Is the amount of physical and occupational therapy received by patients with pneumonia associated with 30-day hospital readmission or death?In this cohort study of 30 746 patients with pneumonia or influenza-related conditions discharged from 12 acute care hospitals in western Pennsylvania, there was a significant inverse association between the amount of therapy received and the risk of 30-day hospital readmission or death.In this study, the amount of therapy received by patients with pneumonia or influenza-related conditions in the acute care setting was associated with decreases in the risk of 30-day hospital readmission or death. This cohort study evaluates whether the use of physical and occupational therapy in the acute care hospital is associated with 30-day hospital readmission risk or death. Pneumonia often leads to functional decline during and after hospitalization and is a leading cause of hospital readmissions. Physical and occupational therapists help improve functional mobility and may be of help in this population.To evaluate whether use of physical and occupational therapy in the acute care hospital is associated with 30-day hospital readmission risk or death.This cohort study included the electronic health records and administrative claims data of 30 746 adults discharged alive with a primary or secondary diagnosis of pneumonia or influenza-related conditions from January 1, 2016, to March 30, 2018. Patients were treated at 12 acute care hospitals in a large health care system in western Pennsylvania. Data for this study were analyzed from September 2019 through March 2020.Number of physical and occupational therapy visits during the acute care stay categorized as none, low (1-3), medium (4-6), or high (>6).Outcomes were 30-day hospital readmission or death. Generalized linear mixed models were estimated to examine the association of therapy use and outcomes, controlling for patient demographic and clinical characteristics. Subgroup analyses were conducted for patients older than 65 years, for patients with low functional mobility scores, for patients discharged to the community, and for patients discharged to a post–acute care facility .Of 30 746 patients, 15 507 (50.4%) were men, 26 198 (85.2%) were White individuals, and the mean (SD) age was 67.1 (17.4) years. The 30-day readmission rate was 18.4% (5645 patients), the 30-day death rate was 3.7% (1146 patients), and the rate of either outcome was 19.7% (6066 patients). Relative to no therapy visits, the risk of 30-day readmission or death decreased as therapy visits increased . The association was stronger in the subgroup with low functional mobility and in individuals discharged to a community setting.In this study, the number of therapy visits received was inversely associated with the risk of readmission or death. The association was stronger in the subgroups of patients with greater mobility limitations and those discharged to the community. Current guidelines for the treatment of pneumonia primarily focus on diagnostic guidelines and pharmacologic interventions24 and do not address early mobility among patients hospitalized with pneumonia.Physical and occupational therapists play key roles in promoting mobility and improving functional status in the acute care setting. They also assist with discharge planning by determining the most appropriate postacute setting for patients leaving the hospital with limitations in physical function and ADLs. Finally, they provide patient and family education on rehabilitation care after discharge. While the evidence base for the effectiveness of physical and occupational therapy in the acute care setting is fairly robust for the treatment of conditions such as stroke, hip fracture, and joint replacement,22 In a retrospective cohort study of adults hospitalized with pneumonia and declining physical function, Kim et al25 reported that patients receiving more physical therapy per day  had a lower risk of 30-day readmission. In a systematic review of 4 studies, Larsen et al26 found that early mobilization by physical therapists (PTs) decreased length of stay for individuals with pneumonia, but had no association with mortality or hospital readmissions. The authors did acknowledge the limited breadth of their review and the need for further research in this area.Limited evidence suggests that early mobilization and rehabilitation decrease morbidity and mortality in patients hospitalized with pneumonia.Our study attempts to add to the evidence base on early mobility for patients with pneumonia. Our objective was to determine whether the amount of physical and occupational therapy received by adults hospitalized with pneumonia or influenza-related conditions is associated with the risk of 30-day readmission or death. We hypothesized that the amount of therapy received would be inversely associated with the risk of readmission or death.STROBE) reporting guideline.We examined electronic health records and administrative claims data from a large health care system in western Pennsylvania. These data were supplemented with US census data to obtain a measure of median household income at the patient zip code and with the Social Security Death Index database to identify individuals who died during follow-up. This study was reviewed and classified as exempt by the University of Pittsburgh’s institutional review board; patient consent was waived by the review board. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  codes  of electronic health records and administrative claims data from a large health care system in western Pennsylvania. We identified adults aged 18 years or older who were discharged from 1 of 12 acute care hospitals with a primary or secondary diagnosis of pneumonia or influenza-related conditions based on We examined billing data to identify the number of PT and occupational therapist (OT) visits during the patient’s inpatient stay. For analytic purposes, we summed the number of therapist visits and created a 4-level categorical variable: no visits, 1 to 3 visits (low), 4 to 6 visits (medium), and more than 6 visits (high). We categorized the number of visits based on the tertile distribution of the data from patients who had at least 1 therapist visit. We also created dichotomous variables to indicate whether only a PT saw the patient, only an OT saw the patient, or both therapist types (PT and OT) saw the patient.27 Death outside the health system may have also been secondary to readmission to another hospital outside the system.We created a dichotomous outcome variable to indicate within-system hospital readmission within 30 days of discharge or death within 30 days of discharge with or without an in-system readmission. We chose this composite outcome to address the competing risk of death with hospital readmission.23 illness severity,23 comorbidities, discharge destination, and measures of mobility and activity). The risk of mortality and illness severity measures were derived from the All Patient Refined Diagnosis Related Group (APR-DRG) algorithm. The algorithm uses a set of diagnosis and procedure codes to come up with a DRG as well as severity of illness and risk of mortality modifiers.29 Severity of illness is defined as the extent to which organ systems lose function or have physiologic decompensation; it is categorized as minor, moderate, major, and extreme.30 Risk of mortality is a measure of the likelihood of in-hospital mortality based on secondary diagnosis, age, principal diagnosis, and certain procedure codes.11 APR-DRGs are calculated for all hospitalizations and have traditionally been used by health systems for cost adjustment, but they are also used by researchers to risk-adjust claims data.37 We also calculated the Elixhauser comorbidity index24 and created indicator variables for the following diagnoses: arrhythmia, pulmonary circulatory disease, neurological disease, kidney failure, liver disease, cancer, coagulopathy, obesity, and weight loss. Discharge destination was classified as home, home with home health, skilled nursing facility, or inpatient rehabilitation facility. Mobility and activity were measured at the time of hospital admission by nursing staff using the 6-Clicks Activity Measure for Postacute Care (AM-PAC). The AM-PAC 6-Clicks, which has established reliability and validity,40 consists of 6 questions regarding patient mobility  and 6 questions regarding patient activity  and is scored on a scale from 6 to 24, with lower scores indicating more difficulty performing the tasks. We created categorical variables for the AM-PAC scores based on their tertile distribution. Complete data variable definitions are provided in eTable 2 in the We created several covariates to represent demographic characteristics  and clinical characteristics , we excluded the AM-PAC activity data from our analysis. We also excluded the severity of illness measure because it was highly correlated with the risk of mortality measure  and moderately correlated with the Elixhauser index . A small amount of data was missing (<5%) for the following measures included in our models: AM-PAC mobility, race, income, and risk of mortality. We imputed mean values for the missing data.2 tests to assess difference in the point estimates of our exposure categories.We first estimated a model to examine the association between therapy use and 30-day readmission or death, controlling for patient demographic and clinical characteristics. We also estimated this model for the following subgroups: individuals older than 65 years; individuals with low mobility scores ; individuals discharged to the community; and individuals discharged to a post–acute care facility . We used χP < .05, and all tests were 2-tailed. All analyses were conducted using Stata version 16 (StataCorp).We conducted several sensitivity analyses. We performed the analysis on the subgroup of individuals who survived for the first 30 days after discharge and on the subgroup of individuals who had complete data. We also conducted an analysis with total visits categorized by quartiles. Finally, we conducted a time-to-event analysis using a Cox regression model. Statistical signficance was set at Our sample consisted of 30 746 adults with pneumonia or influenza-related conditions who survived their inpatient stay . Rates oA total of 12 058 patients (39.2%) were discharged home, 8303 (27.0%) were discharged home with home health, 8386 (27.3%) were discharged to skilled nursing facilities, and 1999 (6.5%) were discharged to inpatient rehabilitation. Overall, 5645 patients (18.4%) had an in-system hospital readmission in 30 days, 1146 patients (3.7%) died within 30 days, and 6066 (19.7%) had an in-system hospital readmission or died within 30 days.2 = 11.25; P = .003). Being seen by both a physical and occupational therapist was not significant for the lower mobility subgroup. The associations among patients older than 65 years followed a pattern similar to the full sample model, but the odds ratios crossed the null. Our full sample model results are presented in eTable 3 in the 2 = 16.2; P < .001). Therapy visits were not associated with readmission or death in the subgroup of patients discharged to a post–acute care facility, although being seen by both a PT and OT in the acute care setting decreased risk of readmission or death .When limiting the sample to individuals who survived the first 30 days or individuals with complete data, we found similar results for therapy visits and for seeing both a physical and occupational therapist, although the findings were not significant in some instances .Several studies have reported an association between functional decline in the acute care hospital and adverse health outcomes, including hospital readmission.Our findings also suggest that it is the number of therapy visits received, more than the types of therapists seen, that was associated with the risk of readmission or death. In some models, being seen by a PT and OT in the acute care setting decreased the risk of readmission or death compared with only being seen by 1 therapist type. Two therapists working on the patient’s case may facilitate a discharge that is more appropriate for the patient’s needs. Furthermore, because OTs tend to focus more on ADLs, while PTs tend to focus more on mobility , there is likely added benefit in addressing both areas.40 recorded by nursing staff. Unfortunately, we did not have mobility measures at hospital discharge. As others have reported, patients with impaired mobility in our study were more likely to be readmitted, independent of therapist visits (eTable 3 in the Our work adds to the evidence on the effectiveness of therapy in the acute care setting for the treatment of pneumonia and influenza-related conditions, medical diagnoses that are less studied than neurologic and orthopedic diagnoses typically treated by therapists. A strength of our study is that we controlled for the degree of difficulty patients had with mobility at hospital admission using AM-PAC scores46 therapy administrators in acute care settings are often challenged to appropriately allocate therapist time. Studies such as this may help inform the prioritization of patients and increase the value add of therapy, which is sometimes viewed by acute care hospital administrators as a cost center only.Our findings have policy relevance given that pneumonia is among the qualifying diagnoses in the Center for Medicare & Medicaid Services Hospital Readmission Reduction Program, which penalizes hospitals for excess 30-day, unplanned readmissions. Due to the general undersupply of OTs and PTs in US acute care hospitals and high turnover rates,This study has several limitations. We examined care in 1 health system in western Pennsylvania, which limits generalizability. We also only captured within-system readmissions. While we were able to capture out-of-system deaths, which may have been preceded by an out-of-system readmission, we did not capture out-of-system readmissions. Our manner of identifying pneumonia patients may also limit generalizability or comparisons with other studies because we included some influenza-related diagnoses. We also only examined all-cause readmissions and not those considered unplanned. Nor did we identify the type of care delivered by therapists. Finally, our study design did not allow for conclusions on causality. Future studies should examine other health systems and geographic populations and explore the amount of time and the specific activities performed during each therapy visit.In this study, we examined the association between therapy visits and the risk of 30-day readmission or death in adults hospitalized with a diagnosis of pneumonia or influenza-related conditions. We found that the number of therapy visits received was inversely associated with the risk of readmission or death. This association was greater in the subgroups of patients with lower mobility and patients discharged to the community.


