bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of Enteropathogenic and Enterohemorrhagic
Escherichia coli
Serogroup O103 Strains by Molecular Typing of Virulence and Housekeeping Genes and Pulsed-Field Gel Electrophoresis
Authors: Beutin Lothar , Kaulfuss Stefan , Herold Sylvia , Oswald Eric , Schmidt Herbert
Published: April 2005

We investigated the genetic relationships of 54
Escherichia coli
O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing
E. coli
(STEC) O103:H2 strains from Australia, Canada, France, Germany, and Northern Ireland that are characterized by the intimin (
eae
) epsilon gene and carry enterohemorrhagic
E. coli
(EHEC) virulence plasmids. MLST profile II groups five human and animal enteropathogenic
E. coli
(EPEC) O103:H2 strains that were positive for intimin (
eae
) beta. Although strains belonging to MLST groups II and I plus III are closely related to each other (92.6% identity), major differences were found in the housekeeping
icdA
gene and in the virulence-associated genes
eae
and
escD. E. coli
O103 strains with MLST patterns IV to VII are genetically distant from MLST I, II, and III strains, as are the non-O103
E. coli
strains EDL933 (O157), MG1655 (K-12), and CFT073 (O6). Comparison of MLST results with those of PFGE and virulence typing demonstrated that
E. coli
O103 STEC and EPEC have recently acquired different virulence genes and DNA rearrangements, causing alterations in their PFGE patterns. PFGE typing was very useful for identification of genetically closely related subgroups among MLST I strains, such as Stx2-producing STEC O103 strains from patients with hemolytic uremic syndrome. Analysis of virulence genes contributed to grouping of
E. coli
O103 strains into EPEC and STEC. Novel virulence markers, such as
efa
(EHEC factor for adherence),
paa
(porcine adherence factor), and
cif
(cell cycle-inhibiting factor), were found widely associated with
E. coli
O103 EPEC and STEC strains.
Loop-Mediated Isothermal Amplification Method Targeting the
lytA
Gene for Detection of
Streptococcus pneumoniae
Authors: Seki Mitsuko , Yamashita Yoshihisa , Torigoe Hirotaka , Tsuda Hiromasa , Sato Setsuko , Maeno Masao
Published: April 2005

It is difficult to separate
Streptococcus pneumoniae
from the genotypically similar species
Streptococcus mitis
and
Streptococcus oralis
, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting
S. pneumoniae
. An
S. pneumoniae
-specific LAMP primer targeting the
lytA
gene was designed. The primer specificity was validated using 10
Streptococcus
and 7 non-
Streptococcus
species. Within 60 min, the assay could detect 10 or more copies of purified
S. pneumoniae
DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3
S. oralis
, 17
S. mitis
, and 1
Streptococcus
species) that harbor virulence-factor-encoding genes (
lytA
or
ply
) were tried to differentiate
S. pneumoniae
. The detection of
S. pneumoniae
in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing
S. pneumoniae
infection.
Characterization of
Borrelia lusitaniae
Isolates Collected in Tunisia and Morocco
Authors: Younsi Hend , Sarih M'Hammed , Jouda Fatima , Godfroid Edmond , Gern Lise , Bouattour Ali , Baranton Guy , Postic Daniele
Published: April 2005

Borrelia lusitaniae
is a species within the complex
Borrelia burgdorferi
sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living
Ixodes ricinus
ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the
ospA
gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that
B. lusitaniae
strains isolated in North Africa constitute a clone of Portuguese origin.
Colonization of Human Immunodeficiency Virus-Infected Outpatients in Taiwan with
Candida
Species
Authors: Hung Chien-Ching , Yang Yun-Liang , Lauderdale Tsai-Ling , McDonald L. Clifford , Hsiao Chin-Fu , Cheng Hsiao-Hsu , Ho Yong An , Lo Hsiu-Jung
Published: April 2005

To understand the
Candida
colonization of human immunodeficiency virus (HIV)-infected outpatients in Taiwan, we have conducted a prospective cohort study of
Candida
colonization and its risk factors at the National Taiwan University Hospital from 1999 to 2002. More than 50% of the patients were colonized with
Candida
species, and 12% developed symptomatic candidiasis. Patients colonized with fluconazole-resistant strains of
Candida
species had a higher prevalence of candidiasis than those colonized with susceptible strains. Our analysis found that antibiotic treatment and lower CD4
+
counts (<200 cells/mm
3
) increased the rate of oropharyngeal candidiasis in HIV-infected patients, while antiretroviral therapy protected patients from the development of candidiasis.
Species-Level Identification of Isolates of the
Acinetobacter calcoaceticus
-
Acinetobacter baumannii
Complex by Sequence Analysis of the 16S-23S rRNA Gene Spacer Region
Authors: Chang Hsien Chang , Wei Yu Fang , Dijkshoorn Lenie , Vaneechoutte Mario , Tang Chung Tao , Chang Tsung Chain
Published: April 2005

