bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Species-Specific PCR-Restriction Fragment Length Polymorphism Analysis Procedure for Identification of
Madurella mycetomatis
Authors: Ahmed Abdalla O. A. , Mukhtar Moawia M. , Kools-Sijmons Marly , Fahal Ahmed H. , de Hoog Sybren , van den Ende Bert Gerrits , Zijlstra Eduard E. , Verbrugh Henri , Abugroun El Sir A. M. , Elhassan Ahmed M. , van Belkum Alex 
Published: December 2019

ABSTRACT

Madurella mycetomatis
is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of
M. mycetomatis
and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of
M. mycetomatis
reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of
M. mycetomatis
, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with
Cfo
I,
Hae
III,
Msp
I,
Sau
3AI,
Rsa
I, and
Spe
I restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.
Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis
Authors: Lucero Nidia E. , Foglia Luis , Ayala Sandra M. , Gall David , Nielsen Klaus 
Published: December 2019

ABSTRACT

The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to
Brucella
is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the conventional tests. Further, the CELISA is simpler to perform that the CFT and may readily be standardized by the use of purified S-LPS antigen and monoclonal antibody for competition.
Australian Isolates of
Legionella longbeachae
Are Not a Clonal Population
Authors: Montanaro-Punzengruber J. C. , Hicks L. , Meyer W. , Gilbert G. L. 
Published: December 2019

ABSTRACT

Legionella longbeachae
is almost as frequent a cause of legionellosis in Australia as
Legionella pneumophila
, but epidemiological investigation of possible environmental sources and clinical cases has been limited by the lack of a discriminatory subtyping method. The purpose of this study was to examine the genetic variability among Australian isolates of
L. longbeachae
serogroup 1. Pulsed-field gel electrophoresis (PFGE) of
Sfi
I fragments revealed three distinct pulsotypes among 57 clinical and 11 environmental isolates and the ATCC control strains of
L. longbeachae
serogroups 1 and 2. Each pulsotype differed by four bands, corresponding to <65% similarity. A clonal subgroup within each pulsotype was characterized by >88% similarity. The largest major cluster was pulsotype A, which included 43 clinical isolates and 9 environmental isolates and was divided into five subgroups. Pulsotypes B and C comprised smaller numbers of clinical and environmental isolates, which could each be further divided into three subgroups. The ATCC type strain of
L. longbeachae
serogroup 1 was classified as pulsotype B, subtype B3, while the ATCC type strain of
L. longbeachae
serogroup 2 was identified as a different pulsotype, LL2.
Sfi
I macrorestriction analysis followed by PFGE showed that the Australian
L. longbeachae
strains are not a single clonal population as previously reported.
Evidence from Molecular Fingerprinting of Limited Spread of Drug-Resistant Tuberculosis in Texas
Authors: Wilson Rebecca W. , Yang Zhenhua , Kelley Michael , Cave M. Donald , Pogoda Janice M. , Wallace Richard J. , Cegielski J. Peter , Dunbar Denise F. , Bergmire-Sweat David , Elliott L. Bruce , Barnes Peter F. 
Published: December 2019

