bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphism in the Gene Coding for the Immunodominant Antigen gp43 from the Pathogenic Fungus
Paracoccidioides brasiliensis
Authors: Morais Flavia V. , Barros Tânia F. , Fukada Márcio K. , Cisalpino Patrícia S. , Puccia Rosana
Published: December 2019

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of
Paracoccidioides brasiliensis
B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17
P. brasiliensis
isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.
Detection of Immunoglobulin M Antibodies to P35 Antigen of
Toxoplasma gondii
for Serodiagnosis of Recently Acquired Infection in Pregnant Women
Authors: Suzuki Yasuhiro , Ramirez Raymund , Press Cindy , Li Shuli , Parmley Stephen , Thulliez Philippe , Remington Jack S.
Published: December 2019

We examined the efficiency of detection of immunoglobulin M (IgM) antibodies to a 35-kDa antigen (P35) of
Toxoplasma gondii
for serodiagnosis of acute infection in pregnant women. A double-sandwich enzyme-linked immunosorbent assay (ELISA) with recombinant P35 antigen (P35-IgM-ELISA) was used for this purpose. On the basis of the clinical history and the combination of results from the toxoplasma serological profile (Sabin-Feldman dye test, conventional IgM and IgA ELISAs, and the differential agglutination test), the patients were classified into three groups: group I, status suggestive of recently acquired infection; group II, status suggestive of infection acquired in the distant past; group III, status suggestive of persisting IgM antibodies. Eighteen (90.0%) of 20 serum samples from group I patients were positive by the P35-IgM-ELISA, whereas none of the 33 serum samples from group II patients were positive. Only 4 (25.0%) of 16 serum samples from group III patients were positive by the P35-IgM-ELISA, whereas all these serum samples were positive by the conventional IgM ELISA. These results indicate that demonstration of IgM antibodies against P35 by the P35-IgM-ELISA is more specific for the acute stage of the infection than demonstration of IgM antibodies by the ELISA that uses a whole-lysate antigen preparation. Studies with sera obtained from four pregnant women who seroconverted (IgG and IgM antibodies) during pregnancy revealed that two of them became negative by the P35-IgM-ELISA between 4 and 6 months after seroconversion, whereas the conventional IgM ELISA titers remained highly positive. The P35-IgM-ELISA appears to be useful for differentiating recently acquired infection from those acquired in the distant past in pregnant women.
Molecular Epidemiological Analysis of
Cryptosporidium
spp. in the United Kingdom: Results of Genotyping
Cryptosporidium
spp. in 1,705 Fecal Samples from Humans and 105 Fecal Samples from Livestock Animals
Authors: McLauchlin J. , Amar C. , Pedraza-Díaz S. , Nichols G. L.
Published: December 2019

Cryptosporidium
present in 1,705 fecal samples from humans and 105 from livestock animals were analyzed by PCR-restriction fragment length polymorphism of the
Cryptosporidium
oocyst wall protein. Overall, genotype 1 (human exclusive type) was detected in 37.8% of the samples from humans, genotype 2 (broad host range) was detected in 61.5%, a third genotype designated genotype 3 (
Cryptosporidium meleagridis
) was detected in 0.3%, and both genotypes 1 and 2 were recovered from 0.4%. All samples from livestock yielded genotype 2. Among 469 patients infected during eight drinking water-related outbreaks, five outbreaks were predominantly due to genotype 1, and three were due to genotype 2. Fifty-four samples were collected from patients involved with five swimming pool-associated outbreaks: two outbreaks were due to genotype 1, one was due to genotype 2, and the remaining two involved both genotypes 1 and 2. Among 26 family outbreaks and 1 children's nursery outbreak (2 to 3 members per group), the same genotype was recovered from the different members of each outbreak: 13 were due to genotype 1, and 14 were due to genotype 2. In eighteen patients reporting contact with animals and/or farms, genotype 1 was recovered from one patient and genotype 2 was recovered from the remaining 17. Among the sporadic cases, there were distinct geographical and temporal variations in the distribution of the genotypes. The spring peak in cases was due to genotype 2. Genotype 1 was significantly more common in patients infected during the late-summer–autumn peak and in those with a history of foreign travel.
Evaluation of an Immunocapture-Agglutination Test (Brucellacapt) for Serodiagnosis of Human Brucellosis
Authors: Orduña Antonio , Almaraz Ana , Prado Ana , Gutierrez M. Purificación , Garcia-Pascual Agustina , Dueñas Ana , Cuervo Milagros , Abad Ramon , Hernández Beatriz , Lorenzo Belen , Bratos Miguel A. , Torres Antonio Rodriguez
Published: December 2019

