bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultrasound-Enhanced Latex Immunoagglutination and PCR as Complementary Methods for Non-Culture-Based Confirmation of Meningococcal Disease
Authors: Gray Stephen J. , Sobanski Michael A. , Kaczmarski Edward B. , Guiver Malcolm , Marsh W. John , Borrow Raymond , Barnes Rosemary A. , Coakley W. Terence 
Published: December 2019

ABSTRACT
Preadmission administration of antibiotics to patients with suspected meningococcal infection has decreased the likelihood of obtaining an isolate and has stimulated development of rapid and reliable non-culture-based diagnostic methods. The sensitivity of the conventional test card latex agglutination test (TCLAT) for detection of capsular polysaccharide has been reported to be suboptimal. In the United Kingdom meningococcal DNA detection by PCR has become readily available and is now used as a first-line investigation. Recently, the performance of latex antigen detection has been markedly improved by ultrasound enhancement. Three tests for laboratory confirmation of meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii) ultrasound-enhanced latex agglutination test (USELAT), were compared in a retrospective study of 125 specimens (serum, plasma, and cerebrospinal fluid specimens) from 90 patients in whom meningococcal disease was suspected on clinical grounds. Samples were from patients with (i) culture-confirmed meningococcal disease, (ii) culture-negative but PCR-confirmed meningococcal disease, and (iii) clinically suspected but non-laboratory-confirmed meningococcal disease. USELAT was found to be nearly five times more sensitive than TCLAT. Serogroup characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by PCR; however, for 12 other specimens for which PCR had failed to indicate the serogroup, USELAT gave a result. USELAT is a rapid, low-cost method which can confirm a diagnosis, identify serogroups, and guide appropriate management of meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a centralized PCR assay service for detection of meningococci would give the benefits of timely information and improved epidemiological data.
PCR-Restriction Enzyme Analysis for Detection of
Candida
DNA in Blood from Febrile Patients with Hematological Malignancies
Authors: Morace Giulia , Pagano Livio , Sanguinetti Maurizio , Posteraro Brunella , Mele Luca , Equitani Francesco , D’Amore Giuseppina , Leone Giuseppe , Fadda Giovanni 
Published: December 2019

ABSTRACT

Blood samples were drawn daily from 72 patients who had hematological malignancies, neutropenia, and fever and who had failed to respond to broad-spectrum antibiotics. Each sample was used for conventional fungal blood cultures and for detection and identification of
Candida
DNA by a PCR method with subsequent restriction enzyme analysis (REA) recently developed in our laboratory. The PCR method was able to detect five CFU of
Candida
spp. per ml of blood, and subsequent REA of the amplicons allowed the identification of the
Candida
species most commonly implicated in cases of candidiasis. Thirty-one patients were PCR-REA positive, and four of these patients were also culture positive. The ultimate diagnosis for 13 of these patients and 1 patient who was PCR-REA negative was disseminated candidiasis (confirmed by clinical data, multiple cultures, histology, autopsy, and/or ultrasonographic evidence of hepatosplenic candidiasis). The molecular method is significantly more sensitive than conventional fungal blood cultures and has a high negative predictive value (97.5%) for the development of disseminated candidiasis in neutropenic patients.
Genetic Features of
Streptococcus agalactiae
Strains Causing Severe Neonatal Infections, as Revealed by Pulsed-Field Gel Electrophoresis and
hylB
Gene Analysis
Authors: Rolland Karine , Marois Corinne , Siquier Veronique , Cattier Blandine , Quentin Roland 
Published: December 2019

ABSTRACT

A collection of 114 independent
Streptococcus agalactiae
strains, including 54 strains isolated from the cerebrospinal fluid (CSF) samples of neonates and 60 strains from asymptomatic patients, was characterized by pulsed-field gel electrophoresis (PFGE) of DNA restricted with
Sma
I and by PCR analysis of the
hylB
gene. All strains were previously studied by multilocus enzyme electrophoresis (MLEE) (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576–2581, 1995). Among these 114 strains, there were 92 PFGE patterns. Eleven genetic groups (A to K) were identified with 38% divergence. A more homogeneous group (PFGE group A) was defined, consisting of 73% of the strains previously identified as belonging to a particular MLEE phylogenetic group. A 162-kb fragment was identified as a marker of strains that invaded the central nervous system of neonates. It was detected in 69% of the PFGE patterns obtained with CSF isolates and in only 1.8% of the PFGE patterns obtained with carrier strains. The
hylB
gene encoding hyaluronate lyase was amplified for all strains in our collection. Ten of 15 isolates belonging to an MLEE subgroup, previously described as being likely to cause invasive infection, had an insertion in the
hylB
gene (IS
1548
).
Phenotypic and Genotypic Characterization of
Mycobacterium africanum
Isolates from West Africa
Authors: Frothingham Richard , Strickland Percy L. , Bretzel Gisela , Ramaswamy Srinivas , Musser James M. , Williams Diana L. 
Published: December 2019

