bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, Autoregulation, and DNA Binding Properties of the
Mycobacterium tuberculosis
TrcR Response Regulator
Authors: Haydel Shelley E. , Benjamin William H. , Dunlap Nancy E. , Clark-Curtiss Josephine E.
Published: July 2002

The TrcRS two-component system of
Mycobacterium tuberculosis
is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the
trcR
and
trcS
two-component system genes are transcribed in broth-grown
M. tuberculosis
. Analysis of the
trcR
and
trcS
genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of
M. tuberculosis
growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a
trcR-lacZ
fusion plasmid and a TrcR expression plasmid were cotransformed into
Escherichia coli
. Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the
trcR-lacZ
fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the
trcR
promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping
trcR
PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the
trcR
promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the
trcR
promoter was mutated and analyzed in
lacZ
transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the
trcR
promoter resulted in the loss of
trcR
transcriptional activation in the presence of TrcR. This report indicates that the
M. tuberculosis
TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the
trcR
promoter.
Identification of [2Fe-2S] Clusters in Microbial Ferrochelatases
Authors: Dailey Tamara A. , Dailey Harry A.
Published: July 2002

The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria
Caulobacter crescentus
and
Mycobacterium tuberculosis
possess [2Fe-2S] clusters. The enzyme from
C. crescentus
is a homodimeric, membrane-associated protein while the enzyme from
M. tuberculosis
is monomeric and soluble. The clusters of the
C. crescentus
and
M. tuberculosis
ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast
Schizosaccharomyces pombe
. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.
Comparison of Fluorescent In Situ Hybridization and Conventional Culturing for Detection of
Helicobacter pylori
in Gastric Biopsy Specimens
Authors: Rüssmann Holger , Kempf Volkhard A. J. , Koletzko Sibylle , Heesemann Jürgen , Autenrieth Ingo B.
Published: July 2002

In this study, we have investigated 201 gastric biopsy specimens obtained from dyspeptic patients for the presence of
Helicobacter pylori
. By means of fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescence-labeled oligonucleotide probes specific for
H. pylori
, this pathogen was detected in 63 biopsy specimens. By using conventional culturing,
H. pylori
was isolated from 49 of these 63 gastric biopsy specimens. In contrast, FISH failed to identify
H. pylori
in four samples from which the pathogen was cultured. The lowest sensitivity was obtained by using the urease test.
H. pylori
was detected indirectly by this method in 43 of 67 biopsy specimens, which were positive for the pathogen as determined by FISH and/or culturing. All 49
H. pylori
isolates that were detected by FISH and culturing underwent antimicrobial susceptibility testing for clarithromycin, a macrolide drug that is a key component in the therapy of peptic ulcer disease caused by this pathogen. Clarithromycin susceptibility testing of cultured isolates was carried out by the E-test, whereas FISH was used on biopsy specimens to detect clarithromycin-resistant mutant strains. No discrepancies were found between these two methods. Thirty-seven strains were clarithromycin sensitive, and eight
H. pylori
isolates were resistant to the macrolide. From another four biopsy specimens, a mixture of clarithromycin-sensitive and -resistant strains was identified by both methods. Thus, FISH is a reliable technique for determining the clarithromycin susceptibility of this pathogen. Taken together, FISH is a more sensitive and rapid technique than culturing for detection of
H. pylori
in gastric biopsy specimens. However, in the microbiology routine diagnostic laboratory, the combination of both FISH and conventional culturing significantly increases the sensitivity in detection of
H. pylori
.
Role of PCR in Diagnosis and Prognosis of Visceral Leishmaniasis in Patients Coinfected with Human Immunodeficiency Virus Type 1
Authors: Pizzuto Massimo , Piazza Manuela , Senese Daniela , Scalamogna Chiara , Calattini Sara , Corsico Laura , Persico Tiziana , Adriani Beatrice , Magni Carlo , Guaraldi Giovanni , Gaiera Giovanni , Ludovisi Alessandra , Gramiccia Marina , Galli Massimo , Moroni Mauro , Corbellino Mario , Antinori Spinello
Published: July 2002

