Cryptococcus neoformans and C. gattii have only recently been reported. We describe a novel C. neoformans × C. gattii hybrid strain (serotype AB) that was previously described as C. gattii and that caused a lethal infection in an AIDS patient from Canada.Interspecies hybrids of  Cryptococcus neoformans and C. gattii are pathogenic yeasts that may cause meningoencephalitis. C. neoformans primarily infects immunocompromised patients and occurs worldwide, whereas C. gattii primarily mainly infects otherwise healthy persons and has been thought to occur in subtropical regions .C. neoformans var. neoformans × C. gattii hybrids were isolated from 2 HIV-negative patients in the Netherlands  , 2 RNA polymerase II subunits (RPB1 and RPB2), and translation elongation factor 1α (TEF1α) (Ten colonies of CBS10496 were used for DNA extraction (A), which indicates that CBS10496 has ≈2× more DNA than haploid strains. We concluded that CBS10496 is diploid or aneuploid. Staining with 4′,6-diamidino-2-phenylindole showed that cells of CBS10496 were monokaryotic (B).DNA content of CBS10496 was compared with that of CBS8710 and CBS10510. The G1 peak of reference strains was located at positions 31.6 (CBS8710) and 31.1 (CBS10510), and the G2 peak was located at positions 65.8 (CBS8710) and 56.4 (CBS10510). The G1 peak of CBS10496 was located at position 57.5, and the G2 peak was located at position 115.7. Thus, the G1 peak of CBS10496 coincided with the G2 peak of the haploid strains , panel Akaryotic , panel BC. neoformans .Two alleles representing AFLP1/VNI and AFLP4/VGI were found when fragments of MATα and the MATα serotype A–specific primer pair resulted in an amplicon. When MATa and the MATa serotype A–specific PCRs were conducted, no amplicon was obtained. These findings indicate that CBS10496 has a MATα serotype A background. All reference strains yielded amplicons with the expected primer pairs. In addition, CBS10510, a MATα serotype B strain, was amplified with the MATα specific primer pair, and E566, a MATa serotype B strain, yielded an amplicon with the MATa-specific PCR. These results indicate that a C. gattii and a MATα serotype A background are present in CBS10496. Because the mating type of the C. gattii background within CBS10496 was unknown, 30 MATα clones of CBS10496 were sequenced to determine whether a MATα serotype B allele could be identified. However, all clones were MATα serotype A; no MATα serotype B clones were found.Amplification of CBS10496 in a PCR with the C. neoformans var. grubii (AFLP1/VNI) and C. gattii (AFLP4/VGI). We conclude that this isolate is a novel aneuploid hybrid of C. neoformans var. grubii  and C. gattii .Our results indicated that CBS10496 is a monokaryotic, diploid, or aneuploid strain with the novel AB serotype. AFLP and sequence analysis showed that the isolate contained fragments of C. gattii on the basis of a weak positive reaction on CGB medium .CBS10496 had been identified as MATα background was observed. Although an amplicon was obtained with C. gattii–specific mating-type primers, the C. gattii background could not be linked to a mating type. We hypothesize that the serotype AB C. neoformans × C. gattii hybrid CBS10496 was formed by mating of a MATa serotype B strain with a MATα serotype A strain and subsequent loss of the MATa serotype B allele. Detection of single ITS and TEF1α alleles in CBS10496 further supports our findings because it indicates that other alleles were also lost. Loss of genetic material has been observed in other hybrids, such as serotype AD and BD hybrids (We expected that CBS10496 would have 2 mating-type loci. However, only a serotype A C. gattii parent of the serotype AB hybrid belongs to the AFLP4/VGI genotype, as was the case for serotype BD hybrids (C. gattii parental sequence of all known serotype BD C. neoformans × C. gattii hybrid isolates was identical to sequences of AFLP4/VGI strains CBS1622 and CBS6992 in all regions studied (C. gattii–AFLP4/VGI subgroup in all isolated C. neoformans × C. gattii hybrids may indicate that this subgroup preferentially forms interspecies hybrids.Our results show that the


Spinal fractures can be an important cause for disabling back pain. Therefore, in judging the cost-effectiveness of nonsurgical or surgical therapy, not only direct costs but also the indirect costs should be calculated. In this prospective randomized study, the costs incurred by nonsurgically and surgically treated patients with a traumatic thoracolumbar spine fracture without neurological involvement were analysed.32 patients with a traumatic thoracolumbar spine fracture were prospectively randomized for operative or nonsurgical treatment. Patients were sent a questionnaire every three months to inquire about work-status, additional health costs and doctor visits. The patients who have minimum followup of two years were included.Of thirty-two patients, 30 met the criterion of the followup period of at least two years. Fourteen patients received nonsurgical therapy, while 16 received surgical treatment. Direct costs of the treatment of nonsurgically treated patients were €10,608 . For the operatively treated group, these costs were €18,769 . Indirect costs resulted in a total of €219,187  per nonoperatively treated patient. In the operatively treated group, these costs were €66,004 .In the treatment of traumatic thoracolumbar spine fractures, the indirect costs exceed the direct costs by far and make up 95.4% of the total costs for treatment in nonsurgically treated patients and 71.6% of the total costs in the operative group. In view of cost-effectiveness, the operative therapy of traumatic thoracolumbar spine fractures is to be preferred. Spinal fractures can be an important cause for disabling back pain. Back pain is a major health problem in industrialized countries. The magnitude of the problem is obvious when expressed as incidence and prevalence figures. The annual incidence of back pain in the United States (US) has been reported to be about 5%.The direct costs for the treatment of traumatic thoracolumbar spine fractures are up to two times higher for surgical therapy compared with nonsurgical therapy.332 patients of traumatic fracture of T10 - L4, AO type A (compression fracture) with no neurological deficit (ASIA/Frankel E) and age 18-60 years from October 1998 until October 2003 were included. Exclusion criteria were AO type A.1.1 fracture, pregnancy, pathologic or osteoporotic fracture, patients with end-stage disease (ASA IV), patients with a history of previous back surgery, patients with a recent psychiatric history, patients using drugs or other illegal substances or patients presenting with any accompanying injury that might interfere with the treatment of the spine fracture or the mobilization scheme after hospital discharge  and any other type of disablement which could interfere with return to work. Patients were randomized between nonsurgical and surgical therapy. All patients were included based on written informed consent. This study was approved by the local ethical committee. Patients were sent a questionnaire every three months to inquire about work-status, additional health costs and doctor visits. Roland Morris Disability Questionnaire-24 (RMDQ-24), VAS Pain and VAS Spine Scores were used to evaluate the patients' well-being. RMDQ-24 is a validated questionnaire to measure self-assessed disability due to back pain. The disability questionnaire was constructed by choosing statements from the Sickness Impact Profile. Patients are given a score of one point for each of the 24 items of the questionnaire that were ticked. A patient's score could thus vary from zero (no disability) to 24 (severe disability).6© (nadroparine) 2850 IU was given as an anticoagulant until the patient was discharged. A Jewett hyperextension orthosis was fitted, and the patients were instructed to wear the brace at all times, except when bathing, for three months.The patients who were randomized to be managed nonsurgically were treated with a course of six weeks on a Rotorest bed .The patients who were randomized to receive surgical therapy were managed with a short-segment posterolateral fixation with an AO titanium Universal Spine System [USS ].910© (cefuroxime) was given intravenously as prophylactic antibiotic. Fraxiparine© (nadroparine) 2850 IU was given as an anticoagulant until the patient was discharged. After operation, the patient was mobilized with a Jewett hyperextension orthosis for three months. A vigorous physiotherapy scheme was given to improve trunk musculature. After 9 till 12 months, the USS was removed.Preoperative 1500 milligrams of ZinacefThe approach most frequently used to estimate the cost of illness (COI) is the human capital approach. AccordinDirect costs consist primarily of medical costs of diagnosis, treatment, continuing care and rehabilitation. It also includes nonmedical expenditures caused by disease, e.g., travel expenses.The hospital-care costs include the costs of clinical care; and additional costs of treatment, diagnostics, paramedical care and operating rooms. The hospital-care costs are divided into outpatient costs and inpatient costs. Since the data concerning medical equipment  were not separately recorded, it was not possible to include these costs into this calculation. The following cost categories were used from a former study performed in our clinic.31. The costs of hospitalization days ; 2. costs of laboratory tests; 3. costs of blood products; 4. costs of radiology (including X rays and CT scans of the thoracolumbar spine); 4. costs of physical therapy (staff and overheads); 5. costs