bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoretic Karyotypes and Genome Sizing of the Pathogenic Fungus
Paracoccidioides brasiliensis
Authors: Nogueira Cano Maria Isabel , Cisalpino Patrícia Silva , Galindo Ivan , Ramírez José Luiz , Mortara Renato Arruda , da Silveira José Franco
Published: December 2019

Here we present the karyotype analysis and genome sizing of
Paracoccidioides brasiliensis
, a pathogen refractory to conventional genetic analysis. We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates of
P. brasiliensis
. Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp. Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found. Different technical approaches were employed to estimate the DNA content and to define the ploidy of
P. brasiliensis
. An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy). The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separated
P. brasiliensis
chromosomal band is diploid.
PCR Methods for Rapid Identification and Characterization of
Actinobacillus seminis
Strains
Authors: Appuhamy S. , Coote J. G. , Low J. C. , Parton R.
Published: December 2019

Twenty-four isolates of
Actinobacillus seminis
were typed by PCR ribotyping, repetitive extragenic palindromic element (REP)-based PCR, and enterobacterial repetitive intergenic consensus (ERIC)-based PCR. Five types were distinguished by REP-PCR, and nine types were distinguished by ERIC-PCR. PCR ribotyping produced the simplest pattern and could be useful for identification of
A. seminis
and for its differentiation from related species. REP- and ERIC-PCR could be used for strain differentiation in epidemiological studies of
A. seminis.
Optochin Revisited: Defining the Optimal Type of Blood Agar for Presumptive Identification of
Streptococcus pneumoniae
Authors: Gardam M. A. , Miller M. A.
Published: December 2019

To determine the optimal media for optochin susceptibility testing of
Streptococcus pneumoniae
, we measured inhibition zones for 72
S. pneumoniae
and 22
Streptococcus viridans
isolates on three blood-containing media. Because 15.3, 0, and 22.2% of
S. pneumoniae
organisms were misidentified on Columbia agar, Trypticase soy agar (TSA), and Mueller-Hinton agar, respectively, each containing sheep blood, we recommend that TSA-sheep blood agar be used.
Molecular Evidence that a Distinct
Vibrio cholerae
O1 Biotype El Tor Strain in Calcutta May Have Spread to the African Continent
Authors: Sharma Charu , Ghosh Amit , Dalsgaard A. , Forslund Anita , Ghosh R. K. , Bhattacharya S. K. , Nair G. Balakrish
Published: December 2019

We present molecular evidence that a distinct genotype of
Vibrio cholerae
O1 which appeared in Calcutta, India, in September 1993 and which is characterized by a unique ribotype that is not found in the standardized ribotyping scheme of
V. cholerae
and that shows a specific pulsed-field gel electrophoresis profile may have spread to the west African country of Guinea-Bissau where it was responsible for an epidemic of cholera which began in October 1994 and continued into 1996.
Culture-Confirmed Reinfection of a Person with Different Strains of
Borrelia burgdorferi
Sensu Stricto
Authors: Golde William T. , Robinson-Dunn Barbara , Stobierski Mary Grace , Dykhuizen Daniel , Wang Ing-Nang , Carlson Vernette , Stiefel Harlan , Shiflett Susan , Campbell Grant L.
Published: December 2019

In recent years, the utility of serum-based diagnostic testing for Lyme disease has improved substantially; however, recovery by culture of the bacterium from skin biopsies of suspected patients is still the only definitive laboratory test. Reinfection of patients has been assumed to occur but as yet has not been documented by serial isolates from the same person. We present a case of culture-confirmed reinfection of a patient in Menominee County, Michigan.
Borrelia burgdorferi
was isolated from the skin punch biopsy specimens during each episode of erythema migrans (EM) and was subjected to molecular strain typing, genetic analysis of two outer surface protein genes, protein profile analysis, and serum antibody response testing. Results show that these isolates are distinct strains of the bacterium and that the two episodes of EM were caused by independent infections. This report describes the documented, culture-confirmed reinfection of a human by two different strains of
B. burgdorferi.
Characterization of Staphylococci with Reduced Susceptibilities to Vancomycin and Other Glycopeptides
Authors: Tenover Fred C. , Lancaster Michael V. , Hill Bertha C. , Steward Christine D. , Stocker Sheila A. , Hancock Gary A. , O’Hara Caroline M. , Clark Nancye C. , Hiramatsu Keiichi
Published: December 2019

