bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic Polymorphisms forMycobacterium aviumsubsp.paratuberculosisDiagnostics
Authors: Semret Makeda , Alexander David C. , Turenne Christine Y. , de Haas Petra , Overduin Pieter , van Soolingen Dick , Cousins Debby , Behr Marcel A. 
Published: August 2005

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences betweenM. aviumsubsp.paratuberculosisand its close relativeM. aviumsubsp.avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains,M. aviumsubsp.aviumstrain 104 andM. aviumsubsp.paratuberculosisstrain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSPAs) and those only present in the latter (LSPPs). We examined the distribution of 3 LSPAs and 17 LSPPs across a panel of 383M. aviumcomplex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSPA8 is 100% specific for the identification ofM. aviumsubsp.paratuberculosis. Of the 17 LSPPs, 10 regions were not specific forM. aviumsubsp.paratuberculosiswhile 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited forM. aviumsubsp.paratuberculosisdiagnostics.
Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for Methicillin-Resistant
Staphylococcus aureus
Authors: Smyth R. W. , Kahlmeter Gunnar 
Published: August 2005

ABSTRACT

In disk diffusion tests, cefoxitin is now considered a better indicator than oxacillin for the presence of the
mecA
gene in
Staphylococcus aureus
. A logical extension of this work is the incorporation of cefoxitin into media selective for methicillin-resistant
Staphylococcus aureus
(MRSA). This paper describes the development and subsequent testing of mannitol salt agar containing 4 mg/liter cefoxitin with a unique collection of well-characterized MRSA strains, including low-level methicillin-resistant strains and an equal number of known
mecA
-negative
S. aureus
strains. The agar supported the growth of 96.6% of the
mecA
-positive strains in the collection and inhibited the growth of 100% of the
mecA
-negative strains. These results suggest that selective media based on cefoxitin are superior to those based on oxacillin for the detection of MRSA.
Diagnosis of Cat Scratch Disease with Detection of
Bartonella henselae
by PCR: a Study of Patients with Lymph Node Enlargement
Authors: Hansmann Yves , DeMartino Sylvie , Piémont Yves , Meyer Nicolas , Mariet Philippe , Heller Rémy , Christmann Daniel , Jaulhac Benoît 
Published: August 2005

ABSTRACT

Cat scratch disease (CSD) is mostly due to
Bartonella henselae
after inoculation of the organism through a skin injury. Since the causative bacteria cannot be easily cultured from human lymph node samples, the diagnosis usually relies on epidemiological, clinical, histological, and serological criteria (classical criteria). A study was performed to determine the diagnostic value of PCR analysis for the detection of
B. henselae
for the diagnosis of CSD and its place in the diagnostic strategy alongside the classical criteria. Over a 7-year period, lymph node biopsy specimens or cytopunctures from 70 patients were systematically tested by PCR for the presence of
B. henselae
DNA (
htrA
gene) in the Bacteriology Laboratory of the Hôpitaux Universitaires de Strasbourg. Serological testing by an immunofluorescence assay for
B. henselae
antibodies was also performed for each patient, and clinical, epidemiological, and histological data were collected. The patients were then divided into two groups according to the number of positive diagnostic criteria for CSD: 29 patients with definite CSD (two or more classical criteria) and 15 patients with possible CSD (less than two classical criteria). The remaining 26 patients for whom another diagnosis was retained were used as a control group. Among all criteria, PCR analysis had the best specificity (100%). The PCR assay for
B. henselae
was positive for 22 (76%; 95% confidence interval [CI
95
], 56.5 to 89.7%) of the 29 definite CSD patients and 3 (20%; CI
95
, 4.3 to 48.1%) of the 15 possible CSD patients. We then studied combinations of diagnostic criteria, including
B. henselae
PCR analysis. The best diagnostic performance was observed if at least two criteria were present among serologic, epidemiologic, histological, and molecular criteria.
Computational Approach Involving Use of the Internal Transcribed Spacer 1 Region for Identification of
Mycobacterium
Species
Authors: Mohamed Amr M. , Kuyper Dan J. , Iwen Peter C. , Ali Hesham H. , Bastola Dhundy R. , Hinrichs Steven H. 
Published: August 2005

