bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Version 2.0 Qualitative and Quantitative AMPLICOR Reverse Transcription-PCR Tests for Hepatitis C Virus RNA: Calibration to International Units, Enhanced Genotype Reactivity, and Performance Characteristics
Authors: Lee Sung C. , Antony Anisha , Lee Nitta , Leibow Jeff , Yang Jian Q. , Soviero Stephen , Gutekunst Karen , Rosenstraus Maurice
Published: December 2019

Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2.0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.
Panfungal PCR and Multiplex Liquid Hybridization for Detection of Fungi in Tissue Specimens
Authors: Hendolin Panu H. , Paulin Lars , Koukila-Kähkölä Pirkko , Anttila Veli-Jukka , Malmberg Henrik , Richardson Malcolm , Ylikoski Jukka
Published: December 2019

A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (
Aspergillus flavus
,
Aspergillus fumigatus
,
Candida albicans
,
Candida krusei
,
Candida glabrata
,
Candida parapsilosis
,
Candida tropicalis
, and
Cryptococcus neoformans
) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.
Application of tRNA Intergenic Spacer PCR for Identification of
Enterococcus
Species
Authors: Baele Margo , Baele Paul , Vaneechoutte Mario , Storms Virginie , Butaye Patrick , Devriese Luc A. , Verschraegen Gerda , Gillis Monique , Haesebrouck Freddy
Published: December 2019

tRNA intergenic spacer PCR (tDNA-PCR) was evaluated for its usefulness in the differentiation of enterococcal species of human and animal origin. This technique was carried out for 124 strains belonging to 17 enterococcal species and generated DNA fragments, which were separated by capillary electrophoresis. tDNA-PCR enabled us to discriminate for all species tested.
Enterococcus faecium
showed minor but reproducible differences with
Enterococcus durans
, while
Enterococcus hirae
was easily distinguishable.
Enterococcus avium
,
Enterococcus malodoratus
, and
Enterococcus raffinosus
generated highly similar though distinctive patterns.
Granulocytic Ehrlichiae in
Ixodes persulcatus
Ticks from an Area in China Where Lyme Disease Is Endemic
Authors: Cao Wu-Chun , Zhao Qiu-Min , Zhang Pan-He , Dumler J. Stephen , Zhang Xi-Tan , Fang Li-Qun , Yang Hong
Published: December 2019

A total of 372 adult
Ixodes persulcatus
ticks were collected from vegetation in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be endemic. The ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with primers derived from the 16S rRNA gene. Of 310 ticks obtained from the Dahe forestry farm, two pools (each containing 5 ticks) were found positive, with a minimum infection rate of 0.6%. Ehrlichial DNA was also detected in one female (1.6%) of 62 ticks collected from the Yulin forestry farm. The overall minimum infection rate of the 372
I. persulcatus
adults was 0.8%. The nucleotide sequences of 919-bp PCR products from the three positive tick specimens were identical to each other and very closely related to the members of the
Ehrlichia phagocytophila
genogroup. This is the first identification of granulocytic ehrlichiae in ticks in Asia and the first report of infection in
I. persulcatus
anywhere.
Frequency of Isolation of
Staphylococcus lugdunensis
among Staphylococcal Isolates Causing Endocarditis: a 20-Year Experience
Authors: Patel Robin , Piper Kerryl E. , Rouse Mark S. , Uhl James R. , Cockerill Franklin R. , Steckelberg James M.
Published: December 2019

Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of
Staphylococcus lugdunensis
among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as
S. lugdunensis
.
Can Pooling Be Used for Seroprevalence Studies of Hepatitis C?
Authors: Stephens G. M. , Raboud J. M. , Karakas L. , Sherlock C. H.
Published: December 2019

Two hepatitis C antibody assays were used to test diluted positive sera. Dilutions of 1 in 5, 1 in 10, and 1 in 20 all resulted in loss of reactivity, with the greatest losses occurring in samples with low and moderate reactivities. These results disqualify pooling as a strategy for seroprevalence studies and screening programs.
Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications
Authors: Georgopoulou Amalia , Markoulatos Panayotis , Spyrou Niki , Vamvakopoulos Nicholas C.
Published: December 2019

