bluuebunny/crossref_metadata_2025 huggingface dataset:
Insufficient Expression of theilv-leuOperon Encoding Enzymes of Branched-Chain Amino Acid Biosynthesis Limits Growth of aBacillus subtilis ccpAMutant
Authors: Ludwig Holger , Meinken Christoph , Matin Anastasija , Stülke Jörg
Published: October 2002

Bacillus subtilis ccpAmutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired. This growth defect is largely due to a lack of expression of thegltABoperon. However, growth is impaired even in the presence of glutamate. Here, we demonstrate that theccpAmutant strain needs methionine and the branched-chain amino acids for optimal growth. The control of expression of theilv-leuoperon by CcpA provides a novel regulatory link between carbon and amino acid metabolism.
Transmission of Drug-Resistant Tuberculosis in Texas and Mexico
Authors: Quitugua Teresa N. , Seaworth Barbara J. , Weis Stephen E. , Taylor Jeffery P. , Gillette J. Seb , Rosas Ishmael I. , Jost, Kenneth C. , Magee D. Mitchel , Cox Rebecca A.
Published: October 2002

To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico,
Mycobacterium tuberculosis
isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS
6110
-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR
M. tuberculosis
strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.
Ca3 Fingerprinting of
Candida albicans
Bloodstream Isolates from the United States, Canada, South America, and Europe Reveals a European Clade
Authors: Pujol Claude , Pfaller Michael , Soll David R.
Published: October 2002

It was previously demonstrated by a cluster analysis that 26 unrelated U.S. isolates of
Candida albicans
separated into three distinct groups (groups I, II, and III) while South African isolates separated into four distinct groups (groups I, II, III, and SA). To verify the absence or underrepresentation of SA isolates in North America, and to identify which groups are represented in Europe and South America, collections of bloodstream isolates from each geographical locale were analyzed by cluster analyses based on genetic fingerprinting with the Ca3 probe. The results verify that North America is almost devoid of SA isolates (2%). However, the results reveal a new clade, designated group E, relatively specific to Europe. While 26% of a European collection of 46 isolates was composed of group E isolates, only 2% of the 164 North American isolates, 5% of 22 South American isolates, and 1% of 361 South African isolates were composed of group E isolates. The North American collection proved to be the least-diverse collection in regard to group representation. In a comparison of collections from the Northeast, Midwest, and Southwest regions of the United States, Canada, and South America, it was demonstrated that both the U.S. Southwest and the South American collections were devoid of group II isolates. Together these results identify for the first time a European-specific clade and demonstrate clear distinctions in the representations of the five demonstrated clades (groups I, II, III, SA, and E) in different geographical locales.
Alveolar Echinococcosis: Characterization of Diagnostic Antigen Em18 and Serological Evaluation of Recombinant Em18
Authors: Sako Yasuhito , Nakao Minoru , Nakaya Kazuhiro , Yamasaki Hiroshi , Gottstein Bruno , Lightowers Marshall W. , Schantz Peter M. , Ito Akira
Published: October 2002

The
Echinococcus multilocularis
protein Em18 is one of the most promising antigens for use in serodiagnosis of alveolar echinococcosis in human patients. Here we identify an antigenic relationship between Em18 and a 65-kDa immunodominant
E. multilocularis
surface protein previously identified as either EM10 or EmII/3. The NH
2
-terminal sequence of native Em18 was determined, revealing it to be a fragment of EM10. Experiments were undertaken to investigate the effect of proteinase inhibitors on the degradation of EM10 in crude extracts of
E. multilocularis
protoscoleces. Em18 was found to be the product of degradation of EM10 by cysteine proteinase. A recombinant Em18 (RecEm18, derived from
349
K to
508
K of EM10) was successfully expressed by using
Escherichia coli
expression system and then evaluated for use in serodiagnosis of alveolar echinococcosis. RecEm18 was recognized by 27 (87.1%) and 28 (90.3%) of 31 serum samples from clinically and/or pathologically confirmed alveolar echinococcosis patients by enzyme-linked immunosorbent assay and immunoblotting, respectively. Of 33 serum samples from cystic echinococcosis patients, 1 was recorded as having a weak positive reaction to RecEm18; however, none of the serum samples which were tested from neurocysticercosis patients (
n
= 10) or healthy people (
n
= 15) showed positive reactions. RecEm18 has the potential for use in the differential serodiagnosis of alveolar echinococcosis.
Genetic Analysis of
Pseudomonas aeruginosa
Isolates from the Sputa of Australian Adult Cystic Fibrosis Patients
Authors: Anthony Mario , Rose Barbara , Pegler Mary Beard , Elkins Mark , Service Helen , Thamotharampillai Keerthi , Watson Jason , Robinson Michael , Bye Peter , Merlino John , Harbour Colin
Published: October 2002

