bluuebunny/crossref_metadata_2025 huggingface dataset:
Ochrobactrum intermedium
Infection after Liver Transplantation
Authors: Möller Lieke V. M. , Arends Jan P. , Harmsen Hermie J. M. , Talens Adriaan , Terpstra Peter , Slooff Maarten J. H.
Published: December 2019

A case of bacteremia due to
Ochrobactrum intermedium
, with concomitant liver abscesses, in an orthotopic liver transplant recipient is presented. Identical microorganisms were isolated from fecal specimens and from an aspirate of a liver abscess that was indicative of invasion of the graft by gastrointestinal spread. 16S DNA sequence analysis of the blood isolate revealed the recovery of the recently proposed new species
O. intermedium
, closely related to
Ochrobactrum anthropi
and
Brucella
spp.
Systemic Infection of an Immunocompromised Patient with
Methylobacterium zatmanii
Authors: Hornei B. , Lüneberg E. , Schmidt-Rotte H. , Maaß M. , Weber K. , Heits F. , Frosch M. , Solbach W.
Published: December 2019

We describe the identification of
Methylobacterium zatmanii
as the causative agent of bacteremia and fever in an immunocompromised patient. The patient, a 60-year-old man, had a 5-month history of acute myeloid leukemia and had been on chemotherapy throughout this period. Seven days after the onset of neutropenia, the patient developed fever. The combination of ciprofloxacin, co-trimoxazole, imipenem, amikacin, and vancomycin led to a complete defervescence. On subculture from six positive blood cultures, the organism grew only on buffered charcoal yeast extract agar and not on standard agars. Identification by universal PCR and subsequent sequence analysis of the amplified 16S rRNA gene segment was achieved. This identification by molecular biology techniques was confirmed by conventional biochemical tests. To our knowledge, this is the first description of
M. zatmanii
isolated from patient material.
Serotyping of
Streptococcus pneumoniae
by Coagglutination with 12 Pooled Antisera
Authors: Lalitha M. K. , Thomas Kurien , Kumar R. Satish , Steinhoff Mark C. ,   the IBIS Study Group
Published: December 2019

We report on the performance of a recently introduced commercial chessboard method using 12 antisera, in comparison with that of the 55-antiserum panel used in determining the serogroups and types (SGTs) of
Streptococcus pneumoniae
, both of which were carried out by a coagglutination technique. Of a total of 150 strains of
S. pneumoniae
studied, 135 (90%) belonged to the SGTs represented in the 23-valent pneumococcal vaccine; of these, 130 (96.3%) were identified as the same SGTs by both typing methods. The remaining five strains showed cross-reactivity with more than two pools by the chessboard method, but could be assigned to a single SGT by the Quellung test. The 96.3% concordance of the chessboard method suggests it can be adopted for determination of the SGTs of
S. pneumoniae
in laboratories.
Klebsiella pneumoniaeLipopolysaccharide O Typing: Revision of Prototype Strains and O-Group Distribution among Clinical Isolates from Different Sources and Countries
Authors: Hansen Dennis S. , Mestre Francesca , Albertí Sebastián , Hernández-Allés Santiago , Álvarez Dolores , Doménech-Sánchez Antonio , Gil José , Merino Susana , Tomás Juan M. , Benedí Vicente J.
Published: December 2019

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide fromKlebsiella pneumoniaewhich overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638K. pneumoniaeclinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+and half O−strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.
Quantitative Detection of Hepatitis B Virus DNA in Two International Reference Plasma Preparations
Authors: Heermann Klaus-Hinrich , Gerlich Wolfram H. , Chudy Michael , Schaefer Stephan , Thomssen Reiner ,   The Eurohep Pathobiology Group
Published: December 2019

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up to a factor of 120. The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype
adw2
) and genotype D (
ayw2/3
), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion-derived DNA reacted much less efficiently. After identification and elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 × 10
9
HBV DNA molecules/ml (range, 2.1 × 10
9
to 3.4 × 10
9
HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 × 10
9
HBV DNA molecules/ml (range, 2.1 × 10
9
to 3.0 × 10
9
HBV DNA molecules/ml in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organization reference sample.
Multicenter Evaluation of the Fully Automated COBAS AMPLICOR PCR Test for Detection of
Chlamydia trachomatis
in Urogenital Specimens
Authors: Vincelette Jean , Schirm Jurjen , Bogard Marc , Bourgault Anne-Marie , Luijt Dirk S. , Bianchi Anne , van Voorst Vader Pieter C. , Butcher Ann , Rosenstraus Maurice
Published: December 2019

