster, with 10 distinct subgenotypes identified, eight of which have not been reported before.
Stable Carbon Isotopic Fractionations Associated with Inorganic Carbon Fixation by Anaerobic Ammonium-Oxidizing Bacteria
Authors: Schouten Stefan , Strous Marc , Kuypers Marcel M. M. , Rijpstra W. Irene C. , Baas Marianne , Schubert Carsten J. , Jetten Mike S. M. , Sinninghe Damsté Jaap S.
Published: June 2004

Isotopic analyses of
Candidatus
“Brocadia anammoxidans,” a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against
13
C; i.e., lipids are depleted by up to 47‰ versus CO
2
. Similar results were obtained for the anammox bacterium
Candidatus
“Scalindua sorokinii,” which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.
Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for
Escherichia coli
O157:H7
Authors: Kudva Indira T. , Griffin Robert W. , Murray Megan , John Manohar , Perna Nicole T. , Barrett Timothy J. , Calderwood Stephen B.
Published: June 2004

Polymorphic amplified typing sequences (PATS) for
Escherichia coli
O157:H7 (O157) was previously based on indels containing XbaI restriction enzyme sites occurring in O-island sequences of the O157 genome. This strain-typing system, referred to as XbaI-based PATS, typed every O157 isolate tested in a reproducible, rapid, straightforward, and easy-to-interpret manner and had technical advantages over pulsed-field gel electrophoresis (PFGE). However, the system was less discriminatory than PFGE and was unable to differentiate fully between unrelated isolates. To overcome this drawback, we enhanced PATS by using another infrequently cutting restriction enzyme, AvrII (also known as BlnI), to identify additional polymorphic regions that could increase the discriminatory ability of PATS typing. Referred to as AvrII-based PATS, the system identified seven new polymorphic regions in the O157 genome. Unlike XbaI, polymorphisms involving AvrII sites were caused by both indels and single-nucleotide polymorphisms occurring in O-island and backbone sequences of the O157 genome. AvrII-based PATS by itself provided poor discrimination of the O157 isolates tested. However, when primer pairs amplifying the seven polymorphic AvrII sites were combined with those amplifying the eight polymorphic XbaI sites (combined PATS), the discriminatory power of PATS was enhanced. Combined PATS matched related O157 isolates better than PFGE while differentiating between unrelated isolates. PATS typed every O157 isolate tested and directly targeted polymorphic sequences responsible for differences in the restriction digest patterns of O157 genomic DNA, utilizing PCR rather than relying on gel electrophoresis. This enabled PATS to resolve the ambiguity in PFGE typing, including that arising from the “more distantly related” and “untypeable” profiles.
Relationship of MIC and Bactericidal Activity to Efficacy of Vancomycin for Treatment of Methicillin-Resistant
Staphylococcus aureus
Bacteremia
Authors: Sakoulas George , Moise-Broder Pamela A. , Schentag Jerome , Forrest Alan , Moellering Robert C. , Eliopoulos George M.
Published: June 2004

