COVID patients had higher BMI , higher prevalence of diabetes mellitus , and lower incidence of coronary artery disease . Both groups had almost similar predicted average survival rates based on the GO-FAR score, but only 1.5% of COVID patients survived to discharge compared to 7% of non-COVID patients (p < 0.05). Conclusion: The rate of survival to hospital discharge in COVID-19 patients who suffer IHCA is worse than in non-COVID patients, and lower than that predicted by the GO-FAR score. This finding may help inform our patient population about risk factors associated with high mortality in COVID-19 infection, as well as educate hospitalized patients and healthcare proxies in the setting of code status designation.Background: There are limited data regarding the outcome of in-hospital cardiopulmonary resuscitation (CPR) in COVID-19 patients. In this study, we compared the outcomes of in-hospital cardiac arrests (IHCA) before and at the peak of the COVID-19 pandemic at Montefiore Medical Center in the Bronx, New York, United States. We also identified the most common comorbidities associated with poor outcomes in our community. Methods: This was a multi-site, single-center, retrospective, observational study. Inclusion criteria for COVID patients were all confirmed positive cases who had in-hospital cardiac arrest (IHCA) between 1 March 2020 and 30 June 2020. The non-COVID cohort included all cardiac arrest cases who had IHCA in 2019. We excluded all out-of-hospital cardiac arrest (OHCA). We compared actual survival to that predicted by the GO-FAR score, a validated prediction model for determining survival following IHCA. Results: There were 334 cases in 2019 compared to 450 cases during the specified period in 2020. Patients who initially survived cardiac arrest but then had their code statuses changed to do not resuscitate (DNR) were excluded. Groups were similar in terms of sex distribution, and both had an average age of about 66 years. Seventy percent of COVID patients were of Black or Hispanic ethnicity. A shockable rhythm was present in 7% of COVID patients and 17% of non-COVID patients ( Since the initial detection of COVID-19 cases in the United States in February 2020, more than 40 million cases have been confirmed, with over 656,000 deaths to date .Patients with COVID-19 requiring mechanical ventilation have poor survival rates ,4. FurthThe first study to report outcomes of in-hospital cardiac arrest (IHCA) in COVID-19 was from Wuhan, China, where the survival rate of patients with severe COVID-19 pneumonia and in-hospital cardiac arrest was very poor, with a return of spontaneous circulation (ROSC) rate of 13.2% and a 30-day survival rate of 2.9% .In this study, we compared the baseline characteristics and survival rates of COVID and non-COVID patients who experienced IHCA at our institution, with the aim of gaining insight into risk factors associated with poor outcomes and to highlight the importance of goals of care discussions for critically ill COVID-19 patients.The electronic medical record (EMR) system from three affiliated hospitals in the Bronx, NY, United States, was used for patient selection and data collection. All patients with COVID-19 confirmed by either nasopharyngeal swab or SARS-CoV-2 who were aged 18 and above, had IHCA and underwent cardiac resuscitation (CPR) between 15 March and 30 June 2020 were included in the COVID cohort. The non-COVID cohort consisted of all patients who had in-hospital cardiac arrests and underwent cardiac resuscitation in the period 1 January to 31 December 2019. We used a longer time period for non-COVID patients to increase the size of the comparison group and to reach better matching between the two groups. Patients with OHCA or who arrived to the hospital with ongoing cardiac resuscitation were excluded. Internal transfers within the three hospitals were considered a single hospitalization. Those who were transferred outside the institution or who had cardiac arrest prior to transfer to our institution were excluded. Patients whose code statuses were changed to do not resuscitate (DNR) after initial survival of cardiac arrest were also excluded. This study was approved by the local institutional review board.All data were obtained from EMRs and cardiac arrest documentation. Patients’ data included age, gender, race, body mass index (BMI), code status as either full code or (DNR), and comorbidities such as hypertension, diabetes mellitus (DM), coronary artery disease, and chronic kidney disease. Other data included location of cardiac arrest, initial electrocardiogram (ECG) rhythm during the cardiac arrest, and length of hospital stay.The GO-FAR score (Good Outcome Following Attempted Resuscitation) is a clinically validated risk score that utilizes pre-arrest variables to predict the probability of survival to hospital discharge following IHCA . These vp-value <0.05 was considered statistically significant. STATA-16 was used for analysis.Descriptive statistics with simple graphic analysis were performed. Numerical variables are reported as mean, median, or range as appropriate. Categorical variables are reported as the percentage of patients in each category. Proportions were compared using the Wald test. A p < 0.05). COVID patients had higher BMIs  and a higher prevalence of diabetes mellitus . Coronary artery disease was more common in non-COVID patients . Despite both groups having almost similar predicted average-to-low survival rates based on the GO-FAR score, only 7 (1.5%) COVID patients survived to discharge compared to 23 (7%) non- COVID patients (p < 0.05).There were 334 patients who had IHCA in 2019. Of the 6500 COVID-positive patients who were admitted between 15 March and 30 June 2020, we found 450 patients who underwent cardiac resuscitation efforts for IHCA. Age and sex distribution were similar in both groups . SeventyA recent systematic review and a meta-analysis that included 40 studies in the period 1985–2018 showed that one-year survival after IHCA was 13.4% on average . The lowMultiple studies during the COIVD-19 pandemic have suggested that HTN, DM, and CAD are the most common comorbidities that affect the survival of COVID-19 patients, while coexisting infection, malignancy, immunodeficiency, and cerebrovascular disease are less common .p < 0.05). The low survival rate among the COVID-19 patients may be attributed to the absence of shockable rhythms during the cardiac arrest event.Numerous studies have found that the survival to hospital discharge is approximately 50% for patients who have a shockable rhythm during cardiac arrest. In contrast, the likelihood of survival from an initially non-shockable rhythm is two to three times lower (about 15–20%) . In our Age has been widely associated with reduced survival after cardiac arrest, especially for patients over the age of 60 . PreviouThe average BMI in COVID patients was higher than in the non-COVID patients. This may contribute to the lower survival rate. The “obesity paradox” is a phenomenon that describes how obesity, as a risk factor for cardiac disease, can lead to improved mortality outcome in severe cardiac illness such as cardiac arrest. One study found high BMI to be associated with lower all-cause mortality in survivors of cardiac arrest . HoweverBesides obesity, coronary artery disease (CAD) has also been associated with improved survival following cardiac arrest . This haDiabetes mellitus (DM) was more common in our COVID cohort than in the non- COVID cohort. DM has been associated with reduced return to spontaneous rhythm and 24-h survival . COVID pStudies investigating the relationship between race and outcomes following in-hospital cardiac arrest found Black and Hispanic patients had lower rates of neurological recovery and survival compared to Caucasians . UnfortuDespite similar predicted average survival rates based on the GO-FAR score, the actual survival for COVID patients was much lower for COVID patients than for non-COVID patients. One reason for this discrepancy may be that clinical features captured by the GO-FAR score may be incomplete or of insufficient magnitude with respect to COVID infection. Black and Hispanic patients, who represented a higher percentage of the COVID than non-COVID cohort, have been shown to have a lower survival following in-hospital cardiac arrest . HoweverCPR is a complex intervention that includes airway management, ventilation, chest compressions, drug therapy, and defibrillation. In the setting of COVID-19 infection, viral particles can remain in the air with a half-life of approximately 1 h, increasing the risk of transmission to resuscitation providers. Multiple studies have demonstrated a significant risk of viral transmission due to aerosol and droplet generation during CPR ,22,23. DIn addition, during this unprecedented time in New York City, the number of critically ill patients exceeded the capacities of ICUs in most hospitals. Operating rooms, general medical floors, and hallways became sites to treat critically ill COVID-19 patients ,27. Pre-This was a multi-hospital system study with a predominantly Black and Hispanic population with a high prevalence of significant comorbidities that explains the lower survival rate following IHCA in our community compared to the rate nationwide (7% vs. 13.4%). The two groups were chosen from different years and over different periods of time to avoid false negative results in the non-COVID group in 2020 and to achieve a better match between the two groups. Ten percent of COVID patients’ records regarding the rhythm during the cardiac arrest were missing due to the high volume of patients undergoing cardiac arrest with a limited number of providers. Undocumented details of cardiac arrest procedures included elements such as time of event, medications administered, ventilator setting at time of arrest, oxygen saturation and other vital signs. Additionally, as this study was carried out retrospectively, selection bias could not be completely avoided.Due to the low number of cardiac arrest survivors at our institution, we could not statistically verify the relation between the risk factors and the outcome of cardiac arrest during the COVID pandemic.The rate of survival to hospital discharge in COVID patients who suffer cardiac arrest is extremely low. The results from this study may inform providers, hospitalized patients, and their relatives when discussing designation of code status. This study also demonstrates the most common risk factors that affect the outcomes of inpatient cardiac arrest in COVID-19 patients. Further research with a larger and more diverse population is needed to better understand survival probability in specific patient subgroups.


