bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences
Authors: Roth Andreas , Fischer Marga , Hamid Mohamed E. , Michalke Sabine , Ludwig Wolfgang , Mauch Harald 
Published: December 2019

ABSTRACT

Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in
Mycobacterium kansasii
and
M. gastri
, or highly similar, for example, in
M. malmoense
and
M. szulgai
. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus
Mycobacterium
representing 13 species (
M. avium
,
M. conspicuum
,
M. gastri
,
M. genavense
,
M. kansasii
,
M. malmoense
,
M. marinum
,
M. shimoidei
,
M. simiae
,
M. szulgai
,
M. triplex
,
M. ulcerans
, and
M. xenopi
). An alignment of these sequences together with additional sequences available in the EMBL database (for
M. intracellulare
,
M. phlei
,
M. smegmatis
, and
M. tuberculosis
) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for
M. simiae
,
M. genavense
, and
M. triplex
, and distinct branches for
M. xenopi
and
M. shimoidei
. Comparisons of
M. gastri
with
M. kansasii
and
M. malmoense
with
M. szulgai
revealed ITS sequence similarities of 93 and 88%, respectively.
M. marinum
and
M. ulcerans
possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.
Use of Ribotyping and Hemolysin Activity To Identify Highly Virulent
Streptococcus suis
Type 2 Isolates
Authors: Staats Jacque J. , Plattner Brandon L. , Nietfeld Jerome , Dritz Steve , Chengappa M. M. 
Published: December 2019

ABSTRACT

Nineteen
Streptococcus suis
type 2 isolates were evaluated for their virulence in pigs and mice. Of these, seven were determined to be highly virulent in pigs on the basis of clinical sign scores and gross pathology and histopathology results. Clinical sign scores correlated with gross pathology and histopathology scores at
P
equal to 0.004 and
P
equal to 0.009, respectively. The virulence of highly virulent isolates in pigs compared somewhat with virulence in mice, but the correlation was not significant. No correlation of virulence was noted among the moderately virulent and avirulent isolates in pigs and mice. Chromosomal DNAs from all
S. suis
isolates were evaluated by
Pst
I,
Pvu
II,
Eco
RI, and
Hae
III restriction enzyme digestion followed by hybridization with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from 16S and 23S rRNAs from
Escherichia coli
. The hybridization patterns (ribotypes) varied depending upon the enzyme used, but a significant number of isolates determined to be highly virulent in pigs had unique hybridization patterns compared with those of the moderately virulent and avirulent isolates (
P
= 0.002). In addition, hemolysin activity showed a high correlation to virulence (
P
= 0.00008) and ribotype (
P
= 0.002).
Comparative Analysis of Infrequent-Restriction-Site PCR and Pulsed-Field Gel Electrophoresis for Epidemiological Typing of
Legionella pneumophila
Serogroup 1 Strains
Authors: Riffard Serge , Presti François Lo , Vandenesch François , Forey Françoise , Reyrolle Monique , Etienne Jerome 
Published: December 2019

ABSTRACT

Two methods were compared for the analysis of 48 unrelated and epidemiologically related
Legionella pneumophila
serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for
Xba
I and
Pst
I restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with
Sfi
I. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of
L. pneumophila
from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, ≥0.996) tool for typing of
Legionella
. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.
Transmission of Ovine Herpesvirus 2 in Lambs
Authors: Li Hong , Snowder Garry , O’Toole Donal , Crawford Timothy B. 
Published: December 2019