We attempted to find a relationship between the microbiological properties of bloodstream isolates of methicillin-resistant
Staphylococcus aureus
(MRSA) and the efficacy of vancomycin in the treatment of bacteremia. Vancomycin susceptibility testing was performed, and bactericidal activity was determined for 30 isolates from 30 different patients with MRSA bacteremia for whom clinical and microbiological outcome data were available. The majority of these patients had been previously enrolled in multicenter prospective studies of MRSA bacteremia refractory to conventional vancomycin therapy. Logistic regression found a statistically significant relationship between treatment success with vancomycin and decreases in both vancomycin MICs (≤0.5 μg/ml versus 1.0 to 2.0 μg/ml;
P
= 0.02) and degree of killing (reduction in 1og
10
CFU/milliliter) by vancomycin over 72 h of incubation in vitro (
P
= 0.03). For MRSA isolates with vancomycin MICs ≤ 0.5 μg/ml, vancomycin was 55.6% successful in the treatment of bacteremia whereas vancomycin was only 9.5% effective in cases in which vancomycin MICs for MRSA were 1 to 2 μg/ml. Patients with MRSA that was more effectively killed at 72 h by vancomycin in vitro had a higher clinical success rate with vancomycin therapy in the treatment of bacteremia (log
10
< 4.71 [
n
= 9], 0%; log
10
4.71 to 6.26 [
n
= 13], 23.1%; log
10
> 6.27 [
n
= 8], 50%). We conclude that a significant risk for vancomycin treatment failure in MRSA bacteremia begins to emerge with increasing vancomycin MICs well within the susceptible range. Elucidating the mechanisms involved in intermediate-level glycopeptide resistance in
S. aureus
should begin by examining bacteria that begin to show changes in vancomycin susceptibility before the development of obvious resistance. Prognostic information for vancomycin treatment outcome in MRSA bacteremia may also be obtained by testing the in vitro bactericidal potency of vancomycin.
Molecular Characterization of Rifampin- and Isoniazid-Resistant
Mycobacterium tuberculosis
Strains Isolated in Poland
Authors: Sajduda Anna , Brzostek Anna , Popławska Marta , Augustynowicz-Kopeć Ewa , Zwolska Zofia , Niemann Stefan , Dziadek Jarosław , Hillemann Doris
Published: June 2004

A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of
Mycobacterium tuberculosis
isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were
rpoB
(RMP),
katG
, and the regulatory region of
inhA
(INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the
rpoB
gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the
katG
gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation −15C→T in the regulatory region of
inhA
. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in
M. tuberculosis
clinical isolates.
Rapid Quantification of Hepatitis B Virus DNA by Automated Sample Preparation and Real-Time PCR
Authors: Stelzl Evelyn , Muller Zsofia , Marth Egon , Kessler Harald H.
Published: June 2004

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLIPREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within ±0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within ±0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time.
Comparison of Quantitative and Semiquantitative Enzyme-Linked Immunosorbent Assays for Immunoglobulin G against
Chlamydophila pneumoniae
to a Microimmunofluorescence Test for Use with Patients with Respiratory Tract Infections
Authors: Hermann Corinna , Gueinzius Katja , Oehme Albrecht , von Aulock Sonja , Straube Eberhard , Hartung Thomas
Published: June 2004

We previously reported a high degree of variation in the sensitivities of serodiagnostic kits for the detection of
Chlamydophila pneumoniae
in sera from healthy donors. Since a low predictive value of a test can impair its diagnostic value, we have extended our studies to samples from patients with pneumonia. We focused on the most promising enzyme-linked immunosorbent assays (ELISAs) (SeroCP and SeroCP Quant; Savyon) identified in our previous study and included a new ELISA (sELISA; Medac). The agreement between all ELISAs for immunoglobulin G (IgG) and a reference microimmunofluorescence (MIF) test for IgG (SeroFIA; Savyon) was ≥90% for a collective of 80 patients. The positive predictive values were all ≥93%. The negative predictive values ranged from 68 to 83%. False-negative results were obtained only for samples that had low titers in the MIF test. The correlation of the IgG antibody titers determined by the MIF and SeroCP Quant tests was high (
r
sp
= 0.9). Since the semiquantitative SeroCP and quantitative SeroCP Quant ELISAs achieved the highest sensitivities, they were evaluated further by using a second batch of sera from 50 patients with predominantly medium and low antibody titers in the MIF test and a control collection of sera from 80 children with negative MIF results. Again, the tests showed a high concordance with the MIF results (96%), and the antibody titers in the SeroCP Quant and MIF tests correlated well (
r
sp
= 0.8). The specificities determined with the negative sera were ≥99% for the SeroCP Quant test and 86% for the SeroCP test. These results show that ELISAs that are fast and objective deliver seroprevalence results, sensitivities, and specificities that are very similar to those of the MIF test.
Dengue Virus Serotyping Based on Envelope and Membrane and Nonstructural Protein NS1 Serotype-Specific Capture Immunoglobulin M Enzyme-Linked Immunosorbent Assays
Authors: Shu Pei-Yun , Chen Li-Kuang , Chang Shu-Fen , Su Chien-Ling , Chien Li-Jung , Chin Chuan , Lin Ting-Hsiang , Huang Jyh-Hsiung
Published: June 2004

Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping.
Molecular Epidemiologic Evaluation of Endocarditis Due to
Oerskovia turbata
and CDC Group A-3 Associated with Contaminated Homograft Valves
Authors: McNeil Michael M. , Brown June M. , Carvalho Michelle E. , Hollis Dannie G. , Morey Roger E. , Reller L. Barth
Published: June 2004

Oerskovia turbata
is an unusual bacterial cause of endocarditis and septicemia in immunocompromised patients. In this study, we compared 12 isolates from a 1975 medical center cluster, 11 originally identified as
O. turbata
(four from the blood of a homograft aortic valve-associated endocarditis patient and seven from contaminated homograft valves) and one CDC group A-3 strain from the blood of a second endocarditis patient with fatal outcome, with eight control strains from unrelated locations. The control strains included type and reference strains of
O. turbata
,
Cellulomonas hominis
, and CDC group A-3. The four blood isolates from the first patient and six of the valve isolates shared identical biochemical, antimicrobial susceptibility, and BglI ribotype patterns that differed from the second patient's isolate and control strains. The blood isolate from the second patient and the remaining valve isolate shared a phenotypic and genotypic profile and were phenotypically identical to, but epidemiologically different from, the CDC group A-3 reference strain with the strain-specific enzyme. Also, these isolates differed from the type strain and the other reference strains of
C. hominis
and
O. turbata
. Our results indicate that the four blood isolates from the first patient and six of the homograft valve isolates represent a single clone of
O. turbata
associated with endocarditis. Additionally, our results indicate that the blood isolate from the second patient and one of the homograft valve isolates differ from
O. turbata
and
C. hominis
and represent a unique clone of CDC group A-3 associated with fatal endocarditis.
Characterization of
Chlamydia trachomatis omp1
Genotypes Detected in Eye Swab Samples from Remote Australian Communities
Authors: Stevens Matthew P. , Tabrizi Sepehr N. , Muller Rosanne , Krause Vicki , Garland Suzanne M.
Published: June 2004

Chlamydia trachomatis
conjunctival samples collected over a 6-month period from individuals with clinical signs of trachoma and located in remote communities in the Australian Northern Territory were differentially characterized according to serovar and variants. The rationale was to gain an understanding of the epidemiology of an apparent increased prevalence of acute trachoma in areas thought to be less conducive to this disease. Characterization was performed through sequencing of a region of the
omp1
gene spanning the four variable domains and encoding the major outer membrane protein. Nucleotide and deduced amino acid sequences were genotyped by using a BLAST similarity search and were examined by phylogenetic analyses to illustrate evolutionary relationships between the clinical and GenBank reference strains. The predominant genotype identified corresponded to that of serovar C (87.1%), followed by the genotype corresponding to serovar Ba (12.9%). All nucleotide and amino acid sequences exhibited minor levels of variation with respect to GenBank reference sequences. The
omp1
nucleotide sequences of the clinical samples best aligned with those of the conjunctival
C
.
trachomatis
reference strains C/TW-3/OT and Ba/Apache-2. All clinical samples (of serovar C) exhibited four or five nucleotide changes compared with C/TW-3/OT, while all serovar Ba samples had one or two nucleotide differences from Ba/Apache-2. Phylogenetic analyses revealed close relationships between these Northern Territory chlamydial samples and the respective reference strains, although the high proportion of sequence variants suggests an evolutionarily distinct
C
.
trachomatis
population causing eye infections in Australia. Given that such genotypic information has gone unreported, these findings provide knowledge and a foundation for trachoma-associated
C
.
trachomatis
variants circulating in the Northern Territory.
Multifocal Detection of Multidrug-Resistant
Pseudomonas aeruginosa
Producing the PER-1 Extended-Spectrum β-Lactamase in Northern Italy
Authors: Pagani Laura , Mantengoli Elisabetta , Migliavacca Roberta , Nucleo Elisabetta , Pollini Simona , Spalla Melissa , Daturi Rossana , Romero Egidio , Rossolini Gian Maria
Published: June 2004

