bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Methicillin-Resistant
Staphylococcus aureus
Directly from Nasal Swab Specimens by a Real-Time PCR Assay
Authors: Warren David K. , Liao Robert S. , Merz Liana R. , Eveland Michael , Dunne W. Michael
Published: December 2004

Screening for colonization with methicillin-resistant
Staphylococcus aureus
(MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories, including molecularly based techniques, require a culture step and the isolation of pure colonies that result in a minimum of 20 to 24 h until a result is known. We describe a qualitative in vitro diagnostic test for the rapid detection of MRSA directly from nasal swab specimens (IDI-MRSA; Infectio Diagnostic, Inc., Sainte-Foy, Québec, Canada), based upon a real-time PCR and direct detection of MRSA via amplicon hybridization with a fluorogenic target-specific molecular beacon probe. Samples from 288 patients were analyzed for the presence of MRSA with the IDI-MRSA assay, compared to detection by either direct plating or enrichment broth selective culture methods. The diagnostic values for this MRSA screening method were 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods. The time from the start of processing of specimen to result was approximately 1.5 h. In our hands, the IDI-MRSA assay is a sensitive and specific test for detection of nasal colonization with MRSA and providing for same-day results, allowing more efficient and effective use of infection control resources to control MRSA in health care facilities.
Phenotypic Profiles of Enterotoxigenic
Escherichia coli
Associated with Early Childhood Diarrhea in Rural Egypt
Authors: Shaheen Hind I. , Khalil Sami B. , Rao Malla R. , Abu Elyazeed Remon , Wierzba Thomas F. , Peruski Leonard F. , Putnam Shannon , Navarro Armando , Morsy Badria Z. , Cravioto Alejandro , Clemens John D. , Svennerholm Ann-Mari , Savarino Stephen J.
Published: December 2004

Enterotoxigenic
Escherichia coli
(ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.
Practical Implementation of a Multiplex PCR for Acute Respiratory Tract Infections in Children
Authors: Gruteke Paul , Glas Afina S. , Dierdorp Mirjam , Vreede Willem B. , Pilon Jan-Willem , Bruisten Sylvia M.
Published: December 2004

Molecular testing for acute respiratory infections (ARIs) has documented value but limited implementation due to questions that typically slow the acceptance of new tests. This study sought to address these questions and achieve implementation. Rhinovirus was added to a nested multiplex PCR (M-PCR), increasing its diagnostic yield. Over one winter, three hospital pediatric departments used the M-PCR to complement their direct fluorescent-antibody assay (DFA) for respiratory syncytial virus (RSV). Clinicians recorded “pretest probability estimates” (using continuous scales for various pathogen groups) for comparison with test results; treatments and test turnaround times were also recorded. Transnasal and throat swabs, with or without nasopharyngeal aspirate (NPA), were M-PCR tested. NPA-containing sample sets found to be RSV positive by DFA were not further tested. Single PCR for human metapneumovirus (hMPV) was performed retrospectively. Of 178 ARI episodes representing 172 patients, NPA was included in 97 sample sets; 54 (56%) were determined to be RSV positive. The other NPA-containing sample sets (
n
= 43) yielded 27 findings (63%), and the swab-only sets (
n
= 81) yielded 47 findings (58%); rhinovirus was found most often. Testing for hMPV yielded seven positive results. M-PCR median turnaround times were 4 days in swab-only samples and 5 days with NPA. Antibiotics were prescribed in 50 episodes, at rates similar for RSV and rhinovirus. Pretest probability estimates of a viral cause were lower in episodes caused by rhinovirus than in episodes caused by RSV. The hospitals continued to use M-PCR for NPA-containing samples found to be RSV negative by DFA. Test implementation is more likely with higher diagnostic yield and a protocol that reflects day-to-day clinical and laboratory operations.
Genetic Profile of an Oka Varicella Vaccine Virus Variant Isolated from an Infant with Zoster
Authors: Sauerbrei Andreas , Rubtcova Elena , Wutzler Peter , Schmid D. Scott , Loparev Vladimir N.
Published: December 2004

