bluuebunny/crossref_metadata_2025 huggingface dataset:
High Rates of Clustering of Strains Causing Tuberculosis in Harare, Zimbabwe: a Molecular Epidemiological Study
Authors: Easterbrook Philippa J. , Gibson Andrea , Murad Shahed , Lamprecht Dianie , Ives Natalie , Ferguson Alex , Lowe Odette , Mason Peter , Ndudzo Angelika , Taziwa Alfred , Makombe Robert , Mbengeranwa Lovemore , Sola Christophe , Rostogi Nalim , Drobniewski Francis
Published: October 2004

We examined the pattern of tuberculosis (TB) transmission (i.e., reactivation versus recent transmission) and the impact of human immunodeficiency virus (HIV) infection in Harare, Zimbabwe. Consecutive adult smear-positive pulmonary TB patients presenting to an urban hospital in Harare were enrolled. A detailed epidemiological questionnaire was completed, and tests for HIV type 1 and CD4 cell counts were performed for each patient. Molecular fingerprinting of the genomic DNA recovered from cultures of sputum was performed by two molecular typing methods: spacer oligonucleotide typing (spoligotyping) and analysis of variable number of tandem DNA repeats (VNTRs). A cluster was defined as isolates from two or more patients that shared the same spoligotype pattern or the same VNTR pattern, or both. DNA suitable for typing was recovered from 224 patients. The prevalence of HIV infection was 79%. Of 187 patient isolates (78.6%) typed by both spoligotyping and analysis of VNTRs, 147 were identified as part of a cluster by both methods. By spoligotyping alone, 84.1% of patient isolates were grouped into 20 clusters. The cluster size was generally <8 patient isolates, although three large clusters comprised 68, 25, and 23 patient isolates. A total of 89.4% of the patient isolates grouped into 12 clusters defined by analysis of VNTRs, with 2 large clusters consisting of 127 and 13 patient isolates, respectively. Thirty-six percent of patient isolates with a shared spoligotype and 17% with a shared VNTR pattern were geographically linked within Harare, but they were not linked on the basis of the patient's home district. In a multivariate analysis, there were no independent predictors of clustering, including HIV infection status. Comparison with the International Spoligotype database (Pasteur Institute, Pointe à Pitre, Guadeloupe) demonstrated that our three largest spoligotype clusters are well recognized and ubiquitous in Africa. In this epidemiologically well characterized urban population with a high prevalence of HIV infection, we identified a very high level of strain clustering, indicating substantial ongoing recent TB transmission. Geographic linkage could be detected in a proportion of these clusters. A small group of actively circulating strains accounted for most of the cases of TB transmission.
Controlled Clinical Comparison of the BacT/ALERT FN and the Standard Anaerobic SN Blood Culture Medium
Authors: Mirrett S. , Petti C. A. , Woods C. W. , Magadia R. , Weinstein M. P. , Reller L. B.
Published: October 2004

To determine the optimal anaerobic companion bottle to pair with the BacT/ALERT (bioMérieux, Durham, N.C.) nonvented aerobic FA (FA) medium for recovery of pathogenic microorganisms from adult patients with bacteremia and fungemia, we compared the BacT/ALERT FN (FN) anaerobic bottle with the standard BacT/ALERT SN (SN) anaerobic bottle. Each bottle, FA, FN, and SN, was filled with 8 to 12 ml of blood. Of 11,498 blood culture sets received in the clinical microbiology laboratories at two university medical centers, 7,945 sets had all three bottles filled adequately and 8,569 had both anaerobic bottles filled adequately. Of 686 clinically important (based on previously published criteria) isolates detected in one or both adequately filled anaerobic bottles, more staphylococci (
P
< 0.001), including
Staphylococcus aureus
(
P
< 0.001); members of the family
Enterobacteriaceae
(
P
< 0.001); and all microorganisms combined (
P
< 0.001) were detected in FN bottles. In contrast, more
Pseudomonas aeruginosa
isolates (
P
< 0.01) and yeasts (
P
< 0.001) were detected in SN bottles. More
Bacteroides fragilis
group bacteremias were detected only in the FN (six) than in the SN (one) anaerobic bottle (
P
= not significant). Overall, the mean time to detection was shorter with FN (16.8 h) than with SN (18.2 h). This difference in time to detection was greatest for the
B. fragilis
group: FN, 28 h, versus SN, 60.0 h. Many of the facultative microorganisms recovered in either FN or SN were also found in the companion FA. When microorganisms found in the companion FA bottle were omitted from the analysis, significantly more staphylococci (
P
< 0.001), including
S. aureus
(
P
< 0.001), and
Enterobacteriaceae
(
P
< 0.005) still were detected in FN bottles, whereas there were no significant differences for
P. aeruginosa
and yeasts, which were found as expected in FA bottles. We conclude that the companion anaerobic FN bottle detects more microorganisms than does the anaerobic SN bottle when used in conjunction with the nonvented aerobic FA bottle in the BacT/ALERT blood culture system.
Comparative Analysis of Gtf Isozyme Production and Diversity in Isolates of
Streptococcus mutans
with Different Biofilm Growth Phenotypes
Authors: Mattos-Graner Renata O. , Napimoga Marcelo H. , Fukushima Kasuo , Duncan Margaret J. , Smith Daniel J.
Published: October 2004

