bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of
Acanthamoeba
Authors: Khan Naveed A. , Jarroll Edward L. , Panjwani Noorjahan , Cao Zhiyi , Paget Timothy A. 
Published: December 2019

ABSTRACT

Acanthamoeba
keratitis is a vision-threatening infection caused by pathogenic species of the genus
Acanthamoeba
. Although not all
Acanthamoeba
spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic
Acanthamoeba
. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in
Acanthamoeba
-conditioned medium and differentiated pathogenic and nonpathogenic
Acanthamoeba
. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic
Acanthamoeba
isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with
Acanthamoeba
-conditioned medium as well as with
Acanthamoeba
- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of
Acanthamoeba
. In pathogenic
Acanthamoeba
isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic
Acanthamoeba
strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic
Acanthamoeba
strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.
Relative Abundance of Oligosaccharides in
Candida
Species as Determined by Fluorophore-Assisted Carbohydrate Electrophoresis
Authors: Goins Tresa L. , Cutler Jim E. 
Published: December 2019

ABSTRACT

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues in
Candida
mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall. The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples. FACE analysis revealed the major oligomannosides released by acid hydrolysis and β-elimination of Fehling-precipitated mannan from
Candida albicans
, which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis. FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells. Whole-cell acid hydrolysis and whole-cell β-elimination of two isolates each of
C. albicans
,
C. glabrata
,
C. krusei
,
C. lusitaniae
,
C. parapsilosis
,
C. rugosa
,
C. stellatoidea
, and
C. tropicalis
resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed. Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings. Differences in the mannoprotein composition of
C. albicans
A9 and four spontaneous cell surface mutants were also detected. Mannan “fingerprints,” or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles. FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.
Nosocomial Transmission of Echovirus 30: Molecular Evidence by Phylogenetic Analysis of the VP1 Encoding Sequence
Authors: Bailly Jean-Luc , Béguet Aline , Chambon Martine , Henquell Cécile , Peigue-Lafeuille Hélène 
Published: December 2019

ABSTRACTWe investigated six cases of enterovirus infection in a neonatal unit. The index patient, a 5-day-old boy, was admitted with aseptic meningitis due to echovirus 30 (E30). Secondary infections with E30 occurred in five babies. Comparison of the complete VP1 sequences showed that the isolates recovered from the index patient and his mother were closely related to those recovered from the five babies with secondary infections, demonstrating a nosocomial transmission of the virus. In the phylogenetic tree reconstructed from the VP1 sequences, the isolates formed a monophyletic cluster related to an E30 strain collected in June 1997 during an outbreak of aseptic meningitis.
Estimation of Minimum Sterol 14α-Demethylation-Inhibitory Concentration of Azoles in
Candida
Yeasts Using Acetate-Mediated Growth Inhibition: Potential Utility in Susceptibility Testing
Authors: Shimokawa Osamu , Nakayama Hiroaki 
Published: December 2019

ABSTRACT

We have recently shown that 14α-demethylation-deficient cells of
Candida albicans
are subject to growth arrest by 0.24 M acetate in a yeast extract-peptone-glucose medium and that the minimum concentration of an azole antifungal agent required for total inhibition of sterol 14α-demethylation (MDIC for minimum demethylation-inhibitory concentration) is practically identical to its MIC determined in the acetate-supplemented medium (O. Shimokawa and H. Nakayama, Antimicrob. Agents Chemother. 43:100–105, 1999). In the present study we estimated the MDICs of three different azoles (fluconazole, ketoconazole, and itraconazole) for strains of various
Candida
species using this method and compared them with the MICs determined in the corresponding acetate-free medium. The results demonstrated that the test strains were divided into two classes. One class of strains was characterized by tolerance to 14α-demethylation deficiency (MIC > MDIC) and consisted of strains of
C. albicans
,
C. guilliermondii
,
C. kefyr
, and
C. tropicalis
. The other class was intolerant to 14α-demethylation deficiency (MIC ≈ MDIC) and comprised strains of
C. glabrata
,
C. krusei
, and
C. parapsilosis
. We also showed that replacement of the yeast extract-peptone-glucose medium with RPMI 1640 medium did not affect the results substantially. Furthermore, the 80% inhibitory concentration (IC
80
) in RPMI 1640 medium, recommended as a substitute for the conventional MIC in susceptibility testing, was found to be close to the MDIC.
Development of a Quantitative Real-Time Detection Assay for Hepatitis B Virus DNA and Comparison with Two Commercial Assays
Authors: Pas Suzan D. , Fries Edwin , De Man Robert A. , Osterhaus Albert D. M. E. , Niesters Hubert G. M. 
Published: December 2019

ABSTRACT

A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV DNA standards. This real-time PCR detection system had a dynamic range of 373 to 10
10
genome copies per ml and showed an excellent correlation with both the commercial HBV Digene Hybrid Capture II microplate assay (Digene Diagnostics) and the HBV MONITOR assay (Roche Diagnostics). To demonstrate its clinical utility, four chronically HBV-infected patients treated with lamuvidine were monitored using the three different assays. From the results we concluded that this assay is an excellent alternative for monitoring of HBV-infected patients in routine diagnostics and clinical practice, enabling the analysis of a large dynamic range of HBV DNA in a single, undiluted sample.
Characterization of
Bartonella clarridgeiae
Flagellin (FlaA) and Detection of Antiflagellin Antibodies in Patients with Lymphadenopathy
Authors: Sander Anna , Zagrosek Anja , Bredt Wolfgang , Schiltz Emile , Piémont Yves , Lanz Christa , Dehio Christoph 
Published: December 2019

