Surgical defects of the popliteal fossa pose a reconstructive challenge to the surgeon, given the intimate relationship of this area with the knee joint and underlying vital neuro-vasculature.An 88-year-old woman was treated for a biopsy proven invasive squamous cell carcinoma of the right popliteal fossa with Mohs micrographic surgery. The resultant defect (4.5 × 4.3 cm) was repaired using a random patterned bilobed flap with an undersized secondary lobe and porcine xenograft with excellent functional and cosmetic outcome.Numerous approaches to reconstruction of the popliteal fossa have been proposed, including primary closure, secondary intent healing, skin grafting, random flaps, muscle flaps, fasciocutaneous flaps, freeflaps, and tissue expansion. Sub-optimal repair of the popliteal fossa can reduce mobility of the knee joint, carry a high risk of wound dehiscence and may result in significant scarring. Here, we present the use of a bilobed flap with an undersized secondary lobe, in a patient with limited tissue laxity, for the repair of the popliteal fossa.Utilizing a random pattern bilobed flap with an undersized secondary lobe is an excellent alternative for the repair of moderate sized popliteal fossa defects. This region is intimately involved with mobility of a large articulation – the knee – and overlies with proximity, vital lower-extremity vessels. Furthermore, the fossa’s location on the lower extremity predisposes procedures at the site to both prolonged healing, secondary to decreased perfusion and a higher infection risk. Considering the aforementioned, a successful and reproducible surgical approach must consider anatomic and functional aspects alongside cosmesis.The authors report a case of a moderately sized Mohs micrographic surgery defect in the right popliteal fossa and subsequent repair, with excellent restoration of function and cosmesis.This work is reported in line with the SCARE criteria .2An 88-year-old woman presented with a biopsy-proven invasive squamous cell carcinoma of the right popliteal fossa . The tum3While there are few publications on reconstruction of the popliteal fossa, mostly in the setting of trauma and burn scars, various techniques have been described and may be employed as dictated by the resultant defect’s characteristics and surgeon preference. The predictability of scar outcome and cosmesis makes primary closure favorable whenever possible, but in the popliteal region the approach is typically not suitable for larger defects due to potential restriction of joint mobility and higher risk of dehiscence. Secondary intention healing is also not an ideal choice for flexion creases at large joints as they can result in contracture that precludes full limb movement, as well as prolonged healing and conspicuous unsightly scarring. Skin grafts reduce the possibility of contracture, however, they require extended immobilization with splinting and meticulous dressing changes at the expense of prolonged patient discomfort. Despite their limitations, primary closure, granulation, and grafts join a host of previously described approaches that have been successfully used to repair defects in the popliteal fossa, including – random flaps, muscle flaps, fasciocutaneous flaps, free-flaps, and tissue expansion. Since it’s description in 1918, the bilobed flap has been a workhorse for nasal reconstruction, and through a series of modifications, has become recognized as an adaptable and dependable reconstructive option at anatomic sites beyond the nose, including the popliteal fossa. For larger defects, an axial-based approach to the bilobed, allowing for larger flap dimensions and enhanced viability, has been described using the sural arteries . HoweverAs with all cutaneous reconstructions, complications associated with bilobed flaps such as hemorrhage, hematoma formation, infection, wound dehiscence, and trap-dooring are all possible. Furthermore, healing by second intention over a joint, may impair mobility and prolong healing. However, with proper patient selection, appropriate flap design, and meticulous surgical execution, all complications mentioned can be minimized or altogether avoided. No complications were observed in our case.4Popliteal tumors and associated post-extirpation defects present the dermatologic surgeon with rare and unique surgical and reconstruction challenges that demand both functional and cosmetic considerations in the setting of lower-extremity-associated perfusion and infection risk. As such, a thorough understanding of anatomy, reconstructive options, and associated complications are crucial for reproducible high-quality patient care. Popliteal fossa reconstruction with a random-pattern based bilobed flap is a favorable alternative to technically cumbersome and time-consuming axial, free-flap, or tissue expansion approaches for small to moderately sized defects in the region. As demonstrated in this case, bilobed flap with an undersized secondary lobe can be successfully applied to the reconstruction of the popliteal fossa with minimal impact on joint function and cosmetic outcome while minimizing prolonged joint immobility that is so often a concern with reconstructions at articulations.Dan C. Filitis, Juliya Fisher and Faramarz H. Samie have no conflicts of interest to disclose.This research is not funded.This case report does not require ethical approval.A written consent was obtained and is available upon request.Dan C. Filitis: case report design, writing, subject research, care of patient.Juliya Fisher: case report design, writing, subject research.Faramarz H. Samie: case report design, care of patient, subject research, review and final approval of manuscript.None.Faramarz H. Samie.Not commissioned, externally peer reviewed.


