bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of
Mycobacterium tuberculosis
Authors: Van Der Zanden A. G. M. , Te Koppele-Vije E. M. , Vijaya Bhanu N. , Van Soolingen D. , Schouls L. M.
Published: March 2003

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the
rpoB
gene using reverse line blot hybridization and DNA sequencing. Analysis of the
rpoB
gene revealed that samples containing rifampin-resistant
Mycobacterium tuberculosis
carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the
rpoB
gene was more accurate in identifying mutations in the core region of the
rpoB
gene. Sequence analysis of the
rpoB
gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.
Molecular Characterization of Gentamicin-Resistant
Enterococci
in the United States: Evidence of Spread from Animals to Humans through Food
Authors: Donabedian S. M. , Thal L. A. , Hershberger E. , Perri M. B. , Chow J. W. , Bartlett P. , Jones R. , Joyce K. , Rossiter S. , Gay K. , Johnson J. , Mackinson C. , Debess E. , Madden J. , Angulo F. , Zervos M. J.
Published: March 2003

We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were ≥128 μg/ml. The
aac(6′)-Ie-aph(2")-Ia
,
aph(2")-Ic
, and
aph(2")-Id
genes were identified by PCR in isolates from animals, food, and humans. The
aph(2")-Ib
gene was not identified in any of the isolates. Two
Enterococcus faecalis
isolates (MICs > 1,024 μg/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork
E. faecalis
isolates from Michigan with the
aac(6′)-Ie-aph(2")-Ia
gene had related PFGE patterns and 2
E. faecalis
isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.
Influence of Culture Conditions on the Fatty Acid Profiles of Laboratory-Adapted and Freshly Isolated Strains of
Helicobacter pylori
Authors: Scherer Christiane , Müller Karl-D. , Rath Peter-M. , Ansorg Rainer A. M.
Published: March 2003

Cellular fatty acids of
Helicobacter pylori
have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions.
H. pylori
is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9
c
), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9
c
and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9
c
and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.
New Types of Toxin A-Negative, Toxin B-Positive Strains among
Clostridium difficile
Isolates from Asia
Authors: Rupnik Maja , Kato Naoki , Grabnar Miklavz , Kato Haru
Published: March 2003

A total of 56
C. difficile
strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of
tcdA
and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A
−
B
+
strain,
C. difficile
8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A
+
B
+
strains. Toxinotypes XVI and XVII represent two new groups of A
−
B
+
strains. Strains of the well-known A
−
B
+
group from toxinotype VIII have a nonsense mutation at the beginning of
tcdA
gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5′-end sequence of
tcdA
in two newly described A
−
B
+
strains does not contain an identical mutation. The prevalence of variant
C. difficile
strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.
In Vitro Susceptibility Testing of
Aspergillus
spp.: Comparison of Etest and Reference Microdilution Methods for Determining Voriconazole and Itraconazole MICs
Authors: Pfaller J. B. , Messer S. A. , Hollis R. J. , Diekema D. J. , Pfaller M. A.
Published: March 2003

The performance of the Etest for voriconazole and for itraconazole susceptibility testing of 376 isolates of
Aspergillus
spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35°C. The isolates included
A. fumigatus
,
A. flavus
,
A. niger
,
A. terreus
,
A. versicolor
,
A. glaucus
,
A. nidulans
,
A. ustus
, and
A. sydowii
. Overall agreement percentages between the Etest and microdilution MICs were 97.6% for voriconazole and 95.8% for itraconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with voriconazole and higher values with itraconazole. The Etest method using RPMI agar appears to be a useful method for determining the voriconazole and itraconazole susceptibilities of
Aspergillus
spp.
Comparison of Four Chromogenic Media and Hektoen Agar for Detection and Presumptive Identification of
Salmonella
Strains in Human Stools
Authors: Perez J. M. , Cavalli P. , Roure C. , Renac R. , Gille Y. , Freydiere A. M.
Published: March 2003

