bluuebunny/crossref_metadata_2025 huggingface dataset:
Antipneumococcal Activity of Telithromycin by Agar Dilution, Microdilution, E Test, and Disk Diffusion Methodologies
Authors: Davies Todd A. , Kelly Linda M. , Jacobs Michael R. , Appelbaum Peter C.
Published: December 2019

Agar dilution and microdilution (both in air) and E test and disk diffusion (both in air and CO
2
) were used to test the activity of telithromycin against 110 erythromycin-susceptible and 106 erythromycin-resistant pneumococci. The MICs at which 50 and 90% of strains are inhibited (MIC
50
s and MIC
90
s, respectively) for erythromycin-susceptible strains varied between 0.008 and 0.016 μg/ml and 0.016 and 0.03 μg/ml when the samples were incubated in air. By comparison, telithromycin MIC
50
s and MIC
90
s for erythromycin-resistant strains were in air 0.03 to 0.125 and 0.125 to 0.5 μg/ml, respectively. When agar dilution was used as the reference method, essential agreement was found for 112 of 216 strains (51.9%) for microdilution, 168 of 216 (77.8%) for E test in air, and 132 of 216 (61.1%) for E test in CO
2
. With the exception of four strains tested by E test in CO
2
, all organisms were susceptible to a proposed telithromycin susceptibility breakpoint of ≤1 μg/ml. By disk diffusion with 15-μg telithromycin disks, all strains but one had zones of inhibition ≥19 mm in diameter when incubated in CO
2
, while all strains had zone diameters of ≥22 mm when incubated in air. Zone diameters in air were generally 4 to 5 mm larger than in CO
2
. By all methods, MICs and zones of all erythromycin-resistant strains occurred in clusters separated from those seen with erythromycin-susceptible strains. The results for macrolide-resistant strains with
erm
and
mef
resistance determinants were similar. The results show that (i) telithromycin is very active against erythromycin-susceptible and -resistant strains irrespective of macrolide resistance mechanism; (ii) susceptibility to telithromycin can be reliably tested by the agar, microdilution, E test, and disk diffusion methods; and (iii) incubation in CO
2
led to smaller zones by disk diffusion and higher MICs by E test, but at a susceptible MIC breakpoint of ≤1 μg/ml and a susceptible zone diameter cutoff of ≥19 mm in CO
2
, 215 of 216 strains were found to be susceptible to telithromycin.
Fractionation of Membrane Components from Tachyzoite Forms ofToxoplasma gondii: Differential Recognition by Immunoglobulin M (IgM) and IgG Present in Sera from Patients with Acute or Chronic Toxoplasmosis
Authors: Giraldo Mónica , Cannizzaro Hélia , Ferguson Michael A. J. , Almeida Igor C. , Gazzinelli Ricardo T.
Published: December 2019

Tachyzoite forms ofToxoplasma gondiiwere subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acute and chronic toxoplasmosis, respectively. Improved scores of specificity (100%) and sensitivity (100%) were achieved when a secondary antibody against human IgG1 instead of total IgG was employed to measure the reactivity of IgG antibodies with the F3 fraction. To purify tachyzoite antigens recognized by human IgM or IgG antibodies, the F2 or F3 fraction was loaded onto an octyl-Sepharose column and eluted with a propan-1-ol gradient. The main antigen(s) recognized by IgM or IgG eluted in a single peak from the octyl-Sepharose resin loaded with either F2 (30 to 50% propan-1-ol) or F3 (15 to 35% propan-1-ol), respectively. These semipurified fractions gave improved scores when used to detectT. gondii-specific IgM (95.7% specificity and 81.8% sensitivity) or IgG (100% specificity and 93.75% sensitivity) in an enzyme-linked immunosorbent assay. Further biochemical and immunological analyses of antigens partially purified from F2 and F3 indicate that glycoinositolphospholipids are preferentially recognized by IgM, whereas proteins of approximately 30 to 40 kDa are recognized by IgG, elicited duringT. gondiiinfection in humans.
Evaluation of Diagnostic Value and Epidemiological Implications of PCR for
Pneumocystis carinii
in Different Immunosuppressed and Immunocompetent Patient Groups
Authors: Sing Andreas , Trebesius Karlheinz , Roggenkamp Andreas , Rüssmann Holger , Tybus Karin , Pfaff Friederike , Bogner Johannes R. , Emminger Christoph , Heesemann Jürgen
Published: December 2019

