bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of
Vibrio alginolyticus
and
Vibrio splendidus
from Aquacultured Carpet Shell Clam (
Ruditapes decussatus
) Larvae Associated with Mass Mortalities
Authors: Gómez-León J. , Villamil L. , Lemos M. L. , Novoa B. , Figueras A.
Published: January 2005

Two episodes of mortality of cultured carpet shell clams (
Ruditapes decussatus
) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain.
Vibrio alginolyticus
was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events.
Vibrio splendidus
biovar II, in addition to
V. alginolyticus
, was isolated as a result of a mixed
Vibrio
infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams.
V. alginolyticus
TA15 and
V. splendidus
biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.
Synergy Tests by E Test and Checkerboard Methods of Antimicrobial Combinations against
Brucella melitensis
Authors: Orhan Gani , Bayram Aysen , Zer Yasemin , Balci Iclal
Published: January 2005

Two different synergy testing methods, the checkerboard and the E test methods, were used to compare the in vitro efficacies of various antimicrobial combinations against 16
Brucella melitensis
strains isolated from blood cultures. The rate of agreement of the E test and checkerboard methods was found to be 55%. The most concordant results were found for the streptomycin-doxycycline combination in 12 (75%) tests, in which four strains showed synergistic activity by E test and antagonistic activity by the checkerboard method and in which one strain showed antagonistic activity by both methods. Even though each of these methods uses different conditions and endpoints, the results of both methods frequently agreed.
Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies
Authors: Yu Jigui , Lin Jisheng , Benjamin William H. , Waites Ken B. , Lee Che-hung , Nahm Moon H.
Published: January 2005

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.
PCR Detection of Bacteria on Cardiac Valves of Patients with Treated Bacterial Endocarditis
Authors: Rovery Clarisse , Greub Gilbert , Lepidi Hubert , Casalta Jean-Paul , Habib Gilbert , Collart Frédéric , Raoult Didier
Published: January 2005

We used broad-range PCR amplification and sequencing to detect and identify bacterial DNA in 156 valves of patients treated for infective endocarditis (IE). Bacterial DNA was found more frequently in patients who underwent valve replacement while on antibiotic treatment for IE (60%) than in patients who had completed antibiotic treatment for IE (37%;P= 0.02). We found specific bacterial DNA in valves removed from 11 of 30 patients who had completed antibiotic treatment for IE. Six had no histological evidence of IE. The presence of DNA was significantly correlated with the presence of histologic lesions (P= 0.001) and with the presence of bacteria detected by Gram staining (P< 0.001).Bartonellaand streptococci were detected for much longer after antibiotic treatment by PCR than other species (P= 0.047 and 0.04, respectively), and coagulase-negative staphylococci were detected for much shorter periods (P= 0.02). The finding that bacterial DNA was more likely to be detected in valves of patients with active IE than in patients who had completed antibiotic treatment for IE shows that bacterial DNA is cleared slowly. There was no significant correlation between the duration of antibiotic therapy and the presence of bacterial DNA in valves. Since the persistence of bacterial DNA in valves does not necessarily indicate the persistence of viable bacteria, the detection of bacterial DNA in valves from IE patients should be interpreted with caution, in particular in those patients with a past history of treated IE.
The Pathogen of Frogs
Amphibiocystidium ranae
Is a Member of the Order Dermocystida in the Class Mesomycetozoea
Authors: Pereira Cristiane N. , Di Rosa Ines , Fagotti Anna , Simoncelli Francesca , Pascolini Rita , Mendoza Leonel
Published: January 2005

The pathogen of frogs
Amphibiocystidium ranae
was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as
Dermocystidium
spp.,
Rhinosporidium seeberi
, and
Sphaerothecum destruens
in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded ≈1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of
A. ranae
from both
Rana esculenta
and
Rana lessonae
was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus
Dermocystidium
and closely related to
Rhinosporidium
. These data strongly supported the placement of the genus
Amphibiocystidium
within the mesomycetozoeans, which is in agreement with the phenotypic features that
A. ranae
shares with the other members of this class. Interestingly, during this study
Dermocystidium percae
did not group within the
Dermocystidium
spp. from fish; rather, it was found to be the sister group to
Sphaerothecum destruens
. This finding suggests that
D. percae
could well be a member of the genus
Sphaerothecum
or perhaps represents a new genus.
How Evolution of Mutations Conferring Drug Resistance Affects Viral Dynamics and Clinical Outcomes of Cytomegalovirus-Infected Hematopoietic Cell Transplant Recipients
Authors: Springer Kathryn L. , Chou Sunwen , Li Shaobing , Giller Roger H. , Quinones Ralph , Shira James E. , Weinberg Adriana
Published: January 2005