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were ≥4 μg/ml or for which the teicoplanin MICs were ≥8 μg/ml and 24 control strains for which the vancomycin MICs were ≤2 μg/ml or for which the teicoplanin MICs were ≤4 μg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 μg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 μg/ml were noted for these isolates. Vitek results were 4 μg/ml for all strains for which the vancomycin MICs were ≥4 μg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either ≤2 or ≥16 μg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 μg of vancomycin per ml consistently differentiated those strains inhibited by 8 μg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains,
Staphylococcus aureus
ATCC 29213, and on occasion,
Enterococcus faecalis
ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.
Immunoglobulin M Capture Assay for Serologic Confirmation of Early Lyme Disease: Analysis of Immune Complexes with Biotinylated
Borrelia burgdorferi
Sonicate Enhanced with Flagellin Peptide Epitope
Authors: Brunner Michael , Stein Stanley , Mitchell Paul D. , Sigal Leonard H.
Published: December 2019

We previously reported on the efficacy of the enzyme-linked immunoglobulin M capture immune complex (IC) biotinylated antigen assay (EMIBA) for the seroconfirmation of early Lyme disease and active infection with
Borrelia burgdorferi
. In earlier work we identified non-cross-reacting epitopes of a number of
B. burgdorferi
proteins, including flagellin. We now report on an improvement in the performance of EMIBA with the addition of a biotinylated form of a synthetic non-cross-reacting immunodominant flagellin peptide to the biotinylated
B. burgdorferi
B31 sonicate antigen source with the avidin-biotinylated peroxidase complex detection system used in our recently developed indirect IgM-capture immune complex-based assay (EMIBA). As in our previous studies, the enzyme-linked immunosorbent assay (ELISA) reactivities of antibodies liberated from circulating ICs (by EMIBA) were compared with those of antibodies in unprocessed serum (antibodies found free in the serum, thus as an IgM-capture ELISA, but not EMIBA, because the antibodies were not liberated from ICs), the sample usually used in standard ELISAs and Western blot assays. The addition of the flagellin epitope enhanced the ELISA signal obtained with untreated sera from many Lyme disease patients but not from healthy controls. In tests with both free antibodies and ICs, with or without the addition of the flagellin epitope to the sonicate, we found the most advantageous combination was IC as the source of antibodies and sonicate plus the flagellin epitope as the antigen. In a blinded study of sera obtained from patients with early and later-phase Lyme disease, EMIBA with the enhanced antigenic preparation compared favorably with other serologic assays, especially for the confirmation of early disease.
Mycobacterium tuberculosis
Infection in a Green-Winged Macaw (
Ara chloroptera
): Report with Public Health Implications
Authors: Washko Rita M. , Hoefer Heidi , Kiehn Timothy E. , Armstrong Donald , Dorsinville Guy , Frieden Thomas R.
Published: December 2019

Mycobacterium tuberculosis
was isolated from the eyelid, skin, tongue, and lungs of a green-winged macaw (
Ara chloroptera
). Two persons living in the same household were culture positive for pulmonary tuberculosis 3 to 4 years before tuberculosis was diagnosed in the bird. Although humans have not been shown to acquire tuberculosis from birds, an infected bird may be a sentinel for human infection.
Intravitam Diagnosis of Human Rabies by PCR Using Saliva and Cerebrospinal Fluid
Authors: Crepin P. , Audry L. , Rotivel Y. , Gacoin A. , Caroff C. , Bourhy H.
Published: December 2019

An optimized reverse transcription (RT)-PCR protocol for the intravitam detection of rabies virus genomic RNA was tested with clinical samples obtained from 28 patients suspected of having rabies, 9 of whom were confirmed to have had rabies by postmortem examination. RT-PCR using saliva combined with an immunofluorescence assay performed with skin biopsy samples allowed detection of rabies in the nine patients.
Development of a Monoclonal Antibody to a
Ureaplasma urealyticum
Serotype 9 Antigen
Authors: Naessens Anne , Cheng Xiaoxing , Lauwers Sabine , Robertson Janet A.
Published: December 2019

We produced a monoclonal antibody (MAb) to
Ureaplasma urealyticum
Vancouver, the serotype 9 standard strain. By immunoblotting, this MAb showed a single, 85-kDa band with the homologous serotype and a minor, 100-kDa band with serotype 2 but did not react with any other serotype standard strain. Clinical isolates of
U. urealyticum
were tested with this MAb and with two sets of polyclonal antisera against the 14 serotype standard strains. The use of MAb 9-2H9 correctly identified certain serotype 9 strains but did not react with wild-type strains lacking the serotype 9 determinant.
Bile-Esculin Test for Presumptive Identification of Enterococci and Streptococci: Effects of Bile Concentration, Inoculation Technique, and Incubation Time
Authors: Chuard C. , Reller L. B.
Published: December 2019

