bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the INNO-LiPA HBV Genotyping Assay for Determination of Hepatitis B Virus Genotype
Authors: Osiowy Carla , Giles Elizabeth 
Published: December 2003

ABSTRACT
Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.
Genetic Heterogeneity in the rRNA Gene Locus of
Trichophyton tonsurans
Authors: Gaedigk Andrea , Gaedigk Roger , Abdel-Rahman Susan M. 
Published: December 2003

ABSTRACT

Trichophyton tonsurans
is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of
T. tonsurans
by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated
T. tonsurans
from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in
T. tonsurans
.
Human Herpesvirus 8 Load in Matched Serum and Plasma Samples of Patients with AIDS-Associated Kaposi's Sarcoma
Authors: Polstra Abeltje M. , van den Burg Remco , Goudsmit Jaap , Cornelissen Marion 
Published: December 2003

ABSTRACT

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 × 10
6
copies of HHV-8 DNA (
r
2
> 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.
Development of a Multitarget Real-Time TaqMan PCR Assay for Enhanced Detection of
Francisella tularensis
in Complex Specimens
Authors: Versage Jessica L. , Severin Darlena D. M. , Chu May C. , Petersen Jeannine M. 
Published: December 2003

ABSTRACT

Tularemia is the zoonotic disease caused by the gram-negative coccobacillus
Francisella tularensis.
Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease.
F. tularensis
is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting
F. tularensis
in complex specimens. Targeted regions included the IS
Ftu2
element and the
23kDa
,
fopA
, and
tul4
genes. Analysis of the four TaqMan assays demonstrated that three (IS
Ftu2
,
23kDa
, and
tul4
) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-
Francisella
bacteria tested and capable of differentially diagnosing both
F. tularensis
and
Francisella philomiragia
. When the multitarget TaqMan assay (IS
Ftu2
,
23kDa
, and
tul4
) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (
P
≤ 0.05). The sensitive and specific nature of this rapid multitarget TaqMan assay provides a valuable new tool that with future evaluations can be used for analyzing clinical specimens, field samples during bioterrorism threat assessment, and samples from outbreaks and for improving our understanding of the ecology and environmental prevalence of
F. tularensis
.
Culture-Independent Identification of Pathogenic Bacteria and Polymicrobial Infections in the Genitourinary Tract of Renal Transplant Recipients
Authors: Domann Eugen , Hong George , Imirzalioglu Can , Turschner Simon , Kühle Johannes , Watzel Corinna , Hain Torsten , Hossain Hamid , Chakraborty Trinad 
Published: December 2003

ABSTRACTRenal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such asCorynebacterium urealyticumandGardnerella vaginalis, but also unusual agents includingAnaerococcus lactolyticus,Bacteroides vulgatus,Dialister invisus,Fusobacterium nucleatum,Lactobacillus iners,Leptotrichia amnionii,Prevotella buccalis,Prevotella ruminicola,Rahnella aquatilis, andStreptococcus intermediuswere detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.
Natural Acquisition of
Helicobacter pylori
Infection in Newborn Rhesus Macaques
Authors: Solnick Jay V. , Chang Kikuko , Canfield Don R. , Parsonnet Julie 
Published: December 2003

ABSTRACT

Helicobacter pylori
infection is usually acquired in childhood, but precise estimates of the age of acquisition are difficult to obtain in young children. Since serial endoscopic biopsies are not feasible in human infants, we examined acquisition of
H. pylori
infection that is known to occur in socially housed nonhuman primates. By 12 weeks of age, 8 of 20 newborns (40%) were culture positive for
H. pylori
, and prevalence reached 90% by 1 year of age. Newborns from infected dams were more commonly infected than those from uninfected dams, particularly during the peripartum period, suggesting that close contact during this time may facilitate transmission. Transient infection was uncommon and occurred only after the first positive culture. These results suggest that in a high-prevalence environment, persistent
H. pylori
infection may be acquired at an earlier age than was previously thought. Since clean, potable water was readily available, contamination of water supply is not essential for widespread infection at an early age in areas where hygiene is otherwise poor. Furthermore, breastfeeding seems to offer little protection, since newborn macaques breastfeed during the first year of life and typically are fully weaned only when another newborn arrives the following spring.
Loop-Mediated Isothermal Amplification for Detection of African Trypanosomes
Authors: Kuboki Noritaka , Inoue Noboru , Sakurai Tatsuya , Di Cello Francescopaolo , Grab Dennis J. , Suzuki Hiroshi , Sugimoto Chihiro , Igarashi Ikuo 
Published: December 2003