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality among hematopoietic cell transplant (HCT) recipients. We describe two pediatric HCT recipients who developed persistent and severe drug-resistant CMV infections. CMV resistance to foscarnet and ganciclovir was detected after only 6 and 11 weeks of therapy, respectively. Viral
pol
mutations associated with drug resistance in these patients included T838A (a novel mutation) and D588N, which were shown by marker transfer to confer foscarnet and multidrug resistance, respectively. Each of these mutations significantly reduced in vitro replication of CMV, suggesting that they may decrease viral fitness. This finding was further supported by the disappearance of mutations upon withdrawal of antiviral pressure in one patient. Novel antivirals or combination therapy may be required for the treatment of drug-resistant CMV in HCT recipients and perhaps in other severely immunocompromised patients.
Dry-Reagent-Based PCR as a Novel Tool for Laboratory Confirmation of Clinically Diagnosed
Mycobacterium ulcerans
-Associated Disease in Areas in the Tropics Where
M. ulcerans
Is Endemic
Authors: Siegmund V. , Adjei O. , Racz P. , Berberich C. , Klutse E. , van Vloten F. , Kruppa T. , Fleischer B. , Bretzel G.
Published: January 2005

After tuberculosis and leprosy, Buruli ulcer (BU), caused by
Mycobacterium ulcerans
, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic “gold standard.” Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of
M. ulcerans
in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.
Evaluation of the COBAS TaqMan HCV Test with Automated Sample Processing Using the MagNA Pure LC Instrument
Authors: Germer Jeffrey J. , Harmsen W. Scott , Mandrekar Jayawant N. , Mitchell P. Shawn , Yao Joseph D. C.
Published: January 2005

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (
R
2
= 0.99) across a wide range of HCV RNA levels (25 to 5 × 10
6
IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (
R
2
= 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.
Epidemiology ofCampylobacter jejuniIsolated from Patients with Guillain-Barré and Fisher Syndromes in Japan
Authors: Takahashi Masaki , Koga Michiaki , Yokoyama Keiko , Yuki Nobuhiro
Published: January 2005

Campylobacter jejuniisolation is the standard for the diagnosis of this type of bacterial infection, but there have been no epidemiological studies of a large number ofC. jejuniisolates from patients with Guillain-Barré syndrome (GBS) and Fisher syndrome (FS). For 13 years, stool specimens from GBS/FS patients have been sent from 378 hospitals throughout Japan to the Tokyo Metropolitan Institute of Public Health. A total of 113 strains (11%) were isolated from the stool specimens from 1,049 patients. The isolation rate did not differ by region. The rates were 22% for 449 patients with a history of diarrhea and 2% for the others. An additional 18 isolates were provided by various hospitals. There was no noticeable seasonal distribution in the onset ofC. jejuniisolated from patients with GBS/FS. The male/female ratios were 1.7:1 for GBS and 2.2:1 for FS. The patient age range showed a peak in 10- to 30-year-old subjects who had GBS and in 10- to 20-year-old subjects who had FS. The predominance of young adults and male patients who hadC. jejuni-associated GBS/FS may be related to the preponderance of young adults and male patients who hadC. jejunienteritis. The median interval from diarrhea onset to neurologic symptom onset was 10 days for GBS/FS. Penner'sC. jejuniserotype HS:19 was more frequently present in GBS (67%) than in enteritis (6%) patients. HS:2 was more frequent in FS (41%) than in enteritis (14%) patients. These findings suggest that certainC. jejunistrains specifically trigger GBS and that others specifically trigger FS.
Molecular Epidemiology of Respiratory Syncytial Virus Infections among Children with Acute Respiratory Symptoms in a Community over Three Seasons
Authors: Sato Mizuho , Saito Reiko , Sakai Takatsugu , Sano Yasuko , Nishikawa Makoto , Sasaki Asami , Shobugawa Yugo , Gejyo Fumitake , Suzuki Hiroshi
Published: January 2005