ABSTRACT

The pattern of acquisition of ovine herpesvirus 2 (OHV-2) infection in lambs was examined by a competitive-inhibition enzyme-linked immunosorbent assay and PCR. Newborn lambs (
n
= 118) did not exhibit antibody at birth. Viral DNA in peripheral blood leukocytes was detected in only 3% (
n
= 77) of newborn lambs before suckling. After nursing, viral DNA was sporadically present in about 10 to 20% of lambs until about 3 months of age. Thereafter, strong DNA signals began to appear in increasing numbers of lambs, reaching 100% by 5.5 months of age. Viral DNA in nasal secretions began to be detectable in about 30% of lambs at 5.5 months of age, achieved significant levels in 88% of lambs by 7.5 months of age, and then declined. The kinetics of the humoral immune response in lambs paralleled those of viral DNA in nasal secretions but did not parallel its presence in blood leukocytes. In the experiment to define the time of infection of OHV-2 in lambs, all five lambs separated from the flock at 2.5 months of age remained uninfected until the termination of the experiment at 1 year of age. In contrast, lambs weaned at 2.5 months of age and returned to the flock had become infected at 3.5 months of age. Weaning and separation from the flock at 3.5 months of age did not prevent infection. The study showed that OHV-2 infection does not commonly occur in perinatal lambs and that OHV-2-free sheep can be established by separation of lambs at the proper time, which has important implications for potential control measures.
Detection and Confirmation of
Mycoplasma pneumoniae
in Urogenital Specimens by PCR
Authors: Sharma S. , Brousseau R. , Kasatiya S. 
Published: December 2019

ABSTRACT

Following the isolation of
Mycoplasma pneumoniae
from urogenital specimens (M. Goulet, R. Dular, J. G. Tully, G. Billows, and S. Kasatiya, J. Clin. Microbiol. 33:2823–2825, 1995), a study was undertaken to confirm the observations by PCR. Specific primers directed to the P1 adhesin gene of
M. pneumoniae
were used. A total of 300 genital specimens were tested for
M. pneumoniae
and
Mycoplasma genitalium
by culture and PCR. Of these, 15 were positive by culture and 17 were positive by PCR for
M. pneumoniae
. No
M. genitalium
was detected in any of the specimens by either method. The present study demonstrates that PCR is sensitive and rapid compared to cumbersome culture methods and can be used to detect
M. pneumoniae
in urogenital specimens in a routine diagnostic laboratory.
Improving the Yield of Blood Cultures for Patients with Early Lyme Disease
Authors: Wormser Gary P. , Nowakowski John , Nadelman Robert B. , Bittker Susan , Cooper Denise , Pavia Charles 
Published: December 2019

ABSTRACT

This study was designed to improve the recovery of
Borrelia burgdorferi
from blood. With the techniques used,
B. burgdorferi
could be recovered from the blood of approximately 25% of patients with early Lyme disease associated with erythema migrans. Serum was a better source of culture material than whole blood. The volume of blood cultured correlated directly with yield, particularly for patients with a single erythema migrans lesion.
Results from 5 Years of Nationwide DNA Fingerprinting of
Mycobacterium tuberculosis
Complex Isolates in a Country with a Low Incidence of
M. tuberculosis
Infection
Authors: Bauer Jeanett , Yang Zhenhua , Poulsen Sigrid , Andersen Åse B. 
Published: December 2019

ABSTRACT

Results from DNA fingerprint analyses of
Mycobacterium tuberculosis
complex isolates from tuberculosis (TB) patients diagnosed during 5 years in Denmark are presented. The lack of success in eradicating TB in this low-incidence country may be explained by an unrecognized high frequency of active TB transmission (57%) among native Danes. Only two strains of
M. tuberculosis
are responsible for 40% of all clustered cases of TB among Danes.
Multicomponent Chimeric Antigen for Serodiagnosis of Canine Visceral Leishmaniasis
Authors: Soto Manuel , Requena Jose M. , Quijada Luis , Alonso Carlos 
Published: December 2019

ABSTRACT

In this work, we describe the assembly of a synthetic gene coding for several antigenic determinants found in different
Leishmania infantum
antigens. Selected epitopes were derived from the ribosomal proteins LiP2a, LiP2b, and LiP0 and from the histone H2A. The resulting gene was overexpressed in
Escherichia coli
either as a fusion protein (with the vector pMAL-c2) or alone (with the vector pQE). In both cases, high-level bacterial production of the recombinant protein was achieved and the products were found to be stable. Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments confirmed that the corresponding epitopes are present in the engineered protein. Finally, a serological evaluation of this multiple-epitope protein by Falcon assay screening test-ELISA revealed a sensitivity of 79 to 93% and a specificity of 96 to 100% in diagnosis of canine visceral leishmaniasis, indicating that this protein represents a valuable tool for serodiagnosis.
Ultrasensitive Reverse Transcription-PCR Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Sun Rita , Ku Joanne , Jayakar Harsha , Kuo Jo-Chi , Brambilla Donald , Herman Steven , Rosenstraus Maurice , Spadoro Joanne 
Published: December 2019

