bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Virulence of Pigeon Isolates of
Salmonella enterica
subsp.
enterica
Serovar Typhimurium Variant Copenhagen for Humans
Authors: Pasmans Frank , Van Immerseel Filip , Hermans Katleen , Heyndrickx Marc , Collard Jean-Marc , Ducatelle Richard , Haesebrouck Freddy
Published: May 2004

Salmonella enterica
serovar Typhimurium variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeons from the city of Ghent (Belgium) and from 26 pooled fecal samples from 114 pigeon lofts (22.8%). These isolates belonged to phage type (PT) 99. Seven of the pigeon isolates were further compared in vitro to five human variant Copenhagen isolates, 2 isolates of PT 208, 1 isolate each of PT 120 and U302, and a nontypeable isolate. No differences in invasiveness in human intestinal epithelial Caco-2 cells were found. The human strains, however, were able to multiply significantly more inside human THP-1 macrophages than the pigeon strains. After inoculation of mice with a pigeon PT 99 strain, high numbers of
Salmonella
bacteria were shed with the feces, the internal organs were heavily colonized, and the animals showed severe clinical symptoms resulting in death. In conclusion, the less-pronounced ability of the pigeon variant Copenhagen strains to multiply inside human macrophages than human strains as well as the lack of human PT 99 isolates during 2002, despite the relatively high frequency of this PT in the pigeon population, suggest these strains to be of low virulence to humans. However, the high virulence for mice of the tested strain implies that rodents may act as reservoirs.
Longitudinal Study of the Excretion Patterns of ThermophilicCampylobacterspp. in Young Pet Dogs in Denmark
Authors: Hald Birthe , Pedersen Karl , Wainø Michael , Jørgensen Jens Christian , Madsen Mogens
Published: May 2004

TheCampylobacterexcretion patterns of 26 domestic pet dogs were described in a longitudinal study. The dogs entered the study between 3 and 8 months of age and were monitored until 2 years of age. They were tested monthly forCampylobactercarriage in stool samples that were cultured on theCampylobacter-selective media CAT and modified CCDA agar at 37 and 42°C. This study comprised 366 fecal swab samples, of which 278 (76.2%) were found to beCampylobacterpositive, with the following distribution of species: 75.0%Campylobacter upsaliensis, 19.4%Campylobacter jejuni, 2.1%Campylobacter lari, 0.7%Campylobacter coli, and 2.8%Campylobacterspp. Isolates were typed by pulsed-field gel electrophoresis (PFGE) to elucidate the strain excretion pattern. All study dogs excretedCampylobacterspp. during the study period. At 3 months of age, 60% of the dogs carriedCampylobacter, increasing to nearly 100% carriers at 1 year of age, whereafter the carriage rate decreased to 67% at 24 months of age. The PFGE types showed that individual dogs were often colonized by unique strains ofC. upsaliensisfor several months, up to 21 months or longer. TheseC. upsaliensisstrains were either clonal (or underwent concurrent minor mutative changes) or independent strains. In contrast, the excretedC. jejuniisolates were much more diverse and, in most cases, only seen in one sample from each dog. A high degree of diversity among different dogs was seen. We conclude that young domestic pet dogs excretedCampylobacterspp. during the majority of their puppyhood and adolescent period. In generalC. upsaliensisstrains were excreted for months, with short-term interruptions by or cocolonization with other transitoryCampylobacterspp., predominantlyC. jejuni. C. jejuniwas more prevalent in dogs between 3 months and 1 year of age than in dogs between 1 and 2 years of age.
Detection and Differentiation of Human Parvovirus Variants by Commercial Quantitative Real-Time PCR Tests
Authors: Hokynar Kati , Norja Päivi , Laitinen Harri , Palomäki Pekka , Garbarg-Chenon Antoine , Ranki Annamari , Hedman Klaus , Söderlund-Venermo Maria
Published: May 2004

Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.
Expertise of French Laboratories in Detection, Genotyping, and Quantification of Hepatitis C Virus RNA in Serum
Authors: Lefrère Jean-Jacques , Roudot-Thoraval Françoise , Lunel Françoise , Alain Sophie , Chaix Marie-Laure , Dussaix Elisabeth , Gassin Michèle , Izopet Jacques , Pawlotsky Jean-Michel , Payan Christopher , Stoll-Keller Françoise , Thibault Vincent , Trabaud Mary-Anne , Bettinger Dominique , Bogard Marc , Branger Michel , Buffet-Janvresse Claudine , Charrois Anne , Defer Christine , Laffont Catherine , Lerable Joëlle , Levayer Thierry , Martinot-Peignoux Michèle , Mercier Bernard , Rosenberg Arielle R.
Published: May 2004

Before initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.
DNA Macroarray for Identification and Typing of
Staphylococcus aureus
Isolates
Authors: Trad Salim , Allignet Jeanine , Frangeul Lionel , Davi Marilyne , Vergassola Massimo , Couve Elisabeth , Morvan Anne , Kechrid Amel , Buchrieser Carmen , Glaser Philippe , El Solh Névine
Published: May 2004