To study the molecular epidemiology of respiratory syncytial virus (RSV) in a community, children with acute respiratory symptoms at a pediatric outpatient clinic in Niigata, Japan, were analyzed over three seasons from November 2001 to July 2004. Of 499 nasopharyngeal aspirate specimens, 185 (37.1%) were RSV positive, and only 8 (4.5%) of 177 patients were shown by the reverse transcription (RT)-PCR method to be reinfected. RSV infection occurred beginning in the early winter, and the rates declined in the spring. The predominant subgroup changed from A to B and returned to A over the three seasons. Phylogenetic analysis also revealed that multiple genotypes cocirculated each year, with genotype GA5 of subgroup A predominating in the 2001-2002 and the 2003-2004 seasons. A new genotype of subgroup B (named BA, according to the nomenclature for viruses) with a 60-nucleotide insertion in the second variable region of the attachment glycoportein protein was predominant as an emerging strain in the 2002-2003 season, but this was not associated with new epidemiological or clinical features, unlike the cases of disease caused by other genotypes in the other seasons. In conclusion, our molecular analysis of RSV confirms that multiple genotypes cocirculate each year and that the genotype predominating may shift with the season. Support for determination of the genotype by RT-PCR as an effective tool for characterization of RSV circulation patterns in the community is provided.
Aspergillus Hyphae in Infected Tissue: Evidence of Physiologic Adaptation and Effect on Culture Recovery
Authors: Tarrand Jeffrey J. , Han Xiang Y. , Kontoyiannis Dimitrios P. , May Gregory S.
Published: January 2005

Microbiologic cultures of fungi are routinely incubated at ambient temperatures in room air, and the rate of recovery of
Aspergillus
species from clinical specimens is poor. Failure of current culture methods to mimic the physiologic temperature and low-oxygen environment found in hypha-laden infected tissue may underlie this poor recovery. Experiments were performed to compare the recovery of
Aspergillus
spp. incubated at 35°C in 6% O
2
-10% CO
2
with that at 25°C in room air. The samples tested included
Aspergillus
-infected tissue specimens from a dog model and human autopsies, experimental anaerobically stressed
Aspergillus
inocula, and 10,062 consecutive clinical specimens. Culture at 35°C in 6% O
2
-10% CO
2
significantly enhanced the recovery of
Aspergillus
spp. from the infected autopsy tissue samples. Incubation at 35°C alone resulted in approximately 10-fold-improved culture recovery from the experimentally stressed hyphae, and the 6% O
2
-10% CO
2
atmosphere independently favored growth under temperature-matched conditions. Finally, incubation at 35°C (in room air) improved the overall recovery of
Aspergillus
spp. from clinical specimens by 31%. Culture at 35°C in a microaerobic atmosphere significantly enhances the recovery of
Aspergillus
spp. from various sources.
Aspergillus
hyphae growing in infected tissue appear to be adapted to the physiologic temperature and hypoxic milieu.
Evaluation of Nested PCR in Diagnosis of Typhoid Fever
Authors: Prakash Pradyot , Mishra Om Prakash , Singh Alok Kumar , Gulati Anil Kumar , Nath Gopal
Published: January 2005

In this study, nested PCR using
H1-d
primers, which is specific for
Salmonella enterica
serovar Typhi, was compared to blood culture and the single-tube Widal test. Results indicate that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal test for diagnosis of typhoid fever.
Evaluation of an Accelerated Protocol for Detection of Extended-Spectrum β-Lactamase-Producing Gram-Negative Bacilli from Positive Blood Cultures
Authors: Navon-Venezia Shiri , Leavitt Azita , Ben-Ami Ronen , Aharoni Yuval , Schwaber Mitchell J. , Schwartz David , Carmeli Yehuda
Published: January 2005