The species
Acinetobacter calcoaceticus
,
A. baumannii
, genomic species 3, and genomic species 13TU included in the
Acinetobacter calcoaceticus
-
Acinetobacter baumannii
complex are genetically highly related and difficult to distinguish phenotypically. Except for
A. calcoaceticus
, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the
A. calcoaceticus
-
A. baumannii
complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of
Acinetobacter
were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the
A. calcoaceticus
-
A. baumannii
complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as
A. calcoaceticus
(18 isolates) or
A. baumannii
(61 isolates) with the API 20 NE system (bioMérieux Vitek, Marcy l'Etoile, France) were identified as
A. baumannii
(46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the
A. calcoaceticus
-
A. baumannii
complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the
A. calcoaceticus
-
A. baumannii
complex.
Clonal Dissemination of Macrolide-Resistant and Penicillin-Susceptible Serotype 3 and Penicillin-Resistant Taiwan 19F-14 and 23F-15
Streptococcus pneumoniae
Isolates in Japan: a Pilot Surveillance Study
Authors: Kasahara Kei , Maeda Koichi , Mikasa Keiichi , Uno Kenji , Takahashi Ken , Konishi Mitsuru , Yoshimoto Eiichiro , Murakawa Koichi , Kita Eiji , Kimura Hiroshi
Published: April 2005

Large-scale surveillance studies using molecular techniques such as pulsed-field gel electrophoresis (PFGE) have revealed that the spread of antibiotic-resistant pneumococci is due to clonal spread. However, in Japan, surveillance studies using such molecular techniques have never been done. Therefore, we conducted a pilot surveillance study to elucidate the present situation in Japan. Among the 145 isolates examined, the most prevalent serotype was type 19F (20%), for which most isolates were not susceptible to penicillin (86.2%) but were positive for the
mef
(A)/
mef
(E) gene (89.7%). The secondmost prevalent was serotype 3 (16.6%), for which most isolates were susceptible to penicillin (87.5%) and positive for the
erm
(B) gene (91.7%). PFGE analysis showed that both serotypes consisted mainly of clonally identical or related isolates and, in particular, 38% of the type 19F isolates were indistinguishable from or closely related to the Taiwan 19F-14 clone. In addition, some of the Japanese type 23F isolates with the
erm
(B) gene were indistinguishable from or related to the Taiwan 23F-15 clone as analyzed by PFGE. Based on the results of our pilot study performed in a single institution, it is likely that international antibiotic-resistant clones have already spread in Japan; therefore, a nationwide surveillance study should be urgently conducted.
Loop-Mediated Isothermal Amplification for Rapid Detection of Newcastle Disease Virus
Authors: Pham Hang Minh , Nakajima Chie , Ohashi Kazuhiko , Onuma Misao
Published: April 2005

We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection.
Laboratory-Based Surveillance and Molecular Epidemiology of Influenza Virus in Taiwan
Authors: Shih Shin-Ru , Chen Guang-Wu , Yang Ching-Chun , Yang Weng-Zhi , Liu Ding-Ping , Lin Jih-Hui , Chiu Shu-Chun , Chen Haur-Young , Tsao Kuo-Chien , Huang Chung-Guei , Huang Ya-Ling , Mok Chee-Keng , Chen Chi-Jene , Lin Tzou-Yien , Wang Jen-Ren , Kao Chuan-Liang , Lin Kwei-Hsian , Chen Li-Kuang , Eng Hock-Liew , Liu Yung-Ching , Chen Po-Yen , Lin Jen-Shiou , Wang Jen-Hsien , Lin Cheng-Wen , Chan Yu-Jiun , Lu Jang-Jih , Hsiung Chao A. , Chen Pei-Jer , Su Ih-Jen
Published: April 2005

A laboratory-based surveillance network of 11 clinical virological laboratories for influenza viruses was established in Taiwan under the coordination of the Center for Disease Control and Prevention (CDC), Taiwan. From October 2000 to March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred frequently in winter in the northern hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B also occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from the year 2002 clustered into different groups, and the data indicate that both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were cocirculating. Sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least 2 years before the vaccine strains were introduced. No clear seasonality of influenza reports in Taiwan occurred in contrast to other more continental regions.
Dynamic Range of Hepatitis C Virus RNA Quantification with the Cobas Ampliprep-Cobas Amplicor HCV Monitor v2.0 Assay
Authors: Gourlain Karine , Soulier Alexandre , Pellegrin Bertrand , Bouvier-Alias Magali , Hézode Christophe , Darthuy Françoise , Rémiré Jocelyne , Pawlotsky Jean-Michel
Published: April 2005