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC
53
-UG
52
primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (
Hae
III,
Dde
I,
Nco
I, and
Ava
I). To compensate for potential genetic drift within the recognition sites of
Hae
III,
Dde
I, or
Nco
I in atypical clinical samples, the RFLP patterns generated with
Hpa
II and
Sty
I as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.
Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for
Staphylococcus
spp
Authors: Yugueros Javier , Temprano Alejandro , Berzal Beatriz , Sánchez Marı́a , Hernanz Carmen , Luengo José Marı́a , Naharro Germán
Published: December 2019

The
gap
gene of
Staphylococcus aureus
, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12
Staphylococcus
spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers.
Alu
I digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in
Staphylococcus epidermidis
,
Staphylococcus hominis
, and
Staphylococcus simulans
which were different from the other species. An identical RFLP pattern was observed for 112
S. aureus
isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for
S. aureus
cells of 20 CFU when cells were suspended in saline. PCR amplification of the
gap
gene has the potential for rapid identification of at least 12 species belonging to the genus
Staphylococcus
, as it is highly specific.
Evaluation of Three Newly Developed Enzyme-Linked Immunosorbent Assays and Two Agglutination Tests for DetectingSalmonella entericasubsp.entericaSerovar Dublin Infections in Dairy Cattle
Authors: Veling J. , van Zijderveld F. G. , van Zijderveld-van Bemmel A. M. , Barkema H. W. , Schukken Y. H.
Published: December 2019

In this study test characteristics of three newly developed enzyme-linked immunosorbent assays (ELISAs) forSalmonella entericasubsp.entericaserovar Dublin were evaluated and compared with two agglutination tests. The ELISAs involved were an indirect ELISA with serovar Dublin lipopolysaccharide (LPS ELISA), an indirect ELISA with serovar Dublin flagellar antigen (GP ELISA), and a double-antibody sandwich blocking ELISA that uses monoclonal antibodies againstS. entericasubsp.entericaserovar Enteritidis flagellin (GM-DAS ELISA). The agglutination tests involved were two routine serum agglutination tests with either somatic (O) or flagellar (H) antigen. Diagnostic specificity of the three ELISAs was determined using 840 serum samples from seven dairy herds without any history of serovar Dublin infection. Cutoff values at a titer of 100, 100, and 10, respectively, for the LPS ELISA, GP ELISA, and GM-DAS blocking ELISA resulted in a specificity of 99.3, 100, and 100%, respectively. Using these cutoff values the LPS ELISA, GP ELISA, and GM-DAS ELISA were able to detect, respectively, 30, 46, and 38% of 50 fecal culture-positive animals from 13 herds with a recent serovar Dublin infection. With the same cutoff values, active carriers (n= 18) were detected for 94.4% with the LPS ELISA and for 100% with the GP and GM-DAS ELISAs. Kappa values determined on the results of all tests from 8 of the 13 serovar Dublin-infected herds and the 7 control herds demonstrated a good correlation between the results of all ELISAs and the H-agglutination test. The results of the O-agglutination test failed to correlate with those of the other tests. Using a set of sera from 170 aborting cows (with 25 abortions due to serovar Dublin), test results of the ELISAs and the H-agglutination test were comparable. The H-agglutination test may be used successfully for single sample testing, especially to diagnose abortion due to serovar Dublin. It is concluded that the ELISAs are useful diagnostic tools in serovar Dublin control programs and that they are preferred to agglutination tests for reasons of automation and costs.
Detection of
Chlamydia pneumoniae
and
Helicobacter pylori
DNA in Human Atherosclerotic Plaques by PCR
Authors: Farsak Bora , Yildirir Aylin , Akyön Yakut , Pinar Ahmet , Öç Mehmet , Böke Erkmen , Kes Sirri , Tokgözoğlu Lale
Published: December 2019

