bluuebunny/crossref_metadata_2025 huggingface dataset:
Snapshot of Moving and Expanding Clones of
Mycobacterium tuberculosis
and Their Global Distribution Assessed by Spoligotyping in an International Study
Authors: Filliol Ingrid , Driscoll Jeffrey R. , van Soolingen Dick , Kreiswirth Barry N. , Kremer Kristin , Valétudie Georges , Anh Dang Duc , Barlow Rachael , Banerjee Dilip , Bifani Pablo J. , Brudey Karine , Cataldi Angel , Cooksey Robert C. , Cousins Debby V. , Dale Jeremy W. , Dellagostin Odir A. , Drobniewski Francis , Engelmann Guido , Ferdinand Séverine , Gascoyne-Binzi Deborah , Gordon Max , Gutierrez M. Cristina , Haas Walter H. , Heersma Herre , Kassa-Kelembho Eric , Ly Ho Minh , Makristathis Athanasios , Mammina Caterina , Martin Gerald , Moström Peter , Mokrousov Igor , Narbonne Valérie , Narvskaya Olga , Nastasi Antonino , Niobe-Eyangoh Sara Ngo , Pape Jean W. , Rasolofo-Razanamparany Voahangy , Ridell Malin , Rossetti M. Lucia , Stauffer Fritz , Suffys Philip N. , Takiff Howard , Texier-Maugein Jeanne , Vincent Véronique , de Waard Jacobus H. , Sola Christophe , Rastogi Nalin
Published: May 2003

The present update on the global distribution of
Mycobacterium tuberculosis
complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for
M. tuberculosis
complex, including
Mycobacterium bovis
, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of
M. tuberculosis
. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS
6110
low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (
Mycobacterium africanum
, Beijing,
M. bovis
, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of
M. tuberculosis
families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.
Improved Detection of Rhinoviruses by Nucleic Acid Sequence-Based Amplification after Nucleotide Sequence Determination of the 5′ Noncoding Regions of Additional Rhinovirus Strains
Authors: Loens K. , Ieven M. , Ursi D. , de Laat C. , Sillekens P. , Oudshoorn P. , Goossens H.
Published: May 2003

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5′ noncoding region (5′ NCR) of the viral genome. The nucleotide sequence of the 5′ NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.
Microbial Population Diversity in the Urethras of Healthy Males and Males Suffering from Nonchlamydial, Nongonococcal Urethritis
Authors: Riemersma W. A. , van der Schee C. J. C. , van der Meijden W. I. , Verbrugh H. A. , van Belkum A.
Published: May 2003

Nonchlamydial, nongonococcal urethritis (NCNGU) is suggested to be a sexually transmitted disease in men. NCNGU patients were compared to control subjects with regard to the presence of potentially infectious bacteria in the first void urine. Patients' pre- and post-antibiotic-treatment urine samples and two samples obtained 2 weeks apart from healthy volunteers, who did not receive antibiotic therapy, were analyzed with broad-spectrum PCR tests aiming at eubacterial small subunit rRNA genes. Restriction fragment length polymorphism analysis of the amplicons cloned from the mixtures of PCR products revealed that many different species of microorganisms were found to be colonizing the male urethra. We document here clear differences in the composition of the resident urethral flora between samples obtained from various individuals and between samples obtained at various points in time for a single individual. No major changes in population complexity were found upon antimicrobial treatment. In two of five patients a previously suggested pathogen (
Mycoplasma genitalium
or
Haemophilus parainfluenzae
) was accurately identified on the basis of DNA sequencing. No ubiquitous, azithromycin-sensitive organism was identified as a common pathogen in all patients, but up to 40% of all clones represented as-yet-unclassified bacterial species. Relatively often
Pseudomonas
spp. or
Pseudomonas-
like organisms were identified in the bacterial flora of patients. Interestingly, an as-yet-uncharacterized microbial species was identified as a negative predictor of NCNGU. This species was identified in all control subjects and was absent from all of the patient' samples (5 of 5 versus 0 of 5,
P
= 0.0079). This suggests that NCNGU might also be diagnosed by assessing the absence rather than the presence of certain bacterial species.
Controlled Comparison of BACTEC 13A, MYCO/F LYTIC, BacT/ALERT MB, and ISOLATOR 10 Systems for Detection of Mycobacteremia
Authors: Crump John A. , Tanner David C. , Mirrett Stanley , McKnight Celeste M. , Reller L. Barth
Published: May 2003