Varicella virus vaccine strain Oka (V-Oka) has in rare cases caused zoster in vaccinated people. Despite broad usage of V-Oka, little is known about varicella-zoster virus genomic sequence variation of strains in vaccine and isolates from patients with vaccine adverse events. Direct sequencing of 20 regions of V-Oka-GSK was compared to the sequences of the original V-Oka-Biken, GlaxoSmithKline Oka vaccine (V-Oka-GSK), and Oka-parental (P-Oka) strains. We analyzed single nucleotide polymorphisms (SNP) differentiating the Oka parental and Oka vaccine strains identified in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 51, 52, 54, 55, and 59 and eight base substitutions within ORF 62. Sixteen of these SNP impose an amino acid change in the corresponding gene product. The genotypic analysis revealed that (i) both V-Oka-GSK and V-Oka-Biken comprise mixtures of strains represented in variable proportion from lot to lot; (ii) V-Oka-GSK/zoster isolated from the zoster patient had six wild-type SNP in ORF 9A, 10, 21, 52, 55, and 62 (mutation 108838); (iii) none of the six revertant SNP would reliably discriminate Oka vaccine from the wild type; and (iv) the genomic variation found in V-Oka/zoster might be associated with changes in the biological behavior of the virus. Further studies will be needed to identify potential virulence factors in variant vaccine strains.
Prevalence and Mechanisms of Erythromycin Resistance in Group A and Group B
Streptococcus
: Implications for Reporting Susceptibility Results
Authors: Desjardins M. , Delgaty K. L. , Ramotar K. , Seetaram C. , Toye B.
Published: December 2004

Increased rates of erythromycin resistance among group B
Streptococcus
(GBS) and group A
Streptococcus
(GAS) have been reported. Cross-resistance to clindamycin may be present, depending on the mechanism of resistance. We determined the prevalence of macrolide-resistant determinants in GBS and GAS isolates to guide the laboratory reporting of erythromycin and clindamycin susceptibility. Susceptibilities were determined by the disk diffusion and broth microdilution methods. Inducible and constitutive resistance to clindamycin was determined by the double-disk diffusion method. The presence of the
ermTR
,
ermB
, and
mefA
genes was confirmed by PCR. Of the 338 GBS isolates, 55 (17%) were resistant to erythromycin, whereas 26 (8%) were resistant to clindamycin. The
erm
methylase gene was identified in 48 isolates, 22 of which had inducible resistance to clindamycin and 26 of which had constitutive resistance to clindamycin. The remaining seven resistant isolates had
mefA
. Of the 593 GAS isolates, 49 (8%) and 6 (1%) isolates were resistant to erythromycin and clindamycin, respectively. Erythromycin resistance was due to
mefA
in 33 isolates, whereas 14 isolates had
erm
-mediated resistance (9 isolates had inducible resistance and 5 isolates had constitutive resistance). In our population, erythromycin resistance in GAS was predominantly mediated by
mefA
and erythromycin resistance in GBS was predominantly mediated by
erm
. Regional differences in mechanisms of resistance need to be taken into consideration when deciding whether to report clindamycin susceptibility results on the basis of in vitro test results. Testing by the double-disk diffusion method would be an approach that could be used to address this issue, especially for GAS.
Detection of Four
Plasmodium
Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays
Authors: Rougemont Mathieu , Van Saanen Madeleine , Sahli Roland , Hinrikson Hans Peter , Bille Jacques , Jaton Katia
Published: December 2004

There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus
Plasmodium
was designed; the primer set was polymorphic enough internally to design four species-specific probes for
P. falciparum
,
P. vivax
,
P. malarie
, and
P. ovale
. Real-time PCR with species-specific probes detected one plasmid copy of
P. falciparum
,
P. vivax
,
P. malariae
, and
P. ovale
specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
Novel 180- and 480-Base-Pair Insertions in African and African-American Strains of
Helicobacter pylori
Authors: McNulty Shannon L. , Mole Beth M. , Dailidiene Daiva , Segal Issy , Ally Reid , Mistry Rajesh , Secka Ousman , Adegbola Richard A. , Thomas Julian E. , Lenarcic Erik M. , Peek Richard M. , Berg Douglas E. , Forsyth Mark H.
Published: December 2004