A group of 76 consecutive human immunodeficiency virus (HIV)-positive patients with fever of unknown origin (
n
= 52) or fever associated with pulmonary diseases was evaluated in order to assess the usefulness of PCR with peripheral blood in the diagnosis and follow-up of visceral leishmaniasis. We identified 10 cases of visceral leishmaniasis among the 52 patients with fever of unknown origin. At the time of diagnosis, all were parasitemic by PCR with peripheral blood. During follow-up, a progressive decline in parasitemia was observed under therapy, and all patients became PCR negative after a median of 5 weeks (range, 6 to 21 weeks). However, in eight of nine patients monitored for a median period of 88 weeks (range, 33 to 110 weeks), visceral leishmaniasis relapsed, with positive results by PCR with peripheral blood reappearing 1 to 2 weeks before the clinical onset of disease. Eight
Leishmania infantum
and two
Leishmania donovani
infections were identified by PCR-restriction fragment length polymorphism analysis. PCR with peripheral blood is a reliable method for diagnosis of visceral leishmaniasis in HIV-infected patients. During follow-up, it substantially reduces the need for traditional invasive tests to assess parasitological response, while a positive PCR result is predictive of clinical relapse.
Methicillin-Resistant, Quinupristin-Dalfopristin-Resistant
Staphylococcus aureus
with Reduced Sensitivity to Glycopeptides
Authors: Werner G. , Cuny C. , Schmitz F.-J. , Witte W.
Published: July 2002

Of 3,052
Staphylococcus aureus
strains collected by the European SENTRY surveillance study, 35 were found to be nonsusceptible to quinupristin-dalfopristin (MIC of ≥2 mg/liter). These isolates originated from four hospitals in France and one in Spain. In isolates from two Parisian hospitals exhibiting the same
Sma
I macrorestriction pattern, streptogramin resistance was based on
vatA
and
vgbA
. One isolate from a hospital in Lyon and 22 from a hospital in Lille were of the
vatB vgaB
streptogramin A resistance genotype and possessed
ermA
and/or
ermC
. As deduced from the loss of either streptogramin A or streptogramin B resistance determinants in particular isolates, resistance to quinupristin-dalfopristin requires mechanisms conferring resistance to both compounds. The
Sma
I macrorestriction patterns of strains from hospitals in Lille and Lyon were different; however, similarity analysis suggested a relatedness of 20 methicillin-resistant
S. aureus
strains from the Lille hospital, a finding confirmed by PCR typing based on three different genomic polymorphisms. These groups of isolates were found to be hetero-glycopeptide-intermediate susceptible
S. aureus
. Information about the failure of glycopeptide chemotherapy has not been available.
Identification of a
p28
Gene in
Ehrlichia ewingii
: Evaluation of Gene for Use as a Target for a Species-Specific PCR Diagnostic Assay
Authors: Gusa Asiya A. , Buller Richard S. , Storch Gregory A. , Huycke Mark M. , Machado Linda J. , Slater Leonard N. , Stockham Steven L. , Massung Robert F.
Published: July 2002

PCR was used to amplify a 537-bp region of an
Ehrlichia ewingii
gene encoding a homologue of the 28-kDa major antigenic protein (P28) of
Ehrlichia chaffeensis
. The
E. ewingii p28
gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from
Ehrlichia
and
Cowdria
spp.:
p30
of
Ehrlichia canis
(≤71.3%),
p28
of
E. chaffeensis
(≤68.3%), and
map1
of
Cowdria ruminantium
(67.3%). The peptide sequence of the
E. ewingii
partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with ≤69.1% identity to P28 of
E. chaffeensis
, ≤67.3% identity to P30 of
E. canis
, and ≤63.1% identity to MAP1 of
C. ruminantium
. Primers were selected from the
E. ewingii p28
sequence and used to develop a species-specific PCR diagnostic assay. The
p28
PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed
E. ewingii
infections that were available. The assay did not produce PCR products with DNA extracted from
E. chaffeensis-
,
E. canis-
, or
E. phagocytophila
-infected samples, confirming the specificity of the
p28
assay for
E. ewingii
. The sensitivity of the
E. ewingii
-specific PCR assay was evaluated and determined to detect as few as 38 copies of the
p28
gene.
In Vitro Activity of Posaconazole against Clinical Isolates of Dermatophytes
Authors: Barchiesi Francesco , Arzeni Daniela , Camiletti Valeria , Simonetti Oriana , Cellini Andreina , Offidani Anna M. , Scalise Giorgio
Published: July 2002

