bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of 16S rRNA Sequencing and Reevaluation of a Short Biochemical Scheme for Identification of Clinically Significant
Bacteroides
Species
Authors: Song Yuli , Liu Chengxu , Bolaňos Mauricio , Lee Julia , McTeague Maureen , Finegold Sydney M.
Published: April 2005

Sequence analysis of the 16S rRNA gene represents a highly accurate and versatile method for bacterial classification and identification, even when the species in question is notoriously difficult to identify by phenotypic means. In this study, we evaluated the utility of 16S rRNA gene sequencing as a means of identifying clinically important
Bacteroides
species. We sequenced 231 clinical isolates that had been identified by a short biochemical scheme. Based on the sequence analysis, 192 clinical isolates were assigned to an established species, with the other 39 clinical strains revealing five unique sequences that may represent five novel species. This is in contrast to identification obtained from a short biochemical scheme, by which only 73.5% (172 of 231) of isolates were correctly identified to species level. Based on the solid identification obtained from 16S rRNA gene sequencing, the short biochemical scheme was modified and improved to provide clinical laboratories with an inexpensive and simple alternative for the identification of isolates of clinically significant
Bacteroides
species.
Multilocus Variable-Number Tandem Repeat Typing of
Mycobacterium ulcerans
Authors: Ablordey Anthony , Swings Jean , Hubans Christine , Chemlal Karim , Locht Camille , Portaels Françoise , Supply Philip
Published: April 2005

The apparent genetic homogeneity of
Mycobacterium ulcerans
contributes to the poorly understood epidemiology of
M. ulcerans
infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related
M. marinum
genome sequence were screened in a collection of 23
M. ulcerans
isolates, one
Mycobacterium
species referred to here as an intermediate species, and five
M. marinum
strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight
M. ulcerans
and five
M. marinum
genotypes. The results from the VNTR analysis corroborated the genetic relationships of
M. ulcerans
isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within
M. ulcerans
and
M. marinum.
Comparison of an Internally Controlled, Large-Volume LightCycler Assay for Detection of
Mycobacterium tuberculosis
in Clinical Samples with the COBAS AMPLICOR Assay
Authors: Burggraf Siegfried , Reischl Udo , Malik Naeem , Bollwein Markus , Naumann Ludmila , Olgemöller Bernhard
Published: April 2005

We present a sensitive and specific assay for reliable and flexible detection of members of the
Mycobacterium tuberculosis
complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-μl glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR
M. tuberculosis
assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.
Determinants for the Occurrence of Acute Exacerbation of Hepatitis B Virus Infection in Chinese Patients after HBeAg Seroclearance
Authors: Yuan He-Jun , Yuen Man-Fung , Wong Danny Ka-Ho , Sum Siu-Man , Doutreloigne Joke , Sablon Erwin , Lai Ching-Lung
Published: April 2005

This study was performed to determine the factors for predicting the occurrence of acute exacerbation of hepatitis B virus infection in HBeAg-negative patients. Two hundred and sixteen patients with known times of HBeAg seroclearance were recruited. Liver biochemistry and virologic markers were monitored. Precore and core promoter mutations were determined by a line probe assay. The median age at HBeAg seroclearance was 34.5 years. The median follow-up duration was 26.4 months. Fifty-six (27.9%) patients had acute exacerbations. By Cox regression analysis, male gender, older age, and core promoter mutations at the time of HBeAg seroclearance were independently associated with the occurrence of acute exacerbation after HBeAg seroclearance (
P
= 0.025, 0.018, and 0.001, respectively). Fourteen (7.0%) patients had HBeAg seroreversion within a median follow-up period of 11.6 months after HBeAg seroclearance. By Cox regression analysis, older age at HBeAg seroclearance was independently associated with the chance of HBeAg seroreversion (
P
= 0.01). We concluded that male patients with core promoter mutations and delayed HBeAg seroclearance had a higher cumulative chance of acute exacerbation in the HBeAg-negative phase. Patients with delayed HBeAg seroclearance had a higher frequency of HBeAg seroreversion.
Discrimination within Phenotypically Closely Related Definitive Types of
Salmonella enterica
Serovar Typhimurium by the Multiple Amplification of Phage Locus Typing Technique
Authors: Ross Ian L. , Heuzenroeder Michael W.
Published: April 2005

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including
Salmonella
. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related
Salmonella
species. The presence of genomic sequences derived from stable temperate phages in
Salmonella
offers an alternative for MLST of
Salmonella
. We have used MLST of prophage loci in
Salmonella enterica
serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.
Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification
Authors: Okafuji Takao , Yoshida Naoko , Fujino Motoko , Motegi Yoshie , Ihara Toshiaki , Ota Yoshinori , Notomi Tsugunori , Nakayama Tetsuo
Published: April 2005