Helicobacter pylori
is a genetically diverse bacterial species that chronically infects human stomachs and sometimes causes severe gastroduodenal disease. Studies of polymorphic DNA sequences can suggest geographic origins of individual strains. Here, we describe a 180-bp insertion (ins180), which is just after the translation stop of a gene of unknown function, near the promoter of
jhp0152-
jhp0151
two-component signal transduction genes in strain J99, and absent from this site in strain 26695. This ins180 insertion was found in 9 of 9 Gambian (West African), 9 of 20 (45%) South African, and 9 of 40 (23%) Spanish strains but in only 2 of 20 (10%) North American strains and none of 20 Lithuanian, 20 Indian, and 20 Japanese strains. Four South African isolates that lacked ins180 and that belonged to an unusual outlier group contained a 480-bp insertion at this site (ins480), whereas none of 181 other strains screened contained ins480. In further tests 56% (10 of 18) of strains from African Americans but only 17% (3 of 18) of strains from Caucasian Americans carried ins180 (
P
< 0.05). Thus, the
H. pylori
strains of modern African Americans seem to retain traces of African roots, despite the multiple generations since their ancestors were taken from West Africa. Fragmentary ins180-like sequences were found at numerous sites in
H. pylori
genomes, always between genes. Such sequences might affect regulation of transcription and could facilitate genome rearrangement by homologous recombination. Apparent differences between African-American and Caucasian-American
H. pylori
gene pools may bear on our understanding of
H. pylori
transmission and disease outcome.
High Prevalence of Natural
Chlamydophila
Species Infection in Calves
Authors: Jee JunBae , Degraves Fred J. , Kim TeaYoun , Kaltenboeck Bernhard
Published: December 2004

We investigated the acquisition and prevalence of
Chlamydophila
sp. infection in calves. Specimens were collected at weekly intervals from birth to week 12 postpartum from 40 female Holstein calf-dam pairs in a dairy herd. Real-time PCR detected, quantified, and differentiated
Chlamydophila
23S rRNA gene DNA from vaginal cytobrush swabs and milk samples. Chemiluminescence enzyme-linked immunosorbent assay with lysed
Chlamydophila abortus
or
Chlamydophila pecorum
elementary body antigens quantified antibodies against
Chlamydophila
spp. in sera.
Chlamydophila
sp. DNA was found in 61% of calves and 20% of dams in at least one positive quantitative PCR. In calves, clinically inapparent
C. pecorum
infection with low organism loads was fivefold more prevalent than
C. abortus
infection and was most frequently detected by vaginal swabs compared to rectal or nasal swabs. In dams,
C. abortus
dominated in milk and
C. pecorum
dominated in the vagina. The group size of calves correlated positively (
P
< 0.01) with
Chlamydophila
infection in quadratic, but not linear, regression. Thus, a doubling of the group size was associated with a fourfold increase in frequency and intensity of
Chlamydophila
infection. For groups of 14 or 28 calves, respectively, logistic regression predicted a 9 or 52% probability of infection of an individual calf and a 52 or 99.99% probability of infection of the group. Anti-
Chlamydophila
immunoglobulin M antibodies in
Chlamydophila
PCR-positive calves and dams and in dams that gave birth to calves that later became positive were significantly higher than in PCR-negative animals (
P
≤ 0.02). Collectively, crowding strongly enhances the frequency and intensity of highly prevalent
Chlamydophila
infections in cattle.
Emergence and Spread of Community-Associated Methicillin-ResistantStaphylococcus aureusin Rural Wisconsin, 1989 to 1999
Authors: Stemper Mary E. , Shukla Sanjay K. , Reed Kurt D.
Published: December 2004

We investigated the emergence and spread of community-associated strains of methicillin-resistantStaphylococcus aureus(CA-MRSA) in central and northern Wisconsin by determining the temporal and clonal relationships and geographic expansion among 581 of 956 clinical isolates of MRSA collected between 1989 and 1999. Based on EcoRI plasmid profiles (PP), two types, PP-11 and PP-13, were highly stable over time and were consistently associated with multidrug-sensitive strains recovered from outpatients treated at Native American community clinics. Pulsed-field gel electrophoresis (PFGE) yielded six major clonal groups (MCGs) and 19 minor clonal groups. The six MCGs represented 82% of the isolates. All strains with either PP-11 or -13 were present in MCG-2. Eighty-nine percent of the isolates in MCG-2 originated from Native American clinics, and 90% belonged to two PFGE types (19 and 20), the types associated with an outbreak of MRSA in a Native American community in 1992. MCG-2 isolates were multidrug sensitive, harbored type IVa staphylococcal cassette chromosomemec, and were very closely related by PFGE to the Midwestern CA-MRSA strain MW2. MCG-2 strains were mostly obtained from skin infections and affected patients with a mean age of 24 (±18.0) years. MCG-2 strains spread to four additional Native American communities and 20 other communities. Our findings suggest that CA-MRSA in Wisconsin likely originated in Native American communities in the early 1990s and since has become widespread throughout the state. Two early CA-MRSA strains (WI-33 and WI-34) in Wisconsin represent progenitors of the MW2 strain, based on their almost indistinguishable genotypic characteristics.
Clinical and Laboratory Features of
Mycobacterium porcinum
Authors: Wallace Richard J. , Brown-Elliott Barbara A. , Wilson Rebecca W. , Mann Linda , Hall Leslie , Zhang Yansheng , Jost Kenneth C. , Brown June M. , Kabani Amin , Schinsky Mark F. , Steigerwalt Arnold G. , Crist Christopher J. , Roberts Glenn D. , Blacklock Zeta , Tsukamura Michio , Silcox Vella , Turenne Christine
Published: December 2004