We evaluated a protocol for the accelerated detection of extended-spectrum β-lactamases (ESBLs) in gram-negative bloodstream pathogens. Two hundred eighty-three blood culture bottles were subjected to direct ESBL testing by inoculating samples directly from blood culture bottles onto agar plates containing cefotaxime and ceftazidime disks, with and without clavulanate. Standard ESBL testing in accordance with the NCCLS guidelines after subculturing on agar plates was performed in parallel. Results of the direct ESBL testing were reported 2.3 days sooner and were comparable to those of the standard NCCLS method with sensitivity, specificity, and positive and negative predictive values of 100, 98, 94, and 100%, respectively.
PCR Amplification with Primers Based on IS
2404
and GC-Rich Repeated Sequence Reveals Polymorphism in
Mycobacterium ulcerans
Authors: Ablordey Anthony , Kotlowski Roman , Swings Jean , Portaels Françoise
Published: January 2005

We describe a simple genotyping method for
Mycobacterium ulcerans
based on PCR amplification of genomic regions between IS
2404
and a frequently repeated GC-rich sequence. Application of this method to a global collection produced 10
M. ulcerans
genotypes corresponding to their geographic origin.
Phenotypic and Molecular Analysis of Tellurite Resistance among Enterohemorrhagic
Escherichia coli
O157:H7 and Sorbitol-Fermenting O157:NM Clinical Isolates
Authors: Bielaszewska Martina , Tarr Phillip I. , Karch Helge , Zhang Wenlan , Mathys Werner
Published: January 2005

A total of 66 (98.5%) of 67 enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 strains had increased potassium tellurite (Te) MICs (32 to 1,024 μg/ml), grew on Te-containing media, and possessed Te resistance (
ter
) genes, whereas 83 (96.5%) of 86 sorbitol-fermenting (SF) EHEC O157:NM strains had Te MICs of ≤4 μg/ml, did not grow on Te-containing media, and lacked
ter
genes. Optimal detection of SF EHEC O157:NM strains requires Te-independent strategies.
Diagnostic Evaluation of a Real-Time Reverse Transcriptase PCR Assay for Detection of Classical Swine Fever Virus
Authors: Risatti G. , Holinka L. , Lu Z. , Kutish G. , Callahan J. D. , Nelson W. M. , Brea Tió E. , Borca M. V.
Published: January 2005

A fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase (RT) PCR for classical swine fever virus (CSFV) was evaluated for diagnostic sensitivity and specificity by using clinical samples obtained from the Dominican Republic, where the disease is enzootic. The sensitivity of this test, using nasal swab samples taken from both symptomatic and asymptomatic animals, exceeded the diagnostic sensitivity of virus isolation (100% versus 72.4%, respectively) with little loss of specificity (98.9% versus 100%, respectively). At the herd level, three of four infected farms were identified by virus isolation, while the CSFV real-time RT-PCR assay identified all four infected premises. This simple and accurate test permits rapid detection of CSFV in affected herds.
Clonal Spread of a
Clostridium difficile
Strain with a Complete Set of Toxin A, Toxin B, and Binary Toxin Genes among Polish Patients with
Clostridium difficile
-Associated Diarrhea
Authors: Pituch Hanna , Kreft Deborah , Obuch-Woszczatyński Piotr , Wultańska Dorota , Meisel-Mikołajczyk Felicja , Łuczak Mirosław , van Belkum Alex
Published: January 2005

Clinically relevant
Clostridium difficile
strains usually produce toxins A and B. Some
C. difficile
strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with
C. difficile
-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented.
Simultaneous Detection of Immunoglobulin A (IgA) and IgM Antibodies against Hepatitis E Virus (HEV) Is Highly Specific for Diagnosis of Acute HEV Infection
Authors: Takahashi Masaharu , Kusakai Shigeyuki , Mizuo Hitoshi , Suzuki Kazuyuki , Fujimura Kuniko , Masuko Kazuo , Sugai Yoshiki , Aikawa Tatsuya , Nishizawa Tsutomu , Okamoto Hiroaki
Published: January 2005

Serum samples collected from 68 patients (age, mean ± the standard deviation [SD], 56.3 ± 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 ± 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 ± 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection.
Assessing the Serodiagnostic Potential of 35
Mycobacterium tuberculosis
Proteins and Identification of Four Novel Serological Antigens
Authors: Weldingh Karin , Rosenkrands Ida , Okkels Limei Meng , Doherty T. Mark , Andersen Peter
Published: January 2005