We evaluated the validity and the usefulness of a new test for the diagnosis of human brucellosis based on an immunocapture-agglutination technique. A total of 315 sera from 82 patients with a diagnosis of brucellosis, 157 sera from patients in whom brucellosis was suspected but not confirmed, and 412 sera from people living in rural areas with endemic brucellosis were studied. The seroagglutination test (SAT), Coombs anti-Brucella test, and Brucellacapt test were evaluated. All the initial sera from the 82 patients proved to be positive in Brucellacapt and Coombs tests, while only 75 (91.4%) were positive in the SAT. If a ≥1/160 diagnostic threshold titer was defined for the Brucellacapt test, Coombs test, and SAT, the sensitivities were 95.1, 91.5, and 65.8%, respectively. Taking the same diagnostic threshold titer for the 157 sera from the unconfirmed but suspected patients, the specificities of the Brucellacapt, Coombs, and SAT were 81.5, 96.2, and 100%, respectively; for the 412 control sera, the specificities were 99.0, 99.8, and 100%. The diagnostic efficiency (area below the receiver operating characteristic curve) of Brucellacapt was 0.987852 (95% confidence interval [CI], 0.95109 to 0.99286), very similar to the diagnostic efficiency of the Coombs test (0.97611; 95% CI, 0.94781 to 0.99146) and higher than that of SAT (0.91013; 95% CI, 0.86649 to 0.94317). The results of the Brucellacapt test were compared with those of the Coombs test (correlation coefficient, 0.956;
P
= 0.000) and SAT (correlation coefficient, 0.866;
P
= 0.000). The study shows very good correlation between the Brucellacapt and Coombs tests, with a high concordance between titers obtained in the two tests. Nevertheless, lower correlation and concordance were found between the Brucellacapt and Coombs tests when the results for titers of ≥1/160 were compared (0.692;
P
= 0.000). In acute brucellosis, the Brucellacapt and Coombs tests render positive titers of ≥1/160. When the titers are lower, they increase significantly in the following 30 days, despite the evolution of SAT titers. In contrast, Brucellacapt and Coombs titers are always high (≥1/640) in brucellosis with long evolution, whether SAT titers are higher or lower than 1/160.
Serological Relationships of
Cryptococcus
spp.: Distribution of Antigenic Factors in
Cryptococcus
and Intraspecies Diversity
Authors: Ikeda Reiko , Sugita Takashi , Shinoda Takako
Published: December 2019

The antigenic formulas of 34 species in the genus
Cryptococcus
were determined by using type strains and eight factor sera prepared from adsorption experiments with
Cryptococcus neoformans
serotypes. These antigenic factors were shared by 19 species. The strains used could be divided into eight serological groups. The patterns of groups 1, 2, 3, 5, and 6 were the same as the patterns of
C. neoformans
serotypes A, D, A-D, B, and C, respectively. The species belonging to group 4 reacted to factor sera 1, 2, and 3. Group 7 contained one species that reacted only to factor serum 1. The 15 species in group 8 did not react to any of the factor sera used. Compared to the reported molecular phylogenetic tree, the serological and phylogenetic data were correlated in the
Filobasidium
lineage. All the members of the albidus clade in the
Filobasidium
lineage had antigens 1, 2, and 3, and all the strains in the magnus clade belonged to serogroup 8. Moreover, intraspecies diversity was examined using strains of
C. curvatus
,
C. humicolus
, and
C. laurentii
. Serological heterogeneity was observed in the species
C. humicolus
and
C. laurentii
, as well as in phylogenetic relationships previously published. Using serological features, similarities and differences between
Cryptococcus
species were demonstrated. Our study contributes to a better description of the genus
Cryptococcus
and related species phenotypically and phylogenetically.
Detection of Influenza A Viruses from Different Species by PCR Amplification of Conserved Sequences in the Matrix Gene
Authors: Fouchier Ron A. M. , Bestebroer Theo M. , Herfst Sander , Van Der Kemp Liane , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E.
Published: December 2019

The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in the detection of influenza A viruses in human as well as animal secretions. Virus isolation alone is unsatisfactory for this purpose because of its inherent limited sensitivity and the lack of host cells that are universally permissive to all influenza A viruses. Previously described PCR methods are more sensitive but are targeted predominantly at virus strains currently circulating in humans, since the sequences of the primer sets display considerable numbers of mismatches to the sequences of animal influenza A viruses. Therefore, a new set of primers, based on highly conserved regions of the matrix gene, was designed for single-tube reverse transcription-PCR for the detection of influenza A viruses from multiple species. This PCR proved to be fully reactive with a panel of 25 genetically diverse virus isolates that were obtained from birds, humans, pigs, horses, and seals and that included all known subtypes of influenza A virus. It was not reactive with the 11 other RNA viruses tested. Comparative tests with throat swab samples from humans and fecal and cloacal swab samples from birds confirmed that the new PCR is faster and up to 100-fold more sensitive than classical virus isolation procedures.
Species-Specific Identification of Human Adenoviruses by a Multiplex PCR Assay
Authors: Xu WanHong , McDonough Mike C. , Erdman Dean D.
Published: December 2019