Bowel cancer is one of the leading cancer-types in both sexes worldwide. Despite that most new cases and deaths occur in people aged 70 years or older, few clinical trials have investigated the best way to administer chemotherapy in older or frail patients. The NORDIC9-study established that moderately dose-reduced combination chemotherapy improved survival without extra side-effects compared to full dose single drug therapy. However, many older patients with incurable cancer seem to prefer preserved quality of life rather than longer survival. Therefore, our aim with the current quality of life analysis of the NORDIC9-study was to assess that the more effective chemotherapy was not at the expense of decreased quality of life. Our analyses showed that moderately dose-reduced combination chemotherapy-maintained quality of life, physical functioning, and resulted in less symptoms than treatment with full dose single drug in older patients not tolerating standard combination chemotherapy usually provided to young and fit patients.not a candidate for standard combination chemotherapy were included and randomly assigned to receive either S1 or SOx. The EORTC QLQ-C30 questionnaire was completed at baseline, after 9, and 18 weeks. The primary endpoint was global Quality of Life (QoL) at 9 weeks. For statistical analysis, a non-inferiority design was chosen applying linear mixed effects models for repeated measurements. The results were interpreted according to statistical significance and anchor-based, clinically relevant between-group minimally important differences (MID). A total of 160 patients aged (median (Interquartile range (IQR))) 78 years (76–81) were included. The QLQ-C30 questionnaire was completed by 150, 100, and 60 patients at baseline, at 9, and 18 weeks, respectively. The difference at 9 weeks in global QoL was 6.85  and 7.37  in the physical functioning domain in favor of SOx exceeding the threshold for MID. At 18 weeks, the between-group MID in physical functioning was preserved. Dose-reduced combination chemotherapy may be recommended in vulnerable older patients with mCRC, rather than full-dose monotherapy.Quality of life data from randomized trials are lacking in older patients with metastatic colorectal cancer (mCRC). In the randomized NORDIC9-study, reduced-dose S1+oxaliplatin (SOx) showed superior efficacy compared to full-dose S1 monotherapy. We hypothesized that treatment with SOx does not result in inferior quality of life. Patients with mCRC aged ≥70 years and that were  The primary results established that SOx resulted in significantly improved progression-free survival (PFS) , p = 0.047). Additionally, patients treated with SOx experienced less toxicity, and fewer hospitalizations, compared to S1 [Colorectal cancer (CRC) predominantly affects older adults ; 56% of When the NORDIC9-study was designed , we aimeOnly little is known about QoL, functional status, and symptom burden in vulnerable older patients with mCRC receiving palliative chemotherapy. Oncologists are usually interested in traditional tumor-centered endpoints used in randomized controlled trials (RCTs) of cancer drugs ,10,11. ODespite that incidence and mortality rates top in adults aged ≥70 years, older patients with cancer are underrepresented in RCTs ,18,19,20RCTs addressing QoL and patient-centered endpoints have long been desired by patients ,25, by rThe overarching objective of the present study was to investigate whether prolonged PFS with reduced-dose combination chemotherapy comes at the expense of poorer QoL and physical functioning. We hypothesized that treatment with reduced-dose SOx might result in inferior QoL and physical functioning compared to treatment with full-dose S1 monotherapy.not candidates for standard combination chemotherapy. Participants were recruited from 23 centers in four Nordic countries. Detailed protocol, study design including CONSORT flow diagram, findings of the primary endpoint, and several secondary endpoints have been published [The NORDIC9-study was a prospective, randomized open-label phase II multi-center study including older vulnerable patients aged ≥70 years with mCRC that were ublished . Nationahttps://portal.findresearcher.sdu.dk/da/publications/statistical-analysis-plan-for-nordic9-study-quality-of-life-analy, accessed on 9 March 2021), thus, securing transparency and reproducibility.The current manuscript conforms to the CONSORT 2010 statement for reporting RCTs with patient-reported outcomes as primary or secondary outcomes ,36. A st2 orally twice daily on days 1–14 + oxaliplatin 100 mg/m2 intravenously on day 1, q3w (once every 3 weeks)) or full-dose S1 . Dose modification was allowed according to the treatment protocol. The addition of bevacizumab  was optional at the discretion of the treating physician, but the decision was required before randomization. Evaluation of response was planned after every three cycles. Patients were treated until disease progression/death or unacceptable toxicities, whichever occurred first.Patients were randomly assigned  to receive either reduced-dose SOx (S1 20 mg/mPatient-reported outcomes (PRO) on QoL were assessed with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 version 3.0 (EORTC QLQ-C30) . The EORPhysician-reported outcomes of physical functioning were assessed through the ECOG PS. The ECOG PS score has six possible values where 0 means that the patient is “fully active, able to carry on all pre-disease performance without restriction” and a score of 5 means that the patient is “dead”. The ECOG PS is the most frequently used clinical score in oncology daily practice and research.Patient-reported and physician-reported data were collected at the time of randomization (baseline), after 9-, and 18-week follow-ups, corresponding to the administration of three and six cycles of chemotherapy. For the EORTC QLQ-C30, patients self-completed the questionnaire in paper-based versions where after it was collected by qualified study nurses and stored in patient documentation at the clinical research units. For the ECOG PS, physicians noted the score at the time of the clinical visit and stored it in the participants’ electronic medical record.Compliance of the patient-reported outcomes was calculated using the proportion of randomized patients with completed questionnaires and the proportion of patients expected to complete questionnaires  at all scheduled visits (at 9-week and 18-week follow-ups).PRO objectives, hypotheses, and analyses were pre-specified in a statistical analysis plan prior to data analyses.The primary endpoint of the current analyses was to test the hypothesis that treatment with reduced-dose SOx is not inferior to full-dose S1 in terms of affecting global QoL. We did this by comparing the impact of SOx vs. S1 on changes in the global QoL domain as assessed by the questionnaire from randomization to the 9-week follow-up. We choose to compare the 9-week follow-up scores to baseline, because in patients with considerable tumor load, the first two months are essential for whether an objective response or disease stabilization is obtained or not. Moreover, toxicity generally occurs at the initiation of the treatment, thus, both factors may alter global health.While recognizing that the value of using the global QoL domain as primary outcome in clinical trials investigating mCRC has been questioned due its low sensitivity to changes , the effThis included explorative analyses of the effects of SOx relative to S1 on other clinically important patient-reported outcome measurements in the following EORTC QLQ-C30 subscales: (1) physical functioning, (2) role functioning, and (3) social functioning from baseline to the 9-, and 18-week follow-ups.We selected domains of functioning as these were considered to play a key role in older patients regarding the maintenance of their general physical and mental well-being. We hypothesized that SOx would not result in inferior outcomes compared to S1.Given the toxicity profile of the applied cytotoxic agents and the adverse events reported in the primary publication of the NORDIC9-study , we alsoWe hypothesized that patients receiving S1 full-dose monotherapy would experience lower toxicity but may be higher symptom burden due to less efficacy compared to SOx.Furthermore, the risk of deterioration in physician-reported physical functioning specified by the ECOG PS score at the 9-week follow-up was established. We hypothesized that the risk of deterioration was higher in patients receiving SOx compared to S1.n = 150), while the physician-reported outcome, risk of deterioration based on ECOG PS was conducted on the intention to treat (ITT) population of the randomized trial (n = 160).Formal sample size calculation for the PRO data was not performed. Hence, the current sample follows the sample size needed for the primary endpoint of the NORDIC9-study . The curThe primary statistical model for the PROs consisted of linear mixed effects model for repeated measures (MMRM) including all PRO assessments . This allowed us to compare the average treatment effect of reduced-dose SOx vs. full-dose S1.These analyses were performed as non-inferiority analyses reporting coefficients with 95% confidence intervals for mean differences and comparing with between-group mean minimal important differences (MID) as reported by Musoro et al. . As it iTo provide easily interpretable estimates of the probabilities of non-inferiority and superiority we provided a sensitivity analysis fitted a Bayesian linear mixed effects model  with weakly informative symmetric priors ) for regression coefficients and weakly informative ) priors