Recent molecular studies have shown
Mycobacterium porcinum
, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the
M. fortuitum
third biovariant complex. We identified 67 clinical and two environmental isolates of the
M. fortuitum
third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the
hsp65
gene of
M. porcinum
(ATCC 33776
T
) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with
M. porcinum
ATCC 33776
T
. Sequencing of 441 bp of the
hsp65
gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake,
d
-mannitol, i-
myo
-inositol, and catalase at 68°C. They were negative for
l
-rhamnose and
d
-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin.
M. porcinum
ATCC 33776
T
gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the
M. fortuitum
third biovariant
d
-sorbitol-negative group and porcine strains of
M. porcinum
and confirmed that they belong to the same species. Identification of
M. porcinum
presently requires
hsp65
gene PRA or 16S rRNA or
hsp65
gene sequencing.
Varicella Seroprevalence and Molecular Epidemiology of Varicella-Zoster Virus in Argentina, 2002
Authors: Dayan Gustavo H. , Panero María S. , Debbag Roberto , Urquiza Ana , Molina Marta , Prieto Susana , del Carmen Perego María , Scagliotti Graciela , Galimberti Diana , Carroli Guillermo , Wolff Cristina , Schmid D. Scott , Loparev Vladimir , Guris Dalya , Seward Jane
Published: December 2004

There is limited data on immunity against varicella-zoster virus (VZV) in adults in different parts of Argentina, and it is not known which VZV strains are circulating in Argentina. The objectives of this study were as follows: (i) to evaluate seroprevalence of varicella among adults, assessing the accuracy of clinical history and determining the sociodemographic factors associated with seropositivity; and (ii) to determine the VZV strains circulating in Argentina. A cross-sectional serological survey enrolling 2,807 women aged 15 to 49 years attending public health-care settings in four cities in Argentina (i.e., Buenos Aires, Salta, Mendoza, and Rosario) and one rural area was conducted from August to November 2002. Specimens for identification of VZV strains were obtained from vesicular lesions from 13 pediatric patients with varicella from different areas of the country. PCR amplification was used for genotyping. The overall seroprevalence of varicella antibodies was 98.5% (95% confidence interval, 98.0 to 98.9), ranging from 97.2% in central Buenos Aires to 99.3% in southern Buenos Aires and Salta. Varicella seroprevalence increased with age. Crowding and length of residence in the same place were associated with seropositivity. The positive predictive value of varicella history for immunity to varicella was 99.4%; however, the negative predictive value was 2.5%. The European genotype was identified in all viral specimens. In Argentina, seroprevalence in women more than 15 years old was high regardless of the area of residence. Negative or uncertain varicella history was not a good predictor of immunity. VZV genotype was stable in all areas of the country.
Detection of Immunoglobulin M Antibodies Specific for
Toxoplasma gondii
with Increased Selectivity for Recently Acquired Infections
Authors: Kaul Ravi , Chen Peilin , Binder Steven R.
Published: December 2004

Toxoplasma gondii
infections can cause serious complications in pregnant women, leading to miscarriage, stillbirth, and birth defects. Definitive diagnosis of
T. gondii
acute infection is therefore critical for the clinical management of a mother and her fetus. Positive immunoglobulin M (IgM) results are not sufficient as evidence of recent infection, as these antibodies are often present for many months. Further, IgG avidity and differential agglutination tests, two tests used by reference laboratories to distinguish between recent and past infections, are not always in agreement, and both methods yield a significant number of indeterminate results. We report the development of a new toxoplasma IgM immunoassay that is performed by using a bead-based immunoassay on an automated analyzer (BioPlex 2200). Initial validation included 204 samples from pregnant women and 198 samples from asymptomatic healthy adults. An overall specificity of 99% was observed. Further, 100% sensitivity for acute infections was observed for 10 well-characterized seroconversion panels. We then examined 50 samples from pregnant women, all of which were IgM positive by ELISA, which had been fully evaluated in a reference laboratory. Of the 50 samples, 34 (68%) tested positive in the BioPlex 2200 toxoplasma IgM assay, of which 32 of 34 (94%) exhibited an acute or equivocal pattern by differential agglutination. Of the 16 negative samples, 15 (94%) showed high-IgG-avidity antibodies. Collectively, these results suggest that this new toxoplasma assay shows a preferential response to IgM antibodies produced by recent infections, reducing the number of positive results for pregnant women that will require extensive additional clinical evaluation.
Phenotypic and Molecular Detection of CTX-M-β-Lactamases Produced by
Escherichia coli
and
Klebsiella
spp
Authors: Pitout Johann D. D. , Hossain Ashfaque , Hanson Nancy D.
Published: December 2004