A multiplex PCR assay was developed by using primers to the fiber gene that could differentiate human adenovirus (Ad) species A through F in a single amplification reaction. The assay correctly identified the species of all 49 recognized Ad prototype strains as well as 180 geographically and temporally diverse Ad field isolates. Ad serotype 6 (Ad6) (species C), Ad16 (species B), Ad31 (species A), and Ad40 and Ad41 (species F) could also be distinguished by amplicon size within each respective species. In comparison, a previously described Ad species-specific multiplex PCR assay that used primers to the Ad hexon gene gave equivocal results with several serotypes of species B, whereas our multiplex assay amplified all species B serotypes equally well. Our multiplex PCR assay will permit rapid, accurate, and cost-effective classification of Ad isolates.
Analysis of Immunoreactivity to aStreptococcus equisubsp.zooepidemicusM-Like Protein To Confirm an Outbreak of Poststreptococcal Glomerulonephritis, and Sequences of M-Like Proteins from Isolates Obtained from Different Host Species
Authors: Nicholson Mary Lou , Ferdinand LaReesa , Sampson Jacquelyn S. , Benin Andrea , Balter Sharon , Pinto Sergio Wyton Lima , Dowell Scott F. , Facklam Richard R. , Carlone George M. , Beall Bernard
Published: December 2019

The etiologic agent of a large 1998 outbreak of poststreptococcal acute glomerulonephritis (PSGN) in Nova Serrana, Brazil, was found likely to be a specific strain ofStreptococcus equisubsp.zooepidemicusfrom contaminated cheese (S. Balter et al., Lancet 355:1776–1780, 2000). In the present study, we used a serologic screen for a known surface-exposed virulence factor to confirm the epidemiologic findings. Using primers flanking a previously characterized M-like protein gene (J. F. Timoney et al., Infect. Immun. 63:1440–1445, 1995), we amplified and sequenced the M-like protein (designated Szp5058) gene and found it to be identical among four independent acute-phase PSGN patient isolates. Convalescent-phase sera from 33 of 44 patients in the PSGN outbreak were found to contain antibodies highly reactive to a purified Szp5058 fusion protein, compared with 1 of 17 control sera (P< 0.0001), suggesting that Szp5058 was expressed during infection and further implicating this strain as the cause of the PSGN outbreak. The predicted signal sequence and cell wall association motif of Szp5058 were highly conserved with the corresponding sequence fromS. equisubsp.zooepidemicusSzpW60, while the predicted surface-exposed portions differed markedly between these two proteins. The 5′ end of theszp5058gene, including its variable region, was identical to theszpgene from another strain associated with a previous PSGN outbreak in England (M. Barham et al., Lancet i:945–948, 1983), and the correspondingszpsequence found from the Lancefield group C type strain isolated from a guinea pig. In addition, the hypervariable (HV) portion ofszp5058was identical to a previously published HV sequence from a horse isolate (J. A. Walker and J. F. Timoney, Am. J. Vet. Res. 59:1129–1133, 1998). Three other strains ofS. equisubsp.zooepidemicus, including another strain previously associated with a PSGN outbreak, were each found to contain a distinctszpgene. Two of theseszpgenes had HV regions identical toszpregions from isolates recovered from different host species.
Detection of
Legionella pneumophila
Using a Real-Time PCR Hybridization Assay
Authors: Ballard A. L. , Fry N. K. , Chan L. , Surman S. B. , Lee J. V. , Harrison T. G. , Towner K. J.
Published: December 2019

A real-time PCR hybridization assay for
Legionella pneumophila
is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample.
Detection ofEhrlichia platysDNA in Brown Dog Ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan
Authors: Inokuma Hisashi , Raoult Didier , Brouqui Philippe
Published: December 2019

Thirty-twoRhipicephalus sanguineusfemales collected from eight free-roaming dogs in Okinawa Island, Japan, were examined for ehrlichial DNA by 16S rRNA-based PCR and subsequent sequencing. Partial sequences ofEhrlichia platys16S rRNA (678 to 679 bp) were detected in three ticks (9.4%) from two dogs. This is the first report of detection ofE. platysin Japan, and also the first report of detection in ticks.
Detection of Extended-Spectrum β-Lactamases in Klebsiellae with the Oxoid Combination Disk Method
Authors: Carter Michael W. , Oakton Karen J. , Warner Marina , Livermore David M.
Published: December 2019

The Oxoid combination disk method for detecting extended-spectrum β-lactamases (ESBLs) depends on comparing the inhibition zones of cefpodoxime (10-μg) and cefpodoxime-plus-clavulanate (10- plus 1-μg) disks. The presence of clavulanate enlarged the zones for all of 180 ESBL-producing klebsiellae by ≥5 mm, whereas zones for cefpodoxime-susceptible isolates and cefpodoxime-resistant isolates with AmpC and K1 β-lactamases were enlarged by ≤1 mm. Good discrimination was achieved with either the NCCLS or the British disk method.
Meningoencephalitis Associated with
Globicatella sanguinis
Infection in Lambs
Authors: Vela Ana I. , Fernández Elena , las Heras Alfonso , Lawson Paul A. , Domínguez Lucas , Collins Matthew D. , Fernandez-Garayzabal Jose F.
Published: December 2019

