bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnostic Approach for Differentiating Infected from Vaccinated Poultry on the Basis of Antibodies to NS1, the Nonstructural Protein of Influenza A Virus
Authors: Tumpey Terrence M. , Alvarez Rene , Swayne David E. , Suarez David L.
Published: February 2005

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in
Escherichia coli
or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.
Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses
Authors: Bronzoni Roberta Vieira de Morais , Baleotti Flávia Graciela , Ribeiro Nogueira Rita Maria , Nunes Márcio , Moraes Figueiredo Luiz Tadeu
Published: February 2005

A new approach was developed for the rapid detection and identification of Brazilian alphaviruses and flaviviruses. The methodology involves the genus-specific detection of
Alphavirus
and
Flavivirus
by a duplex reverse transcription-PCR (D-RT-PCR), followed by multiplex nested PCR (M-N-PCR) or nested PCR (N-PCR) assays for species-specific identification. By this protocol, 25 arboviruses were specifically detected and identified. Detection levels between 10
1.3
and 10
3.5
50% tissue culture infective doses (TCID
50
)/ml of
Flavivirus
and
Alphavirus
strains were achieved by D-RT-PCR, and levels of <1 TCID
50
/ml were achieved by M-N-PCR assays. To assess the suitability and clinical application of this methodology, a total of 101 human or animal stored samples were analyzed. Results obtained suggest that this technique could be applied as a rapid diagnostic tool in clinical samples in which arbovirus infection is suspected and differential diagnosis is required, avoiding the need to test specimens by separate PCR methods.
Characterization of Virus Isolates by Particle-Associated Nucleic Acid PCR
Authors: Stang Alexander , Korn Klaus , Wildner Oliver , Überla Klaus
Published: February 2005

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3′ end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.
Indirect Enzyme-Linked Immunosorbent Assay for Detection ofBrucella melitensis-Specific Antibodies in Goat Milk
Authors: Funk N. D. , Tabatabai L. B. , Elzer P. H. , Hagius S. D. , Martin B. M. , Hoffman L. J.
Published: February 2005

Brucella melitensisis the cause of brucellosis in sheep and goats, which often results in abortion. Few cases ofB. melitensisinfection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection ofB. melitensis-specific antibodies in goat milk.Brucellasalt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection ofB. melitensis-specific antibodies in goat milk.
Mutations within the Hepatitis C Virus Genotype 1b E2-PePHD Domain Do Not Correlate with Treatment Outcome
Authors: Gaudy Catherine , Lambelé Marie , Moreau Alain , Veillon Pascal , Lunel Francoise , Goudeau Alain
Published: February 2005

The hepatitis C virus (HCV) envelope protein 2 (E2) interacts in vitro with the interferon alpha (IFN-α)-inducible double-stranded RNA-activated protein kinase, suggesting a possible mechanism by which HCV may evade the antiviral effects of IFN-α. Variability in the part of the HCV E2 gene encoding the carboxy-terminal part of the protein, which includes the interaction domain (E2-PePHD), was explored in 25 patients infected with HCV genotype 1b and receiving IFN-α therapy. PCR products were generated and sequenced for 15 patients with a sustained response and for 10 patients with no virological response after treatment with IFN-α and ribavirin. PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment, after 2 months in responders, and after 6 months in nonresponders. Quasispecies analysis of the pretreatment PePHD region was performed for isolates from patients displaying amino acid substitutions in this domain on direct sequencing. The E2-PePHD sequence was highly conserved in both resistant and susceptible genotype 1b strains and was identical to the prototype HCV type J sequence. No significant emergence of PePHD mutants during therapy was observed in our clonal analysis, and sporadic mutations and treatment outcomes were not found to be correlated. The PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in HCV type 1b-infected patients.
Detection and Characterization of Diarrheagenic
Escherichia coli
from Young Children in Hanoi, Vietnam
Authors: Nguyen Trung Vu , Le Van Phung , Le Huy Chinh , Gia Khanh Nguyen , Weintraub Andrej
Published: February 2005

