People living with inflammatory bowel disease (IBD) and their caregivers are faced with indirect and out-of-pocket costs that they would not otherwise experience. These costs impact one’s ability to contribute to the economy to their fullest potential. The indirect costs of IBD in Canada are estimated to be at least $1.51 billion in 2023 and include costs associated with lost productivity resulting from a combination of missed work (absenteeism), decreased workplace productivity (presenteeism), unemployment, premature mortality, and caregiving costs. Unemployment is the largest contributor to indirect costs ($1.14 billion), followed by costs of absenteeism and presenteeism ($285 million). Caregiving costs for children with IBD are estimated to be nearly $58 million. Canadians with IBD also pay $536 million every year for care that is not covered by universal or supplemental private health insurance; this includes allied healthcare , medication, and other supportive therapy. Combined, the indirect and out-of-pocket costs of IBD in Canada are estimated at more than $2 billion CAD in 2023. This is substantially higher than the estimate of $1.29 billion in Crohn’s and Colitis Canada’s 2018 Impact of IBD report with differences attributable to a combination of rising prevalence, inflation, and the addition of presenteeism and caregiving costs to the total indirect costs. IBD impacts the ability of people living with the disease (or caring for people living with IBD) from contributing to the economy to their fullest potential.In Canada, the indirect costs of IBD are estimated to be at least $1.51 billion, though this is likely an underestimate.Costs of unemployment are the largest contributor to indirect costs ($1.14 billion), followed by costs of absenteeism and presenteeism ($285 million).Caregiving costs for children with IBD are estimated to be nearly $58 million.Canadians with IBD pay $536 million out of their own pockets to obtain care, medications, and supportive therapy not covered by universal or private health insurance plans.The annual indirect and out-of-pocket costs were estimated at $1.29 billion in 2018. Decreased workplace productivity was reported as a significant contributor to indirect costs, driven by premature retirement ($629 million CAD), absenteeism ($88 million CAD), and premature death ($34 million CAD). Out-of-pocket expenses borne by people living with IBD were estimated at $541 million. Data on the costs of presenteeism, professional advancement, and caregiving were lacking and therefore not included in the estimate of costs in 2018.et al. this volume). In Canada, direct costs are largely born by provincially or territorially administered universal health insurance, supplemented by private extended health benefit plans. However, many people will have to pay for some portion of their direct medical costs out-of-pocket when not covered by governmental or supplemental private insurance, in addition to direct non-medical costs required for individuals to access needed healthcare . Indirect costs are costs to the individual and society because of the impact that IBD has on a person’s ability to contribute to their full potential. This article details recent data on the indirect and out-of-pocket costs experienced by people living with IBD in Canada.The total economic burden of the inflammatory bowel diseases (IBD) comprises both direct and indirect costs . Direct Indirect costs attributable to a disease reflect the impact that living with a disease (such as IBD) has on a person’s ability to achieve their full potential for engaging with the world. In this sense, indirect costs of IBD can be thought of as lost opportunity. On the level of the individual living with IBD, indirect costs refer to the costs of what a person is missing out on, either in work or in life, because of IBD. On a societal level, indirect costs refer to the loss to society resulting from the impact that IBD has on a person’s ability to fully contribute to their community. Indirect costs are often separated into work-related costs  and intangible costs . In addition, the care needs of a person living with IBD can impact the abilities of close family members and friends to contribute to their communities; this is especially true for children, the elderly, and people with severe psychiatric disease or neurocognitive deficits. Therefore, in considering the indirect costs of IBD, the costs incurred by primary caregivers cannot be overlooked.Indirect costs can often be difficult to quantify precisely. Most attempts to measure indirect costs use the human capital approach, where a person’s lost wages are presumed to represent the costs of lost productivity. Further, there are relatively few studies that have focused on the impact that IBD has on one’s ability to be optimally productive at work or otherwise contribute to the well-being of society, especially in the Canadian setting.Absenteeism costs refer to lost wages due to missed work. Costs related to intermittent absences can directly impact an individual’s earnings when they do not have paid sick days or impact employers through costs related to replacing an employee or lost productivity due to an employee’s absence. Among respondents to the Canadian Community Health Survey with IBD, those who were employed missed an average of 1.6 (SD: 4.4) days of work over a three-month period; people without IBD missed an average of 1.0 (SD: 3.5) days of work . This eqSeveral studies have described work-related productivity costs using data from Sweden, where they track healthcare data along with data on access of disability pensions and sick leave pay for the entire population. Sick leave and permanent disability result in 63 days of work missed in people with Crohn’s disease  and 41 days of work missed in people with ulcerative colitis (costing $8219 USD) per year . Over thAmong 540 persons in the WORK-IBD cohort from the Netherlands, 18% reported missing work in the last week, and 4% had missed at least 50% of workdays . An analPresenteeism occurs when an individual is present at work but is unable to work at their full potential. Presenteeism costs estimate the cost associated with this reduction in workplace productivity. Among 762 work-age employed Manitobans with IBD, 37% reported reduced workplace productivity during at least one day in the previous two weeks, with 19% reporting reduced productivity on three or more of the previous 14 days . In the Extrapolating costs estimated in the Finnish study  to the CA Manitoba study reported no delays in graduating from high school among children with IBD and their non-IBD peers . Marks oDisease severity may be related to missed days of school and delays in educational progression in high school. In a study of 675 German and Austrian students with IBD, 10.4% reported repeating a year of school; the repetition rate was highest among persons who had missed more than two weeks of school related to IBD, or those who had active IBD symptoms . A FrencImportantly, childhood IBD may have a long-term influence on earning potential. In a study using population-based Swedish healthcare data, the annual salary at 30 years of age among individuals who were diagnosed with IBD during childhood was 5.4%  less than healthy controls . ChildreAmong parents and caregivers of Canadian children with IBD, the mean wages lost annually was $8367 ; costs were greatest among parents of females and parents with lower educational attainment and income . In a suAmong individuals reporting higher levels of caregiver burden, 65% and 85% reported absenteeism and presenteeism in the previous week, respectively . In a muApplying the most recent Canadian estimate of $8367 in indirect costs per child with IBD per year to the estimated 6068 children currently living with IBD in Canada and adjusting for inflation, we estimate the total cost of lost wages among caregivers to be $58 million annually. This figure does not account for lost wages among individuals caring for adults and seniors with IBD and therefore likely underestimates the total indirect costs to caregivers associated with IBD.Since the 2018 Impact of IBD in Canada report , the attet al. this volume, Age-Related Comorbidities).According to data from Statistics Canada, there were 53 deaths with either Crohn’s disease or ulcerative colitis listed as the primary cause of death in 2020 among individuals of working age (20–64 years) . Using tIncorporating costs of absenteeism, presenteeism, unemployment, premature mortality, and caregiving costs, we estimate the total indirect costs of IBD in Canada to be $1.51 billion. However, this is likely an underestimate of the true indirect costs of living with IBD in Canada as they do not capture costs related to delayed entry into the workforce, failure of individuals living with IBD to reach their maximum educational potential, and caregiving costs for adults and seniors with IBD. Additionally, there may be other intangible costs that cannot be assigned a value that increase the societal costs of IBD.et al. (this volume). Direct costs borne by the healthcare systems and private insurance plans are discussed in Kuenzig et al. (this volume).Non-medical direct out-of-pocket costs include costs that are incurred in the process of obtaining medical care, including the costs of parking during a clinic visit, transportation to a medical appointment, or paying a babysitter to watch children while attending an infusion of a medication. Medical out-of-pocket expenses include anything the individual must pay for directly, including the costs of medications or health services  not covered by insurance, complementary and alternative medicines , ostomy supplies, travel to attend appointments, dietary therapy , and medications for bowel preparation prior to colonoscopy. These costs are a major financial burden for people with IBD  and poseA Canadian study reported mean out-of-pocket expenses for 243 children with IBD at $5236 (SD: $6931) . Travel A survey was conducted in 2022 by Crohn’s and Colitis Canada aimed at understanding unmet needs of the IBD community. Respondents reported mean out-of-pocket costs of $1579 (SD: $2017). Among the 797 people with IBD responding to the survey, the highest reported costs were prescription medications , vitamins and other supplements , acupuncture or massage therapy , transportation to and from medical appointments , mental health care , and over the counter medications . Only 20% of respondents reported decreasing transportation costs to and from clinic appointments with the shift to virtual care because of the COVID-19 pandemic.Using the estimate of $5236 for out-of-pocket costs for IBD among the 6068 children living with IBD in Canada, adjusted for inflation, the total out-of-pocket costs among children with IBD is estimated to be over $36 million. Using the estimate of $1579 for out-of-pocket costs of IBD among the 316,530 adults and seniors living with IBD in