Thirty lambs displayed symptoms of meningoencephalitis. An unusual gram-positive coccus was isolated in pure culture from the blood and brain samples from one of the affected animals, and phenotypic and phylogenetic characterization showed this to be
Globicatella sanguinis
. This is the first report of the isolation of
G. sanguinis
in pure culture from an animal infection.
Fungal Contamination of Food in Hematology Units
Authors: Bouakline Adel , Lacroix Claire , Roux Nicole , Gangneux Jean Pierre , Derouin Francis
Published: December 2019

The prevalence of thermotolerant fungi on non-heat-sterilizable food was determined.
Aspergillus
spp. were noted in 100% of pepper and regular tea samples, 12 to 66% of fruits, 27% of herbal teas, and 20% of freeze-dried soup samples. All soft cheese samples were contaminated by
Geotrichum
and yeast (
Candida norvegensis
) but
Candida albicans
was never identified.
Randomly Amplified Polymorphic DNA Analysis of
Erysipelothrix
spp
Authors: Okatani Alexandre Tomomitsu , Hayashidani Hideki , Takahashi Toshio , Taniguchi Takahide , Ogawa Masuo , Kaneko Ken-ichi
Published: December 2019

The usefulness of randomly amplified polymorphic DNA method (RAPD) to identify each species of genus
Erysipelothrix
and for epidemiological analysis of this genus was studied. Eighty-one strains and 18 random primers were tested. Among the tested primers, the primers NK51 (GGTGGTGGTATC) and NK6 (CCCGCGCCCC) produced noticeable results. The primer NK51 revealed four species-specific RAPD patterns. Of the 66 strains of
E. rhusiopathiae
, 64 had the same unique band of 884 bp. Of the 12 strains of
E. tonsillarum
, 11 produced a 1,265-bp band. In addition, two strains, previously thought to be
E. rhusiopathiae
, produced the 1,265-bp band, suggesting that they had been misclassified. One strain of
E. tonsillarum
produced the 884-bp band, suggesting that it too was
E. rhusiopathiae
. The
E. rhusiopathiae
strain of serovar 13 produced a 650-bp band, and the strain of serovar 18 produced a clear 420-bp band as well as three weak bands of 1,265, 918, and 444 bp. The primer NK6 revealed 14 RAPD patterns that were not serovar specific. However, different patterns were produced among strains of the same serovar showing that the RAPD method is able to identify the genetic variations of strains of this genus and can rapidly and easily differentiate strains of the same serovar. Based on these results, we concluded that the RAPD method with primers NK51 and NK6 is a rapid and reliable method to identify the species of this genus; we also concluded that this method might be a useful tool for the epidemiological analysis of the
Erysipelothrix
species.
Bacterial Colonization of Disposable Soft Contact Lenses Is Greater during Corneal Infiltrative Events than during Asymptomatic Extended Lens Wear
Authors: Sankaridurg Padmaja R. , Sharma Savitri , Willcox Mark , Naduvilath Thomas J. , Sweeney Deborah F. , Holden Brien A. , Rao Gullapalli N.
Published: December 2019

Microorganisms, especially gram-negative bacteria, are considered to play a role in the etiology of certain corneal infiltrative events (CIEs) observed during soft contact lens wear. This study explored the possibility of microbial colonization of soft contact lenses as a risk factor leading to CIEs. In a clinical trial conducted from March 1993 to January 1996, 330 subjects wore disposable soft contact lenses on a 6-night extended-wear and disposal schedule. During this period, 4,321 lenses (118 during CIEs; 4,203 during asymptomatic lens wear) were recovered aseptically and analyzed for microbial colonization. A greater percentage of lenses were free from microbial colonization during asymptomatic wear than during CIEs (42 versus 23%;
P
< 0.0001). The incidence of gram-positive bacteria, gram-negative bacteria and fungi was greater during CIEs than during asymptomatic lens wear (
P
< 0.05). During asymptomatic lens wear, gram-positive bacteria were isolated most frequently and were usually normal external ocular microbiota. Of the gram-positive bacteria, the incidence of
Streptococcus pneumoniae
was greater during CIE than during asymptomatic wear (7.6 versus 0.6%;
P
< 0.0001). While gram-negative bacteria were seen in few cases during asymptomatic wear, their incidence during CIE in comparison to asymptomatic wear was substantial and significant (23.7 versus 3.8%;
P
< 0.0001). Also, the level of colonization was high. Of CIEs, events of microbial keratitis, contact lens acute red eye, and asymptomatic infiltrative keratitis were associated with lens colonization with gram-negative bacteria or
S. pneumoniae
. Colonization of soft contact lenses with pathogenic bacteria, especially gram-negative bacteria and
S. pneumoniae
, appears to be a significant risk factor leading to CIE.
Diagnostic Implications of Human Cytomegalovirus Immediate Early-1 and pp67 mRNA Detection in Whole-Blood Samples from Liver Transplant Patients Using Nucleic Acid Sequence-Based Amplification
Authors: Blok M. J. , Lautenschlager I. , Goossens V. J. , Middeldorp J. M. , Vink C. , Höckerstedt K. , Bruggeman C. A.
Published: December 2019