Genetic investigations were carried out with 50 phenotypically selected strains of
Pseudomonas aeruginosa
from 18 patients attending an Australian cystic fibrosis (CF) center. The isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). Phylogenetic analysis of the macrorestriction patterns showed rates of genetic similarity ranging from 76 to 100%; 24 (48%) of the strains from 11 patients had greater than 90% similarity. A dominant strain emerged: 15 isolates from seven patients had identical PFGE patterns, and 4 other isolates were very closely related. The 50 isolates were grouped into 21 pulsotypes on the basis of visual delineation of a three-band difference. Ten of the 18 (56%) patients were infected with clonal or subclonal strains. Sequence analysis of PCR products derived from the
mucA
gene showed 20 mutations, with the number of mutations in individual isolates ranging from 1 to 4; 19 of these changes are reported here for the first time. Potentially functional changes were found in 22 (44%) isolates. Eight changes (five transversions and three single base deletions) led to premature stop codons, providing support for the presence of
mucA
mutations as one pathway to mucoidy. There was a trend toward an association between the dominant strain and lack of potentially functional
mucA
mutations (
P
= 0.09 by the χ
2
test) but no relationship between genotype and phenotype. This is the first study of genetic variation in
P. aeruginosa
isolates from adult Australian CF patients. The findings highlight the need for further investigations on the transmissibility of
P. aeruginosa
in CF patients.
Combined Use of Two Genetic Fingerprinting Methods, Pulsed-Field Gel Electrophoresis and Ribotyping, for Characterization of
Escherichia coli
O157 Isolates from Food Animals, Retail Meats, and Cases of Human Disease
Authors: Avery S. M. , Liebana E. , Reid C.-A. , Woodward M. J. , Buncic S.
Published: October 2002

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207
Escherichia coli
O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (
n
= 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (
n
= 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease
Xba
I, while for ribotyping, two restriction endonucleases (
Pst
I and
Sph
I) were combined to digest genomic DNAs simultaneously. The 207
E. coli
O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated
E. coli
O157 isolates in the study (Simpson's index of diversity [
D
] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the
E. coli
O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of
E. coli
O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.
Nasopharyngeal Carriage of Potential Bacterial Pathogens Related to Day Care Attendance, with Special Reference to the Molecular Epidemiology of
Haemophilus influenzae
Authors: Peerbooms Paul G. H. , Engelen Marlene N. , Stokman Dominique A. J. , van Benthem Birgit H. B. , van Weert Maria-Lucia , Bruisten Sylvia M. , van Belkum Alex , Coutinho Roel A.
Published: October 2002

Nasopharyngeal carriage of
Haemophilus influenzae
,
Streptococcus pneumoniae
, and
Moraxella catarrhalis
was studied in 259 children attending day care centers (DCC) in Amsterdam, The Netherlands, and in 276 control children. The DCC children were sampled a second time after 4 weeks. Carriage rates for DCC children and controls were 58 and 37% for
S. pneumoniae
, 37 and 11% for
H. influenzae
, and 80 and 48% for
M. catarrhalis
, respectively. No increased antibiotic resistance rates were found in strains isolated from DCC children. All
H. influenzae
isolates were typed by random amplified polymorphic DNA (RAPD) analysis. Evidence for frequent transmission of
H. influenzae
strains within DCC was found. In the control group only two isolates (4%) displayed identical RAPD types versus 38% of strains from DCC children. Colonization with
H. influenzae
appeared to be short-lived in these children; more than half of the children harboring
H. influenzae
in the first sample were negative in the second sample, whereas most children still positive in the second sample had a different genotype than in the first sample. Of the newly acquired strains in the second sample, 40% were identical to a strain that had been found in a child in the same DCC in the first sample. DCC are to be considered epidemiological niches with a high potential for the spread of pathogenic microorganisms.
Analysis of Phylogenetic Relationship of
Cylindrocarpon lichenicola
and
Acremonium falciforme
to the
Fusarium solani
Species Complex and a Review of Similarities in the Spectrum of Opportunistic Infections Caused by These Fungi
Authors: Summerbell R. C. , Schroers H.-J.
Published: October 2002