Canada, the total out-of-pocket costs among adults with IBD is estimated to be $500 million. Combining the pediatric and adult out-of-pocket costs, we estimate the total out-of-pocket costs for IBD to be approximately $536 million.The indirect and out-of-pocket costs of IBD are substantial: $1.51 billion in indirect costs and $536 million in out-of-pocket costs. Combined, indirect and out-of-pocket costs account for more than $2 billion annually. This cost is a substantial increase compared to the estimated $1.29 billion in indirect and out-of-pocket costs estimated in the 2018 report  of providing multidisciplinary care that does not require individuals to pay out of pocket or rely on private insurance.There is a lack of data around the indirect and out-of-pocket costs borne by those acting as caregivers for adults and seniors with IBD, which needs to be described.Additional data are needed to describe the total out-of-pocket costs for people living with IBD, including data on the costs of supplements, ostomy supplies, travel to medical appointments, and dietary therapy.For patient partners, this chapter presents the concerning picture of the significant costs of IBD for individuals living with this condition and their family caregivers. IBD is an expensive chronic condition with costs borne by individuals and Canadian society due to missed work, decrease in productivity, unemployment, and out-of-pocket expenses. Stakeholders must consider how to support persons living with IBD and promote innovative and holistic solutions. As individuals living with IBD face significant barriers to thriving financially, workplace support alternatives  and enhanced benefits and services  need to be promoted to help persons living with IBD and to maximize their full potential in terms of education, earnings, and productivity. Children and young adults living with IBD require early support to be empowered to reach their maximum potential. The indirect healthcare costs of family caregivers  also require the attention of stakeholders to promote strategies that support them. The experiences and insights of individuals with IBD and their families should guide these initiatives. Furthermore, patient partners emphasized the importance of recognizing that individuals living with IBD, and their families, are still dealing with significant out-of-pocket expenses. These issues require the attention, strategizing, and operationalization of different stakeholders to help reduce these costs and diminish the burden of the disease on the mental health of individuals living with IBD and their families.Canadian healthcare should evolve to include multidisciplinary care, including access to mental health professionals, dietitians, and other allied healthcare providers whose care improves the quality of life for people living with IBD. Additionally, extended health benefit providers should be levied to provide additional benefits regarding these costs to reduce overall health expenditures.The costs of supplements, ostomy supplies, and nutritional therapies  should be covered by universal healthcare programs.Virtual healthcare delivery should be continued beyond the COVID-19 pandemic due to its ability to decrease individual and caregiver costs related to travelling and missed work for medical appointments. Provincial health authorities need to continue remuneration for these services, which decrease indirect and out-of-pocket costs to the individual.Crohn’s and Colitis Canada should advocate for workplace programs designed to increase workplace productivity among people living with IBD, including tools to decrease both absenteeism and presenteeism.Crohn’s and Colitis Canada should advocate to government for mandatory paid sick days for all individuals, in all sectors.There is a currently lack of understanding among employees and employers in the workplace around what individual rights and workplace accommodations are, regarding chronic disease. Advocacy should target this educational deficit.Crohn’s and Colitis Canada should advocate for medical travel tax deduction limits be increased (in accordance with inflation), the broadening the scope of national tax benefits to include chronic diseases . Consistent guidelines concerning qualifications for these credits are needed .Crohn’s and Colitis Canada should work to partner with social workers, financial/tax experts, and/or lawyers to provide people with IBD financial or legal advice on their rights as citizens with a potentially disabling chronic disease. Practitioners need to understand the financial burden IBD places on an individual and should provide referrals to these experts.This article appears as part of the supplement “The Impact of Inflammatory Bowel Disease in Canada in 2023”, sponsored by Crohn’s and Colitis Canada, and supported by Canadian Institutes of Health Research Project Scheme Operating Grant (Reference number PJT-162393).


Circovirus, in a patient in France who had acute hepatitis of unknown origin.Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus  The world is regularly exposed to the emergence or re-emergence of known or unknown infectious agents. The COVID-19 pandemic illustrates the massive impact of such emergence on human lives, national economies, and social organizations. Infections of undetermined origin must be diagnosed early so that adapted measures are put in place to prevent the spread of potentially harmful pathogens. New diagnostic technologies such as shotgun metagenomics (SMg), which requires no prior knowledge of the agents sought, have greatly simplified diagnosis of novel pathogens. SMg has become a key tool for surveillance of viral emergence  colitis, parvovirus B19 bicytopenia, and aspergillus bronchitis. At admission, she was receiving multiple therapies, including immunosuppressive and anti-infectious drugs. Serum aminotransferase levels had progressively increased from December 2021 and peaked in April 2022   Figure 1Results of a liver biopsy showed signs of acute hepatitis, without suggestions of a given etiology. The following markers of infection were absent: hepatitis A virus IgM, hepatitis B virus DNA, hepatitis C virus RNA, hepatitis D virus RNA, hepatitis E virus RNA, HIV RNA, herpes simplex virus 1 and 2 DNA, varicella zoster virus DNA, CMV DNA, Epstein-Barr virus DNA, human herpes virus 6 DNA, adenovirus DNA, enterovirus RNA, parvovirus B19 DNA, and markers of leptospirosis. CMV and Epstein-Barr virus DNAs were undetectable at admission but became detectable at the time of the aminotransferase peak; viral levels were 2.9 log IU/mL for CMV and 4.4 log IU/mL for Epstein-Barr virus. There were no markers of autoimmune hepatitis, and withdrawal or diminution of potentially hepatotoxic treatments had no effect on cytolysis. Aminotransferase levels started to decrease spontaneously 7 weeks after admission. SMg testing was prescribed to identify a potential treatable cause of this acute hepatitis. The patient expressed no opposition to the use of her data and samples for this purpose.https://www.qiagen.com/us). We generated DNA libraries using a Nextera XT kit nd generated RNA libraries using a TruSeq Total RNA kit . We sequenced these libraries using NextSeq 500/550 High Output Kit v2.5 300 Cycles (Illumina). We performed metagenomics data analysis using MetaMIC software (https://gitlab.com/mndebi/metamic). The software filters out poor-quality data, identifies sequences by comparison with an nucleotide-based database, reduces background noise by comparison with environmental controls, and establishes a report on the presence or absence of bacteria, viruses, fungi, and parasites.The SMg technique has already been described (https://github.com/lh3/bwa) and by manual checking using the IGV 2.9.4 tool (https://software.broadinstitute.org/software/igv/). We performed phylogenetic analysis using a library of the replicase region and full-length Circovividae genome sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and the nucleotide database from GenBank, and MUSCLE alignment (https://www.megasoftware.net).We performed data reanalysis for genome reconstruction and phylogenetic analysis. We assembled viral DNA sequences and RNA transcripts by using Metaspades 3.15.3 software  , panel AC. parisii clustered with other circoviruses, on the same branch as recently described wolverine circovirus , a replicase gene spanning 6 conserved regions, a capsid protein gene, and an ORF3, the role of which remains unknown.Our shotgun metagenomics approach enabled us to identify a putative new member of the Circoviridae family, provisionally named Porcine circovirus 2 on human cell lines, including liver cells, demonstrates the ability of this virus to replicate in human cells (Porcine circovirus 3, the closest known circovirus, causes respiratory and neurologic diseases, cardiac and multisystemic inflammation, reproductive failure, and porcine dermatitis and nephropathy syndrome (Circoviruses are single-stranded DNA viruses generally transmitted via the fecal–oral route, with a potential pathogenic role in animals. Thus far, no human circovirus infections have been recorded (Additional information for unknown circovirus in immunosuppressed patient with hepatitis, France, 2022


Streptococcus suis (S.suis) is a neglected zoonotic disease that imposes a significant economic burden on healthcare and society. To our knowledge, studies estimating the cost of illness associated with S.suis treatment are limited, and no study focuses on treatment costs and potential key drivers in Thailand. This study aimed to estimate the direct medical costs associated with S.suis treatment in Thailand and identify key drivers affecting high treatment costs from the provider’s perspective.S.suis patients admitted at Chiang Mai University Hospital (CMUH) was conducted. Descriptive statistics were used to summarize the data of patients’ characteristics, healthcare utilization and costs. The multiple imputation with predictive mean matching strategy was employed to deal with missing Glasgow Coma Scale (GCS) data. Generalized linear models (GLMs) were used to forecast costs model and identify determinants of costs associated with S.suis treatment. The modified Park test was adopted to determine the appropriate family. All costs were inflated applying the consumer price index for medical care and presented to the year 2019.A retrospective analysis of the 14-year data from 2005–2018 of confirmed S.suis patients, the average total direct medical cost was 12,4675 Thai baht (THB) , of which the majority of expenses were from the “others” category . Infective endocarditis (IE), GCS, length of stay, and bicarbonate level were significant predictors associated with high total treatment costs. Overall, marginal increases in IE and length of stay were significantly associated with increases in the total costs (standard error) by 132,443 THB  and 5,490 THB , respectively. In contrast, increases in GCS and bicarbonate