Several chromogenic media have been developed to enhance the specificity of
Salmonella
detection. We compared the performance of four commercial chromogenic media—namely, ABC medium (Lab M. Ltd., Bury, United Kingdom), COMPASS Salmonella agar (Biokar Diagnostics, Beauvais, France), CHROMagar Salmonella agar (CHROMagar Company, Paris, France), and SM ID agar (bioMerieux, Marcy l'Etoile, France)—with conventional Hektoen medium. Nine hundred sixteen stool samples from inpatients at three hospitals were cultured, in parallel, on the five media, both by direct inoculation and after selective enrichment in selenite broth. Sixty-four
Salmonella
strains with 12 serotypes were isolated on at least one medium. After 48 h of incubation, sensitivity before and after enrichment was 62.5 and 89.1% with ABC medium, 77.1 and 93.8% with COMPASS agar, 66.7 and 89.1% with CHROMagar, 68.8 and 85.9% with SM ID agar, and 85.4 and 98.4% with Hektoen agar, respectively. Broth enrichment and prolonged incubation (48 versus 24 h) increased the sensitivity of all five media. Only one strain was not isolated on Hektoen agar. The number of false-positive isolates was higher with all five media after enrichment in selenite broth and after incubation for 48 h compared to 24 h. The specificity of the four chromogenic media was better than 91% after incubation for 24 h (77.7% with Hektoen agar) and better than 84% after incubation for 48 h (74.8% with Hektoen agar). This higher specificity reduces the need for confirmatory tests, thereby cutting technical time and reagent requirements. Both COMPASS agar and CHROMagar Salmonella, which after simple additional tests showed close efficiencies (96 and 97%, respectively), can be recommended as single-plate media of choice for the detection and presumptive identification of salmonellae in stools.
Two-Center Collaborative Evaluation of the Performance of the BD Phoenix Automated Microbiology System for Identification and Antimicrobial Susceptibility Testing of
Enterococcus
spp. and
Staphylococcus
spp
Authors: Fahr Anne-Marie , Eigner Ulrich , Armbrust Martina , Caganic Alexandra , Dettori Giuseppe , Chezzi Carlo , Bertoncini Luca , Benecchi Magda , Menozzi Maria Grazia
Published: March 2003

The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was assessed for identification (ID) and antimicrobial susceptibility testing (AST) for the majority of clinically encountered bacterial isolates in a European collaborative two-center trial. A total of 469 bacterial isolates of the genera
Staphylococcus
(275 isolates),
Enterococcus
(179 isolates), and
Streptococcus
(15 isolates, for ID only) were investigated; of these, 367 were single patient isolates, and 102 were challenge strains tested at one center. Sixty-four antimicrobial drugs were tested, including the following drug classes: aminoglycosides, beta-lactam antibiotics, beta-lactam-beta-lactamase inhibitors, carbapenems, cephems, folate antagonists, quinolones, glycopeptides, macrolides-lincosamides-streptogramin B (MLS), and others. Phoenix ID results were compared to those of the laboratories' routine ID systems (API 32 Staph, API 32 Strep, and VITEK 2 [bioMérieux, Marcy l'Etoile, France]); Phoenix AST results were compared to those of frozen standard broth microdilution (SBM) panels according to NCCLS guidelines (NCCLS document M 100-S 9, approved standard M 7-A 4). Discrepant results were repeated in duplicate. Concordant IDs of 97.1, 98.9, and 100% were observed for staphylococci, enterococci, and streptococci, respectively. For AST results the overall essential agreement was 93.3%; the category agreement was 97.3%; and the very major error rate, major error rate, and minor error rate were 1.2, 1.9, and 1.3%, respectively. In conclusion, the Phoenix ID results showed high agreement with results of the systems to which they were being compared; the AST performance was highly equivalent to that of the SBM reference method.
Proficiency Testing Program for Clinical Laboratories Performing Antifungal Susceptibility Testing of Pathogenic Yeast Species
Authors: Ramani Rama , Chaturvedi Vishnu
Published: March 2003