ABSTRACTWhile PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in theTrypanosoma bruceigroup (includingT. brucei brucei,T. brucei gambiense,T. brucei rhodesiense, andT. evansi) andT. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infectiveT. brucei gambiensefurther highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.
Epidemiology of
Aspergillus terreus
at a University Hospital
Authors: Baddley John W. , Pappas Peter G. , Smith Anita C. , Moser Stephen A. 
Published: December 2003

ABSTRACT

Invasive fungal infections due to
Aspergillus
species have become a major cause of morbidity and mortality among immunocompromised patients.
Aspergillus terreus
, a less common pathogen, appears to be an emerging cause of infection at our institution, the University of Alabama hospital in Birmingham. We therefore investigated the epidemiology of
A. terreus
over the past 6 years by using culture data; antifungal susceptibility testing with amphotericin B, voriconazole, and itraconazole; and molecular typing with random amplification of polymorphic DNA-PCR (RAPD-PCR). During the study period, the percentage of
A. terreus
isolates relative to those of other
Aspergillus
species significantly increased, and
A. terreus
isolates frequently were resistant to amphotericin B. Molecular typing with the RAPD technique was useful in discriminating between patient isolates, which showed much strain diversity. Further surveillance of
A. terreus
may better define epidemiology and determine whether this organism is becoming more frequent in relation to other
Aspergillus
species.
Nosocomial Outbreak of Infections by
Proteus mirabilis
That Produces Extended-Spectrum CTX-M-2 Type β-Lactamase
Authors: Nagano Noriyuki , Shibata Naohiro , Saitou Yuko , Nagano Yukiko , Arakawa Yoshichika 
Published: December 2003

ABSTRACT

Nineteen multidrug-resistant
Proteus mirabilis
strains were isolated from 19 patients suffering from infections probably caused by
P. mirabilis
. These strains were recovered from urine or other urogenital specimens of 16 inpatients and three outpatients with a hospitalization history in a urology ward of Funabashi Medical Center, from July 2001 to August 2002. These strains demonstrated resistance to cefotaxime, ceftriaxone, cefpodoxime, and aztreonam, while they were highly susceptible to ceftazidime (MIC, ≤0.5 μg/ml). The resistance level of these strains to cefotaxime was decreased by the presence of clavulanic acid. Therefore, the strains were speculated to produce extended-spectrum class A β-lactamases. These strains were later found to carry
bla
CTX-M-2
genes by both PCR and sequencing analyses. The profiles of
Sma
I-digested genomic DNA of 19 isolates were distinguished into five different clusters by biased sinusoidal field gel electrophoresis. Four of them, consisting of 18 isolates, were suggested to be a clonal expansion. These findings suggested that a nosocomial outbreak of infections by CTX-M-2-producing
P. mirabilis
had occurred in our medical center. Most patients suffered from urogenital malignancies with long-term catheterization. Cefazolin, cefoperazone-sulbactam, and/or levofloxacin were mostly administered to the patients, but these agents seemed ineffective for eradication of CTX-M-2 producers. Early recognition and rapid identification of colonizing antimicrobial-resistant bacteria, including CTX-M-2-producing
P. mirabilis
, would be the most effective measures to cope with further spread of this kind of hazardous microorganism in clinical environments.
Listeria monocytogenes
Serotype Identification by PCR
Authors: Borucki Monica K. , Call Douglas R. 
Published: December 2003

ABSTRACT

Serotyping is a universally accepted subtyping method for
Listeria monocytogenes
. Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a “gold standard” for comparing isolates analyzed in different labs and with different techniques. Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system. Serotyping PCR primers were designed from variable regions of the
L. monocytogenes
genome. Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122
L. monocytogenes
strains into five serotype groups [1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c]. Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.
Antibiotic-Resistant Invasive Pediatric
Streptococcus pneumoniae
Clones in Israel
Authors: Greenberg David , Dagan Ron , Muallem Marie , Porat Nurith 
Published: December 2003

