D-glucose can be helpful in evaluating the extent of disease and response to treatment. The management of CS patients can be challenging, requiring immunosuppression medications, antiarrhythmic drugs, implantable cardiac devices, and cardiac ablation procedures. We report a patient with CS initially presenting with paroxysmal atrial fibrillation who later developed polymorphic ventricular tachycardia, highlighting the complexity of diagnosis and management in patients with multisystem sarcoidosis.Cardiac sarcoidosis (CS) can cause atrial and ventricular arrhythmias, conduction system disease, and congestive heart failure. The use of advanced imaging modalities including cardiac magnetic resonance and positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro- D-glucose positron emission tomography (18F-FDG-PET) can be helpful in evaluating the extent of the disease and the patient’s response to treatment. CS patients often require management via immunosuppression medications, antiarrhythmic drugs, and implantable cardiac devices. We report a patient with CS initially presenting with paroxysmal atrial fibrillation (AF) who later developed polymorphic ventricular tachycardia, highlighting the complexity of diagnosis and management in patients with multisystem sarcoidosis.Sarcoidosis is a multisystem, granulomatous disease of unknown etiology. Patients with cardiac sarcoidosis (CS) can develop atrial and ventricular arrhythmias, conduction system disease, and congestive heart failure. The use of advanced imaging modalities including cardiac magnetic resonance (CMR) imaging and 2-deoxy-2-[fluorine-18]fluoro-A 55-year-old Caucasian male presented to an outside clinic with a two-month history of fatigue and right bundle branch block (RBBB). At the time of presentation, he exhibited AF with a rapid ventricular response. Symptoms included shortness of breath and chest discomfort during episodes of AF, which lasted 15 to 120 minutes. Cardiac computed tomography (CT) angiography revealed a lesion in the left anterior descending coronary artery, but subsequent cardiac catheterization revealed no evidence of obstructive coronary artery disease. Notably, mildly enlarged lymph nodes in the pericardial region and mediastinum were observed on the CT angiogram. Three years prior, his left ventricular ejection fraction (LVEF) was 55% on cardiac catheterization. Two weeks prior to referral, transthoracic echocardiogram revealed an LVEF of 40%, and the newly depressed LVEF was suspected to be due to tachycardia-induced cardiomyopathy related with AF. The patient was initially started on rivaroxaban and propafenone by the referring physician. He continued to have episodes of AF and was switched to amiodarone. He was referred to our center for AF ablation.His past medical history also included hypertension and hyperlipidemia. At the time of presentation to our center, his medications included metoprolol, rivaroxaban, and amiodarone. Due to a suspected amiodarone-induced cough and the patient’s young age, amiodarone was discontinued and changed to dofetilide.. He underwent cryoballoon pulmonary vein isolation (PVI) without complication. The following day, he underwent CMR to evaluate his fractionated RBBB and reduced LVEF seen on transthoracic echocardiogram and intracardiac echocardiogram during the ablation. CMR showed an LVEF of 40% and extensive, patchy delayed enhancement throughout the left ventricular myocardium along with enlarged mediastinal lymph nodes, consistent with cardiac and pulmonary sarcoidosis .Electrocardiogram (ECG) in our clinic showed sinus rhythm with RBBB with a QRS duration of 160 ms and fragmented QRS 1 The patient was reluctant to use corticosteroids and was instead started on methotrexate with folic acid. A baseline 18F-FDG-PET/CT scan showed extensive heterogeneous 18F-FDG uptake throughout the myocardium that corresponded with perfusion abnormalities on the myocardial perfusion imaging scan . However, although his shortness of breath resolved, the patient’s complaint of fatigue remained.One week later, the patient underwent endobronchial ultrasound-guided mediastinal lymph node biopsy, which revealed poorly formed, non-necrotizing granulomas. Based on these findings, he met the 2014 Heart Rhythm Society Expert Consensus Statement criteria for the diagnosis of CS and subsequently underwent dual-chamber implantable cardioverter-defibrillator (ICD) implantation for primary prevention.Based on the active inflammation seen on cardiac PET and the patient’s reduced LVEF, the methotrexate dosage was increased from 15 mg to 25 mg weekly; 400 mg infliximab was intravenously administered at baseline, weeks two and six, and then every six weeks thereafter.. ECG was performed and showed normal sinus rhythm with frequent premature ventricular contractions . The patient was admitted to the hospital for initiation of sotalol. On discharge, the frequency of infliximab treatment was increased to every four weeks. No programming changes were made to the ICD.Seven months after cardiac ablation, the patient experienced multiple presyncopal episodes while at work. ICD remote transmission showed five episodes of non-sustained polymorphic ventricular tachycardia (VT), as well as episodes of monomorphic