Antifungal susceptibility testing is expected to facilitate the selection of adequate therapy for fungal infections. The general availability of antifungal susceptibility testing in clinical laboratories is low, even though a number of standard methods are now available. The objective of the present study was to develop and evaluate a proficiency testing program (PTP) for the antifungal susceptibility testing of pathogenic yeasts in laboratories licensed by the New York State Department of Health. A number of quality control standards, and methods for documenting laboratory performance, were developed in consultation with the laboratory directors. The participating laboratories were provided with five American Type Culture Collection strains of pathogenic yeasts for which the minimum inhibitory concentrations (MICs) of amphotericin B and fluconazole were well defined. A majority of laboratories (14 of 17) used broth microdilution, and these were evenly split between the NCCLS M-27A protocol and the Sensititre YeastOne method. The other three laboratories performed susceptibility testing with Etest. Overall, the levels of agreement between MIC reference ranges and the reported MICs were 85 and 74% for amphotericin B and for fluconazole, respectively. All laboratories except one successfully detected fluconazole resistance in a
Candida krusei
strain. However, amphotericin B resistance in a
Candida lusitaniae
strain was not detected by any of the participating labs. It is concluded that a suitably designed PTP could adequately monitor the competence of clinical laboratories performing antifungal susceptibility testing.
High-Level Expression and Purification of a Truncated Merozoite Antigen-2 ofBabesia equiinEscherichia coliand Its Potential for Immunodiagnosis
Authors: Huang Xiaohong , Xuan Xuenan , Yokoyama Naoaki , Xu Longshan , Suzuki Hiroshi , Sugimoto Chihiro , Nagasawa Hideyuki , Fujisaki Kozo , Igarashi Ikuo
Published: March 2003

The gene encoding a truncated merozoite antigen-2 (EMA-2t) ofBabesia equiwas cloned and highly expressed inEscherichia colias a glutathioneS-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathioneS-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected withB. equifrom sera of horses infected withBabesia caballiand healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies inB. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence ofB. equiwas 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.
Differentiation of
Candida albicans
and
Candida dubliniensis
by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae
Authors: Bliss Joseph M. , Sullivan Mark A. , Malone Jane , Haidaris Constantine G.
Published: March 2003

To identify antigens specific for the filamentous form of
Candida albicans
, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable
C. albicans
filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with
C. dubliniensis
filaments, while scFv12 did not. Neither scFv reacted with
C. glabrata
,
C. parapsilosis
,
C. rugosa
,
C. tropicalis
, or
Saccharomyces cerevisiae
grown under conditions that stimulated filament formation in
C. albicans
and
C. dubliniensis
. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both
C. albicans
and
C. dubliniensis
and another specific only to
C. albicans
adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.
Patterns of Resistance Associated with Integrons, the Extended-Spectrum β-Lactamase SHV-5 Gene, and a Multidrug Efflux Pump of
Klebsiella pneumoniae
Causing a Nosocomial Outbreak
Authors: Gruteke Paul , Goessens Wil , van Gils Jan , Peerbooms Paul , Lemmens-den Toom Nicole , van Santen-Verheuvel Marga , van Belkum Alex , Verbrugh Henri
Published: March 2003