Forty-four nonreplicate clinical isolates of
Pseudomonas aeruginosa
that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum β-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of
bla
PER
ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired
bla
PER-1
gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The
bla
PER-1
gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the
bla
PER-1
locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing
bla
PER-1
revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR
P. aeruginosa
clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.
Characterization of G10P[11] Rotaviruses Causing Acute Gastroenteritis in Neonates and Infants in Vellore, India
Authors: Iturriza Gómara Miren , Kang Gagandeep , Mammen Ajit , Jana Atanu Kumar , Abraham Mary , Desselberger Ulrich , Brown David , Gray Jim
Published: June 2004

Rotavirus G10P[11] strains, which are commonly found in cattle, have frequently been associated with asymptomatic neonatal infections in India. We report the finding of G10P[11] strains associated with severe disease in neonates in Vellore, southern India. Rotavirus strains from 43 fecal samples collected from neonates with or without gastrointestinal symptoms between 1999 and 2000 were genotyped by reverse transcription-PCR. Forty-one neonates (95%) were infected with G10P[11] rotavirus strains, and 63% of the infections were in children who had gastrointestinal symptoms, including acute watery diarrhea. G10P[11] strains were also seen infecting older children with dehydrating gastroenteritis in Vellore. Characterization of the genes encoding VP7, VP4, VP6, and NSP4 of these strains revealed high sequence homology with the corresponding genes of the asymptomatic neonatal strain I321, which in turn is very closely related to bovine G10P[11] strains circulating in India. No significant differences were seen in the sequences obtained from strains infecting symptomatic neonates or children and asymptomatic neonates.
Serological and Genetic Diversity of Capsular Polysaccharides in
Enterococcus faecalis
Authors: Hufnagel Markus , Hancock Lynn E. , Koch Stefanie , Theilacker Christian , Gilmore Michael S. , Huebner Johannes
Published: June 2004

Enterococci possess capsular carbohydrate antigens that are targets of opsonic antibodies. These antigens may be used to develop alternative options for the treatment and prevention of enterococcal infections. The present study was done to analyze the diversity of capsular polysaccharides in
Enterococcus faecalis
. Four type-specific sera were used in an enzyme-linked immunosorbent assay format to detect polysaccharide antigen extracted from bacterial cell walls. A total of 55% of a collection of 29
E. faecalis
strains could be grouped into one of four serogroups. Additional analysis of the strains by opsonophagocytic assays revealed agreement between the results of the two methods for 72% of the isolates. An additional four strains could be assigned to a serogroup on the basis of opsonic killing by sera with antibodies against the four prototypes strains, provisionally named CPS-A to CPS-D. The results of the two methods disagreed for one strain (4%). When the results of both methods were combined, 66% of the strains could be classified. One strain had to be assigned to two serogroups. The assignments to the four serogroups were confirmed by analysis of the genetic organization of the biosynthetic capsular polysaccharide (
cps
) locus. All strains grouped into serotypes CPS-A and CPS-B possessed only the
cpsA
and
cpsB
genes, while all strains grouped into serogroups CPS-C and CPS-D possessed an additional eight or nine genes. Our results suggest the existence of a limited number of
E. faecalis
capsule serotypes, and we provisionally propose four serotypes, named CPS-A to CPS-D, and the respective prototype strains for these families.
Genotypic Variation and Stability of Four Variable-Number Tandem Repeats and Their Suitability for Discriminating Strains ofMycobacterium leprae
Authors: Truman Richard , Fontes Amanda B. , de Miranda Antonio B. , Suffys Philip , Gillis Thomas
Published: June 2004

