bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Analysis of Environmental and Clinical Populations ofCryptococcus neoformans
Authors: Litvintseva Anastasia P. , Kestenbaum Lori , Vilgalys Rytas , Mitchell Thomas G.
Published: February 2005

Cryptococcus neoformansis a major, global cause of meningoencephalitis in immunocompromised patients. Despite advances in the molecular epidemiology ofC. neoformans, its population structure and mode of reproduction are not well understood. In the environment, it is associated with avian guano or vegetation. We collected nearly 800 environmental isolates from three locations in the United States (viz., North Carolina, California, and Texas) and compared them with one another and with clinical isolates from North Carolina. As expected, they consisted of the most prevalent serotypes, serotypes A and D, as well as serotype AD hybrids. The majority of environmental isolates were obtained from pigeon excreta. All environmental and clinical isolates of serotype A or D had theMATα mating-type allele. However, the AD hybrids includedMATaalleles typical of serotypes A and D. Using an amplified fragment length polymorphism fingerprinting technique with two primer pairs, we identified 12 genotypes among the isolates of serotype A. Six of these genotypes were present in both the clinical and the environmental populations. However, one of the most prevalent environmental genotypes was absent from the clinical samples, and two other genotypes were isolated only from patients. The combined molecular data suggest that this environmental population ofC. neoformansis predominantly clonal, although there was evidence for recent or past recombination.
DNA Macrorestriction Analysis of Nontypeable Group B Streptococcal Isolates: Clonal Evolution of Nontypeable and Type V Isolates
Authors: Amundson Nicole R. , Flores Aurea E. , Hillier Sharon L. , Baker Carol J. , Ferrieri Patricia
Published: February 2005

Group B streptococci (GBS) are serotyped according to capsular polysaccharide (CPS) type (Ia to VIII); an isolate is classified as nontypeable (NT) if no detectable CPS is found. Surface-localized protein antigens (α, β, R1, and R4) serve as additional markers to classify GBS isolates, which is particularly useful since NT isolates often express one or more of these proteins. To compare genetic resemblance among isolates with similar protein profiles, we studied 58 NT isolates digested with the SmaI macrorestriction enzyme prior to pulsed-field gel electrophoresis (PFGE). Of these 58, 15.5% expressed α only, 20.7% expressed α+β, 15.5% expressed R4, and 25.8% expressed R1,R4, while 22.4% of the isolates expressed no detectable proteins. The largest PFGE profile group, with 48% of the isolates, was group 4, composed primarily of isolates that expressed R1,R4 or no proteins. The second most common profiles were 3 and 32, each with 13.8% of the isolates. Since NT isolates in profile group 4 were highly related to type V isolates, as demonstrated by PFGE profiles, we investigated 45 type V isolates. Two-thirds of the type V isolates within profile group 4 were classified into subgroup 4a, compared to 28.2% of 39 NT isolates. Only 11% of the V/R1,R4 isolates were identical to the prototype group 4 profile, in contrast to 75% of the NT/R1,R4 isolates. A shift of type V isolates into profile 4 subgroups may be indicative of a genetic change over time. PFGE is a valuable approach for comparison of GBS isolate relatedness and for monitoring of NT and typeable GBS isolates for potential clonal divergence.
Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel
Authors: Hindiyeh Musa , Levy Virginia , Azar Roberto , Varsano Noemi , Regev Liora , Shalev Yael , Grossman Zehava , Mendelson Ella
Published: February 2005

The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories.
Evaluation of the COBAS Amplicor HBV Monitor Assay and Comparison with the Ultrasensitive HBV Hybrid Capture 2 Assay for Quantification of Hepatitis B Virus DNA
Authors: Konnick Eric Q. , Erali Maria , Ashwood Edward R. , Hillyard David R.
Published: February 2005