ABSTRACT

The rapid and reliable identification of clinically significant
Mycobacterium
species is a challenge for diagnostic laboratories. This study evaluates a unique sequence-dependent identification algorithm called
MycoAlign
for the differential identification of
Mycobacterium
species. The
MycoAlign
system uses pan-
Mycobacterium
-specific primer amplification in combination with a customized database and algorithm. The results of testing were compared with conventional phenotypic assays and GenBank sequence comparisons using the 16S rRNA target. Discrepant results were retested and evaluated using a third independent database. The custom database was generated using the hypervariable sequences of the internal transcribed spacer 1 (ITS-1) region of the rRNA gene complex from characterized
Mycobacterium
species. An automated sequence-validation process was used to control quality and specificity of evaluated sequence. A total of 181
Mycobacterium
strains (22 reference strains and 159 phenotypically identified clinical isolates) and seven nonmycobacterial clinical isolates were evaluated in a comparative study to validate the accuracy of the
MycoAlign
algorithm.
MycoAlign
correctly identified all referenced strains and matched species in 94% of the phenotypically identified
Mycobacterium
clinical isolates. The ITS-1 sequence target showed a higher degree of specificity in terms of
Mycobacterium
identification than the 16S rRNA sequence by use of GenBank BLAST. This study showed the
MycoAlign
algorithm to be a reliable and rapid approach for the identification of
Mycobacterium
species and confirmed the superiority of the ITS-1 region sequence over the 16S rRNA gene sequence as a target for sequence-based species identification.
Results of Disk Diffusion Testing with Cefoxitin Correlate with Presence of
mecA
in
Staphylococcus
spp
Authors: Swenson Jana M. , Tenover Fred C. ,   the Cefoxitin Disk Study Group
Published: August 2005

ABSTRACT

The cefoxitin disk diffusion (DD) test for predicting
mecA-
mediated oxacillin resistance in staphylococci was assessed during a three-phase study. In phase 1, one laboratory tested 62 and 53 strains of
Staphylococcus aureus
and coagulase-negative staphylococci (CoNS), respectively. These data were used to choose the provisional cefoxitin DD breakpoints (resistant/susceptible) of ≤19 mm/≥20 mm for
S. aureus
and ≤24 mm/≥25 mm for CoNS for the next phase of testing. In phase 2, 10 laboratories each tested approximately 40 in-house strains of staphylococci (half of which were
S. aureus
) using Mueller-Hinton agar from different manufacturers. In this phase, the sensitivity and specificity, respectively, of the cefoxitin disk test were 98 and 100% for
S. aureus
and 99 and 96% for CoNS. The cefoxitin DD test performed equivalently to oxacillin broth microdilution (BMD) and to oxacillin DD tests among
S. aureus
and
mecA
-positive CoNS strains but gave better results than oxacillin BMD or oxacillin DD for
mecA
-negative strains of CoNS. The cefoxitin DD test also was much easier to read and did not require the use of transmitted light for detection of resistance. Based on data from the first two phases, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) adopted the use of the cefoxitin DD test for predicting
mecA-
mediated oxacillin resistance in staphylococci and revised Table 2C in CLSI document M100-S14 to reflect the change. In the third phase, an additional 61 challenge strains of CoNS for which the oxacillin MICs were 0.5 to 2 μg/ml were tested in a single laboratory to determine the effectiveness of the cefoxitin DD test for this group of borderline-resistant isolates. These data were used to refine the description of the test in CLSI document M100-S15. The cefoxitin DD test is preferred over the oxacillin DD test for predicting
mecA
-mediated oxacillin resistance in
S. aureus
and CoNS.
Nosocomial Outbreak Caused by Multidrug-Resistant
Pseudomonas aeruginosa
Producing IMP-13 Metallo-β-Lactamase
Authors: Pagani Laura , Colinon Céline , Migliavacca Roberta , Labonia Maria , Docquier Jean-Denis , Nucleo Elisabetta , Spalla Melissa , Li Bergoli Michele , Rossolini Gian Maria 
Published: August 2005