It has not been possible to distinguish different strains ofMycobacterium lepraeaccording to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiatingM. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12M. lepraestrains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C16G8) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT17varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA18varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA18locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminateM. lepraestrains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.
Evaluation and Comparison of Two Commercial Enzyme-Linked Immunosorbent Assay Kits for Detection of Antigenically Diverse Human Noroviruses in Stool Samples
Authors: Burton-MacLeod Jonathan A. , Kane Erin M. , Beard Rachel S. , Hadley Leslie A. , Glass Roger I. , Ando Tamie
Published: June 2004

Two recently commercialized enzyme-linked immunosorbent assay kits, the SRSV(II)-AD (Denka Seiken Co. Ltd., Tokyo, Japan) and IDEIA NLV (DakoCytomation Ltd., Ely, United Kingdom) kits, that detect human norovirus (HuNV) antigens in stool samples were evaluated to assess whether they could be used instead of reverse transcription-PCR (RT-PCR) for routine diagnosis. The sensitivities and specificities of the two kits were tested with a panel of 103 stool samples containing HuNVs of 4 and 10 genetic subgroups within genogroups I and II (GI and GII), respectively, and 39 stool samples containing other enteric viruses. The Denka kit had a high sensitivity (>70% for 10 of the 14 subgroups) but a specificity of only 69%, and the Dako kit had a low sensitivity (<30% for 6 GII subgroups) but a high specificity of 100%. Statistical analysis suggests that HuNVs of four subgroups (subgroups GII/2, GII/5, GII/6, and GII/n) are likely to elude detection by the Dako kit. The two kits also demonstrated differences in reactivities. While the Dako kit discriminated between the GI and GII antigens of HuNVs, the Denka kit cross-reacted with samples containing all GI and GII subgroups of HuNVs. Moreover, the Denka kit also reacted with samples containing human sapovirus (HuSV). We demonstrate that the cross-reactivity of the Denka kit is not due to specific reactions with HuNV and HuSV antigens. These results indicate that neither the Denka kit nor the Dako kit has all the performance characteristics required to replace the RT-PCR methods used to detect HuNVs.
Variability of theChlamydia trachomatis omp1Gene Detected in Samples from Men Tested in Male-Only Saunas in Melbourne, Australia
Authors: Lister Nichole A , Tabrizi Sepehr N. , Fairley Christopher K. , Smith Anthony , Janssen Peter H. , Garland Suzanne
Published: June 2004

A recent screening program in Melbourne, Victoria, Australia, has shown thatChlamydia trachomatisis an important infection among men who frequent male-only saunas. To evaluate theC. trachomatisisolates circulating in local saunas, theC. trachomatis-positive samples collected during the program underwent amplification and sequencing of theomp1gene, and the corresponding serovars were deduced. Forty-sevenC. trachomatis-positive samples collected (from October 2001 to September 2002) from 39 men were evaluated. The deduced serovars found, in descending order of prevalence, were D, G, and J; and serovars B, E, F, and H were each found in single samples. The seven different serovars identified in the study sample indicate that local saunas are a reservoir of multipleC. trachomatisstrains, possibly maintained by the introduction of new patrons or regular patrons who have been exposed toC. trachomatiselsewhere. No significant genetic variants were found, as most variable positions were silent and were detected only in single samples.
Rapid and Specific Detection of
Escherichia coli
Clonal Group A by Gene-Specific PCR
Authors: Johnson James R. , Owens Krista , Manges Amee R. , Riley Lee W.
Published: June 2004