An emerging pattern of similarity in medical case reports led to a project to compare the phylogenetic affinities of two well-known tropical fungal opportunistic pathogens,
Cylindrocarpon lichenicola
and
Acremonium falciforme
, to members of the
Fusarium solani
species complex.
C. lichenicola
and
A. falciforme
, despite their deviating conidial morphologies, were shown via sequencing of the ribosomal large subunit to be well instituted within a clade mainly consisting of typical
F. solani
strains and other species until recently considered variants of
F. solani
. The original name
Fusarium lichenicola
C. B. Massalongo is reestablished, and the new combination
F. falciforme
is made. Recognition of these species as fusaria is necessary for correct interpretation of current and future molecular diagnostic tests. Reevaluation of species morphology in light of the molecular findings showed that certain features, especially elongate filiform conidiophores with integrated terminal phialides, facilitate correct microscopic classification of these atypical
Fusarium
species. There is a strong and underrecognized overlap in the spectra of cases caused by members of the
F. solani
clade, particularly ocular infections, mycetomas, and, in the neutropenic host, disseminated and other serious systemic infections. A novel synthesis of case reports shows that patients from areas with warm climates may develop a distinctive fusarial intertrigo caused by
F. solani
,
Fusarium lichenicola
, or
Fusarium oxysporum
.
Comparison of the Etest and the Sensititre Colorimetric Methods with the NCCLS Proposed Standard for Antifungal Susceptibility Testing of
Aspergillus
Species
Authors: Meletiadis Joseph , Mouton Johan W. , Meis Jacques F. G. M. , Bouman Bianca A. , Verweij Paul E.
Published: October 2002

The susceptibilities of 25 clinical isolates of
Aspergillus fumigatus
,
A. flavus
,
A. terreus
,
A. nidulans
, and
A. ustus
to itraconazole and amphotericin B were determined by an agar diffusion-dilution method (the Etest method) and a colorimetric broth microdilution method (the Sensititre method); and the results were compared with those obtained by the NCCLS proposed standard M-38P method for antifungal susceptibility testing of filamentous fungi. Various MIC endpoints for the three methods were determined visually by four different observers in three blinded experiments, and the reproducibilities among the observers (interobserver agreement) and among the replicates (interexperimental agreement) as well as the levels of agreement between the NCCLS, the Etest, and the Sensititre methods were calculated. High levels of reproducibility (within 1 twofold dilution) were found for the NCCLS method (>95%) with the MIC-0 endpoint (complete inhibition of growth) for both drugs and with the MIC-1 endpoint (slight growth) for itraconazole and for the Sensititre method (>90%) with all MIC endpoints, although for the latter the interexperimental agreement for itraconazole was comparatively lower (83 to 93%). The Etest method was less reproducible (67 to 87%) for both drugs. Using the recommended MIC endpoints, high levels of agreement (within one twofold dilution) between the NCCLS and the Sensititre methods for all species were found for amphotericin B (>77%) but not for itraconazole (<66%), for which the MICs by the Sensititre method were up to 3 twofold dilutions lower than the corresponding MICs by the NCCLS method. The use of the first blue well as an endpoint for the Sensititre method and 48 h of incubation improved the levels of agreement with the NCCLS method. Low levels of agreement between the NCCLS and the Etest methods using the recommended MIC endpoints were found for most species, especially after 48 h of incubation (<50%), when the MICs obtained by the Etest method were up to 9 twofold dilutions higher than the corresponding MICs obtained by the NCCLS method. Relatively better agreement was found after 24 h, although it was species dependent, with the highest levels of agreement (>82%) found for
A. terreus
and
A. ustus
for amphotericin B and
A. fumigatus
for both drugs. Overall, better agreement was found when MIC-0 was used as the MIC endpoint for the NCCLS method for both drugs and when the MICs by the Etest method were determined after 48 h of incubation for itraconazole and after 24 h of incubation for amphotericin B.
External Quality Assessment Program for
Chlamydia trachomatis
Diagnostic Testing by Nucleic Acid Amplification Assays
Authors: Land Sally , Tabrizi Sepehr , Gust Anthony , Johnson Elizabeth , Garland Susan , Dax Elizabeth M.
Published: October 2002