levels were associated with decreases in the total costs (standard error) by 13,118 THB  and 7,497 THB , respectively.Among 130 IE, GCS, length of stay, and bicarbonate level were significant cost drivers associated with direct medical costs. Patients’ clinical status during admission significantly impacts the outcomes and total treatment costs. Early diagnosis and timely treatment were paramount to alleviate long-term complications and high healthcare expenditures.The online version contains supplementary material available at 10.1186/s12889-023-15623-w. Streptococcus suis (S.suis) is a gram-positive alpha hemolytic bacterium mainly in pigs which can cause severe infection in humans through exposure or contact with pigs and consumption of raw pork [S.suis infection has been predominantly found among working-age men and imposed substantial health and economic burden, particularly in Southeast Asia [raw pork . S.suis ast Asia –4. The hast Asia . The cliast Asia –8.S.suis infection incurred 769 years of life lost (YLL), 826 quality-adjusted life years (QALYs) lost, and 793 productivity-adjusted life years (PALYs) lost in 2019, which was associated with a loss of US$11.3 million in GDP or US$36,033 lost per person [The disease has been notable, causing substantial losses in the swine industry . In humaS.suis treatment are still limited. Up to date, no study has investigated the cost of illness from S.suis treatment and factors affecting the treatment costs in Thailand. Therefore, this study aimed to estimate the real-world financial burden concerning direct medical costs associated with S.suis treatment in Thailand and identify key drivers affecting high treatment costs from the healthcare provider’s perspective.To our knowledge, studies estimating treatment costs associated with S.suis positive patients admitted from January 2005 to December 2018 with confirmed positive S.suis either by cerebrospinal fluid (CSF) or hemoculture. The index date was the hospital admission date. S.suis cases with specimens taken from CSF or hemoculture were identified from the hospital laboratory records of microbiological proven S.suis patients with their hospital numbers (HNs). All confirmed S.suis cases with complete medical and healthcare resource utilization records who had been admitted from 1 May 2005 to 31 December 2018 were collected and reviewed. All costs were derived from hospital charges provided by the Office of Medical Records and Statistics, Chiang Mai University Hospital, to estimate the direct medical costs associated with treatment during the episode.This was a cross-sectional study of the 14-year data at Chiang Mai University Hospital (CMUH), a 1400-bed tertiary teaching hospital during 2005–2018, the largest hospital in northern Thailand. The hospital’s electronic database was used to retrieve healthcare utilisation of The study was approved by the Research Ethics Committee, Faculty of Medicine, Chiang Mai University (IRB no.010/2018) and Monash University Human Research Ethics Committee (MUHREC) (Project no.12225). Patients’ names, and personal and traceable information were omitted and treated as confidential in all study procedures. The informed consent was waived due to the retrospective nature of the study.S.suis treatment which is generally fully paid by the provider in Thailand’s healthcare system. Direct medical costs included medication, laboratory tests, and others, e.g., room charges, meals, medical staff services, and medical devices during hospital admission. All costs were inflated to the 2019 value using Consumer Price Index with the medical care section [This study considered the healthcare provider’s perspective and included only direct medical costs associated with  section . The ave section .S.suis treatment. In addition, the models had the advantage of displaying both the mean and variance functions directly based on the original scale. Therefore, there would not be any issue concerning retransformation. Potential multicollinearity was examined before building the final model. The modified Parks test was adopted to determine the appropriate family for healthcare expenditures based on the relationship between raw scale means and variance function [Descriptive statistics were used to summarize the data on patients’ characteristics, healthcare utilization and costs. Multiple imputations with predictive mean matching on log-transformed costs were employed to deal with missing data  which wafunction . The intfunction . After tfunction . SignifiS.suis patients with complete medical records and hospital charges data were eligible based on the inclusion criteria. Among these, there were 89 males and 41 females. However, Glasgow Coma Score (GCS) data and length of stay were available only among 101 and 128 patients respectively. The mean age was 56.32 ± 13.77 years. The significant risk behaviors included alcohol drinking (50.77%) and eating raw pork (37.69%). Septicaemia was the most common clinical presentation (56.15%), followed by meningitis (36.92%) and infective endocarditis (IE) (26.15%) (Table S. suis infected patients recovered from their infection (52.31%), while around one-third recovered with sequelae (33.08%). The average admission duration was 18 days. The characteristics of S.suis patients are presented in Table A total of 130 confirmed S.suis was estimated to be 16,207,676  in 2019 , 13,187 (US$ 425), 8,240 (US$ 265), and 72,926 , respectively. Majority of expenses were from “others” category. The average total treatment cost per episode was 124,675 . After removing patients who died at the hospital and rerunning the analysis, the similar trends were observed while the mean length of stay was 19.25 (SD = 17.62) days , valvular heart disease (VHD), recent pigs/pork exposure, having sequelae, GCS, SAPS, meningitis, IE, vomiting, neck stiffness, length of stay, bicarbonate level, serum phosphorus, blood urea nitrogen. After multicollinearity checking, two predictors were removed: VHD and neck stiffness. According to multivariable GLM analyses for costs employing stepwise forward logistic regression at A bootstrap of 1000 sampling with replacement was performed for interval validation. The bootstrap-corrected calibration coefficient of the total cost model was 0.716 with a mean optimism equating to -0.0057 (95%CI -0.202 to 0.289), reflecting low bias and a good model.S.suis infection (Table Infective endocarditis (IE), GCS, length of hospital stay, and bicarbonate were associated with the high total treatment costs of on Table . OverallS.suis treatment among admitted infected patients for a single episode under the healthcare provider’s perspective at the largest university-affiliated hospital in northern Thailand. The average total treatment cost per episode was 12,4675 Thai baht (THB), or $4,016, with the majority of expenses falling under the "others" category . According to GLM multivariable analyses, IE, GCS, length of stay, and bicarbonate level were significant predictors of high treatment costs. Overall, marginal increases in IE and length of stay were significantly associated with increases in the total costs. Conversely, marginal gains in GCS and bicarbonate levels were significantly associated with decreases in the total costs.The study estimated direct medical costs associated with Most patients were male, who are usually the family’s primary breadwinner while the total hospital treatment cost per episode US$4,016) is more than half of the average annual income per capita  [ is more The median length of stay among infected patients in the study was 18 days which was consistent with the average reported in previous literature (17 and 23 days) , 8 The rS.suis patients. This condition usually requires extended hospitalization and leads to long-term sequelae, particularly hearing loss incurring high healthcare expenditure. The benefits of adjunctive corticosteroids in combination with antibiotic therapy in reducing the risk of hearing loss among adults with acute bacterial meningitis was demonstrated in a randomised controlled trial, and a network meta-analysis [S.suis meningitis patients. In this study, meningitis and corticosteroids use were significant in univariable analyses but not significant in the multivariable analyses. This might be due to smaller sample size of S.suis patients in our study.Meningitis is a major clinical presentation accounting for more than one-third of our analysis , 18. Howanalysis . Despiteanalysis , 21, nonanalysis . TherefoBased on our findings, the increase in GCS and a higher level of serum bicarbonate were associated with lower treatment costs. This suggests that patients in stable condition are likely to benefit from treatment with more favourable outcomes at a lower cost. Low serum bicarbonate level was related to multiple detrimental manifestations, including progression of chronic kidney disease, metabolic acidosis, and all-cause mortality –24. ThesS.suis patients were analysed. However, S.suis is a severe disease which usually requires hospitalisation and immediate medical attention. Therefore, direct medical costs associated with the treatment should have been captured. Due to the retrospective nature of the study, there might be potential recalled bias relating to information on risk behaviors and exposure to pigs/raw pork. We did not include long-term treatment costs from complications, mainly IE, hearing impairment, and vestibular dysfunction, which could incur tremendous long-term costs. Additionally, the direct cost estimates were based on data from a university-affiliated tertiary hospital which may not reflect healthcare costs at lower-level setting. Finally, the healthcare provider’s perspective used in the study limited frame of costs included while direct non-medical costs, productivity costs, and out-of-pocket costs were not captured. Further studies considering societal aspects are warranted to provide a clearer picture of S.suis cost of illness and its consequences in society.Some limitations can be noted in our analyses. Only the treatment costs during the first episode of admitted S.suis cost of illness and key drivers. The analyses demonstrate substantial hospitalisation costs incurred from S.suis infection, of which the majority of cases are middle-aged men who are usually the main income generator of families in Thai society. This suggests potential catastrophic medical costs upon acute infection among affected families. According to the cost model in our study, IE, GCS, length of stay, and bicarbonate level were significant predictors associated with high total treatment costs. Patients’ clinical stability, especially GCS and bicarbonate level at admission, are crucial to the outcomes and healthcare costs. This emphasises the importance of timely diagnosis and early treatment in averting high direct medical costs and complications.In conclusion, this study provides consolidating evidence on Additional file 1. Online Supplementary Material.