Performance characteristics of the COBAS Amplicor HBV Monitor test (Roche Diagnostics), which measures hepatitis B virus (HBV) DNA quantitatively, were evaluated and compared with the Ultrasensitive HBV Hybrid Capture 2 (HC2; Digene Corporation) assay. Linearity and within-run precision were assessed for both methods by using eight HBV DNA-positive samples serially diluted to obtain a range of <100 to 500,000 HBV DNA copies/ml and run in triplicate. Agreement between the methods was studied with 100 clinical samples. HC2 assay performance near the limit of detection was investigated through repeat testing of 149 samples with HC2 and testing of 37 samples with HC2 results of <4,700 HBV DNA copies/ml by Amplicor assay and a qualitative PCR assay. The linearity experiment for Amplicor had regression of observed values compared to expected values (y= 1.073x− 0.247;R2= 0.993,n= 32; for HC2,y= 0.855x+ 0.759,R2= 0.729,n= 18). Within-run standard deviation of log HBV DNA copies/ml ranged from 0.003 to 0.348 (Amplicor) and 0.027 to 0.253 (HC2). Agreement assessed by Deming regression was poor [Amplicor = 1.197(HC2) − 0.961;R2= 0.799, standard error of the estimate (SEE) = 0.710,n= 94]. Near the lower limit of detection, 32 of 149 repeat HC2 results were <4,700 HBV DNA copies/ml. Of the 37 samples with HC2 results of <4,700 HBV DNA copies/ml, HBV DNA was not detected in 15 samples, while HBV DNA was detected by at least one PCR method in 12 samples. Amplicor is linear from 200 to 200,000 HBV DNA copies/ml with undiluted samples, and this range can be expanded through dilution. Inconsistent HC2 results near the limit of detection justify use of a grey zone.
Molecular Identification of a Novel Deltaproteobacterium as the Etiologic Agent of Epizootic Bovine Abortion (Foothill Abortion)
Authors: King Donald P. , Chen Ching-I , Blanchard Myra T. , Aldridge Brian M. , Anderson Mark , Walker Richard , Maas John , Hanks Don , Hall Mark , Stott Jeffrey L.
Published: February 2005

Epizootic bovine abortion (EBA) is endemic in California's coastal range and the foothill regions of the Sierra Nevada, where it has been the primary diagnosed cause of abortion in beef cattle for >50 years. Investigation of these losses has defined a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves. Although the unusual clinical presentation and unique fetal pathology associated with EBA have been recognized since the 1950s, the identity of the etiologic agent is unknown. In this study, suppression-hybridization PCR was used to identify a fragment of the 16S rRNA gene of a previously undescribed bacterium in thymus tissue derived from affected fetuses. Phylogenetic analysis revealed that this pathogen was a deltaproteobacterium closely related to members of the orderMyxococcales. A specific PCR was subsequently developed to detect the presence of this bacterium in DNA extracted from fetal thymuses. Using histopathology as the definitive diagnosis for EBA, this PCR demonstrated 100% specificity and 88% sensitivity. The bacterium was also detected in the argasid tickOrnithodoros coriaceus, which is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.
Use of the MicroSeq 500 16S rRNA Gene-Based Sequencing for Identification of Bacterial Isolates That Commercial Automated Systems Failed To Identify Correctly
Authors: Fontana Carla , Favaro Marco , Pelliccioni Marco , Pistoia Enrico Salvatore , Favalli Cartesio
Published: February 2005

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux). However, more and more frequently, microbiologists must isolate “difficult” strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 “difficult” clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems—VITEK 2 and Phoenix—while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.
Development of Colorimetric Microtiter Plate Assay for Assessment of Antimicrobials against
Acanthamoeba
Authors: McBride James , Ingram Paul R. , Henriquez Fiona L. , Roberts Craig W.
Published: February 2005

We have developed and optimized a 96-well microtiter plate assay, based on the reduction of alamarBlue, to assess the efficacies of much needed new antimicrobials against
Acanthamoeba
species. This assay has been optimized for determination of drug efficacy against two potentially pathogenic species,
Acanthamoeba castellanii
and
Acanthamoeba polyphaga
, and has been validated by comparison of their relative susceptibilities to chlorhexidine, a drug widely used to treat
Acanthamoeba
keratitis. The results demonstrate that the assay is comparable to a manual counting assay and that
A. polyphaga
is more resistant to chlorhexidine than
A. castellanii
. Thus, by use of the manual counting assay, 3.125 μM chlorohexidine was almost completely effective against
A. castellanii
, whereas this concentration was less than 20% effective against
A. polyphaga
. Similar results were obtained by the alamarBlue assay. The new assay was used to determine the relative susceptibilities of
A. castellanii
and
A. polyphaga
to the alkylphosphocholines (APCs) hexadecylphosphocholine (hexadecyl-PC; miltefosine) and octadecylphosphocholine (octadecyl-PC) as well as an alkylgycerolphosphocholine, edelfosine. Both APCs studied were equally effective against
A. castellanii
, but octadecyl-PC was less effective than hexadecyl-PC against
A. polyphaga
. Both APCs were more effective than edelfosine against both
Acanthamoeba
species.
A. polyphaga
was found to be significantly less susceptible to each of the phosphocholine analogues. The newly described assay offers a number of advantages over those described previously. It is less labor-intensive than previously described assays and is sensitive and rapid, and the results can be read in a nonsubjective manner. As it is based on a standard 96-well, microtiter plate, it is amenable to automation and high throughput.
Characterization of Cytolethal Distending Toxin ofCampylobacterSpecies Isolated from Captive Macaque Monkeys
Authors: Dassanayake Rohana P. , Zhou You , Hinkley Susanne , Stryker Cynthia J. , Plauche Gail , Borda Juan T. , Sestak Karol , Duhamel Gerald E.
Published: February 2005