The fully automated COBAS AMPLICOR CT/NG test for the detection of
Chlamydia trachomatis
was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the
C. trachomatis
major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98.5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99.1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of
C. trachomatis
exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.
Rapid Identification of Clinically Relevant
Nocardia
Species to Genus Level by 16S rRNA Gene PCR
Authors: Laurent Frederic J. , Provost Frederique , Boiron Patrick
Published: December 2019

Two regions of the gene coding for 16S rRNA in
Nocardia
species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant
Nocardia
isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including
Dietzia
,
Gordona
,
Mycobacterium
,
Rhodococcus
,
Streptomyces
, and
Tsukamurella
. Besides, unlike the latter group of isolates, all
Nocardia
strains exhibited one
Mln
I recognition site but no
Sac
I restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of
Nocardia
spp. isolated from pathogenic samples.
Comparative Evaluation of Ligation-Mediated PCR and Spoligotyping as Screening Methods for Genotyping of
Mycobacterium tuberculosis
Strains
Authors: Bonora Stefano , Gutierrez M. Cristina , Di Perri Giovanni , Brunello Francesca , Allegranzi Benedetta , Ligozzi Marco , Fontana Roberta , Concia Ercole , Vincent Veronique
Published: December 2019

Spoligotyping has been suggested as a screening test in multistep genotyping of
Mycobacterium tuberculosis
strains. Relying on restriction fragment length polymorphism (RFLP) analysis with IS
6110
(IS
6110
RFLP analysis) as a “gold standard,” we performed a comparative evaluation of spoligotyping and ligation-mediated PCR (LMPCR), a recently described PCR-based typing method, as rapid screening tests for fingerprinting of 158
M. tuberculosis
strains collected in Verona, Italy. LMPCR seemed to be comparable to spoligotyping in terms both of feasibility with rapidly extracted DNA and of generation of software-analyzable images. Moreover, LMPCR grouped considerably fewer strains than spoligotyping (38 versus 67%) and was found to reduce the cluster overestimation rate (26.3 versus 58%) and to give a better discriminatory index (0.992 versus 0.970) compared to spoligotyping. In our geographical region, where there was no evidence of clustered strains carrying fewer than six IS
6110
copies, LMPCR was found to be more discriminatory than spoligotyping. We also evaluated two models of three-step typing strategies, involving the use of spoligotyping and LMPCR as screening methods and IS
6110
RFLP analysis as a further supporting test. LMPCR proved to be a more effective first-step test than spoligotyping, significantly reducing the need for subtyping. LMPCR should be considered an alternative to spoligotyping as a rapid screening method for
M. tuberculosis
fingerprinting, particularly in areas with a low prevalence of
M. tuberculosis
strains carrying few copies of IS
6110
.
The
cps
Genes of
Streptococcus suis
Serotypes 1, 2, and 9: Development of Rapid Serotype-Specific PCR Assays
Authors: Smith Hilde E. , Veenbergen Vincent , van der Velde Joeke , Damman Marloes , Wisselink Henk J. , Smits Mari A.
Published: December 2019

We developed three type-specific PCR assays for the rapid and sensitive detection of
Streptococcus suis
serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of
S. suis
serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (
cps
) locus of
S. suis
serotype 2. Here we extended these studies and characterized major parts of the
cps
loci of
S. suis
serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual
cps
genes and chromosomal DNAs from the 35 different serotypes. Four genes of
S. suis
serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of
S. suis
serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of
S. suis
serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.
Molecular Analysis of Riboflavin Synthesis Genes in
Bartonella henselae
and Use of the
ribC
Gene for Differentiation of
Bartonella
Species by PCR
Authors: Bereswill Stefan , Hinkelmann Silke , Kist Manfred , Sander Anna
Published: December 2019

