Arthur Michalek and colleagues explore the consequences of scientific misconduct and, using a single case as an illustration, estimate that direct costs can approach US$525,000, although total costs are likely to be higher. The consequences of scientific misconduct are far-ranging and the costs associated with their investigation are substantial.It is possible to estimate the cost (direct and indirect) of investigating a single case of scientific misconduct.For a specific investigation for which costs were estimated for all phases of the review process, direct cost estimates approached US$525,000.For an individual country, the total costs to associated with the review of all cases of scientific misconduct, both reported and not reported to the Office of Research Integrity, are likely to be exponentially higher.Fallout from scientific misconduct can be pervasive. From the broadest perspective, the public, current and future patients, funding agencies, and even the course of research may be adversely affected by scientific misconduct. At the local level. members of the perpetrator's laboratory, colleagues, trainees, and the financial resources and reputation of the home institution may become tainted. The costs associated with these acts are substantial. This article will present a model we have developed to estimate the monetary costs of scientific misconduct. Estimates are based on a case that occurred at our institution, the Roswell Park Cancer Institute, which is a National Cancer Institute–designated Comprehensive Cancer Center located in the United States. Our experiences will likely not be wholly representative of other institutions, but we feel could be instructional and should serve as a guide in the calculation of costs at other institutions.Scientific misconduct is defined by the US Office of Research Integrity (ORI) as “fabrication, falsification, or plagiarism in proposing, performing, or reviewing research or in reporting research results” Scientific misconduct likely dates back to the earliest days of scientific inquiry. Fanelli The costs associated with scientific misconduct can be divided into three domains: conduct of the fraudulent research, investigation, and remediation.These costs includes all monetary investments  made in the fabricated research as well as intangibles such as loss of productivity of the associated research group, loss of trust, the demoralization of faculty/trainees, and misdirection of the research efforts of other labs. In some cases, the institution may be required to reimburse the funding agency for costs of the fraudulent research as well as pay penalties, and in certain instances, temporarily suspend other studies during the investigation.An aspect frequently overlooked in the discussion of misconduct costs are those directly related to the investigation. These costs vary considerably and are dependent on the nature of the incident  and the associated time required to investigate. However, all investigations share similar elements that need to be considered when calculating overall costs. At our institution, in keeping with the model proposed by the ORI, allegations of misconduct proceed through three levels of review, each assuming escalating responsibilities and costs.At our institution, allegations are initially reviewed by the Vice President for Corporate Ethics and the Dean of Educational Affairs. If the allegation is determined to have merit, an inquiry is initiated. This second level requires review by a committee appointed by the Vice President for Corporate Ethics and the Dean. Membership consists of the Vice President for Corporate Ethics, the Dean, four faculty members, and an attorney. The Inquiry Committee determines whether there is sufficient evidence of possible research misconduct to warrant an investigation. The inquiry is not intended to reach a final conclusion about whether research misconduct definitely occurred or who was responsible. That is the role of the Investigation Committee, which is appointed by the Vice President for Corporate Ethics and the Dean. Membership is broader and includes other professional expertise. Membership consists of the Vice President for Corporate Ethics, the Dean, at least two individuals from outside the unit or department of the complainant(s) who are expert in the subject matter or scientific area, a statistician, a representative from Human Resources, an attorney, and any other members deemed appropriate. The purpose of the investigation is to explore the allegations in detail, to examine the evidence in depth, and to determine specifically whether research misconduct has been committed, by whom, and to what extent.Costs of the investigation may be divided into personnel , material costs, and consultant costs. The most expensive component of any investigation is faculty time. Faculty members engaged in our reviews are usually associate or full professors. Faculty members on investigation committees spend considerable time both in and out of the formal committee meetings. Time spent outside formal meetings is directed at reviewing materials, securing additional information, reanalysis of data, writing, and other preparatory activities. Our experience is that faculty spend anywhere from three to ten times more time working outside of meetings than they do in meetings. Individual time commitments vary based on the individual's expertise as well as committee assignments. Costs associated with witnesses' time must also be considered. The number and frequency of witness interviews varies based on the complexity of the investigation. Witnesses also spend time outside of meetings preparing their testimony. Administrative support costs include secretarial and clerical time needed for transcription of recordings, photocopying, filing, and other related tasks. Most investigations will require sequestration of physical materials including all laboratory notebooks, computers, and other electronic storage devices. At times forensic computer experts are required to analyze hard drives as well as to retrieve e-mail exchanges or other documents that are still resident on the institutional server.These costs include those necessitated by program closure. Not only are funds previously invested in the fraudulent research lost, but so too are funds currently supporting the fraudulent research. Moreover, pending grant applications may be recalled and further funding of existing grants may be delayed or lost. Loss of funding can be devastating to the honest members of the affected laboratory. A myriad of administrative decisions need to be made regarding such things as continuance of trainees  and staff members from the affected lab, impact on trainee's research; and the costs of possibly phasing out bona fide research conducted by the guilty party. Other less-obvious costs include reputational damage to the institution, which may affect competitiveness of future grants as well as fundraising and, for those involved in patient care, there is potential patient harm and loss of patient trust and revenue. Institutional expenses may also include the cost of civil legal action from patients. Extrainstitutional costs may include intellectual corruption of the scientific literature, misdirection of future research, costs to journals in retracting deceptive research, and costs to revise guidelines based on fraudulent research.Very little research has been done to develop methods for formally modeling the cost of scientific fraud. Research in this area has been directed primarily at attempting to model the behavior of the individual scientist with respect to incentives for committing a fraudulent act. It has been our aim to develop a data-based modeling approach aimed at better understanding the factors that contribute to the overall cost of scientific fraud.We are aware of no published methodological research with respect to modeling the factors that contribute to an estimate of the average aggregate cost (AC) of fraud. The purpose of such models would be two-fold: (1) Identifying the most significant factors associated with the cost of the “average” misconduct case in terms of the proportion of variation explained and (2) prediction of the aggregate cost of a misconduct case conditional on measureable factors. A semi-additive model of AC might take the formMC = measurable costsIC = intangible costsε = stochastic error.Examples of some ICs would include loss of future earnings related to a line of research; reputational damage to the institution, which may affect competitiveness for future grants and contracts; negative effects on fundraising; and, for those involved in patient care, loss of patient revenue.1, x2, …, xd represent the d factors for which a given institution or institutions have the ability to measure and collect in an administrative database and ε again represents stochastic error. Based on our experiences at Roswell Park Cancer Institute, a list of factors to be considered for inclusion in a model of cost include, but is not limited to the following:Practically speaking, a more manageable working model would take the form1 = grant direct and indirect dollars returned to the funding agency,x2 = institutional legal costs,x3 = hourly cost of faculty time commitment to an investigation panel,x4 = cost of sequestration of evidentiary materials,x5 = human resource–related costs,x6 = institutional start-up costs for supporting the fraudulent research,x7 = Institutional Review Board–related costs for suspending and closing clinical studies,x8 = Institute Animal Care and Use Committee–related costs for suspending and closing animal studies,x9 = payment of penalties related to tainted research,x10 = hourly costs associated with retracting published research,x11 = hourly costs of specialized consultants needed for advisement to the investigation panel.xTo date we have not gathered cost factor information prospectively or with any degree of precision in order to fit these types of models. Hence, our cost estimates to date amount to a “best guess” scenario, as illustrated in the next section. Ultimately, to apply this model a database will be developed from which we can examine statistically the relative contributions of each factor to the MC. Then, for example, the fitted model then may be utilized for estimating the cost of a future misconduct case in terms of resource management.The following case study was based on an actual investigation. Cost estimates are given in US dollars.An allegation of research misconduct was made against a senior scientist for enhancing and fabricating images and data contained in a federal grant application.The allegation, in accordance with institute policy, was reviewed by the Vice President for Corporate Ethics and the Dean. A determination was made that there was sufficient credible and specific potential evidence of research misconduct to warrant an inquiry. The deliberation and data gathering to support this decision cost approximately $1,000.00.An Inquiry Panel was convened consisting of the aforementioned membership. The Panel reviewed the grant application in question, additional information regarding more than a dozen figures in the grant, as well as e-mail correspondence between the respondent and several staff members. The panel concluded that there was sufficient evidence to support the allegation and that an investigation was warranted. Panel time required to review and discuss data to support this decision cost about $13,000.00.At this point the respondent's laboratory equipment was sequestered as were all lab notebooks, computer hard drives, and other electronic devices. Sequestration involved members of institute security, the Information Technology department, and an outside forensic computer company. All computer and electronic devices were copied and copies supplied to the laboratory personnel so the affected lab could continue working on research other than that related to the questionable project until the investigation was completed and a decision had been reached. These actions cost an estimated $10,000.00.An Investigation Committee was empanelled as described above. Over the course of ten meetings the Committee reviewed all of the questionable lab figures, primary data sources from lab books, electronic data and figures, and e-mail correspondence. The Committee also interviewed the respondent, the complainant, and other members of the laboratory in question. Given the complexity of the case the Investigation Committee was composed of eight individuals who spent well over 100 hours in meetings  and an estimated 700 hours outside of committee . Other related costs included transcriptionist and clerical support for photocopying, filing, scheduling, and correspondence . Moreover, given that the Investigation Committee determined that there was evidence of scientific misconduct, a review of the scientist's other grant applications as well as manuscripts was undertaken. Approximately 50 person-hours were spent reviewing other grants and manuscripts .We estimate that the direct cost of this case approached $525,000. This includes faculty and witness salaries of about $512,000, clerical support costs of ∼$2,500, and other personnel costs  of ∼$10,000. Other significant costs not factored into the above figure (indirect costs) include deliberation time of senior administrative faculty , loss of current grants , withdrawal of two pending grant applications  and one renewal , the cost to the Institute of maintaining affected pre- and postdocs until other laboratories could be found , and the cost of maintaining all the records for at least 6 years after the investigation has been completed.n = 217 cases) in their last reporting year, the direct costs would exceed $110 million. We hope that our work will encourage others to add to our understandings of these costs.The precise prevalence of scientific misconduct is unknown, owing largely to its clandestine nature as well as to the problem of underreporting. Fanelli Scientists are people and subject to the frailties of human nature, so we may never be able to totally eliminate scientific misconduct. However, we can prevent those cases of misconduct more related to “omission” of scientific standards rather than commission of misdeeds. How this can be achieved has not yet been determined. Most academic institutions have, like ours, undertaken a number of efforts to increase awareness through education and training, setting forth and enforcing scientific codes of conduct, providing mentorship training, auditing and monitoring procedures, and implementing procedures for reporting and investigating alleged incidents of misconduct. The ultimate effectiveness of these approaches may take time to discern. What is known, however, is that the costs of these proactive activities pale in comparison to the costs of a single case of scientific misconduct.


