bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Multiplexed Bead Assay for Assessment of Epstein-Barr Virus Immunologic Status
Authors: Klutts J. S. , Liao R. S. , Dunne W. M. , Gronowski A. M.
Published: November 2004

Currently, serological assays using either indirect immunofluorescence assay or enzyme-linked immunosorbent assay (ELISA) are performed to evaluate the status of Epstein-Barr virus (EBV) infection in humans. Although these methods are reliable, they are limited to testing an antibody response to a single viral antigen per reaction, thus necessitating a panel of assays to complete the evaluation. In contrast, a new bead-based method (BioPlex 2200; Bio-Rad Laboratories, Hercules, Calif.) can analyze the humoral response to multiple antigens in a single tube. This approach potentially reduces overall cost, turnaround time, and sample volume. The aim of this study was to evaluate the multiplexed EBV serologic assays performed on the BioPlex 2200 platform compared to results of conventional heterophile and ELISA-based assays. A total of 167 nonconsecutive, stored serum samples from adult and pediatric patients submitted for EBV serologic studies were used in the evaluation. Concordance between results generated by the BioPlex 2200 system and conventional assays was calculated. The anti-EA-D assay had the lowest concordance at 91%. The BioPlex 2200 system showed 97% agreement with conventional heterophile and anti-nuclear antigen assays and 92% agreement with the anti-VCA IgG and immunoglobulin M assays. Agreement between the BioPlex 2200 system and conventional testing was 92% with respect to categorization of acute versus nonacute EBV disease. The correlation between these two systems with regard to assignment into one of four categories of EBV status was also good (82%). In summary, there is excellent correlation between contemporary EBV serologic testing and the BioPlex 2200 system.
Application of Mycobacterial Interspersed Repetitive Unit Typing to Manitoba Tuberculosis Cases: Can Restriction Fragment Length Polymorphism Be Forgotten?
Authors: Blackwood K. S. , Wolfe J. N. , Kabani A. M.
Published: November 2004

Since 1993, allMycobacterium tuberculosisisolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or “types.” Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n= 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (DMIRU= 0.831 to 0.984 versusDRFLP= 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.
Strong Association between Genotype F and Hepatitis B Virus (HBV) e Antigen-Negative Variants among HBV-Infected Argentinean Blood Donors
Authors: França Paulo H. C. , González Jorge E. , Munné M. Silvina , Brandão Larissa H. , Gouvea Vera S. , Sablon Erwin , Vanderborght Bart O. M.
Published: November 2004

A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (
P
< 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles—T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764—were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (
P
< 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.
Use of Multilocus Variable-Number Tandem-Repeat Analysis for TypingMycobacterium aviumsubsp.paratuberculosis
Authors: Overduin Pieter , Schouls Leo , Roholl Paul , van der Zanden Adri , Mahmmod Nofel , Herrewegh Arnold , van Soolingen Dick
Published: November 2004

The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused byMycobacterium aviumsubsp.paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting ofM. aviumsubsp.paratuberculosisisolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity ofM. aviumsubsp.paratuberculosis.
Molecular Characteristics of Strains of the Cameroon Family, the Major Group of
Mycobacterium tuberculosis
in a Country with a High Prevalence of Tuberculosis
Authors: Niobe-Eyangoh Sara Ngo , Kuaban Christopher , Sorlin Philippe , Thonnon Jocelyn , Vincent Veronique , Gutierrez M. Cristina
Published: November 2004

