bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Detection and Identification of Human Adenovirus Species by Adenoplex, a Multiplex PCR-Enzyme Hybridization Assay
Authors: Pehler-Harrington Karen , Khanna Marilyn , Waters Chris R. , Henrickson Kelly J. 
Published: September 2004

ABSTRACT

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10
−2
, 10
−1
, 10
−1
, 10
−2
, 10
−1
, and 10
−2
, respectively. Similarly, the LOD for the six DNA controls ranged from 10
2
to 10
3
copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.
Isoenzymatic Analysis of 712 Strains of
Leishmania infantum
in the South of France and Relationship of Enzymatic Polymorphism to Clinical and Epidemiological Features
Authors: Pratlong Francine , Rioux Jean-Antoine , Marty Pierre , Faraut-Gambarelli Françoise , Dereure Jacques , Lanotte Geneviève , Dedet Jean-Pierre 
Published: September 2004

ABSTRACT

In the south of France, leishmaniasis due to
Leishmania infantum
occurs in the following five foci of endemicity (from west to east): Pyrénées-Orientales, Cévennes, Provence, Côte d'Azur, and Corsica. Between 1981 and 2002, 712
Leishmania
strains obtained from humans, dogs, cats, and sand flies were studied by isoenzyme analysis. In total, seven zymodemes were identified: MON-1, MON-11, MON-24, MON-29, MON-33, MON-34, and MON-108. The Pyrénées-Orientales focus is characterized by a predominance of human cutaneous leishmaniasis and a high enzymatic polymorphism (five zymodemes). In the other foci, where human visceral leishmaniasis is predominant, only two zymodemes are present.
L. infantum
MON-1 is the parasite most frequently found, in patients both with and without concomitant human immunodeficiency virus infection. MON-1 is the only zymodeme present in dogs, which act as the reservoir host in all of the foci. In Cévennes, where the complete life cycle of zymodeme MON-1 has been identified,
Phlebotomus perniciosus
and
Phlebotomus ariasi
are vectors. The enzymatic polymorphism is compared to that of neighboring countries (Spain and Italy). In Pyrénées-Orientales, small variant zymodemes with electromorphs of heterozygote-like and homozygotic patterns can be explained by different genetic hypotheses.
Development and Validation of a Diagnostic DNA Microarray To Detect Quinolone-Resistant
Escherichia coli
among Clinical Isolates
Authors: Yu Xiaolei , Susa Milorad , Knabbe Cornelius , Schmid Rolf D. , Bachmann Till T. 
Published: September 2004

ABSTRACT

The incidence of resistance against fluoroquinolones among pathogenic bacteria has been increasing in accordance with the worldwide use of this drug.
Escherichia coli
is one of the most relevant species for quinolone resistance. In this study, a diagnostic microarray for single-base-mutation detection was developed, which can readily identify the most prevalent
E. coli
genotypes leading to quinolone resistance. Based on genomic sequence analysis using public databases and our own DNA sequencing results, two amino acid positions (83 and 87) on the A subunit of the DNA gyrase, encoded by the
gyrA
gene, have been identified as mutation hot spots and were selected for DNA microarray detection. Oligonucleotide probes directed against these two positions were designed so that they could cover the most important resistance-causing and silent mutations. The performance of the array was validated with 30 clinical isolates of
E. coli
from four different hospitals in Germany. The microarray results were confirmed by standard DNA sequencing and were in full agreement with phenotypic antimicrobial susceptibility testing.
Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic
Escherichia coli
Authors: Reischl Udo , Youssef Mohammad T. , Wolf Hans , Hyytia-Trees Eija , Strockbine Nancy A. 
Published: September 2004

ABSTRACT

To facilitate the diagnosis of enterotoxigenic
Escherichia coli
(ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161
E. coli
isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.
Use of Recombinant E Protein Domain III-Based Enzyme-Linked Immunosorbent Assays for Differentiation of Tick-Borne Encephalitis Serocomplex Flaviviruses from Mosquito-Borne Flaviviruses
Authors: Holbrook Michael R. , Shope Robert E. , Barrett Alan D. T. 
Published: September 2004

