bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Glycopeptide-Resistant
Enterococcus faecium
(GRE) from Broilers and Pigs in Denmark: Genetic Evidence that Persistence of GRE in Pig Herds Is Associated with Coselection by Resistance to Macrolides
Authors: Aarestrup Frank Møller 
Published: December 2019

ABSTRACT

Glycopeptide-resistant enterococci (GRE) from broilers and pigs were characterized to investigate the background for the persistence of GRE in pig herds. All porcine isolates belonged to closely related pulsed-field gel electrophoretic (PFGE) types, with the
ermB
and
vanA
genes located on the same transferable genetic element. Broiler isolates belonged to different PFGE types. The persistence of GRE in Danish pig herds after the ban of glycopeptides may be explained by the genetic link between
ermB
and
vanA
and coselection by use of macrolides for treatment and growth promotion.
Performance of Three Enzyme Immunoassays and Two Direct Fluorescence Assays for Detection of
Giardia lamblia
in Stool Specimens Preserved in ECOFIX
Authors: Fedorko Daniel P. , Williams Esther C. , Nelson Nancy A. , Calhoun Leslie B. , Yan Sizhuang S. 
Published: December 2019

ABSTRACT

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial
Giardia lamblia
-specific enzyme immunoassays (EIAs) (ProSpecT
Giardia
Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier
Giardia lamblia
[Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for
G. lamblia
(Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect
G. lamblia
in 34
G. lamblia
-positive and 44
G. lamblia
-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect
G. lamblia
in the same specimens preserved in formalin as the “gold standard” for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of
G. lamblia
cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.
Clinical and Financial Benefits of Rapid Detection of Respiratory Viruses: an Outcomes Study
Authors: Barenfanger Joan , Drake Cheryl , Leon Nidia , Mueller Tina , Troutt Tammy 
Published: December 2019

ABSTRACT

To assess the expected benefits of rapid reporting of respiratory viruses, we compared patients whose samples were processed using standard techniques such as enzyme immunoassays, shell vial assays, and culture tube assays (year 1) to patients whose samples were processed with the same standard techniques in addition to immunofluorescent testing (FA) directly on cytocentrifuged samples (year 2). The cytospin FA screened for influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3, and adenovirus (DAKO Diagnostics Ltd.). The specificity of the cytospin FA for all viruses was 100%. The sensitivities for influenza A virus and RSV were 90 and 98%, respectively, but the sensitivities for influenza B virus and adenovirus were unacceptable (14.3 and 0%, respectively). However, since the former viruses account for >85% of our isolates from clinical specimens, the cytospin FA is an excellent screening test since the positive result was available within hours. The mean turnaround time for all positive viruses was 4.5 days in year 1 and 0.9 day in year 2 (
P
= 0.001). This rapid reporting resulted in physicians having access to information sooner, enabling more appropriate treatment. The mean length of stay in the hospital for inpatients with respiratory viral isolates was 10.6 days for year 1 versus 5.3 days for year 2. Mean variable costs for these patients was $7,893 in year 1 and $2,177 in year 2. After subtracting reagent costs and technological time, the savings in variable costs was $144,332/year. Summarizing, the cytospin FA markedly decreased turnaround time and was associated with decreased mortality, length of stay, and costs and with better antibiotic stewardship.
Detection of Significant Bacteriuria by Automated Urinalysis Using Flow Cytometry
Authors: Okada Hiroshi , Sakai Yutaka , Miyazaki Shigenori , Arakawa Soichi , Hamaguchi Yukio , Kamidono Sadao 
Published: December 2019

ABSTRACT
A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods.
Enteric Virus Infections and Diarrhea in Healthy and Human Immunodeficiency Virus-Infected Children
Authors: Liste Mary B. , Natera Ivelisse , Suarez José A. , Pujol Flor H. , Liprandi Ferdinando , Ludert Juan E. 
Published: December 2019