Multiresistant
Klebsiella pneumoniae
caused a nosocomial outbreak. Resistance patterns of the presumed outbreak isolates varied among and within patients. In order to control the outbreak, screening for extended-spectrum β-lactamase (ESBL)-producing
K. pneumoniae
was commenced. A number of susceptible
K. pneumoniae
strains were stored to serve as controls in genetic strain typing. Typing by pulsed-field gel electrophoresis proved the clonality of the strains in the recognized outbreak patients. Typing of the control strains by pulsed-field gel electrophoresis showed that at least one patient had been missed by the ESBL screening procedure. Further genetic typing confirmed the presence of the SHV-5 ESBL gene in all but one of the outbreak strains. Variable presence of integrons that carried the aminoglycoside resistance genes
aadB
and
aadA2
was found. A
gyrA
mutation in codon 83 was present in all outbreak strains tested, despite considerable differences in ciprofloxacin MICs. The MICs of ciprofloxacin and the chemically unrelated drug cefoxitin were correlated (
r
= 0.86,
P
< 0.01) and were compatible with the overexpression of an efflux pump in a subset of the outbreak strains. We conclude that outbreak strains that express an ESBL gene only at a low level may pass unnoticed in a screening procedure, when the laboratory is unaware of variable ESBL expression. In this particular outbreak, screening for strains for which ciprofloxacin MICs were ≥0.25 μg/ml would in retrospect have been the most sensitive method for detection of the
K. pneumoniae
outbreak strain.
Identification of a Mutation Associated with Erythromycin Resistance in
Bordetella pertussis
: Implications for Surveillance of Antimicrobial Resistance
Authors: Bartkus J. M. , Juni B. A. , Ehresmann K. , Miller C. A. , Sanden G. N. , Cassiday P. K. , Saubolle M. , Lee B. , Long J. , Harrison, A. R. , Besser J. M.
Published: March 2003

Erythromycin treatment failures and in vitro resistance of
Bordetella pertussis
have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant
B. pertussis
strains tested contained an A-to-G transition mutation at position 2058 (
Escherichia coli
numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.
Genotypes at the Internal Transcribed Spacers of the Nuclear rRNA Operon of
Pneumocystis jiroveci
in Nonimmunosuppressed Infants without Severe Pneumonia
Authors: Totet Anne , Pautard Jean-Claude , Raccurt Christian , Roux Patricia , Nevez Gilles
Published: March 2003

The frequency of
Pneumocystis jiroveci
(human-derived
Pneumocystis
) in immunocompetent infants developing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of
P. jiroveci
organisms was mostly performed with samples from immunosuppressed patients with pneumocystosis (
Pneumocystis
carinii
pneumonia [PCP]). The present report describes the genotypes of
P. jiroveci
organisms in 26 nonimmunosuppressed infants developing a mild
Pneumocystis
infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11
P. jiroveci
ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B
1
a
3
(first score), which corresponds to type Eg (second score). Mixed infections were diagnosed in three infants. The occurrence of such diversity of
P. jiroveci
ITS types, an identical main type, and mixed infections has previously been reported in immunosuppressed patients with PCP. Thus, the
P. jiroveci
ITS genotypes detected in immunocompetent infants and immunosuppressed patients developing different forms of
Pneumocystis
infection share characteristics, suggesting that both groups of individuals make up a common human reservoir for the fungus. Finally, the frequency of
P. jiroveci
in nonimmunosuppressed infants with acute respiratory syndromes and the genotyping results provide evidence that this infant population is an important reservoir for the fungus.
Longitudinal Analysis of
Prototheca zopfii
-Specific Immune Responses: Correlation with Disease Progression and Carriage in Dairy Cows
Authors: Roesler Uwe , Hensel Andreas
Published: March 2003

In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that
Prototheca zopfii
mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.
Detection by PCR and Isolation Assays of the Anaerobic Intestinal Spirochete
Brachyspira aalborgi
from the Feces of Captive Nonhuman Primates
Authors: Munshi M. Arif , Taylor Nyree M. , Mikosza Andrew S. J. , Spencer Peter B. S. , Hampson David J.
Published: March 2003