The American Academy of Orthopaedic Surgeons and American Society for Surgery of the Hand recently proposed three quality measures for carpal tunnel syndrome (CTS): Measure 1 - Discouraging routine use of Magnetic resonance imaging (MRI) for diagnosis of CTS; Measure 2 - Discouraging the use of adjunctive surgical procedures during carpal tunnel release (CTR); and Measure 3 - Discouraging the routine use of occupational and/or physical therapy after CTR. The goal of this study were to 1) Assess the feasibility of using the specifications to calculate the measures in real-world healthcare data and identify aspects of the specifications that might be clarified or improved; 2) Determine if the measures identify important variation in treatment quality that justifies expending resources for their further development and implementation; 3) Assess the facility- and surgeon-level reliability of measures.The measures were calculated using national data from the Veterans Health Administration (VA) Corporate Data Warehouse for three fiscal years . Facility- and surgeon-level performance and reliability were examined. To expand the testing context, the measures were also tested using data from an academic medical center.The denominator of Measure 1 was 132,049 VA patients newly diagnosed with CTS. The denominators of Measures 2 and 3 were 20,813 CTRs received by VA patients. The median facility-level performances on the three measures were 96.5, 100, and 94.7%, respectively. Of 130 VA facilities, none had < 90% performance on Measure 1. Among 111 facilities that performed CTRs, only 1 facility had < 90% performance on Measure 2. In contrast, 21 facilities (18.9%) and 333 surgeons (17.8%) had lower than 90% performance on Measure 3 (Median facility- and surgeon-level reliability for Measure 3 were very high (0.95 and 0.96 respectively).Measure 3 displayed adequate facility- and surgeon-level variability and reliability to justify its use for quality monitoring and improvement purposes. Measures 1 and 2 lacked quality gaps, suggesting they should not be implemented in VA and need to be tested in other healthcare settings. Opportunities exist to refine the specifications of Measure 3 to ensure that different organizations calculate the measure in the same way. Carpal tunnel syndrome (CTS) involves compression of the median nerve at the wrist and can cause debilitating symptoms, such as pain, numbness or tingling in the fingers, loss of sleep, and thumb weakness . Among eIn 2016, the American Academy of Orthopaedic Surgeons (AAOS) published the “Management of Carpal Tunnel Syndrome Evidence-Based Clinical Practice Guideline” . When heRecognizing the need to operationalize consensus standards of care for CTS into feasible and valid quality measures, the ASSH/AAOS Carpal Tunnel Quality Measures Workgroup was convened in January 2017 to review the clinical practice guidelines with the purpose of selecting quality concepts and drafting initial specifications of process-oriented quality measures [Table Once quality measures have been proposed and initial specifications have been developed, it is essential to pilot test them in real-world healthcare data at the intended levels of measurement . Pilot testing of quality measures has several goals including: (a) determining the feasibility of using the measure specifications and available data elements to calculate the measures in healthcare data from diverse settings, and relatedly, identifying aspects of the specification that might be improved or clarified ; (b) assInitial specification and pilot testing of Measures 1–3 was done in 2012–2014 Truven Marketscan data which precluded testing of International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) versions of the measures, and did not include facility or surgeon identifiers, making it impossible to assess if meaningful variation exists between the units to which the measures will be applied  .The goal of this study was to test if the three CTS quality measures selected by ASSH/AAOS Carpal Tunnel Quality Measures Workgroup might be useful for monitoring and improving the quality of CTS treatment. We conducted measure testing in data from the Veterans Health Administration (VA), the largest publicly-funded integrated healthcare system in the US, and secondarily in a private academic medical center. Specifically, we sought to 1) Identify aspects of the specifications that might be clarified or improved; 2) Determine if the measures identify variation in treatment quality that justifies expending resources for their further development and implementation; 3) Assess the facility- and surgeon-level reliability of measures.All three measures were operationalized according to the AAOS/ASSH specifications  Table 11 using nFor measures with adequate variability, facility- and surgeon-level reliability analysis was conducted. Reliability in this context refers to how well a measure distinguishes real differences in quality between measurement units  in the presence of measurement error (noise), and is characterized with beta-binomial signal-to-noise ratio . This isIn order to test the measures in a different healthcare context, they were also calculated for 2014–2016 Stanford Health Care (SHC) data using the STAnford medicine Research data Repository (STARR), a clinical data warehouse containing live electronic medical records, including all diagnoses, procedures, and encounter-level patient data. Overall measure performance was calculated, but SHC surgeon identifiers were not available.As summarized in Table The denominator of the measure is all patients receiving CTR during the measurement period. The numerator was the number of patients who did not receive at least one of the adjunctive treatments at the same time.The denominator of the measure is all patients receiving CTR during the measurement period. The numerator was the number of patients who did not receive in-clinic postoperative hand physical therapy or occupational therapy (OT/PT) within 6 weeks of CTR. To address concerns that the OT/PT visits were for something unrelated to CTS or CTR, we also pilot tested a version of Measure 3 that required that the OT/PT visit include a CTS diagnosis.Of 132,049 VA patients who were diagnosed with CTS in FY16–18, 5066 (3.8%) received an upper extremity MRI (nationwide Measure 1 performance = 96.2%). Performance on Measure 1 across 130 VA facilities ranged from 90 to 100%  per measurement period, corresponding to their first CTS diagnosis. If the intention is to focus on the initial diagnosis of CTS, then a diagnosis-free period before the index encounter should be required. The ICD-10-CM code G56.03  should be included in the specifications. Also, as some VA patients were diagnosed with CTS initially as inpatients, the specification should be specific about whether inpatient diagnoses should be included.Of 20,813 VA patients who received carpal tunnel release in FY16–18, only 31 received an adjunctive procedure  CTR per patient or if all CTRs should be included. For pilot testing, we included the first CTR per patient in each reporting period. It also important to note that measure statement is intended to discourage “internal neurolysis using operating microscope”, but code 64727 is not that specific: “Neuroplasty  Procedures on the Extracranial Nerves, Peripheral Nerves, and Autonomic Nervous System”. Similarly, the measure statement discourages “radical nine-tendon flexor synovectomy” but code 25115 is less specific “Radical excision of bursa, synovia of wrist, or forearm tendon sheaths ”. Therefore, the measures are capturing a somewhat broader range of procedures than suggested by the measure description.Of 20,813 VA patients who received carpal tunnel release, 1814 patients received in-clinic OT/PT in the postoperative 6 weeks after CTR ; 0.95  for facilities with at least 20 cases; and 0.95  for facilities with at least 50 cases. Median surgeon-level reliability for Measure 3 was 0.99 overall ; 0.96  for surgeons with at least 5 cases; and 0.95  for surgeons with at least 30 cases.The median facility- level reliability for Measure 3 was 0.95 overall , and reveal a compelling quality gap that needs to be addressed. In evaluating three recently proposed measures of CTS treatment quality in a national publicly-funded healthcare system and a single private academic healthcare system, we found several aspects of the specifications that can be clarified or improved. Revealing opportunities to improve measure specifications is among the most important reasons to test measures in real-world healthcare data. In VA, we also found that the measures were feasible to implement at the facility and surgeon levels. Although we could not obtain surgeon identifiers in SHC data for research purposes, these data would be available for operational and quality monitoring efforts.Careful pilot testing of quality measures also provides an opportunity to appreciate the inherent tension between accuracy and feasibility when measure developers try to operationalize clinical evidence with billing codes. As noted above, the codes used to capture “internal neurolysis using operating microscope” and “radical nine-tendon flexor synovectomy” capture a somewhat broader range of procedures. Also, because procedure codes do not carry information regarding the indication for the procedure, it is unknown if the MRIs captured by Measure 1 were for CTS