A DNA macroarray containing 465 intragenic amplicons was designed to identify
Staphylococcus aureus
at the species level and to type
S. aureus
isolates. The genes selected included those encoding (i)
S. aureus
-specific proteins, (ii) staphylococcal and enterococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by mobile elements. The macroarray was hybridized with the cellular DNAs of 80
S. aureus
clinical isolates that were previously typed by analyses of their antibiograms and SmaI patterns. The set selected contained unrelated, endemic, and outbreak-related isolates belonging to 45 SmaI genotypes. In a gene content dendrogram, the 80 isolates were distributed into 52 clusters. The outbreak-related isolates were linked in the same or a closely related cluster(s). Clustering based on gene content provided a better discrimination than SmaI pattern analysis for the tested
mecA
+
isolates that were endemic to Europe. All of the antibiotic resistance genes detected could be correlated with their corresponding phenotypes, except for one isolate which carried a
mecA
gene without being resistant. The 16 isolates responsible for bone infections were distinguishable from the 12 isolates from uninfected nasal carriers by a significantly higher prevalence of the
sdrD
gene coding for a putative SD (serine-aspartate) adhesin (in 15 and 7 isolates, respectively). In conclusion, the macroarray designed for this study offers an attractive and rapid typing method which has the advantage of providing additional information concerning the gene content of the isolate of interest.
Comparison of Conventional and Molecular Methods for Identification of Aerobic Catalase-Negative Gram-Positive Cocci in the Clinical Laboratory
Authors: Bosshard P. P. , Abels S. , Altwegg M. , Böttger E. C. , Zbinden R.
Published: May 2004

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of
Streptococcus pneumoniae
and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.
Widespread Skin and Soft-Tissue Infections Due to Two Methicillin-Resistant
Staphylococcus aureus
Strains Harboring the Genes for Panton-Valentine Leucocidin
Authors: Diep Binh An , Sensabaugh George F. , Somboona Naraporn S. , Carleton Heather A. , Perdreau-Remington Françoise
Published: May 2004

Infections caused by community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) are emerging as a major public health problem. CA-MRSA has been associated previously with skin and soft-tissue infection (SSTI) and with carriage of staphylococcal cassette chromosome
mec
(SCC
mec
) type IV and the Panton-Valentine leucocidin (PVL) virulence factor. To assess the clonal distribution of PVL-carrying strains and the association with SSTI in the San Francisco Bay area, we surveyed six collections of
S. aureus
isolates—671 isolates in all—collected between 1997 and 2002 originating from inpatient and outpatient clinical specimens and from a community-based sampling. Isolates were genotyped by pulsed-field gel electrophoresis, multilocus restriction fragment typing, and multilocus sequence typing and assayed for the PVL virulence factor. The
S. aureus
populations showed a high proportion of PVL-carrying strains, with frequencies ranging up to 70% in MRSA isolated from jail inmate patients and 69% in MRSA from patients receiving surgical treatment at an outpatient clinic specializing in treating SSTIs. PVL-carrying isolates were identified in nine clonal groups, but 88.5% of the PVL-carrying MRSA isolates belonged to only two clonal groups. These two clonal groups carried the SCC
mec
type IV resistance determinant and were more likely than other clonal groups to be recovered from SSTI sites than from other sites (
P
< 0.0001). There is evidence of clonal replacement over the period from 1999 to 2002, with one of these two clonal groups being supplanted by the other.
Rapid Quantification of Drug Resistance Gene Expression in
Candida albicans
by Reverse Transcriptase LightCycler PCR and Fluorescent Probe Hybridization
Authors: Frade Joao P. , Warnock David W. , Arthington-Skaggs Beth A.
Published: May 2004

We developed a rapid, sensitive, and reproducible assay to quantify
Candida albicans ACT1
,
CDR1
,
CDR2
,
ERG11
, and
MDR1
mRNA using a two-step reverse transcription and LightCycler real-time PCR (RT-LightCycler PCR) method with sequence-specific hybridization probes. We compared RT-LightCycler PCR with Northern hybridization for quantitative analysis of gene expression in isolates with various fluconazole susceptibilities. Specificity of each LightCycler PCR was verified by LightCycler melting curve analysis and agarose gel electrophoresis of amplified products. Correlation of quantification results between RT-LightCycler PCR and Northern hybridization yielded correlation coefficients of ≥0.91 for all genes except
MDR1
(0.74). In this case, reduced correlation was due to the inability of Northern hybridization to accurately quantify the high
MDR1
expression in a susceptible dose-dependent isolate which was shown by RT-LightCycler PCR to overexpress
MDR1
>200-fold relative to the other isolates tested. In four isolates, low levels of
CDR2
mRNA were detected by RT-LightCycler PCR but were undetectable by Northern hybridization. mRNA quantification by RT-LightCycler PCR correlates with Northern hybridization and offers additional advantages, including increased sensitivity and speed of analysis, along with lower RNA concentration requirements and an increased dynamic range of signal detection.
Comparison of Two Real-Time Quantitative Assays for Detection of Severe Acute Respiratory Syndrome Coronavirus
Authors: Hourfar Michael K. , Roth W. Kurt , Seifried Erhard , Schmidt Michael
Published: May 2004