An association between certainCampylobacterspecies and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examineCampylobacterspecies isolated from captive conventionally raised macaque monkeys for the presence of thecdtBgene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkeyCampylobacterisolates and examinations by light microscopy, confocal microscopy, and flow cytometry. AlthoughcdtBgene sequences were found in bothCampylobacter jejuniandCampylobacter coli, the production of cytolethal distending toxin correlated positively (P< 0.0001) only withC. jejuni. We concluded that cytolethal distending toxin activity is a characteristic ofC. jejuni. OurC. jejuni cdtBgene-specific PCR assay might be of assistance for differentiating toxigenicC. jejunifromC. coliin clinical laboratories.
Seroprevalence of Noroviruses in Swine
Authors: Farkas Tibor , Nakajima Setsuko , Sugieda Masaaki , Deng Xiaoyun , Zhong Weiming , Jiang Xi
Published: February 2005

Noroviruses (NVs) are important human pathogens that cause acute gastroenteritis. Genetically related animal enteric NVs have also been described, but there is no evidence of interspecies transmission of NVs. In this study we characterized antibody prevalence among domestic pigs by using recombinant capsid antigens of two human NVs (Norwalk and Hawaii) and one swine NV (SW918) that is genetically related to GII human NVs. Recombinant SW918 capsid protein expressed in baculovirus self-assembled into virus-like particles (VLPs) that were detected by antibodies against GII (Hawaii and Mexico), but not GI (Norwalk and VA115), human NVs. NVs recognize human histo-blood group antigens as receptors, but SW918 VLPs did not bind to human saliva samples with major histo-blood group types. Seventy-eight of 110 (71%) pig serum samples from the United States and 95 of 266 (36%) pig serum samples from Japan possessed antibodies against SW918. Serum samples from pigs in the United States were also tested for antibodies against human NVs; 63% were positive for Norwalk virus (GI) and 52% for Hawaii virus (GII). These results indicate that NV infections are common among domestic pigs; the finding of antigenic relationships between SW918 and human NVs and the detection of antibodies against both GI and GII human NVs in domestic animals highlights the importance of further studies on NV gastroenteritis as a possible zoonotic disease.
Use of an Immunochromatographic Assay for Rapid Detection of
Trichomonas vaginalis
in Vaginal Specimens
Authors: Huppert Jill S. , Batteiger Byron E. , Braslins Phillip , Feldman James A. , Hobbs Marcia M. , Sankey Heather Z. , Sena Arlene C. , Wendel Karen A.
Published: February 2005

Trichomonas vaginalis
infection is estimated to be the most widely prevalent nonviral sexually transmitted infection in the world. Wet-mount microscopy is the most common diagnostic method, although it is less sensitive than culture. The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, Mass.) (referred to here as OSOM) is a new point-of-care diagnostic assay for
T. vaginalis
that uses an immunochromatographic capillary flow (dipstick) assay and provides results in 10 min. The purpose of this study was to determine the test characteristics of OSOM compared to those of a composite reference standard (CRS) comprised of wet-mount microscopy and
T. vaginalis
culture. This multicenter cross-sectional study enrolled sexually active women ≥18 years of age who presented with symptoms of vaginitis, exposure to
T. vaginalis
, or multiple sexual partners. Vaginal-swab specimens were obtained for
T. vaginalis
culture, wet mount, and rapid testing. The prevalence of
T. vaginalis
in this sample was 23.4% (105 of 449) by the CRS. The sensitivity and specificity of OSOM vaginal-swab specimens were 83.3 and 98.8%, respectively, while wet mount had a sensitivity and specificity of 71.4 and 100%, respectively, compared to the CRS. OSOM performed significantly better than wet mount (
P
= 0.004) and detected
T. vaginalis
in samples that required 48 to 72 h of incubation prior to becoming culture positive. The performance of the rapid test was not affected by the presence of coinfections with chlamydia and gonorrhea. The OSOM Trichomonas Rapid Test is a simple, objective test that can be expected to improve the diagnosis of
T. vaginalis
, especially where microscopy and culture are unavailable.
Evaluation of a Two-Step Approach for Large-Scale, Prospective Genotyping ofMycobacterium tuberculosisIsolates in the United States
Authors: Cowan Lauren S. , Diem Lois , Monson Timothy , Wand Philip , Temporado David , Oemig Tanya V. , Crawford Jack T.
Published: February 2005

