bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Analysis of
Cryptococcus neoformans
Mitochondrial Cytochrome
b
Gene Sequences
Authors: Biswas Swarajit Kumar , Wang Li , Yokoyama Koji , Nishimura Kazuko
Published: December 2003

Mitochondrial cytochrome
b
genes (
cyt b
) of 40 strains of
Cryptococcus neoformans
were partially sequenced to determine the genetic relations. With the exception of the type strain of
C. neoformans
var.
neoformans
, all strains contained introns in their sequences. Analysis of 386 bp of coding sequence from each strain under investigation revealed a total of 27 (6.99%) variable nucleotide sites and categorized isolates of
C. neoformans
into nine
cyt b
types.
C. neoformans
var.
gattii
included
cyt b
types I to V, and
C. neoformans
var.
neoformans
comprised types VI to IX.
cyt b
types were correlated with serotypes. All strains with
cyt b
types I, IV, and V were serotype B. All other strains except IFM 5878 (serotype B) with
cyt b
types II and III were serotype C. Serotype D strains had
cyt b
types VI and IX, and serotype A strains were
cyt b
type VIII. Of four serotype AD strains, one was
cyt b
type VII and the remaining three were type VIII. The phylogenetic tree based on deduced amino acid sequences divided the strains only into
C. neoformans
var.
neoformans
and
C. neoformans
var.
gattii
. These results indicate that
cyt b
sequences are effective for DNA typing as well as phylogenetic analysis of
C. neoformans
.
Borna Disease Virus RNA in Immunocompromised Patients in Southwestern France
Authors: Cotto Emmanuelle , Neau Didier , Cransac-Neau Martine , Auriacombe Marc , Pellegrin Jean-Luc , Ragnaud Jean-Marie , Fillet Anne-Marie , Belnard Magali , Fleury Hervé , Lafon Marie-Edith
Published: December 2003

Borna disease virus (BDV) is a neurotropic RNA virus with a wide host range. Human infections, although controversial, have been described in Europe, Asia, and the United States. The present study investigated the existence of BDV infections in immunocompromised human beings, namely, 82 human immunodeficiency virus (HIV)-infected and 80 therapeutically immunosuppressed patients. BDV p40 RNAs were detected in peripheral white blood cells with reverse transcription-nested PCR and hybridization in, respectively, 11 (13.41%) and 1 (1.25%) of the two groups of patients. BDV p24 RNAs were identified in only one of those. BDV RNA was detected in the absence of any neuropsychiatrical illness, suggesting that BDV infections may occur in asymptomatic carriers. The severity and particularity of cellular immunosuppression could explain the significantly increased detection of BDV RNA in HIV-infected patients.
Comparison of Antibiotic Resistance and Serotype Composition of Carriage and Invasive Pneumococci among Bangladeshi Children: Implications for Treatment Policy and Vaccine Formulation
Authors: Saha Samir K. , Baqui Abdullah H. , Darmstadt Gary L. , Ruhulamin M. , Hanif Mohammed , El Arifeen Shams , Santosham Mathuram , Oishi Kazunori , Nagatake Tsuyoshi , Black Robert E.
Published: December 2003

The nasopharyngeal carriage of
Streptococcus pneumoniae
is thought to pose a risk for invasive pneumococcal diseases, and the evaluation of carriage strains is thus often used to inform antibiotic treatment and vaccination strategies for these diseases. In this study, the age-specific prevalences, resistance to antibiotics, and serotype distributions of 1,340 carriage strains were analyzed and compared to 71 pneumococcal strains isolated from the cerebrospinal fluid of children under 5 years old with meningitis. Overall, the nasal carriage rate was 47%. One-fourth (26%) of the infants under 1 month of age and one-half (48%) of the infants under 12 months of age were colonized with
S. pneumoniae
. Rural children were colonized earlier than those from urban areas. Approximately one-fourth and one-half of the cases of pneumococcal meningitis occurred in the first 3 and 6 months of life, respectively. The respective rates of resistance for carriage and meningitis strains to penicillin (7 and 3%), cotrimoxazole (77 and 69%), and erythromycin (2 and 1%) were similar, whereas chloramphenicol resistance was lower among carriage strains (3%) than among meningitis strains (15.5%). The predominant serogroups of carriage and invasive isolates were variable and widely divergent. Thus, hypothetical 7-, 9-, and 11-valent vaccines, based on the predominant carriage strains of the present study, would cover only 23, 26, and 30%, respectively, of the serotypes causing meningitis. Further, currently available 7-, 9-, and 11-valent vaccines would protect against only 26, 43, and 48%, respectively, of these meningitis cases. In conclusion, while the surveillance of carriage strains for resistance to antibiotics appears useful in the design of empirical treatment guidelines for invasive pneumococcal disease, data on the serotypes of carriage strains have limited value in vaccine formulation strategies, particularly for meningitis cases.
Risk Factors Associated with
Campylobacter jejuni
Infections in Curaçao, Netherlands Antilles
Authors: Endtz Hubert P. , van West Hanneke , Godschalk Peggy C. R. , de Haan Lidewij , Halabi Yaskara , van den Braak Nicole , Kesztyüs Barbara I. , Leyde Ewald , Ott Alewijn , Verkooyen Roel , Price Lawrence J. , Woodward David L. , Rodgers Frank G. , Ang C. Wim , van Koningsveld Rinske , van Belkum Alex , Gerstenbluth Izzy
Published: December 2003