Chlamydia pneumoniae
and
Helicobacter pylori
can cause persistent infections of the respiratory and gastrointestinal tract, respectively. It has been suggested that persistent infection of arteries with these bacteria can contribute to the development of atherosclerosis. The aims of this study were to determine the presence of
C. pneumoniae
and
H. pylori
DNA in atherosclerotic plaque samples by PCR and to evaluate the correlation between clinical status and DNA positivity of these bacteria. Eighty-five consecutive patients (mean age, 59 ± 10; 75 male, 10 female) undergoing coronary artery bypass grafting, carotid endarterectomy, and surgery of the abdominal aorta for atherosclerotic obstructive lesions were included in the study. Forty-six endarterectomy specimens from the atherosclerotic lesions and 39 specimens from healthy regions of the ascending aorta, which were accepted as the control group, were excised. The presence of microorganism DNA in endarterectomy specimens was assessed by PCR.
C. pneumoniae
DNA was found in 12 (26%) of 46 endarterectomy specimens and none of the healthy vascular-wall specimens (
P
< 0.001), while
H. pylori
DNA was found in 17 (37%) of 46 endarterectomy specimens and none of the controls (
P
< 0.001). Either
C. pneumoniae
or
H. pylori
DNA was positive in 23 (50%) of 46 patients and none of the controls (
P
< 0.001). Six of the atherosclerotic lesions showed coexistence of both of the microorganism DNAs. The presence of
C. pneumoniae
and
H. pylori
DNA in a considerable number of atherosclerotic plaques but their absence in healthy vascular wall supports the idea that they may have a role in the development of atherosclerosis, especially in countries where infection is prevalent and where conventional risk factors fail to explain the high prevalence of atherosclerotic vascular disease.
Prevalence of Diarrheagenic
Escherichia coli
in Finns with or without Diarrhea during a Round-the-World Trip
Authors: Keskimäki Markku , Mattila Leena , Peltola Heikki , Siitonen Anja
Published: December 2019

The incidence of diarrhea and the prevalence of bacterial enteropathogens, viruses, and parasites in feces of subjects with and without diarrhea were evaluated in 204 Finns traveling round the world (from Finland to China, Malaysia, Australia, Fiji, Chile, and Brazil and back to Finland). Special emphasis was placed on the finding of diarrheagenic
Escherichia coli
(enterotoxigenic, enteropathogenic, Shiga toxin-producing, and enteroaggregative strains) by PCR from growth on primary culture plates. From the PCR-positive samples, corresponding strains were isolated, confirmed as
E. coli
, and O serotyped. Of all the subjects, 37% experienced a total of 90 episodes of diarrhea. No adenoviruses or rotaviruses were detected, and findings of parasites were insignificant. In contrast, enteropathogenic bacteria were present in 62% of the 65 diarrheal and in 33% of the 127 nondiarrheal samples (
P
< 0.001); diarrheagenic
E. coli
strains were found in 35 and 26% of these, respectively (not statistically significant). As a single pathogen,
E. coli
was found in 20 and 24% of samples (not significant). Of all diarrheagenic
E. coli
strains, enteropathogenic strains were the most commonly found independently of the clinical picture of the subjects, whereas
Salmonella enterica
as a single pathogen was the most common non-
E. coli
organism found in diarrheal samples. Multiple bacterial pathogens were found 10 times more commonly in diarrheal than in nondiarrheal samples (20 versus 2%;
P
< 0.001).
NSP4 Gene Analysis of Rotaviruses Recovered from Infected Children with and without Diarrhea
Authors: Lee Chun-Nan , Wang Yu-Lan , Kao Chuan-Liang , Zao Chih-Ling , Lee Chin-Yun , Chen Hsiao-Neng
Published: December 2019