Variola major, the causative agent of smallpox, has been eradicated from nature. However, stocks still exist; thus, there is a need for relevant decontamination studies, preferably with nonpathogenic simulants. Previous studies have shown a similarity in response of vaccinia virus and variola major to various decontaminants and thermal inactivation. This study compared vaccinia and fowlpox viruses under similar conditions, using disinfectants and temperatures for which variola major data already existed. Most disinfectants showed similar efficacy against vaccinia and fowlpox, suggesting the utility of fowlpox as a decontamination simulant. Inactivation kinetics studies showed that fowlpox behaved similarly to variola major when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride, but differed in its response to 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol. Thermal inactivation studies demonstrated that fowlpox is a suitable thermal simulant for variola major between 40°C and 55°C. Poxviruses are large, double-stranded DNA viruses that infect a wide variety of hosts, ranging fromcaterpillars to humans. They are the largest animal virus, with a brick-shape, ranging in size from 200 to 400 nm long and 170 to 200 nm wide. Due to their large size, poxviruses can be visualized with light microscopy, although the fine details of the virus remain obscure. Poxvirus genomes encode the genes necessary for replication and immune modulation, with replication occurring in the host cell's cytoplasm. The genome is large compared to most viruses, containing 130–300 kb, depending on the virus type. The DNA is A+T rich and linear. Unlike most DNA-containing viruses, poxviruses encode their own polymerase [Poxviruses are classified into two categories: entomopox (insect infecting) and chordopox (vertebrate infecting). The chordopox viruses are further divided into eight categories, including orthopox, which includes variola major and vaccinia virus, and avipox, which includes fowlpox virus . Variola major, the virus that causes the contagious disease smallpox, has been considered one of the most destructive viruses in history, affecting 1/10th of all humankind and killing 300 million people in the 20th century alone , althougVaccina virus, long considered the prototype of variola major, has been used as both a simulant and vaccine for variola major. They show remarkable sequence similarity; however, vaccinia virus does not cause disease in healthy individuals, possibly due to the fact that variola major virus contains complement inhibitors 100 times more potent than those of vaccinia virus . HoweverThe purpose of this paper is to investigate whether fowlpox, also a member of the chordopox family, can be used as a simulant for variola major. Fowlpox virus naturally infects chicken and turkey; however, it is not infectious to humans. Fowlpox virus has a genome consisting of 260 genes. Of the 260 genes, 65 are conserved homologues of chordopox genes. When compared by Hexamer and Glimer computer programs, sixteen fowlpox virus genes have a closest match to variola major. These genes encode for transcription factors, RNA-polymerase subunits, DNA repair, protein modification, structural proteins, and immune evasion proteins. Seventeen fowlpox virus genes closely match those of vaccinia virus .The fowlpox genome is approximately 70–100 kbp larger than chordopox genomes. The large genome is due to several factors. First, the fowlpox genome contains a diverse array of genes. Several genes encoding cellular function show homology with proteins of unknown function from yeast, bacteria, roundworm, plant, and human. Another gene shows similar homology to a protein found in yeast, human, tomato and fruit fly. Secondly, the fowlpox genome contains host range genes, limiting infection to members of the avian family. Thirdly, the virus is so large due to the presence of novel cellular homologues [Vaccinia strain WR (VR-119) and Fowlpox strain FPV-MDVgBH (Merck's Disease vaccine strain) (VR-2330) were obtained from the American Type Culture Collection .BHK-21 (CCL-10), BSC-40 cells (CRL-2761) and UMNSAH/DF-1 cells (chicken embryo fibroblast cells) (CRL-12203) were also obtained from ATCC.Vaccinia virus was propagated in BHK-21 monolayer cultures in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C. Infected cells were harvested and centrifuged at 3,210 × g at 4°C for 10 minutes. Pellets were resuspended in DMEM containing 10% FBS, freeze-thawed for three cycles, sonicated for four minutes on ice and then centrifuged at 3,210 × g at 4°C for 10 minutes. The supernatant was collected and titered in BSC-40 cells and served as the source of virus for all experiments described in this paper.5 cells per well in six well plates for vaccinia plaque assays. Cells were allowed to reach confluence overnight at 37°C. in DMEM containing 10% FBS. Following absorption, medium was removed and replaced with Minimal Essential Medium (MEM) containing 5% FBS and 1% SeaPlaque Agarose. Forty-eight hours after infection, plaques were visualized by staining with 0.01% crystal violet. The plaques were counted, and the results were reported in plaque forming units per milliliter (pfu/mL).BSC-40 cells were plated at a density of 5.0 × 10Fowlpox virus was propagated in UMNSAH/DF-1 monolayer cultures in DMEM containing 10% FBS at 39°C. Infected cells were harvested and centrifuged at 3,210 × g at 4°C for 10 minutes. Pellets were resuspended in DMEM containing 10% FBS, freeze-thawed for three cycles, sonicated for four minutes on ice, and centrifuged at 3,210 × g at 4°C for 10 minutes. The supernatant was collected and titered in UMNSAH/DF-1 cells and served as the source of virus for all experiments described in this paper.6 cells per well in six well plates for the fowlpox plaque assay. Cells were allowed to reach confluence overnight at 39°C in DMEM containing 10% FBS. Following absorption, media was removed and replaced with 50% MEM, 50% DMEM, 2.5% FBS, and 1% SeaPlaque Agarose. Five days postinfection, plaques were visualized by staining with 0.01% crystal violet. Plaques were counted, and the results were reported in pfu/mL.UMNSAH/DF-1 cells were plated at a density of 1.0 × 10μm filter.The following disinfectants were tested for their efficacy against vaccinia and fowlpox viruses: ethanol (70%), isopropyl alcohol (50%), sodium hypochlorite (0.5%), formaldehyde (30%), benzalkonium chloride (10%), a mixture of cetyltrimethylammonium chloride (6.67%) and benzalkonium chloride (3.33%), and a mixture of iodine (1.75%) and polyethyleneglycol nonylphenyl ether (10%). All disinfectants were prepared in distilled, deionized water and then filtered through a 0.25 μm filter.The following concentrations of disinfectants were used to determine the inactivation kinetics of vaccinia and fowlpox viruses: ethanol (40%), sodium hypochlorite (0.05% and 0.025%), a mixture of cetyltrimethylammonium chloride (0.1%) and benzalkonium chloride (0.05%), a mixture of iodine (0.1%) and polyethyleneglycol nonylphenyl ether (5.7%), and a mixture of iodine (0.05%) and polyethyleneglycol nonylphenyl ether (0.3%). All disinfectants were prepared in distilled, deionized water and then filtered through a 0.25 The plaque assays described above for vaccinia and fowlpox viruses were followed to determine the activity of the disinfectants, with minor changes. Vaccinia and fowlpox viruses were diluted 1: 10 in sterile water (control) or disinfectant. To test the infectivity of the treated viruses, the virus was treated with disinfectants for 1, 3, 5, and 10 minutes. To determine the inactivation kinetics of the individual disinfectants, the viruses were treated for 15, 30, 45, 60, 90, 120, 150, and 180 seconds.In order to determine the thermal stability of Vaccinia and fowlpox viruses were diluted in 0.85% saline, pH 4.5, phosphate buffered saline (PBS), pH 7.4, 10% skim milk, and heart infusion broth as described by Hahon and Kozikowski [Aliquots of vaccinia and fowlpox viruses were stored at −80°C and 4°C for one week. After the one week storage period, the viruses were diluted 1: 100 in sterile PBS heated to 56°C as described by Hahon and Kozikowski [To determine the order of the reaction, the following graphs were produced. To determine a first-order reaction, graphs of the natural log of the virus concentration versus time were produced. A linear line with a negative slope indicated a first-order reaction. To determine a second-order reaction, graphs of 1/virus concentration versus time were produced. A linear line with a positive slope indicated a second-order reaction. Vt/V0 = ekt−, where Vt is the concentration of virus at time, t, and V0 is the concentration of virus at the zero timepoint, and k is the reaction rate. The rate of reaction for the second-order reactions was determined using the following equation: (1/Vt) = (1/V0) + kt, where Vt is the concentration of virus at time, t, V0 is the concentration of virus at the zero timepoint, and k is the reaction rate.The rate of reaction for the first-order reactions was determined using the following equation: t1/2) for viruses showing a first-order reaction rate was determined using the following equation: t1/2 = ln  2/k, where k is the rate constant determined using the equations described above. The t1/2 for viruses showing a second-order reaction rate was determined using the following equation: t1/2 = 1/(V0 × k), where V0 is the concentration of virus at the zero timepoint. The virus half-life . A straight line with the slope −ΔH/R is obtained, where R is the universal gas constant, from which the ΔH can be calculated from. The ΔH units were calories per mole. Hahon and Kozikowski [S. The Eyring equation is as follows: k = (KT/h) × e(−ΔH/RT) × e(ΔS/R), where K is Boltzmann's constant, T is the absolute temperature, h is Plank's constant, R is the universal gas constant, and k is the rate constant calculated for the reaction. The ΔS were calculated in Calories per mole per degree Celsius.The rate constant and virus half-life for the thermal inactivation studies were calculated as described above. The calculations of Hahon and Kozikowski  were usezikowski  describeChemical toxicity assays were performed by incubating BSC-40 and UMNSAH/DF-1 cells with the highest dose of chemical for the viral absorption