A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to
Campylobacter jejuni
, and a rise in the incidence of laboratory-confirmed
Campylobacter
enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic
C. jejuni
. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related
C. jejuni
isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of
Campylobacter
infections.
Molecular Evidence for Dissemination of Unique
Campylobacter jejuni
Clones in Curaçao, Netherlands Antilles
Authors: Duim Birgitta , Godschalk Peggy C. R. , van den Braak Nicole , Dingle Kate E. , Dijkstra Jeroen R. , Leyde Ewald , van der Plas Jan , Colles Frances M. , Endtz Hubert P. , Wagenaar Jaap A. , Maiden Martin C. J. , van Belkum Alex
Published: December 2003

Campylobacter jejuni
isolates (
n
= 234) associated with gastroenteritis and the Guillain-Barré syndrome (GBS) in the island of Curaçao, Netherlands Antilles, and collected from March 1999 to March 2000 were investigated by a range of molecular typing techniques. Data obtained by pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), automated ribotyping, and sequence analysis of the short variable region of the flagellin gene (
flaA
) were analyzed separately and in combination. Similar groupings were obtained by all methods, with the data obtained by MLST and AFLP analysis exhibiting the highest degree of congruency. MLST identified 29 sequence types, which were assigned to 10 major clonal complexes. PFGE, AFLP analysis, and ribotyping identified 10, 9, and 8 of these clonal groups, respectively; however, these three techniques permitted subdivision of the clonal groups into more different types. Members of seven clonal groups comprising 107 isolates were obtained from November 1999 to February 2000, and no distinguishing characteristics were identified for two GBS-associated strains. The sequence type 41 (ST-41), ST-508, and ST-657 clonal complexes and their corresponding AFLP types have been rare or absent in the
Campylobacter
data sets described to date. We conclude that several clonal complexes of
C. jejuni
are associated with human disease in Curaçao, and some of these have not been reported elsewhere. Furthermore, given the observation that
C. jejuni
-associated diseases appear to be more severe from November to February, it can be speculated that this may be due to the presence of virulent clones with a limited span of circulation.
Detection of the Hepatitis B Virus X Protein (HBx) Antigen and Anti-HBx Antibodies in Cases of Human Hepatocellular Carcinoma
Authors: Hwang Guang-Yuh , Lin Chuang-Yu , Huang Li-Mei , Wang Yan-Hsiung , Wang Jing-Chyi , Hsu Ching-Ting , Yang Sheng-Shun , Wu Cheng-Chung
Published: December 2003

Hepatitis B virus X protein (HBx) expressed in
Escherichia coli
DH5α by recombinant DNA technology was purified to homogeneity by use of glutathione-Sepharose beads. Immunological characterization of the recombinant HBx protein was performed. Specific binding between the anti-HBx monoclonal antibody and HBx protein showed the specificity of the recombinant HBx protein. The intact HBx protein of the factor Xa-digested glutathione
S
-transferase-HBx fusion protein was further purified and was used as an antigen for screening the titers of anti-HBx antibodies in sera. Titers of anti-HBx in sera from 20 patients with hepatocellular carcinoma (HCC), 20 patients with chronic hepatitis (CH), and 20 healthy individuals were evaluated by Western blotting and a quantitative enzyme-linked immunosorbent assay. The results indicated that 70% of sera from HCC patients and 5% of sera from CH patients contained antibodies with significant binding to the HBx protein. Western blotting of HBx protein in liver extracts from 20 HCC patients was also performed by using the anti-HBx monoclonal antibody. Results showed that 85% of HCC patients' liver tissues contained a specific HBx protein with the same molecular size as the purified intact HBx. Full correlation was found between anti-HBx antibody positivity in serum and HBx protein positivity in HCC tissues. The data demonstrated that the etiology of HCC is involved with hepatitis B virus (HBV) infection and that HBx in particular plays a role in the development of HBV-related HCC.
Concordance ofHelicobacter pyloriStrains within Families
Authors: Kivi Mårten , Tindberg Ylva , Sörberg Mikael , Casswall Thomas H. , Befrits Ragnar , Hellström Per M. , Bengtsson Carina , Engstrand Lars , Granström Marta
Published: December 2003