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries.
Escherichia coli
is an emerging agent among pathogens that cause diarrhea. The development of a highly applicable technique for the detection of different categories of diarrheagenic
E. coli
is important. We have used multiplex PCR by combining eight primer pairs specific for enteroaggregative
E. coli
(EAEC), enteroinvasive
E. coli
(EIEC), enterohemorrhagic
E. coli
, enteropathogenic
E. coli
(EPEC), and enterotoxigenic
E. coli
(ETEC). This facilitates the identification of five different categories of diarrheagenic
E. coli
from stool samples in a single reaction simultaneously. The prevalences of diarrheagenic
E. coli
were 22.5 and 12% in the diarrhea group and the control group, respectively. Among 587 fecal samples from Vietnamese children under 5 years of age with diarrhea, this technique identified 132 diarrheagenic
E. coli
strains. This included 68 samples (11.6%) with EAEC, 12 samples (2.0%) with EIEC, 39 samples (6.6%) with EPEC, and 13 samples (2.2%) with ETEC. Among the 249 age-matched controls, 30 samples were positive for diarrheagenic
E. coli
. The distribution was 18 samples (7.2%) with EAEC, 11 samples (4.4%) with EPEC, and 1 sample (0.4%) with ETEC.
Use of Amplified Fragment Length Polymorphism To Identify and Type Brucella Isolates of Medical and Veterinary Interest
Authors: Whatmore Adrian M. , Murphy Terry J. , Shankster Stephen , Young Emma , Cutler Sally J. , Macmillan Alastair P.
Published: February 2005

Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of
Brucella
isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of
Brucella
isolates. The technique could readily differentiate
Brucella
spp. from all
Ochrobactrum
spp. representing the group of organisms most closely related to
Brucella
spp. Application of AFLP highlighted the genetic homogeneity of
Brucella
. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including
Brucella abortus
,
B. melitensis
,
B. ovis
,
B. neotomae
, marine mammal isolates (no species name),
B. canis
, and
B. suis
, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only
B. suis
and
B. canis
isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to
Brucella
and enhance the value of this approach.
Recurrent Colonization of Successively Implanted Tracheoesophageal Vocal Prostheses by a Member of theFusarium solaniSpecies Complex
Authors: Honraet K. , De Vos M. M. , Summerbell R. C. , van Kempen I. , De Saeger S. , Vermeersch H. , Van Peteghem C. , Nelis H. J.
Published: February 2005

Tracheoesophageal vocal prostheses (TVP) in laryngectomized patients commonly deteriorate due to overgrowth by yeasts, particularlyCandidaspecies. We describe the first case of colonization of such devices by a member of theFusarium solanispecies complex in a patient with a history of glottal carcinoma. Three isolates, from three prostheses, were found morphologically consistent with the traditional picture ofF. solani.Ribosomal sequence analysis showed that the isolates belonged to a distinct, as yet apparently unnamed phylogenetic species within theF. solanispecies complex. This species, one of two distinct genetic types (genotype 2) traditionally considered part of the plant-pathogenic subtaxonFusarium solanif. sp.radicicola, has not previously been identified as an agent of human or animal disease, although it is closely related to a known etiologic agent of mycetoma, anAcremonium-like species recently renamedFusarium falciforme. Sequence and multisatellite M13 polymorphism analysis revealed no distinctions among the case isolates. Production of cyclosporine was detected for all three case isolates.
Self-Assembly of the Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses
Authors: Han M. G. , Wang Q. , Smiley J. R. , Chang K. O. , Saif L. J.
Published: February 2005