for variance components. This model resulted in posterior probabilities of superiority, non-inferiority, equivalence, and inferiority, providing straightforward comparison between possible scenarios of treatment effects among arms.Deterioration of ECOG PS score from baseline to 9-, and 18-week follow-ups was defined as a ≥1-point decline from baseline and was analyzed using logistic regression. In the main analysis, discontinuation of the study participation for any reason was regarded as deterioration of performance. Two sensitivity analyses (A and B) were conducted. In sensitivity analysis “A” only discontinuation due to death was considered as deterioration, while patients who left the study for other reasons were excluded. In sensitivity analysis “B” only observed ECOG PS were used to define deterioration and any patients who left the study were excluded.p-values ≤ 0.05 were considered statistically significant and estimates were reported with 95% confidence intervals (95%CI).All data were analyzed in STATA v16 , As only global QoL was considered as the primary endpoint along with a pre-specified primary data assessment time point, and other outcomes were considered secondary and supportive to the primary outcome, no adjustment for multiplicity was performed.Missing data in the PRO analyses were handled by restricted maximum likelihood estimation in the linear mixed effects model including all answered questionnaires. This analysis assumed missing at random questionnaires, although this assumption could not be checked in our study. Missing data in the ECOG PS analysis were handled by the two pre-specified sensitivity analyses (see above).Acceptable fulfillment of model assumptions was investigated by normal quantile-quantile plots for residuals and random effects in the linear mixed models, in case of deviations, nonparametric bootstrapping with 1000 repetitions was performed.Performance of the MCMC algorithm applied in the Bayesian models was investigated by diagnostic plots .Goodness of fit of the logistic regression model was investigated by Hosmer–Lemeshow’s test.In general, each PRO domain was interpreted according to clinically relevant MID using guidelines for between-group differences and changes over time ,42.For the global QoL domain, the result was interpreted according to the anchor-based MID for between-group differences as reported by Musoro et al. , who estFor functioning domains , symptom domains, and single items  the results were interpreted according to the anchor-based mean MID for deterioration by between-group differences according to Musoro et al. . HoweverRisk of deterioration was compared between the two treatment groups according to the analysis specified above.From March 2015 to October 2017, 160 patients with a median age of 78 years (interquartile range 76–81) were included in the NORDIC9-study, of whom 150 were available for the QoL analysis . PatientThe EORTC QLQ-C30 questionnaire was completed by all 150 (100%) patients at baseline. Patients alive and still on-study at 9-week and 18-week follow-ups, thus constituting the proportion of number of patients expected to return a questionnaire, were 132 and 114, respectively. Questionnaire compliance at 9 weeks was 76% (100/132) and 52% (60/114) at 18 weeks. Compliance was balanced between groups at all assessment time points.Most missing questionnaires were lacking due to a procedural error; one site including 36 patients completed EORTC QLQ-C30 only at baseline.p = 0.127). Despite this being non-significant, the difference exceeded the pre-specified threshold for MID between groups. At 18 weeks, the difference was neither statistically significant nor clinically relevant  at 9 weeks in favor of SOx , S1: −4.22 , relevant .Regarding dyspnea, clinically-relevant MID was defined according to the EORTC evidence-based guidelines for between-group differences by Cocks et al.p = 0.03). This difference exceeded the pre-specified threshold for between-group MID. At 18 weeks, a non-significant, however clinically relevant between-group difference of 7.88  was seen in favor of the SOx group in physical functioning , S1: −10.44 , p = 0.106).In the physical functioning domain, a statistically significant between-group difference of 7.37  at 9 weeks was seen in favor of SOx , S1: −7.52 , Regarding the domains of social and role functioning, neither statistically significant differences, nor clinically relevant MIDs were observed at 9 and 18 weeks .p = 0.024). The between-group difference slightly decreased at 18 weeks, though the trend was not altered  was seen in dyspnea at 9 weeks in favor of the SOx group, which also exceeded the pre-specified threshold for between-group MID , S1: 8.27 ,  altered .p = 0.239)  was observed in favor of SOx , S1: 8.78 , = 0.239) .In terms of diarrhea, nausea and vomiting, and pain, neither statistically significant differences nor clinically relevant MID were observed during follow-up .p = 0.366). Similar result was observed at 18 weeks of treatment , p = 0.676)  = 0.75 , = 0.676) . The resThe sensitivity analyses resulted in a probability of non-inferiority of the reduced-dose combination chemotherapy of at least 89% for all investigated EORTC QLQ-C30 domains at 9 weeks, and at least 76% at 18 weeks. The only deviation from this was the fatigue domain, for which the evidence of non-inferiority was only 64% and 46% at 9 and 18 weeks, respectively . The posOur findings demonstrate that reduced-dose combination chemotherapy may be the treatment of choice in older vulnerable patients with mCRC when preservation of QoL, physical functioning, and symptom relief are important goals of the treatment. Although non-significant, a clinically relevant between-group MID in favor of SOx was observed in the primary endpoint, global QoL at the primary assessment time point at 9 weeks after commencing the treatment. Additionally, a statistically significant and clinically relevant difference in the key secondary endpoint of physical functioning in favor of SOx treatment was seen at the primary data assessment time point. Likewise, our findings indicate that patients treated with SOx experienced less symptom burden with clinically relevant between-group MID in symptoms of dyspnea and fatigue at 9 weeks and at 18 weeks, respectively. The Bayesian sensitivity analyses confirmed our findings of good evidence of non-inferiority of the reduced-dose combination chemotherapy.Patients receiving reduced-dose SOx gained PFS benefit, experienced less toxicity , and prenon-hematological toxicities in the experimental arm. In 86% of these studies, against expectations [Although the global QoL seems to provide beneficial information, its value and sensitivity reflecting to the toxicity issues patients are facing during palliative treatment for mCRC were questioned by Schuurhuizen and colleagues ,44. In tctations ,44, globctations . FurtherAnother option might be the application of risk of deterioration or time to deterioration as endpoint, based on clinician-reported outcomes, like ECOG PS; however, ECOG PS and oncologists’ clinical judgement have shown poor association compared to a systematic in-depth evaluation, called comprehensive geriatric assessment (CGA) in older patients with cancer ,47. HencA major strength of our study is that the statistical analysis plan was developed and made available online before the analyses were conducted securing transparency and reproducibility. In addition, to the best of our knowledge, our study was the first applying the MID for interpreting the EORTC QLQ-C30 for objective comparison and interpretation of QoL domains in older patients with mCRC treated with chemotherapy . Using MIt must be acknowledged, that our dataset has some limitations due to missing items and questionnaires; however, in older vulnerable adults with metastatic cancer the attrition leading to missing data is inevitable considering the advanced cancer and vulnerabilities these patients are presented with. Patients with most symptoms having lowest baseline scores are at highest risk for drop-out, thus, changes in MIDs might be biased at 9 and 18 weeks. It is expected at patient with higher baseline scores or obtaining response will complete the questionnaires during the treatment course, which reflects to the general perception of QoL of patients who were less vulnerable or derived benefit from the treatment. Notably, the number of missing items and questionnaires due to patient drop-out is consistent with previously reported data from a similar population . To dealThe NORDIC9-study established that reduced-dose combination chemotherapy in addition to its increased efficacy did not result in inferior QoL compared to the reference treatment with full-dose monotherapy in older patients not fit for standard combination treatment. Thus, the treatment of choice in older vulnerable patients with mCRC should be reduced-dose combination chemotherapy when preservation of QoL, physical functioning, and symptom relief are important goals of the treatment.Many older patients with cancer tend to prioritize improvement or preservation of QoL rather than overall survival. Therefore, patient-centered endpoints including patient preferences and shared decision-making with a focus on preservation and improvement of physical and mental wellbeing should be the primary or co-primary endpoints of future clinical trials in older patients with cancer, rather than PFS and surrogate outcomes widely applied in medical oncology. Likewise, for appropriate patient stratification, the concept of CGA should be incorporated and guide treatment and supportive care interventions.


Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a form of autoimmune encephalitis. The characteristic clinical features include seizure, psychosis-like symptoms, abnormal movements, and autonomic disturbances. Patients with anti-NMDAR encephalitis can present with various types of movement disorders. Typically, the movement disorders start following intervals of psychiatric and prodromal manifestations in young adults; however, in children, these might be an early presentation of anti-NMDAR encephalitis. The disease is under-recognized and underdiagnosed in Pakistan. Early recognition of the disease is important to commence timely treatment leading to a better prognosis. Here we present a collection of anti-NMDAR encephalitis patients, specifically focussing on the different types of movement disorders and the differences in clinical manifestations between children and adults. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is regarded as one of the most common types of autoimmune encephalitis . Anti-NMAutoimmune encephalitis, especially anti-NMDAR encephalitis is associated with various movement disorders ,7. The mThe clinical presentation of psychotic symptoms, seizures, abnormal movements, and autonomic disturbances is regarded as highly characteristic of NMDA-receptor antibodies in both female and male patients . ClinicaThe phenomenology of the movement disorders associated with anti-NMDAR encephalitis still lacks consensus. Its clearer description will facilitate confident recognition and enable ensuing earlier immunotherapy in anti-NMDAR encephalitis . The movThe basic aim of this study is to characterize the different types of movement disorders and find the differentiating features between the children and adult patients diagnosed with anti-NMDAR encephalitis, which can help in early diagnosis.After approval from Lady Reading Hospital Medical Teaching Institution Ethical Review Board with approval no. 50/LRH/MTI, a total of 17 patients  were enrolled in this observational study. The study was conducted in the neurology unit of Lady Reading Hospital Peshawar from 18/05/2019 to 17/05/2021. An informed written consent (in Urdu) was taken from all participants. All those encephalitic patients were excluded from the study who had proven infective etiology for their presentations.The age of study participants ranged from 15 months to 27 years with a median age of 8 years at symptoms onset. Among these patients, 10 were less than 12 years of age (median age of 6 years and mean age of 5.6 years) whilst seven patients were more than 12 years of age (median age of 18 years and mode of 14 years).The diagnosis of anti-NMDAR encephalitis was made on the basis of history, electroencephalography (EEG), and cerebrospinal fluid (CSF) findings, ruling out other differential diagnosis and positive antibodies testing as proposed by international group diagnostic criteria for anti-NMDAR encephalitis. Data were analyzed through Statistical Package for the Social Sciences (SPSS) version 20 . The percentages, frequency, mean, standard deviation (SD), and p-values were calculated.Features of movement disorderThe characteristics of movement disorders in different age groups are summarized in Table The most common movement disorder noted was stereotypical movement disorder defined as “involuntary, coordinated, repetitive, rhythmic, purposeless but seemingly purposeful or ritualistic movement, posture or utterance”. The stereotypy was either simple or complex stereotypies. The simple stereotypical movements included no-no or yes-yes movements of the head, cycling movements of legs, clapping hands, thrashing of the arm. This could progress to the rest of the body as well. One of the complex stereotypies noted was the patient moving in a circle with clapping hands and uttering sounds.Orofacial dyskinesia was observed in 12 patients. Orofacial dyskinesia was characterized by involuntary and repetitive movements of face and mouth including grimacing, sucking, rolling, and protrusion of the tongue, jaw opening and closing. All patients were noted to have a stereotypical movement disorder. The most common movement disorder noted in children <12 years of age was stereotypical movement disorder followed by orofacial dyskinesia (71%). Among patients >12 years of age, orofacial dyskinesia was still the common movement disorder (71%) noted, followed by catatonia (57%). Comparing the two groups, the frequency of stereotypical movement disorder and orofacial dyskinesia was the same in both groups with no statistical difference. The hyperkinetic movements of dystonia, choreoathetosis were common in patients less than 12 years of age whilst the hypokinetic movements including catatonia and bradykinesia were more prevalent in patients more than 12 years of age. One of the patients presented with hypoventilation requiring ventilatory support.Epileptic seizuresThe majority of patients (12/17) suffered from seizures. Seven out of 12 patients presented with focal seizures with or without loss of awareness. Five patients presented with NORSE (new onset refractory status epilepticus). Two patients who were initially thought to have probable seizures based on the history provided actually had stereotypical movement disorder including clapping and turning around in circles.Associated symptomsThe associated clinical manifestations in different age groups are summarized in Figure Immunomodulatory treatmentAll patients were given IV methylprednisolone for five days. Six out of 17 patients had a good response to pulsed steroids. These patients were started on a steroid-sparing drug with weaning off oral steroids. Due to the financial constraints, some of the patients went on to have second-line immunotherapy i.e. rituximab instead of intravenous immune globulins (IVIGs). Four out of 17 patients required IVIGs. One of the patients had plasmapheresis. Six out of 17 patients received rituximab.Thirteen patients had a good response and resumed normal life with two patients having persistent neuropsychological problems  evident from poor academic performance and impaired social relationship after 18 months of their initial presentation. One of the patients presented and was diagnosed six months post-illness and therefore was left with occipital blindness and behavioral changes. Three out of 17 patients lost to follow-up.The movement disorder is a frequent and emphasized feature of anti-NMDAR encephalitis but lacks an expert-based gold-standard description ,6,12,13.The comparison of clinical presentation between adults and children suffering from anti-NMDAR encephalitis shows a difference ,18. The Frequently convulsive and rarely non-convulsive seizures are manifestations of anti-NMDAR encephalitis . About 2With regards to psychiatric symptoms, a retrospective study found that 2/3 (59%) patients presented with psychiatric symptoms. Among them 40% presented with visual or auditory hallucinations, 23% with acute schizoaffective episodes or depression, 8% with mania, and 6% with addictive or eating disorders . StudiesPatients with anti-NMDAR encephalitis frequently show autonomic instability and respiratory dysfunction which necessitate their management in intensive care units. ,28,29. TA variety of symptoms and presentations have been reported in patients with anti-NMDAR encephalitis. Movement disorders are typical but non-specific. More than one type of movement disorder is present in nearly all cases, a factor that poses a diagnostic challenge to the neurologist and can delay the start of appropriate treatment. Despite its small sample size, our study has shown that pediatric patients are more likely to develop hyperkinetic movement disorders, while hypokinetic movement disorders are more prevalent among adult patients. Early and accurate diagnosis of anti-NMDAR encephalitis and treatment initiation has significant long-term prognostic implications. Recognition of the movement disorders and other symptoms can help in the early diagnosis of anti-NMDAR encephalitis and its timely management.


The objective of the study was to assess the genetic variability, drought tolerance behavior, and identify promising breeding lines for different rice ecologies and drought breeding programs. Restricted maximum likelihood (REML) analysis using the mixed model approach revealed a considerable genetic variation in the population for yield and yield contributing traits in non-stress and drought-stress conditions. We observed very high heritability for all the selected traits under stress 2015 WS (73.8% to 85.3%) and 2016 WS (72.4% to 93.5%) and non-stress 2015 WS (68.2% To 92.9%) and 2016 WS (61.4% to 92.6%) environments, indicating possible selection for grain yield under drought stress and non-stress with the same precision level. None of the secondary traits except harvest index and biomass included in our study showed a positive association with grain yield, indicating indirect selection’s ineffectiveness in improving yield under drought. A total of 48 promising breeding lines were found to have a better yield than donor Chao Khaw (up to 38% advantage) and popular drought-tolerant cultivars Shabhagidhan (up to 48% advantage) in stress conditions and recommended for rainfed upland ecology, 34 breeding lines under the well-watered condition suited for rainfed lowland ecology. Overall, the study found 21 common breeding lines that showed their superiority in non-stress and under drought stress situations, fitting best in rainfed lowland ecology with occasional drought occurrence. The large genetic variation found in this population can be exploited further to develop a few forward breeding high-yielding lines with better drought tolerance ability and used as drought donors in drought breeding programs.Drought is one of the most predominant abiotic stresses in this century, leading to a drastic reduction in the yield of rainfed rice ecosystems. Breeding of drought-resilient rice varieties is very much in demand for sustainable rice production in drought-prone rainfed ecology. An experiment was designed under irrigated non-stress and drought-stress situations involving an exotic drought-tolerant landrace (Chao Khaw) and a high-yielding aromatic rice cultivar (Kasturi), and an F Climate change is not a myth but a fact, which becomes a great threat to society due to its potential to intensify extreme events such as drought around the globe. Increased global temperatures and frequent changes in monsoon primarily cause the occasional droughts and floods, more common in many rice-growing areas in South and Southeast Asia.Rice is the most widely consumed staple food as a cereal grain, is one of the most water-intensive crops, and grows about in the 25.12 million hectares area in irrigated conditions and 42.75 million hectares in lowland conditions in India. Moreover, rice consumes about 80% of irrigated freshwater resources worldwide . HoweverOn the other hand, rainfed lowland ecology covers 33% of the rice area globally but yields only 19% of total global production . This coMany rice varieties grown in rainfed ecology are primarily developed for irrigated conditions and are preferred due to their high-yielding potential and better grain quality. These varieties are high yielding during the favorable season but perform poorly during the occurrence of occasional drought. Since the rainfed rice ecology is highly variable, breeding for drought-prone climate needs varieties that combine high-yielding potential with good drought tolerance . It is aThe evolution of rice cultivars in drought-prone rainfed areas has allowed the development of a large number of drought-tolerant landraces which can withstand drought to many extents. Many of these landraces have also been known to possess deep roots up to 70 cm below the soil surface. Greater root length density at depth has also been reported in drought-tolerant landraces such as