of operating room, overheads, staff and implant; 6. costs of spinal orthosis and cast and 7. the costs of outpatient visits (including costs of radiology).The costs of procedures performed by medical specialists were calculated using the relevant costs for each procedure. The costs incurred were the referring costs for the operation, for anesthesiology and for additional support.After discharge from the clinic, part of the care is also delivered by the general physician . To make an estimation of the costs towards the general physician, patients indicated how often and how many minutes they visited a GP.Indirect costs of disease are defined as production losses and related costs to society due to morbidity and mortality.The social security system in the Netherlands is based on two laws prescribing the obligation to have insurance for the loss of income due to sickness, injury or disability. Under the Sickness Benefits Act (SBA), workers receive ‘sick’ pay during absence, of a maximum of 52 weeks. If the worker is still unable to work after 52 weeks, the patient is entitled to a disability pension covered by the Disablement Insurance Act (DIA).In the present study, costs of absenteeism have been estimated by multiplying the total number of sick days with the mean costs of one sick day. The gross sick pay starts after two qualifying days for the sickness benefit and amounts to 70% of the daily wage. However, due to additional insurances or collective labor agreements, almost every employee receives full wages during absenteeism. We therefore extrapolated the sick pay to 100%. Besides the insurance costs, the Sickness Benefits Act also involves administration costs. In 1999 the total administration costs amounted to 5.5% of the total insurance costs. The administration costs were estimated by adding this proportion to the insurance costs.The total costs of disablement were estimated by multiplying the total days of disability with the mean daily pension in 2000. This was €18,500  per year for men and €12,000  per year for women.Of the 32 included patients, two were lost to followup and could not be contacted. 30 patients with a followup of at least two years were included for analysis.14 patients received nonsurgical therapy and 16 surgical treatment. Mean age was 45.7 years (range 27-59) in the surgically treated patients and 37.3 years (range 18-53) in the nonsurgical group . The male-to-female ratio was 10:7 in the surgical and 10:5 in the nonsurgical group (not significant). More than 80% of the fractures appeared at the levels Th12 and L1, without a significant difference concerning fracture localization in the two treatment groups. The predominant trauma mechanisms leading to the thoracolumbar fracture were motor vehicle accident and fall from height. At hospital admission, 25 fractures (78%) were classified as type A3, five fractures as type A1 and two fractures as type A2 according to the comprehensive classification. There was no significant difference in distribution of various type A fractures between the two groups. During operation, two type B fractures (distraction type) were identified showing evidence of injuries to the posterior ligamentous complex. Both fractures were included in the study, based on the ‘intention to treat’ principle. In these two specific patients, hardware was not removed. MRI studies were not routinely performed; so the number of unrecognized type B fractures in the nonsurgical group is unknown. The mean Load Sharing Classification in the surgically treated patients was 6.5 ; and in the nonsurgical group, 6.1 . Considering the LSC scores, there was no significant difference between the two groups.P = 0.033).The estimated pre-injury pain scores as measured by VAS Pain  averaged 94 mm (range 75-100) for the nonsurgical group and 94 mm (range 78-100) for the surgical group. At final followup, the average scores were 72 mm (15-100) and 87 mm (55-100) for the nonsurgical and surgical groups respectively. The surgically treated patients had significant lesser pains score (P = 0.020).The estimated pre-injury VAS Spine scores  averaged 94 (74-100) in the nonsurgical group and 87 (61-100) in the surgical group. At final followup, the VAS Spine score was 61 (11-100) for the nonsurgical group versus 81 (45-100) for the surgical group. This difference in score in favor of the surgical treatment was significant (P = 0.030).The Roland Morris Disability Questionnnaire-24 (RMDQ-24) pre-injury functional disability scores , which were estimated on hospital admission, show no significant difference between the two treatment groups: an average of 0.1 (0-2) for the nonsurgical group and 0.6 (0-6) for the surgical group. At final followup, the average score was 8.9 (0-24) for the nonsurgical group and 3.1 (0-14) for the surgical group. The patients receiving nonsurgical treatment were found to have a significantly higher RMDQ-24 score in comparison with the surgical group , VAS Spine Score  and VAS pain .In both the groups, the Pearson's rank correlation test found no correlation between the final amount of local and regional kyphotic deformity and disability according to RMDQ-24 . Five of the 11 (46%) working patients in the nonsurgically treated group returned to their job - three of them to the same job and two did return to a job which was less demanding. The average time for returning to work for nonsurgical patients was 14 months (range 6-33). Nine of the 11 (82%) working patients in the surgical group returned to their previous job; none of them changed to a physically less demanding job. Average returning time in the operatively treated group was 7 months (1-18). No significant difference between the nonsurgical and surgical groups could be reached because of the spread in time to return to work from one till 33 months.The surgically treated patients returned to work early and the difference was statistically significant , which were €18,300  for the surgically treated group.General practice care costs: Costs of a GP are €47 ($56) per hour. Patients who were treated nonoperatively visited the GP for 500 minutes. Total costs were €392 ($470), resulting in €28 ($34) per patient. Surgically treated patients spent 214 minutes at their GP. Total costs were €169 ($203), resulting in €11 ($13) per patient.Examples of expenditures made were clothing, education for a job change, physiotherapy and transportation costs. In the nonsurgically treated group, these expenditures totaled €9,525  or €680 ($816) per patient. The surgical group spent €7,322 , resulting in €458 ($550) per patient.In the nonoperatively treated group, 11 patients did have a regular job. The total period of absence of these 11 patients was 115 months. This resulted in total costs of €120,500  or €8,607  per nonsurgically treated patient. Twelve patients in the surgical group had a regular job. The total period of absence was 78 months. This resulted in total costs of €88,400  or €5,525  per operatively treated patient. In Because of our strict inclusion and exclusion criteria , the disablement in our group was caused by the thoracolumbar spine fracture in all cases. Through the questionnaire, we could verify that the disablement was caused by residual back pain or was treatment related. In the nonoperatively treated group of patients, six were disabled. These were all men with a mean age of 39 years (range 18-58). One hundred fifty-nine years have to be compensated. This results in a total compensation of €2,941,500 , or €210,107  per nonoperatively treated patient. In the surgical group, there were two disabled patients, one man aged 48 and one woman aged 36. Thus a total of 46 years have to be compensated. This results in a total compensation of €662,500 , or €41,406  per operatively treated patient.This is the first study which studies prospectively the costs that are made in the treatment of traumatic thoracolumbar spine fractures and in which direct and indirect costs are calculated. We also took this opportunity to compare the costs of nonoperative and operative treatment to see which therapy is more cost-effective. Cost-effectiveness can be used as a rational way of deciding how to get the best health improvement from limited resources - which is a reasonable, even essential, concern.19et al. were higher than the costs in this study - $24,600 versus $12,730 for nonsurgical treatment and $45,300 versus $22,523 for surgically treated patients.34Two studies reported direct costs of treatment of thoracolumbar spine fractures. The results for the direct costs reported in one of these studies are used in this report.Our hypothesis that indirect costs are a more important factor than the direct costs in the treatment of thoracolumbar spine fractures was confirmed. The indirect costs exceed the direct costs by far and make up 95.4% of the total costs for treatment in nonsurgically treated patients and 71.6% of the total costs in the surgical group. The costs of disablement are an important factor. When a patient aged 18 years becomes disabled and is unfit for work, 47 years have to be compensated. This stresses the importance of the indirect costs in judging cost-effectiveness. In comparative studies, these indirect costs are never addressed, which makes a good cost-effectiveness study impossible, resulting in charges for operative therapy to be as much as four times the charges for conservative therapy.It seems justified to state that in view of cost-effectiveness, the operative surgical therapy of traumatic thoracolumbar type A (excluding A.1.1) spine fractures is to be preferred because of the lower indirect costs.