ABSTRACT

Forty-three stool samples from 27 human immunodeficiency virus (HIV)-seropositive children and 38 samples from 38 HIV-negative children, collected during a 15-month period, were examined for enteric viruses. Diagnostic assays included enzyme immunoassays for rotavirus, adenovirus, and Norwalk virus; polyacrylamide gel electrophoresis for picobirnavirus and atypical rotavirus; and PCR for astrovirus and enterovirus. Specimens from HIV-positive children were more likely than those of HIV-negative children to have enterovirus (56 versus 21%;
P
< 0.0002) and astrovirus (12 versus 0%;
P
< 0.02), but not rotavirus (5 versus 8%;
P
> 0.5). No adenoviruses, picobirnaviruses, or Norwalk viruses were found. The rates of virus-associated diarrhea were similar among HIV-positive and HIV-negative children. Enteroviruses were excreted for up to 6 months in HIV-positive children; however, no evidence for prolonged excretion of poliovirus vaccine was observed. These results suggest that although infection with enterovirus and astrovirus may be frequent in HIV-infected children, enteric viruses are not associated with the diarrhea frequently suffered by these children.
Use of the Neisserial Lipoprotein (Lip) for Subtyping
Neisseria gonorrhoeae
Authors: Trees David L. , Schultz Abbigail J. , Knapp Joan S. 
Published: December 2019

ABSTRACT

The pathogenic
Neisseria
species
N. meningitidis
and
N. gonorrhoeae
possess an outer membrane lipoprotein, designated Lip, which is present in all strains tested. The predicted protein sequence of Lip consists of a consensus AAEAP amino acid repeat. The objective of this study was to determine the feasibility of using the Lip repeat number and sequence for subtyping of
Neisseria gonorrhoeae
. The
lip
genes of each isolate were amplified by PCR and sequenced to determine the repeat number and sequence. Among the 46 strains we examined, eight different Lip repeat numbers were identified, with lengths of 11 (1 strain), 12 (14 strains), 13 (2 strains), 14 (10 strains), 15 (5 strains), 16 (10 strains), 17 (3 strains), and 20 (1 strain) repeats. Analysis indicated differences in the sequences within the repeats that resulted in amino acid alterations in repeat classes that contained multiple strains. Among the 46 isolates examined, we were able to identify 17 unique Lip subtyping patterns.
Real-Time PCR for Diagnosis and Follow-Up of Toxoplasma Reactivation after Allogeneic Stem Cell Transplantation Using Fluorescence Resonance Energy Transfer Hybridization Probes
Authors: Costa J.-M. , Pautas C. , Ernault P. , Foulet F. , Cordonnier C. , Bretagne S. 
Published: December 2019

ABSTRACT

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify
Toxoplasma gondii
DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 × 10
6
to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to ≤0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.
Detection of
Paracoccidioides brasiliensis
in Tissue Samples by a Nested PCR Assay
Authors: Bialek Ralf , Ibricevic Aida , Aepinus Christian , Najvar Laura K. , Fothergill Annette W. , Knobloch Jürgen , Graybill John R. 
Published: December 2019

ABSTRACT

A nested PCR assay for the detection of
Paracoccidioides brasiliensis
DNA was evaluated, using a sequence of the immunogenic
gp43
gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of
P. brasiliensis
, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally
P. brasiliensis
-infected and two uninfected BALB/c mice and from 20
Histoplasma capsulatum
-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven
P. brasiliensis
isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the
gp43
gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 × 10
3
to 1.3 × 10
7
CFU of
P. brasiliensis
per g of lung. Uninfected BALB/c mice and
H. capsulatum
-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of
P. brasiliensis
in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.
Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the
rpoB
Gene
Authors: Lee Hyeyoung , Park Hee-Jung , Cho Sang-Nae , Bai Gill-Han , Kim Sang-Jae 
Published: December 2019