The transmembrane glycoprotein NSP4 functions as a viral enterotoxin capable of inducing diarrhea in young mice. It has been suggested that NSP4 may be a key determinant of rotavirus pathogenicity and a target for vaccine development. Twenty two G1P[6] rotaviruses from babies with and without diarrhea were comparatively analyzed along with reference strains and another 22 Taiwanese human rotaviruses of G and P combination types different from the G1P[6] type. The sequence variations in the NSP4 genes were studied by direct sequencing analysis of the amplicons of reverse transcription-PCR. Two genetic groups could be identified in this analysis. While the majority of these strains were closely related to the Wa strain, the G2 viruses were closely related to the S2 strain. Furthermore, phylogenetic analysis of the NSP4 gene among the G2 rotaviruses revealed three distinct lineages associated with DS-1, S2, and E210, respectively, as has been reported previously for the VP7 gene. However, we found no apparent correlation in the deduced amino acid sequences corresponding to the proposed enterotoxic peptide region between the rotaviruses recovered from individuals with and without diarrhea. The NSP4 gene product being a pathogenic determinant may not be a generalized phenomenon.
Rapid-Cycle PCR Method To Detect
Bordetella pertussis
That Fulfills All Consensus Recommendations for Use of PCR in Diagnosis of Pertussis
Authors: Farrell D. J. , McKeon M. , Daggard G. , Loeffelholz M. J. , Thompson C. J. , Mukkur T. K. S.
Published: December 2019

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of
Bordetella pertussis
infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (
Bordetella
spp.). In specimens positive for
Bordetella
spp.,
B. pertussis
was differentiated from
Bordetella parapertussis
and
Bordetella bronchiseptica
by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse β-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS
481
and IS
1001
PCR (∼1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS
481
and IS
1001
. Fifty-one specimens were positive for
B. pertussis
by POR and IS
481
PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS
481
PCR. A total of 652 specimens were negative by both methods.
B. parapertussis
was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of
B. pertussis
.
Identification and Phylogenetic Relationship of the Most Common Pathogenic
Candida
Species Inferred from Mitochondrial Cytochrome
b
Gene Sequences
Authors: Yokoyama Koji , Biswas Swarajit Kumar , Miyaji Makoto , Nishimura Kazuko
Published: December 2019

We sequenced a 396-bp region of the mitochondrial cytochrome
b
gene of the most common clinically important
Candida
species:
Candida albicans
,
C. glabrata
,
C. parapsilosis
,
C. tropicalis
,
C. krusei
, and
C. lusitaniae
. The recently described species of
Candida
,
C. dubliniensis
, associated with mucosal candidiasis in human immunodeficiency virus-infected individuals, was also included. Two to five strains of each species were examined. Some species represented intraspecies variation, which was not more than 1.8% (DNA). However, interspecies variations were more than 10 and 7%, respectively, for DNA and amino acid sequences. Multiple alignments of nucleotide and deduced amino acid sequences revealed species-specific nucleotides and amino acids. Nucleotide- and amino acid-based phylogenetic trees were constructed and are discussed. Using the database, it is possible to identify presumptive
Candida
species within a working day.
Conservation of Type-Specific B-Cell Epitopes of Glycoprotein G in Clinical Herpes Simplex Virus Type 2 Isolates
Authors: Liljeqvist Jan-Åke , Svennerholm Bo , Bergström Tomas
Published: December 2019

Glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion and to a cell-associated, heavily O-glycosylated carboxy-terminal portion that constitutes the mature gG-2 (mgG-2). The mgG-2 protein is commonly used as a type-specific antigen in the serodiagnosis of HSV-2 infection. As the amino acid sequence variability of mgG-2 in clinical isolates may affect the performance of such assays, the gG-2 gene was sequenced from 15 clinical HSV-2 isolates. Few mutations were identified, and these were mostly localized outside the epitope regions described earlier. Five isolates were identical to different laboratory strains, indicating that the gG-2 gene is highly conserved over time. In the search for HSV-2 isolates harboring mutations within the immunodominant region of mgG-2, a pool of 2,400 clinical HSV-2 isolates was tested for reactivity with two anti-mgG-2 monoclonal antibodies (MAbs). Ten MAb escape HSV-2 mutants, which all harbored structurally restricted single- or dual-point mutations within the respective epitopes explaining the loss of binding, were identified. Sera from corresponding patients were reactive to mgG-2, as well as to a peptide representing the immunodominant region, suggesting that the point mutations detected did not diminish seroreactivity to mgG-2. The conservation of the gG-2 gene reported here further supports the use of mgG-2 as a type-specific antigen in the diagnosis of HSV-2 infections.
Characterization of a Coronavirus Isolated from a Diarrheic Foal
Authors: Guy James S. , Breslin Jamie J. , Breuhaus Babetta , Vivrette Sally , Smith Lynda G.
Published: December 2019

