bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Patient Characteristics on Performance of Nucleic Acid Amplification Tests and DNA Probe for Detection of
Chlamydia trachomatis
in Women with Genital Infections
Authors: Marrazzo Jeanne M. , Johnson Robert E. , Green Timothy A. , Stamm Walter E. , Schachter Julius , Bolan Gail , Hook Edward W. , Jones Robert B. , Martin David H. , St. Louis Michael E. , Black Carolyn M. 
Published: February 2005

ABSTRACT

The performance of nucleic acid amplified tests (NAAT) for
Chlamydia trachomatis
at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical
C. trachomatis
over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women ≥20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of
C. trachomatis
in diverse clinical populations of women.
Description of
Campylobacter curvus
and
C. curvus
-Like Strains Associated with Sporadic Episodes of Bloody Gastroenteritis and Brainerd's Diarrhea
Authors: Abbott Sharon L. , Waddington Michael , Lindquist David , Ware Jim , Cheung Wendy , Ely Janet , Janda J. Michael 
Published: February 2005

ABSTRACT

Campylobacter curvus
is a rarely encountered
Campylobacter
species in human, animal, and environmental samples. During the course of two investigations, one involving a search for possible bacterial agents causing bloody gastroenteritis and a second concerning a small outbreak of Brainerd's diarrhea in northern California, 20 strains of
C. curvus
or
C. curvus
-like organisms were isolated by a microfiltration technique and prolonged incubation. The results suggest that
C. curvus
may be an underappreciated
Campylobacter
that may be involved in sporadic and outbreak cases of bloody or chronic diarrhea in humans.
Detection and Identification of
Enterocytozoon bieneusi
and
Encephalitozoon
Species in Stool and Urine Specimens by PCR and Differential Hybridization
Authors: Notermans Daan W. , Peek Ron , de Jong Menno D. , Wentink-Bonnema Ellen M. , Boom René , van Gool Tom 
Published: February 2005

ABSTRACT

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals.
Enterocytozoon bieneusi
and
Encephalitozoon intestinalis
are most commonly associated with chronic diarrhea. All
Encephalitozoon
species, including
E. intestinalis
,
E. hellem
, and
E. cuniculi
, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and
Vittaforma corneae
as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three
Encephalitozoon
species and
V. corneae
. An analytical sensitivity of 3.5 × 10
2
to 3.5 × 10
3
spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different
Encephalitozoon
species.
Production of Herpes B Virus Recombinant Glycoproteins and Evaluation of Their Diagnostic Potential
Authors: Perelygina Ludmila , Patrusheva Irina , Hombaiah Soumya , Zurkuhlen Holley , Wildes Martin J. , Patrushev Nikolai , Hilliard Julia 
Published: February 2005

ABSTRACTB virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n= 40) and -positive (n= 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.
Isolation ofCandida famatafrom a Patient with Acute Zonal Occult Outer Retinopathy
Authors: Carrasco Luis , Ramos Marta , Galisteo Rebeca , Pisa Diana , Fresno Manuel , González María Eugenia 
Published: February 2005

ABSTRACTThe etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined.Candida famatawas isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by usingC. famataas a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patient's blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole,C. famatastill appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment.
Identifying Sources of Human Exposure to Plague
Authors: Lowell Jennifer L. , Wagner David M. , Atshabar Bakyt , Antolin Michael F. , Vogler Amy J. , Keim Paul , Chu May C. , Gage Kenneth L. 
Published: February 2005

ABSTRACT

Yersinia pestis
, the etiologic agent of plague, has shaped the course of human history, killing millions of people in three major pandemics. This bacterium is still endemic in parts of Asia, Africa, and the Americas, where it poses a natural disease threat to human populations.
Y. pestis
has also recently received attention as a possible bioterrorism agent. Thus, rapid methods to distinguish between bioterrorism and naturally occurring plague infections are of major importance. Our study is the first to demonstrate that variable-number tandem repeats (VNTRs) in the
Y. pestis
genome can link human case isolates to those obtained from suspected environmental sources of infection. We demonstrate the valuable utility of VNTR markers in epidemiological investigations of naturally occurring plague and the forensic analysis of possible bioterrorism events.
Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis
Authors: Kumar Manish , Shukla P. K. 
Published: February 2005

ABSTRACT

The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccard's coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by
Aspergillus fumigatus
and was used for the diagnosis of fungal corneal ulcers in patients.
Molecular Characterizations of Human and Animal Group A Rotaviruses in The Netherlands
Authors: van der Heide R. , Koopmans M. P. G. , Shekary N. , Houwers D. J. , van Duynhoven Y. T. H. P. , van der Poel W. H. M. 
Published: February 2005

ABSTRACTTo gain more insight into interspecies transmission of rotavirus group A, human and animal fecal samples were collected between 1997 and 2001 in The Netherlands. A total of 110 human stool samples were successfully P and G genotyped by reverse transcriptase PCR. All strains belonged to the main human rotavirus genotypes G1 to G4, G9, [P4], [P6], [P8], and [P9]. [P8]G1 was predominant, and 5.5% belonged to the G9 genotype. Eleven percent of all P[8] genotypes could be genotyped only by a recently published modified primer. Rotavirus-positive fecal samples from 28 calf herds were genotyped by DNA sequencing. Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus-positive calf herds, respectively. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found. Genotype G6 bovine rotaviruses were divided into three clusters: UK-like, VMRI-29-like, and Hun4-like. DNA sequencing of a part of the VP7 gene was shown to be useful as a quick determination of uncommon or novel strains of which the genotyping cannot be done by genotyping PCR. Of equine strains, both VP4 and VP7 genes could be used for genotyping: two [P12]G3 and four [P12]G14 equine rotaviruses were determined. We did not find indications for rotavirus interspecies transmissions, although the recently published human G6-Hun4 is genetically related to our G6 bovine isolates. All bovine, porcine, and equine rotaviruses were within genotypes previously reported for these animal species.
Diagnostic Approach for Differentiating Infected from Vaccinated Poultry on the Basis of Antibodies to NS1, the Nonstructural Protein of Influenza A Virus
Authors: Tumpey Terrence M. , Alvarez Rene , Swayne David E. , Suarez David L. 
Published: February 2005

