bluuebunny/crossref_metadata_2025 huggingface dataset:
Transesophageal Echocardiographic Diagnosis of Aortic Arch–Left Innominate Vein Fistula
Authors: Vengala Srinivas , Nanda Navin C. , Sidhu Maninder Singh , Dod Harvinder S. , Agrawal Gopal , Singh Vikramjit , Kirklin James K.
Published: January 2005

We present a patient with aortic arch–left innominate vein (LIV) fistula diagnosed by transesophageal echocardiography. Also, an interrupted linear echo was noted within the dilated LIV with flow signals moving across the area of interruption, suggestive of left innominate vein pseudo‐aneurysm or dissection. The patient subsequently underwent successful surgical repair of the fistula.
Assessment of Myocardial Viability with Dobutamine Stress Echocardiography in Patients with Ischemic Left Ventricular Dysfunction
Authors: Yao Siu‐Sun , Chaudhry Farooq A.
Published: January 2005

The noninvasive assessment of myocardial viability has proved clinically useful for distinguishing hibernating and/or stunned myocardium from irreversibly injured myocardium in patients with chronic ischemic heart disease or recent myocardial infarction, with marked regional and/or global left ventricular (LV) dysfunction. Noninvasive techniques utilized for the detection of viability in asynergic myocardial regions include positron emission tomographic imaging of residual metabolic activity, single photon emission tomography (SPECT) of radioisotope uptake with thallium‐201, low‐dose dobutamine echocardiography assessment of inotropic reserve and myocardial contrast echocardiography for evaluation of microvascular integrity. Of these techniques, dobutamine stress echocardiography is a safe, widely available and relatively inexpensive modality for the identification of myocardial viability for risk stratification and prognosis. Low‐dose dobutamine response can accurately predict improvement of dysfunctional yet viable myocardial regions, and thus identify a subset of patients whose LV function will improve following successful coronary revascularization.
Assessment of Myocardial Viability Using Myocardial Contrast Echocardiography
Authors: Wei Kevin
Published: January 2005

Myocardial contrast echocardiography (MCE) is a technique that uses microbubbles as a tracer during simultaneous ultrasound of the heart. The microbubbles can be used to provide quantitative information regarding the adequacy of myocardial blood flow (MBF), as well as the spatial extent of microvascular integrity. In acute myocardial infarction, MCE can identify the presence of collateral flow within the risk area, and can therefore predict preservation of myocardial viability and ultimate infarct size even prior to reperfusion. After reperfusion, the extent of microvascular no‐reflow can be determined, and has significant implications for recovery of left ventricular function. In chronic ischemic heart disease, MCE has also been shown to successfully differentiate viable from necrotic myocardium. This technique can accurately predict recovery of function after revascularization. More importantly, MCE can be used to identify viable segments that may help to prevent infarct expansion and remodeling, and thus improve patient outcomes.
Identification of different bacterial DNAs in human coronary arteries
Authors: Lehtiniemi J. , Karhunen P. J. , Goebeler S. , Nikkari S. , Nikkari S. T.
Published: January 2005

Background  Various studies have suggested a link between infection, atherosclerosis and coronary artery disease. We studied whether bacterial DNA is present in coronary specimens obtained from left anterior descending coronary arteries of subjects having sudden deaths of cardiovascular and other causes, as verified by an autopsy.

Materials and methods  Coronary specimens were obtained from five subjects who died of sudden coronary causes and five controls. Broad‐range 16‐s rDNA PCR (Br‐PCR) amplification, cloning and sequencing were used to detect bacterial rDNA.

Results  Bacterial rDNA sequences of oral pathogens were detected from the coronary samples in all cases regardless of the cause of death.

Conclusions  Br‐PCR is a powerful method to detect bacterial rDNA. By this method we were able to detect wide palette of oral bacteria from coronary tissues. Our findings suggest that atheromas may act as mechanical sieves collecting bacteria from the circulation.
Alpha‐Gal on bioprostheses: xenograft immune response in cardiac surgery
Authors: Z. Konakci K. , Bohle B. , Blumer R. , Hoetzenecker W. , Roth G. , Moser B. , Boltz‐Nitulescu G. , Gorlitzer M. , Klepetko W. , Wolner E. , Ankersmit H. J.
Published: January 2005

Background  The α‐Gal (Galα1,3‐Galβ1–4GlcNAc‐R) epitope is the major xenoantigen causing hyperacute rejection of pig organs transplanted into primates. Porcine bioprostheses are utilized in cardiac surgery. However, premature degeneration of bioprostheses has limited utilization in younger patients and the immune response remains elusive. We sought to investigate whether a specific α‐Gal immune response may play a role in this clinical scenario.