The new severe acute respiratory syndrome (SARS) coronavirus (CoV), described in February 2003, infected a total of 8,439 people. A total of 812 people died due to respiratory insufficiency. Close contact with symptomatic patients appeared to be the main route of transmission. However, potential transmission by blood transfusion could not be definitely excluded. Two real-time SARS-specific PCR assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. Both assays rely on reverse transcription and amplification of extracted RNA. Dilutions of gamma-irradiated cell culture supernatants of SARS CoV-infected Vero E6 cells were prepared to determine the precisions, linear ranges, and accuracies of the assays. The linear range for the Artus RealArt HPA-Coronavirus assay (Artus assay) was 1 × 10
2
to 1 × 10
7
copies/ml, and that for the Roche LightCycler SARS CoV Quantification kit (Roche assay) was 1 × 10
4
to 2 × 10
8
copies/ml. The detection limit of the Roche assay was 3,982.1 copies/ml, whereas that of the Artus assay was 37.8 copies/ml. Detection limits were calculated with a standard preparation that was recommended for use by the World Health Organization. However, quantification of CoV in this preparation may be imprecise. In summary, both assays are suitable for quantitative measurement of SARS CoV at the high concentrations expected in sputum samples. The Artus assay is also suitable for detection of SARS CoV at the low concentrations found in serum samples.
Detection ofCandidain Concentrated Oral Rinse Cultures by Real-Time PCR
Authors: White P. Lewis , Williams David W. , Kuriyama Tomoari , Samad Shamim A. , Lewis Michael A. O. , Barnes Rosemary A.
Published: May 2004

The incidence of oral candidosis has increased in recent years, largely as a result of the emergence of human immunodeficiency virus infection and the more widespread use of immunosuppressive chemotherapy. This development has been associated with a need for more reliable methods for the detection ofCandida. The present study assessed the performance of a real-time PCR and two block-based PCRs for the detection ofCandidain 193 concentrated oral rinse culture (CRC) specimens. A total of 102 CRC specimens were positive by culture forCandida; and 96, 90, and 75 of these were also positive by real-time, N18-specific, and internal transcribed spacer (ITS)-specific PCRs, respectively. The five false-negative results by the real-time PCR were all non-Candida albicanspositive by culture. Of the 91 culture-negative CRC specimens, 20, 41, and 44 were positive by the real-time PCR and the N18- and ITS-specific PCRs, respectively. All three PCRs detected fungal DNA in 8 culture-negative CRC specimens, with a further 30 being positive by two of the three PCRs. A total of 32 CRC specimens wereCandidafree by all methods. In summary, a real-time PCR that provides a sensitive, specific, and rapid alternative technique for detection ofCandidain the mouth is described.
Comparison of Two 3ABC Enzyme-Linked Immunosorbent Assays for Diagnosis of Multiple-Serotype Foot-and-Mouth Disease in a Cattle Population in an Area of Endemicity
Authors: Bronsvoort B. M. de C. , Sørensen K. J. , Anderson J. , Corteyn A. , Tanya V. N. , Kitching R. P. , Morgan K. L.
Published: May 2004

The development of a serological test for foot-and-mouth disease virus (FMDV) which is quick and easy to use, which can identify all seven serotypes, and which can differentiate vaccinated from convalescing or potential virus carriers would be a major advance in the epidemiological toolkit for FMDV. The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the “gold standard.” Diagnostic sensitivity and specificity were examined over a range of test cutoffs by using receiver operating characteristic curves, which allowed comparison of the overall performance of each test. The results indicated that the CHEKIT ELISA kit was 23% sensitive and 98% specific and the Danish C-ELISA was 71% sensitive and 90% specific at the recommended cutoff. These results have important implications if the tests are to be used to screen herds or individual cattle in surveillance programs, at border crossings for import-export clearance, or following emergency vaccination in an outbreak situation.
Serotyping of 800 Strains of
Erysipelothrix
Isolated from Pigs Affected with Erysipelas and Discrimination of Attenuated Live Vaccine Strain by Genotyping
Authors: Imada Yumiko , Takase Ai , Kikuma Reiko , Iwamaru Yoshifumi , Akachi Shigehiro , Hayakawa Yûji
Published: May 2004