To evaluate the value of single and nested PCRs for diagnosis of
Pneumocystis carinii
pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human immunodeficiency virus [HIV]-positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower positive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any additional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent
P. carinii
colonizations in about 17 to 20% of immunocompetent and immunosuppressed non-HIV patients.
Identification of
Campylobacter jejuni
,
C. coli
,
C. lari
,
C. upsaliensis
,
Arcobacter butzleri
, and
A. butzleri
-Like Species Based on the
glyA
Gene
Authors: Al Rashid Shahnaz Tahihra , Dakuna Irene , Louie Helena , Ng David , Vandamme Peter , Johnson Wendy , Chan Voon Loong
Published: December 2019

Currently, the detection and identification of
Campylobacter
and
Arcobacter
species remains arduous, largely due to cross-species phenotypic similarities and a relatively narrow spectrum of biochemical reactivity. We have developed a PCR-hybridization strategy, wherein degenerate primers are used to amplify
glyA
fragments from samples, which are then subjected to species-specific oligodeoxyribonucleotide probe hybridizations, to identify and distinguish between
Campylobacter jejuni
,
C. coli
,
C. lari
,
C. upsaliensis
,
Arcobacter butzleri
, and an
A. butzleri
-like species. Evaluation of this strategy with genomic DNA from different type strains suggests that this approach is both specific and sensitive and thus may be applicable in a diagnostic assay to identify and differentiate these highly related species.
Evaluation of Ciprofloxacin as a Representative of Veterinary Fluoroquinolones in Susceptibility Testing
Authors: Riddle Christa , Lemons Carol L. , Papich Mark G. , Altier Craig
Published: December 2019

Currently in veterinary medicine, ciprofloxacin is often used in susceptibility testing to represent the entire class of fluoroquinolone antimicrobials. Using quality control organisms as well as clinical isolates, we compared the MIC of ciprofloxacin to those of three other fluoroquinolones used in animals and found that ciprofloxacin is not an adequate representative of other members of this class.
Restriction Fragment Length Polymorphism Analysis Using Random Chromosomal Gene Probes for Epidemiological Analysis of
Campylobacter jejuni
Infections
Authors: Fujimoto Shuji , Umene Kenichi , Saito Mitsumasa , Horikawa Kazumi , Blaser Martin J.
Published: December 2019

We have evaluated the ability of a new genotyping method for
Campylobacter jejuni
based on restriction fragment length polymorphisms using random chromosomal gene probes. DNAs from
C. jejuni
strains digested with each of three restriction enzymes,
Hha
I,
Hae
III, and
Hpa
II, were analyzed by Southern hybridization using each of two unrelated cosmid clones, P14 and P15 (respectively containing 30- and 35-kb genomic DNA fragments of
C. jejuni
strain OH4384). The method reported provides a stable and discriminating means for identifying
C. jejuni
strains and should be useful for epidemiological analyses.
Evaluation of a New Commercially Available Immunoglobulin M and Immunoglobulin G Immunochromatographic Test for Diagnosis of Melioidosis Infection
Authors: Cuzzubbo Andrea J. , Chenthamarakshan V. , Vadivelu Jamuna , Puthucheary Savithiri D. , Rowland Denise , Devine Peter L.
Published: December 2019

An immunochromatographic test for the rapid determination of immunoglobulin M (IgM) and IgG antibodies to
Burkholderia pseudomallei
was evaluated by using sera from bacteriologically confirmed melioidosis patients and high-risk and clinically suspected patients, along with disease control groups. The sensitivities were 100 and 93% for the IgG and IgM tests, respectively, while the specificity was 95% for both assays. The test was rapid and simple to perform, with results obtained in 10 min.
Pooling of Urine Specimens for Detection of Asymptomatic
Chlamydia trachomatis
Infections by PCR in a Low-Prevalence Population: Cost-Saving Strategy for Epidemiological Studies and Screening Programs
Authors: Morré Servaas A. , Meijer Chris J. L. M. , Munk Christian , Krüger-Kjaer Susanne , Winther Jeanette F. , Jørgensens Hans O. , van den Brule Adriaan J. C.
Published: December 2019