We report the results from 57 Australian diagnostic laboratories testing two external quality assessment panels using either the Roche Amplicor
Chlamydia trachomatis
test (R-PCR) or the Abbott LCx
Chlamydia trachomatis
assay (A-ligase chain reaction [LCR]). Panel samples were either normal urine spiked with
Chlamydia trachomatis
antigen or clinical urine specimens. There was no significant difference between laboratories or between assays in detection of
C. trachomatis-
positive clinical samples. Only at the lower limit of detection of the assays did the R-PCR demonstrate increased sensitivity over the A-LCR in the detection of
C. trachomatis
antigen. However, it was found that single-sample testing could lead to decreased test sensitivity. Detection of the presence of inhibitors of nucleic acid amplification differed between laboratories.
Identification of
Streptococcus bovis
Biotype I Strains among
S. bovis
Clinical Isolates by PCR
Authors: Songy Wanda B. , Ruoff Kathryn L. , Facklam Richard R. , Ferraro Mary J. , Falkow Stanley
Published: October 2002

Streptococcus bovis
causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both
S. bovis
bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers).
S. bovis
is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify
S. bovis
biotype I strains among
S. bovis
clinical isolates.
Outbreaks of Disease Suspected of Being Due to Human Monkeypox Virus Infection in the Democratic Republic of Congo in 2001
Authors: Meyer Hermann , Perrichot Mathilde , Stemmler Markus , Emmerich Petra , Schmitz Herbert , Varaine Francis , Shungu Robert , Tshioko Florimond , Formenty Pierre
Published: October 2002

Seven outbreaks of disease characterized by a pustular rash and suspected to have been caused by human monkeypox virus were investigated. The outbreaks occurred between February and August 2001 in the province of Equateur in the Democratic Republic of Congo. The outbreaks involved a total of 31 persons and caused five deaths. Specimens from 14 patients were available and were analyzed by electron microscopy, virus isolation, and PCR assays specific for monkeypox virus and varicella-zoster virus. We provide evidence that two outbreaks were indeed caused by monkeypox virus (16 cases, with four deaths), that in two outbreaks both monkeypox and varicella-zoster virus were involved (seven cases, with one death), and that two outbreaks were cases of chickenpox caused by infection with varicella-zoster virus (six cases, with no deaths). In one outbreak, no evidence for either monkeypox or chickenpox was found (two cases, with no deaths).
Clinical and Laboratory Features of
Mycobacterium mageritense
Authors: Wallace Richard J. , Brown-Elliott Barbara A. , Hall Leslie , Roberts Glenn , Wilson Rebecca W. , Mann Linda B. , Crist Christopher J. , Chiu Sher H. , Dunlap Robbie , Garcia Maria J. , Bagwell J. Todd , Jost Kenneth C.
Published: October 2002

Six clinical isolates of the nonpigmented, rapidly growing species
Mycobacterium mageritense
were recovered from sputum, bronchial wash, blood, sinus drainage, and two surgical wound infections from separate patients in Texas, New York, Louisiana, and Florida. The isolates matched the ATCC type strain by PCR restriction enzyme analysis of the 65-kDa
hsp
gene sequence of Telenti, high-performance liquid chromatography, biochemical reactions, and partial 16S rRNA gene sequencing. These are the first isolates of this species to be described in the United States and the first isolates to be associated with clinical disease. Susceptibility testing of all known isolates of the species revealed all isolates to be susceptible or intermediate to amikacin, cefoxitin, imipenem, and the fluoroquinolones and sulfonamides but resistant to clarithromycin. Because of their phenotypic and clinical similarity to isolates of the
Mycobacterium fortuitum
third biovariant complex (sorbitol positive), isolates of
M. mageritense
are likely to go undetected unless selected carbohydrate utilization or molecular identification methods are used.
Bordetella pertussis
Isolates with a Heterogeneous Phenotype for Erythromycin Resistance
Authors: Wilson K. E. , Cassiday P. K. , Popovic T. , Sanden G. N.
Published: October 2002

