bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of the
sodA
Gene from
Corynebacterium melassecola
and Role of Superoxide Dismutase in Cellular Viability
Authors: Merkamm Muriel , Guyonvarch Armel
Published: July 2002

The
sodA
gene encoding the
Corynebacterium melassecola
manganese-cofactored superoxide dismutase (SOD) has been cloned in
Escherichia coli
and sequenced. The gene is transcribed monocistronically; the predicted polypeptide is 200 amino acids long and associates in a homotetrameric, manganese-dependent form, able to complement an SOD-deficient
E. coli
mutant. A second open reading frame, coding for a putative 217-amino-acid protein with high homology to peptide methionine sulfoxide reductases from various origins, has been identified immediately upstream of
sodA
in the opposite transcription orientation. The
sodA
gene was inactivated by insertion of an integrative vector carrying a kanamycin resistance gene. The growth rate of the SOD-deficient integrant was only slightly affected in BHI rich medium as well as in BMCG chemically defined medium, but was strongly affected by the presence of the redox-cycling agent paraquat. The SOD deficiency had, on the other hand, a deleterious effect on viability as soon as the culture entered the stationary phase of growth in BHI medium. Surprisingly, SOD deficiency was able to rescue the dramatic loss of viability observed for the wild-type strain in BMCG synthetic medium when glucose was not the limiting growth factor.
Molecular Typing of
Salmonella
Serotypes Prevalent in Animals in England: Assessment of Methodology
Authors: Liebana Ernesto , Guns Daniel , Garcia-Migura Lourdes , Woodward Martin J. , Clifton-Hadley Felicity A. , Davies Robert H.
Published: July 2002

Salmonella
enterica
serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no “gold standard” fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both
Pst
I/
Sph
I ribotyping and
Xba
I-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by
Xba
I-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.
Helicobacter pylori
Growth and Urease Detection in the Chemically Defined Medium Ham's F-12 Nutrient Mixture
Authors: Testerman Traci L. , McGee David J. , Mobley Harry L. T.
Published: July 2002

Obstacles continue to hinder in vitro studies of the gastric human pathogen
Helicobacter pylori
, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that
H. pylori
grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ∼10
3
CFU/ml. All
H. pylori
strains (
n
= 21), including fresh clinical isolates, grew in serum-free F-12.
H. pylori
grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of
H. pylori
in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), β-cyclodextrin (200 μg/ml), or cholesterol (50 μg/ml).
H. pylori
also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar,
H. pylori
only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 μg/ml), β-cyclodextrin (200 μg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or β-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for
H. pylori
. Several lines of evidence suggested that BSA itself is not responsible for
H. pylori
growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of
H. pylori
using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of
H. pylori
metabolism, virulence factors, and transmission, and result in improved recovery and identification of
H. pylori
from clinical specimens.
Chlamydia trachomatis
Serology: Diagnostic Value of Outer Membrane Protein 2 Compared with That of Other Antigens
Authors: Bas S. , Muzzin P. , Vischer T. L.
Published: July 2002

Different immunoassays using recombinant antigens or synthetic peptides were evaluated for the serodiagnosis of
Chlamydia trachomatis
infections. Antigens used included cysteine-rich outer membrane protein 2 (OMP2), heat shock protein 60, the polypeptide encoded by open reading frame 3 of the plasmid (pgp3), synthetic peptides derived from species-specific epitopes in variable domain IV of the major OMP (MOMP) (Labsystems, Helsinki, Finland), and a fragment of the total lipopolysaccharide (Medac, Hamburg, Germany). Because cross-reactions between chlamydial species have been reported,
Chlamydia pneumoniae
-specific antibodies were also determined by immunoassays (Labsystems). Responses obtained with serum samples from patients with well-defined diseases (i.e., urethral or endocervical samples from which
C. trachomatis
DNA was amplified) were compared to those obtained with samples from healthy blood donors. The best sensitivity (79%) associated with the best specificity (82%) was obtained when immunoglobulin G (IgG) responses to both MOMP and pgp3 were considered. The highest sensitivity (89%) was obtained with anti-OMP2 IgG, but the lowest specificity (57%) was obtained with this antibody, due to probable cross-reactivity with
C. pneumoniae
OMP2.
Quantification of Human Cytomegalovirus DNA in Bone Marrow Transplant Recipients by Real-Time PCR
Authors: Griscelli Frank , Barrois Michel , Chauvin Sylvie , Lastere Stephane , Bellet Dominique , Bourhis Jean-Henri
Published: July 2002