To compare the performance of the BACTEC 13A (Becton Dickinson, Sparks, Md.), BACTEC MYCO/F LYTIC (Becton Dickinson), BacT/ALERT MB (bioMérieux, Durham, N.C.), and ISOLATOR 10 lysis-centrifugation (Wampole Laboratories, Cranbury, N.J.) systems for detection of mycobacteremia in adults, we inoculated 5-ml aliquots of blood from patients with suspected mycobacteremia into the bottle or tube required for each system. Of 600 sets tested, 85 (14%) yielded
Mycobacterium avium
complex (MAC) and 9 (2%) yielded other species of mycobacteria. Of 26 complete (three bottles and one tube) adequately filled (5 ± 1 ml) sets from which MAC was recovered, BACTEC 13A was positive for 19 (73%), BACTEC MYCO/F LYTIC was positive for 21 (81%), BacT/ALERT MB was positive for 22 (85%), and ISOLATOR 10 was positive for 21 (81%). Of the six possible two-way comparisons, the mean times to detection for the recovery of MAC from each bottle in positive adequately paired sets were 15.3 days for BACTEC 13A versus 12.8 days for MYCO/F LYTIC for 33 of 340 pairs, 14.1 days for BACTEC 13A versus 11.6 days for BacT/ALERT MB for 38 of 380 pairs, 12.6 days for BACTEC 13A versus 20.0 days for ISOLATOR 10 for 26 of 261 pairs, 12.8 days for BACTEC MYCO/F LYTIC versus 11.0 days for BacT/ALERT MB for 33 of 340 pairs, 13.2 days for BACTEC MYCO/F LYTIC versus 20.4 days for ISOLATOR 10 for 24 of 230 pairs, and 9.9 days for BacT/ALERT MB versus 19.0 days for ISOLATOR 10 for 24 of 257 pairs. There were no significant differences in yields between the systems. However, the mean time to detection differed significantly among the systems. The time to detection was shortest for BacT/ALERT MB, followed by BACTEC MYCO/F LYTIC and BACTEC 13A and then ISOLATOR 10. Although the numbers were too small for statistical comparison, the time to detection was substantially shorter for MAC than for
Mycobacterium tuberculosis
complex in the liquid systems. The continuously monitored systems (BACTEC MYCO/F LYTIC and BacT/ALERT MB) were as sensitive and, on balance, faster for the detection of MAC bacteremia than were the heretofore standard manual ISOLATOR 10 and radiometric BACTEC 13A systems.
Simple and Fast Lateral Flow Test for Classification of Leprosy Patients and Identification of Contacts with High Risk of Developing Leprosy
Authors: Bührer-Sékula S. , Smits H. L. , Gussenhoven G. C. , van Leeuwen J. , Amador S. , Fujiwara T. , Klatser P. R. , Oskam L.
Published: May 2003

The interruption of leprosy transmission is one of the main challenges for leprosy control programs since no consistent evidence exists that transmission has been reduced after the introduction of multidrug therapy. Sources of infection are primarily people with high loads of bacteria with or without clinical signs of leprosy. The availability of a simple test system for the detection of antibodies to phenolic glycolipid-I (PGL-I) of
Mycobacterium leprae
to identify these individuals may be important in the prevention of transmission. We have developed a lateral flow assay, the ML Flow test, for the detection of antibodies to PGL-I which takes only 10 min to perform. An agreement of 91% was observed between enzyme-linked immunosorbent assay and our test; the agreement beyond chance (kappa value) was 0.77. We evaluated the use of whole blood by comparing 539 blood and serum samples from an area of high endemicity. The observed agreement was 85.9% (kappa = 0.70). Storage of the lateral flow test and the running buffer at 28°C for up to 1 year did not influence the results of the assay. The sensitivity of the ML Flow test in correctly classifying MB patients was 97.4%. The specificity of the ML Flow test, based on the results of the control group, was 90.2%. The ML Flow test is a fast and easy-to-perform method for the detection of immunoglobulin M antibodies to PGL-I of
M.
leprae
. It does not require any special equipment, and the highly stable reagents make the test robust and suitable for use in tropical countries.
Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles
Authors: Woo Patrick C. Y. , Ng Kenneth H. L. , Lau Susanna K. P. , Yip Kam-tong , Fung Ami M. Y. , Leung Kit-wah , Tam Dorothy M. W. , Que Tak-lun , Yuen Kwok-yung
Published: May 2003