Streptococcus mutans
is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of
S. mutans
that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the
gtfB
and
gtfC
genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of
gtf
genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct
S. mutans
genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control
gtf
gene expression.
Aqueous Humor and Serum Immunoblotting for Immunoglobulin Types G, A, M, and E in Cases of Human Ocular Toxoplasmosis
Authors: Garweg Justus G. , Garweg Silvia-Daniela L. , Flueckiger Franziska , Jacquier Patrick , Boehnke Matthias
Published: October 2004

The purpose of this study was to compare the local and systemicToxoplasma-specific humoral immune responses in individuals with ocular toxoplasmosis (OT). To this end, paired aqueous humor and serum samples from 46 individuals with active OT and from 30 individuals without inflammatory eye disease (controls) were analyzed by immunoblotting for anti-Toxoplasmaimmunoglobulin G (IgG), IgA, IgM, and IgE directed against 20- to 120-kDa antigens. The presence in the aqueous humor of a unique band, or of at least three bands that were at least three times more intense in aqueous humor than in serum, was taken as evidence of local antibody production. IgG bands were detected in 98% of the aqueous humor samples, while IgA bands were detected in 76%, IgM bands were detected in 8%, and IgE bands were not detected in any. Evidence of local production of specific antibodies was found in 32 cases (70%) (IgG in 23 [50%]; IgA in 16 [35%]). In 10 instances (22%), routine laboratory tests were not indicative of OT. In 14 cases (30%), no local antibody production was detected by immunoblotting; 3 of these cases yielded evidence of local antibody production according to the Goldmann-Witmer coefficient. Local antibody production was revealed for 7 of the 30 controls (23%). Hence, the sensitivity of immunoblotting for IgG and IgA is 70%, and the specificity is 77%. We conclude that immunoblotting for local specific IgG and IgA supports the clinical diagnosis of OT in 70% of cases. In 22% of these, the diagnosis is not confirmed by other laboratory tests. Hence, immunoblotting increases the sensitivity of routine laboratory tests and should be considered for samples that register negative by such tests.
Seroprevalence of Antibodies against Noroviruses among Students in a Chinese Military Medical University
Authors: Dai Ying-chun , Nie Jun , Zhang Xu-fu , Li Zhi-feng , Bai Yang , Zeng Zhi-rong , Yu Shou-yi , Farkas Tibor , Jiang Xi
Published: October 2004

Noroviruses (NVs) are important causes of nonbacterial gastroenteritis in humans, but the role of NVs as a cause of diseases in the Chinese people, particularly in Chinese military personnel, remains unclear. This study investigated antibody prevalence and factors that associate with the prevalence of antibody to NVs among students attending a military medical university. Serum specimens were tested by an enzyme-linked immunosorbent assay for immunoglobulin G antibody to recombinant capsid antigens of three NVs (rNorwalk, rMxV, and rVA387). Of 588 serum samples tested, the antibody prevalence was 88.9, 54.1, or 90.0% for the three antigens, respectively. There were significant differences in the prevalence of antibody to rMxV between blood types (
P
< 0.05); the prevalence for type O was the highest (62.5%), and the prevalence for type B was the lowest (49.1%). The average optical density values for antibody to rNorwalk and rMxV were lowest among students with type B. The number of students who did not have antibody to any of the three antigens was the highest for blood type B (6.9%) compared to other blood types (0.8 to 3.4% [
P
< 0.006]). The antibody prevalence also varied with the hometown residencies of the students before joining the military, with the highest rates for students from rural areas, lower rates for students from small towns or villages, and the lowest rates for students from large cities. The numbers of students who did not have antibody to any of the three antigens were highest for students from the large cities, lower for students from small towns or villages, and lowest for students from rural areas. The distribution of ABO blood types did not differ among the three groups. These data suggest that NVs are prevalent in China and that both genetic and environmental factors play a role in NV infection.
Longitudinal Study of Transmission, Diversity, and Stability of
Streptococcus mutans
and
Streptococcus sobrinus
Genotypes in Brazilian Nursery Children
Authors: Klein Marlise Inêz , Flório Flávia Martão , Pereira Antonio Carlos , Höfling José Francisco , Gonçalves Reginaldo Bruno
Published: October 2004

