bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Term Effects of Immunization with Recombinant Lipoprotein Outer Surface Protein A on Serologic Test for Lyme Disease
Authors: Fawcett Paul T. , Rose Carlos D. , Maduskuie Victoria
Published: July 2004

Immunization with recombinant lipoprotein outer surface protein A vaccine is known to interfere with some serologic tests for Lyme disease. We tested sera from 152 vaccine recipients by using in-house and commercial Western blot assays and found that vaccination caused interference in up to 25% of recipients and can persist for over 6 years.
Detection of Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in SARS Patients by Enzyme-Linked Immunosorbent Assay
Authors: Lau Susanna K. P. , Woo Patrick C. Y. , Wong Beatrice H. L. , Tsoi Hoi-Wah , Woo Gibson K. S. , Poon Rosana W. S. , Chan Kwok-Hung , Wei William I. , Peiris J. S. Malik , Yuen Kwok-Yung
Published: July 2004

We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His
6
-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities—96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.
Detection of Human Immunodeficiency Virus Type 1 Antiretroviral Resistance Mutations by High-Density DNA Probe Arrays
Authors: Gonzalez R. , Masquelier B. , Fleury H. , Lacroix B. , Troesch A. , Vernet G. , Telles J. N.
Published: July 2004

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.
Prospective Study of a Real-Time PCR That Is Highly Sensitive, Specific, and Clinically Useful for Diagnosis of Meningococcal Disease in Children
Authors: Bryant Penelope A. , Li Hua Yi , Zaia Angelo , Griffith Julia , Hogg Geoff , Curtis Nigel , Carapetis Jonathan R.
Published: July 2004

Due to the early administration of antibiotics, meningococcal disease is increasingly difficult to diagnose by culturing. Laboratory studies have shown PCR to be sensitive and specific, but there have been few clinical studies. The objectives of this study were to determine the diagnostic accuracy and clinical usefulness of meningococcal PCR through a prospective comparison of real-time PCR, nested PCR, and standard culturing of blood and cerebrospinal fluid (CSF). The setting was a tertiary-care pediatric hospital in Australia, and the participans were 118 children admitted with possible septicemia or meningitis. The main outcome measures—sensitivity, specificity, and positive and negative predictive values—were compared to a “gold standard ” fulfilling clinical and laboratory criteria. For 24 cases of meningococcal disease diagnosed by the gold standard, culturing of blood or CSF was positive for 15 (63%), nested PCR was positive for 21 (88%), and real-time PCR was positive for 23 (96%). The sensitivity, specificity, and positive and negative predictive values of real-time PCR (the most sensitive test) for all specimens were, respectively, 96% (95% confidence interval, 79 to 99%), 100% (95% confidence interval, 96 to100%), 100% (95% confidence interval, 85 to 100%), and 99% (95% confidence interval, 94 to 100%). Of 54 patients with suspected meningococcal disease at admission, 23 had positive PCR results. Only one PCR specimen was positive in a patient thought unlikely to have meningococcal disease at admission. Blood PCR remained positive for 33% of patients tested at up to 72 h. Real-time PCR has high positive and negative predictive values in this clinical setting, with better confirmation of cases than nested PCR. Targeting patients for PCR based on admission criteria appears to be practical, and the test may remain useful for several days after the start of antibiotic administration.
Combination of PCR Targeting the VD2 of
omp1
and Reverse Line Blot Analysis for Typing of Urogenital
Chlamydia trachomatis
Serovars in Cervical Scrape Specimens
Authors: Molano Monica , Meijer Chris J. L. M. , Morré Servaas A. , Pol Rene , van den Brule Adriaan J. C.
Published: July 2004

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the
omp1
gene, for the detection and typing of urogenital
Chlamydia trachomatis
infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of
C. trachomatis
. The assay developed was tested with reference strains of
C. trachomatis
serovars and cervical scrapes of 86 Colombian women previously found to be positive for
C. trachomatis
by using plasmid PCR. Two sets of primers directed to the VD2 region of the
omp1
gene of
C. trachomatis
were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of
C. trachomatis
(Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single
C. trachomatis
infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple
C. trachomatis
infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital
C. trachomatis
serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple
C. trachomatis
infections and for use in large epidemiological studies.
Performance of a New, Rapid Assay for Detection of
Trichomonas vaginalis
Authors: Kurth Ann , Whittington William L. H. , Golden Matthew R. , Thomas Katherine K. , Holmes King K. , Schwebke Jane R.
Published: July 2004