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.
Utility of Pooled Urine Specimens for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Men Attending Public Sexually Transmitted Infection Clinics in Mumbai, India, by PCR
Authors: Lindan Christina , Mathur Meenakshi , Kumta Sameer , Jerajani Hermangi , Gogate Alka , Schachter Julius , Moncada Jeanne
Published: April 2005

Pooling urogenital specimens for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
by nucleic acid amplification tests is an attractive alternative to individual testing. As pooling can reduce the costs of testing as well as labor, it has been advocated for use in resource-poor settings. However, it has neither been widely adopted nor evaluated for use in developing countries. We evaluated the practical use of pooling first-catch urine (FCU) specimens for the detection of
C. trachomatis
and
N. gonorrhoeae
from 690 men in Mumbai, India, by PCR. FCU, urethral smears, and swabs were collected from men seen at two sexually transmitted infection (STI) clinics. All laboratory testing was done at the Lokmanya Tilak General Hospital. Gram stain smears and culture isolation for
N. gonorrhoeae
were performed. Each FCU was tested individually and in pools using the Roche Amplicor PCR for
C. trachomatis
and
N. gonorrhoeae
with an internal control for inhibition. Specimen pools consisted of aliquots from five consecutively processed FCUs combined into an amplification tube. An optical density reading of ≥0.20 indicated a pool for which subsequent testing of individual samples was required. Prevalence by PCR on single specimens was 2.2% (15/690) for
C. trachomatis
and 5.4% (37/690) for
N. gonorrhoeae
. Compared to individual FCU results, pooling for
C. trachomatis
and
N. gonorrhoeae
had an overall sensitivity of 96.1% (50/52). Specificity was 96.5% (83/86) in that three pools required single testing that failed to identify a positive specimen. Pooling missed two positive specimens, decreased the inhibition rate, and saved 50.3% of reagent costs. In this resource-limited setting, the use of pooling to detect
C. trachomatis
and
N. gonorrhoeae
by PCR proved to be a simple, accurate, and cost-effective procedure compared to individual testing.
Invasive Group A Streptococcal Disease in Alberta, Canada (2000 to 2002)
Authors: Tyrrell Gregory J. , Lovgren Marguerite , Kress Bertha , Grimsrud Karen
Published: April 2005

Invasive group A streptococcal (iGAS) disease was placed under surveillance in Alberta in August 1999. The purpose of this study was to determine the incidence rates of iGAS infections throughout Alberta over a 3-year period (2000 to 2002) and to better understand the epidemiology of iGAS in this province. There were a total of 441 cases of invasive GAS disease over the 3 years examined (average population over 3 years, 3,055,765) and 47 deaths. The incidence in Alberta was 5.0 (2000), 5.7 (2001), and 3.8 (2002) per 100,000. The two main metropolitan regions (Edmonton and Calgary) had the majority of iGAS disease cases (305 cases), producing incidence rates of 4.8 (Edmonton) and 6.9 (Calgary) in 2000, 6.9 (Edmonton) and 6.6 (Calgary) in 2001, and 4.1 (Edmonton) and 3.9 (Calgary) in 2002, as well as deaths attributable to GAS (31 deaths). The three most prevalent M types were M1 (71 cases), M3 (52 cases), and MPT2967 (44 cases). With respect to age, the highest incidence rates occurred in those less than 1 year old (11.7 per 100,000) and those 65 years or older (11.5 per 100,000). Varicella virus infection preceded iGAS disease in 25% of children 8 years of age and under. A seasonal association was observed during the 3 years studied, with the highest number of cases occurring in the winter months and the lowest occurring during the summer months. The data for years 2000 and 2001 show that the metropolitan regions of Alberta experienced some of the highest incidence rates reported in North America in the past decade.
Use of Variable-Number Tandem Repeats To Examine Genetic Diversity of
Neisseria meningitidis
Authors: Yazdankhah Siamak P. , Lindstedt Bjørn-Arne , Caugant Dominique A.
Published: April 2005

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of
Neisseria meningitidis
and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107
N. meningitidis
isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.
Characterization of the Genetic Lineages Responsible for Pneumococcal Invasive Disease in Portugal
Authors: Serrano I. , Melo-Cristino J. , Carriço J. A. , Ramirez M.
Published: April 2005