The aim of this study was to perform a follow-up evaluation of the
Streptococcus mutans
and
Streptococcus sobrinus
colonization profile of children's oral cavities, which included the pattern of vertical transmission from mother to child, genotypic diversity, and stability of the strains. The subjects were 16 mother-child pairs, who were monitored for 20 months. Samples of saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from the children were collected bimonthly. Saliva samples from the mothers were also collected. After isolation and identification, the arbitrarily primed PCR method was performed for the genotypic characterization of
S. mutans
(968 isolates) and
S. sobrinus
(111 isolates). At the time the strains were acquired, the children harbored one to four distinct genotypes of
S. mutans
and only one genotype of
S. sobrinus
. Although
S. mutans
prevalence and genotypic diversity were greater than those of
S. sobrinus
, the presence of matching genotypes of
S. mutans
and
S. sobrinus
was similar (in 81.25 and 83.33% of mother-child pairs, respectively), suggesting vertical transmission for both species. This longitudinal study showed an increase in genotypic diversity of
S. mutans
in the oral cavity during the follow-up period: most of the initially acquired genotypes persisted, normally those genotypes transmitted by the mother, and some were lost during follow-up; new strains were also acquired. In conclusion,
S. mutans
and
S. sobrinus
genotypes acquired from maternal or alternative sources may show effective persistence in the oral cavity and/or transitory detection in the children's mouths, reflecting the continuous development of oral microbiota in children.
Prospective Study of Human Metapneumovirus Infection in Children Less Than 3 Years of Age
Authors: König Brigitte , König Wolfgang , Arnold Ralf , Werchau Herrmann , Ihorst Gabriele , Forster Johannes
Published: October 2004

Most lower respiratory tract infections (LRTIs) in children under the age of 3 years are due to respiratory syncytial virus (RSV). Epidemiological, host, and viral factors eventually account for the severity of LRTIs, but they do not completely explain it. Human metapneumovirus (hMPV) was recently identified in children with LRTIs. In a population-based prospective multicenter study (the PRI.DE study, conducted in Germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with LRTIs for RSV A and B, influenza viruses (IVs) A and B, and parainfluenza viruses (PIVs) 1 to 3. Of the children requiring intensive care (
n
= 85), 18% had hMPV infections, and 60% of these children were infected with hMPV in combination with RSV. We did not detect hMPV in a randomly selected subset of RSV-positive nasopharyngeal secretions (
n
= 120) from children not requiring intensive care support. hMPV was detected in <1% of virus-negative samples from patients without intensive care support (
n
= 620). Our data support the hypothesis that coinfections with RSV and hMPV are more severe than infections with either RSV or hMPV alone, at least in children younger than 3 years of age.
Performance of Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays Using a West Nile Virus Recombinant Antigen (preM/E) for Detection of West Nile Virus- and Other Flavivirus-Specific Antibodies
Authors: Hogrefe Wayne R. , Moore Ronald , Lape-Nixon Mary , Wagner Michael , Prince Harry E.
Published: October 2004

Focus Technologies developed an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a mu-capture IgM ELISA for the detection of West Nile virus (WNV)-specific antibodies based on a WNV preM/E protein recombinant antigen. Normal and disease state serum panels were used to assess the performance characteristics of the two WNV ELISA kits. Totals of 807 and 1,423 sera were used to assess the IgG ELISA and IgM ELISA kits, respectively. The Focus Technologies IgG ELISA had a sensitivity of 97.6% and a specificity of 92.1% (excluding non-WNV flavivirus sera). The comparative method for WNV IgG may lack sensitivity in detecting IgG in early WNV infection, so the specificity of the Focus IgG ELISA may be higher than 92.1%. When sera from patients either infected with or vaccinated against other flaviviruses were tested on the WNV IgG assay, 35% of the sera reacted as positive for WNV IgG. Yellow fever and Japanese encephalitis vaccinees were less reactive in the IgG ELISA than St. Louis and dengue fever patients. The Focus Technologies IgM ELISA had a sensitivity and a specificity of 99.3% (excluding the non-WNV flavivirus sera). The overall cross-reactivity for the IgM ELISA to flavivirus sera was 12%, with 31% of St. Louis encephalitis patients found to be WNV IgM positive and no yellow fever vaccinees found to be WNV IgM positive. In a selected population of 706 sera, 15 false-positive WNV IgM sera were identified. The use of a background subtraction method for the IgM ELISA eliminated all 15 false-positive results, giving a specificity of 100% for the Focus IgM ELISA.
Characterization of Pathogenic
Vibrio parahaemolyticus
Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates
Authors: Martinez-Urtaza Jaime , Lozano-Leon Antonio , DePaola Angelo , Ishibashi Masanori , Shimada Kanae , Nishibuchi Mitsuaki , Liebana Ernesto
Published: October 2004

