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This article continues the discussions introduced in the earlier article submitted to The Health Care Manager that is titled Epidemic Simulation for Syndromic Surveillance, where a format for analysis of the incidence of a bioterrorist attack was presented. Part 2 of this series provides a discussion of the observed outcomes from the simulation techniques. This simulation was conducted as part of a federal grant award administered through the Center for Biological Defense at the University of South Florida. The disease entity simulated was an attack of anthrax introduced into the Central Florida region. The spread, effects, and eventual control of the disease entity are highlighted. [ABSTRACT FROM AUTHOR]

This article continues the discussions introduced in an earlier article submitted to The Health Care Manager entitled 'Epidemic Simulation for Syndromic Surveillance,' wherein a format for analysis of the incidence of a bioterrorist attack was presented. This article outlines a simulation conducted as part of a federal grant award administered through the Center for Biological Defense at the University of South Florida. The disease entity simulated was an attack of anthrax introduced into the Central Florida region. The spread, effects, and eventual control of the disease entity are highlighted. [ABSTRACT FROM AUTHOR]

This article reports on a project to develop a simulation-based test bed for the BioDefend Syndromic Surveillance System. BioDefend is a system that data mines syndrome reports from emergency rooms and so forth to produce early alerts of epidemic onset. An existing large-scale epidemic simulation will be adapted to provide synthetic reports of syndromes associated with extremely rare events such as pandemics and bioterrorism. The Spatiotemporal Epidemiological Modeler will be used as the basis of the test bed. Results from the much simpler Spatiotemporal Epidemiological Modeler simulation will be validated by comparison against results from the more complex Epidemiological Simulation System. These synthesized reports will be used to test BioDefend's ability to detect epidemic outbreaks and to evaluate its data-mining algorithm. The development of an optimal algorithm for processing syndrome reports to provide reliable epidemic early warnings is a difficult research problem that the test bed should help address. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling is ideally suited to study structure, dynamics, and interactions of intrinsically disordered proteins as alpha-synuclein.Here we describe all steps required for a corresponding study: the spin labeling procedure, sample preparation, spectroscopic experimental procedure, and data analysis.

Human alpha-Synuclein (aS), implicated in Parkinson's disease, adopts a rich variety of different conformations depending on the macromolecular context. In order to unravel its pathophysiological role, monitoring its intracellular conformational state and identifying differences for the disease variants is crucial. Here, we present an intracellular spectroscopy approach based on a systematic spin-labeling site-scan in combination with intracellular electron paramagnetic resonance spectroscopy determining conformations on a molecular scale. A quantitative and model-based data analysis revealed that the vast majority of aS, be it wild-type or disease variants A30P or A53T, exists in the monomeric intrinsically disordered form in the cell.

The intrinsically disordered human protein alpha-Synuclein (αS) has a prominent role in Parkinson's disease (PD) pathology. Several familial variants of αS are correlated with inherited PD. Disease mutations have been shown to have an impact on lipid membrane binding. Here, using electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling, we show that familial PD-associated variants are structurally defective in membrane binding and alter the local binding properties of the protein.

Study Hypothesis: Since the 1990s, Italian hospitals are required to comply with emergency disaster plans known as Emergency Plan for Massive Influx of Casualties. While various studies reveal that hospitals overall suffer from an insufficient preparedness level, the aim of this study was to better determine the preparedness level of Emergency Departments of Italian hospitals by assessing the knowledge-base of emergency physicians regarding basic disaster planning and procedures., Methods: A prospective observational study utilized a convenience sample of Italian Emergency Departments identified from the Italian Ministry of Health website. Anonymous telephone interviews were conducted of medical consultants in charge at the time in the respective Emergency Departments, and were structured in 3 parts: (1) general data and demographics, (2) the current disaster plan and (3) protocols and actions of the disaster plan., Results: Eighty-five Emergency Departments met inclusion criteria, and 69 (81 %) agreed to undergo the interview. Only 45 % of participants declared to know what an Emergency Plan for Massive Influx of Casualties is, 41 % believed to know who has the authority to activate the plan, 38 % knew who is in charge of intra-hospital operations. In Part 3 physicians revealed a worrisome inconsistency in critical content knowledge of their answers., Conclusions: Results demonstrate a poor knowledge-base of basic hospital disaster planning concepts by Italian Emergency Department physicians-on-duty. These findings should alert authorities to enhance staff disaster preparedness education, training and follow-up to ensure that these plans are known to all who have responsibility for disaster risk reduction and management capacity.