Organisms producing CTX-M-β-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-β-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of
Escherichia coli
and
Klebsiella
species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum β-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-β-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for
bla
CTX-M-1-like
genes, 95 (54%) were positive for
bla
CTX-M-14-like
genes, and the remaining 56 (32%) were negative for
bla
CTX-M
genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.
Simultaneous Detection of
Staphylococcus aureus
and Coagulase-Negative Staphylococci in Positive Blood Cultures by Real-Time PCR with Two Fluorescence Resonance Energy Transfer Probe Sets
Authors: Sakai H. , Procop G. W. , Kobayashi N. , Togawa D. , Wilson D. A. , Borden L. , Krebs V. , Bauer T. W.
Published: December 2004

A real-time PCR assay that uses two fluorescence resonance energy transfer probe sets and targets the
tuf
gene of staphylococci is described here. One probe set detects the
Staphylococcus
genus, whereas the other probe set is specific for
Staphylococcus aureus
. One hundred thirty-eight cultured isolates, which contained 41 isolates of staphylococci representing at least nine species, and 100 positive blood cultures that contained gram-positive cocci in clusters were tested. This assay was 100% sensitive and 100% specific for the detection of the
Staphylococcus
genus and of
S. aureus
.
Diarrhea Caused by Rotavirus in Children Less than 5 Years of Age in Hanoi, Vietnam
Authors: Nguyen Trung Vu , Le Van Phung , Le Huy Chinh , Weintraub Andrej
Published: December 2004

Group A rotaviruses are the major cause of diarrhea in young children worldwide. From March 2001 to April 2002, 836 children less than 5 years of age were investigated in Hanoi, Vietnam. This included 587 children with diarrhea and 249 age-matched controls. Group A rotavirus was identified in 46.7% of the children with diarrhea and 3.6% of the controls, which was a significant difference. Within the diarrhea group, the highest prevalence was seen in children from 13 to 24 months of age, and the prevalence was higher in males than in females. The symptoms of acute diarrhea caused by rotavirus were watery diarrhea, vomiting, fever, and dehydration. A higher prevalence of rotavirus detection was obtained for children who had all of these symptoms, followed by those who had diarrhea with vomiting-dehydration, fever-dehydration, and dehydration. The high rates occurred from September to December, although the infection was encountered all year round. In 58 patients (21.2% of the rotavirus-infected children), rotavirus infection was detected in association with either diarrheagenic
Escherichia coli
or
Shigella
spp. The most frequent combinations were rotavirus-enteroaggregative
E. coli
and rotavirus-enteropathogenic
E. coli
. At least one enteropathogen was identified from about 64% percent of the samples. The bacterial infection may not have given rise to clinical symptoms of such severity. The present study demonstrates the burden of rotavirus diarrhea in Hanoi, Vietnam. Continuous surveillance of diarrhea caused by rotavirus in young children would play an important role in diagnosis, treatment, and prophylaxis in order to improve the health of children in Vietnam.
Clonal Expansion of a Globally Disseminated Lineage ofMycobacterium tuberculosiswith Low IS6110Copy Numbers
Authors: Warren R. M. , Victor T. C. , Streicher E. M. , Richardson M. , van der Spuy G. D. , Johnson R. , Chihota V. N. , Locht C. , Supply P. , van Helden P. D.
Published: December 2004