Improved diagnostic reagents are needed for the detection of
Mycobacterium tuberculosis
infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35
M. tuberculosi
s proteins as recombinant proteins in
Escherichia coli
and analyzed their serodiagnostic potentials. By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before. The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97%. The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients. Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect
M. tuberculosis
-specific antibodies.
Extensive Gene Diversity in Septicemic
Escherichia coli
Strains
Authors: Mokady Daphna , Gophna Uri , Ron Eliora Z.
Published: January 2005

Extraintestinal pathogenic
Escherichia coli
strains (ExPEC) are the cause of a diverse spectrum of invasive infections in humans and animals, and these infections often lead to septicemia. Strains of serogroups O2 and O78 of
E. coli
are involved in human urinary tract infections and newborn meningitis and also constitute the major serotypes involved in avian colisepticemia. In the present study we compared the unique genomic sequences of two such septicemic strains, strains O2-1772 and O78-9, obtained by suppression subtractive hybridization. Evaluation of the degree of similarity between these two strains, which cause the same disease, revealed a high degree of diversity, with only a few shared genes. Subsequently, additional strains of each serogroup of human and animal origin were screened by PCR, and the results provided further evidence for the existence of a high degree of genome plasticity. These results were unexpected, in view of data showing that the two O157:H7 strains that have been sequenced are nearly identical in terms of virulence factors. Furthermore, the data obtained for the septicemic strains suggest that each step in the infection can be mediated by a number of alternative virulence factors, indicating the existence of a mix-and-match combinatorial system. Although whole-genome comparisons of
E. coli
strains causing different diseases have shown great differences in gene contents, we show that such differences exist even within strains that cause the same disease and that target the same host tissues. Moreover, in addition to the high level of genome plasticity, we show that the large pool of virulence genes in the septicemic strains is independent of the host, implying a high degree of zoonotic risk.
Should physicians be allowed to use alcohol while on call?: Table 1
Authors: Peterman J F , Desbiens N A
Published: January 2005

Although physician alcohol use that leads to impairment has been extensively discussed, few statements address the issue of alcohol use of physicians who are on call. In this paper the authors review recent information on physicians’ perceptions of alcohol use by themselves and their colleagues while on call. It is argued that conflicts in physicians’ perceptions are due to the fact that the larger society has not addressed the question of whether drinking on call is public or private behaviour. The authors argue that when medicine is understood as a practice defined partly in terms of standards of excellence, the present approach of the American Medical Association to prohibit practicing medicine under the influence of alcohol requires a prohibition of drinking alcohol while on call, unless studies determine a clear threshold for drinking alcohol without placing patients at risk.
Of souls, selves, and cerebrums: a reply to Himma
Authors: Beckwith F J
Published: January 2005

Ken Himma argues that a human being becomes a moral person at the commencement of brain activity. In response to Himma, the author offers (1) brief comments on Himma’s project, (2) an alternative account of the human person that maintains that a human being is a human person by nature as long as it exists, and (3) a counterexample to Himma’s position that shows it cannot account for the wrongness of the purposeful creation of anencephalic-like children. The author concludes with replies to two challenges to his position.
Improving hospital doctors’ working lives: online questionnaire survey of all grades
Authors: Dornhorst A , Cripps J , Goodyear H , Marshall J , Waters E , Boddy S-A
Published: January 2005

Background

In 2001, the Department of Health produced the Improving Working Lives (IWL) for Doctors document. This is the first national survey which asks hospital doctors what changes are needed to improve their working lives.

Methods

An online questionnaire was run over a period of six weeks and was open to all doctors of all grades. Doctors were asked to choose their top five factors from a list of 35 diverse choices or to provide alternatives in free text. Demographic data were also collected.

Results

1603 hospital doctors working in the UK completed the online questionnaire. Improved secretarial or managerial support was the first IWL choice for consultants, with different aspects of clinical and non-clinical support representing their top four choices. Junior hospital doctors and staff and associate specialist grades (staff grades, associate specialists, and clinical assistants) identified improved support for education and training as their first choice, while among the female specialist registrars, it was improved support for childcare.

Greater opportunities to develop new skills was an important issue for doctors in the surgical specialties and improved access to mentoring was important for all junior doctors, staff and associate specialist grades, and doctors from black and ethnic minority groups.

