bluuebunny/crossref_metadata_2025 huggingface dataset:
ccfA
, the Genetic Determinant for the cCF10 Peptide Pheromone in
Enterococcus faecalis
OG1RF
Authors: Antiporta Michelle H. , Dunny Gary M.
Published: July 2002

The nosocomial pathogen
Enterococcus faecalis
has a unique pheromone-inducible conjugative mating system. Conjugative transfer of the
E. faecalis
plasmid pCF10 is specifically induced by the cCF10 peptide pheromone (LVTLVFV). Genomic sequence information has recently allowed the identification of putative structural genes coding for the various enterococcal pheromones (D. B. Clewell et al., Mol. Microbiol. 35:246-247, 2000). The cCF10 pheromone sequence LVTLVFV was found within an open reading frame designated
ccfA
, encoding a putative lipoprotein precursor. Several other pheromone sequences were found in similar locations within other predicted lipoproteins. CcfA shows significant sequence relatedness to the
Escherichia coli
protein YidC, an inner membrane protein translocase, as well as to a large number of homologs identified in gram-positive and in gram-negative bacteria. Analysis of the deduced CcfA amino acid sequence suggested that mature cCF10 peptide could be formed from the proteolytic degradation of its signal peptide. Expression of the cloned
ccfA
gene with an inducible expression vector dramatically increased cCF10 production by
E. faecalis
and also resulted in cCF10 production by
Lactococcus lactis
, a non-pheromone producer. Site-directed mutagenesis of the
ccfA
sequence encoding the cCF10 peptide confirmed that
ccfA
was a functional genetic determinant for cCF10.
Demonstration that
fbiC
Is Required by
Mycobacterium bovis
BCG for Coenzyme F
420
and FO Biosynthesis
Authors: Choi Kwang-Pil , Kendrick Nathan , Daniels Lacy
Published: July 2002

Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated
Mycobacterium bovis
strain BCG (
M. bovis
) mutants that could not make coenzyme F
420
were identified. Four independent mutants that could not make F
420
or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the
M. bovis
homologue of the
Mycobacterium tuberculosis
gene Rv1173, which we have named
fbiC
. Complementation of an
M. bovis
FbiC
−
mutant with
fbiC
restored the F
420
phenotype. These data demonstrate that
fbiC
is essential for F
420
production and that FbiC participates in a portion of the F
420
biosynthetic pathway between pyrimidinedione and FO. Homologues of
fbiC
were found in all 11 microorganisms that have been fully sequenced and that are known to make F
420
. Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the
M. bovis
FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the
M. bovis
FbiC. Histidine-tagged FbiC overexpressed in
Escherichia coli
produced a fusion protein of the molecular mass predicted from the
M. bovis
BCG sequence (∼95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.
Identification of an Akinete Marker Gene in
Anabaena variabilis
Authors: Zhou Ruanbao , Wolk C. Peter
Published: July 2002

Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.
Multilocus Sequence Typing System for
Campylobacter jejuni
Authors: Dingle K. E. , Colles F. M. , Wareing D. R. A. , Ure R. , Fox A. J. , Bolton F. E. , Bootsma H. J. , Willems R. J. L. , Urwin R. , Maiden M. C. J.
Published: July 2002

The gram-negative bacterium
Campylobacter jejuni
has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194
C. jejuni
isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (
http://mlst.zoo.ox.ac.uk
), forming a virtual isolate collection which could be continually expanded. These data indicated that
C. jejuni
is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of
C. jejuni
epidemiology to be established and will permit more detailed studies of the population genetics of this organism.
Epidemiology and Infection Control Implications of
Acinetobacter
spp. in Hong Kong
Authors: Houang Elizabeth T. S. , Chu Y. W. , Leung C. M. , Chu K. Y. , Berlau J. , Ng K. C. , Cheng A. F. B.
Published: July 2002

