bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Saponin Hydrolase from
Neocosmospora vasinfecta
var.
vasinfecta
Authors: Watanabe Manabu , Sumida Naomi , Yanai Koji , Murakami Takeshi
Published: February 2004

We isolated a soybean saponin hydrolase from
Neocosmospora vasinfecta
var.
vasinfecta
PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (
sdn1
) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The
sdn1
gene was successfully expressed in
Trichoderma viride
under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [α-
l
-rhamnopyranosyl (1→2)-β-
d
-galactopyranosyl (1→2)-
d
-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates.
Phenazines and Other Redox-Active Antibiotics Promote Microbial Mineral Reduction
Authors: Hernandez Maria E. , Kappler Andreas , Newman Dianne K.
Published: February 2004

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction.Pseudomonas chlororaphisPCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures ofP. chlororaphisimplies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacteriumShewanella oneidensisMR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.
Shifts in the Membrane Fatty Acid Profile ofStreptococcus mutansEnhance Survival in Acidic Environments
Authors: Fozo Elizabeth M. , Quivey Robert G.
Published: February 2004

Acid adaptation ofStreptococcus mutansUA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of theS. mutansacid-adaptation arsenal.
Highly Stable
l
-Lysine 6-Dehydrogenase from the Thermophile
Geobacillus stearothermophilus
Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression
Authors: Heydari Mojgan , Ohshima Toshihisa , Nunoura-Kominato Naoki , Sakuraba Haruhiko
Published: February 2004

l
-Lysine dehydrogenase, which catalyzes the oxidative deamination of
l
-lysine in the presence of NAD, was found in the thermophilic bacterium
Geobacillus stearothermophilus
UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ
1
-piperideine-6-carboxylate, indicating that the enzyme is
l
-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM
l
-lysine. The enzyme was relatively selective for
l
-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The

K
m

values for
l
-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in
Escherichia coli
, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of
G. stearothermophilus
. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.
Development of Additional Selectable Markers for the Halophilic ArchaeonHaloferax volcaniiBased on theleuBandtrpAGenes
Authors: Allers Thorsten , Ngo Hien-Ping , Mevarech Moshe , Lloyd Robert G.
Published: February 2004

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry.Haloferax volcaniioffers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation ofH. volcanii leuBandtrpAgenes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuBand ΔtrpAmutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying theleuBortrpAgene under control of a constitutive promoter. The use of these selectable markers in deletion of thelhrgene ofH. volcaniiis described.
Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe
Authors: Eiden-Plach Antje , Zagorc Tatjana , Heintel Tanja , Carius Yvonne , Breinig Frank , Schmitt Manfred J.
Published: February 2004

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1andTPI1) or from inducible and/or repressible (GAL1,AOX1, andNMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.
Selection of Unusual Actinomycetal Primary σ
70
Factors by Plant-Colonizing
Frankia
Strains
Authors: Lavire Céline , Blaha Didier , Cournoyer Benoit
Published: February 2004

Functional adaptations of σ
70
transcriptional factors led to the emergence of several paralogous lineages, each one being specialized for gene transcription under particular growth conditions. Screening of a
Frankia
strain EaI-12 gene library by σ
70
DNA probing allowed the detection and characterization of a novel actinomycetal primary (housekeeping) σ
70
factor. Phylogenetic analysis positioned this factor in the RpoD cluster of proteobacterial and low-G+C-content gram-positive factors, a cluster previously free of any actinobacterial sequences. σ
70
DNA probing of
Frankia
total DNA blots and PCR screening detected one or two
rpoD
-like DNA regions per species.
rpoD
matched the conserved region in all of the species tested. The other region was found to contain
sigA
, an alternative primary factor.
sigA
appeared to be strictly distributed among
Frankia
species infecting plants by the root hair infection process. Both genes were transcribed by
Frankia
strain ACN14a grown in liquid cultures. The molecular phylogeny of the σ
70
family determined with
Frankia
sequences showed that the alternative actinomycetal factors and the essential ones belonged to the same radiation. At least seven distinct paralogous lineages were observed among this radiation, and gene transfers were detected in the HrdB actinomycetal lineage.
Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (
fabH
) and Polyhydroxyalkanoate Synthase (
phaC
) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in
Escherichia coli
JM109
Authors: Nomura Christopher T. , Taguchi Kazunori , Taguchi Seiichi , Doi Yoshiharu
Published: February 2004