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and
Mycobacterium
species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (
Granulicatella adiacens
was misidentified as
Abiotrophia defectiva
,
Helcococcus kunzii
was misidentified as
Clostridium hastiforme
,
Olsenella uli
was misidentified as
Atopobium rimae
,
Leptotrichia buccalis
was misidentified as
Fusobacterium mortiferum
, and
Bergeyella zoohelcum
was misidentified as
Rimerella anatipestifer
), and two (5.4%) were misidentified at the species level (
Actinomyces odontolyticus
was misidentified as
Actinomyces meyeri
and
Arcobacter cryaerophilus
was misidentified as
Arcobacter butzleri
). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.
Detection of Anti-West Nile Virus Immunoglobulin M in Chicken Serum by an Enzyme-Linked Immunosorbent Assay
Authors: Johnson Alison J. , Langevin Stanley , Wolff Katherine L. , Komar Nicholas
Published: May 2003

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.
DNA-Level Characterization ofHelicobacter pyloriStrains from Patients with Overt Disease and with Benign Infections in Bangladesh
Authors: Rahman Motiur , Mukhopadhyay Asish K. , Nahar Shamsun , Datta Simanti , Mashhud Ahmad Milan , Sarker Safique , Masud Ibna M. , Engstrand Lars , Albert M. John , Nair G. Balakrish , Berg Douglas E.
Published: May 2003

The complex relation between the genotype ofHelicobacter pyloriand its association with clinical outcome is not well understood. Studies in the West have showed that strains expressing certain virulence factors (vacAs1,vacAm1, andcagA) are associated with duodenal ulcer disease. However, theH. pylorigenotype is known to vary with geographic region. In the present study, we compared several virulence markers (cagA,vacA, andiceA) and neutral markers (IS605, IS606, and IS608) inH. pyloristrains isolated from 65 adult patients with peptic ulcer (PU) and 50 patients with nonulcer dyspepsia (NUD). PCR tests indicated thatcagAis present in 75% of the strains from patients with PU compared to 55% in patients with NUD, and 80% of the isolates from patients with PU carried potentially toxigenicvacAs1alleles of the vacuolating cytotoxin gene (vacA) compared to 60% in isolates from patients with NUD. However, no significant difference in any other virulence marker was observed in isolates from both groups. Phylogenetic analysis of thevacAmiddle region and the 5′ end of thecagAgene indicates that Bangladeshi isolates are more closely related toH. pyloriisolates from India and are different from isolates from East Asia.
Molecular Epidemiology of
Mycobacterium avium
subsp.
paratuberculosis
: Evidence for Limited Strain Diversity, Strain Sharing, and Identification of Unique Targets for Diagnosis
Authors: Motiwala Alifiya S. , Strother Megan , Amonsin Alongkorn , Byrum Beverly , Naser Saleh A. , Stabel Judith R. , Shulaw William P. , Bannantine John P. , Kapur Vivek , Sreevatsan Srinand
Published: May 2003