Knowledge of the clonal expansion ofMycobacterium tuberculosisand accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170M. tuberculosisisolates with ≤6 IS6110insertions identified in Cape Town, South Africa, were characterized by principal genetic grouping, restriction fragment length polymorphism analysis, spoligotyping, IS6110insertion site mapping, and variable-number tandem repeat (VNTR) typing. These analyses indicated that all but one of the isolates analyzed were members of principal genetic group 2 and of the same low-IS6110-copy-number lineage. The remaining isolate was a member of principal genetic group 1 and a different low-IS6110-copy-number lineage. Phylogenetic reconstruction suggests clonal expansion through sequential acquisition of additional IS6110copies, expansion and contraction of VNTR sequences, and the deletion of specific direct-variable-repeat sequences. Furthermore, comparison of the genotypic data of 91 representative low-IS6110-copy-number isolates from Cape Town, other southern African regions, Europe, and the United States suggests that certain low-IS6110-copy-number strain spoligotypes and IS6110fingerprints were acquired in the distant past. These clones have subsequently become widely disseminated and now play an important role in the global tuberculosis epidemic.
Genome Diversity of
Pseudomonas aeruginosa
Isolates from Cystic Fibrosis Patients and the Hospital Environment
Authors: Finnan Shirley , Morrissey John P. , O'Gara Fergal , Boyd E. Fidelma
Published: December 2004

Pseudomonas aeruginosa
is a gram-negative rod that is ubiquitous in nature.
P. aeruginosa
is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts. In cystic fibrosis patients,
P. aeruginosa
is the leading cause of death. In this study, the evolutionary genetic relationships among 17
P. aeruginosa
isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone
groEL
. The
P. aeruginosa
isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere. Phylogenetically, clinical and environmental isolates clustered together with one another on the
mdh
gene tree. At the
groEL
locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments. Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase. Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions. Among the clinical isolates examined, the 15 virulence regions were variably present. The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions. Of the four genomic islands (the flagellum island and PAGI
-
1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI
-
1, -2, and -3 were absent from all isolates tested. Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen.
Detection of
gyrA
and
parC
Mutations Associated with Ciprofloxacin Resistance in
Neisseria gonorrhoeae
by Use of Oligonucleotide Biochip Technology
Authors: Zhou Wenming , Du Weidong , Cao Huimin , Zhao Jianlong , Yang Sen , Li Wei , Shen Yujun , Zhang Shumei , Du Wenhui , Zhang Xuejun
Published: December 2004

An oligonucleotide biochip that specifically detects point mutations in the
gyrA
and
parC
genes of
Neisseria gonorrhoeae
was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the
N. gonorrhoeae
strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in
gyrA
and
parC
and to confirm the biochip data. The study demonstrates that all of the point mutations in the
gyrA
and
parC
genes of
N. gonorrhoeae
were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in
N. gonorrhoeae
isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of
N. gonorrhoeae.
Real-Time PCR Assay for a Unique Chromosomal Sequence of
Bacillus anthracis
Authors: Bode Elizabeth , Hurtle William , Norwood David
Published: December 2004

Real-time PCR has become an important method for the rapid identification of
Bacillus anthracis
since the 2001 anthrax mailings. Most real-time PCR assays for
B. anthracis
have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the
rpoB
gene and the
gyrA
gene. In the present study, subtraction-hybridization with a plasmid-cured
B. anthracis
tester strain and a
Bacillus cereus
driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published
B. anthracis
Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from
B. anthracis
strain A2012. The importance of this chromosomal assay will be to verify the presence of
B. anthracis
independently of plasmid occurrence.
Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application
Authors: Rodríguez-Lázaro David , D'Agostino Martin , Pla Maria , Cook Nigel
Published: December 2004

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of
Mycobacterium avium
subsp.
paratuberculosis
is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results.
Evaluation of Multiplex PCRs for Diagnosis of Infection with Diarrheagenic
Escherichia coli
and
Shigella
spp
Authors: Aranda K. R. S. , Fagundes-Neto U. , Scaletsky I. C. A.
Published: December 2004

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic
Escherichia coli
(EPEC) isolates, enteroaggregative
E. coli
(EAEC) isolates, enterotoxigenic
E. coli
(ETEC) isolates, enteroinvasive
E. coli
(EIEC) isolates, Shiga toxin-producing
E. coli
(STEC) isolates, and
Shigella
spp. The targets selected for each group were
eae
and
bfpA
for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates,
stx
1
and
stx
2
for STEC isolates, and
ipaH
for EIEC isolates and
Shigella
spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had
Shigella
spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and
Shigella
spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.
Detection of the
van
Alphabet and Identification of Enterococci and Staphylococci at the Species Level by Multiplex PCR
Authors: Depardieu Florence , Perichon Bruno , Courvalin Patrice
Published: December 2004