Trichomonas vaginalis
infection is highly prevalent, may have serious health consequence, and is readily treatable. However, screening has been limited by currently available tests, which tend to be insensitive, expensive, or require a delay before results are reported. The XenoStrip-Tv (Xenotope Diagnostics, Inc., San Antonio, Tex.) was evaluated on vaginal swab specimens from 936 women attending sexually transmitted disease clinics in Seattle, Wash. (
n
= 497), and Birmingham, Ala. (
n
= 439).
T. vaginalis
prevalence by culture (InPouch; Biomed) was 8.7% in Seattle and 21.0% in Birmingham. Compared to culture, the XenoStrip assay in Seattle was 76.7% (95% confidence interval [95% CI] = 61.4 to 88.2) sensitive and 99.8% (95% CI = 98.8 to 99.9) specific, and in Birmingham it was 79.4% (95% CI = 69.6 to 87.1) sensitive and 97.1% (95% CI = 94.8 to 98.6) specific. The positive predictive values were 97.1% in Seattle and 87.9% in Birmingham; the negative predictive values were 97.8 and 94.7%, respectively. Rapid test performance did not vary by vaginal symptoms or by the presence of other vaginal or cervical syndromes or infections. The sensitivity did vary by day of culture-positive result, with a 71% decline in XenoStrip sensitivity for every additional day delay until
T. vaginalis
was first detected in cultures (odds ratio = 0.29, 95% CI = 0.18 to 0.49). The rapid assay was more sensitive than wet preparation microscopy (78.5% versus 72.4% [
P
= 0.04]) but was less specific (98.6% versus 100% [
P
= 0.001]). The XenoStrip rapid assay is well suited for use in settings with a moderately high prevalence of
T. vaginalis
infection, particularly when microscopy is not practical.
Isolation of Recombinant Antibodies against EspA and Intimin of
Escherichia coli
O157:H7
Authors: Kühne Sarah A. , Hawes William S. , La Ragione Roberto M. , Woodward Martin J. , Whitelam Garry C. , Gough Kevin C.
Published: July 2004

Intimin, Tir, and EspA proteins are expressed by attaching-effacing
Escherichia coli
, which include enteropathogenic and enterohemorrhagic
E. coli
pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic
E. coli
O157:H7 were produced by using
E. coli
expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from
E. coli
O157 also recognized the protein from the
eae
-deficient O157 mutant DM3 and from
E. coli
O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of γ intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.
Intra- and Interlaboratory Study of a Method for Testing the Antifungal Susceptibilities of Dermatophytes
Authors: Ghannoum M. A. , Chaturvedi V. , Espinel-Ingroff A. , Pfaller M. A. , Rinaldi M. G. , Lee-Yang W. , Warnock D. W.
Published: July 2004

The National Committee for Clinical Laboratory Standards (NCCLS) M38-A standard for the susceptibility testing of conidium-forming filamentous fungi does not explicitly address the testing of dermatophytes. This multicenter study, involving six laboratories, investigated the MIC reproducibility of seven antifungal agents tested against 25 dermatophyte isolates (5 blinded pairs of five dermatophyte species per site for a total of 300 tests), using the method of dermatophyte testing developed at the Center for Medical Mycology, Cleveland, Ohio. The dermatophytes tested included
Trichophyton rubrum
,
Trichophyton mentagrophytes
,
Trichophyton tonsurans
,
Epidermophyton floccosum
, and
Microsporum canis
. Seven antifungals with activity against dermatophytes were tested, including ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Interlaboratory MICs for all isolates were in 92 to 100% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 88 to 99% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Intralaboratory MICs between blinded pairs were in 97% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 96% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Data from this study support consideration of this method as an amendment to the NCCLS M38-A standard for the testing of dermatophytes.
Coexistence of Multiple Genotypes, Including Newly Identified Genotypes, in Outbreaks of Gastroenteritis Due to
Norovirus
in Japan
Authors: Kageyama Tsutomu , Shinohara Michiyo , Uchida Kazue , Fukushi Shuetsu , Hoshino Fuminori B. , Kojima Shigeyuki , Takai Reiko , Oka Tomoichiro , Takeda Naokazu , Katayama Kazuhiko
Published: July 2004