A preliminary investigation of the genetic biodiversity of
Mycobacterium tuberculosis
complex strains in Cameroon, a country with a high prevalence of tuberculosis, described a group of closely related
M. tuberculosis
strains (the Cameroon family) currently responsible for more than 40% of smear-positive pulmonary tuberculosis cases. Here, we used various molecular methods to study the genetic characteristics of this family of strains. Cameroon family
M. tuberculosis
strains (i) are part of the major genetic group 2 and lack the TbD1 region like other families of epidemic strains, (ii) lack spacers 23, 24, and 25 in their direct repeat (DR) region, (iii) have an identical number of repeats in 8 of 12 variable-number tandem repeats of mycobacterial interspersed repetitive unit (MIRU-VNTR) loci, (iv) have similar IS
6110
-restriction fragment length polymorphism (RFLP) multiband patterns (10 to 15 copies) with seven common IS
6110
bands, (v) do not have an IS
6110
element in their DR locus, and (vi) have four IS
6110
elements in open reading frames (adenylate cyclase, phospholipase C,
moeY
, and ATP binding genes). Analysis by spoligotyping, MIRU-VNTR, and IS
6110
-RFLP typing methods revealed differences not observed in previous studies; polymorphism as assessed by MIRU-VNTR typing was lower than suggested by spoligotyping, and in rare cases, strains with identical IS
6110
-RFLP patterns had spoligotypes differing by as much as 15 spacers. Our findings confirm the recent expansion of this family in Cameroon and indicate that the interpretation of molecular typing results has to be adapted to the characteristics of the strain population within each setting. The knowledge of this particular genotype, with its large involvement in tuberculosis in Cameroon, allows greater refinement of tuberculosis transmission studies by interpreting data in the context of this geographic area.
Longitudinal Study of Hepatitis Activity and Viral Replication before and after HBeAg Seroconversion in Chronic Hepatitis B Patients Infected with Genotypes B and C
Authors: Yuen Man-Fung , Fung Scott K. , Tanaka Yasuhito , Kato Takanobu , Mizokami Masashi , Yuen John Chi-Hang , Wong Danny Ka-Ho , Yuan He-Jun , Sum Siu-Man , Chan Annie On-On , Wong Benjamin Chun-Yu , Lai Ching-Lung
Published: November 2004

The aims of this study were to compare chronic hepatitis B (CHB) patients with genotypes B and C for the probability of HBeAg seroconversion, hepatitis activity, and viral replication before and after HBeAg seroconversion and to compare the prevalence of core promoter and precore mutations. A total of 180 asymptomatic Chinese patients with CHB were monitored for a median of 53.8 months, and 38 patients with cirrhosis-related complications were studied. Hepatitis B virus (HBV) DNA levels were measured in 16 patients with HBeAg seroconversion at 3 months before, during, and 3 months after HBeAg seroconversion and in all patients at the last follow-up. Hepatitis B genotypes and core promoter and precore mutations were determined. Compared to patients with genotype C (
n
= 109), patients with subtype Ba (
n
= 69) had a higher rate of anti-HBe positivity on presentation (
P
= 0.05). HBeAg-positive patients with subtype Ba had a higher cumulative rate of HBeAg seroconversion than patients with genotype C (
P
= 0.02). However, there were no differences between the two groups with regard to the median HBV DNA levels before, during, and after HBeAg seroconversion; the probability of having persistently normal or elevated aminotransferase levels; and the median HBV DNA levels and liver biochemistry at the last follow-up. There was no difference in the prevalence of genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications. Though patients with subtype Ba had earlier HBeAg seroconversion, the liver biochemistry, HBV DNA levels in different phases of the disease, and the probability of development of cirrhosis-related complications were the same with genotypes Ba and C.
Evaluation of the BD PHOENIX Automated Microbiology System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci
Authors: Horstkotte Matthias A. , Knobloch Johannes K.-M. , Rohde Holger , Dobinsky Sabine , Mack Dietrich
Published: November 2004

The new BD PHOENIX automated microbiology system (Becton Dickinson Diagnostic Systems, Sparks, Md.) is designed for automated rapid antimicrobial susceptibility testing and identification of clinically relevant bacteria. In our study, the accuracy and speed of the BD PHOENIX oxacillin MIC determination for detecting methicillin resistance was evaluated for 200 clinical isolates of coagulase-negative staphylococci (CoNS). Compared to
mecA
PCR, the BD PHOENIX system detected methicillin resistance with a sensitivity of 99.2%. According to the actual NCCLS oxacillin MIC breakpoint of ≥0.5 μg/ml, the specificity was only 64.9%, attributable to false-positive results in 26
mecA
-negative strains, including 16 non-
Staphylococcus epidermidis
strains. Alternative oxacillin breakpoints of ≥1, ≥2, and ≥4 μg/ml resulted in increased specificities of 83.8, 94.6, and 100% and high sensitivities of 99.2, 99.2, and 96.7%, respectively. Similarly, NCCLS broth microdilution oxacillin MICs exhibited a sensitivity of 100% but a low degree of specificity. However, the previous oxacillin MIC breakpoint of ≥4 μg/ml performed with a sensitivity of 98.4% and a specificity of 98.7%. BD PHOENIX oxacillin MIC results were available after 9 h for 40.5% of the examined CoNS strains and were completed after 17 h. Our results revealed the high reliability of the BD PHOENIX system as a phenotypic method for detection of resistance to oxacillin in
mecA
-positive CoNS. However, for the improvement of specificity, reevaluation of the optimal oxacillin MIC breakpoint for CoNS appears to be necessary.
Mycobacterium africanumGenotyping UsingNovel Spacer Oligonucleotides in the Direct RepeatLocus
Authors: Brudey Karine , Gutierrez M. Cristina , Vincent Véronique , Parsons Linda M. , Salfinger Max , Rastogi Nalin , Sola Christophe
Published: November 2004