None of the enteric caliciviruses except Po/Sapo/GIII/Cowden/80/US replicates in cell culture, which complicates efforts to develop control strategies or to study viral replication. To develop serological assays for bovine noroviruses (BoNVs) and to determine the cross-reactivity of BoNV with human noroviruses, we generated two recombinant baculoviruses, rCV186-OH and rJNCV, to express the capsid genes of Bo/CV186-OH/00/US (
Norovirus
genogroup III [GIII], genotype 2 [GIII/2]). rCV186-OH expressed the expected 57-kDa capsid protein, but rJNCV expressed a truncated capsid protein of 35 kDa. Sequence analysis of rJNCV identified a single nucleotide deletion in the P domain of the capsid gene, which introduced a stop codon at amino acid 323. The recombinant capsid protein produced by rCV186-OH but not that produced by rJNCV self-assembled into virus-like particles (VLPs) similar to native BoNV. An antibody-capture enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA (Ag-ELISA) detected serum antibody and antigen, respectively, from calves infected with Bo/CV186-OH/00/US but not antibodies or antigens to other enteric viruses. In other tests of the GIII/2 BoNV Ag-ELISA, no cross-reactivity was observed with VLPs from one GI and four GII human noroviruses and porcine sapovirus Cowden strain. Because, like human noroviruses, BoNVs do not grow in cell culture, the BoNV VLPs will be useful in the serological assays described for the detection of BoNV antibody and antigen. Consistent with the phylogenetic analysis of the capsid genes of bovine and human noroviruses (M. G. Han, J. R. Smiley, C. Thomas, and L. J. Saif, J. Clin. Microbiol. 42:5214-5224, 2004), the results suggest that GIII/2 BoNV does not share significant antigenic relationships with the five characterized human noroviruses tested.
Tyrosine Phosphorylation of CagA from Chinese
Helicobacter pylori
Isolates in AGS Gastric Epithelial Cells
Authors: Zhang Youli , Argent Richard H. , Letley Darren P. , Thomas Rachael J. , Atherton John C.
Published: February 2005

Helicobacter pylori
strains possessing the
cag
pathogenicity island (PaI) are associated with the development of gastroduodenal diseases, including gastric cancer.
cag
PaI products induce the secretion of interleukin-8 (IL-8) from epithelial cells and facilitate the translocation of CagA into the cell cytosol. In East Asia, where the incidence of gastric cancer is high, most strains possess the
cag
PaI. To date, however, no
cag
PaI phenotypic data have been provided for strains isolated in mainland China. Here we used 31 Chinese strains to determine the genotypic and phenotypic status of the
cag
PaI. All strains possessed
cagA
and
cagE
, and we observed a variation in the length of
cagA
variable regions. Nucleotide sequencing of the
cagA
variable region revealed that CagA was of two types, a short “Western” form with two tyrosine phosphorylation sites and a longer “East Asian” form with three tyrosine phosphorylation sites. Coculture of strains with AGS epithelial cells showed that strains could induce IL-8 secretion from the cells and that CagA with three phosphorylation sites became more phosphorylated than that with two and could induce significantly (
P
< 0.001) more cells to elongate. We hypothesize that the preponderance of the more active East Asian form of
cagA
may underlie the high rate of gastric cancer in China.
Six-Month Multicenter Study on Invasive Infections Due to
Streptococcus pyogenes
and
Streptococcus dysgalactiae
subsp.
equisimilis
in Argentina
Authors: Lopardo Horacio A. , Vidal Patricia , Sparo Monica , Jeric Paola , Centron Daniela , Facklam Richard R. , Paganini Hugo , Pagniez N. Gaston , Lovgren Marguerite , Beall Bernard
Published: February 2005

During a 6-month period, 95 invasive infections due to
Streptococcus pyogenes
and group C or group G
Streptococcus dysgalactiae
subsp.
equisimilis
were recorded from 40 centers of 16 cities in Argentina. We describe here epidemiologic data available for 55 and 19 patients, respectively, associated with invasive infections due to
S. pyogenes
and
S. dysgalactiae
subsp.
equisimilis
. The associated isolates and 58 additional pharyngeal isolates were genotyped and subjected to serologic and/or antibiotic susceptibility testing. Group A streptococcal
emm
type distribution and strain association with toxic shock appeared to differ somewhat from results found within the United States; however, serologic characterization and
sof
sequence typing suggested that
emm
types found in both countries are reflective of shared clonal types.
Identification of Genetic Evidence for Dobrava Virus Spillover in Rodents by Nested Reverse Transcription (RT)-PCR and TaqMan RT-PCR
Authors: Weidmann Manfred , Schmidt P. , Vackova M. , Krivanec K. , Munclinger P. , Hufert F. T.
Published: February 2005

A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in
Apodemus sylvaticus
and
Mus musculus
rodents. The identity of
A. sylvaticus
was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from
Apodemus flavicollis
to other rodents.
Sensitivity and Specificity of the ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Detection of HIV-1 Drug Resistance Mutations by Use of an ABI PRISM 3100 Genetic Analyzer
Authors: Eshleman Susan H. , Crutcher Gillian , Petrauskene Olga , Kunstman Kevin , Cunningham Shawn P. , Trevino Christina , Davis Cheryl , Kennedy John , Fairman Jeff , Foley Brian , Kop JoAnn
Published: February 2005