Over the last 15 years, Undiagnosed Diseases Programs have emerged to address the significant number of individuals with suspected but undiagnosed rare genetic diseases, integrating research and clinical care to optimize diagnostic outcomes. This narrative review summarizes the published literature surrounding Undiagnosed Diseases Programs worldwide, including thirteen studies that evaluate outcomes and two commentary papers. Commonalities in the diagnostic and research process of Undiagnosed Diseases Programs are explored through an appraisal of available literature. This exploration allowed for an assessment of the strengths and limitations of each of the six common steps, namely enrollment, comprehensive clinical phenotyping, research diagnostics, data sharing and matchmaking, results, and follow-up. Current literature highlights the potential utility of Undiagnosed Diseases Programs in research diagnostics. Since participants have often had extensive previous genetic studies, research pipelines allow for diagnostic approaches beyond exome or whole genome sequencing, through reanalysis using research-grade bioinformatics tools and multi-omics technologies. The overall diagnostic yield is presented by study, since different selection criteria at enrollment and reporting processes make comparisons challenging and not particularly informative. Nonetheless, diagnostic yield in an undiagnosed cohort reflects the potential of an Undiagnosed Diseases Program. Further comparisons and exploration of the outcomes of Undiagnosed Diseases Programs worldwide will allow for the development and improvement of the diagnostic and research process and in turn improve the value and utility of an Undiagnosed Diseases Program. Rare diseases, although individually uncommon, affect an estimated 1 in 16 people in the general population –4. The UThe UDN gained international recognition and informed the development of several other programs worldwide . ReflectThe US UDN, like most UDPs subsequently created, has a diagnostic pipeline based on several key stages, outlined in Fig. The current global landscape of UDPs includes state-wide, national and multi-national initiatives that characterize themselves as continuous programs  rather than discrete research studies, and offer an ongoing pathway for individuals who have previously received standard clinical care yet remain undiagnosed. What constitutes ‘standard’ clinical care, as well as the components of an individual UDP, varies considerably and is influenced by resources, funding, and staff expertise. However, for the purpose of this review we have defined the key features of a UDP as a regional program that is disease-agnostic—open to undiagnosed individuals with heterogeneous presentations—and has a formalized methodology that incorporates research and advanced testing. This contrasts with the multitude of phenotype-specific research initiatives, such as diagnostic programs for genetic epilepsies . UDPs alThis review aims to summarize the published, peer-reviewed literature that is available surrounding UDPs globally to contextualize and inform the implementation of future UDPs.This narrative review was conducted by using search terms such as ‘undiagnosed disease program’, ‘undiagnosed genetic disease’, ‘research program’, and ‘diagnostic program’ on databases including Medline, Scopus, and PubMed. This allowed for the identification of several publications on UDPs. In addition, countries with UDPs were further identified through membership in the UDNI, and available publications by these UDPs were retrieved. Included articles were English language, and where multiple studies were available for a single UDP, the most seminal was selected. This was a narrative review, without systematic protocol, and as such may reflect biases or perceptions of the authors.The nature of recruiting individuals into a UDP means that literature is restricted to retrospective observational cohort studies, rather than prospective case–control studies. Table Although there is no international agreement on the components of a UDP, the steps presented earlier in Fig. Step 1: EnrollmentThe enrollment process differs among UDPs. For example, some programs accept direct applications from individuals , 28, whiMost UDPs require individuals to have high suspicion for a monogenic condition in the face of a lack of diagnosis following standard clinical investigations. An important limitation of these criteria is the lack of a quantifiable metric for the extent of these investigations, aside from the Japanese inclusion criteria which specifies the lack of a diagnosis for at least 6 months  and the Step 2: Comprehensive clinical phenotypingAll fifteen UDPs recognize the use of phenotypic information as a key component of the UDP, consistent with the identification of deep phenotyping as a critical step for many diagnostic approaches to rare disease . HoweverStep 3: Research diagnosticsThe diagnostic process within a UDP is also variable, but typically begins with the completion and review of prior testing, and comprehensive unbiased genomic sequencing (ES or whole genome sequencing [WGS]), if not recently completed. This is followed by reanalysis of the genomic data and, if required, the application of advanced technologies such as RNA sequencing . CommonlOne common feature of all UDPs examined is the option of unbiased genomic sequencing, either ES –31, 34, Ongoing research is needed to better understand the diagnostic yield of ES/WGS, which a 2021 systematic review found has a wide range between 13 and 70% in a cohort with suspected monogenic disease, with a slight increase in yield from WGS . Much ofIf ES is non-diagnostic, the alternative pathways UDPs may take to increase the diagnostic yield are summarized in Fig. There is also a role for ‘multiomic’ technologies in diagnosis, such as RNA sequencing, methylation profiling, and metabolomics. These can be beneficial in assessing the pathological consequences of genomic variants . RNA seqStep 4: Data sharing and matchmakingDuring research genomics, deleterious variants in candidate genes may be identified, suggestive of a novel disease-gene association. In this setting, data sharing is vitally important in determining whether there are additional individuals with variants in the same gene and an overlapping phenotype . MatchmaData sharing plays a role in most UDPs examined, and methods of sharing variant and phenotypic data are summarized in Table In evaluating the role of data-sharing in UDPs, guidelines such as the FAIR principles should be considered . These gMost UDPs share genomic data at the variant level, but standardizing the interpretation of these variants can be a complex task. To mitigate inconsistencies in variant interpretation, sharing platforms like ClinVar can prove to be valuable . When deAn illustrative example of effective data-sharing can be found in the European UDP. With numerous European Reference Networks involved, the UDP integrates data from multiple sites and employs a standardized pipeline and a common workflow for the analysis of NGS data. These data are then shared through platforms such as the European Genome-Phenome Archive and the RD-Connect Genome-Phenome Analysis Platform, with controlled access measures in place . The sucData and metadata are also important more generally in research, since population level genomic data from a diverse range of the global population assists in the interpretation of test results and understanding of the clinical validity of variants . UDPs maStep 5: Results and follow-upUltimately, the UDP provides a diagnosis or an inconclusive result. As UDPs focus on the diagnosis of a broad range of conditions, treatment is generally not integrated into the program. In eight of the thirteen UDPs examined, results and recommendations are provided to the referring clinician or clinical center responsible for follow-up , 32, 33.The primary outcome of all UDPs examined was molecular diagnosis, with diagnostic yield ranging between 3 and 53% and summarized in Table Another factor contributing to the variability of diagnostic yield is what each UDP classifies as a ‘diagnosis’. Some studies only report individuals reaching a (likely) pathogenic diagnosis according to standards such as the American College of Medical Genetics diagnostic criteria . Others,Several UDPs also reported additional research-based outcomes, e.g., focusing on research impact beyond diagnostic yield or on patient-based outcomes relating to how the UDP impacted the individual. Additional research-based outcomes included novel gene discovery , 31–35, Patient-based outcomes included time to diagnosis , 31, 32,There is increasing recognition of the role of UDPs in providing a pathway for those undiagnosed, with this narrative review summarizing the outcomes of thirteen UDPs worldwide who have published their findings in peer-reviewed journals. More UDPs exist, based on involvement with the UDNI , but theThere is a need for ongoing evaluation to facilitate a better understanding of the utility of each component of a UDP, in order to inform best practice. Data on the impact and cost effectiveness of a UDP is needed to drive the policy change required to implement sustainable UDPs within health care systems and improve global equity of access to diagnostic technologies. Further research is also needed to understand how to best support undiagnosed families in the context of their diagnostic journey. For families remaining without a diagnosis, nonprofit organizations such as the international Wilhelm Foundation and national Syndrome Without A Name (SWAN) programs provide avenues for support, but comprehensive care navigation should be integrated into national health systems.As UDPs continue to be developed internationally, future research should incorporate an understanding of the limitations and successes of existing UDPs. An important part of this is characterizing the goals and key components of each step within a UDP that maximize the likelihood of a successful diagnosis. We outline proposed goals and components in Table