This study involves a first evaluation of 25 novel spacer oligonucleotides in addition to the 43 routine spacers for molecular characterization of a panel of 65 isolates of tubercle bacilli from different geographic origins that were initially classified asMycobacterium africanumbased on phenotypic characters. The 68-spacer format defined four additional patterns, and three groups were identified. The relatively homogeneous groups A1 and A2 included strains from West Africa, and A3-1 included strains from East Africa. The presence of deletion region RD9 confirmed the reclassification of theM. africanumsubtype II spoligopattern within group A3-1 asMycobacterium tuberculosis.These isolates may represent a diverging branch ofM. tuberculosisin Africa. The use of new spacers also suggested an undergoing evolution ofM. africanumsubtype I in West Africa. Our results showed that the strain differentiation within theM. tuberculosiscomplex is improved by using novel spacers, and extensive studies using new-generation spoligotyping may be helpful to better understand the evolution ofM. africanum.
Characterization of AncestralMycobacterium tuberculosisby Multiple Genetic Markers and Proposal of Genotyping Strategy
Authors: Sun Yong-Jiang , Lee Ann S. G. , Ng Sze Ta , Ravindran Sindhu , Kremer Kristin , Bellamy Richard , Wong Sin-Yew , van Soolingen Dick , Supply Philip , Paton Nicholas I.
Published: November 2004

Sixty-eight ancestralMycobacterium tuberculosisisolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol.42:1986-1993, 2004). These TbD1+ancestral isolates were further characterized and typed in this study by IS6110restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS6110RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS6110RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS6110RFLP is proposed to achieve maximal discrimination for unrelated TbD1+strains. This typing strategy may be useful in areas where TbD1+ancestral strains are prevalent.
Multilocus Microsatellite Typing System for
Penicillium marneffei
Reveals Spatially Structured Populations
Authors: Fisher Matthew C. , Aanensen David , de Hoog Sybren , Vanittanakom Nongnuch
Published: November 2004

For eukaryotic pathogens that have low levels of genetic variation, multilocus microsatellite typing (MLMT) offers an accurate and reproducible method of characterizing genetic diversity. Here, we describe the application of an MLMT system to the emerging pathogenic fungus
Penicillium marneffei
. Isolates used for this study were those held in the culture collections of the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and the Chiang Mai University Department of Microbiology, Chang Mai, Thailand. High genetic diversity and extensive spatial structure were observed among clinical isolates, with the geographical area of origin for each isolate strongly correlating with the occurrence of two deeply divided clades. Within each clade, multilocus linkage associations were highly significant and could be explained by genetically differentiated populations or by an exclusively clonal reproductive mode, or both. Our results show that southeast Asian penicilliosis is caused by a fungus with a complex population genetic structure. Furthermore, this MLMT system generates digital data that can be easily queried against a centrally held database via the internet (
http://pmarneffei.multilocus.net/
); this provides a powerful epidemiological tool for analyzing the underlying parameters that are responsible for the emergence of
P. marneffei
in human immunodeficiency virus-positive populations.
Role of
Cannomys badius
as a Natural Animal Host of
Penicillium marneffei
in India
Authors: Gugnani Harish , Fisher Matthew C. , Paliwal-Johsi Anubha , Vanittanakom Nongnuch , Singh Irabanta , Yadav Pratap Singh
Published: November 2004