The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.
In Vitro Method To Study Antifungal Perfusion in
Candida
Biofilms
Authors: Samaranayake Y. H. , Ye J. , Yau J. Y. Y. , Cheung B. P. K. , Samaranayake L. P.
Published: February 2005

Antimycotic perfusion through
Candida
biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different
Candida
species (i.e.,
Candida albicans
,
Candida parapsilosis
, and
Candida krusei
) that were developed on microporous filters. Scanning electron microscopy revealed that
C. albicans
formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with
C. parapsilosis
and
C. krusei
, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by
C. albicans
biofilms in comparison to
C. parapsilosis
and
C. krusei
. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in
Candida
is dependent both on the antimycotic and the
Candida
species in question, and in clinical terms, these phenomena could contribute to the failure of
Candida
biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.
Outbreak of Infection Caused by
Enterobacter cloacae
Producing the Novel VEB-3 Beta-Lactamase in China
Authors: Jiang Xiaofei , Ni Yuxing , Jiang Yanqun , Yuan Feiyi , Han Lizhong , Li Ming , Liu Hong , Yang Li , Lu Yuan
Published: February 2005

Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive
Enterobacter cloacae
isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related
E. cloacae
isolates possessed a novel
bla
VEB
-type beta-lactamase,
bla
VEB-3
.
Bla
VEB-3
was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the
bla
VEB-3
and the
bla
CTX-M-3
-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.
Sequence Variation in the
porB
Gene from B:P1.4 Meningococci Causing New Zealand's Epidemic
Authors: Dyet Kristin H. , Martin Diana R.
Published: February 2005

Since mid-1991, New Zealand has experienced an epidemic of meningococcal disease. The epidemic has been caused by serogroup B meningococci expressing PorA type P1.7-2,4, belonging to the ST-41/ST-44 complex, lineage III. Most B:P1.7-2,4 meningococci express type 4 PorB (87.0%), although case isolates with
porB
other than type 4 have been identified throughout the duration of the epidemic. To assess the genetic relatedness of case isolates with an alternative
porB
gene, multilocus restriction typing validated against multilocus sequence typing was used. This determined that B:P1.7-2,4 meningococci with a
porB
gene that was other than type 4 had the same clonal origin. It was concluded that strains with alternative
porB
genes had diverged from the original type 4
porB
. Variation in
porB
was also shown to be associated with the uptake of DNA encoding one or two of the PorB variable regions leading to mosaic
porB
. Point mutation rather than horizontal transfer and recombination was implicated as the mechanism of sequence variation in some strains. This work will serve as a reference point to determine if the administration of a strain-specific vaccine increases the level of
porB
divergence and variation already observed in New Zealand case isolates. It also complements the study undertaken of PorA stability which showed that variation in P1.7-2,4 PorA was almost exclusively due to deletions in the P1.4 epitope of the epidemic strain.
Molecular Analysis of Microbial Diversity in Advanced Caries
Authors: Chhour Kim-Ly , Nadkarni Mangala A. , Byun Roy , Martin F. Elizabeth , Jacques Nicholas A. , Hunter Neil
Published: February 2005

Real-time PCR analysis of the total bacterial load in advanced carious lesions has shown that the total load exceeds the number of cultivable bacteria. This suggests that an unresolved complexity exists in bacteria associated with advanced caries. In this report, the profile of the microflora of carious dentine was explored by using DNA extracted from 10 lesions selected on the basis of comparable total microbial load and on the relative abundance of
Prevotella
spp. Using universal primers for the 16S rRNA gene, PCR amplicons were cloned, and approximately 100 transformants were processed for each lesion. Phylogenetic analysis of 942 edited sequences demonstrated the presence of 75 species or phylotypes in the 10 carious lesions. Up to 31 taxa were represented in each sample. A diverse array of lactobacilli were found to comprise 50% of the species, with prevotellae also abundant, comprising 15% of the species. Other taxa present in a number of lesions or occurring with high abundance included
Selenomonas
spp.,
Dialister
spp.,
Fusobacterium nucleatum
,
Eubacterium
spp., members of the
Lachnospiraceae
family,
Olsenella
spp.,
Bifidobacterium
spp.,
Propionibacterium
sp., and
Pseudoramibacter alactolyticus
. The mechanisms by which such diverse patterns of bacteria extend carious lesions, including the aspect of infection of the vital dental pulp, remain unclear.
Long-Term Molecular Analysis of Tuberculosis Strains in Alabama, a State Characterized by a Largely Indigenous, Low-Risk Population
Authors: Kempf Mirjam-Colette , Dunlap Nancy E. , Lok Kerry H. , Benjamin William H. , Keenan Nancy B. , Kimerling Michael E.
Published: February 2005