ABSTRACT

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the
rpoB
gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of
rpoB
, and the amplified DNAs were subsequently digested with restriction enzymes such as
Msp
I and
Hae
III. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as
Mycobacterium gordonae
,
M. kansasii
,
M. celatum
, and
M. fortuitum
, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the
rpoB
gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species.
Genetic Characterization of Toxin A-Negative, Toxin B-Positive
Clostridium difficile
Isolates by PCR
Authors: Moncrief J. Scott , Zheng Limin , Neville Laurie M. , Lyerly David M. 
Published: December 2019

ABSTRACT

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive
Clostridium difficile
isolates from various geographical sites around the world. All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST. A deletion of approximately 1.7 kb was found at the 3′ end of the
toxA
gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g.,
C. difficile
serotype F 1470). Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463. This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from
Clostridium sordellii
and toxin B from toxin A-negative, toxin B-positive strain F 1470. Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.
A Molecular Epidemiological Study of
Mycobacterium simiae
Isolated from AIDS Patients in Guadeloupe
Authors: Legrand Eric , Devallois Anne , Horgen Lionel , Rastogi Nalin 
Published: December 2019

ABSTRACT

A molecular epidemiological study of
Mycobacterium simiae
strains isolated from AIDS patients in Guadeloupe was performed by the random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of
Dra
I- or
Xba
I-digested bacterial DNAs. A comparison of RAPD profiles suggested a similarity of banding patterns within a group of patients (two clusters of two and three patients), but the available epidemiological and clinical information did not support this finding. PFGE, on the other hand, showed that all the patients were contaminated with individual isolates. Combined numerical analysis performed by compiling the PFGE patterns obtained after
Xba
I and
Dra
I digestions of bacterial DNAs suggested the occurrence of polyclonal infection in three of nine patients. Our results do not support a common source of
M. simiae
infection in Guadeloupe.
Simple and Reliable Multiplex PCR Assay for Surveillance Isolates of Vancomycin-Resistant Enterococci
Authors: Kariyama Reiko , Mitsuhata Ritsuko , Chow Joseph W. , Clewell Don B. , Kumon Hiromi 
Published: December 2019

ABSTRACT

Conditions have been optimized for the use of a multiplex PCR for the detection of vancomycin-resistant enterococci in nosocomial surveillance specimens. Seven primer sets targeting the genes
vanA
,
vanB
,
vanC1
,
vanC2/C3 Enterococcus faecalis
-specific,
Enterococcus faecium
-specific, and
rrs
(16S rRNA) were used in one reaction tube. The PCR method developed in the present study is simple and reliable for the rapid characterization of vancomycin-resistant enterococci.
Use of 5-Bromo-4-Chloro-3-Indolyl-α-
d
-
N
-Acetylneuraminic Acid in a Novel Spot Test To Identify Sialidase Activity in Vaginal Swabs from Women with Bacterial Vaginosis
Authors: Wiggins R. , Crowley T. , Horner P. J. , Soothill P. W. , Millar M. R. , Corfield A. P. 
Published: December 2019

ABSTRACT

The validity of measuring vaginal sialidase activity to identify bacterial vaginosis (BV) was determined by using 5-bromo-4-chloro-3-indolyl-α-
d
-
N
-acetylneuraminic acid in a near-patient test. The sensitivity and specificity of the test for prediction of BV were 95.6 and 96.3%, respectively. Positive and negative predictive values were 95.6 and 96.3%, respectively. This test may be an alternative to Gram staining.
A Lymph Node-Derived Cytopathic Simian Immunodeficiency Virus Mne Variant Replicates in Nonstimulated Peripheral Blood Mononuclear Cells
Authors: Kimata Jason T. , Mozaffarian Afsaneh , Overbaugh Julie 
Published: December 2019