In a previous study, we showed that
Acinetobacter
genomic DNA group 3 was the most common species among blood culture isolates and was commonly found on superficial carriage sites of the healthy and the sick, which are different findings from those reported in Europe and North America. We used amplified ribosomal DNA restriction analysis and pulsed-field gel electrophoresis to study further the molecular epidemiology of acinetobacters in our region. Over a study period of 6 weeks with 136 consecutive routine clinical isolates (1.33% of all specimens), genomic DNA groups 2 (
Acinetobacter baumannii
), 3, and 13TU were obtained from 59 of 69 positive patients. There is a significant difference in the specimen sources of the three genomic DNA groups, with group 13TU being significantly associated with the respiratory tract (chi-square exact test,
P
= 0.0064). Settle plates showed a significantly heavier environmental load from the intensive care unit (ICU) than from the four surgical wards examined (22 of 70 versus 76 of 120 plates with <5 colonies; chi-square test,
P <
0.0001). Genomic group 3 accounted for 6 of 12 clusters of possibly related strains among patients, between patients and the ICU environment, and in the ICU environment. Genomic groups 2 and 3 accounted for 21% of the 132 genomically identified isolates recovered from 21 of 41 local vegetables, 53 of 74 fish and meat samples, and 22 of 60 soil samples. Group 13TU was present only in patients' immediate surroundings. The role played by the environment and by human carriage should be evaluated in order to devise a cost-effective infection control program pertinent to our situation of acinetobacter endemicity.
Evaluation of the Triage Micro Parasite Panel for Detection of
Giardia lamblia
,
Entamoeba histolytica/Entamoeba dispar
, and
Cryptosporidium parvum
in Patient Stool Specimens
Authors: Sharp Susan E. , Suarez Clarisa A. , Duran Yolanda , Poppiti Robert J.
Published: July 2002

A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for
Giardia lamblia
, four were positive for
Entamoeba histolytica/E. dispar
, and three were positive for
Cryptosporidium parvum
as determined by both methods. There was one false positive by Triage (
C. parvum
) and four false negatives by O&P (two
G. lamblia
, one
E. histolytica/E. dispar
, and one
C. parvum
). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of
G. lamblia
,
E. histolytica/E. dispar
, and
C. parvum
in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than
G. lamblia
,
E. histolytica/E. dispar
, or
C. parvum
are suspected.
Automated High-Throughput Genotyping for Study of Global Epidemiology of
Mycobacterium tuberculosis
Based on Mycobacterial Interspersed Repetitive Units
Authors: Supply Philip , Lesjean Sarah , Savine Evgueni , Kremer Kristin , van Soolingen Dick , Locht Camille
Published: July 2002

Large-scale genotyping of
Mycobacterium tuberculosis
is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of
M. tuberculosis
is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607–2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for
M. tuberculosis
complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of
M. tuberculosis
complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of
M. tuberculosis
MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.
PCR Fingerprinting of
Candida albicans
Associated with Chronic Hyperplastic Candidosis and Other Oral Conditions
Authors: Bartie Kerry L. , Williams David W. , Wilson Melanie J. , Potts A. John C. , Lewis Michael A. O.
Published: July 2002

The purpose of this study was to genotype strains of
Candida albicans
to determine whether specific types were associated with chronic hyperplastic candidosis (CHC). A total of 67 candidal isolates from CHC patients (
n
= 17) and from patients with other oral conditions (
n
= 21) were genotyped by PCR fingerprinting employing two interrepeat primer combinations (1245 and 1246 primers or 1251 primer) and a single minisatellite-specific M13 primer. The most suitable primer for fingerprint analysis was found to be primer 1251, yielding well-resolved banding patterns. For the 67 isolates tested, PCR fingerprinting delineated 25 (1245 and 1246 primers), 27 (1251 primer), and 25 (M13 primer) profiles. The majority of
C. albicans
isolates from multiple sites within the mouth produced identical profiles (six out of nine subjects examined). For patients for whom a series of longitudinal isolates was available, strain persistence for up to 7 years was evident for five out of eight individuals, despite episodes of antifungal therapy. Computer-assisted comparison of the interrepeat PCR fingerprints identified seven distinct profiles that were shared among isolates from different individuals. However, no association was evident among isolates of
C. albicans
from specific clinical conditions. Eight isolates that were initially identified as
C. albicans
but having atypical PCR profiles were later confirmed as
Candida dubliniensis
. In conclusion, the genotypic data do not indicate clonal restriction of
C. albicans
with respect to CHC. Furthermore, these results have demonstrated that in the majority of individuals, colonizing populations of
C. albicans
are clonal in nature and exhibit strain persistence.
Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of
Babesia microti
-Like Parasite
Authors: Tsuji Masayoshi , Wei Qiang , Zamoto Aya , Morita Chiharu , Arai Satoru , Shiota Tsunezo , Fujimagari Masato , Itagaki Asao , Fujita Hiromi , Ishihara Chiaki
Published: July 2002