ABSTRACT

The
Mycobacterium tuberculosis
complex includes
M. tuberculosis
,
M. bovis
,
M. africanum
, and
M. microti
. Most clinical isolates are
M. tuberculosis
or
M. bovis
. These species can be distinguished by phenotypes and genotypes. However, there is no simple definition of
M. africanum
, and some authors question the validity of this species. We analyzed 17 human isolates from Sierra Leone, identified as
M. africanum
by biochemical and growth characteristics. We sequenced polymorphic genes and intergenic regions. We amplified DNA from six loci with variable numbers of tandem repeats (VNTRs) and determined the exact number of repeats at each locus in each strain. All
M. africanum
isolates had the ancestral CTG Leu at
katG
codon 463. Drug-resistant
M. africanum
isolates had
katG
and
rpoB
mutations similar to those found in drug-resistant
M. bovis
and
M. tuberculosis
. Fourteen Sierra Leone
M. africanum
isolates (designated group A) had
katG
codon 203 ACC Thr, also found in
M. africanum
T
(the T indicates type strain) from Senegal. Group A isolates clustered with
M. africanum
T
by VNTR analysis. Three
M. africanum
isolates (group B) had
katG
codon 203 ACT Thr, found in
M. tuberculosis
T
, and clustered with
M. tuberculosis
T
by VNTR analysis. Phenotypic identification of
M. africanum
yielded a heterogeneous collection of strains. Genotypic analyses identified a cluster (
M. africanum
group A) which included
M. africanum
T
and was distinct from the rest of the
M. tuberculosis
complex. Future studies of
M. africanum
should include both phenotypic and genotypic analyses.
Use of Base Excision Sequence Scanning for Detection of Genetic Variations in St. Louis Encephalitis Virus Isolates
Authors: Charrel Rémi N. , Lévy Nicolas , Tesh Robert B. , Chandler Laura J. 
Published: December 2019

ABSTRACT
Twenty-two isolates of St. Louis encephalitis (SLE) virus of various geographical origins (Brazil, Argentina, Panama, Texas, Missouri, Maryland, California, and Florida) were examined for genetic variation by the base excision sequence scanning (BESS T-scan) method. A fragment was amplified in the envelope gene with the forward primer labeled in the PCR. The BESS T-scan method determined different clusters according to the profiles generated for the isolates and successfully grouped the isolates according to their geographical origins. Two major clusters, the North American cluster (cluster A) and the South and Central American cluster (cluster B), were defined. Two subgroups, the Texas-California subgroup (subgroup A1) and the Missouri-Maryland-Florida subgroup (subgroup A2), were distinguished within group A. Similarly, group B strains were subclustered to a South American subgroup (subgroup B1) and a Central American subgroup (subgroup B2). These results were consistent with those obtained by DNA sequencing analysis. The ability of the BESS T-scan method to discriminate between strains that present with high degrees of nucleotide sequence similarity indicated that this method provides reliable results and multiple applications for other virus families. The method has proven to be suitable for phylogenetic comparison and molecular epidemiology studies and may be an alternative to DNA sequencing.
Species-Specific Identification of
Mycobacterium leprae
by PCR-Restriction Fragment Length Polymorphism Analysis of the
hsp65
Gene
Authors: Rastogi Nalin , Goh Khye Seng , Berchel Mylene 
Published: December 2019

ABSTRACT

PCR-restriction fragment length polymorphism analysis (PRA) of the
hsp65
gene present in all mycobacteria was used in the present investigation to characterize
Mycobacterium leprae
. Bacilli were extracted and purified from different organs from experimentally infected armadillos and nude mice (Swiss mice of
nu/nu
origin). A total of 15 samples were assayed in duplicate, and the results were compared with those obtained for a total of 147 cultivable mycobacteria representing 34 species. Irrespective of its origin or viability,
M. leprae
strains from all the samples were uniformly characterized by two fragments of 315 and 135 bp upon
Bst
EII digestion and two fragments of 265 and 130 bp upon
Hae
III digestion. PRA is a relatively simple method and permits the conclusive identification of
M. leprae
to the species level.
Tick-Borne Relapsing Fever Imported from West Africa: Diagnosis by Quantitative Buffy Coat Analysis and In Vitro Culture of
Borrelia crocidurae
Authors: van Dam Alje P. , van Gool Tom , Wetsteyn José C. F. M. , Dankert Jacob 
Published: December 2019