To assess the performance of the Sensa fecal occult blood test (FOBT) in a population-based screening program.Manitoba, Canada.This historical cohort study included individuals 52 to 74 years of age diagnosed with colorectal cancer (CRC) from 2008 to 2013. CRCs were categorized by detection following a screening program FOBT (Sensa), non-program FOBT (non-Sensa), or no FOBT. Screening program CRCs were classified as program-detected, interval program, or non-compliant. Logistic regression was used to compare characteristics by detection mode. Cox regression adjusted for lead-time was used to examine the effect of detection mode on survival.1,498 individuals were diagnosed with CRC; 132 (8.8%) had a screening program FOBT, 626 (41.8%) had a non-program FOBT, and 740 (49.4%) had no FOBT. Of the screening program FOBT CRCs, 72 were program-detected (54.5%), 42 were interval program (31.8%), and 18 were non-compliant (13.6%). Sensa interval cancer rate was 37.4% and sensitivity was 63.1% : 54.3%-72.0%). The risk of death for individuals that had a non-program (Hazard ratio (HR) = 0.57, 95% CI:0.44–0.75) or a screening program FOBT  was lower than no FOBT. There was no significant difference in the risk of death for interval, non-compliant, and non-program CRCs compared to program-detected CRCs. Adjusting for lead time bias, sex, income quintile, tumour location, and age at diagnosis did not appreciably change the risk estimates.More than one-third of CRCs may not be detected by Sensa. There may be no difference in survival between CRC detected by Sensa and non-Sensa FOBTs. Several observational studies have evaluated the impact of colorectal cancer (CRC) screening on population mortality and reported that that survival for screen-detected cancers is higher than symptomatically-detected cancers –7. HowevAll Canadian provinces have implemented organized, population-based CRC programs . ManitobThe province of Manitoba, located in central Canada, has a population of approximately 1.34 million (as of 2016) . Two-thiFive data sources were used: the Manitoba Cancer Registry (MCR), the Manitoba Health Population Registry (MHPR), the Medical Claims Database (MCD), the ColonCheck registry, and Statistics Canada 2006 census data. The MCR is a population-based database that is legally mandated to collect, classify, and maintain accurate, comprehensive information about cancer cases including diagnosis date, histology, topography, stage, and treatment. The MCR was used to identify individuals diagnosed with CRC, area of residence, diagnosis age, tumour location, and death date.The MHPR includes all provincial residents and is maintained by Manitoba Health, the publically-funded provincial health insurance agency. The MHPR contains demographic and migration information and a personal health identification number (PHIN) for all individuals which can be used to link provincial databases. The MHPR was used to determine provincial health coverage duration. The MCD, which includes claims filed by physicians and laboratories for payment of services, was used to identify individuals who had non-program FOBTs and colonoscopies after abnormal screening program FOBTs. The MHPR and the MCD have been validated for accuracy and have been used to study many health outcomes .Since FOBTs provided by the screening program are not included in MCD, the ColonCheck Registry was used to identify individuals who completed a screening program FOBT and the FOBT result. Statistics Canada 2006 census data were used to determine neighbourhood-level average household income (21). Average household income was categorized into quintiles from Q1 (the lowest income quintile) to Q5 (the highest income quintile) based on each individual’s area of residence. Previous reports have found that census-derived area-based income measures are reasonable proxies for individual-level income (22–24). To protect confidentiality, linkages were performed via scrambled PHINs using anonymous versions of all databases and no patients were contacted. The study was approved by the University of Manitoba’s Health Research Ethics Board and Manitoba Health’s Information Privacy Committee.CRCs were classified by detection mode as screening program FOBT, non-program FOBT, or no FOBT. Screening program FOBT detected CRCs included individuals that completed a screening program FOBT (Sensa) performed within two years preceding the CRC diagnosis. Non-program (non-Sensa) FOBTs included individuals that had a non-program (non-Sensa) FOBT performed in the two years preceding a CRC diagnosis and no screening program FOBT in the same time period . Individuals diagnosed with CRC that had no FOBT in the two years prior to the CRC diagnosis date were classified as no FOBT.We further classified individuals that had a screening program FOBT as either program-detected, interval program, or non-compliant. Program detected CRCs included individuals that had an abnormal screening program FOBT, subsequent colonoscopy within a year, and then a CRC diagnosis within 90 days of the colonoscopy. Interval program CRCs included individuals that had 1) a normal screening program FOBT within the 2 years preceding a CRC diagnosis or 2) an abnormal screening program FOBT followed by a subsequent colonoscopy within a year of FOBT, and then a CRC diagnosed greater than 90 days after the colonoscopy . Non-compliant CRCs included individuals that had 1) a normal screening program FOBT more than 2 years preceding a CRC diagnosis and no other FOBT within those 2 years, 2) an abnormal screening program FOBT with no colonoscopy within the subsequent year and CRC diagnosed within the subsequent 2 years, or 3) an indeterminate screening program FOBT with no repeat FOBT and a CRC diagnosed within 2 years of the indeterminate FOBT.Right-sided CRCs were defined as those occurring in the cecum, ascending colon, hepatic flexure, and transverse colon. Left-sided CRCs were defined as those occurring in the splenic flexure, descending colon, and sigmoid colon.Fisher’s exact tests using Monte Carlo estimation, Kruskal-Wallis test, and logistic regression were used to compare the characteristics of individuals diagnosed with CRC by detection mode. Cox regression was used to examine the impact of detection mode on the risk of death using bootstrapping with 1,000 resamples to obtain more reliable estimates. We examined models adjusted for income quintile, sex, tumour location, and age at diagnosis. Income quintile and sex were included to attempt to control for selection bias , 17. Ageet al. to all screening program detected and to 80% of non-program detected CRCs [Follow-up time was defined as the time from the CRC diagnosis date to death date, migration date, or the end of the follow-up period . To factor in lead-time bias, we applied the correction developed by Duffy ted CRCs . Althougted CRCs , 20. Theted CRCs .Since the mean sojourn time (when the tumour is asymptomatic but screen-detectable) has been estimated to range from three to six years, we calculated survival for 0, 2 and 5 year sojourn times –26. SchoFrom 2008 to 2013, 1,498 individuals 52 to 74 years of age who lived in Winnipeg were diagnosed with CRC. Overall, 626 people (41.8%) diagnosed with CRC had a non-program FOBT, 740 (49.4%) had no FOBT, and 132 (8.8%) had a screening program FOBT. Of the individuals that completed a screening program FOBT, 72 were classified as program-detected (54.5%), 42 were interval program (31.8%), and 18 were non-compliant (13.6%) .The interval CRC proportion was 0% in 2008, 10.0% in 2009, 16.7% in 2010, 12.9% in 2011, 52.8% in 2012, and 41.5% in 2013. Excluding non-compliant CRCs, the Sensa FOBT interval CRC rate was 36.8%  29.0%, 45.7%) and sensitivity for CRC detection was 63.1% .The characteristics of the individuals diagnosed with CRC by detection mode are shown in A higher percentage of individuals diagnosed with a program-detected CRC were diagnosed at stage I (43.1%) and were more likely to be diagnosed in the left colon (48.6%). The proportion of program-detected CRCs and those that occurred following a non-program FOBT remained fairly stable over time. A higher proportion of CRCs that had no FOBT occurred