We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species,
Apodemus speciosus
,
Apodemus argenteus
,
Clethrionomys rufocanus
,
Eothenomys smithii
,
Crocidura dsinezumi
, and
Sorex unguiculatus
, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring
Babesia microti
-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from
A. speciosus
, and the other 1 was from
C. rufocanus
. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States
B. microti
(U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of
B. microti
-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that
A. speciosus
serves as a major reservoir for both Kobe- and Hobetsu-type
B. microti
-like parasites, and that
C. rufocanus
may also be an additional reservoir on Hokkaido Island.
European Proficiency Testing Program for Molecular Detection and Quantitation of Hepatitis B Virus DNA
Authors: Valentine-Thon Elizabeth , van Loon Anton M. , Schirm Jurjen , Reid Jim , Klapper Paul E. , Cleator Graham M.
Published: July 2002

External quality control of hepatitis B virus (HBV) DNA detection remains an important issue. This study reports and compares the results obtained from two different proficiency panels for both the qualitative and quantitative assessment of HBV DNA. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica, Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each contained two negative samples and six positive samples with 10
3
to 10
7
copies/ml (panel 1) or 10
3
to 2 × 10
6
copies of HBV DNA per ml (panel 2). For panel 1, 42 laboratories submitted 20 qualitative (all in-house PCRs) and 37 quantitative (87% commercial assays) data sets. For panel 2, 51 laboratories submitted 25 qualitative (all in-house PCRs) and 47 quantitative (94% commercial assays) data sets. Five data sets (8.8%) in panel 1 and two data sets (2.8%) in panel 2 contained totals of six and two false-positives, respectively, corresponding to false-positive result rates of 5.3% for panel 1 and 1.4% for panel 2. The false-negative result rates of 10.5% for panel 1 and 17.4% for panel 2 were dependent on the detection levels of the assays employed as well as panel composition. In the qualitative analysis of all data sets, 47.4% (panel 1) and 51.4% (panel 2) had all samples correct. An adequate or better score (all correct or only the weak-positive sample missed) was obtained with 77.2% of the panel 1 samples and 68.1% of the panel 2 samples. In the quantitative analysis, 57.1% (panel 1) and 42.6% (panel 2) of the data sets achieved an adequate or better score (positive results within the acceptable range of the geometric mean ± 0.5 log
10
of all positive results). These results demonstrate that while the qualitative performance of HBV detection has considerably improved compared to that of a previously published HBV proficiency study, the detection levels of many commercial quantitative assays are still too high to allow adequate quantitation of all relevant clinical samples.
PCR-Based Diagnosis of
Helicobacter pylori
Infection and Real-Time Determination of Clarithromycin Resistance Directly from Human Gastric Biopsy Samples
Authors: Chisholm Stephanie A. , Owen Robert J. , Teare E. Louise , Saverymuttu Seth
Published: July 2002

A novel PCR detection assay that amplifies the
Helicobacter pylori
-specific vacuolating cytotoxin gene (
vacA
) and thus enables rapid diagnosis of infection is described. Additionally, a real-time probe hybridization melting point analysis assay to detect all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S
rrn
gene showed that the
vacA
assay was sensitive and specific when tested on biopsy samples from 121 patients. Clarithromycin susceptibilities could be determined in the majority (92.3%) of culture-positive gastric biopsy samples analyzed, four of which generated melting peaks indicative of clarithromycin resistance by either an A→G or A→C mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of matched cultures. This PCR-based system was simple to perform and could be completed in 3 to 4 h, thereby overcoming the delays associated with conventional culture methods for
H. pylori
identification and susceptibility testing.
Evaluation of PCR Methods for Rapid Identification and Differentiation of
Streptococcus uberis
and
Streptococcus parauberis
Authors: Hassan A. A. , Khan I. U. , Abdulmawjood A. , Lämmler C.
Published: July 2002