Infection by
Penicillium marneffei
in human immunodeficiency virus-positive patients in India has recently been described; the aim of our study was to survey wild rodents and their associated environment in order to identify the natural populations of this fungus. Surveys recovered
P. marneffei
from the internal organs of 10 (9.1%) of 110 bamboo rats (
Cannomys badius
) examined from Manipur state, India, an area endemic for penicilliosis marneffei. Identification of the isolates was based on a detailed study of their morphological characteristics, in vitro conversion to fission yeast form, and exoantigen tests. Multilocus microsatellite typing (MLMT) of the isolates revealed five genotypes. No genotypes were shared between sample sites, and all bamboo rats were infected with a single genotype within sample sites, demonstrating spatial genetic heterogeneity. One MLMT genotype was identical to that seen in a human isolate, suggesting that either coinfection from a common source or host-to-host transmission had occurred. This demonstrates the utility of an MLMT-based approach to elucidating the epidemiology of
P. marneffei
.
Borrelia
Species in Host-Seeking Ticks and Small Mammals in Northern Florida
Authors: Clark Kerry
Published: November 2004

The aim of this study was to improve understanding of several factors related to the ecology and environmental risk of
Borrelia
infection in northern Florida. Small mammals and host-seeking adult ticks were collected at several sites, and specimens were tested for the presence of
Borrelia
species, primarily by PCR amplification. Tissues from some vertebrates and ticks were initially cultured in BSK-H medium to isolate spirochetes, but none were recovered. However, comparison of partial flagellin (
flaB
), 66-kDa protein (
p66
), and outer surface protein A (
ospA
) gene sequences from DNAs amplified from small mammals and ticks confirmed the presence of several
Borrelia
species.
Borrelia lonestari
DNA was detected among lone star ticks (
Amblyomma americanum
) at four sites.
Borrelia burgdorferi
sensu stricto strains were detected in all small mammal species tested and in
A. americanum
,
Ixodes affinis
, and
Ixodes scapularis
ticks.
Borrelia bissettii
was found in a cotton mouse and cotton rats and in
I. affinis
ticks. The study findings extend the known geographic distributions of
B. lonestari
in
A. americanum
and of
B. burgdorferi
sensu lato in
A. americanum
,
I. affinis
,
I. scapularis
, and small mammals to new sites in Florida. The presence of
B. burgdorferi
sensu stricto strains in host-seeking lone star ticks at two sites in Florida suggests that
A. americanum
should still be considered a possible vector of
B. burgdorferi
sensu lato.
Genetic Diversity of Human Pathogenic Members of the
Fusarium oxysporum
Complex Inferred from Multilocus DNA Sequence Data and Amplified Fragment Length Polymorphism Analyses: Evidence for the Recent Dispersion of a Geographically Widespread Clonal Lineage and Nosocomial Origin
Authors: O'Donnell Kerry , Sutton Deanna A. , Rinaldi Michael G. , Magnon Karen C. , Cox Patricia A. , Revankar Sanjay G. , Sanche Stephen , Geiser David M. , Juba Jean H. , van Burik Jo-Anne H. , Padhye Arvind , Anaissie Elias J. , Francesconi Andrea , Walsh Thomas J. , Robinson Jody S.
Published: November 2004

Fusarium oxysporum
is a phylogenetically diverse monophyletic complex of filamentous ascomycetous fungi that are responsible for localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients, respectively. Although members of this complex were isolated from patients during a pseudoepidemic in San Antonio, Tex., and from patients and the water system in a Houston, Tex., hospital during the 1990s, little is known about their genetic relatedness and population structure. This study was conducted to investigate the global genetic diversity and population biology of a comprehensive set of clinically important members of the
F. oxysporum
complex, focusing on the 33 isolates from patients at the San Antonio hospital and on strains isolated in the United States from the water systems of geographically distant hospitals in Texas, Maryland, and Washington, which were suspected as reservoirs of nosocomial fusariosis. In all, 18 environmental isolates and 88 isolates from patients spanning four continents were genotyped. The major finding of this study, based on concordant results from phylogenetic analyses of multilocus DNA sequence data and amplified fragment length polymorphisms, is that a recently dispersed, geographically widespread clonal lineage is responsible for over 70% of all clinical isolates investigated, including all of those associated with the pseudoepidemic in San Antonio. Moreover, strains of the clonal lineage recovered from patients were conclusively shown to genetically match those isolated from the hospital water systems of three U.S. hospitals, providing support for the hypothesis that hospitals may serve as a reservoir for nosocomial fusarial infections.
Diagnostic Relevance of Immunoglobulin G Avidity for Hepatitis A Virus
Authors: Roque-Afonso Anne-Marie , Grangeot-Keros Liliane , Roquebert Bénédicte , Desbois Delphine , Poveda Jean-Dominique , Mackiewicz Vincent , Dussaix Elisabeth
Published: November 2004