Materials and methods  We investigated the presence of α‐Gal‐epitope on native and fixed porcine valves by means of confocal laser scanning microscope (CLSM). ELISA was utilized to evidence whether implantation of bioprostheses elicits augmentation of pre‐existing cytotoxic anti α‐Gal IgM antibodies within 10 days of surgery. Patients who underwent coronary artery bypass grafting (CABG) or mechanical valve replacement served as controls (each group, n = 12). To corroborate the clinical relevance of the α‐Gal immune response in vivo, we studied serum obtained before and after implantation of bioprostheses and its potency to lyse porcine α‐Gal‐bearing PK15 cells.

Results  We found the immunogenic α‐Gal‐epitope on fibrocytes interspersed in the connective tissue of porcine valves as determined by vimentin/IB4 lectin binding. Moreover, patients who were provided with a bioprostheses had developed a significant increase of naturally occurring cytotoxic IgM antibodies directed towards α‐Gal after surgical intervention as compared with control patients (P < 0·0001, respectively). Sera obtained from the patients after the implantation of bioprostheses demonstrated an increased cytotoxicity against α‐Gal‐bearing PK‐15 cells as compared with preoperative sera (P < 0·001). The specificity of the cytotoxic effects was proven as soluble Galα1–3Galβ1–4GlcNAc markedly inhibited cell death of α‐Gal‐bearing PK15 cells (P < 0·001).

Conclusion  Our data suggest that implantation of bioprostheses in cardiac surgery induces a xenograft‐specific immune response. Procedures diminishing the presence of α‐Gal on bioprostheses, such as utilization of genetically manipulated α‐Gal‐deficient xenograft or pretreatment with α‐Galactosidase, might diminuate the immune response against bioprostheses and extend durability.
Neurohormonal risk stratification for sudden death and death owing to progressive heart failure in chronic heart failure
Authors: Berger R. , Huelsmann M. , Strecker K. , Moertl D. , Moser P. , Bojic A. , Pacher R.
Published: January 2005

Background  This study tested various neurohormones for prediction of heart failure death (death owing to progressive deterioration of ventricular function; HFD). Moreover, B‐type natriuretic peptide (BNP) as a predictor of sudden death (SD; as reported previously) and the best predictor of HFD were combined for a simple risk stratification model.

Design  BNP, the N‐terminal fragment of BNP (N‐BNP), and of the atrial natriuretic peptide (N‐ANP) and big endothelin levels were obtained from 452 patients with a left ventricular ejection fraction ≤ 35%. Outcome was documented during an observation period up to 3 years.

Results  Two hundred and ninety‐eight patients survived without heart transplantation, 65 patients underwent heart transplantation and 89 patients died (SD 44 patients, HFD 31 patients, other causes 14 patients). The only independent predictor of SD was BNP (as reported), and the best independent predictor of HFD was N‐ANP (P = 0·0001). Analyzing 293 survivors and 44 patients with SD, fewer patients with BNP < 130 pg mL−1 (Group A, n = 110) died [1%] as compared with patients with BNP > 130 pg mL−1 and N‐ANP < 6300 fmol mL−1 (Group B, n = 177; 18%; P = 0·0001) and patients with BNP > 130 pg mL−1 and N‐ANP > 6300 fmol mL−1 (Group C, n = 50; 19%; P = 0·0001). Analyzing 293 survivors and 31 patients with HFD, fewer patients died in Group A (n = 109; 0%; P = 0·0001) and Group B (n = 153; 6%; P = 0·0001) as compared with patients of Group C (n = 62; 34%).

Conclusion  Prognostic power of neurohormones depends on the mode of death. The combined determination of BNP and N‐ANP identifies patients with minimal risk of death, elevated SD but low HFD risk as well as elevated SD and HFD risk.
Photoelectric Dye‐coupled Polyethylene Film as a Prototype of Retinal Prostheses
Authors: Uji Akihito , Matsuo Toshihiko , Ishimaru Sanae , Kajiura Akiko , Shimamura Kaoru , Ohtsuki Hiroshi , Dan‐oh Yasufumi , Suga Sadaharu
Published: January 2005

Abstract:  Photoelectric dyes, which absorb light and convert photon energy to electric potentials, have been previously shown to stimulate retinal neurons in culture. In this study, a photoelectric dye was coupled to a polyethylene film surface and tested in vitro using retinal tissues from chick embryos at the 12‐day embryonic stage, at which time outer segments of retinal photoreceptor cells have not yet developed. Carboxyl moieties were introduced to a polyethylene film surface by fuming nitric acid, and then a photoelectric dye, 2‐[2‐[4‐(dibutylamino)phenyl]ethenyl]‐3‐carboxymethylbenzothiazolium bromide, was coupled to the film through amide linkage. Intracellular calcium elevation was observed with Fluo‐4 in retinal tissues placed on the dye‐coupled polyethylene film, in contrast to retinal tissues which had no contact with the film. The response was inhibited by calcicludine, a voltage‐gated calcium channel blocker, and also by extracellular calcium depletion. The photoelectric dye, coupled to the polyethylene film surface, absorbed light under a dissecting microscope and stimulated neurons in retinal tissues, showing that the dye‐coupled film could be used as a prototype of retinal prostheses.
A Novel Bioartificial Liver With Culture of Porcine Hepatocyte Aggregates Under Simulated Microgravity
Authors: Hochleitner Boris , Hengster Paul , Duo Li , Bucher Herwig , Klima Günter , Margreiter Raimund
Published: January 2005