time (one hour for BSC-40 and two hours for UMNSAH/DF-1). Following incubation, the cell morphology was observed using a light microscope. Toxicity effects were noted to ensure that cells were healthy enough to produce viral protein, allowing the infection process to occur. Additionally, the determinations of toxicity effects were important to ensure that any cell death was due to viral infection and not the treatment chemical.The ethanol, isopropyl alcohol, and sodium hypochlorite had no observable effects on either cell line. The formaldehyde and the surface-active detergents  had some toxic effects on cells at the highest two dilutions, but cells that recovered and grew had normal morphology.All the chemicals tested disinfected both vaccinia virus and fowlpox virus Tables and 2. DiThe four chemicals chosen for the inactivation kinetic studies were shown by Tanabe and Hotta to demonstrate high viracidal properties at minimum concentrations . As showThe 40% ethanol inactivation kinetics in this study vary somewhat from the data reported by Tanabe and Hotta . They reFowlpox virus had an inactivation profile similar to that of vaccinia virus. Like vaccinia virus, 0.05% and 0.025% sodium hypochlorite inactivated the fowlpox virus within 15 seconds of treatment, as shown in Inactivation rate constants were calculated for vaccinia and fowlpox viruses treated with ethanol (40%), sodium hypochlorite (0.05% and 0.025%), a mixture of cetyltrimethylammonium chloride (0.1%) and benzalkonium chloride (0.05%), a mixture of iodine (0.1%) and polyethyleneglycol nonylphenyl ether (5.7%), and a mixture of iodine (0.05%) and polyethyleneglycol nonylphenyl ether (0.3%). The calculated rate constants for vaccinia and fowlpox viruses are shown in Overall, vaccinia virus exhibited greater inactivation rate constants than fowlpox virus treated with the same chemicals. For both viruses, the order of the inactivation reaction when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether was a second-order reaction. The viruses showed different reaction orders when treated with 40% ethanol. Vaccinia virus exhibited a first-order reaction, while fowlpox virus exhibited a second-order reaction. A first-order reaction was observed in both vaccinia and fowlpox virus treated with 0.05% and 0.025% sodium hypochlorite, a mixture of 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride, and 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether.Vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.05% sodium hypochlorite, 0.1% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride had a calculated half-life of less than 8.41 seconds . The dif5 times greater than the half-life of vaccinia virus treated with the same chemical. However, vaccinia virus treated with 40% ethanol had a virus half-life over 20 times greater than fowlpox virus treated with the same disinfectant.For two chemicals, the virus half-life of vaccinia and fowlpox virus differed greatly. Fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether had a half-life 2.4 × 10H for vaccinia virus is 44,138 calories per mole in 0.85% saline  for vaccinia virus in 0.85% saline ranged from 67.61 Cal/°C at 40°C to 65.85 Cal/°C at 55°C (see S) for fowlpox virus in 0.85% saline ranged from 71.70 Cal/°C at 40°C to 63.93 Cal/°C at 55°C  for vaccinia virus in PBS, pH 7.4, ranged from 69.08 Cal/°C at 40°C to 63.17 Cal/°C at 55°C (see S) for fowlpox virus in PBS, pH 7.4 ranged from 68.34 Cal/°C at 40°C to 62.45 Cal/°C at 55°C  for vaccinia virus in skim milk ranged from 70.04 Cal/°C at 40°C to 62.82 Cal/°C at 55°C (see S) for fowlpox virus in skim milk ranged from 67.74 Cal/°C at 40°C to 65.12 Cal/°C at 55°C  titers only dropped by one-log unit for both vaccinia and fowlpox viruses. The calculated Δmole see . The ent55°C see . The ent55°C see .Vaccina and fowlpox viruses also behaved similarly in heart infusion broth, both in their inactivation curves, but also in the fact that both viruses showed decreased aggregation at lower temperatures in this test liquid. Both viruses, especially noted in vaccinia as the time course progressed, plaque size dramatically decreased and titers actually increased between the 30- and 45- minute timepoints. At the 30- and 45-minute timepoints, both vaccinia and fowlpox plaques were approximately 10 times smaller than those observed during plaque assay optimization procedures, during which the virus was not treated with chemicals or heated. This could be indicative of decreased viral aggregation. However, by the 60-minute timepoint, titers had decreased to below or similar to the starting titer and plaque size returned to the normal sizes observed during plaque assay optimization.H for vaccinia virus in heart infusion broth is 95,204 calories per mole; while the calculated ΔH fowlpox virus in heart infusion broth is 65,229 calories per mole (see S) for vaccinia virus in heart infusion broth ranged from 63.12 Cal/°C at 40°C to 64.86 Cal/°C at 55°C (see S) for fowlpox virus in heart infusion broth ranged from 73.95 Cal/°C at 40°C to 63.55 Cal/°C at 55°C  cells. Due to the more difficult nature of maintaining primary cell lines, it has been demonstrated in this work that fowlpox can be grown in an immortalized CEF line, the UMNSAH/DF-1 cell line. Although the concentration of fowlpox virus was on the order of 1-2 log units less than the concentration of vaccinia virus, the minimum concentration of fowlpox virus produced by these cells was approximately 7 × 10Data from this study suggest that fowlpox virus is a suitable decontamination simulant for variola major when using disinfectants at full strength . Fowlpox virus and vaccine virus are both inactivated within 1 minute when using the following disinfectants: 70% ethanol, 50% isopropyl alcohol, 0.5% sodium hypochlorite, 30% formaldehyde, 10% benzalkonium chloride, a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride, and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether. Viral inactivation with minimal concentrations of the same chemicals was also examined in the present study, for the purpose of determining the inactivation kinetics of both vaccinia and fowlpox. To first principles, the inactivation curves for each virus and chemical appear similar; however mathematical analysis of the data reveals distinct differences for vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol.The reaction orders described in this study are both first- and second-order decay reactions. First-order reactions were observed with vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% and 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride. Vaccinia virus treated with 40% ethanol also showed a first-order reaction, while a second-order reaction was observed when the virus was treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether. Fowlpox virus exhibited second-order reactions when treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol. β-chloroethyl) methylamine (nitrogen mustard), a chemical known for virus inactivation, can survive treatment with this agent when the particles are aggregated [The first-order decay reaction is typically expected when observing viral kinetics. However, further analysis of the unique properties of poxviruses suggests possible reasons for the observed second-order reaction of fowlpox virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol and the vaccinia virus treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether. First, vertebrate poxviruses have the unique ability to nongenetically reactivate. In nongenetic reactivation, an infectious poxvirus particle is able to reactivate a second inactivated poxvirus particle . Secondlgregated . Based ok, is calculated from the starting concentration of virus and the concentration of virus at time, t. The k values differed by no more than 0.7 1/s for the vaccinia and fowlpox viruses treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride. However, the vaccinia and fowlpox viruses treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol show large differences in k values. These observed differences could be due to the fact that when treated with 40% ethanol, vaccinia undergoes a first-order reaction and fowlpox undergoes a second-order reaction. When treated with 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether, the data suggests that fowlpox virus is more stable in that disinfectant.Kinetic analysis further elucidated the inactivation similarities and differences between vaccinia and fowlpox viruses. The rate constant, k value is an important variable for comparison, for the purpose of this work, the virus half-life (t1/2) value is a better indicator of simulant suitability. The purpose of this work is to determine if fowlpox is a suitable decontamination simulant for variola minor, thus the amount of time the virus will survive is more important than the rate of the reaction. Based on the data presented in Although the The studies presented in this paper suggest that fowlpox is a suitable decontamination simulant for variola major when using the following full strength disinfectants: 70% ethanol, 50% isopropyl alcohol, 0.5% sodium hypochlorite, 30% formaldehyde, 10% benzalkonium chloride, a mixture of 6.67% cetyltrimethylammonium chloride and 3.33% benzalkonium chloride and a mixture of 1.75% iodine and 10% polyethyleneglycol nonylphenyl ether. Fowlpox virus is also a suitable inactivation kinetics simulant when treated with the following chemicals: 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride. Fowlpox virus is not a suitable inactivation kinetics simulant for variola major when treated with the following chemicals: 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol. Knowing the genetic and decontamination similarities of variola major and vaccinia from previous studies, fowlpox virus has been shown to be a suitable variola major simulant through identical decontamination results using vaccinia virus as the common link between the studies.H and ΔS of variola major virus in various buffers at temperatures from 40°C to 55°C. Previous studies with vaccinia virus demonstrated that vaccinia virus has a range of ΔH values from 20,000 to 90,000 calories per mole [H values in the range described by Kaplan [H values for the vaccinia virus and