ABSTRACT

To determine the contribution of recent transmission to spread of drug-resistant tuberculosis in Texas, we performed IS
6110
-based and pTBN12-based restriction fragment length polymorphism (RFLP) analyses on
Mycobacterium tuberculosis
isolates. Isolates collected from 201 patients in Texas between 1992 and 1994 were studied. The distribution of cases was strikingly focal. All cases were reported from 35 of the 254 counties in Texas, and 74% (148 of 201) were reported from only 9 counties. One hundred sixty-one (80%) of the patients had
M. tuberculosis
isolates with unique RFLP patterns, and 41 (20%) patients were in 20 clusters, each comprising 2 to 3 patients. The largest number of cases of drug-resistant tuberculosis were reported in counties bordering Mexico, but the percentage of clustered cases was highest in northeast Texas and in counties that included the cities of Dallas, Fort Worth, and Houston. Compared to nonclustered patients, clustered patients were more likely to be African American and to have been born in the United States. Clustered patients were significantly more likely to be from the same geographic area, and clustered patients from the same geographic area were more likely to have isolates with identical drug susceptibility patterns, suggesting that they were linked by recent transmission. In 11 of 20 clusters, clustered patients were from geographically separate regions, and most isolates did not have identical drug susceptibility patterns, suggesting that tuberculosis was contracted from a common source in the remote past. Based on the low percentage of clustered cases and the small cluster size, we conclude that there is no evidence for the extensive transmission of drug-resistant tuberculosis in Texas.
Epidemiology of Hepatitis B, C, E, and G Virus Infections and Molecular Analysis of Hepatitis G Virus Isolates in Bolivia
Authors: Konomi Nami , Miyoshi Chiaki , La Fuente Zerain Carlos , Li Tian-Cheng , Arakawa Yasuyuki , Abe Kenji 
Published: December 2019

ABSTRACT
Prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis G virus (HGV), and hepatitis E virus (HEV) was investigated among 574 healthy blood donors in Bolivia. HCV RNA and HGV RNA in the serum were identified by a nested reverse transcription-PCR using primers derived from the 5′ untranslated region (5′ UTR). We also tested for hepatitis B surface antigen (HBsAg) and for the antibody to HEV. The results revealed that HGV RNA was present in 84 of 574 (14.6%) tested blood donors, whereas HBsAg was detected in only 2 (0.3%) donors, and no individuals positive for HCV RNA were found. Anti-HEV immunoglobulin G (IgG) was detected in 93 (16.2%) individuals and anti-HEV IgM was found in 10 (1.7%) individuals among the same population. Phylogenetic analysis of 44 HGV isolates in the 5′ UTR showed that 27 (61%) isolates were genotype 3 (Asian type) and the remaining 17 (39%) isolates were genotype 2 (United States and European type). Moreover, we obtained a full-length nucleotide sequence of the HGV genome (designated HGV-BL230) recovered from a Bolivian blood donor. The BL230 was composed of 9,227 nucleotides and had a single open reading frame, encoding 2,842 amino acid residues. Interestingly, the BL230 belonged to genotype 2 of HGV at the level of a full-length sequence, although this was classified as genotype 3 by a phylogenetic analysis based on the 5′ UTR sequence. The BL230 differed from previously reported HGV/hepatitis GB virus type C isolates by 12 to 13% of the nucleotide sequence and 4% of the amino acid sequence. Our data indicate a high prevalence of HGV in native Bolivians, and the major genotype of HGV was type 3.
Detection of Multiple Human Papillomavirus Types in Condylomata Acuminata Lesions from Otherwise Healthy and Immunosuppressed Patients
Authors: Brown Darron R. , Schroeder Jill M. , Bryan Janine T. , Stoler Mark H. , Fife Kenneth H. 
Published: December 2019

ABSTRACT
Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.
Improved Detection of
Streptococcus pneumoniae
in Middle-Ear Fluid Cultures by Use of a Gentamicin-Containing Medium
Authors: Peled Nechama , Yagupsky Pablo 
Published: December 2019

ABSTRACT

The performance of Columbia agar medium with added sheep blood and gentamicin (CAG) for isolation of
Streptococcus pneumoniae
from middle-ear fluid cultures was compared to that of routine blood agar medium (BA). Of 238 pneumococcal isolates recovered, CAG plates detected 233 (97.9%) but BA plates detected only 208 (87.4%) (
P
< 0.001).
Phenotypic and Phylogenetic Characterization of a New
Corynebacterium
Species from Dogs: Description of
Corynebacterium auriscanis
sp. nov
Authors: Collins Matthew D. , Hoyles Lesley , Lawson Paul A. , Falsen Enevold , Robson Robert L. , Foster Geoffrey 
Published: December 2019