The availability of a conjugate vaccine has the potential to reduce the disease burden of pneumococci and to alter the serotype frequency in the disease-causing population through immunoselection. These changes will probably be reflected in the distributions of individual genetic lineages within the population. We present a characterization of a collection of recent (1999 to 2002) invasive isolates from Portugal (
n
= 465) by macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. During this time, serotypes 14, 1, 3, 4, 8, 9V, 23F, 7F, 19A, and 12B were the 10 most prevalent overall by decreasing rank order. By combining the PFGE data with the sequence types (STs) of 104 isolates, we were able to identify the genetic lineages of the majority of the isolates. We found 66 STs, including 20 novel STs, corresponding to 47 different lineages by e-BURST analysis. We found in our collection a number of previously identified internationally disseminated lineages, especially among macrolide-resistant and penicillin-resistant isolates, and these accounted for most of the isolates. Most of the major lineages (17 of 25) were identified in all years of the study, suggesting that the pneumococcal population associated with invasive disease was stable. This study provides a characterization of the pneumococcal population associated with invasive disease that will be useful for detecting potential selective effects of the novel conjugate vaccine.
Testing for Induction of Clindamycin Resistance in Erythromycin-Resistant Isolates of
Staphylococcus aureus
Authors: Steward Christine D. , Raney Patti M. , Morrell Allison K. , Williams Portia P. , McDougal Linda K. , Jevitt Laura , McGowan John E. , Tenover Fred C.
Published: April 2005

Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on 128 selected nonduplicate isolates of
Staphylococcus aureus
. Disk diffusion testing involved placing CLI and erythromycin (ERY) disks approximately 12 mm apart (measured edge to edge) on a Mueller-Hinton agar plate that had been inoculated with an
S. aureus
isolate; the plate was then incubated for 16 to 18 h. Two distinct induction phenotypes (labeled D and D
+
) and four noninduction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) were observed in disk diffusion results. A clear, D-shaped zone of inhibition around the CLI disk was designated as the D phenotype and was observed for 21 isolates while a D-shaped zone containing inner colonies growing up to the CLI disk was designated as D
+
(17 isolates). In addition, 10 isolates were CLI susceptible and ERY resistant but were not inducible and showed no blunting of the CLI zone (Neg phenotype). Isolates that were CLI and ERY resistant (constitutive macrolide-lincosamide-streptogramin B resistance) demonstrated either a double zone of inhibition with an inner ring of reduced growth up to the edge of the disks (HD phenotype; 33 isolates) or solid growth around the CLI and ERY disks (R phenotype; 16 isolates). Finally, 31 isolates were susceptible by disk testing to both CLI and ERY (S phenotype). PCR results showed that isolates with a D phenotype harbored
ermA
, isolates with a D
+
phenotype contained either
ermC
(16 isolates) or
ermA
and
ermC
(one isolate), and all 10 isolates with a Neg phenotype contained
msrA
. All isolates with an HD or R phenotype harbored at least one
erm
gene. Isolates showing the D
+
phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing
S. aureus
isolates for inducible CLI resistance.
CHROMagar Candida Medium for Direct Susceptibility Testing of Yeast from Blood Cultures
Authors: Tan Grace L. , Peterson Ellena M.
Published: April 2005

An evaluation was performed on 95 blood cultures positive for
Candida
spp. to determine the correlation of direct susceptibility testing of fluconazole versus both standardized disk diffusion and MIC methods. For direct testing, an aliquot taken from BD BACTEC Plus and/or BD BACTEC Lytic/10 bottles (Becton Dickinson [BD], Sparks, MD) positive by gram stain for yeast was subcultured to CHROMagar Candida (BD), and a 25-μg fluconazole disk (BD) was placed on the plate. The area of growth inhibition surrounding the disk was measured at 24 and 48 h. In addition, a subculture of the isolate was tested by a microdilution MIC using YeastOne (TREK Diagnostics Systems Inc., OH) and disk diffusion (NCCLS M44-A) using a standardized inoculum plated onto CHROMagar Candida as well as Mueller-Hinton agar to which 2% glucose and 0.5 μg/ml methylene blue dye was added (MH-GMB). The categorical interpretation derived from the MIC was used as the reference to which the disk diffusion results were compared. There were a total of 41
Candida albicans
, 23
Candida glabrata
, 20
Candida parapsilosis
, 9
Candida tropicalis
, and 1 each of
Candida krusei
and
Candida lusitaniae
tested. At 24 h there was full agreement among the methods for all
C. albicans
,
C. tropicalis
,
C. lusitaniae
, and
C. krusei
isolates. For the
C. parapsilosis
isolates at 24 h there was one very major discrepancy using the direct CHROMagar and one major error with the standardized MH-GMB. The majority of the errors were seen at 24 h with the
C. glabrata
isolates. Of the 23
C. glabrata
isolates at 24 h by direct CHROMagar, there were 10 minor and 1 very major error; by MH-GMB there were 12 minor and 2 very major errors; and by standardized CHROMagar Candida there were 13 minor and 2 major errors. There were no very major errors with
C. glabrata
when all plates were read at 48 h. At 24 h by the direct and standardized CHROMagar the majority of
C. glabrata
isolates were more resistant, whereas by MH-GMB they were more susceptible than the reference MIC interpretation. In summary, subculturing yeast directly from blood cultures onto CHROMagar to which a fluconazole disk has been added may provide a presumptive identification at 24 h and, with the exception of
C. glabrata
, was able to predict the susceptibility to fluconazole with the majority of
Candida
isolates examined in this evaluation.
Internally Controlled Real-Time PCR Monitoring of Adenovirus DNA Load in Serum or Plasma of Transplant Recipients
Authors: Claas Eric C. J. , Schilham Marco W. , de Brouwer Caroline S. , Hubacek Petr , Echavarria Marcela , Lankester Arjan C. , van Tol Maarten J. D. , Kroes Aloys C. M.
Published: April 2005