The biosynthesis pathway for riboflavin (vitamin B
2
), the precursor of the essential cofactors flavin mononucleotide and flavin adenine dinucleotide, is present in bacteria and plants but is absent in vertebrates. Due to their conservation in bacterial species and their absence in humans, the riboflavin synthesis genes should be well suited either for detection of bacterial DNA in human specimens or for the differentiation of pathogenic bacteria by molecular techniques. A DNA fragment carrying the genes
ribD
,
ribC
, and
ribE
, which encode homologues of riboflavin deaminase (RibD) and subunits of riboflavin synthetase (RibC and RibE), respectively, was isolated from a plasmid-based DNA library of the human pathogen
Bartonella henselae
by complementation of a
ribC
mutation in
Escherichia coli
. Sequence analysis of the
ribC
gene region in strains of
B. henselae
, which were previously shown to be genetically different, revealed that the
ribC
gene is highly conserved at the species level. PCR amplification with primers derived from the
ribC
locus of
B. henselae
was used to isolate the corresponding DNA regions in
B. bacilliformis
,
B. clarridgeiae
, and
B. quintana
. Sequence analysis indicated that the riboflavin synthesis genes are conserved and show the same operon-like genetic organization in all four
Bartonella
species. Primer oligonucleotides designed on the basis of localized differences within the
ribC
DNA region were successfully used to develop species-specific PCR assays for the differentiation of
B. henselae
,
B. clarridgeiae
,
B. quintana
, and
B. bacilliformis
. The results obtained indicate that the riboflavin synthesis genes are excellent targets for PCR-directed differentiation of these emerging pathogens. The PCR assays developed should increase our diagnostic potential to differentiate
Bartonella
species, especially
B. henselae
and the newly recognized species
B. clarridgeiae.
Evaluation of the Tuberculin Gamma Interferon Assay: Potential To Replace the Mantoux Skin Test
Authors: Pottumarthy Sudha , Morris Arthur J. , Harrison Adrian C. , Wells Virginia C.
Published: December 2019

We evaluated an in vitro test of cell-mediated immunity, the tuberculin gamma interferon assay,
Quanti
FERON-TB (QIFN), in 455 individuals from three groups: group I, 237 immigrants from high-risk countries; group II, 127 health care workers undergoing Mantoux testing; group III, 91 patients being investigated for possible active tuberculosis (79 patients) or
Mycobacterium avium-Mycobacterium intracellulare
complex infection (12 patients). The QIFN results were compared either to those of the Mantoux test or to microbiological and clinical diagnosis, as appropriate. In each group the correlation between the diameter of induration for the skin test and the magnitude of QIFN response was significant and of moderate strength (Spearman’s rank correlation coefficient; ρ = 0.59 to 0.61;
P
< 0.001). For group I, the agreement between QIFN and Mantoux results was 89% for Mantoux-negative and 64% for Mantoux-positive individuals. For group II, when ≥10-mm-diameter induration was taken as positive, the agreement was 81% for Mantoux-negative and 67% for Mantoux-positive individuals. For group III, agreement was 81% for Mantoux-negative and 86% for Mantoux-positive patients. For patients being evaluated for active tuberculosis, the performance of the Mantoux test was not statistically different from that of the QIFN assay. In patients with active tuberculosis, the assay had a sensitivity of 77%, not significantly higher for extrapulmonary than pulmonary cases (83% versus 74%). QIFN sensitivity was not significantly different for smear-negative or smear-positive cases (80% versus 71%). The QIFN assay is a potential replacement for the Mantoux test. The acceptability of these performance values and those of similar evaluations will determine the place this test will have in detecting evidence of mycobacterial infection.
Clinical Comparison of the
Treponema pallidum
CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing
Authors: Halling V. W. , Jones M. F. , Bestrom J. E. , Wold A. D. , Rosenblatt J. E. , Smith T. F. , Cockerill F. R.
Published: December 2019

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of ≥0.650 and ≤0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the
Treponema pallidum
microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.
Slime Production and Expression of the Slime-Associated Antigen by Staphylococcal Clinical Isolates
Authors: Ammendolia M. G. , Di Rosa R. , Montanaro L. , Arciola C. R. , Baldassarri L.
Published: December 2019

The ability to produce slime and to express a slime-associated antigen was examined in a collection of staphylococcal clinical isolates. Slime-producing strains were found among coagulase-negative staphylococci in percentages comparable to those reported in other studies; surprisingly, a high percentage of
Staphylococcus aureus
strains also were able to produce this extracellular material. In the latter case, this ability was strongly dependent on the presence of an additional carbohydrate source in the growth medium. Expression of the slime-associated antigen appeared to be species specific and confined to the
Staphylococcus epidermidis
sensu stricto isolates; its strong association with the ability of these strains to produce thicker biofilms indicated slime-associated antigen as a possible virulence marker for
S. epidermidis
.
Ciprofloxacin Resistance in
Campylobacter jejuni
Isolates: Detection of
gyrA
Resistance Mutations by Mismatch Amplification Mutation Assay PCR and DNA Sequence Analysis
Authors: Zirnstein Gerald , Li Yu , Swaminathan Bala , Angulo Frederick
Published: December 2019