Norovirus
(NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology
299:
225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol.
41:
1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.
Comparison of Three Nucleic Acid Amplification Tests for Detection of
Chlamydia trachomatis
in Urine Specimens
Authors: Gaydos Charlotte A. , Theodore Mellisa , Dalesio Nicholas , Wood Billie Jo , Quinn Thomas C.
Published: July 2004

Traditionally, culture and immunoassays have been performed for the detection of sexually transmitted diseases, including
Chlamydia trachomatis
. However, these assays may often require invasive specimen collection methods, such as female cervical and male urethral swabs. Recently, nucleic acid amplification tests (NAATs) have been approved for testing for the presence of
C. trachomatis
in urine samples. Our objective was to compare the sensitivities and specificities of
C. trachomatis
detection in urine samples with three NAATs: the Abbott LCx (LCx), BD ProbeTec ET (ProbeTec), and Gen-Probe APTIMA Combo 2 (AC2). Urine specimens (
n
= 506) were collected from both symptomatic and asymptomatic males and females from various high school health clinics. Specimens were tested for
C. trachomatis
with the three NAATs, and a true-positive result was defined as any two positive NAATs. The
C. trachomatis
prevalence was 14.8% (75 of 506 samples). Of the 75 urine samples defined as true positives, LCx detected 72, ProbeTec 72, and AC2 detected 75. The sensitivities of LCx, ProbeTec, and AC2 for
C. trachomatis
detection were 96.0, 96.0, and 100%, and the specificities were 99.1, 100, and 98.8%, respectively. Four of five samples that were positive with AC2 and negative with LCx and ProbeTec were found to be positive with an alternative target TMA-based NAAT, APTIMA
C. trachomatis
, suggesting that they may have been true positives. Two of four uniquely positive LCx samples available for subsequent testing were both found to be positive by Roche PCR. We found that the LCx, ProbeTec, and AC2 NAATs are highly sensitive and specific methods for the detection of
C. trachomatis
in urine specimens and can be recommended for noninvasive screening of
C. trachomatis
in urine.
Studies on the Involvement of the Exopolysaccharide Produced by Cystic Fibrosis-Associated Isolates of the
Burkholderia cepacia
Complex in Biofilm Formation and in Persistence of Respiratory Infections
Authors: Cunha Mónica V. , Sousa Sílvia A. , Leitão Jorge H. , Moreira Leonilde M. , Videira Paula A. , Sá-Correia Isabel
Published: July 2004

Bacteria belonging to the
Burkholderia cepacia
complex (BCC) are important opportunistic pathogens that lead to respiratory infections in patients with cystic fibrosis (CF). The clinical outcome following colonization with BCC bacteria is highly variable, and so far, unpredictable. A large percentage (80 to 90%) of BCC isolates from CF patients produce the exopolysaccharide (EPS) cepacian, which has been hypothesized to play a role in the colonization and persistence of these bacteria in the CF lung. In this work, we demonstrate that although it is not required for the initiation of biofilm formation, cepacian plays a role in the establishment of thick biofilms. This conclusion was based on a comparison of the abilities of EPS-defective mutants derived from a
B. cepacia
mucoid CF isolate by random plasposon insertion mutagenesis and the ability of the parental strain to form biofilms. However, the systematic characterization of 108 CF isolates, corresponding to 15 distinct strains, indicated that other strain-dependent factors are also involved in the development of thick, mature biofilms. The isolates examined belonged to the species
B. cepacia
,
B. multivorans
,
B. cenocepacia
, and
B. stabilis
and were obtained during a 7-year period of surveillance from 21 CF patients receiving care at the major Portuguese CF center. Most of them (90%) were serial isolates from 12 persistently infected patients. In spite of the concept that bacteria growing in biofilms display more resistance to antibiotics and to host phagocyte killing than do planktonically growing cells, no clear correlation could be established between the ability of the various strains examined to produce EPS and/or to form biofilms in vitro and the persistence or virulence of the respiratory infections they caused in different patients.
Prevalence of the
hifBC
,
hmw1A
,
hmw2A
,
hmwC
, and
hia
Genes in
Haemophilus influenzae
Isolates
Authors: Ecevit I. Zafer , McCrea Kirk W. , Pettigrew Melinda M. , Sen Ananda , Marrs Carl F. , Gilsdorf Janet R.
Published: July 2004