ABSTRACT
The serological diagnosis of infection by flaviviruses is complicated by the presence of flavivirus cross-reactive antibodies that produce false-positive results for flavivirus infections, especially in regions where more than one virus is endemic. Current diagnostic reagents for tick-borne flavivirus infection have been found to cross-react with yellow fever- or dengue virus-positive sera. This study utilized recombinant flavivirus E protein domain 3 (rE-D3) as a diagnostic reagent to differentiate between infection by mosquito- and tick-borne flaviviruses. This study found that the use of rE-D3 in an enzyme-linked immunosorbent assay (ELISA)-based format allowed the differentiation between serum specific for either mosquito- or tick-borne flaviviruses, but not among the members of the tick-borne encephalitis (TBE) serocomplex of flaviviruses. Sera derived against several TBE serocomplex rE-D3 were found to cross-react with heterologous rE-D3 within the TBE serocomplex, but not with those from mosquito-borne flaviviruses, in both Western blots and ELISAs. Mouse hyperimmune sera generated against TBE serocomplex viruses were also found to react specifically with TBE serocomplex rE-D3, but not with rE-D3 from mosquito-borne viruses and vice versa. When a similar test using virus-derived antigen was performed, a loss of both specificity and sensitivity was observed. These results indicate that flavivirus rE-D3 would be a useful reagent for the detection of infection by TBE serocomplex flaviviruses, several of which are potential biothreat agents, but would not provide the ability to differentiate among infections by separate members of the serocomplex.
Virulence Characteristics and Molecular Epidemiology of Enteroaggregative
Escherichia coli
Isolates from Hospitalized Diarrheal Patients in Kolkata, India
Authors: Kahali Soumen , Sarkar Bhaswati , Rajendran K. , Khanam Jasmina , Yamasaki Shinji , Nandy Ranjan K. , Bhattacharya S. K. , Ramamurthy T. 
Published: September 2004

ABSTRACT

Enteroaggregative
Escherichia coli
(EAEC) is an important diarrheal enteropathogen defined by aggregative adherence to cultured epithelial cells. We have detected EAEC from 121 (6.6%) of 1,826 hospitalized patients admitted with diarrhea to the Infectious Diseases Hospital in Kolkata, India. Watery diarrhea was recorded significantly (
P
= 0.0142) more often in children. The majority of the EAEC isolates were not serotypeable (62%) and showed resistance to five or more antibiotics (76%). We studied different virulence genes and the molecular epidemiology of 121 EAEC isolates recovered from diarrheal patients. A PCR assay for detection of virulence genes, an assay for determination of clump formation in liquid culture, and a HeLa cell adherence assay were carried out to characterize the EAEC isolates. Investigations were also conducted to correlate the virulence gene profiles with diarrheal symptoms and molecular epidemiology by pulsed-field gel electrophoresis (PFGE). Two or more virulence genes were detected in 109 (90.1%) EAEC isolates. In the cluster analysis, some isolates with specific gene profiles and phenotypes formed a group or subcluster. This study highlights the comparative distributions of three fimbrial adhesins and other virulence genes among EAEC isolates. The diverse virulence gene and PFGE profiles, along with the existence of diverse serotypes and antibiograms, suggests that the EAEC isolates are genetically heterogeneous in Kolkata.
Correlation of
wbiI
Genotype, Serotype, and Isolate Source within Species of the
Burkholderia cepacia
Complex
Authors: Vinion-Dubiel Arlene D. , Spilker Theodore , Dean Charles R. , Monteil Henri , LiPuma John J. , Goldberg Joanna B. 
Published: September 2004

ABSTRACT

Gram-negative bacteria of the
Burkholderia cepacia
complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene
wbiI
. Homologues of
wbiI
are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc
wbiI
gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this
wbiI
PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single
wbiI
restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple
wbiI
RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between
wbiI
RFLP type and species. There was also no apparent correlation between
wbiI
RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same
wbiI
RFLP type, indicating that
wbiI
RFLP typing may be a useful tool to help track Bcc outbreaks.
Isolation of Metronidazole-Resistant
Bacteroides fragilis
Carrying the
nimA
Nitroreductase Gene from a Patient in Washington State
Authors: Schapiro Jeffrey M. , Gupta Rachna , Stefansson Eric , Fang Ferric C. , Limaye Ajit P. 
Published: September 2004