Genotyping ofMycobacterium tuberculosisisolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping.M. tuberculosisisolates (n= 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.
Sequence Analysis of the
Escherichia coli
O15 Antigen Gene Cluster and Development of a PCR Assay for Rapid Detection of Intestinal and Extraintestinal Pathogenic
E. coli
O15 Strains
Authors: Beutin Lothar , Tao Jiang , Feng Lu , Krause Gladys , Zimmermann Sonja , Gleier Kerstin , Xia Qiuyu , Wang Lei
Published: February 2005

A collection of 33
Escherichia coli
serogroup O15 strains was studied with regard to O:H serotypes and virulence markers and for detection of the O-antigen-specific genes
wzx
and
wzy
. The strains were from nine different countries, originated from healthy or diseased humans and animals and from food, and were isolated between 1941 and 2003. On the basis of virulence markers and clinical data the strains could be split into different pathogroups, such as uropathogenic
E. coli
, enteropathogenic
E. coli
, Shiga toxin-producing
E. coli
, and enteroaggregative
E. coli
. H serotyping and genotyping of the flagellin (
fliC
) gene revealed 11 different H types and a close association between certain H types, virulence markers, and pathogroups was found. Nucleotide sequence analysis of the O-antigen gene cluster revealed putative genes for biosynthesis of O15 antigen. PCR assays were developed for sensitive and specific detection of the O15-antigen-specific genes
wzx
and
wzy.
The high pathotype diversity found in the collection of 33 O15 strains contrasted with the high level of similarity found in the genes specific to the O15 antigen. This might indicate that the O15 determinant has been spread by horizontal gene transfer to a number of genetically unrelated strains of
E. coli.
Diagnosis of Congenital Toxoplasmosis by Two-Dimensional Immunoblot Differentiation of Mother and Child Immunoglobulin G Profiles
Authors: Nielsen Henrik Vedel , Schmidt Dorte Remmer , Petersen Eskild
Published: February 2005

Differentiation between the specific immunoglobulin G (IgG) response to
Toxoplasma gondii
by a mother and her newborn child is helpful in the diagnosis of congenital infection with
T. gondii
in newborns without
T. gondii
-specific IgM and/or IgA antibodies at birth. Previous methods include immunoblotting and complexing
T. gondii
antigen with the sera from the mother and child and comparing the bands after electrophoresis. We developed a two-dimensional immunoblotting (2DIB) method with
T. gondii
RH strain tachyzoite antigen and validated the method with sera from 11 children identified through the neonatal screening program for congenital toxoplasmosis in Denmark. The children were identified by using
Toxoplasma
-specific IgM antibodies at the screening test, but the presence of
T. gondii
-specific IgM and/or IgA antibodies could not be confirmed at the subsequent serum sample tested. The children were monitored for at least 12 months, and in seven of eight patients monitored for 12 months the results of the 2DIB-predicted congenital infection were confirmed by the presence of persistent
Toxoplasma
-specific IgG antibodies. 2DIB is a sensitive technique that allows early differentiation between passively transferred maternal
T. gondii
-specific IgG antibodies and antibodies synthesized by the newborn child.
Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years
Authors: Ariga Toshihide , Shimada Yasushi , Shiratori Kenji , Ohgami Kazuhiro , Yamazaki Shudo , Tagawa Yoshitsugu , Kikuchi Masayuki , Miyakita Yoshie , Fujita Kozo , Ishiko Hiroaki , Aoki Koki , Ohno Shigeaki
Published: February 2005

Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.
Comparison of Hepatitis C Virus NS5b and 5′ Noncoding Gene Sequencing Methods in a Multicenter Study
Authors: Laperche Syria , Lunel Françoise , Izopet Jacques , Alain Sophie , Dény Paul , Duverlie Gilles , Gaudy Catherine , Pawlotsky Jean-Michel , Plantier Jean-Christophe , Pozzetto Bruno , Thibault Vincent , Tosetti François , Lefrère Jean-Jacques
Published: February 2005

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5′ noncoding (5′ NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5′ NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5′ NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.
A Novel
Shigella dysenteriae
Serovar Isolated in Canada
Authors: Melito P. L. , Woodward D. L. , Munro J. , Walsh J. , Foster R. , Tilley P. , Paccagnella A. , Isaac-Renton J. , Ismail J. , Ng L. K.
Published: February 2005