PCR primers specific for the recently described antimicrobial resistance-associated
Escherichia coli
clonal group A (CGA), a widespread cause of drug-resistant urinary tract infections in the United States, were devised on the basis of a novel single-nucleotide polymorphism identified within the housekeeping gene
fumC
, i.e., C288T. In comparison with two reference PCR-based fingerprinting methods, ERIC2 PCR and random amplified polymorphic DNA (RAPD) analysis, a PCR assay incorporating the new primers provided 100% sensitivity and 100% specificity for the detection of CGA among 138 diverse clinical and reference
E. coli
isolates.
E. coli
reference (ECOR) strain 47 was shown to be a member or a close relative of CGA (by ERIC2 PCR and RAPD analysis, respectively) and yielded a positive assay result. The new CGA-specific PCR assay, which exhibited interlaboratory reproducibility and stability under various experimental conditions, should allow the rapid and specific detection of CGA by any laboratory equipped for diagnostic PCR.
Comparison of the Roche LightCycler
vanA
/
vanB
Detection Assay and Culture for Detection of Vancomycin-Resistant Enterococci from Perianal Swabs
Authors: Sloan L. M. , Uhl J. R. , Vetter E. A. , Schleck C. D. , Harmsen W. S. , Manahan J. , Thompson R. L. , Rosenblatt J. E. , Cockerill F. R.
Published: June 2004

We compared the performance characteristics of a real-time PCR method, the LightCycler
vanA
/
vanB
detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 μg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 μg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 μg/ml (
n
= 25 versus
n
= 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 μg/ml (
n
= 31 versus
n
= 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 μg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 μg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (∼3.5 versus ≥72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.
Real-Time PCR Assay Using Fine-Needle Aspirates and Tissue Biopsy Specimens for Rapid Diagnosis of Mycobacterial Lymphadenitis in Children
Authors: Bruijnesteijn van Coppenraet E. S. , Lindeboom J. A. , Prins J. M. , Peeters M. F. , Claas E. C. J. , Kuijper E. J.
Published: June 2004

A real-time PCR assay was developed to diagnose and identify the causative agents of suspected mycobacterial lymphadenitis. Primers and probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of the genus
Mycobacterium
and the species
Mycobacterium avium
and
M
.
tuberculosis
. The detection limit for the assay was established at 1,100 CFU/ml of pus, and the specificity tests showed no false-positive reaction with other mycobacterial species and other pathogens causing lymphadenitis. From 67 children with suspected mycobacterial lymphadenitis based on a positive mycobacterial skin test, 102 samples (58 fine-needle aspirates [FNA] and 44 tissue specimens) were obtained. The real-time PCR assay detected a mycobacterial infection in 48 patients (71.6%), whereas auramine staining and culturing were positive for 31 (46.3%) and 28 (41.8%) of the patients. The addition of the real-time PCR assay to conventional diagnostic tests resulted in the recognition of 13 more patients with mycobacterial disease. These results indicate that the real-time PCR is more sensitive than conventional staining and culturing techniques (
P
= 0.006). The
M
.
avium
-specific real-time PCR was positive for 38 patients, and the
M
.
tuberculosis
-specific real-time PCR was positive for 1 patient. Analysis of 27 patients from whom FNA and tissue biopsy specimens were collected revealed significantly more positive real-time PCR results for FNA than for tissue biopsy specimens (
P
= 0.003). Samples from an age-matched control group of 50 patients with PCR-proven cat scratch disease were all found to be negative by the real-time PCR. We conclude that this real-time PCR assay with a sensitivity of 72% for patients with lymphadenitis and a specificity of 100% for the detection of atypical mycobacteria can provide excellent support for clinical decision making in children with lymphadenitis.
Identification of Clinically Relevant Viridans Group Streptococci by Sequence Analysis of the 16S-23S Ribosomal DNA Spacer Region
Authors: Chen Chao Chien , Teng Lee Jene , Chang Tsung Chain
Published: June 2004