fowlpox virus described in this paper fall into the range described Kaplan, with the exception of vaccinia virus heated in brain heart infusion broth. The difference in ΔH values in this paper with vaccinia virus in brain heart infusion broth might be due to the decreased virus aggregation, which might have affected the inactivation and plaque counts. Hahon and Kozikowski [H values calculated for vaccinia virus in 0.85% saline, pH 4.5, PBS, pH 7.4, and 10% skim milk were in the range described for both vaccinia [H values calculated for fowlpox virus in 0.85% saline, pH 4.5, PBS, pH 7.4, 10% skim milk, and brain heart infusion broth were in the range described for both vaccinia (Kaplan) and variola major virus [S of the order of 10 to 100 calories per mol per degree” for the denaturation of proteins. The ΔS values described in this work for both vaccinia and fowlpox viruses fall well within the 10–100 calories per mol per degree Celsius range. Like the decontamination studies, vaccinia virus served as the common link between variola virus and fowlpox virus for the thermal stability studies. Hahon and Kozikowski  determinper mole . Variolay Kaplan . The calzikowski  did not vaccinia  and variThese studies suggest that fowlpox can serve as a thermal stability simulant for variola major in 0.85% saline, pH 4.5, PBS, pH 7.4, 10% skim milk, and heart infusion broth at temperatures ranging from 40°C to 55°C. At temperatures above, 55°C, fowlpox does not appear to be as stable as vaccinia virus. As the storage conditions studies demonstrate, even when stored at −80°C, fowlpox quickly denatures when heated at 56°C when compared to vaccinia virus.


We isolated and characterized St. Louis encephalitis virus (SLEV) from cloacal swabs of naturally exposed adult sentinel chickens in 2006. Phylogenetic analysis of SLEV strains isolated in Florida indicated that Brazilian SLEV circulated in 1972 and 2006; lineages were VA and VB. Flavivirus, Flaviviridae) in 1999, St. Louis encephalitis virus  was the most important agent of epidemic viral encephalitis  and high (epidemic) activity. SLEV was detected by sentinel chickens every year before introduction of WNV (1988–2007) . Since 2In 2006, a total of 2,901 adult sentinel chickens were maintained at 275 sites of potential enzootic arbovirus transmission in 34 Florida counties. Blood was collected weekly from each chicken during peak transmission months (July–December) and tested with hemagglutination inhibition assay, immunoglobulin M antibody-capture ELISA, or plaque reduction neutralization test, as previously described  of sentinel chicken sites with recent confirmed arbovirus transmission activity for cloacal swab collection from 95 chickens. During the weekly scheduled bleeding of the flocks, 1,338 cloacal swabs were collected in viral culturettes ; 529 swabs were retrospectively processed for molecular detection assays and virus isolation in Vero cells, as previously described .To characterize SLEV strains, we amplified the envelope region using previously described primers  , our datPhylogram of the complete envelope region of St. Louis encephalitis virus (SLEV) strains, inferred using the maximum parsimony method in MEGA4 software (


To the Editor: Accumulating evidence suggests that the degree of serologic cross-reactivity to pandemic (H1N1) 2009 virus varies between populations worldwide. To assess potential serologic cross-reactivity in Singapore, we obtained serum samples during May–June 2009 from 50 randomly recruited, healthy volunteers born mostly before 1958  before widespread transmission of pandemic (H1N1) 2009 virus in Singapore. Standard serologic hemagglutination-inhibition (HI) tests ; 31 (62%) were women, 42 (84%) were born in Singapore , and 26 (52%) had not traveled outside Singapore. None of the 50 participants had HI or MN titers /59/2007 . Use of /59/2007 . Thus, o>80 years of age) in western populations (United States and United Kingdom) /1918–like viruses were widespread in Asia, these contrasting results are a stimulus for additional large-scale studies to assess the effect of these viruses in these populations.Although differences in population demographics and laboratory methods used make comparisons between studies difficult, one of the most striking observations from various studies has been the higher levels of cross-reactive antibody titers in prepandemic serum samples from older persons  2009 virus. Chen et al.  2009 virus originated mainly from swine viruses in North America and Europe (In conclusion, partial cross-immunity and cell-mediated immunity may be present but not detected by HI or MN assays. Thus, results of standard serologic assays may not be providing all relevant data (


Heterocapsa circularisquama DNA virus  is a giant virus (girus) with a ~356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments , one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments. Heterocapsa circularisquama forms blooms causing massive death of shellfish such as pearl oysters and mussels, and is one of the most intensively studied dinoflagellate species [Dinoflagellates (Dinophyceae) are one of the highly abundant and ubiquitous unicellular eukaryotic ("protistan") components in marine environments . They co species .H. circularisquama [H. circularisquama blooms [Phycodnaviridae [HcDNAV is a marine giant virus  contarisquama ,9. HcDNAa blooms . HcDNAV a blooms ,11. Duria blooms . Based oaviridae . HoweverPhycodnaviridae includes intensively-studied algal virus members such as chlorella viruses and Emiliania huxleyi viruses [Mimiviridae, Poxviridae, Iridoviridae, and Asfarviridae. Mimiviridae is represented by the freshwater amoeba-infecting mimivirus [Mimiviridae [Poxviridae include a number of successful pathogens known to infect a tremendous variety of terrestrial animals, such as insects, reptiles, birds, and mammals [Asfarviridae [Ornithodoros). In these hosts, ASFV infection is usually asymptomatic [ viruses -17, and  viruses . NCLDVs imivirus  and its imivirus . Based oiviridae . Poxviri mammals . Iridovi mammals . The lasrviridae ,25 is curviridae . ASFV isptomatic . HoweverH. circularisquama on 6 dpi), virus particles were collected as described in [In an attempt to further characterize HcDNAV, we performed a low coverage shotgun sequencing of its genome. Specifically, from 4 liters of HcDNAV suspension . A multiple sequence alignment of the HcDNAV PolB and its close homologs confirmed the presence of conserved residues for exonuclease and polymerase activities [SDTDS- instead of the YGDTDS- sequence usually found in dsDNA viruses. In addition, we identified two ORFs in the upstream region of the PolB ORF in a divergent orientation. Their products were respectively predicted to be 245 and 194 aa in length . The former showed a significant similarity to HNH endonucleases with its BLASTP best hit to mimivirus L245 ; the latter showed a significant similarity to hypothetical proteins from NCLDVs with its best hit to mimivirus R325 . Incidentally, R325 is located near the PolB gene (R322) in the mimivirus genome [HcDNAV PolB gene was found to be 3,675 bp long , punctuated by normal start and stop codons, and no intron or intein-like sequence was observed. The predicted protein product is 1,225 amino acids (aa) long. Unexpectedly, the translated amino acid sequence showed the closest BLASTP hits against PolB sequences from different ASFV isolates, with the best homolog being DPOL_ASFL6  in the NCBI non-redundant sequence database. The best non-ASFV hit corresponded to the PolB sequence of tivities  . Next, we used a wider range of viral homologs including those of bacteriophages. The resulting tree based on 320 amino acid residues again supported the grouping of HcDNAV/ASFV with a 98% bootstrap value , for which we obtained a similar result. The 892 bp sequence showed BLASTX best hit against ASFV RNA polymerase sequence . A monophyletic grouping between the HcDNAV sequence (97 aa) and the ASFV RNA polymerase sequence was again received a high bootstrap value of 87%  expedition  revealedPhycodnaviridae family [PolB is one of the most reliable phylogenetic markers for large eukaryotic DNA viruses ,33. The e family . It is cOur finding now establishes an evolutionary link between a terrestrial pathogen and a marine girus. A recent metagenomic analysis of corals provided evidence for the existence of viruses related to herpesviruses , which hThe authors declare that they have no competing interests.NK conceived the study. YS, KT and NN conducted purification and sequencing of HcDNAV and RT-PCR experiment. HO designed and carried out bioinformatics analyses. HO, JMC, YT and NK contributed to the interpretation of data and wrote the manuscript. All authors read and approved the final manuscript.Transcription of the PolB gene of HcDNAV. To verify the transcription of the HcDNAV PolB gene, reverse transcription-PCR (RT-PCR) experiment was conducted. The total RNA samples were isolated from HcDNAV-inoculated Heterocapsa circularisquama cells collected at 0, 1, 6, 12, and 24 hpi, then reverse-transcribed according to the method given by Nagasaki et al. [i et al. . PCR ampClick here for fileConserve blocks from the multiple sequence alignment of PolB sequences from NCLDVs. The data provided shows the presence of conserved residues for exonuclease and polymerase activities in the HcDNAV PolB and its close homologs. Species abbreviation is followed by a database sequence identifier. Intein sequences were removed from the sequences prior to alignment. The alignment was generated by T-Coffee [Amsacta moorei entomopoxvirus 'L'; APMV: Acanthamoeba polyphaga mimivirus; ASFV: African swine fever virus; CeV: Chrysochromulina ericina virus; EhV: Emiliania huxleyi virus 86; EsV: Ectocarpus siliculosus virus 1; FsV: Feldmannia species virus; HaV: Heterosigma akashiwo virus 01; HcDNAV: Heterocapsa circularisquama DNA virus; HvAv: Heliothis virescens ascovirus 3e; IIV: Invertebrate iridescent virus 6; LCDV: Lymphocystis disease virus 1; OtV: Ostreococcus virus OsV5; PBCV: Paramecium bursaria Chlorella virus 1; PoV: Pyramimonas orientalis virus; VAR: Variola virus.T-Coffee  and ClusT-Coffee . AmEPV: Click here for fileMaximum likelihood tree of RNA polymerase large subunit amino acid sequences. The tree is based on an alignment containing 66 amino acid residues with no gaps. Alignment and tree construction method is the same as those used for the tree in Fig. Click here for file


