bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Partial 16S Ribosomal DNA Sequencing for Identification of
Nocardia
Species by Using the MicroSeq 500 System with an Expanded Database
Authors: Cloud Joann L. , Conville Patricia S. , Croft Ann , Harmsen Dag , Witebsky Frank G. , Carroll Karen C. 
Published: February 2004

ABSTRACT

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate
Nocardia
species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i) conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii) molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii) when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of
Nocardia
species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens
N. brasiliensis
,
N. cyriacigeorgica
,
N. farcinica
,
N. nova
,
N. otitidiscaviarum
, and
N. veterana
.
Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments
Authors: Leb Victoria , Stöcher Markus , Valentine-Thon Elizabeth , Hölzl Gabriele , Kessler Harald , Stekel Herbert , Berg Jörg 
Published: February 2004

ABSTRACT
We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.
Comparison of Mismatch Amplification Mutation Assay with DNA Sequencing for Characterization of Fluoroquinolone Resistance in
Neisseria gonorrhoeae
Authors: Sultan Zafar , Nahar Shamsun , Wretlind Bengt , Lindback Emma , Rahman Motiur 
Published: February 2004

ABSTRACT

A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of
gyrA
at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of
gyrA
in 117
Neisseria gonorrhoeae
isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the
gyrA
genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in
N. gonorrhoeae
.
Smallpox and
pan
-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms
Authors: Kulesh David A. , Baker Robert O. , Loveless Bonnie M. , Norwood David , Zwiers Susan H. , Mucker Eric , Hartmann Chris , Herrera Rafael , Miller David , Christensen Deanna , Wasieloski Leonard P. , Huggins John , Jahrling Peter B. 
Published: February 2004

ABSTRACT

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and
pan
-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the
pan
-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 10
7
, 1.24 × 10
5
, 1.24 × 10
3
, and 1.24 × 10
1
genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence.
Epidemiology and Etiology of Sexually Transmitted Infection among Hotel-Based Sex Workers in Dhaka, Bangladesh
Authors: Nessa Khairun , Waris Shama-A , Sultan Zafar , Monira Shirajum , Hossain Maqsud , Nahar Shamsun , Rahman Habibur , Alam Mahbub , Baatsen Pam , Rahman Motiur 
Published: February 2004

ABSTRACT

The prevalence of sexually transmitted infections (STIs) and reproductive tract infections (RTIs) among hotel-based sex workers (HBSWs) in Dhaka, Bangladesh, was studied. A total of 400 HBSWs were enrolled in the study during April to July 2002. Endocervical swabs, high vaginal swabs, and blood samples from 400 HBSWs were examined for
Neisseria gonorrhoeae
(by culture),
Chlamydia trachomatis
(by PCR),
Trichomonas vaginalis
(by microscopy), antibody to
Treponema pallidum
(by both rapid plasma reagin and
Treponema pallidum
hemagglutination tests), and antibody to herpes simplex virus type 2 (HSV-2) (by enzyme-linked immunosorbent assay). Sociodemographic information as well as gynecological and obstetric information was collected. Among the HBSWs, 228 women (57%) were symptomatic and 172 (43%) were asymptomatic, 35.8% were positive for
N. gonorrhoeae
, 43.5% were positive for
C. trachomatis
, and 4.3% were positive for
T. vaginalis
. A total of 8.5% had syphilis, 34.5% were positive for HSV-2, and 86.8% were positive for at least one RTI or STI. There was no significant difference between the prevalences of STIs among the symptomatic and asymptomatic HBSWs. These data suggested a high prevalence of STIs, particularly gonorrhea and chlamydia, among HBSWs in Dhaka.
Direct Amplification and Genotyping of
Dientamoeba fragilis
from Human Stool Specimens
Authors: Peek Ron , Reedeker Freek R. , van Gool Tom 
Published: February 2004

ABSTRACT

Dientamoeba fragilis
is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect
D. fragilis
DNA directly from human stool samples. The genetic diversity of
D. fragilis
from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that
D. fragilis
sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that
D. fragilis
shows remarkably little variation in its small-subunit rRNA gene.
Genetic Diversity of Cell-Invasive Erythromycin-Resistant and -Susceptible Group A Streptococci Determined by Analysis of the
RD2
Region of the
prtF1
Gene
Authors: Spinaci Cinzia , Magi Gloria , Zampaloni Claudia , Vitali Luca A. , Paoletti Claudia , Catania Maria R. , Prenna Manuela , Ferrante Luigi , Ripa Sandro , Varaldo Pietro E. , Facinelli Bruna 
Published: February 2004