Nucleic acid sequence-based amplification (NASBA) was used for detection of the human cytomegalovirus (CMV) immediate early-1 (IE) and the late pp67 mRNA in 353 blood samples collected from 34 liver transplant patients. The diagnostic value of these assays was compared to that of the pp65 antigenemia assay. Overall, 95 and 42% of the antigenemia-positive samples were IE NASBA and pp67 NASBA positive, respectively. Although the results from pp67 NASBA and the antigenemia assay appeared to correspond poorly, a clear correlation was seen between pp67 NASBA-negative results and low numbers of pp65 antigen-positive cells. Twenty patients (59%) were treated with ganciclovir after the diagnosis of symptomatic CMV infection. Before initiation of the antiviral therapy, the antigenemia assay detected the onset of symptomatic infection in all patients, whereas 95 and 60% of these patients were IE NASBA and pp67 NASBA positive, respectively. Although the sensitivity of IE NASBA was very high, the positive predictive value (PPV) of this assay for the onset of a symptomatic infection was only 63%. The PPV of the antigenemia assay as well as pp67 NASBA was considerably higher (80 and 86%, respectively). Thus, the detection of IE mRNA using NASBA appears to be particularly useful as a marker for early initiation of antiviral therapy in patients at high risk for the development of a symptomatic infection. Also, IE NASBA was found to be more sensitive than the antigenemia assay for monitoring CMV infection during antiviral therapy. On the contrary, pp67 NASBA did not appear to have additional diagnostic value compared to the antigenemia assay.
Acrophialophora fusisporaBrain Abscess in a Child with Acute Lymphoblastic Leukemia: Review of Cases and Taxonomy
Authors: Al-Mohsen I. Z. , Sutton D. A. , Sigler L. , Almodovar E. , Mahgoub N. , Frayha H. , Al-Hajjar S. , Rinaldi M. G. , Walsh T. J.
Published: December 2019

A 12-year-old girl with acute lymphoblastic leukemia was referred to King Faisal Specialist Hospital and Research Center. The diagnosis without central nervous system (CNS) involvement was confirmed on admission, and chemotherapy was initiated according to the Children Cancer Group (CCG) 1882 protocol for high-risk-group leukemia. During neutropenia amphotericin B (AMB) (1 mg/kg of body weight/day) was initiated for presumed fungal infection when a computed tomography (CT) scan of the chest revealed multiple nodular densities. After 3 weeks of AMB therapy, a follow-up chest CT revealed progression of the pulmonary nodules. The patient subsequently suffered a seizure, and a CT scan of the brain was consistent with infarction or hemorrhage. Because of progression of pulmonary lesions while receiving AMB, antifungal therapy was changed to liposomal AMB (LAMB) (6 mg/kg/day). Despite 26 days of LAMB, the patient continued to have intermittent fever, and CT and magnetic resonance imaging of the brain demonstrated findings consistent with a brain abscess. Aspiration of brain abscess was performed and the Gomori methenamine silver stain was positive for hyphal elements. Culture of this material grewAcrophialophora fusispora. Lung biopsy showed necrotizing fungal pneumonia with negative culture. The dosage of LAMB was increased, and itraconazole (ITRA) was added; subsequently LAMB was discontinued and therapy was continued with ITRA alone. The patient demonstrated clinical and radiological improvement. In vitro, the isolate was susceptible to low concentrations of AMB and ITRA.A. fusisporais a thermotolerant, fast-growing fungus with neurotropic potential. We report the first case of human infection involving the CNS.AcrophialophoraresemblesPaecilomycesbut differs in having colonies that become dark and in the development of phialides along the sides or at the tips of echinulate brown conidiophores. Conidia are borne in long chains and are smooth or ornamented with fine-to-coarse echinulations, sometimes in spiral bands. The taxonomy of the genusAcrophialophorais reviewed, andAcrophialophora nainianaandAcrophialophora levisare considered as synonyms ofA. fusispora.
Development of Reverse Transcription-PCR (Oligonucleotide Probing) Enzyme-Linked Immunosorbent Assays for Diagnosis and Preliminary Typing of Foot-and-Mouth Disease: a New System Using Simple and Aqueous-Phase Hybridization
Authors: Alexandersen Soren , Forsyth Morag A. , Reid Scott M. , Belsham Graham J.
Published: December 2019