Erythromycin is currently being used for both prophylaxis and treatment of pertussis infections. Erythromycin resistance was first recognized in
Bordetella pertussis
in Arizona in 1994, and since then, three additional resistant isolates have been identified in the United States. To better assess the potential public health impact of erythromycin-resistant
B. pertussis
, we used the disk diffusion assay to evaluate the frequency of erythromycin resistance among 1,030 recently circulating U.S. isolates and found the rate of occurrence to be <1%. We also describe a novel heterogeneous phenotype, with erythromycin-resistant colonies appearing only after a 7-day incubation period. To optimize patient management, we recommend that clinicians be alert to potential treatment failures and that laboratorians use a 7-day incubation period when screening for resistance. Our ongoing national surveillance will continue to monitor for resistant
B. pertussis
isolates and their potential association with changing pertussis epidemiology.
Comparison of CHROMagar Salmonella Medium and Xylose-Lysine-Desoxycholate and Salmonella-Shigella Agars for Isolation of
Salmonella
Strains from Stool Samples
Authors: Maddocks Susan , Olma Tom , Chen Sharon
Published: October 2002

The growth and appearance of 115 stock
Salmonella
isolates on a new formulation of CHROMagar Salmonella (CAS) medium were compared to those on xylose-lysine-desoxycholate agar (XLD), Salmonella-Shigella agar (SS), and Hektoen enteric agar (HEA) media. CAS medium was then compared prospectively to XLD and SS for the detection and presumptive identification of
Salmonella
strains in 500 consecutive clinical stool samples. All stock
Salmonella
isolates produced typical mauve colonies on CAS medium. Nine
Salmonella
strains were isolated from clinical specimens. The sensitivities for the detection of salmonellae after primary plating on CAS medium and the combination of XLD and SS after enrichment were 100%. The specificity for the detection of salmonellae after primary plating on CAS medium (83%) was significantly (
P
< 0.0001) higher than that after primary plating on the combination of SS and XLD media (55%) (a 28% difference in rates; 95% confidence interval, 23.0 to 34%). Twenty-nine non-
Salmonella
organisms produced mauve colonies on CAS medium, including 17
Candida
spp. (59%) and 8
Pseudomonas
spp. (28%). These were easily excluded as salmonellae by colony morphology, microscopic examination of a wet preparation, or oxidase testing. One biochemically inert
Escherichia coli
isolate required further identification to differentiate it from
Salmonella
spp. The use of plating on CAS medium demonstrated high levels of sensitivity and specificity and reduced the time to final identification of
Salmonella
spp., resulting in substantial cost savings. It can be recommended for use for the primary isolation of
Salmonella
spp. from stool specimens. Other media (e.g., XLD) are required to detect
Shigella
spp. concurrently.
Phenotyping and Genotyping of
Sporothrix schenckii
Isolates According to Geographic Origin and Clinical Form of Sporotrichosis
Authors: Mesa-Arango Ana Cecilia , del Rocío Reyes-Montes María , Pérez-Mejía Amelia , Navarro-Barranco Hortensia , Souza Valeria , Zúñiga Gerardo , Toriello Conchita
Published: October 2002

Sporothrix schenckii
isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37°C, median lethal dose [LD
50
]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (
P
< 0.01) in the mean conidial length of
S. schenckii
clinical isolates from CO (
x̅
= 4.03 ± 1.04 μm) compared with those of clinical isolates from MX (
x̅
= 2.06 ± 0.53 μm) and GT (
x̅
= 2.68 ± 0.83 μm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35°C (
x̅
= 50.1% ± 15.9%) and 37°C (
x̅
= 72.7% ± 10.9%). In general, the highest virulence, as determined by measurement of the LD
50
for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44
Sporothrix
isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37°C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.
Evaluation of the Phenol Ammonium Sulfate Sedimentation Smear Microscopy Method for Diagnosis of Pulmonary Tuberculosis
Authors: Selvakumar N. , Rahman Fathima , Garg Renu , Rajasekaran S. , Mohan Nalini Sunder , Thyagarajan K. , Sundaram V. , Santha T. , Frieden Thomas R. , Narayanan P. R.
Published: October 2002