Pooling, in groups of five, of urine specimens from asymptomatically infected men in a population with 4% prevalence, as determined by case finding, is 100% sensitive and specific and results in a 60.5% reduction in the number of tests needed. Pooling of urine specimens in groups of 10 for the estimation of population-based prevalence is 96.1% sensitive and 100% specific and saves 90% of the test costs.
Disseminated Infection Caused by
Scedosporium prolificans
in a Patient with Acute Multilineal Leukemia
Authors: de Batlle J. , Motjé M. , Balanzà R. , Guardia R. , Ortiz R.
Published: December 2019

In this report, we describe a case of disseminated infection caused by
Scedosporium prolificans
(
S. inflatum
) in a patient affected by chemotherapy-induced acute multilineal leukemia and neutropenia. For the fungus isolated in four blood cultures, high MICs of currently available antifungal agents were found. Postmortem examination revealed multiorgan involvement.
Bartonella vinsonii
subsp.
berkhoffii
as an Agent of Afebrile Blood Culture-Negative Endocarditis in a Human
Authors: Roux Véronique , Eykyn Susannah J. , Wyllie Sarah , Raoult Didier
Published: December 2019

We report a case of endocarditis in a human infected with
Bartonella vinsonii
subsp.
berkhoffii
, which causes bacteremia and endocarditis in dogs. Bacterial identification was established by PCR amplification and sequencing of an intergenic spacer region (ITS1), 16S ribosomal DNA, and a gene encoding citrate synthase (
gltA
).
Bartonella
antibodies were detected by immunofluorescence.
Detection of
Legionella
Species in Respiratory Specimens Using PCR with Sequencing Confirmation
Authors: Cloud J. L. , Carroll K. C. , Pixton P. , Erali M. , Hillyard D. R.
Published: December 2019

Legionella
spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of
Legionella pneumophila
. We can specifically detect the clinically significant
Legionella
species including
L. pneumophila
,
L. micdadei
,
L. longbeachae
,
L. bozemanii
,
L. feeleii
, and
L. dumoffii
. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for
Legionella
species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for
Legionella
species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.
Molecular Epidemiology of
Staphylococcus epidermidis
in a Neonatal Intensive Care Unit over a Three-Year Period
Authors: Villari P. , Sarnataro C. , Iacuzio L.
Published: December 2019

Coagulase-negative staphylococci, especially
Staphylococcus epidermidis
, are increasingly important nosocomial pathogens, particularly in critically ill neonates. A 3-year prospective surveillance of nosocomial infections in a neonatal intensive care unit (NICU) was performed by traditional epidemiologic methods as well as molecular typing of microorganisms. The aims of the study were (i) to quantify the impact of
S. epidermidis
on NICU-acquired infections, (ii) to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species, (iii) to evaluate the use of different methods for the epidemiologic typing of the isolates, and (iv) to characterize the occurrence and the spread of staphylococci with decreased glycopeptide susceptibility. Results confirmed that
S. epidermidis
is one of the leading causes of NICU-acquired infections and that the reduced glycopeptide susceptibility, if investigated by appropriate detection methods such as population analysis, is more common than is currently realized. Typing of isolates, which can be performed effectively through molecular techniques such as pulsed-field gel electrophoresis but not through antibiograms, showed that many of these infections are due to clonal dissemination and, thus, are potentially preventable by strict adherence to recommended infection control practices and the implementation of programs aimed toward the reduction of the unnecessary use of antibiotics. These strategies are also likely to have a significant impact on the frequency of the reduced susceptibility of staphylococci to glycopeptides, since this phenomenon appears to be determined either by more resistant clones transmitted from patient to patient or, to a lesser extent, by strains that become more resistant as a result of antibiotic pressure.
Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR
Authors: Corless C. E. , Guiver M. , Borrow R. , Edwards-Jones V. , Kaczmarski E. B. , Fox A. J.
Published: December 2019

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure
Taq
DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.
Zinc Salts Inactivate Clinical Isolates of Herpes Simplex Virus In Vitro
Authors: Arens Max , Travis Sharon
Published: December 2019

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37°C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.
Identification of Enterotoxigenic
Escherichia coli
Harboring Longus Type IV Pilus Gene by DNA Amplification
Authors: Gutiérrez-Cázarez Zita , Qadri Firdausi , Albert M. John , Girón Jorge A.
Published: December 2019