Streptococcus uberis
and
Streptococcus parauberis
reference strains and isolates obtained from routine diagnostics were investigated by PCR with oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene, the 23S rRNA gene, and the 16S-23S rRNA intergenic spacer region of both species. All three primer pairs allowed an identification of 67 isolates as
S. uberis
and 4 isolates as
S. parauberis
.
Characterization of AfaE Adhesins Produced by Extraintestinal and Intestinal Human
Escherichia coli
Isolates: PCR Assays for Detection of Afa Adhesins That Do or Do Not Recognize Dr Blood Group Antigens
Authors: Le Bouguénec Chantal , Lalioui Lila , du Merle Laurence , Jouve Mabel , Courcoux Pascale , Bouzari Saeid , Selvarangan Rangaraj , Nowicki Bogdan J. , Germani Yves , Andremont Antoine , Gounon Pierre , Garcia Marie-Isabelle
Published: July 2002

Operons of the
afa
family are expressed by pathogenic
Escherichia coli
strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048–5059, 1999) was used to develop a new PCR assay for detecting all the operons of the
afa
family with a single genetic tool. This PCR approach was validated by investigating three collections of human
E. coli
isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the
afa
operons are variable, particularly with respect to the primary sequence encoded by the
afaE
gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr
+
adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr
−
adhesins) do not. Thus, the
afa
operons detected in this study were characterized by subtyping the
afaE
gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The
afaE8
subtype (Afa/Dr
−
adhesin) was found to predominate in
afa
-positive isolates from sepsis patients (75%); it was frequent in
afa
-positive pyelonephritis
E. coli
(55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr
+
strains (regardless of the
afaE
subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in
afa
-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by
afa
-positive strains.
Evaluation of the Roche
Neisseria gonorrhoeae
16S rRNA PCR for Confirmation of AMPLICOR PCR-Positive Samples and Comparison of Its Diagnostic Performance According to Storage Conditions and Preparation of Endocervical Specimens
Authors: Van Dyck E. , Smet H. , Van Damme L. , Laga M.
Published: July 2002

The AMPLICOR PCR was used to detect
Neisseria gonorrhoeae
in endocervical specimens. A 16S rRNA PCR performed on
N. gonorrhoeae-
positive samples showed sensitivities of 73.2, 64.3, and 94.4% for samples treated directly with AMPLICOR lysis buffer, samples suspended in 2-sucrose phosphate, and samples suspended in diluted phosphate-buffered saline, respectively.
Biofilm Formation by Gram-Negative Bacteria on Central Venous Catheter Connectors: Effect of Conditioning Films in a Laboratory Model
Authors: Murga R. , Miller J. M. , Donlan R. M.
Published: July 2002

Human blood components have been shown to enhance biofilm formation by gram-positive bacteria. We investigated the effect of human blood on biofilm formation on the inner lumen of needleless central venous catheter connectors by several gram-negative bacteria, specifically
Enterobacter cloacae, Pseudomonas aeruginosa
, and
Pantoea agglomerans
. Results suggest that a conditioning film of blood components promotes biofilm formation by these organisms in an in vitro system.
Basidiobolus ranarum
as an Etiologic Agent of Gastrointestinal Zygomycosis
Authors: Khan Z. U. , Khoursheed M. , Makar R. , Al-Waheeb S. , Al-Bader I. , Al-Muzaini A. , Chandy R. , Mustafa A. S.
Published: July 2002