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C
3
to C
5
, medium-chain-length (MCL) PHAs consist of monomer units of C
6
to C
14
, and SCL-MCL PHAs consist of monomers ranging in size from C
4
to C
14
. Although previous studies using recombinant
Escherichia coli
have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant
E. coli
. The 3-ketoacyl-acyl carrier protein synthase III gene (
fabH
) from
E. coli
was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the
Aeromonas caviae
PHA synthase gene (
phaC
), or the pPPAC plasmid, which harbors the
Pseudomonas
sp. strain 61-3 PHA synthase gene (
phaC1
), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant
fabH
genes enabled recombinant
E. coli
to induce the production of monomers of C
4
to C
10
and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.
rpoB
Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera
Streptococcus
,
Enterococcus
,
Gemella
,
Abiotrophia
, and
Granulicatella
Authors: Drancourt Michel , Roux Véronique , Fournier Pierre-Edouard , Raoult Didier
Published: February 2004

We developed a new molecular tool based on
rpoB
gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete
rpoB
gene for
Streptococcus anginosus
,
S. equinus
, and
Abiotrophia defectiva
. Sequences were aligned with these of
S. pyogenes
,
S. agalactiae
, and
S. pneumoniae
available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30
Streptococcus
,
Enterococcus
,
Gemella
,
Granulicatella
, and
Abiotrophia
species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and
Bacillus cereus
, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the
S. pneumoniae
-
S. oralis
-
S. mitis
group and the
S. agalactiae
-
S. difficile
group. These signatures allowed us to design a
S. pneumoniae
-specific PCR and sequencing primer pair.
Acquisition of High-Risk Human Papillomavirus Infections and Pap Smear Abnormalities among Women in the New Independent States of the Former Soviet Union
Authors: Syrjänen Stina , Shabalova Irena , Petrovichev Nicolay , Kozachenko Vladimir , Zakharova Tatjana , Pajanidi Julia , Podistov Jurij , Chemeris Galina , Sozaeva Larisa , Lipova Elena , Tsidaeva Irena , Ivanchenko Olga , Pshepurko Alla , Zakharenko Sergej , Nerovjna Raisa , Kljukina Ludmila , Erokhina Oksana , Branovskaja Marina , Nikitina Maritta , Grunberga Valerija , Grunberg Alexandr , Juschenko Anna , Tosi Piero , Cintorino Marcella , Santopietro Rosa , Syrjänen Kari
Published: February 2004

The rates of acquisition and the times of incident high-risk (HR) human papillomavirus (HPV) infections and Pap smear abnormalities and their predictive factors were analyzed in women participating in a multicenter screening study in three countries of the New Independent States of the former Soviet Union. The 423 patients were prospectively monitored for a mean of 21.6 months. At the baseline, 118 women were HR HPV DNA negative (Hybrid Capture II assay) and Pap smear negative (group 1), 184 were HPV DNA positive and Pap smear negative (group 2), and 121 were HPV DNA negative and Pap smear positive (group 3). The time to the acquisition of an incident abnormal Pap smear (19.4 months) was significantly longer in group 1 than in group 2 (9.2 months) (
P
= 0.0001). The times of acquisition of incident HR HPV infection were 16.6 and 11.0 months in group 1 and group 3, respectively (
P
= 0.006). The monthly rates of acquisition of incident HR HPV infections were very similar in group 1 (1.0%) and group 3 (0.8%), whereas the rate of acquisition of an abnormal Pap smear was significantly higher in group 2 (3.1%) than in group 1 (1.5%) (
P
= 0.0001). The acquisition of HR HPV infection (but not a positive Pap smear result) was significantly (
P
= 0.0001) age dependent. The only significant independent (
P
= 0.001) predictor of the incidence of an abnormal Pap smear result was a high HR HPV load of >20 relative light units/control value (CO) (rate ratio, 2.050; 95% confidence interval, 1.343 to 3.129). Independent predictors of incident HR HPV infection were patient category (a sexually transmitted disease) and ever having been pregnant. The time of acquisition of HR HPV infection was 3 months shorter than that of an abnormal Pap smear. At the baseline the high load of a particular HR HPV type is the single most important predictor of an incident Pap smear abnormality, whereas young age and having a sexually transmitted disease predict incident HR HPV infections.
High Rate of Transfer of
Staphylococcus aureus
from Parental Skin to Infant Gut Flora
Authors: Lindberg Erika , Adlerberth Ingegerd , Hesselmar Bill , Saalman Robert , Strannegård Inga-Lisa , Åberg Nils , Wold Agnes E.
Published: February 2004