Over the past 6 years, a number of zoonotic and vectorborne viral diseases have emerged in Southeast Asia and the Western Pacific. Vectorborne disease agents discussed in this article include Japanese encephalitis, Barmah Forest, Ross River, and Chikungunya viruses. However, most emerging viruses have been zoonotic, with fruit bats, including flying fox species as the probable wildlife hosts, and these will be discussed as well. The first of these disease agents to emerge was Hendra virus, formerly called equine morbillivirus. This was followed by outbreaks caused by a rabies-related virus, Australian bat lyssavirus, and a virus associated with porcine stillbirths and malformations, Menangle virus. Nipah virus caused an outbreak of fatal pneumonia in pigs and encephalitis in humans in the Malay Peninsula. Most recently, Tioman virus has been isolated from flying foxes, but it has not yet been associated with animal or human disease. Of nonzoonotic viruses, the most important regionally have been enterovirus 71 and HIV.


Encephalomyocarditis virus was identified in the serum of 2 febrile patients in Peru. Etiologic studies of acute febrile disease were conducted in sites across South America, including Cusco and Iquitos, Peru. Patients’ clinical signs and symptoms were recorded, and acute- and convalescent-phase serum samples were obtained for serologic examination and virus isolation in Vero E6 and C6/36 cells. Virus isolated in Vero E6 cells was identified as encephalomyocarditis virus (EMCV) by electron microscopy and by subsequent molecular diagnostic testing of samples from 2 febrile patients with nausea, headache, and dyspnea. The virus was recovered from acute-phase serum samples from both case-patients and identified with cardiovirus-specific reverse transcription–PCR and sequencing. Serum samples from case-patient 1 showed cardiovirus antibody by immunoglobulin M ELISA  and by neutralization assay . Serum samples from case-patient 2 did not contain antibodies detectable by either assay. Detection of virus in serum strongly supports a role for EMCV in human infection and febrile illness. Picornaviridae, genus Cardiovirus) is a group of closely related virus strains belonging to 1 serotype with a wide host range , was reported in association with fever of unknown origin in a child 8 months of age  M ELISA for flaviviruses, alphaviruses, bunyaviruses, arenaviruses, and rickettsia, as well as virus isolation, reverse transcription–PCR (RT-PCR), and sequencing. The study protocol was approved by the Naval Medical Research Center Institutional Review Board , in compliance with all federal regulations governing the protection of human patients; the protocol also was reviewed by the Peruvian Ministry of Health.Clinical diagnoses were considered confirmed if laboratory testing resulted in isolation of virus from the specimen, virus detection by RT-PCR, or a 4-fold or greater increase in IgM antibody titers. Diagnoses were considered presumptive if the IgM titer was elevated in the acute-phase sample only or a 4-fold or greater increase was discerned upon comparison of acute- and convalescent-phase titers. In the absence of laboratory evidence of a specific pathogen, cases were classified as negative.2 flasks. Upon observation of viral cytopathic effect, or 10 days postinoculation (dpi) if no cytopathic effect was observed, cells were removed from the flasks and placed on 12-well glass spot-slides for examination by immunofluorescence assay using group- and virus-specific polyclonal and monoclonal antibodies. The antibodies used were reactive with dengue viruses 1–4, yellow fever virus, St. Louis encephalitis virus, Rocio virus, Ilheus virus, West Nile virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, Mayaro virus, Trocara virus, Oropouche virus, Caraparu virus, Murtucu virus, Guaroa virus, hantavirus, Machupo virus, and Tacaribe virus; after the cardiovirus isolation, EMCV antibody also was used.Viruses were isolated by using a modification of a published protocol , a master seed stock was prepared from each virus and working stocks were made by passage in Vero E6 cells. Cytopathic effect developed rapidly, and the cultures were harvested at 3–4 dpi; some infected cells were fixed in 2.5% glutaraldehyde for electron microscopy examination. For virus reisolation at CDC, acute-phase serum samples were injected into Vero E6 cells. Virus isolates were obtained in Vero E6 cells from acute-phase serum of both case-patients. The isolates, IQD6726 (Iquitos) and FSC575 (Cusco), were passaged in Vero E6 cells to obtain stocks for further analysis. Litters of suckling mice were inoculated intracerebrally with both strains of virus. Brain, spleen, and liver tissues were harvested and fixed in 2.5% glutaraldehyde for electron microscopic examination.2 , 2.5 U of Fast Start Taq DNA Polymerase (Roche), and forward and reverse primers , in a total volume of 50 μL. Cycling parameters were 42ºC for 50 min, 50°C for 10 min, and 95ºC for 5 min, followed by 45 cycles of 95ºC for 30 sec, 45ºC for 40 sec, and 60ºC for 1 min. All PCR products were analyzed by electrophoresis in 2% agarose gels stained with ethidium bromide.RNA was extracted from the virus isolates by using the QIAamp Viral RNA Mini Kit . cDNA reactions consisted of 2 μL of extracted viral RNA, 50 ng of random hexamers , 4 μL of 5× first strand buffer , 200 μmol/L of each dNTP , 40 U of RNasin ribonuclease inhibitor , and 200 U of SuperScript II reverse transcriptase (Invitrogen) in a total volume of 20 μL. The cDNA reactions were incubated for 10 min at 25°C, followed by 45 min at 42°C and 4 min at 95°C. For the PCR assays, 3 genome regions were targeted, the 5′-NTR, VP1, and 3D regions . Two setAmplicons were purified from an agarose gel (QIAGEN gel extraction kit) before sequencing. Both strands were sequenced by using the PCR primers, a Prism BigDye Terminator version 1.1 or 3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems), and a model 3100 or 3130 Genetic Analyzer (Applied Biosystems). The sequences determined in this study were deposited in the GenBank sequence database, accession nos. EU979543–EU979548.www.ncbi.nlm.nih.gov/blast). For phylogenetic reconstruction, sequences were aligned with those of other cardioviruses by using PileUp , and trees were generated with the neighbor-joining method implemented in ClustalX version 1.83 (Sequences were identified in GenBank by using BLAST to compare sequences (50) of isolate FSC575 and serial dilutions of serum  were incubated together at 37°C for 2.5 h before Vero E6 cells were added to the wells. After incubation for 5 days at 37°C, each plate was stained with 0.05% crystal violet in 25% ethyl alcohol and, after drying, the optical density in each well was measured at 570 nm. Each specimen was run in triplicate; the final titer was estimated by use of the Spearman-Karber method  is situated at an elevation of 120 m and has a population of ≈350,000. The city is surrounded by the Nanay, Itaya, and Amazon rivers and is accessible only by air or river. The climate is tropical, mean temperature is 25°C, and annual rainfall is 2.7 m. Rodents are common in urban and rural areas; swine and primates are abundant in rural zones.Case-patient 1 was a 59-year-old housewife from Iquitos. She sought treatment at Hospital Apoyo Iquitos after 3 days of fever, pallor, poor appetite, malaise, nausea, and headache. She recovered completely from fever after 6 days of treatment as an outpatient. Acute-phase (3 days postonset), convalescent-phase (15 days postonset), and late convalescent–phase (8 months later) blood samples were obtained. She had no identifiable focus of infection, negative thick and thin smears for malaria, and a negative tourniquet test. She reported no yellow fever or hepatitis B vaccination and no travel outside of Iquitos within 2 weeks before onset. The patient was interviewed 8 months after seeking treatment when virus isolation results were completed. She reported only limited contact with rats and cats in her house and a neighbor’s house where she played bingo frequently. After 7 days and nights of trapping in both houses and in a sewer adjacent to the neighbor’s house, no rodents were collected.The second case-patient was identified in the Cusco Regional Hospital but had traveled from Quebrada, a small rural village on the Yanatile River, about 8 hours travel time from the city of Cusco. Quebrada  is located in Yanatile district, Calca Province, in the Department of Cusco. The weather in Quebrada is warmer than that in Cusco (temperate climate) at an altitude of 2,926 m. Malaria and leishmaniasis are endemic to the area. The city is accessible by air and land from Cusco.Bartonella spp. were negative, as were urine culture and a tourniquet test for dengue hemorrhagic fever. The patient was treated with oral ciprofloxacin (750 mg 2×/d for 7 days), oral paracetamol , and intravenous fluids. He recovered completely after 7 days as an inpatient. At the time of his convalescent-phase sample (15 days postonset), his physical examination was within normal limits. He reported tuberculosis in a family member contact (treated for 5 months), yellow fever vaccination in 1994, and no vaccination against hepatitis B. No case investigation was carried out for this patient to determine risk factors for disease. However, the typically rural populations near Cusco have contact with a variety of animal species such as mules, dogs, cats, swine, rodents, rabbits, llamas, alpacas, and