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29 reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species were
Streptococcus anginosus
,
S. constellatus
,
S. gordonii
,
S. intermedius
,
S. mitis
,
S. mutans
,
S. oralis
,
S. parasanguinis
,
S. salivarius
,
S. sanguinis
, and
S. uberis
. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for the score for
S. gordonii
strains. The interspecies similarity scores for the ITS sequences varied from 0.31 to 0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMérieux Vitek, Marcy l'Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing for identification of VS was verified by 16S rDNA sequencing for all strains except strains of
S. oralis
and
S. mitis
, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.
Comparative Evaluation of Three Serological Methods for Detection of Human Herpesvirus 8-Specific Antibodies in Canadian Allogeneic Stem Cell Transplant Recipients
Authors: Sergerie Yan , Abed Yacine , Roy Jean , Boivin Guy
Published: June 2004

Human herpesvirus 8 (HHV-8) has been associated with all types of Kaposi's sarcoma (KS), including posttransplantation KS. However, little is known regarding HHV-8 infections in hematopoietic stem cell transplant (SCT) recipients. In this study, we used a variety of serological assays, including in-house-developed enzyme immunoassays (EIAs) utilizing synthetic peptides corresponding to lytic viral antigens (ORFs 65 and K8.1) and a commercial EIA kit based on a whole virus lysate (Advanced Biotechnologies Inc.), as well as latent- and lytyc-antigen-based immunofluorescence assays (IFAs) to determine the seroprevalence of HHV-8 in 42 allogeneic SCT recipients from Canada. Using the two peptide-based EIA methods as for screening, HHV-8-specific antibodies were detected in five (12%) patients between days 21 and 91, although only one (2%) subject was positive for HHV-8-specific antibodies before transplantation. All positive results from these five patients were confirmed by at least one of the IFAs, with an additional patient showing seropositivity before transplantation. However, the commercial EIA was negative at all time points (days −7, 21, and 91) in those five patients. The episodes of seroconversion or reactivation were not associated with sustained viremia, since HHV-8 DNA was not detected by real-time PCR in the corresponding leukocytes and plasma of the seropositive patients. No clinical or laboratory abnormalities were clearly associated with HHV-8 seropositivity. This study confirms the utility of simple peptide-based EIA methods to assess the presence of HHV-8-specific antibodies in immunocompromised patients and emphasizes the need of conducting prospective studies to determine the source of HHV-8 infection in SCT recipients.
Prevalence of
Helicobacter pylori vacA
and
cagA
Genotypes in Ethiopian Dyspeptic Patients
Authors: Asrat Daniel , Nilsson Ingrid , Mengistu Yohannes , Kassa Endale , Ashenafi Senait , Ayenew Kiros , Wadström Torkel , Abu-Al-Soud Waleed
Published: June 2004

A total of 300 gastric biopsy samples and 50
Helicobacter pylori
isolates were collected from Ethiopian adult dyspeptic patients. The
vacA
and
cagA
genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among
vacA
genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The
vacA
and
cagA
positive
H. pylori
strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (
P
< 0.05).
Evaluation of a High-Throughput Repetitive-Sequence-Based PCR System for DNA Fingerprinting of
Mycobacterium tuberculosis
and
Mycobacterium avium
Complex Strains
Authors: Cangelosi Gerard A. , Freeman Robert J. , Lewis Kaeryn N. , Livingston-Rosanoff Devon , Shah Ketan S. , Milan Sparrow Joy , Goldberg Stefan V.
Published: June 2004