Many Swedish infants carry
Staphylococcus aureus
in their intestinal microflora. The source of this colonization was investigated in 50 families. Infantile
S. aureus
strains were isolated from rectal swabs and stool samples at 3 days and at 1, 2, 4, and 8 weeks of age. The strains were identified by using the random amplified polymorphic DNA method and compared to strains from swab cultures of the mothers' hands, nipples, and nares and from the fathers' hands and nares. Maternal stool samples were also obtained at a later stage to compare infant and adult intestinal
S. aureus
colonization. Although 60% of 1-month-old children had
S. aureus
in the stools, this was true of only 24% of the mothers. The median population numbers in colonized individuals also differed: 10
6.8
CFU/g of feces among infants at 2 weeks of age versus 10
3.2
CFU/g of feces in the mothers. Of
S. aureus
strains in the stools of 3-day-old infants, 90% were identical to a parental skin strain. A total of 96% of infants whose parents were
S. aureus
skin carriers had
S. aureus
in their feces and 91% had the same strain as at least one of the parents. In comparison, only 37% of infants to
S. aureus
-negative parents had
S. aureus
in the stool samples. Thus, infantile intestinal
S. aureus
colonization was strongly associated with parental skin
S. aureus
carriage (
P
= 0.0001). These results suggest that
S. aureus
on parental skin establish readily in the infantile gut, perhaps due to poor competition from other gut bacteria.
International Clones of Methicillin-Resistant
Staphylococcus aureus
in Two Hospitals in Miami, Florida
Authors: Chung Marilyn , Dickinson Gordon , de Lencastre Herminia , Tomasz Alexander
Published: February 2004

A total of 202 methicillin-resistant
Staphylococcus aureus
(MRSA) single-patient isolates recovered between January and June 1998 in two hospitals in Miami, Florida, were characterized by a combination of several molecular typing techniques: multilocus sequence typing,
spaA
typing, pulsed-field gel electrophoresis, and determination of the structure of the SCC
mec
element. The overwhelming majority of the isolates—187of 202, or 93%—belonged to one of three internationally spread epidemic clones which were identified on the basis of their multilocus sequence type (ST) as E-MRSA-16 (ST36), the New York clone V (ST8), and the New York/Japan clone (ST5; SCC
mec
II) and its single- and double-locus variants. The rest of the isolates (15 of 202, or 7%) were more genetically diverse and were each recovered from a few patients only. Of the 23 MRSA strains isolated from confirmed human immunodeficiency virus-positive patients, as many as 17 (or 70%) belonged to a single ST8 clone carrying SCC
mec
type IV. The data provide further evidence for the conclusion of earlier studies that most MRSA disease in hospitals is caused by relatively few pandemic clones.
Polymorphism and Drug-Selected Mutations in the Protease Gene of Human Immunodeficiency Virus Type 2 from Patients Living in Southern France
Authors: Colson P. , Henry M. , Tourres C. , Lozachmeur D. , Gallais H. , Gastaut J. A. , Moreau J. , Tamalet C.
Published: February 2004

The susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease inhibitors (PI) is largely unknown. We studied HIV-2 protease genes from 21 HIV-2-infected patients who were exposed or not exposed to PI. The aim of this study was (i) to characterize the polymorphism of HIV-2 protease in the absence of drug, (ii) to know whether the HIV-2 protease gene naturally harbors HIV-1 drug resistance codons, and (iii) to identify mutations emerging under PI-selective pressure. Sixty-five HIV-2 RNA or proviral DNA samples were directly sequenced from the plasma or peripheral blood mononuclear cells of 8 patients who had received PI and 13 patients who had never received any antiretroviral. In untreated patients, the highest amino acid variability in HIV-2 protease was observed at positions 14, 40, 43, 46, 65 and 70, and seven codons (10V, 32I, 36I, 46I, 47V, 71V, and 73A) associated with drug resistance in HIV-1 were highly prevalent. In addition, at six positions (positions 7, 46, 62, 71, 90, and 99), the amino acid variability or the amino acid frequencies or both differed significantly in PI-treated and untreated patients, suggesting that mutations 7K→R, 46V→I, 62V→A/T, 71V→I, 90L→M and 99L→F were occurring under PI-selective pressure. At these positions, at least one sample simultaneously harbored both wild-type and mutated codons, while substitutions at positions 62, 71, 90, and 99 were confirmed in a longitudinal analysis. Moreover, the presence of codons 46I and 99F in the absence of drug in HIV-2 subtype B proteases may reflect natural resistance to PI. In conclusion, the present study revealed that HIV-2 strains harbor specific patterns of natural polymorphism and resistance.
Antigenic and Genetic Characterization of Serotype G2 Human Rotavirus Strains from the African Continent
Authors: Page N. A. , Steele A. D.
Published: February 2004

Serotype G2 rotavirus strains were isolated in seven countries on the African continent during 1999 and 2000. To investigate the associated DS-1 genogroup characteristics, subgroup (VP6) enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and P genotyping were performed on 10 G2 strains. The antigenic and genetic variation of the gene encoding the major neutralization glycoprotein (VP7) was also investigated by using G2-specific monoclonal antibodies and sequence analysis. Alterations in the characteristic DS-1 genogroup gene constellations were more likely to occur in the VP4 gene, and three genotypes were observed: P[4], P[6], and a dual P[4]-P[6] type. The failure of G2-specific monoclonal antibodies to type African G2 strains was more likely due to improper storage of the original stool, although G2 monotypes were detected. Phylogenetic analyses revealed clusters of serotype G2 strains that were more commonly associated with seasons during which G2 was predominant. No rotavirus vaccine trials have been conducted in an area where G2 strains were the predominant circulating serotype, and the continued surveillance of rotavirus epidemics in Africa will be preparation for future vaccine implementation in an area that clearly needs these preventative medicines.
Laboratory Evaluation of a Fully Automated Chemiluminescence Immunoassay for Rapid Detection of HBsAg, Antibodies to HBsAg, and Antibodies to Hepatitis C Virus
Authors: Ismail Nahed , Fish Geoffrey E. , Smith Michael B.
Published: February 2004