ABSTRACT

Antibiotic-resistant international clones of
Streptococcus pneumoniae
are increasingly reported in different parts of the world. We investigated the spread of these clones through an active surveillance performed at the Israeli Streptococcal National Center during 1998 and 1999. Isolates were tested for antibiotic susceptibility, serotyped, and genotyped by random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis. Of 437 isolates, 276 (63.4%) were antibiotic resistant and 156 (35%) were penicillin nonsusceptible (PNS). The PNS isolates were less frequently encountered in southern Israel (27 of 136 [20%]) than in other regions (127 of 301 [42%]). Among 276 antibiotic-resistant isolates, 43 fingerprint patterns were observed. The most common clones were 9V/14-a (19.2%), 5-a (17.8%), and 1-a (10%). The 9V/14-a clone was less common, while the 1-a clone was more frequent in the south than in other regions. The 5-a clone was more common in Jerusalem than in other regions. Among the Jewish and Arab populations the most frequent clones were 9V/14-a (20%) and 1-a (25%), respectively. Three international clones, 9V/14-a-Spain
9V
-3, 6B-a-Spain
6B
-2, and 5-a-Colombia
5
-19, comprised 40% of all antibiotic-resistant isolates and 56% of all PNS isolates. The seven-valent conjugate vaccine covers 58% of the most common clones, all highly PNS clones, and 94% of the multidrug-resistant clones in Israel, while the nine-valent vaccine covers all of them. The most common antibiotic-resistant invasive pediatric
S. pneumoniae
clones—mainly the three international ones—contribute significantly to increases in antibiotic resistance. Their geographic distribution varies within the country and between the different populations.
Evaluation of Methodology for Serotyping Invasive and Nasopharyngeal Isolates of
Haemophilus influenzae
in the Ongoing Surveillance in Brazil
Authors: Bokermann Sérgio , Zanella Rosemeire C. , Lemos Ana Paula S. , de Andrade Ana Lúcia S. S. , Brandileone Maria Cristina de C. 
Published: December 2003

ABSTRACT

To assess the magnitude of discrepant results obtained by routine
Haemophilus influenzae
serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (
n
= 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (
n
= 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of
H. influenzae
serotypes in the
H. influenzae
type b vaccine era.
Quantitative Validation of Media for Transportation and Storage of
Streptococcus pneumoniae
Authors: Charalambous B. M. , Batt S. L. , Peek A. C. , Mwerinde H. , Sam N. , Gillespie S. H. 
Published: December 2003

ABSTRACT

The need to design effective
Streptococcus pneumoniae
vaccines and to monitor resistance means that it is essential to have efficient methods to determine carriage rates. Two liquid media, consisting of skim milk, glycerol, glucose, and tryptone soya broth (STGG) or skim milk, glycerol, and glucose (SGG) alone, were evaluated for their ability to maintain pneumococcal viability. Optimal recovery of
S. pneumoniae
was achieved when swabs were transferred to STGG medium prior to plating onto blood agar-gentamicin selective plates (22%) compared to 7% when plated out directly (
P
< 0.0001 by Fisher's exact test). Both STGG and SGG media are appropriate for the long-term storage of pneumococci and primary swab samples at −70°C, with no decrease in viable count observed following repeated freeze-thaw cycles. Samples could be stored refrigerated for up to 3 days in either STGG or SGG medium with no significant loss of viability. Viability decreased progressively in storage at 20 to 30°C, with greater losses of viability occurring at the higher temperatures. There were no significant differences in viability between isolates in the two media. STGG preserved pneumococci significantly better (about twofold) than SGG medium at 21°C (
P
< 0.0001) and 30°C (
P
< 0.0001). Samples can be stored for 4 and 2.5 days at 6 to 8°C, 28 and 17 h at 21°C, and 15 and 7 h at 30°C in STGG and SGG media, respectively. For field studies undertaken in resource-limited environments, SGG medium can be prepared by using locally available materials. The quantitative data reported in this study will enable researchers to plan appropriate transport and storage protocols.