In spite of the potential risk involved with contamination of seafood with
Vibrio parahaemolyticus
, there is a lack of information on the occurrence of pathogenic
V. parahaemolyticus
in Europe. This organism was isolated in 1999 from a large outbreak (64 cases admitted to a single hospital) associated with raw oyster consumption in Galicia, Spain, one of the most important regions in shellfish production worldwide. Two
V. parahaemolyticus
isolates from the 1999 Galicia outbreak, three additional clinical isolates obtained in the same period from hospitals in Spain, two reference strains from clinical sources, and five Spanish environmental isolates were examined. Seventeen isolates belonging to the pandemic clone isolated in Asia and North America were included in the study for comparison. All isolates were characterized by serotyping, PCR for virulence-related genes, pulsed-field gel electrophoresis (PFGE), and plasmid analysis. Four of the five clinical isolates from hospitals in Spain belonged to serotype O4:K11; the remaining isolate was O4:K untypeable (KUT). All five isolates were positive for
V. parahaemolyticus toxR
and
tlh
(species-specific genes) and
tdh
and negative for
trh
and group-specific PCR (a PCR method for detection of the pandemic clone). PFGE analysis with NotI and SfiI discriminated the European isolates in two closely related PFGE types included in a homogeneous cluster, clearly differentiated from the Asian and North American isolates. The five environmental isolates belonged to serotypes O2:K28, O2:KUT, O3:K53, O4:KUT, and O8:K22 and were negative for all virulence genes. The five isolates were discriminated into five different PFGE types unrelated to any other isolate included in the study. While the virulence characteristics (
tdh
positive,
trh
negative) of the Spanish clinical isolates matched those of the O3:K6 clone from Asia and North America, they were clearly excluded from this clone by group-specific PCR, PFGE, and serotyping. The results of this study suggest that a unique and specific clone could be related to the
V. parahaemolyticus
infections in Europe.
Evaluation and Validation of Real-Time Reverse Transcription-PCR Assay Using the LightCycler System for Detection and Quantitation of Norovirus
Authors: Pang Xiaoli , Lee Bonita , Chui Linda , Preiksaitis Jutta K. , Monroe Stephan S.
Published: October 2004

We developed an assay for the detection and quantitation of norovirus with the LightCycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR) and previously published primers in the capsid and the polymerase gene. One hundred thirty-two stool specimens from the Provincial Laboratory for Public Health (Microbiology), Alberta, Canada, and the Centers for Disease Control and Prevention, Atlanta, Ga., were used to validate the new assay. The samples were collected from patients involved in outbreaks of acute gastroenteritis or children who presented with sporadic gastroenteritis. The real-time LC RT-PCR assay detected norovirus strains from three genogroup I (G-I) clusters (G-I/1, G-I/2, and G-I/3) and 10 genogroup II (G-II) clusters (G-II/1, G-II/2, G-II/3, G-II/4, G-II/6, G-II/7, G-II/10, G-II/12, G-II/15, and G-II/16). There was 100% concordance with the results from 58 stool specimens which tested positive by conventional RT-PCR assays. By dilution analysis, the real-time LC RT-PCR was 10,000 times more sensitive than the conventional RT-PCR. The new assay increased the number of samples in which noroviruses were detected by 19%. The real-time LC RT-PCR had a wide dynamic range, detecting from 5 to 5 × 10
6
copies of RNA per reaction, resulting in a theoretical lower limit of detection of 25,000 copies of RNA per g of stool. No cross-reactions were found with specimens containing sapovirus, rotavirus, astrovirus, and adenovirus. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of norovirus infection.
Accuracy of Phenotypic and Genotypic Testing for Identification of
Streptococcus pneumoniae
and Description of
Streptococcus pseudopneumoniae
sp. nov
Authors: Arbique Judy C. , Poyart Claire , Trieu-Cuot Patrick , Quesne Gilles , Carvalho Maria da Glória S. , Steigerwalt Arnold G. , Morey Roger E. , Jackson Delois , Davidson Ross J. , Facklam Richard R.
Published: October 2004

We have identified an unusual group of viridans group streptococci that resemble
Streptococcus pneumoniae
. DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the
S. oralis-
S. mitis
group of viridans group streptococci. We suggest that this novel species be termed
Streptococcus pseudopneumoniae
. A combination of phenotypic and genetic reactions allows its identification.
S. pseudopneumoniae
strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO
2
but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of
S. pneumoniae
. Genetic tests for pneumolysin (
ply
) and manganese-dependent superoxide dismutase (
sodA
) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and
S. pneumoniae
.
Application of Molecular Fingerprinting for Qualitative Assessment of Small-Intestinal Bacterial Diversity in Dogs
Authors: Suchodolski Jan S. , Ruaux Craig G. , Steiner Jörg M. , Fetz Kathrin , Williams David A.
Published: October 2004