Accurate quantification of hepatitis C virus (HCV) RNA is needed in clinical practice to decide whether to continue or stop pegylated interferon-α-ribavirin combination therapy at week 12 of treatment for patients with chronic hepatitis C. Currently the HCV RNA quantification assay most widely used worldwide is the Amplicor HCV Monitor v2.0 assay (Roche Molecular Systems, Pleasanton, Calif.). The HCV RNA extraction step can be automated in the Cobas Ampliprep device. In this work, we show that the dynamic range of HCV RNA quantification of the Cobas Ampliprep/Cobas Amplicor HCV Monitor v2.0 procedure is 600 to 200,000 HCV RNA IU/ml (2.8 to 5.3 log IU/ml), which does not cover the full range of HCV RNA levels in infected patients. Any sample containing more than 200,000 IU/ml (5.3 log IU/ml) must thus be retested after dilution for accurate quantification. These results emphasize the need for commercial HCV RNA quantification assays with a broader range of linear quantification, such as real-time PCR-based assays.
Identification of Genotype 3 Hepatitis E Virus (HEV) in Serum and Fecal Samples from Pigs in Thailand and Mexico, Where Genotype 1 and 2 HEV Strains Are Prevalent in the Respective Human Populations
Authors: Cooper K. , Huang F. F. , Batista L. , Rayo C. D. , Bezanilla J. C. , Toth T. E. , Meng X. J.
Published: April 2005

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. Increasing evidence indicates that hepatitis E is a zoonotic disease. There exist four major genotypes of HEV, and HEV isolates identified in samples from pigs belong to either genotype 3 or 4. Genotype 1 and 2 HEVs are found exclusively in humans. To determine whether genotype 1 and 2 HEVs also exist in pigs, a universal reverse transcription-PCR assay that is capable of detecting all four HEV genotypes was used to test for the presence of HEV RNA in serum and/or fecal samples from pigs in Thailand, where genotype 1 human HEV is prevalent, and from pigs in Mexico, where genotype 2 human HEV was epidemic. In Thailand, swine HEV RNA was detected in sera from 10/26 pigs of 2 to 4 months of age but not in sera from 50 pigs of other ages. In Mexico, swine HEV RNA was detected in 8/125 sera and 28/92 fecal samples from 2- to 4-month-old pigs. Antibodies to swine HEV were also detected in about 81% of the Mexican pigs. A total of 44 swine HEV isolates were sequenced for the open reading frame 2 gene region. Sequence analyses revealed that all swine HEV isolates identified in samples from pigs in Thailand and Mexico belong to genotype 3. Phylogenetic analyses revealed that minor branches associated with geographic origin exist among the swine HEV isolates. The results indicated that genotype 1 or 2 swine HEV does not exist in pigs from countries where the respective human HEV genotype 1 or 2 is prevalent. It is likely that only genotype 3 and 4 HEV strains have zoonotic potential.
Analysis of the Genetic Structure of Nontypeable Pneumococcal Strains Isolated from Conjunctiva
Authors: Berrón Sonsoles , Fenoll Asunción , Ortega Montserrat , Arellano Noemí , Casal Julio
Published: April 2005

More than 50% of the nontypeable (NT) pneumococcal strains received in our laboratory for reference purposes are isolated in sporadic cases of conjunctivitis. To determine the genetic structure of the population of these NT conjunctival strains, we analyzed 75 pneumococci (40 NT and 35 typeable) isolated from conjunctivas and 30 (15 NT and 15 typeable) isolated from other sources. The NT and typeable conjunctival strains grouped in separate clusters, whereas NT and typeable pneumococci isolated from other sources were similarly distributed. NT conjunctival strains belonged to two well-differentiated clonal lineages. The first, represented by three newly described sequence types, featured fully antibiotic susceptible strains and appeared to be characteristic of conjunctival tissue; the second, represented by the previously described ST344, had a pattern of multiresistance to penicillin, tetracycline, and erythromycin and shared a genetic background with some NT strains isolated from other sources.
Usefulness of the Hepatitis C Virus Core Antigen Assay for Screening of a Population Undergoing Routine Medical Checkup
Authors: Gaudy Catherine , Thevenas Catherine , Tichet Jean , Mariotte Nicole , Goudeau Alain , Dubois Frédéric
Published: April 2005