A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.
Fluorescent Amplified-Fragment Length Polymorphism Genotyping of
Neisseria meningitidis
Identifies Clones Associated with Invasive Disease
Authors: Goulding Jonathan N. , Hookey John V. , Stanley John , Olver Will , Neal Keith R. , Ala'Aldeen Dlawer A. A. , Arnold Catherine
Published: December 2019

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of
Neisseria meningitidis
. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of
N. meningitidis
. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of
N. meningitidis
as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of
N. meningitidis
for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.
PCR Detection and Evidence of Shedding of Porcine Circovirus Type 2 in Boar Semen
Authors: Larochelle Renée , Bielanski Andrzej , Müller Peter , Magar Ronald
Published: December 2019

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.
Apparent Culture-Negative Prosthetic Valve Endocarditis Caused by
Peptostreptococcus magnus
Authors: van der Vorm Eric R. , Dondorp Arjen M. , van Ketel Ruud J. , Dankert Jacob
Published: December 2019

In two patients with prosthetic valve endocarditis due to
Peptostreptococcus magnus
, blood cultures in the BacT/Alert and BACTEC 9240 systems were signal negative. The capability of the BacT/Alert system to detect various
Peptostreptococcus
species was assessed.
P. magnus
and
P. anaerobius
could not be detected, and subcultures remained negative. The growth in conventional media of these two species and other
Peptostreptococcus
species was similar.
Characterization of Non-Type B
Haemophilus influenzae
Strains Isolated from Patients with Invasive Disease
Authors: Cerquetti Marina , Ciofi degli Atti Marta Luisa , Renna Giovanna , Tozzi Alberto Eugenio , Garlaschi Maria Laura , Mastrantonio Paola ,   The Hi Study Group
Published: December 2019

Forty-one non-type b
Haemophilus influenzae
isolates from cases of invasive disease were characterized. By PCR capsular genotyping, 33 nonencapsulated strains, 4 type f isolates, and 4 b
−
strains were identified. By pulsed-field gel electrophoresis, the nonencapsulated isolates exhibited great genetic heterogenicity, whereas the type f and the b
−
strains seemed to have a clonal spread. Occurrence of the
hifA
gene was found by PCR in 18% of the nonencapsulated, 50% of the b
−
, and all of the type f strains. Hemagglutinating fimbriae were generally expressed by nonencapsulated isolates when fimbrial gene
hifA
was present. Two nonencapsulated isolates not susceptible to ampicillin were detected; no strains were positive for β-lactamase production.
Identification of
Corynebacterium glucuronolyticum
Strains from the Urogenital Tract of Humans and Pigs
Authors: Devriese Luc A. , Riegel Philippe , Hommez Jozef , Vaneechoutte Mario , de Baere Thierry , Haesebrouck Freddy
Published: December 2019

Bacterial strains isolated from the genital tracts of humans (predominantly males), semen of boars, and uterine and vaginal secretions of sows were identified as
Corynebacterium glucuronolyticum
and were compared with the type strains of the recently proposed species
Corynebacterium glucuronolyticum
and
Corynebacterium seminale
. The two type strains as well as the clinical strains were shown by DNA-DNA hybridization and sequencing of the 16S rRNA gene to be related at the species level. All strains were classified as
C. glucuronolyticum
, because this name has nomenclatural priority over
C. seminale
.
Comparison of Susceptibility Testing of
Mycobacterium tuberculosis
Using the ESP Culture System II with That Using the BACTEC Method
Authors: Ruiz P. , Zerolo F. J. , Casal M. J.
Published: December 2019

The ESP Culture System II was evaluated for its capacity to test the susceptibility of 389 cultures of
Mycobacterium tuberculosis
to streptomycin, rifampin, ethambutol, and isoniazid. Good agreement with results with the BACTEC TB 460 was found. ESP II is a reliable, rapid, and automated method for performing susceptibility testing.
Comparison of Iodophor and Alcohol Pledgets with the Medi-Flex Blood Culture Prep Kit II for Preventing Contamination of Blood Cultures
Authors: Wilson Michael L. , Weinstein Melvin P. , Mirrett Stanley , Reimer Larry G. , Fernando Cha , Meredith Frances T. , Reller L. Barth
Published: December 2019