DNA amplification of
lngA
, the structural gene of longus type IV pilus produced by human enterotoxigenic
Escherichia coli
(ETEC) was achieved by the use of specific oligonucleotide primers designed from the nucleotide sequence of
lngA
. A 630-bp fragment representing the entire
lngA
gene was amplified in eight prototype strains previously characterized as longus positive. Five ETEC strains producing colonization factor antigen III (CFA III) (also a type IV pilus) were also positive by PCR, confirming the DNA homology between CFA III and longus. None of the non-ETEC and non-
E. coli
enteropathogens studied showed the 0.63-kbp amplicon. The procedure thus detected only ETEC strains harboring type IV pili genes with or without other colonization factors. Except for five
lngA
PCR-positive, probe-positive strains, all
lngA
PCR-positive strains produced the pilin as demonstrated by immunoblotting. To test the amplification procedure in a clinical setting, a collection of 264 fresh clinical
E. coli
strains isolated from 88 Mexican children with diarrhea was screened by PCR. Among 82 ETEC isolates found, 30 (36.5%) were
lngA
PCR-positive. Twenty-seven percent of the children shed ETEC that possessed
lngA
. In parallel with DNA probes or PCR protocols to detect enterotoxin genes, the
lngA
PCR method may prove useful for detection of ETEC harboring type IV pilus genes in epidemiological studies.
A PCR-Colorimetric Microwell Plate Hybridization Assay for Detection of
Mycobacterium tuberculosis
and
M. avium
from Culture Samples and Ziehl-Neelsen-Positive Smears
Authors: Rossi Maria Cristina , Gori Andrea , Zehender Gianguglielmo , Marchetti Giulia , Ferrario Giulio , De Maddalena Chiara , Catozzi Lidia , Bandera Alessandra , Esposti Anna Degli , Franzetti Fabio
Published: December 2019

Differentiation between
Mycobacterium tuberculosis
and
M. avium
is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-
Mycobacterium
primers followed by hybridization of the amplification products to biotinylated
M. tuberculosis
and
M. avium
-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of
M. tuberculosis
and
M. avium
. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.
Evaluation of the VITEK 2 System for Rapid Identification of Yeasts and Yeast-Like Organisms
Authors: Graf Barbara , Adam Thomas , Zill Edith , Göbel Ulf B.
Published: December 2019

The new VITEK 2 system is a fully automated system dedicated to the identification and susceptibility testing of microorganisms. In conjunction with the VITEK ID-YST card the VITEK 2 system allows the identification of clinically important yeasts and yeast-like organisms in 15 h due to a sensitive fluorescence-based technology. The ID-YST card consists of 47 biochemical reactions. The database comprises 51 taxa, including newly described species. In this study we evaluated the reliability of the VITEK ID-YST card for the identification of yeasts and yeast-like organisms encountered in a clinical microbiology laboratory. A total of 241 strains representing 21 species were studied. The strains were isolated from clinical samples within a period of 60 days prior to the identification. The tests were performed using 24-h to 55-h subcultures on Sabouraud-gentamicin-chloramphenicol agar. Each strain was tested in parallel using the ID 32C strip as a comparison method combined with microscopic morphology and an agglutination test for
C. krusei
. Overall, 222 strains (92.1%) were unequivocally identified including 11 isolates (4.6%) identified with low discrimination resolved by simple additional tests. Ten strains (4.1%) for which results were given with low discrimination could not be unequivocally identified with supplemental tests, 4 strains (1.7%) were misidentified and 5 strains (2.1%) could not be identified. In conclusion, we found that the VITEK 2 system is a rapid and accurate method for the identification of medically important yeasts and yeast-like organisms.
Effects of Bovine Herpesvirus Type 1 Infection in Calves with Maternal Antibodies on Immune Response and Virus Latency
Authors: Lemaire Mylène , Weynants Vincent , Godfroid Jacques , Schynts Frédéric , Meyer Gilles , Letesson Jean-Jacques , Thiry Etienne
Published: December 2019