diagnosis or something else. While this is a well-known, and to some extent unavoidable, limitation of quality measure design, it is always important to evaluate how much the decisions to enhance feasibility distort the underlying evidence and clinical practice guidelines. Some noise is acceptable as long as it is more or less randomly distributed among accountable entities .Of the three proposed measures, only Measure 3 displayed adequate facility- and surgeon-level variability to justify its use for quality monitoring and improvement purposes, at least in the VA and SHC. The extent to which these results generalize to other settings is unknown. The overall facility- and surgeon-level reliability for Measure 3 was excellent. Limiting the measure to surgeons with some minimum number of cases  in the measurement period improved the range of reliability.The underlying rationale for Measure 3 is to avoid routine use of postoperative in-clinic, instead of unsupervised, OT/PT. As with many quality measures, a baseline rate of OT/PT use is expected and will be warranted in some cases . A measure’s usefulness comes from identifying facilities or surgeons whose routine practice is suboptimal given the underlying evidence and the performance of their peers. For example, Fig. At least in the healthcare systems studied here, Measures 1 and 2 did not identify significant quality gaps that need to be addressed, and may not be useful as quality measures. Its good news that these clinical practice guidelines are already being implemented to a high degree. It is possible that testing in other data sources and health care systems would reveal lower performance and/or more variability. These results also suggest that future quality measure workgroups should focus on processes of care that not only have strong evidence and are feasible to specify, but that also have more undesirable variability in practice. The purpose of quality measures is to drive improvement, which presupposes room to improve.The most important limitation in our evaluation is the reliance on data from only two healthcare systems, making unknown the generalizability of these results. The main reason we supplemented our VA analysis with data from a private academic institution was to explore whether differences exist between public and privately funded systems. While the results were very similar, Stanford is only one institution. The prior testing of these measures in Truven Marketscan data was unable to examine variability at the facility and surgeon levels, however, the overall estimates in performance for Measures 1–3 were 99, 98, and 86% respectively - consistent with our results . NonetheWe found that Measure 3  displayed adequate facility- and surgeon-level variability and reliability to justify its use for monitoring and quality improvement purposes. Measures 1 and 2 displayed lack of a quality gap, suggesting they should not be implemented unless their importance and measurement characteristics can be demonstrated in other healthcare systems. Opportunities exist to refine the specifications of Measure 3 to ensure different organizations calculate the measure in the same way. The results of this study highlight the importance of testing proposed quality measures in real-world healthcare data before widespread implementation.


The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including real-time polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam.Seven positive confirmed samples  were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE.Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe .We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam. Asfarviridae and genus Asfivirus [African swine fever (ASF) is a pernicious and devastating disease of the world swine industry. It has been demonstrated that this disease is caused by an enveloped DNA virus, and this virus replicates in the cytoplasm of infected cells in pigs ,2. It issfivirus . Many prAdditionally, as a highly contagious virus, pigs exposure to African swine fever virus (ASFV) resulted in up to 100% of morbidity in pigs, and the mortality of ASF depended on the virulence of the virus, the host, and transmission cycles ,5. The fUntil now, a vaccine or treatment is not yet available for ASFV. Therefore, rapid and reliable diagnosis plays a vital role in control measures to reduce the spread of ASFV ,7. AccorAdditionally, the HAD test is “gold standard” method and a positive HAD test is definitive for ASFV diagnosis. Although, virus isolation in combination with HAD test using porcine alveolar macrophages (PAMs) cells is a prestigious method for ASFV diagnosis, the performance of this test requires excellent laboratory skills and takes more than a week to obtain the result ,7,8. OneHowever, recent studies on real-time PCR system has shown that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false negative result. It was indicated that a single mismatch on primer or probe binding site causes false-negative results, and up to 33% were recognized . Mainly,In the current study, a novel probe with slight modification based on our data and some references have been tested in comparison with the original OIE recommended probe to bring the best understanding underlying the adverse effects of a mismatch in probe binding site on accurate diagnosis of ASFV in field samples in Vietnam.The study was conducted in compliance with the institutional rules for the care and use of laboratory animals and using a protocol approved by the Ministry of Agriculture and Rural Development (MARD) Vietnam (TCVN 8402:2010).2 and adherent cell populations were selected after overnight culture according to the OIE guidelines.Primary PAMs were collected from the lung of pigs cultured in RPMI 1640 medium  supplemented with 10% fetal cattle serum, at 37°C with 5% CO2, the first passage virus was harvested by freezing and thawing. For the second passage, 50 μL of this virus harvest was added to 2 mL fresh PAMs, then incubated and harvested as described for the first passage. For the third passage, 50 μL of this virus harvest were added to 2 mL fresh PAMs, then incubated at 37°C, in an atmosphere with 5% CO2. After one-day incubation, a 20-μL preparation of 1% homologous red blood cells (RBC) in buffered saline was added to each well. The plates were examined for HAD and cytopathic effects over four days in PAM cells according to the recommendation by OIE [Seven ASFV positive spleen samples checked by conventional PCR using PPA1/PPA2 primer sets were further examined by virus isolation in combination with haemadsorption (HAD) assay, and subsequently confirmed by conventional PCR and sequencing analysis were used for this study. Briefly, these cells were obtained and resuspended in Dulbecco’s modified Eagle’s medium  supplemented with streptomycin , ampicillin  and 5% fetal calf serum  to a final concentration of 1×106 cells/mL. Six-well culture plates , containing 2 mL of the cell suspension, were inoculated with 50 μL of a clarified 10% spleen homogenate. After three days of incubation at 37°C, in an atmosphere with 5% COASFV genomic DNA was extracted by using the QIAamp DNA Mini Kit  from cell supernatants or spleen homogenate. Conventional PCR was performed with specific primers, PPA1/PPA2, and King’s primers as shown in The real-time PCR was carried out on an Agilent AriaMx Real-Time PCR System  according to the OIE-recommended procedure described in King et al  using tw5.5 to 106.5 HAD50/mL. To verify these data, we performed conventional PCR and then sequencing analysis using PPA1 and PPA2 primers to confirm positive HAD as recommended by the OIE. These results are shown in Seven positive samples checked by conventional PCR (data do not show) were further analyzed by virus isolation combined with HAD test according to OIE protocol. Subsequently, positive HAD was verified by conventional PCR and sequencing analysis using PPA1 and PPA2 primers. The results are shown in To recognize the mutation in probe binding site, seven positive confirmed samples were further checked by sequencing analysis using King’s primer set. The region sequences for probe binding are shown in A new degenerate probe based on our data and some references was designed as seen in ASF is a highly contagious viral disease of swine, and the mortality and morbidity were up to 100% in domestic pigs . ASF is Real-time PCR and conventional PCR assay are the most widely used assay and also recommended by OIE for virological and molecular diagnosis of ASF ,8. Both Additionally, it indicated that the lower sensitivity of the OIE-recommended probe or virulent of ASFV might be due to the presence of a nucleotide mismatch of the probe with the target sequences of some ASFV isolates, which is consistent with what we presented in our sequence alignment. As shown in In summary, we improved successfully the real-time PCR system by probe modification to enable the detection of the circulating ASFV strains in Vietnam. The novel modified probe based on our current data and some references in GenBank has shown higher sensitivity than the original one, recognizing clinical ASFVs with and without a mutation in probe binding regions. Additionally, a combination of the real-time PCR with the OIE modified probe and the virus isolation in PAMs cell with HAD test is powerful tool for rapid and reliable diagnosis as well as genomic surveillance in Vietnam.