The research aimed to isolate, adapt to cell culture, and characterize the lumpy skin disease virus (LSDV) from clinically infected cattle in Bangladesh.From September 2019 to June 2020, 37 skin nodules and skin swabs were aseptically collected from afflicted cattle in the outbreak regions of Jhenaidah and Kishoreganj in Bangladesh. The LSDV was isolated from embryonated specific pathogen-free (SPF) chicken eggs along the chorioallantoic membrane (CAM) route and the Vero cell line after several blind passages. The viral attachment protein was targeted for molecular detection using polymerase chain reactions (PCR). For phylogenetic analysis, PCR-positive products were partially sequenced.The virus was evident in the cell line, showed cytopathic effects after the 13 blind passage, and on the CAM of SPF chicken eggs, exhibited thickening of the CAM with pock-like lesions. A total of 12 samples (32.43%) tested positive for LSDV by PCR. Phylogenetic analysis of the present isolates (accession numbers MN792649 and MN792650) revealed 100% similarity with strains from India (MN295064), Kenya , Greece (KY829023), Serbia (KY702007), and Kazakhstan (MN642592); moreover, 99.43% to 100% similarity to the sheep pox virus.Partially sequenced LSDV was developed as a vaccine seed and was first isolated in Bangladesh and characterized at the molecular level. Lumpy skin disease (LSD) is a contagious viral infection, transmitted through vectors, capable of crossing international borders, and by the lumpy skin disease virus (LSDV) ,2. The oThe first outbreaks of LSDV were reported in Zambia, in 1929, and have since been recognized in several African countries . The illLSDV is a virus composed of double-stranded DNA with an entire genome of about 151 kilobase pairs (kbp) and 156 probable genes in the central coding region .LSD constitutes one of Bangladesh‘s most economically significant emergent livestock diseases due to its swift and unusual spread ,14,18,19All laboratory work was conducted at the “Virology Laboratory, Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh, following the standard procedures. The Institutional Ethical Committee [approval number AWEEC/BAU/2019 (52)] approved using live animals and cell cultures in the laboratory experiment.The research was done in the various regions of Jhenaidah  and Kishoreganj  districts in Bangladesh from September 2019 to March 2020. Regular monitoring and discussion with veterinary professionals at the particular district-level veterinary hospital were used to evaluate outbreaks.A total of 37 samples  of skin biopsies from cutaneous nodules and nodule swabs were taken from cattle with clinical signs suspected of infection with the LSD virus. The sample collection and processing procedures were done per standard procedure . After cThe obtained samples from skin biopsies were thawed at 25°C and then cleaned in an aseptic manner with PBS (pH 7.2) . A tissuChicken egg embryos around 10 days old were used for virus isolation and propagation . The spe2 (5%) incubator. At confluent cell growth, a 500-µl of virus inoculum was injected into cells supplemented with MEM and 5% FBS and incubated at 37°C with 5% CO2 for virus adaptation, and several consecutive passages were carried out until the cell‘s cytopathic effect (CPE) was observed [Vero and MDBK cell lines were utilized to isolate and adapt viruses in continuous cells via numerous blind passages ,26. Inceobserved ,22. Afteobserved ,24.® Genomic DNA Purification Kit, Promega, USA) was used for the extraction of the LSD virus DNA following the manufacturer‘s instructions. PCR was done to amplify the target sequence of the viral attachment protein gene in the DNA of LSDV [From a 20% tissue suspension of the field samples, infected embryonated eggs, and infected cell culture fluid, a commercially available kit  and deposited onto an agarose gel well. Ethidium bromide was used to stain DNA in the gel after electrophoresis completion, and a UV-transilluminator  was used to visualize stained DNA .p-distance technique and are represented as base differences per site unit [PCR products were commercially sequenced for molecular characterization and uploaded to GenBank. Sequenced data were assembled and aligned using Codon Code Aligner and Nucleotide basic local alignment search tools. Using the neighbor-joining approach, phylogenetic analysis was done using MEGA X software against related GenBank sequences ,29. The ite unit ,29. All Field isolates and re-isolated viruses from adapted cell lines and SPF eggs were used for molecular characterization using their extracted DNA with specific primers that showed bands at the 192-bp region after gel electrophoresis. Real-time PCR was performed to verify the positive outcomes . The virAfter 3 to 5 days following inoculation, the embryonated eggs die. The CAM was expanded by hemorrhagic lesions encompassing the whole membrane and uneven congestion. The LSDV lesion on CAM ranged from membrane thickening in the first passage to multiple white foci, more evident in the second and third passages . BesidesTo produce the desired titer of the virus, an adaptation of the virus should be carried out in established cell lines to overcome the inadequacy of SPF eggs and the short-term survival of primary cell lines. LSDV was developed by inoculating MDBK and Vero cell lines with naturally infected cattle samples. The Vero cell demonstrated a characteristic CPE within 72 h of infection from 5 to 6 passages . AnotherThe commercially sequenced PCR products were analyzed, assembled, and uploaded to GenBank (accession numbers MN792649 and MN792650). Phylogenetic analysis of LSDV showed 100% similarity with MN295064 (India); AF325528, MN072619, and KX683219 (Kenya); KY829023 (Greece); AF409137 (South Africa); KY702007 (Serbia); KX894508 (Israel); MN642592 (Kazakhstan); and 98.85%–99.93% similarity with the sequence data published from South Africa , Croatia (MG972412), and Russia (MH646674) . In addiThe LSDV pandemic is inextricably entwined with food safety and cattle rearing because it threatens food security and causes financial losses to the livestock sector . This stFor LSD virus isolation, SPF egg  and two continuous cell lines, such as Vero and MDBK , were used for the first time in Bangladesh. Except for primary cell lamb testis described in Bangladesh, no available data about LSD virus isolation in SPF eggs or cell lines was found during this study . The priCPE, as described by Kumar et al. , was obsIn the phylogenetic analysis, it was observed that the present isolates (MN792649 and MN792650) assembled with 100% similarity with the isolates from India, Kenya ,34, GreeLSD is a problem in many countries around the world. This study identified LSDV in cattle through clinical signs and isolated it by virus culture in SPF eggs and continuous cell lines, namely Vero and MDBK, for the first time in Bangladesh. These local isolates could be vaccine candidates for developing an effective LSD vaccine in Bangladesh. The low-level genetic variability could indicate cross-border transmission of the virus and the sustainability of its virulence.


In the late 1970s, 52,000 pregnant women died in Kashmir, India. This tragic and disconcerting event, especially given the precarious economic and social reality of the affected community, was the result of a large-scale, common-source, water-borne epidemic of hepatitis. The limited infrastructure and resources in the region challenged researchers investigating the epidemic. Since all patients lacked markers of acute hepatitis A virus and hepatitis B virus, the existence of a new disease called non-A non-B hepatitis (NANBH) was announced [The investigators in Kashmir, headed by Dr. Mohammed Khuroo, collaborated with Dr. Robert Purcell at NIH to identify the new virus causing the outbreak. Time passed, and in Russia, Dr. Balayan inoculated himself with samples recovered from a hepatitis outbreak in a regiment of Russian soldiers in Afghanistan. This led to the first identification of virus-like particles in 1983 and represented a decisive step toward the characterization of this etiological agent of hepatitis . Viral particles in stool samples recovered during outbreaks in Mexico, together with isolates from Pakistan and Burma, allowed the description and classification of the molecular characteristics of the new hepatitis E virus (HEV) in early 1990. Later, the identification of swine HEV led to the characterization of viral diversity and underscored the importance of zoonosis in the transmission of this virus. Very importantly, the discovery of HEV as the causative agent of an intriguing chronic liver disease in transplant patients in 2010 highlighted its ability to cause chronic infection in immunosuppression settings . More reHepeviridae family and the Paslahepevirus genus. The Paslahepevirus balayani species includes genotypes (gts) HEV-gt1 and HEV-gt2, which are restricted to infecting humans, while HEV-gt3 and HEVgt-4 infect both humans and diverse animal species, with pigs being the main source from which zoonosis has been studied. HEV-gt7 infects camels and can also infect humans. All genotypes can be transmitted via the fecal–oral route or through contaminated blood derivatives and through the vertical route from mother to fetus. HEV-gt1 and HEV-gt2 are responsible for large, waterborne, fecal–oral outbreaks in low-income regions; HEV-gt3 and HEV-gt4 are responsible for most of the sporadic cases of hepatitis in industrialized countries. Recently, an HEV zoonosis via rat was demonstrated; this HEV was shown to belong to the Rocahepevirus ratti species [Currently, it is accepted that HEV infection, initially considered a poverty predictor, is also a major problem in high-income regions because of its zoonotic transmission. Indeed, HEV circulates practically worldwide, due, in part, to the diversity in its transmission routes, which in turn are related to specific viral variants. This RNA virus belongs to the  species .HEV can be present either as a naked particle or as a quasienveloped virion . Both foThe diverse forms of HEV that can infect humans make it a pathogen of global relevance. Indeed, the spread of this virus is not dictated by wealth or poverty. However, despite the milestone in the discovery of HEV, the infection is still neglected in multiple countries. There is no approved drug for the treatment of hepatitis E, and although the benefits of a vaccine licensed in China in 2011 have been shown in recent outbreaks in Africa, it is not widely available .Pathogens aims to collect the most recent advancements in the study of HEV. The valuable involvement of scientists worldwide with distinct points of view regarding this underestimated pathogen is expected.Considering the WHO’s recommendations for eliminating infectious hepatitis by 2030, joint efforts are required to improve the diagnosis and therapy of hepatitis E with viral eradication as the goal. The characterization of immune signatures associated with disease progression, extrahepatic manifestations, the design of experimental models, and the epidemiology of HEV is necessary. This Special Issue of