Abstract:  An extracorporeal bioartificial liver device could provide vital support to patients suffering from acute liver failure. We designed a novel, customized bioreactor for use as a bioartificial liver (patent pending). The Innsbruck Bioartificial Liver (IBAL) contains aggregates of porcine hepatocytes grown under simulated microgravity. The culture vessel rotates around its longitudinal axis and is perfused by two independent circuits. The circuit responsible for exchange of plasma components with the patient consists of a dialysis tube winding spirally around the internal wall of the culture vessel. IBAL was evaluated in vitro. Viability tests showed sufficient viability of hepatocytes for up to 10 days. Cytologic examination of samples from the bioreactor showed liver cell aggregates. These were also examined by electron microscopy. A number of biochemical parameters were analyzed. In conclusion, cell culture is possible for at least 10 days in the IBAL system, organoid hepatocyte aggregates are formed and synthetic activity of the hepatocytes was demonstrated.
The allele interaction between apolipoprotein ɛ2 and ɛ4 in Taiwanese Alzheimer's disease patients
Authors: Lung F.‐W. , Yen Y.‐C. , Chou L.‐J. , Hong C.‐J. , Wu C.‐K.
Published: January 2005

Objective:  This study aimed to determine the impact of the present of apolipoprotein ɛ (Apoɛ) 2 on the relationship between Apoɛ4 and Alzheimer's disease (AD).

Method:  We examined ApoE genotypes in 428 Taiwanese patients with AD and 807 controls; all participants were older than 65 years.

Results:  The allele frequency of Apoɛ4 was greater in AD patients than controls, but significantly lower than in Caucasians. The presence of an ɛ2 allele alone was not associated with lower risk for AD, but the presence of an ɛ2 allele was associated with an ɛ4 allele frequency similar to that of controls.

Conclusion:  The low allele frequency of ɛ4 in persons with an ɛ2 allele suggests that this may be part of the protective effect of ɛ2 against AD.
Structured assessment of current mental state in clinical practice: an international study of the reliability and validity of the Current Psychiatric State interview, CPS‐50
Authors: Falloon I. R. H. , Mizuno M. , Murakami M. , Roncone R. , Unoka Z. , Harangozo J. , Pullman J. , Gedye R. , Held T. , Hager B. , Erickson D. , Burnett K. ,   the Optimal Treatment Project Collaborators
Published: January 2005

Objective:  To develop a reliable standardized assessment of psychiatric symptoms for use in clinical practice.

Method:  A 50‐item interview, the Current Psychiatric State 50 (CPS‐50), was used to assess 237 patients with a range of psychiatric diagnoses. Ratings were made by interviewers after a 2‐day training. Comparisons of inter‐rater reliability on each item and on eight clinical subscales were made across four international centres and between psychiatrists and non‐psychiatrists. A principal components analysis was used to validate these clinical scales.

Results:  Acceptable inter‐rater reliability (intra‐class coefficient > 0.80) was found for 46 of the 50 items, and for all eight subscales. There was no difference between centres or between psychiatrists and non‐psychiatrists. The principal components analysis factors were similar to the clinical scales.

Conclusion:  The CPS‐50 is a reliable standardized assessment of current mental status that can be used in clinical practice by all mental health professionals after brief training.
Fifteen years of research with the Staff Observation Aggression Scale: a review
Authors: Nijman H. L. I. , Palmstierna T. , Almvik R. , Stolker J. J.
Published: January 2005

Objective:  Research on the prevalence and causes of in‐patient aggression has been hindered by the use of different methods for measuring aggression. Since Palmstierna and Wistedt presented the Staff Observation Aggression Scale (SOAS) in 1987, this data collection method has been used in various studies, which may make comparisons more useful.

Method:  Studies with SOAS aggression data were compiled using MEDLINE, the Internet, and references from SOAS papers.

Results:  Reviews of studies on psychometric properties suggest fair to good inter‐rater reliability and validity for SOAS assessments. The number of aggressive incidents per patient per year found on acute admissions wards (n = 38) considerably varied, with a range of 0.4–33.2 incidents (mean = 9.3).

Conclusion:  Although the aggression data included in the present review were obtained in highly comparable ways, substantial differences in aggression rates between wards were still found. Some countries (e.g. the Netherlands) appear to have a relatively high incidence of aggression on acute wards.
The prevalence of child‐psychiatric disorders among 8–9‐year‐old children in Danish mainstream schools
Authors: Bilenberg N. , Petersen D. J. , Hoerder K. , Gillberg C.
Published: January 2005

Objective: Prevalence rates for child psychopathology in Denmark are unknown; and efficient screening and non‐respondent analyses are essential for epidemiological purposes.