ABSTRACT

An outbreak of
Pseudomonas aeruginosa
showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-β-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a
bla
IMP-13
gene carried on a gene cassette inserted in a class 1 integron which also contained an
aacA4
aminoglycoside resistance cassette encoding an AAC(6′)-Ib enzyme. The
bla
IMP-13
-containing integron and its genetic environment appeared to be similar to those found in
P. aeruginosa
isolates producing IMP-13 from a hospital in Rome. The
bla
IMP-13
gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.
Genetic Characteristics of Human Enterovirus 71 and Coxsackievirus A16 Circulating from 1999 to 2004 in Shenzhen, People's Republic of China
Authors: Li Linlin , He Yaqing , Yang Hong , Zhu Junping , Xu Xingye , Dong Jie , Zhu Yafang , Jin Qi 
Published: August 2005

ABSTRACT
The genetic and phylogenetic characteristics of human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) sampled from children with hand, foot, and mouth disease in Shenzhen, People's Republic of China, over a 6-year period (1999 to 2004) were examined with reverse transcription-PCR and DNA sequencing. Out of 147 stool specimens, 60 showed positive signals when screened with EV71- and CA16-specific primers. EV71 was identified in 19 specimens, and CA16 was identified in 41 specimens; coinfection by EV71 and CA16 was not observed. Phylogenetic analysis of all EV71 strains isolated from the mainland Chinese samples established C4 as the predominant genotype. Only one other known strain (3254-TAI-98; AF286531), isolated in Taiwan in 1998, was identified as belonging to genotype C4. Phylogenetic analysis of CA16 strains allowed us to identify three new genetic lineages (A, B, and C), with lineage C recently predominating in Asian countries, such as the People's Republic of China, Malaysia, and Japan. These new observations indicate that CA16 circulating in the People's Republic of China is genetically diverse, and additional surveillance is warranted.
New Microsatellite Multiplex PCR for
Candida albicans
Strain Typing Reveals Microevolutionary Changes
Authors: Sampaio Paula , Gusmão Leonor , Correia Alexandra , Alves Cíntia , Rodrigues Acácio G. , Pina-Vaz Cidália , Amorim António , Pais Célia 
Published: August 2005

ABSTRACT

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of
Candida albicans
strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114
C. albicans
strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of
C. albicans
strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.
Phylogenetic and Pathotypic Comparison of Concurrent Urine and RectalEscherichia coliIsolates from Men with Febrile Urinary Tract Infection
Authors: Johnson James R. , Scheutz Flemming , Ulleryd Peter , Kuskowski Michael A. , O'Bryan Timothy T. , Sandberg Torsten 
Published: August 2005

ABSTRACTAmong men with febrile urinary tract infection (FUTI), whether the host's fecal flora is the source for the urine strain (“fecal-urethral” hypothesis), and whether pathogenesis is driven by prevalence versus special pathogenicity, are unknown. Accordingly, pretherapy urine isolates from 65 men with FUTI were compared with concurrent rectal isolates from the same hosts according to serotype, genomic profile, phylogenetic group, and virulence genotype. The host's multiple rectal colonies included only the urine clone in 25% of subjects, the urine clone plus additional clones in 22%, and only nonurine clones in 54%. Compared with the 67 unique rectal clones, the 65 urine isolates were significantly enriched for phylogenetic group B2, virulence-associated serotypes, and specific virulence genes and contained more virulence genes (median, 10 versus 6:P< 0.001). In multivariable models, phylogenetic group B2,hlyD(hemolysin),cnf1(cytotoxic necrotizing factor),iroN(siderophore receptor),ompT(outer membrane protease), andmalX(pathogenicity island marker) most strongly predicted urine source. These findings challenge the fecal-urethral and prevalence hypotheses for FUTI pathogenesis and instead strongly support the possibility of alternate infection routes in some men and the special pathogenicity hypothesis. They also identify specific bacterial traits as potential targets for anti-FUTI interventions.
Detection and Quantification of Human Immunodeficiency Virus Type 1 p24 Antigen in Dried Whole Blood and Plasma on Filter Paper Stored under Various Conditions
Authors: Li Chung-Chen , Seidel Kristy D. , Coombs Robert W. , Frenkel Lisa M. 
Published: August 2005