The objectives of this study were to understand the molecular diversity of animal and human strains of
Mycobacterium avium
subsp
. paratuberculosis
isolated in the United States and to identify
M. avium
subsp.
paratuberculosis
-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS
900
integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint
M. avium
subsp.
paratuberculosis
isolates recovered from animals (
n
= 203) and patients with Crohn's disease (
n
= 7) from diverse geographic localities. Six hundred bacterial cultures, including
M. avium
subsp.
paratuberculosis
(
n
= 303), non-
M. avium
subsp.
paratuberculosis
mycobacteria (
n
= 129), and other nonmycobacterial species (
n
= 168), were analyzed to evaluate the specificity of two IS
900
integration loci and a newly described
M. avium
subsp.
paratuberculosis
-specific sequence (locus 251) as potential targets for the diagnosis of
M. avium
subsp.
paratuberculosis
. MPIL fingerprint analysis revealed that 78% of bovine origin
M. avium
subsp.
paratuberculosis
isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the
M. avium
subsp.
paratuberculosis
isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between
M. avium
subsp.
paratuberculosis
strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among
M. avium
subsp.
paratuberculosis
isolates recovered from human and ovine sources.
Two International Methicillin-Resistant
Staphylococcus aureus
Clones Endemic in a University Hospital in Patras, Greece
Authors: Aires de Sousa M. , Bartzavali C. , Spiliopoulou I. , Santos Sanches I. , Crisóstomo M. I. , de Lencastre H.
Published: May 2003

Pulsed-field gel electrophoresis (PFGE) of
Sma
I macrofragments and hybridization of
Cla
I digests with the
mecA
- and Tn
554
-specific DNA probes were used to define the endemic clones of methicillin-resistant
Staphylococcus aureus
(MRSA) among strains collected in 1993 and 1998 to 2000 at the University Hospital of Patras, Patras, Greece. Representatives of each clonal type were analyzed by
spaA
typing, multilocus sequence typing (MLST), and staphylococcal chromosomal cassette
mec
(SCC
mec
) typing. The results indicated the existence of two successive international MRSA clones: (i) a clonal type with PFGE type A, sequence type (ST) 30 (ST30), and SCC
mec
type IV, which was very similar to a clone widely spread in the United Kingdom, Mexico, and Finland, and (ii) a clonal type with PFGE type B, ST239, and SCC
mec
III, which was related to the Brazilian clone. Both clones seem to be widespread in Greece as well. A novel MRSA clone is also described and is characterized by a new MLST type (ST80) associated with SCC
mec
type IV and with the presence of Panton-Valentine leukocidin genes.
Characterization of Atypical Enteropathogenic
Escherichia coli
Strains Harboring the
astA
Gene That Were Associated with a Waterborne Outbreak of Diarrhea in Japan
Authors: Yatsuyanagi Jun , Saito Shioko , Miyajima Yoshimichi , Amano Ken-Ichi , Enomoto Katsuhiko
Published: May 2003

The virulence traits of the
Escherichia coli
strain associated with a waterborne diarrhea outbreak were examined. Forty-one of 75 students (ages 12 to 15) in Akita Prefecture, Japan, showed clinical symptoms. Seven
E. coli
Ouk:K-:H45 isolates were isolated from the patients as the causative agent of this outbreak. One isolate (EC-3605) showed the presence of
E. coli
attaching-and-effacing (
eaeA
) and enteroaggregative
E. coli
heat-stable enterotoxin-1 (
astA
) genes and the absence of Shiga toxin (
stx1
and
stx2
) genes. A polymorphic enteropathogenic
E. coli
(EPEC) adherence factor plasmid was detected in EC-3605 with a major structural gene deletion and a regulatory gene frameshift mutation, revealing that EC-3605 represents an atypical EPEC strain harboring the
astA
gene. The role that atypical EPEC strains harboring the
astA
gene play in human disease is unclear. Our results, along with those of others, present a possibility that these strains comprise a distinct category of diarrheagenic
E. coli
and that
astA
affects the age distribution of atypical-EPEC infection.
Monitoring Cytomegalovirus Infection in Adult and Pediatric Bone Marrow Transplant Recipients by a Real-Time PCR Assay Performed with Blood Plasma
Authors: Leruez-Ville Marianne , Ouachée Marie , Delarue Richard , Sauget Anne-Sophie , Blanche Stéphane , Buzyn Agnès , Rouzioux Christine
Published: May 2003