The purpose of this study was to investigate the presence of the anaerobic intestinal spirochetes
Brachyspira aalborgi
and
Brachyspira pilosicoli
in the feces of captive nonhuman primates (
n
= 35) from 19 species housed at the Zoological Gardens, Perth, Western Australia. Both spirochete species are known to infect human beings. DNA was extracted from freshly collected feces with a commercially available QIAamp DNA stool minikit and subjected to PCR protocols amplifying portions of the 16S rRNA genes of the two spirochete species. The feces were also subjected to selective culture for the spirochetes. Subsequently, feces from 62 other captive animals or birds representing 39 species at the zoo were examined by PCR to determine whether they were reservoirs of infection. Six fecal samples from individuals from four primate species (two vervet monkeys, two Tonkean macaques, one Japanese macaque, and one hamadryas baboon) tested positive in the
B. aalborgi
PCR.
B. aalborgi
was not detected by PCR in any of the other animal or bird species tested, and
B. pilosicoli
was not detected in the primates or any of the other animals or birds.
B. aalborgi
was isolated from both PCR-positive vervet monkeys. This is the first time that
B. aalborgi
has been isolated from nonhuman primates and the first time that it has been isolated from the feces of any species.
Molecular Epidemiology of
Pseudomonas aeruginosa
Colonization in a Burn Unit: Persistence of a Multidrug-Resistant Clone and a Silver Sulfadiazine-Resistant Clone
Authors: Pirnay Jean-Paul , De Vos Daniel , Cochez Christel , Bilocq Florence , Pirson Jean , Struelens Marc , Duinslaeger Luc , Cornelis Pierre , Zizi Martin , Vanderkelen Alain
Published: March 2003

To study the epidemiology of
Pseudomonas aeruginosa
colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental
P. aeruginosa
isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with
P. aeruginosa
, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal
P. aeruginosa
colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD
r
). The MDR strain was found at a higher frequency in sputum samples than the SSD
r
strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (
oprI
,
oprL
, and
oprD
) and the pyoverdine type revealed that all predominant strains except the SSD
r
strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.
Community-Acquired Poliovirus Infection in Children with Primary Immunodeficiencies in Tunisia
Authors: Triki Hinda , Barbouche Mohamed Ridha , Bahri Olfa , Bejaoui Mohamed , Dellagi Koussay
Published: March 2003

The global polio eradication program recommends the use of massive vaccination campaigns with live vaccine through National Immunization Days (NIDs) to displace the wild virus from the community. Immunodeficient patients may be indirectly infected and become chronic excretors and potential reservoirs of polioviruses, a concern for the posteradication era. This prospective study aimed to assess the risk of community-acquired infection of immunodeficient patients following NIDs, the dynamics of viral excretion and the genetic variation of excreted viruses. Sixteen children with various primary immunodeficiencies, who did not receive the vaccine during the campaign, were investigated. Stool samples were collected weekly, shortly after the NIDs, during at least 3 months, and were processed for viral isolation. Isolates were characterized by three intratypic differentiation methods and partial sequencing of the VP1/2A region. Polioviruses were detected in 4 out of 16 patients (serotype 1 in 3 patients and serotype 3 in 1 patient). Sequencing revealed more than 99% homology with homotypic Sabin strains, suggesting recent infection. Duration of viral excretion ranged from 1 to 7 weeks. Nine out of eleven isolates from the three poliovirus serotype 1-infected patients disclosed a non-Sabin-like phenotype by enzyme-linked immunosorbent assay and had recurrent mutations within or close to the neutralizing antigenic sites. In summary, the risk of secondary infection in immunodeficient patients is within the range previously reported for the general population. Although none of the four infected patients developed prolonged viral excretion, particular viral variants were selected and may be of epidemiological significance.
Bacillus anthracis
Virulence in Guinea Pigs Vaccinated with Anthrax Vaccine Adsorbed Is Linked to Plasmid Quantities and Clonality
Authors: Coker Pamala R. , Smith Kimothy L. , Fellows Patricia F. , Rybachuck Galena , Kousoulas Konstantin G. , Hugh-Jones Martin E.
Published: March 2003

Bacillus anthracis
is a bacterial pathogen of great importance, both historically and in the present. This study presents data collected from several investigations and indicates that
B. anthracis
virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2. Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20
B. anthracis
isolates representative of worldwide genetic diversity. These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of
B. anthracis
from environmental samples. We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports. By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence. This model was validated by using a randomly chosen set of 12 additional
B. anthracis
isolates. Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen.
Five-Test Simple Scheme for Species-Level Identification of Clinically Significant Coagulase-Negative Staphylococci
Authors: De Paulis Adriana N. , Predari Silvia C. , Chazarreta Carlos D. , Santoianni Jorge E.
Published: March 2003