ABSTRACT

The quantification of human immunodeficiency virus type 1 (HIV-1) by an assay measuring heat-dissociated (HD) p24 antigen (Ag) in specimens of whole blood and plasma stored on filter paper, and of plasma stored in tubes, was compared to HIV-1 RNA plasma levels determined by real-time reverse transcription (RT)-PCR. The stability of p24 Ag on filter paper under conditions simulating specimen transport was also evaluated. The HD p24 Ag in both plasma and whole-blood specimens stored on filter paper correlated with plasma HIV-1 RNA levels (Spearman rank ρ = 0.74 [
P
< 0.0001] and ρ = 0.56 [
P
= 0.0001], respectively). The sensitivity of the HD p24 Ag assay was similar when plasma and whole blood on filter paper were contrasted to the real-time RT-PCR assay (80% versus 82.5% and 78.6% versus 83.3%, respectively). However, while the specificity of the HD p24 Ag assay of plasma on filter paper was 100%, the specificity was diminished in whole-blood specimens. The storage of specimens on filter paper for 2 weeks at 37°C, 24°C, or 0°C did not alter the detection or quantification of HD p24 Ag. These results suggest that transport and storage of plasma on filter paper and quantification of HD p24 Ag may be a reliable method for HIV-1 load monitoring.
Identification of Candidate Periodontal Pathogens and Beneficial Species by Quantitative 16S Clonal Analysis
Authors: Kumar Purnima S. , Griffen Ann L. , Moeschberger Melvin L. , Leys Eugene J. 
Published: August 2005

ABSTRACT

Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted DNA approaches, and so it is likely that pathogens remain undiscovered. The purpose of this study was to use culture-independent, quantitative analysis of biofilms associated with chronic periodontitis and periodontal health to identify pathogens and beneficial species. Samples from subjects with periodontitis and controls were analyzed using ribosomal 16S cloning and sequencing. Several genera, many of them uncultivated, were associated with periodontitis, the most numerous of which were gram positive, including
Peptostreptococcus
and
Filifactor
. The genera
Megasphaera
and
Desulfobulbus
were elevated in periodontitis, and the levels of several species or phylotypes of
Campylobacter
,
Selenomonas
,
Deferribacteres
,
Dialister
,
Catonella
,
Tannerella
,
Streptococcus
,
Atopobium
,
Eubacterium
, and
Treponema
were elevated in disease.
Streptococcus
and
Veillonella
spp. were found in high numbers in all samples and accounted for a significantly greater fraction of the microbial community in healthy subjects than in those with periodontitis. The microbial profile of periodontal health also included the less-abundant genera
Campylobacter
,
Abiotrophia
,
Gemella
,
Capnocytophaga
, and
Neisseria
. These newly identified candidates outnumbered
Porphyromonas gingivalis
and other species previously implicated as periodontopathogens, and it is not clear if newly identified and more numerous species may play a more important role in pathogenesis. Finally, more differences were found in the bacterial profile between subjects with periodontitis and healthy subjects than between deep and shallow sites within the same subject. This suggests that chronic periodontitis is the result of a global perturbation of the oral bacterial ecology rather than a disease-site specific microbial shift.
Model for Assessment of Proficiency of Human Immunodeficiency Virus Type 1 Sequencing-Based Genotypic Antiretroviral Assays
Authors: Huang Diana D. , Bremer James W. , Brambilla Donald J. , Palumbo Paul E. ,   for the Pediatric ACTG Sequencing Working Group
Published: August 2005

ABSTRACT
Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of ≥98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.
Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles
Authors: Gafan Gavin P. , Lucas Victoria S. , Roberts Graham J. , Petrie Aviva , Wilson Michael , Spratt David A. 
Published: August 2005

ABSTRACT

Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (
n
= 30) and from those with gingivitis (
n
= 30). Extracted DNA from gingival margin plaque was subjected to PCR targeting the 16S rRNA gene using universal primers. DGGE profiles were analyzed in three ways. (i) Bacterial diversity was compared between cohorts by using the Shannon-Wiener index (also known as the Shannon-Weaver index). (ii) A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. (iii) Individual DGGE bands and their intensities for both cohorts were compared using a logistic regression analysis. The Shannon-Wiener indices demonstrated a greater bacterial diversity associated with no-gingivitis plaque (
P
= 0.009). Dendrograms demonstrated that seven clades associated with gingivitis and five clades associated with no gingivitis. The logistic regression demonstrated that one band was significantly associated with no gingivitis (
P
= 0.001), while two bands were significantly associated with gingivitis (
P
= 0.005 and
P
= 0.042). In conclusion, this study demonstrates that the development of gingivitis might be accompanied by a decrease in bacterial diversity. Furthermore, we have demonstrated that logistic regression is a good statistical method for analyzing and characterizing DGGE profiles.