Dular, Azucena, and Rayada compared with high-yielding drought-susceptible varieties such as IR64 . HoweverMost traditional drought-tolerant donors are not used directly in breeding programs because of the linkage drag of several undesirable traits they possess. Hence, identifying and introgression of the genes or QTLs from them to existing elite varieties is the primary step for developing new generation drought-tolerant rice cultivars for rainfed lowland areas.In the past, many drought-tolerant lines were developed by successful crosses between a drought-tolerant donor and a high-yielding drought-sensitive cultivar . This stExperiments were conducted at the International Rice Research Institute (IRRI), South Asia Hub (IRRI-SAH), ICRISAT Campus, Hyderabad in 2015 WS (wet season/rainy season) and 2016 WS. The experimental plots are situated . The soil type of the conducted experimental plots was clay loam in texture. The non-stress experiments were sown during June and drought stress experiments were sown in July in 2015 WS and 2016 WS. To avoid the monsoon rain and maintain the drought cycle imposition, the sowings of the drought-stress experiment were delayed for one month from the non-stress experiment to get the severe stress cycles throughout the crop cycle. This drought imposition and drought screening method have been highly successful in the earlier study .Kasturi (RP 2144-108-5-3-2), long slender, improved aromatic variety for the irrigated lowland ecosystem variety, which has developed by selection from the cross-Basmati 370/CRR 88-17-1-5 at Indian Institute of Rice Research (IIRR), Rajendra Nagar, Hyderabad, was released for commercial cultivation in the year 1994. This variety is semidwarf in stature (95 cm), matures at 135 to 140 days, and yields about 30 to 35 quintals under the well-watered situation with an optimum nutrient application, which shows sensitivity during the reproductive stage drought situation. On the other hand, the donor parent, Chao Khaw, is a long bold Laos landrace, collected by the International Rice Research Institute germplasm collection and identified as a promising drought-tolerant genotype with blast-resistant having low grain quality traits, semitall in stature (110 cm) with medium duration (120 to 125 days) (IRRI unpublished data).1 seeds were generated. True single plants were harvested in 2014 WS, and 200 F2 individual plants were harvested in the dry season of 2015. Finally, in our study, 156 breeding lines in the F2:3 and F2:4 generations were included for screening for two main rice seasons in two conditions. IR 64, a high-yielding but drought-sensitive variety, and Sahabagidhan, a drought-tolerant variety, were planted along with the population in non-stress and reproductive stage drought-stress conditions.The population was developed by crossing between Kasturi and Chao Khaw in the dry season (postrainy) of 2014, and 34 F2:3 and F2:4 population was conducted under well-watered and reproductive stage drought conditions. The field selected for the study was upland in topography with good drainage and loamy clay soil. Seeds were sown in a nursery bed and transplanted in the main field after 25 days of sowing under non-stress (well water condition) and reproductive stage drought-stress condition. Under non-stress and reproductive stage drought-stress conditions, each family was planted in two-row of 2 m with 20 cm × 15 cm spacing. The experiments were laid out with an augmented RCBD design with checks and parents repeated five times, and a standard package of practices was followed to raise the good crop. The total rainfall received during the crop cycle was 421.8 mm in 2015 WS and 834.6 mm in 2016 WS. The weekly rainfall pattern for 2015 WS and 2016 WS has been given in The phenotypic evaluation of the FWater table depth was measured by installing a 1.1-m unplasticized polyvinyl chloride carbonate (UPVC) pipe in experimental fields at regular intervals. Depletion in the water table was measured through a meter scale daily after the onset of the stress. This irrigation regime resulted in stress levels that caused LR and tip burning on most entries at the end of each drying cycle. The repeated stress cycles ensured that all entries experienced stress during the sensitive stage of 15 days before and after flowering. On average, this protocol has been found to reduce yield by 50%–70% relative to fully irrigated controls .In all the trials, observation was recorded for days to 50% flowering (DTF), plant height (PHT), number of productive tillers (NPT), panicle length (PL), total number of grains per panicle (TGP), number of filled grains per panicle (TFGP), spikelet fertility percentage (SPF%), plot yield (PY), biomass (BM), and harvest index (HI). Data were recorded as per the standard evaluation system .1 (2g) for each trait and their respective standard errors were determined. Best linear unbiased predictors (BLUPs) were estimated for each line in each experiment along with standard errors to test the significance of variance components. Further phenotypic correlations were estimated to determine trait associations in GenStat 17.The trait data in both stress and non-stress experiments were analyzed using restricted maximum likelihood (REML) methods for each season considering genotypes as a random effect and blocks as a fixed effect in the mixed-model procedure using GenStat for Windows 17th Edition, VSN International, Hemel Hempstead, United Kingdom1 . The varDrought treatments in both years resulted in soil drying and subsequent yield reductions in parents and population compared with the well-watered control. The yield reduction in drought-tolerant parent Chao Khaw under drought-stress environments (DSE), DSE-2015, and DSE-2016 was 61.72% and 54.12%, respectively, whereas the reduction was much higher in drought-sensitive parent Kasturi in DSE-2015 (80.03%) and DSE-2016 (82.78%). The drought stress reduced 74.87% yield in the drought-tolerant cultivar Shabhagidhan in DSE-2015 and 70.35% in DSE-2016. Overall, the imposed stress caused mean reductions in yield of over 60% (60.24% in DSE-2015 and 64.86% in DSE-2016) in drought-stress trials than well-watered control. The population means for all the traits in the drought-stress environments were lower than the population means in the non-stress environment in both the years .The mean values for DTF of the population in DSE-2015 and DSE-2016 were 4–10 days less than NSE-2015 and NSE-2016. As expected, the population mean for other traits, that is, PHT, NPT, PL, PY, BM, HI, TGP, TFGP, and SPF% in both stress environments, was lower than the non-stress environments. It is worth mentioning that the reduction from non-stress to stress environment was higher in TFGP than TGP, which indicates the role of fertility breakdown in the breeding lines under stress conditions.A single frame violin plot was drawn to visualize the phenotypic distribution of different traits across the environments for all the traits studied. The violin plots showed clear differentiation among stress and non-stress environments . The dis2g) were higher in NSE-2016 WS, followed by NSE-2015 WS than in DSE-2015 WS and DSE-2016 WS  and HI (21% & 23% and 29% & 30%) in DSE-2015 and DSE-2016, respectively.Restricted maximum likelihood analysis for different components revealed that genotype variances were significant for all the traits studied based on the performance in individual drought-stress environments (2015 WS and 2016 WS) and the non-stress environments (2015 WS and 2016 WS). PY’s genotypic variance components  and HI , showed a very high genetic advance of mean (GAM).The Pearson’s correlation analysis for different yield contributing traits under stress and non-stress conditions revealed that only BM and HI (0.22–0.65) were associated with grain yield in stress and non-stress environments . Days toIn this experimental screening, we have found some transgressive segregants, which yielded much better than the popular mega variety, IR 64. These breeding lines may or may not have the drought-tolerant capacity but showed their yield superiority under well-watered conditions. A total of 34 breeding lines were found to have a significantly higher yield than the mega variety, IR 64. More than half of the breeding lines (21 lines) yielded more than 4,000 kg/ha. These breeding lines flowered earlier than the female parent Kasturi with 92 to 100 days falling in the medium maturity group and at par with IR 64 .Some drought-tolerant breeding lines for the areas in rainfed upland and highly drought-prone ecologies were identified from this study irrespective of their performance in the well-watered condition. We found 48 breeding lines with an average yield of 1,650 kg/ha and spans from 1,446 to 1,934 kg/ha. In contrast, the drought-tolerant popular variety Shabhagidhan and donor parent Chao Khaw yielded an average of 940 kg/ha and 1,210 kg/ha, respectively, over two years. The female parent Kasturi yielded just above half-ton per hectare. All the breeding lines under the drought stress condition were in the medium maturity group (86–96 days) with semidwarf plant type (73–91 cm). In contrast, the female parent Kasturi flowered in 104 days and the tolerant donor flowered in 91 days. The spikelet fertility of those breeding lines was 49%–58% under severe drought conditions .This study identified 21 breeding lines common across the non-stress, and stress environments of 2015 WS and 2016 WS showed better yield performance than the tolerant check Shabhagidhan and tolerant donor Chao Khaw. These 21 breeding lines were selected from the 48 drought-tolerant promising breeding lines and 34 promising breeding lines in non-stress conditions. The top 14 breeding lines yielded more than 4,000 kg/ha , and the average yield of the same 14 breeding lines, under drought stress, was 1,537 kg/ha  .Overall, all the 21 breeding lines yielded more than 3,500 kg/ha  in non-stress conditions and more than 1,000 kg/ha . In contrast, drought-tolerant Shabhagidhan had 3,407 kg/ha and 940 kg/ha in non-stress and drought stress conditions, respectively, whereas the donor parent Chao Khaw yielded 1,210 kg/ha in stress 2,864 kg/ha in a non-stress environment. Though Kasturi is a high-yielding variety, the yield was 640 kg/ha, which indicates its drought sensitivity under stress conditions. Interestingly, all the identified breeding lines flowered between 92 and 100 days in well-watered conditions and 83–96 days under stress conditions indicating they fall into medium-maturity groups. We also found that all the 21 breeding lines are semidwarf plant types (92–109 cm in non-stress and 75–86 cm in stress days). These breeding lines also have high tillering capacity with 6–8 productive tillers per hill.Erratic rainfall distribution in rainfed areas and depleting the ground water level in irrigated areas threaten rice production globally and demand the urgent need to develop rice cultivars capable of producing more from diminishing water resources. The drought-tolerant rice varieties are believed to reduce rice production risk in rainfed areas, increase productivity in the changing climatic scenario, and alleviate poverty in rice-growing areas. Breeders need to adopt adapted solutions and strategies to respond to a different kind of drought situations based on the precise characteristics of different rice ecologies. For instance, the rice ecology of Eastern India receives sufficient rainfall, and so, rice is grown in rainfed conditions. However, due to erratic rain once in a few years, occasional drought leads to drastic yield reduction. Hence, rice drought breeding includes diverse approaches suitable for various rice ecologies and needs. For decades, traditional farmers have grown many landraces that may have low-yielding capacity but can mitigate the abiotic stress. Modern breeding strategies of elite x elite crosses for developing the high