The objective of this study was to evaluate the advantages of cytomegalovirus (CMV) real-time PCR in blood plasma to monitor CMV infection in a population of adult and pediatric bone marrow recipients in comparison with the pp65 antigenemia method. Fifty allogeneic bone marrow transplant recipients from our center, including 23 adults and 27 children, were enrolled. A CMV real-time PCR designed to amplify a well-conserved region of the UL123 gene was evaluated for its results with whole blood and blood plasma. The CMV real-time PCR assay and the CMV antigenemia method were performed in parallel with 558 blood samples. The results obtained by the two techniques were significantly correlated (
r
= 0.732;
P
< 0.0001). Twenty patients developed at least one episode of CMV replication, with a total of 24 episodes detected by CMV PCR; antigenemia assays were positive in 17 of these 24 episodes. The first positive PCR test preceded the first positive antigenemia by a median of 8 days. The median time interval necessary to obtain a negative CMV PCR test after implementation of preemptive treatment was 28 days. CMV PCR of plasma was positive in two children with CMV disease (one with early CMV pneumonia and one with CMV gastroenteritis), while CMV antigenemia remained negative. The use of CMV PCR with plasma to guide both implementation and discontinuation of CMV preemptive therapy might reduce the risk of occurrence of CMV disease since patients would be treated earlier, and it might also help to reduce the duration of treatment, which could attenuate the side effects of antiviral drugs.
Epidemiology of
Cyclospora cayetanensis
and Other Intestinal Parasites in a Community in Haiti
Authors: Lopez Adriana S. , Bendik Jean M. , Alliance Jean Y. , Roberts Jacquelin M. , da Silva Alexandre J. , Moura Iaci N. S. , Arrowood Michael J. , Eberhard Mark L. , Herwaldt Barbara L.
Published: May 2003

We conducted an exploratory investigation in a community in Haiti to determine the prevalence of
Cyclospora cayetanensis
infection and to identify potential risk factors for
C. cayetanensis
infection. In 2001, two cross-sectional stool surveys and a nested case-control study were conducted. In 2002, a follow-up cross-sectional stool survey was conducted among children ≤10 years of age. Stool specimens from study participants and water samples from their wells were examined for
Cyclospora
and other intestinal parasites. In stools, the prevalence of infection with
Cyclospora
in persons of all ages decreased from 12% (20 of 167 persons) in February 2001 to 1.1% (4 of 352 persons) in April 2001, a 90.8% decrease. For children ≤10 years of age, the prevalence rates were 22.5% (16 of 71 children) in February 2001, 3.0% (4 of 135 children) in April 2001, and 2.5% (2 of 81 children) in January 2002. Use of the water from the artesian well in the northern region of the community versus the one in the south was the only risk factor associated with
Cyclospora
infection in multivariate analyses (odds ratio, 18.5; 95% confidence interval, 2.4 to 143.1). The water sample from one of the nine wells or water sources tested (one sample per source) in January 2001, shortly before the investigation began, was positive for
Cyclospora
by UV fluorescence microscopy and PCR. None of the water samples from the 46 wells or water sources tested during the investigation (one sample per source per testing period, including the artesian wells) were positive for
Cyclospora
. Further studies are needed to assess the role of water as a possible risk factor for
Cyclospora
infection in Haiti and other developing countries.
Applications of Competitor RNA in Diagnostic Reverse Transcription-PCR
Authors: Kleiboeker Steven B.
Published: May 2003

Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.
Development and Verification of an Automated Sample Processing Protocol for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Lee Brenda G. , Fiebelkorn Kristin R. , Caliendo Angela M. , Nolte Frederick S.
Published: May 2003

We developed and verified an automated sample processing protocol for use with the AMPLICOR HIV-1 MONITOR test, version 1.5 (Roche Diagnostics, Indianapolis, Ind.). The automated method uses the MagNA Pure LC instrument and total nucleic acid reagents (Roche Applied Science, Indianapolis, Ind.) to extract human immunodeficiency virus type 1 (HIV-1) RNA from plasma specimens. We compared the HIV-1 load results for a dilution series (1 to 5 nominal log
10
copies/ml) and 175 clinical specimens processed by the automated method to those for the same samples processed by the manual methods specified by the manufacturer. The sensitivity, dynamic range, and precision of the viral load assay obtained by automated processing of specimens were similar to those obtained by an ultrasensitive manual processing method. The results were highly correlated (
R
2
, 0.95), and were in close agreement, with a mean difference of 0.09 log
10
(standard deviation, 0.292). The limits of agreement were ±0.58 log
10
for results for samples processed by both the manual and the automated methods. These performance characteristics were achieved with a smaller sample volume (200 versus 500 μl) and without a high-speed centrifugation step and required only 15 min of labor for a batch of 32 samples. In conclusion, the automated sample preparation protocol can replace both the standard and the ultrasensitive manual methods used with the AMPLICOR HIV-1 MONITOR test and can substantially reduce the labor associated with this test.
Multilocus Sequence Typing and Evolutionary Relationships among the Causative Agents of Melioidosis and Glanders,
Burkholderia pseudomallei
and
Burkholderia mallei
Authors: Godoy Daniel , Randle Gaynor , Simpson Andrew J. , Aanensen David M. , Pitt Tyrone L. , Kinoshita Reimi , Spratt Brian G.
Published: May 2003