The purpose of this study was to identify genetic variants on chromosome X associated with intraocular pressure (IOP) and determine if they possess any sex-specific effects.Association analyses were performed across chromosome X using 102,407 participants from the UK Biobank. Replication and validation analyses were conducted in an additional 6599 participants from the EPIC-Norfolk cohort, and an independent 331,682 participants from the UK Biobank.P < 5 × 10−8), located within or near the following genes: MXRA5 , GPM6B , NDP, and EFHC2 . Alleles associated with increased IOP were also associated with increased risk for primary open-angle glaucoma in an independent sample. Finally, our results indicate that chromosome X genetics most likely do not illicit sex-specific effects on IOP.We identified three loci associated with IOP at genomewide significance (In this study, we report the results of genomewide levels of association of three loci on chromosome X with IOP, and provide a framework to include chromosome X in large-scale genomewide association analyses for complex phenotypes. Reduced IOP is associated with estrogen treatment in post-menopausal womenThe unique nature of chromosome X's sex-specific ploidy and differences in the gene expression mechanisms make it an ideal candidate when investigating traits that have, or may have, differences between sexes. However, variants on chromosome X are regularly excluded from GWAS analysesAll participants provided full informed consent in accordance with ethical approval granted and overseen by the UK Biobank Ethics and Governance Council. Inclusion and exclusion criteria for participants have been described in a previous study,,,,Participants were measured once per eye using the Ocular Response Analyzer . Of the two measures of IOP provided by the ORA, we selected the IOPcc measure as it is believed to be a more accurate representation of true IOP, less influenced by corneal properties. The mean of right and left eyes was used as the outcome variable for participants with information for both eyes. If information was only available for a single eye, that measure was used for that individual. POAG disease status was determined using a combination of International Classification of Diseases (ICD-10) codes and self-reported answers to the question “Has a doctor told you that you have any of the following problems with your eyes? (You can select more than one answer)” with “Glaucoma” as one of the possible options. IOP measures were adjusted for participants treated with nonsurgical IOP-lowering medications by dividing the measured IOP by 0.7 (the mean IOP reduction achieved by medication),,Full details for DNA extraction, genotyping, and imputation procedures have been fully described elsewhere.IOPcc was used as the outcome variable in the initial male/female combined analysis in a linear mixed model regression, with an additive model assumption for allelic effects. Male genotypes were coded as homozygous diploid to account for dosage compensation. Adjustments were made for age, sex, and the first five principal components, in addition to a random effects variable to correct for cryptic relatedness and population structure. All IOPcc association analyses in the UK Biobank cohort were performed using BOLT-LMM (version 2.3.2).SNP heritability was calculated using a restricted maximum likelihood (REML) analysis with the GCTA software.,Conditional analysis was performed using the GCTA software.,–The Norfolk arm of the European Prospective Investigation into Cancer (EPIC) cohort formed the replication cohort for this study. The recruitment, phenotypic assessment, and genotyping procedures of this cohort have been previously described.P = 2.7 × 10−9). A total of 590,896 genotyped and imputed SNPs were tested for association  explained by directly genotyped SNPs  on chromosome X was 0.7%  overlaps with the genomic region coding for the matrix remodeling associated 5 (MXRA5) gene. Evidence for association has previously been identified between MXRA5 and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis,MXRA5 is expressed in both adult and fetal samples of multiple ocular tissues: trabecular meshwork, cornea, and ciliary body.MXRA5 was measured in the trabecular meshwork, which accounts for 75% of the resistance to aqueous humour outflow,MXRA5 is expressed in fetal optic nerve tissue and continues to be expressed in adult optic nerve tissue at a relatively lower level.The first associated region  is within an intergenic region, downstream of norrin cystine knot growth factor (NDP) and upstream of EF-hand domain containing 2 (EFHC2). This region has a low recombination rate in European populations, so the associated SNPs are in linkage disequilibrium with both genes. NDP is expressed in several relevant tissues, including the retina and choroid,EFHC2 has also been identified in the trabecular meshwork and cornea.NDP with eye development ,NDP could be the more likely functional candidate.The second associated locus  was within and around the glycoprotein M6B (GPM6B) gene. GPM6B is expressed within the trabecular meshwork, cornea and ciliary bodyGPM6B is incomplete, but it likely performs a cellular housekeeping role.GPM6B is required before any conclusions can be drawn on likely mechanisms underlying this association.The third associated region  are significant expression quantitative trait locus (eQTLs) for their respective genes, although rs66819623 is also an eQTL for the nearby gem nuclear organelle associated protein 8 (GENIM8) gene. Neither the conditional SNP nor any other associated SNPs at the NDP locus are known eQTLs.Data from the Genotype-Tissue Expression (GTEx) consortiumP < 0.017 (0.05/3). Despite the sample size of the replication cohort being less than 6.5% of the discovery cohort, the SNP for the MXRA5 locus replicated at Bonferroni adjusted significance , whereas the SNPs for the GPM6B and NDP loci were nominally replicated with the same direction of effect  whereas the SNP at the NDP locus was nominally associated , although no significant association was identified for the SNP at the MXRA5 locus . However, the direction of effect for all three was consistent with their direction of effect on IOPcc  were similar between men and women and fell well within each other's 95% confidence interval. Meanwhile, the effect estimate for women at the GPM6B locus was half of the effect estimate for men , whereas significance for the other two loci was similar between the two analyses despite the large difference in sample size , which accounts for approximately 2.8% of the total SNP heritability, increasing the total proportion of IOPcc variation explained by common SNPs to 0.254.NDP and EFHC2. Based on all currently available information, there is evidence for NDP as a candidate gene for association at that locus. A key component of this evidence is NDP's role in eye development and the Mendelian diseases Norrie disease and FEVR2,,NDP locus in our study further supports evidence that some mechanisms underlying PACG also contribute to normal variation in IOP.Three loci were found for IOP on the X chromosome, and the strongest locus was between Replication had limitations due to sample size, IOPcc was tested in a sample size less than 6.5% of the discovery cohort and the POAG validation dataset had 4607 POAG cases providing an effective sample size of 9086, less than 10% of the discovery sample. Despite this, by using a combination of direct IOPcc replication and POAG validation tests in two independent samples, we provide evidence that the three loci identified in this study are true positive associations.NDP and MXRA5 loci there are not sex-specific effects. The similarity in betas with differences in P values for the MXRA5 and NDP loci suggests these loci have the same effect in men and women, but that there is more uncertainty or statistical “noise” in the female analysis. Men have only one copy of the X chromosome, resulting in the same allelic effects being present across all cells within a tissue; women have two copies, although for the majority of X genes the copy on one of the chromosomes is silenced by X-inactivation.P value, whereas the slope/estimate of effect size will be relatively unchanged as the homozygotes at each end of the slope will be the same. This indicates that there are not sex-differences for the association between the MXRA5 and NDP loci with IOPcc, but that greater statistical power is available in men, due to skewed X-inactivation in heterozygous women. Therefore, when studying chromosome X for complex traits, it is to be expected that the male participants will drive the associations in an XWAS and be the primary source for identifying associated loci, but female samples are still required to determine whether there are any sex-specific effects in allele effect size.Results from our sex-stratified analyses indicate that for the GPM6B locus, with the allelic effect size for homozygous women being approximately half that of the effect size in men. GPM6B escapes X-inactivation in women,There is a difference in allelic effect size for associated variants at the ,,–,,,Although there is evidence for sex-specific effects for IOP variation and POAG risk,The initial combined XWAS begins with the assumption that men have full dosage compensation for chromosome X genes, allowing the use of “off the shelf” analysis software such as BOLT-LMMIn summary, our analysis identified three novel loci associated with IOPcc and POAG risk. One of these loci might provide supportive evidence of some shared genetic architecture between PACG and normal IOP variation. This analysis indicates that despite sex differences in chromosome X ploidy, this does not result in sex differences in the genetic risk from chromosome X variants for this trait. Finally, we demonstrate that chromosome X can be included in GWAS conducted in very large datasets, with minimal effects to the analysis time and computational burden.


APC) gene mutation in Japan.Ductal carcinoma of the prostate is a histological subtype with a higher mortality than acinar adenocarcinoma. The number of cases is small and there are no treatment guidelines. We believe that this is the first report of ductal carcinoma of the prostate with an adenomatosis polyposis coli (An 85-year-old man presented with gross hematuria, and a papillary tumor in the prostatic urethra that was diagnosed as ductal carcinoma of the prostate following transurethral resection. Genetic analysis found an APC mutation with loss of heterozygosity. Immunostaining revealed focal nuclear translocation of β-catenin. APC mutations associated with loss of β-catenin degradation in the Wnt signaling pathway and result in over accumulation of β-catenin are thought to increase mortality. In this patient, β-catenin migrated into tumor cell nuclei.To the best of our knowledge, this is the first report of ductal carcinoma of the prostate with an APC mutation in Japan. The development of a therapeutic Wnt inhibitor is discussed. Ductal carcinoma of the prostate is a histologic subtype that was first described in 1967. It has an estimated incidence of 0.5 to 6% of all diagnosed prostate cancers and clinical implications that are not well understood . ApproxiAPC (p.T1556Nfs*3) mutation with loss of heterozygosity (LOH), variant allele frequency of 83%, and a copy number of 1.1 , and are found in more than 80% of sporadic colorectal tumors [APC gene mutations were first reported in FAP in 1991, and the progression from adenoma to cancer is thought to involve multistage carcinogenesis with accumulation of APC, KRAS and TP53 gene mutations [APC gene function. We believe that this APC mutation affected Wnt signal transduction.l tumors . APC genutations . WNT is utations . APC proWnt signaling was shown to be involved in the progression of prostatic intraepithelial neoplasia (PIN) to prostate adenocarcinoma in mouse prostate cancer models and to induce high-grade PIN in differentiated luminal cells . AbnormaAnticancer drugs that block the Wnt signaling pathway are not currently available, but some are in development. OMP-18R5 (Vantictumab) targets the FZD1, FZD2, FZD5, FZD7, and FZD8 frizzled protein receptors to block WNT signaling are being investigated in phase I clinical trials in breast, pancreatic, and non-small cell lung cancer. LGK974, which blocks WNT signaling by targeting porcupine, is currently in phase I and II clinical trials in colorectal cancer [To the best of our knowledge, this is the first report of ductal carcinoma of the prostate with an APC mutation in Japan. Genetic analysis may be help to guide the development of candidate targeted drugs for ductal carcinoma of the prostate.