The performance of a fully automated, random access, enhanced chemiluminescence immunoassay (Ortho/ECi) for the detection of antibody to hepatitis C virus (HCV) (anti-HCV), HBsAg, and antibody to HBsAg (anti-HBsAg), in human serum was compared to a Abbott second-generation enzyme immunoassay (EIA 2.0). The Ortho/ECi assays employ an immunometric technique with enhanced chemiluminescence for optimal assay performance. With regard to the study of clinical laboratory performance, six groups of sera prescreened with Abbott EIAs were assayed: anti-HCV-negative samples (
n
= 318), anti-HCV-positive samples (
n
= 177), anti-HBsAg-negative samples (
n
= 241), anti-HBsAg-positive samples (
n
= 239), HBsAg-positive samples (
n
= 158), and HBsAg-negative samples (
n
= 312). Sera with discrepant results in the two serological assays were resolved by confirmatory tests. Sera with indeterminate results by one or more confirmatory tests were evaluated by reviewing medical records. The overall concordance between the Ortho/ECi assay and the Abbott EIA were 97.78, 93.54, and 97.66% for anti-HCV antibodies, anti-HBsAg antibodies, and HBsAg, respectively. After resolving the discrepancies, the specificities of the new assay for anti-HCV and anti-HBsAg antibodies and HBsAg were 98.1, 92.8, and 100%, respectively. The sensitivities of the new assay for anti-HCV, anti-HBsAg, and HBsAg were 100, 98.8, and 97.4%, respectively. In conclusion, The Ortho/ECi assays for diagnosis of HCV and hepatitis B virus (HBV) infections are highly specific and sensitive assays. The rapid turnaround time, random access, full automation, and high throughput make it an effective assay system for clinical laboratory diagnosis of HCV and HBV infections.
Enzyme-Linked Immunosorbent Assay forHelicobacter pyloriNeeds Adjustment for the Population Investigated
Authors: Hoang Thi Thu Ha , Wheeldon Thuc-Uyen , Bengtsson Carina , Phung Dac Cam , Sörberg Mikael , Granström Marta
Published: February 2004

Helicobacter pyloriinfection and peptic ulcer disease are common in developing countries, e.g., Vietnam. An enzyme-linked immunosorbent assay (ELISA) for screening of patients and for seroepidemiology is a useful tool but needs to be validated in the population studied. We used in-house ELISA with sonicated Swedish and Vietnamese strains as antigens to measure immunoglobulin G antibodies after absorption with sonicatedCampylobacter jejuniin sera from 270H. pyloriculture-confirmed peptic ulcer patients, 128 Swedish urea-breath test and immunoblot-positive healthy controls, and 432 Vietnamese immunoblot-positive population controls. Sonicated whole-cell antigen based on the local strains showed a significantly better performance. Immunoblot-positive peptic ulcer patients had significantly higher antibody concentrations than immunoblot-positive population controls, necessitating a lower cutoff level if serology is used for screening or epidemiological purposes. The study shows that the parameters of ELISA forH. pylorineed to be adjusted for the population being investigated.
Detection of Species-Specific
Helicobacter
Ribosomal DNA in Intestinal Biopsy Samples from a Population-Based Cohort of Patients with Ulcerative Colitis
Authors: Streutker C. J. , Bernstein C. N. , Chan V. L. , Riddell R. H. , Croitoru K.
Published: February 2004

The inflammatory bowel diseases are considered an abnormal host immune response to an environmental stimulus. Evidence suggests a role for intestinal bacteria in initiating and/or providing an ongoing stimulus for inflammation in inflammatory bowel disease.
Helicobacter pylori
is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma. Studies in various animal models, particularly mice, have identified enterohepatic
Helicobacter
species that are capable of causing hepatitis and enterocolitis. We hypothesize that
Helicobacter
species may have a role in maintaining inflammation in humans with inflammatory bowel disease. In order to investigate this, biopsy specimens were obtained from patients with and without inflammatory bowel disease. DNA was extracted from the tissues and subjected to PCR with primers designed to detect the ribosomal DNA of members of the
Helicobacter
species. DNA from six biopsy samples from 60 inflammatory bowel disease patients tested positive. This included 5 of 33 ulcerative colitis patients that were positive compared to 0 of 29 age-matched controls (
P
< 0.04). Sequencing of the bands produced by PCR amplification revealed ≥99% homology with
H. pylori
. These results indicate that a member of the
Helicobacter
species may be involved in some cases of ulcerative colitis.
Multicenter Comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A2 Reference Method for Testing New Antifungal Agents against Clinical Isolates of
Candida
spp
Authors: Espinel-Ingroff A. , Pfaller M. , Messer S. A. , Knapp C. C. , Holliday N. , Killian S. B.
Published: February 2004