A broth macrodilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was used to compare the in vitro activity of posaconazole (PCZ) with that of itraconazole (ITC) against 30 clinical isolates of dermatophytes belonging to six different species. In terms of MICs, PCZ showed an activity equal to that of ITC. MICs of PCZ at which 50% (MIC
50
) and 90% (MIC
90
) of the isolates were inhibited were 0.5 and >4.0 μg/ml, respectively. The MIC
50
and MIC
90
of ITC were 1.0 and >4.0 μg/ml, respectively. However, PCZ showed a more potent fungicidal activity than that of ITC against isolates belonging to the genus
Microsporum
(
P
= 0.03). PCZ merits further investigation as a potentially useful agent for treatment of dermatophytosis.
Kinetics of Dengue Virus-Specific Serum Immunoglobulin Classes and Subclasses Correlate with Clinical Outcome of Infection
Authors: Koraka P. , Suharti C. , Setiati T. E. , Mairuhu A. T. A. , Van Gorp E. , Hack C. E. , Juffrie M. , Sutaryo J. , Van Der Meer G. M. , Groen J. , Osterhaus A. D. M. E.
Published: July 2002

The kinetics of dengue virus (DEN)-specific serum immunoglobulin classes (immunoglobulin M [IgM] and IgA) and subclasses (IgG1 to IgG4) were studied in patients suffering from dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Serum samples from non-DEN febrile patients were included as controls. IgM, IgG1, and IgG3 serum antibodies were the predominant immunoglobulins throughout the course of illness in all three patient groups. In contrast, IgA antibodies were significantly higher in the acute phase in DSS patients compared to those in DF patients (P< 0.05). The levels of IgG1 differed significantly between patients with DF and those with DHF and DSS (P< 0.05). A significant difference was also found in IgG3 levels between DF patients and DHF patients (P< 0.05) but not between DF patients and DSS patients. Finally, levels of IgG4 antibodies differed significantly between DF patients and DSS patients (P< 0.05). Collectively, these data show that increased levels of DEN-specific IgA, IgG1, and IgG4 serum antibodies are risk markers for the development of DHF and DSS and that their measurement may provide valuable guidance for early therapeutic intervention.
Laboratory Diagnosis of Common Herpesvirus Infections of the Central Nervous System by a Multiplex PCR Assay
Authors: Markoulatos Panayotis , Georgopoulou Amalia , Siafakas Nikolaos , Plakokefalos Elias , Tzanakaki Georgina , Kourea-Kremastinou Jenny
Published: July 2002

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of α-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.
Rapid Identification of Methicillin-Resistant
Staphylococcus aureus
from Positive Blood Cultures by Real-Time Fluorescence PCR
Authors: Tan Thean Yen , Corden Sally , Barnes Rosemary , Cookson Barry
Published: July 2002

Methicillin-resistant
Staphylococcus aureus
septicemia is associated with significant morbidity and mortality and requires treatment with intravenous glycopeptides. For blood cultures positive for gram-positive cocci, 24 to 48 h is required for the detection of
S. aureus
bacteremia and the provision of antibiotic susceptibility testing results. We describe a molecular biology-based assay that requires 2 h from the time of initial positivity of blood cultures. The assay correctly detected 96% of the
S. aureus
isolates including all methicillin-resistant
S. aureus
isolates. Clinical data collected during the study suggest that 28% of patients with
S. aureus
bacteremia do not receive early and appropriate treatment and that 10% of patients may initially be receiving inappropriate glycopeptide treatment.
Risk ofMycobacterium tuberculosisTransmission in a Low-Incidence Country Due to Immigration from High-Incidence Areas
Authors: Lillebaek Troels , Andersen Åse B. , Bauer Jeanett , Dirksen Asger , Glismann Steffen , de Haas Petra , Kok-Jensen Axel
Published: July 2002