We compared the sensitivity and specificity of the phenol ammonium sulfate (PhAS) sediment smear microscopy method for detection of acid-fast bacilli with those of direct smear microscopy, using culture results for
Mycobacterium tuberculosis
as the “gold standard.” The sensitivities of the PhAS and direct smear methods were 85% (465 of 547) and 83% (454 of 547), respectively, and the specificity of each method was 97%. The PhAS method was better accepted by the laboratory technicians and safer but necessitates an overnight sedimentation, which delays reporting of results until 1 day after sputum collection.
Inefficiency of 0.3% Carbol Fuchsin in Ziehl-Neelsen Staining for Detecting Acid-Fast Bacilli
Authors: Selvakumar N. , Rahman Fathima , Rajasekaran S. , Narayanan P. R. , Frieden Thomas R.
Published: October 2002

We compared the sensitivity and specificity of a modified Ziehl-Neelsen (modified-ZN) staining method for acid-fast bacilli (AFB) with that of the standard Ziehl-Neelsen (standard-ZN) staining method, using culture results with
Mycobacterium tuberculosis
as the “gold standard.” The sensitivity (72%; 101 of 140) of the modified-ZN staining method, which uses 0.3% carbol fuchsin, was significantly lower than that of the standard-ZN staining method (84%; 117 of 140); the modified-ZN method missed 21% of cases detected by the standard-ZN method and 11% more of culture-positive samples than the standard-ZN method. The World Health Organization recommendation of 0.3% carbol fuchsin in the ZN method for staining AFB needs to be reconsidered.
Enterotoxigenic Profiles of Food-Poisoning and Food-Borne
Bacillus cereus
Strains
Authors: Guinebretière Marie-Hélène , Broussolle Véronique , Nguyen-The Christophe
Published: October 2002

The enterotoxigenic profiles of 51
B. cereus
food-related strains were compared to those of 37
B. cereus
food-poisoning strains.
cytK
and association of
hbl-nhe-cytK
enterotoxin genes were more frequent among diarrheal strains (73 and 63%) than among food-borne strains (37 and 33%). Unlike diarrheal strains, food-borne strains showed frequent
nhe
and
hbl
gene polymorphisms and were often low toxin producers.
DNA Hybridization Test: Rapid Diagnostic Tool for Excluding Bacterial Vaginosis in Pregnant Women with Symptoms Suggestive of Infection
Authors: Witt Armin , Petricevic Ljubomir , Kaufmann Ulrike , Gregor Hubertus , Kiss Herbert
Published: October 2002

This prospective comparative study evaluated a DNA hybridization test (Affirm VPIII) as an alternative to Gram stain for the diagnosis of bacterial vaginosis. We examined vaginal smears from 1,725 pregnant women between the 12th and 36th weeks of gestation with clinical signs of vaginal infection. The DNA hybridization test compared well with Gram stain and can be used as a rapid diagnostic tool to exclude bacterial vaginosis.
Preliminary Evaluation of an Immunochromatographic Strip Test for SpecificTreponema pallidumAntibodies
Authors: Zarakolu Pinar , Buchanan Ian , Tam Milton , Smith Kim , Hook Edward W.
Published: October 2002

We evaluated a prototype immunochromatographic strip (ICS) test for qualitative detection ofTreponema pallidumantibodies in 353 sera from 157 patients. For sera from 43 syphilis patients, the ICSs were reactive, while for sera from 114 patients without syphilis, including 22 with biologically false-positive Rapid Plasma Reagin test results, the ICSs were nonreactive. The ICS test may expand the available options for serological testing for syphilis.
Emergence of an Influenza B Virus with Antigenic Change
Authors: Nakagawa Naoko , Nukuzuma Souichi , Haratome Shigekazu , Go Shosan , Nakagawa Toshimasa , Hayashi Kozaburo
Published: October 2002

Influenza B virus Yamagata group strains, isolated in the 2000 to 2001 influenza epidemic season, reacted poorly to the polyclonal ferret sera prepared against strains isolated earlier. The results of genetic analysis clarified that a point mutation of the nucleotide at position 126 in the HA1 region and the corresponding one-amino-acid substitution altered viral antigenicity.
Performance of an IS
6110
-Based PCR Assay and the COBAS AMPLICOR MTB PCR System for Detection of
Mycobacterium tuberculosis
Complex DNA in Human Lymph Node Samples
Authors: Rimek Dagmar , Tyagi Sachin , Kappe Reinhard
Published: October 2002