ABSTRACT

The
RD2
region of the internalization-associated gene
prtF1
, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the
RD2
region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the
RD2
region were consistent with a number of
RD2
repeats ranging from one to five, more frequently four (
n
= 30), three (
n
= 27), and one (
n
= 18). A new method to type cell-invasive GAS (
RD2
typing) was developed by combining PCR analysis of the
RD2
region and restriction analysis of PCR products with endonucleases
Hae
III,
Dde
I, and
Hin
fI. Overall, 10
RD2
types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of
RD2
typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and
Sma
I macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of
RD2
type and
emm
type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology,
RD2
typing may be especially helpful in typing cell-invasive GAS.
Serotypes, Virulence Genes, and Intimin Types ofShiga Toxin (Verotoxin)-ProducingEscherichia coliIsolatesfrom Cattle in Spain and Identification of a New Intimin VariantGene(eae-ξ)
Authors: Blanco M. , Blanco J. E. , Mora A. , Dahbi G. , Alonso M. P. , González E. A. , Bernárdez M. I. , Blanco J. 
Published: February 2004

ABSTRACTA total of 514 Shiga toxin-producingEscherichia coli(STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carriedstx1genes, 278 (54%) possessedstx2genes, and 135 (26%) possessed bothstx1andstx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7stx2eae-γ1ehxA(46 isolates) was the most common, followed by O157:H7stx1stx2eae-γ1ehxA(34 isolates), O113:H21stx2(25 isolates), O22:H8stx1stx2ehxA(15 isolates), O26:H11stx1eae-β1ehxA(14 isolates), and O77:H41stx2ehxA(14 isolates). Forty-one (22 of serotype O26:H11) isolates had intiminβ 1, 82 O157:H7 isolates possessed intimin γ1, three O111:H- isolates had intimin type γ2, one O49:H- strain showed intimin type δ, 13 (six of serotype O103:H2) isolates had intimin type ε and eight (four of serotype O156:H-) isolates had intimin ζ. We have identified a new variant of theeaeintimin gene designated ξ (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.
Prevalence, Risk Factors, and Genetic Diversity of
Bartonella henselae
Infections in Pet Cats in Four Regions of the United States
Authors: Guptill L. , Wu C.-C. , HogenEsch H. , Slater L. N. , Glickman N. , Dunham A. , Syme H. , Glickman L. 
Published: February 2004

ABSTRACT

Blood was collected from a convenience sample of 271 pet cats aged 3 months to 2 years (mean age, 8 months, median and mode, 6 months) between May 1997 and September 1998 in four areas of the United States (southern California, Florida, metropolitan Chicago, and metropolitan Washington, D.C.). Sixty-five (24%) cats had
Bartonella henselae
bacteremia, and 138 (51%) cats were seropositive for
B. henselae
. Regional prevalences for bacteremia and seropositivity were highest in Florida (33% and 67%, respectively) and California (28% and 62%, respectively) and lowest in the Washington, D.C. (12% and 28%, respectively) and Chicago (6% and 12%, respectively) areas. No cats bacteremic with
B. clarridgeiae
were found. The 16S rRNA type was determined for 49
B. henselae
isolates. Fourteen of 49 cats (28.6%) were infected with 16S rRNA type I, 32 (65.3%) with 16S rRNA type II, and three (6.1%) were coinfected with 16S rRNA types I and II. Flea infestation was a significant risk factor for
B. henselae
bacteremia (odds ratio = 2.82, 95% confidence interval, 1.1 to 7.3). Cats ≥13 months old were significantly less likely to be bacteremic than cats ≤6 months old (odds ratio = 0.18, 95% confidence interval, 0.05 to 0.61). Flea infestation, adoption from a shelter or as a stray cat, hunting, and being from Florida or California were significant risk factors for
B. henselae
seropositivity. DNA fingerprint was significantly associated with region (
P
= 0.03) and indoor/outdoor status of cats (
P
= 0.03).
GenotypicAnalysis ofMycobacterium tuberculosisin Bangladesh andPrevalence of the BeijingStrain
Authors: Banu Sayera , Gordon Stephen V. , Palmer Si , Islam Reazul , Ahmed Shakeel , Alam Khan Mashrequl , Cole Stewart T. , Brosch Roland 
Published: February 2004

ABSTRACTGenotypic analysis was performed on 48Mycobacterium tuberculosiscomplex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were allM. tuberculosis;13 of them were found to be of the “ancestral” type, while 35 were of the “modern” type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.
Genetic Characterization and Antibiotic Resistance of
Campylobacter jejuni
Isolated from Meats, Water, and Humans in Sweden
Authors: Lindmark H. , Harbom B. , Thebo L. , Andersson L. , Hedin G. , Osterman B. , Lindberg T. , Andersson Y. , Westöö A. , Olsson Engvall E. 
Published: February 2004