VT 18F-FDG-PET scan performed 10 months after the initial 18F-FDG-PET scan revealed an overall decrease in 18F-FDG uptake, with a new focus in the mid-inferolateral myocardial wall . On the current regimen, the patient has remained clinically stable without any further atrial or ventricular arrhythmias.Transthoracic echocardiogram revealed stable LVEF of 40%. A cardiac 2 Young and middle-aged African-American females are at greatest risk, followed by African-American males and Caucasian females, in order of prevalence.3 In one retrospective study, cardiac involvement in sarcoidosis was diagnosed more frequently in males.4 Although sarcoidosis is typically thought of as a disease of African-American females, a high degree of suspicion is required to diagnose CS, regardless of the presenting patient’s sex or race.The prevalence of sarcoidosis is estimated to be 4.7 to 64 in 100,000 people, with a higher incidence in African-American and northern European populations.5 Despite its high contribution to mortality, CS is often clinically silent, and only 40% to 50% of patients with CS at autopsy are correctly diagnosed with such during the patient’s lifetime.6Myocardial involvement in sarcoidosis occurs in up to 25% of cases in the United States and may be the cause of 13% to 25% of deaths from the disease.7 In one cohort of patients with CS, 32% were reported to have supraventricular arrhythmias with the following types: AF, 18%; atrial tachycardia, 7%; atrial flutter, 5%; and atrioventricular nodal reentrant tachycardia, 2%.8 Atrial arrhythmias in CS are common but occur less frequently than ventricular arrhythmias.9 Mechanisms of atrial arrhythmias in CS include triggered activity, abnormal automaticity, and reentry.We described a male Caucasian patient with CS who presented with AF. The precise incidence of atrial arrhythmias in CS patients remains unknown. AF can be the first presenting manifestation.10 Although atrial dilation secondary to left ventricular dysfunction is known to contribute to the onset of atrial arrhythmias, atrial granulomas may also lead to scarring and atrial arrhythmias such as AF.10 Delayed gadolinium enhancement technology exists to visualize atrial scarring on CMR, but to our knowledge, the use of atrial delayed enhancement has not been studied in CS patients and should be a focus of future research.11Granulomas on necropsy in CS patients have been found as histologic evidence of the disease in the left ventricular free wall, basal ventricular septum, right ventricle, papillary muscles, and right and left atria, in the order of decreasing frequency.12 In another study of nine CS patients with atrial arrhythmias, two patients with paroxysmal AF and three with persistent AF underwent PVI; one individual with persistent AF later experienced reccurence.13 Our patient had no recurrence of AF on ICD interrogation following cryoballoon PVI, although immunosuppression was initiated several months after ablation on the basis of extensive 18F-FDG uptake on PET. Evidence supporting the use of immunosuppression to treat atrial arrhythmias in sarcoidosis is limited to case reports.14 Many experts state that it would be useful to test the utility of immunosuppression to treat AF in patients with CS.1There are limited data on whether catheter-based PVI is effective in CS patients with AF. In a prior case in which AF was the initial presentation of CS, the patient had recurrent AF after PVI but experienced a decreased AF burden following immunosuppressive treatment.15 CS patients with preserved or mildly reduced left ventricular function may still be at risk for ventricular arrhythmias.16 The 2014 HRS expert consensus statement attempted to further risk stratify CS patients in terms of sudden cardiac death (SCD) based on reduced right or LVEF, indication for pacemaker implantation, unexplained syncope or near-syncope, or late gadolinium enhancement on CMR.1 The present case illustrates that early diagnosis of CS and SCD risk stratification, with ICD implantation when appropriate, are critical in this patient population. A dual-chamber ICD was implanted in this particular case because the patient had significant sinus bradycardia and RBBB. The capacity to pace the atrium allowed for b-blocker titration to treat the cardiomyopathy, and for antiarrhythmic drug titration as needed to treat AF and/ or other arrhythmias, should they occur. On follow-up device interrogation, the patient was paced in the atrium up to 60% of the time, suggesting an atrial lead was needed to provide an adequate chronotropic response.Despite presenting with AF and having a moderately depressed LVEF at diagnosis, our patient subsequently developed poly- and monomorphic VT. The 2008 American College of Cardiology/American Heart Association/Heart Rhythm Society (HRS)’s device-based guidelines support the performance of ICD implantation in patients with CS, regardless of left ventricular function, as a class IIA recommendation (level of evidence: C).2DS2-VASc score. Although no data exist on the risk of thromboembolism in sarcoidosis patients with AF or on the effect of anticoagulation in this group, CS patients with AF should receive thromboprophylaxis in accordance with guidelines for non-valvular AF.1 