Adherence of
Haemophilus influenzae
to respiratory epithelial cells is the first step in the pathogenesis of
H. influenzae
infection and is facilitated by the action of several adhesins located on the surface of the bacteria. In this study, prevalences of
hifBC
, which represent the pilus gene cluster;
hmw1A
,
hmw2A
, and
hmwC
, which represent high-molecular-weight (HMW) adhesin genes; and
hia
, which represents
H. influenzae
adhesin (Hia) genes were determined among clinical isolates of encapsulated type b (Hib) and nonencapsulated (NTHi)
H. influenzae. hifBC
genes were detected in 109 of 170 (64%) Hib strains and in 46 of 162 (28%) NTHi isolates (
P
= 0.0001) and were more prevalent among the invasive type b strains than invasive NTHi strains (
P
= 0.00003). Furthermore,
hifBC
genes were significantly more prevalent (
P
= 0.0398) among NTHi throat isolates than NTHi middle ear isolates.
hmw1A
,
hmw2A
,
hmwC
, and
hia
genes were not detected in Hib strains. Among NTHi isolates, the prevalence of
hmw1A
was 51%, the prevalence of
hmw2A
was 23%, the prevalence of
hmwC
was 48%, and the prevalence of
hia
was 33%. The
hmw
genes were significantly more prevalent among middle ear than throat isolates, while
hia
did not segregate with a respiratory tract site. These results show the variability of the presence of adhesin genes among clinical
H. influenzae
isolates and suggest that hemagglutinating pili may play a larger role in
H. influenzae
nasopharyngeal colonization than in acute otitis media whereas the HMW adhesins may be virulence factors for acute otitis media.
Clonal Relatedness of EnterotoxigenicEscherichia coliStrains Isolated from a Cohort of Young Children in Guinea-Bissau
Authors: Steinsland Hans , Valentiner-Branth Palle , Aaby Peter , Mølbak Kåre , Sommerfelt Halvor
Published: July 2004

In a cohort study of 200 young children in Guinea-Bissau, it was previously found that some enterotoxigenicEscherichia coli(ETEC) strains were more pathogenic than others, depending on the type of toxin that they could produce, and that natural ETEC infections induced substantial protection against new infections with ETEC strains that had the same combination of toxins and colonization factors (CFs). We wanted to describe the clonal relatedness of the ETEC strains isolated during this study and to investigate whether the protective antigens and the virulence factors that were responsible for the pathogenic traits were common to strains that were clonally closely related or whether they were more likely to be encoded by the ETEC virulence plasmids that normally encode the toxins and the CFs. By performing repetitive sequence-based PCR analysis of strains representing 452 infections, we found that strains that had the same toxin-CF profile were usually closely related, although a few were unrelated. Strains that did not have the same toxin-CF profiles but that were positive for a given toxin or for a given CF were not consistently more closely related to each other than to strains that were negative for the same toxin or CF. Our results indicate that the pathogenic traits of ETEC were mainly attributed to genes carried on the ETEC virulence plasmids. Because most strains that had the same toxin-CF profile were closely related, it seems likely that the toxin-CF-specific protection was clonal and was not targeting antigens encoded by the virulence plasmids. These results are of relevance to the ETEC vaccine development effort.
Diversity among Community Isolates of Methicillin-Resistant
Staphylococcus aureus
in Australia
Authors: O'Brien F. G. , Lim T. T. , Chong F. N. , Coombs G. W. , Enright M. C. , Robinson D. A. , Monk A. , Saïd-Salim B. , Kreiswirth B. N. , Grubb W. B.
Published: July 2004