In addition to human encephalitis and meningitis cases, the West Nile (WN) virus outbreak in the summer and fall of 1999 in New York State resulted in bird deaths in New York, New Jersey, and Connecticut. From August to December 1999, 295 dead birds were laboratory-confirmed with WN virus infection; 262 (89%) were American Crows (Corvus brachyrhynchos). The New York State Department of Health received reports of 17,339 dead birds, including 5,697 (33%) crows; in Connecticut 1,040 dead crows were reported. Bird deaths were critical in identifying WN virus as the cause of the human outbreak and defining its geographic and temporal limits. If established before a WN virus outbreak, a surveillance system based on bird deaths may provide a sensitive method of detecting WN virus.


Diabetes costs represent a large burden to both patients and the health care system. However, few studies that examine the economic consequences of diabetes have distinguished between the two major forms, type 1 and type 2 diabetes, despite differences in underlying pathologies. Combining the two diseases implies that there is no difference between the costs of type 1 and type 2 diabetes to a patient. In this study, we examine the costs of type 1 diabetes, which is often overlooked due to the larger population of type 2 patients, and compare them to the estimated costs of diabetes reported in the literature.Using a nationally representative dataset, we estimate yearly and lifetime medical and indirect costs of type 1 diabetes by implementing a matching method to compare a patient with type 1 diabetes to a similar individual without the disease. We find that each year type 1 diabetes costs this country $14.4 billion (11.5–17.3) in medical costs and lost income. In terms of lost income, type 1 patients incur a disproportionate share of type 1 and type 2 costs. Further, if the disease were eliminated by therapeutic intervention, an estimated $10.6 billion (7.2–14.0) incurred by a new cohort and $422.9 billion (327.2–519.4) incurred by the existing number of type 1 diabetic patients over their lifetime would be avoided.We find that the costs attributed to type 1 diabetes are disproportionately higher than the number of type 1 patients compared with type 2 patients, suggesting that combining the two diseases when estimating costs is not appropriate. This study and another recent contribution provides a necessary first step in estimating the substantial costs of type 1 diabetes on the U.S. Type 1 diabetes (T1D) is an autoimmune disease, often diagnosed early in life, characterized by the destruction of the insulin-secreting β cells in the pancreas. As a consequence, patients become insulin-dependent and must follow a rigid, daily regimen of exogenous insulin replacement. In contrast, type 2 diabetes (T2D) is typically a disease of adulthood , where a relative insulin deficiency arises due to insulin resistance and abnormal pancreatic β cell function With the increasing focus on the T2D epidemic, the impact of T1D in this country is often overlooked, particularly from an economic perspective. Many cost-of-illness (COI) studies have been performed on diabetes, but most combine the costs for T1D and T2D over just one year. The Agency for Health Research Quality (AHRQ), using the Medical Expenditures Panel Survey (MEPS), a nationally representative data set These studies do not break down diabetes costs by type in their estimates. Rather, they attempt to apply the proportion of patients with type 1 to obtain the fraction of costs attributed to type 1, an assumption that does not account for the distinct nature and differing disease progressions of the two forms of the disease. Furthermore, there is wide variation in results among the cost studies. For example, a study by the Milken Institute calculates combined T1D and T2D indirect costs to be over $105 billion in 2003 Patients with T1D typically suffer from the disease for a longer period of time. Regular maintenance of T1D requires daily insulin shots and constant monitoring, representing a significant lifelong cost and time requirement. For T1D, these long-term effects are likely to spill over to other aspects of their lives with resulting economic impacts particularly in indirect costs. Milton et al. To address these deficiencies and provide a comprehensive first step in identifying the annual and lifetime costs of T1D, this study was designed to estimate the direct medical expenditures and indirect costs associated with T1D in the U.S. Together with more traditional biomedical research into the causes and treatments of T1D, this study allows for a comprehensive understanding of the costs and benefits associated with the disease. Doctors, patients, researchers, policy makers, and drug developers can use these results to more fully understand the impact of T1D on individuals and society.In this paper, we present the results of this analysis that show, as might be expected given the different disease pathologies that underlie the forms of diabetes, that the cost breakdown in T1D is different than T2D, with indirect costs being disproportionately larger for T1D. Furthermore, we show that over a lifetime, the economic costs of T1D are in excess of $10 billion for a new yearly cohort of approximately 30,000 patients.Using nationally representative data sets, we identify T1D patients and examine direct medical expenditures and indirect costs associated with the disease by matching a T1D patient to a counterpart without diabetes. Since finding an exact match becomes increasingly difficult as more covariates are used, we implement the propensity score matching (PSM) technique first introduced by Rosenbaum and Rubin Although matching is not new to cost studies, few match on more than simply age, gender, and race. However, to obtain an unbiased and accurate measure of costs attributed to T1D, the matched control individuals must be as similar to the population with T1D as possible so that the only difference between the two can be attributed to T1D. This is not likely to be the case if matching is based on only a few broad demographic variables.The matching method compares with other cost estimation methods such as those used by the ADA, which require detailed and accurate knowledge of the relationship between diabetes and its comorbidities Ideally, one would like to observe the diabetic counterfactual—the life of a patient with diabetes had he not had diabetes—and attribute all differences to diabetes. Of course, the counterfactual does not exist in real life, and therefore the notion of a nondiabetic diabetic-type is pertinent. This is equivalent to the nonsmoker smoker-type, which is used in the smoking literature to determine the cost of smoking Matching each diabetic with a nondiabetic diabetic-type and attributing any differences to T1D requires several assumptions 0y be the outcome  for a person without T1D.Let 1y be the outcome for a patient with T1D.Let D be an indicator for having T1D.Let X are the covariates used for matching a patient with T1D to a nondiabetic diabetic-type and ⊥ indicates independence. The outcomes 0y and 1y are independent of whether or not the individual has T1D, after conditioning on all the covariates in X. In other words, after matching on X, having T1D is the only difference between a T1D patient and his matched counterpart that affects the outcome. The assumption is strong and requires careful consideration in determining the appropriate covariates that affect both the probability of T1D and the outcome of interest.Then,Including more observable characteristics for matching improves the results of the matching estimator, but makes it increasingly difficult to find an exact match for each individual with diabetes. Matching based on propensity scores addresses this problem by providing a natural weighting scheme for potential matches. The propensity score is the probability of receiving “treatment” conditional on the observable characteristics D given X. Individuals with the same X have positive probability of having T1D.It is written as T1D patients are matched using the propensity score to individuals in a control group of those without diabetes. The difference in outcomes between the patients and their matched counterparts is called the average treatment on the treated. It is the portion that can be attributed to T1D. It is written asiy is the outcome for an individual with T1Djy is the outcome for a nondiabetic diabetic-typedN is the number of people with T1DjC is the set of matched control individuals for the T1D patientsw is the weighting function that depends on the PSM method.The simplest weighting method is a 1 to 1 nearest neighbor matching. The difference between the two groups is then the average difference between the outcome of a T1D patient and his matched counterpart. The average treatment cost difference between a patient with T1D and a person without diabetes is:A difficult part of the matching process is the determination of appropriate covariates for matching. There is no hard and fast rule for choosing covariates, but there should be a theoretical reason for why the covariate affects both the probability of having T1D and the outcome of interest After matching on propensity scores, the match quality is evaluated by comparing the means of the T1D group and the matched control group. Since matching is made on propensity score, which is weighted naturally by the importance of each variable, the means are not expected to all be exactly the same, but should be close. In general, sample differences in the unmatched case exceed those in the matched case. To quantify the differences between the unmatched and matched samples, we define the following One likely cause of the small amount of T1D cost studies is the difficulty in distinguishing patients with T1D from those with T2D in secondary data. Most population surveys simply ask whether a person has ever been told by a physician that he has diabetes, and data with detailed medical tests are not nationally representative. One exception is the National Health and Nutrition Examination Survey (NHANES) Other studies also categorize