Diagnosis of acute hepatitis A virus (HAV) infection is based on the detection of HAV immunoglobulin M (IgM). However, IgM could be detected due to nonspecific polyclonal activation of the immune system. An avidity test for anti-HAV IgG was developed to distinguish acute infection, where low-avidity antibodies are detected, from immune reactivation. The assay was tested on 104 samples, including 11 sera from patients with past infection, 15 sera from patients with acute infection and 4 collected after recovery, 10 sera from vaccinated subjects, 4 sera from patients with suspected immune reactivation, and 60 unselected HAV-IgM positive sera, collected over 1 year in a routine laboratory. The avidity index (AI) was expressed as percentage. The results were provided as the mean ± one standard deviation. Patients with a history of prior infection had AIs of >70% (mean, 86% ± 10), whereas the mean AI was 36% ± 16 during acute HAV infection (
P
< 0.001). Within the first month after the onset of hepatitis, avidity was either noncalculable due to a very low IgG titer or <50%. In patients with immune reactivation, avidity was >70% (88% ± 10%), a finding consistent with a prior infection. Among the 60 unselected sera, 35 (58%) had a noncalculable or <50% avidity, and most of them had a detectable HAV RNA, confirming HAV infection. In contrast, 16 (27%) had an avidity of >70%, and none was reverse transcription-PCR positive, suggesting immune reactivation. These 16 patients were significantly older than the others (50 ± 16 years versus 26 ± 14 years). The new anti-HAV IgG avidity assay we developed could improve HAV infection diagnosis, particularly in elderly patients.
Detection of
Campylobacter
spp. in ChickenFecal Samples by Real-TimePCR
Authors: Lund Marianne , Nordentoft Steen , Pedersen Karl , Madsen Mogens
Published: November 2004

A
real-time PCR assay for detecting thermophilic
Campylobacter
spp. directly in chicken feces has been developed. DNA was isolated
from fecal material by using magnetic beads followed by PCR with a
prealiquoted PCR mixture, which had been stored at−
18°C.
Campylobacter
could be detected in less
than 4 h, with a detection limit of 100 to 150 CFU/ml, in a
fecal suspension. A bacterial internal control was added before DNA
extraction to control both DNA isolation and the presence of PCR
inhibitors in the samples. The assay was performed on 111 swab samples
from a Danish surveillance program and compared to conventional
culturing using selective enrichment. There was no statistically
significant difference in performance between real-time PCR and culture
by selective enrichment, and the diagnostic specificity was 0.96 with
an agreement of 0.92. Therefore, the assay should be useful for
screening poultry flocks for the presence of
Campylobacter
.
Nocardia kruczakiae
sp. nov., a Pathogen in Immunocompromised Patients and a Member of the “
N. nova
Complex”
Authors: Conville Patricia S. , Brown June M. , Steigerwalt Arnold G. , Lee Judy W. , Anderson Victoria L. , Fishbain Joel T. , Holland Steven M. , Witebsky Frank G.
Published: November 2004

Molecular methodologies have become useful techniques for the identification of pathogenic
Nocardia
species and for the recognition of novel species that are capable of causing human disease. Two isolates recovered from immunocompromised patients were characterized as
Nocardia nova
by biochemical and susceptibility testing results. The restriction fragment length polymorphism (RFLP) patterns obtained by restriction endonuclease analysis (REA) of an amplified portion of the heat shock protein gene were identical to those obtained with the type strain of
N. nova
. REA of an amplified portion of the 16S rRNA gene showed RFLP patterns that were unlike those obtained for the type strain of
N. nova
but that were similar to those obtained for the type strains of
N. africana
and
N. veterana
. Subsequent sequencing of a portion of the 16S rRNA gene produced identical results for the two patient isolates. Sequence analysis of 1,352-bp portions of the 16S rRNA gene indicated that these isolates were 99.8% similar to the recently described species
N. veterana
but were only 99.3, 98.1, and 98.1% similar to the type strains of
N. africana
,
N. nova
, and
N. vaccinii
, respectively. DNA-DNA hybridization studies confirmed that the two patient isolates belonged to the same species but were not closely related to
N. africana
,
N. nova
,
N. vaccinii
, or
N. veterana
. The patient isolates have been designated
N. kruczakiae
sp. nov. Because
N. africana
,
N. veterana
, and the new species are not readily differentiated from
N. nova
by phenotypic methods alone, the designation “
N. nova
complex” can be used to designate isolates such as these that phenotypically resemble
N. nova
but that have not been definitively characterized by 16S rRNA gene sequencing or DNA-DNA hybridization.
Distribution of Serogroups and Genotypes among Disease-Associated and Carried Isolates of
Neisseria meningitidis
from the Czech Republic, Greece, and Norway
Authors: Yazdankhah Siamak P. , Kriz Paula , Tzanakaki Georgina , Kremastinou Jenny , Kalmusova Jitka , Musilek Martin , Alvestad Torill , Jolley Keith A. , Wilson Daniel J. , McCarthy Noel D. , Caugant Dominique A. , Maiden Martin C. J.
Published: November 2004