ABSTRACTLymph nodes (LNs) are sites of active human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication and disease at both early and late stages of infection. Consequently, variant viruses that replicate efficiently and subsequently cause immune dysfunction may be harbored in this tissue. To determine whether LN-associated SIVs have an increased capacity to replicate and induce cytopathology, a molecular clone of SIV was isolated directly from DNA extracted from unpassaged LN tissue of a pig-tailed macaque (Macaca nemestrina) infected with SIVMne. The animal had declining CD4+T-lymphocyte counts at the time of the LN biopsy. In human CD4+T-cell lines, the LN-derived virus, SIVMne027, replicated with relatively slow kinetics and was minimally cytopathic and non-syncytium inducing compared to other SIVMne clones. However, in phytohemagglutinin-stimulated pig-tailed macaque peripheral blood mononuclear cells (PBMCs), SIVMne027 replicated efficiently and was highly cytopathic for the CD4+T-cell population. Interestingly, unlike other SIVMne clones, SIVMne027 also replicated to a high level in nonstimulated macaque PBMCs. High-level replication depended on the presence of both the T-cell and monocyte/macrophage populations and could be enhanced by interleukin-2 (IL-2). Finally, the primary determinant governing the ability of SIVMne027 to replicate in nonstimulated and IL-2-stimulated PBMCs mapped togag-pol-vif. Together, these data demonstrate that LNs may harbor non-syncytium-inducing, cytopathic viruses that replicate efficiently and are highly responsive to the effects of cytokines such as IL-2.
Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding
Authors: Dudas Kathleen C. , Ruyechan William T. 
Published: December 2019

ABSTRACT
We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation of 1 mol of FM per mol of ICP8 have been established. The binding properties of the modified protein were analyzed by polyacrylamide gel shift analysis with model oligonucleotides. This analysis indicates that while intrinsic binding is similar to that observed with unmodified protein, the cooperative binding of the modified protein to single-stranded DNA is significantly altered. Helix-destabilizing assays, whose results are a reflection of cooperative binding, also indicate that this property of ICP8 is decreased upon modification with FM. Mapping of the site of modification by cyanogen bromide cleavage and peptide sequencing has shown that the major site of modification is cysteine 254. This position in the primary structure of ICP8 is distinct from the regions previously shown to be involved in the interaction of this protein with single-stranded DNA.
Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2
Authors: Rutledge Elizabeth A. , Halbert Christine L. , Russell David W. 
Published: December 2019

ABSTRACT
Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.
Characterization of Replication-Competent Hepatitis A Virus Constructs Containing Insertions at the N Terminus of the Polyprotein
Authors: Zhang Yuan , Kaplan Gerardo G. 
Published: December 2019

ABSTRACT

To determine whether hepatitis A virus (HAV) could tolerate the insertion of exogenous sequences, we constructed full-length HAV cDNAs containing in-frame insertions at the N terminus of the polyprotein and transfected the derived T7 RNA polymerase in vitro transcripts into FRhK-4 cells. Replication of HAVvec1, a construct containing an insertion of 60 nucleotides coding for a polylinker, a 2B/2C cleavage site for HAV protease 3C
pro
, and two initiation codons that restored the sequence of the N terminus of the polyprotein, was detected 2 weeks after transfection by indirect immunofluorescence analysis using anti-HAV monoclonal antibodies. Western blot analysis of HAVvec1-infected cells using anti-VP2 and anti-VP4 antibodies failed to detect the expression of the inserted sequences. Insertion of a 24-mer oligonucleotide coding for a FLAG epitope into HAVvec1 resulted in its HAV-mediated expression which was retained upon deletion of a Gln residue from the inserted 2B/2C cleavage site. Western blot analysis using anti-FLAG and anti-VP2 antibodies showed that the FLAG epitope accumulated in infected cells fused to VP0. Replacement of the FLAG epitope with an epitope of the circumsporozoite protein (CSP) of
Plasmodium falciparum
resulted in its stable HAV-mediated expression for at least six serial passages in FRhK-4 cells. Sedimentation analysis in sucrose density gradients showed that the CSP epitope accumulated in infected cells fused to VP0, forming 80S empty capsids which also contained native VP0. Our data suggest that the HAV internal ribosome entry site can efficiently direct dual initiation of translation of the polyprotein from AUG codons separated by 66 to 78 nucleotides and show that HAV can tolerate insertions at the N terminus of the polyprotein.