Repetitive-sequence-based PCR (rep-PCR) is useful for generating DNA fingerprints of diverse bacterial and fungal species. Rep-PCR amplicon fingerprints represent genomic segments lying between repetitive sequences. A commercial system that electrophoretically separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been adapted for use with
Mycobacterium
species. The ability of this system to type
M. tuberculosis
and
M. avium
complex (MAC) isolates was evaluated.
M. tuberculosis
strains (
n
= 56) were typed by spoligotyping with rep-PCR as a high-resolution adjunct. Results were compared with those generated by a standard approach of spoligotyping with IS
6110
-targeted restriction fragment length polymorphism (IS
6110
-RFLP) as the high-resolution adjunct. The sample included 11 epidemiologically and genotypically linked outbreak isolates and a population-based sample of 45 isolates from recent immigrants to Seattle, Wash., from the African Horn countries of Somalia, Eritrea, and Ethiopia. Twenty isolates exhibited unique spoligotypes and were not analyzed further. Of the 36 outbreak and African Horn isolates with nonunique spoligotypes, 23 fell into four clusters identified by IS
6110
-RFLP and rep-PCR, with 97% concordance observed between the two methods. Both approaches revealed extensive strain heterogeneity within the African Horn sample, consistent with a predominant pattern of reactivation of latent infections in this immigrant population. Rep-PCR exhibited 89% concordance with IS
1245
-RFLP typing of 28
M. avium
subspecies
avium
strains. For
M. tuberculosis
as well as
M. avium
subspecies
avium
, the discriminative power of rep-PCR equaled or exceeded that of RFLP. Rep-PCR also generated DNA fingerprints from
M. intracellulare
(
n
= 8) and MAC
x
(
n
= 2) strains. It shows promise as a fast, unified method for high-throughput genotypic fingerprinting of multiple
Mycobacterium
species.
Detection and Prevalence of Active Drug Efflux Mechanism in Various Multidrug-Resistant
Klebsiella pneumoniae
Strains from Turkey
Authors: Hasdemir Ufuk Over , Chevalier Jacqueline , Nordmann Patrice , Pagès Jean-Marie
Published: June 2004

The prevalence of active drug efflux pump and porin alterations was investigated in Turkish nosocomial strains of
Klebsiella pneumoniae
exhibiting a multidrug-resistant phenotype. MICs of various antibiotics, including quinolones, chloramphenicol, tetracycline, and β-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine β-naphthylamide (PAβN). Thirty-nine percent of the strains exhibited a PAβN-modulated resistance for quinolones, chloramphenicol, and tetracycline. In these strains, a significant increase of chloramphenicol accumulation was gained in the presence of the efflux pump inhibitor PAβN or with the energy uncoupler carbonyl cyanide
m
-chlorophenylhydrazone. Moreover, high-level expression of the membrane fusion protein AcrA, which was immunodetected in most of those isolates, suggests that the AcrAB/TolC efflux machinery contributed to their antibiotic resistance. Studies of
K. pneumoniae
porins indicated that the majority of the strains, including extended-spectrum β-lactamase producers and efflux-positive ones, presented an alteration in their sorbitol-sensitive porin (OmpK35) expression. This is the first report showing the prominent role of active drug efflux in the antibiotic resistance of nosocomial
K. pneumoniae
strains from Turkey.
Prevalence of and Risk Factors for FelineTritrichomonas foetusandGiardiaInfection
Authors: Gookin Jody L. , Stebbins Martha E. , Hunt Emily , Burlone Karen , Fulton Merritt , Hochel Robin , Talaat Miriam , Poore Matthew , Levy Michael G.
Published: June 2004

Data were gathered for 117 cats from 89 catteries at an international cat show to examine prevalence and risk factors for felineTritrichomonas foetusandGiardiainfection. Prevalence ofT. foetuswas 31% among cats (36 out of 117) and catteries (28 out of 89) based on results of fecal smear examination (5 out of 36), fecal culture in modified Diamond's medium (9 out of 36), fecal culture in In Pouch TF medium (20 out of 36), or PCR amplification of the ribosomal RNA gene from feces withT. foetus-specific primers (34 out of 36). Catteries in whichT. foetuswas identified were more likely to have had a recent history of diarrhea, historical diagnosis of coccidia infection in adult cats, and a decreased number of square feet of facility per cat. Evidence did not exist for the ongoing transmission ofT. foetusby water, food, or contact with other species.