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (
P
< 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.
Polymorphism of
Bordetella pertussis
Isolates Circulating for the Last 10 Years in France, Where a Single Effective Whole-Cell Vaccine Has Been Used for More than 30 Years
Authors: Weber Christian , Boursaux-Eude Caroline , Coralie Gilberte , Caro Valérie , Guiso Nicole
Published: July 2002

We compared
Bordetella pertussis
isolates collected in France over the last 10 years, the vaccine strains used for more than 30 years, and isolates collected before the introduction of generalized vaccination. The analysis included serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with
Xba
I and
Spe
I, and sequencing of the
pt
S1 gene, encoding the S1 subunit of pertussis toxin, and the
prn
gene, encoding the adhesin pertactin. We found that the incidence of infection increases every 3 years. Ninety-five per cent of the isolates analyzed express type 3 fimbriae. Most of the isolates circulating since 1991, unlike the vaccinal strains, express a type A pertussis toxin and a type 2 pertactin. The isolates could be classified into five major groups by pulsed-field gel electrophoresis. Most of these groups correlated with the pertactin type expressed by the isolates. Pulsed-field gel electrophoresis is more discriminative than sequencing particular genes since it could differentiate isolates expressing type 2 pertactin into two subgroups: those circulating in 1993 to 1997 and those circulating in 1997 to 2001. This observation suggests that there has been continuous evolution of the
B. pertussis
population.
Nosocomial Fungemia Due to
Exophiala jeanselmei
var.
jeanselmei
and a
Rhinocladiella
Species: Newly Described Causes of Bloodstream Infection
Authors: Nucci Marcio , Akiti Tiyomi , Barreiros Gloria , Silveira Fernanda , Revankar Sanjay G. , Sutton Deanna A. , Patterson Thomas F.
Published: July 2002

Fungi have become increasingly important causes of nosocomial bloodstream infections. The major cause of nosocomial fungemia has been
Candida
spp, but increasingly molds and other yeasts have caused disease.
Exophiala jeanselmei
and members of the genus
Rhinocladiella
are dematiaceous moulds, which have been infrequently associated with systemic infection and have not been described as causes of fungemia. In this paper, the occurrence of 23 cases of fungemia due to these organisms over a 10-month period is reported and the clinical characteristics of patients and outcomes are described. The majority of patients were immunosuppressed; 21 of 23 (91%) had received blood products and 78% had a central venous catheter. All patients had at least one manifestation of fever, but only one patient had signs or symptoms suggesting deep-seated infection. Antifungal therapy was given to 19 of the 23 patients; of those who did not receive therapy, 3 died prior to the culture result and 1 had been discharged without therapy. Antifungal susceptibility of the organisms showed activity of amphotericin B, itraconazole, and the new triazole antifungals voriconazole and posaconazole.
E. jeanselmei
and
Rhinocladiella
species are potential causes of nosocomial fungemia and may be associated with systemic infection.
Genetic Variability and Prevalence of
Bartonella henselae
in Cats in Berlin, Germany, and Analysis of Its Genetic Relatedness to a Strain from Berlin That Is Pathogenic for Humans
Authors: Arvand Mardjan , Klose Alexander J. , Schwartz-Porsche Dorothea , Hahn Helmut , Wendt Constanze
Published: July 2002