Method: A multi‐method strategy involving a two‐step design: first screening of 621 children between the ages of 8–9 years attending mainstream education using the Child Behaviour Checklist (CBCL). Second, screen‐positive probands and a sample of screen‐negative probands were given in‐depth child‐psychiatric assessment. In cases of non‐respondents, children were assessed by teachers.

Results: CBCL screening results were compared with case status in clinical assessment. A positive predictive value of 0.25 and a correct classification rate (CCR) of 0.72 were obtained. The teacher interviews, used in cases of non‐respondents, had a CCR of 0.87. According to DSM‐IV criteria, the prevalence rate of psychiatric disorders was 10.l% [95% CI; 7.0–13.2].

Conclusions: The CBCL proved an effective screening tool. The brief interview with teachers is a valid assessment of psychiatric disorders among non‐respondents.
Myths and plain truths about schizophrenia epidemiology – the NAPE lecture 2004
Authors: McGrath J. J.
Published: January 2005

Objective:  Science needs to constantly match research models against the data. With respect to the epidemiology of schizophrenia, the widely held belief that the incidence of schizophrenia shows little variation may no longer be supported by the data. The aims of this paper are (i) to explore data‐vs.‐belief mismatch with respect to the incidence of schizophrenia, and (ii) to speculate on the causes and consequences of such discrepancies.

Method:  Based on a recently published systematic review of the incidence of schizophrenia, the distribution of incidence rates around the world was examined. In order to examine if the incidence of schizophrenia differed by sex, male vs. female risk ratios were generated.

Results:  The distribution of incidence rates for schizophrenia is asymmetrical with many high rates skewing the distribution. Based on the central 80% of rates, the incidence of schizophrenia varies in a five‐fold range (between 7.7 and 43.0 per 100 000). Males have a significantly higher incidence of schizophrenia compared with females (median male to female risk ratio = 1.4), and this difference could not be accounted for by diagnostic criteria or age range.

Conclusion:  The beliefs that (i) the incidence of schizophrenia does not vary between sites and (ii) males and females are equally affected, may have persisted because of an unspoken deeper belief that schizophrenia is an egalitarian and exceptional disorder. Our ability to generate productive hypotheses about the aetiology of schizophrenia rests on an accurate appraisal of the data. Beliefs not supported by data should be identified and relabelled as myths.
Ability of New APTIMA CT and APTIMA GC Assays To Detect
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Male Urine and Urethral Swabs
Authors: Chernesky M. A. , Martin D. H. , Hook E. W. , Willis D. , Jordan J. , Wang S. , Lane J. R. , Fuller D. , Schachter J.
Published: January 2005

A clinical evaluation was conducted in six North American centers to determine the ability of APTIMA CT (ACT) and APTIMA GC (AGC) nucleic acid amplification assays to detect
Chlamydia trachomatis
and
Neisseria gonorrhoeae
infections in 1,322 men by testing their urethral swabs and first-catch urine (FCU). The results obtained with ACT and AGC assays were compared to an infected patient status determined by testing the specimens with the APTIMA Combo 2 and the BD ProbeTec energy transfer multiplex assays. Symptoms did not influence the values. Positive and negative agreements of the ACT and AGC assays for individual specimens were high, with each comparator assay ranging between 94.3 and 100% for positives and 93.9 and 99.4% for negatives. The ACT and AGC assays performed on noninvasive specimens such as FCU effectively identified
C. trachomatis
or
N. gonorrhoeae
infections in symptomatic and asymptomatic men and should be suitable for screening male populations.
Comparison of
emm
Typing and Ribotyping with Three Restriction Enzymes To Characterize Clinical Isolates of
Streptococcus pyogenes
Authors: Doktor Stella Z. , Beyer Jill M. , Flamm Robert K. , Shortridge Virginia D.
Published: January 2005

A total of 336
Streptococcus pyogenes
isolates recently recovered from patients with pharyngitis from 13 countries were characterized by
emm
typing and riboprinting using an automated Riboprinter (Dupont/Qualicon) based on the patterns produced by three restriction enzymes, EcoRI, PstI, and HindIII. Three enzymes were necessary to increase the discrimination of ribogroups formed by each enzyme. A total of 40 ribogroups and 38
emm
sequences (not counting allelic variations) were identified. Multilocus sequence typing was performed on a sampling of the isolates, and those results were consistent with those of both
emm
typing and ribotyping. Correlations were observed among all three methods.
Detection and Identification of
Mycobacterium tuberculosis
in Joint Biopsy Specimens by
rpoB
PCR Cloning and Sequencing
Authors: Yun Yeo-Jun , Lee Keun-Hwa , Haihua Lin , Ryu Yoon-Jong , Kim Bum-Joon , Lee Young-Ho , Baek Goo-Hyun , Kim Hee-Joong , Chung Moon-Sang , Lee Myung-Chul , Lee Sang-Hoon , Choi In-Ho , Cho Tae-Jun , Chang Bong-Soon , Kook Yoon-Hoh
Published: January 2005