Eight hundred
Erysipelothrix
strains isolated between 1992 and 2002 from swine with erysipelas in Japan were serotyped. Thirty-seven, 47, 73, and 643 strains were isolated from animals with acute septicemia, urticaria, chronic endocarditis, and chronic arthritis, respectively, of which 381, 146, 254, and 19 isolates belonged to serotypes 1a, 1b, and 2b and other serotypes, respectively. All serotype 1a isolates were further examined for acriflavine resistance and their genotypes to discriminate them from the attenuated live vaccine strain, defined as serotype 1a, which is resistant to 0.02% acriflavine and which shows low levels of pathogenicity in mice. Of the serotype 1a isolates, 64.6% were acriflavine resistant, with 98.4% of these acriflavine-resistant strains having been isolated from animals with chronic arthritis. By randomly amplified polymorphic DNA (RAPD) analysis, almost all the acriflavine-resistant serotype 1a strains showed the 253-bp band characteristic of vaccine strains and were easily discriminated from all 113 strains of acriflavine-sensitive serotype 1a strains from animals with acute and subacute swine erysipelas. The incidence of acriflavine-resistant strains of the distinctive RAPD type 1-2 was markedly higher than that of the other RAPD types and serotypes. RAPD type 1-2 strains also included a specific group identifiable by restriction fragment length polymorphism DNA analysis. Furthermore, the pathogenicities of 29 isolates of RAPD type 1-2 for mice were lower than those of the 21 isolates of other RAPD types. Our results indicate that RAPD type 1-2 strains are live vaccine strains and that 37% of the cases of chronic swine erysipelas detected in the past 11 years in Japan have occurred as a side effect of live vaccine use.
Simultaneous Analysis of Multiple Staphylococcal Enterotoxin Genes by an Oligonucleotide Microarray Assay
Authors: Sergeev Nikolay , Volokhov Dmitriy , Chizhikov Vladimir , Rasooly Avraham
Published: May 2004

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many
Staphylococcus aureus
isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (
ent
) genes. The proposed typing method is based on PCR amplification of the target region of the
ent
genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each
ent
gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16
ent
genes. The microarray assay revealed that some of these isolates contained additional previously undetected
ent
genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different
ent
genes in one
S. aureus
strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among
S. aureus
strains, and for the analysis of microbial pathogens in general.
Evaluation of “Helicobacter heilmannii” Subtypes in the Gastric Mucosas of Cats and Dogs
Authors: Priestnall Simon L. , Wiinberg Bo , Spohr Anette , Neuhaus Britta , Kuffer Manuela , Wiedmann Martin , Simpson Kenneth W.
Published: May 2004

Infection with candidatus “Helicobacter heilmannii” is associated with gastritis and mucosa-associated lymphoid tissue lymphoma in people. Infection with “H. heilmannii” type 1 predominates (80%) and is thought to be acquired from dogs, cats, or pigs. We further examined the zoonotic potential of dogs and cats by amplifying gastric DNA from cats (n= 45) and dogs (n= 10) with primers against “H. heilmannii”ureBand 16S rRNA genes and sequencing the products. Fluorescence in situ hybridization (FISH) with eubacterial and “H. heilmannii”-specific probes was employed to directly visualize “H. heilmannii” types and their intragastric distribution.ureBsequences of “H. heilmannii” amplicons clustered with human and feline isolates of “H. heilmannii” and were distinct from the “H. heilmannii”-like organisms (HHLO)H. felis,H. salomonis, andH. bizzozeronii. 16S ribosomal DNA sequences in 20 “H. heilmannii”-infected cats and dogs were distinct from “H. heilmannii” type 1 and “H. suis” and clustered with “H. heilmannii” types 2 and 4. FISH confirmed the presence of “H. heilmannii” types 2 and 4 in dogs but failed to definitively characterize the “H. heilmannii” types present in cats. In infected dogs, “H. heilmannii” inhabited the gastric mucus and glands, and in dogs coinfected with other HHLO it shared the same gastric niche. The results indicate that dogs and cats are predominantly colonized by “H. heilmannii” bacteria that are distinct from type 1 and from “H. suis.” As “H. heilmannii” type 1 predominates in people, the zoonotic risk posed by dogs and cats is likely small.
Hyperinvasive Neonatal Group B Streptococcus Has Arisen from a Bovine Ancestor
Authors: Bisharat Naiel , Crook Derrick W. , Leigh James , Harding Rosalind M. , Ward Phil N. , Coffey Tracey J. , Maiden Martin C. , Peto Tim , Jones Nicola
Published: May 2004

The genetic relatedness and evolutionary relationships between group B streptococcus (GBS) isolates from humans and those from bovines were investigated by phylogenetic analysis of multilocus sequence typing data. The collection of isolates consisted of 111 GBS isolates from cows with mastitis and a diverse global collection of GBS isolates from patients with invasive disease (
n
= 83) and carriers (
n
= 69). Cluster analysis showed that the majority of the bovine isolates (93%) grouped into one phylogenetic cluster. The human isolates showed greater diversity and clustered separately from the bovine population. However, the homogeneous human sequence type 17 (ST-17) complex, known to be significantly associated with invasive neonatal disease, was the only human lineage found to be clustered within the bovine population and was distinct from all the other human lineages. Split decomposition analysis revealed that the human isolate ST-17 complex, the major hyperinvasive neonatal clone, has recently arisen from a bovine lineage.
Correlation between Viral Loads of Cytomegalovirus in Blood and Bronchoalveolar Lavage Specimens from Lung Transplant Recipients Determined by Histology and Immunohistochemistry
Authors: Chemaly Roy F. , Yen-Lieberman Belinda , Castilla Elias A. , Reilly Amy , Arrigain Susana , Farver Carol , Avery Robin K. , Gordon Steven M. , Procop Gary W.
Published: May 2004