in the lower two income groups.After adjusting for sex, income quintile, and age, right-sided CRCs were more likely to be non-program FOBT detected (Odds Ratio (OR) = 2.09, 95% CI 1.11, 3.90), no FOBT , or interval  compared to program-detected CRCs . Rectal There were 167 deaths in the non-program FOBT group, 367 in the no FOBT group, 13 in the program-detected group, 9 in the interval program group, and fewer than six deaths in the non-compliant group. The median follow-up time was 3.5 years. After adjusting for lead-time bias (sojourn time of 2 years), the risk of death for individuals that had a non-program or a screening program FOBT was half of those that had no FOBT . At a sojourn time of five years, the HR of death for individuals who had completed a non-program FOBT was 0.71  and the HR for those that had a screening program FOBT was 0.78 .After adjusting for lead-time bias using a sojourn time of two years, the risk of death was non-significantly higher for individuals with an interval program , non-compliant , or non-program FOBT . The risk of death for individuals who did not have a FOBT was twice as high as program-detected CRCs . Adjustment for sex, income quintile, tumour location, and age at diagnosis did not appreciably change the risk estimates. At a sojourn time of five years, the risk of death was not significantly higher for individuals diagnosed through the other detection modes compared to program-detected CRCs.et al. found that the sensitivity from 19 studies of fecal immunochemical tests (FIT) was 79%  [This study shows the beneficial impact on survival for individuals who had a screening program or non-program FOBT compared to individuals who had no FOBT after adjustment for lead time bias. However, the Sensa interval CRC rate in this study was 36.8% with a sensitivity of 63.1%. The guaiac Hema-screen FOBT (an older guaiac) pilot in Scotland found an interval CRC rate  of 31.2% in the first round of screening, 47.7% in the second round, and 58.9% in the third round . A summa9%, 86%) .et al. found a 46% reduction in overall survival among FOBT program-detected CRCs and a 50% reduction among colonoscopy-detected CRC patients 50–79 years of age compared to those diagnosed symptomatically in Germany from 2003 to 2010 [Prior observational studies that have examined the impact of FOBT screening on survival have also found improved survival for screen-detected CRCs. Brenner In the present study, the risk of death for individuals that had no FOBT prior to diagnosis compared to program-detected CRC was three times higher when unadjusted for lead-time bias. The risk decreased when adjusted for lead-time bias using a sojourn time of two years and five years. Therefore, the results of observational studies that did not adjust for lead-time bias likely overestimated the benefits of FOBT screening in real-world practice.et al. examined overall survival for individuals diagnosed with CRC in Denmark from 2005 to 2008 [Two prior observational analyses of screening survival did adjust for lead-time bias. Lindegjerg  to 2008 . The riset al. found a significantly higher risk of death for CRCs diagnosed after a negative FOBT  compared to program-detected CRCs adjusted for lead-time bias [Our study also found no significant difference in risk of death for interval program CRCs compared to program-detected CRCs after controlling for lead-time bias. The Australian National Bowel Cancer Program also found that the survival for individuals with an interval program CRC was not significantly different than program-detected CRCs after adjusting for lead-time bias . Howeverime bias . The lacWe found no significant difference in risk of death for individuals diagnosed after a non-program FOBT compared to those diagnosed with a program-detected CRC. However, program-detected CRCs were more likely to be left-sided than those diagnosed after a non-program FOBT. Additionally, CRCs diagnosed after a non-program FOBT had similar characteristics to CRCs diagnosed after no FOBT . Some of the difference between program-detected CRCs and those found after a non-program FOBT could be related to use of non-program FOBTs for symptom evaluation. We assumed that 20% of non-programs were completed by symptomatic individuals , 20. In Unlike previous studies, we did not include stage at diagnosis in the survival analyses , 3. SincThis study has strengths and weaknesses. Although we used data from previously validated population-based administrative health databases, this was an observational study and the potential for residual confounding by unmeasured or unrecognized factors exists –36. The In conclusion, this study found a significant survival advantage for individuals diagnosed with CRC following a screening or a non-program FOBT compared to those who had no FOBT. The results of this study reinforce the importance of screening for CRC regardless of whether an FOBT is performed through an organized program or through an individual’s PCP. However, the sensitivity of Sensa in a real-world setting is at the lower end of the range previously reported and similar to that reported earlier for older guaiac FOBTs. This finding should prompt existing screening programs that use Sensa or older guaiac FOBTs to consider using alternate tests such as FIT and provide further justification for emerging screening programs to choose a FIT.


We developed a web-based cost assessment tool (CAT) to collect cost data as an improvement from a desktop instrument to perform economic evaluations of the Centers for Disease Control and Prevention’s (CDC’s) Colorectal Cancer Control Program (CRCCP) grantees. We describe the development of the web-based CAT, evaluate the quality of the data obtained, and discuss lessons learned.We developed and refined a web-based CAT to collect 5 years (2009–2014) of cost data from 29 CRCCP grantees. We analyzed funding distribution; costs by budget categories; distribution of costs related to screening promotion, screening provision, and overarching activities; and reporting of screenings for grantees that received funding from non-CDC sources compared with those grantees that did not.CDC provided 85.6% of the resources for the CRCCP, with smaller amounts from in-kind contributions (7.8%), and funding from other sources (6.6%) . Grantees allocated, on average, 95% of their expenditures to specific program activities and 5% to other activities. Some non-CDC funds were used to provide screening tests to additional people, and these additional screens were captured in the CAT.A web-based tool can be successfully used to collect cost data on expenditures associated with CRCCP activities. Areas for future refinement include how to collect and allocate dollars from other sources in addition to CDC dollars. Centers for Disease Control and Prevention and RTI International designed a web-based cost assessment tool (CAT) to collect cost and resource data. The design of the CAT was based on published methods of collecting cost data for program evaluation.We describe the development of the web-based CAT, evaluate the quality of the data obtained, and discuss lessons learned. We found that grantees were successfully able to collect and report cost data across years by using the web-based CAT.Data on activity-based expenditures and funding sources, collected using the web-based CAT, are essential in planning for the allocation of limited health care resources.Colorectal cancer (CRC) screening can detect early-stage CRC and adenomatous polyps ,2. HowevCDC and RTI International conducted economic evaluations as part of the Demo and Program 1. To help improve cost evaluation of CRC screening, we developed a cost assessment tool (CAT) to collect cost data and to perform economic evaluations. Cost assessments allow program planners and policy makers to determine optimal allocation of limited health care resources, identify the most efficient approach to implementing screening programs, and assess annual budget implications . HoweverThe design of the web-based CAT for Program 1 (OMB no. 0920–0745) was based on published methods of collecting cost data for program evaluation . We collFor the Demo, we initially