ABSTRACT

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in
Escherichia coli
or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.
Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses
Authors: Bronzoni Roberta Vieira de Morais , Baleotti Flávia Graciela , Ribeiro Nogueira Rita Maria , Nunes Márcio , Moraes Figueiredo Luiz Tadeu 
Published: February 2005

ABSTRACT

A new approach was developed for the rapid detection and identification of Brazilian alphaviruses and flaviviruses. The methodology involves the genus-specific detection of
Alphavirus
and
Flavivirus
by a duplex reverse transcription-PCR (D-RT-PCR), followed by multiplex nested PCR (M-N-PCR) or nested PCR (N-PCR) assays for species-specific identification. By this protocol, 25 arboviruses were specifically detected and identified. Detection levels between 10
1.3
and 10
3.5
50% tissue culture infective doses (TCID
50
)/ml of
Flavivirus
and
Alphavirus
strains were achieved by D-RT-PCR, and levels of <1 TCID
50
/ml were achieved by M-N-PCR assays. To assess the suitability and clinical application of this methodology, a total of 101 human or animal stored samples were analyzed. Results obtained suggest that this technique could be applied as a rapid diagnostic tool in clinical samples in which arbovirus infection is suspected and differential diagnosis is required, avoiding the need to test specimens by separate PCR methods.
Characterization of Virus Isolates by Particle-Associated Nucleic Acid PCR
Authors: Stang Alexander , Korn Klaus , Wildner Oliver , Überla Klaus 
Published: February 2005

ABSTRACT
Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3′ end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.
Indirect Enzyme-Linked Immunosorbent Assay for Detection ofBrucella melitensis-Specific Antibodies in Goat Milk
Authors: Funk N. D. , Tabatabai L. B. , Elzer P. H. , Hagius S. D. , Martin B. M. , Hoffman L. J. 
Published: February 2005

ABSTRACTBrucella melitensisis the cause of brucellosis in sheep and goats, which often results in abortion. Few cases ofB. melitensisinfection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection ofB. melitensis-specific antibodies in goat milk.Brucellasalt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection ofB. melitensis-specific antibodies in goat milk.
Mutations within the Hepatitis C Virus Genotype 1b E2-PePHD Domain Do Not Correlate with Treatment Outcome
Authors: Gaudy Catherine , Lambelé Marie , Moreau Alain , Veillon Pascal , Lunel Francoise , Goudeau Alain 
Published: February 2005

ABSTRACT
The hepatitis C virus (HCV) envelope protein 2 (E2) interacts in vitro with the interferon alpha (IFN-α)-inducible double-stranded RNA-activated protein kinase, suggesting a possible mechanism by which HCV may evade the antiviral effects of IFN-α. Variability in the part of the HCV E2 gene encoding the carboxy-terminal part of the protein, which includes the interaction domain (E2-PePHD), was explored in 25 patients infected with HCV genotype 1b and receiving IFN-α therapy. PCR products were generated and sequenced for 15 patients with a sustained response and for 10 patients with no virological response after treatment with IFN-α and ribavirin. PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment, after 2 months in responders, and after 6 months in nonresponders. Quasispecies analysis of the pretreatment PePHD region was performed for isolates from patients displaying amino acid substitutions in this domain on direct sequencing. The E2-PePHD sequence was highly conserved in both resistant and susceptible genotype 1b strains and was identical to the prototype HCV type J sequence. No significant emergence of PePHD mutants during therapy was observed in our clonal analysis, and sporadic mutations and treatment outcomes were not found to be correlated. The PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in HCV type 1b-infected patients.
Detection and Characterization of Diarrheagenic
Escherichia coli
from Young Children in Hanoi, Vietnam
Authors: Nguyen Trung Vu , Le Van Phung , Le Huy Chinh , Gia Khanh Nguyen , Weintraub Andrej 
Published: February 2005

ABSTRACT

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries.
Escherichia coli
is an emerging agent among pathogens that cause diarrhea. The development of a highly applicable technique for the detection of different categories of diarrheagenic
E. coli
is important. We have used multiplex PCR by combining eight primer pairs specific for enteroaggregative
E. coli
(EAEC), enteroinvasive
E. coli
(EIEC), enterohemorrhagic
E. coli
, enteropathogenic
E. coli
(EPEC), and enterotoxigenic
E. coli
(ETEC). This facilitates the identification of five different categories of diarrheagenic
E. coli
from stool samples in a single reaction simultaneously. The prevalences of diarrheagenic
E. coli
were 22.5 and 12% in the diarrhea group and the control group, respectively. Among 587 fecal samples from Vietnamese children under 5 years of age with diarrhea, this technique identified 132 diarrheagenic
E. coli
strains. This included 68 samples (11.6%) with EAEC, 12 samples (2.0%) with EIEC, 39 samples (6.6%) with EPEC, and 13 samples (2.2%) with ETEC. Among the 249 age-matched controls, 30 samples were positive for diarrheagenic
E. coli
. The distribution was 18 samples (7.2%) with EAEC, 11 samples (4.4%) with EPEC, and 1 sample (0.4%) with ETEC.