Epidemiological and genetic characteristics of norovirus outbreaks in long-term care facilities, 2003-2006.

To identify the epidemiological and genetic characteristics of norovirus (NoV) outbreaks and estimate the impact of NoV infections in an older population, we analysed epidemiological and laboratory data collected using standardized methods from long-term care facilities (LTCFs) during 2003-2006. Faecal specimens were tested for NoV by real-time reverse transcriptase-polymerase chain reaction. NoV strains were genotyped by sequencing. Of the 234 acute gastroenteritis (AGE) outbreaks reported, 163 (70%) were caused by NoV. The annual attack rate of outbreak-associated NoV infection in LTCF residents was 4%, with a case-hospitalization rate of 3·1% and a case-fatality rate of 0·5%. GII.4 strains accounted for 84% of NoV outbreaks. Median duration of illness was longer for GII.4 infections than non-GII.4 infections (33 vs. 24 h, P<0·001). Emerging GII.4 strains (Hunter/2004, Minerva/2006b, Terneuzen/2006a) gradually replaced the previously dominant strain (Farmington Hills/2002) during 2004-2006. NoV GII.4 strains are now associated with the majority of AGE outbreaks in LTCFs and prolonged illness in Oregon.

Salmonella infections associated with mung bean sprouts: epidemiological and environmental investigations.

We investigated an outbreak of Salmonella Enteritidis (SE) infections linked to raw mung bean sprouts in 2000 with two case-control studies and reviewed six similar outbreaks that occurred in 2000-2002. All outbreaks were due to unusual phage types (PT) of SE and occurred in the United States (PT 33, 1, and 913), Canada (PT 11b and 913), and The Netherlands (PT 4b). PT 33 was in the spent irrigation water and a drain from one sprout grower. None of the growers disinfected seeds at recommended concentrations. Only two growers tested spent irrigation water; neither discarded the implicated seed lots after receiving a report of Salmonella contamination. We found no difference in the growth of SE and Salmonella Newport on mung beans. Mung bean sprout growers should disinfect seeds, test spent irrigation water, and discontinue the use of implicated seed lots when pathogens are found. Laboratories should report confirmed positive Salmonella results from sprout growers to public health authorities.

Risk factors for sporadic Shiga toxin-producing Escherichia coli O157 infections in FoodNet sites, 1999-2000.

To monitor risk factors for illness, we conducted a case-control study of sporadic Shiga toxin-producing Escherichia coli O157 (STEC O157) infections in 1999-2000. Laboratory-confirmed cases of STEC O157 infection were identified through active laboratory surveillance in all or part of seven states. Patients and age-matched controls were interviewed by telephone using a standard questionnaire. Information was collected on demographics, clinical illness, and exposures to food, water, and animals in the 7 days before the patient's illness onset. During the 12-month study, 283 patients and 534 controls were enrolled. STEC O157 infection was associated with eating pink hamburgers, drinking untreated surface water, and contact with cattle. Eating produce was inversely associated with infection. Direct or indirect contact with cattle waste continues to be a leading identified source of sporadic STEC O157 infections.

Contaminated drinking water in one town manifesting as an outbreak of cryptosporidiosis in another.

In early 1992 we identified an outbreak of cryptosporidiosis in Oregon and sought to identify and control its source. We used a series of studies to identify risk factors for illness: (i) a case-control study among employees of a long-term-care facility (LTCF); (ii) a matched case-control study of the general community; (iii) a cohort study of wedding attendees; and (iv) a cross-sectional survey of the general community. Drinking Talent water was associated with illness in the LTCF (OR = 22.7, 95 % CI = 2.7-1009.0), and in the community (matched OR = 9.5, 95% CI 2.3-84.1). Drinking Talent water was associated with illness only among non-Talent residents who attended the wedding (P < 0.001) and in the community (RR = 6.5, 95 % CI 3.3-12.9). The outbreak was caused by contaminated municipal water from Talent in the absence of a discernible outbreak among Talent residents, suggesting persons exposed to contaminated water may develop immunity to cryptosporidiosis.

Where's the beef? The role of cross-contamination in 4 chain restaurant-associated outbreaks of Escherichia coli O157:H7 in the Pacific Northwest.