A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of
Enterococcus faecium
,
Enterococcus faecalis
,
Staphylococcus aureus
, and
Staphylococcus epidermidis
at the species level. Primers targeting the genes
vanA
,
vanB
,
vanC
,
vanD
,
vanE
,
vanG
, and
ddl
of
E. faecium
and
E. faecalis
and
nuc
of
S. aureus
and a chromosomal portion specific to
S. epidermidis
were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant
S. aureus
, that may escape phenotype-based automated rapid methods.
Evaluation of Decontamination Methods and Growth Media for Primary Isolation of
Mycobacterium ulcerans
from Surgical Specimens
Authors: Yeboah-Manu Dorothy , Bodmer Thomas , Mensah-Quainoo Ernestina , Owusu Samuel , Ofori-Adjei David , Pluschke Gerd
Published: December 2004

We evaluated four decontamination methods and one nondecontamination procedure in combination with four egg-based media for the primary isolation of
Mycobacterium ulcerans
from tissue specimens. With mycobacterial recovery and contamination rates of 75.6 and 2.4%, respectively, the combination of the oxalic acid decontamination method with Lowenstein-Jensen medium supplemented with glycerol yielded the best results.
Vancomycin Resistance,
esp
, and Strain Relatedness: a 1-Year Study of Enterococcal Bacteremia
Authors: Harrington S. M. , Ross T. L. , Gebo K. A. , Merz W. G.
Published: December 2004

The prevalence of
esp,
a gene associated with infection-derived and outbreak strains, in enterococcal blood isolates from 2002 was determined. Fifty-five of 137 (40.1%)
Enterococcus faecalis
isolates, 30 of 58 (51.7%)
E. faecium
isolates, 1 of 1
E. raffinosus
isolate, 0 of 4
E. gallinarum
isolates, and 0 of 1
E. casseliflavus
isolate were positive.
esp
wasn't associated with vancomycin resistance (VR) or clinical service. VR
E. faecium
isolates were less genetically diverse than vancomycin-susceptible strains. A large cluster of VR isolates, belonging to
esp
-positive
E. faecium
, was revealed. These data support the hypothesis that
esp
and VR may contribute to dissemination of particular clones.
Study of Molecular Epidemiology of Candidiasis in Portugal by PCR Fingerprinting of
Candida
Clinical Isolates
Authors: Correia Alexandra , Sampaio Paula , Almeida Judite , Pais Célia
Published: December 2004

PCR fingerprinting was used to type 177 yeast isolates obtained from two medical institutions.
Candida albicans
was the predominant species found, followed by
C. tropicalis
,
C. glabrata
,
C. parapsilosis
,
C. guilliermondii
, and
C. krusei
, which accounted for over 20% of the strains isolated. This survey represents the first study of molecular epidemiology of candidiasis in Portugal.
Identification of a Novel Mycoplasma Species from an Oriental White-Backed Vulture (
Gyps bengalensis
)
Authors: Oaks J. Lindsay , Donahoe Shannon L. , Rurangirwa Fred R. , Rideout Bruce A. , Gilbert Martin , Virani Munir Z.
Published: December 2004

An intracellular organism was isolated from the tissues of an Oriental white-backed vulture (
Gyps bengalensis
) in chicken embryo fibroblast cell cultures. Biochemical and physical properties, ultrastructural features, and 16S ribosomal DNA sequencing classified this organism as a new taxon of mycoplasma, for which the name “
Mycoplasma vulturii
” is proposed.
Histidine Decarboxylase in
Enterobacteriaceae
Revisited
Authors: Wauters Georges , Avesani Véronique , Charlier Jacqueline , Janssens Michèle , Delmée Michel
Published: December 2004

With a modification of Taylor's decarboxylation broth, histidine decarboxylase was detected in
Enterobacter aerogenes
,
Morganella morganii
,
Raoultella ornithinolytica
, and some strains of
Citrobacter youngae
and
Raoultella planticola
. This method provides a useful confirmatory test for identification of
E. aerogenes
strains.
Development and Evaluation of Fluorogenic TaqMan Reverse Transcriptase PCR Assays for Detection of Dengue Virus Types 1 to 4
Authors: Ito Mikako , Takasaki Tomohiko , Yamada Ken-Ichiro , Nerome Reiko , Tajima Shigeru , Kurane Ichiro
Published: December 2004