Basidiobolus ranarum
is a known cause of subcutaneous zygomycosis. Recently, its etiologic role in gastrointestinal infections has been increasingly recognized. While the clinical presentation of the subcutaneous disease is quite characteristic and the disease is easy to diagnose, gastrointestinal basidiobolomycosis poses diagnostic difficulties; its clinical presentation is nonspecific, there are no identifiable risk factors, and all age groups are susceptible. The case of gastrointestinal basidiobolomycosis described in the present report occurred in a 41-year-old Indian male who had a history of repair of a left inguinal hernia 2 years earlier and who is native to the southern part of India, where the subcutaneous form of the disease is indigenous. Diagnosis is based on the isolation of
B. ranarum
from cultures of urine and demonstration of broad, sparsely septate hyphal elements in histopathologic sections of the colon, with characteristic eosinophilic infiltration and the Splendore-Hoeppli phenomenon. The titers of both immunoglobulin G (IgG) and IgM antibodies to locally produced antigen of the fungus were elevated. The patient failed to respond to 8 weeks of amphotericin B therapy, and the isolate was later found to be resistant to amphotericin B, itraconazole, fluconazole, and flucytosine but susceptible to ketoconazole and miconazole. One other noteworthy feature of the fungus was that the patient's serum showed raised levels of Th2-type cytokines (interleukins 4 and 10) and tumor necrosis factor alpha. The present report underscores the need to consider gastrointestinal basidiobolomycosis in the differential diagnosis of inflammatory bowel diseases and suggests that, perhaps, more time should be invested in developing standardized serologic reagents that can be used as part of a less invasive means of diagnosis of the disease.
Evaluation of BACTEC MYCO/F Lytic Medium for Recovery of Mycobacteria, Fungi, and Bacteria from Blood
Authors: Fuller DeAnna D. , Davis Thomas E. , Denys Gerald A. , York Mary K.
Published: July 2002

MYCO/F Lytic medium (MFL), a liquid medium developed for use with the BACTEC 9240 blood culture system, was compared to the Isolator system (IS) for the recovery of fungi and to the BACTEC 13A medium for the recovery of mycobacteria. Recovery of bacteria was compared to routine BACTEC Plus Aerobic/F (AF) blood cultures. Microbial growth was detected in 203 (17%) of 1,166 blood cultures. Fifty-seven specimens were positive for fungi: 35 were positive with both IS and MFL; six were positive with IS only (three
Candida albicans
, one
Histoplasma capsulatum
, one
Candida glabrata
, and one
Fusarium
species isolate); three were positive with AF only (two
C. albicans
and one
Candida parapsilosis
isolate); and 13 were positive with MFL only (five
C. glabrata
, three
C. albicans
, two
Candida krusei
, two
Candida tropicalis
, and one
C. parapsilosis
isolate;
P
> 0.05 versus IS). Eighteen of 19 blood cultures positive for
H. capsulatum
grew in both IS and MFL, although the time to detection for MFL was greater. The mean time to detection for all fungi was 8.15 days for IS and 12.07 days for MFL. Seven hundred forty specimens were also cultured for mycobacteria with MFL and 13A. Forty-four grew mycobacteria; 38 were positive with both 13A and MFL; and 16 were positive with MFL only.
Mycobacterium avium
was recovered from 41 specimens; 36 were positive for both systems and 5 were positive for MFL alone. MFL was also compared to the AF bottle for the same 740 specimens. MFL and AF both detected 34 of the 40 clinically significant bacteria, while IS detected only 15 of 40. In summary, MFL is an excellent medium for the recovery of fungi, mycobacteria, and bacteria; however, the time to detection of
H. capsulatum
is increased.
Seven Human Immunodeficiency Virus (HIV) Antigen-Antibody Combination Assays: Evaluation of HIV Seroconversion Sensitivity and Subtype Detection
Authors: Ly Thoai Duong , Martin Lynn , Daghfal David , Sandridge Arnold , West Daniel , Bristow Richard , Chalouas Laurence , Qiu Xiaoxing , Lou Sheng C. , Hunt Jeffrey C. , Schochetman Gerald , Devare Sushil G.
Published: July 2002

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at ∼25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was ∼125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.
Antibiotic-Resistant Cell-Detaching
Escherichia coli
Strains from Nigerian Children
Authors: Okeke Iruka N. , Steinrück Hartmut , Kanack Kristen J. , Elliott Simon J. , Sundström Lars , Kaper James B. , Lamikanra Adebayo
Published: July 2002

The properties of 23 cell-detaching
Escherichia coli
strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding α-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching
E. coli
strains may represent an important reservoir for antibiotic resistance genes.
Frequency of Isolation of
Staphylococcus lugdunensis
in Consecutive Urine Cultures and Relationship to Urinary Tract Infection
Authors: Haile D. T. , Hughes J. , Vetter E. , Kohner P. , Snyder R. , Patel R. , Cockerill F. R.
Published: July 2002