The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of bovine herpesvirus type 1 (BHV-1) was performed to determine whether they could become seronegative after the disappearance of maternal antibodies while latently infected with BHV-1. Four uninfected calves were controls. All calves were monitored serologically for 13 to 18 months. In addition, the development of a cell-mediated immune response was assessed by an in vitro antigen-specific gamma interferon (IFN-γ) production assay. All calves had positive IFN-γ responses as early as 7 days until at least 10 weeks after infection. However, no antibody rise was observed after infection in the three calves with the highest titers of maternal antibodies. One of the three became seronegative by virus neutralization test at 7 months of age like the control animals. This calf presented negative IFN-γ results at the same time and was classified seronegative by enzyme-linked immunosorbent assay at around 10 months of age. This calf was latently infected, as proven by virus reexcretion after dexamethasone treatment at the end of the experiment. In conclusion, this study demonstrated that BHV-1-seronegative latent carriers can be obtained experimentally. In addition, the IFN-γ assay was able to discriminate calves possessing only passively acquired antibodies from those latently infected by BHV-1, but it could not detect seronegative latent carriers. The failure to easily detect such animals presents an epidemiological threat for the control of BHV-1 infection.
Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and HumanStaphylococcus aureusIsolates
Authors: Zadoks Ruth , van Leeuwen Willem , Barkema Herman , Sampimon Otlis , Verbrugh Henri , Schukken Ynte Hein , van Belkum Alex
Published: December 2019

Thirty-eight bovine mammaryStaphylococcus aureusisolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) afterSmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized humanS. aureusisolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison ofS. aureusstrains, including methicillin-resistantS. aureus, derived from different host species and from different databases. For 28 bovineS. aureusisolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis ofS. aureusisolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovineS. aureus.
Development and Evaluation of a Molecular Viability Assay for
Pneumocystis carinii
Authors: Maher Nancy , Vermund Sten , Lasbury Mark , Lee C.-H. , Bartlett Marilyn , Unnasch Thomas R.
Published: December 2019

Despite recent declines in incidence,
Pneumocystis carinii
pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While
P. carinii
DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for
P. carinii
. This method is based upon the detection of
P. carinii
mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 10
6
trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing
P. carinii
from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable
P. carinii
collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.
Prevalence ofCryptococcus neoformansvar.neoformans(Serotype D) andCryptococcus neoformansvar.grubii(Serotype A) Isolates in New York City
Authors: Steenbergen Judith N. , Casadevall Arturo
Published: December 2019

Analysis of 40 New York CityCryptococcus neoformansisolates revealed that 39 were typeable, of which 85 and 12.5% wereCryptococcus neoformansvar.grubii(serotype A) andCryptococcus neoformansvar.neoformans(serotype D), respectively. The prevalence of serotype D isolates in New York City appears to be significantly higher than indicated by previous studies of North American isolates.
Prevalence of Polyclonal
mefA
-Containing Isolates among Erythromycin-Resistant Group A Streptococci in Southern Taiwan
Authors: Yan Jing-Jou , Wu Hsiu-Mei , Huang Ah-Huei , Fu Hsiu-Mei , Lee Chen-Ting , Wu Jiunn-Jong
Published: December 2019

A total of 204 nonrepetitive isolates of group A streptococci (GAS), including 107 randomly collected between 1992 and 1995 and 66 and 31 consecutively collected in 1997 and 1998, respectively, from a university hospital in southern Taiwan were examined to determine the prevalence and mechanisms of erythromycin resistance among these isolates. Resistance to erythromycin was detected in 129 isolates (63.2%) by the agar dilution test. Of these, 42 isolates (32.6%) were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and all carried the
ermB
gene; 4 (3.1%) were assigned to the inducible MLS resistance (iMLS) phenotype, and all harbored the
ermTR
gene; and 83 (64.3%) were erythromycin resistant but susceptible to clindamycin (M phenotype), and all possessed the
mefA
gene. Distributed by years, the rates of erythromycin resistance and different phenotypes were 61.7% (53.0% cMLS, 6.1% iMLS, and 40.9% M phenotype) between 1992 and 1995, 62.1% (12.2% cMLS and 87.8% M phenotype) in 1997, and 71.0% (9.1% cMLS and 90.9% M phenotype) in 1998. Pulsed-field gel electrophoresis showed that all but 2 cMLS isolates were clonal in origin, and 17 clones were detected among the M-phenotype isolates. These results indicate that the high incidence and increasing rate of erythromycin-resistant GAS in southern Taiwan are due to the prevalence of multiple M-phenotype clones and that clindamycin may be the drug of choice for the treatment of infections with GAS in penicillin-hypersensitive patients in this area.
Genomic Variability of
Haemophilus influenzae
Isolated from Mexican Children Determined by Using Enterobacterial Repetitive Intergenic Consensus Sequences and PCR
Authors: Gomez-De-Leon Patricia , Santos Jose I. , Caballero Javier , Gomez Demostenes , Espinosa Luz E. , Moreno Isabel , Piñero Daniel , Cravioto Alejandro
Published: December 2019