Helicobacter pyloriinfection is typically acquired in early childhood, and a predominantly intrafamilial transmission has been postulated. To what extent family members share the same strains is poorly documented. Our aim was to explore patterns of shared strains within families by using molecular typing. Family members ofH. pylori-infected 10- to 12-year-old index children identified in a school survey were invited to undergo gastroscopy. Bacterial isolates were typed with random amplified polymorphic DNA and PCR-restriction fragment length polymorphism of the genesureA-B,glmM, orflaA. The presence or absence of thecagpathogenicity island, a bacterial virulence factor, was determined by PCR. GelCompar II software, supplemented with visual inspection, was used in the cluster analysis. In 39 families, 104 individuals contributed 208 bacterial isolates from the antrum and corpus. A large proportion, 29 of 36 (81%) of the offspring in a sibship, harbored the same strain as at least one sibling. Mother-offspring strain concordance was detected in 10 of 18 (56%) of the families. Of 17 investigated father-offspring relations in eight families, none were strain concordant. Spouses were infected with the same strains in 5 of 23 (22%) of the couples. Different strains in the antrum and corpus were found in 8 of 104 (8%) of the subjects. Our family-based fingerprinting study demonstrates a high proportion of shared strains among siblings. Transmission between spouses seems to be appreciable. The data support mother-child and sib-sib transmission as the primary transmission pathways ofH. pylori.
Persistence of Two Genotypes of
Neisseria gonorrhoeae
during Transmission
Authors: Martin I. M. C. , Ghani A. , Bell G. , Kinghorn G. , Ison C. A.
Published: December 2003

Isolates of
Neisseria gonorrhoeae
were tested using a highly discriminatory typing method,
opa
typing, to examine the genetic diversity over a 2-year study period of isolates from all consecutive patients with gonorrhea attending the Genitourinary Medicine clinic in Sheffield, United Kingdom. Two
opa
genotypes were detected throughout the 2-year time period and comprised 41% of all strains tested. The persistence of two
opa
types was investigated further to determine the apparent genetic stability, by examining the ability of isolates to undergo intragenic and intergenic recombination and mutation in vitro. Intragenic recombination or mutation involving the
opa
genes of
N. gonorrhoeae
in the selected isolates was not detected, but intergenic recombination did occur.
opa
genes of
N. gonorrhoeae
in vivo appear to diversify primarily through intergenic recombination. Intergenic recombination in vivo would require the presence of a mixed gonococcal infection, in which an individual is concurrently colonized with more than one strain of
N. gonorrhoeae
. We propose that the level of diversity of
opa
genotypes in a population is linked to the degree of sexual mixing of individuals and the incidence of mixed infections of
N. gonorrhoeae
.
Association of the Presence of
Helicobacter
in Gallbladder Tissue with Cholelithiasis and Cholecystitis
Authors: Silva Cíntia Presser , Pereira-Lima Júlio Carlos , Oliveira Adriana Gonçalves , Guerra Juliana Becattini , Marques Daniela Lemos , Sarmanho Letícia , Cabral Mônica Maria Demas Álvares , Queiroz Dulciene Maria Magalhães
Published: December 2003

The presence of
Helicobacter
DNA species has been investigated in the biliary epithelium of patients with biliary diseases. However, conflicting results have been observed that may have been due to the small number of subjects studied, difficulty in obtaining a healthy control group, absence of controlling for confounding factors, or differences among populations. Therefore, we investigated the presence of
Helicobacter
species by culture and nested PCR of 16S rRNA genes in gallbladder tissue and bile from 46 Brazilian subjects with and 18 without cholelithiasis. The control group was mainly composed of liver donors and of patients who had submitted to cholecystectomy as part of the surgical treatment for morbid obesity. No
Helicobacter
species were grown from the bile or gallbladder tissues.
Helicobacter
DNA was detected in the gallbladder tissue and bile from 31.3 and 42.9% of the patients, respectively. In a logistic regression model, cholelithiasis was positively and independently associated with the female gender (
P
= 0.02), increasing age (
P
= 0.002), and the presence of
Helicobacter
DNA in the gallbladder tissue (
P
= 0.009). The presence of
Helicobacter
DNA in the bile was not associated with cholelithiasis (
P
= 0.8). A significant association between the presence of
Helicobacter
DNA in the gallbladder epithelium and histological cholecystitis, even after adjusting for gender and age (
P
= 0.002), was also observed. The sequences of the 16S rRNA genes were >99% similar to that of
Helicobacter pylori
. In conclusion, our results support the hypothesis that
Helicobacter
is associated with the pathogenesis of human cholelithiasis and cholecystitis.
Use of the Roche LightCycler Instrument in a Real-Time PCR for
Trichomonas vaginalis
in Urine Samples from Females and Males
Authors: Hardick Justin , Yang Samuel , Lin Shin , Duncan Della , Gaydos Charlotte
Published: December 2003

Trichomonas vaginalis
is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of
T. vaginalis
DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for
T. vaginalis
DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the “gold standard” and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of
T. vaginalis
and may prove useful in clinical settings and for large-scale screening programs.
Molecular Epidemiology of Penicillin-Susceptible Non-β-Lactam-Resistant
Streptococcus pneumoniae
Isolates from Greek Children
Authors: Bogaert D. , Hermans P. W. M. , Grivea I. N. , Katopodis G. S. , Mitchell T. J. , Sluijter M. , de Groot R. , Beratis N. G. , Syrogiannopoulos G. A.
Published: December 2003