Iodophor and alcohol pledgets were compared with the Medi-Flex Prep Kit II for skin disinfection before venipuncture. Of 12,367 blood cultures collected, 6,362 were done with conventional pledgets and 6,005 were done with Medi-Flex kits. Contamination occurred in 351 of 6,362 blood cultures (5.5%; range, 3.7 to 8.1%) with conventional pledgets versus 328 of 6,005 (5.5%; range, 3.5 to 7.5%) with Medi-Flex kits.
Detection of Bovine Herpesvirus 4 (BoHV-4) DNA in the Cell Fraction of Milk of Dairy Cattle with History of BoHV-4 Infection
Authors: Donofrio Gaetano , Flammini Cesidio Filippo , Scatozza Franco , Cavirani Sandro
Published: December 2019

We have demonstrated, by PCR and restriction enzyme analysis of the PCR product, the presence of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk from dairy cattle with a history of BoHV-4 infection. We next evaluated the infectious nature of BoHV-4 DNA in those cells. Cocultivation of a BoHV-4-sensitive cell line with BoHV-4 DNA-positive milk cell samples produced cytopathic effects. The same result was obtained from frozen and thawed milk cell fraction coming from the cell milk fraction PCR-positive cows, ensuring that cells were killed and only infectious virus could be recovered after cocultivation with sensitive cells. This report shows that infectious BoHV-4 can be present in milk cells and that therefore nursing may be one of the transmission routes of BoHV-4.
Salmonella enterica
Serovar Virchow with CTX-M-Like β-Lactamase in Spain
Authors: Simarro Encarna , Navarro Ferrán , Ruiz Joaquı́n , Miró Elisenda , Gómez Joaquı́n , Mirelis Beatriz
Published: December 2019

Four
Salmonella enterica
serovar Virchow strains resistant to broad-spectrum cephalosporins were isolated from patients with gastroenteritis in 1997 and 1998 in Murcia and Barcelona, Spain. The isolates expressed a β-lactamase with a pI of about 8 and a positive PCR when specific primers for CTX-M-9 were used. These results suggest the presence of a CTX-M-9 β-lactamase in these strains.
Molecular Identification of
Pasteurella dagmatis
Peritonitis in a Patient Undergoing Peritoneal Dialysis
Authors: Wallet Frédéric , Touré Fatouma , Devalckenaere Annie , Pagniez Dominique , Courcol René J.
Published: December 2019

Pasteurella dagmatis
was identified as the etiologic agent of peritonitis in a continuous ambulatory peritoneal dialysis patient by utilizing a molecular kit in our hospital's clinical laboratory. This method would appear a useful approach to identify a species of
Pasteurella
not included in the existing database of commercial identification kits when discrepancies exist between phenotypic tests.
Fluorometric Assessment of In Vitro Antidermatophytic Activities of Antimycotics Based on Their Keratin-Penetrating Power
Authors: Okeke C. N. , Tsuboi R. , Kawai M. , Ogawa H.
Published: December 2019

Keratin particles impregnated with amorolfine or clotrimazole in serial doubling dilutions (64 to 0.125 μg/ml) were used to evaluate the activities of these agents against 20 isolates each of
Trichophyton mentagrophytes
and
Trichophyton rubrum
in a yeast carbon broth medium incorporating Alamar Blue dye. The proposed MIC with keratin impregnation (MIC
K
) is defined as the lowest concentration of an agent used to impregnate keratin particles that effects a fluorescence-based fungal growth quotient of 0.05 or less. The conventional colorimetric and visual MICs of amorolfine for the dermatophytes, ≤0.03 μg/ml for
T. mentagrophytes
and ≤0.063 μg/ml for
T. rubrum
, were approximately half of those of clotrimazole for the same isolates. The superiority of the MIC
K
s of amorolfine for isolates of
T. mentagrophytes
(2.0 μg/ml; range, 0.5 to 8.0 μg/ml) and
T. rubrum
(4.0 μg/ml; range, 2.0 to 8.0 μg/ml) over those of clotrimazole (32 μg/ml [range, 8.0 to >64 μg/ml] and 64 μg/ml [range, 16 to >64 μg], respectively) may indicate the strong in vivo antidermatophytic activity of amorolfine as a topical agent. The new antidermatophytic susceptibility testing procedure has potential clinical utility for the in vitro screening of agents for use in the topical treatment of superficial mycoses.
Production and Characterization of Recombinant
Aspergillus fumigatus
Cu,Zn Superoxide Dismutase and Its Recognition by Immune Human Sera
Authors: Holdom M. D. , Lechenne B. , Hay R. J. , Hamilton A. J. , Monod M.
Published: December 2019