The
gyrA
quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible
Campylobacter jejuni
isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this
gyrA
mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the
gyrA
mutation without the need for sequencing the
gyrA
gene.
Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene
Authors: Tunney Michael M. , Patrick Sheila , Curran Martin D. , Ramage Gordon , Hanna Donna , Nixon James R. , Gorman Sean P. , Davis Richard I. , Anderson Neil
Published: December 2019

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for
Propionibacterium acnes
and polyclonal antiserum specific for
Staphylococcus
spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.
Comparison of Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methods for Testing Activity of Cefditoren against
Streptococcus pneumoniae
Authors: Kelly Linda M. , Jacobs Michael R. , Appelbaum Peter C.
Published: December 2019

This study evaluated the susceptibility of pneumococci to cefditoren by agar dilution and microdilution methods (both in air) and by E-test (AB Biodisk, Solna, Sweden) and disk diffusion methods (both in CO
2
). By the three MIC tests, the MICs at which 50 and 90% of isolates were inhibited (MIC
50
s and MIC
90
s) were, respectively, as follows (in micrograms per milliliter): for the 65 penicillin-susceptible strains tested, 0.016 and 0.03 (by agar dilution), 0.016 and 0.03 (by microdilution), and 0.016 and 0.03 (by E test); for the 68 penicillin-intermediate strains tested, 0.125 and 0.5 (by agar dilution), 0.125 and 0.5 (by microdilution), and 0.25 and 0.5 (by E test); and for the 67 penicillin-resistant strains tested, 1.0 and 1.0 (by agar dilution), 0.5 and 1.0 (by microdilution), and 1.0 and 1.0 (by E test). With tentative cefditoren breakpoints (in micrograms per milliliter) of ≤2.0 (susceptible), 4.0 (intermediate), and ≥8.0 (resistant), all strains were susceptible to cefditoren by agar, microdilution, and E-test results; with breakpoints of ≤1.0, 2.0, and ≥4.0 μg/ml, 97% of strains were cefditoren susceptible by agar dilution results, 98% were susceptible by microdilution results, and 99% were susceptible by E-test results. When microdilution and E-test results were compared to those from the reference agar dilution method, 191 (95.5%) and 183 (91.5%) of strains gave essential agreement (±1 log
2
dilution); 8 (2.7%) minor discrepancies were found for both methods with a breakpoint of ≤1.0 μg/ml, and no discrepancies were found with a breakpoint of ≤2.0 μg/ml. Disk test results (breakpoint, ≤1.0 μg/ml) produced 2 major and 30 minor errors, with corresponding zone diameters (in millimeters) of ≥20 (susceptible), 17 to 19 (intermediate), and ≤16 (resistant); a ≤2.0-μg/ml breakpoint yielded zone diameters of ≥16 mm (susceptible). All three methods for testing the MIC of cefditoren showed excellent correlation.
Is There an Ecological Relationship between Rates of Antibiotic Resistance of Species of the Genus
Streptococcus
?
Authors: Gómez-Lus Rafael , Granizo Juan J. , Aguilar Lorenzo , Bouza Emilio , Gutierrez Avelino , García-de-Lomas Juan ,   the Spanish Surveillance Group for Respiratory Pathogens
Published: December 2019

The relationship between resistance to antibiotics on the part of
Streptococcus pneumoniae
and
Streptococcus pyogenes
was studied by comparing different prevalences of resistance among hospitals obtained from a recent microbiological surveillance of community-acquired respiratory tract infections. A high correlation for erythromycin resistance was found between
S. pneumoniae
isolates from lower respiratory tract infections and
S. pyogenes
isolates collected from pharyngeal swabs.
Elimination of Bacterial DNA from
Taq
DNA Polymerases by Restriction Endonuclease Digestion
Authors: Carroll Nora M. , Adamson Peter , Okhravi Narciss
Published: December 2019

The incidence of false positives due to the presence of bacterial DNA in
Taq
DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.
Prevalence of
Borrelia burgdorferi
and Granulocytic and Monocytic Ehrlichiae in
Ixodes ricinus
Ticks from Southern Germany
Authors: Baumgarten Birgit U. , Röllinghoff Martin , Bogdan Christian
Published: December 2019