Osteoarticular tuberculosis (OAT) is an extrapulmonary tuberculosis and accounts for 1 to 3% of all tuberculosis cases. We used an
rpoB
PCR-plasmid TA cloning-sequencing method to detect and identify tubercle bacilli in surgical specimens from patients suspected of having OAT. By comparing the similarities of the
rpoB
sequences determined with those in GenBank,
Mycobacterium tuberculosis
was detected in 23 of 43 samples. Three of the 23 positive samples had mutations at codon 531, which are commonly observed in rifampin-resistant
M. tuberculosis
strains. Our results suggest that the
rpoB
PCR-TA cloning-sequencing method developed, which detects
M. tuberculosis
and which simultaneously determines its rifampin susceptibility, can also be used efficiently for the diagnosis of OAT.
First Report of Vancomycin-Resistant Staphylococci Isolated from Healthy Carriers in Brazil
Authors: Palazzo I. C. V. , Araujo M. L. C. , Darini A. L. C.
Published: January 2005

Reduced susceptibility or resistance to vancomycin has been reported among clinical isolates of staphylococci in previous studies. In the present study we report on the isolation of four vancomycin-resistant staphylococcal strains from healthy carriers inside and outside the hospital environment. These carriers did not receive treatment with any antibiotic. All coagulase-negative staphylococcal strains showed variable levels of resistance to several antimicrobial agents, including oxacillin, and unstable resistance to vancomycin, with decreased vancomycin MICs (<4 mg/liter) after 10 days of passage in a nonselective medium. However, exposure of these revertants to vancomycin selected staphylococcal strains resistant to vancomycin at very high frequencies (10
−2
and 10
−3
). The vancomycin resistance in these staphylococcal strains was not mediated by the
van
gene. The cell wall of the staphylococcal strains studied became thickest after culture in medium containing vancomycin, and the differences in cell wall thickness were statistically significant (
P
< 0.001). Thus, the thickening of the cell wall in these staphylococcal strains may be an important contributor to vancomycin resistance.
Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR
Authors: Healy Mimi , Huong Joe , Bittner Traci , Lising Maricel , Frye Stacie , Raza Sabeen , Schrock Robert , Manry Janet , Renwick Alex , Nieto Robert , Woods Charles , Versalovic James , Lupski James R.
Published: January 2005

Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of
Neisseria meningitidis
infections.
N. meningitidis
outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.
Rapid, High-Throughput, Multiplex, Real-Time PCR for Identification of Mutations in the
cyp51A
Gene of
Aspergillus fumigatus
That Confer Resistance to Itraconazole
Authors: Balashov Sergey V. , Gardiner Rebecca , Park Steven , Perlin David S.
Published: January 2005

Aspergillus fumigatus
is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in
A. fumigatus
is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type
cyp51A
gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48
A. fumigatus
clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The
cyp51A
allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G
54
W (
n
= 1), G
54
E (
n
= 12), G
54
K (
n
= 3), G
54
R (
n
= 3), and G
54
V (
n
= 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance.
Molecular Characterization of Diphtheria Toxin Repressor (dtxR) Genes Present in NontoxigenicCorynebacterium diphtheriaeStrains Isolated in the United Kingdom
Authors: De Zoysa Aruni , Efstratiou Androulla , Hawkey Peter M.
Published: January 2005

Nontoxigenic strains ofCorynebacterium diphtheriaerepresent a potential reservoir for the emergence of toxigenicC. diphtheriaestrains if they possessed functional diphtheria toxin repressor (dtxR) genes. We studied the predominant strain of nontoxigenicC. diphtheriaecirculating in the United Kingdom to see if they possesseddtxRgenes and ascertain whether they were functional. A total of 26 nontoxigenicC. diphtheriaestrains isolated in the United Kingdom during 1995 and 4 nontoxigenic strains isolated in other countries were analyzed by PCR and direct sequencing to determine the presence and intactness of thedtxRgenes. The functionality of the DtxR proteins was assayed by testing for the production of siderophore in medium containing high and low concentrations of iron. PCR amplification and sequence analysis of thedtxRgenes revealed four variants of the predicted DtxR protein among the nontoxigenic strains isolated in the United Kingdom. Production of siderophore in medium containing a low concentration of iron and repression of siderophore production in medium containing a high concentration of iron demonstrated that in all the strains thedtxRgenes were functional. These findings demonstrate that, if lysogenised by a bacteriophage, nontoxigenic strains circulating in the United Kingdom could produce toxin and therefore represent a potential reservoir for toxigenicC. diphtheriae.
Comparison ofHelicobacterspp. in Cheetahs (Acinonyx jubatus) with and without Gastritis
Authors: Terio K. A. , Munson L. , Marker L. , Aldridge B. M. , Solnick J. V.
Published: January 2005