The aims of this study were to evaluate the use of molecular fingerprinting for assessment of bacterial diversity in canine duodenal juice and to evaluate the variation in the small intestinal microflora at repeated sampling. Two groups of dogs were used. Duodenal juice was collected from eight dogs euthanized for an unrelated project (group 1). Duodenal juice was also collected endoscopically from six dogs at weekly intervals for a total of 3 weeks (group 2). The variable V6-V8 region of bacterial 16S ribosomal DNA was amplified, and PCR amplicons separated by denaturing gradient gel electrophoresis (DGGE). The reproducibility of DGGE profiles and variations in bacterial diversity between dogs were evaluated by comparing similarity indices (Dice's coefficient; 100% represents complete identity) of DGGE profiles from group 1 dogs. Weekly variations in the flora of the small intestine were evaluated by comparison of DGGE profiles from different time points within the same individuals in group 2. The mean (± standard deviation) similarity of DGGE profiles of duodenal juice between the dogs in group 1 was 38.3 ± 15.7% (range, 12.5 to 76.65%). There was a significantly higher variation in DGGE profiles between different dogs than between duplicates obtained from the same dog (P< 0.0001). DGGE profiles from samples collected at different time points varied within individuals, possibly due to variation over time or slight variation in sampling location. DGGE profiles indicate that dogs have a highly diverse microflora of the small intestine, with marked differences between individual dogs.
Noninfectious Recombinant Antigen for Detection of St. Louis Encephalitis Virus-Specific Antibodies in Serum by Enzyme-Linked Immunosorbent Assay
Authors: Purdy David E. , Noga Amanda J. , Chang Gwong-Jen J.
Published: October 2004

Proper surveillance of virus activity and a timely response to viral outbreaks depend upon the rapid diagnosis of viral infections. The immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a fast, sensitive test routinely used for the diagnosis of the medically important West Nile and St. Louis encephalitis flaviviruses. However, the suckling mouse brain-derived (SMB) antigen used in this assay is tedious to prepare and has a risk of exposing personnel to live virus and hazardous chemicals. We report the development of a St. Louis encephalitis virus (SLEV) noninfectious recombinant antigen that is a safe and easily produced alternative antigen for use in diagnostic assays. The expression plasmid pCB8SJ2, containing the premembrane and envelope structural protein-encoding regions of SLEV, was constructed to express secreted extracellular virus-like particles (VLPs) from CHO cells. Blind-coded human serum panels were assembled from patients having recent SLEV, West Nile virus (WNV), Powassan virus, or La Crosse encephalitis virus infections to assess the sensitivity and specificity of assays with SLEV VLP or SMB antigen. MAC-ELISAs with either antigen had comparable sensitivity for the detection of IgM antibodies against SLEV. Importantly, when these two antigens were tested against a human serum panel from patients having recent WNV or Powassan virus infections, the SLEV VLPs were less likely than SMB antigen to detect flavivirus cross-reactive IgM antibodies. An optimized IgG antibody capture ELISA (GAC-ELISA) with both WNV and SLEV VLPs was developed to circumvent the frequently observed higher background in the antigen-capture IgG-ELISA (ACG-ELISA). For the detection of IgG antibodies against WNV, the GAC-ELISA resulted in a statistically significant higher performance accuracy (P= 0.003) than the ACG-ELISA when the WNV VLP antigen was used in both assays. However, no statistical difference was observed in the assay performance of the GAC-ELISA with SLEV VLP or the ACG-ELISA with SLEV SMB antigen.
Type C Botulism Due to Toxic Feed Affecting 52,000 Farmed Foxes and Minks in Finland
Authors: Lindström Miia , Nevas Mari , Kurki Joanna , Sauna-aho Raija , Latvala-Kiesilä Annikki , Pölönen Ilpo , Korkeala Hannu
Published: October 2004

The largest reported outbreak of type C botulism in fur production animals is described. Epidemiological investigation of 117 out of 157 (response rate, 74.5%) farms revealed that 44,130 animals died or were euthanized, while 8,033 animals with milder symptoms recovered. The overall death rate in all animals at risk was 21.7%. The death rates were significantly higher in blue and shadow foxes (24.2 and 27.8%, respectively) than in silver and blue silver foxes and minks (below 4%). All minks had been immunized against botulinum toxin type C. Deaths were associated with feed manufactured by a local processor, 83 of whose customer farms (70.9%) reported dead or sick animals. Five feedlots out of 19 delivered to the farms on the day preceding the onset of the outbreak (day 2) were associated with a death rate higher than 40%. These feedlots consisted of fresh feed processed on day 2 and feed processed 1 day earlier (day 1). In laboratory analysis, the day 2 feed contained botulinum toxin type C (>600 minimum lethal doses/g), while the day 1 feed did not contain toxin. Toxin was not detected in feed raw-material samples.
Clostridium botulinum
type C was detected by PCR in some feed components and in feed. However, as the feed temperature was continuously 8°C or below and the pH was continuously 5.6 or below according to the manufacturer, it seems unlikely that spore germination and toxin formation occurred during overnight storage. Hence, the events leading to toxin formation were not determined.
Detection of Galactomannan Antigenemia in Patients Receiving Piperacillin-Tazobactam and Correlations between In Vitro, In Vivo, and Clinical Properties of the Drug-Antigen Interaction
Authors: Walsh Thomas J. , Shoham Shmuel , Petraitiene Ruta , Sein Tin , Schaufele Robert , Kelaher Amy , Murray Heidi , Mya-San Christine , Bacher John , Petraitis Vidmantas
Published: October 2004