The fluorogenic TaqMan reverse transcriptase PCR (RT-PCR) assay was developed for detecting each of the dengue virus (DV) types 1 to 4. DV genome was detected in all the 35 serum samples from confirmed dengue cases by the TaqMan RT-PCR, although it was not detected in 13 and 21% by conventional type-specific and cross-reactive RT-PCR, respectively.
Molecular Characterization of Fluconazole Resistance in a Case of
Candida albicans
Ocular Infection
Authors: Pancholi Preeti , Park Steven , Perlin David , Kubin Christine , Della-Latta Phyllis
Published: December 2004

Ocular yeast infections in diabetics are a therapeutic challenge. Drug resistance and reduced azole susceptibility are major concerns. The case we describe characterizes a
Candida albicans
strain from a vitrectomy specimen that was susceptible to fluconazole by in vitro testing but recalcitrant to therapy. Molecular studies revealed transient overexpression of
CDR1
and
ERG11
mRNA in the presence of fluconazole that may have contributed to poor clinical response in this patient.
Early Reinfection with Human Metapneumovirus in an Infant
Authors: Ebihara Takashi , Endo Rika , Ishiguro Nobuhisa , Nakayama Tsuguyo , Sawada Hiroyuki , Kikuta Hideaki
Published: December 2004

Human metapneumovirus (hMPV) is one of the major pathogens of respiratory illness. Reinfection with hMPV occurs frequently throughout life. We describe an infant who was infected with two different hMPV strains during a period of only 1 month.
Diagnosis based injury severity scaling: investigation of a method using Australian and New Zealand hospitalisations
Authors: Stephenson S , Henley G , Harrison J E , Langley J D
Published: December 2004

Objective:To assess the performance of theInternational Classification of Diseases(ICD) based injury severity score, ICISS, when applied to two versions of the 10th edition of ICD, ICD-10 and ICD-10-AM.

Design:ICISS was assessed on its ability to predict threat to life using logistic regression modelling. Models used ICISS and age as predictors and survival as the outcome.

Setting:Australia and New Zealand.

Patients or subjects:Hospitalisations with an ICD-10-AM principal diagnosis in the range S00–T89 from 1 July 1999 to 30 June 2001 (Australia) or 1 July 1999 to 31 December 2001 (New Zealand).

Interventions:None.

Main outcome measures:The models were assessed in terms of their discrimination, measured by the concordance score, and calibration, measured using calibration curves and the Hosmer-Lemeshow statistic.

Results:523 633 Australian and 124 767 New Zealand hospitalisations were selected, including 7230 and 1565 deaths respectively. Discrimination was high in all the fitted models with concordance scores of 0.885 to 0.910. Calibration results were also promising with all calibration curves being close to linear, though ICISS appeared to underestimate mortality somewhat for cases with an ICISS score less than 0.6. Overall ICISS performed better when applied to the Australian than the New Zealand hospitalisations. Australian and New Zealand hospitalisations were very similar. ICISS was also only a little more successful when ICD-10-AM rather than mapped ICD-10 was used.

Conclusions:ICISS appears to be a reasonable way to estimate severity for databases using ICD-10 or ICD-10-AM. It is also likely to work well for other clinical variants of ICD-10.
Child and family safety device affordability by country income level: an 18 country comparison
Authors: Hendrie D , Miller T R , Orlando M , Spicer R S , Taft C , Consunji R , Zaloshnja E
Published: December 2004

Objective:To compare availability, urban price, and affordability of child/family safety devices between 18 economically diverse countries.

Design:Descriptive: urban price surveys by local safety organisations or shoppers.

Setting:Retail stores and internet vendors.

Main outcome measures:Prices expressed in US dollars, and affordability measured by hours of factory work needed to buy a child safety seat, a belt-positioning booster seat, a child bicycle helmet, and a smoke alarm.

Results:Prices of child and family safety devices varied widely between countries but the variation for child safety seats and bicycle helmets did not relate strongly to country income. Safety devices were expensive, often prohibitively so, in lower income countries. Far more hours of factory work were required to earn a child safety device in lower income than middle income, and middle income than higher income, countries. A bicycle helmet, for example, cost 10 hours of factory work in lower income countries but less than an hour in higher income countries. Smoke alarms and booster seats were not available in many lower income countries.

Conclusions:Bicycles and two-axle motor vehicles were numerous in lower and middle income countries, but corresponding child safety devices were often unaffordable and sometimes not readily available. The apparent market distortions and their causes merit investigation. Advocacy, social marketing, local device production, lowering of tariffs, and mandatory use legislation might stimulate market growth. Arguably, a moral obligation exists to offer subsidies that give all children a fair chance of surviving to adulthood.