Nineteen
Bartonella henselae
strains and one
Bartonella clarridgeiae
strain were isolated from blood samples of 97 pet cats and 96 stray cats from Berlin, Germany, indicating prevalence rates of 1 and 18.7%, respectively, for
B. henselae
and 0 and 1%, respectively, for
B. clarridgeiae
. Eighteen of 19
B. henselae
isolates corresponded to 16S rRNA type II. Pulsed-field gel electrophoresis (PFGE) analysis revealed seven different PFGE types among the feline
B. henselae
strains. Interestingly, all feline isolates displayed low genetic relatedness to
B. henselae
strain Berlin-1, which is pathogenic for humans.
Molecular Epidemiology of
Mycobacterium tuberculosis
Infection in Israel
Authors: Ravins M. , Bercovier H. , Chemtob D. , Fishman Y. , Rahav G.
Published: July 2002

Restriction fragment length polymorphism analysis with IS
6110
and DR-r probes was used to study 69
Mycobacterium tuberculosis
isolates obtained from Israeli patients and new immigrants from the former Soviet Union and Ethiopia. DNA fingerprinting identified unique patterns for almost all isolates, indicating that most patients were infected with a unique strain imported from their country of origin and that their latent infection was reactivated in Israel.
Phenotypic and Genotypic Characterization of
Pediococcus
Strains Isolated from Human Clinical Sources
Authors: Barros Rosana R. , Carvalho Maria Da Glória S. , Peralta José Mauro , Facklam Richard R. , Teixeira Lúcia M.
Published: July 2002

Seventy-two strains of pediococci isolated from human clinical sources were characterized by conventional physiological tests, chromogenic enzymatic tests, analysis of whole-cell protein profiles (WCPP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of chromosomal DNA restriction profiles by pulsed-field gel electrophoresis (PFGE). Conventional tests allowed identification of 67 isolates: 52 strains were identified as
Pediococcus acidilactici
, 15 strains were identified as
Pediococcus pentosaceus
, and 5 strains were not identified because of atypical reactions. Analysis of WCPP identified all isolates since each species had a unique WCPP. By the WCPP method, the atypical strains were identified as
P. acidilactici
(two strains) and
P. pentosaceus
(three strains). The chromogenic substrate test with
o
-nitrophenyl-β-
d
-glucopyranoside differentiated all 54 strains of
P. acidilactici
(negative reactions) and 13 (72%) of 18 strains of
P. pentosaceus
(positive reactions). Isolates of both species were shown to be nonclonal as revealed by the genetic diversity when chromosomal DNA was analyzed by PFGE. Using WCPP as the definitive identification procedure,
P. acidilactici
(28 of 54 strains; 51.8%) was more likely than
P. pentosaceus
(4 of 18 strains; 22.3%) to be isolated from blood cultures.
Molecular Characteristic-Based Epidemiology of Hepatitis B, C, and E Viruses and GB Virus C/Hepatitis G Virus in Myanmar
Authors: Nakai Kazuhiko , Win Khin Maung , Oo San San , Arakawa Yasuyuki , Abe Kenji
Published: July 2002

We carried out a molecular characteristic-based epidemiological survey of various hepatitis viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and GB virus C (GBV-C)/hepatitis G virus (HGV), in Myanmar. The study population of 403 subjects consisted of 213 healthy individuals residing in the city of Yangon, Myanmar, and the surrounding suburbs and 190 liver disease patients (155 virus-related liver disease patients and 35 nonviral disease patients). The infection rates of the viruses among the 213 healthy subjects were as follows: 8% for HBV (16 patients), 2% for HCV (4 patients), and 8% for GBV-C/HGV (17 patients). In contrast, for 155 patients with acute hepatitis, chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma, the infection rates were 30% for HBV (46 patients), 27% for HCV (41 patients), and 11% for GBV-C/HGV (17 patients). In the nonviral liver disease group of 35 patients with alcoholic liver disease, fatty liver, liver abscess, and biliary disease, the infection rates were 6% for HBV (2 patients), 20% for HCV (7 patients), and 26% for GBV-C/HGV (9 patients). The most common viral genotypes were type C of HBV (77%), type 3b of HCV (67%), and type 2 of GBV-C/HGV (67%). Moreover, testing for HEV among 371 subjects resulted in the detection of anti-HEV immunoglobulin G (IgG) in 117 patients (32%). The age prevalence of anti-HEV IgG was 3% for patients younger than 20 years and 30% or more for patients 20 years of age or older. Furthermore, a high prevalence of anti-HEV IgG (24%) was also found in swine living together with humans in Yangon. These results suggest that these hepatitis virus infections are widespread in Myanmar and have led to a high incidence of acute and chronic liver disease patients in the region.
B-Cell Epitope Mapping of the VapA Protein of
Rhodococcus equi
: Implications for Early Detection of
R. equi
Disease in Foals
Authors: Vanniasinkam Thiru , Barton Mary D. , Heuzenroeder Michael W.
Published: July 2002