Genomic fingerprints from 92 capsulated and noncapsulated strains of
Haemophilus influenzae
from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint patterns were detected in the
H. influenzae
strains. Isolates from patients with different diseases were represented by a variety of patterns, which clustered into two major groups. Of the 37 strains isolated from cases of meningitis, 24 shared patterns and were clustered into five groups within a similarity level of 1.0. One fragment of 1.25 kb was common to all meningitis strains.
H. influenzae
strains from healthy carriers presented fingerprint patterns different from those found in strains from sick children. Isolates from healthy individuals were more variable and were distributed differently from those from patients. The results show that ERIC-PCR provides a powerful tool for the determination of the distinctive pathogenicity potentials of
H. influenzae
strains and encourage its use for molecular epidemiology investigations.
Differentiation of
Borrelia burgdorferi
Sensu Lato on the Basis of RNA Polymerase Gene (
rpoB
) Sequences
Authors: Lee Seung-Hyun , Kim Bum-Joon , Kim Jong-Hyun , Park Kyung-Hee , Kim Seo-Jeong , Kook Yoon-Hoh
Published: December 2019

We determined the nucleotide sequences (329 bp) of the
rpoB
DNAs from 22 reference strains of
Borrelia
. No insertions or deletions were observed. Deduced amino acid sequences of amplified
rpoB
DNA comprised 109 amino acid residues (N
450
to M
558
[
Escherichia coli
numbering]). All amino acid sequences were identical with the exception of those of
Borrelia lusitaniae
PotiB2 (T
461
→A) and
B. bissettii
DN127 (I
498
→V). Each species of
B. burgdorferi
sensu lato was differentiated as a distinct entity in the phylogenetic tree constructed by a neighbor-joining method.
B. burgdorferi
sensu lato could be distinguished from
B. turicatae
and
B. hermsii
, which are associated with relapsing fever. Seventeen Korean isolates could be identified by PCR-linked direct sequencing and restriction analysis of the
rpoB
DNA. These results suggest that
rpoB
DNA is useful for identification and characterization of
Borrelia
. In addition, we developed the rapid species identification method using the species-specific primer sets based on
rpoB
gene sequences.
Antibodies against Specific Proteins of and Immobilizing Activity against Three Strains ofBorrelia burgdorferiSensu Lato Can Be Found in Symptomatic but Not in Infected Asymptomatic Dogs
Authors: Hovius Joppe W. R. , Hovius K. Emil , Oei Anneke , Houwers Dirk J. , van Dam Alje P.
Published: December 2019

In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected byBorrelia burgdorferisensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source:B. burgdorferistrain B31,Borrelia gariniistrain A87S, andBorrelia afzeliistrain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P< 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogsBorreliaDNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity againstBorrelia, as shown by both Western blotting and immobilization assays.
Quantitative Approach for the Serodiagnosis of Canine Lyme Disease by the Immunoblot Procedure
Authors: Guerra Marta A. , Walker Edward D. , Kitron Uriel
Published: December 2019

Serum samples obtained from healthy, asymptomatic dogs in areas of Wisconsin and northern Illinois where Lyme disease is endemic or nonendemic were assayed for antibodies toBorrelia burgdorferiby enzyme-linked immunosorbent assay (ELISA), and positive results were confirmed by immunoblot assay. We found that 56.9% (562 of 1,077) of the samples were positive by ELISA and 82.0% (461 of 562) were positive by immunoblotting. A logistic regression model was developed to distinguish between nonvaccinated dogs naturally infected withB. burgdorferifrom areas where the disease is endemic and dogs from areas where the disease is nonendemic that were vaccinated against Lyme disease. Of the 18 protein bands analyzed, 8 were found to be significantly different (P< 0.05) between the two groups. p93, p34, p31, and p28 occurred with increased frequency in vaccinated dogs, while p58, p37, p35, and p30 occurred more frequently in naturally infected dogs. The logistic regression equation obtained was used to determine the probability of natural infection among vaccinated dogs residing in areas where the disease is endemic. Of 125 samples, 87.2% had a very low probability of natural infection and only 2.4% were highly likely to be infected. Logistic regression is a useful method for distinguishing between vaccinated and naturally infected dogs and predicting the serological status of vaccinated dogs from areas where Lyme disease is endemic.
Slaughter Pigs Are Commonly Infected by Closely Related but Distinct Gastric Ulcerative Lesion-Inducing Gastrospirilla
Authors: Roosendaal Robert , Vos Jan H. , Roumen Thijs , van Vugt René , Cattoli Giovanni , Bart Aldert , Klaasen Henricus L. B. M. , Kuipers Ernst J. , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G.
Published: December 2019