ABSTRACT

The incidence of
Campylobacter jejuni
has increased during the last decade, and today it is the leading cause of bacterial enteritis in most developed countries. Still, there is a lack of knowledge about infection routes and to what extent identified sources are responsible for spreading the bacterium to humans. The major objective of this work was to explore the genetic similarity between
C. jejuni
isolated from different sources.
C. jejuni
isolated from patients (
n
= 95), five types of meat (
n
= 71), and raw water (
n
= 11) during the year 2000 were subtyped by pulsed-field gel electrophoresis (PFGE). The pulsotypes obtained after digestion with
Sma
I revealed not only that
C. jejuni
is genetically diverse but also that specific pulsotypes occur frequently. Five clusters comprising 88 of the 162
Sma
I-digested isolates were obtained. After digestion with
Kpn
I most isolates in four of the five clusters were still indistinguishable, while the fifth cluster was strongly dissolved. The clusters comprised high frequencies of human and meat isolates, while only one of nine water isolates belonged to a cluster. The largest cluster comprised 21 human isolates, one raw water isolate, and seven chicken meat isolates, originating from at least six different broiler flocks. Low frequencies of antibiotic resistance were revealed when the meat and water isolates were tested for sensitivity to six antibiotics. Interestingly, the five isolates resistant to quinolones displayed similar or identical pulsotypes. The results showed that PFGE has proved useful in identifying clones and will be used in future work focusing on identification and eradication of the major reservoirs for common clones.
Use of theTrypanosoma cruziRecombinant Complement Regulatory Protein To Evaluate Therapeutic Efficacy following Treatment of Chronic Chagasic Patients
Authors: Meira Wendell S. F. , Galvão Lúcia M. C. , Gontijo Eliane D. , Machado-Coelho George L. L. , Norris Karen A. , Chiari Egler 
Published: February 2004

ABSTRACTOne of the greatest concerns in Chagas' disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The tests available to evaluate cure after the specific treatment are the complement-mediated lysis (CoML) and flow cytometry tests, but they are not feasible for routine clinical use. In this study, we evaluated an enzyme-linked immunosorbent assay (ELISA) based on the recombinantTrypanosoma cruzicomplement regulatory protein (rCRP) as a method to determine parasite clearance in comparison to the CoML and other methods such as conventional serology, hemoculture, and PCR in serum samples of 31 patients collected before and after the treatment, monitored for an average of 27.7 months after chemotherapy. The results showed that the percentage of patient samples that were positive by rCRP ELISA was reduced from 100 to 70.3, 62.5, 71.4, and 33.4% in the first, second, third, and fourth years after treatment, respectively, while the samples positive by CoML were reduced to 85.2, 81.2, 71.4, and 33.4% during the same period, demonstrating the same significant tendency in the reduction of positive samples. On the other hand, the conventional serology (CS) tests did not present this reduction. The percentage of samples positive by PCR was initially 77.4% and decreased to 55.5, 68.7, 47.7, and 50.0% at the fourth year after treatment, confirming the drastic clearance of circulating parasites after treatment. Our results strongly suggest that the rCRP ELISA was capable of detecting the early therapeutic efficacy in treated patients and confirmed its superiority over the CS tests and parasitologic methods.
Identification and H
2
O
2
Production of Vaginal Lactobacilli from Pregnant Women at High Risk of Preterm Birth and Relation with Outcome
Authors: Wilks Mark , Wiggins Rebecca , Whiley Angela , Hennessy Enid , Warwick Simon , Porter Helen , Corfield Anthony , Millar Michael 
Published: February 2004

ABSTRACT

Lactobacilli, principally the strains that are hydrogen peroxide (H
2
O
2
) producing, may have a protective effect against vaginal colonization by pathogenic species such as those that cause bacterial vaginosis. Previous reports have also suggested that H
2
O
2
-producing lactobacilli in the vagina may protect pregnant women against ascending infection of the chorioamniotic membranes and uterine cavity. We report the identification and H
2
O
2
production of lactobacilli isolated from vaginal swabs collected at 20 weeks' gestation from a population of pregnant women at high risk of preterm birth. We also report the correlation between identification and H
2
O
2
production in relation to the outcomes of chorioamnionitis and preterm birth. Lactobacilli were identified by partial 16S rRNA gene sequencing. H
2
O
2
production by isolates was determined by a semiquantitative method. The most commonly isolated species were
L. crispatus
,
L. gasseri
,
L. vaginalis
and
L. jensenii
. Amounts of H
2
O
2
produced by lactobacilli varied widely. The presence of lactobacilli producing high levels of H
2
O
2
in the vagina of this population of pregnant women was associated with a reduced risk of bacterial vaginosis at 20 weeks' gestation and subsequent chorioamnionitis.
L. jensenii
and
L. vaginalis
produced the highest levels of H
2
O
2
. We postulate that H
2
O
2
-producing lactobacilli are able to reduce the incidence of ascending infections of the uterus and the subsequent production of proinflammatory molecules which are important in the pathogenesis of chorioamnionitis and preterm birth.