A collection of 147 isolates of
Burkholderia pseudomallei
,
B. mallei
, and
B. thailandensis
was characterized by multilocus sequence typing (MLST). The 128 isolates of
B. pseudomallei
, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of
B. thailandensis
and
B. pseudomallei
was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the
B. pseudomallei
isolates formed a cluster of closely related lineages that were fully resolved from the cluster of
B. thailandensis
isolates, confirming their separate species status. However, isolates of
B. mallei
, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the
B. mallei
isolates clustered within the
B. pseudomallei
group of isolates. Alleles at six of the seven loci in
B. mallei
were also present within
B. pseudomallei
isolates, and
B. mallei
is a clone of
B. pseudomallei
that, on population genetics grounds, should not be given separate species status.
pgi
Genotyping Is a Surrogate for Serotyping of Encapsulated
Haemophilus influenzae
Authors: Anyanwu Juliana N. , Rodriguez Carina A. , Fleming Katherine E. , Adderson Elisabeth E.
Published: May 2003

Study of the epidemiology of invasive infections caused by encapsulated
Haemophilus influenzae
has been complicated by the poor sensitivity and specificity of the serologic assays used to identify specific capsular polysaccharides. The population structure of these bacteria is highly clonal, however, and serotype is highly correlated with other genetic characteristics. We sought to determine if alleles of the highly conserved phosphoglucose isomerase (
pgi
) gene correspond to the serotypes of encapsulated
H. influenzae
strains.
pgi
alleles of 52 well-characterized encapsulated
H. influenzae
isolates were amplified by PCR, sequenced, and compared to one another and to additional previously reported
H. influenzae pgi
alleles. Overall, 83% of the strains possessed
pgi
alleles associated with the major serotype a, b, e, and f clonotypes that cause the most invasive disease in the United States. Six strains (four type a and two type f) had unusual
pgi
alleles, which suggested that these strains belonged to less common clonotypes of encapsulated bacteria or were actually nontypeable strains.
pgi
genotyping may provide a simple and stable surrogate for capsular serotyping. Further studies correlating
pgi
typing with the expression of capsule are likely to increase our understanding of the epidemiology and pathogenesis of these infections.
Evaluation of a New Serotyping Assay for Detection of Anti-Hepatitis C Virus Type-Specific Antibodies in Serum Samples
Authors: Gault Elyanne , Soussan Patrick , Morice Yoann , Sanders Lara , Berrada Assia , Rogers Brian , Dený Paul
Published: May 2003

The performance of a new version (HC03) of the hepatitis C virus (HCV) serotyping 1-6 assay (Abbott Murex Laboratories), a specific test for serological determination of HCV types, was evaluated using a selected panel of 180 HCV RNA-positive sera. HC03 was more sensitive than the current HC02 version, typing 53 (37.6%) of 141 samples which were not typable with HC02. Furthermore, the HC03 specificity was 94.1% as evaluated with a panel of 22 genotyped samples. This new version of the test improves the quality of the serological approach to HCV type determination.
Genetic Characterization of a Novel, Naturally Occurring Recombinant Human G6P[6] Rotavirus
Authors: Rahman Mustafizur , De Leener Karolien , Goegebuer Truus , Wollants Elke , Van der Donck Ingrid , Hoovels Lieve Van , Van Ranst Marc
Published: May 2003