ABSTRACT

Members of the
Bacteroides fragilis
group are among the most common anaerobic bacterial isolates in clinical specimens. Metronidazole, a 5-nitroimidazole, is often used as empirical therapy for anaerobic infections. Susceptibility testing is not routinely performed because of nearly universal susceptibility of
Bacteroides
spp. to this agent. We report a case of metronidazole-resistant
Bacteroides fragilis
in the United States and demonstrate the presence of the
nimA
gene, encoding a nitroreductase previously shown to mediate resistance to 5-nitroimidazole antimicrobial agents in
B. fragilis
strains from Europe and Africa. Because clinical failures in
Bacteroides
infections have been associated with the use of inactive antimicrobial agents, clinicians need to be aware of the possibility of metronidazole-resistant
B. fragilis
strains in the United States and the importance of susceptibility testing in selected situations.
Use of the MagNA Pure LC Automated Nucleic Acid Extraction System followed by Real-Time Reverse Transcription-PCR for Ultrasensitive Quantitation of Hepatitis C Virus RNA
Authors: Cook Linda , Ng Ka-Wing , Bagabag Arthur , Corey Lawrence , Jerome Keith R. 
Published: September 2004

ABSTRACT

Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay for HCV RNA. The assay amplifies a portion of the 5′ untranslated region of HCV, which is then quantitated using the TaqMan 7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche). Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least 10
9
IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible, with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively. The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for routine HCV viral load testing.
Identification of the Emerging Pathogen
Vibrio vulnificus
Biotype 3 by Commercially Available Phenotypic Methods
Authors: Colodner Raul , Raz Raul , Meir Irit , Lazarovich Tsilia , Lerner Larisa , Kopelowitz June , Keness Yoram , Sakran Waheeb , Ken-Dror Shifra , Bisharat Naiel 
Published: September 2004

ABSTRACT

Identification of the emerging pathogen
Vibrio vulnificus
biotype 3 has become a challenge for clinical laboratories in the last few years. In this study, the abilities of five commercial systems to identify this new species have been evaluated for the first time, using a unique collection of strains. Fifty-one well-documented wild strains of
V. vulnificus
biotype 3 were processed using API 20 NE, GNI+ Vitek 1 cards, ID-GNB Vitek 2 cards, Neg Combo 20 Microscan panels, and NMIC/ID-5 BD Phoenix panels. The numbers of strains identified as
V. vulnificus
by ID-GNB, NMIC/ID-5, and GNI+ were 50 (98.0%), 46 (90.2%), and 7 (13.7%), respectively. Neg Combo 20 Microscan panels and API 20 NE were unable to identify any of the strains of this emerging pathogen to the species level and mostly misidentifies them as other species of the
Vibrionaceae
family. Data on the phenotypic pattern of
V. vulnificus
biotype 3 when processed in all five systems as presented here could help clinical laboratories in identifying this new pathogen.
Prevalence of Periodontal Pathogens in Dental Plaque of Children
Authors: Gafan Gavin P. , Lucas Victoria S. , Roberts Graham J. , Petrie Aviva , Wilson Michael , Spratt David A. 
Published: September 2004