Recent reports associate
Staphylococcus lugdunensis
with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as
S. lugdunensis
. In no case was
S. lugdunensis
isolated in pure culture; 29 (94%) of 31
S. lugdunensis
isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against
S. lugdunensis. S. lugdunensis
may be an unrecognized yet infrequent cause of urinary tract infection.
Genomic Variation of
Bartonella henselae
Strains Detected in Lymph Nodes of Patients with Cat Scratch Disease
Authors: Zeaiter Zaher , Fournier Pierre-Edouard , Raoult Didier
Published: July 2002

Bartonella henselae
is the primary agent of cat scratch disease (CSD). In order to study the genetic variation of
B. henselae
and the correlation of the various genotypes with epidemiological and clinical findings, two seminested,
groEL
- and
pap31
-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative
Bartonella
species. Compared to our reference intergenic spacer region-based PCR, the
groEL
- and
pap31
-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with
B. henselae
; neither
Bartonella clarridgeiae
nor other species were detected. Based on the
groEL
and
pap31
sequences,
B. henselae
amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of
B. henselae
in patients with CSD.
Food-Borne Outbreak of Gastroenteritis Associated with Genogroup I Calicivirus
Authors: Johansson P. J. Hugo , Torvén Maria , Hammarlund Ann-Christin , Björne Ulla , Hedlund Kjell-Olof , Svensson Lennart
Published: July 2002

An outbreak of gastroenteritis affecting 158 of 219 (72%) guests and employees at a hotel is described. Food served at the hotel restaurant is believed to have been the source of the outbreak and to have been contaminated by sick employees working in the restaurant. A secondary attack rate of 22% was seen involving 43 persons in all. In stool specimens from seven of eight patients, Norwalk-like viruses (NLVs) were detected by electron microscopy. While NLV-specific PCR using primers JV12 and JV13 were negative, all specimens examined with primers NVp69 and NVp110 were positive. The failure of primers JV12 and JV13 was attributed to several mismatches in the JV12 primer. Genotyping and sequence analysis revealed that all samples had identical sequences and clustered with genogroup I, and the most closely related well-characterized genotype is Desert Shield. This is the first described food-borne outbreak associated with genogroup I virus in Sweden.
Evaluation of INNO-LIA Syphilis Assay as a Confirmatory Test for Syphilis
Authors: Hagedorn Hans-Jochen , Kraminer-Hagedorn Anna , De Bosschere Katrien , Hulstaert Frank , Pottel Hans , Zrein Maan
Published: July 2002

We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from
Treponema pallidum
proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the
T. pallidum
hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent
T. pallidum
absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (
P
= 0.021 and
P
< 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques.
Automated Extraction of Genomic DNA from Medically Important Yeast Species and Filamentous Fungi by Using the MagNA Pure LC System
Authors: Loeffler Juergen , Schmidt Kathrin , Hebart Holger , Schumacher Ulrike , Einsele Hermann
Published: July 2002

A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (
n
= 28) of serially diluted
Aspergillus fumigatus
conidia (10
5
to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p ≤ 0.004). DNA from all fungal species analyzed could be extracted and amplified by real-time PCR. Negative controls from all MagNA Pure isolations remained negative. Sixty-three clinical samples showed identical results by both methods, whereas in 4 of 67 samples, discordant results were obtained. Thus, the MagNA Pure LC technique offers a fast protocol for automated DNA isolation from numerous fungi, revealing high sensitivity and purity.
Botulism Due to
Clostridium baratii
Type F Toxin
Authors: Harvey Sydney M. , Sturgeon Joan , Dassey David E.
Published: July 2002

Botulism results from consumption of preformed toxin or in vivo toxin elaboration in wounds or intestine. Of U.S. food-borne botulism cases since 1950, the majority were due to toxin A, but a significant number of suspect cases were never confirmed by culture or toxin detection. We report here a possible case of food-borne botulism attributed to toxin F production by a
Clostridium baratii
organism isolated from food consumed by the patient. The isolation of a toxin-producing
Clostridium
species other than
Clostridium botulinum
from food and stool requires deviation from the usual laboratory protocols, which may account for the lack of complete laboratory confirmation of clinically diagnosed cases.
Asynchronous Accumulation of
Lettuce Infectious Yellows Virus
RNAs 1 and 2 and Identification of an RNA 1
trans
Enhancer of RNA 2 Accumulation
Authors: Yeh Hsin-Hung , Tian Tongyan , Rubio Luis , Crawford Brett , Falk Bryce W.
Published: July 2002