ABSTRACT
With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This “ultrasensitive” method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.
Genetic Analysis of
Histoplasma capsulatum
Strains Isolated from Clinical Specimens in Thailand by a PCR-Based Random Amplified Polymorphic DNA Method
Authors: Poonwan Natteewan , Imai Tamae , Mekha Nanthawan , Yazawa Katsukiyo , Mikami Yuzuru , Ando Akikazu , Nagata Yoshiho 
Published: December 2019

ABSTRACT

Thirteen strains of
Histoplasma capsulatum
were isolated from clinical specimens, including those from AIDS patients, in Thailand. Random amplified polymorphic DNA (RAPD) analysis with three different PCR primers showed that the DNA fingerprint patterns of the Thai isolates were very similar to each other and homogeneous, with only one exceptional strain, although the patterns were clearly different from those of a reference North American strain with all primers tested. Although the difference in the DNA fingerprinting patterns was minor, Thai isolates could be classified into two to four groups. A common PCR band (about 700 bp) in the patterns of all
H. capsulatum
strains was extracted, and its DNA sequence was determined. A new PCR primer set for the identification of
H. capsulatum
species was developed based on this sequence information. This primer set was 100% successful in the identification of the reference strain as well as all Thai isolates. The results of specificity tests of the primer set for the identification of the fungus are also discussed.
Molecular Epidemiology of an Outbreak of
Enterobacter cloacae
in the Neonatal Intensive Care Unit of a Provincial Hospital in Gauteng, South Africa
Authors: van Nierop W. H. , Duse A. G. , Stewart R. G. , Bilgeri Y. R. , Koornhof H. J. 
Published: December 2019

ABSTRACT

An outbreak of
Enterobacter cloacae
in the neonatal intensive care unit of a provincial hospital in Gauteng, South Africa, resulting in nine deaths was investigated. Macrorestriction analysis using pulsed-field gel electrophoresis revealed that three isolates of
E. cloacae
from blood cultures of patients, six from environmental sources, and one from the hands of a staff member belonged to the same genotypic cluster.
Description of
Gemella sanguinis
sp. nov., Isolated from Human Clinical Specimens
Authors: Collins Matthew D. , Hutson Roger A. , Falsen Enevold , Sjöden Berit , Facklam Richard R. 
Published: December 2019

ABSTRACT

Six strains of a hitherto undescribed gram-positive, catalase-negative, facultatively anaerobic coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains were genealogically identical and constitute a new subline within the genus
Gemella
. The unknown bacterium was readily distinguished from
Gemella haemolysans
,
Gemella bergeriae
, and
Gemella morbillorum
by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as
Gemella sanguinis
sp. nov. The type strain is CCUG 37820
T
.
Diagnosis of
Mycoplasma pneumoniae
Pneumonia in Children
Authors: Waris Matti E. , Toikka Pia , Saarinen Taina , Nikkari Simo , Meurman Olli , Vainionpää Raija , Mertsola Jussi , Ruuskanen Olli 
Published: December 2019

ABSTRACT

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of
Mycoplasma pneumoniae
infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for
M. pneumoniae
-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of
M. pneumoniae
DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of
M. pneumoniae
pneumonia in children of any age.
Prevalence of Human Calicivirus Infections in Kenya as Determined by Enzyme Immunoassays for Three Genogroups of the Virus
Authors: Nakata Shuji , Honma Shinjiro , Numata Kazuko , Kogawa Keiko , Ukae Susumu , Adachi Noriaki , Jiang Xi , Estes Mary K. , Gatheru Zippora , Tukei Peter M. , Chiba Shunzo 
Published: December 2019