Patients with sarcoidosis are at higher risk for pulmonary embolism, raising the concern that systemic inflammation may increase the risk of hypercoagulability in this population.17 Medications such as b-blockers, calcium channel blockers, sotalol, dofetilide, and amiodarone can also be used in the course of treatment, though there are no comparative data to guide antiarrhythmic drug selection.1 Sotalol is often a safe and effective choice in patients with CS as it treats both atrial and ventricular arrhythmias and avoids the risks of pulmonary and hepatic toxicities that can occur with amiodarone. This is advantageous, as many patients may have concomitant pulmonary and hepatic sarcoidosis.The risk of stroke in CS patients with AF is not known. From the time of diagnosis, our patient was anticoagulated with a novel oral anticoagulant based on his CHAWe presented the case of a 55-year-old male with paroxysmal AF and fractionated RBBB who was diagnosed with CS. Despite having only a moderately depressed LVEF, the patient subsequently developed ventricular arrhythmias. This complex case illustrates several critical teaching points about CS: (1) regardless of a patient’s sex or race, a high index of suspicion is needed to diagnose CS; (2) the risk of SCD should be considered, and an ICD implanted when indicated; and (3) the management of these patients, which may include the use of immunosuppression medication, antiarrhythmic therapy, cardiac device implantation, and cardiac ablation, is challenging and requires ongoing vigilance. Further research should be conducted to improve the handling of these individuals.


The neurotoxicity of general anesthesia to the developing human brains is controversial. We assessed the associations between surgery under general anesthesia in infancy and development at age 1 year using the Japan Environment and Children’s Study (JECS), a large-scale birth cohort study.In the JECS, 103,062 pregnancies and 104,065 fetuses were enrolled between January 2011 and March 2014. Of the 100,144 registered live births, we excluded preterm or post-term infants, multiple births, and infants with chromosomal anomalies and/or anomalies of the head or brain. Data on surgical procedures under general anesthesia in infancy were collected from self-administered questionnaires by parents at the 1-year follow-up. Developmental delay at age 1 year was assessed using the Japanese translation of the Ages and Stages Questionnaires, Third Edition (J-ASQ-3), comprising five developmental domains.Among the 64,141 infants included, 746 infants had surgery under general anesthesia once, 90 twice, and 71 three or more times. The percentage of developmental delay in the five domains of the J-ASQ-3 significantly increased with the number of surgical procedures. After adjusting for potential confounding factors, the risk of developmental delays in all five domains was significantly increased in infants who had surgery under general anesthesia three times or more .Surgery under general anesthesia in infancy was associated with an increased likelihood of developmental delay in all five domains of the J-ASQ-3, especially the gross motor domain at age 1 year. The neurodevelopment with the growth should be further evaluated among the children who had surgery under general anesthesia.UMIN000030786)UMIN Clinical Trials Registry (number:  Recently, the influence of anesthesia on children’s neurodevelopment has attracted attention. Numerous animal studies have indicated that exposing the developing brain to anesthetics may lead to widespread apoptotic neurodegeneration, interference with synaptogenesis during brain development, and long-term neurocognitive impairment –6. HowevAlthough numerous studies have been conducted, there is no large-scale Japanese study investigating the neurotoxicity of anesthesia. The Japan Environment and Children’s Study (JECS) is an ongoing prospective nationwide birth cohort study in Japan, and the participating children will be followed up until they reach 13 years of age. In this study, we investigated the association of surgery under general anesthesia in infancy with developmental outcomes at age 1 year using data from the JECS.The data were obtained from the ongoing JECS, for which pregnant women were recruited between January 2011 and March 2014 from 15 regions throughout Japan. The JECS was designed to investigate mothers and children until the latter is 13 years old. Scheduled to continue until 2027, the JECS measures the effect of environmental factors on children’s health. This study was registered in the UMIN Clinical Trials Registry (number: UMIN000030786). The details of the JECS have been described elsewhere [After registration, the women answered self-administered questionnaires twice: in the first trimester and the second/third trimester. The father’s information was also collected using a self-administered questionnaire at a time between the mother’s recruitment and the child’s first-month check-up. Children’s medical records were transcribed by physicians, midwives, nurses, and/or research coordinators after delivery, and 1 month after birth. Children were followed up mainly through self-administered questionnaires completed by their mothers or the