yielding ignore the need for landraces in the breeding programs. The underutilized landraces and their hidden treasures of tolerant genes/QTLs may help impart the maximum water-use efficiency in the elite modern breeding lines under alleviating the drought stress.Though many studies have been conducted over the last two decades to develop drought-tolerant lines in rice, limited reports are in the public domain, where drought breeding programs involve exotic drought-tolerant landrace and popular aromatic cultivars. One such exotic landrace is Chao Khaw from Laos, which is considered an excellent drought-tolerant line with hidden drought-stress tolerant QTLs or genes. On the other hand, Kasturi, an aromatic rice variety, has high-yielding potential, excellent milling quality, blast resistance, and stem borer tolerance but is sensitive to water stress.Drought screening, differentiating high-yielding and drought-sensitive lines from drought-tolerant ones and their yield gaps under non-stress and stress conditions, is considered adequate for selecting drought-tolerant higher-yielding lines. In this study, the drought stress experiments with imposed three cycles of stress and adjustment of sowing to avoid heavy rainfall, could accurately distinguish the drought-tolerant and drought-sensitive lines. The literature cites that if a lowland drought screening experiment reduces yield in the range of 30%, 31%–65%, and 65%–85%, then the drought should be considered mild, moderate, and severe types, respectively, and appropriate for drought tolerance breeding . Yield rAlong with the grain yield, other traits such as plant height, harvest index, spikelet fertility percentage, and the total number of filled grains showed reduced values under drought stress compared to the non-stress conditions. The reduction of spikelet fertility percentage under drought stress is considered crucial for yield reduction in rice under stress . In thisThe mixed model REML analysis of variance revealed a significant variation among the breeding lines for all the traits across the environments. Furthermore, this confirms the fact that a good amount of segregation exists for the studied traits. Parents differed significantly for all the traits in both the drought-stress environments, which was also observed by previous studies . ParentsFrom the ranges of various traits across the environments and years, it is evident that most of the traits were higher in NSE-2016. The comparably high rainfall during 2016 non-stress conditions may be the reason for that. Under the drought stress conditions, except TFGP, the range of all traits was higher in DSE-2016 than in DSE-2015. The high groundwater level of the trial fields in all the three stress cycles in DSE-2016 was high compared to DSE-2015 which further confirms the large variability in DSE-2016 than DSE-2015. In short, the comparable favorable conditions of DSE-2016 were helpful in the expression of the breeding lines variably. It seems that the range of PHT and NPT was least affected across the trials except for DSE-2015. The relatively higher range of HI in drought-stress conditions indicates that there must be few breeding lines with a superior capacity of both sink and source and convert the photosynthates into usable economic yield.All the breeding lines are grouped into the early to mid-early groups. Both the parents were from the early to medium category, and none of the parents was in the late category, which may be the reason for low variability in the population for flowering. Similarly, the low difference between parents for PH and PL may be responsible for the low variability of those traits. As expected, for PY, comparatively low variability was observed under non-stress conditions than the drought-stress conditions. High variability under the stress condition implicates stress-induced additional variability among the breeding lines through upregulation or downregulation of genes responsible for drought mitigation in plants. Like PY, HI also showed higher stress conditions than non-stress conditions reflecting the stress-induced variability in the population under drought-stress conditions.The heritability estimates were found more or less similar in stress and non-stress environments and high for all the traits studied. No consistent reductions in heritability under severe drought stress were visible; in some cases, the heritability was higher in the stress environment. Heritability values of >70% for phenological traits and grain yield were also reported earlier . The relper se of those breeding lines than drought escape. There was no advantage of tall, semidwarf, or dwarf plants in influencing the yield under both non-stress and stress conditions as no association of plant height and grain yield was observed in both the conditions.The significant positive association observed between grain yield and harvest index under stress indicates that the yield differences we observed under drought stress were primarily due to the vast variation in plants’ capacity to maintain seed set under stress, apart from accumulating biomass. This result is consistent with previous studies, which reported that the reduction in yield when the drought stress is applied during flowering time is mainly due to spikelet sterility . As therSimilarly, in high yield, NPT and PL had no role, which indicate that breeding lines with more productive tillers and panicle length could have a more significant number of chaffy seeds or the weight of the seed was lower compared to other breeding lines with less productive tillers or panicle length but with high seed weight. As expected, in this studied population, TGP had no association with grain yield under both the conditions across the season, pointing out that the breeding lines behaved differently in producing filled grains. Many breeding lines might have many grains but many chaffy grains, whereas breeding lines may have few grains but more filled grains. This phenomenon is also reflected by spikelet fertility percentage as SPF% also showed no correlation with grain yield. There is no correlation between SPF% and grain yield which indicates that many low-yielding breeding lines are present in the population with high spikelet fertility. In other words, due to the smaller number of grains per panicle, these breeding lines could translocate the photosynthates to the majority of the grains. Except for biomass and harvest index, the absence of correlation with other traits with grain yield inferred that the breeding lines showed a very linear relationship with biomass and harvest index, but different breeding lines behaved differently with other traits. Hence, more biomass accumulation and plants’ capacity to maintain the good source-sink relationship under both conditions are beneficial to get a higher yield. No correlation between the number of filled grains and grain yield indicates the seed weight and the seed length, which may play an essential role in determining the grain yield rather than filled grains. The secondary traits included in this study were ineffective toward grain yield under stress and confirmed the earlier studies .Two breeding lines yielded almost two times more than the tolerant check, Shabhagidhan, and 1.5 times the tolerant parent, Chao Khaw. This confirms that it is possible to select superior stress-tolerant breeding lines directly selecting grain yield under stress conditions from tolerant or sensitive crosses. These results have similarities with , where tIn conclusion, this study revealed the availability of large genetic variation with high heritability in the studied population under both the drought-stress and non-stress conditions. The sufficient variation under drought stress confirms that the population was perfect for studying rice drought tolerance behavior. The imposed drought condition, which made the water depth level below the recommended level, provides the ideal condition for drought tolerance study. The studied population needs to be evaluated in multiseason and locations to know the significant genotype–environment interactions. The promising breeding lines identified suitable for different ecologies should extensively be evaluated under multilocation trials (MLTs) for immediate release as varieties or use as a donor in breeding programs.The original contributions presented in the study are included in the article/AK, PK, CV, and VS involved in conceptualization and methodology. CV contributed in field trial establishment and data collecting. CV, PP, and AS involved in statistical analysis. PP and VS contributed in writing original draft preparations. VS, AK, and PK involved in writing, reviewing, and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics.The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public. The coronavirus disease 2019 (COVID-19) pandemic has brought about an unprecedented need for population-scale pathogen testing. Nucleic acid-based tests, particularly reverse transcription-quantitative PCR (RT-qPCR) tests, are the most frequently used and are currently the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics  of reverse-transcribed, noninfectious, and nonhazardous SARS-CoV-2 sequences. This laboratory also did not work with infectious virus or patient samples. The target sequence included the CDC “N2” locus that is now widely used in RT-qPCR diagnostics  and C 1. It was We later learned that anomalous SARS-CoV-2 test results among researchers were not limited to a single laboratory. At a second research institute, 10 researchers in three separate research groups tested positive for SARS-CoV-2, with one testing positive on two separate occasions . These rTo determine whether amplified DNA products that could affect test results were widely present in the laboratory, we collected swabs from various surfaces and equipment throughout the laboratory, eluted any captured DNA, and performed qPCR using the CDC N2 primer/probe set. The qPCR did not include reverse transcriptase and therefore only detected amplified cDNA, not viral RNA. Although the researchers had followed standard practices for working with amplified nucleic acids, including physical separation of pre- and postamplification workspaces, we found that nearly every surface had detectable quantities of N2 amplicon . The higSampling of the laboratory space in the second research institute revealed that there was also widespread N2 amplicon contamination in the space , which wFollowing the discovery of extensive laboratory contamination, work using the N2 amplicon in the first laboratory ceased. Repeated efforts to decontaminate the laboratory were met with limited success. We performed iterative cycles of cleaning, swabbing, and qPCR detection of remaining DNA. These efforts relied on chemical agents, including bleach, DNA Away, and hydrogen peroxide solutions. After five rounds, we were able to reduce, but not eliminate, the LAMP DNA . Some inThe SARS-CoV-2 pandemic has brought about an era during which, for the first time in history, humans are subject to recurrent, population-scale nucleic acid testing. It has also prompted laboratories around the world to research SARS-CoV-2 biology and improved nucleic acid diagnostics. Therefore, nucleic acids with the capacity to trigger positive diagnostic test results are likely present at nearly every research institute and many clinical laboratories. We have presented instances in which positive SARS-CoV-2 tests were likely triggered by laboratory-generated DNA, and we identified widespread laboratory DNA contamination. Anomalous tests among researchers have been reported at other research institutes as well, with one report confirming lack of infection through negative serology tests , 12. WhiThe rapid spread and persistence of contaminating DNA throughout a laboratory and beyond raises important issues regarding the stewardship of nucleic acids