Chronic gastritis causes significant morbidity and mortality in captive cheetahs but is rare in wild cheetahs despite colonization by abundant spiral bacteria. This research aimed to identify theHelicobacterspecies that were associated with gastritis in captive cheetahs but are apparently commensal in wild cheetahs.Helicobacterspecies were characterized by PCR amplification and sequencing of the 16S rRNA, urease, andcagAgenes and by transmission electron microscopy of frozen or formalin-fixed paraffin-embedded gastric samples from 33 cheetahs infected withHelicobacterorganisms (10 wild without gastritis and 23 captive with gastritis). Samples were screened for mixed infections by denaturant gel gradient electrophoresis of the 16S rRNA gene and by transmission electron microscopy. There was no association betweenHelicobacterinfection and the presence or severity of gastritis. Eight cheetahs had 16S rRNA sequences that were most similar (98 to 99%) toH. pylori. Twenty-five cheetahs had sequences that were most similar (97 to 99%) to “H. heilmannii”orH. felis. No cheetahs had mixed infections. The ultrastructural morphology of all bacteria was most consistent with “H. heilmannii,” even when 16S rRNA sequences wereH. pylori-like. The urease gene fromH. pylori-like bacteria could not be amplified with primers for either “H. heilmannii”orH. pyloriurease, suggesting that this bacteria is neitherH. pylorinor “H. heilmannii.” ThecagAgene was not identified in any case. These findings question a direct role forHelicobacterinfection in the pathogenesis of gastritis and support the premise that host factors account for the differences in disease between captive and wild cheetah populations.
Identification of Clinically Relevant
Enterococcus
Species by Direct Sequencing of
groES
and Spacer Region
Authors: Tsai Jui-Chang , Hsueh Po-Ren , Lin Hsiao-Mann , Chang Hui-Jen , Ho Shen-Wu , Teng Lee-Jene
Published: January 2005

We determined the
groESL
sequences (
groES
,
groEL
, and the intergenic spacer) of 10 clinically relevant
Enterococcus
species and evaluated the feasibility of identifying
Enterococcus
species on the basis of these sequences. Seven common clinical
Enterococcus
species,
E. faecalis
,
E. faecium
,
E. casseliflavus
,
E. gallinarum
,
E. avium
,
E. raffinosus
, and
E. hirae
, and three less common
Enterococcus
species,
E. cecorum
,
E. durans
, and
E. mundtii
, were examined in this study. We found that the
groES
genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the
groES
and
groEL
genes) are quite variable (17 to 57 bp in length) among
Enterococcus
species but are conserved in strains within each species, with only a few exceptions. Considerable variation of
groES
or
groEL
sequences was also observed. The evolutionary trees of
groES
or
groEL
sequences revealed similarities among
Enterococcus
species. However, the overall intraspecies variation of
groES
was less than that of
groEL
. The high interspecies variation and low intraspecies variation indicate that the
groES
and spacer sequences are more useful than
groEL
for identification of clinically relevant
Enterococcus
species. The sequences of these two genetic traits,
groES
and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of
Enterococcus
.
High Frequency of Human Enterovirus Species C Circulation in Madagascar
Authors: Rakoto-Andrianarivelo Mala , Rousset Dominique , Razafindratsimandresy Richter , Chevaliez Stéphane , Guillot Sophie , Balanant Jean , Delpeyroux Francis
Published: January 2005

Four poliomyelitis outbreaks caused by vaccine-derived polioviruses have been reported recently, including one in Madagascar in 2002. In all cases, the viral strains involved were recombinant between poliovirus vaccine strains and nonpoliovirus strains, probably enterovirus species C. Nevertheless, little is known about the circulation and epidemiology of enteroviruses in the regions where these outbreaks occurred. To assess the circulation of enteroviruses (particularly enterovirus species C) in Madagascar, we genetically characterized 55 enterovirus strains isolated between 1994 and 2002. The strains were identified and compared by partially sequencing the region encoding the VP1 capsid protein. Phylogenetic analysis and pairwise comparison with prototype enterovirus strains distinguished two different species: 25 isolates belonged to human enterovirus B species, and 30 isolates were identified as coxsackievirus A13, A15, A17, A18, A20, A21, and A24, belonging to the human enterovirus species C. The relatively high frequency and the wide distribution of species C coxsackie A viruses in different regions of Madagascar suggest that they had been silently and widely circulating in the country during the whole study period. The circulation of coxsackie A viruses, combined with the low routine oral polio vaccine coverage, may have played a role in the emergence of the recent outbreak in Madagascar.
Human Cytomegalovirus UL144 Gene Polymorphisms in Congenital Infections
Authors: Picone Olivier , Costa Jean-Marc , Chaix Marie-Laure , Ville Yves , Rouzioux Christine , Leruez-Ville Marianne
Published: January 2005