Their obvious advantages derive from both, the possibility to realize very high ratios of emitter complexes to target molecules, and the intrinsically long decay times of the Eu3+-complexes, which allows an almost complete discrimination against bothersome autofluorescence via time-gated measuring techniques; the narrow line emission in conjunction with large apparent Stokes shifts are additional benefits with regard to spectral separation of excitation and emission with optical filters. Last but not least, a reasonable strategy to couple the beads to the analytes is mandatory. We have thus screened a variety of complexes and ancillary ligands; the four most promising candidates evaluated and compared to each other were β-diketonates ; highest solubilities in polystyrene were obtained with trioctylphosphine co-ligands. All beads had overall quantum yields in excess of 80% as dried powders and lifetimes well beyond 600 µs. Core-shell particles were devised for the conjugation to model proteins . Their applicability was tested in biotinylated titer plates using time gated measurements and a Lateral Flow Assay as practical examples.Efficiently luminescing spherical polymer particles (beads) in the nanoscale regime of up to approximately 250 nm have become very valuable tools in bioanalytical assays. Eu As the rare earth molecular markers and the bead-labels discussed in the following live off the same luminescence principles, we first give a brief introduction on rare earth complexes followed by an outline on rare earth containing beads and applications. Knowledgeable readers may decide to leap over some of the introductory paragraphs.The luminescence of aromatic β−diketonates of Europium has caught the attention of scientists more than 80 years ago; a plethora of complexes and properties has been investigated in the meanwhile. The impetus of these on biomedical research is particularly impressive; dating back to the early eighties, rare earth complexes and applications have been commercialized as very sensitive immunoassays, in which the inherently long decay times of numerous complexes could be exploited to detect antibodies or hormones in minute concentrations .3+ and Tb3+ via (intrashell) f-f transitions is possible but very inefficient due to their quantum mechanically forbidden nature. One attractive workaround is the use of rare earth complexes, in which a strongly absorbing ligand is attached to the central ion and acts as an antenna. Hence, after singlet excitation of the ligand (1S → 1S*) the ligand undergoes intersystem crossing into a triplet , from which intramolecular energy transfer occurs typically to (5D0 (Eu3+) or 5D4 (Tb3+), respectively, also termed resonance levels. The energetic difference (ΔE) between 3T and 5D0 or 5D4 is most significant: if the ligand triplet is less than roughly 2000 cm-1 above 5D0 , trioctylphosphinoxide (TOPO) or bidentate phenanthroline and bipyridine are well known examples 2, see below] would contain nominally 2000 luminescing molecules. In practice, more than one analyte molecule per bead will be needed to enable ELISA analyses for instance, of course. However, along with very high efficiencies of beads, including extinction coefficents of the complexes in excess of 50,000 M−1·cm−1, a tremendous intensity increase is possible and observed. Thus, luminescent polymer beads comprising rare earth complexes have been developed in the most interesting size range of approximately 10 to a few hundred nm; as bead matrix materials predominantly polystyrene but also PMMA, or both, are employed . Howeve3(TOPO)2-beads in 0.1, 0.01, and 0.001% wt aqueous dispersions. The excitation maxima shift from 374 nm in powderous beads to 345 nm on diluting the 0.1% dispersion to 0.01%, further dilution does not alter the excitation maximum. At the same time, the intensity drops by a factor of 1.3 only rather than a factor of 10, while the second dilution step (0.01–0.001) affords an intensity decrease by a factor of 8. These factors are clearly due to inner filter effects at too strong absorbances and scattering by the beads. This behaviour is hard to unravel quantitatively in turbid media: next to the inner filter effect the spatial directions of scattering of both, excitation and emission are wavelength-dependent (Rayleigh-Gans-Debye scattering). Thus, we were not able to determine the efficiencies in dispersions reproducibly and refrain from reporting these. However, the decay times (3(TOPO)2 here too exhibiting the highest efficiency. It is worth pointing out that the high positive ζ-potentials of the beads  and others were used. All attempts of exploiting the ease of one-pot-syntheses failed insofar as they were notoriously accompanied by losses in efficiency. Although for our purposes, negatively charged carboxylate-surfaces proved to be more successful, in principle, positively charged amine-surfaces can be obtained as well, of course; experimental details for a succesive core-shell procedure for amino-beads are provided in the ESI for completeness.3(TOPO)2 was chosen for its obvious suitability with respect to physical and optical properties as evident from For the investigations to follow, Eu(ttfa)The somewhat lower quantum yield and absorption from method 1 prompted us to develop method 2, which was able to carry 20% wt of the complex in the synthesis and led to beads containing 6.08% wt. For method 2 we employed a mixture of acrylic acid, styrene-4-sulfonic acid sodium salt (NaSS), and methylmethacrylate for the shell, strictly avoiding acidic pH-values by neutralization and reaction in MOPS-buffer. The main properties of the beads obtained by the two methods are collected in 3+-chelate imbedded particles of 200 nm diameter and roughly 500,000 surface carboxylate groups per bead: the so called “parking area” (area available for one carboxy group) varies from batch to batch between 22 and 32 Å2 (2). The procedure for analysis involved EDC/NHS activation, followed by reaction with hexylamine and titration of hexylamine remaining in the mother liquor with o-phthalaldehyde (3(TOPO)2 (see 3(TOPO)2-beads without shell, core-shell beads after “methods 1 and 2” and Fluoro-Max in 0.0025% dispersions, where inner filter effects are absent.It is at this stage useful to compare the bead’s properties with commercially available particles. Among the commercial beads, the reference gold standard for our purposes was Fluoro-Max with Eund 32 Å2 . Determialdehyde  (detailsPO)2 see ; Table 1Finally, we should mention that the commercial dispersion may contain larger particle due to agglomeration, as suggested by fluorescence microscopy  beads, with which the very strong bonding to Biotin can be exploited. Neutravidine was chosen for the lower isoelectric point (i.e.p. = 6.4), as Avidin (i.e.p. = 9.4) might give rise to stronger adsorptive interaction rather than covalent coupling to the negatively charged beads and falsify the general picture for proteins with lower, i.e.p. The coupling routine proceeded via EDC-NHS activation as laid out in detail in the ESI and subsequent coupling with the proteins. A parallel experiment was run with non-activated beads onto which the proteins can only be adsorbed rather than covalently linked, thus informing on non-specific interaction, which would compromise the analysis. The number of Neutravidin molecules coupled to the surface was determined by titration with 4′-hydroxyazobenzene-2-carboxylic acid (HABA) as described in the ESI. HABA forms a weak complex with Neutravidin; on addition of Biotin it is released and can be determined photometrically in solution. This analysis showed that roughly 2300 Neutravidin molecules resided on the beads corresponding to 88% coupling efficiency. This number is fairly close to the maximum amount of ca., 2,700 molecules/bead as estimated from the hydrodynamic diameter of 7.4 nm for Neutravidin , even th−4% wt beads , which conservatively recalculates into a 3 × 10−18 mol detection limit and even lower, if less than complete coverage of the bead surface with protein is required—which is most likely. Additionally, the amount of unspecifically adhering protein is between 11% (beads method 1) and 6% (beads method 2) only.We tested two core-shell beads prepared by employing method 1 and method 2 (see previous paragraph). After covalent coupling and (unspecific) adsorptive coupling with Avidin or Neutravidin, to the shelled beads were tested in biotinylated titer plates by time-resolved determination in a Victor 4 spectrometer with time windows set between 300 and 1,100 µs. Last but not least, an important test method in this context is the performance in Lateral Flow Immunoassays. Using the fluorescence microscope, we were able to follow the chromatography in LFIA strips. For this purpose, we used strips having a biotinylated test line to capture the Neutravidin-equipped beads; the more even and intense the response, the more useful the beads will be in LFIA. After “loading” of the strips with beads dispersed in a dedicated flow medium (running buffer), washing and drying, they were inspected microscopically and their relative lightness evaluated . Core-shell beads according to methods 1 and 2 and Fluoro-Max reference beads are reproduced in 3+ with respect to their efficiencies as co-coordinated TOPO-complexes and in polymer beads; a novel aromatic system comprising 3-phenathrytrifluorodieketonate was also included. For methods with additive- and emulsifier-free polymer synthesis we found Eu(ttfa)3(TOPO)2 to be most suitable. We have therefore taken beads with occluded Eu(ttfa)3(TOPO)2 and elaborated two methods for applying shells in order to preserve the cores’ high efficiencies in aqueous dispersions and concurrently to provide anchoring groups for subsequent protein conjugation. The beads thus obtained were exemplarily tested for ELISA-like analyses and for Lateral Flow Immunoassyas; high-end commercial beads were compared as contol. The beads, taking advantage of a large amplification factors and ultimate brightnesses, enabled determinations in the attomol regime and beyond; in Lateral Flow experiments they proved to be superior to existing commercial materials accessible to us. With respect to efficiency, the beads are close to the physical limit. Further improvements in dispersions will presumably be restricted to the optimization of their scattering behaviour.A plethora of rare earth complexes useful for immuno-assays have emerged during the last three decades, often with considerable preparative effort; originally employed, relatively simple β-diketonate complexes have long been outperformed by recent developments. Bottlenecks in the pursuit of suitable complexes were the screening against water and excitation energies compatible with suitable light sources, like high power UV-LEDs. While β-diketonates show a good match with, e.g., present 365 nm LEDs, their functionalization for subsequent conjugation to obtain biomarkers and protection from ambient water at the same time is not a trivial task. This had been realized at an early stage in this research already. Imbeddings into submicron-polymer particles soon proved to be an elegant solution to circumvent the drawbacks. Nowadays the toolbox for immunological beads contains various complexes and strategies for functionalization. We have ventured to directly compare very popular and efficient trifluoro-substituted, aromatic β-diketonates of Eu