Adenoviruses have been recognized as important pathogens in immunocompromised hosts. Particularly in pediatric allogeneic stem cell transplant recipients, the morbidity of the patients and mortality in those patients with disseminated infections have been found to increase over the last few years. Severe infections are predominantly but not exclusively caused by subgroup C adenoviruses. A multiplex real-time PCR assay using molecular beacons as probes was developed to enable monitoring of adenovirus DNA in those patients with simultaneous identification of subgroups. An internal control was coamplified in the multiplex PCR to check for the DNA isolation procedure as well as the presence of inhibitors in the clinical samples. The assay has been applied retrospectively in patient groups with different clinical outcomes of infection. In fatal cases, significantly higher adenovirus loads developed, exceeding even 10
11
copies/ml of serum or plasma. Patients with viral loads over 10
6
copies/ml appear to have an increased risk for fatal complications. This quantitative real-time PCR assay has been prospectively used clinically since 2002 to study the course of adenovirus infection. In addition, the assay provides objective start and end points of therapeutic interventions, including the clinically important evaluation of antiviral drugs.
Use of an Electronic Nose To DiagnoseMycobacterium bovisInfection in Badgers and Cattle
Authors: Fend R. , Geddes R. , Lesellier S. , Vordermeier H.-M. , Corner L. A. L. , Gormley E. , Costello E. , Hewinson R. G. , Marlin D. J. , Woodman A. C. , Chambers M. A.
Published: April 2005

It is estimated that more than 50 million cattle are infected withMycobacterium bovisworldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs ofM. bovisthat are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers withM. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection withM. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosingM. bovisinfection in cattle and badgers.
Species of
Phaeoacremonium
Associated with Infections in Humans and Environmental Reservoirs in Infected Woody Plants
Authors: Mostert Lizel , Groenewald Johannes Z. , Summerbell Richard C. , Robert Vincent , Sutton Deanna A. , Padhye Arvind A. , Crous Pedro W.
Published: April 2005

To date, three species of
Phaeoacremonium
have been associated with phaeohyphomycosis. These are
P. parasiticum
(formerly
Phialophora parasitica
),
P. inflatipes
, and
P. rubrigenum
. Numerous unknown isolates resembling
Phaeoacremonium
spp. have in recent years been isolated from human patients as well as from woody plants that appear to be the main environmental source of these fungi. Nine new
Phaeoacremonium
species, of which six were obtained as etiologic agents of human opportunistic infection, are reported. They can be identified based on their cultural and morphological characters, and the identifications are strongly supported in phylogenetic analyses of partial sequences of the actin, β-tubulin, and calmodulin genes. A multiple-entry electronic key based on morphological, cultural, and β-tubulin sequence data was developed to facilitate routine species identification. Reexamination of all isolates of
P. inflatipes
associated with human disease showed them to be misidentified and to belong to the new taxa described here.
Reemergence of
emm
1 and a Changed Superantigen Profile for Group A Streptococci Causing Invasive Infections: Results from a Nationwide Study
Authors: Ekelund Kim , Skinhøj Peter , Madsen Jesper , Konradsen Helle Bossen
Published: April 2005

