bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow Cytometry Susceptibility Testing for the Antifungal Caspofungin
Authors: Mitchell M. , Hudspeth M. , Wright A. 
Published: June 2005

ABSTRACT

Rapid antifungal susceptibility testing for the antifungal agent caspofungin can be performed using flow cytometry (FC). An FC procedure using acridine orange provided minimum inhibitory concentration (MIC) results within 7 to 9 h which were compared with results obtained using the NCCLS M27-A2 protocol. To evaluate the consistency of this method, susceptibility testing using caspofungin was performed using 73 isolates of eight different species of
Candida
from various clinical samples in Central California. Macrotiter or microdilution tests were performed according to the NCCLS M27-A2 protocol, and the MICs were compared to those provided by our flow cytometry method. All isolates tested had results within the sensitive interpretive category, and 90% of the results compared within 1 dilution, showing excellent agreement between the methods. The MIC at which 50% of the isolates tested were inhibited (MIC
50
) and the MIC
90
of caspofungin for all eight
Candida
species were within 1 dilution. This flow cytometer 7-h protocol for testing the antifungal susceptibility of
Candida
species to caspofungin provided results equivalent to those obtained with the M27-A2 protocol.
Outbreak of Dysentery Associated with Ceftriaxone-Resistant
Shigella sonnei
: First Report of Plasmid-Mediated CMY-2-Type AmpC β-Lactamase Resistance in
S. sonnei
Authors: Huang I-Fei , Chiu Cheng-Hsun , Wang Mei-Hui , Wu Chan-Yao , Hsieh Kai-Sheng , Chiou Christine C. 
Published: June 2005

ABSTRACT

We document the first report of plasmid-encoded CMY-2-type AmpC β-lactamase identified among
Shigella sonnei
isolates resistant to ceftriaxone and obtained after an outbreak of bacillary dysentery in Taiwan. One hundred eighty-two children in two elementary schools in Yu-Li, Taiwan, where an outbreak occurred after a typhoon hit this area in 2001, were enrolled in this study. Clinical and epidemiologic data on the infected children were collected. Pulsed-field gel electrophoresis (PFGE) was performed on the isolates to determine the genetic relatedness of outbreak strains. Plasmid analysis and PCR were performed to identify β-lactamase genes responsible for ceftriaxone resistance. Forty-seven children from the two elementary schools were culture positive for
S. sonnei
in this outbreak. Twenty-three children were asymptomatic. Of the total isolates 55.3% were resistant to ampicillin. One hundred percent of the isolates obtained from children in school A were initially susceptible to both ampicillin and ceftriaxone. Of isolates obtained from school B 96.2% were nonsusceptible to ceftriaxone. However, two isolates from school A developed resistance to ampicillin during the course of treatment. All 18 available isolates showed closely related PFGE patterns (4, 4a, 4b, and 4c). CMY-2-type AmpC β-lactamase was responsible for ceftriaxone resistance in ceftriaxone-nonsusceptible isolates; Southern blot hybridization confirmed that such a resistance gene was located on the plasmid. This is the first report of plasmid-mediated CMY-2-type AmpC β-lactamase in
S. sonnei
. Ampicillin-resistant isolates can develop during the course of antibiotic treatment.
High Frequency of Gastric Colonization with MultipleHelicobacter pyloriStrains in Venezuelan Subjects
Authors: Ghose C. , Perez-Perez G. I. , van Doorn L. J. , Domínguez-Bello M. G. , Blaser M. J. 
Published: June 2005

ABSTRACTMultipleHelicobacter pyloristrains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixedH. pyloricolonization in 127 subjects from Venezuela, a country of highH. pyloriprevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121H. pylori-positive persons, the prevalence ofcagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of thevacA s(mean, 49%) andm(mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization.vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that theH. pyloriEast AsianvacAgenotype has survived in all three populations tested.
Novel PCR-Restriction Fragment Length Polymorphism Method for Determining Serotypes or Serogroups of
Streptococcus pneumoniae
Isolates
Authors: Batt Sarah L. , Charalambous Bambos M. , McHugh Timothy D. , Martin Siobhan , Gillespie Stephen H. 
Published: June 2005