A total of 287 adult
Ixodes ricinus
ticks, collected in two regions of southern Germany (Frankonia and Baden-Württemberg) where
Borrelia burgdorferi
infections are known to be endemic, were examined for the presence of 16S ribosomal DNA specific for the
Ehrlichia phagocytophila
genogroup,
E. chaffeensis
,
E. canis
, and
B. burgdorferi
by nested PCR. Totals of 2.2% (6 of 275) and 21.8% (65 of 275) of the ticks were positive for the
E. phagocytophila
genogroup and
B. burgdorferi
, respectively. Two ticks (0.7%) were coinfected with both bacteria. Of 12 engorged
I. ricinus
ticks collected from two deer, 8 (67%) were positive for the
E. phagocytophila
genogroup and one (8%) was positive for
B. burgdorferi
. There was no evidence of infection with
E. canis
or
E. chaffeensis
in the investigated tick population. The nucleotide sequences of the 546-bp
Ehrlichia
PCR products differed at one or two positions from the original sequence of the human granulocytic ehrlichiosis (HGE) agent (S.-M. Chen, J. S. Dumler, J. S. Bakken, and D. H. Walker, J. Clin. Microbiol. 32:589–595, 1994). Three groups of sequence variants were detected; two of these were known to occur in other areas in Europe or the United States, whereas one has not been reported before. Thus, in the German
I. ricinus
tick population closely related granulocytic ehrlichiae are prevalent, which might represent variants of
E. phagocytophila
or the HGE agent.
Value of PCR for Detection of
Toxoplasma gondii
in Aqueous Humor and Blood Samples from Immunocompetent Patients with Ocular Toxoplasmosis
Authors: Bou Germán , Figueroa Marta S. , Martí-Belda Paloma , Navas Enrique , Guerrero Antonio
Published: December 2019

Toxoplasma gondii
infection is an important cause of chorioretinitis in the United States and Europe. Most cases of
Toxoplasma
chorioretinitis result from congenital infection. Patients are often asymptomatic during life, with a peak incidence of symptomatic illness in the second and third decades of life. Diagnosis is mainly supported by ophthalmological examination and a good response to installed therapy. However, establishment of a diagnosis by ophthalmological examination alone can be difficult in some cases. To determine the diagnostic value of PCR for the detection of
T. gondii
, 56 blood and 56 aqueous humor samples from 56 immunocompetent patients were examined. Fifteen patients with a diagnosis of ocular toxoplasmosis had increased serum anti-
T. gondii
immunoglobulin G levels but were negative for anti-
T. gondii
immunoglobulin M (group 1), and 41 patients were used as controls (group 2). Samples were taken before antiparasitic therapy was initiated, and only one blood sample and one aqueous humor sample were obtained for each patient. Single nested PCRs and Southern blot hybridization were performed with DNA extracted from these samples. The results obtained showed sensitivity and specificity values of 53.3 and 83%, respectively. Interestingly, among all patients with ocular toxoplasmosis, a positive PCR result with the aqueous humor sample was accompanied by a positive PCR result with the blood sample. This result suggests that ocular toxoplasmosis should not be considered a local event, as PCR testing of blood samples from patients with ocular toxoplasmosis yielded the same result as PCR testing of aqueous humor samples. PCR testing may be useful for discriminating between ocular toxoplasmosis and other ocular diseases, and also can avoid the problems associated with ocular puncture.
Biotyping and Virulence Properties of Skin Isolates of
Candida parapsilosis
Authors: De Bernardis Flavia , Mondello Francesca , San Millàn Rosario , Pontòn Josè , Cassone Antonio
Published: December 2019

The biotype and virulence of skin isolates of
Candida parapsilosis
were compared with blood isolates of the same fungus. Morphotype, resistotype, and electrophoretic karyotype determinations did not reveal any special cluster with a unique or dominant pathogenic feature among all of the isolates, regardless of their source. However, all cutaneous isolates had uniformly elevated secretory aspartyl-protease (Sap) activity, more than four times higher than the enzyme activity of the blood isolates. They were also highly vaginopathic in a rat vaginitis model, being significantly more virulent than blood isolates in this infection model. In contrast, skin isolates were nonpathogenic in systemic infection of cyclophosphamide-immunodepressed mice, while some blood isolates were, in this model, highly pathogenic (median survival time, 2 days, with internal organ invasion at autopsy). Finally, skin isolates did not differ, as a whole, from blood isolates in their adherence to plastic. This property was associated with a morphotype, as defined by a colony with continuous fringe, which was present among both skin and blood isolates. While confirming the genetic heterogenicity of
C. parapsilosis
, our data strongly suggest that the potential of this fungus to cause mucosal disease is associated with Sap production and is substantially distinct from that of systemic invasion.
Detection of Specific Immunoglobulin E during Maternal, Fetal, and Congenital Toxoplasmosis
Authors: Villena I. , Aubert D. , Brodard V. , Quereux C. , Leroux B. , Dupouy D. , Remy G. , Foudrinier F. , Chemla C. , Gomez-Marin J. E. , Pinon J. M.
Published: December 2019