With a tuberculosis case detection rate of 5.9 per 100,000 population in 2001, Alabama ranked twelfth highest in the United States. However, cases among foreign-born and human immunodeficiency virus-infected individuals remain low in Alabama. To understand the endemic statewide disease pattern, tuberculosis strains were studied for clustering in a long-term population-based study from January 1994 to May 2000. IS6
110
restriction fragment length polymorphism analysis was performed for 1,834 strains. Spoligotyping was used as a secondary typing method for the 37% of isolates displaying a restriction fragment length polymorphism pattern with <6 IS
6110
copies. A total of 721 (41%) patients provided isolates that composed 114 clusters, each containing isolates from 2 to 136 patients, suggesting that recent transmission accounted for 35% of tuberculosis cases. Demographic, behavioral, and clinical characteristics of patients with clustered versus nonclustered isolates stratified by low-copy-number strains (<6 IS
6110
copies) versus high-copy-number strains (≥6 IS
6110
copies) were evaluated. Younger age, black race, a history of alcohol abuse, and homelessness were predictors of clustering of low-copy-number, strains and younger age, urban residency, alcohol abuse, homelessness, noninjection drug use, and a history of incarceration and/or cavitary disease were predictors of clustering of high-copy-number strains. By identifying local characteristics of tuberculosis clustering through molecular fingerprinting, control programs can distribute their limited resources to impact the transmission of tuberculosis in high-risk populations and evaluate strain distribution across geographical areas.
Novel Replicon-Based Reporter Gene Assay for Detection of Rubella Virus in Clinical Specimens
Authors: Tzeng Wen-Pin , Zhou Yumei , Icenogle Joseph , Frey Teryl K.
Published: February 2005

Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination.
Endocarditis Due to a New Rod-Shaped
Neisseria
sp
Authors: Michaux-Charachon Sylvie , Lavigne Jean-Philippe , Le Flèche Anne , Bouziges Nicole , Sotto Albert , Grimont Patrick A. D.
Published: February 2005

We report the first case of pacemaker endocarditis due to a new rod-shaped
Neisseria
sp. isolated from blood culture. On the basis of
rrs
sequencing, the isolate was found to be most closely related to an uncultured organism from human subgingival plaque and was identified as
Neisseria
sp. group AK105. A cure was achieved after a combination of surgical and antibiotic treatment. Oral flora-induced pacemaker endocarditis is a rare condition that reinforces the need for good oral hygiene as an important preventive measure.
Reliability of the E-Test Method for Detection of Colistin Resistance in Clinical Isolates of
Acinetobacter baumannii
Authors: Arroyo L. A. , García-Curiel A. , Pachón-Ibañez M. E. , Llanos A. C. , Ruiz M. , Pachón J. , Aznar J.
Published: February 2005

We compared the E-test to the broth microdilution method for testing the susceptibility of 115 clinical isolates of
Acinetobacter baumannii
to colistin. Twenty-two (19.1%) strains were resistant to colistin and 93 (80.8%) strains were susceptible according to the reference broth microdilution method. A categorical agreement of 98.2% was found, with only two (1.7%) very major errors. Agreement within 1 twofold dilution between the E-test and the broth microdilution was 16.5%. Complete agreement was found for the strains for which MICs fell within the range of 0.25 to 1 μg of colistin/ml. However, there was poor concordance, particularly in extreme dilutions with higher MICs by the E-test method.
Detection of
Aspergillus fumigatus
and a Mutation That Confers Reduced Susceptibility to Itraconazole and Posaconazole by Real-Time PCR and Pyrosequencing
Authors: Trama Jason P. , Mordechai Eli , Adelson Martin E.
Published: February 2005