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite
Crinivirus
,
Lettuce infectious yellow virus
(LIYV; family
Closteroviridae
). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the
trans
accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a
trans
enhancer for RNA 2 accumulation.
Isolation of Human Immunodeficiency Virus Type 1 Cores: Retention of Vpr in the Absence of p6

gag
Authors: Accola Molly A. , Öhagen Åsa , Göttlinger Heinrich G.
Published: July 2002

Mature human immunodeficiency virus type 1 (HIV-1) virions contain a typically cone-shaped core that encases the viral genome. In this study, we established conditions which allowed the efficient isolation of morphologically intact HIV-1 cores from virions. The isolated cores consisted mostly of cones which appeared uniformly capped at both ends but were heterogeneous with respect to the shape of the broad cap as well as the dimensions and angle of the cone. Vpr, a nonstructural virion component implicated in the nuclear import of the viral genome, was recovered in core preparations of HIV-1 and simian immunodeficiency viruses from African green monkeys. Unexpectedly, p6

gag

, a structural protein required for the incorporation of Vpr, was absent from HIV-1 core preparations. Taken together, our results indicate that the incorporation of Vpr into the virion core is a conserved feature of primate lentiviruses and that the interactions required for the uptake of Vpr into assembling particles differ from those which confine Vpr within the core.
Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes
Authors: Molinier-Frenkel Valérie , Gahery-Segard Hanne , Mehtali Majid , Le Boulaire Christophe , Ribault Sébastien , Boulanger Pierre , Tursz Thomas , Guillet Jean-Gérard , Farace Françoise
Published: July 2002

We previously demonstrated that a single injection of 109PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.
Identification of a Minimal Size Requirement for Termination of Vesicular Stomatitis Virus mRNA: Implications for the Mechanism of Transcription
Authors: Whelan Sean P. J. , Barr John N. , Wertz Gail W.
Published: July 2002

The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3′-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5′ modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with
Vesicular stomatitis virus
, the prototypic
Rhabdovirus
, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.
Development of Minimal Lentivirus Vectors Derived from Simian Immunodeficiency Virus (SIVmac251) and Their Use for Gene Transfer into Human Dendritic Cells
Authors: Mangeot Philippe-Emmanuel , Nègre Didier , Dubois Bertrand , Winter Arend J. , Leissner Philippe , Mehtali Majid , Kaiserlian Dominique , Cosset François-Loïc , Darlix Jean-Luc
Published: July 2002

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5′ cytomegalovirus early promoter, the 5′ viral sequences up to the 5′ end ofgagrequired for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3′ long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 107transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, asinvector was designed by deleting 151 nucleotides in the 3′ LTR U3 region, and this SIVsinvector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34+cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.
Endocytosis and Nuclear Trafficking of Adeno-Associated Virus Type 2 Are Controlled by Rac1 and Phosphatidylinositol-3 Kinase Activation
Authors: Sanlioglu Salih , Benson Peter K. , Yang Jusan , Atkinson E. Morrey , Reynolds Thomas , Engelhardt John F.
Published: July 2002

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that causes no currently known pathology in humans. Despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. In this study, we have examined endocytic and intracellular trafficking of AAV-2 using fluorescent (Cy3)-conjugated viral particles and molecular techniques. Our results demonstrate that internalization of heparan sulfate proteoglycan-bound AAV-2 requires αVβ5 integrin and activation of the small GTP-binding protein Rac1. Following endocytosis, activation of a phosphatidylinositol-3 (PI3) kinase pathway was necessary to initiate intracellular movement of AAV-2 to the nucleus via both microfilaments and microtubules. Inhibition of Rac1 using a dominant N17Rac1 mutant led to a decrease in AAV-2-mediated PI3 kinase activation, indicating that Rac1 may act proximal to PI3 kinase during AAV-2 infection. In summary, our results indicate that αVβ5 integrin-mediated endocytosis of AAV-2 occurs through a Rac1 and PI3 kinase activation cascade, which directs viral movement along the cytoskeletal network to the nucleus.