The bile-esculin test is used to differentiate enterococci and group D streptococci from non-group D viridans group streptococci. The effects on test performance of the concentration of bile salts, inoculum, and duration of incubation were examined with 110 strains of enterococci, 30 strains of
Streptococcus bovis
, and 110 strains of non-group D viridans group streptococci. Optimal sensitivity (>99%) and specificity (97%) of the bile-esculin test can be obtained with a bile concentration of 40%, a standardized inoculum of 10
6
CFU, and incubation for 24 h.
Molecular Identification of
Gemella
Species from Three Patients with Endocarditis
Authors: La Scola Bernard , Raoult Didier
Published: December 2019

Gemella morbillorum
and
Gemella haemolysans
are opportunistic pathogens which cause endocarditis and other severe infections. We report on three patients with endocarditis, one with endocarditis caused by
G. haemolysans
and two with endocarditis caused by
G. morbillorum
. The paucity of reports concerning these bacteria is probably related to the difficulties associated with their identification. For example, one of the strains reported in this study was originally sent to our laboratory with a preliminary characterization as a short “gram-negative” coccobacillus, highlighting the specific problem associated with Gram staining of these bacteria. The usefulness of 16S rRNA gene amplification, partial sequencing, and comparison of the nucleotide sequence to those in databases when standard phenotypic identification schemes are not helpful is emphasized. We also suggest that the use of simple tests, such as testing susceptibility to vancomycin for gram-negative bacteria and colistin for gram-positive bacteria, could prevent misinterpretation of Gram staining in gram-variable bacteria such as
Gemella
spp.
Differentiation of
Mycobacterium ulcerans
,
M. marinum
, and
M. haemophilum
: Mapping of Their Relationships to
M. tuberculosis
by Fatty Acid Profile Analysis, DNA-DNA Hybridization, and 16S rRNA Gene Sequence Analysis
Authors: Tønjum T. , Welty D. B. , Jantzen E. , Small P. L.
Published: December 2019

Although
Mycobacterium ulcerans
,
M. marinum
, and
M. haemophilum
are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to
M. tuberculosis. M. ulcerans
and
M. marinum
were most closely related to one another, and each displayed very strong genetic affinities to
M. tuberculosis
; they are actually the two mycobacterial species outside the
M. tuberculosis
complex most closely related to
M. tuberculosis
.
M. haemophilum
was more distinct from
M. ulcerans
and
M. marinum
, and it appeared to be as related to these two species as to
M. tuberculosis
. These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for
M. ulcerans
,
M. marinum
, and
M. haemophilum
. In addition, the finding that
M. ulcerans
and
M. marinum
are more closely related to
M. tuberculosis
than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of
M. tuberculosis
.
M. marinum
may be particularly useful in this regard since strains of this species grow much more rapidly than
M. tuberculosis
and yet can cause systemic disease in immunocompromised hosts.
Comparative Evaluation of FUNGITEST and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of
Candida
Species and
Cryptococcus neoformans
Authors: Davey Kate G. , Holmes Ann D. , Johnson Elizabeth M. , Szekely Adrien , Warnock David W.
Published: December 2019

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of
Candida
spp. (50
C. albicans
, 50
C. glabrata
, 10
C. kefyr
, 20
C. krusei
, 10
C. lusitaniae
, 20
C. parapsilosis
, and 20
C. tropicalis
isolates) and 20 isolates of
Cryptococcus neoformans
. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of
C. glabrata
(71% agreement). The poorest agreement between the results for individual agents was seen with
C. glabrata
(38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and
C. tropicalis
(50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several
Candida
spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of
Candida
spp. or
C. neoformans.
Identification of a New DNA Region Specific for Members of
Mycobacterium tuberculosis
Complex
Authors: Magdalena Juana , Vachée Anne , Supply Philip , Locht Camille
Published: December 2019