We studied the usefulness of the recently designed Trak-C assay for the detection and quantification of the hepatitis C virus (HCV) core antigen (Ag) for the screening of HCV infection in 4,201 subjects selected from 74,150 consecutive volunteers undergoing routine medical checkups. Subjects were selected for screening because they had risk factors (group II,
n
= 321) and/or elevated alanine transaminase activity (group I,
n
= 3847). Initially, the anti-HCV antibody assay and the Trak-C assay were performed on each patient. Subsequently, the Trak-C assay was performed only when the anti-HCV enzyme immune assay (EIA) was positive. Positive samples were further evaluated for anti-HCV antibodies by a third-generation strip immunoblot assay and for HCV RNA. Four samples (1.2%) from group II and 113 (2.9%) from group I were anti-HCV EIA positive. We also tested 33 subjects who previously tested positive for anti-HCV in our medical center. Among the 150 anti-HCV EIA-positive samples, the HCV core Ag result was in accord with the HCV RNA result in 146 cases (97.3%). When the EIA result was positive, the HCV core Ag concentration and the HCV RNA load were correlated (
r
2
= 0.78;
P
< 0.001). Four samples with low viral loads were Trak-C negative but HCV RNA positive. Among the 2,395 anti-HCV EIA-negative serum samples collected during the first part of the study, 17 (0.7%) were found to contain very low levels of HCV core Ag (<8.5 pg/ml, the cutoff value being 1.5 pg/ml). All these samples were HCV RNA negative and considered to be false positives. This was confirmed by HCV core Ag neutralization analysis. The HCV core Ag assay is a useful method in the screening strategy of HCV infection and provides a reliable means of distinguishing between current and cleared HCV infections that is well correlated with HCV RNA testing.
Multilocus Sequence Typing versus Pulsed-Field Gel Electrophoresis for Characterization of Extended-Spectrum Beta-Lactamase-Producing
Escherichia coli
Isolates
Authors: Nemoy Lucia L. , Kotetishvili Mamuka , Tigno Justine , Keefer-Norris Ananda , Harris Anthony D. , Perencevich Eli N. , Johnson Judith A. , Torpey Dave , Sulakvelidze Alexander , Morris J. Glenn , Stine O. Colin
Published: April 2005

Extended-spectrum beta-lactamase (ESBL)-producing
Escherichia coli
strains are emerging pathogens. Molecular typing of ESBL-producing
E. coli
is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current “gold standard” for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing
E. coli
isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing
E. coli
. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing
E. coli
, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.
Analysis ofMycobacterium tuberculosisGenotypes in Madrid and Identification of Two New Families Specific to Spain-Related Settings
Authors: García de Viedma Darío , Bouza Emilio , Rastogi Nalin , Sola Christophe
Published: April 2005

In Spain, tuberculosis (TB) patterns are changing because of the recent increase in the number of cases among immigrants. To establish the composition of circulatingMycobacterium tuberculosisstrains before the effects of foreign strains appear, this study focused on molecular characterization of 233 patient isolates using spoligotyping. The spoligotyping data were further analyzed using an international database, SpolDB4. The results obtained showed that the general features of theM. tuberculosispopulation in Spain are coherent with those of other European countries, with the Latin American and Mediterranean group, and with the Haarlem 3 and T1 families as the most prevalent genotypes. The Spanish isolates clustered mostly with genotypes which had previously been isolated in countries linked with Spain. We also describe and fully characterize two novelM. tuberculosisfamilies, Madrid1 and Madrid2, which are specific to Spain-related settings. The data reported here provide a solid reference when monitoring changes in the composition of theM. tuberculosispopulation in Spain as a consequence of the increasing rate of TB in the foreign population.
System To Assess Genome Sequencing Needs for Viral Protein Diagnostics and Therapeutics
Authors: Gardner Shea N. , Kuczmarski Thomas A. , Zhou Carol E. , Lam Marisa W. , Slezak Tom R.
Published: April 2005

Computational analyses of genome sequences may elucidate protein signatures unique to a target pathogen. We constructed a Protein Signature Pipeline to guide the selection of short peptide sequences to serve as targets for detection and therapeutics. In silico identification of good target peptides that are conserved among strains and unique compared to other species generates a list of peptides. These peptides may be developed in the laboratory as targets of antibody, peptide, and ligand binding for detection assays and therapeutics or as targets for vaccine development. In this paper, we assess how the amount of sequence data affects our ability to identify conserved, unique protein signature candidates. To determine the amount of sequence data required to select good protein signature candidates, we have built a computationally intensive system called the Sequencing Analysis Pipeline (SAP). The SAP performs thousands of Monte Carlo simulations, each calling the Protein Signature Pipeline, to assess how the amount of sequence data for a target organism affects the ability to predict peptide signature candidates. Viral species differ substantially in the number of genomes required to predict protein signature targets. Patterns do not appear based on genome structure. There are more protein than DNA signatures due to greater intraspecific conservation at the protein than at the nucleotide level. We conclude that it is necessary to use the SAP as a dynamic system to assess the need for continued sequencing for each species individually and to update predictions with each additional genome that is sequenced.
Epidemiology and Predictors of Mortality in Cases of
Candida
Bloodstream Infection: Results from Population-Based Surveillance, Barcelona, Spain, from 2002 to 2003
Authors: Almirante Benito , Rodríguez Dolors , Park Benjamin J. , Cuenca-Estrella Manuel , Planes Ana M. , Almela Manuel , Mensa Jose , Sanchez Ferran , Ayats Josefina , Gimenez Montserrat , Saballs Pere , Fridkin Scott K. , Morgan Juliette , Rodriguez-Tudela Juan L. , Warnock David W. , Pahissa Albert
Published: April 2005