vicuñas.Case-patient 2 was a 39-year-old male farmer from Cusco-Acomayo/Quebrada Calca. He sought treatment at the local hospital after 7 days of fever. Additional symptoms were headache, malaise, retro-ocular pain, sweats, weight loss, arthralgia, photophobia, poor appetite, myalgia, chills, pallor, nausea, vomiting, and abdominal pain. He was hospitalized with a diagnosis of febrile syndrome and urinary tract infection (UTI). Urinalysis showed 20–25 leukocytes per high-power field. The patient had elevated values for alkaline phosphatase , total bilirubin , and direct bilirubin . Thick and thin smears for malaria and Virus isolates were obtained from acute-phase serum specimens of both patients by inoculation of Vero E6 cell cultures. Electron microscopic studies of infected Vero E6 cells and mouse tissues demonstrated cytoplasmic accumulations of particles consistent with the features of picornaviruses. Virions averaged 24 nm in diameter and were occasionally found in paracrystalline arrays , panel ATheilovirus genus, respectively. The Peru strains were also most closely related to the European pig strains in the 5′-NTR and 3D regions  to >1,024 in the convalescent-phase sample (day 15). The neutralization assay yielded similar results; acute-phase titer was <10 and a convalescent-phase titer was >1,280. Serum samples from case-patient 2, collected on days 7 and 15, were negative in both assays , despite the presence of virus in the day 7 serum sample.,,Our ongoing febrile surveillance studies identified and documented 2 cases of human EMCV disease, each of which was diagnosed by virus isolation from acute-phase serum. One patient had an undifferentiated fever with complete recovery after 6 days of fever. The second patient’s disease was complicated by concomitant UTI, which presumably prolonged the course of disease (11 days of fever). Even though EMCV was isolated from this patient, we cannot be certain whether specific symptoms resulted from the viral infection or from the concurrent UTI. At the time of hospitalization, the urine culture was negative, but the patient had received a course of antimicrobial drugs before his hospitalization. However, the urinary tract symptoms also could be consistent with EMCV infection because orchitis has been observed in male laboratory male mice and hamsters  with aseptic meningitis, poliomyelitis-like paralysis, encephalomyelitis, Guilláin-Barré syndrome, and fever of unknown origin (,Because few clinical or public health laboratories are capable of identifying cardiovirus infection, the effect on human health is unknown. The original detection and identification of SAFV used a combination of traditional virus isolation in cell culture and creation and characterization of a cDNA library by DNase sequence–independent single-primer amplification (


Objective: The involvement of the genital mycoplasmas Ureaplasma urealyticum               and Mycoplasma hominis in complications of pregnancy has remained             controversial especially because these microorganisms are frequent colonizers of the             lower genital tract. Recovery of bacteria from the placenta appears to be the sole technique             to represent a true infection and not vaginal contamination. Therefore, we investigated the             presence of genital mycoplasmas, aerobic and anaerobic bacteria, and fungi in human             placentas and evaluated their association with morbidity and mortality of pregnancy.					Methods: We cultured placentas from 82 women with complicated             pregnancies. One hundred placentas from women with uncomplicated pregnancies were             evaluated as controls. When possible, placentas were examined histologically for presence             of chorioamnionitis.					Results: Microorganisms were recovered from 52% of the placentas             of complicated pregnancies and U. urealyticum was the microorganism isolated most             frequently from the placenta. A significant association between positive mycoplasma             culture of the placenta and complication of pregnancy was found, and chorioamnionitis             was positively related to isolation of mycoplasmas.					Conclusions: These data suggest that genital mycoplasmas are             able to infect the human placenta where they can cause chorioamnionitis.             This infection of the placenta by genital mycoplasmas is related to preterm birth and             fatal outcome of pregnancy.


Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family.  Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late.  While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus.  Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells.  Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection.  Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.   The family  Before using the Ambion Amino Allyl MessageAmp II kit, add the recommended volume of 100% ethanol to the wash buffers.In PCR reaction tube, add between 100ng to 5µg of total RNA and 1µl of T7 oligo (dT) primer.  Bring the volume up to 12µl with nuclease-free water.Incubate samples at 70°C for 10 min in a thermocycler.Remove RNA samples from 70°C and centrifuge briefly.  Place on ice.st Strand Synthesis master mix and keep at room temperature (with 10% overage for pipetting error). (Table 1)Prepare the 1Gently pipette Master Mix or flick to mix, and then centrifuge briefly.Transfer 8µl of the Master Mix to each RNA sample.  Mix by pipetting up and down 3-4 times.Incubate at 42°C for 2 hours in thermocycler.Centrifuge samples briefly and place on ice.  Immediately proceed to the next step of dsDNA synthesis.nd Strand Synthesis master mix on ice (with 10% overage for pipetting error). (Table 2)Prepare the 2Gently pipette Master Mix or flick to mix, and then centrifuge briefly.Transfer 80µl to each sample and gently mix by pipetting up and down 3-4 times.Incubate at 16°C for 2 hours in thermocycler.After the 2 hour incubation, proceed with the cDNA clean-up step or freeze immediately at –20°C.Remove the cDNA Pure from the refrigerator and allow it to equilibrate to room temp for 30 minutes before use.  Shake the bottle to fully resuspend the magnetic cDNA binding beads before use.Aliquot nuclease-free water into a 1.5mL tube and incubate at 50-60°C for at least 10 minutes during the previous 2hr incubation.Add 180µl of cDNA Pure to each sample, and thoroughly mix by pipetting up and down.  Transfer the samples to a 96-well round-bottom plate.Continue to mix the samples by gently shaking the plate on an orbital shaker for at least 2 minutes.Move the plate to a magnetic stand to capture the magnetic beads.  Leave plate on the stand for approximately 6 minutes, or until the mixture becomes transparent and the binding beads have pelleted.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.Remove the plate from the magnetic stand.Add 150µl cDNA Wash Buffer to each well and shake the plate for 1 minute on the orbital shaker at moderate speed.  Beads will NOT disperse at this step, due to the low surface tension of the wash buffer.Move the plate to a magnetic stand to capture the magnetic beads.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.Remove the plate from the magnetic stand.nd time with 150µl cDNA Wash Buffer.Repeat the wash a 2nd wash, dry the beads by shaking the plate for 2 minutes on the orbital shaker at the maximum speed.  Do not overdry!After the 2Elute the cDNA from the beads by adding 18µl of the preheated nuclease-free water to each sample.Vigorously shake the plate for 3 minutes on the orbital shaker, then check to make sure the magnetic beads are fully dispersed.  If not, continue shaking.Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.Carefully transfer the eluted cDNA (~16µl) to a new PCR plate (or PCR tubes).Proceed directly to the next step, or freeze the cDNA at -20°C. (Table 3)Prepare the IVT master mix at room temperature. Gently pipette the Master Mix or flick to mix, and centrifuge briefly.Add 24µl to each sample, and gently mix by pipetting up and down 3-4 times.Incubate at 37°C for 14 hours in a thermocycler, then hold at 4°C until ready for the next step.Vortex the RNA binding beads briefly to obtain an even mixture before use.(Table 4) This can be done ahead of time--the prepared binding mix can be stored at room temperature for up to one week.Prepare the aRNA Binding Mix at room temperature. Mix well by vortexing.Aliquot the aRNA Elution Buffer into a 1.5mL tube and incubate at 50-60°C for at least 10 minutes.Add 70µl of the aRNA binding mix to each sample and mix well by pipetting up and down 3-4 times.Transfer the samples from the PCR plate to a 96-well round-bottom plate.Add 50µl of 100% isopropanol to each sample and mix well by pipetting up and down 3-4 times.Gently shake the plate on an orbital shaker for at least 2 minutes to thoroughly mix the samples.Move the plate to a magnetic stand to capture the magnetic beads.  Leave the plate on the stand until the mixture becomes transparent and the binding beads have pelleted.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.Remove the plate from the magnetic stand.Add 100µl aRNA Wash Solution to each well and shake the plate for 1 minute on the orbital shaker at moderate speed.  Beads may not fully disperse at this step.Move the plate to a magnetic stand to capture the magnetic beads.