collected all resource use data via a Microsoft Excel-based tool. However, in 2009 we piloted a web-based tool. The web-based tool allowed us to embed data checks within the Program 1 CAT; therefore, fewer mistakes would be made during data entry and the quality of the data received would be improved. Examples of the embedded checks included asking grantees to allocate at least 95% of the total amount of annual funding to specific program activities in their reporting. By using an algorithm indicating that total allocation to spending activities was equal to or greater than 95% of total funding, the grantee could submit the CAT; if total allocation was less than 95%, the grantee needed to revise inputs. Grantees also had to confirm that 100% of staff time spent was allocated to specific activities and that the amount of funding received and the amount of carryover funding from previous years were accurate. If any of these checks failed, the grantee would need to review and revise inputs before submitting. Because of the ease of use of the web-based tool and the success of embedded checks, we implemented the web-based tool for Program 1.In the Demo version of the CAT, we had an overall “other” category for activities that were not easily placed in existing categories. The activities that were placed in this “other” category were activities where grantees received lump-sum amounts that could not be easily divided among existing activities, or activities that were not included in the existing list. In the Program 1 web-based CAT, we added 2 “other” categories: an “other” category specifically for screening promotion activities, and an “other” category specifically for screening provision activities. In analyzing the second year of Program 1 CAT data, we found commonalities in activities that were included in the “other” categories, leading us to add more activities to the CAT, including patient navigation for both screening promotion and screening provision components and mass media for screening promotion activities.In addition to collecting Program 1 cost data, we collected data on the number of screenings conducted by the grantee. We asked grantees to report on total number of individuals screened, screening tests performed by test type, follow-up colonoscopies, adenomatous polyps/lesions detected, and cancers detected. We also asked grantees to report total number of people previously diagnosed with CRC who were undergoing follow-up surveillance for recurrence or development of CRC and total number of people enrolled in insurance programs. We used this data to supplement the Colorectal Cancer Clinical Data Elements (CCDEs) that Program 1 grantees provided CDC. While the CCDEs collected data only on screenings funded by CDC, the Program 1 CAT collected the same information for all screenings facilitated by the grantee regardless of the source of funding.To maintain systematic and standardized data collection, we provided all grantees with a data user’s guide and provided technical assistance via teleconferences and email. We hosted webinars about how to collect and input data using the Program 1 web-based CAT. The information in the Program 1 CAT was collected retrospectively; however, to improve the accuracy of the data, grantees were encouraged to track and log information required prospectively when feasible. Cost data were collected and analyzed on an annual basis for 5 years (2009–2014). In the first year of Program 1, we collected data from 26 grantees; in years 2 through 5, we collected data from 29 grantees annually.On completion of the annual submission of the Program 1 CAT, we conducted data quality checks. We confirmed that data were entered into each of the broad categories of personnel, contracts, screening provision, and administration/overhead. If no costs were reported in any of these categories, we followed up with grantees to understand why. When funding amounts were reported for screening provision, we verified that screening numbers were also provided in the tool (or to CDC in year 5). Each year, before releasing the Program 1 annual CAT summary data to grantees, CDC also compared the data reported in the CAT with information in the fiscal database on approved, expended, and carryover funds. We generally found high levels of concordance.To analyze the Program 1 CAT, we calculated staff cost per activity by using salary information and hours spent by staff members on each activity. We also calculated cost of contracts by activity and prepared a summary of cost data for each submission for each grantee’s review and approval. The summary provided grantees with information on the labor and nonlabor cost per activity and costs by budget category and in-kind contributions.We performed 4 different analyses. First, we examined the distribution of funding from CDC, in-kind, and other sources. This evaluation of funding was essential to identify the extent to which assessments based on CDC funding alone would provide valid results and whether all funding sources needed to be considered. Second, we analyzed the cost by budget categories because certain types of data, such as contracts, tend to be more difficult to allocate to specific activities. Third, we examined the distribution of expenditures for screening promotion activities, screening provision activities, and overarching activities for each year of the program. On the basis of qualitative feedback from grantees, we anticipated that the overarching component would be high in year 1 because a large proportion of resources was initially allocated to planning activities; similarly, screening provision costs would be low because of the contracts that needed to be executed before initiating screenings of eligible individuals. Fourth, we evaluated the percentage of total expenditures that was not allocated to specific activities by year  and summarized the “other” activity category across all program years. As a quality measure, we required that at least 95% of the total funding be allocated to specific budget categories.We compared the number of people screened based on data from both the Program 1 CAT and from the CCDEs. For grantees reporting other funding sources in addition to CDC dollars and where the other sources were earmarked for screening provision, we anticipated higher reported numbers of people screened. To reduce the reporting burden, in year 5 we did not require grantees to report the number of people screened if they received only CDC funding. We asked only grantees who received funds from non-CDC sources to report screens in the Program 1 CAT and used the screens reported in the CCDEs for grantees who reported only CDC funding (some of these grantees continued to voluntarily report the screens performed in the CAT). Because we could not verify additional funding for all grantees for each year in sufficient detail, we excluded selected grantees from this comparative analysis. We excluded grantees if they had not yet begun screening  and if they reported extra funding inconsistently across years . Overall, we had a total of 45 program years with only CDC funds for screening, and 57 program-years with other funding for screening.On average, most funding (85.6%) was from CDC . A smallIn year 1, most costs (43.8%) were for overarching activities, which decreased to 37.2% by year 5 . ScreeniThe largest total cost category in all years was contracts, materials, and supplies . This caOverall, grantees were able to allocate more than 95% of their costs to specific program activities. Across years, grantees were unable to allocate approximately 2.4% of costs among promotion activities, 0.8% of costs among provision activities, and 1.8% of costs among other activities .Grantees who received funding from other sources reported higher average screening numbers in the Program 1 CAT compared with the CCDEs . Across We described how we developed and used a standardized cost data collection instrument to support an economic evaluation of grantees participating in Program 1. We collected data from grantees annually for 5 years. We found that, in addition to CDC, in-kind cost contributions and funding from other sources were