Cytomegalovirus (CMV) is an important pathogen in lung transplant recipients. Early detection of CMV end-organ disease should help with treatment management. We determined the CMV viral load by hybrid capture in bronchoalveolar lavage (BAL) fluid samples from patients who had undergone lung transplantation. For 39 of these samples (from 25 patients), corresponding transbronchial biopsy samples were available for CMV immunohistochemistry (IHC). The CMV IHC results were interpreted and categorized as positive or negative, and the positive results were subcategorized as typical if cells with both significant nuclear enlargement or Cowdry A-type inclusions and positive staining were present or as atypical if definitive nuclear staining was seen but significant nuclear enlargement was not. Diagnostic CMV viral inclusions were reported in the anatomic diagnosis, based on hematoxylin-eosin staining alone, for three (8%) of the biopsy samples. CMV was detected by IHC in 13 (33%) samples (5 typical, 8 atypical). The median CMV viral load in BAL samples was 0 copies/ml for BAL samples from patients with IHC-negative biopsy samples; 47,678 copies/ml for BAL samples from patients with biopsy samples with positive, atypical staining; and 1,548,827 copies/ml for BAL samples from patients with biopsy samples with positive, typical staining (
P
< 0.001). Compared to routine pathology of biopsy samples, the use of IHC increased the diagnostic yield of CMV. Also, the CMV viral load in BAL fluid samples increased along with immunoreactivity from negative to positive, atypical staining to positive, typical staining. The CMV viral load determined with the end-organ sample, the BAL fluid sample, was higher than the corresponding viral load determined with blood. Both IHC and determination of the CMV viral load in BAL samples may be useful for the detection of individuals at risk for the development of fulminant invasive CMV disease.
Systematic 16S rRNA Gene Sequencing of Atypical Clinical Isolates Identified 27 New Bacterial Species Associated with Humans
Authors: Drancourt M. , Berger P. , Raoult D.
Published: May 2004

Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a ≥1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (≤10 cases reported in the literature) human pathogens. Eleven new species, “Actinobaculum massiliae,” “CandidatusActinobaculum timonae,”Paenibacillus sanguinis, “CandidatusBacteroides massiliae,”Chryseobacterium massiliae, “CandidatusChryseobacterium timonae,”Paenibacillus massiliensis, “CandidatusPeptostreptococcus massiliae,” “CandidatusPrevotella massiliensis,”Rhodobacter massiliensis, and “CandidatusVeillonella atypica” were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases.
Use of β-Lactamase Inhibitors in Disk Tests To Detect Plasmid-Mediated AmpC β-Lactamases
Authors: Black Jennifer A. , Thomson Kenneth S. , Pitout Johann D. D.
Published: May 2004

Seeking a simple disk test for detection of organisms producing plasmid-mediated AmpC β-lactamases, we evaluated the diagnostic utility of the β-lactamase inhibitors 48-1220 (Ro 48-1220) and LN-2-128. Using NCCLS disk methodology, inhibition zone diameters were determined for five β-lactam antibiotics tested alone and in combination with 20 μg of either 48-1220 or LN-2-128. Using an increase of ≥4 mm in zone diameter in the presence of an inhibitor as a positive test, cefotetan with LN-2-128 and 48-1220 was adequate for the detection of organisms producing plasmid-mediated AmpCs (specificity of 90% and sensitivity of 100%).
Human Papillomavirus Type 16 Viral Load Is Higher in Human Immunodeficiency Virus-Seropositive Women with High-Grade Squamous Intraepithelial Lesions Than in Those with Normal Cytology Smears
Authors: Lefevre Jonas , Hankins Catherine , Money Deborah , Rachlis Anita , Pourreaux Karina , Coutlée François
Published: May 2004

Human papillomavirus type 16 (HPV-16) viral load in cervicovaginal lavage samples collected from 66 human immunodeficiency virus-seropositive women was inversely correlated with blood CD4 count (
P
= 0.002). HPV-16 viral load was 81-fold higher in women with cervical smears suggestive of high-grade lesions (median, 4,425,883 copies/μg of DNA) than in women with normal smears (median, 54,576), controlling for age (
P
= 0.006).
Comparison of Chlorhexidine and Tincture of Iodine for Skin Antisepsis in Preparation for Blood Sample Collection
Authors: Barenfanger Joan , Drake Cheryl , Lawhorn Jerry , Verhulst Steven J.
Published: May 2004