Toxoplasma immunoglobulin E (IgE) antibodies in 664 serum samples were evaluated by using an immunocapture method with a suspension of tachyzoites prepared in the laboratory in order to evaluate its usefulness in the diagnosis of acute
Toxoplasma gondii
infection during pregnancy, congenital infection, and progressive toxoplasmosis. IgE antibodies were never detected in sera from seronegative women, from patients with chronic toxoplasma infection, or from infants without congenital toxoplasmosis. In contrast, they were detected in 86.6% of patients with toxoplasmic seroconversion, and compared with IgA and IgM, the short kinetics of IgE was useful to date the infection precisely. For the diagnosis of congenital toxoplasmosis, specific IgE detected was less frequently than IgM or IgA (25 versus 67.3%), but its detection during follow-up of children may be interesting, reflecting an immunological rebound. Finally, IgE was detected early and persisted longer in progressive toxoplasmosis with cervical adenopathies, so it was also a good marker of the evolution of toxoplasma infection.
Degenerate and Nested PCR: a Highly Sensitive and Specific Method for Detection of Human Papillomavirus Infection in Cutaneous Warts
Authors: Harwood Catherine A. , Spink Patricia J. , Surentheran T. , Leigh Irene M. , de Villiers Ethel-Michele , McGregor Jane M. , Proby Charlotte M. , Breuer Judith
Published: December 2019

The role of human papillomavirus (HPV) in anogenital carcinogenesis is firmly established, but evidence that supports a similar role in skin remains speculative. Immunosuppressed renal transplant recipients have an increased incidence of viral warts and nonmelanoma skin cancer, and the presence of HPV DNA in these lesions, especially types associated with the condition epidermodysplasia verruciformis (EV), has led to suggestions that HPV may play a pathogenic role. However, differences in the specificities and sensitivities of techniques used to detect HPV in skin have led to wide discrepancies in the spectrum of HPV types reported. We describe a degenerate nested PCR technique with the capacity to detect a broad spectrum of cutaneous, mucosal, and EV HPV types. In a series of 51 warts from 23 renal transplant recipients, this method detected HPV DNA in all lesions, representing a significant improvement over many previously published studies. Cutaneous types were found in 84.3% of warts and EV types were found in 80.4% of warts, whereas mucosal types were detected in 27.4% of warts. In addition, the method allowed codetection of two or more distinct HPV types in 94.1% of lesions. In contrast, single HPV types were detected in all but 1 of 20 warts from 15 immunocompetent individuals. In summary, we have established a highly sensitive and comprehensive degenerate PCR methodology for detection and genotyping of HPV from the skin and have demonstrated a diverse spectrum of multiple HPV types in cutaneous warts from transplant recipients. Studies designed to assess the significance of these findings to cutaneous carcinogenesis are under way.
Prevalence of
Shigella
Enterotoxins 1 and 2 among
Shigella
Strains Isolated from Patients with Traveler's Diarrhea
Authors: Vargas Martha , Gascon Joaquim , De Anta Maria Teresa Jimenez , Vila Jordi
Published: December 2019

Shigella
spp. are known primarily as a cause of bacillary dysentery. However, in an initial phase, numerous patients exhibit watery diarrhea that may or may not be followed by dysentery. New virulence factors associated with the species of
Shigella
have recently been described. These are enterotoxins 1 and 2 of
Shigella
(ShET-1 and ShET-2, respectively). The aim of the present study was to determine the prevalence of ShET-1 and ShET-2 in species of
Shigella
isolated from patients with traveler's diarrhea. During the period from 1993 to 1998, stool samples from 500 travelers with diarrhea were cultured for the isolation of
Shigella
spp. and other enteropathogens. The detection of ShET-1 and ShET-2 was performed by a PCR technique with specific primers. Among a total of 51 strains of
Shigella
isolated during this period (22
S. flexneri
, 26
S. sonnei
, and 3
S. dysenteriae
strains), at least one enterotoxin was detected in 31 (60.78%) strains; 2 (9.09%; both of which were
S. flexneri
strains) produced only ShET-1, while 21 (41.17%; 3
S. flexneri
, 15
S. sonnei
, and 3
S. dysenteriae
strains) produced ShET-2. Furthermore, 8 (15.69%) of 22
S. flexneri
strains presented both enterotoxins. Our results show that the prevalence of ShET-2 was high in all the
Shigella
species studied and confirm that ShET-1 is detected only in
S. flexneri.
Bartonella vinsoniisubsp.berkhoffiiand Related Members of the Alpha Subdivision of theProteobacteriain Dogs with Cardiac Arrhythmias, Endocarditis, or Myocarditis
Authors: Breitschwerdt Edward B. , Atkins Clarke E. , Brown Talmage T. , Kordick Dorsey L. , Snyder Patti S.
Published: December 2019