Rapid isolation of Salmonella from food is essential for faster typing and source tracking in an outbreak. The objective of this study was to investigate a rapid isolation method that would augment the standard U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) method. Food samples with low microbial load, including egg salad and ice cream, moderately high-microbial-load tomatoes, and high-microbial-load ground beef were intentionally inoculated with 2 to 48 CFU of Salmonella enterica serotype Typhimurium. The samples were preenriched in buffered peptone water for 6 h, and then selectively concentrated by immunomagnetic separation and plated for isolation on xylose-lysine-desoxycholate agar: the 6IX method. Salmonella Typhimurium was presumptively identified from approximately 97% of the low-microbial-load and moderately high-microbial-load samples by the 6IX method 2 days before the BAM standard method for isolation of Salmonella. In 49% of the beef samples, Salmonella Typhimurium was presumptively identified 1 or 2 days earlier by the 6IX method. Given the inocula used, our data clearly indicated that for most of the food samples tested, with the exception of ground beef, Salmonella Typhimurium could be isolated two laboratory days earlier with the 6IX method compared with the BAM method. In conclusion, this 6IX method may expedite Salmonella isolation and, therefore, has the potential to accelerate strain tracking for epidemiological analysis in a foodborne outbreak.

A reported loss of mecA prompted us to monitor 360 cryostocked methicillin-resistant Staphylococcus aureus strains for stability. Concurrently, 14 well-characterized strains were stored in a Microbank preservation system and subjected to multiple freeze-thaw events. There were no significant declines in the methicillin-resistant populations with either method over a two-year period.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-based illness in the United States. Real-time PCR, pandemic group-specific PCR, ribotyping, and multilocus sequence typing were used to characterize 30 strains of V. parahaemolyticus including 11 strains associated with foodborne outbreaks in Florida and 6 known pandemic strains. Thirteen strains were positive for four pandemic group-specific PCR markers, including 5 strains associated with outbreaks in Florida. Molecular typing methods were used to further define the pandemic status of these strains because the current PCR markers are not sufficient to identify pandemic isolates. Nine of the Florida strains clustered with a majority of the known pandemic strains, based on ribotyping patterns using PvuII, but no isolated pandemic branch was formed. Using multilocus sequence typing, it was determined that 14 strains possess a previously determined pandemic sequence type. This study identified 13 novel sequence types and seven to nine novel alleles for each locus. Furthermore, the results indicate that seven of the strains from recent foodborne outbreaks in Florida are pandemic strains, and that multilocus sequence typing was the most accurate molecular method to identify these strains.

Research at the Center for Biological Defense identified plasmid-borne forms of Bacillus anthracis pXO2 genes in a Gram-positive, endospore-forming rod, isolated from a forensic specimen considered a credible threat of harbouring anthrax. Conventional, commercial and molecular-based methods indicated that the isolate (CBD 119(T)) was not B. anthracis and considered not to be a member of the Bacillus cereus group. Based on the 16S rRNA gene sequence similarities, strain CBD 119(T) was most closely related to Bacillus luciferensis LMG 18422(T) (99.3 %). Phenotyping and fatty acid methyl ester analysis of the isolate were conducted alongside B. luciferensis JCM 12212(T). The major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), and >7 iso or anteiso forms) supported inclusion of the isolate in the genus Bacillus. Strain CBD 119(T) was inconsistent with B. luciferensis JCM 12212(T) for 18 of 96 traits evaluated including motility, degree of endospore-driven swelling and pH optimum; the two were linked by fatty acid methyl ester analysis as separate but closely related species. DNA-DNA relatedness between strain CBD 119(T) and B. luciferensis JCM 12212(T) resulted in less than 20 % hybridization. The results of biochemical and physiological characterization, chemotaxonomic analysis and DNA-DNA hybridization differentiated strain CBD 119(T) both phenotypically and genotypically from the only species with validly published name with greater than 97 % 16S rRNA gene sequence similarity. The isolate has an accelerated doubling time when grown in aerated broth at pH 5.9 relative to that at pH 7.1. Therefore, it is proposed that strain CBD 119(T) represents a novel species, Bacillus acidiceler sp. nov. The type strain is strain CBD 119(T) (=NRRL B-41736(T)=DSM 18954(T)).