important sources of assistance to the programs, although not all grantees indicated that they were recipients of additional contributions. Among those who did, many did not receive or report additional contributions consistently. This may account for the slight variation in distribution of funding sources across years, particularly in the last year of the program. To collect this data more accurately in the future, grantees need to be provided guidance on how to collect this information.Spending for overarching activities, those that supported both screening promotion and screening provision activities, were a significant portion of grantees’ expenditures, particularly in the early years. This was not surprising because programs were in their start-up phases and had not yet begun in earnest to promote or provide CRC screening. Grantees needed time, for example, to hire their staff and form partnerships to make the programs viable. As the programs were implemented, the proportion of funding used for these overarching activities generally decreased, as expected.All grantees reported cost data by budget category, and more than half of expenditures was allocated to labor and to screening and diagnostic services. On the basis of previous experience in using a cost tool , labor and clinical service costs are typically captured accurately . HoweverThe Guide to Community Preventive Services by the Community Preventive Services Task Force , grantees also conducted screening promotion activities that were not evidence-based interventions  that we ultimately included in the Program 1 CAT . We attempted through trainings and the user’s guide to define the program activities for the grantees and designate how to allocate time. Although in most cases we were able to attribute most of the costs to program activities, some inconsistencies are likely in how grantees ultimately defined the activities. We also anticipated recall bias related to time inputs, although we tried to alleviate that through training and by encouraging tracking of data prospectively. Lastly, some grantees were unable to disaggregate contracts and other inputs into activities and could only report them as a lump sum. These were allocated into the “other” categories.We provide details on the methods used to collect data on activity-based expenditures and funding sources. These data are necessary to plan for optimal use of resources, and the additional details in these data are advantageous compared with previously existing resources such as budget or funding information. Although the focus of the CAT is specifically on cost and resource collection for the Demo and for Program 1, the CAT can be customized. For example, the CAT has been used with success in estimating cost and resource use for both national and international cancer registries and for other cancer screening programs –26. On t


Rosa hybrida) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial B. cinerea inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose genes involved in B. cinerea resistance could provide useful genetic and genomic resources, we devised a virus-induced gene silencing (VIGS) procedure for the functional analysis of B. cinerea resistance genes in rose petals. We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar ‘Samantha’ showed the greatest decrease in RhPR10.1 expression among the cultivars tested. To determine whether jasmonic acid and ethylene are required for B. cinerea resistance in rose petals, we used VIGS to silence the expression of RhLOX5 and RhEIN3  and found that petal susceptibility to B. cinerea was affected. Finally, a VIGS screen of B. cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B. cinerea resistance. Collectively, our data show that the combination of the DPDA and VIGS is a reliable and high-throughput method for studying B. cinerea resistance in rose.Fresh-cut roses ( The supply of cut roses is heavily reliant on the logistics of long-distance transportation, as cut roses are produced mainly in developing countries with low labor costs and suitable climates, such as Kenya, Colombia, Ecuador, and China, whereas the consumer market is mainly in the developed countries of North America, Europe, and Japan. Globally, the average transport distance for each rose, from farm to customer, is more than 1500 km2. During this long-distance transportation, cut roses are challenged by various biotic and abiotic stresses, among which gray mold disease caused by Botrytis cinerea causes the most severe postharvest losses.Fresh cut roses are among the most popular ornamental plants, generating billions of dollars in trade annually and accounting for more than one-third of the total cut flower industry worldwideB. cinerea is among the world’s most notorious plant pathogens, causing gray mold disease in over 200 dicotyledonous and monocotyledonous species3. Germinated B. cinerea conidia produce secondary metabolites and phytotoxic proteins that induce host cell death during the penetration of the host epidermis4. In rose, B. cinerea infection leads to necrotic lesions on petals, and symptoms develop rapidly during postharvest transport, during which flowers are packed in boxes with high relative humidity5.B. cinerea interaction has been limited compared to research on the pathogen’s behavior in other plants, such as the model plant Arabidopsis thaliana (Arabidopsis) and the Solanaceous species Nicotiana benthamiana and tomato (Solanum lycopersicum), which are also susceptible to B. cinerea. However, research on B. cinerea infection in Arabidopsis and N. benthamiana has focused on the infection of leaves and, in tomato, that of fruits. In ornamental crops such as gerbera6 and rose7 by contrast, B. cinerea mainly infects flower petals, damaging the most economically important organ of these plants, while the leaves, fruits, stems, and sepals are rarely infected or of little importance. Petal cells begin to senesce immediately after harvest8, which facilitates infection by necrotrophic fungi such as B. cinerea. In addition, petal epidermal cells are a unique plant tissue with specialized conical structures. The valleys between conical cells may facilitate the survival of airborne B. cinerea conidia.Despite the economic importance of this pathogen in roses, research on the rose–B. cinerea and rose, both for fundamental research and for breeding purposes. Previously, rose was inoculated with B. cinerea via the inoculation of whole flowers with fungal conidia; disease severity was scored according to a “disease index” based on a scale of 0–5 (or more), ranging from no infection to the fungus covering the whole flower10. However, this conventional method cannot be used to accurately quantify disease resistance. We therefore aimed to develop an improved method for artificial inoculation and disease quantification in rose petals. To this end, we designed and optimized a detached petal disc assay (DPDA) for artificial inoculation and accurate symptom quantification of B. cinerea infection in rose.Artificial inoculation is a critical technique in disease phenotyping and thus in studies of the interaction between B. cinerea resistance is limited by the low efficiency and long timeframe of genetic transformation (2 years from transformation to flower production), we used an alternative molecular approach involving virus-induced gene silencing (VIGS). This method, in which target genes are knocked down based on double-stranded RNA-triggered RNA degradation, has been widely exploited for gene functional analysis11. In VIGS, when a recombinant virus carrying the sequence of a host gene\spreads throughout the plant, the host target gene transcripts are degraded together with the viral transcripts, and the gene of interest in the host plant is therefore silenced. Previously, a VIGS approach using recombinant tobacco rattle virus (TRV) was established for the silencing of genes in rose flowers12. Once young plantlets or sprouts are vacuum infiltrated with Agrobacterium tumefaciens (Agrobacterium) carrying recombinant TRV-derived vectors, the infected plantlets/sprouts are grown in soil or grafted to a root stock until flowering (which takes approximately 