A total of 128
Streptococcus pneumoniae
isolates that were susceptible to penicillin but resistant to non-β-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee
23F
-4, England
14
-9, and Greece
6B
-22) representing 30% of the isolates. The
erm
(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the
mef
(A) and
mef
(E) genes, respectively. The pneumococci representing
erm
(B),
erm
(A), and
mef
genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the
tet
(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-β-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-β-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.
Use of Genetic Distance as a Measure of Ongoing Transmission of
Mycobacterium tuberculosis
Authors: van der Spuy G. D. , Warren R. M. , Richardson M. , Beyers N. , Behr M. A. , van Helden P. D.
Published: December 2003

The stability of the genotypic marker IS
6110
, used to define the epidemiology of
Mycobacterium tuberculosis
, is one of the most important factors influencing the interpretation of DNA fingerprint data. We propose that evolved strains should be considered together with clustered strains to represent chains of ongoing transmission. For the present study we used a large set of fingerprint data for strains collected between 1992 and 1998 from residents of a community with a high incidence of tuberculosis in Cape Town, South Africa. Interstrain genetic distances were calculated by counting the banding pattern mismatches in the IS
6110
DNA fingerprints of different isolates. These data demonstrate that the propensity to change by one or two bands is independent of the IS
6110
copy number. Hence, the genetic distance between pairs of isolates can be simply expressed as the number of differences in the banding patterns. From this foundation, a data set which identifies newly evolved strains has been generated. Inclusion of these evolved strains into various molecular epidemiological calculations significantly increased the estimate of ongoing transmission in this study setting. The indication is that nearly all cases of tuberculosis in this community are due to ongoing transmission. This has important implications for tuberculosis control, as it indicates that the control measures used at present are unable to reduce the level of transmission. This technique may also be applicable to the study of low-incidence tuberculosis outbreaks as well as the analysis of epidemiological data from other disease epidemics.
Improved Outcomes Associated with Limiting Identification of
Candida
spp. in Respiratory Secretions
Authors: Barenfanger Joan , Arakere Pushpalatha , Cruz Rafael Dela , Imran Adil , Drake Cheryl , Lawhorn Jerry , Verhulst Steven J. , Khardori Nancy
Published: December 2003

Pneumonia due to infection with
Candida
spp. is extremely rare even though these yeasts are commonly cultured from respiratory secretions. The diagnosis of pneumonia due to
Candida
spp. should be made only by demonstrating tissue invasion of a biopsy specimen. Physicians might misinterpret the presence of
Candida
spp. in respiratory secretions as being the etiological agent of pneumonia. This study describes the practice of limiting identification (ID) of rapidly growing yeasts (i.e.,
Candida
spp.) in respiratory secretions and its impact on patients. Before November 2001, rapidly growing yeasts found in respiratory secretions were identified to the species level. After November, rapidly growing yeasts were reported as “yeasts, not
Cryptococcus
.” The group of patients with respiratory secretions processed before November 2001 is called the full ID group (
n
= 267); the group with samples processed after that date is called the limited ID group (
n
= 77). Full ID patients had an average length of hospital stay of 12.1 days/patient; that of limited ID patients was 10.1 days/patient, a decrease of 2 days/patient (
P
= 0.02). The full ID patients had an average cost of $9,407/patient; that of limited ID patients was $6,973/patient, a decrease of $2,434/patient (
P
= 0.03). Antifungal medications were used in 103 of 267 (39%) full ID patients and in 16 of 77 (21%) limited ID patients, a decrease of 18% (
P
= 0.004). Limited ID patients had a mortality rate of 14.3%; that of full ID patients was 18.7%, a decrease of 4.4% (
P =
0.37). This policy of limiting yeast ID did not impair the diagnosis of pneumonia. Rather, decreases in lengths of stay, costs, and administration of unnecessary antifungal therapy were observed after instituting this policy.
Characterization of a Novel Rapidly Growing
Mycobacterium
Species Associated with Sepsis
Authors: Hong Tao , Butler W. Ray , Hollis Frank , Floyd Margaret M. , Toney Sean R. , Tang Yi-Wei , Steele Cindy , Leggiadro Robert J.
Published: December 2003

A rapidly growing mycobacterium was isolated five times from blood cultures from a 6-year-old female patient with relapsed pre-B-cell acute lymphocytic leukemia. All five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA gene, indicative of a single species. High-performance liquid chromatography analysis of mycolic acids indicated that the species was similar to
Mycobacterium smegmatis.
Sequence analysis of the 16S rRNA gene (1,455 bp) for one isolate demonstrated that the species was closely related to
Mycobacterium diernhoferi
. Based on the phenotypic features and phylogenetic analysis, it was concluded that the isolates represented a novel rapidly growing
Mycobacterium
species. The name “
Mycobacterium hackensackense
” is proposed for this unique strain, 147-0552
T
, which was deposited in the American Type Culture Collection as ATCC BAA-823
T
.
Accuracy of Six Commercially Available Systems for Identification of Members of the Family
Vibrionaceae
Authors: O'Hara Caroline M. , Sowers Evangeline G. , Bopp Cheryl A. , Duda Sarah B. , Strockbine Nancy A.
Published: December 2003

