bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Real-Time PCR for Detection of Members of the
Ehrlichia phagocytophila
Genogroup in Host Animals and
Ixodes ricinus
Ticks
Authors: Pusterla Nicola , Huder Jon B. , Leutenegger Christian M. , Braun Ueli , Madigan John E. , Lutz Hans 
Published: December 2019

ABSTRACT

A TaqMan PCR was established for identification and quantitation of members of the
Ehrlichia phagocytophila
group in experimentally infected cows and in
Ixodes ricinus
ticks. The TaqMan PCR identified a 106-bp section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for members of the
E. phagocytophila
group, which include
E. phagocytophila
,
Ehrlichia equi
, and the agent of human granulocytic ehrlichiosis. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of
E. phagocytophila
. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The numbers of ehrlichiae in leukocytes of the two cows experimentally infected with
E. phagocytophila
were measured daily by TaqMan PCR and had a course similar to that of the percentages of infected leukocytes determined daily by light microscopy. The prevalence of infected free-living ticks, which were collected from areas where bovine ehrlichiosis is endemic and from regions with sporadic occurrences of granulocytic ehrlichiosis in dogs and horses, was identical as determined by nested PCR and TaqMan PCR.
Evidence of the Human Granulocytic Ehrlichiosis Agent in
Ixodes ricinus
Ticks in Switzerland
Authors: Pusterla Nicola , Leutenegger Christian M. , Huder Jon B. , Weber Rainer , Braun Ueli , Lutz Hans 
Published: December 2019

ABSTRACT

A total of 1,667
Ixodes ricinus
ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the
Ehrlichia phagocytophila
group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of
Ehrlichia
. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in
I. ricinus
in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.
Laboratory Diagnosis of Common Viral Infections of the Central Nervous System by Using a Single Multiplex PCR Screening Assay
Authors: Read Steven J. , Kurtz John B. 
Published: December 2019

ABSTRACT
A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF.
Discrimination of Subtype B and Non-Subtype B Strains of Human Immunodeficiency Virus Type 1 by Serotyping: Correlation with Genotyping
Authors: Murphy Gary , Belda Francisco J. , Pau Chou-Pong , Clewley Jonathan P. , Parry John V. 
Published: December 2019

ABSTRACT

The ability of a peptide-based serotyping assay to differentiate human immunodeficiency virus (HIV) type 1 (HIV-1) subtype B infections from non-subtype B infections was investigated with 166 anti-HIV-1- and HIV RNA-positive (by PCR) serum or plasma specimens. The specimens were divided genetically into those infected with subtype B and non-subtype B by application of a screening heteroduplex mobility assay (HMA) that used plasmids for subtypes A and B alone. Specimens that were not clearly infected with HIV-1 subtype B by HMA or for which the two methods had discordant results in distinguishing those infected with subtype B from those infected with non-subtype B were then investigated with a full HMA plasmid panel and, for selected specimens,
env
sequencing. For the 141 genotyped and serotypically reactive specimens, the correlation between genotyping and serotyping (all subtypes) was 69%. Of the 67 specimens that reacted monotypically as serotype B, 64 were shown to be infected with genotype B (positive predictive value, 96%). Of the 82 specimens that contained genotype B nucleic acid, 64 reacted monotypically as serotype B (sensitivity, 78%), and 4 specimens reacted with a single non-subtype B peptide; the viruses in 14 specimens could not be assigned a serotype. Initial screening results had indicated that 12 samples had results discordant between restricted HMA and serotyping. The V3 loop amino acids of the infecting HIV strains from the seven specimens with discordant serology results were analyzed. For five specimens discordance occurred when the amino acid sequence of the infecting virus closely resembled those of more than one consensus peptide antigen or when the observed V3 crown motif of the strain was atypical for the genetic subtype present. For the other two specimens no explanation for the discordance was identified. Five specimens gave unclear or discordant results in the initial HMA screen, but the results were resolved when the full plasmid panel was used. Serotyping, although of limited sensitivity, distinguishes between subtype B and non-subtype B infections with a high degree of specificity. However, it poorly differentiates the major non-subtype B subtypes, particularly subtypes A and C. When HIV-1 subtype B predominates, serological typing and/or subtype-restricted HMA screening usefully distinguishes between subtype B and non-subtype B infections.
Relatedness Analyses of
Histoplasma capsulatum
Isolates from Mexican Patients with AIDS-Associated Histoplasmosis by Using Histoplasmin Electrophoretic Profiles and Randomly Amplified Polymorphic DNA Patterns
Authors: Reyes-Montes M. R. , Bobadilla-Del Valle M. , Martínez-Rivera M. A. , Rodríguez-Arellanes G. , Maravilla E. , Sifuentes-Osornio J. , Taylor M. L. 
Published: December 2019