Cardiac arrhythmias, endocarditis, or myocarditis was identified in 12 dogs, of which 11 were seroreactive toBartonella vinsoniisubspeciesberkhoffiiantigens. Historical abnormalities were highly variable but frequently included substantial weight loss, syncope, collapse, or sudden death. Fever was an infrequently detected abnormality. Cardiac disease was diagnosed following an illness of short duration in most dogs, but a protracted illness of at least 6 months' duration was reported for four dogs. Valvular endocarditis was diagnosed echocardiographically or histologically in eight dogs, two of which also had moderate to severe multifocal myocarditis. Four dogs lacking definitive evidence of endocarditis were included because of seroreactivity toB. vinsoniiantigens and uncharacterized heart murmurs and/or arrhythmias. Alpha proteobacteria were not isolated from the blood by either conventional or lysis centrifugation blood culture techniques. Using PCR amplification and DNA sequencing of a portion of the 16S rRNA gene,B. vinsoniiwas identified in the blood or heart valves of three dogs. DNA sequence alignment of PCR amplicons derived from blood or tissue samples from seven dogs clustered among members of the alpha subdivision of theProteobacteriaand suggested the possibility of involvement of one or more alpha proteobacteria; however, because of the limited quantity of sequence, the genus could not be identified. Serologic or molecular evidence of coinfection with tick-transmitted pathogens, includingEhrlichia canis,Babesia canis,Babesia gibsonii, or spotted fever group rickettsiae, was obtained for seven dogs. We conclude thatB. vinsoniisubsp.berkhoffiiand closely related species of alpha proteobacteria are an important, previously unrecognized cause of arrhythmias, endocarditis, myocarditis, syncope, and sudden death in dogs.
Genotypic and Phenotypic Characterization of “
Streptococcus milleri
” Group Isolates from a Veterans Administration Hospital Population
Authors: Clarridge Jill E. , Osting Cheryl , Jalali Mehri , Osborne Janet , Waddington Michael
Published: December 2019

Because identification of the species within the “Streptococcus milleri” group is difficult for the clinical laboratory as the species share overlapping phenotypic characteristics, we wished to confirm biochemical identification with identification by 16S rRNA gene sequence analysis. Ninety-four clinical isolates previously identified as the “Streptococcus milleri” group were reclassified as
S. anginosus
,
S. constellatus
, or
S. intermedius
with the API 20 Strep system (bioMerieux Vikek, Hazelton, Mo.) and the Fluo-card (Key Scientific, Round Rock, Tex.). In addition, we determined the Lancefield group, hemolysis, colony size, colony texture, repetitive extragenic palindromic PCR (rep-PCR) pattern, and cellular fatty acid (CFA) profile (MIDI, Newark, Del.). 16S rRNA gene sequence analysis with 40 selected representative strains showed three distinct groups, with
S. constellatus
and
S. intermedius
found to be more closely related to each other than to
S. anginosus
, and further distinguished a biochemically distinct group of urogenital isolates within the
S. anginosus
group of isolates. Except for strains unreactive with the Fluo-card (8%), all
S. anginosus
and
S. intermedius
strains identified by sequencing were similarly identified by biochemical testing. However, 23% of the selected
S. constellatus
isolates identified by sequencing (9% of all
S. constellatus
isolates) would have been identified as
S. anginosus
or
S. intermedius
by biochemical tests. Although most
S. anginosus
strains formed one unique cluster by CFA analysis and most
S. constellatus
strains showed similar rep-PCR patterns, neither method was sufficiently dependable for identification. Whereas Lancefield group or lactose fermentation did not correspond to sequence or biochemical type,
S. constellatus
was most likely to be beta-hemolytic and
S. intermedius
was most likely to have a dry colony type. The most frequent isolate in our population was
S. constellatus
, followed by
S. anginosus
. There was an association of
S. anginosus
with a gastrointestinal or urogenital source, and there was an association of
S. constellatus
and
S. intermedius
with both the respiratory tract and upper-body abscesses.
Importance of Selective Media for Recovery of Yeasts from Clinical Specimens
Authors: Sandven Per , Lassen Jørgen
Published: December 2019