that can confound test results. The contamination in these laboratories was likely exacerbated by the use of LAMP reactions, which generate concatenated copies of the amplified target sequence . A singlThe widespread presence of amplified SARS-CoV-2 DNA products in both research institutes had impacts that extended beyond the research teams that generated the amplicons. The CDC currently recommends that individuals who test positive not receive additional diagnostic tests for 90 days, given the long residence time of residual SARS-CoV-2 RNA in infected patients . A subseLaboratory-generated nucleic acids have the potential to affect the community beyond those directly associated with the laboratory. Wastewater surveillance has been implemented as an early warning system to detect community spread that has fallen outside the framework of individual testing , 16. SimIn order to reduce the risks involved with working with nucleic acids that can compromise testing, we have devised a series of common-sense recommendations for laboratory workers. These nucleic acids include not only amplicons from LAMP, recombinase polymerase amplification (RPA), PCR, or other methods but also plasmids and any other nucleic acid-containing SARS-CoV-2 sequences. Steps to prevent laboratory contamination, such as engineering controls, good work practices, and proper personal protective equipment (PPE), should be coordinated in advance of the initiation of research. In the cases outlined here, the researchers used basic engineering controls of separating pre- and postamplification workspaces. More stringent controls would include physical separation of these spaces in different rooms and unidirectional workflow from pre- to postamplification spaces. The affected researchers wore lab coats, gloves, and surgical masks. Unidirectional workflow would also ensure that PPE worn in postamplification spaces was not worn in other laboratory or clinical spaces. These controls would all reduce the likelihood of contaminating personal items belonging to researchers and technicians and are indeed standard practice in many clinical diagnostic laboratories. Periodic environmental surveys of laboratory surfaces and personal items reduced the frequency of anomalous positive test in the affected laboratories and should be included in experimental/diagnostic design.A plan for how to respond to a presumptive positive test and how to verify it should be in place before initiation of work with these nucleic acids. When designing experiments, attempts should be made to limit the likelihood that a contaminant in the laboratory would interfere with the result for every single approved test. In the research laboratory described here, it was possible to follow up on the initial positive result using another test provider that used the N1 and N3 primer-probe sets.N-glycosylase (UNG) system, should also be used . Watermarks have been used in engineered microorganisms to differentiate recombinant viruses from circulating viruses , 18. Whe be used . This syLaboratories should declare and post notices if they produce problematic nucleic acids and indicate their presence to those who handle waste streams. This would help direct those with positive test results to the resources for verification. Rigorous engineering controls and standard operating procedures for working with these DNA products should be in place, including proper use of fume hoods or biosafety cabinets when working with amplified nucleic acids . These pThe advent of at-home testing poses another challenge in preventing environmental contamination with interfering DNA. The first completely at-home test to receive emergency use authorization from the Food and Drug Administration is a LAMP-based test . It is iPCR-based testing is the current gold standard for SARS-CoV-2 testing, and these tests are highly sensitive and accurate. Even in these reported instances, the tests are performing as intended to detect SARS-CoV-2 sequences. We emphasize that research-produced nucleic acids triggering a SARS-CoV-2-positive diagnostic test result is a rare circumstance. For those engaged in research that generates nucleic acids with the capacity to interfere with testing or surveillance, we have a responsibility to not contaminate our environments in ways that will undermine trust and impede life-saving public health initiatives, for SARS-CoV-2 or otherwise.Test results from individual researchers were volunteered and directly communicated. The Institutional Review Board of the Harvard Faculty of Medicine determined that this did not constitute research on human subjects.Laboratory surfaces were swabbed for contamination using clean Q-tips brand cotton swabs. Swabs were prepared for sampling in a clean biosafety cabinet that had never been used for SARS-CoV-2 work. First, the working area and equipment were cleaned with a solution of either 2% bleach or 1% hydrogen peroxide, followed by 70% ethanol. Swabs were cut to fit inside the tubes, which were closed and placed in a clean box. Gloves were worn while sampling and changed between samplings. To sample a surface, a clean swab was removed from its tube, rubbed over the surface to be tested, and placed back in the tube. Two swabs were left unopened to serve as negative controls. Once swabbing was complete, 300 μl of TE buffer   was added to each tube. The tubes were then incubated at 37°C for 30 min, vortexed briefly, and then centrifuged at high speed for 30 s. In a biosafety cabinet, 50 μl of the swab eluate was transferred from each tube to a corresponding well of a sterile 96-well PCR plate, sealed, and stored at −20°C until analysis.TC] value) were set at a TC of 45 for purposes of visualization.The qPCR mixture with primers and probe was prepared for analysis of the CDC N2 amplicon, using N2 primer and probe  and Luna universal probe qPCR master mix . To analyze 96 samples, including controls, 300 μl of water and 200 μl N2 of primer/probe mixture were added to a tube containing 1 ml of 2× Luna probe mixture. A 96-well qPCR plate was placed on a cold block on ice, and 15 μl of this mixture was added to each well. Using a multichannel pipette, 5 μl of each swab eluate was added into a corresponding well of the plate containing the qPCR mix. On each plate, negative-control swabs, wells containing only TE buffer, and positive controls known to contain the N2 DNA amplicon were also analyzed. The plate was sealed with optical film and placed in a LightCycler 96 qPCR machine (Roche), which was run for 45 cycles. Data were analyzed using Prism 8 software (GraphPad). Values for samples with undetectable DNA (no cycle threshold [


The three-armed cluster-randomized trial ARena  aimed to foster appropriate antibiotic use and reduce overprescribing in German ambulatory care to counter antibiotic resistance. Multi-faceted interventions targeted primary care physicians, teams and patients. This study examined the effectiveness of the implementation program. ARena was conducted in 14 primary care networks with 196 practices. All arms received data-based feedback on antibiotics prescribing and quality circles. Arms II and III received different add-on components each. Primary outcome examined is the prescribing rate for systemic antibiotics for cases with non-complicated acute infections . Secondary outcomes refer to the prescribing of quinolones and guideline-recommended antibiotics. Based on pseudonymized quarterly claims data, mixed logistic regression models examined pre-post intervention antibiotic prescribing rate changes and compared to matched standard care. A significant rate reduction  and significantly lower prescribing rates were observed for all arms  compared to matched standard care (29.4%). Fluoroquinolone prescribing was reduced in all intervention arms and rates for recommended substances generally increased. No significant post-interventional difference between intervention arms was detected. Findings indicate implementation program impact compared to standard care. On a global and on national scale, the development and increase of antimicrobial resistance are advanced by over-consumption of antibiotics in human and veterinary healthcare and by inappropriate prescribing. In Germany, about 85% of the used antibiotics are prescribed during consultations in the primary care sector ,2. CompaIn the context of the national strategy to reduce antibiotic consumption and counteract antimicrobial resistance , the AReThe ARena implementation program used multi-faceted interacting components to address knowledge, attitudes and experiences of primary care physicians , care teams and patients about the use of antibiotics in PCNs across two German federal states . The program used different strategies to reduce undesirable prescribing rates. All three intervention arms received a base module of intervention components comprising e-learning on communication for physicians to limit misinterpretation of patient preferences, moderated quality circles (QCs) with data-based feedback on antibiotics prescribing for physicians to facilitate appropriate prescribing, public information campaigns including social media, performance-based additional reimbursement, and culture-sensitive print information material for patients. Based on program developers’ assessment of barriers for implementation, arm II received e-learning on communication and separate QCs addressing the team of medical assistants in participating practices, and tablet pcs providing patient information in waiting areas. Including the medical assistants was assumed to be supportive for rational prescribing since sensitizing practice teams for a stronger interaction with patients might facilitate a relieve of the physician’s workload and contribute to optimized decision-making ,14. ConsThe primary objective of this study was to examine and compare the change of prescribing rates for targeted index diagnoses in the ARena intervention arms and compare prescribing rates to non-participating standard care, matched to participating PCNs, based on quarterly claims data.A total of 87,377 patient cases with ARena index diagnoses during the intervention period (Q4 2017-Q2 2019) were included for analysis, which exceeded the target of 79,685 cases calculated for the evaluation. Sample sizes differ between primary and secondary outcomes, because they focus on subgroups of the primary outcome population. One patient might represent several cases.Physicians working in general practice were the largest group of prescribers in PCNs (76.5%) and in standard care (75.8%) (p < 0.001) and in intervention arm III . Additionally, in intervention arm I, the pre-post-comparison showed a significant reduction of antibiotic prescriptions . No significant difference was detected between intervention arms regarding within-group change in the RCT. Intervention arms showed different baseline prescribing rates and post-intervention prescribing rate reductions. A significant reduction of antibiotic prescriptions was observed in intervention arm II ). The comparison of changes in each of the three intervention arms to the matched standard care was evaluated in logistic mixed regression models and considered pre/post intervention as covariate. For all intervention arms, a larger reduction of antibiotic prescribing compared to the matched standard care was observed  of fluoroquinolone prescribing, low pre-interventional rates were observed for trial participants, and these were further reduced during the intervention period  in matched standard care. At the post-intervention measuring point, prescribing of guideline-recommended antibiotics in the intervention arms had increased and was higher than in matched standard care .For bronchitis, an increase in prescribing of recommended substances was recognized in all intervention arms and in matched standard care. Intervention arms I and III reached a