Secondary bacterial infections and pneumonia are major mortality causes of respiratory viruses, and the disruption of the upper respiratory tract (URT) microbiota is a crucial component of this process. However, whether this URT dysbiosis associates with the viral species  is unclear.Veillonella was identified as a prominent biomarker for any type of respiratory viral infections. Influenza virus infections are significantly correlated with increased oropharynx microbiota diversity and enrichment of functional metabolic pathways such as L-arginine biosynthesis and tetracycline resistance gene tetW. We used the GRiD algorithm and found the predicted growth rate of common respiratory pathogens was increased upon influenza virus infection, while commensal bacteria, such as Streptococcus infantis and Streptococcus mitis, may act as a colonization resistance to the overgrowth of these pathogens.Here, we recruited 735 outpatients with upper respiratory symptoms, identified the infectious virus types in 349 participants using multiplex RT-PCR, and profiled their upper respiratory microbiome using the 16S ribosomal RNA gene and metagenomic gene sequencing. Microbial and viral data were subsequently used as inputs for multivariate analysis aimed at revealing viral type-specific disruption of the upper respiratory microbiota. We found that the oropharyngeal microbiota shaped by influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), and human rhinovirus (HRV) infections exhibited three distinct patterns of dysbiosis, and We found that respiratory viral infections are linked with viral type-specific disruption of the upper respiratory microbiota, particularly, influenza infections uniquely associated with increased microbial diversity and growth rates of specific pathogens in URT. These findings are essential for clarifying the differences and dynamics of respiratory microbiota in healthy participants and acute respiratory viral infections, which contribute to elucidating the pathogenesis of viral-host-bacterial interactions to provide insights into future studies on effective prevention and treatment of respiratory tract infections.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01597-9. Respiratory viruses, such as influenza (Flu) and human respiratory syncytial virus (RSV), are major threats to global public health . RespiraStreptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae in the lung  pac (v0.55)  package (v0.6.0)  and R st(v0.6.0)  package,(v0.6.0)  package From March to August 2019, a total of 735 individuals with influenza-like symptoms were included in this study. The multiplex RT-PCR was used for FluA, FluB, HRV, or RSV verification  of the FluB (AUC up to 92.64%) group was more extensive than that in the FluA (AUC = 85.51%), RSV (AUC = 85.08%), and HRV (AUC = 89.28%) groups Fig. A, B, indWe analyzed the microbial characteristics of populations with gender, age, BMI, and viral infection types and found that only FluA-positive patients exhibited higher alpha diversity of URT microbiota Fig. A. HowevetetW   algorithm was used to estimate the growth rates of pathogens, including nce Fig. B.Fig. 5Sr| > 0.5, P < 0.05). The results showed that the high growth rate of T. denticola was significantly correlated to formaldehyde assimilation III (dihydroxyacetone cycle) and other pathways, and the high growth rate of T. medium correlates to the enrichment of biotin biosynthesis II, 1,4-dihydroxy-2-naphthoate biosynthesis II, while H. sputorum is related to CMP-3-deoxy-D-manno-octulosonate biosynthesis I  and influenza B infection [S. infantis was positively associated with influenza H1 immunoglobulin A (IgA) titers [Streptococcus may have probiotic potential against respiratory virus infections.In addition to identifying the characteristics of respiratory dysbiosis associated with different viral infections, we further proposed that the increased growth rate of pathogenic bacteria is the crucial process of influenza viral infections, which may directly mediate secondary bacterial infections in the lower respiratory tract. Bioinformatic tools such as MetaPhlAn  and Kraknfection . Among h) titers . These rVeillonella was identified as a prominent biomarker for any type of the four respiratory virus infections. Intriguingly, influenza infections are uniquely associated with enhanced microbial diversity and growth rates of specific pathogens in URT. We proposed that pre-changes in the oropharyngeal microbiome are potentially indicative of respiratory viruses-induced secondary bacterial infections, which provides a better understanding of complications of upper respiratory tract infection. While further investigations are needed to translate these results into potential clinical and public health applications, our findings demonstrated a critical role of microbiome perturbations in bridging the link between upper respiratory viral infections and severe secondary infections.We found that respiratory viral infections correlate to viral type-specific disruption of the upper respiratory microbiota, and Additional file 1: Table S1. List of primers used for multiplex qPCR. Table S2. The differential genera between virus-positive and negative groups. Table S3. In the random forest model with 100 resamplings, the Mean Decrease Accuracy from group 1 and group 2 was used for the Wilcoxon test. Table S4. The Spearman interaction of differential pathways, differential ARGs, ARGs count, and differential species in the FluA group and healthy controls. Table S5. The growth rate of potential pathogens in situ calculated by GRID algorithm. Table S6. The Spearman interaction of all pathways, ARGs, and pathogens.Additional file 2: Figure S1. Multiple qPCR standard curves for FluA, FluB, RSV, and HR. Figure S2. Reads count and proportions of microbiota in sequencing profiles. (A) Microbial reads count in 16S rRNA gene sequencing data. (B) The proportions of microbiota in 16S rRNA gene sequencing data. (C) Microbial and human reads count in metagenomic sequencing data. Figure S3. The variation of microbial composition between virus-positive and negative groups. PCoA based on Bray–Curtis distances was analyzed, and PERMANOVA was used to evaluate the variation. Figure S4. The differential genera, pathway, and ARGs between health and FluA, FluB or FluAB group. (A) Compared to healthy controls, the functional pathways (MetaCyc database) with significant differences in virus infection were calculated. The number and color represent the fold change relative to the control group. Blank blocks indicate no significant difference. (B) The number of antibiotic resistance genes (ARGs) based on metagenomic data was used to evaluate the diversity of ARGs, and the variation between the infected and healthy groups was counted by Kruskal–Wallis test and Wilcoxon test with subsequent Bonferroni correction. (C) Significantly different resistance genes compared to healthy controls were shown. (D) LEfSe was used to calculate the different genera between healthand FluA, FluB, orFluAB participants, respectively. Figure S5. The relative abundance difference of potential pathogens between infected and healthy subjects. (A) Nine pathogenic species (in the blue circle) were identified in the oropharyngeal samples based on metagenomics sequencing analysis. (B) Kruskal-Wallis test and Wilcoxon test was used to calculate the relative abundance difference of nine pathogens between the infected and the healthy subjects. Figure S6. The growth rate of potential pathogens (in the blue circle) was detected based on metagenomics sequencing analysis.


A new novel virus, Langya virus (LayV), was detected in China in August 2022, 3 years after the COVID-19 pandemic. LayV is similar to the previously discovered Mojiang henipavirus. Other zoonotic henipaviruses include the Hendra and Nipah viruses. The emergence of the zoonotic Langya virus is attributed to climate change and wildlife encroachment, as LayV is detected in shrews. Those who are infected in China showed various symptoms, but no deaths have been recorded yet. This review aims to shed light on the current state of Langya virus outbreak, its infection control efforts, and the remaining challenges that need to be addressed to curb the outbreak.We utilized online publication databases such as PubMed, Google Scholar, and Scopus in writing this review article.A surveillance study on thirty-five febrile patients in Eastern China identified the Langya virus outbreak. The current efforts from the Chinese government and health authorities to reduce the transmission and spread of Langya virus such as isolation and characterization of LayV, challenges associated with the increase in cases of LayV, and trackable recommendations such as strengthening the healthcare system in China, sensitization of people about risks associated with Langya virus outbreaks, creating an intensive surveillance system network, etc. were discussed.It is germane and pertinent that the Chinese government and health authorities continue to intensify efforts against Langya virus and address the challenges to effectively reduce transmission. Paramyxoviridae. Studies conducted by a group of scientists show that the LayV genome is closely related to the previously discovered Mojiang henipavirus. Hendra and Nipah viruses are other zoonotic henipaviruses (Chakraborty et al. The world has been struggling to avert the spread of infectious diseases that seriously threaten global health since 2019, following the outbreak of COVID-19 in Wuhan, China (Tabari et al. Langya virus affects multiple systems. Clinical symptoms and signs of the virus include fever, cough, headache, myalgia, nausea, and vomiting, as well as other blood abnormalities including leukopenia and thrombocytopenia. Apart from that, liver and kidney function are also affected (Lee et al. In this article, we discuss the outbreak of the LayV, current efforts of the Chinese government and health authority, challenges encountered, and recommendations to overcome the challenges.The Langya virus is an RNA virus comprising 18,402 nucleotides (Zhang et al. Crocidura lasiura shrews commonly found in Northeast Asia. Out of the 121 tested Crocidura lasiura shrews, 52.1% had a positive result for LayV. The virus is also found in 20% of the Crocidura shantungensis samples, 5% of dogs and 2% of domestic goats surveyed, suggesting multiple potential hosts, with shrews potentially being the natural reservoir of LayV (Fig. The transmission is thought to occur from animals to humans, with no human-to-human transmission reported. A serosurvey of domestic animals found that the viral RNA of LayV was predominantly detected in shrews, specifically the ayV Fig. .Fig. 1TrA surveillance study on thirty-five febrile patients in Eastern China identified the Langya virus outbreak. Further investigation reported that among these thirty-five patients, twenty-six were isolated LayV-positive cases (Sah et al. During the LayV outbreak, researchers characterized the virus genome from one of the symptomatic patients, who are mostly farmers and factory workers (Zhang et al. Currently, very few reports on the Langya virus have reached the public, likely shadowed by the continuous, though subsiding, nationwide attention on COVID-19. Though specific national challenges faced by the Chinese authorities and people remain unknown, other challenges from the limited reports can be identified.Nationally, the coordination framework of Chinese healthcare may hamper the outbreak response. Looking back at the early stages of the COVID-19 pandemic response, inflexible coordination and inadequate national administrative mandates impeded effective control. Local reports were suppressed and redundantly analyzed across the multi-level health system when they did reach the wider community (Mallapaty Locally, health workers and authorities find it difficult to detect the source and prevent the transmission of LayV (Chakraborty et al. Apart from public health challenges, scientifically, the biology of the LayV is still poorly understood despite its similarities to the previously discovered henipaviruses. It is imperative for researchers to gain an understanding of LayV’s molecular structure and characteristics. This is crucial in providing essential information for the development of effective therapy and vaccines against the infection as well as to design preventive measures and controls nationally and globally.Learning from previous outbreaks will allow for more robust emergency responses. Scrutinizing responses to previous henipaviruses outbreaks allow best practices to be identified, which then can be utilized for LayV control.The first case of HeV was discovered on August 1st, 1994 in Australia in a 10-year-old mare (Sun et al. The first outbreak of NiV was detected from 1998 to 1999 in Malaysia and Singapore (Mazzola and Kelly-Cirino A One Health approach was employed during the Hendra and Nipah outbreak to develop preventive measures. The approach is