mothers’ partners at 1 month, 6 months, and 1 year after birth.This study is based on the “jecs-an-20180131” dataset, released in March 2018. The JECS enrolled 103,062 pregnancies, 104,065 fetuses, and 100,144 live births. Among the live births, we excluded preterm (< 37 weeks) or post-term (> 41 weeks) infants, multiple births, and infants with congenital diseases, all of which can affect development. The information on gestational age and multiple births were collected using medical record transcripts at birth. The congenital diseases excluded were chromosomal anomalies and anomalies of the head or brain. Information regarding chromosomal anomalies  was collected from medical records at the 1-month check-up and self-administered questionnaires at 1 year. Information regarding anomalies of the head or brain  was obtained from medical records at the first-month check-up. Information on whether the child underwent surgery under general anesthesia before age 1 year was obtained from the question “Number of surgical procedures under general anesthesia” in the questionnaire administered at the 1-year follow-up. This questionnaire did not cover the date of surgery under general anesthesia, the disease that necessitated the procedure, surgery name, or anesthesia type.The Ages and Stages Questionnaire-Third Edition (ASQ-3) is a parent-completed developmental screening measure whose utility has been demonstrated –18. ThisInfants with missing information regarding gestational age, pregnancy outcome, chromosomal anomalies, anomalies of the head or brain, number of surgical procedures under general anesthesia, or J-ASQ-3 results were excluded. Thus, the 64,141 infants remained to be included in our analysis. The subject selection process is depicted in Fig. We examined the association between surgery under general anesthesia in infancy and development at age 1 year. We divided the subjects into four groups, depending on the number of surgical procedures under general anesthesia: none, once, twice, and three times or more. Infants below and above the cutoff for each domain were termed “delayed” and “normal,” respectively. In univariate analysis, the association between the delay in each J-ASQ-3 domain and the number of surgical procedures under general anesthesia was evaluated using the Cochran–Armitage test. Subsequently, multiple logistic regression analyses were performed adjusting for confounders . The information regarding sex, gestational age, birth weight, 5-min Apgar score, delivery method, and maternal age at birth was collected from the medical record transcription at birth, while information on congenital diseases was obtained from the self-administered questionnaires at 1 year of age. For sensitivity analyses, the models were re-run using maternal age as a categorical variable. Additional analyses adjusting for paternal age at registration, parents’ educational level, and household income were conducted. Besides, we also conducted the analyses excluded infants with congenital heart diseases, because infants who had such diseases are known to be at high risk for developmental delay . Furtherp value of < 0.05 was considered statistically significant. Multiple logistic regression analyses were performed using SPSS Statistics ver. 24 (IBM), and the Cochran–Armitage test was performed using the R software ver. 3.5.0.A Subject characteristics are shown in Table Table p < 0.001 in all domains).Table Figure The above analyses were conducted using maternal age as a continuous variable. When it was used as a categorical variable, the results were much the same  Additional file 2. Adjusted odds ratios of developmental delay among infants who had surgical procedures under general anesthesia compared with infants who did not have surgery, for each of the five domains  Additional file 3. Adjusted odds ratios of developmental delay among infants who had surgical procedures under general anesthesia compared with infants who did not have surgery, for each of the five domains  Additional file 4. Cutoff scores of each J-ASQ-3 domain reported for Japanese children, and numbers of normal and delayed infantsAdditional file 5. Number of delayed infants for each J-ASQ-3 domain, according to the number of surgery under general anesthesia using the cutoff scores reported for Japanese childrenAdditional file 6. Adjusted odds ratios of developmental delay among infants who had surgical procedures under general anesthesia compared with infants who did not have surgery, for each of the five domains using the cutoff scores of the J-ASQ-3 reported for Japanese children