A working scheme developed in our laboratory for identification (by species group and species) of coagulase-negative staphylococci (CNS) was evaluated with 201 consecutive isolates and then validated by using the reference method of Kloos and Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol.
1:
82-88, 1975). This five-test simple scheme (referred to here as the simple scheme) combines the novobiocin susceptibility test with tests for urease, pyrrolidonyl arylamidase, ornithine decarboxylase, and aerobic acid from mannose. The addition of one or two tests within a particular species group could then positively identify the isolate. Two commercial systems, Staph-Zym (Rosco) and API-Staph (bioMérieux), along with results obtained by using Rosco diagnostic tablets (nongrowth tests), were also compared with the reference method. One isolate could not be identified even by the reference method. Of the remaining 200 strains, 191 (95.5%) strains were correctly identified with Staph-Zym and 171 strains (85.5%) were correctly identified with API-Staph. The most frequent clinical CNS species isolated were
Staphylococcus epidermidis
(50.5%),
S. haemolyticus
(18.5%),
S. saprophyticus
subsp.
saprophyticus
(16.0%),
S. lugdunensis
(6.0%), and
S. warneri
(2.5%). The simple scheme validated with the reference method has demonstrated an excellent correlation in the identification of the three most frequent species isolated:
S. epidermidis
,
S. haemolyticus
, and
S. saprophyticus
subsp.
saprophyticus
. With the simple scheme, identification of CNS was possible within 24 h after the enzymatic tests were used, whereas up to 72 h is necessary for the growth tests. This methodology would be very useful in any clinical microbiology laboratory for the presumptive identification of CNS species groups and species.
Characterization of Monkey Enteropathogenic
Escherichia coli
(EPEC) and Human Typical and Atypical EPEC Serotype Isolates from Neotropical Nonhuman Primates
Authors: Carvalho Vania M. , Gyles Carlton L. , Ziebell Kim , Ribeiro Marcela A. , Catão-Dias José L. , Sinhorini Idércio L. , Otman Jamile , Keller Rogéria , Trabulsi Luiz R. , Pestana de Castro Antônio F.
Published: March 2003

Enteropathogenic
Escherichia coli
(EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of
E. coli
is unclear. This study was designed to characterize diarrheagenic
E. coli
recovered from the feces of 56 New World nonhuman primates, primarily marmosets (
Callithrix
spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis.
E. coli
recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic
E. coli
and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the
eae
gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of
eae
+
E. coli
were serotyped and examined by PCR for genes encoding EPEC virulence markers. The
eae
+
E. coli
isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.
Susceptibility Testing with the Manual Mycobacteria Growth Indicator Tube (MGIT) and the MGIT 960 System Provides Rapid and Reliable Verification of Multidrug-Resistant Tuberculosis
Authors: Ådjers-Koskela Kati , Katila Marja-Leena
Published: March 2003

The objective of the study was to compare the manual Mycobacteria Growth Indicator Tube (MGIT) method and the BACTEC MGIT 960 system to the BACTEC 460 method for susceptibility testing of
Mycobacterium tuberculosis
. The evaluation was based on testing of 36
M. tuberculosis
strains with various susceptibilities to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM). In addition, five of the strains generating discrepant results in testing for EMB were analyzed for heteroresistance. For INH, the susceptibility test results obtained by the MGIT 960 and the manual MGIT systems agreed with the BACTEC 460 results in 94 and 97% of the cases, respectively. The results of susceptibility to RMP were all in agreement. For SM, 78 and 72% of the results obtained by the MGIT 960 and the manual MGIT systems, respectively, agreed with the BACTEC 460 results. In contrast, less than 80% of the results for susceptibility to EMB obtained by the two MGIT methods agreed with the BACTEC 460 results. All five strains analyzed for EMB heteroresistance were found to consist of resistant and susceptible subpopulations. The average turnaround times were 6.4 days for the MGIT 960 system, 6.5 for the manual MGIT system, and 8.7 days for the BACTEC 460 method. Both MGIT methods can be regarded as accurate and rapid alternatives to the BACTEC 460 method for detection of strains resistant to INH and RMP. However, more studies are needed for solving the problems associated with susceptibility testing to EMB and SM.
Clinical Implications of
Mycobacterium kansasii
Species Heterogeneity: Swiss National Survey
Authors: Taillard Caroline , Greub Gilbert , Weber Rainer , Pfyffer Gaby E. , Bodmer Thomas , Zimmerli Stefan , Frei Reno , Bassetti Stefano , Rohner Peter , Piffaretti Jean-Claude , Bernasconi Enos , Bille Jacques , Telenti Amalio , Prod'hom Guy
Published: March 2003