A multicenter (three centers) study compared MICs obtained by the Sensititre YeastOne Colorimetric Antifungal plate to reference microdilution broth (NCCLS M27-A2 document) MICs of three new triazoles (posaconazole, ravuconazole, and voriconazole) and the echinocandin caspofungin acetate for 100 isolates of
Candida
spp. In addition, amphotericin B and fluconazole were tested as control drugs. Colorimetric MICs of caspofungin and amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first slightly purple or blue well. Two comparisons of MIC pairs by the two methods were evaluated: 24-h colorimetric MICs were compared to NCCLS MICs at 24 and at 48 h. The interlaboratory reproducibility of YeastOne and reference MICs was also examined. The best performance of the YeastOne plate was with 24-h MICs (overall, 95 to 99% agreement) for all the species and antifungal agents. These results suggest the potential value of the YeastOne plate for use in the clinical laboratory for the four new antifungal agents evaluated.
Evaluations of Commercial West Nile Virus Immunoglobulin G (IgG) and IgM Enzyme Immunoassays Show the Value of Continuous Validation
Authors: Malan Annette K. , Martins Thomas B. , Hill Harry R. , Litwin Christine M.
Published: February 2004

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.
Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting
Authors: Schuurman Tim , de Boer Richard F. , Kooistra-Smid Anna M. D. , van Zwet Anton A.
Published: February 2004

We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 × 10
2
to 2 × 10
2
CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.
Determination of Infectious Load of
Mycoplasma genitalium
in Clinical Samples of Human Vaginal Cells
Authors: Blaylock Mark W. , Musatovova Oxana , Baseman Janet G. , Baseman Joel B.
Published: February 2004

Mycoplasma genitalium
is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of
M. genitalium
has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of
M. genitalium
infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma
gyrA
and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of
M. genitalium
infections in clinical and experimental samples.
Molecular Epidemiology of Multiresistant
Streptococcus pneumoniae
with Both
erm
(B)- and
mef
(A)-Mediated Macrolide Resistance
Authors: Farrell David J. , Morrissey Ian , Bakker Sarah , Morris Louise , Buckridge Sylvie , Felmingham David
Published: February 2004

Of a total of 1,043 macrolide-resistant
Streptococcus pneumoniae
isolates collected from 24 countries as part of PROTEKT 1999-2000, 71 isolates tested positive for both the
mef
(A) and
erm
(B) genes. Of 69 isolates subjected to further molecular investigations, all were resistant to tetracycline, 63 (91.3%) were resistant to penicillin, and 57 (82.6%) were resistant to trimethoprim-sulfamethoxazole. One isolate was also fluoroquinolone resistant, and another was resistant to quinupristin-dalfopristin. The ketolide telithromycin retained activity against all of the isolates. Of the 69 of these 71 isolates viable for further testing, 46 were from South Korea, 13 were from the United States, 8 came from Japan, and 1 each came from Mexico and Hungary. One major clonal complex (59 [85.5%] of 69 isolates) was identified by serotyping (with 85.5% of the isolates being 19A or 19F), pulsed-field gel electrophoresis, and multilocus sequence typing. The remaining isolates were less clonal in nature. Representative isolates were shown to carry the mobile genetic elements Tn
1545
and mega, were negative for Tn
1207.1
, had tetracycline resistance mediated by
tet
(M), and contained the
mef
(E) variant of
mef
(A). All isolates were positive for
mel
, a homologue of the
msr
(A) efflux gene. These clones are obviously very efficient at global dissemination, and hence it will be very important to monitor their progress through continued surveillance. Telithromycin demonstrated high levels of activity (MIC for 90% of the strains tested, 0.5 μg/ml; MIC range, 0.06 to 1 μg/ml) against all isolates.
Molecular Characteristics and Global Spread of
Mycobacterium tuberculosis
with a Western Cape F11 Genotype
Authors: Victor Thomas C. , de Haas Petra E. W. , Jordaan Annemarie M. , van der Spuy Gian D. , Richardson Madalene , van Soolingen D. , van Helden Paul D. , Warren Robin
Published: February 2004