A real-time PCR and pyrosequencing method was developed to detect
Aspergillus fumigatus
in whole blood by amplifying the
cyp51A
gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole.
Evaluation of Amplified Fragment Length Polymorphism for Differentiation of Avian Mycoplasma Species
Authors: Hong Yang , García Maricarmen , Levisohn Sharon , Lysnyansky Inna , Leiting Victoria , Savelkoul P. H. M. , Kleven S. H.
Published: February 2005

Amplified fragment length polymorphism (AFLP) was used for typing avian mycoplasma species. Forty-four avian mycoplasma strains were successfully typed into eight distinct groups, with each representing a different species. Homology of AFLP patterns of 35% or less was used as a cutoff value to differentiate avian mycoplasma strains into different species.
Lot Quality Assurance Sampling of Sputum Acid-Fast Bacillus Smears for Assessing Sputum Smear Microscopy Centers
Authors: Selvakumar N. , Murthy B. N. , Prabhakaran E. , Sivagamasundari S. , Vasanthan Samuel , Perumal M. , Govindaraju R. , Chauhan L. S. , Wares Fraser , Santha T. , Narayanan P. R.
Published: February 2005

Assessment of 12 microscopy centers in a tuberculosis unit by blinded checking of eight sputum smears selected by using a lot quality assurance sampling (LQAS) method and by unblinded checking of all positive and five negative slides, among the slides examined in a month in a microscopy centre, revealed that the LQAS method can be implemented in the field to monitor the performance of acid-fast bacillus microscopy centers in national tuberculosis control programs.
Ecoepidemiology of
Laribacter hongkongensis
, a Novel Bacterium Associated with Gastroenteritis
Authors: Teng Jade L. L. , Woo Patrick C. Y. , Ma Shirley S. L. , Sit Thomas H. C. , Ng Lip-tet , Hui Wai-ting , Lau Susanna K. P. , Yuen Kwok-yung
Published: February 2005

In a territory-wide surveillance study,
Laribacter hongkongensis
was isolated solely from freshwater fish (60% of grass carps, 53% of bighead carps, and 25% of mud carps). Comparing the pulsed-field gel electrophoresis patterns of fish and patient isolates revealed that most patient isolates were clustered together, suggesting that some clones could be more virulent.
Quality Control Guidelines for Testing Gram-Negative Control Strains with Polymyxin B and Colistin (Polymyxin E) by Standardized Methods
Authors: Jones Ronald N. , Anderegg Tamara R. , Swenson Jana M.
Published: February 2005

An eight-laboratory study addressed the urgent need for quality control (QC) ranges for susceptibility determination when testing colistin (polymyxin E) and polymyxin B, two polycationic peptide antimicrobial agents, against multidrug-resistant gram-negative bacilli. For
Escherichia coli
ATCC 25922l, the QC ranges were as follows: for colistin, 0.25 to 1 μg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 μg/ml (13 to 19 mm). For
Pseudomonas aeruginosa
ATCC 27853, the QC ranges were as follows: for colistin, 0.25 to 2 μg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 μg/ml (14 to 18 mm). More than 97% of all reported QC results were within these proposed ranges.
Specimen Storage in Transport Medium and Detection of Group B Streptococci by Culture
Authors: Rosa-Fraile Manuel , Camacho-Muñoz Enrique , Rodríguez-Granger Javier , Liébana-Martos Carmen
Published: February 2005

Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21°C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4°C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21°C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4°C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration.
Prevalence and Serotype Distribution of Encapsulated
Haemophilus influenzae
Isolates from Patients with Lower Respiratory Tract Infections in Poland
Authors: Skoczyńska Anna , Lewandowska Magdalena , Klarowicz Anna , Hryniewicz Waleria
Published: February 2005

Among 1,272
H. influenzae
isolates obtained from patients with lower respiratory tract infections, 5.6% were found to be encapsulated. The most common were serotypes b (40.3%) and e (38.9%) followed by serotypes f (16.7%) and d (4.1%). All encapsulated isolates within a particular serotype showed high genetic homogeneity and did not produce β-lactamases.
Bacterial Genotype Affects the Manifestation and Persistence of BovineStaphylococcus aureusIntramammary Infection
Authors: Haveri M. , Taponen S. , Vuopio-Varkila J. , Salmenlinna S. , Pyörälä S.
Published: February 2005