Linear B-cell epitopes of the
Rhodococcus equi
virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current
R. equi
disease (51 sera), as well as from foals that had either recovered from
R. equi
infection 10 months previously (3 sera) or that had no known history of
R. equi
disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with
R. equi
disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for
R. equi
infection in foals.
Antifungal Susceptibilities, Varieties, and Electrophoretic Karyotypes of Clinical Isolates of
Cryptococcus neoformans
from Brazil, Chile, and Venezuela
Authors: Calvo Belinda M. , Colombo Arnaldo L. , Fischman Olga , Santiago A. , Thompson L. , Lazera Marcia , Telles Flavio , Fukushima Kazutaka , Nishimura Kazuko , Tanaka Reiko , Myiajy Makoto , Moretti-Branchini M. Luiza
Published: July 2002

One hundred clinical isolates of
Cryptococcus neoformans
from human immunodeficiency virus (HIV)-infected and non-HIV-infected patients from Brazil, Chile, and Venezuela were separated according to varieties and tested for antifungal susceptibility. A high susceptibility to antifungal agents was observed among all the isolates. The electrophoretic karyotyping of 51 strains revealed good discrimination among
Cryptococcus neoformans
var.
neoformans
strains.
Diagnosis of Oropouche Virus Infection Using a Recombinant Nucleocapsid Protein-Based Enzyme Immunoassay
Authors: Saeed Mohammad F. , Nunes Marcio , Vasconcelos Pedro F. , Travassos Da Rosa Amelia P. A. , Watts Douglas M. , Russell Kevin , Shope Robert E. , Tesh Robert B. , Barrett Alan D. T.
Published: July 2002

Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.
Evaluation of COBAS AMPLICOR (Roche): Accuracy in Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
by Coamplification of Endocervical Specimens
Authors: Livengood Charles H. , Wrenn Jane W.
Published: July 2002

We evaluated further the accuracy of the COBAS AMPLICOR (Roche) (CA) PCR-based system in detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in endocervical specimens. Endocervical specimens collected for any indication for testing for
C. trachomatis
and
N. gonorrhoeae
among a university hospital health system population were included. Testing for
C. trachomatis
was done by two PCR methods, CA and manual microwell AMPLICOR (Roche) (MWA), and by culture; testing for
N. gonorrhoeae
was done by CA and culture. Discrepancy resolution was performed. Reproducibility testing and hands-on labor time measurements for CA were done. Among 654
C. trachomatis
samples, the prevalence of true positivity was 9.2%, and among the 618
N. gonorrhoeae
samples, the prevalence of true positivity was 4.4%. For detection of
C. trachomatis
, the sensitivity, specificity, and negative and positive predictive values were, respectively, as follows for each test: CA, 93.3, 99.7, 99.3, and 96.4%; MWA, 91.7, 99.7, 99.2, and 96.5%; and culture, 65.0, 100, 96.6, and 100%. For detection of
N. gonorrhoeae
those values were as follows: CA, 96.3, 100, 99.8, and 100%; and culture, 92.6, 100, 99.7, and 100%. Hands-on labor time for each clinical result was estimated to be at 7.5 min. The prevalence of inhibitory specimens was 3.5%, including two positive
C. trachomatis
samples which would have been missed otherwise. The direct cost of each clinical result with CA was estimated to be $9.09. Our methods include a diverse range of indications for testing among women, using endocervical swabbing samples, 2 M sucrose phosphate transport medium, and discrepancy resolution for comparison. Under our test conditions, the CA system is an accurate, rapid, and cost- and labor-efficient method for detection of
C. trachomatis
and
N. gonorrhoeae
.
Identification of
Mycobacterium
Species by Multiple-Fluorescence PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene
Authors: Gillman Lawrence M. , Gunton James , Turenne Christine Y. , Wolfe Joyce , Kabani Amin M.
Published: July 2002