In order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic.
Mycobacterium tuberculosis
strain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific
rrs491
polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.
PCR Assays for Identification of
Coccidioides posadasii
Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen
Authors: Bialek Ralf , Kern Jan , Herrmann Tanja , Tijerina Rolando , Ceceñas Luis , Reischl Udo , González Gloria M.
Published: February 2004

A conventional nested PCR and a real-time LightCycler PCR assay for detection of
Coccidioides posadasii
DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family
Onygenaceae
remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be
C. posadasii
(accession number
AF013256
), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as
C. posadasii
. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.
Emergence of Nonencapsulated and Encapsulated Non-b-Type Invasive
Haemophilus influenzae
Isolates in Portugal (1989-2001)
Authors: Bajanca Paula , Caniça Manuela
Published: February 2004

Phenotypes and genetic relatedness of invasive
Haemophilus influenzae
strains were evaluated from 1989 through 2001. Among 119 isolates, multidrug resistance decreased (from 50 to 0%), the level of
H. influenzae
serotype b (Hib) strains declined (from 81 to 16%), the level of noncapsulated strains rose (from 19 to 80%), and the first invasive
H. influenzae
serotype f strain was described. This study documents changes in invasive
H. influenzae
infections in Portugal, i.e., the emergence of non-type-b strains that are genetically diverse and unrelated to Hib.
Simultaneous Presence of Multiple
Campylobacter
Species in Dogs
Authors: Koene M. G. J. , Houwers D. J. , Dijkstra J. R. , Duim B. , Wagenaar J. A.
Published: February 2004

The prevalence of coinfection of
Campylobacter
species in dogs was determined using four isolation methods. In 26% of the positive-testing stools, multiple
Campylobacter
species were identified. The use of multiple isolation methods as well as the time lapse between sampling and processing are important for detection of coinfection.
Gamma Interferon-Based Immunodiagnosis of Tuberculosis: Comparison between Whole-Blood and Enzyme-Linked Immunospot Methods
Authors: Schölvinck Elisabeth , Wilkinson Katalin A. , Whelan Adam O. , Martineau Adrian R. , Levin Michael , Wilkinson Robert J.
Published: February 2004

The gamma interferon secretion levels of 52 adults whose T cells had been stimulated with the
Mycobacterium tuberculosis
antigens CFP-10 and ESAT-6 were measured by enzyme-linked immunospot methods and a whole-blood-based assay. The test results correlated well (
r
= 0.689,
P
< 0.0001). Its simple format and use of only small volumes of blood make the whole-blood assay suitable for pediatric practice and field trials.
Primer Pair p289-p290, Designed To Detect Both Noroviruses and Sapoviruses by Reverse Transcription-PCR, Also Detects Rotaviruses by Cross-Reactivity
Authors: Ludert Juan E. , Alcalá Ana C. , Liprandi Ferdinando
Published: February 2004

A primer pair (p289-p290) designed to detect both noroviruses and sapoviruses by reverse transcription-PCR (Jiang et al., J. Virol. Methods
83:
145, 1999) cross-reacts with rotaviruses. The rotavirus amplicon corresponds to genome segment 1. Furthermore, primer pair p289-p290 detected rotaviruses as efficiently as rotavirus-specific primers directed to rotavirus gene 4.
Critical Use of Nucleic Acid Amplification Techniques To Test for
Mycobacterium tuberculosis
in Respiratory Tract Samples
Authors: Van den Wijngaert S. , Dediste A. , VanLaethem Y. , Gerard M. , Vandenberg O. , Zissis G.
Published: February 2004

The usefulness of employing Belgian selection criteria before performing nucleic acid amplification techniques (NAT) was evaluated. The results of this study show that for smear-negative patients with an abnormal chest radiology result in the absence of a respiratory tract infection by bacterial pathogens, testing with NAT is of major benefit.
Development of a Serum-Based Taqman Real-Time PCR Assay for Diagnosis of Invasive Aspergillosis
Authors: Challier Svetlana , Boyer Stéphanie , Abachin Eric , Berche Patrick
Published: February 2004