The human cytomegalovirus (HCMV) UL144 gene is a tumor necrosis factor-like receptor with the potential to affect HCMV virulence. HCMV strains display genetic variability in the UL144 region, and the analysis of a potential link between UL144 gene polymorphisms and disease severity has scarcely been studied. However, a correlation between the UL144 genotype and congenital-disease outcome has been reported in one previous study, with the observation that all asymptomatic infants had a single UL144 genotype. In order to confirm or refute this finding, we determined the UL144 polymorphisms of HCMV strains recovered from the amniotic fluids of 38 infected fetuses and compared them to HCMV strains obtained from 30 viremic adult controls. The UL144 sequences were distributed among five genotypes (A, B, C, AC, and AB), as previously described. We observed similar percentages of the three major genotypes A (37%), B (33%), and C (27%) in our population. The UL144 genotype distributions were similar among the group of infected adults and the group of infected fetuses and among symptomatic and asymptomatic fetuses (P< 0.05). In our series, all five UL144 genotypes could be vertically transmitted from mothers to fetuses, and all could cause symptomatic congenital infection. We concluded that determination of UL144 polymorphisms in cases of congenital infection is not relevant, since it is unlikely to help predict the outcome of the infection.
Susceptibility of Filamentous Fungi to Voriconazole Tested by Two Microdilution Methods
Authors: Linares Maria José , Charriel Guadalupe , Solís Francisco , Rodriguez Fernando , Ibarra Ana , Casal M.
Published: January 2005

The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by two microdilution techniques: the National Committee for Clinical Laboratory Standards reference method M38-A and a colorimetric method, Sensititre YeastOne. The study tested a total of 244 isolates: 223
Aspergillus
(136
Aspergillus fumigatus
, 37
A. niger
, 26
A. terreus
, 16
A. flavus
, 7
A. nidulans
, and 1
A. ustus
), 14
Fusarium
(8
Fusarium moniliformis
, and 6
F. oxysporum
), 6
Scedosporium apiospermum
, and 1
Rhizomucor pusillus
strain and four control strains (
Candida parapsilosis
ATCC 22019,
C. krusei
ATCC 6258,
A. fumigatus
ATCC 204305, and
A. flavus
ATCC 204304). For all tested species except one
F. moniliforme
strain and
R. pusillus
, the MIC, the MIC at which 50% of the isolates are inhibited (MIC
50
), and MIC
90
ranges of ≤1 μg/ml were obtained for voriconazole, indicating excellent activity against these species. The high rate of agreement between the two methods used (97 to 99%) suggests that the Sensititre YeastOne colorimetric method may be a valuable tool for determining the susceptibility of filamentous fungi to voriconazole.
Development of a Molecular-Beacon Assay To Detect the G
1896
A Precore Mutation in Hepatitis B Virus-Infected Individuals
Authors: Waltz Therese L. , Marras Salvatore , Rochford Gemma , Nolan John , Lee Eugenia , Melegari Margherita , Pollack Henry
Published: January 2005

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >10
6
copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies.
Molecular Analysis of
Malassezia sympodialis
-Related Strains from Domestic Animals
Authors: Cabañes F. J. , Hernández J. J. , Castellá G.
Published: January 2005

Recently, several new lipid-dependent species belonging to the genus
Malassezia
have been described. Some of them, such as
Malassezia dermatis
,
Malassezia nana
, and the tentatively named “
Malassezia equi
,” have similar phenotypes and are genetically close to
Malassezia sympodialis
Simmons et Guého 1990. DNA characterization by D1/D2 26S rRNA gene and internal transcribed spacer (ITS)-5.8S rRNA gene sequencing analysis of lipid-dependent strains from different animal species close to
M. sympodialis
is described and illustrated. Phylogenetic analysis of both the D1/D2 regions of 26S rRNA gene and ITS-5.8S rRNA gene sequences showed four distinct clusters. Cluster I included isolates from different animal species (horse, pig, and lamb) and the type culture of
M. sympodialis
. Cluster II included isolates from horses grouping close to the “
M. equi
” AJ305330 sequence. Cluster III comprised isolates mainly from goats. Cluster IV contained isolates mainly from cats grouping together with the
M. nana
AB075224 sequence. This last cluster included isolates from healthy and external otitic ears. All of these strains had identical 26S rRNA gene and ITS regions. It is not clear whether the value of these genetic differences is for the definition of species or whether they only demonstrate genetic variation among strains from different origins within
M. sympodialis
, which are in the course of differentiation and probably adaptation to specific animal hosts.
Contribution of (1→3)-β-
d
-Glucan Chromogenic Assay to Diagnosis and Therapeutic Monitoring of Invasive Aspergillosis in Neutropenic Adult Patients: a Comparison with Serial Screening for Circulating Galactomannan
Authors: Pazos Carmen , Pontón José , Palacio Amalia Del
Published: January 2005