Between 1999 and 2002, 496 invasive group A streptococcal (GAS) isolates from clinical microbiological departments in Denmark and subsequently 487 (98%) questionnaires from the clinicians treating the patients were received as part of a national surveillance.
emm
types and streptococcal superantigen (SAg) genes were determined. The incidence of invasive GAS infections was on average 2.3 per 100,000 per year. Bacteremia with no focal symptoms (27%) was together with erysipelas (20%) the most prevalent clinical diagnoses. Streptococcal toxic shock syndrome occurred in 10% of patients, of which 56% died. The overall case fatality rate within 30 days was 23%. In total, 47 different
emm
types were identified, of which
emm
1,
emm
3,
emm
4,
emm
12,
emm
28, and
emm
89 were identified in 72% of the 493 available isolates. During the 4-year period the presence of
emm
1 increased from 16% in 1999 to 40% in 2002. Concurrently, the presence of
emm
3 decreased from 23% in 1999 to 2% in 2002. The
emm
1 isolates predominantly carried
speA
, although the frequency decreased from 94% in 1999 to 71% in 2002, whereas the
emm
1-specific prevalence of
speC
increased from 25 to 53%. In a historical perspective, this could be interpreted as a reemergence of
emm
1 and could indicate a possible introduction of a new
emm
1 subclone. However, this reemergence did not result in any significant changes in the clinical manifestations during the study period. Our results show the complexity of invasive GAS infections, with time-dependent variations in the incidence and distribution of
emm
and SAg genes, which emphasizes the need for continuous epidemiological and molecular investigations.
Invasive Infections Caused byTrichosporonSpecies andGeotrichum capitatumin Patients with Hematological Malignancies: a Retrospective Multicenter Study from Italy and Review of the Literature
Authors: Girmenia Corrado , Pagano Livio , Martino Bruno , D'Antonio Domenico , Fanci Rosa , Specchia Giorgina , Melillo Lorella , Buelli Massimo , Pizzarelli Giampaolo , Venditti Mario , Martino Pietro
Published: April 2005

Trichosporonosis is an uncommon but frequently fatal mycosis in immunocompromised patients. A multicenter retrospective study was conducted to characterize cases of proven or probable invasive trichosporonosis diagnosed over the past 20 years in Italian patients with hematological diseases. Of the 52 cases identified, 17 were classified asTrichosporonsp. infections and 35 were attributed toGeotrichum capitatum. Acute myeloid leukemia accounted for 65.4% of the cases. The incidence rates ofTrichosporonsp. andG. capitatuminfections in acute leukemia patients were 0.4 and 0.5%, respectively. Overall, 76.9% of cases had positive blood cultures. Pulmonary involvement was documented in 26.9% of cases. Death was reported for 57.1% ofG. capitatuminfections and for 64.7% ofTrichosporonsp. infections. A literature review on trichosporonosis in patients with any underlying disease or condition revealsG. capitatumas a predominantly European pathogen, particularly in certain Mediterranean areas, whileTrichosporonsp. infections are seen with similar frequencies on all continents. The majority of publishedTrichosporonsp. andG. capitatuminfections occurred in patients with hematological diseases (62.8 and 91.7%, respectively). Well over half of these were suffering from acute leukemia (68 and 84% of patients withTrichosporonsp. andG. capitatuminfections, respectively). Crude mortality rates were 77% forTrichosporonspp. and 55.7% forG. capitatum. The optimal therapy for trichosporonosis has yet to be identified; however, in vitro experiences are providing encouraging evidence of the potential role of the new triazoles, in particular, voriconazole.
Epidemiology of Emerging Methicillin-Resistant
Staphylococcus aureus
(MRSA) in Denmark: a Nationwide Study in a Country with Low Prevalence of MRSA Infection
Authors: Faria Nuno A. , Oliveira Duarte C. , Westh Henrik , Monnet Dominique L. , Larsen Anders R. , Skov Robert , de Lencastre Hermínia
Published: April 2005