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.Remove the plate from the magnetic stand.nd time with 100µl aRNA Wash Solution.Repeat the wash a 2nd wash, dry the beads by shaking the plate for 1 minute on the orbital shaker at the maximum speed.  Do not overdry the samples!After the 2Elute the aRNA from the beads by adding 40µl preheated aRNA Elution Buffer to each sample.Vigorously shake the plate on the orbital shaker for 3 minutes, then check to make sure the magnetic beads are fully dispersed.  If not, continue shaking.Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.  The supernatant contains the cleaned-up amino-allyl incorporated aRNA.Carefully transfer the eluted aRNA to a new PCR plate (or PCR tubes). Check the RNA concentration of the samples by measuring 1.5µl on a NanoDrop spectrophotometer.Immediately continue onto the next step, or store the aRNA at –80°C.* Mix the RNA binding beads with the bead resuspension solution first.** Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.st and 2nd strand cDNA synthesis, IVT) is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature.  A PCR cycler with adjustable heated lid is preferred – even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridization can significantly affect yield of amplified product.A second round of amplification is not advised as biases in array data have been observed.  Mixing carefully at each enzymatic step  variation as to the exact timing of transcription.Using a custom designed poxvirus array2,3,4.The methods utilized in this work are able to predict virus genes transcribed early or late in the replication cycle, but have more difficulty distinguishing early-only versus genes with an early and late promoter since transcripts with a dual early/late promoter may persist and be detected at late times. In addition, run-through transcription of late viral genes may affect signal at a given probe/spot on the array, as the RNA hybridizing to the array may have come from the designated ORF or an upstream ORF.  Tiling arrays have attempted to resolve this issue, however challenges remain in detecting run through transcription using hybridization based approaches5,6,7,8.  Since the expression of many genes involved in host defense is altered after infection, the contribution of viral genes that counteract host immune responses should therefore be taken into consideration.Host transcriptional patterns can also be assessed using these methods.  However, vaccinia encodes a variety of mechanisms to inhibit host responses, and host transcriptional responses may be diminished compared to other stimuliUtilizing these methods, a map of the transcriptional timing of all viral genes can be identified and used to interrogate functions of unknown viral genes.  In addition, these methods can be utilized to dissect the intricate dialogue between virus and host.  These methods are broadly applicable to other host-pathogen infection systems.  If the pathogen of interest does not have polyadenylated mRNAs, alternative methods can be used to directly label the total RNA, without linear amplification.  By analyzing both host and virus gene expression during synchronous infection, these methods allow us to gain insight into virus interaction with the host cellular environment as well as host counter-defenses against virus infection.


To the Editor:Ilheus virus (ILHV) (genus Flavivirus in the Ntaya antigenic complex) is most closely related to Rocio virus. However, antibodies produced during ILHV infection cross-react in serologic assays to other flavivirus antigens, and ILHV was originally classified in the Japanese encephalitis antigenic complex  and Vero cells. After 3 days, cytopathic effects were observed, and cells were screened by an immunofluorescence assay for reactivity against alphaviruses and flaviviruses by using polyclonal antibodies. The cells gave positive results with yellow fever, Saint Louis encephalitis, Rocio, and Ilheus antisera. Viral RNA was then extracted from the patient’s acute-phase serum and cell supernatants and processed for virus sequencing. Amplification of an ≈250-bp product by SyBRgreen real-time reverse transcriptase PCR and subsequent sequencing of the amplicon were conducted with the flavivirus consensus primers FU1 and cFD2 (www.ncbi.nlm.nih.gov/blast) and alignment to GenBank sequences. FSE800 had 96% identity (182 of 188 bp) with the nonstructural (NS) 5 region of the original ILHV strain AY632539 (A blood specimen (containing isolate FSE800) was collected, and an acute-phase serum sample was processed for virus isolation in C6/36 , 1 aa change only in Brazilian isolates , and 1 only in Peruvian isolates . With the exception of the Ala-Val change, all were nonconservative changes. The effect of the amino acid changes has yet to be determined. However, nucleotide sequence comparison between the 6 ILHV isolates has shown that these gene regions are highly conserved geographically and temporally.Ilheus virus is not usually associated with human disease, and human ILHV infections may not be correctly identified without a virus isolate because of the similar clinical symptoms and cross-reactivity in serologic assays to other flaviviruses. As laboratory surveillance is enhanced to monitor the emergence of West Nile virus in Central and South America, detection of ILHV and other flaviviruses may increase.


Culex spp. mosquitoes collected in Xinjiang, People’s Republic of China. In 2007, to determine whether this virus was infecting humans, we tested serum from febrile patients. We found immunoglobulin (Ig) M and IgG against the virus, which suggests human infection in this region.In 2006, Tahyna virus was isolated from  Bunyaviridae, genus Orthobunyavirus, California serogroup) was first isolated in the former Czechoslovakia in 1958 from a pool of Aedes caspius mosquitoes (Tahyna virus (TAHV) has been reported to cause human illness throughout much of Europe and Asia, including countries adjacent to the border of northwestern China  –21 and Vero cells in 6-well plates, which were incubated at 37°C (1′51′′E) , panel BRNA was extracted from an XJ0625 RNA lysate and subjected to reverse transcription–PCR (RT-PCR) amplification by using genus primer sets designed for the detection of flavivirus, alphavirus, and bunyavirus RNA , sequences were aligned with those of viruses belonging to the California virus group. The phylogenic status of XJ0625 isolate was assessed by using MEGA version 3.1 software (www.megasoftware.net), and phylogenetic trees were constructed by using the neighbor-joining algorithm with 1,000 bootstrap replicates.The nucleotide sequence of the small (S) (EU622820) and medium (M) (EU622819) segments of XJ0625 were sequenced by using the primers SF (5′-AGTAGTGTACCCCACTTGAAT AC-3′), SR (5′-CAAATGGATTTGATCCTGATGC-3′), M1F (5′-CACAAGTTCCAAGA TGATGTT-3′), M 1R (5′-CTGTGCCTTCTGCTTGGACTA-3′), M2F (5′-GTCCAAGC AGAAGGCACAGAT-3′), M2R (5′-GTGGTCACTGTACATTCTCCTGAA-3′), M3F (5′-CACACTTCTGTTTAGCAGATACC-3′), M 3R (5′-CTCTAGTCTATAGCTTGCTG GTGTT-3′), M4F (5′-GCACCAATCTGAACGCAATAACAC-3′), and M4R (5′-AGTAG TGTGCTACCAAGTATA-3′). By using Clustal X version 1.8  in all patients and headache with no other specific symptoms in a few. The serum specimens were screened by indirect immunofluorescence assay by using XJ0625-infected BHK-21 cells. Infected and uninfected cell suspensions were applied to Teflon-coated, 10-well slides, air dried, and fixed. The serum specimens were applied to the spot slides at dilutions of 1:40 for IgM detection and 1:80 for IgG detection. After incubation and washing, the spot slides were treated with fluorescein-conjugated antihuman IgM or IgG, dried, and examined by fluorescent microscope. Of the 323 samples, 42 (13.0%) were IgG positive and 17 (5.3%) of 323 were positive for both IgM and IgG against XJ0625 .To determine whether the initial illness of the patients was associated with TAHV infection, in December 2007 (95–127 days after collection of the first sample) we obtained a second serum sample from 10 of the IgM-positive patients. These paired serum specimens were tested by serum dilution neutralization test with XJ0625 virus on BHK-21 cells. Serial 2-fold dilutions of serum were added to equal volumes of culture medium containing XJ0625 virus (50% tissue culture infective dose on a 96-well microtiter plate and incubated at 37°C for 1 h; 100 μL of each virus plus diluted serum was added to 4 wells of a 96-well tissue culture plate containing confluent monolayer of BHK-21 cells. After the cultures were incubated for 48 h at 37°C, we calculated the antibody titer as the highest dilution at which cytopathic effects were completely inhibited in the well. Most of the samples contained neutralizing antibody at a titer of 40 or 80 , though We isolated TAHV from mosquitoes in China and detected IgG consistent with TAHV antibodies in 13.0% of the samples, which suggests that human infection with TAHV is common in the area. We recognize that further testing is necessary to determine whether this serologic response was due to TAHV or to another California serogroup orthobunyavirus.These results indicate that in northwestern China, TAHV should be investigated as a possible etiologic agent in cases of febrile illness with pulmonary involvement and in cases with central nervous system involvement not attributable to other causes. Research should be conducted to identify the primary mosquito vectors and vertebrate amplifier hosts in this region. Additionally, TAHV should be investigated as a cause of illness in other areas of China.