A binary classification system has been established for group A rotaviruses, with the viral capsid protein VP7 defining G types and VP4 defining P types. At least 15 G types and 21 P types have been isolated globally with various G and P combinations. Most of the currently circulating human rotaviruses belong to G1P[8], G2P[4], G3P[8], and G4P[8]. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P[6]. This unique rotavirus was isolated from a 13-month-old human immunodeficiency virus (HIV)- negative child of an HIV-seropositive Malian mother that was hospitalized with severe diarrhea in Belgium after returning from a trip to Mali. The VP7 and VP4 genes of the rotavirus strain were sequenced, and phylogenetic trees were constructed. Nucleotide and amino acid sequence comparisons with 15 known G genotypes indicated that the VP7 sequence of strain B1711 was most closely related to an American (Se584) and an Italian (PA151) human G6 strain (95 to 96% nucleotide and 98% amino acid identity). Comparison of the VP4 sequence with 21 P types showed the closest similarity to P[6] genotypes, with greatest similarity to a G8P[6] Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P[6] genotypic combination. The discovery and surveillance of novel human and nonhuman rotavirus G or P types or of novel G/P combinations is essential for the design of future rotavirus vaccines and for our understanding of rotavirus diversity and evolution.
Evaluation of the Vitek 2 ID-GNB Assay for Identification of Members of the Family
Enterobacteriaceae
and Other Nonenteric Gram-Negative Bacilli and Comparison with the Vitek GNI+ Card
Authors: O'Hara Caroline M. , Miller J. Michael
Published: May 2003

We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family
Enterobacteriaceae
and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at −70°C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive
Salmonella
spp. and were identified as
Escherichia coli
; another was a lactose-positive, malonate-negative
Salmonella enterica
subsp.
arizonae
strain that was identified as
E. coli
. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.
Recombinant VP7-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Colorado Tick Fever Virus
Authors: Mohd Jaafar Fauziah , Attoui Houssam , Gallian Pierre , Biagini Philippe , Cantaloube Jean-François , de Micco Philippe , de Lamballerie Xavier
Published: May 2003

VP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genusColtivirus, familyReoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol.22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral generaOrbivirus,Orthoreovirus, andRotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus.
Identification and Characterization of a New Variant of Shiga Toxin 1 in
Escherichia coli
ONT:H19 of Bovine Origin
Authors: Bürk Christine , Dietrich Richard , Açar Gabriele , Moravek Maximilian , Bülte Michael , Märtlbauer Erwin
Published: May 2003

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an
Escherichia coli
strain, ONT:H19, isolated from bovine feces. The complete
stx
1
gene of this strain was amplified and sequenced. Nucleotide sequence homology with
stx
1
from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for
stx
2
by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.
Rapid Identification of
Escherichia coli
Pathotypes by Virulence Gene Detection with DNA Microarrays
Authors: Bekal Sadjia , Brousseau Roland , Masson Luke , Prefontaine Gabrielle , Fairbrother John , Harel Josée
Published: May 2003

One approach to the accurate determination of the pathogenic potential (pathotype) of isolated
Escherichia coli
strains would be through a complete assessment of each strain for the presence of all known
E. coli
virulence factors. To accomplish this, an
E. coli
virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different
E. coli
pathotypes was developed. Fluorescently labeled genomic DNAs from
E. coli
strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation. Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged. Virulence factors belonging to two different pathotypes were detected in one human
E. coli
isolate (strain H87-5406). The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (
espA
,
espB
,
tir
). After hybridization with these probes, we were able to distinguish
E. coli
strains harboring
espA
,
espB
, and
tir
sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1). Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping. Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes.
Real-Time PCR Assay of Individual Human Immunodeficiency Virus Type 1 Variants in Coinfected Human Lymphoid Tissues
Authors: Ito Yoshinori , Grivel Jean-Charles , Margolis Leonid
Published: May 2003