Strict infection control measures introduced during the 1970s have kept the incidence of methicillin-resistant
Staphylococcus aureus
(MRSA) infections extremely low in Denmark. Nevertheless, similarly to other countries, MRSA infections began to appear in the community in the late 1990s. A nationwide surveillance program has collected and stored all MRSA isolates since 1988 and, since 1999, clinical information has been also recorded. We used this information and isolates in a detailed epidemiological and molecular analysis of the 81 MRSA infections identified in Denmark in 2001. MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE),
spa
typing, multilocus sequence typing, and SCC
mec
typing. Comparison of the 45 community-onset MRSA (CO-MRSA) infections with the 36 hospital-acquired MRSA (HA-MRSA) infections showed several striking contrasts. Most CO-MRSA were recovered from skin and soft tissue infections caused by isolates carrying the Panton-Valentine leucocidin toxin genes, and the majority (84%) of isolates belonged to a single clonal type, ST80-IV, which has been found in the community in other European countries. Clone ST80-IV could be traced in Denmark back to 1993. ST80-IV was rarely found in HA-MRSA infections, which belonged to a large number of clonal types, including some pandemic MRSA clones. The low number of HA-MRSA infections and the diversity of MRSA clones in Danish hospitals may be the result of successful infection control measures that prevent spread of clones in hospitals. The mechanism of spread of the ST80-IV clone in the Danish community is not known, and new control measures are needed to control further spread of this and other CA-MRSA clones.
Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Authors: Palacios G. , Jabado O. , Cisterna D. , de Ory F. , Renwick N. , Echevarria J. E. , Castellanos A. , Mosquera M. , Freire M. C. , Campos R. H. , Lipkin W. I.
Published: April 2005

A sensitive nested reverse transcription-PCR assay, targeting a short fragment of the gene encoding the small hydrophobic protein (SH gene), was developed to allow rapid characterization of mumps virus in clinical samples. The sensitivity and specificity of the assay were established using representative genotypes A, B, C, D, E, and F. Mumps virus RNA was characterized directly from cerebrospinal fluid (CSF) samples and in extracts of mumps virus isolates from patients with various clinical syndromes. Direct sequencing of products and subsequent phylogenetic analysis enabled genetic classification. A simple web-based system of sequence analysis was established. The study also allowed characterization of mumps virus strains from Argentina as part of a new subgenotype. This PCR assay for characterization of mumps infections coupled to a web-based analytical program provides a rapid method for identification of known and novel strains.
Borrelia burgdorferi ospC
Heterogeneity among Human and Murine Isolates from a Defined Region of Northern Maryland and Southern Pennsylvania: Lack of Correlation with Invasive and Noninvasive Genotypes
Authors: Alghaferi Muneera Y. , Anderson Jennifer M. , Park Jinho , Auwaerter Paul G. , Aucott John N. , Norris Douglas E. , Dumler J. Stephen
Published: April 2005

B. burgdorferi
invasiveness correlates with
ospC
genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites,
B. burgdorferi ospC
heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3′
ospC
sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same
ospC
were seen in only one locality, and five of six sites contained two or more
B. burgdorferi ospC
clones. Four invasive patient isolates and six small mammal isolates clustered with “noninvasive” reference
ospC
genotypes. A high degree of
ospC
diversity exists among
B. burgdorferi
isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive
ospC
types contradicts the
ospC
invasiveness hypothesis. Alternative genetic markers for
B. burgdorferi
disseminated disease should be investigated.
Clinical Manifestations of Staphylococcal Scalded-Skin Syndrome Depend on Serotypes of Exfoliative Toxins
Authors: Yamasaki Osamu , Yamaguchi Takayuki , Sugai Motoyuki , Chapuis-Cellier Colette , Arnaud François , Vandenesch François , Etienne Jerome , Lina Gerard
Published: April 2005

We sought a possible correlation between the clinical manifestations of staphylococcal scalded-skin syndrome (SSSS) and the serotype of exfoliative toxins (ET) by PCR screening of the
eta
and
etb
genes in
Staphylococcus aureus
strains isolated from 103 patients with generalized SSSS and 95 patients with bullous impetigo. The
eta
gene and the
etb
gene were detected in, respectively, 31 (30%) and 20 (19%) episodes of generalized SSSS and 57 (60%) and 5 (5%) episodes of bullous impetigo. Both genes were detected in 52 (50%) episodes of generalized SSS and 33 (35%) episodes of bullous impetigo. To explain this link between
etb
and generalized SSSS, we examined the distribution of ETA- and ETB-specific antibodies in the healthy population (
n
= 175) and found that the anti-ETB antibody titer was lower than the anti-ETA titer. Thus, ETA is associated with bullous impetigo and ETB is associated with generalized SSSS, possibly owing to a lower titer of anti-ETB neutralizing antibodies in the general population.
env
Gene Typing of Human Immunodeficiency Virus Type 1 Strains on Electronic Microarrays
Authors: Saunders N. A. , Alexander S. , Tatt I.
Published: April 2005