BACKGROUND: From March through August 1993, outbreaks of Escherichia coli O157:H7 occurred at 4 separate Oregon and Washington steak and salad bar restaurants affiliated with a single national chain. OBJECTIVE: To determine the cause of outbreaks of E coli O157:H7 at 4 chain restaurants. METHODS: Independent case-control studies were performed for each outbreak. Available E coli O157:H7 isolates were subtyped by pulse-field gel electrophoresis and by phage typing. RESULTS: Infection was not associated with beef consumption at any of the restaurants. Implicated foods varied by restaurant but all were items served at the salad bar. Among the salad bar items, no single item was implicated in all outbreaks, and no single item seemed to explain most of the cases at any individual restaurant. Molecular subtyping of bacterial isolates indicated that the first 2 outbreaks, which occurred concurrently, were caused by the same strain, the third outbreak was caused by a unique strain, and the fourth was multiclonal. CONCLUSIONS: Independent events of cross-contamination from beef within the restaurant kitchens, where meats and multiple salad bar items were prepared, were the likely cause of these outbreaks. Meat can be a source of E coli O157:H7 infection even if it is later cooked properly, underscoring the need for meticulous food handling at all stages of preparation.

Multinational outbreak of Salmonella enterica serotype Newport infections due to contaminated alfalfa sprouts.

CONTEXT: In December 1995, reported Salmonella enterica serotype Newport (SN) infections increased sharply in Oregon and British Columbia but not elsewhere in North America. Similar unexplained increases had been noted in 6 other states in the fall of 1995. OBJECTIVE: To determine the source of the outbreak(s). DESIGN: Case-control studies, environmental investigations, bacterial subtyping, and surveillance information review. SETTINGS: Oregon and British Columbia communities (winter 1995-1996) and Georgia, Oklahoma, Pennsylvania, Vermont, Virginia, and West Virginia (fall 1995). PARTICIPANTS: Oregon and British Columbia residents with culture-confirmed SN infections and onset from December 1, 1995, through February 29, 1996, and healthy community controls. MAIN OUTCOME MEASURES: Odds ratio (OR) of illness associated with exposures; distribution patterns and culture of alfalfa seeds and sprouts; subtyping of SN isolates. RESULTS: We identified 133 cases in Oregon and British Columbia; 124 (93%) occurred in patients older than 18 years; 87 (65%) were female. Case patients were more likely than community control subjects to report having eaten alfalfa sprouts in the 5 days preceding illness (41% [17/41] vs 4% [3/75]; OR, 17.0; 95% confidence interval, 4.3-96.0). Case isolates shared a distinctive pulsed-field gel electrophoresis (PFGE) pattern. The SN was grown from seeds and alfalfa sprouts. The distribution of 1 seed lot to multiple growers corresponded to the distribution of cases. Distribution of a second seed lot from the same European wholesaler corresponded to the location of the fall outbreak, which was characterized by a similar demographic profile. The PFGE pattern of fall outbreak isolates and confiscated sprouts and seeds was indistinguishable from the Oregon and British Columbia outbreak and differed from background isolates. CONCLUSIONS: The SN-contaminated alfalfa seeds were distributed to multiple growers across North America in 1995 and resulted in a protracted international outbreak scattered over many months. Current sprouting methods are inadequate to protect consumers from such events. Alfalfa sprouts may be an elusive but important vehicle for salmonellosis and other enteric infections.

A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk.

A protracted outbreak of Escherichia coli O157:H7 infections was caused by consumption of unpasteurized ("raw") milk sold at Oregon grocery stores. Although it never caused a noticeable increase in reported infections, the outbreak was recognized because of routine follow-up interviews. Six of 16 Portland-area cases reported between December 1992 and April 1993 involved people who drank raw milk from dairy A. By pulsed-field gel electrophoresis (PFGE), E. coli O157:H7 isolates from these cases and from the dairy A herd were homologous (initially, 4 of 132 animals were E. coli O157:H7-positive). Despite public warnings, new labeling requirements, and increased monitoring of dairy A, retail sales and dairy-associated infections continued until June 1994 (a total of 14 primary cases). Seven distinguishable PFGE patterns in 3 homology groups were identified among patient and dairy herd E. coli O157:H7 isolates. Without restrictions on distribution, E. coli O157:H7 outbreaks caused by raw milk consumption can continue indefinitely, with infections occurring intermittently and unpredictably.

An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat.