Recent case reports describe patients receiving piperacillin-tazobactam who were found to have circulating galactomannan detected by the double sandwich enzyme-linked immunosorbent assay (ELISA) system, leading to the false presumption of invasive aspergillosis. Since this property of piperacillin-tazobactam and galactomannan ELISA is not well understood, we investigated the in vitro, in vivo, and clinical properties of this interaction. Among the 12 reconstituted antibiotics representing four classes of antibacterial compounds that are commonly used in immunocompromised patients, piperacillin-tazobactam expressed a distinctively high level of galactomannan antigen in vitro (
P
= 0.001). After intravenous infusion of piperacillin-tazobactam into rabbits, the serum galactomannan index (GMI) in vivo changed significantly (
P
= 0.0007) from a preinfusion mean baseline value of 0.27 to a mean GMI of 0.83 by 30 min to slowly decline to a mean GMI of 0.44 24 h later. Repeated administration of piperacillin-tazobactam over 7 days resulted in accumulation of circulating galactomannan to a mean peak GMI of 1.31 and a nadir of 0.53. Further studies revealed that the antigen reached a steady state by the third day of administration of piperacillin-tazobactam. Twenty-six hospitalized patients with no evidence of invasive aspergillosis who were receiving antibiotics and ten healthy blood bank donors were studied for expression of circulating galactomannan. Patients (
n
= 13) receiving piperacillin-tazobactam had significantly greater mean serum GMI values (0.74 ± 0.14) compared to patients (
n
= 13) receiving other antibiotics (0.14 ± 0.08) and compared to healthy blood bank donors (0.14 ± 0.06) (
P
< 0.001). Five (38.5%) of thirteen patients receiving piperacillin-tazobactam had serum GMI values > 0.5 compared to none of thirteen subjects receiving other antibiotics (
P
= 0.039) and to none of ten healthy blood bank donors (
P
= 0.046). These data demonstrate that among antibiotics that are commonly used in immunocompromised patients, only piperacillin-tazobactam contains significant amounts of galactomannan antigen in vitro, that in animals receiving piperacillin-tazobactam circulating galactomannan antigen accumulates in vivo to significantly increased and sustained levels, and that some but not all patients receiving this antibiotic will demonstrate circulating galactomannan above the threshold considered positive for invasive aspergillosis by the recently licensed double sandwich ELISA.
Use of Multiple Nucleic Acid Amplification Tests To Define the Infected-Patient “Gold Standard” in Clinical Trials of New Diagnostic Tests for
Chlamydia trachomatis
Infections
Authors: Martin David H. , Nsuami Malanda , Schachter Julius , Hook Edward W. , Ferrero Dennis , Quinn Thomas C. , Gaydos Charlotte
Published: October 2004

Nucleic acid amplification tests (NAATs) can be used to define the infected-patient “gold standard” for the purpose of designing studies of the performance of
Chlamydia trachomatis
diagnostic tests. It is unclear how many test results run by different NAATs and what combinations of specimens comprise the best infected-patient gold standard. We approached this question with data from a large study of the performance of a new NAAT. Data were available from three endocervical swabs and a urine specimen collected from each of 1,412 women and tested by three different NAATs. Results from all three assays were used equally in a rotating fashion to define the infected-patient gold standard. Multiple different infected-patient gold standards for estimating swab and urine specimen sensitivity and specificity for one NAAT method were created by varying the number and combinations of swab and urine comparator results with two different NAATs, The effect of changing the infected-patient gold standard definition was determined by constructing receiver-operator-like curves with calculated sensitivities and specificities for each test. The one-positive-of-two-results or two-positive-of-two-results (same or two different assays) infected-patient gold standard definitions produced low sensitivity and low specificity estimates, respectively. If four comparator NAAT results were used, the any-three-positive-of-four-results definition or the at-least-one-specimen-positive-by-each-of-two-comparator-assays definition appeared to provide better combinations of sensitivity and specificity estimates. The any-two-positive-out-of-three-results definition resulted in estimates that were as good as produced with the former two definitions. This analytic approach provides a means of clearly visualizing the effects of changing NAAT-based infected-patient gold standards and should be helpful in designing future studies of new
C. trachomatis
diagnostic tests.
PCR Assay Using Cerebrospinal Fluid for Diagnosis of Cerebral Toxoplasmosis in Brazilian AIDS patients
Authors: Vidal José E. , Colombo Fabio Antonio , Penalva de Oliveira Augusto C. , Focaccia Roberto , Pereira-Chioccola Vera Lucia
Published: October 2004