Conclusions

Hospital doctors in the UK need more support to improve their working lives. The principle needs are better secretarial and managerial support for consultants; education, training, and mentoring for junior doctors and staff and associate specialist grades; and improved opportunities to develop new skills for those in surgical specialties. Support with childcare is an important issue for female specialist registrars. The Department of Health, NHS trusts, deaneries, and Royal Colleges need to endorse policies that promote a training and working environment that will improve working lives for all hospital doctors, ensuring that appropriate and continuing support is available from the time doctors enter the new foundation programmes and proposed run-through grades, to their time spent as consultants in today’s NHS.
The Bitter Brew
Authors: Elankumaran S.
Published: January 2005

Assam, a state in Northeast India, has been experiencing the problem of militancy for over two decades. Tea was a major industry in the state and due to its pivotal position, militants had been excessively harassing it. Tata Tea Limited (TTL), a subsidiary of a 125-year old and well respected conglomerate, the House of Tatas, was one of the largest players in the tea industry in the region. The company had never succumbed to militants' pressure of parting with money but had acted as a responsible corporate citizen by involving itself in substantial community development initiatives. Under one such initiative — the Special Medical Assistance Scheme — a leader of a militant outfit has been treated for pregnancy and accompanying life-threatening health problem in a hospital in Mumbai. After childbirth and treatment, when she was about to board a flight to Delhi, the Assam government with the help of Mumbai police arrested her on a tip off. The arrest led to the controversial allegation that TTL had been funding terrorists in Assam. Based on press and published reports, this case unfolds the whole episode with relevant background and raises debatable ethical issues that are universally relevant for business in today's environments.
Diecraft Australia
Authors: Sethuraman Kannan , Tirupati Devanath
Published: January 2005

Diecraft Australia Pty. Ltd, a subsidiary of Tupperware, manufactured high-quality, high-precision, close tolerance plastic injection moulds for houseware products. Diecraft's major customer has always been Tupperware, which accounted for more than 90% of its turnover. In 2001, the company had sales in excess of A$23 million and was experiencing problems in meeting the targeted due dates. The case provides a detailed, step-by-step description of the order processing, engineering design and manufacturing process. It also discusses the procedures adopted for plant loading, scheduling and control. It identifies other problems which may explain its poor adherence to scheduled due dates. It brings out several key features of job shops and provides an opportunity to understand and appreciate issues relating to capacity planning, developing quotes for delivery and the detailed workings of the shop.
Chronic cardiotrophin-1 stimulation impairs contractile function in reconstituted heart tissue
Authors: Zolk Oliver , Engmann Sven , Münzel Felix , Krajcik Rasti
Published: January 2005

Cardiotrophin-1 (CT-1) is known to promote survival but also to induce an elongated morphology of isolated cardiac myocytes, leading to the hypothesis that CT-1, which is chronically augmented in human heart failure, might induce eccentric cardiac hypertrophy and contractile failure. To address this, we used heart tissues reconstituted from neonatal rat cardiac myocytes (engineered heart tissue, EHT) as multicellular in vitro test systems. CT-1 dose-dependently affected contractile function in EHTs. After treatment with 0.1 nM CT-1 (corresponds to plasma levels in humans) for 10 days, twitch tension significantly decreased to 0.30 ± 0.04 mN ( n = 15) vs. 0.45 ± 0.04 mN ( n = 16) in controls. Furthermore, positive inotropic effects of cumulative concentrations of Ca2+and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension amounted to 0.27 ± 0.04 mN ( n = 15) vs. 0.47 ± 0.06 mN ( n = 16) in controls ( P < 0.001). When EHTs were treated for only 5 days, qualitatively similar results were obtained but changes were less pronounced. Immunostaining of whole mount EHT preparations revealed that after CT-1 treatment, the number of nonmyocytes significantly increased by 98% (1 nM, 10 days), and myocytes did not form compact, longitudinally oriented muscle bundles. Interestingly, expression of the Ca2+-handling protein calsequestrin was markedly reduced (69 ± 7% of control) by treatment with CT-1 (0.1 nM, 10 days). In summary, long-term exposure to CT-1 induces contractile dysfunction in EHTs. Structural changes due to impaired differentiation and/or remodeling of heart tissue may play an important role.
Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart
Authors: Frederich Markus , Zhang Li , Balschi James A.
Published: January 2005