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The
senX3-regX3
IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116
Mycobacterium tuberculosis
strains characterized by different IS
6110
restriction fragment length polymorphisms, 2
Mycobacterium africanum
strains, 3
Mycobacterium bovis
strains (including 2 BCG strains), and 1
Mycobacterium microti
strain, a specific PCR fragment was amplified in all cases. This collection included
M. tuberculosis
strains that lack IS
6110
or
mtp40
, two target sequences that have previously been used for the detection of
M. tuberculosis
. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU.
Epidemiological Analysis of
Salmonella enteritidis
Isolates from Humans and Broiler Chickens in Thailand by Phage Typing and Pulsed-Field Gel Electrophoresis
Authors: Boonmar Sumalee , Bangtrakulnonth Aroon , Pornrunangwong Srirat , Terajima Jun , Watanabe Haruo , Kaneko Ken-Ichi , Ogawa Masuo
Published: December 2019

To determine the phage types (PT) of
Salmonella enteritidis
found in Thailand and to clarify the potential for human infection by
S. enteritidis
in broiler chicken meat, human and poultry isolates taken from Thailand between 1990 and 1997 were phage typed and analyzed by pulsed-field gel electrophoresis (PFGE). Ten different PT were found among the 302 isolates phage typed, with PT 4 being the most frequent in human (73.9%) and poultry (76.2%) isolates, followed by PT 1 (8.0%), 8 (3.6%), and 7a (2.2%) in human isolates and by PT 7a (4.9%), 1 (3.7%), and 12 (2.4%) in poultry isolates. Of the 53 isolates analyzed by PFGE, 45 showed an indistinguishable pattern (pattern A) by
Bln
I-digested PFGE and the other 8 isolates showed a very similar pattern that differed by only a few bands. These results indicate the spread of a genetically identical clone of
S. enteritidis
in humans and poultry in Thailand.
Gemella bergeriae
sp. nov., Isolated from Human Clinical Specimens
Authors: Collins Matthew D. , Hutson Roger A. , Falsen Enevold , Sjöden Berit , Facklam Richard R.
Published: December 2019

Six strains of a hitherto-undescribed gram-positive, catalase-negative, facultatively anaerobic coccus from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains are genealogically homogeneous and constitute a new subline within the genus
Gemella
. The unknown bacterium was readily distinguished from
Gemella haemolysans
, the type species of the genus
Gemella
, and from
Gemella morbillorum
by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from clinical specimens be classified as
Gemella bergeriae
sp. nov. The type strain of
G. bergeriae
is CCUG 37817 (= strain 617-93).
Diagnostic Value of Monitoring Human Cytomegalovirus Late pp67 mRNA Expression in Renal-Allograft Recipients by Nucleic Acid Sequence-Based Amplification
Authors: Blok Marinus J. , Goossens Valere J. , Vanherle Sabina J. V. , Top Bert , Tacken Nicole , Middeldorp Jaap M. , Christiaans Maarten H. L. , van Hooff Johannes P. , Bruggeman Cathrien A.
Published: December 2019

The diagnostic value of monitoring human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) after renal-allograft transplantation was evaluated. RNAs were isolated from 489 whole-blood specimens of 42 patients for the specific amplification of the late pp67 (UL65) mRNA. NASBA results were compared to results from the pp65 antigenemia assay, virus isolation by cell culture, and serology. The sensitivity value for NASBA proved to be higher than that for the antigenemia assay (50 versus 35%) for the detection of HCMV infection, while the sensitivity values of cell culture and NASBA were comparable (54 and 50%, respectively). NASBA detected the onset of HCMV infection simultaneously with cell culture and the antigenemia assay. Both the antigenemia assay and NASBA are very specific (100%) and highly predictive (100%) for the onset of HCMV infection. Antiviral therapy with ganciclovir resulted in negative results for cell culture, the antigenemia assay, and NASBA. In conclusion, monitoring HCMV pp67 mRNA expression by NASBA is a highly specific method for the detection of HCMV infection in renal-allograft recipients and is more sensitive than the antigenemia assay. Furthermore, NASBA can be used to monitor the progression of HCMV infections and the effect of antiviral therapy on viral activity.
Nosocomial Outbreak of
Klebsiella pneumoniae
Producing SHV-5 Extended-Spectrum β-Lactamase, Originating from a Contaminated Ultrasonography Coupling Gel
Authors: Gaillot Olivier , Maruéjouls Christophe , Abachin Éric , Lecuru Fabrice , Arlet Guillaume , Simonet Michel , Berche Patrick
Published: December 2019