defined as a collaborative and integrated approach to attaining optimal health for people, animals, and the environment. For instance, in Australia, a program to vaccinate horses against the Hendra virus to prevent transmission to humans is currently in place (Manyweathers et al. These approaches should be considered for the control of the Langya virus. However, the One Health approach faces challenges of its own: for instance, the uptake of the 2012 Hendra vaccine has been slow due to concerns about safety, cost, and effectiveness (Manyweathers et al. One Health is a major approach that is being used presently to control zoonotic diseases globally. It is crucial for experts in public health, animal, human, and environmental health as well as other pertinent disciplines and industries to collaborate in order to use a holistic idea to combat LayV in China. We recommend the Chinese government work closely with the China CDC and other stakeholders and employ swift responses based on their recommendations. The reports of redundancy, which was due to miscommunication and underreporting, seen during the COVID-19 pandemic should be avoided by utilizing national emergency notifications. We also recommend the government actively support research, surveillance, monitoring, and health system strengthening. Further recommendations are discussed below to tackle LayV outbreak in a timely manner Fig. .Fig. 2ReStrengthening of healthcare system in China: The healthcare system is one of the major parastatals that are important in preventing and controlling infections. This includes the provision of adequate personal protective equipment to healthcare workers, the education and training of professionals involved in handling and managing samples, and improving health system capacity.Sensitization of people about risks associated with LayV outbreak: Community health workers should be engaged in educating people on the effects of wildlife encroachment, climate change, disease prevention and control, and one health approach and its importance. This will help in pre-informing the public about risks associated with LayV outbreaks and will prevent further spread of the disease.Creation of an intensive surveillance system network in China: The health authorities should create a stronger community-based LayV surveillance network within the country. This will reduce the transmission of the virus and may also reduce other infectious diseases in China. Other countries in the southeastern part of Asia will also benefit positively from the surveillance network to keep track of henipaviruses outbreaks.Provision of immune boosters by the health practitioners: During this period of LayV outbreak, health practitioners should include immune boosters such as zinc, selenium, vitamin C and D in the supportive care given to the patient with LayV to alleviate the progression of the disease. A study reported that immunocompromised patients infected with LayV showed more severe symptoms compared to non-immunocompromised patients (Shakoor et al. Engagement in long-term LayV research: We recommend more research on the mode of transmission of LayV. Researchers can investigate already discovered companion viruses, HeV and NiV, to know more about LayV. This will contribute to the development of specific preventive measures against LayV. After the intensive research work on the characterization of LayV, pathophysiology studies, and vaccine research should not be left behind. Vaccine development should be considered. At present, a clinical trial is being conducted by the National Institute of Allergy and Infectious Disease to evaluate a Nipah vaccine’s ability to prevent infection (Sah et al. Enforcement of strict regulatory measures on wild encroachment: As the virus was identified and isolated in shrews, the government should enact strict regulatory measures for wildlife hunting to prevent further LayV outbreaks and the potential outbreak of another novel virus. As the source of the virus remains unknown, the reservoir of the virus should also be closely regulated.Monitoring and screening of live animal markets for LayV: We recommend that animal markets should be carefully monitored in cities and in rural communities where farmers may practice subsistence agriculture. In addition, during this outbreak, the public should avoid places where shrews, the currently identified reservoir, are located, such as markets, zoos, and game reserves. Apart from China, all neighboring countries should monitor the spread of disease on the border to prevent regional transmission of LayV.The current efforts from the Chinese government and health authorities to reduce the transmission and spread of LayV such as isolation and characterization of LayV are in place but not enough to eradicate the virus. Trackable recommendations such as strengthening the healthcare system in China, sensitization of people about risks associated with LayV outbreaks, creating an intensive surveillance system network, provision of immune boosters by health practitioners, engaging in long-term LayV research, enforcement of strict regulatory measures on wild encroachment, and monitoring and screening of live animal markets for LayV are provided in this piece to intensify the current efforts provided by the Chinese government and health authorities. It is crucial and germane for the Chinese government and health authorities to continue intensifying efforts with the recommendations provided in this piece and previous lessons learned from the control of other henipaviruses, to effectively reduce the spreading of the Langya virus and avoid further regional outbreaks.


Considering the current global crisis of COVID-19, another viral pandemic might exacerbate concerns as well as the healthcare and socioeconomic repercussions. The present outbreak of monkeypox on a worldwide scale is unprecedented. Monkeypox is a zoonotic viral disease caused by a double-stranded DNA virus MPXV of the orthopox genus. This was first identified as a pox-like viral disease in monkeys in 1958, and the first case of human monkeypox was detected in the Democratic Republic of the Congo in 1970. Since then, occasional cases and minor outbreaks have been reported in non-endemic countries, with their re-emergence in 2022. On July 23, 2022, the World Health Organization (WHO) declared monkeypox a global public health emergency.Shah et al. delineates the journey of monkeypox since the first human case, focusing on the reasons for the growing instances and methods to prevent the virus from spreading. The evolution of MPXV is governed by positive Darwinian selection that drives the adaptation of host interaction . The study using 156 coding genes encompassing more than 95% of the MPXV genome isolated from 1,500 samples found that the genes involved in host interaction and host determination  evolved under positive Darwininan selection. Three recently reported missense substitution mutations  were found to be critical for the virus’ adaptation to humans. Thus, monitoring the mutational landscape of the viral genes, particularly those undergoing positive selection for predicting viral transmission and virulence, is crucial.A perspective review by Khattak et al.; Rampogu et al.). A meta-analysis by Rani et al. confirms that skin lesions contain the viral DNA that raises the risk of infection and potential transmission via direct skin-to-skin contact. The virus can spread to people by contact with the body fluids, skin lesions, and droplets of infected animals. Additionally, eating undercooked meat, being exposed to contaminated fomites, infected animals, eating bush meat, or wild game may all result in animal-to-human transmission. Human-to-human transmission can also occur when patients share the same household and consumables, or through healthcare contact .As there are not many treatment options available for monkeypox disease, understanding the mechanisms of transmission and symptoms might help prevent the disease’s spread. Common disease symptoms include fever, swollen lymph nodes, muscular pains, headache, backache, and weariness. The appearance of rashes with blisters and crusts follows these symptoms. Although the symptoms typically develop 10 to 14 days after infection, they can appear up to 21 days. One to four days following the prodrome, there is a severe, deep-seated, vesicular, or pustular rash with central distribution. The face is most commonly afflicted, followed by the palms, soles, oral mucous membrane, and occasionally the genitalia and conjunctivae . Unfortunately, most of these techniques are equivocal and cannot distinguish MPXV disease from other poxvirus infections. However, an accurate diagnosis can be made via PCR analysis. A DNA oligonucleotide microarray that involves the TNF receptor gene crmB has been designed as an additional rapid method for identifying orthopoxviruses .Identifying a disease at the right time is crucial for prevention and treatment. There are several methods available for the diagnosis of MPXV that include assays for IgM and IgG, Enzyme-Linked Immunosorbent Assay (ELISA), Polymerase Chain Reaction (PCR), electron microscopy, virus isolation, Immunofluorescence antibody test, and histopathology . They identified twelve possible natural curcumin compounds with antiviral potential. Al Mashud et al. conducted a similar in silico investigation to evaluate the ability of O-rhamnosides and Kaempferol-o-rhamnosides derivatives to inhibit MPXV and Marburg virus. They discovered that the compounds L07, L08, and L09 exhibited better affinity for the target protein than the FDA-approved antiviral drug Cidofovir, suggesting their potential for antiviral treatment against MPXV and Marburg virus. However, before these molecules can be utilized as drugs, they must be thoroughly investigated in real-world situations and put through clinical trials.Fast-track therapeutic development is crucial for combating a disease with a high transmission rate. To this end, computational drug discovery and repurposing of existing drugs could be a viable tool for developing therapies against MPXV. In an attempt to identify a potential drug against the virus, Khattak et al.). However, the earlier generation of ACAM2000 may also be used off-label for the same purpose. Vaccinia immune globulin may be alternative post-exposure preventive measures if smallpox vaccination is unavailable. Translation of a novel drug or vaccine from laboratory to clinic requires time and should be tested in clinical trials. According to the study by Alorfi et al., only 10 interventional trials are registered at ClinicalTrials.gov. Of these, 4 trials were registered in Europe, 3 in America, and 3 in Africa. Forty percent of the registered trials were conducted for the JYNNEOS vaccine and 30% for Tecovirimat. The effectiveness and safety of the medications and vaccinations used to treat MPXV must thus be evaluated immediately through large-scale randomized clinical trials. Global concerted efforts must be made continuously to prepare for another epidemic.Vaccines are the most potent protective measures against viral infections. The smallpox vaccines are effective against MPXV. The ACAM2000, a newer-generation smallpox vaccination, has received FDA approval for treating MPXV (MH: Writing – original draft, Writing – review & editing. SS: Writing – review & editing. SK: Writing – original draft, Writing – review & editing.