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.
Use of a Heteroduplex Mobility Assay To Detect Differences in the Fusion Protein Cleavage Site Coding Sequence among Newcastle Disease Virus Isolates
Authors: Berinstein Analia , Sellers Holly S. , King Daniel J. , Seal Bruce S.
Published: July 2002

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.
Evaluation of Four Commercial Immunoglobulin G (IgG)- and IgM-Specific Enzyme Immunoassays for Diagnosis of
Mycoplasma pneumoniae
Infections
Authors: Petitjean J. , Vabret A. , Gouarin S. , Freymuth F.
Published: July 2002

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting
Mycoplasma pneumoniae
immunoglobulin G (IgG)- and IgM-specific antibodies Platelia EIA, ImmunoWELL
M. pneumoniae
ELISA IgG and IgM, ETI-MP-IgG and IgM EIAs and Biotest anti-
M. pneumoniae
IgG and IgM ELISA (referred to herein as EIA-Platelia, EIA-BMD, EIA-Sorin, and EIA-Biotest). Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections who tested positive by PCR for
M. pneumoniae
in respiratory specimens (group I; 52 serum samples), 61 healthy children and adults (group II; 61 serum samples), and 20 patients with rheumatoid factor or antinuclear antibodies, or who tested positive for antiviral IgM (group III; 20 serum samples). In group III, the IgM specificity for EIA-Platelia, EIA-BMD, EIA-Biotest, and EIA-Sorin was 100, 90, 65, and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivities (89 to 92%); the sensitivity for IgG was greater with EIA-BMD and EIA-Biotest than with EIA-Platelia and EIA-Sorin (66 and 78% versus 55 and 52%, respectively). In adult patients from group I, 9 to 10 serum samples were positive for IgG with a concordant sensitivity of 75 to 83% between the four EIAs but a striking difference in IgM sensitivity: 16% by EIA-Platelia and EIA-BMD, 50% by EIA-Biotest, and 58% by EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIAs and making them inaccurate for routine diagnosis. A good concordance of IgG seroprevalence in healthy adults was found between the four EIAs (66 to 70%), though this concordance was lower with EIA-Platelia (43%). In healthy children, EIA-BMD and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former compared to 17 and 20%, respectively, for the latter). These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of
M. pneumoniae
infections in children, as long as the EIA used is specific. In adults, the difficult interpretation of EIAs suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.
Evaluation of a Strand Displacement Amplification Assay (BD ProbeTec-SDA) for Detection of
Neisseria gonorrhoeae
in Urine Specimens
Authors: Akduman Deniz , Ehret Josephine M. , Messina Kimberly , Ragsdale Sharron , Judson Franklyn N.
Published: July 2002

The performance of a strand displacement amplification assay (the BDProbeTec-SDA assay) in detecting
Neisseria gonorrhoeae
in urine specimens was evaluated. When performed under stringent quality control conditions, the BDProbeTec-SDA assay is a sensitive, specific, and efficient method for the screening of large numbers of noninvasively obtained specimens. Because the predictive value of an assay is a function of the prevalence of the disease, culture confirmation is needed for samples with positive results from populations in which the prevalence of a disease is low or in situations in which false-positive results may have important medical, psychosocial, or medicolegal consequences.
Paracoccidioides
brasiliensis
87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry
Authors: Díez Soraya , Gómez Beatriz L. , Restrepo Angela , Hay Rod J. , Hamilton Andrew J.
Published: July 2002