Six commercially available bacterial identification products were tested with
Vibrio alginolyticus
(12 strains),
V. cholerae
(30 strains),
Photobacterium
(
Vibrio
)
damselae
(10 strains), V.
fluvialis
(10 strains),
V. furnissii
(4 strains),
V. hollisae
(10 strains),
V. metschnikovii
(9 strains),
V. mimicus
(10 strains),
V. parahaemolyticus
(30 strains), and
V. vulnificus
(10 strains) to determine the accuracy of each system for identification. The products included API 20E, Crystal E/NF, MicroScan Neg ID2 and Rapid Neg ID3, and Vitek GNI+ and ID-GNB. Each product was tested only with those species that were listed in its database. Overall, the systems correctly identified 63.9, 80.9, 63.1, 73.6, 73.5, and 77.7% of the isolates to species level, respectively. Error rates ranged from 0.8% for the API 20E to 10.4% for the Rapid Neg ID3. The API 20E gave “no identification” for 13.1% of the isolates, while the Neg ID2, GNI+, ID-GNB, and Crystal were unable to identify 1.8, 2.9, 5.0, and 6.9%, respectively. For
V. cholerae
, specifically, accuracy ranged from 50.0 to 96.7%, with the API 20E having the worst performance and Crystal having the best.
V. fluvialis
presented the biggest challenge for the API 20E and the GNI+, with probabilities averaging 10%, while
V. mimicus
was a major problem with the Crystal E/NF, which identified none of the strains correctly. With the Neg ID2, correct answers were often obtained only after a modified inoculation of the panel with a bacterial suspension prepared with 0.85% NaCl. Additional tests required for identification often included growth in the absence of NaCl, which is not readily available in most clinical laboratories. The only product to correctly identify at least 90% of
V. cholerae
strains was the Crystal E/NF, and only three of the six products, the API 20E and both of the Vitek cards, correctly identified more than 90% of the
V. parahaemolyticus
strains. Thus, extreme care must be taken in the interpretation of answers from these six commercially available systems for the identification of
Vibrio
species.
Rapid Identification of Commonly EncounteredCandidaSpecies Directly from Blood Culture Bottles
Authors: Selvarangan Rangaraj , Bui Uyen , Limaye Ajit P. , Cookson Brad T.
Published: December 2003

We report a rapid-cycle, real-time PCR method for identifying sixCandidaspp. directly from BACTEC blood culture bottles. Target sequences in the noncoding internal transcribed spacer regions of the rRNA operon were simultaneously amplified and interrogated with fluorescent probes to identifyCandida albicans,C. glabrata,C. parapsilosis,C. tropicalis,C. krusei, andC. lusitaniae; these account for 88% of the yeast species isolated from positive blood cultures in our laboratory. Any of the first four species can be identified in a single reaction using two fluorescent hybridization probe sets. The antifungal-resistant speciesC. kruseiandC. lusitaniaeare detected in a second reaction, also with two probe sets. The assay was validated with DNA extracted from BACTEC blood culture bottles positive for yeasts (n= 62) and was 100% concordant with culture identification based on biochemical and morphological features ofC. albicans(n= 22),C. parapsilosis(n= 10),C. tropicalis(n= 1)C. glabrata(n= 22),C. krusei(n= 2), andC. lusitaniae(n= 1). No cross-reactivity was observed in blood culture samples growing yeasts other than the above-mentioned species (n= 4), in those growing bacteria (n= 12), or in the absence of microbial growth. Our assay allows rapid (≤2 h) and specific detection of the most commonCandidaspp. directly from positive blood cultures and may facilitate delivery of optimal antifungal therapy.
Molecular Analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 Genes of a Buffalo Rotavirus Strain: Identification of the Rare P[3] Rhesus Rotavirus-Like VP4 Gene Allele
Authors: Martella V. , Ciarlet M. , Pratelli A. , Arista S. , Terio V. , Elia G. , Cavalli A. , Gentile M. , Decaro N. , Greco G. , Cafiero M. A. , Tempesta M. , Buonavoglia C.
Published: December 2003

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.
Development and Validation of a Quantitative Real-Time PCR Assay Using Fluorescence Resonance Energy Transfer Technology for Detection of
Aspergillus fumigatus
in Experimental Invasive Pulmonary Aspergillosis
Authors: O'Sullivan Cathal E. , Kasai Miki , Francesconi Andrea , Petraitis Vidmantas , Petraitiene Ruta , Kelaher Amy M. , Sarafandi Alia A. , Walsh Thomas J.
Published: December 2003