Campylobacter jejuni isolated from cases (n = 24) and water samples (n = 4) had identical core genome MLST profiles. Contamination through cracks in the reservoir most probably occurred during heavy rainfall. Water supply systems are susceptible to contamination, particularly to certain weather conditions. This highlights the importance of water safety planning and risk-based surveillance to mitigate risks.On 6 June 2019, the Norwegian Institute of Public Health was notified of more than 50 cases of gastroenteritis in Askøy. A reservoir in a water supply system was suspected as the source of the outbreak because of the acute onset and geographical distribution of cases. We investigated the outbreak to confirm the source, extent of the outbreak and effect of control measures. A case was defined as a person in a household served by Water Supply System A (WSS-A) who had gastroenteritis for more than 24 h between 1 and 19 June 2019. We conducted pilot interviews, a telephone survey and an SMS-based cohort study of residents served by WSS-A. System information of WSS-A was collected. Whole genome sequencing on human and environmental isolates was performed. Among 6,108 individuals, 1,573 fulfilled the case definition. Residents served by the reservoir had a 4.6× higher risk of illness than others.  Campylobacter jejuni is the most common species in human infections [Campylobacteriosis is a common cause of bacterial diarrhoeal illness worldwide  and Campfections . Patientfections . Immunocfections . Faecal-fections . In Norwfections -6.Despite advances in water management and sanitation in high-income countries, waterborne outbreaks still occur and may acutely infect many people simultaneously . SeveralCampylobacter. Staff of the OPHS noted that many patients presenting with gastroenteritis had home addresses near each other, which led to a suspicion that drinking water could be the source of the outbreak. A joint investigation was carried out by the municipal services, the Norwegian Food Safety Authority and the Norwegian Institute of Public Health (NIPH), with the aim to confirm the source, extent of the outbreak and effect of control measures.On the evening of 6 June 2019, the Medical Officer in Askøy reported an outbreak of gastroenteritis to the NIPH. In a 24 h period, 10 people had been hospitalised with fever, abdominal pain and diarrhoea, and ca 30 individuals had sought medical attention from out-of-hours primary healthcare services (OPHS). At least one person had tested positive for The 29,500 inhabitants of the island municipality Askøy receive water from three different water supply systems, of which the largest, Water Supply System A (WSS-A) from the 1950s, serves ca 12,000 people in the south of the island. WSS-A has nine reservoirs, including three built as unlined mountain caverns. Its Reservoir X was early on suspected to be the source of the outbreak because of the geographical distribution of cases that clustered in two areas.In order to determine the extent of the outbreak, we collected data on in-person and telephone consultations with the International Classification of Primary Care (ICPC-2) codes for diarrhoea (D11), gastrointestinal infection (D70) and gastroenteritis (D73) that occurred at the OPHS and general practitioners’ (GP) offices in Askøy between 3 June and 15 June. We mapped all consultations by household address and water supply zone.Campylobacter infections were interviewed using a standardised 19-page trawling questionnaire in order to exclude possible exposures other than drinking water. The questionnaire included detailed questions about food consumption and purchases, animal contact and environmental exposures in the week before the onset of symptoms, as well as clinical and demographical information.Several of the initial cases with confirmed In order to rapidly ascertain the start and geographical areas of the outbreak, we contacted all childcare centres in the municipality on 11 and 12 June to document absence for illness. As children normally attend childcare centres close to their homes, it was likely that the household and childcare centre water supply zones would be the same. For the childcare centre survey, a case was defined as any person absent from the childcare centre (child or employee) because of the symptoms diarrhoea or vomiting between 28 May and 7 June. We then mapped the childcare centres by water supply zones served by the different reservoirs and compared the attack rates in childcare centres served by Reservoir X against those served by other reservoirs.We included all residents who received water from WSS-A in a retrospective cohort study, and identified eight different water supply zones .People in households that partially or exclusively received water from Reservoir X were defined as exposed. We defined a case as a person with gastroenteritis (defined as having symptoms of (i) diarrhoea only or (ii) vomiting and at least one of the following: abdominal distention, fever, stomach pain or nausea, with duration of illness of more than 24 h), with symptom onset between 1 and 19 June 2019.We sent all households served by WSS-A an SMS with a link to our questionnaire on 13 June 2019 and posted the link on the municipality’s website. We requested that one person should respond on behalf of all household members. We asked about household illness, clinical presentation, tap water consumption and whether the household had received and followed the boil water advisory (BWA). The survey was closed on 20 June.We used R version 3.6.0 (2019–04–26) for statistical analyses.Campylobacter isolates from patients to the National Reference Laboratory for Enteropathogenic Bacteria (NRL) for confirmation and genotyping.The primary diagnostic laboratory sent We extracted DNA on MagNAPure 96 ) and used KAPA HyperPlus  for library preparation and Agencourt AMPure XP  for removal of adaptor dimers. Whole genome sequencing was performed as paired-end (250 bp × 2) sequencing on the MiSeq platform  aiming for coverage of > 50×. Quality control of the raw reads was done through FastQC.Campylobacter isolates was performed using the seven-gene scheme developed by Keith Jolly at PubMLST [Campylobacter of 637 core and 958 accessory genome targets modified from Cody et al. [We used SeqSphere+ software, version 5.1  for analysis. Briefly, multilocus sequence typing (MLST) of the  PubMLST . Core gey et al. ,20.Escherichia coli and coliform bacteria and for heterotrophic plate count, and monthly for intestinal enterococci and Clostridium perfringens, according to standard methods described in the national drinking water legislation [According to the routine sampling and analysis plan for WSS-A, water samples are collected and tested for faecal indicator bacteria: weekly for islation .Campylobacter using semiquantitative and quantitative determination in foods and drinking water  with pre-incubation on enrichment broth of filtered sample followed by plating on a selective medium [Campylobacter was confirmed by phase contrast microscopy.After the outbreak was detected, we started extra sampling of the water in WSS-A and analysed it for faecal indicator bacteria at an accredited laboratory using standard methods. On 6 June, we also took water samples from Reservoir X and several other points along the distribution system and immediately analysed it for e medium . The prewww.yr.no).We reviewed operational system information and historical data from WSS-A, including results from drinking water routine monitoring schemes before the outbreak. We assessed critical points and possible sources of contamination  through interviews of WSS-A staff and visual inspections of selected areas of WSS-A, including Reservoir X. Local rainfall was assessed using data from the national meteorological website  on Thursday 6 June . The conThe gastroenteritis patients’ residences were geographically concentrated in two areas of the municipality, which coincide with three water supply zones served by Reservoir X. These zones had higher incidence rates (IR) for consultations than other supply zones in WSS-A at the time of the outbreak detection .Campylobacter infection. They reported diarrhoea, stomach pain and fever with onset on 4 June (n = 1) or 5 June (n = 4). They lived in areas that received drinking water from WSS-A and had consumed tap water at home or used tap water for brushing teeth in the week before symptom onset. Other reported exposures, such as attendance at common dinners or events, consumption of food items, contact with animals or recreational water exposure, were not common to all five cases.We interviewed five of the first cases with confirmed All 27 childcare centres in the municipality participated in the study; eight (with 769 children and employees) were in areas supplied by Reservoir X and 19  were in areas supplied by other reservoirs. The overall attack rate was 20% for the childcare centres in affected areas and 2% for the childcare centres in unaffected areas. Absences started to increase at the childcare centres in affected areas on Monday 3 June (n = 26) and peaked on Friday 7 June with 81 absences (11%).The SMS with the questionnaire was sent to 4,409 persons with mobile phone numbers registered to residences in the supply area of WSS-A. Data from 6,192 