A reverse transcription-PCR (RT-PCR)–enzyme-linked immunosorbent assay system that detects a relatively conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease virus (FMDV) has been developed. The high specificity of the assay is achieved by including a rapid hybridization step with a biotin-labeled internal oligonucleotide. The assay is highly sensitive, fast, and easy to perform. A similar assay, based on a highly variable region of the FMDV genome and employing a single asymmetric RT-PCR and multiple hybridization oligonucleotides, was developed to demonstrate the method's ability to type FMDV. Based on our theoretical and practical knowledge of the methodology, we predict that similar assays are applicable to diagnosis and strain differentiation in any system amenable to PCR amplification.
Genotyping of Verocytotoxin-Producing
Escherichia coli
O157: Comparison of Isolates of a Prevalent Phage Type by Fluorescent Amplified-Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Analyses
Authors: Smith David , Willshaw Geraldine , Stanley John , Arnold Catherine
Published: December 2019

We applied the high-resolution genotyping technique fluorescent amplified-fragment length polymorphism (FAFLP) analysis to 71 isolates of a single phage type (PT8) of pulsed-field gel electrophoresis (PFGE)-characterized verocytotoxin-producing
Escherichia coli
O157. Twenty-seven similar, but not identical, groupings were defined by both FAFLP analysis and the PFGE profiles. Given the FAFLP analysis conditions described here, these two methods exhibited equivalent discriminatory powers.
Comparison of the Rodac Imprint Method to Selective Enrichment Broth for Recovery of Vancomycin-Resistant Enterococci and Drug-Resistant
Enterobacteriaceae
from Environmental Surfaces
Authors: Hacek Donna M. , Trick William E. , Collins Susan M. , Noskin Gary A. , Peterson Lance R.
Published: December 2019

We compared the Rodac imprint technique to selective enrichment broth for detecting vancomycin-resistant enterococci (VRE) and multidrug-resistant
Enterobacteriaceae
(MDRE) on surfaces. Rodac plates contained tryptic soy agar with 5% sheep blood, vancomycin (6 μg/ml), ceftazidime (2 μg/ml), amphotericin B (2 μg/ml), and clindamycin (1 μg/ml). Two types of broth were used: brain heart infusion (BHI) and BHI plus vancomycin (6 μg/ml) and ceftazidime (2 μg/ml) (BHIVC). Of the 46 surfaces cultured for VRE, 12 (26%) were positive. Of the 12 VRE-positive surfaces, 11 (92%) grew from Rodac, 8 (67%) grew from BHIVC, and 7 (58%) grew from BHI. A larger study is needed for MDRE, as only 4 of 43 surfaces were MDRE positive. The Rodac imprint technique successfully recovered VRE from environmental surfaces.
Detection of
Ehrlichia chaffeensis
DNA in
Amblyomma americanum
Ticks in Connecticut and Rhode Island
Authors: Ijdo Jacob W. , Wu Caiyun , Magnarelli Louis A. , Stafford Kirby C. , Anderson John F. , Fikrig Erol
Published: December 2019

Ehrlichia chaffeensis
, the causative agent of human monocytic ehrlichiosis, is transmitted by
Amblyomma americanum
ticks, which are most abundant in the southern United States. Because serologic evidence suggests that residents of Connecticut are exposed to
E. chaffeensis
,
A. americanum
ticks were collected in Connecticut and Rhode Island for PCR analysis to detect
E. chaffeensis
DNA. Eight of 106 (7.6%)
A. americanum
ticks from Connecticut and 6 of 52 (11.5%) from Rhode Island contained
E. chaffeensis
DNA. Thus,
E. chaffeensis
is present in ticks in southern New England and transmission of
E. chaffeensis
may occur there.
Species Identification of
Mycobacterium avium
Complex Isolates by a Variety of Molecular Techniques
Authors: Beggs Marjorie L. , Stevanova Rossina , Eisenach Kathleen D.
Published: December 2019

Organisms in the
Mycobacterium avium
complex (MAC;
M. avium
,
M. intracellulare
, and “nonspecific or X” MAC) are emerging pathogens among individual organisms of which significant genetic variability is displayed. The objective of the present study was to evaluate various molecular methods for the rapid and definitive identification of MAC species. Isolates were obtained from both human immunodeficiency virus (HIV)-positive patients and HIV-negative patients with and without known predisposing conditions. The isolates were initially hybridized with nucleic acid probes complementary to the rRNA of the respective mycobacterial species (AccuProbe Culture Confirmation kits for
M. avium
,
M. intracellulare
, and MAC species; Gen-Probe). Isolates were also examined by PCR and in some cases by Southern blot hybridization for the insertion element IS
1245
. Two other techniques included a PCR assay that amplifies the
mig
gene, a putative virulence factor for MAC, and
hsp65
gene amplification and sequencing. This study led to the following observations. Eighty-five percent of the isolates from HIV-positive patients were
M. avium
and 86% of the isolates from HIV-negative patients were
M. intracellulare
. Fifteen of the
M. avium
isolates did not contain IS
1245
and 7% of the
M. intracellulare
isolates were found to carry IS
1245
. All of the
M. avium
strains were
mig
positive, and all of the
M. intracellulare
strains were
mig
negative.
Rapid Identification of Bacteria from Positive Blood Cultures by Fluorescence-Based PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene
Authors: Turenne Christine Y. , Witwicki Evelyn , Hoban Daryl J. , Karlowsky James A. , Kabani Amin M.
Published: December 2019