Does immigration from a high-prevalence area contribute to an increased risk of tuberculosis in a low-incidence country? The tuberculosis incidence in Somalia is among the highest ever registered. Due to civil war and starvation, nearly half of all Somalis have been forced from their homes, causing significant migration to low-incidence countries. In Denmark, two-thirds of all tuberculosis patients are immigrants, half from Somalia. To determine the magnitude ofMycobacterium tuberculosistransmission between Somalis and Danes, we analyzed DNA fingerprint patterns of isolates collected in Denmark from 1992 to 1999, comprising >97% of all culture-positive patients (n= 3,320). Of these, 763 were Somalian immigrants, 55.2% of whom shared identical DNA fingerprint patterns; 74.9% of these were most likely infected before their arrival in Denmark, 23.3% were most likely infected in Denmark by other Somalis, and 1.8% were most likely infected by Danes. In the same period, only 0.9% of all Danish tuberculosis patients were most likely infected by Somalis. The Somalian immigrants in Denmark could be distributed into 35 different clusters with possible active transmission, of which 18 were retrieved among Somalis in the Netherlands. This indicated the existence of some internationally predominant Somalian strains causing clustering less likely to represent recent transmission. In conclusion,M. tuberculosistransmission among Somalis in Denmark is limited, and transmission between Somalis and Danes is nearly nonexistent. The higher transmission rates between nationalities found in the Netherlands do not apply to the situation in Denmark and not necessarily elsewhere, since many different factors may influence the magnitude of active transmission.
Typing of
Candida albicans
Isolates by Sequence Analysis of the Cytochrome
b
Gene and Differentiation from
Candida stellatoidea
Authors: Biswas Swarajit Kumar , Yokoyama Koji , Wang Li , Nishimura Kazuko , Miyaji Makoto
Published: July 2002

Including type strains, mitochondrial cytochrome
b
genes of 32 strains of
Candida albicans
and 6 strains of
Candida stellatoidea
, presently treated as a synonym for
C. albicans
, were partially sequenced. Analysis of 396-bp nucleotide sequences of the strains under investigation divided
C. albicans
isolates into three types: type I, type II, and type III; however, strains of
C. stellatoidea
represented distinct type IV isolates. Deduced amino acid sequences of type I, type II, and type III were identical and differed from that of type IV by one amino acid. Genotypes (rDNA type) of the test strains were also checked. Cytochrome
b
typing did not correlate with genotyping, and different genotypes occurred for one cytochrome
b
type. This study shows that cytochrome
b
gene sequences are useful for analyzing the genetic relatedness of
C. albicans
isolates and effective for differentiating
C. stellatoidea
from
C. albicans
.
Genotyping
Encephalitozoon hellem
Isolates by Analysis of the Polar Tube Protein Gene
Authors: Xiao Lihua , Li Lixia , Moura Hercules , Sulaiman Irshad , Lal Altaf A. , Gatti Simonetta , Scaglia Massimo , Didier Elizabeth S. , Visvesvara Govinda S.
Published: July 2002

To develop an alternative genotyping tool, the genetic diversity of
Encephalitozoon hellem
was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24
E. hellem
isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate
E. hellem
genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of
E. hellem
in clinical samples.
Clinical Isolates of Non-O157 Shiga Toxin-Producing
Escherichia coli
: Serotypes, Virulence Characteristics, and Molecular Profiles of Strains of the Same Serotype
Authors: Eklund Marjut , Scheutz Flemming , Siitonen Anja
Published: July 2002

All human Shiga toxin-producing
Escherichia coli
(STEC) non-O157 strains (
n
= 56) isolated in Finland from 1990 to August 2000 were characterized for the O:H serotype,
stx
1
and
stx
2
genes, production of enterohemolysin, and sensitivity to 12 antimicrobial agents. Strains of the same serotype were genotyped by pulsed-field gel electrophoresis (PFGE) after
Xba
I restriction of total DNA. The 56 non-O157 isolates belonged to 29 serotypes. Two of the serotypes (O102:H7 and OX181:H49) have not previously been described as being associated with STEC infections in humans or isolated from animals. Thirty-four strains (61%) within seven serotypes (O103:H2 [14 isolates], O26:H11 [6 isolates], O145:H28 [4 isolates], O145:HNM [3 isolates], O15:HNM [3 isolates], OX174:H21 [2 isolates], and O Rough:HNM [2 isolates]) were represented by more than one isolate. Of these strains, O103:H2 isolates were divided into seven, O26:H11 isolates were divided into four, and the rest within a serotype were divided into two genotypes in PFGE. In PCR, 31 (55%) of the 56 strains were positive for the
stx
2
gene only and 24 strains (43%) were positive for
stx
1
only. One strain (O43:H2) carried both
stx
1
and
stx
2
. Forty-two strains (75%) produced enterohemolysin, and 39 strains (70%) possessed the
eae
gene. Of the latter 39 strains, 36 (92%) were enterohemolytic, whereas only 6 (35%) of the 17 isolates lacking the
eae
gene were enterohemolytic (
P
< 0.001). The majority of the strains (44 strains, 79%) were sensitive to all 12 antimicrobials tested. Of the 56 strains, 20 (36%) were associated with small family outbreaks in nine families and 14 (25%) were associated with recent travel abroad.
Detection of Ebola Viral Antigen by Enzyme-Linked Immunosorbent Assay Using a Novel Monoclonal Antibody to Nucleoprotein
Authors: Niikura Masahiro , Ikegami Tetsuro , Saijo Masayuki , Kurane Ichiro , Miranda Mary E. , Morikawa Shigeru
Published: July 2002