The 87-kDa antigen derived from the fungal pathogen
Paracoccidioides brasiliensis
can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a
Histoplasma capsulatum
80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with
P. brasiliensis
confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.
Evaluation of
Chlamydia pneumoniae
and
Mycoplasma pneumoniae
as Etiologic Agents of Persistent Cough in Adolescents and Adults
Authors: Wadowsky Robert M. , Castilla Elias A. , Laus Stella , Kozy Anita , Atchison Robert W. , Kingsley Lawrence A. , Ward Joel I. , Greenberg David P.
Published: July 2002

Chlamydia pneumoniae
and
Mycoplasma pneumoniae
were evaluated as agents of persistent cough in adolescents and adults (
n
= 491). Tests of 473 respiratory specimens by culture or PCR or both identified four episodes (0.8%) of
M. pneumoniae
-associated illness and no episodes of
C. pneumoniae
illness, suggesting that these bacteria do not frequently cause persistent cough.
Soft Tissue Infection with
Absidia corymbifera
in a Patient with Idiopathic Aplastic Anemia
Authors: Cloughley R. , Kelehan J. , Corbett-Feeney G. , Murray M. , Callaghan J. , Regan P. , Cormican M.
Published: July 2002

We describe a case of primary cutaneous mucormycosis (zygomycosis) in a patient with idiopathic aplastic anemia which responded to surgical debridement and therapy with liposomal amphotericin B. The tissue removed at surgery showed dense infiltration with fungal hyphae on histopathological examination. Primary cultures of tissue on solid media were negative, but
Absidia corymbifera
was isolated from unprocessed tissue placed in brain heart infusion broth.
Nine-Year Surveillance of Methicillin-ResistantStaphylococcus aureusin a Hospital Suggests Instability ofmecADNA Region in an Epidemic Strain
Authors: Donnio Pierre-Yves , Louvet Laurence , Preney Laure , Nicolas Denis , Avril Jean-Loup , Desbordes Laurent
Published: July 2002

The distributions of the antibiotic resistance patterns in a population ofStaphylococcus aureusisolates from a teaching hospital were studied over a 9-year period. The results indicate the existence of successive major epidemic methicillin-resistant strains and the emergence of a methicillin-susceptible strain with an unusual resistance pattern. Our findings suggest that this methicillin-susceptibleS.aureusstrain could be derived from the dominant gentamicin-susceptible methicillin-resistantS.aureusstrain with the loss of a 40-kb DNA fragment.
Imported Visceral Leishmaniasis: Diagnostic Dilemmas and Comparative Analysis of Three Assays
Authors: Iqbal Jamshaid , Hira Parsotam R. , Saroj Grover , Philip Reeni , Al-Ali Faiza , Madda Patrick J. , Sher Ali
Published: July 2002

The present study evaluates the performances of three noninvasive serological assays for the detection of immunoglobulin G antibodies to leishmania antigen for the diagnosis of imported cases of kala azar (visceral leishmaniasis [VL]) in a country, Kuwait, where the disease is not endemic. A total of 323 individuals including 21 patients with documented cases of VL, 72 individuals with suspected cases of VL, 155 patients with other parasitic infections, and 75 healthy control individuals were tested by indirect hemagglutination assay (IHA; Behring Diagnostics GmbH, Marburg, Germany), indirect fluorescent-antibody assay (IFA; bioMerieux sa, Marcy l'Etoile, France), and a qualitative membrane-based immunoassay with recombinant leishmania antigen K39 (strip-test; Intersep Ltd, Berkshire, United Kingdom). Our data show that IHA is the most sensitive test (100%), followed by IFA (86.6%) and the strip-test (80.0%). The strip-test was the most specific (100%) of the three assays, followed by IFA (93.0%) and IHA (86.0%). However, the strip-test failed to detect at least three confirmed cases of VL. We conclude that IHA is preferred over IFA and the strip-test for the screening of individuals with suspected cases of VL, especially in a country where VL is not endemic and where the number of cases is regular but limited. The details about some of the patients with VL are presented to highlight the diversity of clinical presentations and problems encountered in the diagnosis of VL in a country where VL is not endemic.
Genetic and Phenotypic Differences between
Legionella pneumophila
Strains
Authors: Samrakandi Mustapha M. , Cirillo Suat L. G. , Ridenour Dennis A. , Bermudez Luiz E. , Cirillo Jeffrey D.
Published: July 2002

Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species
Legionella
pneumophila
, although more than 40 other species are known. Certain
L
.
pneumophila
subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of
L
.
pneumophila
. We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of
Legionella
and found a correlation between the presence of two of these loci,
rtxA
and
lvh
, and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in
Legionella
pathogenesis.
DNA Microarray-Based Typing of an Atypical Monophasic
Salmonella enterica
Serovar
Authors: Garaizar Javier , Porwollik Steffen , Echeita Aurora , Rementeria Aitor , Herrera Silvia , Wong Rita Mei-Yi , Frye Jonathan , Usera Miguel A. , McClelland Michael
Published: July 2002

A multidrug-resistant
fljB
-lacking
Salmonella enterica
serovar [4,5,12:i:−] emerged in Spain in 1997. We analyzed the genome from four strains of this serovar using a microarray containing almost all the predicted protein coding regions of serovar Typhimurium strain LT2, including the pSLT plasmid. Only a few differences from serovar Typhimurium LT2 were observed, suggesting the serovar to be Typhimurium as well. Six regions of interest were identified from the microarray data. Cluster I was a deletion of 13 genes, corresponding to part of the regulon responsible for the anaerobic assimilation of allantoin. Clusters II and IV were associated with the absence of the Fels-1 and Fels-2 prophage. Cluster III was a small group of Gifsy-1 prophage-related genes that appeared to be deleted or replaced. Cluster V was a deletion of 16 genes, including
iroB
and the operon
fljAB
, which is reflected in the serovar designation. Region VI was the gene STM2240, which appears to have an additional homologue in these strains. The regions spanning the deletions involving the allantoin operon and the
fljAB
operon were PCR amplified and sequenced. PCR across these regions may be an effective marker for this particular emergent serovar. While the microarray data for all isolates of the new serovar were essentially identical for all LT2 chromosomal genes, the isolates differed in their similarity to pSLT, consistent with the heterogeneity in plasmid content among isolates of the new serovar. Recent isolates have acquired a more-complete subset of homologues to this virulence plasmid. In general, microarrays can provide useful complementary data to other typing methods.
Identification by Phage Display and Characterization of Two Neutralizing Chimpanzee Monoclonal Antibodies to the Hepatitis E Virus Capsid Protein
Authors: Schofield D. J. , Glamann J. , Emerson S. U. , Purcell R. H.
Published: July 2002

Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) γ1/κ antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants,Kd) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.
Reovirus Protein ςNS Binds in Multiple Copies to Single-Stranded RNA and Shares Properties with Single-Stranded DNA Binding Proteins
Authors: Gillian Anne Lynn , Schmechel Stephen C. , Livny Jonathan , Schiff Leslie A. , Nibert Max L.
Published: July 2002

Reovirus nonstructural protein ςNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of ςNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of ςNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The ςNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, ςNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, ςNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl
2
, distinguishing it from a classical ATP-dependent helicase. These properties of ςNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for ςNS in replication of the reovirus RNA genome.
Human Immunodeficiency Virus Type 1 Virion Density Is Not Determined by Nucleocapsid Basic Residues
Authors: Cimarelli Andrea , Luban Jeremy
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is sufficient for assembly and release of virion-like particles from the plasma membrane. To promote assembly, the Gag polyprotein must polymerize to form a shell that lines the inner membrane of nascent virions. Several techniques have been used to functionally map the domain required for Gag polymerization (the I domain). Among these methods, isopycnic centrifugation has been used under the assumption that changes in virion density reflect impairment in Gag-Gag interaction. If virion density is determined by efficient Gag-Gag interaction, then mutation of basic residues in the nucleocapsid (NC) domain should disrupt virion density, since these residues constitute the I domain. However, we have previously shown that simultaneous disruption of up to 10 HIV-1 NC basic residues has no obvious effect on virion density. To rule out the possibility that HIV-1 NC basic residues other than those previously mutated might be important for virion density, mutations were introduced at the remaining sites and the ability of these mutations to affect Gag-Gag interaction and virion density was analyzed. Included in our analysis is a mutant in which all NC basic residues are replaced with alanine. Our results show that disruption of HIV-1 NC basic residues has an enormous effect on Gag-Gag interaction but only a minimal effect on the density of those virions that are still produced. Therefore, the determinants of the I domain and of virion density are genetically distinguishable.
Effective Vaccine for Lassa Fever
Authors: Fisher-Hoch S. P. , Hutwagner L. , Brown B. , McCormick J. B.
Published: July 2002