Klebsiella pneumoniae
resistant to ceftazidime was isolated from six adult women and two neonates hospitalized between July and November 1993 in the Department of Obstetrics and Gynecology of Boucicaut Hospital (Paris, France). The epidemiological investigation revealed a notably short delay (less than 48 h) between admission and contamination of the six adults and peripartum transmission to the neonates. The only environmental source of ceftazidime-resistant
K. pneumoniae
was the ultrasonography coupling gel used in the emergency room. Phenotypic (biotyping and antibiotyping) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analysis of all the clinical isolates indicated the spread of a single strain. It produced SHV-5 and TEM-1 β-lactamases, as demonstrated by isoelectric focusing and gene sequencing. The risk of cross-contamination in ultrasonography procedures is usually low and had not been associated so far with bacteria producing an extended-spectrum β-lactamase (ESBL). Furthermore, this is the first time an epidemic of an SHV-5 ESBL-producing member of the family
Enterobacteriaceae
has been reported from a French hospital.
Identification of
Corynebacterium amycolatum
and Other Nonlipophilic Fermentative Corynebacteria of Human Origin
Authors: Wauters Georges , Van Bosterhaut Bernard , Janssens Michèle , Verhaegen Jan
Published: December 2019

Four identification tests, proposed in addition to conventional methods, were evaluated with 320 fermentative nonlipophilic
Corynebacterium
strains: growth at 20°C, glucose fermentation at 42°C, alkalinization of sodium formate, and acid production from ethylene glycol. These tests were highly discriminant.
Corynebacterium amycolatum
displayed a unique profile, allowing it to be distinguished from similar species, such as
C. xerosis
,
C. striatum
, and
C. minutissimum.
Evaluation of PCR in Detection of
Mycobacterium tuberculosis
from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays
Authors: Marchetti Giulia , Gori Andrea , Catozzi Lidia , Vago Luca , Nebuloni Manuela , Rossi M. Cristina , Esposti Anna Degli , Bandera Alessandra , Franzetti Fabio
Published: December 2019

We compared the sensitivities and specificities of four nested PCR assays for the detection of
Mycobacterium tuberculosis
from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were
M. tuberculosis
positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of
M. tuberculosis
. Three genomic sequences (
mtp40
, 65-kDa antigen gene, and IS
6110
) with different molecular masses and numbers of repetitions within the
M. tuberculosis
genome were targeted. On the IS
6110
sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS
6110
insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the
mtp40
sequence present in a single copy in the
M. tuberculosis
genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 μg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect
M. tuberculosis
from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.
Detection of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Pools of Sera Negative for Antibodies to HIV-1 and HIV-2
Authors: Morandi Pierre-Alain , Schockmel Gérard A. , Yerly Sabine , Burgisser Philippe , Erb Peter , Matter Lukas , Sitavanc Radan , Perrin Luc
Published: December 2019

A total of 234 pools were prepared from 10,692 consecutive serum samples negative for antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 collected at five virological laboratories (average pool size, 45 serum samples). Pools were screened for the presence of HIV-1 RNA by a modified commercial assay (Amplicor HIV-1 Monitor test) which included an additional polyethylene glycol (PEG) precipitation step prior to purification of viral RNA (PEG Amplicor assay). The sensitivity of this assay for HIV-1 RNA detection in individual serum samples within pools matches that of standard commercial assays for individual serum samples, i.e., 500 HIV-1 RNA copies per ml. Five pools were identified as positive, and each one contained one antibody-negative, HIV-1 RNA-positive serum sample, corresponding to an average of 1 infected sample per 2,138 serum samples. Retrospective analysis revealed that the five HIV-1 RNA-positive specimens originated from individuals who had symptomatic primary HIV-1 infection at the time of sample collection and who were also positive for p24 antigenemia. We next assessed the possibility of performing the prepurification step by high-speed centrifugation (50,000 ×
g
for 80 min) of 1.5-ml pools containing 25 μl of 60 individual serum samples, of which only 1 contained HIV-1 RNA (centrifugation Amplicor assay). The sensitivity of this assay also matches the sensitivities of standard commercial assays for HIV-1 RNA detection in individual serum samples. The results demonstrate that both assays with pooled sera can be applied to the screening of large numbers of serum samples in a time- and cost-efficient manner.
Characteristics of
Arthrobacter cumminsii
, the Most Frequently Encountered
Arthrobacter
Species in Human Clinical Specimens
Authors: Funke Guido , Pagano-Niederer Maja , Sjödén Berit , Falsen Enevold
Published: December 2019