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR–single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.
Genomic Variations in Echovirus 30 Persistent Isolates Recovered from a Chronically Infected Immunodeficient Child and Comparison with the Reference Strain
Authors: Bailly Jean-Luc , Chambon Martine , Henquell Cécile , Icart Josette , Peigue-Lafeuille Hélène
Published: December 2019

Seven sequential isolates of echovirus type 30 (EV30) were recovered over 22 months from a child with severe combined immune deficiency syndrome. The nucleotide sequences of the 5′ halves of the genomes (4,400 nucleotides) of the first (S1) and last (S7) isolates were determined and compared with that of the EV30 Bastianni reference strain, also determined in this study. In genome regions P1 and P2, 101 variations were identified between the two isolates. Synonymous differences far outnumbered nonsynonymous differences. Amino acid changes affected both capsid and nonstructural polypeptides (particularly 2B). The VP1 nucleotide sequences of the seven isolates were determined to analyze genome evolution during the chronic infection. In the phylogenetic tree, the seven isolates were directly related to the prototype strain in an individual monophyletic group, strongly suggesting that the chronic infection in the child arose from a single persistent EV30 isolate. Four lineages were observed in the persistent isolates. Isolates S2, S4, S5, and S6 were close relatives of one another, whereas isolates S1 and S3 formed individual lineages. Isolate S7, distantly related to all other isolates, formed the fourth lineage. These findings suggest the quasispecies nature of the genomes of the seven sequential EV30 isolates. Grouping of persistent isolates on the basis of replicative capacities was consistent with phylogenetic relationships. Overall, the results indicate that genetically related EV30 variants with different replicative capacities coexisted in a carrier state, probably in the gastrointestinal tract, during the infection of the child.
Pandemic Spread of an O3:K6 Clone of
Vibrio parahaemolyticus
and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and
toxRS
Sequence Analyses
Authors: Matsumoto Chiho , Okuda Jun , Ishibashi Masanori , Iwanaga Masaaki , Garg Pallavi , Rammamurthy Thandavarayan , Wong Hin-Chung , Depaola Angelo , Kim Yung Bu , Albert M. John , Nishibuchi Mitsuaki
Published: December 2019

Vibrio parahaemolyticus
O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the
tdh
gene but not the
trh
gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150–3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the
tdh
and
trh
typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the
toxRS
sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus
Vibrio
. The
toxRS
sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The
tdh
-positive and
trh
-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their
toxRS
sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of
V. parahaemolyticus
and reports a novel
toxRS
-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.
Quantification of Fungal DNA by Using Fluorescence Resonance Energy Transfer and the Light Cycler System
Authors: Loeffler Juergen , Henke Norbert , Hebart Holger , Schmidt Diethard , Hagmeyer Lars , Schumacher Ulrike , Einsele Hermann
Published: December 2019

The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens,
Candida albicans
and
Aspergillus fumigatus
. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of
C. albicans
and
A. fumigatus
could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 10
1
and 10
4
Aspergillus
conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR–enzyme-linked immunosorbent assay analysis.
Pig and Goat Blood as Substitutes for Sheep Blood in Blood-Supplemented Agar Media
Authors: Anand Chandar , Gordon Rhonda , Shaw Helene , Fonseca Kevin , Olsen Merle
Published: December 2019

In many developing countries sheep and horse blood, the recommended blood supplements in bacteriological media, are not readily available, whereas pig and goat blood are. Therefore, this study examined the use of pig and goat blood as potential substitutes for sheep blood in blood-supplemented bacteriologic media commonly used in clinical microbiology laboratories. In general, the growth characteristics and colony morphologies of a wide range of aerobic and anaerobic bacteria and
Candida albicans
were similar on media containing pig, goat, and sheep blood, although differences were found.
Enterococcus
sp. uniformly produced alpha-hemolysis when incubated in CO
2
, but in anaerobic conditions the hemolysis varied. In contrast, beta-hemolytic streptococci produced identical hemolytic reactions on all three media. Synergistic hemolysis was not observed on pig blood agar in the CAMP test nor on goat blood agar in the reverse CAMP test. The preparation of chocolate agar (heated) with pig blood required heating to a higher temperature than with sheep or goat blood to yield suitable growth of
Haemophilus
species. In general, we conclude that pig and goat blood are suitable alternatives to sheep blood for use in bacteriological media in settings where sheep and horse blood are not readily available.
High Levels of Epstein-Barr Virus DNA in Blood of Solid-Organ Transplant Recipients and Their Value in Predicting Posttransplant Lymphoproliferative Disorders
Authors: Baldanti Fausto , Grossi Paolo , Furione Milena , Simoncini Lavinia , Sarasini Antonella , Comoli Patrizia , Maccario Rita , Fiocchi Roberto , Gerna Giuseppe
Published: December 2019