Invasive pulmonary aspergillosis (IPA) is a frequently fatal infection in immunocompromised patients that is difficult to diagnose. Present methods for detection of
Aspergillus
spp. in bronchoalveolar lavage (BAL) fluid and in tissue vary in sensitivity and specificity. We therefore developed an
A. fumigatus
-specific quantitative real-time PCR-based assay utilizing fluorescent resonance energy transfer (FRET) technology. We compared the assay to quantitative culture of BAL fluid and lung tissue in a rabbit model of experimental IPA. Using an enzymatic and high-speed mechanical cell wall disruption protocol, DNA was extracted from samples of BAL fluid and lung tissues from noninfected and
A. fumigatus
-infected rabbits. A unique primer set amplified internal transcribed spacer regions (ITS) 1 and 2 of the rRNA operon. Amplicon was detected using FRET probes targeting a unique region of ITS1. Quantitation of
A. fumigatus
DNA was achieved by use of external standards. The presence of PCR inhibitors was determined by use of a unique control plasmid. The analytical sensitivity of the assay was ≤10 copies of target DNA. No cross-reactivity occurred with other medically important filamentous fungi. The assay results correlated with pulmonary fungal burden as determined by quantitative culture (
r
= 0.72, Spearman rank correlation;
P
≤ 0.0001). The mean number of genome equivalents detected in untreated animals was 3.86 log
10
(range, 0.86 to 6.39 log
10
) in tissue. There was a 3.53-log
10
mean reduction of
A. fumigatus
genome equivalents in animals treated with amphotericin B (AMB) (95% confidence interval, 3.38 to 3.69 log
10
;
P
≤ 0.0001), which correlated with the reduction of residual fungal burden in lung tissue measured in terms of log
10
CFU/gram. The enhanced quantitative sensitivity of the real-time PCR assay was evidenced by detection of
A. fumigatus
genome in infarcted culture-negative lobes, by a greater number of mean genome equivalents compared to the number of CFU per gram in tissue and BAL fluid, and by superior detection of therapeutic response to AMB in BAL fluid compared to culture. This real-time PCR assay using FRET technology is highly sensitive and specific in detecting
A. fumigatus
DNA from BAL fluid and lung tissue in experimental IPA.
Evaluation of S. aureus ID, a New Chromogenic Agar Medium for Detection of
Staphylococcus aureus
Authors: Perry John D. , Rennison Claire , Butterworth Lynne A. , Hopley Andrew L. J. , Gould F. Kate
Published: December 2003

S. aureus ID (bioMérieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of
Staphylococcus aureus. S. aureus
produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains formed green colonies on S. aureus ID compared with 91.1% of strains forming mauve colonies on CHROMagar Staph. aureus. A total of 94.3% of strains were recovered within 18 to 20 h with conventional media. The sensitivity was increased after 48 h of incubation to 98.7, 96.2, and 95.6% with S. aureus ID, CHROMagar Staph. aureus, and conventional media, respectively. A total of 97.4% of green colonies on S. aureus ID were confirmed as
S. aureus
compared with 94.4% of mauve colonies on CHROMagar Staph. aureus. We conclude that S. aureus ID is a highly sensitive and specific medium for the isolation and identification of
S. aureus
from wound swabs.
rpoB
-Based Identification of Nonpigmented and Late-Pigmenting Rapidly Growing Mycobacteria
Authors: Adékambi Toïdi , Colson Philippe , Drancourt Michel
Published: December 2003

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the
rpoB
gene, which encodes the bacterial β subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial
rpoB
gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level.
Mycobacterium abscessus
and
Mycobacterium mucogenicum
isolates were clearly distinguished from
Mycobacterium chelonae
;
Mycobacterium mageritense
isolates were clearly distinguished from “
Mycobacterium houstonense
.” Four isolates were not identified at the species level because they exhibited >3% partial
rpoB
gene sequence divergence from the corresponding type strain; they belonged to three taxa related to
M. mucogenicum
,
Mycobacterium smegmatis
, and
Mycobacterium porcinum
. For
M. abscessus
and
M. mucogenicum
, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp
rpoB
sequence is a rapid and accurate tool for identification of RGM.
Multilocus Sequence Typing of
Candidaglabrata
Reveals Geographically EnrichedClades
Authors: Dodgson Andrew R. , Pujol Claude , Denning David W. , Soll David R. , Fox Andrew J.
Published: December 2003

The
haploid pathogenic yeast
Candida glabrata
is the second most
common
Candida
species isolated from cases of bloodstream
infection. The clinical relevance of
C. glabrata
is enhanced
by its reduced susceptibility to fluconazole. Despite this, little is
known of the epidemiology or population structure of this species. We
developed a multilocus sequence typing (MLST) scheme for
C.
glabrata
and used it to fingerprint a geographically diverse
collection of 107 clinical isolates and 2 reference strains.
Appropriate loci were identified by amplifying and sequencing fragments
of the coding regions of 11
C. glabrata
genes in 10 unrelated
isolates. The 6 most variable loci (
FKS
,
LEU2
,
NMT1
,
TRP1
,
UGP1
, and
URA3
) were
sequenced in the collection of 109 isolates. From the 3,345 bp
sequenced in each isolate, 81 nucleotide sites were found to be
variable. These defined 30 STs among the 109 strains. The technique was
validated by comparison with random amplified polymorphic DNA and the
complex DNA fingerprinting probes Cg6 and Cg12. MLST identified 5 major
clades among the isolates studied. Three of the clades exhibited
significant geographical bias. Our data demonstrate for the first time,
with such a large geographically diverse strain collection, that
distinct genetic clades of
C. glabrata
prevail in different
geographical
regions.
New Reverse Transcription-PCR Assay for Rapid and Sensitive Detection of Enterovirus Genomes in Cerebrospinal Fluid Specimens of Patients with Aseptic Meningitis
Authors: Jacques Jérôme , Carquin Jocelyne , Brodard Véronique , Moret Hélène , Lebrun Dorine , Bouscambert Maude , Motte Jacques , Rémy Gérard , Andréoletti Laurent
Published: December 2003