ABSTRACT

Porphyromonas gingivalis
,
Actinobacillus actinomycetemcomitans
, and
Tannerella forsythensis
have been implicated as the main etiological agents of periodontal disease. The purpose of this work was to estimate the prevalence of these organisms in plaque from children without gingivitis (group 1;
n
= 65) and from those with gingivitis (group 2;
n
= 53). Extracted DNA from plaque was subjected to two rounds of PCR targeting the 16S rRNA gene using both universal primers and species-specific primers. The results were as follows: group 1,
P. gingivalis
, 49%;
A. actinomycetemcomitans
, 55%; and
T. forsythensis
, 65%; group 2,
P. gingivalis
, 47%;
A. actinomycetemcomitans
, 59%; and
T. forsythensis
, 45%.
T. forsythensis
was detected more frequently in children with no gingivitis than in those with gingivitis (
P
= 0.03). There was no significant difference between the two groups with respect to the presence of
P. gingivalis
or
A. actinomycetemcomitans
in either group (
P
> 0.05). Logistic regression analysis revealed that the odds of a patient having gingivitis were 2.3 times greater in the absence of
T. forsythensis
. In conclusion, the results of this study have shown that the three pathogens can be detected in the dental plaque of healthy children and of those with gingivitis and that
T. forsythensis
is associated with dental plaque at sites with no gingivitis.
Field Evaluation of a Rapid Human Immunodeficiency Virus (HIV) Serial Serologic Testing Algorithm for Diagnosis and Differentiation of HIV Type 1 (HIV-1), HIV-2, and Dual HIV-1-HIV-2 Infections in West African Pregnant Women
Authors: Rouet François , Ekouevi Didier K. , Inwoley André , Chaix Marie-Laure , Burgard Marianne , Bequet Laurence , Viho Ida , Leroy Valériane , Simon François , Dabis François , Rouzioux Christine 
Published: September 2004

ABSTRACT
We evaluated a two-rapid-test serial algorithm using the Determine and Genie II rapid assays, performed on-site in four peripheral laboratories during the French Agence Nationale de Recherches sur le SIDA (ANRS) 1201/1202 Ditrame Plus cohort developed for prevention of mother-to-child transmission of human immunodeficiency virus (HIV) infection in Côte d'Ivoire. A total of 1,039 specimens were retested by two commercial enzyme-linked immunosorbent assays (ELISAs). The following specimens were tested: 315 specimens found on-site to be infected with HIV type 1 (HIV-1), 8 specimens found on-site to be infected with HIV-2, 71 specimens found on-site to be infected with both HIV-1 and HIV-2, 40 specimens found on-site to have indeterminate results for HIV infection, and 605 specimens taken during a quality assurance program. For HIV discrimination, 99 positive serum samples (20 with HIV-1, 8 with HIV-2, and 71 with HIV-1 and HIV-2 on the basis of our rapid test algorithm) were retested by the Peptilav test, Western blot (WB) assays, and homemade monospecific ELISAs. Real-time DNA PCRs for the detection of HIV-1 and HIV-2 were performed with peripheral blood mononuclear cells from 35 women diagnosed on-site with HIV-1 and HIV-2 infections. Compared to the results of the ELISAs, the sensitivities of the Determine and Genie II assays were 100% (95% lower limit [95% LL], 99.1%) and 99.5% (95% confidence interval [95% CI], 98.2 to 99.9%), respectively. The specificities were 98.4% (95% CI, 96.9 to 99.3%) and 100% (95% LL, 99.3%), respectively. All serological assays gave concordant results for infections with single types. By contrast, for samples found to be infected with dual HIV types by the Genie II assay, dual reactivity was detected for only 37 samples (52.1%) by WB assays, 34 samples (47.9%) by the Peptilav assay, and 23 samples (32.4%) by the monospecific ELISAs. For specimens with dual reactivity by the Genie II assay, the rates of concordance between the real-time PCR assays and the serological assays were 25.7% for the Genie II assay, 82.9% for the Peptilav assay, 74.3% for WB assays, and 80% for the homemade ELISAs. Our algorithm provided high degrees of sensitivity and specificity comparable to those of ELISAs. Even if they are rare, women identified by the Genie II assay as being infected with HIV-1 and HIV-2 mostly appeared to be infected only with HIV-2.
Diagnosing Invasive Aspergillosis during Antifungal Therapy by PCR Analysis of Blood Samples
Authors: Lass-Flörl Cornelia , Gunsilius Eberhard , Gastl Günther , Bonatti Hugo , Freund Martin C. , Gschwendtner Andreas , Kropshofer Gabriele , Dierich Manfred P. , Petzer Andreas 
Published: September 2004

ABSTRACT

We evaluated the value of
Aspergillus
PCR as a tool for diagnosing invasive aspergillosis from whole-blood samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens. A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoalveolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had probable, and 12 had possible invasive
Aspergillus
infection according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive PCR results for
Aspergillus
were associated with fatal outcome, as three of six patients died. For patients with probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has been started. Performance of
Aspergillus
PCR using tissue samples is recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.