Several subtypes of
Mycobacterium kansasii
have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of
M. kansasii
strains (
n
= 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the
hsp65
gene. Subtype 1 comprised 67% of the isolates (
n
= 128), while subtypes 2 and 3 comprised 21% (
n
= 40) and 8% (
n
= 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (
n
= 8).
M. kansasii
subtype 1 was considered pathogenic in 81% of patients, while
M. kansasii
subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with
M. kansasii
subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping
M. kansasii
may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.
Changes in the Human Immunodeficiency Virus p7-p1-p6
gag
Gene in Drug-Naive and Pretreated Patients
Authors: Gallego Oscar , de Mendoza Carmen , Corral Angélica , Soriano Vincent
Published: March 2003

Resistance to antiretroviral agents often results from mutations within the human immunodeficiency virus (HIV)
pol
gene. Moreover, insertions within the p6
gag-pol
region have recently been found to be involved with resistance to nucleoside analogs. Overall, we found that 21% of 156 specimens collected from HIV-infected individuals (17.6% from 74 drug-naive patients and 24.4% from 82 pretreated patients) harbored these insertions. Insertions around the KQE (Lys-Gln-Glu) motif were found in 12.2% of the pretreated patients but in none of the drug-naive subjects (
P
= 0.002). In contrast, insertions around the PTAP (Prol-Thre-Ala-Prol) motif were seen at similar rates (∼15%) among drug-naive and pretreated patients, which supports the idea that they may be natural polymorphisms.
Entirely Automated Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma by Using the Ultrasensitive COBAS AMPLICOR HIV-1 Monitor Test and RNA Purification on the MagNA Pure LC Instrument
Authors: Hölzl Gabriele , Stöcher Markus , Leb Victoria , Stekel Herbert , Berg Jörg
Published: March 2003

The ultrasensitive COBAS AMPLICOR HIV-1 Monitor test was complemented with automated RNA purification on the MagNA Pure LC instrument. This enabled entirely automated ultrasensitive assessment of viral loads in human immunodeficiency virus type 1 (HIV-1)-infected individuals. The detection limit of the fully automated assay and the viral load measurements in 80 clinical samples were found to be in good agreement with those of the conventional ultrasensitive COBAS AMPLICOR HIV-1 Monitor test. The fully automated assay showed markedly reduced hands-on time and was found to be suitable for the routine assessment of HIV-1 viral loads in a clinical diagnostic laboratory.
Novel Sample Preparation Method for Safe and Rapid Detection of
Bacillus anthracis
Spores in Environmental Powders and Nasal Swabs
Authors: Luna Vicki A. , King Debra , Davis Carisa , Rycerz Tony , Ewert Matthew , Cannons Andrew , Amuso Philip , Cattani Jacqueline
Published: March 2003

Bacillus anthracis
spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects
B. anthracis
in samples containing <10 spores.
Detection and Species-Level Identification of Primate Herpesviruses with a Comprehensive PCR Test for Human Herpesviruses
Authors: Johnson Grant , Dick Daryl , Ayers Melissa , Petric Martin , Tellier Raymond
Published: March 2003