The Cu,Zn superoxide dismutase (SOD) of
Aspergillus fumigatus
has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a λEMBL3
A. fumigatus
genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an
A. fumigatus
λgt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The
A. fumigatus
Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu,Zn SODs in general. A recombinant
A. fumigatus
Cu,Zn SOD has been expressed in
Pichia pastoris
, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native
A. fumigatus
Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with
A. fumigatus
infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of
A. fumigatus
infections.
Ability of the VITEK 2 Advanced Expert System To Identify β-Lactam Phenotypes in Isolates of
Enterobacteriaceae
and
Pseudomonas aeruginosa
Authors: Sanders Christine C. , Peyret Michel , Moland Ellen Smith , Shubert Carole , Thomson Kenneth S. , Boeufgras Jean-Marc , Sanders W. Eugene
Published: December 2019

The Advanced Expert System (AES) was used in conjunction with the VITEK 2 automated antimicrobial susceptibility test system to ascertain the β-lactam phenotypes of 196 isolates of the family
Enterobacteriaceae
and the species
Pseudomonas aeruginosa
. These isolates represented a panel of strains that had been collected from laboratories worldwide and whose β-lactam phenotypes had been characterized by biochemical and molecular techniques. The antimicrobial susceptibility of each isolate was determined with the VITEK 2 instrument, and the results were analyzed with the AES to ascertain the β-lactam phenotype. The results were then compared to the β-lactam resistance mechanism determined by biochemical and molecular techniques. Overall, the AES was able to ascertain a β-lactam phenotype for 183 of the 196 (93.4%) isolates tested. For 111 of these 183 (60.7%) isolates, the correct β-lactam phenotype was identified definitively in a single choice by the AES, while for an additional 46 isolates (25.1%), the AES identified the correct β-lactam phenotype provisionally within two or more choices. For the remaining 26 isolates (14.2%), the β-lactam phenotype identified by the AES was incorrect. However, for a number of these isolates, the error was due to remediable problems. These results suggest that the AES is capable of accurate identification of the β-lactam phenotypes of gram-negative isolates and that certain modifications can improve its performance even further.
The Alleles of the
bft
Gene Are Distributed Differently among Enterotoxigenic
Bacteroides fragilis
Strains from Human Sources and Can Be Present in Double Copies
Authors: d'Abusco Anna Scotto , Del Grosso Maria , Censini Stefano , Covacci Antonello , Pantosti Annalisa
Published: December 2019

Enterotoxigenic
Bacteroides fragilis
(ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated
bft-1
,
bft-2
, and
bft-3
, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms
bft-1
and
bft-2
or
bft-3
borne by ETBF. By sequencing the portion of the
bft
gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as
bft-2
or
bft-3
, we found in our collection two strains harboring
bft-3
, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that
bft-1
is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while
bft-2
is present in isolates from feces of children. This association is statistically significant (
P
, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the
bft
and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring
bft-1
(three strains) or
bft-2
(one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring
bft-3
, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.
Prevalence of Vancomycin-Resistant Enterococci in Fecal Samples from Hospitalized Patients and Nonhospitalized Controls in a Cattle-Rearing Area of France
Authors: Gambarotto Karine , Ploy Marie-Cécile , Turlure Pascal , Grélaud Carole , Martin Christian , Bordessoule Dominique , Denis Francois
Published: December 2019

Vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens over the last decade, but little is known about their epidemiology. We report on the prevalence of VRE fecal colonization on the basis of a prospective study among patients hospitalized in a hematology intensive care unit and among nonhospitalized subjects living in the local community. A total of 243 rectal swabs from hematology patients and 169 stool samples from the control group were inoculated onto bile-esculin agar plates with and without 6 mg of vancomycin per liter and into an enrichment bile-esculin broth supplemented with 4 mg of vancomycin per liter. A total of 37% of the hospitalized patients and 11.8% of the subjects from the community were found to be VRE carriers. A total of 65 VRE strains were isolated: 12 (18.5%)
E. faecium
, 46 (70.7%)
E. gallinarum
, and 7 (10.8%)
E. casseliflavus
strains. No
E. faecalis
strains were detected. All the
E. faecium
strains were of the
vanA
genotype. Molecular typing by pulsed-field gel electrophoresis revealed a different pattern for each
vanA
VRE strain that originated from an individual subject. To our knowledge, this is the first study to be carried out in a cattle-rearing region of France. It reports a higher VRE prevalence than that reported in previous European or U.S. studies. A partial explanation is the use of an enrichment broth step which enabled detection of strains which would otherwise have been missed, but the fact that subjects and patients were recruited from a predominantly agricultural area where vancomycin-related antibiotics have recently been used in animal husbandry could also contribute to the high levels of VRE in patients and subjects alike.
PCR-Based Assay To Quantify Human Immunodeficiency Virus Type 1 DNA in Peripheral Blood Mononuclear Cells
Authors: Christopherson Cindy , Kidane Yorda , Conway Brian , Krowka John , Sheppard Haynes , Kwok Shirley
Published: December 2019

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (
P
, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (
P
, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.
Line Probe Assay for Monitoring Drug Resistance in Hepatitis B Virus-Infected Patients during Antiviral Therapy
Authors: Stuyver Lieven , Van Geyt Caroline , De Gendt Sija , Van Reybroeck Georges , Zoulim Fabien , Leroux-Roels Geert , Rossau Rudi
Published: December 2019

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.
Application of 16S rRNA Gene Sequencing To Identify
Bordetella hinzii
as the Causative Agent of Fatal Septicemia
Authors: Kattar M. M. , Chavez J. F. , Limaye A. P. , Rassoulian-Barrett S. L. , Yarfitz S. L. , Carlson L. C. , Houze Y. , Swanzy S. , Wood B. L. , Cookson B. T.
Published: December 2019

We report on the first case of fatal septicemia caused by
Bordetella hinzii
. The causative organism exhibited a biochemical profile identical to that of
Bordetella avium
with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either
B. avium
or previously reported strains of
B. hinzii
. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing
B. hinzii
from
B. avium
were production of alkali from malonate and resistance to several antimicrobial agents.
Genotypic Stability of Cold-Adapted Influenza Virus Vaccine in an Efficacy Clinical Trial
Authors: Cha Tai-An , Kao Kevin , Zhao Jackie , Fast Patricia E. , Mendelman Paul M. , Arvin Ann
Published: December 2019

An investigational live influenza virus vaccine, FluMist, contains three cold-adapted H1N1, H3N2, and B influenza viruses. The vaccine viruses are 6/2 reassortants, in which the hemagglutinin (HA) and neuraminidase (NA) genes are derived from the circulating wild-type viruses and the remaining six genes are derived from the cold-adapted master donor strains. The six genes from the cold-adapted master donor strains ensure the attenuation, and the HA and NA genes from the wild-type viruses confer the ability to induce protective immunity against contemporary influenza strains. The genotypic stability of this vaccine was studied by employing clinical samples collected during an efficacy trial. Viruses present in the nasal and throat swab specimens and in supernatants after culturing the specimens were detected and subtyped by multiplex reverse transcriptase (RT)-PCR. Complete genotypes of these detected viruses were determined by a combination of RT-PCR and restriction fragment length polymorphism, multiplex RT-PCR and fluorescent single-strand conformation polymorphism, and nucleic acid sequencing analysis. The FluMist vaccine appeared to be genotypically stable after replication in the human host. All viruses detected during the 2-week postvaccination period were shed vaccine viruses and had maintained the 6/2 genotype.