Two-hundred seventeenStaphylococcus aureusisolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.
Detection of Severe Acute Respiratory Syndrome Coronavirus RNA in Plasma during the Course of Infection
Authors: Wang Wei-Kung , Fang Chi-Tai , Chen Hui-Ling , Yang Chao-Fu , Chen Yee-Chun , Chen Mei-Ling , Chen Shey-Ying , Yang Jyh-Yuan , Lin Jih-Hui , Yang Pan-Chyr , Chang Shan-Chwen
Published: February 2005

We examined severe acute respiratory syndrome-associated coronavirus (SARS-CoV) RNA in plasma of 32 patients (probable SARS cases) by a quantitative real-time reverse transcription-PCR assay and reported that the highest detection rate, 75%, was found between day 5 and day 7 of illness, followed by rates of 64, 50, and 38% found between day 8 and day 11, day 2 and day 4, and day 12 and day 16, respectively. Analysis of sequential SARS-CoV load in plasma from six cases revealed different patterns of viremia, with the peak between day 4 and day 8. Our findings of the high detection rate of SARS-CoV RNA in plasma before day 11, together with the relative convenience of collecting and handling plasma, suggest that plasma can be used for early diagnosis of SARS.
Molecular Characterization of Foot-and-Mouth Disease Virus Type C of Indian Origin
Authors: Nagendrakumar Singanallur Balasubramanian , Reddy Guddeti Srinivas , Chandran Dev , Thiagarajan Dorairajan , Rangarajan Pundi Narasimha , Srinivasan Villuppanoor Alwar
Published: February 2005

Comparison of nucleotide sequences of the partial 1D region of foot-and-mouth disease type C viruses of Indian origin with those of European, South American, and Southeast Asian viruses revealed that the Indian viruses form a distinct genotype. The vaccine strain C IND/51/79 belongs to this genotype and may be a prototype strain of this genotype.
Cutaneous Melioidosis in a Man Who Was Taken as a Prisoner of War by the Japanese during World War II
Authors: Ngauy Viseth , Lemeshev Yan , Sadkowski Lee , Crawford George
Published: February 2005

Melioidosis, an infection caused by the gram-negative bacillus
Burkholderia pseudomallei
, is endemic to Southeast Asia and Northern Australia. Human infection is acquired through contact with contaminated water via percutaneous inoculation. Clinical manifestations range from skin and soft tissue infection to pneumonia with sepsis. We report a case of a man who was taken as a prisoner of war by the Japanese during World War II who presented with a nonhealing ulcer on his right hand 62 years after the initial exposure.
Scedosporium apiospermumSoft Tissue Infection Successfully Treated with Voriconazole: Potential Pitfalls in the Transition from Intravenous to Oral Therapy
Authors: Schaenman Joanna M. , DiGiulio Daniel B. , Mirels Laurence F. , McClenny Nancy M. , Berry Gerald J. , Fothergill Annette W. , Rinaldi Michael G. , Montoya Jose G.
Published: February 2005

An immunocompromised patient with an invasive soft tissue infection due toScedosporium apiospermumwas successfully treated with voriconazole and surgical debridement. After transition from intravenous to oral therapy, successive adjustments of the oral dose were required to achieve complete resolution. For soft tissue infections due to molds characterized by thin, septate hyphae branching at acute angles, voriconazole should be considered a first-line antifungal agent. The potential usefulness of plasma voriconazole levels for guiding optimal therapy should be investigated.
Diagnostic and Therapeutic Pitfalls Associated with Primaquine-Tolerant
Plasmodium vivax
Authors: Spudick Jeanne M. , Garcia Lynne S. , Graham David M. , Haake David A.
Published: February 2005

We describe a U.S. Army Ranger returning from duty in Afghanistan and Iraq with life-threatening infection due to
Plasmodium vivax
. Morphological variants were observed in blood films prepared using samples collected by venipuncture. The patient's multiple relapses indicate infection with primaquine-tolerant
P. vivax
. Strategies for relapse prevention using primaquine are reviewed.
Tenosynovitis of the Wrist Due to ResistantMycobacterium tuberculosisin a Heart Transplant Patient
Authors: Le Meur Alexandre , Arvieux Cédric , Guggenbuhl Pascal , Cormier Michel , Jolivet-Gougeon Anne
Published: February 2005