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.

The genus Salmonella is composed of more than 2,400 serotypes, many of which cause enteric diseases in humans and animals. Several Salmonella serotypes are multidrug resistant, and there is evidence of the clonal spread of these strains from animals to humans. Salmonella enterica serotype Newport is one of the serotypes that increasingly present a multidrug-resistant phenotype. Source tracking and antibiotic resistance testing are important considerations for identifying the outbreak strain. The first goal of this study was to examine the antibiotic susceptibility patterns of clinical and environmental Salmonella Newport isolates from various geographic locations and to compare the discriminatory ability of two DNA fingerprinting techniques. The second goal was to determine whether the antibiotic resistance profiles and typing patterns correlated. Thirty Salmonella Newport isolates, including environmental and human clinical strains, were subjected to pulsed-field gel electrophoresis (PFGE), ribotyping, and antibiotic susceptibility testing. Eighty percent of the isolates showed total or intermediate resistance to one or more drugs; 75% of the isolates were multidrug resistant. Ribotyping with the EcoRI enzyme and PFGE with the XbaI enzyme each divided the isolates into 14 groups. Cluster analysis based on antibiotic susceptibility patterns generated 23 profiles. The susceptible and resistant isolates were not differentiated on the basis of either of the molecular typing techniques. Hence, no correlation was observed between the antibiotic resistance profiles and the DNA subtyping patterns. In conclusion, ribotyping is as discriminatory as PFGE and, when used in combination with antibiotic resistance profiles, provides a powerful tool for the source tracking of Salmonella Newport.

We examined 299 methicillin-resistant, community-associated Staphylococcus aureus isolates from Florida and Washington State for the presence of the USA300 epidemic clone. Pulsed-field gel electrophoresis demonstrated the epidemic clone in 43% of our S. aureus strains and in isolates from both states. The majority of the USA300 isolates (88%) were from wound infections.

During the anthrax attack of 2001, the Florida Department of Health (FDOH) Bureau of Laboratories in Tampa received hundreds of isolates suspected of being Bacillus anthracis. None were confirmed to be B. anthracis since most isolates were motile and not even in the Bacillus cereus group. Although the sentinel laboratories now send fewer isolates to FDOH laboratories, should another attack occur the number of isolates submitted would likely increase dramatically, and this upsurge would seriously challenge personnel who are expected to be busy examining an increased number of environmental samples. We examined two selective and differential growth media and alternative motility methods that could be used to streamline the processing of suspicious isolates. Of 60 isolates previously sent to the FDOH laboratory, 56 were endospore-forming gram-positive rods and only 7 grew on mannitol-egg yolk-polymyxin B agar and/or the Anthracis chromogenic agar. Microscopic observation of early-log-phase growth (2 to 3 h) in a shaking broth was the best method to detect motility in 40 isolates that appeared nonmotile in the motility media investigated. One of these growth media and microscopic examination of shaken broth cultures can be used to show that an isolate is not B. anthracis before expensive molecular and antibody-based tests are performed. By doing so, costs could be reduced and analysis time shortened.

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing <10 spores.

Four recently completed large-scale randomized controlled trials have assessed the impact of insecticide-treated bednets and curtains on overall child mortality in Africa. These results have sparked numerous discussions among implementing agency representatives and researchers about the public health significance of the results. For the interpretation of impact, most of the arguments have been based on the observed decrease in protective efficacy (a relative measure of impact) with increasing malaria transmission (range: 14-29%, regression for trend: F = 245 on 1,2 DF, P < 0.003). However, an analysis of the absolute measure of impact (the risk difference) showed a different pattern. The impact ranges from 3.8 to 6.9 lives saved per 1000 children protected per year, without a significant trend (F = 2.8 on 1,2 DF, P = 0.2) and with equally high values in both low and high transmission sites. When assessing the public health importance of an intervention, both relative and absolute decrease in risk should be considered.

Antibody responses to malarial antigens were determined in 614 serum samples collected from the Wopkaimin population of the Star Mountains of Papua New Guinea. In point prevalence surveys made in 1982-1983, 33.7% of the persons examined were infected with Plasmodium falciparum, P. vivax, or P. malariae. Of these, 72.9% were infected with P. falciparum. In a standard fluorescent antibody test, highest level responses were to P. falciparum, followed by P. malariae, P. vivax, and P. ovale. A strong correlation was found between results of the fluorescent antibody tests and those obtained in an enzyme-linked immunosorbent assay using P. falciparum antigens. The failure of immune responses to eliminate these species of Plasmodium in this highly isolated population is discussed.