individuals were reported through the online questionnaire, of whom 1,913 reported illness. We excluded data for 79 household members who reported onset of illness before 1 June and for five who reported onset of illness after 19 June. After this exclusion, data were available from 2,526 persons who responded on behalf of 6,108 household members, which yields a coverage of 51%  of the residents supplied by WSS-A. Mean age of the included household members was 34 years (range: 0 to 93 years) and 50% were female.A total of 1,829 persons reported at least one of the following symptoms: diarrhoea , abdominal pain , headache , nausea , fever , abdominal distention , vomiting  and bloody stool . In total, 1,573 respondents met the case definition, leading to an attack rate of 26%. The number of cases by date of symptom onset peaked on 6 June and decreased gradually thereafter . The meaResidents who were supplied from Reservoir X had a 4.6× higher risk of illness than residents served by other reservoirs in WSS-A .Among residents supplied by Reservoir X, the risk of illness increased with the amount of daily consumption of tap water  .Regarding the notification of BWA via SMS, 88% of households  reported that they had received an SMS with the BWA, while 179 (7%) did not receive the BWA and 124 (5%) were unsure.A total of 2,384 households reported having complied with the BWA . Reasons for non-compliance were reported by 142 of the households; the main reasons were purchasing bottled water (n = 76), considering that the risk of becoming ill was low (n = 9) and drinking little or no tap water (n = 4). For the remaining 53 households, reasons for non-compliance to the BWA were not reported. C. jejuni from persons served by Reservoir X. All isolates were sequence-typed as ST1701, which is part of the clonal complex ST-45, and all had identical cgMLST profiles, cluster type 97 . Two representative sequences have been submitted to the European Nucleotide Archive (ENA) . When comparing publicly available ST1701 C. jejuni sequences to the outbreak strain, we did not observe any close genetic relationships, although poultry or birds were identified as a possible source.The Norwegian Surveillance System for Communicable Diseases (MSIS) registered 181 laboratory-confirmed cases of campylobacteriosis linked to this outbreak. The NIPH received 24 isolates of In the 3 years before 3 June 2019, the routine monitoring programme for WSS-A did not detect any faecal indicator bacteria in WSS-A or in Reservoir X, except for occasional findings of coliform bacteria. The samples collected during routine monitoring for WSS-A on 3 June were also negative for faecal indicator bacteria, including samples located near Reservoir X.Of eight water samples taken on 6 June, five were collected from Reservoir X and areas supplied by Reservoir X and were faecally contaminated, while the remaining three samples from other areas were not. Seven water samples collected from the distribution system at the same locations on 9 June, after Reservoir X was taken out of service on 7 June, were negative for faecal indicator bacteria. One sample taken inside Reservoir X on 9 June was positive for faecal indicator bacteria, as were all samples collected in Reservoir X during the week of the outbreak (until 13 June).Campylobacter (7 June). The positive samples had been taken from Reservoir X, two households and one school supplied by Reservoir X.Four of seven water samples were positive for Under normal conditions, Reservoir X supplies a defined zone (zone 6) of 1,350 residents. However, before the outbreak, a valve had been opened from Reservoir X to ensure replacement of water in response to customer complaints about the water quality in the area. This led to a connection between zone 6 and zones 7 and 8 , serving3 of water and is located above a residential area in mountainous terrain. The inflow and outflow of Reservoir X is through the same pipe. Because of the design of the reservoir, the chosen sampling location for routine monitoring samples did not capture the water flowing out of the reservoir. Potential contamination introduced to the reservoir would therefore not necessarily not be detected.Reservoir X is a basin constructed as an unlined rock cavern with an entrance sealed by a locked door. It holds ca 400 mDuring the visual inspection of Reservoir X, we observed natural cracks located in the back of the reservoir. There were leaks in the concrete construction and we observed water running from inside the roof. A large antenna is also located above the reservoir, with power lines running over the closed cavern, were birds could potentially gather and increase the risk of bird faeces contaminating the area below. Although varied wildlife is reported in the area, we did not observe this during our inspection of the area.No unusual malfunctions in the distribution system were reported before the outbreak. In weeks 21 and 22, three episodes of extreme rainfall  occurred in the area after a long dry period from 1 April to 20 May. Weather data obtained from a nearby weather station  indicated heavy rainfall which coincided with registered consultations of gastroenteritis in the Norwegian Syndromic Surveillance System (NorSySS) .In addition to issuing a BWA and closure of Reservoir X , emergenThe results of epidemiological, microbiological and environmental investigations support that contaminated water from Reservoir X caused an outbreak of more than 1,500 cases of campylobacteriosis in Askøy in June 2019. The triangulation of epidemiological, genomic, geographical and water systems data was essential for confirming the role of Reservoir X in the outbreak and determining the extent of exposure within the community. The rationale for the decisions early in the phase of the outbreak was based on local knowledge and mapping of cases rather than epidemiological studies. This observation gave a hint towards the drinking water supply system as the source to the outbreak. The use of multiple approaches and use of mixed methods also allowed us to identify contributing factors, such as inclement weather conditions, and ensure that the implemented control measures had successfully stopped the outbreak. While we were unable to conclusively determine how the reservoir became contaminated, the diversity of data sources used to investigate this outbreak support the hypothesis that environmental contamination through cracks in Reservoir X most likely occurred during heavy rainfall following a long dry period.Campylobacter has frequently been identified as the cause of waterborne outbreaks [utbreaks ,23, ofteutbreaks ,25 and iutbreaks -30 or inutbreaks ,32. In 2utbreaks  and servutbreaks . Continuutbreaks .There was no indication of contamination with faecal indicator bacteria before the outbreak from routine sampling conducted on 3 June. This is a common finding in many waterborne outbreaks in which routine monitoring was neither the source of early detection nor able to prevent the occurrence of an outbreak . AlthougTimely detection of waterborne outbreaks is crucial in preventing widespread exposure of the population and limiting the negative health consequences. Although the exposure to contaminated drinking water may be almost instantaneous for the supplied residents, the detection may be delayed, particularly in large areas served by the same water supply ,39, whilThis investigation has several limitations. Initially, it was difficult to determine the scale of the rapidly developing outbreak. Although campylobacteriosis is a notifiable disease in Norway, only a small proportion of sick residents were tested and laboratory-confirmed cases were not reported rapidly enough to follow the progression of the outbreak. For this reason, consultations for gastroenteritis were tracked manually through the GP and OPHS offices on a daily basis for outbreak monitoring until monitoring with NorSySS was possible. The SMS-based cohort study corroborated the onset and duration of the outbreak, but since only one person in the household was asked to respond on behalf all members of the family, this may have led to an underestimation of cases. In addition, the specific case definition excluded more than 500 people who reported illness possibly linked to the outbreak. On the other hand, the widespread media attention may have affected the results of the SMS-based cohort study by overestimating illness among the respondents. However, we chose to use a specific case definition to avoid including people that were ill for other reasons.A large waterborne outbreak leading to more than 1,500 cases of campylobacteriosis occurred in Askøy in Norway in June 2019. Through multiple data sources, we were able to determine that contamination of drinking water occurred through cracks in a mountain reservoir, probably because of heavy rainfall after an extended dry period. This is a reminder that water supply systems, in particular ageing infrastructure, are generally vulnerable and susceptible to contamination that may result in an outbreak, especially as external risks such as climate factors are changing. This investigation highlights the importance of conducting water safety planning, updating the infrastructure and performing risk-based surveillance to mitigate risks.