ABSTRACT

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus
Corynebacterium
sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus
Corynebacterium
; this subline is close to but distinct from
C. falsenii
,
C. jeikeium
, and
C. urealyticum
. The unknown bacterium from dogs was distinguished from all currently validated
Corynebacterium
species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species,
Corynebacterium auriscanis
. The type strain of
C. auriscanis
is CCUG 39938
T
.
Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of
Actinobacillus actinomycetemcomitans
,
Bacteroides forsythus
, and
Porphyromonas gingivalis
Authors: Tran Simon Dangtuan , Rudney Joel D. 
Published: December 2019

ABSTRACT

Among putative periodontal pathogens,
Actinobacillus actinomycetemcomitans
,
Bacteroides forsythus
, and
Porphyromonas gingivalis
are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects
A. actinomycetemcomitans
and
P. gingivalis
. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six
A. actinomycetemcomitans
, one
B. forsythus
, and four
P. gingivalis
strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10
A. actinomycetemcomitans
, 10
P. gingivalis
, and 100
B. forsythus
cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
strains tested, and (iii) no amplicons were detected for the eight other bacterial species.
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.
Evaluation of Reverse Transcription-PCR and a Bacteriophage-Based Assay for Rapid Phenotypic Detection of Rifampin Resistance in Clinical Isolates of
Mycobacterium tuberculosis
Authors: Eltringham I. J. , Drobniewski F. A. , Mangan J. A. , Butcher P. D. , Wilson S. M. 
Published: December 2019

ABSTRACT

New rapid phenotypic assays for the detection of rifampin resistance in
Mycobacterium tuberculosis
have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of
M. tuberculosis
. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of
M. tuberculosis
to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible
dnaK
mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 μg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries.
Monitoring Treatment of Patients with Pulmonary Tuberculosis: Can PCR Be Applied?
Authors: Thomsen Vibeke Østergaard , Kok-Jensen Axel , Buser Mauro , Philippi-Schulz Sabine , Burkardt H.-J. 
Published: December 2019

ABSTRACT

To assess whether PCR is applicable for monitoring the efficacy of antituberculous treatment, respiratory specimens obtained during treatment and follow-up from sputum smear-positive tuberculosis (TB) patients were examined. First, results of smear, culture, and PCR for
Mycobacterium tuberculosis
complex (MTB) and an internal inhibition control (MCC) were correlated retrospectively on 1,601 respiratory specimens from patients with no previous cultures of MTB. MTB optical density (OD) values increased to a maximum level of 3.5 to 4.0, with both increasing numbers of acid-fast bacilli and CFU. MTB/MCC OD ratios also increased with both smear and culture grading and correlated significantly better with both than the MTB OD value. Second, changes in MTB OD values and MTB/MCC OD ratios were compared with microscopy and culture for MTB in monthly sputa obtained during treatment and follow-up in 22 smear-positive pulmonary TB patients. Declines in MTB/MCC OD ratios during antituberculous treatment and follow-up were observed. Patients with moderate disease reached the baseline after 6 to 8 months of standard antituberculous treatment regimen, whereas patients with extensive disease were predicted to reach the baseline 1 year or more after the initiation of treatment. Although PCR detects both dead and live bacteria, we believe that PCR can be used to assess the efficacy of antituberculous treatment since increases or slow reductions in MTB/MCC OD ratios would indicate nonoptimal treatment, noncompliance, reduced bioavailability of drugs, or resistant strains of MTB and thereby would identify patients at risk for treatment failure or reactivation.
Excretion of Hepatitis A Virus (HAV) in Adults: Comparison of Immunologic and Molecular Detection Methods and Relationship between HAV Positivity and Infectivity in Tamarins
Authors: Polish Louis B. , Robertson Betty H. , Khanna Bhawna , Krawczynski Krzysztof , Spelbring John , Olson Fred , Shapiro Craig N. 
Published: December 2019

ABSTRACT
Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools.
Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper
Authors: Frisk A. L. , König M. , Moritz A. , Baumgärtner W. 
Published: December 2019

ABSTRACT
Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.