bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genotypic Determination of
Mycobacterium tuberculosis
Antibiotic Resistance Using a Novel Mutation Detection Method, the Branch Migration Inhibition
M. tuberculosis
Antibiotic Resistance Test
Authors: Liu Y. P. , Behr M. A. , Small P. M. , Kurn N. 
Published: December 2019

ABSTRACT

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413–424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in
M. tuberculosis
, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of
M. tuberculosis
isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426–5430, 1994). RMP and PZA resistances are associated with multiple mutations within the
rpoB
and
pncA
genes, respectively.
M. tuberculosis
genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a “blind study.” Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the
rpoB
gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of
M. tuberculosis
clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).
Helicobacter aurati
sp. nov., a Urease-Positive
Helicobacter
Species Cultured from Gastrointestinal Tissues of Syrian Hamsters
Authors: Patterson M. M. , Schrenzel M. D. , Feng Y. , Xu S. , Dewhirst F. E. , Paster B. J. , Thibodeau S. A. , Versalovic J. , Fox J. G. 
Published: December 2019

ABSTRACT

A novel helicobacter with the proposed name
Helicobacter aurati
(type strain MIT 97-5075c) has been isolated from the inflamed stomachs and ceca of adult Syrian hamsters. The new species is fusiform with multiple bipolar sheathed flagella and periplasmic fibers; it contains urease and gamma-glutamyl transpeptidase. By 16S rRNA sequencing and repetitive element PCR-based DNA fingerprinting, it was found that
H. aurati
represents a distinct taxon and clusters with
Helicobacter muridarum
,
Helicobacter hepaticus
, and
Helicobacter
sp. MIT 94-022.
H. aurati
was recovered from hamsters housed in various research and vendor facilities. Further studies are necessary to define its association with disease and other microbiota in hamsters, as well as its impact on research projects involving hamsters.
H. aurati
(GenBank accession number
AF297868
) can be used in animal experiments to define the factors that are important for gastric helicobacter pathogenesis.
Class 1 Integron-Borne, Multiple-Antibiotic Resistance Encoded by a 150-Kilobase Conjugative Plasmid in Epidemic
Vibrio cholerae
O1 Strains Isolated in Guinea-Bissau
Authors: Dalsgaard Anders , Forslund Anita , Petersen Andreas , Brown Derek J. , Dias Francisco , Monteiro Serifo , Mølbak Kåre , Aaby Peter , Rodrigues Amabelia , Sandström Anita 
Published: December 2019

ABSTRACT

In the 1996–1997 cholera epidemic in Guinea-Bissau, surveillance for antimicrobial resistance showed the emergence of a multidrug-resistant strain of
Vibrio cholerae
O1 during the course of the epidemic. The strain was resistant to ampicillin, erythromycin, tetracycline, furazolidone, aminoglycosides, trimethoprim, and sulfamethoxazole. Concomitant with the emergence of this strain, we observed a resurgence in the number of registered cholera cases as well as an increase in the case fatality rate from 1.0% before the emergence of the multiple-drug-resistant strain to 5.3% after the emergence of the strain. Our study shows that the strain contained a 150-kb conjugative multiple-antibiotic resistance plasmid with class 1 integron-borne gene cassettes encoding resistance to trimethoprim (
dhfrXII
) and aminoglycosides [
ant
(
3"
)-
1a
]). The finding of transferable resistance to almost all of the antibiotics commonly used to treat cholera is of great public health concern. Studies should be carried out to determine to what extent the strain or its resistance genes have been spread to other areas where cholera is endemic.
Prevalence of Enterotoxin Genes in
Aeromonas
spp. Isolated From Children with Diarrhea, Healthy Controls, and the Environment
Authors: Albert M. John , Ansaruzzaman M. , Talukder Kaisar A. , Chopra Ashok K. , Kuhn Inger , Rahman Motiur , Faruque A. S. G. , Islam M. Sirajul , Sack R. Bradley , Mollby Roland 
Published: December 2019