Tubercular tenosynovitis is now rare, which can delay diagnosis of this disease. We report a case of tenosynovitis of the wrist in a heart transplant patient caused by an isoniazid- and streptomycin-resistantMycobacterium tuberculosisstrain. Despite immunosuppression therapy, which can lead to a smoldering evolution, molecular biology analysis of biopsies allowed a rapid diagnosis.
Nalidixic Acid-Resistant
Salmonella enterica
Serotype Typhi Presenting as a Primary Psoas Abscess: Case Report and Review of the Literature
Authors: Shakespeare William A. , Davie Daniel , Tonnerre Claude , Rubin Michael A. , Strong Michael , Petti Cathy A.
Published: February 2005

We report an unusual case of
Salmonella enterica
serotype Typhi presenting as a primary psoas abscess. The isolate tested susceptible to ciprofloxacin but resistant to nalidixic acid in vitro, a pattern associated with fluoroquinolone therapeutic failures. We review the literature for serovar Typhi psoas abscess in the absence of bacteremia and discuss the importance of identifying isolates with reduced susceptibility to fluoroquinolones.
Corynebacterium ulcerans
in an Immunocompromised Patient with Diphtheria and Her Dog
Authors: Lartigue Marie-Frédérique , Monnet Xavier , Le Flèche Anne , Grimont Patrick A. D. , Benet Jean-Jacques , Durrbach Antoine , Fabre Monique , Nordmann Patrice
Published: February 2005

Corynebacterium ulcerans
causes zoonotic infections, such as diphtheria and extrapharyngeal infections. We report here the first case of a diphtheria-like illness caused by
C. ulcerans
in France and transmitted likely by a dog to an immunocompromised woman.
Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts
Authors: Sun Jianjun , Ahmad Shoab , Chen Shanping , Tang Wenxue , Zhang Yanping , Chen Ping , Lin Xi
Published: February 2005

The importance of connexins (Cxs) in cochlear functions has been demonstrated by the finding that mutations in Cx genes cause a large proportion of sensorineural hearing loss cases. However, it is still unclear how Cxs contribute to the cochlear function. Recent data ( 33 ) obtained from Cx30 knockout mice showing that a reduction of Cx diversity in assembling gap junctions is sufficient to cause deafness suggest that functional interactions of different subtypes of Cxs may be essential in normal hearing. In this work we show that the two major forms of Cxs (Cx26 and Cx30) in the cochlea have overlapping expression patterns beginning at early embryonic stages. Cx26 and Cx30 were colocalized in most gap junction plaques in the cochlea, and their coassembly was tested by coimmunoprecipitation. To compare functional differences of gap junctions with different molecular configurations, homo- and heteromeric gap junctions composed of Cx26 and/or Cx30 were reconstituted by transfections in human embryonic kidney-293 cells. The ratio imaging technique and fluorescent tracer diffusion assays were used to assess the function of reconstituted gap junctions. Our results revealed that gap junctions with different molecular configurations show differences in biochemical coupling, and that intercellular Ca2+ signaling across heteromeric gap junctions consisting of Cx26 and Cx30 was at least twice as fast as their homomerically assembled counterparts. Our data suggest that biochemical permeability and the dynamics of intercellular signaling through gap junction channels, in addition to gap junction-mediated intercellular ionic coupling, may be important factors to consider for studying functional roles of gap junctions in the cochlea.
SRC family kinases in cell volume regulation
Authors: Cohen David M.
Published: February 2005

SRC family kinases are a group of nine cytoplasmic protein tyrosine kinases essential for many cell functions. Some appear to be ubiquitously expressed, whereas others are highly tissue specific. The ability of members of the SRC family to influence ion transport has been recognized for several years. Mounting evidence suggests a broad role for SRC family kinases in the cell response to both hypertonic and hypotonic stress, and in the ensuing regulatory volume increase or decrease. In addition, members of this tyrosine kinase family participate in the mechanotransduction that accompanies cell membrane deformation. Finally, at least one SRC family member operates in concert with the p38 MAPK to regulate tonicity-dependent gene transcription.