The NanoChip system was used for subtyping human immunodeficiency virus type 1 (HIV-1) strains using probes complementary to the V1 region of the
env
gene. Probes for six subtypes (A to D, F, and G) and two circulating recombinant forms (AG and AE) of HIV-1 group M were included. The specificity of these oligonucleotides had been evaluated previously in a DNA enzyme immunoassay. Samples from 112 patient sera were used as templates in a nested reverse transcription-PCR to produce amplicons that were applied to the array. The array was then hybridized successively to pairs of oligonucleotide probes. The strains were assigned a subtype on the basis of their probe hybridization patterns. One strain gave a contradictory pattern and was designated as untypeable by the NanoChip assay. Eighty-eight strains gave hybridization patterns that allowed a correct subtype designation to be made by the NanoChip assay compared to either the sequence or the heteroduplex mobility assay (HMA)-determined subtypes. Thirteen strains that reacted with the subtype A probe (SA2) were incorrectly assigned to subtype A, or to one of the related circulating recombinant types (AE or AG), on the basis of reactions with probe SAE1 or SAG1. The results indicate that these oligonucleotides have relatively low specificities. The probe subtypes of three strains matched the subtypes determined for the
gag
and
pol
genes but not the
env
gene, suggesting that a recombination event may have occurred within the
env
gene. Overall, the NanoChip assay gave results comparable to those for HMA and sequencing and provides a convenient and cost-effective means by which to subtype HIV-1.
Performance of CHROMagar MRSA Medium for Detection of Methicillin-Resistant
Staphylococcus aureus
Authors: Diederen Bram , van Duijn Inge , van Belkum Alex , Willemse Piet , van Keulen Peter , Kluytmans Jan
Published: April 2005

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant
Staphylococcus aureus
(MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible
Staphylococcus aureus
isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.
Misidentification of
Mycobacterium leprae
as
Mycobacterium intracellulare
by the COBAS AMPLICOR
M. intracellulare
Test
Authors: Lefmann M. , Moter A. , Schweickert B. , Göbel U. B.
Published: April 2005

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR
Mycobacterium intracellulare
test led to false- positive results. Correct identification of
Mycobacterium leprae
was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.
Susceptibilities of
Mycobacterium tuberculosis
to Isoniazid and Rifampin on Blood Agar
Authors: Coban Ahmet Yilmaz , Bilgin Kemal , Uzun Meltem , Fisgin Nuriye Tasdelen , Akgunes Alper , Cihan Cigdem Cekic , Birinci Asuman , Durupinar Belma
Published: April 2005

In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of
Mycobacterium tuberculosis
to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a “gold standard.” Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of
M. tuberculosis
to INH and RIF.
Comparison betweenrpoBand 16S rRNA Gene Sequencing for Molecular Identification of 168 Clinical Isolates ofCorynebacterium
Authors: Khamis Atieh , Raoult Didier , La Scola Bernard
Published: April 2005

Higher proportions (91%) of 168 corynebacterial isolates were positively identified by partialrpoBgene determination than by that based on 16S rRNA gene sequences. This method is thus a simple, molecular-analysis-based method for identification of corynebacteria, but it should be used in conjunction with other tests for definitive identification.
Human Immunodeficiency Virus Type 1 in Illicit-Drug Solutions Used Intravenously Retains Infectivity
Authors: Bobkov Aleksei F. , Selimova Ludmila M. , Khanina Tatyana A. , Zverev Sergey Y. , Pokrovsky Vadim V. , Weber Jonathan N. , Bobkov Eugene N. , Rylkov Andrey V.
Published: April 2005

The stability of the human immunodeficiency virus type 1 (HIV-1) strain IIIB in drug solutions was studied. The data demonstrate that HIV-1 infectivity can be retained in drug solutions (e.g. , heroin, “Khanka,” and “Vint”) for long periods of time. This fact must be taken into account when designing health education programs for the prevention of HIV and AIDS in Eastern Europe.
Direct Identification of Major Blood Culture Pathogens, Including
Pseudomonas aeruginosa
and
Escherichia coli
, by a Panel of Fluorescence In Situ Hybridization Assays Using Peptide Nucleic Acid Probes
Authors: Søgaard M. , Stender H. , Schønheyder H. C.
Published: April 2005

Rapid identification of four major pathogens from 1,231 positive blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes (AdvanDx Inc., Woburn, Mass.) was evaluated. For
Escherichia coli
,
Staphylococcus aureus
, and
Candida albicans
results agreed with conventional identification. The lower sensitivity of the
Pseudomonas aeruginosa
assay should not compromise the utility of the four assays.
Application of a Molecular Panel To Demonstrate Enterotropic Virus Shedding by Healthy and Human Immunodeficiency Virus-Infected Patients
Authors: Minosse Claudia , Zaniratti Maria S. , Calcaterra Silvia , Carletti Fabrizio , Muscillo Michele , Pisciotta Marina , Pillitteri Letizia , Corpolongo Angela , Lauria Francesco Nicola , Narciso Pasquale , Anzidei Gianfranco , Capobianchi Maria R.
Published: April 2005