substantially higher rate compared to standard care . For sinusitis, all intervention arms showed higher post-interventional prescribing of recommended antibiotics . Patients suffering from tonsillitis in intervention arm I had the highest chance of receiving a recommended substance for the pre timepoint (33.5% recommended antibiotics), while the highest absolute increase was observed in intervention arm III . Comparing post-interventional prescribing, only intervention arm I shows a higher prescribing rate of recommended antibiotics compared to matched standard care . The highest prescribing rates of antibiotics recommended for otitis media are 41.3% of observed cases in intervention arm II post the intervention period (pre: 33.3%) and 37.9% in intervention arm I (pre: 27.7%), compared to 29.3% in matched standard care (pre: 23.6%).The implementation program focused on the reduction of antibiotic prescribing in primary care in Germany for non-complicated self-limiting infections without indication for antibiotics. A significant reduction was observed in the pre-post comparison of all intervention arms. No significant difference between the randomized intervention arms was found, so add-on intervention components in arms II and III did not increase the effect. However, the implementation program was associated with lower antibiotic prescribing than in matched standard care, suggesting that it had impact on professional practice.Different strategies were used to aim for a reduction of undesirable prescribing including enabling measures such as feedback reports, thematically focused quality circles and e-learning modules on physician-patient communication. Enabling measures have been shown to be associated with better acceptance and combining them with restricting interventions was associated with sustainability of the latter . For theIn intervention arm III, the add-on components related to interprofessional care and the increased relevance of information technology solutions in healthcare. PCNs participating in arm III offered cross-sectoral QCs on own initiative and due to internal factors, not all initially planned meetings could take place. The CDSS offered in arm III was implemented into administrative practice software to provide adequate prompts when index diagnoses were coded, which could be ignored or turned off. Its implementation was delayed due to technical difficulties, resulting in a shortened usage period for less practices than expected . The proThe trial was conducted in the particular setting of PCNs. Prior research had concluded that such collaborative health professional networks can contribute to improving healthcare quality and safety  and inteFor the SO of fluoroquinolone prescribing, moderate prescribing still declined in all interventions arms as well as in the matched standard care. However, the chance of inappropriate antibiotic prescribing was substantially lower for cases in all intervention arms compared to cases in matched standard care. Prescribing of indication-specific guideline-recommended antibiotics for the observed index diseases  improved in the intervention arms and was higher than in matched standard care, with the exception of prescribing for otitis media in intervention arm III. Otitis media is a very common disease in early childhood ,28,29,30For patients with co-morbidities, current guidelines in Germany recommend considering antibiotic treatment for non-complicated infections ,40. PresThe particular strengths of this study are the randomized evaluation design, high validity of claims data, low drop out of participating physicians, thorough consideration of excluded ICD-10-coded diagnoses and the additional detailed analysis referring to the type of antibiotics . All cases meeting the inclusion/exclusion criteria were considered. The main limitation of the randomized trial was the randomization at a high aggregation level (14 PCNs) with a risk of by-chance differences. In the observational comparison, the intervention arms were compared to matched standard care, not to a control group of practices from the participating PCNs, since the network structure and peer exchange likely would have led to contamination. At pre-intervention, observed prescribing rates differed between the arms and were lower than expected. Limitations also include the absence of blinding and health-related outcome measures, and the restriction on ICD-10 codes and claims-related health insurance data where disease, patient, and practice information were limited. Direct connection between ICD-10 codes and date of antibiotic prescription was not possible via the provided claims data. Therefore, ICD-10 codes and antibiotic prescriptions were matched at quarter level, which introduces a potential bias.ARena was conducted as a prospective (non-blinded) three-armed cluster randomized trial in two German federal states , with fourteen PCNs as units of randomization. The study was conducted with an intervention period of 21 months, and a two-part evaluation: (a) an outcome evaluation based on quarterly claims-data provided by the statutory health insurance provider AOK and (b) a process evaluation based on surveys . ExamineFourteen PCNs in Bavaria (12 PCNs) and North Rhine-Westphalia (2 PCNs) were recruited by the aQua Institut, Göttingen, Germany, for participation in the ARena trial, and randomized into arm I (4 PCNs), arm II (5 PCNs), and arm III (5 PCNs) by a statistician at the Institute for Medical Biometry, University Hospital Heidelberg, Germany. Randomization considered the number of practices in each PCN, and based on the expected number of practices, aimed for comparable numbers of practices in each intervention arm. Due to intervention components to be offered on PCN level, randomization on practice level was not feasible. For administrative reasons, the focus was on cases in patients insured by AOK health insurance and registered within a specific healthcare delivery program (defined by German law § 140a SGB V a.F. and § 140a Abs. 1. S. 2 Alt. 1 SGB V n.F). At baseline, approximately 40,000 patients with AOK health insurance were registered in 196 participating primary care practices in the 14 PCNs. Medical specialties covered by participating physicians included general practice (largest group), otolaryngology, paediatrics, urology and gynaecology. Observed cases comprised patients who consulted a physician in participating PCNs or in the matched standard care for one of the following reasons: acute upper respiratory tract infection (URTI), acute bronchitis, acute sinusitis, tonsillitis, otitis media, urinary tract infection (UTI), community acquired pneumonia. All diagnoses were based on physician-recorded ICD-10 codes and prescribing information in the administrative data provided by the health insurer for quarterly reimbursement periods, which were linked by the pseudonymized individual insurance number. Cross-validation of recorded codes with patient charts was not possible due to data confidentiality regulations. Each physician-recorded ICD-10 code for defined index diagnoses in the observed quarters represents a case, and each patient could produce multiple cases. Diagnoses for other diseases and diagnoses that warrant an antibiotic therapy were not considered.  , which wSOs examined in this study refer to the percentage of all observed cases with (SO 1) acute non-complicated infections receiving a quinolone prescription when consulting primary care practices (cases with acute bronchitis (age 18–75), sinusitis (>18), otitis media (>2), URTI (>1 year), or tonsillitis (age >1)); (SO 2) acute URTI (>1 year) receiving a prescription for recommended antibiotics (amoxicillin); (SO 3) acute bronchitis (age 18–75) receiving a prescription for recommended antibiotics ; (SO 4) sinusitis (>18 years) receiving a prescription for recommended antibiotics ; (SO 5) tonsillitis (>1 year) receiving a prescription for recommended antibiotics ; and (SO 6) otitis media (>2 years) receiving a prescription for recommended antibiotics .The ARena study protocol also defined SOs referring to cystitis and community-acquired pneumonia which were not considered here due to a very small numbers of cases. Specifications for patient age in all observed sub-groups adhere to study protocol . All outThe following patient-related sociodemographic, disease, and treatment characteristics were provided in the claims data and, as indicated by the study protocol , includeStep 1: Percentage of cases in intervention arms II and III are each lower post-intervention.Step 2: Percentage of cases in intervention arm I is lower post-intervention.Step 3: Compared to intervention arm I, the percentage is lower in intervention arms II and III each.Data were analysed using the statistical software R version 3.6.3 to compare four baseline quarters (Q3 2016–Q2 2017) to four quarters at the end of the intervention (Q3 2018–Q2 2019) for the intervention arms, between arms, and in comparison to matched standard care regarding the defined outcomes. The primary and secondary outcomes, documented data referring to patient and disease characteristics, treatment data, and distribution of medical specialty group were first analysed descriptively stratified by the intervention arm. For categorical variables, absolute and relative frequencies are provided. Note that patient and disease characteristics, treatment data and practice characteristics differ between outcomes, as considered cases are defined for each outcome by the respective disease and prescribing of antibiotics. Confirmatory analysis of the primary outcome was conducted based on the Intention-To-Treat (ITT) population. Since the pre/post reduction was of primary interest and reduction was assumed to be different in each intervention arm, a structured three-step, hierarchical test procedure was applied  to ensurIn Step 1, the pre-post-comparisons in intervention arms II and III were tested using a 2.5% significance level (Bonferroni correction). If at least one pre-post-comparison was significant, Step 2 was conducted . In case the pre-post-comparison in Step 2 was significant, the pre-post-comparisons between intervention arms were tested in Step 3 to examine intervention effectiveness. Logistic mixed effects models were used to explore the primary objective in the hierarchical procedure. The models considered the nested structure of the data with multiple cases per patient and patients nested in practices by including a random effect in the logistic mixed effects model for patients and practices. As fixed effects, timepoint (pre/post), gender, and age group  were included in step 1 and 2. In step 3, the intervention arm was added as fixed effect.p-values for secondary analysis are of explorative nature. Findings reported here focus on the prescribing of antibiotics for upper respiratory tract infections.The primary outcome was additionally analysed comparing the three intervention arms to matched standard care, using a logistic mixed effects model and taking the clustered structure into account. Standard care was matched on practices level by using a propensity score matching based on a logistic regression model under consideration of the matching variables specialist group, baseline number of cases, postal code (first 3 digits), and region. Considered fixed effects in that model are group (intervention arms vs. matched standard care), time-point (pre/post), gender, age, CCI as indication of health status, nationality, and region, town/countryside. Secondary outcomes were analysed using mixed logistic regression models comparing intervention arms with the matched standard care. All Significantly reduced antibiotic prescribing for non-complicated infections in the intervention arms, compared to matched standard care show the impact of the implementation program. Further research should explore which interventions can increase the effects.