Lassa fever has been estimated to cause 5,000 deaths annually in West Africa. Recently, war in the zone where Lassa fever is hyperendemic has severely impeded control and treatment. Vaccination is the most viable control measure. There is no correlation between antibody levels and outcome in human patients, and inactivated vaccines produce high titers of antibodies to all viral proteins but do not prevent virus replication and death in nonhuman primates. Accordingly, we vaccinated 44 macaques with vaccinia virus-expressed Lassa virus structural proteins separately and in combination, with the object of inducing a predominantly TH1-type immune response. Following Lassa virus challenge, all unvaccinated animals died (0% survival). Nine of 10 animals vaccinated with all proteins survived (90% survival). Although no animals that received full-length glycoprotein alone had a high titer of antibody, 17 of 19 survived challenge (88%). In contrast, all animals vaccinated with nucleoprotein developed high titers of antibody but 12 of 15 died (20% survival). All animals vaccinated with single glycoproteins, G1 or G2, died, but all those that received both single glycoproteins (G1 plus G2) at separate sites survived, showing that both glycoproteins are independently important in protection. Neither group had demonstrable antibody levels prior to challenge. We demonstrate that in primates, immune responses to epitopes on both glycoproteins are required to protect against lethal challenge with Lassa virus without having untoward side effects and that this protection is likely to be primarily cell mediated. We show that an effective, safe vaccine against Lassa virus can and should be made and that its evaluation for human populations is a matter of humanitarian priority.
Short- and Long-Term Clinical Outcomes in Rhesus Monkeys Inoculated with a Highly Pathogenic Chimeric Simian/Human Immunodeficiency Virus
Authors: Endo Yasuyuki , Igarashi Tatsuhiko , Nishimura Yoshiaki , Buckler Charles , Buckler-White Alicia , Plishka Ronald , Dimitrov Dimiter S. , Martin Malcolm A.
Published: July 2002

A highly pathogenic simian/human immunodeficiency virus (SHIV), SHIV
DH12R
, isolated from a rhesus macaque that had been treated with anti-human CD8 monoclonal antibody at the time of primary infection with the nonpathogenic, molecularly cloned SHIV
DH12
, induced marked and rapid CD4
+
T cell loss in all rhesus macaques intravenously inoculated with 1.0 50% tissue culture infective dose (TCID
50
) to 4.1 × 10
5
TCID
50
s of virus. Animals inoculated with 650 TCID
50
s of SHIV
DH12R
or more experienced irreversible CD4
+
T lymphocyte depletion and developed clinical disease requiring euthanasia between weeks 12 and 23 postinfection. In contrast, the CD4
+
T-cell numbers in four of five monkeys receiving 25 TCID
50
s of SHIV
DH12R
or less stabilized at low levels, and these surviving animals produced antibodies capable of neutralizing SHIV
DH12R
. In the fifth monkey, no recovery from the CD4
+
T cell decline occurred, and the animal had to be euthanized. Viral RNA levels, subsequent to the initial peak of infection but not at peak viremia, correlated with the virus inoculum size and the eventual clinical course. Both initial infection rate constants,
k
, and decay constants,
d
, were determined, but only the latter were statistically correlated to clinical outcome. The attenuating effects of reduced inoculum size were also observed when virus was inoculated by the mucosal route. Because the uncloned SHIV
DH12R
stock possessed the genetic properties of a lentivirus quasispecies, we were able to assess the evolution of the input virus swarm in animals surviving the acute infection by monitoring the emergence of neutralization escape viral variants.