ABSTRACT

The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus
Histoplasma capsulatum
isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as
H. capsulatum
isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis,
H. capsulatum
isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most
H. capsulatum
isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated
H. capsulatum
isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing
H. capsulatum
from different sources and geographic origins.
Distribution of
Porphyromonas gingivalis
Strains with
fimA
Genotypes in Periodontitis Patients
Authors: Amano Atsuo , Nakagawa Ichiro , Kataoka Kosuke , Morisaki Ichijiro , Hamada Shigeyuki 
Published: December 2019

ABSTRACT

Fimbriae (FimA) of
Porphyromonas gingivalis
are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues. We previously demonstrated that
fimA
can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the
fimA
gene. In the present study, we attempted to detect the four different
fimA
genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method. Four sets of
fimA
type-specific primers were designed for the PCR assay. These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the
fimA
gene. Positive PCR results were observed with reference strains of
P. gingivalis
in a type-specific manner. All other laboratory strains of oral and nonoral bacteria gave negative results. The sensitivity of the PCR assay for
fimA
type-specific detection was between 5 and 50 cells of
P. gingivalis
. Clinical samples were obtained from saliva and subgingival plaque from deep pockets (≥4 mm) of 93 patients with periodontitis. Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR. The presence of
P. gingivalis
was demonstrated in 73 patients (78.5%), and a single
fimA
gene was detected in most patients. The distribution of the four
fimA
types among the
P. gingivalis
-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6.8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8%.
P. gingivalis
with type II
fimA
was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (≥8 mm) pockets. These results suggest that
P. gingivalis
strains that possess type II
fimA
are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.
Latent
Pneumocystis carinii
Infection in Commercial Rat Colonies: Comparison of Inductive Immunosuppressants plus Histopathology, PCR, and Serology as Detection Methods
Authors: Weisbroth Steven H. , Geistfeld James , Weisbroth Stephanie P. , Williams Bruce , Feldman Sanford H. , Linke Michael J. , Orr Sally , Cushion Melanie T. 
Published: December 2019

ABSTRACT

Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of
Pneumocystis carinii
in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of
P. carinii
in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce
P. carinii
infection in rats from an enzootically infected commercial barrier colony. The presence of
P. carinii
was detected by histopathologic methods and by amplification of a targeted region of the
P. carinii
thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of
P. carinii
-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to
P. carinii
did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of
P. carinii
infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-
P. carinii
antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a
P. carinii
carrier state in rodent breeding colonies.
Molecular Cloning and Characterization of the
Ehrlichia chaffeensis
Variable-Length PCR Target: an Antigen-Expressing Gene That Exhibits Interstrain Variation
Authors: Sumner John W. , Childs James E. , Paddock Christopher D. 
Published: December 2019

ABSTRACT

A clone expressing an immunoreactive protein with an apparent molecular mass of 44 kDa was selected from an
Ehrlichia chaffeensis
Arkansas genomic library by probing with anti-
E. chaffeensis
hyperimmune mouse ascitic fluid. Nucleotide sequencing revealed an open reading frame (ORF) capable of encoding a 198-amino-acid polypeptide. The ORF contained four imperfect, direct, tandem 90-bp repeats. The nucleotide and deduced amino acid sequences did not show close homologies to entries in the molecular databases. PCR with primers whose sequences matched the sequences flanking the ORF was performed with DNA samples extracted from cell cultures infected with nine different isolates of
E. chaffeensis
, blood samples from seven patients with monocytic ehrlichiosis, and
Amblyomma americanum
ticks collected in four different states. The resulting amplicons varied in length, containing three to six repeat units. This gene, designated the variable-length PCR target, is useful for PCR detection of
E. chaffeensis
and differentiation of isolates.
Invasive Aspergillosis Caused by
Aspergillus ustus
: Case Report and Review
Authors: Verweij Paul E. , van den Bergh Marjolein F. Q. , Rath Peter M. , de Pauw Ben E. , Voss Andreas , Meis Jacques F. G. M. 
Published: December 2019

ABSTRACT

A case of invasive pulmonary aspergillosis in an allogeneic bone marrow transplant recipient caused by
Aspergillus ustus
is presented.
A. ustus
was also recovered from the hospital environment, which may indicate that the infection was nosocomially acquired. A literature review revealed seven cases of invasive infections caused by
A. ustus
, and three of these were primarily cutaneous infections. In vitro susceptibility testing of 12
A. ustus
isolates showed that amphotericin B and terbinafine had fungicidal activity and that itraconazole and voriconazole had fungistatic activity.
Septicemia Caused by Dysgonic Fermenter 3 in a Severely Immunocompromised Patient and Isolation of the Same Microorganism from a Stool Specimen
Authors: Grob René , Zbinden Reinhard , Ruef Christian , Hackenthal Matthias , Diesterweg Ingrid , Altwegg Martin , von Graevenitz Alexander 
Published: December 2019