ABSTRACT

West African tick-borne relapsing fever (TBRF) is difficult to diagnose due to the low number of spirochetes in the bloodstream of patients. Previously, the causative microorganism,
Borrelia crocidurae
, had never been cultured in vitro. TBRF was rapidly diagnosed for two patients returning from western Africa with fever of unknown origin by quantitative buffy coat (QBC) analysis. Diagnosis was confirmed by intraperitoneal inoculation of blood specimens from patients into laboratory mice. In vitro experiments showed that QBC analysis may be as much as 100-fold more sensitive than thick smear. Spirochetes were also cultured from blood samples from both patients in modified Kelly’s medium and were identified as
B. crocidurae
by partial sequencing of the PCR-amplified
rrs
gene.
Use of Dorset Egg Medium for Maintenance and Transport of
Neisseria meningitidis
and
Haemophilus influenzae
Type b
Authors: Wasas Avril D. , Huebner Robin E. , Klugman Keith P. 
Published: December 2019

ABSTRACT

Studies of bacterial meningitis are hampered by the inability to maintain the viability of etiological agents during transport to reference laboratories. The long-term survival rate of 20 isolates of
Neisseria meningitidis
and
Haemophilus influenzae
type b (Hib) on Dorset egg medium, supplemented Columbia agar base medium, chocolate agar, and Amies medium was compared with that on 70% GC agar (chocolate) transport medium.
N. meningitidis
isolates were also inoculated onto 5% horse blood agar, and Hib was inoculated onto
Haemophilus
test medium. All of the
N. meningitidis
isolates remained viable on Dorset egg medium for 21 days; viability on the other media was poor after only 7 days. Recovery rates of Hib isolates were similar on Dorset egg and
Haemophilus
test media (100% after 21 days) and significantly better than on the other media. Dorset egg medium is inexpensive and easy to make and may be invaluable for studies of bacterial meningitis in developing countries.
Disk with High Oxacillin Content Discriminates between Methicillin-Resistant and Borderline Methicillin-Susceptible
Staphylococcus aureus
Strains in Disk Diffusion Assays Using a Low Salt Concentration
Authors: Petersson Ann Cathrine , Kamme Carl , Miörner Håkan 
Published: December 2019

ABSTRACT

A separation between
mecA
+
strains of
Staphylococcus aureus
and strains lacking
mecA
was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 μg of oxacillin and inocula of approximately 5 × 10
5
CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30°C were employed. The 5-μg oxacillin disks clearly discriminated between borderline methicillin-susceptible and
mecA
+
strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 μg/ml were obtained for strains lacking
mecA
. Significantly higher levels of β-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking
mecA
, in particular strains of phage group V, when agar with ≥2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance.
Infection of Laboratory Mice with the Human Granulocytic Ehrlichiosis Agent Does Not Induce Antibodies to Diagnostically Significant
Borrelia burgdorferi
Antigens
Authors: Bunnell Joseph E. , Magnarelli Louis A. , Dumler J. Stephen 
Published: December 2019

ABSTRACT

Laboratory diagnosis of
Borrelia burgdorferi
is routinely made by an enzyme-linked immunosorbent assay, with positive results confirmed by Western blot analysis. Concern has been raised that false-positive diagnoses may be made on the basis of serologic cross-reactivity with antibodies directed against other bacterial pathogens, in particular the agent of human granulocytic ehrlichiosis (HGE). The present study made use of a mouse model to ascertain the validity of these concerns. Two different strains of mice were inoculated with the HGE agent and assayed for production of polyclonal and monoclonal antibodies to antigens of both of these bacteria. Infection of mice with the HGE agent does not induce diagnostically significant
B. burgdorferi
serologic cross-reactions.
Isolation of DNA after Extraction of RNA To Detect the Presence of
Borrelia burgdorferi
and Expression of Host Cellular Genes from the Same Tissue Sample
Authors: Amemiya Kei , Schaefer Henry , Pachner Andrew R. 
Published: December 2019

ABSTRACT

We are investigating the neuropathogenesis of Lyme disease caused by
Borrelia burgdorferi
in a nonhuman primate model. In the past, two separate pieces of tissue had to be used when both analyzing for the presence of the spirochete and examining the host response to infection. We have modified a procedure to purify DNA from the same sample after the extraction of RNA. The remaining material containing the DNA was precipitated, and residual organic reagent was removed prior to deproteinization and extraction of the DNA. This procedure now allows us to both assay for the presence of the Lyme microorganism and analyze the host response in the same tissue preparation.
Detection of Clinically Relevant Genotypes of Vancomycin-Resistant Enterococci in Nosocomial Surveillance Specimens by PCR
Authors: Jayaratne P. , Rutherford C. 
Published: December 2019