We compared the performance of two PCR assays, an IS
6110
-based in-house protocol and the COBAS AMPLICOR MTB PCR (COBAS MTB) system, for the detection of
Mycobacterium tuberculosis
complex in 43 human lymph node samples from 40 patients. For the in-house PCR and the COBAS MTB assays, respectively, sensitivities were 87.5% versus 45.5% (
P
< 0.05), specificities were 100.0% versus 91.3% (
P
> 0.05), and inhibition rates were 4.8% versus 19.5% (
P
< 0.05). For the COBAS MTB system, additional
N
-acetyl-
l
-cysteine-NaOH pretreatment of the samples changed neither the inhibition rate nor the sensitivity significantly.
Genotyping of Methicillin-Resistant
Staphylococcus aureus
by Assaying for the Presence of Variable Elements Associated with
mecA
Authors: Huygens Flavia , Nimmo Graeme R. , Schooneveldt Jacqueline , Munckhof Wendy J. , Giffard Philip M.
Published: October 2002

The region surrounding
mecA
in methicillin-resistant
Staphylococcus aureus
(MRSA) is highly variable. We describe an approach for the rapid genotyping of MRSA by assaying for the presence or absence of variable or mobile elements previously shown to be associated with the
mecA
region.
Sicca Syndrome Associated with
Tropheryma whipplei
Intestinal Infection
Authors: Bosman Cesare , Boldrini Renata , Borsetti Giuliana , Morelli Sergio , Paglia Maria Grazia , Visca Paolo
Published: October 2002

The case of a 61-year-old woman with Whipple's disease-associated sicca complex is reported.
Tropheryma whipplei
infection was diagnosed by histological and ultrastructural examination of the jejunal mucosa and sequence analysis of the bacterial 16S ribosomal DNA. The role of vitamin A malabsorption in sicca complex secondary to Whipple's disease is discussed.
Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells
Authors: Green Kim Y. , Mory Aaron , Fogg Mark H. , Weisberg Andrea , Belliot Gaël , Wagner Mariam , Mitra Tanaji , Ehrenfeld Ellie , Cameron Craig E. , Sosnovtsev Stanislav V.
Published: October 2002

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative “3A-like” protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.
Immunogenicity and Tolerogenicity of Hepatitis B Virus Structural and Nonstructural Proteins: Implications for Immunotherapy of Persistent Viral Infections
Authors: Kakimi Kazuhiro , Isogawa Masanori , Chung JoSan , Sette Alessandro , Chisari Francis V.
Published: October 2002

Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8+T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8+cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.
Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
Authors: Nishino Yoshii , Kobasa Darwyn , Rubin Steven A. , Pletnikov Mikhail V. , Carbone Kathryn M.
Published: October 2002

Borna disease virus (BDV) infection produces a variety of clinical diseases, from behavioral illnesses to classical fatal encephalitis (i.e., Borna disease [BD]). Since the genomes of most BDV isolates differ by less than 5%, host factors are believed responsible for much of the reported variability in disease expression. The contribution of BDV genomic differences to variation in BD expression is largely unexplored. Here we compared the clinical outcomes of rats infected with one of two related BDV variants, CRP3 or CRNP5. Compared to rats inoculated with CRP3, adult and newborn Lewis rats inoculated with CRNP5 had more severe and rapidly fatal neurological disease, with increased damage to the hippocampal pyramidal neurons and rapid infection of brain stem neurons. To identify possible virus-specific contributions to the observed variability in disease outcome, the genomes of CRP3 and CRNP5 were sequenced. Compared to CRP3, there were four nucleotide changes in the CRNP5 variant, two each in the G protein and in the L polymerase, resulting in four amino acid changes. These results suggest that small numbers of genomic differences between BDV variants in the G protein and/or L polymerase can contribute to the variability in BD outcomes.
Cleavage of Influenza A Virus Hemagglutinin in Human Respiratory Epithelium Is Cell Associated and Sensitive to Exogenous Antiproteases
Authors: Zhirnov Oleg P. , Ikizler Mine R. , Wright Peter F.
Published: October 2002

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [
35
S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 (“cleavage from within”) and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions (“cleavage from without”), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.
Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin
Authors: Lei Pedro , Bajaj Bharat , Andreadis Stelios T.
Published: October 2002

Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of Polybrene (PB) in these interactions. We found that gp70 is not involved in retrovirus binding to FN. Immobilization of the virus, however, does not overcome its receptor requirement, and gp70 is still needed for successful gene transfer. Our results clearly show that retrovirus binds FN through virus-associated heparan sulfate (HS) and that binding is necessary for transduction without PB. Two distinct modes of gene transfer occur depending on PB: (i) in the presence of PB, retrovirus interacts directly with the target cells; and (ii) in the absence of PB, retrovirus binds to FN and target cells interact with the immobilized virus. PB may promote the former mode by interacting with the virus HS and reducing the negative charge of the viral particles. Interestingly, the latter mode is more efficient, leading to significantly enhanced gene transfer. A better understanding of these interactions may provide insight into virus-cell interactions and lead to a more rational design of transduction protocols.
Role of an Essential Triloop Hairpin and Flanking Structures in the 3′ Untranslated Region of
Alfalfa Mosaic Virus
RNA in In Vitro Transcription
Authors: Olsthoorn René C. L. , Bol John F.
Published: October 2002

The minus-strand promoter of
Alfalfa mosaic virus
(AMV), a tripartite plant virus belonging to the family
Bromoviridae
, is located within the 3′-terminal 145 nucleotides (nt), which can adopt a tRNA-like structure (TLS). This contrasts with the subgenomic promoter for RNA4 synthesis, which requires ∼40 nt and forms a single triloop hairpin. Detailed analysis of the minus-strand promoter now shows that a similar triloop hairpin, hairpin E (hpE), is crucial for minus-strand synthesis. The loop sequence of hpE appeared to not be essential for RNA synthesis, whereas the identity and base-pairing capability of bases below the triloop were indeed essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site similar to the process of subgenomic transcription. Similar effects were observed when deleting (part of) the TLS, suggesting that tertiary contacts between hpE and the TLS prevent internal initiation. The data indicate that the minus-strand promoter hpE and the subgenomic promoter hairpin are equivalent in binding the viral polymerase. We propose that the major role of the TLS is to enforce the initiation of transcription by polymerase at the very 3′ end of the genome.
Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes
Authors: Ritzenthaler C. , Laporte C. , Gaire F. , Dunoyer P. , Schmitt C. , Duval S. , Piéquet A. , Loudes A. M. , Rohfritsch O. , Stussi-Garaud C. , Pfeiffer P.
Published: October 2002

Infection by
Grapevine fanleaf nepovirus
(GFLV), a bipartite RNA virus of positive polarity belonging to the
Comoviridae
family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.
Enhanced Cytomegalovirus Infection of Developing Brain Independent of the Adaptive Immune System
Authors: van den Pol Anthony N. , Reuter Jon D. , Santarelli Justin G.
Published: October 2002

Cytomegalovirus (CMV) has been suggested as the most prevalent infectious agent causing neurological dysfunction in the developing brain; in contrast, CMV infections are rare in the adult brain. One explanation generally given for the developmental susceptibility to the virus is that the developing immune system is too immature to protect the central nervous system from viral infection, but as the immune system develops it can protect the brain. We suggest an alternate view: that developing brain cells are inherently more susceptible to CMV infection, independent of the immune system. We used a recombinant mouse CMV that leads to green fluorescent protein expression in infected cells. Control experiments demonstrated a high correlation between the number of cells detected with the viral GFP reporter gene and with immunocytochemical detection of the virus. After intracerebral inoculation, the number of CMV-infected cells in neonatal brains was many times greater than in mature control or mature immunodepressed SCID mice, and the mortality rate of neonates was substantially greater than SCID or control adults. Parallel experiments with live brain slices inoculated in vitro, done in the absence of the systemic immune system, generated similar data, with immature hippocampus, hypothalamus, cortex, striatum, and cerebellum showing substantially greater numbers of infected cells (100-fold) than found in adult slices in these same regions. Interestingly, in the cerebellar cortex, CMV-infected cells were more prevalent in the postmitotic Purkinje cell layer than in the mitotic granule cell layer, suggesting a selective infection of some cell types not dependent on cell division. Together, these data support the view that CMV has an intrinsic preference for infection of developing brain cells, independent, but not mutually exclusive, of the developmental status of the systemic immune system in controlling CMV infection.