Recent human trials of candidate vaccines against the sporozoite and asexual blood stages of the malaria parasite have produced encouraging evidence that synthetic peptides and recombinant proteins can stimulate an antibody response and provide at least limited protection from malaria infection. Current research is focused on the continued evaluation of asexual blood stage antigens and on the improvement of the immunogenicity of candidate antigens through stimulation of more effective cellular and humoral responses. Efforts are also being directed toward the development of transmission-blocking vaccines based on antigens of sexual stages of the parasite.

Extremely high frequencies of the deletion form of alpha(+)-thalassemia (-alpha/), as studied by the DNA mapping technique, were found in the population of Madang, a coastal province in the north of Papua New Guinea (PNG) and in the population of Kar Kar, an island situated near Madang. Ninety-seven percent of the population tested from Madang and 89% of that from Kar Kar Island were either alpha(+)-thalassemia heterozygotes or homozygotes. By contrast, no examples of the deletion form were detected in the Eastern Highlands of PNG. The haplotype frequencies of alpha(+)-thalassemia (-alpha/) in Madang and Kar Kar Island were found to be 81.33% and 66.67%, respectively. A more detailed analysis of the gene deletion revealed that in both populations 96% were of the 4.2 kilobase (kb) type and 4% were of the 3.7-kb type. Thus, this group is the only example in which the 4.2-kb deletion is predominant over 3.7-kb defect. The presence in high frequencies of alpha(+)-thalassemia in the coastal area of Madang and on the neighboring island, where malaria has long been holoendemic or hyperendemic, and its virtual absence from the nonmalarious highlands of PNG suggest the role of malaria as the selective factor in maintaining alpha(+)-thalassemia. If this selective pressure is still operating, and since alpha(+)-thalassemia has no apparent homozygous disadvantage, the abnormal haplotype (-alpha/) will be in the process of fixation in this population.

The malaria incidence and prevalence rates among children who slept under permethrin-impregnated mosquito nets in four villages near Madang, Papua New Guinea, were compared with the rates among children who slept under unimpregnated nets in four paired control villages. Immediately following a parasitological survey in the eight villages, malaria parasites were cleared from the children with chemotherapy, and the mosquito nets in the four experimental villages were impregnated with permethrin. Follow-up parasitological surveys were performed 4 and 10 weeks later. Sporozoite rates in female mosquitos of the Anopheles punctulatus complex decreased significantly in two of the experimental villages after impregnation. Also, the incidence of Plasmodium falciparum between the 4-week and 10-week surveys was significantly lower among the 0-4-year olds in villages with impregnated nets than in those with unimpregnated nets, leading to reduced prevalence of P. falciparum in this age group. Use of permethrin-impregnated nets had no effect on the incidence or prevalence of P. falciparum among 5-9-year olds or on that of P. vivax among the 0-4- or 5-9-year olds.

Ovalocytosis, an hereditary condition in which most erythrocytes are oval in shape, is a polymorphism that occurs in up to 20% or more of the population in Papua New Guinea and Malaysia. Due to the geographical correlation of the trait with endemic malaria, the possibility of a selective advantage in resistance to malaria has been raised. In a study of 202 individuals with greater than or equal to 50% oval red cells matched by age, sex and village of residence with controls having less than or equal to 30% oval cells, ovalocytic subjects had blood films negative for Plasmodium vivax (P = 0.009), for P. falciparum (P = 0.044), and for all species of malaria parasites (P = 0.013), more often than controls. Among individuals parasitaemic at any time there were no clear differences in density of parasitaemia. However, in children 2 to 4 years old, parasite densities of both species were lower in ovalocytic subjects than in controls (0.01 less than P less than 0.025). The differential susceptibility to malaria infection suggested by this study has implications for the evaluation of interventions, including possible future vaccine field trials, in populations where high-frequency ovalocytosis is present.