In the course of infection, human immunodeficiency virus type 1 (HIV-1) mutates, diverging into a “swarm” of viral quasispecies, and the predominance of CCR5- or CXCR4-utilizing quasispecies is strongly associated with the pattern of disease progression. Quantification of CCR5- and CXCR4-utilizing viruses in viral swarms is important in the investigation of the mechanisms of this phenomenon. Here, we report on a new real-time PCR-based methology for the evaluation of replication of individual CCR5- and CXCR4-utilizing variants. The assay is highly reproducible, with a coefficient of variation of <3%, and it accurately estimates the numbers of virus-specific RNA copies even when their difference in the mixture is 2 orders of magnitude. We demonstrate that replications of CCR5- and CXCR4-utilizing variants can be evaluated and distinguished in experimentally coinfected human lymphoid tissue. The assay we developed may facilitate study of the mechanisms of the R5-to-X4 switch in viral swarms in human tissues infected with HIV-1.
Comparison of Lateral-Flow Immunoassay and Enzyme Immunoassay with Viral Culture for Rapid Detection of Influenza Virus in Nasal Wash Specimens from Children
Authors: Cazacu Andreea C. , Greer Jewel , Taherivand Moe , Demmler Gail J.
Published: May 2003

The performance of two commercially available rapid test kits for influenza virus detection was compared to that of viral culture by using 356 nasal wash specimens collected during the 2001 to 2002 influenza season. Overall, the two rapid tests were easy to perform and showed comparable sensitivities (70.4 and 72.2%) and specificities (97.7 and 98.3%); for both test kit groups, most of the specimens that yielded false-negative results were found to be growing influenza B virus.
Failure To Genotype Herpes Simplex Virus by Real-Time PCR Assay and Melting Curve Analysis Due to Sequence Variation within Probe Binding Sites
Authors: Anderson Trevor P. , Werno Anja M. , Beynon Kirsten A. , Murdoch David R.
Published: May 2003

Real-time PCR with melting curve analysis of PCR products is a rapid procedure for detecting and genotyping herpes simplex virus (HSV). When testing mucocutaneous samples for HSV by a real-time PCR assay targeting the DNA polymerase gene, we found that some PCR products had atypical melting curves that did not conform to the expected melting temperatures for HSV type 1 or 2. Sequence analysis showed that these strains had base-pair mismatches over the probe binding sites. An alternative assay is required to type such atypical isolates.
Multiplex PCR for Detection of Three Plasmid-Borne Genes of Enteroaggregative
Escherichia coli
Strains
Authors: Cerna Jorge F. , Nataro James P. , Estrada-Garcia Teresa
Published: May 2003

We developed a novel multiplex PCR assay for enteroaggregative
Escherichia coli
(EAEC) detection, by using three plasmid-borne genes (the aggregative adherence [AA] probe,
aap
, and
aggR
). One or more of the loci were detected in 24 (86%) of 28 patient isolates analyzed. The multiplex PCR assay is a fast, convenient, and sensitive molecular test to detect EAEC.
Detection of Fluconazole-Resistant Isolates of
Candida glabrata
by Using an Agar Screen Assay
Authors: Nelson Susan M. , Cartwright Charles P.
Published: May 2003

The ability of a fluconazole-containing agar screen assay to accurately detect isolates of
Candida glabrata
resistant to the azole antifungal agent fluconazole was evaluated on a collection of 100 clinical isolates of this organism. Results were correlated with the MIC of fluconazole for these isolates and compared with the results of a previously published disk diffusion-based fluconazole resistance screening test. Agar screen assay results were in categorical agreement with MIC-based determinations for 97% (97/100) of the isolates tested. This correlation was higher than that obtained with the disk diffusion technique, which categorized only 87% (87/100) of isolates correctly, and suggests that the agar screening approach can effectively expedite fluconazole susceptibility testing of
C. glabrata
isolates.
Kinetics and Significance of Serum Hepatitis C Virus Core Antigen in Patients with Acute Hepatitis C
Authors: Cividini Agostino , Cerino Antonella , Muzzi Alba , Furione Milena , Rebucci Chiara , Segagni Laura , Gatti Marta , Barnaba Vincenzo , Mondelli Mario U.
Published: May 2003

An immunoassay detecting hepatitis C virus core antigen was evaluated for its ability to predict clinical outcome in a series of patients with acute hepatitis C. In subjects who cleared the virus, core antigen was no longer detectable within 16 weeks of onset, whereas considerable fluctuations were noted among patients progressing to chronic hepatitis, one of whom showed consistently negative values despite the intermittent presence of viral RNA.