Community methicillin-resistant
Staphylococcus aureus
(CMRSA) strains are being isolated with increasing frequency around the world. In Western Australia CMRSA are endemic in geographically remote communities and have been found to belong to five different contour-clamped homogeneous electric field (CHEF) electrophoretic patterns. Representatives of each of these CHEF patterns have been compared to CMRSA representative of CHEF patterns from other Australian states and New Zealand. With one exception, all of the isolates were nonmultiresistant and were not resistant to many antimicrobial agents other than the β-lactams. With one exception, which is not believed to be a CMRSA, all of the isolates harbored a β-lactamase plasmid. Erythromycin resistance was associated with a 2-kb plasmid. One of the β-lactamase plasmids was found to be able to acquire additional resistance determinants to become a multiple resistance plasmid. There were 10 multilocus sequence types belonging to eight distantly related clonal complexes of
S. aureus
. One new sequence type was found. Although most of the CMRSA harbored the type IVa SCC
mec
, a type IV structural variant was found and two new SCC
mec
types were identified. Protein A gene (
spa
) typing revealed two new
spa
types and, with two exceptions, corresponded to multilocus sequence typing. In contrast to other reports on CMRSA, most of the CMRSA strains studied here did not contain the Panton-Valentine leukocidin genes. The results also demonstrate that nonmultiresistant hospital strains such as UK EMRSA-15 may be able to circulate in the community and could be mistaken for CMRSA based on their resistance profiles.
LightCycler-Based Differentiation of
Mycobacterium abscessus
and
Mycobacterium chelonae
Authors: Sedlacek L. , Rifai M. , Feldmann K. , Bange F. C.
Published: July 2004

In this study we introduce a rapid procedure to identify
Mycobacterium abscessus
(types I and II) and
M. chelonae
using LightCycler-based analysis of the
hsp65
gene. Results from 36 clinical strains were compared with
hsp65
gene restriction analysis and biochemical profiles of bacilli. As all three methods yielded identical results for each isolate, this procedure offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnosis of mycobacterial diseases.
Influenza B Virus Victoria Group with a New Glycosylation Site Was Epidemic in Japan in the 2002-2003 Season
Authors: Nakagawa Naoko , Kubota Ritsuko , Maeda Akiko , Okuno Yoshinobu
Published: July 2004

In the 2002-2003 season, influenza B virus Victoria strains were epidemic after a 6-year absence in Kobe City, Japan. They reacted poorly to the immune ferret sera prepared for use against the previous strain. An amino acid substitution in the HA1 region caused them to acquire an N-linked glycosylation site.
Cloning and Characterization of
Trichophyton rubrum
Genes Encoding Actin, Tri r2, and Tri r4
Authors: Gao Jimin , Takashima Akira
Published: July 2004

The three structural genes of
Trichophyton rubrum
encoding actin (3,429 bp) and two antigens, Tri r2 (2,950 bp) and Tri r4 (3,988 bp), were cloned and characterized. They contained six, four, and five exons, respectively. The
T. rubrum
actin protein sequence revealed extremely high homology to other fungal actins.
Serotypes and Genotypes of Erythromycin-Resistant Group B Streptococci in Korea
Authors: Uh Young , Jang In Ho , Hwang Gyu Yel , Lee Mi Kyung , Yoon Kap Jun , Kim Hyo Youl
Published: July 2004

Among 78 erythromycin-resistant group B streptococcus (GBS) isolates from Korea,
ermB
was detected in 58 (74.4%),
mefA
was detected in 14 (17.9%), and
ermTR
was detected in 6 (7.7%). The most prevalent serotypes of erythromycin-resistant GBS were V (detected in 34 isolates [43.6%]) and III (detected in 33 isolates [42.3%]). All serotype V erythromycin-resistant GBS harbored the
ermB
gene.
Application of Oxidation-Reduction Assay for Monitoring Treatment of Patients with Pulmonary Tuberculosis
Authors: Farnia Parissa , Mohammadi Foroazan , Mirsaedi Mehdi , Zarife Abolhasan Zia , Tabatabee Javad , Bahadori Katayoon , Bahadori Muslem , Masjedi Mohammad Reza , Velayati Ali Akbar
Published: July 2004

By oxidation-reduction assay, the viability of
Mycobacterium tuberculosis
in sputum specimens was evaluated. The technique is based on the Alamar Blue and Malachite Green dyes, which change their color in response to
M. tuberculosis
growth. The method is simple, permits visual reading of results, and is applicable for laboratories with limited resources.
Differences in Clinical Manifestation of
Streptococcus pneumoniae
Infection Are Not Correlated with In Vitro Production and Release of the Virulence Factors Pneumolysin and Lipoteichoic and Teichoic Acids
Authors: Spreer Annette , Lis Astrid , Gerber Joachim , Reinert Ralf René , Eiffert Helmut , Nau Roland
Published: July 2004