ABSTRACT

Serotyping
Streptococcus pneumoniae
is a technique generally confined to reference laboratories, as purchasing pneumococcal antisera is a huge investment. Many attempts have been made to modify serological agglutination techniques to make them more accessible, and more recently developments in serotyping have focused on molecular techniques. This paper describes a PCR assay which amplifies the entire capsulation locus between
dexB
and
aliA
. Amplicons are digested to produce serotype-specific patterns. We have shown, using 81 epidemiologically unrelated strains representing 46 different serotypes, that the patterns correlate with a 90 to 100% similarity range for the same serotype or serogroup. Prospective testing of 73 isolates of unknown serotype confirmed reliable serotype attribution, and serotype profiles are reproducible on repeated testing. Once our database contains all 90 serotypes, this technique should be fully portable, cost-effective, and useful in any laboratory with sufficient molecular experience.
Use of the Duplex TaqMan PCR System for Detection of Shiga-Like Toxin-Producing
Escherichia coli
O157
Authors: Hsu Ching-Fang , Tsai Tsung-Yu , Pan Tzu-Ming 
Published: June 2005

ABSTRACT

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the
rfb

Escherichia coli
O157

and
stx
2
genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the
Escherichia coli
O157 serotype from non-
E. coli
O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10
3
to 10
9
CFU/ml of
E. coli
O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of
E. coli
O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10
4
to 10
9
CFU/g (or 10
4
to 10
9
CFU/ml) for feces and apple juice and 10
5
to 10
9
CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10
0
to 10
3
CFU/ml. The real-time PCR assays for

rfb
E. coli

O157
and
stx
2
proved to be rapid tests for the detection of
E. coli
O157 in food matrices and could also be used for the quantification of
E. coli
O157 in foods or fecal samples.
Molecular Epidemiology of EndemicClostridium difficileInfection and the Significance of Subtypes of the United Kingdom Epidemic Strain (PCR Ribotype 1)
Authors: Fawley Warren N. , Parnell Peter , Verity Paul , Freeman Jane , Wilcox Mark H. 
Published: June 2005

ABSTRACTWe previously identified two subtypes of the epidemic strainClostridium difficilePCR ribotype 1, one clindamycin-sensitive strain (arbitrarily primed PCR [AP-PCR] type Ia) and a closely related clindamycin-resistant strain (AP-PCR type Ib) in our institution. We have now carried out prospective epidemiological surveillance for 4 years, immediately following the relocation of two acute medicine wards for elderly patients (wards A and B), to determine the clinical epidemiology of subtypes of the epidemicC. difficilePCR ribotype 1 group. To maximize the chance of strain discrimination, we used three DNA fingerprinting methods, AP-PCR, ribospacer PCR (RS-PCR), and pulsed-field gel electrophoresis (PFGE), to analyzeC. difficileisolates recovered from symptomatic patients and from repeated environmental samplings. On ward B the incidence ofC. difficileinfection correlated significantly with the prevalence of environmentalC. difficileboth in ward areas closely associated with patients and health care personnel (r= 0.53;P< 0.05) and in high-reach sites (r= 0.85;P< 0.05). No such relationships were found on ward A. Seventeen distinctC. difficilegenotypes were identified, 17 by AP-PCR, 12 by PFGE, and 11 by RS-PCR, but only 4 of 17 genotypes caused patient infection. Isolates recovered from the hospital ward environment were much more diverse (14 genotypes). AP-PCR type Ia represented >90% of theC. difficileisolates. In addition to this genotype, only two others were isolated from both patient feces and environmental surfaces. AP-PCR type Ib (clindamycin-resistant PCR ribotype 1 clone) was not associated with any cases ofC. difficileinfection and was isolated from the environment on only two occasions, after having been implicated in a cluster of sixC. difficileinfections 5 months before this study. The disappearance of this strain implies that differences in virulence and/or selective pressures may exist for this strain and the closely related, widespreadC. difficileAP-PCR type Ia strain. Our findings emphasize the need to understand the epidemiology and virulence of clinically significant strains to determine successful control measures forC. difficileinfections.
Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing
Authors: Schoder D. , Schmalwieser A. , Schauberger G. , Hoorfar J. , Kuhn M. , Wagner M. 
Published: June 2005