Rates of contamination of blood cultures obtained when skin was prepared with iodine tincture versus chlorhexidine were compared. For iodine tincture, the contamination rate was 2.7%; for chlorhexidine, it was 3.1%. The 0.41% difference is not statistically significant. Chlorhexidine has comparable effectiveness and is safer, cheaper, and preferred by staff, so it is an alternative to iodine tincture.
Outbreak of
Burkholderia cepacia
Bacteremia in a Pediatric Hospital Due to Contamination of Lipid Emulsion Stoppers
Authors: Doit C. , Loukil C. , Simon A.-M. , Ferroni A. , Fontan J.-E. , Bonacorsi S. , Bidet P. , Jarlier V. , Aujard Y. , Beaufils F. , Bingen E.
Published: May 2004

We describe a 7-month outbreak of nosocomial
Burkholderia cepacia
bacteremia involving eight children in a pediatric hospital and the results of epidemiological investigations. A
B. cepacia
strain genotypically identical to the blood isolates was recovered from the upper surface of capped rubber stoppers of bottles of a commercial lipid emulsion used for parenteral nutrition.
CorynebacteriumSpecies Isolated from Bone and Joint Infections Identified by 16S rRNA Gene Sequence Analysis
Authors: Roux Véronique , Drancourt Michel , Stein Andreas , Riegel Philippe , Raoult Didier , La Scola Bernard
Published: May 2004

By the use of 16S rRNA gene sequence analysis we identified 28 of 31Corynebacteriumspp. isolated from bone and joint infections, including species never before isolated in such infections. Phenotypic analysis led to the correct identification of 8 of 31. 16S rRNA gene sequence analysis appears to be a good technique for identification of clinical strains ofCorynebacteriumspp.
Further Evaluation of a Rapid Diagnostic Test for Melioidosis in an Area of Endemicity
Authors: O'Brien Mathew , Freeman Kevin , Lum Gary , Cheng Allen C. , Jacups Susan P. , Currie Bart J.
Published: May 2004

Immunochromatographic test (ICT) kits for the rapid detection of immunoglobulin G (IgG) and IgM antibodies to
Burkholderia pseudomallei
were compared to the indirect hemagglutination (IHA) assay. In 138 culture-confirmed melioidosis cases, sensitivities were 80, 77, and 88% for IHA, ICT IgG, and ICT IgM, respectively. In a prospective study of 160 consecutive sera samples sent for melioidosis serology, respective specificities were 91, 90, and 69, positive predictive values were 41, 32, and 18, and negative predictive values were 99, 98, and 100%. ICT IgM kits are unreliable for diagnosis of melioidosis, but ICT IgG kits may be useful for diagnosing travelers presenting with possible melioidosis who return from regions where melioidosis is endemic.
Collaborative Study of the NCCLS and Flow Cytometry Methods for Antifungal Susceptibility Testing of
Candida albicans
Authors: Chaturvedi Vishnu , Ramani Rama , Pfaller Michael A.
Published: May 2004

One hundred clinical isolates of
Candida albicans
were tested for amphotericin B and fluconazole susceptibilities by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution test at center 1 (C1). The same isolates were tested blinded at center 2 (C2) by NCCLS and flow cytometry (FC) methods. The agreement between NCCLS and FC methods ranged from 96 to 99%.
Collaborative Study of Antibiotic Medium 3 and Flow Cytometry for Identification of Amphotericin B-Resistant
Candida
Isolates
Authors: Chaturvedi Vishnu , Ramani Rama , Rex John H.
Published: May 2004

Center 1 used the National Committee for Clinical Laboratory Standards M27-A2 method and antibiotic medium 3 (AM3) test to determine amphotericin B resistance in 5 of 30
Candida
isolates. These isolates were tested at center 2 by AM3 test and flow cytometry (FC). The agreements (C1-C2) were 90% for AM3 test and FC and 73% for the AM3 tests.
Validated 5′ Nuclease PCR Assay for Rapid Identification of the Genus
Brucella
Authors: Bogdanovich T. , Skurnik M. , Lübeck P. S. , Ahrens P. , Hoorfar J.
Published: May 2004

A real-time, genus-specific 5′ nuclease PCR assay for amplification of a 322-bp fragment of the
per
gene was developed for rapid (<2 h) identification of
Brucella
spp. from agar plates. The assay, including an internal amplification control (116 bp), identified
Brucella
strains (
n
= 23) and did not detect non-
Brucella
strains (
n
= 174), indicating its usefulness, particularly for laboratories with stringent quality assurance programs.
Use of the Vitek-1 and Vitek-2 Systems for Detection of Constitutive and Inducible Macrolide Resistance in Group B Streptococci
Authors: Tang Patrick , Ng Peter , Lum Ming , Skulnick Martin , Small Glen W. , Low Donald E. , Sarabia Alicia , Mazzulli Tony , Wong Katherine , Simor Andrew E. , Willey Barbara M.
Published: May 2004