Two TB control strategies appropriate for South Asia (a community-based DOTS [CBD] strategy and a family-based DOTS [FBD] strategy) have been shown to be effective in Nepal in meeting the global target for the proportion of registered patients successfully treated. Here we estimate the costs and cost-effectiveness of the two strategies. This information is essential to allow meaningful comparisons between these and other strategies and will contribute to the small but growing body of knowledge on the costs and cost-effectiveness of different approaches to TB control.In 2001–2, costs relating to TB diagnosis and care were collected for each strategy. Structured and semi-structured questionnaires were used to collect costs from health facility records and a sample of 10 patients in each of 10 districts, 3 using CBD and 2 using FBD. The data collected included costs to the health care system and social costs (including opportunity costs) incurred by patients and their supervisors. The cost-effectiveness of each strategy was estimated.Total recurrent costs per patient using the CBD and FBD strategies were US$76.2 and US$84.1 respectively. The social costs incurred by patients and their supervisors represent more than a third of total recurrent costs under each strategy (37% and 35% respectively). The CBD strategy was more cost-effective than the FBD strategy: recurrent costs per successful treatment were US$91.8 and US$102.2 respectively.Although the CBD strategy was more cost-effective than the FBD strategy in the study context, the estimates of cost-effectiveness were sensitive to relatively small changes in underlying costs and treatment outcomes. Even using these relatively patient-friendly approaches to DOTS, social costs can represent a significant financial burden for TB patients. Tuberculosis is a leading cause of death worldwide , South AIn Nepal, over 14,000 new cases of smear positive (i.e. infectious) tuberculosis are notified each year . Many paIn the RCT, the treatment success rates of the CBD and FBD strategies were 85% and 89% respectively . Both stCosting was undertaken in five districts of Nepal, namely Palpa, Syangja, Doti, Baglung and Dolakha. The first three implemented the CBD strategy and the remaining two the FBD strategy. The findings from Palpa district are discussed separately, as the district TB control programme in Palpa was supported by an NGO , which may have affected the study results.The study used a comprehensive approach to estimating costs and cost-effectiveness, to allow a more holistic assessment of the long-term sustainability of the different strategies ,21. The The data for this study were collected in 2001–2002. All costing data was collected in Nepali Rupees (NRs) and then converted into US$ at the official exchange rate of the National Bank of Nepal for 2000–2001 of 1US$ = 74.65NRs.Both start-up and recurrent costs were obtained. Start-up costs (primarily training and communication) were collected from health facilities using a structured questionnaire. Recurrent (ongoing) costs were collected across two main categories: costs to the health system and social costs. The costs to the health system were gathered from district health facilities' financial records using a structured proforma. Social costs (costs to the patients and their supervisors) were collected using semi-structured questionnaires. A sample of 50 patients (10 from each district) and their treatment supervisors was used. In each district, these 10 patients were selected randomly from patients under treatment during the period planned for data collection, using the district TB treatment register. Only patients who had completed the intensive phase of treatment  and one month of the continuation phase of treatment were included, to ensure a wide coverage of costs.In the analysis of recurrent costs, health system-related costs were allocated to six broad types, namely staff salaries, training (including monitoring and supervision), medicines, transportation, utilities and others , and to five levels: national, regional, district, treatment centre and treatment sub-centre. At each level only costs attributed to the organisation and management of the DOTS scheme were included. We also report start-up costs to allow an assessment of the feasibility of implementing such strategies.Social costs were divided into those incurred directly by patients themselves and costs incurred by their treatment supervisors (community members in the CBD strategy and family members in the FBD strategy). Measures to minimise recall bias by patients and their supervisors included triangulation of data between patients and their treatment supervisors, triangulation methods applied during interviews, and informal discussion with local health care providers and community gate-keepers, to confirm details such as travel distances and number of visits to treatment centres. The social costs considered in this study included direct costs, opportunity costs and other (miscellaneous) expenses. Opportunity costs of time lost by patients and their supervisors were estimated as the actual time (in days) lost due to TB by patients and their supervisors for travel to health facility and for visits to the treatment supervisor/patient. The focus of this study was on costs of seeking treatment for TB and not on the economic impact of TB itself; therefore opportunity costs did not include the time lost due to patients being too ill to work.A more detailed description of cost identification methods for each type of cost is given in Table Patients' and supervisors' costs were estimated per patient registered and then scaled up to the actual numbers of TB patients enrolled for treatment in each district according to NTP records Table . MediansThe effectiveness of each scheme was estimated using the number of patients for whom the treatment was successful . To estiA sensitivity analysis of cost-effectiveness was performed to determine how the cost-effectiveness was sensitive to variations in the costs per patient and treatment success rates.In the 5 districts used in the economic study, the total numbers of new sputum smear positive TB patients registered in the districts implementing the CBD and FBD strategies were 460 and 253 respectively. The total numbers of patients who were successfully treated were 382 and 208 respectively, giving treatment success rates of 83% and 82% respectively. A detailed breakdown of costs per patient is given in Table Total recurrent costs per patient were lower using CBD (US$76.2) than using FBD (US$84.1). In both CBD and FBD, social costs incurred by patients and their supervisors represented more than a third of total recurrent costs (37% in CBD and 35% in FBD).Most health system-related costs were salaries  and medicines (26% and 24% respectively). A separate analysis of health system costs by programme level showed that about half the costs were incurred at the national level (US$22.3 and US$24.6 per patient in CBD and FBD respectively). The breakdown of health system-related costs by level . The majority of patients' costs were the opportunity costs of time lost due to seeking treatment for TB (46% and 79% respectively) followed by direct costs (29% and 21% respectively) and other costs (24% in CBD only). Likewise, most supervisors' costs were opportunity costs (73% and 84% respectively).Start-up costs per patient registered were estimated as US$20 under the CBD strategy and US$31.7 under the FBD strategy.The cost-effectiveness of the two strategies is given in Table Unexpectedly, the costs and cost-effectiveness estimated for Palpa district were found to be lower than in the other two districts using the CBD strategy Tables  and 5. TThe effect of changes in costs per patient and treatment success rates in the CBD strategy on the balance of cost-effectiveness between CBD and FBD strategies is shown in Figure The study results show that for both strategies, social costs make up more than a third of total costs. This fits with our expectation that social costs, particularly opportunity costs and direct (travel) costs are dependent on the distance between the patient and treatment supervisor, and is in line with findings from a study conducted in Bangladesh which found facility-based DOTS to be more expensive than a strategy involving Community Health Workers . Thus, eWe found that the CBD strategy was more cost-effective than the FBD strategy: recurrent costs per successful treatment were lower. This is in contrast to findings in Pakistan where family-based DOTS was found to be more cost-effective than a health centre/community-based scheme . One expOnly recurrent costs have been considered in estimating cost-effectiveness, as the primary focus of this study is to assess the economic sustainability of the two approaches in the long term. We recognise, however, that start-up costs are also important, particularly to the health system. For example, the cost-effectiveness of developing similar initiatives elsewhere would depend on the availability of health staff at nearby health facilities .Health systems costs per successful treatment under the CBD strategy were lower than under the FBD strategy. Similar results were found with start-up costs. These findings suggest that that FBD schemes are more expensive to establish and are likely to require larger recurrent resources from the health system than community-based schemes in the long-term. Start-up costs in the CBD strategy are effectively fixed costs and as such are unlikely to be very sensitive to the number of patients treated. On the contrary, start-up costs in the FBD scheme will generally be correlated with the number of patients.An analysis of health systems costs by level showed a significant share of the costs was incurred at the national level. This indicates that budget absorption at the national level is high even in decentralised service delivery strategies such as CBD and FBD. We consider this an important finding, particularly when considering service delivery schemes at the local level as a means of reducing the financial burden to programmes at the national level.Analysis of social costs per successful treatment reveals marginally lower costs for the CBD strategy than for the FBD strategy, supporting an earlier conclusion that it is more cost-effective to administer DOTS through community-based schemes. It is worth noting that these results are generally consistent with the difference in costs incurred by the health system. Within each arm, overall costs incurred by patients represent the largest share of overall social costs – an important finding if cost-minimisation strategies for economically disadvantaged populations are to be explored.An unexpected finding is that the cost per successful treatment to family member treatment supervisors ($11.3) is three times that of community volunteer supervisors ($3.7). This is however mirrored by the finding that costs per successful treatment to patients on the FBD strategy $25.0) are lower than those on the CBD strategy ($30.1)  at relatively low costs per successful treatment and both strategies or variants thereof are worthy of consideration by NTPs contemplating moving towards more patient-friendly supervision and patient support as recommended in the new Stop TB strategy  and the Social costs can represent a significant financial burden for TB patients, particularly if they are already poor. Cost-minimisation strategies should include consideration of social costs, to avoid situations where cost reductions merely shift costs from NTPs to patients. On the other hand, it is important to recognise the burden to the health system at the national level when planning decentralised strategies such as one assessed here.Finally, despite the growing number of publications on the topic, further research using a consistent costing framework is needed to assess cost-effectiveness of similar forms of DOT (CBD and FBD) in other contexts as well as other forms of TB care (eg. self-administered treatment).The authors declare that they have no competing interests.TNM led the data analysis and coordinated the preparation of the manuscript; SCB participated in the design of the study, coordinated the data collection and participated in the data analysis and the preparation of the manuscript; DKK participated in the study design, led the data collection, participated in the analysis and preparation of the manuscript; ATG designed the study, advised on and participated in the data collection and analysis and participated in the preparation of the manuscript; JNN conceived of the study, participated in the design of the study, advised on and participated in the data collection and analysis and participated in the preparation of the manuscript. All authors have seen and approved the final version of the manuscript.


Alcohol abuse results in problems on various levels in society. In terms of health, alcohol abuse is not only an important risk factor for chronic disease, but it is also related to injuries. Social harms which can be related to drinking include interpersonal problems, work problems, violent and other crimes. The scope of societal costs related to alcohol abuse in principle should be the same for both economic evaluations and cost-of-illness studies. In general, economic evaluations report a small part of all societal costs. To determine the cost- effectiveness of an intervention it is necessary that all costs and benefits are included. The purpose of this study is to describe and quantify the difference in societal costs incorporated in economic evaluations and cost-of-illness studies on alcohol abuse.To investigate the economic costs attributable to alcohol in cost-of-illness studies we used the results of a recent systematic review (June 2009). We performed a PubMed search to identify economic evaluations on alcohol interventions. Only economic evaluations in which two or more interventions were compared from a societal perspective were included. The proportion of health care costs and the proportion of societal costs were estimated in both type of studies.The proportion of healthcare costs in cost-of-illness studies was 17% and the proportion of societal costs 83%. In economic evaluations, the proportion of healthcare costs was 57%, and the proportion of societal costs was 43%.The costs included in economic evaluations performed from a societal perspective do not correspond with those included in cost-of-illness studies. Economic evaluations on alcohol abuse underreport true societal cost of alcohol abuse. When considering implementation of alcohol abuse interventions, policy makers should take into account that economic evaluations from the societal perspective might underestimate the total effects and costs of interventions. Alcohol abuse results in problems on various levels in society. In terms of health, alcohol abuse is not only an important risk factor for chronic disease, but it is also related to unintentional and intentional injuries -3. On thFrom the economic point of view, the estimated tangible costs of alcohol in the European Union were €125 billion in 2003, including €59 billion worth of lost productivity through absenteeism, unemployment and lost working years due to premature death . AnotherTo reduce the negative effect of alcohol abuse it is necessary for countries to develop an alcohol policy and implement prevention programs. An alcohol policy can be defined as a set of measures in a jurisdiction or society aimed at minimizing the health and social problems from alcohol consumption . The alcBefore an intervention may be implemented, a firm evidence base for its effectiveness is needed. The effects of an intervention should be both statistically significant and clinically relevant.In light of increasing health care costs and limited resources, interventions aimed at reducing alcohol abuse should not only be effective, but efficient or cost-effective too. Nowadays, policy makers require information about the effectiveness of an intervention in relation to its costs. The perspective of an economic evaluation of an alcohol abuse intervention should be taken into account in the decision making process. If a study is performed from a health care perspective only costs are included which are related to health care use. A study performed from a social perspective includes both healthcare costs and all relevant societal costs. In the case of an alcohol abuse intervention performed from the societal perspective, costs due to productivity losses, crime and law enforcement costs also have to be included, in addition to health care costs. Taking into account the public interest, it is important for a policy maker that all costs related to excessive alcohol use are included in an economic evaluation. In other words: to be able to make a clear decision whether getting good value for the money when introducing a preventive intervention, a policymaker needs economic evaluations that include all related societal costs.Cost-of illness studies which are performed according to the international guidelines of the WHO report all type of healthcare cost and societal costs comprehensively . From ecA recently published review summarizes the costs attributable to alcohol abuse as involved in cost-of illness studies . One of To identify economic evaluations on alcohol interventions, we performed a PubMed search. The search contained the terms: alcohol abuse, alcohol misuse, economic evaluation, cost-effectiveness, cost-utility, life year gained, QALY, prevention, preventive intervention and societal perspective. We searched for economic evaluations that were reported between 2000 and 2009 in English. We only included full economic evaluations performed from the societal perspective in which two or more interventions on alcohol abuse were compared with each other or with usual care . Of the To be able to compare the outcomes of cost-of-illness studies and economic evaluations with different base years and different currency units, all local currencies were first transferred to the euro currency values of that time, following the advice of the Organization for Economic Co-operation and Development, and then recalculated to 2008, the year that was chosen as base year for the current study .Table Averaged over all the cost-of-illness studies, the proportion of the healthcare costs was 17% of the total cost due to alcohol abuse. The societal costs attributed to 83% of the total alcohol abuse costs.Our literature search on economic evaluations of preventive interventions directed at alcohol abuse conducted in July 2009, identified 116 potentially relevant studies. After having read the abstracts, we selected 34 articles to read in full, of those we included 5 studies because these studies satisfied our inclusion criterion ,18. ReasTable Averaged over all economic evaluations the proportion of the healthcare costs at baseline was 57% of the total cost, and of the societal costs amounted for 43%.Our study shows that the scope of societal costs included in five economic evaluations with a societal perspective was smaller than the scope of societal costs as incorporated in four cost-of-illness studies. We found only 5 full-economic evaluation studies on preventive interventions of alcohol abuse. None of the economic evaluations included all types of societal costs that were represented in the cost-of-illness studies. This means that, although it is claimed that the societal perspective is taken, the societal costs as reported in economic evaluations are incomplete in general. In relation to alcohol abuse it is remarkable that none of the studies included drink-driving costs. Only two studies included work-related costs due to productivity losses. These results corresponds with the findings of Barbosa et al. that a societal perspective has never been taken into full account in economic evaluations . The fouAveraged over all economic evaluations, the proportion of healthcare costs (57%) and societal cost (43%) does not correspond with the proportion as reported in cost-of-illness studies . So, the incremental cost-effectiveness ratios (ICER) that are reported in economic evaluations might be underestimations of the true societal costs and effects of preventive interventions directed at alcohol abuse.It is known that economic evaluations of alcohol abuse interventions performed from a healthcare perspective underestimate true costs, because only healthcare costs are involved in this kind of studies. It will be useful to policy makers if studies make clear how a life-style intervention directed towards alcohol abuse affects both the health care costs and the societal costs . StudiesComparing productivity costs between studies is hampered by the existence of two 'schools' that address the issue of valuation of productivity losses in an entirely different manner. Advocates of the friction cost approach come-up with relatively modest estimates of productivity losses, whereas those using the human capital approach may easily reach estimates that are ten to hundred times higher than those generated by frictionists. There is no easy solution to this problem, and both approaches may hold best arguments, depending on the local circumstances of the analysis. In the meantime, absolute clarity about methodology used to derive productivity costs estimates is indispensable.Two studies presented the QALY as an outcome. In studies done from a healthcare perspective the QALY as an outcome measure is almost standard practice. Although economic evaluations presenting the QALY as outcome measure are more or less comparable with each other, the way the QALY is measured in a public-health intervention performed from a societal perspective is debatable. The questionnaires to estimate the QALY are directed on health status, while in a societal perspective also other benefits besides healthcare, like social welfare, should be involved; these are not included in those questionnaires . In geneSome limitations of this study have to be taken into account. We calculated per-study averaged total healthcare costs and societal costs, but it is quite difficult to compare costs as reported in different economic evaluations. This was due to differences in study design, outcome measures, discount rates, et cetera. For example, three studies used 'preventing the decrease of drinking' and 'abstinence' as a surrogate outcome measure. Another limitation is that the cost-of-illness studies showed also differences in the types of costs involved in the studies. That means that a full comparison of the studies is not really possible, although these studies are more complete than the economic evaluations. Furthermore, in the original studies, it is not always clear which costs are placed under the types of costs. It will be possible that 'property damage' costs for instance are placed under the topic 'criminal damage'.The costs included in economic evaluations on alcohol abuse interventions performed from a societal perspective do not correspond with those included in cost-of-illness studies on alcohol abuse. It is obvious that alcohol abuse not only affects health care costs, but has also a big influence on societal costs. Productivity losses account for the largest proportion of the total costs. Therefore, it is important that all economic evaluations report productivity costs in the same way, to ensure comparability among studies.Policy makers need to have data based on real life, i.e. all societal costs  should be included in economic evaluations from the societal perspective. It can be concluded from the results of this study, that this is not the case in most economic evaluations. In considering whether good value for the money is achieved when introducing a preventive intervention, policy makers should take into account that the result from an economic evolution from the societal perspective might be an underestimation of the true costs and effects of the intervention.The authors declare that they have no competing interests.PFVG and MVDB developed the method of the study. All the authors helped to draft the manuscript, read and approved the final manuscript.


Flaviviridae viral family and constitutes an important pathogen. The viral helicase is an ideal target for inhibitor design, since this enzyme is essential for the survival, proliferation and transmission of the virus.Usutu virus belongs to the in silico scoring tools, including Procheck and Verify3D.Towards a drug-design approach, the 3D model of the Usutu virus helicase structure has been designed, using conventional homology modelling techniques and the known 3D-structure of the Murray Valley Encephalitis virus helicase, of the same viral family, as template. The model was then subjected to extended molecular dynamics simulations in a periodic box, filled with explicit water molecules for 10 nanoseconds. The reliability of the model was confirmed by obtaining acceptable scores from a variety of in silico all known structural characteristics of the Flaviviridae viral family helicase enzymes and could provide the platform for further de novo structure-based design of novel anti-Usutu agents.The 3D model of the Usutu virus helicase exhibits  Flaviviridae comprises the genera Flavivirus, Pestivirus and Hepacivirus and includes numerous important human and animal pathogens [Flaviviridae family contain a single-stranded, positive-sense RNA genome of about 9.5–12.5 kb. The genome consists of a single, long open reading frame (ORF), which is flanked by untranslated regions (UTRs) at the 5' and 3' ends.The viral family athogens . The smaPestivirus and Flavivirus RNA replicons have revealed that the non-structural (NS) proteins, which are encoded by the C-terminal part of the polyprotein, play a crucial role in viral RNA replication [Recent studies on sub-genomic lication ,3. AccorSequence alignments of the Usutu viral helicase identified several conserved sequence motifs that are important for biological functions. So far, the crystal structures of helicases from various RNA viruses have been determined, including the helicases from hepatitis C virus, Dengue virus, Yellow Fever Virus and Kunjin virus .Culex pipiens mosquito [Turdus merula) and great grey owls (Strix nebulosa) initially in Austria, but then spread to other central European countries such as Hungary, Switzerland, and Northern Italy  [Usutu virus is the cause of one of the most important arthropod-borne viral (arbovirus) diseases, transmitted mainly by the mosquito . Usutu vl order) .Flaviviridae family as template.In the present work, the three-dimensional structure of the helicase enzyme of Usutu virus was modelled by applying homology modelling techniques and using the crystal structure of the Murray Valley Encephalitis virus helicase of the NC_006551, entry name: Usutu virus, complete genome). Using the Gapped-BLAST [The amino acid sequence of Usutu viral helicase was obtained from the GenBank database  web-server iate one .The homology modelling of the Usutu viral helicase was carried out using the MOE  package and its built-in homology modelling application . The RCSElectrostatic potential surfaces were calculated by solving the nonlinear Poisson-Boltzmann equation using finite difference method as implemented in the PyMOL Software . The potEnergy minimisation was done in MOE initially using the Amber99  forcefieThe produced models were initially evaluated within the MOE package by a residue packing quality function, which depends on the number of buried non-polar side chain groups and on hydrogen bonding. Moreover, the suite PROCHECK  was emplFlaviviridae contains both the protease and the helicase coding regions. For this study, the Usutu virus helicase sequence was aligned with the Murray Valley Encephalitis virus helicase template sequence and all the major helicase motifs, characteristic of the Flaviviridae family were found to be conserved . Among the available helicase structureswithin this family, the Murray Valley Encephalitis virus enzyme presented the highest identity percentage to the Usutu virus helicase. That is also supported by the analysis of the helicase structures available for the whole Flaviviridae family that revealed that the overall 3D structure for the Murray Valley Encephalitis virus helicase ishighly conserved [The Murray Valley Encephalitis virus helicase structure has been established by X-ray crystallography at 1.9 Å resolution . The Muronserved ,18. Moreonserved -22.Flaviviridae family of viruses. The Murray Valley Encephalitis virus, Hepatitis C virus, Dengue virus, West Nile virus and Usutu virus helicases all belong to the superfamily II of helicases and share seven common motifs within their domains, all of which have been structurally conserved on the Usutu virus helicase model [The model was firstly structurally superimposed and subsequently compared to its template, where exhibited an alpha-carbon RMSD less than 0,65 angstroms. Furthermore it was evaluated with MOE and PROCHECK for its geometry [see additional file se model .Flaviviridae helicases. Namely, the three distinct domains of helicases as well as the various motifs  to its template Figures  and 3. T Figures  and 5 [3++ were transferred to the model from the Hepatitis C (1A1V) and the Dengue virus helicase structure (2JLS) respectively, for this purpose. Invariant residues of various motifs in the vicinity of either substrate in the Murray Valley Encephalitis virus template structure were conserved structurally in the Usutu virus helicase model  simulations in the presence of the helicase substrates. The coordinates of the ssRNA and ADP/Mntructure JLS respe Figures . Most in Figures . The recα RMSd of the Usutu virus helicase model from the equivalent domains of the template structures was less than 0.65Ǻ. The low RMSd value indicated that this model remains conformationally close to the template structure upon the minimization and the molecular dynamics simulation course that followed, reflecting its high quality.The 10 ns MD simulations revealed that soon the Usutu virus helicase model reached a conformational equilibrium similar to that of Murray Valley Encephalitis virus helicase structure Figure  and 5C. In order to analyze the molecular surface of the produced Usutu virus helicase model, the electrostatic potential surface was calculated. For direct comparison with the template structures used in this study, electrostatic potential surfaces were also calculated for the Murray Valley Encephalitis virus helicase Figure . The twoFlaviviridae viral family. It is therefore proposed that the Usutu virus helicase model will be suitable for further in silico structure-based de novo drug design experiments. These computer-based methodologies are now becoming integral part of the drug discovery process that may eventually lead to the development of potential inhibitor structures of the Usutu virus helicase enzyme in the future.The 3D model of the Usutu virus helicase was designed using the homologous X-ray crystal structure of the Murray Valley Encephalitis viral helicase as template. The model was successfully evaluated both in terms of its geometry, fold recognition and compliance to the criteria required as a member to the The author declares that they have no competing interests.DV is the sole author of this paper and is responsible for developing the concepts and for writing and revising the manuscript.Extended Procheck Results for both the Usutu virus helicase model and the Murray Valley Encephalitis virus helicase template. Extended Procheck Results for both the Usutu virus helicase model (LEFT COLUMN) and the Murray Valley Encephalitis virus helicase template . A: Ramachandran plot, B: Bond Length Plot, C: Bond Angles plot, D: Dihedrals plot, E: Rotamers plot and F: Contact Energies Plot. It is therefore concluded that the Usutu virus helicase model has inherited all structural characteristics of its Murray Valley Encephalitis virus helicase template.Click here for file