With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.
Evaluation of Recombinant
Leptospira
Antigen-Based Enzyme-Linked Immunosorbent Assays for the Serodiagnosis of Leptospirosis
Authors: Flannery Brendan , Costa Dirceu , Carvalho Fernanda Pinheiro , Guerreiro Hygia , Matsunaga James , Da Silva Emilson Domingos , Ferreira Antonio Gomes Pinto , Riley Lee W. , Reis Mitermayer G. , Haake David A. , Ko Albert I.
Published: July 2002

There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (
P
> 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis.
Hemorrhagic Fever with Renal Syndrome: Diagnostic Problems with a Known Disease
Authors: Wichmann Dominic , Slenczka Werner , Alter Peter , Boehm Stephan , Feldmann Heinz
Published: July 2002

Hemorrhagic fever with renal syndrome (HFRS), caused by different hantaviruses, is a distinct clinical syndrome endemic in several parts of Asia and Europe. However, the clinical picture can sometimes be indistinguishable from that of other infectious or noninfectious diseases. In this report we describe a clinical case, which is a rare occurrence but is a prime example of the difficulties in the diagnosis of HFRS in areas with a low prevalence of the disease.
Phenotypic Variation ofHelicobacter pyloriIsolates from Geographically Distinct Regions Detected by Lectin Typing
Authors: Hynes Sean O. , Broutet Nathalie , Eurohepygast Study Group  , Wadström Torkel , Mikelsaar Marika , O’Toole Paul W. , Telford John , Engstrand Lars , Kamiya Shigeru , Mentis Andreas F. , Moran Anthony P.
Published: July 2002

A total of 309Helicobacter pyloriisolates from 18 different countries were analyzed with a previously developed lectin typing system. The system was developed by using a proteolytic pretreatment to enhance the carbohydrate fraction of the sample. Four lectins fromUlex europaeus,Lotus tetragonolobus,Erythrina cristigali, andTriticum vulgariswere used to type the strains. The lectins were chosen for their specificities for sugars commonly encountered in the lipopolysaccharide ofH. pylori. The isolates were received from their parent institutions as pellets of biomass and were typed at one of three centers (in Ireland, Sweden, and Estonia). All 16 possible lectin reaction patterns were observed in the study, with the isolates with the predominant pattern exhibiting reactions with all the lectins in the panel. For European patients suffering from gastritis, an association was noted between lectin reaction pattern MH4 and atrophic chronic gastritis; isolates with lectin reaction pattern MH4 were isolated from patients with atrophic chronic gastritis, whereas isolates with this pattern were not isolated from patients with chronic gastritis (P= 0.0006). In addition, statistically significant relationships were noted between the lectin reaction pattern and the associated pathology of isolates from the Swedish population. Isolates with patterns MH13 and MH16, which had low lectin reactivities, correlated with nonulcer disease (P= 0.0025 andP= 0.0002, respectively), and all four isolates from adenocarcinoma patients were characterized as possessing reaction pattern MH16. In contrast, isolates with lectin reaction patterns MH1 and MH10, which had high lectin reactivities, were associated with ulcer disease (P= 0.046 andP= 0.0022, respectively).
Molecular Typing of
Treponema pallidum
in South Africa: Cross-Sectional Studies
Authors: Pillay A. , Liu H. , Ebrahim S. , Chen C. Y. , Lai W. , Fehler G. , Ballard R. C. , Steiner B. , Sturm A. W. , Morse S. A.
Published: July 2002