Highly active antiretroviral therapy has decreased the incidence of opportunistic infections in the central nervous system in AIDS patients. However, neurological abnormalities still remain important causes of mortality and morbidity in developing countries. In Brazil, cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients. For these reasons, early, inexpensive, and sensitive diagnostic tests must be evaluated. The aim of this study was to evaluate PCR, using cerebrospinal fluid (CSF) samples to detectToxoplasma gondiiDNA, and to determine if the association of PCR with immunological assays can contribute to a timely diagnosis. We studied two sample groups. First, we analyzed stored CSF samples from 29 newborns and from 39 adults with AIDS without a definitive diagnosis of toxoplasmosis. The goal of this step was to standardize the methodology with a simple and economical procedure to recover theT. gondiiDNA. Next, we prospectively evaluated CSF samples from 12 AIDS patients with a first episode of cerebral toxoplasmosis and 18 AIDS patients with other neurological opportunistic diseases and without previous cerebral toxoplasmosis. In all PCR samples, an indirect immunofluorescent assay and an enzyme-linked immunosorbent assay were performed. Samples from all patients with cerebral toxoplasmosis presented positive PCR results (sensitivity, 100%), and a sample from one of the 18 AIDS patients with other neurological diseases also presented positive PCR results (specificity, 94.4%). These findings suggest the clinical utility of PCR in the diagnosis of cerebral toxoplasmosis in developing countries.
Dramatic Increase in Prevalence of Fecal Carriage of Extended-Spectrum β-Lactamase-Producing
Enterobacteriaceae
during Nonoutbreak Situations in Spain
Authors: Valverde Aránzazu , Coque Teresa M. , Sánchez-Moreno M. Paz , Rollán Azucena , Baquero Fernando , Cantón Rafael
Published: October 2004

The occurrence of extended-spectrum β-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 μg/ml) and cefotaxime (1 μg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (
P
< 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of
Escherichia coli
. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-β-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.
Adenovirus-Mediated Gene Therapy Enhances Parainfluenza Virus 3 Infection in Neonatal Lambs
Authors: Meyerholz David K. , Grubor Branka , Gallup Jack M. , Lehmkuhl Howard D. , Anderson Richard D. , Lazic Tatjana , Ackermann Mark R.
Published: October 2004

Parainfluenza viruses are a common cause of seasonal respiratory disease, but in high-risk individuals (e.g., young children) these viruses can cause severe clinical manifestations that require hospitalization. Beta-defensins are a subclass of antimicrobial peptides with antiviral activity. Use of adenovirus-mediated beta-defensin gene expression has been proposed as therapy for chronic bacterial infections commonly seen in cystic fibrosis patients; however, its use during parainfluenza virus 3 (PIV3) infection has not been evaluated. The hypothesis in this experiment was that adenovirus expression of human beta-defensin 6 (HBD6) would diminish concurrent PIV3 infection in neonatal lambs. The group infected with adenovirus HBD6 and PIV3 had increased levels of pulmonary neutrophil recruitment compared to those for the group infected with PIV3 or PIV3 and adenovirus, with an increased respiration rate and body temperature late in the course of the PIV3-adenovirus HBD6 infection. Interestingly, the adenovirus-treated groups had higher levels of immunohistochemical staining for PIV3 and syncytial cell formation than the group infected with PIV3, suggesting that treatment with the adenovirus vector, regardless of whether it was carrying a target gene, exacerbated the PIV3 infection. The levels of expression of mRNA for antimicrobial surfactant proteins A and D and sheep beta-defensin 1 were increased by PIV3 and adenovirus treatment, and the increased levels of expression roughly corresponded to the degree of inflammation. While pulmonary administration of a high-dose adenovirus vector has been associated with undesirable inflammation, this is the first study to show that it can exacerbate concurrent viral infection, a concern that needs to be addressed for future studies of adenovirus in the lung. Additionally, this study showed that adenovirus-mediated HBD6 expression increases neutrophil recruitment, a recently described attribute of beta-defensins, with mild accentuation of PIV3 activity and inflammation.
Pyrosequencing for Detection of Lamivudine-Resistant Hepatitis B Virus
Authors: Lindström Anna , Odeberg Jacob , Albert Jan
Published: October 2004

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.
Survival of
Neisseria gonorrhoeae
Isolates of Different Auxotypes in Six Commercial Transport Systems
Authors: Graver M. A. , Wade J. J.
Published: October 2004

We determined the rates of survival of six clinical and two control strains of
Neisseria gonorrhoeae
in six commercial transport systems, using NCCLS standard M40-A methodology. Differences in strain recovery and system performance were marked. A strain less robust than the recommended ATCC 43069 would provide a more exacting standard.
Use of Lung Resection and Voriconazole for Successful Treatment of Invasive Pulmonary
Aspergillus ustus
Infection
Authors: Azzola A. , Passweg J. R. , Habicht J. M. , Bubendorf L. , Tamm M. , Gratwohl A. , Eich G.
Published: October 2004