ABSTRACT

Aeromonads are causative agents of a number of human infections. Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear. In spite of a number of virulence factors produced by
Aeromonas
species, their association with diarrhea has not been clearly linked. Recently, we have characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate of
Aeromonas hydrophila
. Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities. We studied the distribution of the
alt
,
ast
, and
act
enterotoxin genes in 115 of 125 aeromonads isolated from 1,735 children with diarrhea, in all 27 aeromonads isolated from 830 control children (
P
= 7 × 10
−4
for comparison of rates of isolation of aeromonads from cases versus those from controls), and in 120 randomly selected aeromonads from different components of surface water in Bangladesh.
Aeromonas
isolates which were positive only for the presence of the
alt
gene had similar distributions in the three sources; the number of isolates positive only for the presence of the
ast
gene was significantly higher for the environmental samples than for samples from diarrheal children; and isolates positive only for the presence of the
act
gene were not found in any of the three sources. Importantly, the number of isolates positive for both the
alt
and
ast
genes was significantly higher for diarrheal children than for control children and the environment. Thus, this is the first study to indicate that the products of both the
alt
and
ast
genes may synergistically act to induce severe diarrhea. In 26 patients,
Aeromonas
spp. were isolated as the sole enteropathogen. Analysis of clinical data from 11 of these patients suggested that isolates positive for both the
alt
and
ast
genes were associated with watery diarrhea but that isolates positive only for the
alt
gene were associated with loose stools. Most of the isolates from the three sources could be classified into seven phenospecies and eight hybridization groups. For the first time,
Aeromonas eucrenophila
was isolated from two children, one with diarrhea and another without diarrhea.
Distribution of a Specific 500-Base-Pair Fragment in
Mycobacterium bovis
Isolates from Sardinian Cattle
Authors: Sechi Leonardo A. , Duprè Ilaria , Leori Guido , Fadda Giovanni , Zanetti Stefania 
Published: December 2019

ABSTRACT

Amplification of a specific, 500-bp fragment from
Mycobacterium bovis
isolates and use of the fragment to differentiate between
Mycobacterium tuberculosis
and
M. bovis
was previously reported (J. G. Rodriguez, G. A. Meja, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995). In the present study, 30
M. bovis
isolates from Sardinian cattle were examined for the presence of this 500-bp fragment; 4 of the 30 isolates lacked the fragment. This result indicates that identification of
M. bovis
strains by amplification of the 500-bp sequence may lead to false-negative results.
G Protein Variation in Respiratory Syncytial Virus Group A Does Not Correlate with Clinical Severity
Authors: Brandenburg A. H. , van Beek R. , Moll H. A. , Osterhaus A. D. M. E. , Claas E. C. J. 
Published: December 2019

ABSTRACT
Respiratory syncytial virus group A strain variations of 28 isolates from The Netherlands collected during three consecutive seasons were studied by analyzing G protein sequences. Several lineages circulated repeatedly and simultaneously during the respective seasons. No relationships were found between lineages on the one hand and clinical severity or age on the other.
Absence of Serotype-Specific Surface Antigen and Altered Teichoic Acid Glycosylation among Epidemic-Associated Strains of
Listeria monocytogenes
Authors: Clark Elizabeth E. , Wesley Irene , Fiedler Franz , Promadej Nattawan , Kathariou Sophie 
Published: December 2019

ABSTRACT
Outbreaks of food-borne listeriosis have often involved strains of serotype 4b. Examination of multiple isolates from three different outbreaks revealed that ca. 11 to 29% of each epidemic population consisted of strains which were negative with the serotype-specific monoclonal antibody c74.22, lacked galactose from the teichoic acid of the cell wall, and were resistant to the serotype 4b-specific phage 2671.
Identification of
Enterococcus
Species and Phenotypically Similar
Lactococcus
and
Vagococcus
Species by Reverse Checkerboard Hybridization to Chaperonin 60 Gene Sequences
Authors: Goh Swee Han , Facklam Richard R. , Chang Michelle , Hill Janet E. , Tyrrell Gregory J. , Burns Emma C. M. , Chan David , He Cheng , Rahim Tazim , Shaw Carol , Hemmingsen Sean M. 
Published: December 2019