A comprehensive assay for the identification of all eight human herpesviruses has been previously reported. This assay was extended to the detection and species-level identification of herpes B virus (
Cercopithecine herpesvirus 1
) and African green monkey cytomegalovirus (
Cercopithecine herpesvirus 5
), two herpesviruses of relevance to the clinical virology laboratory.
Casein Agar: a Useful Medium for Differentiating
Candida dubliniensis
from
Candida albicans
Authors: Mosca Christian O. , Moragues María D. , Llovo José , Al Mosaid Asmaa , Coleman David C. , Pontón José
Published: March 2003

Production of chlamydospores on casein agar at 24°C for 48 h provides a simple means for differentiating
Candida dubliniensis
from
Candida albicans
based on chlamydospore production. Of 109
C. dubliniensis
isolates tested on this medium, 106 (97.2%) produced abundant chlamydospores and three produced few chlamydospores. In contrast, of the 120
C. albicans
isolates tested, 111 (92.5%) failed to produce any chlamydospores, whereas the remaining nine isolates produced few chlamydospores. These findings indicate that abundant chlamydospore production on casein agar is a useful test for discriminating between
C. dubliniensis
and
C. albicans.
Detection ofEhrlichiaspp. in the Blood of Wild White-Tailed Deer in Missouri by PCR Assay and Serologic Analysis
Authors: Arens Max Q. , Liddell Allison M. , Buening Gerald , Gaudreault-Keener Monique , Sumner John W. , Comer James A. , Buller Richard S. , Storch Gregory A.
Published: March 2003

Blood samples collected from wild deer in Missouri in November of 2000 and 2001 were positive by PCR assays forEhrlichiachaffeensis(50 of 217; 23%),Ehrlichiaewingii(44 of 217; 20%), andAnaplasmaspecies (214 of 217; 99%). Nucleotide sequences of selected amplicons from the assay for anaplasma matched sequences of the white-tailed deer agent. Serologic analysis of 112 deer sampled in 2000 showed a very high prevalence of antibodies toE.chaffeensis(97 of 112; 87%) and a low prevalence of antibodies reactive withAnaplasmaphagocytophila(2 of 112; 2%).
Conventional and Molecular Methods for Verification of Results Obtained with BacT/Alert Nonvent Blood Culture Bottles
Authors: Marlowe Elizabeth M. , Gibson Linda , Hogan James , Kaplan Shannon , Bruckner David A.
Published: March 2003

A strategy comparing molecular and conventional methods for verification of the BacT/Alert nonvent blood culture bottles (Organon Teknika, Durham, N.C.) was performed with seeded isolates. The bottles were evaluated with 12 common organisms from bloodstream infections. Overall, the bottles were equivalent as determined by conventional and molecular methods.
Persistence of Colonization of Intestinal Mucosa by a Probiotic Strain,
Lactobacillus casei
subsp.
rhamnosus
Lcr35, after Oral Consumption
Authors: De Champs Christophe , Maroncle Nathalie , Balestrino Damien , Rich Chantal , Forestier Christiane
Published: March 2003

The colonization by the probiotic
Lactobacillus casei
subsp.
rhamnosus
Lcr35 of the gastrointestinal tracts of mice and humans was studied. The mice were orally given 10
9
CFU of Lcr35 either once or three times at 24-h intervals. A 16S ribosomal nucleic probe used in hybridization assays detected Lcr35 in the feces of mice for up to 3 days after the feeding, at a level of 10
8
to 10
9
CFU/g of feces. In the human assay, 12 healthy volunteers were enrolled in a randomized trial and ingested Lcr35 at a dosage of 10
8
or 10
10
or 10
12
CFU every day for 7 days. Then, after a 3-week posttreatment period, there was a second intake period similar to the first one. Analysis of fecal samples showed significant increases in the number of lactobacilli during the first intake period, whatever the dose given. The greatest increases were observed in subjects harboring the lowest indigenous population of Lcr35-like bacteria. During the 3-week posttreatment period, the number of CFU slightly decreased over time, and an increase, although not a statistically significant one, was observed during the second test period. These findings suggest that Lcr35 is able to survive within the gastrointestinal tract.