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2content, α-subunit Thr172phosphorylation increased at O2≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172by an upstream kinase or reduction in the A0.5for [AMP].
Expression of NTPDase1 and NTPDase2 in murine kidney: relevance to regulation of P2 receptor signaling
Authors: Kishore Bellamkonda K. , Isaac Jorge , Fausther Michel , Tripp Sheryl R. , Shi Huihui , Gill Pritmohinder S. , Braun Norbert , Zimmermann Herbert , Sévigny Jean , Robson Simon C.
Published: January 2005

The regulation of renal function by extracellular nucleotides encompasses alterations in glomerular hemodynamics, microvascular function, tubuloglomerular feedback, tubular transport, cell growth or apoptosis, and transport of water and solutes in the medullary collecting duct. Nearly all cells can release ATP or other nucleotides that are then rapidly hydrolyzed in the extracellular milieu. However, little information is available on the cellular expression of ectoenzymes that hydrolyze extracellular nucleotides within the kidney. Nucleoside triphosphate diphosphohydrolases (NTPDases) are plasma membrane-bound ectonucleotidases. NTPDase1 has identity with CD39, a B lymphocyte activation marker, and hydrolyzes extracellular ATP and ADP to AMP within the vasculature, whereas NTPDase2/CD39L(ike)1 preferentially converts ATP to ADP outside of blood vessels. Using immunohistochemical and in situ hybridization approaches, we localized the protein and mRNA of NTPDase1 and 2 in murine renal tissues. In the renal cortex, NTPDase1 is expressed by vascular smooth muscle cells and endothelium in interlobular arteries, afferent glomerular arterioles, and peritubular capillaries. In the inner medulla, NTPDase1 is expressed in ascending thin limbs of Henle's loop, ducts of Bellini, and in the pelvic wall. In contrast, NTPDase2 is expressed in Bowman's capsule, glomerular arterioles, adventitia of blood vessels, and pelvic wall. Thus the distribution patterns of NTPDases have parallels to the known distribution of P2 receptors within the kidney. NTPDases may modulate regulatory effects of ATP and degradation products within the vasculature and other sites and thereby potentially influence physiological as well as multiple pathological events in the kidney.
Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure
Authors: Basile David P. , Fredrich Katherine , Alausa Morufu , Vio Carlos P. , Liang Mingyu , Rieder Mark R. , Greene Andrew S. , Cowley Allen W.
Published: January 2005

Recovery from ischemic acute renal failure (ARF) involves a well-described regenerative process; however, recovery from ARF also results in a predisposition to a progressive renal disease that is not well understood. This study sought to identify alterations in renal gene expression in postischemic, recovered animals that might play important roles in this progressive disorder. RNA isolated from sham-operated control rats or rats 35 days after recovery from bilateral ischemia-reperfusion (I/R) injury was compared using a cDNA microarray containing ∼2,000 known rat genes. A reference hybridization strategy was utilized to define a 99.9% interval and to identify 16 genes that were persistently altered after recovery from I/R injury (12 were upregulated and 4 were downregulated). Real-time PCR verified the altered expression of six of eight genes that had been positively identified. Several genes that were identified had not previously been evaluated within the context of ARF. S100A4, a specific marker of fibroblasts, was identified in a population of interstitial cells that were present postischemic injury. S100A4-positive cells were also identified in tubular cells at earlier time points postischemia. Genes associated with calcification, including osteopontin and matrix Gla protein, were also enhanced postischemic injury. Several proinflammatory genes were identified, including complement C4, were enhanced in postischemic tissues. Conversely, renal kallikrein expression was specifically reduced in the postischemic kidney. In summary, genes with known inflammatory, remodeling, and vasoactive activities were identified in rat kidneys after recovery from ARF, some of which may play a role in altering long-term renal function after recovery from ARF.
The adaptive immune system and long-term outcome in patients with stable coronary disease
Authors: Graf Senta , Nikfardjam Mariam , Lehr Stephan , Maurer Gerald , Wojta Johann , Huber Kurt , Niessner Alexander
Published: January 2005