Production and release of the pneumococcal virulence factors pneumolysin and lipoteichoic and teichoic acid in 75 clinical isolates were investigated. No difference was found between strains causing systemic infection or localized respiratory infection and isolates from asymptomatic carriers. This suggests that the presence of pneumolysin and lipoteichoic and teichoic acid is a necessary but not a sufficient condition for pneumococcal infection and development of invasive disease.
Extended-Spectrum-β-Lactamase (TEM-52)-Producing Strains of
Salmonella enterica
of Various Serotypes Isolated in France
Authors: Weill François-Xavier , Demartin Marie , Fabre Laëtitia , Grimont Patrick A. D.
Published: July 2004

From 2002 to 2003, four isolates of
Salmonella enterica
serotypes Typhimurium, Enteritidis, Blockley, and Panama, isolated in France from patients with gastroenteritis, were found to produce extended-spectrum β-lactamase TEM-52. The study showed the
bla
TEM-52
gene to be located in a Tn
3
-like structure and carried by 100- or 32-kb conjugative plasmids.
Bacteriology of a Bear Bite Wound to a Human: Case Report
Authors: Kunimoto Dennis , Rennie Robert , Citron Diane M. , Goldstein Ellie J. C.
Published: July 2004

Human contact with bears has become more frequent, as has the resultant bear maulings and bite injuries. We report the bacteriology of a patient bitten by a grizzly bear (
Ursus arctos
) from the Rocky Mountains foothills area east of Banff National Park, Alberta, Canada. The patient received field care, including metronidazole and cefazolin. Subsequent deep-wound cultures grew
Serratia fonticola
,
Serratia marcescens
,
Aeromonas hydrophila
,
Bacillus cereus
, and
Enterococcus durans
but no anaerobes.
Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin αvβ3
Authors: Lawler Karen , Meade Gerardene , O'Sullivan Gerald , Kenny Dermot
Published: July 2004

Interaction of tumor cells with the vascular wall is required for metastasis from the bloodstream. The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPS-treated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s−1. At a venous shear rate of 400 s−1, 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor “activation” occurred at a significantly lower shear rate of 50 s−1. This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s−1. The integrin αvβ3has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of αvβ3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s−1. Collectively, our data suggest that αvβ3promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions.
Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells
Authors: Nicoletta Julie A. , Ross Jonathan J. , Li Guangmu , Cheng Qingzhang , Schwartz Jonathon , Alexander Edward A. , Schwartz John H.
Published: July 2004

Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.
Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle
Authors: Bowles D. K. , Maddali K. K. , Ganjam V. K. , Rubin L. J. , Tharp D. L. , Turk J. R. , Heaps C. L.
Published: July 2004

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current ( ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7–8 mo; 35–45 kg) were obtained from the breeder. Males were left intact (IM, n = 8), castrated (CM, n = 8), or castrated with testosterone replacement (CMT, n = 8; 10 mg/day Androgel). Females remained gonad intact ( n = 8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.
Comparison of the inhibitory effects of PYY(3-36) and PYY(1-36) on gastric emptying in rats
Authors: Chelikani Prasanth K. , Haver Alvin C. , Reidelberger Roger D.
Published: July 2004

We compared the effects of the two molecular forms of the brain-gut peptide YY (PYY), PYY(1-36) and PYY(3-36), on gastric emptying. Unanesthetized rats received 20-min intravenous infusions of rat PYY(1-36) (0, 1.7, 5, 17, 50, 100, 170 pmol·kg−1·min−1) and rat PYY(3-36) (0, 0.5, 1.7, 5, 17, 50, 100, 170 pmol·kg−1·min−1), either alone or combined, and gastric emptying of saline was measured during the last 10 min of infusion. For comparison, human PYY(3-36) was administered at 0, 17, and 50 pmol·kg−1·min−1. Gastric emptying was decreased by 11, 24, 26 and 38% in response to 17, 50, 100, and 170 pmol·kg−1·min−1 of rat PYY(1-36); by 10, 26, 41, 53, and 57% in response to 5, 17, 50, 100, and 170 pmol·kg−1·min−1 of rat PYY(3-36); and by 35 and 53% in response to 17 and 50 pmol·kg−1·min−1 of human PYY(3-36), respectively. Estimated ED50s were 470 and 37 pmol·kg−1·min−1 for rat PYY(1-36) and PYY(3-36), respectively. In general, within an experiment, coadministration of PYY(1-36) and PYY(3-36) inhibited gastric emptying by an amount that was comparable to that produced when either peptide was given alone. We conclude that 1) intravenous infusion of PYY(1-36) and PYY(3-36) each produces a dose-dependent inhibition of gastric emptying in rats, 2) PYY(3-36) is an order of magnitude more potent than PYY(1-36) in inhibiting gastric emptying, 3) human PYY(3-36) and rat PYY(3-36) inhibit gastric emptying similarly, and 4) PYY(1-36) and PYY(3-36) do not appear to interact in an additive or synergistic manner to inhibit gastric emptying.
Reduced baroreflex control of heart period after bed rest is normalized by acute plasma volume restoration
Authors: Iwasaki Ken-ichi , Zhang Rong , Perhonen Merja A. , Zuckerman Julie H. , Levine Benjamin D.
Published: July 2004