We compared an
Aspergillus fumigatus
-specific Taqman real-time PCR assay for the diagnosis of invasive aspergillosis (IA) with the enzyme-linked immunosorbent assay Platelia
Aspergillus
method. Patients with proven or probable IA had positive results with at least one of the two methods. The PCR method seems to be more specific, and a combination of the two should improve the diagnosis of IA.
Different Viral Rebound following Discontinuation of Antiretroviral Therapy in Cases of Infection with Viruses Carrying L74V or Thymidine-Associated Mutations
Authors: de Mendoza Carmen , Paxinos Ellen , Barreiro Pablo , Camino Nuria , Núñez Marina , Soriano Vincent
Published: February 2004

A total of 76 patients discontinued treatment with didanosine plus hydroxyurea after 1 year of maintenance therapy. The greatest human immunodeficiency virus (HIV)-RNA rebounds were seen in 10 patients harboring an L74V mutation, and the presence of viruses with this mutation rapidly waned. In contrast, viral rebounds were significantly less pronounced (
P
< 0.01) in 12 subjects harboring thymidine-associated mutations; these mutations persisted in all instances. Thus, selection of an L74V mutation during didanosine therapy may compromise HIV replication in vivo.
Absence of Mucosa-Associated Colonic Helicobacters in an Australian Urban Population
Authors: Grehan Martin , Danon Stephen , Lee Adrian , Daskalopoulos George , Mitchell Hazel
Published: February 2004

Application of nested PCR for
Helicobacter
species to 416 samples obtained at colonoscopy from 15 patients with Crohn's disease, 12 with ulcerative colitis, and 43 controls revealed
H. pylori
DNA in only 6 individuals with no disease association. No other
Helicobacter
species were detected in ileal or colonic samples.
Genetic Analysis of an Ambler Class A Extended-Spectrum Beta-Lactamase from
Capnocytophaga ochracea
Authors: Jolivet-Gougeon Anne , Tamanai-Shacoori Zohreh , Desbordes Laurent , Burggraeve Nathalie , Cormier Michel , Bonnaure-Mallet Martine
Published: February 2004

A beta-lactamase gene (
cfxA3
, 966 bp) was isolated from a beta-lactam-resistant
Capnocytophaga ochracea
clinical isolate and amplified using primers from the
cfxA
gene of
Bacteroides vulgatus
. The MICs of third-generation cephalosporins were much higher than those of the transconjugant
Escherichia coli
strain. The deduced protein sequence, by comparison with CfxA2 of
Prevotella intermedia
, had a Y239D substitution and possessed the characteristics of a class A, group 2e beta-lactamase.
IS
6110
-Mediated Deletion Polymorphism in Isogenic Strains of
Mycobacterium tuberculosis
Authors: Sampson S. L. , Richardson M. , van Helden P. D. , Warren R. M.
Published: February 2004

Previous studies have described IS
6110
-mediated polymorphism as an important driving force in
Mycobacterium tuberculosis
genome evolution and have provided indirect evidence for IS
6110
-driven deletion events. This study provides the first description of an IS
6110
-mediated deletion event in truly isogenic strains. We also provide further support for the hypothesis that the region from Rv1754 to Rv1765 is a hot spot for IS
6110
insertion and deletion events.
Comparison of Two Microdilution Methods for Testing Susceptibility of
Candida
spp. to Voriconazole
Authors: Linares Maria José , Charriel Guadalupe , Solís Francisco , Casal Manuel
Published: February 2004

The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by using two microdilution techniques: the reference method described in National Committee for Clinical Laboratory Standards document M27-A2 and a colorimetric method, Sensititre YeastOne. A total of 272
Candida
isolates (132 of
Candida albicans
, 62 of
C. parapsilosis
, 33 of
Candida glabrata
, 21 of
C. krusei
, 15 of
C. tropicalis
, and 9 of
C. lusitaniae
) and two control strains (
C. parapsilosis
ATCC 22019 and
C. krusei
ATCC 6258) were tested. There was a high rate of agreement between the two methods used (97 to 100%).