Enterovirus (EV) detection by a new commercially available reverse transcription (RT)-PCR assay (Penter RT-PCR test) was compared with EV isolation from cell cultures and with EV detection by an in-house RT-PCR assay. Of the 54 cerebrospinal fluid specimens collected during a summer outbreak of aseptic meningitis, 52% were positive by cell culture versus 76% by in-house RT-PCR assay and 80% by the new RT-PCR test (52 versus 76 versus 80%;
P
= 0.003). This new reliable EV RNA detection test is suitable for clinical diagnosis of EV-related meningitis and may improve the management of EV-related neurological syndromes.
Caspofungin Activity against Clinical Isolates of Fluconazole-Resistant
Candida
Authors: Pfaller Michael A. , Messer Shawn A. , Boyken Linda , Rice Cassie , Tendolkar Shailesh , Hollis Richard J. , Diekema Daniel J.
Published: December 2003

A total of 7,837 clinical isolates of
Candida
were tested against fluconazole, and 351 resistant (fluconazole MIC ≥ 64 μg/ml) isolates were identified (4% of the total tested). All fluconazole-resistant isolates were inhibited by caspofungin at concentrations that can be exceeded by standard doses (MIC at which 90% of the isolates were inhibited, 1 μg/ml; 99% of the MICs were ≤2 μg/ml).
Evaluation of Two Miniaturized Systems, MicroScan W/A and BBL Crystal E/NF, for Identification of Clinical Isolates of
Aeromonas
spp
Authors: Soler L. , Marco F. , Vila J. , Chacón M. R. , Guarro J. , Figueras M. J.
Published: December 2003

Fifty-two clinical strains and 22 type and reference
Aeromonas
strains, previously genetically characterized by 16S rRNA gene restriction fragment length polymorphism, were identified in parallel with the MicroScan Walk/Away and BBL Crystal Enteric/Nonfermenter systems. The former identified only 14.8% of the isolates correctly, and the latter identified only 20.3% correctly, which indicates that neither of these systems is useful for this purpose.
Comparison of the Sequences of the Internal Transcribed Spacer Regions and
PbGP43
Genes of
Paracoccidioides brasiliensis
from Patients and Armadillos (
Dasypus novemcinctus
)
Authors: Hebeler-Barbosa Flavia , Morais Flavia V. , Montenegro Mario R. , Kuramae Eiko E. , Montes Beatriz , McEwen Juan G. , Bagagli Eduardo , Puccia Rosana
Published: December 2003

Paracoccidioides brasiliensis
isolates from 10 nine-banded armadillos (
Dasypus novemcinctus
) were comparable with 19 clinical isolates by sequence analysis of the
PbGP43
gene and ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and by random amplified polymorphic DNA. In this original ITS study, eight isolates differed by one or three sites among five total substitution sites.
Most Human Isolates ofMycobacterium aviumMav-A and Mav-B Are Strong Producers of Hemolysin, a Putative Virulence Factor
Authors: Rindi Laura , Bonanni Daniela , Lari Nicoletta , Garzelli Carlo
Published: December 2003

Hemolysin was quantified in 58 isolates ofMycobacterium aviumfrom human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.
Pseudomonas
Cross-Infection from Cystic Fibrosis Patients to Non-Cystic Fibrosis Patients: Implications for Inpatient Care of Respiratory Patients
Authors: Robinson P. , Carzino R. , Armstrong D. , Olinsky A.
Published: December 2003

A 14-year-old boy with bronchiectasis secondary to chronic aspiration developed multiresistant
Pseudomonas aeruginosa
lower respiratory disease following several inpatient periods where accommodation and physiotherapy services were shared with cystic fibrosis (CF) patients known to be infected with the genetically identical strain of
P. aeruginosa
. Cross-infection with
P. aeruginosa
between CF patients and non-CF patients has not previously been described, and this finding raises significant issues relevant to the treatment of patients with non-CF suppurative lung disease.
Detection of Enterococcal Surface Protein Gene (
esp
) and Amplified Fragment Length Polymorphism Typing of Glycopeptide-Resistant
Enterococcus faecium
during Its Emergence in a Greek Intensive Care Unit
Authors: Routsi C. , Platsouka E. , Willems R. J. L. , Bonten M. J. M. , Paniara O. , Saroglou G. , Roussos C.
Published: December 2003

The emergence of glycopeptide-resistant
Enterococcus faecium
(GREF) in a Greek intensive care unit was studied by amplified fragment length polymorphism analysis and
esp
gene detection. Three GREF clones harboring the
esp
gene were recovered from 17 out of 21 patients, indicating the dissemination of genetically homogenous and virulent strains of GREF.
DNA Fingerprinting of
Ralstonia paucula
by Infrequent-Restriction-Site PCR and Randomly Amplified Polymorphic DNA Analysis
Authors: Moissenet Didier , Vu-Thien Hoang , Benzerara Yahia , Arlet Guillaume
Published: December 2003