We conducted population-based surveillance for
Candida
bloodstream infections in Spain to determine its incidence, the extent of antifungal resistance, and risk factors for mortality. A case was defined as the first positive blood culture for any
Candida
spp. in a resident of Barcelona, from 1 January 2002 to 31 December 2003. We defined early mortality as occurring between days 3 to 7 after candidemia and late mortality as occurring between days 8 to 30. We detected 345 cases of candidemia, for an average annual incidence of 4.3 cases/100,000 population, 0.53 cases/1,000 hospital discharges, and 0.73 cases/10,000 patient-days. Outpatients comprised 11% of the cases, and 89% had a central venous catheter (CVC) at diagnosis. Overall mortality was 44%.
Candida albicans
was the most frequent species (51% of cases), followed by
Candida parapsilosis
(23%),
Candida tropicalis
(10%),
Candida glabrata
(8%),
Candida krusei
(4%), and other species (3%). Twenty-four isolates (7%) had decreased susceptibility to fluconazole (MIC ≥ 16 μg/ml). On multivariable analysis, early death was independently associated with hematological malignancy (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.1 to 10.4). Treatment with antifungals (OR, 0.05; 95% CI, 0.01 to 0.2) and removal of CVCs (OR, 0.3; 95% CI, 0.1 to 0.9) were protective factors for early death. Receiving adequate treatment, defined as having CVCs removed and administration of an antifungal medication (OR, 0.2; 95% CI, 0.08 to 0.8), was associated with lower odds of late mortality; intubation (OR, 7.5; 95% CI, 2.6 to 21.1) was associated with higher odds. The incidence of candidemia and prevalence of fluconazole resistance are similar to other European countries, indicating that routine antifungal susceptibility testing is not warranted. Antifungal medication and catheter removal are critical in preventing mortality.
Sequencing and Resolution of Amplified Herpes Simplex Virus DNA with Intermediate Melting Curves as Genotype 1 or 2 by LightCycler PCR Assay
Authors: Issa Nicolas C. , Espy Mark J. , Uhl James R. , Smith Thomas F.
Published: April 2005

DNA from 101 specimens containing herpes simplex virus (HSV) produced atypical intermediate melting curves compared with those expected for HSV type 1 or HSV type 2 subsequent to real-time PCR. Nucleic acid sequence analysis of amplified target DNA revealed 1- or 3-bp polymorphisms in the probe region which allowed designation of these viruses as HSV genotype 1 or HSV genotype 2. These two subpopulations of HSV were also identified as HSV genotype 1 or HSV genotype 2 using another commercially available PCR method. Amplified HSV target DNA producing intermediate melting curves could be designated as HSV genotype 1 or HSV genotype 2 without performing sequencing or another PCR method with 96/101 (95%) specimens by adding known intermediate HSV DNA characteristic for the two subpopulations as controls.
Detection of Human Immunodeficiency Virus Type 1 DNA in Dried Blood Spots by a Duplex Real-Time PCR Assay
Authors: Luo Wei , Yang Hua , Rathbun Kimberly , Pau Chou-Pong , Ou Chin-Yih
Published: April 2005

A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.
Molecular Evidence for Multiple Host-Specific Strains in the Genus
Rhinosporidium
Authors: Silva Victor , Pereira Cristiane N. , Ajello Libero , Mendoza Leonel
Published: April 2005

The taxonomic relationship of
Rhinosporidium seeberi
with other organisms remained controversial for over a century. Recently, molecular studies have shown
R. seeberi
to be a protistal microbe in the newly described class
Mesomycetozoea
at the animal-fungal boundary. Phylogenetic analyses of
R. seeberi
using 18S small-subunit (SSU) rRNA genes from several hosts suggested
Rhinosporidium
as a monotypic genus. To test this hypothesis, the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 from eight humans, two swans, and a dog with rhinosporidiosis were sequenced. The ITS regions were amplified by PCR using a primer designed from a unique region of
R. seeberi
's 18S SSU rRNA genes in combination with the ITS4 universal primer. In addition, the universal ITS4 and ITS5 primers were also used.
R. seeberi
's ITS sequences showed differences in the numbers of nucleotides among strains. For instance, the eight human ITS sequences were uniformly similar with only a few mismatches and ∼1,060 bp long. In contrast, sequences from one of the swans and the dog were 1,356 bp and ∼1,147 bp long, respectively. Clustal analysis of all of the ITS sequences showed multiple 50- to 60-bp gaps and several mismatches among them. Parsimony analysis placed the
Rhinosporidium
ITS sequences in three well-supported sister groups according to the hosts' identities. This analysis strongly suggests that the genus
Rhinosporidium
may possess multiple host-specific strains. No correlation was found between this finding and the phenotypic features of
R. seeberi
in the studied samples.
Nucleic Acid Amplification Assays for Detection of La Crosse Virus RNA
Authors: Lambert Amy J. , Nasci Roger S. , Cropp Bruce C. , Martin Denise A. , Rose Becky C. , Russell Brandy J. , Lanciotti Robert S.
Published: April 2005