OBJECTIVE: To investigate a 1995 outbreak of Escherichia coli O157:H7 infections and to assess the safety of meat dehydration methods. DESIGN: Survey subsequent to routine surveillance report, environmental investigations, and laboratory experimentation. SETTING: Oregon community. PARTICIPANTS: Members of an extended household and their social contacts with confirmed or presumptive E coli O157:H7 infections. RESULTS: A total of 6 confirmed and 5 presumptive cases were identified. Homemade venison jerky was implicated as the source of transmission. E coli O157:H7 with the same distinctive, pulsed-field gel electrophoresis pattern seen in the case isolates was recovered from leftover jerky, uncooked meat from the same deer, a saw used to dismember the carcass, and fragments of the deer hide. In a subsequent survey, E coli O157:H7 was recovered from 3 (9%) of 32 deer fecal pellets collected in nearby forest land. In the laboratory, inoculated venison was dried at several time and temperature combinations, ranging up to 10 hours at 62.8 degrees C. Viable organisms were recovered under all conditions tested. CONCLUSIONS: Deer can be colonized by E coli O157:H7 and can be a source of human infections. Conditions necessary to ensure the safety of dried meat deserve further review. Game should be handled with the same caution indicated for commercially slaughtered meat.

A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei.

BACKGROUND: In the summer of 1991, simultaneous outbreaks of bloody diarrhea and hemolytic-uremic syndrome caused by Escherichia coli O157:H7 and of bloody diarrhea caused by Shigella sonnei were traced to a lakeside park near Portland, Oregon. METHODS: We identified cases primarily from routine surveillance reports. In case-control studies, the activities of persons with park-associated E. coli O157:H7 or S. sonnei infections were compared independently with those of three sets of controls. We also evaluated environmental conditions at the park and subtyped the bacterial isolates. RESULTS: We identified 21 persons with park-associated E. coli O157:H7 infections (all of them children; median age, six years) and 38 persons with S. sonnei infections (most of them children). These 59 people had visited the park over a 24-day period. Their illnesses were not associated with food or beverage consumption. All the case patients reported swimming, however, and in case-control studies swimming was strongly associated with both types of infection (P = 0.015 or less). The case patients were more likely than the controls to report having swallowed lake water, and they had spent more time in the lake. Numbers of enterococci indicative of substantial fecal contamination (geometric mean, > 50 per deciliter) were detected in the swimming area during some but not all of the outbreak period. Park-associated E. coli O157:H7 isolates were identical by pulsed-field gel electrophoresis and were distinguishable from other isolates in the Portland area. CONCLUSIONS: Lake water that was fecally contaminated by bathers was the most likely vehicle for the transmission of both the E. coli O157:H7 and the S. sonnei infections. The unusually prolonged outbreak suggests both the survival of these enteric organisms in lake water and a low infectious dose.

Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase.

Work from several laboratories suggests a correlation between expression of cysteine proteinase activity and the cytopathic effect of virulent HM1 strain Entamoeba histolytica trophozoites on cultured cell monolayers. Consistent with this relationship, we find that L-6 trophozoites, mutants cloned from the HM1 parent strain, are deficient in both proteinase expression and cytopathic effect. Three other clones, with proteinase expression equal to or greater than that of the HM1 strain, express the cytopathic effect. Furthermore, a nontoxic specific proteinase inhibitor, Z-phenylalanyl-alanyl-CH2F, inhibits the cytopathic effect of live trophozoites in a dose-dependent manner. These results support the hypothesis that expression and release of the cysteine proteinase is an important factor in producing the cytopathic effect, presumably by its degradation of cell anchoring proteins.

Thiol proteinase expression and pathogenicity of Entamoeba histolytica.

Expression of the 56-kilodalton (kDa) neutral thiol proteinase has been shown to correlate with the potential of clinical isolates of Entamoeba histolytica to produce invasive disease. A 56-kDa band was identified by gelatin substrate gel electrophoresis in 10 of 10 isolates from patients with colitis or amebic liver abscesses, but in only 1 of 10 isolates from asymptomatic patients. Pathogenic isolates appear capable of releasing significantly larger quantities of the proteinase, as measured by cleavage of a synthetic peptide substrate, ZRR-AMC (benzyloxy-carbonyl-arginine-arginine-4-amino-7-methylcoumarin). We have also shown that the proteinase is released during the course of clinical invasive amebic disease, as demonstrated by the presence of circulating antibodies detectable by enzyme-linked immunosorbent assay. These studies support the importance of the 56-kDa thiol proteinase in the pathogenesis of invasive amebiasis.