ABSTRACT

Cat scratch disease (CSD) is a frequent clinical outcome of
Bartonella henselae
infection in humans. Recently, two case reports indicated
Bartonella clarridgeiae
as an additional causative agent of CSD. Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir.
B. clarridgeiae
and
B. henselae
can be distinguished phenotypically by the presence or absence of flagella, respectively. Separation of the protein content of purified flagella of
B. clarridgeiae
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa. N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the
flaA
gene cloned from
B. clarridgeiae
. The flagellin of
B. clarridgeiae
is closely related to flagellins of
Bartonella bacilliformis
and several
Bartonella
-related bacteria. Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of
B. clarridgeiae
are found in patients with CSD or lymphadenopathy. Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls.
B. clarridgeiae
FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing. Our results further indicate that
B. clarridgeiae
might be a possible etiologic agent of CSD or lymphadenopathy. However, it remains to be clarified whether antibodies to the FlaA protein of
B. clarridgeiae
are a useful indicator of acute infection.
Urinary Hydatid Antigen Detection by Coagglutination, a Cost-Effective and Rapid Test for Diagnosis of Cystic Echinococcosis in a Rural or Field Setting
Authors: Ravinder P. T. , Parija S. C. , Rao K. S. V. K. Subba 
Published: December 2019

ABSTRACT
We describe here coagglutination (Co-A), a rapid slide agglutination test for the detection of hydatid antigen in the urine for the diagnosis of cystic echinococcosis (CE). Paired urine and serum samples were collected from 16 patients with surgically confirmed CE, 10 patients with ultrasound-proven CE, 14 patients with clinically diagnosed CE, 24 patients with various parasitic diseases other than CE, and 25 healthy control subjects. Co-A detected excreted hydatid antigen in the concentrated urine of 7 of 16 (43.75%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 8 of 14 (57.14%) clinically diagnosed cases of CE. A false-positive reaction was observed with 12.50% of control urine specimens from patients with parasitic diseases other than CE and 12% of urine samples from healthy controls. The circulating antigen was detected in the serum in 13 of 16 (81.25%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 13 of 14 (92.86%) clinically diagnosed cases of CE. False-positive reactions were observed with three sera (12.5%) from controls with other parasitic diseases. The low sensitivity of Co-A for detection of antigen in the urine of a patient whose serum was positive for the antigen is possibly due to low levels of antigen in the urine. Unlike the collection of blood for serum, which is an invasive procedure and also requires technical expertise and disposable syringes, urine can be collected easily and frequently without causing any inconvenience to the patient. Urine as a clinical specimen alternative to serum would be immensely useful in the diagnosis of CE, particularly in a rural or field setting. In such situations as well as in poorly equipped laboratories, Co-A has the potential to be used as a simple, rapid, and economical slide agglutination test for detection of urinary hydatid antigen in the diagnosis of CE.
Molecular Epidemiology of Metronidazole Resistance in a Population of
Trichomonas vaginalis
Clinical Isolates
Authors: Snipes Lauren J. , Gamard Pascale M. , Narcisi Elizabeth M. , Beard C. Ben , Lehmann Tovi , Secor W. Evan 
Published: December 2019

ABSTRACT

Trichomonas vaginalis
, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA
T. vaginalis
virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to
T. vaginalis
resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for
T. vaginalis
infections.
Differentiation of Pathogenic
Bartonella
Species by Infrequent Restriction Site PCR
Authors: Handley Scott A. , Regnery Russell L. 
Published: December 2019

ABSTRACT

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments.
Bartonella
isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease
Hha
I in conjunction with an infrequently cutting endonuclease,
Eag
I,
Sma
I, or
Xba
I. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an
Eag
I,
Sma
I, or
Xba
I site in combination with an
Hha
I site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3′ ends of the
Eag
I-,
Sma
I-, or
Xba
I-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations
Eag
I-
Hha
I and
Sma
I-
Hha
I. However, the combination of
Xba
I-
Hha
I produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic
Bartonella
species.
Evaluation of LightCycler PCR for Implementation of Laboratory Diagnosis of Herpes Simplex Virus Infections
Authors: Espy Mark J. , Ross Teri K. , Teo Rosaline , Svien Kathleen A. , Wold Arlo D. , Uhl James R. , Smith Thomas F. 
Published: December 2019

ABSTRACT

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (
n
= 197; 23.1%) than cell cultures (
n
= 150) for the routine laboratory detection of herpes simplex virus infections.
Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin
Authors: Schroth-Diez Britta , Ponimaskin Evgeni , Reverey Helmut , Schmidt Michael F. G. , Herrmann Andreas 
Published: December 2019

ABSTRACTThe role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.
Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect
Authors: Wang Xi , Hutt-Fletcher Lindsey M. 
Published: December 2019

ABSTRACTThe Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.
Administration of an Anti-CD8 Monoclonal Antibody Interferes with the Clearance of Chimeric Simian/Human Immunodeficiency Virus during Primary Infections of Rhesus Macaques
Authors: Matano Tetsuro , Shibata Riri , Siemon Christine , Connors Mark , Lane H. Clifford , Martin Malcolm A. 
Published: December 2019

ABSTRACTParenteral administration of a mouse anti-human CD8 monoclonal antibody (MAb) to rhesus macaques resulted in a transient depletion of CD8+cells in both the peripheral blood and lymphoid tissues. When administered during primary chimeric simian/human immunodeficiency virus infections, the CD8 MAb caused marked elevations of plasma and cell-associated virus levels in both the peripheral blood and lymphoid tissues and led to prolonged depletion of CD4 cells. Taken together, these results directly demonstrate that CD8+T lymphocytes are actively involved in controlling the acute phase of primate lentivirus infections.
An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro
Authors: Coulon Patrice , Ternaux Jean-Pierre , Flamand Anne , Tuffereau Christine 
Published: December 2019

ABSTRACT
An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.