During a 2-year period, 10 strains of
Arthrobacter cumminsii
were isolated in or received by a Swiss routine clinical bacteriology laboratory, and 5 further isolates were referred to a Swedish bacteriology reference center over a 5-year period, making
A. cumminsii
the most frequently encountered
Arthrobacter
species in these two laboratories. The present report outlines the clinical features of the 15
A. cumminsii
strains and presents an extended biochemical characterization of this microorganism.
A. cumminsii
exhibits a unique cellular fatty acid pattern with the consistent presence of C

14:0
i

and C
14:0
fatty acids as well as relatively large amounts of C

16:0
i

and C
16:0
fatty acids usually not seen in other
Arthrobacter
spp. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to be a useful tool for confirmation of the identification of
A. cumminsii
. The MICs of 39 antimicrobial agents were determined, and it was demonstrated that aminoglycosides and quinolones had only weak activities against
A. cumminsii
strains, in contrast to their activities against most other coryneform bacteria. As a result of the extended characterization of
A. cumminsii
, an emended description of this species is presented. Due to the lack of
A. cumminsii
in established identification systems, it is most likely that this species is underdiagnosed in many routine clinical bacteriology laboratories.
Variables Affecting Results of Sodium Chloride Tolerance Test for Identification of Rapidly Growing Mycobacteria
Authors: Conville Patricia S. , Witebsky Frank G.
Published: December 2019

The sodium chloride tolerance test is often used in the identification of rapidly growing mycobacteria, particularly for distinguishing between
Mycobacterium abscessus
and
Mycobacterium chelonae
. This test, however, is frequently unreliable for the identification of some species. In this study we examined the following variables: medium manufacturer, inoculum concentration, and atmosphere and temperature of incubation. Results show that reliability is improved if the test and control slants are inoculated with an organism suspension spectrophotometrically equal to a 1 McFarland standard. Slants should be incubated at 35°C in ambient air and checked weekly for 4 weeks. Growth on control slants should be critically evaluated to determine the adequacy of the inoculum; colonies should number greater than 50. Salt-containing media should be examined carefully to detect pinpoint or tiny colonies, and colonies should number greater than 50 for a positive reaction. Concurrent use of a citrate slant may be helpful for distinguishing between
M. abscessus
and
M. chelonae
. Molecular methodologies are probably the most reliable means for the identification of rapidly growing mycobacteria and should be used, if possible, when unequivocal species identification is of particular importance.
Sequence-Based Classification Scheme for the Genus
Legionella
Targeting the
mip
Gene
Authors: Ratcliff Rodney M. , Lanser Janice A. , Manning Paul A. , Heuzenroeder Michael W.
Published: December 2019

The identification and speciation of strains of
Legionella
is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the
mip
gene and utilizing gene amplification and direct amplicon sequencing. With the exception of
Legionella geestiana
, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39
Legionella
species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.
Differentiation and Characterization of Enteroviruses by Computer-Assisted Viral Protein Fingerprinting
Authors: Holland Diane T. , Senne Jill , Peter C. R. , Urmeneta Connie , Connor J. D.
Published: December 2019

We have developed and standardized a computerized method for the typing and characterization of enteroviruses with radiolabeled viral protein fingerprints. Enteroviral proteins were radiolabeled with [35S]methionine during growth in cell culture and were then separated by polyacrylamide gel electrophoresis. The dried gel was scanned, and from the resulting computer image (which resembled an autoradiogram) protein patterns were computer extracted and stored in a database. The enterovirus database contained community and prototype strains belonging to 20 different enteroviral serotypes. Each serotype has a discrete protein pattern, and the most important pattern differences for determining each type are in the region of the viral capsid proteins VP1, VP2, and VP3. When the database was challenged with 148 clinical enterovirus strains, 144 (97%) were correctly identified by using the correlation coefficient as a quantitative measure of relatedness between two patterns. This method can identify a type in a single test and represents a practical alternative to virus neutralization because it is less expensive, is much faster (3 rather than 10 days), and does not rely on any virus-specific reagents. The results also show that most of the strains currently isolated from the community have protein patterns different from those of their older prototype strains. Viral protein fingerprinting is an evolving, dynamic system for the typing and characterization of enteroviruses. The method is appropriate for use in clinical virology and reference laboratories for the typing of enteroviruses, for the study of the epidemiology of enteroviruses, and for surveillance of enteroviruses.
Virulence Properties of Shiga Toxin-Producing
Escherichia coli
(STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves
Authors: Wieler L. H. , Schwanitz Anja , Vieler Elke , Busse Barbara , Steinrück H. , Kaper J. B. , Baljer G.
Published: December 2019