ABSTRACT
As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 μl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was ±0.3°C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer′s specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5°C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20°C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.
Increase in Genetic Diversity of
Haemophilus influenzae
Serotype b (Hib) Strains after Introduction of Hib Vaccination in The Netherlands
Authors: Schouls Leo M. , van der Ende Arie , van de Pol Ingrid , Schot Corrie , Spanjaard Lodewijk , Vauterin Paul , Wilderbeek Dorus , Witteveen Sandra 
Published: June 2005

ABSTRACT

Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by
Haemophilus influenzae
serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype
H. influenzae
isolates. The MLVA enabled the differentiation of
H. influenzae
serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated
H. influenzae
and
H. influenzae
serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520
H. influenzae
serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains.
Comparative Analysis of the Diagnostic Performance of Six Major
Echinococcus granulosus
Antigens Assessed in a Double-Blind, Randomized Multicenter Study
Authors: Lorenzo Carmen , Ferreira Henrique B. , Monteiro Karina M. , Rosenzvit Mara , Kamenetzky Laura , García Hector H. , Vasquez Yessika , Naquira Cesar , Sánchez Elizabeth , Lorca Myriam , Contreras María , Last Jerry A. , González-Sapienza Gualberto G. 
Published: June 2005

ABSTRACT

The serodiagnosis of hydatid disease is a valuable instrument for clinical diagnosis and epidemiological surveillance of high-risk populations. In the past decade a wealth of reports on the diagnostic performance of numerous antigens have been produced. However, their diagnostic value has been estimated under different conditions, using different serum collection, therefore precluding their direct comparison. Here we report an unbiased comparison of the same batch of six major
E. granulosus
antigens, namely, hydatid cyst fluid (HCF), native antigen B (AgB), two recombinant AgB subunits, an AgB-derived synthetic peptide, and recombinant cytosolic malate dehydrogenase from
E. granulosus
(EgMDH), against the same serum collection. The double-blind analysis was performed using a standardized protocol and receiver operating characteristic (ROC) data analysis by a network of six South American laboratories. High intercenter reproducibility was attained, and the intralaboratory analysis allowed the comparative ranking of the antigen panel. HCF, AgB, and its AgB8/1 subunit exhibited equivalent diagnostic efficiencies, 81.4% ± 0.5%, 81.3% ± 0.6%, and 81.9% ± 2.0%, respectively; with a more favorable balance toward specificity in the case of the last antigen. The diagnostic efficiencies for the other three antigens were 76.8% ± 6.8%, 69.1% ± 2.7%, and 66.8% ± 2.1%, for the peptide, the AgB8/2 subunit, and the EgMDH, respectively. The study also included an analysis of batch-to-batch variation in the diagnostic performance of different HCF regional preparations. Based on these results, a suggested recommendation on the use of these antigens was drawn.
Discrimination between Primary and Secondary Dengue Virus Infection by an Immunoglobulin G Avidity Test Using a Single Acute-Phase Serum Sample
Authors: Matheus Séverine , Deparis Xavier , Labeau Bhety , Lelarge Josiane , Morvan Jacques , Dussart Philippe 
Published: June 2005