The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes.
Real-Time Quantitative PCR Assays for Detection andMonitoring of Pathogenic Human Viruses in ImmunosuppressedPediatricPatients
Authors: Watzinger F. , Suda M. , Preuner S. , Baumgartinger R. , Ebner K. , Baskova L. , Niesters H. G. M. , Lawitschka A. , Lion T.
Published: November 2004

A
panel of 23 real-time PCR assays based on
Taq
Man
technology has been developed for the detection and monitoring of 16
different viruses and virus families including human polyomaviruses BK
virus and JC virus, human herpesviruses 6, 7, and 8, human
adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus,
cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B
viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory
syncytial virus. The test systems presented have a broad dynamic range
and display high sensitivity, reproducibility, and specificity.
Moreover, the assays allow precise quantification of viral load in a
variety of clinical specimens. The ability to use uniform PCR
conditions for all assays permits simultaneous processing and detection
of many different viruses, thus economizing the diagnostic work. Our
observations based on more than 50,000 assays reveal the potential of
the real-time PCR tests to facilitate early diagnosis of infection and
to monitor the kinetics of viral proliferation and the response to
treatment. We demonstrate that, in immunosuppressed patients with
invasive virus infections, surveillance by the assays described may
permit detection of increasing viral load several days to weeks prior
to the onset of clinical symptoms. In virus infections for which
specific treatment is available, the quantitative PCR assays presented
provide reliable diagnostic tools for timely initiation of appropriate
therapy and for rapid assessment of the efficacy of antiviral treatment
strategies.
Assessment of the Target-Capture PCR Hepatitis B Virus (HBV) DNA Quantitative Assay and Comparison with Commercial HBV DNA Quantitative Assays
Authors: Shyamala Venkatakrishna , Arcangel Phillip , Cottrell Joshua , Coit Doris , Medina-Selby Angelica , McCoin Colin , Madriaga Dennis , Chien David , Phelps Bruce
Published: November 2004

Recent clinical studies suggest that hepatitis B virus (HBV) load and genotype may be independent predictors of responses to antiviral therapies. However, it is difficult for clinicians to accurately determine viral loads in patient samples because results—both the values and the units of measure—can vary greatly among different tests. Accordingly, the World Health Organization (WHO) has produced the first international standard for HBV DNA for nucleic acid amplification technology (NAT) assays. In the present study, we describe the performance of the target-capture PCR HBV DNA quantitative assay for the quantitation of HBV DNA in clinical samples and reference panels. The range of quantitation was between 50 and 10
10
IU/ml. The sensitivity and accuracy of the target-capture PCR assay were demonstrated by using the HBV panel from Quality Control for Medical Diagnostics (QCMD) and the WHO HBV DNA standard. The target-capture PCR assay quantitated the six genotype A members of the QCMD panel and dilutions of the WHO HBV DNA standard within an accuracy of 74 to 142%. Compared to current serological methods, the assay offers window period reductions of 19 days prior to HBV surface antigen and 26 days prior to HBV e antigen detection. The target-capture PCR assay was also compared with four commercially available NAT assays, and the various units of measure were standardized with respect to the international units of the WHO HBV DNA standard. The target-capture PCR assay is a sensitive, accurate, high-throughput, rapid, and reproducible assay for the determination of HBV loads.
Development of a PCR-Restriction Fragment Length Polymorphism Assay for the Epidemiological Analysis of Shiga Toxin-Producing
Escherichia coli
Authors: Shima Kensuke , Terajima Jun , Sato Toshio , Nishimura Kazuhiko , Tamura Kazumichi , Watanabe Haruo , Takeda Yoshifumi , Yamasaki Shinji
Published: November 2004

Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing
Escherichia coli
(STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control
E. coli
strains carrying six different Stx phages such as
E. coli
strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.
Carious Dentine Provides a Habitat for a Complex Array of Novel
Prevotella
-Like Bacteria
Authors: Nadkarni Mangala A. , Caldon C. Elizabeth , Chhour Kim-Ly , Fisher Ilana P. , Martin F. Elizabeth , Jacques Nicholas A. , Hunter Neil
Published: November 2004

Previous analysis of the microbiology of advanced caries by culture and real-time PCR emphasized the high incidence and abundance of gram-negative anaerobic species, particularly
Prevotella
-like bacteria. The diversity of
Prevotella
-like bacteria was further explored by analyzing pooled bacterial DNA from lesions of carious dentine. This was achieved by amplification of a region of the 16S ribosomal DNA with a
Prevotella
genus-specific forward primer and a universal bacterial reverse primer, followed by cloning and sequencing. Cultured
Prevotella
species commonly associated with oral tissues constituted only 12% of the
Prevotella
clones isolated from advanced carious lesions. The remaining 88% consisted of a diverse range of phylotypes. These included five clusters of previously recognized but uncultured oral
Prevotella
spp. and a major cluster containing
Prevotella
-like bacteria most closely related to uncharacterized rumen bacteria. Cluster-specific primers were designed, and the numbers of bacteria within clusters were quantified by real-time PCR, confirming the abundance of these organisms. The data indicated that advanced dental caries provides a unique environment for a complex array of novel and uncultured
Prevotella
and
Prevotella
-like bacteria which, in some cases, may dominate the diverse polymicrobial community associated with the disease.
Quantification of
Leishmania infantum
DNA by a Real-Time PCR Assay with High Sensitivity
Authors: Mary Charles , Faraut Françoise , Lascombe Laurie , Dumon Henri
Published: November 2004

A real-time PCR was developed to quantify
Leishmania infantum
kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the
Leishmania
DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different
L. infantum
and
Leishmania donovani
strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for
Leishmania
quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 10
6
nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of
Leishmania
DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.
Metallo-β-Lactamase-Producing Gram-Negative Bacilli: Laboratory-Based Surveillance in Cooperation with 13 Clinical Laboratories in the Kinki Region of Japan
Authors: Nishio Hisaaki , Komatsu Masaru , Shibata Naohiro , Shimakawa Kouichi , Sueyoshi Noriyuki , Ura Toshiro , Satoh Kaori , Toyokawa Masahiro , Nakamura Tatsuya , Wada Yasunao , Orita Tamaki , Kofuku Tomomi , Yamasaki Katsutoshi , Sakamoto Masako , Kinoshita Shohiro , Aihara Masanori , Arakawa Yoshichika
Published: November 2004

A total of 19,753 strains of gram-negative rods collected during two 6-month periods (October 2000 to March 2001 and November 2001 to April 2002) from 13 clinical laboratories in the Kinki region of Japan were investigated for the production of metallo-β-lactamases (MBLs). MBLs were detected in 96 (0.5%) of the 19,753 isolates by the broth microdilution method, the 2-mercaptopropionic acid inhibition test, and PCR and DNA sequencing analyses. MBL-positive isolates were detected in 9 of 13 laboratories, with the rate of detection ranging between 0 and 2.6% for each laboratory. Forty-four of 1,429 (3.1%)
Serratia marcescens
, 22 of 6,198 (0.4%)
Pseudomonas aeruginosa
, 21 of 1,108 (1.9%)
Acinetobacter
spp., 4 of 544 (0.7%)
Citrobacter freundii
, 3 of 127 (2.4%)
Providencia rettgeri
, 1 of 434 (0.2%)
Morganella morganii
, and 1 of 1,483 (0.1%)
Enterobacter cloacae
isolates were positive for MBLs. Of these 96 MBL-positive strains, 87 (90.6%), 7 (7.3%), and 2 (2.1%) isolates carried the genes for IMP-1-group MBLs, IMP-2-group MBLs, and VIM-2-group MBLs, respectively. The class 1 integrase gene,
intI1
, was detected in all MBL-positive strains, and the
aac
(
6
′)
-Ib
gene was detected in 37 (38.5%) isolates. Strains with identical PCR fingerprint profiles in a random amplified polymorphic DNA pattern analysis were isolated successively from five separate hospitals, suggesting the nosocomial spread of the organism in each hospital. In conclusion, many species of MBL-positive gram-negative rods are distributed widely in different hospitals in the Kinki region of Japan. The present findings should be considered during the development of policies and strategies to prevent the emergence and further spread of MBL-producing bacteria.
Occurrence of Extended-Spectrum β-Lactamases in Members of the Genus
Shigella
in the Republic of Korea
Authors: Kim Seonghan , Kim Junyoung , Kang Yeonho , Park Yongkeun , Lee Bokkwon
Published: November 2004