The etiological agent most commonly associated with bacillary dysentery is
Shigella
. As part of its mandate, the Bacteriology and Enteric Disease Program of Health Canada identifies and serotypes unusual isolates of
Shigella
received from provincial laboratories of public health. In this report, six unusual isolates from three provinces were analyzed biochemically and serologically using slide and tube agglutinations and molecularly using standard pulsed-filed gel electrophoresis (PFGE), PCR, and PCR-restriction fragment length polymorphism (RFLP) techniques. All six isolates were identical. PFGE analysis grouped these strains; biochemically, they were mannitol negative and consistent with the profile of
Shigella
. Serologically, these strains produced weak reactions in
Shigella dysenteriae
serovars 4 and 16 and
Escherichia coli
O159 and O173 antisera. Molecular serotyping by PCR-RFLP of the
rfb
gene produced an
S. dysenteriae
serovar 2/
E. coli
O112ac pattern. They were positive by PCR for
ipaH
and
ial
enteroinvasive genes but negative for all other genes tested. Antiserum was prepared from one of the isolates and tested against
Shigella
and
E. coli
reference strains as well as the other isolates. The antiserum reacted with the five remaining isolates and showed cross-reactivity with
S. dysenteriae
serovars 1, 4, and 16;
Shigella flexneri
type 3; and
E. coli
O118, O159, O168, O172, and O173 antigens. Absorbing the sera with
E. coli
O159 and
S. dysenteriae
serovar 4 antigen removed all cross-reactions and only slightly reduced the homologous titer. Based on biochemical, molecular, and complete serological analysis, we propose that these six isolates represent a new provisional serovar of
S. dysenteriae
, type strain BEDP 02-5104.
High Rate of Fatal Cases of Pediatric Septicemia Caused by Gram-Negative Bacteria with Extended-Spectrum Beta-Lactamases in Dar es Salaam, Tanzania
Authors: Blomberg Bjørn , Jureen Roland , Manji Karim P. , Tamim Bushir S. , Mwakagile Davis S. M. , Urassa Willy K. , Fataki Maulidi , Msangi Viola , Tellevik Marit G. , Maselle Samwel Y. , Langeland Nina
Published: February 2005

Extended-spectrum
beta-lactamases (ESBLs) were present in high proportions of
Escherichia coli
(25% [9 of 36]) and
Klebsiella pneumoniae
isolates (17% [9 of
52]) causing pediatric septicemia at a tertiary hospital in
Tanzania. Patients with septicemia due to ESBL-producing organisms had
a significantly higher fatality rate than those with non-ESBL isolates
(71% versus 39%,
P
= 0.039). This is the
first report of the CTX-M-15 genotype of ESBLs on the African continent
and the first observation of SHV-12 genotype in an isolate of
Salmonella enterica
serotype
Newport.
Simple Method for Determination of the Number ofHelicobacter pyloriCagA Variable-Region EPIYA Tyrosine Phosphorylation Motifs by PCR
Authors: Argent Richard H. , Zhang Youli , Atherton John C.
Published: February 2005

Helicobacter pyloristrains possessing thecagpathogenicity island are associated with the development of gastric cancer. The CagA protein is translocated into epithelial cells and becomes phosphorylated on tyrosine residues within EPIYA motifs, which may be repeated within the variable region of the protein. Strains possessing CagA with greater numbers of these repeats have been more closely associated with gastric carcinogenesis. Phosphorylated CagA leads to epithelial cell elongation, which is dependent on the number of variable-region EPIYA motifs. Thus, determination of the degree of CagA phosphorylation and the number of EPIYA motifs appears to be more important than detection ofcagAalone. Determination of the number of EPIYA motifs by nucleotide sequencing, however, is a laborious and expensive process. We describe here a novel and rapid PCR method for determination of the pattern of repeats containing the EPIYA motif. This will aid in the identification of those strains that may be more likely to cause disease.
Genetic Variants of
Anaplasma phagocytophilum
Infecting Dogs in Western Washington State
Authors: Poitout Florence M. , Shinozaki Joanne K. , Stockwell Patrick J. , Holland Cynthia J. , Shukla Sanjay K.
Published: February 2005