Shiga toxin-producing
Escherichia coli
(STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [
n
= 38] and O118:H− [
n
= 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing
hly
EHEC
(
hly
from enterohemorrhagic
E. coli
). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%;
P
< 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (
stx
,
hly
EHEC
, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.
Diverse and Related 16S rRNA-Encoding DNA Sequences in Prostate Tissues of Men with Chronic Prostatitis
Authors: Riley Donald E. , Berger Richard E. , Miner David C. , Krieger John N.
Published: December 2019

Treatment of chronic prostatitis/chronic pelvic pain syndrome is often empirical because clinical culture methods fail to detect prostate-associated pathogens in >90% of patients. Previously, we tested a variety of specific-microorganism PCRs and began a DNA sequence study after we found that 77% of prostatitis patients were PCR positive for prokaryotic rRNA-encoding DNA sequences (rDNAs) despite negative cultures using optimal techniques. In the present study, 36 rDNA clones from 23 rDNA-positive patients were sequenced. This study represents more than twice the total rDNA sequence and more than twice the number of patients in the previous study. The increased number of patients and clones sequenced allowed enhanced phylogenetic analyses and refinements in our view of rDNA species inhabiting the prostate. A continuum of related rDNAs that might be arbitrarily described as two major groups of rDNAs and several minor groups was found. Sequences termed Pros A, identified in 8 (35%) of 23 rDNA-positive patients, grouped with
Aeromonas
spp. in phylogenetic studies. Sequences termed Pros B, identified in 17 (74%) of 23 rDNA-positive patients, were distinct from previously reported sequences, although all were >90% similar to known gram-negative bacteria. Of the nine patients for whom multiple rDNAs were sequenced, six had biopsy specimens containing rDNAs from more than one species. Four (17%) patients had rDNAs different from those of the Pros A and Pros B groups. Of these four, one patient had rDNA similar to that of
Flavobacterium
spp., another had rDNA similar to that of
Pseudomonas testosteroni
, and two patients had rDNAs <70% similar to known rDNAs. These findings suggest that the prostate can harbor bacteria undetectable by traditional approaches. Most of these diverse sequences are not reported in environments outside the prostate. The sequence similarities suggest adaptation of limited groups of bacteria to the microenvironment of the prostate. Further studies may elucidate the relationship of prostate-associated bacteria to chronic prostatitis/chronic pelvic pain syndrome.
Evaluation of North American Antibody Detection Tests for Diagnosis of Brucellosis in Goats
Authors: Mikolon Andrea B. , Gardner Ian A. , Hietala Sharon K. , Hernandez de Anda Jorge , Chamizo Pestaña Elpidio , Hennager Steven G. , Edmondson Anita J.
Published: December 2019

The sensitivities and specificities of 17 antibody detection tests for brucellosis in goats were estimated. Tests evaluated included the U.S. Department of Agriculture (USDA) card test with 8% cell concentration (8%Card), USDA rapid automated presumptive test (RAP), Mexican rose bengal plate tests with 8 and 3% cell concentrations (8%RB and 3%RB), French rose bengal plate test with 4.5% cell concentration (4.5%RB), USDA standard plate test (SPT), USDA buffered acidified plate agglutination test (BAPA), USDA and Mexican rivanol tests (URIV and MRIV), USDA standard tube tests with
Brucella abortus
and
Brucella melitensis
antigens (SATA and SATM), serum enzyme-linked immunosorbent assay (ELISA), USDA cold-fixation complement fixation tests with
B. abortus
and
B. melitensis
antigens (CFA and CFM), USDA and Mexican milk ring tests (UBRT and MBRT), and a milk ELISA. Test sensitivity was evaluated by using two groups of 10 goats experimentally infected with
B. melitensis
or
B. abortus
and monitored for 24 weeks. Specificity was evaluated by using 200 brucellosis-free nonvaccinated goats from 10 California herds. The 3%RB was considered a good screening test because of high sensitivity at week 24 postinfection (90%), ease of performance, and low cost. The cold-fixation CFA and CFM had 100% specificity in the field study and were considered appropriate confirmatory tests. The milk ELISA was significantly more sensitive (
P
< 0.05) than the UBRT and significantly more specific (
P
< 0.05) than the MBRT. The milk ELISA also had the advantage of objectivity and ease of interpretation.
Geographic Discrimination of
Paracoccidioides brasiliensis
Strains by Randomly Amplified Polymorphic DNA Analysis
Authors: Calcagno Ana María , Niño-Vega Gustavo , San-Blas Felipe , San-Blas Gioconda
Published: December 2019