ABSTRACT

For clinical and epidemiological purposes, it is necessary to be able to classify serological responses during dengue virus infection. Thus, it is important to develop a test that can distinguish between primary and secondary serological responses. The hemagglutination inhibition (HI) test, which is currently recommended by the World Health Organization, is complicated to perform. We developed an enzyme-linked immunosorbent assay based on changes in the avidity of immunoglobulin G during the infectious episode. This test can discriminate between primary and secondary infections by using a single serum sample collected during the acute phase of infection. We took 1,140 avidity measurements with 118 pairs of serum samples or sequential samples taken from patients classified as having primary or secondary infection according to World Health Organization laboratory criteria. The mean percent avidity was significantly lower during primary infection (25.9%) than during secondary infection (66.3%) (Student
t
test,
P
< 0.001). The test had a sensitivity of 82.7% (95% confidence interval [CI] = 79.0 to 86.6) and a specificity of 77.5% (95% CI = 73.3 to 81.7). Based on analysis of only blood samples collected between the third and seventh days of the illness, during which most clinical complications occur, the sensitivity and specificity of the test were 95.1% (95% CI = 92.6 to 97.7) and 80.0% (95% CI = 75.3 to 84.7), respectively. This rapid and simple test appears to be an excellent alternative to the HI test for discriminating between primary and secondary dengue virus infections during the acute phase of dengue.
Molecular Epidemiology of Community- and Health Care-Associated Methicillin-Resistant
Staphylococcus aureus
in Manitoba, Canada
Authors: Wylie John L. , Nowicki Deborah L. 
Published: June 2005

ABSTRACT

Recently, acquisition of methicillin-resistant
Staphylococcus aureus
(MRSA) has been increasingly seen in community settings. Beginning in 1995, we have routinely conducted pulsed-field gel electrophoresis (PFGE) of MRSA isolates received at Cadham Provincial Laboratory (CPL) in Manitoba, Canada. Our diverse collection of isolates coupled with molecular subtype information allowed us to assess the extent to which MRSA isolates in general were associated with community acquisition and whether specific PFGE types were more likely to be found in community settings. Forty percent of the MRSA isolates in our analysis were designated community associated (CA), with two of the six most common PFGE types showing a greater likelihood to be CA-MRSA. Overall, CA-MRSA were more likely to show multiple sensitivity to antibiotics and to be associated with younger age groups. Mapping of specific CA-MRSA types over successive 5-year periods showed rapid temporal shifts in prevalence in different parts of the province.
Optimized Diagnosis of Acute Dengue Fever in Swedish Travelers by a Combination of Reverse Transcription-PCR and Immunoglobulin M Detection
Authors: Lindegren Gunnel , Vene Sirkka , Lundkvist Åke , Falk Kerstin I. 
Published: June 2005

ABSTRACT

The dengue viruses (genus
Flavivirus
, family
Flaviviridae
) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset.
Shifts of
Bordetella pertussis
Variants in Sweden from 1970 to 2003, during Three Periods Marked by Different Vaccination Programs
Authors: Hallander Hans O. , Advani Abdolreza , Donnelly Declan , Gustafsson Lennart , Carlsson Rose-Marie 
Published: June 2005

ABSTRACT

The Swedish population of
Bordetella pertussis
strains was characterized from 1,247 isolates covering a whole-cell vaccine program up to 1979, a 17-year period without vaccination (1979 to 1996), and a period after the introduction of general vaccination among newborns with acellular pertussis vaccines (1997 to 2003). Strains were characterized by serotyping and genotyping of pertactin and
ptxA
and by means of pulsed-field gel electrophoresis (PFGE). With emphasis on vaccine-related markers, the vast majority of circulating strains were of nonvaccine type. There were shifts of serotype connected with shifts of vaccination program. Serotype Fim3 was most frequent during the periods with general vaccination schedules, whereas serotype Fim2 was predominant during the 17-year vaccine-free period. Pertactin 1 was predominant during the pertussis whole-cell (Pw) vaccine period but was thereafter replaced by
prn2
and has not reappeared after the introduction of acellular pertussis (Pa) vaccines.
ptxA
(1) was predominant over all three decades. There was a significant difference in the distribution of serotypes between vaccinated and unvaccinated individuals, but not for pertactin. A few PFGE profiles were predominant over the years: BpSR25 (serotype Fim3
prn1/7
) and BpSR18 (serotype Fim3
prn2
) during the Pw period, BpSR1 (serotype Fim2
prn
2) during the 17 years without general vaccination, and BpSR11 (serotype Fim3
prn
2) after the reintroduction of general vaccination in 1996. Despite differences between the pertactin and toxin types of Pa vaccines and circulating strains, there is no evidence that there is a threat, i.e., the vaccination program so far has been effective against whooping cough, and there seems to be no impact on the effectiveness of the vaccination program from the bacterial polymorphism.