We evaluated a molecular subtyping system for
Treponema pallidum
for its ability to differentiate between strains obtained from male patients with primary syphilis in South Africa. Of 201
T
.
pallidum
-positive specimens, 161 were typeable, revealing 35 subtypes. The unique subtypes identified in Durban, Cape Town, and Carletonville and the total number of subtypes suggested that the strain population was very diverse and varied geographically.
Immunochromatographic Strip Test for Rapid Detection of Diphtheria Toxin: Description and Multicenter Evaluation in Areas of Low and High Prevalence of Diphtheria
Authors: Engler K. H. , Efstratiou A. , Norn D. , Kozlov R. S. , Selga I. , Glushkevich T. G. , Tam M. , Melnikov V. G. , Mazurova I. K. , Kim V. E. , Tseneva G. Y. , Titov L. P. , George R. C.
Published: July 2002

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of
Corynebacterium
spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.
Comparative Evaluation of the BD Phoenix and VITEK 2 Automated Instruments for Identification of Isolates of the
Burkholderia cepacia
Complex
Authors: Brisse Sylvain , Stefani Stefania , Verhoef Jan , Van Belkum Alex , Vandamme Peter , Goessens Wil
Published: July 2002

We evaluated two new automated identification systems, the BD Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux), for identification of isolates of the
Burkholderia cepacia
complex (BCC). The test sample included 42 isolates of the highly virulent and epidemic genomovar III, 45 isolates of
B. multivorans
, and 47 isolates of other members of the BCC. Rates of correct identification by the BD Phoenix and VITEK 2 were similar when all BCC isolates were considered (50 and 53%, respectively) but differed markedly for genomovar III (71 and 38%;
P
< 0.01) and for
B. multivorans
(58 and 89%;
P
< 0.001). For the BD Phoenix as well as the VITEK 2, taking all 134 isolates of the BCC together, rates of correct identification of clinical isolates (56 and 55%, respectively;
n
= 85) were higher than those of environmental isolates (21 and 39%, respectively;
n
= 28). Clinical isolates of genomovar III (
n
= 27) showed correct identification rates of 81% (BD Phoenix) and 48% (VITEK 2) (
P
< 0.01). Rates of misidentification for BD Phoenix and VITEK 2 were 9 and 17% for genomovar III, 22 and 7% for
B. multivorans
, and 36 and 13% for the other BCC members (
P
< 0.01), respectively. More than half of the isolates misidentified by each instrument were identified as
Ralstonia pickettii
,
Ralstonia paucula
(CDC IV C-2 group),
Alcaligenes faecalis
,
Achromobacter
spp., or, for the VITEK 2, “various nonfermenters.” This study reemphasizes that confirmatory identification of BCC, preferably by molecular methods, is highly recommended.
Evaluation of the Subtype Specificity of Monoclonal Antibodies Raised against H1 and H3 Subtypes of Human Influenza A Virus Hemagglutinins
Authors: Varečková Eva , Cox Nancy , Klimov Alexander
Published: July 2002

Three previously described monoclonal antibodies (MAbs) specific for influenza A(H1) hemagglutinins (HA) revealed high sensitivity (98.2 to 99.1%) and specificity (100%) when tested against 245 strains of different subtypes. One of them was included in the World Health Organization's influenza reagent kit for 2001 to 2002. In contrast, two MAbs raised against human influenza A(H3) HA revealed cross-reactivity with viruses of other subtypes.
Differences between C57BL/6 and BALB/cBy Mice in Mortality and Virus Replication after Intranasal Infection with Neuroadapted Sindbis Virus
Authors: Thach Dzung C. , Kimura Takashi , Griffin Diane E.
Published: July 2002