Eight dogs from western Washington State suspected of being infected with
Anaplasma phagocytophilum
because of the finding of morulae in peripheral blood neutrophils were studied for determination of the etiologic agent of disease. All cases were diagnosed between April 2003 and April 2004. Six of the eight dogs had no travel history during the 6 months prior to presentation. Two dogs had traveled within the Northwest United States and Canada. Fever, lethargy, and anorexia were the most common clinical signs in the dogs. Lymphopenia, thrombocytopenia, and an elevated activity of alkaline phosphatase in the serum were the most common laboratory findings. All dogs tested during the acute phase of clinical signs were seropositive for
A. phagocytophilum
antibodies but negative for
Ehrlichia canis
antibodies. PCR amplification and direct sequencing of portions of the 16S rRNA gene from the whole blood of all seven dogs that were tested yielded
A. phagocytophilum
after a comparison to bacterial sequences available in the GenBank database. Five genetic variants were identified based on one or two nucleotide differences in the 16S rRNA gene sequences at nucleotide positions 54, 84, 86, and 120. Individual dogs were infected with more than one variant. Treatment with doxycycline or tetracycline resulted in a rapid resolution of clinical signs. The occurrence of canine granulocytic anaplasmosis in western Washington State suggests that
A. phagocytophilum
infection should be considered in differential diagnoses of dogs presenting with lethargy, anorexia, fever, and lameness, particularly in the context of lymphopenia, thrombocytopenia, and increased serum alkaline phosphatase. The zoonotic importance of
A. phagocytophilum
should support an increase in surveillance for horses and people residing in this area.
Stability of PorA during a Meningococcal Disease Epidemic
Authors: Devoy A. F. , Dyet K. H. , Martin D. R.
Published: February 2005

Meningococci causing New Zealand's epidemic, which began in 1991, are defined as group B, serosubtype P1.4 (subtype P1.7-2,4), belonging to the ST-41/ST-44 complex, lineage III. Of the 2,358 group B isolates obtained from disease cases from 1991 through 2003, 85.7% (2,021 of 2,358) were determined to be serosubtype P1.4. Of the remaining isolates, 156 (6.6%) were not serosubtypeable (NST). Molecular analysis of the
porA
gene from these B:NST meningococcal isolates was used to determine the reason. Most NST isolates (156, 88.5%) expressed a PorA that was distinct from P1.7-2,4 PorA. Fifteen isolates expressed variants of P1.7-2,4 PorA, and a further three expressed P1.7-2,4 PorA without any sequence variation. These three isolates expressed P1.7-2,4 PorA at very low levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and showed variation in the
porA
promoter region. Among the 15 meningococcal isolates expressing variants of P1.7-2,4 PorA, 11 different sequence variations were found. Compared with the P1.7-2,4 PorA sequence, the sequences of these variants contained deletions, insertions, or single-nucleotide substitutions in the VR2 region of the protein. Multilocus restriction typing was used to assess the clonal derivations of B:NST case isolates. Meningococcal isolates expressing distinct PorA proteins belonged mostly to clonal types that were unrelated to the epidemic strain, whereas all meningococcal isolates expressing variants of P1.7-2,4 PorA belonged to the ST-41/ST-44 complex, lineage III. These results, together with those obtained serologically, demonstrate that the P1.7-2,4 PorA protein of meningococci responsible for New Zealand's epidemic has remained relatively stable over 13 years and support the use of a strain-specific outer membrane vesicle vaccine to control the epidemic.
Evidence ofBorreliaAutoimmunity-Induced Component of Lyme Carditis and Arthritis
Authors: Raveche Elizabeth S. , Schutzer Steven E. , Fernandes Helen , Bateman Helen , McCarthy Brian A. , Nickell Steven P. , Cunningham Madeleine W.
Published: February 2005

We investigated the possibility that manifestations of Lyme disease in certain hosts, such as arthritis and carditis, may be autoimmunity mediated due to molecular mimicry between the bacteriumBorrelia burgdorferiand self-components. We first compared amino acid sequences ofStreptococcus pyogenesM protein, a known inducer of antibodies that are cross-reactive with myosin, andB. burgdorferiand found significant homologies with OspA protein. We found thatS. pyogenesM5-specific antibodies and sera fromB. burgdorferi-infected mice reacted with both myosin andB. burgdorferiproteins by Western blots and enzyme-linked immunosorbent assay. To investigate the relationship between self-reactivity and the response toB. burgdorferi, NZB mice, models of autoimmunity, were infected. NZB mice infected withB. burgdorferideveloped higher degrees of joint swelling and higher anti-B. burgdorferiimmunoglobulin M cross-reactive responses than other strains with identical major histocompatibility complex (DBA/2 and BALB/c). These studies reveal immunological cross-reactivity and suggest thatB. burgdorferimay share common epitopes which mimic self-proteins. These implications could be important for certain autoimmunity-susceptible individuals or animals who become infected withB. burgdorferi.
Evaluation of Two Commercially Available, Inexpensive Alternative Assays Used for Assessing Viral Load in a Cohort of Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients from South Africa
Authors: Stevens G. , Rekhviashvili N. , Scott L. E. , Gonin René , Stevens W.
Published: February 2005