diabetes based on their patterns of drug use during the study period Yearly costs are those incurred in an average year between 1999 and 2005. The medical expenditures considered in this study are total expenditures and therefore include self-payments and any third-party payments. In particular, they consist of hospital inpatient, ambulatory visits , emergency room and prescription drug/medical supplies, home health provider, and vision and dental utilization expenditures.Indirect, or productivity costs, are not as straightforward to measure, and income is frequently used to measure productivity losses due to illness To estimate productivity losses attributed to early mortality caused by T1D, we compute the present value of future productivity (PVFP) Propensity score matching ensures that we account for all observed variables in determining the control sample and the subsequent effects on outcomes. However, the results will be biased if an unknown, unobserved factor affects whether an individual has T1D and also the outcomes we observe. A Rosenbaum bounds test can be performed to verify that our findings are not driven by a potential omitted variable Since T1D patients are typically diagnosed at an early age compared to typical T2D patients and T1D patients therefore suffer from the disease for a longer portion of their life, lifetime costs are better for capturing the full burden of the disease and providing a fairer, more accurate, comparison between diseases. We use two approaches for measuring lifetime costs. First, we estimate the lifetime costs of a cohort of newly diagnosed patients by generating a longitudinal profile of costs from the cross-sections of patients in our sample stratified by current age and age of diagnosis. Second, using this longitudinal profile we estimate the amount incurred by the current population over the rest of its lifetime. The first approach seeks to measure the amount saved by a future cohort of newly diagnosed patients if onset of T1D could be prevented. The second method finds the savings by the current patient population if a cure  were discovered today. These calculations admittedly neglect improvements in health care technology over time, but similar methods have been used to estimate the lifetime costs associated with smoking and obesity To obtain expected expenditures, we divide the sample into seven age groups, starting with age group 3 to 18 (age 3 is the age of the youngest T1D patient in the sample). Sample size limitations prevented us from breaking down the 18 and under age group into smaller groups. We multiply total expenditures at each age by the probability of surviving to that age using published survival probabilities We calculate the present value of expected costs by discounting the stream of expenditures at a rate of three percent, as recommended by the World Health Organization Expected lifetime expenditures are described byEt = expenditures during age interval if the individual survives through ttP = probability of surviving through age intervalt = 10, 25, 35, 45, 55, 65, 70+i = discount rate.To estimate the size of the yearly cohort of new T1D patients, we use published T1D incidence rates and total population counts from the 2000 Census We examine the joint significance of the regressors in the logit estimation before and after matching. They are jointly significant before matching with an F-statistic of 19.3. When we rerun the estimation using just the T1D patients and the matched controls, we cannot reject the hypothesis that the covariates have no explanatory power, which indicates that the covariates are able to predict whether the individual has T1D. In other words, conditional on the propensity score, T1D is “randomly” assigned to either group.To confirm the validity of T1D sample of 1.1 million patients in the MEPS, we apply the same T1D clinically-derived definitions that were described above to the NHANES data. We find a sample representing 1.4 million individuals nation-wide who have T1D, slightly larger than the MEPS sample. This figure is based on NHANES data for an average year between 1999 and 2004. The actual number of T1D patients is 140, weighted to represent 1.4 million persons.NHANES collects information on fasting plasma glucose levels and c-peptide levels of a subset of their sample. A fasting plasma glucose level of 126 mg/dl or over indicates diabetes While some patients with T1D may not experience any serious complications, complications that do arise can be quite severe and are likely to lead to more intensive medical care. We attempt to examine the medical use of patients with T1D with complications by using hospital nights as an indicator for complications. Of the 817 person-years in the T1D sample, 605 report zero hospital nights. Limiting the results to the 212 who do report at least one hospital night and comparing them with their matched counterparts yields results shown in Indirect costs for patients with T1D and their matched counterparts during an average year are listed in The CDC finding that T1D accounts for 5 to 10 percent of all diabetes cases remains a popular benchmark We find total yearly medical expenditure attributable to T1D to be $6.9 billion (5.9 billion to 7.9 billion) over the medical expenditures of a matched group of nondiabetic individuals see . Per capWe report mean values, but the underlying population is heterogeneous in terms of costs. There is a small subpopulation of T1D patients who rarely experience any complications associated with the disease. They are currently the focus of scientific studies, though a common factor has not yet been found We compare our figures to the ADA papers, noting that our methodology differs from that of the ADA Per capita total medical expenditures by people with diabetes are generally comparable between our study and the ADA papers. However, the matched control group of individuals without diabetes in our analysis spend less on medical care than the age-sex adjusted individuals without diabetes in the ADA paper  in our paper compared with $4,464 (2007 dollars) in the ADA paper). We use a matching technique to match individuals without diabetes as closely as possible to T1D patients based on a number of factors. Therefore, our T1D control group may in general be healthier than the age-sex adjusted control group used in the ADA papers , indicating the costs we estimate are a lower bound of the cost attributed to T1D.Total income loss estimated for the 1.1 million T1D patients in our sample for an average year between 1999 and 2005 is $7.5 billion (4.6 billion to 10.2 billion) in 2005 dollars, as shown in T1D individuals miss 5.5 (3.2 to 8.4) more work days, experience 7.6 (6.4 to 9.5) more bed days, and miss 3.3 (2.1 to 4.0) more school days per year than their matched counterparts. We do not find that educational attainment is significantly affected by T1D. These findings are consistent with other published findings The ADA papers measure indirect costs by estimating and valuing lost productivity through lost work days and bed days, disability, and increased mortality The per capita figure for the number of lost work and bed days reported by the ADA is similar to our findings. The ADA finds total indirect costs of $58 billion in 2007 dollars. Our estimate of $7.5 billion (4.6 billion to 10.2 billion) in income loss is over 10 percent of the ADA estimation of indirect total diabetes costs. However, mortality costs in our study are not directly comparable to the ADA number due to important differences in methodologies. The ADA studies do not account for the possibility of early mortality for those individuals without diabetes. Removing mortality costs from both figures, we find that non-mortality related indirect costs of the T1D population make up over 25 percent of the total diabetes costs . Thus, tDall et al. It is not surprising that indirect costs make up a large portion of total costs of T1D, given the typical early onset of the disease. Once on a lower earnings trajectory, it may not be possible to make up for the income difference; instead the differences may become even more severe as time passes. The calculation of lifetime costs of T1D, presented here for the first time, is particularly relevant for a chronic, childhood disease such as T1D, where costs are incurred over a long lifetime. Expected discounted lifetime income losses of a new cohort of 30,000 T1D are estimated to be $7.3 billion (4.5 billion to 10.1 billion). The expected discounted lifetime medical costs patients of a new cohort of 30,000 T1D are $3.3 billion (2.1 billion to 4.5 billion). For the current 1.1 million T1D patients, the present value of their expected lifetime medical and indirect costs is $133.7 billion (97.0 billion to 170.4 billion) and $289.2 (211.7 billion to 366.7 billion), respectively.Lifetime costs for several diseases associated with obesity are estimated by Thompson et al. Until now, the economic costs specific to T1D have not been adequately measured. Together with the recent work of Dall et al. It is important to note that our analysis is likely to provide an underestimate of the true societal costs of T1D. Due to data limitations, we necessarily omitted costs such as nursing home expenditures and quality-of-life effects, which may be substantial. Dall et al. While continued work is required to better understand the burden of T1D, COI studies can be used by policy makers, researchers, advocates, doctors, patients to assess the economic impact of the diseases on the individual and society. Used in conjunction with more traditional biomedical research studies, COI studies allow a more complete picture of the impacts of a particular disease to be developed. They can add to a thorough understanding of the costs and benefits of a particular disease treatment or to a better comparison among various diseases. This study is a crucial step forward in capturing the full costs, both yearly and over a lifetime, of T1D. We have shown that the disease has a substantial and disproportionate economic impact, particularly in the indirect costs. Over their lifetime, a patient with T1D incurs substantial medical and indirect costs as a result of their disease, understanding these costs is a crucial component of understanding the full impact of the disease.


Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy.Myxoma virus is a member of the An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain  were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature.It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis. Myxomatosis is a disease primarily of European rabbits and is caused by infection with myxoma virus which can result in very high levels of mortality nearly 100%). In consequence, the virus has been deliberately introduced into some countries as a control measure for wild rabbit populations. Although effective initially, co-evolution of virus and the rabbit population has resulted in the generation of attenuated field strains of the virus and also virus-resistant populations of wild rabbits 00%. In c.Infection by myxoma virus of pet rabbits (and presumably wild rabbits too) within Denmark occurs on a regular basis particularly during September and October of each year and it is a notifiable disease. When infection has been suspected clinically by veterinarians, tissue samples from dead or euthanized animals have been submitted to the Danish National Veterinary Institute (DTU Vet) and laboratory diagnosis has been performed using ELISA based systems and/or electron microscopy. The number of cases and suspicions in each year is normally small but, unexpectedly, in 2007 there was a large increase in the number of clinical cases of myxomatosis observed within Denmark (see below and Table Leporipoxvirus genus within the Poxviridae (see [Myxoma virus is a member of the dae (see ). The vidae . Other dae (see . Recentldae (see . The M13dae (see . HoweverNC_001132]) enabled the design of a sensitive and specific real-time PCR-based assay for this virus. This assay has been used to identify the presence of myxoma virus DNA from within laboratory and clinical samples. Furthermore, sequence analysis of selected regions of these myxoma viruses has been performed to explore the relationship between current Danish viruses and other virulent and attenuated virus strains. Surprisingly, a high proportion of the Danish samples, obtained from clinically diseased animals, contained a frame-shift mutation within the M135R gene, which severely truncates the expressed protein product.The availability of the myxoma virus genome sequence , swinepox and Orf virus or rabbit tissue homogenates were used to prepare samples of viral DNA for analysis. The tissues  from dead or euthanized pet rabbits which had exhibited clinical disease and had been provided for laboratory diagnosis, were homogenized in physiological saline (1 ml/g of tissue) and then clarified by centrifugation. The DNA was isolated using the QIAamp DNA Blood Mini kit (Qiagen) using the "Blood and Body Fluid Spin protocol" as described by the manufacturer. Briefly, supernatant samples (200 μl) were incubated with Qiagen protease at 56°C for 10 mins, mixed with an equal volume of ethanol and applied to the QIAamp Spin columns. Bound DNA was eluted in water (50 μl) and then assayed directly. It was found that using 1 μl of the eluted DNA was optimal in the real time PCR assay described below, for some samples higher amounts could produce a lower signal, presumably due to the presence of some inhibitor.In addition, viral DNA was isolated, as described above, from a sample of Cunivak vaccine  which contains a live attenuated strain of myxoma virus.7 to 101 copies per μl) was prepared and assayed as above.The genome sequence of the Lausanne strain of myxoma virus  was usedNC_001132] using ClustalW2 [A standard PCR was performed using the isolated viral DNA preparations from a variety of different myxoma virus samples with the primers M135Rfor and M135Rrev Table  which halustalW2 .Analysis of M135R protein expression was achieved by making fusion constructs between the coding sequence for chloramphenicol acetyl transferase (CAT) and the M135R coding sequence. The CAT sequence was amplified in a standard PCR using primers CATBamfor and CATXSrev Table  using a These plasmids were assayed by transfection into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3  essentiaIn addition to the M135R gene, five other regions of the myxoma virus genome were amplified by PCR using primers flanking the insertions and deletions identified within a nonpathogenic myxoma virus strain (6918) by Morales et al. . The priLeporipoxvirus) genome together with the homologous sequences from vaccinia virus (Orthopoxvirus), swinepox (Suipoxvirus) and Orf virus (Parapoxvirus) are shown in Figure A real time quantitative PCR (qPCR) assay to detect specifically myxoma virus DNA was designed. The targeted region of the myxoma virus .To characterize the system for the detection of myxoma virus DNA, viral nucleic acid was prepared from cell culture grown samples of myxoma virus, vaccinia virus, swinepox and Orf virus and tested in the qPCR assay. As expected the assay efficiently detected the myxoma virus DNA but did not produce a signal using nucleic acid preparations from cells infected with the other poxviruses tested  proved to be positive (see Table The region of the myxoma virus M029L gene amplified in the diagnostic qPCR assay is quite short (119 bp). In order to obtain some indication of whether all the cases of myxomatosis in Denmark during 2007 were caused by the same strain of virus, the sequence of a larger region of the genome, from 13 selected samples, was examined. For this purpose, the M135R gene was chosen as a target since it had recently been reported to be an important virulence factor . The DNAOn the basis of the sequencing data, it was apparent that DNA fragments containing the insertion of a single nucleotide within the M135R coding sequence should encode a very different protein than the wt sequence  or the frame-shift variant (+G) were constructed and then assayed along with a plasmid encoding the normal CAT coding sequence  and parUsing this assay for myxoma virus, we tested samples from Danish rabbits with clinically determined myxomatosis and we confirmed the diagnosis of the unusually large number of cases of myxomatosis in Denmark during 2007  A sensitive and specific qPCR assay has been established for myxoma virus.2) An unusually high number of clinical cases of myxoma occurred in Denmark in 2007.3) A high proportion of the Danish samples that were analysed had a frame-shift mutation within the M135R protein coding sequence which results in the expression of a severely truncated product.4) The presence of this same frame-shift mutation within an attenuated myxoma virus (6918) is not sufficient to explain its attenuation.5) Analysis of the effect of the frame-shift mutation within the M135R gene, in isolation, on the virulence of myxoma virus in European rabbits is warranted.The authors declare that they have no competing interests.GJB designed the assay, planned the experiments and drafted the manuscript.CP performed the transient expression assays and critically reviewed the manuscript.SØB performed the sequence analysis of the M135R genes and identified the frame-shift mutation within the M135R gene and critically reviewed the manuscript.LAEL provided the myxoma vaccine, supervised some of the sequencing studies and critically reviewed the manuscript.ANEB initiated the work, supervised the provision of the clinical samples, had responsibility for the laboratory diagnosis and critically reviewed the manuscript.All authors read and approved the final manuscript.


The economic impact of mental disorders is wide-ranging, long-lasting and huge. These disorders impose a range of costs on individuals, families and communities as a whole.Schizophrenia is a burdensome illness, with implications not only for patients and families, but also for the health services and other care agencies.There are several methods of economic evaluation. These include cost-minimization analysis, cost-effectiveness analysis, cost–benefit analysis, cost-utility analysis, and cost of illness (COI) analysis. Among the various methods, COI studies describe the economic burden of disease on society. COI studies also allow for comparison between different illnesses. From the health system perspective, only direct medical costs are relevant. From a societal perspective, all costs are relevant including direct medical costs, direct non-medical costs and indirect costs. COI studies emphasize the issue of cost containment as well as the benefits of total elimination of the disease. COI studies can draw attention to disorders with an overall high burden; disorders with poor investment in services and other resources used in their treatment; the possible impact of preventive measures on primary, secondary and tertiary prevention; comparison with other disorders in a common currency. COI studies are also are important in estimating the economic burden in that costs of health service intervention reflect the existing pattern of service delivery.2A COI study can be based on either prevalence or incidence rates, and employs either a top–down or bottom–up approach. The underlying rationale of the prevalence-based method is that costs are assigned to the year in which they are borne, or with which they are directly associated. This type of costing identifies the major contributors to the current expenditure. Expected future earnings lost as a result of premature death are assigned to the year of death. If cost control is the aim of the exercise, this approach allows for the identification of possible targets for economizing. A prevalence-based COI study gives an idea of the magnitude of the problem, and can be useful for priority-setting on a macro level. If a change in service delivery is being implemented, the prevalence approach allows comparison of the balance sheets before and after the change. It can also be of great help in comparing the societal burden of two diseases. However, there is no link to benefit from a possible intervention to reduce the burden of the disease. The incidence approach is based on the principle that the flow of costs associated with the disease should be assigned to the year in which that flow begins. All future direct and indirect costs are estimated and discounted so that they can be measured in monetary terms of the year in which the illness first occurs, because once the illness has occurred the society at one level or another is committed to meet the streams of cost that will be associated with that illness. This approach is useful because it can predict the likely long-term impact of programmes that reduce incidence, make treatment less expensive or improve outcome. An incidence-based COI study is helpful when one has to decide between alternative methods of interventions, because an intervention will affect all future treatment costs and productivity losses.11While estimating the COI, most studies use either of two computational methods to determine the direct costs of disease: a ‘top–down’ or a ‘bottom–up’ approach. In the top–down approach, the entire health care expenditure is calculated and the share attributable to a particular illness is then determined.212Parts of this economic burden are obvious and measurable, while other parts are almost impossible to measure. Among the measurable components of the economic burden are health and social service needs, lost employment and reduced productivity, impact on families and caregivers, levels of crime and public safety, and the negative impact of premature mortality. The parts that cannot be measured in monetary terms are called intangible costs and includes effects on the patients in the form of stigma, stress and side-effects of treatment, and on the caregivers in the form of stress, psychiatric morbidity and stigma.Direct costs are the actual monetary expenditure related to treating an illness or disorder, i.e. these include costs associated with hospitalization, outpatient services, nursing care, drugs, services of a range of professionals, residential care, day care, and domiciliary care and rehabilitation.22Indirect costs concern the monetary value of lost output due to reduced or lost productivity of patients and caregivers, caused by illness, disability or injury of patients,15Intangible costs cannot be expressed in monetary terms, and include effects on the patient in the form of stigma, stress, and side-effects of treatment; and on the caregiver in terms of stress, stigma and psychiatric morbidity.2A number of studies have attempted to calculate the cost of care of schizophrenia in developed countries. The findings of these studies vary widely because of methodological dissimilarities. In contrast, there are only a few studies of COI from developing countries in which comprehensive costing has been undertaken.11316173111634et al.33182233404849On the whole, however, different authors have claimed that either the proportion of direct costs and indirect costs are nearly equal,35162128233514253317253333et al.Unfortunately, information from developing countries is scarce although the illness is as common and perhaps as disabling. The few studies that are available suggest that there could be differences in the types of costs incurred, which is expected, given the vast differences in sociocultural and treatment milieus. Suleiman et al.55, an equivalent dose of trifluperazine amounted to Rs 25 per month, risperidone Rs 60 and clozapine Rs 225 per month. They also found that there was a marked price difference across brands. They concluded that although antipsychotic drugs are affordable, the other costs associated with treatment make them more expensive; these could be co-prescribed antiparkinsonian agents, antidepressants, anxiolytics, etc. Sarma showed that cost of one outpatient visit was Rs 201 in which the contribution of the management was 68% and the patient's contribution 32%;et al., screened four rural populations in India and Pakistan for psychiatric morbidity.700 per month and in the Rawalpindi site it was more than Rs 3000 per month. The total cost was equivalent to between 7 and 14 days of an agricultural worker's wages in India, and approximately 20 days' wages in Pakistan. These total costs decreased appreciably by the follow-up assessment point in 3 of the 4 localities.There are very few studies which have evaluated the cost of mental illnesses in India. Girish We conducted a study on COI of schizophrenia with the hope that such evaluation would increase awareness of the costs incurred by patients, families and treating agencies caring for patients with schizophrenia.Employment status was the only sociodemographic parameter that had a significant influence on the costs of care. Total, as well as direct, costs were significantly higher among the unemployed in this group. The number of visits made to the hospital demonstrated significant positive correlations with total, direct, indirect and provider's costs in both groups of patients. Duration of illness did not correlate significantly with costs. The association of costs with clinical severity and levels of disability were by far the most impressive. There was a strong correlation between the Positive and Negative Symptom Scale (PANSS) scores and Schedule for Assessment of Psychiatric Disability (SAPD) scores with total as well as various other components of costs.This study demonstrates that schizophrenia is an expensive illness in monetary terms, even in developing country settings. Some important conclusions can be drawn from the above study. First, the cost of treatment of schizophrenia in India is different from that reported from many developed countries and hence there is a definite need for such studies to plan health services. Second, the cost of care of schizophrenia is no different from that of other chronic physical illnesses such as diabetes mellitus. There is a great need to generate awareness of this fact among all manner of professionals involved in the care of patients with schizophrenia, as well as health policy-makers. Third, the study also demonstrates that the main brunt of the financial burden caused by schizophrenia is borne by the impoverished family; hence the financial burden on the family is much higher compared with western figures. The recent World health reportThis study has not answered all the issues. First, about three fourths of the patients in this study were urban-based, which meant that they did not have to spend much on travelling to a hospital located in the city. Most of the subjects in the study were local inhabitants and did not have to spend anything on stay near the hospital for treatment. However, since mental health facilities are unevenly distributed in developing countries, patients often have to travel long distances and arrange for accommodation near the treating agency. This inevitably increases the amount spent on such ventures. Second, the costs of inpatient treatment were not covered by the study, which can increase the cost manifold. Third, since indirect costs are often more difficult to compute, they have not been measured in totality. Mortality costs and other costs, e.g. those of caregiving, were not measured in the current study. Therefore, indirect costs were likely to have been underestimated. Moreover, it has been proposed that the human capital approach used in this study does not adequately measure indirect costs; alternative approaches have been suggested.Changing economic trends have meant that evaluation of treatment costs is no longer a subject of mere academic interest, even in the developing world. The need for such studies is obvious, relevant and practical. This is because pharmacoeconomics is likely to become an important basis for health policy decisions as a number of important dynamics evolve in the marketplace. These include consumers acting on their growing access to information and becoming more actively involved in treatment decisions; payers, providers and patients deepening their interaction and overcoming their traditional focus on either costs or benefits alone; and manufacturers being challenged by other health care constituencies as sponsors of cost-based outcome studies. In the same vein, several authors have pointed out that since costs of schizophrenia are context-bound, these might differ from country to country (even in the developing world), based on differences in economic indices, e.g. per capita income, local demography, as well diversity in cultures, social structures, and health care services. Hence, a cost analysis of the existing health care system can help to identify inefficiencies in the system and the expenditure pattern. The need for information on costs in India assumes greater importance as the Government of India is now considering implementing user fees for health care services. In India, information on costs of health services usually comes from budgetary statements. The actual costs of various components of the health care delivery system are often not available. This is because budgetary provisions cover only recurrent expenditures such as salary and drugs but do not reflect the investments already made in the form of infrastructure and equipment, costs borne by the family and the work hours lost due to absence from duty and disability. Hence, there is a need for more studies on the pharmaco-economics of mental illnesses. It is hoped that in the long run such studies will facilitate improved allocation of the resources available for the treatment of mental illness in India.