ABSTRACT
An epidemiological survey on human calicivirus (HuCV) infections and associated gastroenteritis in infants was conducted to clarify the prevalence of HuCV infections in infants and adults in Kenya. Enzyme immunoassays (EIAs) for three genogroups of HuCVs, Norwalk virus (NV), Mexico virus (MXV), and Sapporo virus (SV), were used to detect antigen or antibody. We tested 1,431 stool samples obtained from children younger than 6 years old with acute gastroenteritis who visited outpatient clinics in three districts in Kenya from August 1991 to July 1994. Thirty-two (2.2%) of these stool samples were positive for SV antigen. Only one (0.1%) of 1,186 samples was positive for NV antigen and none of 246 samples was positive for MXV antigen. One hundred ninety-three serum samples were tested for antibodies to NV and MXV, and 64 of them were examined for antibody to SV. The pattern of the age-related prevalence of serum antibody to NV was different from that of antibodies to MXV and SV. The acquisition of serum antibodies to HuCVs in the three genogroups appeared in early childhood, at about 1 to 2 years of age. The prevalence of serum antibody to NV was low (about 60%) throughout adulthood compared with a high prevalence of antibody (∼80 to 90%) to MXV and SV. These data indicate that infections with viruses in the three genogroups of HuCVs are common in Kenya, and immunological responses to NV may be different from those to MXV and SV. The EIAs for the detection of NV and MXV antigens appear to be quite specific for prototype NV and MXV strains, respectively, so that they can detect only a few strains of HuCVs related to them. Alternatively, NV and MXV caused less severe infections that did not bring children to the outpatient clinics for gastroenteritis in Kenya.
Nucleic Acid Sequence-Based Amplification, a New Method for Analysis of Spliced and Unspliced Epstein-Barr Virus Latent Transcripts, and Its Comparison with Reverse Transcriptase PCR
Authors: Brink Antoinette A. T. P. , Vervoort Marcel B. H. J. , Middeldorp Jaap M. , Meijer Chris J. L. M. , van den Brule Adriaan J. C. 
Published: December 2019

ABSTRACT

Nucleic acid sequence-based amplification (NASBA) assays were developed for direct detection of Epstein-Barr virus (EBV) transcripts encoding EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and
Bam
HIA rightward frame 1 (BARF1) and for the noncoding EBV early RNA 1 (EBER1). The sensitivities of all NASBAs were at least 100 copies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA assays. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clinical material. BARF1 expression was found in 6 of 7 nasopharyngeal carcinomas (NPC) but in 0 of 22 Hodgkin’s disease (HD) cases, whereas LMP2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For detection of EBNA1 transcripts in HLs (
n
= 12) and T- and B-cell non-Hodgkin’s lymphomas (
n
= 3 and
n
= 2, respectively), NASBA was compared with reverse transcriptase (RT) PCR. Two samples were positive only with NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We conclude that NASBA is suitable for sensitive and specific detection of the above-mentioned EBV transcripts, regardless of their splicing patterns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs developed in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.
Diagnostic Potential of Toscana Virus N Protein Expressed in
Escherichia coli
Authors: Valassina Marcello , Soldateschi Dario , Dal Maso Grazia Maria , Santini Laura , Bianchi Silvia , Valensin Pier Egisto , Cusi Maria Grazia 
Published: December 2019

ABSTRACT

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in
Escherichia coli
by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (
r
= 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.
Detection and Characterization of Novel Rotavirus Strains in the United States
Authors: Ramachandran M. , Gentsch J. R. , Parashar U. D. , Jin S. , Woods P. A. , Holmes J. L. , Kirkwood C. D. , Bishop R. F. , Greenberg H. B. , Urasawa S. , Gerna G. , Coulson B. S. , Taniguchi K. , Bresee J. S. , Glass R. I. ,   the National Rotavirus Strain Surveillance System Collaborating Laboratories
Published: December 2019

ABSTRACT
We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0.3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.