We compared the recovery of yeasts from clinical specimens cultured on routine bacteriological media to the recovery of yeast from specimens cultured on a selective fungal medium (Sabouraud agar). The use of Sabouraud agar was especially important in cases of mixed cultures, since in such cases yeast was recovered on bacteriological media from only 50% of 44 yeast-positive pus specimens and from 22.5% of 22 yeast-positive throat specimens. The use of a selective fungal medium is therefore necessary to ensure the detection of yeast in specimens containing a mixture of bacteria and yeasts. As a result, clinicians must request yeast isolation when clinically indicated, and the microbiological laboratory must add a selective fungal medium when clinically significant yeasts are likely to be encountered. It is also important that selective fungal media be used in clinical studies of yeast infections.
Evaluation of a New Commercially Available Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Japanese Encephalitis Infections
Authors: Cuzzubbo Andrea J. , Endy Timothy P. , Vaughn David W. , Solomon Tom , Nisalak Ananda , Kalayanarooj Siripen , Dung Nguyen Minh , Warrilow David , Aaskov John , Devine Peter L.
Published: December 2019

A new commercial enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Japanese encephalitis virus infections showed a sensitivity of 88% with sera and 81% with cerebrospinal fluid and a specificity of 97% with sera from patients with primary and secondary dengue virus infections. Specificity was 100% when samples from nonflavivirus infections were tested.
Cloning and Characterization of a Nonhemolytic Phospholipase C Gene from
Burkholderia pseudomallei
Authors: Korbsrisate Sunee , Suwanasai Nuttiga , Leelaporn Amornrut , Ezaki Takayuki , Kawamura Yoshiaki , Sarasombath Suttipant
Published: December 2019

We cloned and characterized a phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) gene from
Burkholderia pseudomallei
. DNA sequence analysis of the gene indicated an open reading frame coding for 700 amino acids with a 34-amino-acid signal peptide. When cleaved, this yields a secreted 73-kDa mature protein. The deduced amino acid sequence exhibited 48% similarity to that of a nonhemolytic PLC from
Pseudomonas aeruginosa
. The expressed PC-PLC was heat stable, nonhemolytic for sheep erythrocytes, and active between pH 2 and 8. Western blot analysis with sera from melioidosis patients indicated that they produced immunoglobulin M antibodies against this PC-PLC protein.
Viability of
Trichomonas vaginalis
in Transport Medium
Authors: Beverly A. L. , Venglarik M. , Cotton B. , Schwebke J. R.
Published: December 2019

The ability of Amies gel agar transport medium to maintain the viability of
Trichomonas vaginalis
was determined by comparing transported vaginal specimens to specimens immediately inoculated into culture medium. The prevalence of trichomonosis in the study population was 26% (68 of 260 women). The immediate inoculation method detected infections in 64 of 68 infected women (sensitivity of 94.1%). The transport method detected 62 of 68 infections (sensitivity of 91.2%). There was no significant difference between the two methods.
Recovery of Mycobacteria from Patients with Cystic Fibrosis
Authors: Bange Franz-Christoph , Kirschner Philip , Böttger Eric C.
Published: December 2019

Despite decontamination, overgrowth by pseudomonads renders cultural isolation of mycobacteria from respiratory specimens of patients with cystic fibrosis (CF) difficult or impossible. We performed a prospective study by comparing levels of reduction of overgrowth and recovery of mycobacteria using either pretreatment with
N
-acetyl-
l
-cysteine (NALC)–NaOH alone or pretreatment with NALC-NaOH and then with oxalic acid. From 406 specimens of 148 CF patients, 11 specimens were positive for mycobacteria, 5 of which grew mycobacteria after decontamination by either procedure. Three specimens grew mycobacteria only after decontamination with NALC-NaOH, whereas three specimens grew mycobacteria only after treatment with NALC-NaOH followed by oxalic acid but were overgrown after decontamination with NALC-NaOH. Thus, inactivation of mycobacteria by the more aggressive oxalic acid treatment offsets its beneficial effect of reducing the proportion of cultures overgrown with microorganisms other than mycobacteria.
Transport and Storage of Fresh and Frozen Gastric Biopsy Specimens for Optimal Recovery of
Helicobacter pylori
Authors: Heep Markus , Scheibl Karl , Degrell Andrea , Lehn Norbert
Published: December 2019

The viability of
Helicobacter pylori
in vitro and in gastric biopsy specimens was determined. Recovery rates were 94, 87, and 77% from biopsy specimens in Portagerm pylori in cooled containers after 1, 2, and 3 days of transport, respectively (
n
= 307), and 97% if stored and shipped in glycerol broth at −70°C (
n
= 232).