Due to strong Coulomb interactions, two-dimensional (2D) semiconductors can support excitons with large binding energies and complex many-particle states. Their strong light-matter coupling and emerging excitonic phenomena make them potential candidates for next-generation optoelectronic and valleytronic devices. The relaxation dynamics of optically excited states are a key ingredient of excitonic physics and directly impact the quantum efficiency and operating bandwidth of most photonic devices. Here, we summarize recent efforts in probing and modulating the photocarrier relaxation dynamics in 2D semiconductors. We classify these results according to the relaxation pathways or mechanisms they are associated with. The approaches discussed include both tailoring sample properties, such as the defect distribution and band structure, and applying external stimuli such as electric fields and mechanical strain. Particular emphasis is placed on discussing how the unique features of 2D semiconductors, including enhanced Coulomb interactions, sensitivity to the surrounding environment, flexible van der Waals (vdW) heterostructure construction, and non-degenerate valley/spin index of 2D transition metal dichalcogenides (TMDs), manifest themselves during photocarrier relaxation and how they can be manipulated. The extensive physical mechanisms that can be used to modulate photocarrier relaxation dynamics are instrumental for understanding and utilizing excitonic states in 2D semiconductors. Electrons and positively charged regions in semiconductors known as holes can form tightly bound states called excitons, offering new opportunities in optoelectronics and in ‘valleytronics,’ which exploits the effects of valleys and peaks in an energy landscape. The excitons display particle-like properties, so are described as quasiparticles. Fengqiu Wang and colleagues at Nanjing University, in China, review many technical aspects of the creation and control of excitons in single layer (2D) semiconductor sheets. The restrictions of 2D materials promote unique exciton behavior and strong light-matter interactions. The authors focus on the ability of excitons to act as photocarriers of absorbed light energy, and on the relaxation processes that can release the energy. Learning how to control exciton formation, interactions and relaxation will be crucial for using them to develop novel optoelectronic and photonic devices. As a result, 2D semiconductors can host a rich set of excitonic species, which exhibit large oscillator strengths and strong light-matter interactions4. Moreover, the flexibility in constructing van der Waals (vdW) heterostructures further enriches the excitonic physics in 2D semiconductors by enabling various interlayer species and introducing exotic effects such as moiré potential patterns5. In addition, the broken inversion symmetry of 2D transition metal dichalcogenides (TMDs) renders the electronic “valleys” non-degenerate, giving the excitons another “valley” degree of freedom with direct optical accessibility7.Due to the reduced dielectric screening and enhanced quantum confinement, the Coulomb interaction experienced by carriers in 2D semiconductors is substantially increased compared to three-dimensional (3D) semiconductors, leading to the formation of tightly bound excitons and strong many-body effects8, an increase in the radiative lifetime of photocarriers has been found to coincide with the enhancement of the luminescence quantum yield (QY), which is beneficial for light-emitting devices9. In the case of photoconductors, the photocarrier lifetime can be a knob to balance the response time and quantum efficiency: shorter lifetimes of photocarriers can lead to a faster response speed at the expense of quantum efficiency, as more photocarriers recombine before entering the circuit10. Hence, exploring deterministic tuning strategies for the dynamic characteristics of photocarriers in 2D semiconductors is of both fundamental relevance and practical significance.Typically, excitons are generated by photoexcitation and dominate the optical properties of 2D semiconductors. Thus, probing the dynamics of the optically generated excited states represents an important aspect of excitonic physics in 2D semiconductors. Moreover, because of the low dimensionality, the excitonic dynamics of 2D semiconductors can be more susceptible to various external stimuli, opening up ways for flexible excited-state lifetime control. This is beneficial for practical applications, as excited-state lifetimes are linked to key figures of merit of multiple optoelectronic and photonic devices. For example, while a short photocarrier lifetime is favorable for the operating bandwidth of ultrafast optical switches12, composition control13, morphology control of nanostructures15, external field application16, etc. Taking GaAs, one of the most widely applied conventional semiconductors, for example, modulation of its photocarrier lifetime has been achieved over a large range, from nanoseconds to sub-picoseconds, by means of low-temperature growth18 or ion implantation20. Combined with post-growth thermal annealing, these methods can effectively tune the photocarrier lifetimes without obviously degrading the crystallinity, making GaAs highly competitive in the application of ultrafast optoelectronic and photonic devices. However, due to the atomic thickness feature, in many cases, the approaches for bulk samples are difficult to adapt to or become ineffective for 2D semiconductors. For instance, the difficulty in introducing point defects or dopants into the lattice in a controllable manner is significantly enhanced. On the other hand, the unique properties of 2D semiconductors, such as robust excitonic states, sensitivity to external environmental factors, and flexibility in constructing vdW heterostructures, promise modulation strategies different from those for conventional materials.To identify robust and deterministic approaches for photocarrier lifetime control, experiences from conventional bulk semiconductor research can be considered, including element doping23, in this review, we focus on summarizing recent efforts in identifying methods to modulate the photocarrier relaxation behavior. We start with a brief introduction to the photocarrier relaxation dynamics in 2D semiconductors and then devote a section to discussing modulation of Coulomb interactions and the resulting effects on the transient properties of 2D semiconductors. Subsequently, we discuss the factors that can influence photocarrier relaxation in 2D semiconductors and corresponding modulation methods, according to the related relaxation pathways or mechanisms. The tailoring approaches discussed include both those arising from the experience with bulk semiconductors, such as doping and applying external fields, and those utilizing the unique properties of 2D materials, such as modulating the surrounding environment and constructing heterostructures. After that, modulation of the spin/valley polarization dynamics is discussed as an important complement to the case of 2D TMDs. Finally, we provide a summary and an outlook on the research directions in the future.Since there have been a number of review articles on the ultrafast photocarrier dynamics in 2D semiconductors24. The optical and optoelectronic properties of 2D semiconductors are dominated by the excitonic states even at room temperature.As the sample thickness is reduced from the bulk to the atomic level, electrons and holes are tightly bound together, forming excitons, due to the greatly enhanced Coulomb interactions compared to the 3D counterparts Fig. . The res23. Once optically injected into the samples, the non-equilibrium photocarriers typically undergo relaxation processes, including rapid thermalization through carrier-carrier scattering, cooling to the band edges through interaction with phonons, and electron–hole recombination either directly or via the assistance of defects or phonons  recombination and Langevin recombination31. SRH recombination is a monomolecular process mediated by tunneling of majority carriers to trap states, and Langevin recombination is a bimolecular process dominated by the Coulomb interaction, whose rate increases with the carrier mobility33.In a vdW heterostructure formed by 2D materials, generally, after interband photoexcitation, the photocarriers in the vdW heterostructure relax the following two main steps, as illustrated in Fig. 34. Moreover, due to the strong spin-orbit coupling of charge carriers at the band-edge, the spin index is locked with the valley index and thus can be accessed by helicity-resolved optical approaches. For recent advances in valley-contrasting physics and applications, readers are referred to refs. 38.In addition, as a unique property of monolayer TMDs, charge carriers can carry another degree of freedom—valley polarization. Because of the broken inversion symmetry of monolayer TMDs, the valleys of energy-momentum dispersion at the corner of the hexagonal Brillouin zone, labeled +K and −K valleys, are no longer equivalent Fig. . The int40. These two effects partially cancel each other out and give rise to a comparatively small shift of the excitonic resonance, and the actual shift of the resulting optical bandgap may vary for different materials and conditions41.The enhanced Coulomb interactions in 2D semiconductors can be modulated by introducing additional screening from the external dielectric environment or injected charge carriers, leading to a modification of quasiparticle bandgaps  and a decrease in the exciton binding energyhBN)43 or using solvents45 or substrates with different dielectric constants48. Fig. 2 and WSe2) using graphene and hBN with different thicknesses, tuning of the electronic bandgap and exciton binding energy by as much as several hundred millielectronvolts has been achieved49. The energy modification realized by this strategy is large enough to drive the directional in-plane motion of excitons. In the study by Hao et al., a lateral heterostructure was formed by covering part of monolayer MoSe2 with hBN. The energy offset caused by the difference in the local dielectric screening was large enough to drive the transport of excitons across the lateral junction, resulting in an initial transport speed of ~10 nm/ps50.Research efforts have been made to alter the dielectric environment of 2D semiconductors by encapsulating 2D semiconductors with boron nitride 47, and recombination47. It has been reported that as the substrate dielectric constant increases from 1.45 (quartz) to 2.5 (SrTiO3), the EEA rate of MoS2 decreases by 2 orders of magnitude, from 0.8 cm2/s for quartz to 0.005 cm2/s for SrTiO3. This modulation has been attributed to a change in the energy distribution of defect states induced by the screening from substrates with different permittivities, affecting the interaction between the defect-trapped excitons and mobile excitons in a defect-assisted Auger-like process46. Similarly, hBN encapsulation has also been reported to suppress the EEA process, where the EEA rate of WS2 has been reduced by ~20 times  tuning of the electronic bandgap and exciton binding energy could be achieved in a ReSe2 monolayer placed on a back-gated graphene device, which was attributed to the tuning of Coulomb interactions by gate-controlled free carriers in graphene54. A decreased exciton binding energy could result in a reduction of the exciton oscillator strength. This effect has been observed in a WS2 field-effect transistor (FET) embedded in a microcavity, where 6–7 times tuning of the exciton oscillator strength was realized by varying the electrostatic doping level, manifesting as changes in PL and reflection intensities55.Coulomb interactions can be appreciably screened by the presence of high-density charge carriers. Modification of the quasiparticle electronic bandgap and exciton binding energy can be observed when the carrier density reaches ~1012/cm2 could lead to an appreciable reduction of the electronic bandgap and exciton binding energy, manifesting as bleaching of excitonic features and redshifted absorption sidebands at all excitonic resonances regardless of the excitation photon energy  included a transition from an asymmetric to symmetric profile and a blueshift of the zero differential reflectance position  × 10rgy Fig. 56. In a ion Fig. . It has 13–1014/cm2, 2D semiconductors undergo a so-called Mott transition from the semiconducting to the metallic state. A threshold carrier density of several 1013 cm2 for complete ionization (Mott transition) was estimated by extrapolating the variation in the exciton binding energy determined from reflectance contrast spectra in a study on electrostatic doping of WS240. The signatures of the Mott