Invasive pulmonary aspergillosis (IPA) is a common infection in neutropenic patients and is associated with high mortality.
Aspergillus ustus
is a species that has only rarely been implicated in human disease. All reported cases of IPA due to
A. ustus
have been fatal. Here, we describe a case of invasive pulmonary
A. ustus
infection successfully treated with lung resection and voriconazole. A 43-year-old man with acute myeloid leukemia underwent two courses of chemotherapy and experienced prolonged neutropenia. Treatment with amphotericin B was given for persistent fever. While he was receiving amphotericin B, a progressive opacity developed in the upper right lobe. Lung tissue obtained through pulmonary wedge resection for histology showed a mold with septate hyphae, consistent with IPA due to
Aspergillus. A. ustus
was grown in culture. The patient was then treated with voriconazole and remained in remission of the mold infection in spite of additional chemotherapy and a leukemic relapse. In summary, this report describes the successful treatment of invasive pulmonary
A. ustus
infection by lung resection and antifungal treatment with voriconazole in a neutropenic patient.
Isolation of Shiga Toxin-ProducingEscherichia colifrom a South American Camelid (Lama guanicoe) with Diarrhea
Authors: Mercado E. C. , Rodríguez S. M. , Elizondo A. M. , Marcoppido G. , Parreño V.
Published: October 2004

Shiga toxin-producingEscherichia colibelonging to serotype O26:H11 was isolated from a 2-month-old guanaco with severe watery diarrhea.E. colicolonies carried thestx1andeaegenes, showed localized adherence to HEp-2 cells, and produced enterohemolysin. A serological response to lipopolysaccharide O26 was observed at the onset of diarrhea.
Comparison of an Immunochromatography Test with Multiplex Reverse Transcription-PCR for Rapid Diagnosis of Respiratory Syncytial Virus Infections
Authors: Kuroiwa Yuki , Nagai Kazushige , Okita Lisa , Ukae Susumu , Mori Toshihiko , Hotsubo Tomoyuki , Tsutsumi Hiroyuki
Published: October 2004

A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the “gold standard” for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.
Diagnostic Value of Reverse Transcription-PCR of Amniotic Fluid for Prenatal Diagnosis of Congenital Rubella Infection in Pregnant Women with Confirmed Primary Rubella Infection
Authors: Macé Muriel , Cointe Denis , Six Caroline , Levy-Bruhl Daniel , Parent du Châtelet Isabelle , Ingrand Didier , Grangeot-Keros Liliane
Published: October 2004

The diagnostic value of RT-PCR of amniotic fluid (AF) for prenatal diagnosis of congenital rubella virus infection in 45 pregnant women with confirmed primary infection was assessed. Specificity of RT-PCR was 100% and sensitivity ranged between 83 and 95%.
Rapid and Economical Method for Biochemical Screening of Stool Isolates for
Salmonella
and
Shigella
Species
Authors: Wilson G.
Published: October 2004

The LOUIS test is a screening protocol for
Salmonella
and
Shigella
that uses rapid enzyme tests. It had a sensitivity of 100% and a specificity of 94% and achieved presumptive reporting of
Salmonella
3 h after colony isolation with savings of time and money compared to commercial identification systems.
First Case of
Arthrographis kalrae
Ethmoid Sinusitis and Ophthalmitis in the People's Republic of China
Authors: Xi Liyan , Fukushima Kazutaka , Lu Changming , Takizawa Kayoko , Liao Ruiduan , Nishimura Kazuko
Published: October 2004

We present here the first case in the People's Republic of China of human disease caused by the fungus
Arthrographis kalrae
. The male patient had fungal panophthalmitis and invasive sinusitis involving the maxillary and ethmoid sinuses. He was an apparently healthy man before receiving trauma to his left eye. He complained of pain and loss of visual acuity in the injured eye, which displayed redness and edema and eventually discharged pus. His symptoms became more severe after he was treated with steroids and several antibacterial agents. A computed tomography scan of the left eye revealed that the maxillary and ethmoid sinuses were involved. A smear of purulent material from the left eye orbit revealed fungal elements, and cultures of the material grew a fungus. The isolate was identified as
A. kalrae
based on gross and microscopic morphologies, biochemical assays, and DNA sequence analysis. The patient received amphotericin B intravenously, itraconazole orally, and atomized allitridum by nebulizing allitridum therapy. The patient's wound healed following surgical intervention, but the patient lost the use of his left eye. This case should remind ophthalmologists and other clinicians to consider the possibility of infections being fungal when antibacterial agents have no effect and the patient's condition worsens.
Universal High-Level Primary Metronidazole Resistance in
Helicobacter pylori
Isolated from Children in Egypt
Authors: Sherif May , Mohran Zaynab , Fathy Hanan , Rockabrand David M. , Rozmajzl Patrick J. , Frenck Robert W.
Published: October 2004

Antimicrobial susceptibility testing was performed on 48 isolates of
Helicobacter pylori
recovered from Egyptian children undergoing routine endoscopies. The isolates were universally highly resistant to metronidazole, but resistance to other tested antimicrobial agents was rare (4% for clarithromycin, erythromycin, and azithromycin resistance versus 2% for ciprofloxacin and ampicillin resistance). Use of metronidazole for the treatment of
H. pylori
in Egypt should be avoided.