Neuroadapted Sindbis virus (NSV), given intranasally, caused fatal encephalitis in 100% of adult C57BL/6 mice and 0% of BALB/cBy mice. Most C57BL/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while BALB/cBy mice did not. In situ hybridization for detecting NSV RNA and immunohistochemistry for detecting NSV antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. Virus antigen was more abundant and infectious virus increased more rapidly and reached higher levels in C57BL/6 mice than in BALB/cBy mice. Surprisingly, infectious virus was cleared faster in C57BL/6 mice, and this was associated with more rapid production of neutralizing antibody. However, viral RNA was cleared more slowly in C57BL/6 mice. In both mouse strains, more infectious virus was present in the lumbar spinal cord than in the cervical spinal cord. These data suggest that genetic susceptibility to fatal NSV encephalomyelitis is determined at least in part by the efficiency of viral replication and spread in the central nervous system. The differences identified in this study provide possible phenotypes for mapping genetic loci involved in susceptibility.
CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland
Authors: Zhu Quan , Gregg Keqin , Lozano Mary , Liu Jinqi , Dudley Jaquelin P.
Published: July 2002

Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland.
Gamma Interferon Impedes the Establishment of Herpes Simplex Virus Type 1 Latent Infection but Has No Impact on Its Maintenance or Reactivation in Mice
Authors: Lekstrom-Himes Julie A. , LeBlanc Rona A. , Pesnicak Lesley , Godleski Matthew , Straus Stephen E.
Published: July 2002

Murine models of gamma interferon (IFN-γ) deficiency demonstrate the role of this cytokine in attenuating acute herpes simplex virus (HSV) disease; however, the effect of IFN-γ on the establishment and maintenance of neuronal latency and viral reactivation is not known. Using the IFN-γ knockout (GKO) model of IFN-γ deficiency and sensitive quantitative PCR methods, we show that IFN-γ significantly reduces the ganglion content of latent HSV-1 in BALB/c mice, which in turn delays viral time to reactivation following UV irradiation. Similar effects were not seen in the C57BL/6 strain. These results indicate that IFN-γ significantly attenuates latent HSV infection in the mouse model of ocular infection but has no impact on the maintenance of latency or virus reactivation.
Potential Dengue Virus-Triggered Apoptotic Pathway in Human Neuroblastoma Cells: Arachidonic Acid, Superoxide Anion, and NF-κB Are Sequentially Involved
Authors: Jan Jia-Tsrong , Chen Bor-Horng , Ma Shiou-Hwa , Liu Chiu-I , Tsai Hui-Ping , Wu Han-Chung , Jiang Shian-Yuan , Yang Kuen-Der , Shaio Men-Fang
Published: July 2002

Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A2(PLA2) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA2activity by the PLA2inhibitors, AACOCF3and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (SOD) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF3. In addition, the transcription factors, NF-κB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-κB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF3and SOD significantly block activation of NF-κB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA2activation to superoxide anion generation and subsequently to NF-κB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-κB.
A Human Milk Factor Susceptible to Cathepsin D Inhibitors Enhances Human Immunodeficiency Virus Type 1 Infectivity and Allows Virus Entry into a Mammary Epithelial Cell Line
Authors: El Messaoudi Kamal , Thiry Lise F. , Liesnard Corinne , Van Tieghem Nicole , Bollen Alex , Moguilevsky Nicole
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells.
The Murine Double-Stranded RNA-Dependent Protein Kinase PKR Is Required for Resistance to Vesicular Stomatitis Virus
Authors: Stojdl David F. , Abraham Ninan , Knowles Shane , Marius Ricardo , Brasey Ann , Lichty Brian D. , Brown Earl G. , Sonenberg Nahum , Bell John C.
Published: July 2002

Interferon (IFN)-induced antiviral responses are mediated through a variety of proteins, including the double-stranded RNA-dependent protein kinase PKR. Here we show that fibroblasts derived from PKR−/−mice are more permissive for vesicular stomatitis virus (VSV) infection than are wild-type fibroblasts and demonstrate a deficiency in alpha/beta-IFN-mediated protection. We further show that mice lacking PKR are extremely susceptible to intranasal VSV infection, succumbing within days after instillation with as few as 50 infectious viral particles. Again, alpha/beta-IFN was unable to rescue PKR−/−mice from VSV infection. Surprisingly, intranasally infected PKR−/−mice died not from pathology of the central nervous system but rather from acute infection of the respiratory tract, demonstrating high virus titers in the lungs compared to similarly infected wild-type animals. These results confirm the role of PKR as the major component of IFN-mediated resistance to VSV infection. Since previous reports have shown PKR to be nonessential for survival in animals challenged with encephalomyocarditis virus, influenza virus, and vaccinia virus (N. Abraham et al., J. Biol. Chem. 274:5953–5962, 1999; Y. Yang et al., EMBO J. 14:6095–6106, 1995), our findings serve to highlight the premise that host dependence on the various mediators of IFN-induced antiviral defenses is pathogen specific.
Persistent Infection of Human Pancreatic Islets by Coxsackievirus B Is Associated with Alpha Interferon Synthesis in β Cells
Authors: Chehadeh Wassim , Kerr-Conte Julie , Pattou François , Alm Gunar , Lefebvre Jean , Wattré Pierre , Hober Didier
Published: July 2002