We report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.
Molecular Epidemiology of Hepatitis C Virus Genotype 4 Isolates in Egypt and Analysis of the Variability of Envelope Proteins E1 and E2 in Patients with Chronic Hepatitis
Authors: Genovese D. , Dettori S. , Argentini C. , Villano U. , Chionne P. , Angelico M. , Rapicetta M.
Published: April 2005

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.
Comparison of the Quantitative Formalin Ethyl Acetate Concentration Technique and Agar Plate Culture for Diagnosis of Human Strongyloidiasis
Authors: Intapan P. M. , Maleewong W. , Wongsaroj T. , Singthong S. , Morakote N.
Published: April 2005

The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of
Strongyloides stercoralis
larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.
Multiplex PCR for Typing Strains of
Toxoplasma gondii
Authors: Ajzenberg Daniel , Dumètre Aurélien , Dardé Marie-Laure
Published: April 2005

A multiplex PCR assay was designed for multilocus strain typing of
Toxoplasma gondii
based on length polymorphism of five microsatellite markers. Eight
T. gondii
strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.
Improvement of the Specificity of an Enzyme-Linked Immunosorbent Assay for Diagnosis of Paracoccidioidomycosis
Authors: Albuquerque C. F. , Silva S. H. Marques da , Camargo Z. P.
Published: April 2005

In an attempt to improve the specificity of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of paracoccidioidomycosis (PCM), sera from patients with PCM were tested using various approaches, such as sodium metaperiodate antigen (gp43) treatment, a serum absorption process withCandida albicansorHistoplasma capsulatumantigens, and dilution of serum in galactose, the main common epitope among pathogenic fungi. The maximum specificity found in this ELISA was 84%. All of these procedures proved inefficient for eliminating all cross-reacting antibodies and obtaining an ELISA specific for PCM diagnosis.
Evaluation of Two Chromogenic Agar Media for Recovery and Identification of
Staphylococcus aureus
Small-Colony Variants
Authors: Kipp Frank , Kahl Barbara C. , Becker Karsten , Baron Ellen J. , Proctor Richard A. , Peters Georg , von Eiff Christof
Published: April 2005

To identify the most rapid and reliable technique for recovery and identification of
Staphylococcus aureus
small-colony variants (SCVs), the colonial appearance of 106 isolates representing SCVs and the normal phenotype were evaluated on two newly described chromogenic agar media. Although almost all of the SCVs grew on the chromogenic agar media, they did not exhibit a change of color. In comparison with conventional media,
S. aureus
ID agar (SAID; bioMérieux, La Balme Les Grottes, France) showed the most reliable results, with 49 of 53 SCVs tested growing either as an SCV colony or with a normal phenotype after only 24 h of incubation. Growth of SCVs was often not detected before 72 h of incubation on some of the media tested. In conclusion, the most accurate and rapid method to detect both the species
S. aureus
and the SCV phenotype is to inoculate specimens onto both Columbia blood agar and SAID.
Controlled Clinical Comparison of Plastic and Glass Bottles of BacT/ALERT FA Medium for Culturing Organisms from Blood of Adult Patients
Authors: Petti Cathy A. , Mirrett Stanley , Woods Christopher W. , Reller L. Barth
Published: April 2005

A new, clear-plastic nonvented aerobic FA bottle, designed to prevent breakage, has been developed for the BacT/ALERT blood culture system. We assessed the new plastic FA bottle by comparing its performance with that of the current glass FA bottle for recovery of microorganisms and time to detection of growth in blood samples obtained for culture from adult patients with suspected bloodstream infections. We conclude that the BacT/ALERT plastic and glass FA bottles are comparable for recovery of microorganisms and that the safety advantage of plastic bottles can be achieved without compromising performance.
Evidence for
icaADBC
-Independent Biofilm Development Mechanism in Methicillin-Resistant
Staphylococcus aureus
Clinical Isolates
Authors: Fitzpatrick Fidelma , Humphreys Hilary , O'Gara James P.
Published: April 2005