5–15 weeks), at which point it is possible to evaluate their phenotypes, such as flower color, scent, and floral development12. Although it has not been reported previously, it is likely that this VIGS protocol could be used to study rose resistance to pathogens. However, the full exploitation of VIGS assays for investigating B. cinerea resistance in rose petals could be hampered by the time (5–15 weeks) and labor costs of VIGS. In addition, a large volume of the Agrobacterium suspension is needed for the immersion and vacuum infiltration of plantlets, and a large area in a greenhouse or climate-controlled chamber is required to support the growth of rose plants from agroinfiltration until flowering. These limitations have prevented the rapid and high-throughput screening of B. cinerea resistance genes in rose flowers.Furthermore, as the functional characterization of rose genes thought to be involved in B. cinerea resistance genes.To address these challenges, we developed a VIGS method for silencing candidate genes in detached rose petal discs, which allows the rapid identification of their function. We demonstrate that this VIGS method, combined with the DPDA, can be used for high-throughput screening of candidate Rosa hybrida) flowers at stage 2 of flower opening13 and punched out 15-mm discs from the center of the petals. We placed the petal discs on a wet filter paper in a 9-cm petri dish and inoculated the abaxial or adaxial surface of the discs with 2 μL of B. cinerea conidia at 1 × 104/mL. However, we observed only tiny disease spots up to 72 h postinoculation (hpi). This may have been due to the difficulty of controlling the wetness of the filter paper under this method, which resulted in insufficient humidity for B. cinerea infection  agar and inoculated the discs using the same method. Through this approach, we observed clear, extended, and consistent lesions on all inoculated discs on either the adaxial or abaxial side at 48 hpi Fig. . We subs4/mL, 1 × 105/mL, and 1 × 106/mL conidia and measured disease lesions at 12, 24, 36, 48, 60, and 72 hpi. At 60 and 72 hpi, we observed statistically significant differences in the disease symptoms (lesion diameter) of discs inoculated with the three different conidia concentrations  and negative regulators  of defense signaling. Third, dark flowers are more suitable for the analysis than pale flowers because the fading of dark-colored petals caused by the silencing of anthocyanin-related genes can be used as a reporter of silencing efficiency (similar to the Phytoene Desaturase (PDS) gene, used as a reporter of silencing efficiency in plant leaves)14.It was previously demonstrated that TRV-mediated VIGS can be used in rose as a tool for the functional analysis of genes involved in flower development and the determination of petal color, and fragrance15. To test the efficiency of gene silencing in the four candidate cultivars, we used the RhDFR1 (Dihydroflavonol 4-reductase 1) gene as a reporter. RhDFR1 is required for the accumulation of anthocyanin in rose flowers, and its silencing has been shown to lead to bleached flower color symptoms throughout rose flowers12. To determine whether the silencing of RhDFR1 induced similar bleaching in rose petal discs, we vacuum infiltrated a 1:1 mixture of Agrobacterium carrying pTRV1 and pTRV2::RhDFR1 into rose petal discs. However, we did not observe increased color fading in the petals up to 10 days postinfiltration , RhDFR1 cannot be used as a silencing reporter gene for VIGS in petal discs.We therefore selected four rose varieties with clear lesions, moderate resistance, and dark color—‘Samantha’, ‘Carola’, ‘Black Magic’, and ‘Red Naomi’—as candidates for VIGS assays. Different cultivars may show different efficiencies of TRV-mediated gene silencing16. Knockdown of RhPR10.1 expression accelerates senescence in rose petals, resulting in a color-fading phenotype. To compare the silencing efficiency of this gene in the four candidate cultivars, we vacuum infiltrated Agrobacterium bacteria containing the recombinant TRV vectors into the petal discs of the four cultivars to silence their RhPR10.1 genes. At 2 weeks postinfiltration, RhPR10.1-silenced ‘Samantha’ and ‘Red Naomi’ petal discs showed significantly increased fading compared with control petals, while the other two cultivars showed no increase in fading  and the response to mechanical damage, while salicylic acid (SA) often acts antagonistically to JA17.Previous studies have shown that in the model plant Arabidopsis, the phytohormone jasmonic acid (JA) plays key roles in resistance to necrotrophic pathogens  at 48 hpi. We found that petal discs infected with B. cinerea exhibited a JA content that was almost 10 times higher than that of mock-inoculated discs, whereas the SA content was significantly reduced  is an important signaling component in plant defense against necrotrophic fungi, such as B. cinerea resistance in rose, we applied various concentrations (from 50 to 400 μM) of 1-aminocyclopropane-1-carboxylic acid (ACC), the direct synthetic precursor of ET, to rose petal discs and then inoculated the discs with B. cinerea 24 h later. All four concentrations of ACC used increased rose resistance to B. cinerea  and inoculated the discs at 24 h posttreatment. As expected, ET-treated petal discs showed significantly increased resistance, while 1-MCP-treated petal discs showed compromised resistance, confirming the involvement of ET in B. cinerea resistance signaling in rose  or with the empty TRV vector as a control (pTRV::00). As expected, RhEIN3-silenced plants showed significantly compromised resistance  genes were identified via comparative RNA-seq analysis of rose flowers infected with B. cinerea2. To illustrate the potential application of our methods to a high-throughput reverse screen of gene function in rose, we performed VIGS to knock down the expression of 10 randomly selected BIT genes. To this end, we cloned fragments of selected BIT genes varying in length from 200 to 400 bp into the TRV vector and inoculated rose petal discs with these fragments via vacuum infiltration. To test the involvement of the selected BIT genes in B. cinerea resistance, we subsequently challenged the TRV::BIT-inoculated rose petal discs with B. cinerea. The TRV::RhMYB44- and TRV::RhTGA2-inoculated plants showed severely compromised resistance, as evidenced by significantly increased lesion size  can fit in a petri dish of this size.B. cinerea. However, we circumvented this concern by including proper controls. For example, we found that petal discs inoculated with B. cinerea accumulated significantly higher levels of JA  and requires large-scale facilities for growing many plants and a great deal of work. By contrast, the silencing of candidate genes in petal discs can be accomplished in a matter of days and uses fewer resources. In the present study, by silencing RhLOX5 and EIN3, which are genes required for JA- and ET-mediated resistance to B. cinerea, respectively, we demonstrated that the DPDA in combination with VIGS in petal discs can be used to elucidate defense signaling against B. cinerea in rose flowers. In addition, although the silencing of RhDFR1 induces bleached petals in complete rose flowers12, this gene is not a suitable VIGS reporter for petal discs. However, we showed that the transient silencing of RhPR10.1 promoted accelerated color-fading phenotypes of petal discs, suggesting that this gene can be used as a visible reporter for VIGS in petal discs.Grafting VIGS in intact rose flowers can certainly also be used to study N. benthamiana, in which it was originally developed21. Considering that agroinoculated petal discs can be conveniently and efficiently silenced within several days and require relatively little space in a climate room, the VIGS and DPDA methods described herein can potentially be used not only for the functional analysis of a single gene but also for high-throughput screening of a large number of candidate genes involved in B. cinerea resistance. Indeed, in this study, we used the DPDA combined with VIGS to identify two novel transcription factor genes not previously known to be involved in B. cinerea