We used a molecular panel, targeting seven enteric viruses, to explore the advantage of using molecular methods to establish the etiology of enteric diseases and to evaluate the prevalence of enteric viruses in asymptomatic human immunodeficiency virus-infected patients. This approach favors rapidity and sensitivity of laboratory diagnosis of viral enteric syndromes.
Development and Application of a
Saccharomyces cerevisiae
-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus
Authors: Gibertoni Aliandra M. , Montassier Maria de Fátima S. , Sena Janete A. D. , Givisiez Patrícia E. N. , Furuyama Cibele R. A. G. , Montassier Hélio J.
Published: April 2005

A
Saccharomyces cerevisiae
-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.
Use of Unmodified Starches and Partial Removal of Serum To Improve Granada Medium Stability
Authors: Rosa-Fraile Manuel , Rodríguez-Granger Javier , Camacho-Muñoz Enrique , Sampedro Antonio
Published: April 2005

The use of 1% unmodified rice starch and 1% horse serum instead of 2% soluble starch and 5% serum in Granada medium is described. These components result in a medium of increased stability, preventing spoilage after a few days of storage at room temperature.
Long-Lasting CD3
+
T-Cell Deficiency after Cord Blood Stem Cell Transplantation in a Human Herpesvirus 6-Infected Child
Authors: Comar Manola , D'Agaro Pierlanfranco , Horejsh Douglas , Galvan Monica , Fiorentini Simona , Andolina Marino , Caruso Arnaldo , Di Luca Dario , Campello Cesare
Published: April 2005

We report a long-lasting (8-month) reactivation of human herpesvirus 6 (HHV-6) infection in child who had undergone cord blood stem cell transplantation. The reactivation was characterized by high viral loads and by immediate-early mRNA positivity. HHV-6 infection was associated with a deep depletion of CD3, while the CD4/CD8 ratio remained substantially unchanged.
The Polypore Mushroom
Irpex lacteus
, a New Causative Agent of Fungal Infections
Authors: Buzina Walter , Lass-Flörl Cornelia , Kropshofer Gabriele , Freund Martin C. , Marth Egon
Published: April 2005

Irpex lacteus
, a wood-decaying basidiomycete, was isolated from a pulmonary abscess of an immunosuppressed child. This medical strain was compared morphologically and by sequencing of the ribosomal intergenic spacers with specimens from both culture collections and herbarium desiccated material. The patient was treated successfully with amphotericin B.
Surgery and Treatment with High-Dose Liposomal Amphotericin B for Eradication of Craniofacial Zygomycosis in a Patient with Hodgkin's Disease Who Had Undergone Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Barron Michelle A. , Lay Margaret , Madinger Nancy E.
Published: April 2005

This case report describes craniofacial zygomycosis in a 24-year-old male with Hodgkin's disease who underwent chemotherapy and autologous hematopoietic stem cell transplantation, followed by a nonmyeloablative allogeneic transplant. Empirical therapy with itraconazole and amoxicillin-clavulanate failed to resolve the infection. Postdiagnosis, surgery and treatment with high-dose liposomal amphotericin B eradicated the disease.
Misidentification of
Mycobacterium peregrinum
, the Causal Organism of a Case of Bacteremia and Automatic Implantable Cardioverter Defibrillator-Associated Infection, Due to Its Unusual Acid-Fast Staining Characteristics
Authors: Short William R. , Emery Christopher , Bhandary Mallika , O'Donnell Judith A.
Published: April 2005

We report an unusual case of
Mycobacterium peregrinum
bacteremia and infection of an automatic implantable cardioverter defibrillator that was originally misidentified as a
Nocardia
sp. due, in part, to its partially acid-fast staining characteristic, morphology, and odor. The misdiagnosis had a direct effect on patient care, though the patient was subsequently successfully treated.
Chryseobacterium indologenes
Non-Catheter-Related Bacteremia in a Patient with a Solid Tumor
Authors: Christakis George B. , Perlorentzou Stavroula P. , Chalkiopoulou Irene , Athanasiou Athanasios , Legakis Nikolas J.
Published: April 2005

A case of non-catheter-related bacteremia caused by
Chryseobacterium indologenes
in a nonneutropenic man with a solid tumor is described. The patient was successfully treated with piperacillin-tazobactam.