Adaptation to spaceflight or head-down-tilt bed rest leads to hypovolemia and an apparent abnormality of baroreflex regulation of cardiac period. In a previous study, we demonstrated that both chronic (2 wk) head-down-tilt bed rest and acute induced hypovolemia led to similar impairments in spontaneous baroreflex control of cardiac period, suggesting that a reduction in plasma volume may be responsible for this abnormality after bed rest. Therefore we hypothesized that this reduced “baroreflex function” could be restored by intravenous volume infusion equivalent to the reduction in plasma volume after bed rest. Six healthy subjects underwent 2 wk of −6° head-down bed rest. Beat-by-beat arterial blood pressure and ECG were recorded during 6 min of spontaneous respiration and fixed-rate breathing (0.2 Hz), and transfer function analysis between systolic blood pressure and R-R interval was performed. Plasma volume was measured with Evans blue dye, and cardiac filling pressures were directly measured (Swan-Ganz catheter). After bed rest, studies were repeated before and after plasma volume restoration, with which both plasma volume and left ventricular end-diastolic pressure were restored to pre-bed rest levels by intravenous dextran40 infusion (288 ± 31 ml). Transfer function gain in the high-frequency range, used as an index of vagally mediated arterial-cardiac baroreflex function, decreased significantly (13.4 ± 3.1 to 8.1 ± 2.9 ms/mmHg, P < 0.05) after bed rest. However, reduced transfer function gain was normalized to the pre-bed rest level (12.2 ± 3.6 ms/mmHg) after precise plasma volume restoration. This result confirms that reductions in plasma volume, rather than a unique autonomic nervous system adaptation to bed rest, are largely responsible for the observed changes in spontaneous arterial-cardiac baroreflex function after bed rest.
Enhanced Striatal NR2B-Containing N-Methyl-d-Aspartate Receptor-Mediated Synaptic Currents in a Mouse Model of Huntington Disease
Authors: Li Lijun , Murphy Timothy H. , Hayden Michael R. , Raymond Lynn A.
Published: July 2004

Huntington disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract near the N terminus of the protein huntingtin, leading to dramatic loss of striatal medium-sized spiny GABAergic projection neurons (MSNs). Evidence suggests overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors (NMDARs) contributes to selective degeneration of MSNs in HD. Striatal MSNs are enriched in NR2B, and whole cell current and excitotoxicity mediated predominantly by the NR2B subtype of NMDARs is increased with expression of mutant huntingtin in transfected cell lines and striatal MSNs from mice models. To test whether synaptic NMDAR current is altered by mutant huntingtin expression, we recorded striatal MSN excitatory postsynaptic currents (EPSCs) evoked by stimulation of cortical afferents in corticostriatal slices from YAC72 mice and their wild-type (WT) littermates at age 21–31 days. The ratio of NMDAR- to AMPAR-mediated EPSC amplitude was significantly increased in YAC72 compared to WT mice. Furthermore, using a paired-pulse stimulation protocol as a measure of presynaptic glutamate release probability, we found no significant differences between YAC72 and WT striatal MSN responses. These data suggest selective potentiation of postsynaptic NMDAR activity at corticostriatal synapses in YAC72 mice. Measurements of EPSC decay kinetics, as well as the effects of NR2B-subtype selective antagonists and glycine concentration on EPSC amplitude, are consistent with the majority of postsynaptic NMDARs being triheteromers of NR1/NR2A/NR2B in both WT and YAC72 mice. Together with previous results, our data suggest that enhanced activity of NR2B-containing NMDARs is one of the earliest changes leading to neuronal degeneration in HD.
Engagement of Rat Striatal Neurons by Cortical Epileptiform Activity Investigated With Paired Recordings
Authors: Bracci Enrico , Centonze Diego , Bernardi Giorgio , Calabresi Paolo
Published: July 2004