Although human immunodeficiency virus type 1 (HIV-1) RNA is the acknowledged “gold standard” marker for monitoring disease activity in patients receiving highly active antiretroviral therapy (HAART), it remains unaffordable in resource-constrained settings. The present study investigated two commercially available kits for the detection of HIV-1 viral load markers as more affordable alternatives to HIV-1 RNA quantitation. The greatly improved heat-denatured, signal-boosted HiSens HIV-1 p24 Ag Ultra kit (Perkin-Elmer) and the ExaVir Load Quantitative HIV-RT kit (Cavidi Tech AB) were compared with the Amplicor HIV-1 Monitor (version 1.5) assay (Roche Molecular Systems Inc.). A total of 117 samples containing HIV-1 subtype C were analyzed by all three methodologies. Eighty-nine of these samples represented serial measurements from 20 patients receiving HAART. The remaining samples analyzed were from a group of treatment-naïve patients. The association between the p24 antigen assay and the RNA assay was fairly strong (
R
2
= 0.686). The association between the reverse transcriptase (RT) quantitation assay and the RNA assay was strong (
R
2
= 0.810). Both alternative assays seemed most useful for the serial monitoring of patients receiving HAART (
n
= 89 plasma samples from 20 patients), as all assays showed a statistically significant downward trend over time, with the trend being either linear or curvilinear. In addition, all three assays showed negative correlations with the CD4 count (CD4 count versus RNA load,
r
= −0.336 and
P
= 0.001; CD4 count versus p24 antigen level,
r
= −0.541 and
P
< 0.0001; CD4 count versus RT level,
r
= −0.358 and
P
= 0.0006). Still of major concern are both the lack of sensitivity and the wide degrees of variability of both assays. However, both assays provide a less expensive alternative to the Roche viral load assay and demonstrate the same trends during treatment.
Population-Level Retrospective Study of Neurologically Expressed Disorders in Ruminants before the Onset of Bovine Spongiform Encephalopathy (BSE) in Belgium, a BSE Risk III Country
Authors: Saegerman C. , Berkvens D. , Claes L. , Dewaele A. , Coignoul F. , Ducatelle R. , Cassart D. , Brochier B. , Costy F. , Roels S. , Deluyker H. , Vanopdenbosch E. , Thiry E.
Published: February 2005

A retrospective epidemiological study (
n
= 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (
n
= 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.
Growth of
Mycobacterium tuberculosis
in Conventional BacT/ALERT FA Blood Culture Bottles Allows Reliable Diagnosis of Mycobacteremia
Authors: Hanscheid T. , Monteiro C. , Cristino J. Melo , Lito L. Marques , Salgado M. J.
Published: February 2005

The conventional BacT/ALERT FA blood cultures supported the ample growth of
Mycobacterium tuberculosis
in seeding experiments and appeared to perform as reliably as the BACTEC Myco/F-Lytic vials in the recovery of
M. tuberculosis
from blood in HIV-infected patients. Overall, blood cultures were positive in 39% of patients with tuberculosis.
Mycobacterium marinum
Infections in Fish and Humans in Israel
Authors: Ucko M. , Colorni A.
Published: February 2005

Israeli
Mycobacterium marinum
isolates from humans and fish were compared by direct sequencing of the 16S rRNA and
hsp65
genes, restriction mapping, and amplified fragment length polymorphism analysis. Significant molecular differences separated all clinical isolates from the piscine isolates, ruling out the local aquaculture industry as the source of human infections.
Evaluation of a New Selective Enrichment Broth for Detection of Group B Streptococci in Pregnant Women
Authors: Heelan Judith S. , Struminsky Judith , Lauro Patricia , Sung C. James
Published: February 2005

Studies at two Brown Medical School-affiliated hospitals were undertaken to evaluate a new selective broth medium (GBS broth) and to compare it to the LIM broth currently used to culture for group B streptococci. Beta-hemolytic group B streptococci produce a carotenoid pigment that turns GBS broth an orange color. From a total of 580 pregnant women, duplicate vaginal-rectal swabs were collected at 35 to 37 weeks of gestation and cultured for group B streptococci, using either LIM broth (a selective broth containing antibiotics) or GBS broth for enrichment. Specimens were either transported to the laboratory or immediately placed in the respective enrichment broths and delivered to the laboratory. GBS broth medium had sensitivity, specificity, and positive and negative predictive values of 87.8, 100, 100, and 95.1% when planted in the laboratory and 90.3, 100, 100 and 97.6%, respectively, when inoculated at bedside. Use of GBS broth would satisfy Centers for Disease Control and Prevention requirements and would provide faster, more-sensitive, and cost-effective detection of group B streptococci in pregnant women.
Persistence of
Escherichia coli
O157 Isolates on Bovine Farms in England and Wales
Authors: Liebana Ernesto , Smith Richard P. , Batchelor Miranda , McLaren Ian , Cassar Claire , Clifton-Hadley Felicity A. , Paiba Giles A.
Published: February 2005