A prospective study of erythromycin and clindamycin resistance was performed with 304 consecutive group B streptococci (GBS) isolates. According to two automated susceptibility testing systems, Vitek-1 and Vitek-2, and double-disk agar diffusion, 79.9% were susceptible to both erythromycin and clindamycin. However, for macrolide-lincosamide-streptogramin B-inducible isolates, the accuracies of the Vitek-1 and Vitek-2 systems were 5.6 and 94.4%. In light of these results, we recommend that GBS be routinely tested using the double-disk diffusion method.
Molecular Evidence of
Anaplasma phagocytophilum
in
Ixodes ricinus
Ticks and Wild Animals in Austria
Authors: Polin Helene , Hufnagl Peter , Haunschmid Reinhard , Gruber Florian , Ladurner Gunther
Published: May 2004

Real-time PCR analysis of a
groESL
heat shock operon segment showed the presence of two genetic lineages of
Anaplasma phagocytophilum
in
Ixodes ricinus
ticks as well as one variant in wild red and roe deer, the latter supposedly representing the natural reservoir of one variant of
A. phagocytophilum.
AFMP2
Encodes a Novel Immunogenic Protein of the Antigenic Mannoprotein Superfamily in
Aspergillus fumigatus
Authors: Chong Ken T. K. , Woo Patrick C. Y. , Lau Susanna K. P. , Huang Yi , Yuen Kwok-yung
Published: May 2004

We cloned the
Aspergillus fumigatus
mannoprotein 2 (
AFMP2
) gene, which encodes a novel immunogenic protein (Afmp2p) of the antigenic mannoprotein superfamily, in
A. fumigatus
. Sequence analysis revealed that Afmp2p has 510 amino acid residues, with a predicted molecular mass of 51.5 kDa. Afmp2p has a putative N-terminal signal peptide, a putative C-terminal glycosylphosphatidylinositol membrane attachment signal sequence, and an upstream GAA cleavage site commonly used for cytoplasmic membrane attachment and implicated in fungal cell wall assembly. Upstream of the GAA cleavage site, Afmp2p contains a 302-amino-acid serine- and threonine-rich region as a site for potential O-glycosylation. Within this serine- and threonine-rich region, 13 repeats of ETSTPCE(T)

n

were observed. Western blot analysis of Afmp2p in
A. fumigatus
fungal cell lysate and culture supernatant and immunogold staining and electron microscopy showed that Afmp2p is predominantly secreted into the culture supernatant, whereas only minimal amounts can be detected in the cell lysate and cell wall. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to
A. fumigatus
develop a specific antibody response against recombinant Afmp2p. The abundance of Afmp2p in secreted form, its minimal cross-reactivity with Afmp1p, and the presence of an antibody response against Afmp2p in patients with
A. fumigatus
infections suggest that Afmp2p is a good candidate for complementing Afmp1p in serodiagnosis of
A. fumigatus
infections.
Use of a Panfungal PCR Assay for Detection of Fungal Pathogens in a Commercial Blood Culture System
Authors: Iwen Peter C. , Freifeld Alison G. , Bruening Tricia A. , Hinrichs Steven H.
Published: May 2004

A panfungal PCR assay was used to evaluate the ability of the ESP blood culture system to detect fungemia. The results showed that the ESP system is reliable for the detection of fungi and showed the applicability of using a molecular-based assay as a potential rapid and reliable method for the identification of fungi.
Culture-Independent Species Typing of Neotropical
Leishmania
for Clinical Validation of a PCR-Based Assay Targeting Heat Shock Protein 70 Genes
Authors: Garcia Lineth , Kindt Ann , Bermudez Hernan , Llanos-Cuentas Alejandro , De Doncker Simonne , Arevalo Jorge , Quispe Tintaya Kelly Wilber , Dujardin Jean-Claude
Published: May 2004

PCR-restriction fragment length polymorphism analysis of heat shock protein 70 genes discriminates most neotropical
Leishmania
species, as well as
Trypanosoma cruzi.
The assay, combined with capillary electrophoresis in a microchip device, may be applied directly on clinical samples with a high sensitivity, hence supporting clinical and epidemiological monitoring of leishmaniasis.
Use of the
omp50
Gene for Identification of
Campylobacter
Species by PCR
Authors: Dedieu Luc , Pagès Jean-Marie , Bolla Jean-Michel
Published: May 2004

We studied the prevalence of the
omp50
gene and the Omp50 protein in
Campylobacter
strains. Immunodetection assays and DNA-DNA hybridizations showed that most
C. coli
strains tested were negative and most
C. jejuni
and
C. lari
strains tested were positive. A PCR assay was developed, using the
omp50
gene as a species-specific target. We propose a combination of a hippurate test and an
omp50
assay to perform identification of
Campylobacter
species.
Detection of Specific Antibodies to Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein for Serodiagnosis of SARS Coronavirus Pneumonia
Authors: Woo Patrick C. Y. , Lau Susanna K. P. , Wong Beatrice H. L. , Tsoi Hoi-wah , Fung Ami M. Y. , Chan Kwok-hung , Tam Victoria K. P. , Peiris J. S. Malik , Yuen Kwok-yung
Published: May 2004