A nationwide survey was carried out in Korea to assess the prevalence of
Shigella
strains producing extended-spectrum β-lactamases (ESBLs). From 1991 to 2002, 5,911 clinical strains were isolated and screened for resistance to extended-spectrum cephalosporins. Twenty of the
Shigella
isolates were ESBL positive, based on the synergistic effects between clavulanate and selected β-lactams (ceftazidime and cefotaxime). Nucleotide sequence analysis of these isolates revealed that they harbored
bla
TEM-19
(eight isolates),
bla
TEM-15
(five isolates),
bla
TEM-52
(six isolates),
bla
TEM-17
(one isolate),
bla
TEM-20
(one isolate), and
bla
CTX-M-14
(three isolates). All the ESBL-encoding genes in this study were carried in conjugable plasmids. Thus, TEM-19, TEM-15, TEM-52, and CTX-M-14 β-lactamases can be considered common Korean ESBL types in
Shigella sonnei
and are probably transmitted through interspecies spread between medical facilities and the community in Korea. This is the first report of the presence of TEM-17, TEM-19, and TEM-20 in Korea and in
S. sonnei
.
Dual-Probe Assay for Rapid Detection of Drug-Resistant
Mycobacterium tuberculosis
by Real-Time PCR
Authors: Wada Takayuki , Maeda Shinji , Tamaru Aki , Imai Shigeyoshi , Hase Atsushi , Kobayashi Kazuo
Published: November 2004

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in
Mycobacterium tuberculosis
. For rapid detection of drug-resistant
M. tuberculosis
in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the
rpoB
gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the
rpoB
gene, and one probe was designed as a tuberculosis (TB) control outside the
rpoB
gene hot-spot. We also designed probes to examine codon 315 of
katG
and codon 306 of
embB
for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was
M. tuberculosis
complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the
rpoB
,
katG
, and
embB
genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant
M. tuberculosis
in clinical laboratories.
Porphyromonas uenonis
sp. nov., a Pathogen for Humans Distinct from
P. asaccharolytica
and
P. endodontalis
Authors: Finegold Sydney M. , Vaisanen Marja-Liisa , Rautio Merja , Eerola Erkki , Summanen Paula , Molitoris Denise , Song Yuli , Liu Chengxu , Jousimies-Somer Hannele
Published: November 2004

Three
Porphyromonas
species (
Porphyromonas asaccharolytica
,
P. endodontalis
, and the novel species that is the subject of the present report,
P. uenonis
) are very much alike in terms of biochemical characteristics, such as enzyme profiles and cellular fatty acid contents.
P. asaccharolytica
is distinguished from the other two species by virtue of production of α-fucosidase and glyoxylic acid positivity. The novel species is difficult to differentiate from
P. endodontalis
phenotypically and was designated a
P. endodontalis
-like organism for some time. However,
P. endodontalis
is recovered almost exclusively from oral sources and also grows poorly on Biolog Universal Agar, both characteristics that are in contrast to those of the other two organisms. Furthermore,
P. uenonis
is glycerol positive in the Biolog AN Microplate system. Both
P. asaccharolytica
and
P. uenonis
are positive by 13 other tests in the Biolog system, whereas
P. endodontalis
is negative by all of these tests.
P. asaccharolytica
grew well in both solid and liquid media without supplementation with 5% horse serum, whereas the other two species grew poorly without supplementation. Sequencing of 16S rRNA revealed about 10% divergence between the novel species and
P. endodontalis
but less than 2% sequence difference between the novel species and
P. asaccharolytica.
Subsequent DNA-DNA hybridization studies documented that the novel organism was indeed distinct from
P. asaccharolytica.
We propose the name
Porphyromonas uenonis
for the novel species. We have recovered
P. uenonis
from four clinical infections in adults, all likely of intestinal origin, and from the feces of six children.