ABSTRACT

This study evaluated a PCR method for the rapid detection of clinically significant genotypes of vancomycin-resistant enterococci (VRE) in nosocomial surveillance specimens. Detection of the
vanA
and
vanB
genes by multiplex PCR using 657 specimens that showed presumptive growth of VRE on bile esculin azide agar containing 6 mg of vancomycin/liter was compared to the conventional method. The diagnostic values for the PCR compared to the phenotypic method were as follows: 99.8% specificity, 95.4% sensitivity, 98.8% positive predictive value, and 99.3% negative predictive value. The average cost per test for PCR is $8.26, compared to $9.45 for the phenotypic method. The average turnaround time for detecting a VRE is 48 h for PCR, compared to 96 h for the conventional method.
Subtyping of
Haemophilus influenzae
Strains by Pulsed-Field Gel Electrophoresis
Authors: Saito Mitsumasa , Umeda Akiko , Yoshida Shin-ichi 
Published: December 2019

ABSTRACT

A total of 200 isolates of
Haemophilus influenzae
were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of
H. influenzae
demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive
H. influenzae
disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with
H. influenzae
infection revealed that the strain that caused the
H. influenzae
infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of
H. influenzae
infection, even when the invasive disease is caused by serotype b strains.
Most
Enterobacter aerogenes
Strains in France Belong to a Prevalent Clone
Authors: Bosi Claude , Davin-Regli Anne , Bornet Charleric , Mallea Monique , Pages Jean-Marie , Bollet Claude 
Published: December 2019

ABSTRACT

The aim of this study was to determine the distribution in France of the
Enterobacter aerogenes
prevalent clone isolated in the hospitals of the Marseille area (A. Davin-Regli, D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bollet, J. Clin. Microbiol. 34:1474–1480, 1996). A total of 123
E. aerogenes
isolates were collected from 23 hospital laboratories and analyzed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR to determine their epidemiological relatedness. Molecular typing revealed that 21 of the 23 laboratories had isolated this prevalent clone harboring the plasmid encoding for extended-spectrum β-lactamase of the TEM-24 type. Most isolates were susceptible only to imipenem and gentamicin. Their dissemination seems to be clonal and was probably the result of the general use of broad-spectrum cephalosporins and quinolones. Four isolates showed an alteration of their outer membrane proteins, causing decrease of susceptibility to third-generation cephalosporins and imipenem and leading to the critical situation of having no alternative therapeutic. The large dissemination of the
E. aerogenes
prevalent clone probably results from its good adaptation to the antibiotics administered in France and the hospital environment, particularly in intensive care units.
Evaluation of the Discriminatory Power of Typing Methods forNeisseria gonorrhoeae
Authors: Van Looveren M. , Ison C. A. , Ieven M. , Vandamme P. , Martin I. M. , Vermeulen K. , Renton A. , Goossens H. 
Published: December 2019

ABSTRACTA panel of 18 strains ofNeisseria gonorrhoeae, known to be temporally and geographically diverse, was used to evaluate a number of typing systems, including conventional auxotyping and serotyping and the molecular methods of arbitrarily primed PCR (AP-PCR), amplified ribosomal-DNA restriction analysis (ARDRA),opatyping, and pulsed-field gel electrophoresis (PFGE). The discriminatory power of the different typing methods were determined with a collection of 87 clinical isolates from commercial sex workers in Indonesia, and Simpson’s index of diversity was calculated. Of the two traditional techniques, auxotyping and serotyping, the latter gives the highest discriminatory index (DI) (DI, 0.846). The combination of auxotyping and serotyping yields a high DI (DI, 0.928). D11344- and D8635-primed PCR showed low DIs of 0.608 and 0.622, respectively, but a combination of the two primers had a DI of 0.849. The combination of serotyping with D11344-primed or D8635-primed PCR resulted in DIs of 0.936 and 0.937, respectively. ARDRA revealed a low DI of 0.743 alone but a DI of 0.955 in combination with serotyping. PFGE using the restriction enzymeBglII andopatyping produced the highest discrimination (DIs, 0.997 and 0.996, respectively) for isolates ofN. gonorrhoeae.
Multiple Types of
Legionella pneumophila
Serogroup 6 in a Hospital Heated-Water System Associated with Sporadic Infections
Authors: Visca Paolo , Goldoni Paola , Lück P. Christian , Helbig Jürgen H. , Cattani Lorena , Giltri Giuseppe , Bramati Simone , Pastoris Maddalena Castellani 
Published: December 2019

ABSTRACT

Five sporadic cases of nosocomial Legionnaires’ disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes.
Legionella pneumophila
serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of
L. pneumophila
serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of
rrn
operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of
Legionella
infection. The long-term persistence in the hot-water system of different clones of
L. pneumophila
serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.