Cross-sectional and longitudinal village-based studies of the transmission dynamics of an S-antigen serotype of the asexual erythrocyte stages of Plasmodium falciparum have been carried out in Madang, Papua New Guinea (PNG). Sera collected from village residents were screened for circulating S-antigen of the FC27 serotype by enzyme-linked immunosorbent assay (ELISA). The prevalence of the FC27 S-antigen was found to vary between villages at a given point in time, as well as within a village over time. Residents of villages 2-5 km apart were infected with P. falciparum of different S-antigen serotypes. This study documents the periodic nature of transmission of a sub-population of P. falciparum defined by the FC27 S-antigen. The variation in a small area in the prevalence of this serotype of P. falciparum in Madang illustrates the complexities of malaria transmission which must be considered in the design of malaria vaccine trials.

Preliminary findings of a study of the role of microspatial behavior and local ecological circumstances in the persistence of Schistosoma haematobium infection in El Ayaisha village, Upper Egypt, are presented. Twelve types of water contact activities were studied in three cohorts of 274,324 and 392 male students aged 5-16 during a 2-year period. Swimming and playing resulted in more frequent and intensive contact with potentially infective water and in more pollution of snail habitats with schistosome eggs than any other type of activity. Irrigation was probably not a major cause of S. haematobium infection in schoolboys and drinking water and fishing carried the smallest risk for this age group. Mapping and discriminate analysis of infection and transmission indicators revealed spatial associations between water contact, schistosome transmission and infection among the various age groups. The highest egg excretion rates in individuals and the highest prevalence rates were noted in the neighborhoods nearest to the canals. Water contact and contamination of water by the heavily infected school age boys is seasonally concentrated during the summer, when intermediate host snails and infective cercariae are also most common in the water. Frequency, type and duration of water contact change with age together with types of water bodies used, causing corresponding changes in exposure risk. Nevertheless, other factors, including acquired immunity, preexisting infections, chemotherapy and the effect of the study on the normal water contact behavior of the study population must also be evaluated. Contact with the Nile and the canals by the general nonschool population of El Ayaisha was studied to verify the observations among the schoolboys and to study socially linked activities and the transmission potential of the various transmission sites. Results are evaluated in regard to the feasibility of various schistosomiasis control measures in El Ayaisha.

A survey was carried out to establish baseline anthropometric and health data in the Wopkaimin, a small group of Mountain Ok-speaking hunter-horticulturalists who live in a remote and isolated part of the Highland fringe of Papua New Guinea, where a major gold and copper mine is being developed. Malaria is hyperendemic in the region and infant mortality has been estimated at approximately 230 per thousand. A 79% sample of the population was examined and the results of the anthropometric survey are reported. Wopkaimin childrens' heights and weights are well below the 50th percentile of British standards throughout growth; at maturity boys' weights and girls' heights and weights are at or below the 3rd percentile, and boys' weights are just above the 10th percentile. Wopkaimin children lag far behind British children in percentage of adult height achieved at all ages, and appear to attain maturity at least four years later. This relatively slow growth and short adult stature may represent an adaptive response to chronic nutritional deprivation, for which there is evidence from skinfold thickness and arm circumference measurements. Younger adults are significntly taller, heavier, fatter and more muscular than older adults; possible explanations for this are discussed. Within the sample, individuals living in villages less than a day's walk from the mine town are significantly taller, heavier and fatter than those living further away, and there are also similar significant anthropometric differences between employed and unemployed men. Ready food availability which has come with the mine development appears to be the most likely explanation for these differences. A continuing study of changing patterns of health and nutrition in the area has been initiated.

Malaria and filariasis surveys were carried out as part of a broader general health survey between December, 1982 and May, 1983 in the Ok Tedi region of the Star Mountains, Western Province. Malaria, tropical splenomegaly syndrome (TSS) and anaemia were identified as significant health problems. Malaria slide positivity rates of 64.9% in children 2 to 9 years of age and 19.5% in adults 15 years and older indicate high levels of stable malaria transmission. Infections with Plasmodium falciparum were the most common (75.2%), but P. vivax (17.4%) and P. malariae (7.4%) were also encountered. Palpable splenomegaly occurred in 79.2% of adults and children over two years of age with more than 50% of the enlarged spleens grade III or greater (Hackett). Microfilariae (Wuchereria bancrofti) were present in 34.3% of night blood films, and estimated haemoglobin values were considerably below WHO standards. Data from the surveys provide a baseline against which to monitor changes in health status which might be expected to occur in conjunction with the development of a major mining project in the area.