We report the evaluation of recombinant severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) nucleocapsid protein enzyme-linked immunosorbent assay (ELISA)-based antibody tests for serodiagnosis of SARS-CoV pneumonia and compare the sensitivities and specificities of this ELISA for detection of immunoglobulin G (IgG), IgM, IgA, and their combinations with serum samples from 149 healthy blood donors who donated blood 3 years ago as controls and 106 SARS-CoV pneumonia patients in Hong Kong. The specificities of the ELISA for IgG, IgM, and IgA detection were 95.3, 96.6, and 96.6%, respectively, with corresponding sensitivities of 94.3, 59.4, and 60.4%, respectively. The present ELISA appears to be a sensitive test for serodiagnosis of SARS-CoV pneumonia, is much more economical and less labor-intensive than the indirect immunofluorescence assay, and does not require cultivation of SARS-CoV.
First Serologic Evidence of Human Spotted Fever Group Rickettsiosis in Korea
Authors: Jang Won-Jong , Kim Jong-Hyun , Choi Yeon-Joo , Jung Kwang-Don , Kim Yang-Gyu , Lee Seung-Hyun , Choi Myung-Sik , Kim Ik-Sang , Walker David H. , Park Kyung-Hee
Published: May 2004

To investigate the prevalence of spotted fever group rickettsioses in Korea, a serosurvey of Japanese spotted fever rickettsiosis in patients with acute febrile illness was conducted with an indirect immunofluorescence assay. Overall, 19.88% of the patients were found to have polyvalent antibody against
Rickettsia japonica
. This study is the first documentation of spotted fever group rickettsiosis in Korea.
Brain Abscess Due to
Gemella haemolysans
Authors: Lee Mi Ra , Lee Sang-Oh , Kim Sue-Yun , Yang Sun Mee , Seo Yiel-Hae , Cho Yong Kyun
Published: May 2004

We present a case of brain abscess due to
Gemella haemolysans
and
Bacteroides
species in a 60-year-old-immunocompetent man who underwent dental procedures. The patient completely recovered following intravenous therapy with ampicillin and metronidazole for 6 weeks.
Clinical Case of Endocarditis due toTrichosporon inkinand Antifungal Susceptibility Profile of the Organism
Authors: Ramos Jose M. , Cuenca-Estrella Manuel , Gutierrez Felix , Elia Matilde , Rodriguez-Tudela Juan L.
Published: May 2004

A fatal case ofTrichosporon inkinprosthetic endocarditis is reported. The isolation sites and susceptibility profiles of 10 other isolates are also reviewed. Four strains were recovered from cutaneous or subcutaneous samples, four were recovered from urine, one was recovered from peritoneal liquid, and one was recovered from bone. Voriconazole and amphotericin B had the most potent activities in vitro against the isolates, with MIC geometric means of 0.11 and 0.30 μg/ml, respectively.
Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl−
Authors: Lang Karl S. , Myssina Swetlana , Lang Philipp A. , Tanneur Valerie , Kempe Daniela S. , Mack Andreas F. , Huber Stephan M. , Wieder Thomas , Lang Florian , Duranton Christophe
Published: May 2004

Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca2+entry through stimulation of cation channels. Both ceramide and Ca2+activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 ± 4% ( n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl−with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl−and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl−is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide.
Evidence for epidermal growth factor receptor as negative-feedback control in aldosterone-induced Na+ reabsorption
Authors: Grossmann Claudia , Freudinger Ruth , Mildenberger Sigrid , Krug Alexander W. , Gekle Michael
Published: May 2004

Aldosterone enhances Na+ reabsorption via epithelial Na+ channels (ENaC). Aldosterone also stimulates the protein kinase ERK1/2- and the epidermal growth factor (EGF) receptor (EGFR)-signaling pathway. Yet EGF and ERK1/2 are known inhibitors of ENaC-mediated Na+ reabsorption. In the present study, using the well-established Madin-Darby canine kidney C7 cell line, we tested the hypothesis that EGFR represents a negative-feedback control for chronic aldosterone-induced Na+ reabsorption [amiloride-inhibitable short-circuit current ( Isc)]. Mineralocorticoid receptor expression was confirmed by RT-PCR and Western blot analysis. Aldosterone enhanced ERK1/2 phosphorylation in an EGFR-dependent way. Furthermore, aldosterone stimulated EGFR expression. Aldosterone (10 nmol/l) induced a small transient increase in Isc under control conditions. Inhibition of ERK1/2 phosphorylation with U-0126 (10 μmol/l) stimulated Isc, indicating constitutive ENaC inhibition. Aldosterone exerted a significantly larger effect in the presence of U-0126 than without U-0126. EGF (10 μg/l) inhibited Isc, whereas inhibition of EGFR kinase by tyrphostin AG-1478 (100 nmol/l) enhanced Isc. Aldosterone was more effective in the presence of AG-1478 than without AG-1478. In summary, we propose that the EGFR-signaling cascade can serve as a negative-feedback control to limit the effect of aldosterone-induced Na+ reabsorption.