ABSTRACT
Dysgonic fermenter 3 (DF-3)-associated bacteremia occurred in a febrile patient with acute myelocytic leukemia during aplasia. Another DF-3 isolate, identical by ribotyping, was grown 10 weeks later from stool collected in the absence of diarrhea. This is the first case in which DF-3 was isolated from blood and stool specimens from the same patient.
Survey of Antibiotic Resistance among Enterococci in North Rhine-Westphalia, Germany
Authors: Reinert R. R. , Conrads G. , Schlaeger J. J. , Werner G. , Witte W. , Lütticken R. , Klare I. 
Published: December 2019

ABSTRACT

A surveillance study on antibiotic resistance of enterococcal isolates (
n
= 730) was carried out in North Rhine-Westphalia, Germany, in 1997. Resistance rates to ampicillin (7.4%), high-level gentamicin (15.0%), high-level streptomycin (27.9%), ciprofloxacin (37.9%), vancomycin (1.5%), and teicoplanin (1.5%) were determined. All vancomycin-resistant enterococci (VRE) carried the
vanA
gene.
Sma
I and
Apa
I macrorestriction patterns indicated an intra- and interhospital spread of VRE.
Controlled Clinical Comparison of bioMérieux VITAL and BACTEC NR-660 Blood Culture Systems for Detection of Bacteremia and Fungemia in Adults
Authors: Wilson Michael L. , Mirrett Stanley , McDonald L. Clifford , Weinstein Melvin P. , Fune Jose , Reller L. Barth 
Published: December 2019

ABSTRACT

A total of 9,446 blood cultures were collected from adult patients at three university-affiliated hospitals. Of these, 8,943 cultures were received with both aerobic bottles filled adequately; 885 yielded 1,016 microorganisms, including 622 isolates (61%) that were the cause of sepsis, 337 isolates (33%) that were contaminants, and 57 isolates (6%) that were indeterminate as the cause of sepsis. With the exception of
Staphylococcus aureus
, which was recovered more often from VITAL aerobic bottles, more pathogenic microorganisms were recovered from BACTEC NR6 (aerobic) bottles than from VITAL aerobic bottles. Growth of pathogenic microorganisms was detected earlier in VITAL aerobic bottles. A total of 8,647 blood cultures were received with both anaerobic bottles filled adequately; 655 yielded 740 microorganisms, including 486 isolates (66%) that were the cause of sepsis, 215 isolates (29%) that were contaminants, and 39 isolates (6%) that were indeterminate as the cause of sepsis. More pathogenic microorganisms were recovered from VITAL anaerobic bottles than from BACTEC NR7 (anaerobic) bottles. Growth of pathogenic microorganisms was detected earlier in VITAL anaerobic bottles. In 8,500 sets all four bottles were received adequately filled. When paired aerobic and anaerobic bottle sets (systems) were compared, more pathogenic microorganisms (again with the exception of
S. aureus
) were recovered from the BACTEC system. For the 304 septic episodes (253 unimicrobial and 51 polymicrobial), significantly more were detected by the BACTEC system. We conclude that VITAL requires modification to improve recovery of pathogenic microorganisms to make it competitive with other commercially available blood culture systems.
Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (
rpoB
)
Authors: Kim Bum-Joon , Lee Seung-Hyun , Lyu Mi-Ae , Kim Seo-Jeong , Bai Gill-Han , Kim Sang-Jae , Chae Gue-Tae , Kim Eui-Chong , Cha Chang-Yong , Kook Yoon-Hoh 
Published: December 2019

ABSTRACT

For the differentiation and identification of mycobacterial species, the
rpoB
gene, encoding the β subunit of RNA polymerase, was investigated.
rpoB
DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of
rpoB
DNAs provided the basis for species differentiation within the genus
Mycobacterium
. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic
Mycobacterium kansasii
was easily differentiated from nonpathogenic
M. gastri
; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified
rpoB
DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of
M. tuberculosis
, rifampin resistance can be simultaneously determined.
CDC Group IV c-2: a New
Ralstonia
Species Close to
Ralstonia eutropha
Authors: Moissenet Didier , Goujon Christophe P. , Garbarg-Chenon Antoine , Vu-Thien Hoang 
Published: December 2019

ABSTRACT

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus
Ralstonia
, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus
Ralstonia
. The closest matches were obtained with
Ralstonia eutropha
. However, four differences in 32 biochemical tests separated
R. eutropha
from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus
Ralstonia
.
Rapid Identification of Thermotolerant
Campylobacter jejuni
,
Campylobacter coli
,
Campylobacter lari
, and
Campylobacter upsaliensis
from Various Geographic Locations by a GTPase-Based PCR-Reverse Hybridization Assay
Authors: van Doorn Leen-Jan , Verschuuren-van Haperen Anita , Burnens André , Huysmans Michael , Vandamme Peter , Giesendorf Belinda A. J. , Blaser Martin J. , Quint Wim G. V. 
Published: December 2019

ABSTRACT

Recently, a gene from
Campylobacter jejuni
encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from
C. jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant
Campylobacter
species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant
Campylobacter
species.