Synthesis of a polysaccharide adhesin by
icaADBC
-encoded enzymes is currently the best-understood mechanism of staphylococcal biofilm development. In four methicillin-resistant
Staphylococcus aureus
isolates, environmental activation of
icaADBC
did not always correlate with increased biofilm production. Moreover, glucose-mediated biofilm development in these isolates was
icaADBC
independent. Apparently, an environmentally regulated,
ica
-independent mechanism(s) of biofilm development exists in
S. aureus
clinical isolates.
Evaluation of a Rapid Assay as an Alternative to Conventional Enzyme Immunoassays for Detection of Hepatitis C Virus-Specific Antibodies
Authors: Daniel Hubert Darius J. , Abraham Priya , Raghuraman Sukanya , Vivekanandan Perumal , Subramaniam Thenmozhi , Sridharan Gopalan
Published: April 2005

A rapid membrane flow-through immunoassay to detect antibodies to hepatitis C virus was compared with a commercial enzyme immunoassay (EIA) and microparticle enzyme immunoassay (MEIA) using 2,590 serum samples. Sensitivity and specificity of the “rapid assay” in comparison to the EIA/MEIA were 99.3 and 99.0%; the correlation coefficient being 0.91. This assay is suitable where infrastructure and laboratory expertise are limited.
First Report of Infection with Community-Acquired Methicillin-Resistant
Staphylococcus aureus
in South America
Authors: Ribeiro Apoena , Dias Cícero , Silva-Carvalho Maria Cícera , Berquó Laura , Ferreira Fabienne Antunes , Santos Raquel Neves Soares , Ferreira-Carvalho Bernadete Teixeira , Figueiredo Agnes Marie
Published: April 2005

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) has recently emerged in the southwestern Pacific, North America, and Europe. These
S. aureus
isolates frequently shared some genetic characteristics, including the SCC
mec
type IV and
lukS-lukF
genes. In this paper we show that typical CA-MRSA isolates have spread to South America (Brazil).
Characterization of a
Bacillus anthracis
Isolate Causing a Rare Case of Fatal Anthrax in a 2-Year-Old Boy from Hong Kong
Authors: Cheung Danny T. L. , Kam Kai Man , Hau Kong Lung , Au Tak Kong , Marston Chung K. , Gee Jay E. , Popovic Tanja , Van Ert Matthew N. , Kenefic Leo , Keim Paul , Hoffmaster Alex R.
Published: April 2005

We used multiple-locus variable-number tandem repeat analysis (MLVA) and
pagA
sequencing to genotype a
Bacillus anthracis
isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.
Use of Single Nucleotide Polymorphisms in theplcRGene for Specific Identification ofBacillus anthracis
Authors: Easterday W. Ryan , Van Ert Matthew N. , Simonson Tatum S. , Wagner David M. , Kenefic Leo J. , Allender Christopher J. , Keim Paul
Published: April 2005

A TaqMan-minor groove binding assay designed around a nonsense mutation in theplcRgene was used to genotypeBacillus anthracis,B. cereus, andB. thuringiensisisolates. The assay differentiatedB. anthracisfrom these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverseB. anthracisstrains.
Fusarium verticillioides
Abscess of the Nasal Septum in an Immunosuppressed Child: Case Report and Identification of the Morphologically Atypical Fungal Strain
Authors: Dornbusch Hans Jürgen , Buzina Walter , Summerbell Richard C. , Lass-Flörl Cornelia , Lackner Herwig , Schwinger Wolfgang , Sovinz Petra , Urban Christian
Published: April 2005

Morphologically atypical
Fusarium verticillioides
causing a nasal abscess in a severely immunosuppressed child was successfully treated with repeated surgical intervention and liposomal amphotericin B, despite amphotericin B resistance in vitro. Definitive identification was achieved by sequencing the translation elongation factor α gene after ribosomal sequencing proved inadequate.
Multilocus Sequence Typing Directly on DNA from Clinical Samples and a Cultured Isolate To Investigate Linked Fatal Pneumococcal Disease in Residents of a Shelter for Homeless Men
Authors: Birtles Andrew , McCarthy Noel , Sheppard Carmen L. , Rutter Harry , Guiver Malcolm , Haworth Elizabeth , George Robert C.
Published: April 2005

Two apparently linked fatal cases of pneumococcal meningitis were investigated. Pneumolysin PCR performed on blood and cerebrospinal fluid was positive in a culture-negative case. A second case yielded
Streptococcus pneumoniae
from blood culture. Multilocus sequence typing performed on DNA extracted from case 1 (specimens) and case 2 (isolate) revealed identical sequence types (ST53) substantiating the link between them.
Bacteremia Caused by
Clostridium intestinale
Authors: Elsayed Sameer , Zhang Kunyan
Published: April 2005

We describe a case of
Clostridium intestinale
bacteremia in a previously healthy adolescent female presenting with fever and abdominal pain. The bacterium was definitively identified via 16S rRNA gene sequencing. This is the first report, in the world literature, of human infection caused by this microorganism.
ON THE NUMBER OF POSITIVE PERIODIC SOLUTIONS OF FUNCTIONAL DIFFERENTIAL EQUATIONS AND POPULATION MODELS
Authors: JIANG DAQING , O'REGAN DONAL , AGARWAL RAVI P. , XU XIAOJIE
Published: April 2005