Epstein-Barr virus (EBV) DNA was quantitated in peripheral blood mononuclear cells (PBMC) from 25 healthy subjects, 105 asymptomatic solid-organ transplant (SOT) recipients, and 15 SOT recipients with symptomatic EBV infections by using a newly developed quantitative-PCR technique. Patients with symptomatic EBV infections had significantly higher (P< 0.001) median EBV DNA levels than asymptomatic SOT recipients and immunocompetent individuals. In SOT recipients, the positive predictive value of EBV DNA levels of >1,000 genome equivalents (GE)/0.5 μg of total PBMC DNA was 64.7% for symptomatic EBV infection, while the negative predictive value was 96.1%. In 19 of 32 (59.3%) asymptomatic SOT recipients, EBV DNA levels were consistently below 1,000 GE for as long as 18 months, while 10 of 32 (31.2%) patients had 1,000 to 5,000 EBV GE at least once during follow-up. In a minority of patients (3 of 32; 9.3%), ≥5,000 GE could be detected at least once during follow-up. Reduction of immunosuppressive treatment decreased EBV DNA levels by ≥1 log10unit in patients with symptomatic EBV infections. Quantification of EBV DNA is valuable for the diagnosis and monitoring of symptomatic EBV infections in SOT recipients.
Multisite Reproducibility of Etest for Susceptibility Testing of
Mycobacterium abscessus
,
Mycobacterium chelonae
, and
Mycobacterium fortuitum
Authors: Woods Gail L. , Bergmann John S. , Witebsky Frank G. , Fahle Gary A. , Boulet Betty , Plaunt Marianne , Brown Barbara A. , Wallace Richard J. , Wanger Audrey
Published: December 2019

A multicenter study was conducted to assess the inter- and intralaboratory reproducibility of the Etest for susceptibility testing of the rapidly growing mycobacteria. The accuracy also was evaluated by comparing Etest results to those obtained by broth microdilution. Ten isolates (four of the
Mycobacterium fortuitum
group, three of
Mycobacterium abscessus
, and three of
Mycobacterium chelonae
) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole in each of four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) using common lots of media and Etest strips. Interlaboratory agreement among MICs (i.e., mode ± 1 twofold dilution) varied for the different drug-isolate combinations and overall was best for trimethoprim-sulfamethoxazole (75% for one isolate and 100% for all others), followed by doxycycline and ciprofloxacin. Interlaboratory agreement based on interpretive category also varied and overall was best for doxycycline (100% for all isolates), followed by trimethoprim-sulfamethoxazole and ciprofloxacin. Interlaboratory reproducibility among MICs was most variable for imipenem, and agreement by interpretive category was lowest for imipenem and amikacin. Modal Etest MICs agreed with those by broth microdilution only for doxycycline and the sulfonamides. For all other drugs, the modal MICs by the two methods differed by more than ± 1 twofold dilution for one or more isolates. In all cases, the Etest MIC was higher and would have caused reports of false resistance. In summary, the Etest in this evaluation did not perform as well as broth microdilution for susceptibility testing of the rapidly growing mycobacteria. It was problematic for most species and drugs, primarily because of a trailing endpoint and/or high MICs compared to broth. Its use will necessitate further investigation, including determination of the optimal medium and incubation conditions and clarification of endpoint interpretation.
Problems Related to Determination of MICs of Oximino-Type Expanded-Spectrum Cephems for
Proteus vulgaris
Authors: Ohno Akira , Ishii Yoshikazu , Ma Ling , Yamaguchi Keizo
Published: December 2019

During in vitro susceptibility testing of clinical isolates of
Proteus vulgaris
, we noted that the MICs of several expanded-spectrum cephems were much higher in the broth microdilution method than in the agar dilution method (termed the MIC gap phenomenon). Here we investigated the mechanism of the MIC gap phenomenon. Cephems with the MIC gap phenomenon were of the oximino type, such as cefotaxime, cefteram, and cefpodoxime, which serve as good substrates for inducible class A β-lactamase (CumA) enzymes produced by
P. vulgaris
; this finding suggests a relationship between the MIC gap phenomenon and CumA. Since peptidoglycan recycling shares a system common to that inducing CumA, we analyzed the mechanism of the MIC gap phenomenon using
P. vulgaris
B317 and isogenic mutants with mutations in the peptidoglycan recycling and β-lactamase induction systems. The MIC gap phenomenon was observed in the parent strain B317 but not in B317G (
cumG
-defective mutant; defective peptidoglycan recycling) and B317R (
cumR
-defective mutant; defective CumA transcriptional regulator). No β-lactamase activity was detected in B317G and B317R. β-Lactamase activity and the MIC gap phenomenon were restored in B317G/pMD301 (strain transcomplemented by a cloned
cumG
gene) and B317R/pMD501 (strain transcomplemented by a cloned
cumR
gene). MICs determined by the agar dilution method increased when lower agar concentrations were used. Our results indicated that the mechanism of the MIC gap phenomenon is related to peptidoglycan recycling and CumA induction systems. However, it remains unclear how β-lactamase induction of
P. vulgaris
is suppressed on agar plates.