Population-level estimates of prevalence of anti-SARS-CoV-2 antibody positivity  is a crucial epidemiological indicator for tracking the Covid-19 epidemic. Such data are in short supply, both internationally and in South Africa. The South African blood services  are coordinating a nationally representative survey of blood donors, which it is hoped can become a cost-effective surveillance method with validity for community-level seroprevalence estimation.th, 10th, 12th, 15th, 20th, 23th and 25th January) for anti-SARS-CoV-2 antibodies, using the Roche Elecsys Anti-SARS-CoV-2 assay on the cobas e411 platform currently available in the blood services’ donation testing laboratories. Using standard methods, prevalence analysis was done by province, age and race, allowing age to be regarded as either a continuous or categorical variable. Testing was performed in the Eastern Cape (EC), Free State (FS), KwaZulu Natal (ZN) and Northern Cape (NC) provinces.Leveraging existing arrangements, SANBS human research ethics committee permission was obtained to test blood donations collected on predefined days  are: EC-63%(2.8%), NC-32%(2.2%), FS-46%(2.4%), and ZN-52%(2.4%).We report on data from 4858 donors - 1457 in EC; 463 in NC; 831 in FS and 2107 in ZN. Prevalence varied substantially across race groups and between provinces, with seroprevalence among Black donors consistently several times higher than among White donors, and the other main population groups (Coloured and Asian) not consistently represented in all provinces. There is no clear evidence that seroprevalence among donors varies by age. Weighted net estimates of prevalence (in the core age range 15–69) by province Our study demonstrates substantial differences in dissemination of SARS-CoV-2 infection between different race groups, most likely explained by historically based differences in socio-economic status and housing conditions. As has been seen in other areas, even such high seroprevalence does not guarantee population-level immunity against new outbreaks – probably due to viral evolution and waning of antibody neutralization. Despite its limitations, notably a ‘healthy donor’ effect, it seems plausible that these estimates are reasonably generalisable to actual population level anti-SARS-CoV-2 seroprevalence, but should be further verified. Owing to the extraordinary public interest in all meaningful information about the COVID-19 pandemic, we here report on an initial analysis of an ongoing study. A fuller analysis will be described in the near future.the prevalence of anti-SARS-CoV-2 antibody positivity , at the community level  investigating how representative this prevalence is likely to be of community-level prevalence, 2) gathering data about the spectrum of disease associated with SARS-CoV-2 infection, and 3) considering options for leveraging ongoing surveillance of prevalence into real-time incidence estimates. In the present work, we focus on early seroprevalence estimates.th, 10th, 12th, 15th, 20th, 23th and 25th January); which were announced to blood centre staff, but without prior notice to potential donors. All donors underwent routine screening through a self-administered questionnaire, one-on-one assessment and a mini-health screening by blood centre staff. Donors who did not meet the routine donor eligibility criteria were excluded from donation and therefore from the study. To date, testing has been performed in the Eastern Cape (EC), Free State (FS), KwaZulu Natal (ZN) and Northern Cape (NC) provinces.The South African National Blood Service (SANBS – serving 8 of 9 provinces in South Africa) and Western Cape Blood Service (WCBS – servicing the Western Cape) obtained ethics clearance from the SANBS Human Research Ethics Committee to perform a SARS-CoV-2 seroprevalence study among South African blood donors. The protocol allowed for the testing of routinely collected donor screening samples on predefined ‘collection days’  and 50 samples confirmed as positive in a COVID-19 convalescent plasma study protocol .For epidemiological interpretation, we take seroprevalence as a close proxy of the prevalence of the condition of having been infected with SARS-CoV-2 at some point. The Elecsys Anti-SARS-CoV-2 assay appears to have particularly good durability of antibody detection for months post PCR reversion and symptom resolution, with no evidence of antibody waning and seroreversion over more than four months in a US COVID-19 convalescent plasma cohort .We ignore, for now, the effects of 1) the donor deferral rule that people with confirmed SARS-CoV-2 infection, or COVID-19-like symptoms, are precluded from donation for a period of two weeks after PCR test and symptom resolution, and 2) deferral of regular donors who were in quarantine due to a positive contact who would not have presented for their routine donation. Given the high rate of asymptomatic infection, this is a relatively minor limitation.Samples collected at the time of donation, were tested for anti-SARS-CoV-2 antibodies, using the Roche Elecsys Anti-SARS-CoV-2 total immunoglobulin nucleocapsid assay on the cobas e411 platform already in use in the blood services. This assay, according to the package insert, has diagnostic specificity in excess of 99.5%, and near perfect sensitivity (point estimate of 100%) at 16 days post PCR positivity. We do not here explore various interesting nuances of how to define and estimate test performance characteristics by distribution of cases (defined primarily by severity of infection and time since infection/symptoms/PCR detection) but note that:We did not perform structured sampling in the sense of a primary or secondary unit of analysis, with steps of random selection of primary sampling units, households or individuals – the study merely observed donors who happen to present themselves at donation facilities on collection days. Prevalence was estimated by typical