In this paper, we employ the fixed point index on cones to study the existence, multiplicity and nonexistence of positive periodic solutions to a system of infinite delay equations, [Formula: see text] in which λ > 0 is a parameter. We prove some general theorems and establish new periodicity conditions for several population growth models.
A HIERARCHY OF APPROXIMATIONS TO THE RADIATIVE HEAT TRANSFER EQUATIONS: MODELLING, ANALYSIS AND SIMULATION
Authors: SCHÄFER MATTHIAS , FRANK MARTIN , PINNAU RENÉ
Published: April 2005

We thoroughly investigate a hierarchy of approximations to the radiative heat transfer equations which is intermediate between moment models and the direct solution. We discuss the modelling of this hierarchy, prove well-posedness and physically expected bounds, and show numerical results validating our approach.
EXPONENTIAL CONVERGENCE OF hp-FEM FOR MAXWELL EQUATIONS WITH WEIGHTED REGULARIZATION IN POLYGONAL DOMAINS
Authors: COSTABEL MARTIN , DAUGE MONIQUE , SCHWAB CHRISTOPH
Published: April 2005

The time-harmonic Maxwell equations do not have an elliptic nature by themselves. Their regularization by a divergence term is a standard tool to obtain equivalent elliptic problems. Nodal finite element discretizations of Maxwell's equations obtained from such a regularization converge to wrong solutions in any non-convex polygon. Modification of the regularization term consisting in the introduction of a weight restores the convergence of nodal FEM, providing optimal convergence rates for the h version of finite elements. We prove exponential convergence of hp FEM for the weighted regularization of Maxwell's equations in plane polygonal domains provided the hp-FE spaces satisfy a series of axioms. We verify these axioms for several specific families of hp finite element spaces.
UNIQUENESS AND COLLAPSE OF SOLUTION FOR A MATHEMATICAL MODEL WITH NONLOCAL TERMS ARISING IN GLACIOLOGY
Authors: MUÑOZ ANA ISABEL , TELLO J. IGNACIO
Published: April 2005

In this paper we study a nonlinear system of differential equations which arises from a stationary two-dimensional Ice-Sheet Model describing the ice-streaming phenomenon. The system consists of a multivalued nonlinear PDE of parabolic type coupled with a first-order PDE and an ODE involving a nonlocal term. We study the uniqueness of weak solution under suitable assumptions (physically reasonable). We also establish that the ice thickness collapses at a finite distance (by employing a comparison principle).
FLUCTUATIONS AND NOISE IN TIME-RESOLVED LIGHT SCATTERING EXPERIMENTS: MEASURING TEMPORALLY HETEROGENEOUS DYNAMICS
Authors: DURI A. , BALLESTA P. , CIPELLETTI L. , BISSIG H. , TRAPPE V.
Published: April 2005

We use Time Resolved Correlation (TRC), a recently introduced light scattering method, to study the dynamics of a variety of jammed, or glassy, soft materials. The output of a TRC experiment is cI(t,τ), the time series of the degree of correlation between the speckle patterns generated by the light scattered at time t and t+τ. We characterize the fluctuations of cIby calculating their Probability Density Function, their variance as a function of the lag τ, and their time autocorrelation function. The comparison between these quantities for a Brownian sample and for jammed materials indicate unambiguously that the slow dynamics measured in soft glasses is temporally heterogeneous. The analogies with recent experimental, numerical and theoretical work on temporal heterogeneity in the glassy dynamics are briefly discussed.
TIME-CLUSTERING ANALYSIS OF RAINFALL FLUCTUATIONS
Authors: TELESCA LUCIANO , LAPENNA VINCENZO , SUMMA DONATO , SCALCIONE EMANUELE
Published: April 2005

Point processes, like other geophysical phenomena, such as earthquakes or avalanches, are well suited to model rain event sequences. Time-clustering analysis has been performed over rainfall sequences by using the statistical approaches of Fano Factor and Allan Factor. The obtained results suggest the presence of clustering in the time distribution of the sequence of rain events, with scaling exponent α ranging between ~0.35 and ~0.42.
INTEGRO-DIFFERENTIAL STOCHASTIC RESONANCE
Authors: TÖRÖK LEVENTE , KISH LÁSZLÓ B.
Published: April 2005

A new class of stochastic resonator (SRT) and Stochastic Resonance (SR) phenomena are described. The new SRT consist of a classical SRT, one or more time derivative circuits and the same number of time integrators. The incoming signal with additive noise is first time derivated, then passes through the classical SRT and finally it is time integrated. The resulting SR phenomena show a well defined SR. Moreover the signal transfer and SNR are the best at the high frequency end. A particular property of the new system is the much smoother output signal due to the time integration.