An association between (unculturable) gastrospirillum-like organisms (GLO) and ulcerative lesions in the pars oesophagea in stomachs of swine has been claimed. In dogs GLO detected by microscopy may represent several
Helicobacter
species or subspecies. Therefore we investigated which
Helicobacter
spp. are present in stomachs of swine and their possible association with ulcerative lesions of the pars oesophagea. The presence of
Helicobacter
spp. in the antrum and pars oesophagea in 122 stomachs of slaughter swine was determined by microscopy (
n
= 122), by culture on selective and nonselective media (
n
= 112), and by a genus-specific 16S ribosomal DNA (rDNA) PCR (
n
= 80). GLO could not be cultured. Phylogenetic analysis of 43 16S rDNA fragments (out of 54 PCR-positive biopsy specimens) revealed the presence of
Helicobacter heilmannii
type 1 in 42 of them. This correlated with the presence of bacteria with GLO morphology.
Helicobacter bilis
16S rDNA was amplified directly from one sample harboring bacteria with
H. bilis
morphology. The association between
Helicobacter
spp. and gastric lesions was investigated with a second group of 41 pigs with (
n
= 21 cases) or without (
n
= 20 controls) gastric lesions. Fifteen of the 21 cases were positive by PCR or microscopy, compared to 7 of 20 of the controls (
P
= 0.03). 16S rDNA sequence analysis of 7 of 14 PCR-positive cases revealed the presence of
H. heilmannii
type 1. Microscopy showed bacteria with GLO morphology. One sample (cases) was culture negative but PCR positive for
Helicobacter pullorum
-related 16S rDNA. In conclusion, our findings indicate that
H. heilmannii
type 1 is the predominant
Helicobacter
spp. in the stomachs of pigs and that its presence is associated with ulcerative lesions in the pars oesophagea.
Evaluation of a Commercially Available Recombinant-Protein Enzyme-Linked Immunosorbent Assay for Detection of Antibodies Produced in Scrub Typhus Rickettsial Infections
Authors: Land Meagan V. , Ching Wei-Mei , Dasch Gregory A. , Zhang Zhiwen , Kelly Daryl J. , Graves Stephen R. , Devine Peter L.
Published: December 2019

The 56-kDa major outer membrane protein antigen of
Orientia tsutsugamuchi
is the immunodominant antigen in human scrub typhus (ST) infections. An enzyme-linked immunosorbent assay (ELISA) using a recombinant 56-kDa protein (r56) to detect specific immunoglobulin M (IgM) produced in ST infections was developed, and its performance was evaluated using sera from patients with active ST (n = 59), spotted fever (SF) (n = 31), and murine typhus (MT) (n = 6) and from those without rickettsial infection (n = 52). The r56 ELISA was compared to an ELISA using native whole cell lysate of
O. tsutsugamushi
Karp or
O. tsutsugamushi
Gilliam as antigens. The performance of the assays using r56 was similar to that of those using native antigens. Using indirect immunoperoxidase (IIP) as the reference test, sensitivities were 86, 88, and 88% while specificities were 84, 90, and 87% in the three assays. Furthermore, cross-reactivity in confirmed cases of SF and MT was low (5.4, 2.7, and 2.7% respectively). The additional use of IgG in the r56 ELISA gave improved performance (sensitivity, 80%; specificity, 96%; cross-reactivity in SF and MT, 2.7%). The detection of high levels of IgG in some IgM-negative patients illustrates the importance of including a test for IgG in the detection of secondary or reactivated infections, since many of these patients were from regions in Thailand where these infections are endemic.
Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR
Authors: Nitsche Andreas , Steuer Nina , Schmidt Christian Andreas , Landt Olfert , Ellerbrok Heinz , Pauli Georg , Siegert Wolfgang
Published: December 2019