The striatum is thought to play an important role in the spreading of epilepsy from cortical areas to deeper brain structures, but this issue has not been addressed with intracellular techniques. Paired recordings were used to assess the impact of cortical epileptiform activity on striatal neurons in brain slices. Bath-application of 4-amynopyridine (100 μM) and bicuculline (20 μM) induced synchronized bursts in all pairs of cortical neurons (≤5 mm apart) in coronal, sagittal, and oblique slices (which preserve connections from the medial agranular cortex to the striatum). Under these conditions, striatal medium spiny neurons (MSs) displayed a strong increased spontaneous glutamatergic activity. This activity was not correlated to the cortical bursts and was asynchronous in pairs of MSs. Sporadic, large-amplitude synchronous depolarizations also occurred in MSs. These events were simultaneously detected in glial cells, suggesting that they were accompanied by considerable increases in extracellular potassium. In oblique slices, cortically driven bursts were also observed in MSs. These events were synchronized to cortical epileptiform bursts, depended on non– N-methyl-d-aspartate (NMDA) glutamate receptors, and persisted in the cortex, but not in the striatum, after disconnection of the two structures. During these bursts, MS membrane potential shifted to a depolarized value (59 ± 4 mV) on which an irregular waveform, occasionally eliciting spikes, was superimposed. Thus synchronous activation of a limited set of corticostriatal afferents can powerfully control MSs. Cholinergic interneurons located <120 μm from simultaneously recorded MSs, did not display cortically driven bursts, suggesting that these cells are much less easily engaged by cortical epileptiform activity.
Metabotropic Glutamate Receptor-Mediated Depression of the Slow Afterhyperpolarization Is Gated by Tyrosine Phosphatases in Hippocampal CA1 Pyramidal Neurons
Authors: Ireland David R. , Guevremont Diane , Williams Joanna M. , Abraham Wickliffe C.
Published: July 2004

Group I metabotropic glutamate receptor (mGluR) agonists increase the excitability of hippocampal CAl pyramidal neurons via depression of the postspike afterhyperpolarization. In adult rats, this is mediated by both mGluR1 and -5, but the signal transduction processes involved are unknown. In this study, we investigated whether altered levels of tyrosine phosphorylation of proteins are involved in the depression of the slow-duration afterhyperpolarization (sAHP) by the Group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in CA1 pyramidal neurons of rat hippocampal slices. Preincubation with the tyrosine kinase inhibitors lavendustin A or genistein, or the Src-specific inhibitor 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1 H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2), did not inhibit the DHPG-mediated depression of the sAHP. However, preincubation with the tyrosine phosphatase inhibitor orthovanadate reduced the effects of DHPG. This effect of orthovanadate was prevented by simultaneous inhibition of tyrosine kinases with lavendustin A. Selective activation of either mGluR1 or -5 by application of DHPG plus either the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) or the mGluR1 antagonist (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) demonstrated that the effect of inhibiting tyrosine phosphatases is not specific to either subtype of mGluR. These results suggest that the depression of the sAHP induced by activation of mGluR1 and -5 is gated by a balance between tyrosine phosphorylation and dephosphorylation.
Optical Imaging of the Heart
Authors: Efimov Igor R. , Nikolski Vladimir P. , Salama Guy
Published: July 2004

Optical techniques have revolutionized the investigation of cardiac cellular physiology and advanced our understanding of basic mechanisms of electrical activity, calcium homeostasis, and metabolism. Although optical methods are widely accepted and have been at the forefront of scientific discoveries, they have been primarily applied at cellular and subcellular levels and considerably less to whole heart organ physiology. Numerous technical difficulties had to be overcome to dynamically map physiological processes in intact hearts by optical methods. Problems of contraction artifacts, cellular heterogeneities, spatial and temporal resolution, limitations of surface images, depth-of-field, and need for large fields of view (ranging from 2×2 mm
2
to 3×3 cm
2
) have all led to the development of new devices and optical probes to monitor physiological parameters in intact hearts. This review aims to provide a critical overview of current approaches, their contributions to the field of cardiac electrophysiology, and future directions of various optical imaging modalities as applied to cardiac physiology at organ and tissue levels.