Components of the adaptive immune system, in particular lymphocytes and immunoglobulin, play a major role in advanced atherosclerotic lesions. We sought to determine whether routine measurements of the relative number of circulating lymphocytes (%L) and γ -globulin (%G) reflecting immunoglobulin are related to event-free survival in patients with stable coronary artery disease (CAD). We prospectively studied the combined endpoint all-cause mortality, myocardial infarction and coronary revascularization procedures in 141 patients after successful percutaneous coronary intervention during a median follow-up time of 13.2 years. Using Cox regression, we found a significant influence of %L on event-free survival (P = 0.007) with a relative risk of 2.21 comparing third to first tertile. Subjects with higher %G values likewise had a shorter event-free survival (P = 0.008) with a relative risk of 1.67 comparing third to first tertile. The predictive value of %L and %G remained significant after adjustment for demographic data, cardiovascular risk factors, extent of CAD and other inflammatory markers. We conclude that the fraction of γ -globulin and in particular the relative lymphocyte cell count may serve as readily available and reliable prognostic tools for the long-term outcome in patients with stable CAD.
Influence of factor VII gene polymorphisms and environmental factors on plasma coagulation factor VII concentrations in middleaged women with and without manifest coronary heart disease
Authors: Eriksson-Berg Margita , Deguchi Hiroyuki , Hawe Emma , Scanavini Daniela , Orth-Gomér Kristina , Schenck-Gustafsson Karin , Humphries Steve E. , Silveira Angela , Hamsten Anders
Published: January 2005

Plasma concentrations of coagulation factorVII (FVII) are determined by environmental and genetic factors. The influence of functional polymorphisms in the FVII gene (-670A>C, –402G>A, –401G>T and R353Q) and of established cardiovascular risk factors on plasma concentrations of FVII were investigated in a representative sample of middle-aged women with (n=238) and without (n=220) coronary heart disease (CHD). Specific and sensitive assays were used to measure FVII antigen (VIIag) and activated factorVII (VIIa).The effect of genotypes was markedly stronger on VIIa than on VIIag, with the percentage variation in FVII levels accounted for by genotypes being greater in controls than in patients. Of the four polymorphisms examined, only the R353Q contributed to the variation inVIIa (24.1% in patients and 30.3% in controls). The –401G>T and –670A>C promoter polymorphisms together accounted for 12.2% of the variation in VIIag amongst patients whereas the –401G>T polymorphism alone contributed 19.7% of the variation in VIIag in controls. Serum triglycerides exerted a major influence onVIIag in both patients (13.0%) and controls (7.2%).Three main haplotypes emerged from the four polymorphisms which accounted for 98% of all haplotypes. Large-scale prospective studies of CHD including FVII haplotypes and sensitive and specific FVII measurements are needed in women.
Identification of Unique Lipoprotein Subclasses for Visceral Obesity by Component Analysis of Cholesterol Profile in High-Performance Liquid Chromatography
Authors: Okazaki Mitsuyo , Usui Shinichi , Ishigami Masato , Sakai Naohiko , Nakamura Tadashi , Matsuzawa Yuji , Yamashita Shizuya
Published: January 2005

Objective—

The contribution of visceral fat accumulation to the development of coronary heart disease was previously reported, but the relation between visceral fat accumulation and serum lipoprotein subclasses was unknown.

Methods and Results—

We examined the relation of lipoprotein subclasses with visceral fat accumulation in 62 male subjects (aged 22 to 67 years) with visceral fat syndrome or obesity. Cholesterol levels in very low–density, low-density, and high-density lipoprotein subclasses (VLDL, LDL, and HDL) were determined by computer-assisted high-performance liquid chromatography. Subcutaneous fat area and visceral fat area were measured by computed tomographic scanning. There was no significant correlation between the subcutaneous fat area and the cholesterol levels in all lipoprotein subclasses. In contrast, the visceral fat area was correlated positively (
P
<0.002) with VLDL and LDL subclasses, except for large LDL, but negatively (
P
<0.001) with those in large and medium HDL subclasses. The observed positive correlations of small and very small LDL subclasses remained significant (
P
<0.005) after adjustment for serum cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, respectively, but a significant negative correlation (
P
<0.005) of large LDL was obtained after adjustment for LDL cholesterol.

Conclusion—

These findings indicate that this simple high-performance liquid chromatography method may be applied for easy detection and evaluation of abnormal distribution of lipoprotein subclasses.