Two noninvasive diagnostic tests, (1→3)-β-
d
-glucan (BG) (Glucatell) and galactomannan (GM) (Platelia
Aspergillus
), were used retrospectively in a twice-weekly screening for the diagnosis of invasive aspergillosis (IA) in 40 treatment episodes (one hospital visit per patient) in 40 neutropenic adult patients at high risk for IA. Five proven IA cases, three probable IA cases, and three possible IA cases were diagnosed. Diagnostic levels of both BG and GM were detected in 100% of patients with proven IA cases and in 66% of patients with probable IA cases. The kinetics of both markers in patients with IA were similar. The sensitivity, specificity, and positive and negative predictive values for GM and BG were identical, namely, 87.5, 89.6, 70, and 96.3%, respectively. False-positive reactions occurred at a rate of 10.3% in both tests, but the patients showing false-positive results were different in each test. Both tests anticipated the clinical diagnosis, computed tomography abnormalities, and the initiation of antifungal therapy in most patients, but BG tended to become positive earlier than GM. A combination of the two tests improved the specificity (to 100%) and positive predictive value (to 100%) of each individual test without affecting the sensitivity and negative predictive values. In conclusion, BG and GM detection are useful tests for the diagnosis of IA in high-risk hematological patients, but a combination of the two tests was very useful to identify false-positive reactions by each test.
External Quality Assessment for Molecular Detection of
Bordetella pertussis
in European Laboratories
Authors: Muyldermans G. , Soetens O. , Antoine M. , Bruisten S. , Vincart B. , Doucet-Populaire F. , Fry N. K. , Olcén P. , Scheftel J. M. , Senterre J. M. , van der Zee A. , Riffelmann M. , Piérard D. , Lauwers S.
Published: January 2005

Although the PCR for the detection of
Bordetella pertussis
is routinely performed in diagnostic laboratories, no quality assessment program has so far been described. We report on the results obtained with two external quality assessment proficiency panels sent to European laboratories. The first proficiency panel contained a series of dilutions of three previously characterized
B. pertussis
clinical isolates and two negative controls. No false-positive results were reported by six laboratories providing seven data sets. The reported limits of detection of the three
B. pertussis
strains varied between 4 and 4,000, 9 and 9,000, and 3 and 30,000 CFU/ml, respectively. The second proficiency panel, composed of a series of dilutions of reference strains of
B. pertussis
,
B. holmesii
,
B. hinzii
, and
B. bronchiseptica
, as well as negative controls, was sent to nine laboratories. One laboratory reported a negative result for a sample and reported a
B. parapertussis
-positive sample to be positive for
B. pertussis
. By using the
B. pertussis
-specific target gene pertactin, one laboratory detected
B. pertussis
with 100% specificity. All other laboratories, which used IS
481
-based assays, reported positive results for the samples containing
B. holmesii
and
B. bronchiseptica
, species that have occasionally been recovered from human respiratory samples. These data show that the choice of the target gene is particularly critical for the species specificity of
B. pertussis
PCR assays.
Utility of Mycobacterial Interspersed Repetitive Unit Typing for Differentiating Multidrug-ResistantMycobacterium tuberculosisIsolates of the Beijing Family
Authors: Kam Kai Man , Yip Chi Wai , Tse Lai Wa , Wong Kin Lai , Lam Tak Kam , Kremer Kristin , Au Betty Kam Yan , van Soolingen Dick
Published: January 2005

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping ofMycobacterium tuberculosisand may represent a feasible approach to study globalM. tuberculosismolecular epidemiology. To evaluate the use of MIRU typing in discriminating drug-resistantM. tuberculosisstrains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110restriction fragment length polymorphism (RFLP) typing. Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype. MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index [HGI] of 0.8827) compared with the IS6110RFLP method (HGI = 0.9979). For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961). There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains ofM. tuberculosis. Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype. Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%). This supported the potential use of this method to analyze the global genetic diversity of MDRM. tuberculosisstrains that may be at different levels of evolutionary divergence.
Use of Variable-Number Tandem-Repeat Typing To DifferentiateMycobacterium tuberculosisBeijing Family Isolates from Hong Kong and Comparison with IS6110Restriction Fragment Length Polymorphism Typing and Spoligotyping
Authors: Kremer Kristin , Au Betty Kam Yan , Yip Peter Chi Wai , Skuce Robin , Supply Philip , Kam Kai Man , van Soolingen Dick
Published: January 2005

TheMycobacterium tuberculosisBeijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family,M. tuberculosisisolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110RFLP for maximal resolution.
Expression and Immunological Characterization of the Carboxy-Terminal Region of the P1 Adhesin Protein of
Mycoplasma pneumoniae
Authors: Chaudhry Rama , Nisar Nazima , Hora Bhavna , Chirasani Sridhar Reddy , Malhotra Pawan
Published: January 2005

Mycoplasma pneumoniae
is the causative agent of primary atypical pneumonia in humans. Adherence of
M. pneumoniae
to host cells requires several adhesin proteins, such as P1, P30, and P116. A major limitation in developing a specific diagnostic test for
M. pneumoniae
is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in
Escherichia coli
. In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in
E. coli
. On screening these recombinant proteins with sera from
M. pneumoniae
-infected patients, only the P1-C1 protein was found to be immunogenic. This protein can be used as an antigen for immunodiagnosis of
M. pneumoniae
infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease.