Exposure to infected poultry is a suspected cause of avian influenza (H5N1) virus infections in humans. We detected infectious droplets and aerosols during laboratory-simulated processing of asymptomatic chickens infected with human- (clades 1 and 2.2.1) and avian-  origin H5N1 viruses. We detected fewer airborne infectious particles in simulated processing of infected ducks. Influenza virus–naive chickens and ferrets exposed to the air space in which virus-infected chickens were processed became infected and died, suggesting that the slaughter of infected chickens is an efficient source of airborne virus that can infect birds and mammals. We did not detect consistent infections in ducks and ferrets exposed to the air space in which virus-infected ducks were processed. Our results support the hypothesis that airborne transmission of HPAI viruses can occur among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry. For expGallus domesticus from the US Department of Agriculture Southeast Poultry Research Laboratory, Athens, GA, USA) and 8-week-old domestic Pekin ducks . All birds were serologically negative for influenza A virus infection by hemagglutinin inhibition (HI) test  were used as the mammalian model for HPAI virus transmission to humans  white Leghorn chickens (3/h) at 0.046 m/s from the processing area toward the air samplers or the naive animals  enclosure  1.5 m wide × 6.7 m long × 2.1 m high with unidirectional and single-pass airflow of 8.3 air changes/h  cyclone air sampler  collected particles and sorted them by their aerodynamic diameters into >4 µm, 1–4 µm, and <1 µm fractions at a flow rate of 0.0035 m5.3–106.5 mean egg infectious dose (EID50)0.1 mL per virus and housed in negative-pressure isolators with HEPA-filtered ventilation. We moved chickens at 24 h after inoculation and ducks at 2.5 days after inoculation, which corresponded to times of peak shedding titers, to the processing enclosure while they were still asymptomatic. We anesthetized them by intramuscular injection of ketamine (10 g/kg) and xylazine (1 g/kg) and collected oral swab samples to confirm infection. The anesthetized birds were processed following 5 steps   was repeated at least twice for reproducibility. Chickens  and ducks (5 per virus) were inoculated intranasally with 105.9–106.1 EID50/0.1 mL per virus  using GraphPad Prism 6 . >1.5 log10 EID50/mL. We isolated VN/04, VN/878/11, WJ/07, and Eg/11 viruses from air samples collected when processing virus-infected chickens, with highest virus quantity in >4 µm particles, moderate quantities in 1–4 µm particles, and no virus in <1 µm particles. We did not detect Eg/10, VN/67511, or VN/09 viruses in air samples  1.2 m above ground, in a holding cage similar to those used in LPMs. Regardless of the distance from the processing area, all exposed chickens died between 3 and 6 dpe. All oral and cloacal swab samples collected at time of death were positive by virus isolation. We found lesions typical of those caused by HPAI and AI viral antigen in multiple internal organs of all exposed chickens, indicating infection after droplet/aerosol infection .10 EID50/mL), and the ferret that was euthanized on 12 dpe seroconverted  infections suggests that LPM slaughter processing of infected poultry could provide sufficient exposure to cause transmission to humans  were higher than those from similar sampling methods in LPMs (The simulated slaughter of infected poultry generated viable virus predominantly in droplets (>4 µm) and aerosols (1–4 µm) but none in particles (<1 µm). Our findings align with those of previous studies that used air samplers in LPMs  in 2-week-old ducks than in 5-week-old ducks  in asymptomatic infected chickens than in ducks. Chickens are highly susceptible to HPAI viruses and in particular to Gs/GD lineage H5N1 viruses, which usually causes multiple organ failure associated with systemic virus replication and high mortality rates  compared with chickens (n = 10) per airborne exposure group, which implies a shorter exposure time for naive ducks.< 1 h because of the need to mimic time-limited exposure events  virus infections of humans.


Recently there has been a large outbreak of Zika virus infections in Colombia, South America. The epidemic began in September 2015 and continued to April 2017, for the total number of Zika cases reported of 107,870. For those confirmed Zika cases, there were nearly 20,000 (18.5%) suspected to be pregnant women, resulting in 157 confirmed cases of microcephaly in newborns reported by their health government agency. There is a clear under-estimation of the total number of cases and in addition no prior publications have been published to demonstrate the clinical aspects of the Zika infection in Colombia. We characterized one Zika presentation to be able to compare and contrast with other cases of Zika infection already reported in the literature.In this case report, we demonstrate congenital microcephaly at week 19 of gestation in a 34-year-old mother who showed symptoms compatible with Zika virus infection from Sincelejo, State of Sucre, in the Colombian Caribbean. Zika virus RNA was detected in the placenta using real-time reverse transcriptase polymerase chain reaction (RT-PCR). At week 25, the fetus weigh estimate was 770 g, had a cephalic perimeter of 20.2 cm (5th percentile), ventriculomegaly on the right side and dilatation of the fourth ventricle. At week 32, the microcephaly was confirmed with a cephalic perimeter of 22 cm, dilatation of the posterior atrium to 13 mm, an abnormally small cerebellum (29 mm), and an augmented cisterna magna. At birth (39 weeks by cesarean section), the head circumference was 27.5 cm, and computerized axial tomography  confirmed microcephaly with calcifications.We report a first case of maternal Zika virus infection associated with fetal microcephaly in Colombia and confirmed similar presentation to those observed previous in Brazil, 2015–2016. Aedes aegypti is as well established vector. Similar to other countries in the Americas affected by Zika virus, the increases of congenital syndromes including microcephaly cases and Guillan Barre syndrome appeared abruptly coincident with the Zika virus disease (ZVD) outbreak. In 20 May 2017, there was a calculation of 0.22% of Colombian population that has been affected by ZVD including almost 20,000 cases of infected pregnant women [Zika, Chikungunya and dengue viruses are tropical vector borne viral diseases that are all endemic in Colombia, South America. During the last 16 months, Colombia has experienced a large outbreak in Zika infection. From September 2015 through May 20th 2017, 107,870 reported cases. However, only 9.5% of these were laboratory confirmed at the Colombian National Institute of Health (INS) [nt women . The numnt women , 4. MostWe describe a case of microcephaly associated with Zika virus infection in an infant born on 17 July 2016. The 34-year-old mother showed symptoms compatible with Zika infection: rash at week 19 of gestation, living in an endemic area during the last 5 years and with no immunodeficiency or autoimmune diseases. Laboratory tests showed no evidence of diabetes or blood-pressure-related disorders. In her medical history, the mother indicated no drug or alcohol use or smoking during pregnancy. No active infection against toxoplasma, rubella, cytomegalovirus, herpes, syphilis, Chikungunya, dengue, and human immune deficiency virus (HIV) was detected in the mother’s serum as per Hospital laboratory testing report.At 16 weeks, ultrasound showed a head circumference of 11.5 cm and fetal weight estimate of 219 g. At 25 weeks, the fetus weight was 770 g, with a cephalic perimeter of 20.2 cm (5th percentile), ventriculomegaly on the right side and dilatation of the fourth ventricle. A third ultrasound at week 32 showed a cephalic perimeter of 22 cm and confirmed the microcephaly diagnosis with dilatation of the posterior atrium to 13 mm, an abnormally small cerebellum (29 mm), and an augmented cisterna magna. At birth, the anterior fontanelle was closed; the facies were symmetrical; normal ocular apertures; normal spleen and urinary size determined via ultrasound; good respiratory pattern; normal palate; normal heart rate with no alterations and no megalia detected. At birth (39 weeks by cesarean section), the infant’s head circumference was 27.5 cm and the computerized axial tomography scan confirmed microcephaly with calcifications  according to Zika specific PCR previously published –6. DetecWe report a first case in Colombia of fetal Zika virus infection via maternal transmission resulting in microcephaly. It is important to note that during the previous five years the mother lived in Sincelejo, Sucre State, an endemic area for Zika in the Colombian Caribbean. As reported by the INS, Zika virus has presumably infected more than 1655 cases in Sucre State, representing 1.53% of the total Colombian population, this data would confirm the high prevalence of Zika in the area where the clinical case derives from. Since the Zika outbreak began in Colombia neurologic anomalies including microcephaly 157 accumulated total cases have been reported to the INS . The datDuring pregnancy, infection with Zika virus has been associated with microcephaly and other fetal brain abnormalities –10. The Between 2010 and 2017, neurologic complications  in French Polynesia Brazil and Colombia have increased dramatically , 9, 10. The 19,935 cases of pregnant women with clinically compatible Zika virus infection suggest that Colombia may experience a dramatic increase in the number of microcephaly cases. Out of a total of 19,935 pregnant women, 157 (0.79%) cases of congenital microcephaly associated with Zika virus infection have been reported; it is unknown whether this number will increase in the near future. It is also unknown the reasons why some pregnant women infected by Zika virus give birth to healthy babies and others do not. Individual genetic background or modulation of the immune system can perhaps be involved in the pathways for the virus to reach trophoblasts. In Colombia, before microcephaly cases appeared, an etiologic relationship between Zika virus and microcephaly was not demonstrated and a relationship between Zika virus and central nervous system anomalies was doubted.In the Colombian Caribbean, we have witnessed an increase in microcephaly associated with Zika virus prevalence and exposure during pregnancy –4. Some n = 157) associated with fetal microcephaly (0.79%). Zika virus related microcephaly needs to be monitored closely in those countries experiencing outbreaks so as to provide accurate information on risk to the unborn fetus. The experience in Colombia as well as other countries provides an important epidemiologic example to predict risk for other countries. This and other reports provide critical information for world medical and community public health.Here, we report the for the first time a case in Colombia, South America of fetal microcephaly associated with maternal Zika virus infection. There have been 19,935 suspected cases of Zika infection of pregnant women in Colombia and these numbers must with a relatively small number (


N = 25,826): corresponding to 373 episodes in the CYD-TDV group  and 528 episodes in the control group . Fewer episodes in the CYD-TDV group resulted in hospitalization than in the control group , but both had a similar average length of stay of 4 days. Overall, a two-thirds reduction in resource consumption and missed school/work days was observed in the CYD-TDV group relative to the control group. The estimated direct and indirect cost (2014 I$) associated with dengue episodes per participant in the CYD-TDV group was 73% lower than in the control group (I$6.72 versus I$25.08); representing a saving of I$I8.36  per participant with vaccination. This is the first study providing information on dengue costs among vaccinated individuals and direct confirmation that vaccination has the potential to reduce dengue illness costs.A tetravalent dengue vaccine (CYD-TDV) has recently been approved in 12 countries in southeast Asia and Latin America for individuals aged 9–45 years or 9–60 years  living in endemic areas. Data on utilization of medical and nonmedical resources as well as time lost from school and work were collected during the active phase of two phase III efficacy studies performed in 10 countries in the Asia-Pacific region and Latin America . We compared dengue-related resource utilization and costs among vaccinated and nonvaccinated participants. Country-specific unit costs were derived from available literature. There were 901 virologically confirmed dengue episodes among participants aged ≥ 9 years ( There are 128 countries where the disease is endemic, encompassing a population of over 3.9 billion people—about 50% of the global population—at potential risk of infection and ensuring disease.®, Sanofi Pasteur, Lyon, France) has recently been approved in Mexico, Brazil, Philippines, El Salvador, Paraguay, Indonesia, Singapore, Guatemala, Peru, Thailand, Bolivia, and Costa Rica. These approvals were based in part on data obtained during two large-scale pivotal phase III studies (CYD14 and CYD15) undertaken in countries in Asia-Pacific and Latin America.9 The studies are still on going to better define the efficacy and safety of CYD-TDV over the longer term. Alongside these two pivotal clinical trials, data on resource use related to dengue management were prospectively collected to estimate the impact of vaccination on the cost burden attributed to the illness. Although a number of studies have suggested that a dengue vaccine has the potential to be cost-effective or even cost-saving,15 none have used prospectively collected resource use and health outcome data associated with vaccination.A recombinant yellow fever-17D–dengue virus, live, attenuated, tetravalent dengue vaccine  and five countries in Latin America  during the first 25 months of the active phase surveillance. Full details of the design, conduct, and main clinical findings of these trials have been reported elsewhere .N = 20,762) or placebo control . Participants were given three doses of CYD-TDV or placebo at 0, 6, and 12 months, and actively monitored over 25 months after the first study injection for acute febrile illness (temperature ≥ 38°C on ≥ 2 consecutive days), and those who presented with fever were screened for signs and symptoms of dengue. The suspected febrile episodes were classified as virologically confirmed dengue if any of the following tests on acute and convalescent blood samples were positive: dengue nonstructural protein (NS1) antigen enzyme-linked immunosorbent assay , and serotype-specific dengue reverse transcriptase polymerase chain reaction.The two studies recruited 31,126 healthy children aged 2–16 years (2–14 years in Asia-Pacific and 9–16 years in Latin American) and randomly assigned them in a 2:1 ratio to receive CYD-TDV  as well as productivity losses for participants and parents/caregivers due to time lost from school and work were prospectively documented in Case Report Forms during each study visit for those with suspected dengue episodes during the first 25 months of the active phase surveillance. Data included information on where the participant consulted , hospitalizations , medications, inpatient and outpatient tests, type of travel used to get to the health center and absenteeism (number of days missed from work or school by the participant or their caregivers).Supplemental Appendices 1–10). All country-specific costs were expressed in 2014 US$ and included direct  and indirect costs . If country-specific cost estimates were only available for years earlier than 2014, these were updated by initially adjusting the estimates to 2014 local currency values accounting for the inflation rate of the country before converting to 2014 US$. If country-specific unit costs were not available, then unit costs were extrapolated from data available from other countries in the region. To assess average unit costs across the 10 countries included in this study , country-specific unit costs were further converted to 2014 international dollars (I$), which adjust for differences in the relative price of the resources used across countries based on purchasing power parity at official exchange rates, and thereby allows cost information from different countries to be combined.16 Inflation rates, exchange rates, and purchasing power parity used for these calculations were based on International Monetary Fund statistics .17Estimates of country-specific unit costs were derived from available literature for the individual countries . Lost productivity due to work days lost by the parent/caregiver was valued by using the average daily wage in each country.A macrocosting method was used for assessing hospitalization costs,Supplemental Appendices 1–10) summed across all items, divided by the total number of episodes. The cost per participant was calculated by multiplying the cost per dengue episode by the resource use incidence. For the purpose of our analysis, we focused mainly on virologically confirmed dengue in those aged ≥ 9 years corresponding to the age indication for this vaccine.19 Cost data for participants aged ≥ 9 years were also compared with that for those aged < 9 years.The cost per dengue episode was calculated by multiplying the resource quantities used with the assigned unit costs identified from the reference sources for the individual countries  was assessed with the F test. Naive bootstrap was used for the calculation of 95% confidence intervals for percentage reductions with CYD-TDV .Chi-squared tests were used to detect significant differences in frequency observed between groups with a 5% threshold for rejecting the independence between groups. A bilateral Student’s N = 31,126): 571 in the CYD-TDV group  and 708 episodes in the control group . The baseline characteristics of the virologically confirmed dengue cases are summarized in N = 25,826): 373 in the CYD-TDV group  and 528 in the control group .20There were 1,279 virologically confirmed dengue episodes , but the average length of stay was the same at 4.0 days in both groups. Dengue episodes in the control group resulted in significantly more missed school days  and there was also a trend toward more work days lost by parents/caregivers in this group .Resource consumption and missed school/work days attributed to virologically confirmed dengue as a percentage of total number of participants in each study group and overall is summarized in Supplemental Appendix 13 for resource use among those aged < 9 years).Overall, dengue episodes in those aged ≥ 9 years generally consumed fewer resources and resulted in fewer missed school/work days than in those aged < 9 years  (see SupP = 0.002). The cost of work and school-related absenteeism were also higher in the control group (P = 0.004). There were no significant differences in consultation costs (P = 0.085) or travel-associated costs (P = 0.42) between the two groups. Country-specific estimated costs associated with hospitalized and nonhospitalized dengue episodes are summarized in Supplemental Appendices 1–10.The estimated average costs associated with virologically confirmed dengue episodes in those aged ≥ 9 years are summarized in Supplemental Appendices 1–10.The estimated costs of virologically confirmed dengue per participant aged ≥ 9 years are summarized in 20 Our study confirms the results of previous modeling studies suggesting that a dengue vaccine could be of economic value in dengue-endemic setting.21This is the first study to show directly that CYD-TDV has the potential to reduce dengue illness costs among participants aged ≥ 9 years, the age group in which vaccine use has been approved. In this age group, we showed that breakthrough dengue episodes in the CYD-TDV group (versus control group) lead to fewer hospitalizations, consumed fewer resources, and resulted in fewer missed school/work days. The associated costs were therefore also lower per participant in the CYD-TDV groups than in the control group. This result is mainly driven by a lower proportion of hospitalized dengue episodes in the CYD-TDV group during the active phase surveillance. Overall, pooled vaccine efficacy rates against symptomatic virologically confirmed dengue during the first 25 months were 60.3% (95% CI: 55.7–64.5) for all participants and 65.6% (95% CI: 60.7–69.9) for those aged ≥ 9 years (Cox Regression).22: length of stay (4.0 days in our study versus 5.5 days in the later); number of consultations (3.0 versus 3.6); workdays lost (2.2 versus 4.5 days); and school days missed (4.9 versus 5.9 days). A similar length of stay was reported for hospitalized dengue patients aged < 15 years in Brazil; mean 5 (standard deviation: 5.2) days, with a trend toward shorter stays among those hospitalized in the private sector.23 Several other studies have reported median/mean stays in the range 3–6 days among the general population hospitalized for dengue.28 Moreover, the mean length of hospital stay was shown to increase with severity of the disease.23Resource use among participants hospitalized for dengue across both groups in our study, in terms of length of stay, number of consultations, workdays and school days missed, appear broadly consistent with those reported in another study undertaken in Thailand among hospitalized dengue patients aged 2–15 years29 Another study that did not differentiate between hospitalized and ambulatory patients reported an average of 1.9 days of schools days lost.28 Similarly, the number of work days lost associated with hospitalized participants in our study (2.2 days) is also consistent with other studies that reported an average 3.1–14.4 days.29 However, the average number of work days lost associated with nonhospitalized participants (0.3 days) in our study appears somewhat lower than that reported elsewhere (1.7–6.0 days of work lost).29 Two other studies that did not differentiate between hospitalized and ambulatory patients reported 4.2 and 5.3 days of work missed, respectively.30The number of school days missed in our study  is also consistent with other studies that reported an average 3.8–6.9 and 2.0–5.2 days of school lost among hospitalized and ambulatory patients, respectively.6 our estimated costs are within the ranges for previous estimates for countries in Latin America and Asia. A study of dengue illness that included 46 countries in Latin America and the Caribbean (excluding North America) reported median cost (2010 US$) per hospitalized and ambulatory cases of US$1,213  and US$451 , respectively.5 Other single-country studies in Latin America have also reported similar per dengue episode costs.38 Similarly, in southeast Asia, another multicountry study of dengue illness (12 countries) reported median cost (2010 US$) per hospitalized and ambulatory cases as US$210  and US$63 , respectively.6 Other individual country studies in Asia have also reported similar cost per dengue episode costs.45The observed total costs per hospitalized dengue episode in the vaccine group  were similar to those in the control group , as were nonhospitalized dengue episodes . Although there is substantial variability in the costs per dengue episode by country,Our study has a number of limitations that should be considered when evaluating our results and their relative generalizability. A weakness of this study relates to the potential inclusion of protocol-driven costs as the trial involves activities/contacts with the study investigators/health staff that may not be otherwise available in routine care. The exclusion criteria may lead to the selection of participants in specific geographic locations and may not necessarily be representative of or generalizable to the “typical” patient population or other regions. In addition, our analysis was based on studies undertaken in countries with high dengue endemicity and as such, may not be representative of countries with low dengue endemicity where disease treatment/management and practice may differ substantially. A major limitation, however, is that the vaccination costs were not included in the cost per episode calculations. In addition, sensitivity analyses on unit costs were not undertaken. Nonetheless, the unit costs would apply to both the CYD-TDV and control groups, and as such would not affect the relative cost ratio between the two groups.The major strength of our study is its prospective design and standardized collection of resource use information as part of two randomized clinical trials, which also facilitates the comparisons between countries. The inclusion of a control group provided an opportunity to directly estimate the impact of vaccination on dengue-related costs. We also included indirect costs related to travel and time lost from school and work for patients and parents/caregivers. Our results suggest that CYD-TDV has the potential to reduce dengue illness costs among participants aged ≥ 9 years, the age group in which vaccine use has been approved.Supplemental Appendix.


Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B).Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus.Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi.Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology. Nipah virus is a highly fatal paramyxovirus that causes respiratory and neurologic disease with widespread vascular damage. Although end stage disease has been characterized in humans and in animal models, the early phase of infection has yet to be described. Thus, it is known where the virus replicates during the final stages of infection, but not how the virus is transported to these end stage tissues or which tissues are targeted first. Using Syrian hamsters that were intranasally inoculated with virus as a model of Nipah virus infection, we show here that Nipah virus initially targets the respiratory tract, specifically the nasal cavity and lung, with virus replication being detected as early as 8 hours after inoculation. During the first 48 hours of infection, the virus slowly penetrates into the underlying tissues of the respiratory tract, but viremia and virus replication in the brain are not detected. In the lung, Nipah virus initially infects epithelial cells lining airways, followed by spread to arterial smooth muscle cells, suggesting that localized spread of the virus within an organ can lead to viral dissemination into the vasculature. Likewise, replication in the olfactory epithelium likely precedes spread of Nipah virus along the olfactory nerve into the brain. Understanding how Nipah virus spreads through the host to cause encephalitis is important for developing effective countermeasures that can target Nipah virus replication and prevent viral dissemination. Nipah virus, a highly virulent paramyxovirus, was first identified in an outbreak in Malaysia and Singapore in 1998 and has since caused almost yearly outbreaks in humans in Bangladesh . Nipah vThe pathology of Nipah virus in humans has only been described in fatal cases from the Malaysia outbreak; lesions were noted in the central nervous system, lung, vasculature and to a lesser extent the spleen and lymph nodes , 7, 9. NHere, we identified the initial target tissues and cell types for Nipah virus during the first 48 hours post intranasal inoculation of Syrian hamsters. Syrian hamsters were used to model Nipah virus infection since they develop late stage lesions similar to those in humans –25. Althhttp://grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf), and the Guide for the Care and Use of Laboratory Animals . All infectious work with Nipah virus was performed in a biosafety level 4 (BSL4) laboratory in the Integrated Research Facility at RML. Sample inactivation and removal of samples from the BSL4 laboratory was performed according to established standard operating procedures [All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories ) and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility, according to the institution’s guidelines for animal use, and followed the guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals  of either NiV-B or NiV-M in a total volume of 80 μl (40 μl per nostril). All hamsters were evaluated daily for clinical signs of disease. Four hamsters from each group were euthanized at 4, 8, 16, 24, 32 and 48 hpi. A terminal heart blood sample was collected from each hamster before necropsy. The nasal cavity, larynx, trachea, lung, cervical lymph nodes, spleen, brain and spinal cord were collected for histologic and virologic analysis.Two groups of twenty-four 6- to 8-week-old female Syrian hamsters  were intranasally inoculated with 5 x 10Necropsies and tissue sampling were performed according to a standard protocol approved by the IBC. Tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and embedded in paraffin. The sections through the nasal turbinates, which were contained within the nasal cavity, and the spinal cord, which was contained within the vertebrae, were decalcified using a 20% EDTA solution in sucrose prior to paraffin embedding.Leukocytes were isolated from terminal blood samples using centrifugation over a histopaque gradient  in conjunction with erythrolysis using ACK lysing buffer  according to manufacturer instructions. The resulting leukocyte pellets were fixed for a minimum of 24 hours in 10% neutral-buffered formalin. Leukocytes from hamsters inoculated with the same Nipah virus isolate and which were euthanized at the same time point were pooled, then processed in HistoGel (Thermo Fisher Scientific) according to manufacturer instructions and embedded in paraffin to form a cell block.in situ hybridization (ISH) were performed on tissue sections and cell blocks. Nipah virus antigen was detected by IHC; tissue sections were labeled with a rabbit polyclonal antiserum against Nipah virus nucleoprotein  [Routine hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and ngapore) . Nipah vngapore) . All sli50 equivalents in the samples.Viral RNA was isolated from hamster tissues using the RNeasy Mini kit  or from hamster blood using the QIAamp Viral RNA Mini kit (Qiagen), according to manufacturer instructions. 5 μl of RNA was used in a one-step real-time RT-PCR targeting the Nipah virus nucleoprotein, as described previously , using t50 was calculated from 4 replicates by the Spearman-Karber method [Virus titrations were performed by end-point titration in Vero C1008 cells. Vero C1008 cells were inoculated with tenfold serial dilutions of tissue homogenates. One hour after inoculation of cells with tissue homogenates, the inoculum was removed and replaced with 200 μl DMEM (Sigma-Aldrich) supplemented with 2% fetal bovine serum , 1 mM L-glutamine , 50 U/ml penicillin and 50 μg/ml streptomycin (Thermo Fisher Scientific). Five days after inoculation with tissue homogenates from NiV-B inoculated hamsters and three days after inoculation with tissue homogenates from NiV-M inoculated hamsters, when full cytopathic effect (CPE) was reached in Vero cells, CPE was scored and the TCIDr method .6 TCID50 of NiV-M and analyzed tissues from the respiratory tract, central nervous system and immune system for the presence of Nipah virus RNA and infectious virus at multiple time points up to 48 hpi. Although viral RNA was detected by qRT-PCR in the lung, nasal turbinates, trachea, larynx, cervical lymph nodes and brain, but not the spleen and spinal cord at 4 hpi, infectious virus could be isolated only from the lung and nasal turbinates by 8 hpi, and from the trachea and larynx by 16 hpi , larynx (B), trachea (C), lung (D), cervical lymph nodes (E), spleen (F), brain (G) and spinal cord (H) at 4, 8, 16, 24, 32 and 48 hpi in Syrian hamsters intranasally inoculated with NiV-B. Viral loads in the tissues were determined as TCID(TIF)Click here for additional data file.S2 FigISH was used to detect positive sense viral RNA, indicating virus replication, in the nasal cavity and lung at 4, 8 and 16 hpi in Syrian hamsters intranasally inoculated with NiV-B. Viral RNA is labeled brown in all images. Virus replication was not present in the nasal cavity at 4 or 8 hpi , yet was observed at 16 hpi, as shown here in the olfactory epithelium lining a nasal turbinate (C). Virus replication was not identified at 4 hpi in the lung (D), yet was detected in pneumocytes at 8 hpi (arrow) and 16 hpi . All images were taken at 400x.(TIF)Click here for additional data file.S3 FigIHC was used to detect the presence of viral antigen in the nasal cavity and lung at 4, 8, 16, 24, 32 and 48 hpi in Syrian hamsters intranasally inoculated with NiV-B. Viral antigen is labeled brown in all images. Viral antigen was not detected in the nasal cavity at 4 or 8 hpi . Viral antigen was first observed in the nasal cavity at 16 hpi, as shown here in the olfactory epithelium lining a nasal turbinate (C). Increasing amounts of viral antigen were detected in olfactory and respiratory epithelium (D-F). The spread of viral antigen to the submucosal glands (arrow) was first detected at 48 hpi (F). Nipah virus antigen was not observed at 4 hpi in the lung (G). Viral antigen was first detected at 8 hpi in the lung; arrow indicates antigen in pneumocytes (H). Increasing amounts of viral antigen were typically detected in pneumocytes at subsequent time points in the lung (I-L). Viral antigen was also detected in bronchiolar respiratory epithelium (arrow) (K) and bronchial respiratory epithelium . The inset in image L depicts an airway which is not visible in the main image. All images were taken at 400x, except for (H) which was taken at 630x.(TIF)Click here for additional data file.S4 FigPositive sense viral RNA, indicating virus replication, was detected by ISH, while IHC was used to detect viral antigen in Syrian hamsters intranasally inoculated with NiV-B. Virus replication was identified in epithelial cells lining the larynx (A), although viral antigen was not detected (B). Virus replication was not detected in the trachea of any hamster (C); however, viral antigen was observed in epithelial cells lining the trachea (D). All images were taken at 400x.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Potyvirus species in sweet potatoes have been detected mostly by serology. Here, we report the complete genome sequences of two Potyvirus species, Sweet potato feathery mottle virus strain (SPFMV-UNB-01) and Sweet potato virus G strain (SPVG-UNB-01).In Brazil,  Ipomoea batatas Lam.)   is a tubas Lam.)  belonginood crop .Potyvirus (family Potyviridae), Sweet potato feathery mottle virus (SPFMV), Sweet potato latent virus (SPLV), Sweet potato mild speckling virus (SPMSV), and Sweet potato virus G (SPVG)  .Ipomoea setosa and kept in a greenhouse at the Experimental Station of Biology of the Universidade de Brasília (EEB-UnB). The virus enrichment process with semipurification of particles was carried out using leaves of I. setosa. Samples were weighed individually, to 1 g of each plant, and then pooled, forming a sample composed of 10 plants (10 g). From the purified particles, RNA extraction was performed using Trizol reagent following the manufacturer’s instructions. The composite sample was sent for sequencing on the Illumina MiSeq platform at the Universidade Católica de Brasília (UCB).In this study, 40 sweet potato plants from distinct producing regions in Brazil were grafted on de novo assembled using CLC Genomics Workbench version 8.0. The resulting contigs were compared to the viral RefSeq database available in GenBank using a BLASTx search  were trimmed and MF185715 (strain SPFMV-UNB-01) and MF185716 (strain SPVG-UNB-01).The sequences reported here have been deposited in GenBank under the accession numbers


Undesirable outcomes in health care are associated with patient harm and substantial excess costs. Coarctation of the aorta (CoA), one of the most common congenital heart diseases, can be repaired with stenting but requires monitoring and subsequent interventions to detect and treat disease recurrence and aortic wall injuries. Avoidable costs associated with stenting in patients with CoA are unknown.We developed an economic model to calculate potentially avoidable costs in stenting treatment of CoA in the United Kingdom over 5 years. We calculated baseline costs for the intervention and potentially avoidable complications and follow-up interventions and compared these to the costs in hypothetical scenarios with improved treatment effectiveness and complication rates.Baseline costs were £16 688 ($25 182) per patient. Avoidable costs ranged from £137 ($207) per patient in a scenario assuming a 10% reduction in aortic wall injuries and reinterventions at follow-up, to £1627 ($2455) in a Best-case scenario with 100% treatment success and no complications. Overall costs in the Best-case scenario were 90.2% of overall costs at Baseline. Reintervention rate at follow-up was identified as most influential lever for overall costs. Probabilistic sensitivity analysis showed a considerable degree of uncertainty for avoidable costs with widely overlapping 95% confidence intervals.Significant improvements in the treatment effectiveness and reductions in complication rates are required to realize discernible cost savings. Up to 10% of total baseline costs could be avoided in the best-case scenario. This highlights the need to pursue patient-specific treatment approaches which promise optimal outcomes.The online version of this article (doi:10.1186/s12913-017-2215-2) contains supplementary material, which is available to authorized users. Trea surgery .Costs associated with undesirable outcomes, including treatment complications and reinterventions, can be quantified as avoidable costs. A clinical area where the economic impact of adverse effects can be studied is coarctation of the aorta (CoA), one of the most common CHD. CoA significantly reduces life expectancy and is associated with morbidity even after successful repair –8. ImpleThe costs associated with undesirable outcomes including treatment-related complications of stenting among individuals with CoA are unknown. Our objective in this paper is to quantify the costs associated with such potentially avoidable outcomes.We developed an economic model to calculate the expected costs associated with stenting for CoA in the United Kingdom on a per patient basis. We included important costs of the initial intervention and monitoring, as well as costs of complications and follow-up interventions that are potentially amenable to improvements in stenting repair of CoA. We specified four hypothetical scenarios of improved treatment outcomes and compared the costs of each scenario to the baseline costs to obtain estimates of avoidable costs. Three scenarios were based on potential increases in stenting treatment success and reductions in complications and reintervention rates. The last scenario represents the ‘ideal’ scenario in which the probability of treatment success is 100% and no complications and reinterventions occur.The analytical approach of comparing expected costs in the Baseline scenario, Scenarios 1–3, and Best-case scenario, is presented in Fig. Figure The first chance node in the model is success at the initial stenting intervention. Stenting success is defined as achieving a post-treatment blood pressure gradient ≤20 mmHg, which represents the threshold for intervention indication in patients with CoA , 11. AorIn the lower branch of the initial intervention period model, we assumed that patients who did not achieve a post-intervention treatment gradient ≤20 mmHg underwent a second intervention, which could be either repeat stenting or surgery. The branch for repeat stenting then leads to a chance node of experiencing an aortic wall injury, and further to the percutaneous or surgical treatment of the injury.The model includes the same events for the short-term and mid-term follow-up period, although different probabilities are assigned to them according to the specific period. The branches emanating from the first chance node at follow-up are either no reintervention or reintervention due to aortic wall injuries or recurrence of coarctation. The branch for reintervention for re-coarctation is identical to that after successful stenting at the initial intervention. The branch for reintervention due to aortic wall injury leads to a chance node of percutaneous or surgical treatment.We applied a 5-year timeframe for our analysis, distinguishing between costs and events during or immediately after the initial treatment; during short-term follow-up (3–18 months); and mid-term follow-up (18 months–5 years). Expected costs were obtained for each of these three time periods separately. Discounting the results at short- and mid-term follow-up, we also calculated expected overall costs over a 5-year period. The time frame was chosen due to limited availability of reliable data beyond 5 years of follow-up.Actuarial survival of non-infant patients after CoA stenting repair is almost 100% even 10 years after treatment . We assuBaseline values for event probabilities and costs are presented in Tables Estimates of event probabilities were derived primarily from a meta-analysis comparing the effectiveness of stenting and balloon dilatation in CoA patients . For eveWe made assumptions about the proportion of patients undergoing repeat stenting (as opposed to surgery) after unsuccessful initial stenting repair; the proportion of patients receiving percutaneous treatment (as opposed to surgery) after aortic wall injury; and the proportion of patients requiring reintervention due to aortic wall injury (as opposed to recurrent CoA) in both short-term and mid-term follow-up periods of the model. Sensitivity of our findings to assumptions was extensively tested, as described below.Our model adopted the health care provider’s perspective and included direct costs only.Cost estimates for the treatment of CoA and complications arising from the initial treatment are based primarily on United Kingdom National Health Service (NHS) reference costs 2013-14. The NHS provides the average unit treatment costs for every fiscal year, collected from all NHS trusts. Reference costs reflect the full costs of providing services [All costs were discounted at the standard NHS discount rate of 3.5% per year. Where necessary we adjusted costs to 2014 Great British Pounds. We assumed health care costs to increase 2 percentage points above annual inflation rate [The primary outcome of our analysis was expected avoidable cost associated with stenting in patients with CoA. In our base case analysis, we calculated the expected costs for the immediate treatment period, expected costs at short-term follow-up, and expected costs at mid-term follow-up by multiplying the event probabilities with their associated costs in each branch of the model. We then summed the expected costs from the three periods to obtain an estimate of the total expected costs. We discounted the expected costs at short-term and mid-term follow-up at 3.5% p.a.We compared the costs of this Baseline scenario, including costs incurred by complications and other undesired events, with the costs that would incur if no or fewer undesired events occurred , 16. We In the first scenario, the proportion of patients with initial treatment success remains constant at the baseline value of 96.7%. Proportion of patients with aortic wall injuries decreases by 10% to 0.7%. Proportion of patients with reinterventions at follow-up decreases by 10% to 8.2% at short-term and 16.7% at mid-term follow-up. Hypertension medication is required by the baseline proportion of 43.0% of patients at short-term and 38.9% at mid-term follow-up.Scenario 2 assumes a constant initial treatment success rate. Both aortic wall injuries and follow-up reinterventions are assumed to be reduced by 25% from baseline. Proportion of patients with aortic wall injuries is 0.6% in this scenario. Reintervention rates at short-term and mid-term follow-up are 6.8% and 13.9%, respectively. Furthermore, a 10% reduction in the proportion of patients requiring anti-hypertensive medication to 38.7% at short-term and 35.0% at mid-term follow-up is assumed.In the third scenario, the initial treatment success rate increases to 100%. Aortic wall injuries are reduced by 50% from baseline to a proportion of 0.4% of patients. Reinterventions at follow-up are also reduced by 50% from baseline, leading to values of 4.6% at short-term and 9.3% at mid-term follow-up. The proportion of patients requiring anti-hypertensive medication is assumed to be reduced by 25% to 32.3% at short-term and 29.2% at mid-term follow-up.Finally, we calculate the costs of stenting for CoA under ideal conditions. In this scenario, treatment success is 100%, with no complications during the initial treatment and at follow-up. The scenario assumes that no reinterventions are necessary and that no patient requires anti-hypertensive medication.We conducted one-way sensitivity analyses. We varied key probability and cost inputs by 10% and plotted the effect on costs avoided compared to baseline, in a tornado diagram. The tornado diagram presents the relative impact that each input factor has on the outcome, holding all other variables constant.We then varied input variables for all five scenarios to assess the sensitivity of the model with respect to (1) the overall level of input values, and (2) the expected avoidable costs in the scenarios relative to the Baseline scenario. Holding all other parameters constant, we inspected the extent to which each parameter had an impact on the difference in avoidable costs in the four scenarios. We did not include the Best-case scenario in one-way sensitivity analyses of parameters that were assumed to be ideal (i.e. 100% treatment success and 0% complication rates) in this scenario.We chose a range from 0 to twice the initial input value for event probabilities and half to twice the initial value for costs in most cases.We conducted probabilistic sensitivity analysis (PSA) to simultaneously take into account the uncertainty associated with all input factors in our model.We generated 1000 random values for each input parameter based on its point estimate and standard deviation (SD). We assigned a beta distribution to event probabilities and a gamma distribution to cost estimates. The values were therefore restricted to between 0 and 1 for probabilities, and to non-negative values for costs. SD for cost estimates were derived from published NHS reference costs for procedures at the aggregate level, which did not show much variation, potentially underestimating SD. We assessed the impact of potentially underestimated SD of cost parameters on our primary outcome, avoidable costs, by rerunning PSA with inflated SD .We then ran 1000 iterations of the model. Each iteration used a different value of the random value distribution of input parameters. We summarized the results of PSA using the mean value of the 1000 iterations and its 95% confidence interval (CI). We also present all 1000 PSA estimates of total costs for each of the scenarios (including Baseline) in a single diagram, which demonstrates the uncertainty surrounding estimates of total costs in the model.The model and sensitivity analyses were implemented in Microsoft Excel .Expected costs per patient at initial treatment, short-term, and mid-term, as well as overall expected costs are displayed for Baseline, Scenarios 1 to 3, and Best-case scenario in Table We calculated the overall expected costs per patient as £16 688 . Expected overall costs in Scenario 1 were £16 551 ($24 975) with avoidable costs amounting to £137 ($207).Scenario 2 showed slightly lower overall costs (£16 338/$24 654) and conversely higher savings from avoidable costs (£350/$528). Optimistic assumptions in Scenario 3, including a 100% initial treatment success rate, brought overall expected costs down to £15 790 ($23 827) with expected avoidable costs at an estimated £898 ($1355).In the Best-case scenario, overall expected costs were £15 061 ($22 727), translating into £1627 ($2455) in avoidable costs. The most substantial cost savings compared to Baseline were accumulated at follow-up (£488/$736 at short-term and £858/$1295 at mid-term), while avoidable costs at the initial treatment amounted to £282 ($426).The tornado diagram Fig.  shows thWe assessed the sensitivity of expected avoidable costs in the four scenarios with respect to all model input parameters. We only display results of univariate analysis for stenting treatment success and reintervention rates. Sensitivity analyses of all parameters are available in the Additional file Figure At the extremes of the sensitivity analysis for reinterventions at follow-up, we observed significant differences between the magnitude of avoidable costs associated with the four scenarios Fig. . CompareAfter 1000 iterations of the model using random values drawn from relevant distributions for each input parameter, mean estimated avoidable costs were similar to expected avoidable costs in the deterministic base case analysis Fig. . This isThe results of PSA show a wide overlap of 95% CIs between avoidable costs in Scenarios 1 and 2, and to some extent between Scenarios 2 and 3. There was minimal overlap in the 95% CIs between Scenario 1 and Best-case  projects have the potential to move clinical practice towards increasingly individualized and patient-centered treatment decisions with better patient outcomes. Just as in other areas of personalized medicine, potential benefits are manifold and include improving mortality and morbidity, and reducing health care expenditures. In patients with CoA, image-based modelling of the aorta was recently used to create a virtual stenting tool which could aid clinical decision-making by simulating post-treatment blood flows in various scenarios , 25. GivOverall, the overall scope for cost savings from improved treatment outcomes in patients undergoing stenting for CoA is small. Cost savings per patient were £137, £350, and £898 in Scenarios 1, 2, and 3, respectively. In the United States, an estimated 521 stenting procedures are conducted annually in patients with CoA . Under tOur analysis was limited by the data available to model undesirable outcomes at follow-up. We included reinterventions due to aortic wall injuries and recurrent CoA as well as antihypertensive medication therapy but did not account for other complications that might occur late after CoA repair due to limited data availability. Similarly, it was not always possible to include estimates of event probabilities from meta-analyses or large trials due to limited availability of such studies and some of the parameters of our model were therefore based on assumptions informed by reports in the literature. Our model was also restricted to a 5-year period. Long-term follow-up studies of patients undergoing stenting are still relatively rare and often conducted retrospectively in small cohorts. For example, long-term studies are required to assess the consequences of exercise-induced hypertension after CoA repair .It is unclear through which mechanisms the improvements in the quality of care for patients with CoA included in our scenario analysis would be achieved and whether such improvements are realistic. For example, whether or not CoA repair can resolve hypertension in patients with CoA remains elusive. Perfect outcomes in all patients, as modeled the Best-case scenario, might never be accomplished. However, our study was designed not only to demonstrate what could realistically be achieved, but also to demonstrate the absolute maximum in cost savings associated with stenting repair of CoA.We focused on costs and events that were related to stenting repair of CoA. We did not aim to obtain estimates of all costs and events associated with CoA. Expected costs of stenting for CoA repair therefore do not represent a true estimate of all costs associated with the disease.Cost estimates were restricted to aggregate level information from the United Kingdom NHS reference costs. It is possible that mean costs of procedures are higher for patients with CHD than other patients due to comorbidities. A potential systematic underestimation of all cost parameters included in the model has implications for the overall cost level but should not affect relative comparisons of avoidable costs in the specified scenarios. PSA results demonstrated a considerable degree of uncertainty associated with the results of the model, which stems from relatively small potential cost savings compared to total costs as well as from uncertainty in input parameters. Total costs in the four scenarios ranged from 98.5 to 90.2% of Baseline costs.We derived SD for cost estimates from aggregate cost items in the NHS reference costs. It is therefore possible that SD were underestimated for the procedures included in the model. Apart from the expected increase in variance, inflating SD to twice their initial value did not change our results.We examined the potential cost savings associated with improvement in effectiveness and reduction of complications in stenting treatment of aortic coarctation. The biggest potential for cost savings lies in improving the endurance of therapies, and thus the reduction of reintervention rates at follow-up. Significant improvements in the treatment effectiveness and reductions in complication rates are required to realize discernible cost savings. Up to 10% of total baseline costs could be avoided in the best-case scenario. This highlights the need to pursue patient-specific treatment approaches which promise optimal outcomes. The individualization of treatment procedures with the help of disease specific modeling can already help to select the most relevant stenosis and to find an optimal location for interventional treatment. Further advances in modelling will potentially enable physicians to identify patients with high risk of treatment failure and prevent costly complications and follow-up treatment in those.


Patients with distal radius fractures (DRF) often have limited range-of-motion (ROM) in multiple planes of movement. No studies have comprehensively examined the impact of various ROM limitations on physical function.We performed a multi-center, longitudinal study of 138 patients with conservatively managed DRF. ROM measures were taken at initial evaluation, and at 4 and 8 weeks later. Self-reported physical function was indexed by the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH).Wrist extension, active thumb opposition and a full composite grip were amongst the strongest ROM measures associated with functional scores over time. However, wrist radial deviation and forearm pronation were non-significantly associated with functional scores.Given that ROM is potentially modifiable, the identification of important ROM measures associated with QuickDASH scores can potentially facilitate patient education and refine interventions to optimize functional recovery. Well-designed randomized intervention studies are however needed to confirm these association findings. Distal radius fractures (DRFs) are a common occurrence in clinical practice and account for 20% of all fractures seen in emergency departments , 2. Due n = 29) examined ROM measures as a total (combined) arc-of-motion in the planes of wrist flexion/extension and forearm supination/pronation and thus, could not tease apart the isolated associations of the specific ROM measures with physical function [n = 20) examined only one ROM measure (wrist extension) [To our knowledge, the published literature on the relationship between the various ROM measures and physical function amongst patients with conservatively managed DRFs is limited to 2 small cross-sectional studies , 8. Kilifunction . The othtension) , these sAs ROM is potentially modifiable, the identification of important ROM measures associated functional scores can facilitate patient education and help refine rehabilitation interventions to optimize functional outcomes. Thus, the aim of this study was to examine the longitudinal associations of 8 forearm, wrist and hand ROM measures with self-reported functional scores amongst patients with non-complicated, conservatively managed DRFs. Specifically, the ROM measures include wrist flexion and extension; wrist radial and ulnar deviation; forearm supination and pronation; active thumb opposition and the ability to make a full composite grip.This study was a retrospective, longitudinal, multi-center study involving four major outpatient occupational therapy hand clinics in Singapore. Patients were evaluated at three time points. Specifically, the baseline time point was taken to be at the commencement of active mobilization, and the subsequent two time points were set at the commencement of passive and strengthening exercises, respectively.All patients with conservatively managed, unilateral DRFs who were referred to four outpatient occupational therapy hand clinics during the period of April to June 2015 were included in the study. Patients were excluded if there were other upper limb fractures, bilateral injuries, or associated complicating injuries such as tendon, ligament or nerve injuries. Patients who had been treated by other institutions as well as those who developed complex regional pain syndrome (CRPS) subsequently were also excluded. The study received ethics approval from all participating institutions. Demographic and clinical information such as age, gender, injured side, handedness , edema,n = 17), and those who had less than 5 years (n = 17). Across the four hand clinics, the average number of years of clinical experience was 6.78 years. 8 therapists were masters/postgraduate level occupational therapists.Patients included in this study were treated by 34 occupational therapists across four hand clinics. There was an equal distribution of therapists who had 5 or more years of clinical experience in hand therapy  were used to measure active range of motion (ROM) of wrist flexion, extension, radial and ulnar deviation as well as forearm supination and pronation in the injured upper limb. Wrist flexion and extension were measured with the forearm in neutral on a stable surface, and the goniometer on the radial aspect of the wrist, aligned along the third metacarpal. Wrist radial and ulnar deviation were measured with the forearm in pronation on a flat surface and elbow slightly flexed. Goniometer measurements were taken along the line of the third metacarpal and the radius bone. Forearm supination and pronation measurements were performed with elbow flexed at 90 degrees, at the side of the body and goniometer readings aligned to the imaginary line between the radial and ulnar styloids. Active thumb opposition was recorded using the modified Kapandji score . MeasureThe QuickDASH is a shortened version of the original 30-item Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire . DevelopContinuous variables were presented as means with standard deviations (SD) for normally distributed data, and medians with interquartile ranges (IQRs) for non-normally distributed data, whilst categorical variables were presented as frequencies with percentages. We modeled the time course of the QuickDASH scores using a generalized least-squares model that included all available observations from all time points, and we accounted for within-patient correlation over time using a first order autoregressive covariance structure , 20. To To assess the independent associations of QuickDASH with the various ROM measures, these measures were each used as a time-varying independent variable in separate models and the covariates included age, sex, edema, hand dominance, time since fracture, and treatment center. To evaluate whether the associations of the ROM measures with QuickDASH changed over time, we considered their first-order interactions with time. To facilitate interpretation and comparison of the results, we scaled the regression coefficients of the ROM measures by their IQRs such that the coefficients may be interpreted as comparing patients with relatively high (75th percentile) versus low (25th percentile) ROM on the QuickDASH scores . BesidesR software, version 3.2.3 (http://www.r-project.org), for all analyses.We assessed the appropriateness of all models using residual plots and we used A total of 138 patients were included in the study. Table Table Table In a sample of 138 patients with non-complicated, conservatively managed distal radius fracture, we examined the longitudinal associations of 8 forearm, wrist and hand ROM measures with QuickDASH scores. Adjusting for covariates, we found that wrist extension, active thumb opposition (using modified Kapandji scores) and the ability to make a full composite grip  were among the strongest ROM measures associated with QuickDASH scores. In contrast, forearm pronation and wrist radial deviation were weakly and non-significantly associated with QuickDASH scores. To our knowledge, this study is the first comprehensive evaluation of the ROM measures associated with changes in self-reported functional recovery in a relatively large cohort of patients with DRF.Previous studies evaluating outcomes of distal radius fractures were often limited to the examination of wrist and forearm ROM measures, as well as functional outcomes such as DASH or the Patient-Rated Wrist Evaluation (PRWE) –9, 22–24In our study, we found that wrist flexion, wrist extension, ulnar deviation, and forearm supination were statistically significantly associated with QuickDASH scores over time. Specifically, on average, an IQR increase in these ROM measures was associated with 4.1 to 9.0 points lower QuickDASH scores Table . Using aOn the other hand, our results appear to contrast with those reported by Karnezis & Fragkiadakis (2002), who found that neither the wrist flexion-extension arc nor the forearm supination-pronation arc movements were significantly associated with functional scores . HoweverIn our study, forearm pronation and wrist radial deviation were weakly associated with QuickDASH scores, and these findings were not reported in previous studies. Nevertheless, to explain this null finding, we reason that limited forearm pronation may be compensated by elbow and shoulder movements during daily activities. Supporting this, Sardelli and colleagues (2011) reported a maximal forearm pronation range of 65 degrees required for typing on a keyboard which was still lesser than the maximal amount of supination (77 degrees) required in opening a door .Our study has limitations. First, the retrospective nature of our study implies that we were unable to standardize (i) the timing of referral from injury to the first occupational therapy session and (ii) the time intervals for the follow-up assessments Table . AlthougTo summarize, in 138 patients with conservatively managed DRFs, we found that the various ROM measures have differential influence on upper limb physical function, with wrist extension, active thumb opposition and ability to make a full composite grip amongst the strongest ROM measures associated with QuickDASH over time. The results of this study have potential implications on efforts in providing treatment and outcome assessments amongst people with DRFs. They also highlight the need for intervention and education of patients with DRFs to maintain hand-related ROM during the immobilization phase, to optimize function post DRF. That said, well-designed randomized intervention studies are needed to confirm these association findings, and future studies should explore how ROM measures interplay with muscle strength and pain levels to influence function post DRF.


Eukaryotic virus–bacteria interactions have recently become an emerging topic of study due to multiple significant examples related to human pathogens of clinical interest. However, such omnipresent and likely important interactions for viruses and bacteria relevant to the applied and agricultural sciences have not been reviewed or compiled. The fundamental basis of this review is that these interactions have importance and deserve more investigation, as numerous potential consequences and applications arising from their discovery are relevant to the applied sciences. The purpose of this review is to highlight and summarize eukaryotic virus–bacteria findings in the food/water, horticultural, and animal sciences. In many cases in the agricultural sciences, mechanistic understandings of the effects of virus–bacteria interactions remain unstudied, and many studies solely focus on co-infections of bacterial and viral pathogens. Given recent findings relative to human viral pathogens, further research related to virus–bacteria interactions would likely result in numerous discoveries and beneficial applications. Bacteria and eukaryotic viruses have long been known to co-exist; however, identification of their relationships related to promoting or inhibiting each other’s presence in their eukaryotic hosts has only relatively recently garnered attention ,2. NumerEnterobacter cloacae or a synthesized norovirus putative carbohydrate receptor for infection of animal tissue, in this case human B cells. It had previously been reported that E. cloacae expresses carbohydrates very similar or equivalent to the human forms of carbohydrates traditionally suspected of being human norovirus receptors/co-receptors. In addition, E. cloacae was reported to bind human noroviruses in a specific manner, with the binding interaction confirmed by ELISA, electron microscopy, loss of binding of a mutant virus, and loss of binding with enzymatic cleavage [E. cloacae and other enteric bacteria present in unfiltered human norovirus-containing stool were necessary for human norovirus replication in B cells, but also presented some evidence suggesting potential mechanisms behind the interaction. Specifically, using unfiltered stool significantly increased norovirus attachment to cells as compared to filtered stool. When synthetic receptor carbohydrates were added to filtered stool attachment was restored comparable to unfiltered, enteric bacteria-containing stool. Furthermore, evidence from co-cultures consisting of a top layer of intestinal epithelial cells with B cells below suggested that enteric bacteria may assist viral translocation across epithelial cells and into B cells, as only considerable increases in virus were seen in the B cell layer if unfiltered stool was used [Escherichia coli Nissle 1917). Interestingly, Rubio-del-Campo et al. [Food and waterborne pathogens impose a serious public health burden, and a number of food and waterborne viruses have been found to interact with host native enteric bacteria . Likewiscleavage . Jones ecleavage  further was used . Furtherwas used  characteo et al.  observedo et al. , supporto et al. ’s observIn another study characterizing the scope of enteric bacteria-norovirus binding, Almand et al.  examinedE. cloacae—have been reported to inhibit human norovirus infection in a gnotobiotic pig model [E. cloacae, Lactobacillus rhamnosus GG, or Escherichia coli Nissle 1917 and then infected with human norovirus. In one case, both L. rhamnosus and E. coli were mixed in a cocktail formulation and experimentally fed to the animals. In both sets of experiments, human norovirus shedding was markedly reduced and better maintenance of intestinal morphology was observed when the animals were pre-colonized with the bacteria [Enterobacter cloacae, which was shown to generally inhibit norovirus infection in another study that did not follow IFN response [E. cloacae) in the context of norovirus infection would likely be valuable. Undoubtedly, more work is needed to better understand human norovirus pathogenesis and the role(s) different enteric bacteria may play.Conversely some bacteria—including ig model ,13. In tbacteria ,13. Othebacteria . It is pbacteria  utilize bacteria ,16; althresponse . Future Bifidobacterium bifidum and Streptococcus thermophilus were significantly less likely to shed rotavirus during their admission than infants given formula alone. It should be noted that the sample size of the study was fairly small, however. Subsequent study in gnotobiotic pigs has also suggested a potential protective effect against rotavirus infection in a bacterial species-dependent manner. Specifically, colonization of pigs with E. coli Nissle 1917 appeared to reduce rotavirus shedding and confer greater protection by increasing plasmacytoid dendritic cells and natural killer (NK)-cell activity in vivo as well as interleukin (IL)-12, IFN-α, and IL-10 in vitro, while Lactobacillus rhamnosus GG had no effect [E. coli Nissle 1917 in gnotobiotic pigs [Similar discrepancies as norovirus have also been observed with rotavirus infection. Some studies have suggested that colonization with certain bacteria has a protective effect on rotavirus infection. In a placebo-controlled, double blind study, Saavedra et al.  found tho effect . Alternao effect  found tho effect . Treatmeo effect , which itic pigs . It shoutic pigs  was testEnterococcus faecium CRL35, was demonstrated to reduce replication of herpes simplex virus 1 and 2 by about 1 log10 in vitro. Evidence suggested that the bateriocin inhibited synthesis of a viral glycoprotein necessary for infection and replication [In addition to the studies discussed above on bacterial protection against norovirus and rotavirus infection, multiple other examples of probiotics demonstrating antiviral activity through different mechanisms are reviewed by Al Kassa et al. . For thelication . The abiN-acteylglucosamine-containing bacterial surface polysaccharides  at an elevated temperature (42 °C) compared to PBS-treated poliovirus. This work was further advanced by Robinson et al. [N-acteylglucosamine-containing polysaccharides enhanced poliovirus stability to bleach treatment. Additionally, the temperature at which poliovirus undergoes conformational change and releases RNA was found to be elevated in a dose-dependent manner when exposed to bacterial lipopolysaccharide. Interestingly, a mutant poliovirus strain with reduced bacteria binding ability was isolated, and found to be less environmentally stable than wild type poliovirus [E. coli strains increased the receptor-binding ability of the capsids when treated with heat  compared to a strain of E. coli that did not bind human norovirus. Because cooking and bleach treatment are common control points for pathogens in food systems, the observation that some bacteria—likely also present along with virus in fecally-contaminated foods—may increase enteric virus stability is worth serious consideration. This is because nearly all studies on the efficacy of different heat and chemical treatments are performed with only virus or artificial “soil” loads that do not contain bacterial polysaccharides as a component. Future work that re-evaluates potential enhancement of enteric virus resistance to some of these treatments may be worth consideration.Another group of food and waterborne virus–bacteria interactions has implications for control of these viruses and offers new considerations in investigating treatments. As mentioned above, enteric bacteria have been reported to assist infection in numerous ways by multiple enteric viruses ,2; inclun et al. , who fouliovirus . Similarliovirus , who shoAlthough a lot of mechanistic research related to eukaryotic-virus bacteria interactions has been performed in the food/water field, less work has been performed related to the horticultural sciences—specifically regarding plant virus–bacteria interactions. A large amount of literature exists studying the effects of both the abiotic  and biotic  stresses that affect plants—including interactions and the effects within and between these different stresses—but little work has been performed investigating bacteria–virus interactions within this field (see review by Lamichhane and Venturi ). HoweveOryza spp.) in Africa because it is being rapidly planted to meet a growing demand. The rationale for this work was that evidence exists suggesting that plants may simultaneously host multiple pathogens, and this could affect the dynamics of host (plant) resistance and pathogen evolution in a possibly different way than studying one plant–one pathogen interaction , and investigated potential mechanisms for any observed effects that the two pathogens may have on each other. Not surprisingly, both the virus and bacteria were found in a large number of plants, and nearly a fifth of all plants tested (18.8%) were positive for both virus and bacteria. When observing the phenotypes of the plants, generally bacterial pathogenic symptoms were visible and viral symptoms reduced. Experimental co-infection of plants was conducted using two different virus strains isolated from different areas. Quantitative PCR was used to determine the relative levels of the pathogens in co-infected plants, with bacterial load being significantly increased when co-infecting with a virus. The levels of one strain of virus were reduced in the presence of X. oryzae but not the other strain. Tollenaere et al. [X. oryzae was linked to promotion of RNA silencing and targeting of viral genomes by the plants. This effect was observed by experimental co-infection of plants with virus, wild type bacteria, and two mutant versions of bacteria lacking either a Transcription Activator-like Effector (TALE) that interacts with the promoter of an important Oryza spp. protein in the anti-viral RNA silencing pathway or a Type III secretor used to inject TALEs into the plant. As expected, the wild type X. oryzae considerably reduced viral levels by over 60% in the plant relative to a virus-only infected plant, while significantly less reduction of viral levels was seen in the two bacterial mutant co-infected plants [A study by Tollenaere et al.  chose toeraction . In the e et al.  present d plants .Erwinia tracheiphila but not E. tracheiphila alone. Infection of the plants by virus slightly delayed onset of bacterial wilt caused by E. tracheiphila by one day in inoculation experiments. However, additional experiments suggested that virus infection resulted in reduced visitation by cucumber beetles, which are vectors for E. tracheiphila. Thus, the mechanism by which zucchini yellow mosaic virus infection reduces the rates of bacterial wilt is likely indirect in part by reducing visitation by bacterial beetle vectors. The specific mechanism by which the virus reduces visitation by beetles was not reported but suspected to be induction of plant traits that make them less desirable to beetles, and likely future research into the mechanism may be of interest for the purposes of pesticide alternatives [In a study by Shapiro et al. , connectrnatives .N-acetylglucosamine units, and has been shown to interact and specifically increase the stability of polioviruses discussed above [Another interaction in agronomy involves the use of a compound that has beneficial effects on plants, but may have peripheral consequences for animal viruses based on observations of animal virus–bacteria interactions. Specifically, chitin and modified chitin compounds have been reported to stimulate plant growth, assist nutrition, promote beneficial microorganisms, and control different plant pathogens and pests (reviewed in Sharp ). Chitined above ,27. Use ed above ,45,46, aed above ,47,48,49ed above .Bacillus species have been shown to have an antiviral effect on cucumber mosaic virus in both a model plant  [NPR1 that elicit resistance against bacteria and fungi [Pseudomonas aeruginosa were cloned into vectors and then transformed into a Pseudomonas fluorescens strain whose wild type did not produce salicyclic acid and was not efficient at inducing systemic resistance in plants. Cloning of the genes resulted in production of salicyclic acid by the bacteria, and induced systemic resistance against tobacco necrosis virus [Bacillus amyloliquefaciens strain isolated from the leaf of a cherry tree was found to decrease the levels and severity of cucumber mosaic virus when sprayed onto pepper and tobacco plants. Evidence that the bacterial application induced systemic resistance in the plants was observed via upregulation of genes in pepper associated with plant defenses. Reduced levels of the naturally-circulating pepper mottle virus and broad bean wilt virus were observed in bacteria-treated peppers compared to controls, further supporting induction of systemic resistance [A number of other indirect interactions between plant viruses and bacteria have been investigated in the case of bacterial protection against viral pathogens. Specifically, a good deal of work has been reported on how different rhizobacteria protect against multiple plant pathogens, including viruses. Generally, the mechanism by which this occurs is through induction of systemic resistance in the plants . Thihaliana) ,37 and phaliana) . Some rend fungi . Howeveris virus . In anotis virus . Althougsistance .10 in less than 10 days. The mechanism of the inactivation was not determined, though evidence suggested that it was not due to an increase in pH or enzymatic action [In addition to having an effect on plant viruses, some evidence has been reported that native plant bacteria can affect animal viruses. In an interesting study by Esseili et al. , counts c action . AlthougCurvularia protuberate is required for the Dichanthelium lanuginosum grass to grow in Yellowstone National Park. However, a novel dsRNA virus was discovered in C. protuberate, and removal of the virus from the grass resulted in loss of heat tolerance, while reintroduction of the virus with a marker rescued heat resistance of the grass [Musa spp.), and multiple instances of the virus capable of doing this have been reported [Drosophila melanogaster, Wolbachia spp. have been demonstrated to reduce the load and increase D. melanogaster’s resistance to Drosophila C virus, Flock House virus, and Nora virus [Wolbachia spp. also exists and may warrant investigation. Multiple different areas of investigation regarding bacteria–virus interactions in horticulture may offer better understanding of plants as holobionts and offer numerous avenues of potential beneficial application.There are still many additional potential areas of future investigation that may be valuable to the horticultural community. A recent body of work related to the potential positive roles of some eukaryotic viruses as symbionts in their hosts has been convincingly argued for both animal and plant viruses ,51. One he grass . On the reported ,54. The reported . Once exreported ; the potreported , but thera virus . The posMuch like the horticultural sciences, a number of papers noting interactions between bacteria and viruses of concern in aquaculture and livestock have been reported . General10 cfu/kg) Lactobacillus plantarum had survival rates increased as much has 26.7% when challenged with grouper iridovirus. Some evidence indicated that L. plantarum stimulated the immune system of the fish, which was one of the suspected mechanisms of protection. Interestingly, an increase in fish survival rate corresponded to higher L. plantarum doses from 0–108 cfu/kg, but the survival rate goes back down at 1010 cfu/kg [Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10 on two salmonid fish viruses, infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV). Commercial, purified dextrans were also tested, with only the largest dextran (T2000) displaying antiviral activity. An antiviral effect for EPS was observed in vitro via 50% tissue culture infective dose (TCID50) by addition of EPS after viral introduction to cells, as well as in vivo in rainbow trout with L. sakei EPS. Although the specific mechanism was not conclusively determined, evidence suggested that introduction of the EPS activated an antiviral immune response, as first levels of poly I:C then IFN-1 spiked in treated fish relative to controls (innate response) followed by a spike in IFN-γ (adaptive response) [Like was observed by Lei et al.  in gnoto0 cfu/kg . Anotheresponse) .Edwardsiella tarda and/or Streptococcus iniae. Fish that were co-infected with both the birnavirus and a bacteria had a 20–35% higher mortality rate than those infected with a bacterium alone [Vibrio harveyi or E. tarda in Olive flounder when intraperitoneally injected. A survey of birnaviruses in Olive flounder in Korean waters by Jung et al. [Vibrio salmonicida [Vibrio carchariae [Vibrio campbellii resulted in accelerated mortality and significantly higher levels of V. campbellii in virus-infected shrimp compared to uninfected shrimp [A number of studies describing bacterial and viral pathogens interacting in co-infections in aquaculture have been reported, and are reviewed by Kotob et al. . For theum alone . A similum alone , as aquag et al.  presentsmonicida  and Vibrrchariae  resultedd shrimp .Mannheimia haemolytica (formerly Pasteurella haemolytica) [M. haemolytica produces a leukotoxin that binds the host leukocytes and activates cytolysis of the cells. Infection with BHV-1 in cattle caused increased expression of the host receptors the leukotoxin targets, and thus resulted in increased leukotoxin binding and leukocyte death. Additionally, BHV-1 infection increased the number of leukocytes in the cattle [M. haemoltyica infection [Virus–bacteria interactions in livestock have also been reported, with similar observations of synergy between bacterial and viral pathogens. One of the more historically documented instances of this is Bovine Respiratory Disease Complex, sometimes called “shipping fever”. This disease is multifactorial, involving multiple potential etiological agents, both viral and bacterial. One specific combination of these that has been studied is the relationship between bovine herpesvirus-1 (BHV-1) and olytica) ,72. Genee cattle . Anothernfection ,68.As can be seen, a number of studies have demonstrated the importance of virus–bacteria interactions in the agricultural and applied sciences. However, many of the studies either lack specific mechanistic insight that may prove valuable for multiple applications in the fields discussed. In these fields, a lot of work has been directed on interactions between viruses and bacteria in co-infected hosts; however, less work has been performed on the role of the host and environmental microbiota. One exception has been the beneficial impact of lactic acid bacteria and rhizobacteria in reducing the severity of viral disease, generally through indirect routes like immune activation. Given the numerous recent findings presented of more direct interactions between viruses and bacteria in humans, future exploratory work of additional direct virus-bacteria interactions in the applied sciences may prove valuable for functional application. As the rapidly growing field of eukaryotic virus–bacteria interactions evolves, multiple potential discoveries are likely to be made that will be valuable for both understanding fundamental processes and have functional applications in the agricultural sciences.