ABSTRACT

Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164–2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818–823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116–3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181–1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and
Streptococcus iniae
, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17
Enterococcus
species (
Enterococcus asini
,
Enterococcus rattus
,
Enterococcus dispar
,
Enterococcus gallinarum
,
Enterococcus hirae
,
Enterococcus durans
,
Enterococcus cecorum
,
Enterococcus faecalis
,
Enterococcus mundtii
,
Enterococcus casseliflavus
,
Enterococcus faecium
,
Enterococcus malodoratus
,
Enterococcus raffinosus
,
Enterococcus avium
,
Enterococcus pseudoavium
,
Enterococcus
new sp. strain Facklam, and
Enterococcus saccharolyticus
), and
Vagococcus fluvialis
,
Lactococcus lactis
, and
Lactococcus garvieae
. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731–734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were
Enterococcus
new sp. strain Facklam (ATCC 700913), 3;
E. asini
, 1;
E. rattus
, 4;
E. dispar
, 2;
E. gallinarum
, 20;
E. hirae
, 9;
E. durans
, 9;
E. faecalis
, 12;
E. mundtii
, 3;
E. casseliflavus
, 8;
E. faecium
, 25;
E. malodoratus
, 3;
E. raffinosus
, 8;
E. avium
, 4;
E. pseudoavium
, 1; an unknown
Enterococcus
clinical isolate, sp. strain R871;
Vagococcus fluvialis
, 4;
Lactococcus garvieae
, 3;
Lactococcus lactis
, 3;
Leuconostoc
sp., 1; and
Pediococcus
sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of
Enterococcus
and related organisms.
Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR
Authors: Schaade Lars , Kockelkorn Peter , Ritter Klaus , Kleines Michael 
Published: December 2019

ABSTRACT

Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (
n
= 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (
n
= 95 [47.5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (
n
= 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (
r
= 0.73;
P
< 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.
Multicenter Comparison of Roche COBAS AMPLICOR MONITOR Version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex Version 3.0 for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Murphy Donald G. , Côté Louise , Fauvel Micheline , René Pierre , Vincelette Jean 
Published: December 2019

ABSTRACT

The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1.5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (
P
< 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log
10
RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log
10
lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.
Real-Time PCR for Quantitative Detection of
Toxoplasma gondii
Authors: Lin Mei-Hui , Chen Tse-Ching , Kuo Tseng-tong , Tseng Ching-Chung , Tseng Ching-Ping 
Published: December 2019

ABSTRACT

The protozoan
Toxoplasma gondii
is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of
T. gondii
. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the
T. gondii
B1 gene. After 40 PCR cycles, the cycle threshold values (C
T
) indicative of the quantity of the target gene were determined. Typically, a C
T
of 25.09 was obtained with DNA from 500 tachyzoites of the
T. gondii
RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05
T. gondii
tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C
T
of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of
T. gondii
. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.
Genetic Diversity and Clonal Patterns among Antibiotic-Susceptible and -Resistant
Streptococcus pneumoniae
Colonizing Children: Day Care Centers as Autonomous Epidemiological Units
Authors: Sá-Leão Raquel , Tomasz Alexander , Sanches Ilda Santos , Nunes Sónia , Alves C. Rute , Avô António Brito , Saldanha Joana , Kristinsson Karl G. , de Lencastre Hermínia 
Published: December 2019

ABSTRACT

Characterization by antibiotype of the 1,096
Streptococcus pneumoniae
recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread
S. pneumoniae
clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.
Characterization of aBorrelia burgdorferiVlsE Invariable Region Useful in Canine Lyme Disease Serodiagnosis by Enzyme-Linked Immunosorbent Assay
Authors: Liang Fang Ting , Jacobson Richard H. , Straubinger Reinhard K. , Grooters Amy , Philipp Mario T. 
Published: December 2019

ABSTRACTSera collected from dogs experimentally infected withBorrelia burgdorferiby tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR1to IR6) of VlsE, the variable surface antigen ofB. burgdorferi. Six synthetic peptides (C1to C6), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR2and IR6, were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR6appears earlier and is stronger than that to IR2. Thus, the IR6sequence alone appeared to be sufficient for serodiagnosis. When C6alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n= 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C6ELISA, compared to 20 positives detected by immunoblot analysis using culturedB. burgdorferilysates as antigen. The sensitivity of using C2and C6together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C6alone, confirming that C6suffices as a diagnostic probe. Moreover, the C6ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other thanB. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C6ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.
Marked Differences in Quantity of Infectious Human Immunodeficiency Virus Type 1 Detected in Persons with Controlled Plasma Viremia by a Simple Enhanced Culture Method
Authors: Tremblay Cécile L. , Giguel Françoise , Merrill Debra P. , Wong Johnson T. , Rosenberg Eric , Kalams Spyros , Walker Bruce D. , D'Aquila Richard T. , Hirsch Martin S. 
Published: December 2019

ABSTRACT
Culture of autologous CD4 lymphocytes from peripheral blood mononuclear cells compared favorably with two other methods for the measurement of cell-associated human immunodeficiency virus type 1 (HIV-1). For subjects with undetectable HIV-1 RNA levels in plasma, there was a 10,000-fold range of cell-associated virus detected. This method provides a simple and reproducible means for monitoring cell-associated HIV-1.