We performed pulsed-field gel electrophoresis on
Escherichia coli
O157 isolates (
n
= 318) from 199 healthy animals in a longitudinal study carried out on nine farms. Investigation of the restriction types proved that at the farm level, the same clones can be detected on sampling occasions separated by as much as 17 months. The cohort animals were repeatedly sampled, and for some of these, the same clones were obtained on sampling occasions separated by as much as 8 months.
Emergence of a Nephropathogenic Avian Infectious Bronchitis Virus with a Novel Genotype in India
Authors: Bayry Jagadeesh , Goudar Mallikarjun S. , Nighot Prashant K. , Kshirsagar Supriya G. , Ladman Brian S. , Gelb Jack , Ghalsasi Govind R. , Kolte Gopal N.
Published: February 2005

We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).
Detection of
Burkholderia pseudomallei
in Soil within the Lao People's Democratic Republic
Authors: Wuthiekanun Vanaporn , Mayxay Mayfong , Chierakul Wirongrong , Phetsouvanh Rattanaphone , Cheng Allen C. , White Nicholas J , Day Nicholas P. J. , Peacock Sharon J.
Published: February 2005

Clinical cases of melioidosis caused by the saprophyte
Burkholderia pseudomallei
were first noted in the Lao People's Democratic Republic (PDR) in 1999. In this study, 36% of 110 soil samples in northern Lao PDR were positive for
B. pseudomallei
, providing further evidence for the presence of melioidosis in this country.
Use of the Restriction Enzyme EcoRI for Pulsed-Field Gel Electrophoretic Analysis of
Helicobacter pylori
Authors: Hosaka Yoshio , Irinoda Kazuhiko , Nakano Ryuichi , Tanabe Satosi , Koizumi Wasaburo , Saigenji Katunori , Inoue Matsuhisa
Published: February 2005

Pulsed-field gel electrophoretic (PFGE) analysis of
Helicobacter pylori
isolates is not commonly employed because of the inability to compare the typing with other typing systems. We adapted the PFGE analysis for
H. pylori
by using EcoRI and slightly modified our laboratory methods to improve the typing of isolates (typeability was 97%).
Identification and Characterization of Sporadic Isolates of
Streptococcus iniae
Isolated from Humans
Authors: Facklam Richard , Elliott John , Shewmaker Lynn , Reingold Art
Published: February 2005

Seven reference strains and seven clinical isolates of
Streptococcus iniae
, submitted to the Centers for Disease Control and Prevention Streptococcus Reference Laboratory between 2001 and 2004, were successfully identified by a conventional identification system. The seven randomly submitted clinical isolates were sensitive to β-lactams, macrolides, quinolones, and vancomycin. Two of the seven clinical isolates were resistant to tetracycline. All seven strains grew well and multiplied in a phagocytosis assay. One of the seven randomly submitted strains was more similar to the type strain of
S. iniae
than to the other six strains. The latter six strains were similar if not identical to representative strains from a cluster of disease in Canada (M. R. Weinstein et al., N. Engl. J. Med.
337:
589-594, 1997).
Evaluation of Etest MBL for Detection of
bla
IMP-1
and
bla
VIM-2
Allele-Positive Clinical Isolates of
Pseudomonas
spp. and
Acinetobacter
spp
Authors: Lee Kyungwon , Yong Dongeun , Yum Jong Hwa , Lim Yong Sik , Bolmström Anne , Qwärnström Anette , Karlsson Åsa , Chong Yunsop
Published: February 2005

The Etest MBL (AB BIODISK, Solna, Sweden) correctly differentiated all 57 isolates of
Acinetobacter
spp. and
Pseudomonas aeruginosa
with the
bla
IMP-1
allele and 135 of 137 (98.5%)
Acinetobacter
spp. and
Pseudomonas
spp. isolates with the
bla
VIM-2
allele. The Etest MBL was reliable for detecting the IMP-1- and VIM-2-producing
Pseudomonas
and
Acinetobacter
isolates.
Bartonella vinsonii
subsp.
arupensis
as an Agent of Blood Culture-Negative Endocarditis in a Human
Authors: Fenollar Florence , Sire Stéphane , Raoult Didier
Published: February 2005

We report the case of a patient hospitalized with endocarditis. The etiological diagnosis of
Bartonella
was suggested by detection of high titers of antibodies by immunofluorescence and Western blotting. Two different nested PCRs performed on sera identified
Bartonella vinsonii
subsp.
arupensis
by sequencing.