Cleavage of C3 by a neutral cysteine proteinase of Entamoeba histolytica.

The major secreted proteinase of Entamoeba histolytica, a 56-kDa neutral cysteine proteinase, activates C by cleaving C3. The action of the proteinase is similar to C-derived C3 convertases because it produces a single cleavage of the alpha-chain in a dose- and time-dependent manner and cleaves C3 between residues 78 and 79, only one amino acid residue distal to the natural site acted on by the C3 convertases. C3a generation was detected by RIA. The 105-kDa fragment produced by the cleavage of the alpha-chain was structurally and functionally equivalent to the alpha'-chain of C3b, as demonstrated by susceptibility to the action of factors I and H and participation in the activation of the alternative pathway of C. Activation of C by the 56-kDa neutral cysteine proteinase may play a role in the early inflammatory response in amebic lesions and thus contribute to the pathogenesis of invasive amebiasis.

The major neutral proteinase of Entamoeba histolytica.

FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.

Degradation of extracellular matrix by larvae of Schistosoma mansoni. I. Degradation by cercariae as a model for initial parasite invasion of host.

We have examined the ability of cercariae of Schistosoma mansoni to degrade a model extracellular connective tissue matrix produced by rat vascular smooth muscle cells in culture. In this model, connective tissue macromolecules are present in the interactive framework that characterizes their structure in vivo. Cercariae were stimulated to degrade the matrix by skin lipid or linoleic acid. At the maximally stimulating concentration of linoleic acid (25 micrograms/cm2), 68% of the total matrix was degraded, including 57% of the glycoprotein, 79% of the elastin, and 8% of the collagen. Degradation of the matrix and transformation of cercariae to schistosomula began within minutes of exposure to maximally stimulating concentrations of linoleic acid. Degradation continued for 24 hours and was dependent on the number of cercariae. Some degradation occurred without exogenous stimulants but at a slower rate than with skin lipid or linoleic acid. Degradation of matrix was inhibited by alpha 1-proteinase inhibitor and soybean trypsin inhibitor. Ethylenediaminetetraacetic acid inhibited degradation by unstimulated but not linoleic acid-stimulated cercariae. Preacetabular gland secretions collected from cercariae also degraded the matrix with an activity 86% of that of live cercariae. Preacetabular gland proteolytic activity was also inhibited by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, and ethylenediaminetetraacetic acid. The similar characteristics of matrix degradation by both live cercariae and cercarial preacetabular gland secretions support the idea that a proteinase secreted from cercarial preacetabular glands facilitates invasion of skin and connective tissue by these larvae. Degradation of elastin and glycoprotein constituents of extracellular matrix is probably essential for skin penetration.

Degradation of extracellular matrix by larvae of Schistosoma mansoni. II. Degradation by newly transformed and developing schistosomula.

We have studied the ability of schistosomula of Schistosoma mansoni to degrade an extracellular connective tissue matrix synthesized by rat vascular smooth muscle cells in culture. Six to 12% of the total matrix was degraded by schistosomula from the time of transformation from cercariae to adult development in vitro. Most matrix degradation occurred during the first 24 hours of incubation and was dependent on the number of schistosomula and the type of medium in which they were incubated. The use of proteinase inhibitors indicated that schistosomula activity was distinctly different from that of cercariae. Newly transformed schistosomula expressed one activity that was similar in inhibition characteristics to that of cercarial preacetabular gland secretions and another activity that was unique to schistosomula. From 1 day after transformation to adulthood, the schistosomula-derived activity was the predominant activity detected. Schistosomula degraded a smaller percentage of the total matrix than did cercariae and showed a different substrate profile. Schistosomula degraded glycoprotein components of extracellular matrix but showed little or no activity against elastin or collagen. Matrix-degrading activity was also detected in schistosomula-conditioned medium. Sedimentation of the activity and lack of permeability through filter barriers suggest that the enzyme may be initially associated with membrane and then sloughed with membrane fragments. Since the schistosomula-derived activity initially overlaps with cercarial preacetabular gland proteolytic activity, the two activities may act in concert to facilitate skin penetration by newly transformed schistosomula. However, schistosomula activity probably serves some, as yet undetermined, function later in development.