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.
Identification of
Ehrlichia chaffeensis
by Nested PCR in Ticks from Southern China
Authors: Cao Wu-Chun , Gao Yu-Min , Zhang Pan-He , Zhang Xi-Tan , Dai Qing-Hua , Dumler J. Stephen , Fang Li-Qun , Yang Hong
Published: December 2019

A total of 717 ticks collected from southern China were examined by nested PCR for the presence of
Ehrlichia chaffeensis
. Sixteen (55.2%) of 29 adult
Amblyomma testudinarium
ticks and 28 (11.7%) of 240 adult and at least 4.2% of 215 nymphal (pooled specimens)
Haemaphysalis yeni
ticks tested positive. Four other species of ticks were negative. Selected positive amplicons were confirmed by DNA sequencing.
Evaluation of
recA
Sequences for Identification of
Mycobacterium
Species
Authors: Blackwood Kym S. , He Cheng , Gunton James , Turenne Christine Y. , Wolfe Joyce , Kabani Amin M.
Published: December 2019

16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus
Mycobacterium
. Previous studies have shown that
Mycobacterium
species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e.,
M. gastri
and
M. kansasii
. In the present study, we have used the
recA
gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The
recA
genes of 30
Mycobacterium
species were amplified by PCR, sequenced, and compared with the published
recA
sequences of
M. tuberculosis
,
M. smegmatis
, and
M. leprae
available from GenBank. By
recA
sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between
M. gastri
and
M. kansasii
to 75.7% between
M. aurum
and
M. leprae
. Exceptions to this were members of the
M. tuberculosis
complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new
Mycobacterium
species that contain a protein intron in their
recA
genes, similar to
M. tuberculosis
and
M. leprae
. We propose that
recA
gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the
Mycobacterium
species.
PCR Fragment Length Polymorphism Analysis of Vancomycin-Resistant
Enterococcus faecium
Authors: Donabedian Susan , Hershberger Ellie , Thal Lee Ann , Chow J. W. , Clewell Don B. , Robinson-Dunn Barbara , Zervos Marcus J.
Published: December 2019

In this study, the glycopeptide resistance element, Tn
1546
, in 124 VanA
Enterococcus faecium
clinical isolates from 13 Michigan hospitals was evaluated using PCR fragment length polymorphism. There were 26 pulsed-field gel electrophoresis (PFGE) types, which consisted of epidemiologically related and unrelated isolates from separate patients (1992 to 1996). Previously published oligonucleotides specific for regions in the
vanA
gene cluster of Tn
1546
were used to amplify
vanRS
,
vanSH
,
vanHAX
,
vanXY
, and
vanYZ
. The glycopeptide resistance element, Tn
1546
, of
E. faecium
228 was used as the basis of comparison for all the isolates in this study. Five PCR fragment length patterns were found, as follows. (i) PCR amplicons were the same size as those of EF228 for all genes in the
vanA
cluster in 19.4% of isolates. (ii) The PCR amplicon for
vanSH
was larger than that of EF228 (3.7 versus 2.3 kb) due to an insertion between the
vanS
and
vanH
genes (79.2% of isolates). (iii) One isolate in a unique PFGE group had a
vanSH
amplicon larger than that of EF228 (5.7 versus 2.3 kb) due to an insertion in the
vanS
gene and an insertion between the
vanS
and
vanH
genes. (iv) One isolate did not produce a
vanSH
amplicon, but when
vanS
and
vanH
were amplified separately, both amplicons were the same size as those as EF228. (v) One isolate had a
vanYZ
PCR product larger than that of EF228 (2.8 versus 1.6 kb). This study shows that in a majority of the VanA
E. faecium
isolates, Tn
1546
is altered compared to that of EF228. A total of 79.2% of the study isolates had the same-size insertion between the
vanS
and
vanH
genes. The results of this study show dissemination of an altered Tn
1546
in heterologous VanA
E. faecium
in Michigan hospitals.
Rapid Detection and Identification ofCandida,Aspergillus, andFusariumSpecies in Ocular Samples Using Nested PCR
Authors: Jaeger Emma E. M. , Carroll Nora M. , Choudhury Sarah , Dunlop Anthony A. S. , Towler Hamish M. A. , Matheson Melville M. , Adamson Peter , Okhravi Narciss , Lightman Susan
Published: December 2019

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species,Candida albicans,Aspergillus fumigatus, andFusarium solani, has been developed. Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed. Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and twoC. albicansorganisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR. Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.