Randomly amplified polymorphic DNA (RAPD) analysis of 33
Paracoccidioides brasiliensis
strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of
P. brasiliensis.
Randomly Amplified Polymorphic DNA Genotyping of Serogroup A Meningococci Yields Results Similar to Those Obtained by Multilocus Enzyme Electrophoresis and Reveals New Genotypes
Authors: Bart Aldert , Schuurman Ilse G. A. , Achtman Mark , Caugant Dominique A. , Dankert Jacob , van der Ende Arie
Published: December 2019

Randomly amplified polymorphic DNA (RAPD) genotyping was applied to one representative strain of each of the 84 electrophoretic types (ETs) of
Neisseria meningitidis
serogroup A previously defined by multilocus enzyme electrophoresis (MEE) (J.-F. Wang et al., Infect. Immun. 60:5267–5282, 1992). Twenty-seven additional isolates comprising six ETs were also tested. MEE and RAPD genotyping yielded similar dendrograms at the subgroup level. Similar results were obtained by both methods for 18 serogroup A meningococci isolated in The Netherlands between 1989 and 1993. Ten of these isolates defined a new subgroup, designated subgroup IX. One isolate belonged to the ET-5 complex, normally associated with serogroup B strains (D. A. Caugant et al., Proc. Natl. Acad. Sci. USA 83:4927–4931, 1986). By RAPD genotyping, meningococci can be linked to previously characterized genotypes by using a computerized database, and dendrograms based on cluster analyses can easily be generated. RAPD analysis offers advantages over MEE since intermediate numbers of isolates of serogroup A meningococci can quickly be assigned to known subgroups and new subgroups can be defined.
Monoclonal Antibody F89/160.1.5 Defines a Conserved Epitope on the Ruminant Prion Protein
Authors: O’Rourke Katherine I. , Baszler Timothy V. , Miller Janice M. , Spraker Terry R. , Sadler-Riggleman Ingrid , Knowles Donald P.
Published: December 2019

The transmissible spongiform encephalopathies are a heterogeneous group of fatal neurodegenerative disorders occurring in humans, mink, cats, and ruminant herbivores. The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests with standardized reagents. Postmortem diagnosis is performed by histologic examination of brain sections from affected animals. The histopathological criteria for transmissible spongiform encephalopathies include gliosis, astrocytosis, neuronal degeneration, and spongiform change. These lesions vary in intensity and anatomic location depending on the host species and genetics, stage of disease, and infectious agent source. Diagnosis by histopathology alone may be ambiguous in hosts with early cases of disease and impossible if the tissue is autolyzed. Deposition of the prion protein (an abnormal isoform of a native cellular sialoglycoprotein) in the central nervous system is a reliable marker for infection, and immunohistochemical detection of this marker is a useful adjunct to histopathology. In the present paper we describe monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the transmissible spongiform encephalopathies in these ruminant species.
Typing of Human
Campylobacter jejuni
Isolates in Finland by Pulsed-Field Gel Electrophoresis
Authors: Hänninen Marja-Liisa , Pajarre Sini , Klossner Marja-Liisa , Rautelin Hilpi
Published: December 2019

A total of 69 pulsed-field gel electrophoresis (PFGE) types were identified among 176
Campylobacter jejuni
isolates from Finnish patients. In two geographic areas studied, five predominant PFGE types comprised over 40% of the isolates. One-third of the isolates had unique PFGE types. In small outbreaks, identical PFGE patterns were demonstrated, indicating a common source of infection.
Positive Result by Serology Indicates Active
Helicobacter pylori
Infection in Patients with Atrophic Gastritis
Authors: Kokkola Arto , Rautelin Hilpi , Puolakkainen Pauli , Sipponen Pentti , Färkkilä Martti , Haapiainen Reijo , Kosunen Timo U.
Published: December 2019

Patients with atrophic corpus gastritis and elevated
Helicobacter pylori
antibody titers but
13
C-urea breath test (
13
C-UBT) and histology results negative for
H. pylori
were randomized into eradication therapy or follow-up only. Antibody levels decreased significantly in six out of seven patients in the eradication group, while in the follow-up group, the titers declined in only one out of eight patients. In patients with atrophic corpus gastritis, positive serology results may indicate an ongoing infection in spite of negative
13
C-UBT and histology results.