transition in transient optical responses have been reported in monolayer TMDs39 and in TMD heterostructures where electrons and holes are confined in different TMD layers after the transition58. The features of excitonic resonance disappear, and a broad range of absorption emerges below the renormalized bandgap along with an optical gain just above the bandgap due to the presence of an electron–hole plasma and induced population inversion  of selenium and sulfur compared to transition metals71. By using electron-beam irradiation, Moody et al.69 demonstrated that Se vacancies could be selectively induced in WSe2 monolayers, giving rise to a greatly prolonged exciton radiative lifetime of ~200 ns and a valley polarization lifetime of ~1 µs. However, it is important to control the species of generated defects, which is rather technically challenging. While many species of defects can accelerate the relaxation process of photocarriers, certain kinds of defects may work differently and inhibit the relaxation process. A first-principle calculation on monolayer WSe2 revealed that Se vacancies could help suppress the phonon (mainly A1g mode)-assisted recombination by breaking the lattice symmetry and reducing the intensity of the A1g mode, giving rise to an increase in the recombination time constant from ~400 ps to ~3.1 ns72. Due to the difficulty in controlling not only the amount but also the species of point defects, it remains challenging to use particle irradiation as a practical method for tuning photocarrier dynamics.Considerable efforts have been devoted to defect engineering in 2D semiconductors. Irradiation by different particle sources, such as electrons3As2 films, which have been widely studied as 3D topological Dirac semimetals, a long-lived relaxation component ranging from 200 ps to 2.8 ns was recently observed in Mn-doped samples, which was attributed to the relaxation of photocarriers from the impurity band to the valence band73. However, the research on element doping of 2D systems and the resulting impact on photocarrier dynamics remains limited, probably hindered by the difficulty in doping atomically thin samples. In a recent study on Re-doped MoS2, it was shown that the exciton lifetime was reduced by ~20 times (from ~22 ps to ~1 ps) at a low doping level of 0.6%, which was attributed to the efficient Auger recombination assisted by Re dopants74.In addition to bombarding samples to generate intrinsic defects, introducing extrinsic defects by element doping is another approach that could be adapted from the experience with bulk semiconductors. The advantage of this approach is the potential to achieve better crystallinity of the samples. As an example, for Cd2 is increased from 3 to 8 ps at 0.6% strain  nanoplatelets was reduced from 1.7 ps to 240 fs as the thickness was reduced from ~15 to ~2 nm. In addition to the enhanced electron–phonon interaction, the more efficient heat transfer to the environment due to the high surface-to-volume ratio also contributes to the accelerated relaxation76. The electron–phonon coupling can also be modulated by modifying the sample lattice. In a recent study, a shortening of the photocarrier relaxation time by an order of magnitude was achieved for Cd3As2 films with 2% Cr doping. The theoretical simulation found that the tuning was due to the opening of the bandgap produced by doping-induced changes in the lattice symmetry, which activated an additional phonon scattering channel for relaxation77.For polar semiconductors, the Fröhlich electron–LO–phonon interaction mediated by Coulomb interactions is much less screened as the thickness of the sample is reduced from the bulk to the atomic level. As a consequence, the relaxation of hot carriers to the band edges is more efficient in 2D semiconductors than in bulk semiconductors, despite the reduced number of phonons and density of states with the reduction of thickness. Such phenomena have been observed in the relaxation of photocarriers in 2D halide perovskite nanoplatelets. As shown in Fig. 80. It has been observed that the excitons of monolayer WSe2 samples interact strongly with phonons in surrounding hBN layers or dielectric substrates 80. As shown in Fig. 6b79 and c80, the coupling manifests as pronounced resonance features in the PL spectra of WSe2 excitons or in the Raman spectra of environmental phonons when the energy difference between excitation and emission matches the energy of phonons.Moreover, owing to the atomic thickness, phonons from the surrounding environment can interact with photocarriers in the 2D samples directly. The efficient interlayer coupling between excitons in 2D semiconductors and phonons in the environment has also been revealed by various optical and electrical measurements2 monolayers on three kinds of dielectric layers , wide-range tuning of the non-radiative recombination lifetime of excitons was achieved, from 160 ± 10 ps for MoSe2/SiO2 to 20 ± 4 ps for MoSe2/HfO2  by suppressing the charge transfer and doping effect from the substrate. In addition, the extracted diffusion coefficient of the excitons was reduced by approximately four times from the bare WS2 monolayer to the fully encapsulated monolayer due to the scattering by additional phonons introduced by hBN42. In a few studies on graphene, it has been found that the photocarrier lifetimes of CVD-grown graphene transferred to hBN are shorter than those of graphene transferred to SiO281, and photocarriers in graphene epitaxially grown on SiC substrates relax faster than those in graphene transferred to SiC substrates83. These discrepancies have been attributed to the difference in the coupling between photocarriers in the graphene layers and phonons in the substrates. The shorter distances between graphene and the substrates due to smoother hBN and direct synthesis enable more efficient coupling, leading to faster relaxation of non-equilibrium photocarriers.In the same study by Fu et al., it was found that the dynamics of exciton formation also varied under different conditions of 2/WSe2 heterostructure, when an out-of-plane electric field was applied across the heterostructure antiparallel to the interlayer exciton dipole moment, the radiative recombination lifetime of the interlayer exciton was increased  with holes residing in MoSe2. In contrast, photocarriers generated in graphene recombine on an ultrashort timescale (~0.9 ps). In this way, the lifetime of carriers in graphene is enhanced by two orders of magnitude86.By carefully designing the layered structure and the resulting band alignment, wide-range tuning (mostly elongation) of photocarrier lifetimes can be achieved. In a study on TMD heterostructures, WSe2, separated photoexcited electrons and holes existed as (quasi)free charge carriers across the vdW interface. As shown in Fig. 2 layers. It has been demonstrated that the recombination dynamics can be well described by a Langevin recombination model, which is mediated by the Coulomb interactions between electrons and holes across the interface. The higher mobility and larger density of photocarriers in BP increase the chance that the electrons and holes meet within the Coulomb capture radius and recombine with each other87.On the other hand, when electrons and holes are less tightly bound, the motion of carriers in real space can also result in the modification of their relaxation dynamics. In a recent study employing black phosphorus (BP), which has a small exciton binding energy (<40 meV), to form a type-II heterostructure with MoS88. Therefore, when the ratios between different quasiparticles in samples are altered by means of doping of free carriers, the relative portions of different relaxation pathways, and thus the transient optical responses of the whole sample, can be modified.In addition to neutral excitons, in real samples and devices, it is inevitable that other types of quasiparticles, such as trions and biexcitons, as well as free carriers, coexist and undergo transitions among each other, which further complicates the relaxation dynamics of non-equilibrium photocarriers. For example, combining with free carriers to form trions represents an important non-radiative decay channel of neutral excitons, which reduces the radiative lifetime of neutral excitons and limits the QY90 or chemical doping9 or the combination of both to achieve a broader controllable regime91. In the work by Lien et al., effective suppression of non-radiative recombination in MoS2 was achieved using an electrostatic doping strategy. As shown in Fig. 89. In the chemical doping case, exfoliated monolayer MoS2 samples were immersed in a non-oxidizing organic superacid, a bis (trifluoromethane) sulfonimide (TFSI) solution, and a hundred-fold enhancement in the PL intensity was achieved, which was attributed to hole doping through surface charge transfer9. The radiative lifetime of excitons has been increased by over two orders of magnitude by TFSI treatment, even larger than what has been achieved by a negative gate bias of −20 V89.Tuning the background free carrier density is an effective approach to modulate these transitions, which can be done by electrostatic2/WSe2 heterostructure, by controlling the gate voltage applied to each TMD layer, the electrostatic doping conditions in these two layers could be tuned independently. As shown in Fig. 84.In a work on a MoSe2 monolayer92.In addition to radiative recombination, it has recently been found that the non-radiative recombination dynamics of neutral and charged excitons can also exhibit discrepant features, as shown in a back-gated WS94 and theoretically96 that for neutral excitons in monolayer TMDs, it is rather challenging to maintain a long lifetime of valley/spin polarization due to the strong electron–hole exchange interaction. Hence, efforts to prolong the lifetime of carriers’ valley/spin polarization mainly involve reducing the Coulomb electron–hole exchange interaction and, more importantly, exploring carrier species with non-zero momentum/spin.To be exploited as an alternative to charges for information storage and processing, the valley/spin polarization of carriers in monolayer TMDs needs to have a sufficiently long lifetime. However, it has been demonstrated both experimentally97, interlayer excitons in vdW heterostructures are also able to store valley/spin polarization. Rivera et al. investigated the valley polarization dynamics of bright interlayer excitons in a MoSe2/WSe2 heterostructure by circular polarization-resolved PL. As illustrated in Fig. σ+ light generates photocarriers in the +K valleys of the MoSe2 and WSe2 layers, which can form interlayer excitons in the +K valleys after ultrafast charge transfer and can recombine to emit photons with σ+ helicity. The degree of polarization demonstrates a lifetime as long as ~40 ns under a gate voltage of +60 V  owing to the suppressed intervalley scattering103.On the other hand, the depolarization of charge carriers with non-zero total momentum/spin, such as  trions2 monolayers, time-resolved Kerr rotation measurements demonstrated an ~130 ns lifetime for negatively charged trions and an ~2 µs lifetime for positively charged trions under n- and p-doped regimes, respectively 110, are of great potential for realizing significantly modified ultrafast relaxation dynamics. It has been predicted that the Janus-MoSTe would exhibit an ultralong lifetime (~1.3 ns) due to the large spatial separation and reduced binding energy111. These advances in sample preparation techniques, while still limited, hold great promise for leading to approaches that can meet the requirements of real optoelectronic and photonic devices. In recent years, the moiré pattern, which is formed in vdW heterostructures with small lattice mismatches and twist angles, has been shown to modify the physical properties of interlayer excitons in TMD-based heterostructures over a long translation period5. Modulation of properties such as the diffusivity and spin/valley polarization through the moiré potential has been demonstrated112. However, studies on modulating the photocarrier relaxation in 2D semiconductors are still at a relatively early stage, and robust approaches to realize reliable and wide-range tuning of the photocarrier relaxation behavior, including both relaxation pathways and temporal dynamics, remain limited. Tremendous research efforts are still needed in both improvement of the fundamental understanding and practical modulation of the photocarrier relaxation in 2D semiconductors.Moreover, advances in synthesis and processing techniques, as well as the discovery of exotic phenomena in 2D semiconductors, continue to suggest new strategies to this end. Recently, by using chemical vapor transport (CVT) and the self-flux growth method, single crystals of MoSe