Ralstonia paucula
(formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of
R. paucula
(4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates.
Fine-Structure Molecular Typing of Irish
Helicobacter pylori
Isolates and Their Genetic Relatedness to Strains from Four Different Continents
Authors: Carroll Ian M. , Ahmed Niyaz , Beesley Sarah M. , Khan Aleem A. , Ghousunnissa Sheikh , Moráin Colm A. O , Smyth Cyril J.
Published: December 2003

Genotyping of 74 Irish
Helicobacter pylori
isolates was performed at four different loci (
vacA
signal sequence and mid-region, insertion-deletion polymorphisms at the 3′ end of the
cag
pathogenicity island, and
cagA
). The predominant
vacA
alleles and insertion-deletion motifs suggest an ancestral relationship between Irish isolates and either specific East Asian or Northern European strains. In addition, fluorescent amplified fragment length polymorphism-PCR genotyping and phylogenetic analysis of 32 representative Irish
H. pylori
isolates and 22 isolates from four different continents demonstrated that the Irish
H. pylori
isolates examined were weakly clonal and showed some association with both European and Asian isolates. These three genotyping techniques show that Irish
H. pylori
isolates have distinctive features that may have evolved in this insular European population.
Evaluation of Enzyme-Linked Immunosorbent Assays and a PCR Test for Detection of Shiga Toxins for Shiga Toxin-Producing
Escherichia coli
in Cattle Herds
Authors: Segura-Alvarez Monserrat , Richter Heinz , Conraths Franz J. , Geue Lutz
Published: December 2003

Antigen capture enzyme-linked immunosorbent assays (ELISAs) for the detection of Stx1 and/or Stx2 in cattle feces were validated in comparison to the Vero cell cytotoxicity neutralization test (as a “gold standard”) applied in the course of a monitoring program for Shiga toxin-producing
Escherichia coli
in German cattle herds as a prescreening test and compared to MK1/MK2 PCR as an alternative prescreening test.
Serologic and Genomic Characterization of a G12 Human Rotavirus in Thailand
Authors: Wakuda M. , Nagashima S. , Kobayashi N. , Pongsuwanna Y. , Taniguchi K.
Published: December 2003

The G and P type specificity of the human rotavirus strain T-152 (G12P[9]) isolated in Thailand was serologically confirmed with G12-specific monoclonal antibodies prepared in this study by using a reference G12 strain, L26, as an immunizing antigen and a P[9]-specific monoclonal antibody, respectively. The genomic relationship of strain T-152 with representative human rotavirus strains was examined by means of Northern blot analysis. The results showed that T152 is closely related to strain AU-1 (G3P[9]). Gene 5 (NSP1 gene) of T152, which did not hybridize with those of any other strains examined, was characterized by sequence determination. The T152 NSP1 gene is 1,652 nucleotides in length, encodes 493 amino acids, and exhibits low identity to those of representative human and animal rotaviruses.
Cocirculation and Evolution of Two Lineages of Influenza B Viruses in Europe and Israel in the 2001-2002 Season
Authors: Chi X. Sherry , Bolar Trentice V. , Zhao Ping , Rappaport Ruth , Cheng Sheau-Mei
Published: December 2003

Forty-nine influenza B virus isolates collected in Belgium, Finland, Spain, and Israel during the 2001-2002 winter season were categorized into either of two lineages, B/Yamagata/16/88 or B/Victoria/2/87, based on the phylogenetic studies of HA1 sequences. The data trace the geographic spread of B/Victoria/2/87-like viruses and support the emergence of B/Hong Kong/1351/02-like viruses, possibly due to selective advantages of reassortment.
Evaluation of a PCR-Based Universal Heteroduplex Generator Assay as a Tool for Rapid Detection of Multidrug-ResistantMycobacterium tuberculosisin Peru
Authors: Mayta Holger , Gilman Robert H. , Arenas Fanny , Valencia Teresa , Caviedes Luz , Montenegro Sonia H. , Ticona Eduardo , Ortiz Jaime , Chumpitaz Rosa , Evans Carlton A. , Williams Diana L.
Published: December 2003

Multidrug-resistant tuberculosis is an increasing health problem worldwide, especially in developing countries. The PCR-UHG-Rif assay, which detects mutations within therpoBgene associated with rifampin resistance, was evaluated for its ability and reliability to detect and identify drug-resistantMycobacterium tuberculosisin a developing country where tuberculosis is highly endemic.
False Identification of
Coccidioides immitis
: Do Molecular Methods Always Get It Right?
Authors: Millar B. Cherie , Jiru Xu , Walker Michael J. , Evans James P. , Moore John E.
Published: December 2003

rRNA sequence analysis of a partial region of the 18S and 5.8S-internal transcribed spacer 2 (ITS2) region of
Chrysosporium keratinophilum
highlights its potential molecular misidentification as
Coccidioides immitis
. Molecular identification of medically important fungi should not be based solely on sequence analysis of the 18S rRNA gene but should be confirmed by sequence analysis of an additional rRNA gene locus, such as the ITS region(s).