resistance in rose.TRV-derived VIGS has been successfully applied to many plant species. However, the high-throughput screening of gene function using VIGS remains challenging in many plant species owing to time, labor, and space requirements. A high-throughput screen of gene function by VIGS has been reported only in Rosa hybrida ‘Samantha’ plants were grown in soil in the greenhouse under the following conditions: 20 to 25 °C with 50% to 70% relative humidity and 16 h/8 h day/night periods. Cut roses were harvested from the greenhouse at stage 2 of flower opening and immediately placed in water. All other rose cultivars were ordered from the Kunming International Flora Auction Trading Center and delivered to the lab within 24 h. The flower stems were recut to 20 cm in length under water and placed in deionized water. After treatment, the flowers were placed in a vase with deionized water under controlled conditions of 23–25 °C with 30–40% relative humidity and 12 h/12 h day/night periods to await further processing.B. cinerea strain B05.10 or CAU8324 was grown on potato dextrose agar at 22 °C. B. cinerea conidia were harvested from 10- to 14-day-old fungal plates and washed with tap water. The conidia were suspended at the desired final concentration in half-strength potato dextrose broth (PDB). For inoculation, rose petals were punched into 16 mm discs and rinsed with deionized water. Then, 2 μL of a conidial suspension was dropped onto the adaxial surface of the punched petals. As a control, petals were mock-inoculated with half-strength PDB. After (mock) inoculation, the petals were transferred to agar plates. The inoculated petals were evaluated by observing disease symptoms at 60 hpi. Each inoculation was repeated at least three times using a total of at least 48 discs. Student’s t test was used for statistical analysis. For the measurement of ion leakage, petal discs from each treatment were placed in 10 mL of 0.4 m mannitol and shaken at 40 rpm for 3 h. A conductivity meter  was used to measure the conductivity of the solution.2O/HCl ) was added, and the samples were vortexed for 10 s and shaken for 30 min at 4 °C. Next, 1 mL of CHCl3 was added, and the solution was vortexed for 10 s, shaken for 30 min at 4 °C, and then centrifuged at 14,000 rpm for 5 min at 4 °C. Finally, 1.2 mL of the lower layer of the liquid was transferred to a fresh test tube and dried at room temperature under nitrogen. The dried sample was resuspended in 0.1 mL of methanol and assayed by liquid chromatography-mass spectrometry .The plant samples were ground to a powder in liquid nitrogen. Then, 500 μL of extraction buffer , salicylic acid (50 and 100 µM), or 1-aminocyclopropane-1-carboxylic acid , or in deionized water as a control, for 24 h. The flower petals were then punched into discs for inoculation.13. Briefly, rose flowers were exposed to ethylene (10 μL/L), 1-MCP (2 μL/L), or regular air as a control for 24 h. A 1 M NaOH solution was added to the chambers to prevent CO2 accumulation. The flower petals were then punched into discs for inoculation.Ethylene and 1-MCP were applied to the roses as described previouslyTRV::RhLOX5 construct, a 461-bp fragment from the 3’ UTR of RhLOX5 was cloned into the TRV vector pTRV2 and then electroporated into Agrobacterium strain GV3011. The TRV constructs TRV::00, TRV::RhPR10.1, and TRV::RhEIN3 have been described previously22. To establish VIGS in rose petals, detached petals were obtained from the outermost whorls of rose flowers at stage 2 of flower opening. Then, a 15-mm disc was punched from the center of each petal. The petal discs were vacuum infiltrated with Agrobacterium carrying TRV constructs as described by Zhang and Thomma14. Briefly, an overnight culture of Agrobacterium was harvested at an OD600 of 0.8 by centrifugation at 4000 rpm for 1 min and then resuspended in the infiltration buffer  to a final OD600 of 2. Agrobacterium cultures containing constructs expressing TRV1 and TRV2 were mixed in a 1:1 ratio and vacuum infiltrated into petal discs. The petal discs were immersed in the bacterial suspension, and infiltration was performed under vacuum at 0.7 MPa. After the release of the vacuum, infiltrated petal discs were washed with deionized water. After TRV infection, the petal discs were left under dark conditions at 8 °C for 3 days. Then, the discs were placed in a climate chamber at 22 °C/19 °C with 16 h/8 h day/night periods for 3 days. At 6 days post-TRV infection, the petal discs were inoculated with B. cinerea. VIGS was repeated at least three times using at least 16 discs in each experiment. After B. cinerea inoculation, Student’s t-test was conducted. All primers are listed in Supplemental Table To obtain the 13. First-strand cDNA was synthesized from 1 μg of total RNA using the Takara Reverse Transcriptase M-MLV cDNA Synthesis Kit  according to the manufacturer’s instructions. One microliter of first-strand cDNA was used as a template in the reaction with the KAPA SYBR Quantitative PCR Kit (Kapa Biosystems), which was run in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). RhACT5 was used as an internal housekeeping gene control as described previously12. All primers used for qPCR are listed in Supplemental Table Total RNA was extracted from rose petal discs as described previouslySupplemental Figure S1Supplemental Figure S2Supplemental Figure S3Supplemental Figure S4Supplemental Figure S5Supplementary Table S1. Primers used in this studySupplementary Table S2. Accession numbers of the sequences included in this study


Limited work has been performed in helping hospice agencies to care for persons with dementia (PWD) and their caregivers in an evidence-based manner despite the increasing number of PWD cared for in this setting. To change the culture of care for PWD and their caregivers receiving hospice, we adapted Aliviado Dementia Care, an evidenced-based interdisciplinary quality improvement program, for use in hospice. The purpose of this pilot study is to examine feasibility and applicability of implementing the Aliviado Dementia Care-Hospice Edition sequentially in 2 hospice agencies in preparation for a nation-wide pragmatic trial. In the first pilot, concluded in March 2019, seven hospice interdisciplinary clinicians were trained as program champions and completed a two-day in-person intensive training on dementia symptom assessment and management, and quality improvement processes. Additionally, 47 interdisciplinary team (IDT) members were provided training via a 5-hour, online program covering dementia symptom assessment and management. All champions trained (100%) reported being satisfied/very satisfied with the program and agreed that the training is applicable to hospice practices. All IDT members who completed the online training (100%) reported being satisfied/very satisfied with the program quality, or agreed/strongly agreed that the content was relevant. The high rates of satisfaction and applicability, reported by the hospice champions and IDT members, provided preliminary evidence supporting the feasibility and applicability of the Aliviado Dementia Care-Hospice Edition.


We report on a study designed to integrate the convoy model of social relations with the successful aging model. The combined approach draws on the natural resources of interpersonal relations to promote successful aging thus minimizing risk of disease and disability, enhancing\maintaining mental and physical functioning, and facilitating continued engagement in life. We describe the MacHouse Affordable Housing Study, a field experiment conducted in an affordable senior housing community. The experiment involved a resident driven intensive intervention program designed to increase social activities and engagement. Based on an extensive needs assessment and interviews with the resident council as well as individual residents a program of activities was developed. This included group meals, an exercise program that included a walking group and an exercise class, communal gardens, computer classes and resident outings. Results indicate that more frequent participation in these activities was associated with increased life satisfaction over time.


Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020.Visceral Leishmaniasis (VL) is caused by a protozoan parasite This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients’ blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts.p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients’ samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal.Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020. Leishmania spp. and transmitted by bite of infected female sandfly (Phlebotomus spp. and Lutzomyia spp.) [Leishmania donovani and is the most common clinical presentation in the Indian sub-continent including Nepal [Leishmania parasite to additional vectors and mammalian hosts [Leishmaniasis, one of the neglected tropical diseases, is caused by a protozoan parasite, ia spp.) . Globallia spp.) . Leishmaia spp.) , 4. VL ing Nepal , 6. Contng Nepal . The disan hosts .Leishmania spp. by employing a molecular approach in VL suspected patients’ samples from non-program districts of Nepal.VL has been identified by WHO as a public health problem and target for its elimination  from the Indian sub-continent including Nepal has been set for 2020 , 10. As This was an exploratory study conducted at Sukraraj Tropical and Infectious Disease Hospital (STIDH), Kathmandu, which serves as a central referral hospital for diagnosis and treatment of infectious and tropical diseases in Nepal. STIDH receives VL patients from both program and non- program districts throughout the country. Suspected VL patients were defined as patients with history of fever for more than two weeks, weight loss, splenomegaly, hepatomegaly, and sign of anemia, according to the National guidelines . Those wApart from the laboratory investigation, a standard questionnaire was also used to gather demographic information such as, gender, age, address and travel history. Blood samples (3–5 mL) were collected from the suspected VL patients  following the manufacturer’s protocol. DNA was eluted to a final volume of 20 μL, and stored at − 20 °C until used for nested-PCR.Leishmania specific primer. The primers information and nested-PCR protocols have been detailed elsewhere [Leishmania spp. were used as positive controls in all assays while PCR-grade water (Milli-Q) was used as negative control. The sizes of the first and second PCR product were approx. 750 bp and 720 bp respectively.For molecular identification of the parasite, the variable regions of the kinetoplast minicircles DNA were amplified using lsewhere . BrieflyThe PCR product was purified using ExoSAP-IT Express  according to the manufacturer’s manual. For this, 5 μl of PCR product and 2 μl of Exosap was mixed and incubated at 37 °C for 4 min followed by 80 °C for 1 min. The purified product was subjected to sequencing reactions using BigDye™ Terminator v3.1 Cycle Sequencing Kit  following the instructions provided by the manufacturer. These single stranded sequences were further purified by using Big Dye Xterminator™ Kit (Applied Biosystems). Finally, the purified amplicons were sequenced in an automated 3500XL Genetic Analyzer (Applied Biosystems).Leishmania spp. sequences deposited on National Center for Biotechnology Information (NCBI) database and identify the Leishmania spp.The quality of raw sequences were checked through base calling in sequencer v. 4.1.4  and consensus sequences were generated using both strands. Subsequently, the final sequence obtained from each sample was subjected to Nucleotide Basic Local Alignment Search Tool (BLAST) to search similarity with the P-value < 0.05 was considered statistically significant (i.e. significantly decreasing or increasing VL trend).Data on VL confirmed cases (2008–2017) was retrieved from Annual Health Reports, Department of Health Services, Ministry of Health and Population, Government of Nepal. Data were analysed for disease trends by using non parametric Mann-Kendall trend tests in XLSTAT software. This study was approved by the Ethical Committee of Nepal Health Research Council. Blood samples were collected after obtaining written informed consent from patients and the concerned hospital. In the case of minors, consent was given by parents or guardians.p = 0.001), while a significantly increasing trend of VL cases was observed in non-program districts (p = 0.002) . Although the number of cases attended at the STIDH is smaller than the national totals (2013–2017 and study year 2016), the trend observed in this hospital was also quite similar to that of the country’s national scenario. Furthermore, STIDH is the only central specialized hospital for treatment of tropical diseases including leishmaniasis in the country and these 14 cases represent all of the cases at the central hospital (STIDH) over the study period (2016). National reports consist of all the VL cases reported throughout the country  including the cases from STIDH.To understand the trend of VL in past 10 years in Nepal, we plotted the number of VL cases from both program and non-program districts  including the cases from STIDH during 2013 to 2017 . Travel history indicated that none of patients had visited to program districts of Nepal and endemic areas of India.Analysis of VL cases from STIDH showed that the age of suspected VL patients ranged from 10 to 70 years, with a median age 24, (47–23) years and all these cases were male (L. donovani)  Fig. . A case Together, our data suggest that - 1) the number of VL cases was found to be increasing in non-program districts of Nepal despite the successful reduction in program districts, and 2) VL cases from these non-program districts were confirmed by a molecular approach (sequencing). It is high time to focus the VL control program in non-program districts to achieve the elimination goals.All four districts  with cases positive by molecular tools are located in the VL non-program districts. Chitwan and Dang districts are located in the lowland areas (250–2500 asl) of Nepal while Sindhuli is in the hilly region (300–3000 asl) and Dolpa is in the mountain region (1500–7500 asl)  [The VL control program has greatly benefited from rK39 test, however it is inadequate when it comes to elimination phase due to declining positive predictive values. Despite the claimed sensitivity and specificity , the rK3complex) , 27, andcomplex) , AlthougOur study provides important insights into the VL expansion to non-program districts which is strongly supported by use of PCR/sequence homology-based method in VL confirmation. However, due to the limited resources in our exploratory study, we could not collect larger number of samples and carry out necessary diagnostic tests for more conclusive results. Similarly, distribution of vector and parasites prevalence in all non-program districts was not studied. Not all the patients underwent bone marrow aspiration for diagnosis since the rK39 had discordant results with PCR for some patients. A small amount of blood was collected on filter paper and used for DNA extraction which might compromise the DNA concentration although we did not confirm it.With the rising number of VL cases reported from non-program districts of Nepal, it has become imperative for formulating new strategies that can overcome the present hassle of elimination obstacles as well as prevent an impending epidemic in low to high mountainous areas. Therefore, these findings should be considered as a signal for inception of a robust strategic plan for accommodation of non-program districts in the elimination program. Moreover, innovative strategies including molecular investigation should also be institutionalized to understand the origin and spread of these strains especially at this pre-elimination phase of VL.