The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing α cells and insulin-containing β cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-α), as evidenced by the detection of IFN-α mRNA and immunoreactive IFN-α with antiviral activity. By double IF staining, IFN-α was detected in insulin-producing β cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by α and β cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-α expression in β cells. The viral replication and the expression of IFN-α in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-α significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human β cells can harbor a persistent CVB infection, and CVB-induced IFN-α plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.
Impaired Gamma Interferon Responses against Parvovirus B19 by Recently Infected Children
Authors: Corcoran Amanda , Doyle Sean , Waldron David , Nicholson Alfred , Mahon Bernard P.
Published: July 2002

Parvovirus B19 is the causative agent of “fifth disease” of childhood. It has been implicated in a variety of conditions, including unsuccessful pregnancy and rheumatoid arthritis, and is a potential contaminant of blood products. There has been little study of immunity to parvovirus B19, and the exact nature of the protective humoral and cell-mediated immune response is unclear. Immune responses to purified virus capsid proteins, VP1 and VP2, were examined from a cohort of recently infected children and compared with responses from long-term convalescent volunteers. The results demonstrate that antibody reactivity is primarily maintained against conformational epitopes in VP1 and VP2. The unique region of VP1 appears to be a major target for cell-mediated immune responses, particularly in recently infected individuals. We confirm that antibody reactivity against linear epitopes of VP2 is lost shortly after infection but find no evidence of the proposed phenotypic switch in either the subclass of parvovirus B19-specific antibody or the pattern of cytokine production by antigen-specific T cells. The dominant subclass of specific antibody detected from both children and adults was immunoglobulin G1. No evidence was found for interleukin 4 (IL-4) or IL-5 production by isolated lymphocytes from children or adults. In contrast, lymphocytes from convalescent adults produced a typical type 1 response associated with high levels of IL-2 and gamma interferon (IFN-γ). However, we observed a significant (P< 0.001) deficit in the production of IFN-γ in response to VP1 or VP2 from lymphocytes isolated from children. Taken together, these results imply that future parvovirus B19 vaccines designed for children will require the use of conformationally preserved capsid proteins incorporating Th1 driving adjuvants. Furthermore, these data suggest novel mechanisms whereby parvovirus B19 infection may contribute to rheumatoid arthritis and unsuccessful pregnancy.
Identification of a Membrane Targeting and Degradation Signal in the p42 Protein of Influenza C Virus
Authors: Pekosz Andrew , Lamb Robert A.
Published: July 2002

Two mRNA species are derived from the influenza C virus RNA segment six, (i) a colinear transcript containing a 374-amino-acid residue open reading frame (referred to herein as the seg 6 ORF) which is translated to yield the p42 protein, and (ii) a spliced mRNA which encodes the influenza C virus matrix (CM1) protein consisting of the first 242 amino acids of p42. The p42 protein undergoes proteolytic cleavage at a consensus signal peptidase cleavage site after residue 259, yielding the p31 and CM2 proteins. Translocation of p42 into the endoplasmic reticulum membrane occurs cotranslationally and requires the hydrophobic internal signal peptide (residues 239 to 259), as well as the predicted transmembrane domain of CM2 (residues 285 to 308). The p31 protein was found to undergo rapid degradation after cleavage from p42. Addition of the 26S proteasome inhibitor lactacystin to influenza C virus-infected or seg 6 ORF cDNA-transfected cells drastically reduced p31 degradation. Transfer of the 17-residue C-terminal region of p31 to heterologous proteins resulted in their rapid turnover. The hydrophobic nature, but not the specific amino acid sequence of the 17-amino-acid C terminus of p31 appears to act as the signal for targeting the protein to membranes and for degradation.