categorical and continuous predictors  by standard methods, using the R platform for statistical computation. Although we are not aware of any biological basis for expecting racial differences – in South Africa, as elsewhere, race is, for historical reasons, a strong correlate of socio-economic status, living conditions, and social circumstances, and therefore a suspected predictor of prevalence. Provincial weighted seroprevalence estimates are based on population size estimates from Machemedze et al , and racDuring the seven collection days held throughout January 2021, 4858 voluntary non-remunerated blood donors from four provinces  of South Africa were tested for anti-SARS-CoV-2 antibodies. The majority of blood donors were from ZN at 2107 (43%), followed by EC at 1457 (30%), FS at 831 (17%) and NC at 463 (10%).There were slightly more male donors 53%). The large majority of donors in our study were White (47%) and Black (34%) with the remainder distributed relatively evenly between the Asian and ‘Coloured’ – a uniquely South African racial label indicating persons with a significant mix of ancestry from, amongst other lineages, South Asia, Indonesia, Southern Africa and Europe , earlier in the epidemic found: 1) a very high prevalence (30–40 percent) among pregnant women attending state sector antenatal care, and people living with HIV presenting for routine viral load assessment ; and 2) For an indication of the meaning of such high seroprevalence values, in a one year old epidemic, consider: A prevalence of 50%, accumulated over 50 weeks, of a condition with a duration of infectiousness of 1 week, implies an average ‘prevalence of infectiousness’ of 1% of the population, with inevitable significant elevations above this average value during peaks. For people reliant on public transport, or working in public spaces, it will be difficult to limit close encounters to fewer than 100 people on any given day – i.e. difficult to encounter fewer than one infectious person per day.We do not claim that blood donors are a perfectly representative of the South African population. Firstly, Black and White donors each account for roughly half the total participants of this study, though South Africa’s population is about 80 percent Black African and only 8 percent White/European . Other pSurvey dates represented in this analysis are barely past South Africa’s ‘second peak’ in COVID-19 incidence. Deferral rules based on confirmed infection or COVID-19-like symptoms should slightly depress seroprevalence estimates relative to ‘true’ prevalence. The age weighting we adopted to estimate total infections also produces a face value underestimate for population totals as it assigns no cases in the age range 0–14, which accounts for about 30 percent of the population. The Elecsys Anti-SARS-CoV-2 antibody assay appears to have particularly good detection sensitivity for months post PCR reversion (The ordering of the prevalence-by-province and prevalence-by-race is what one would expect, based on official case counts, and an understanding of South African society.The particularly high attack rates in majority Black communities points to the limitations, thus far, of non-pharmaceutical interventions in the context of economic deprivation and high population density.after the observation of very high seroprevalence (The high seroprevalence amongst Black donors also raises interesting and important questions about the level of collective immunity thus far obtained through the two primary infection waves to date – but we caution against simplistic interpretations, given that substantial outbreaks have been seen in cities evalence , and theThe low seroprevalence amongst White donors suggests that predominantly White suburban communities lack meaningful collective immunity, and should take infection control measures very seriously for the foreseeable future.Given the relatively low marginal cost of leveraging the infrastructure of the blood services, we are keen to further probe the representativeness of blood-donor-based seroprevalence surveys, and to see to what extent surveillance in the blood services can be a valuable and efficient ongoing activity during major infectious disease outbreaks.With due consideration to both the patent and latent limitations of our study, the key observations we wish to make at this point are:We are mindful of the incompleteness of this project, and the potential to misconstrue the wider ramifications of these findings. We endeavour to proceed rapidly from data gathering to publication, and to offer realistic and grounded interpretations of the findings.


Recycling tenofovir and lamivudine/emtricitabine with dolutegravir (TLD) after failure of non-nucleoside transcriptase inhibitor (NNRTI) first-line antiretroviral therapy (ART) is more tolerable and scalable than dolutegravir plus optimized nucleoside reverse transcriptase inhibitors. Studies have demonstrated TLD’s efficacy as second-line, but long term follow-up is limited.ARTIST is a single arm, prospective, interventional study conducted in Khayelitsha, South Africa, which switched 62 adults with two viral loads (VL) >1000 copies/mL from tenofovir, lamivudine/emtricitabine and an NNRTI to TLD. We report efficacy to 72 weeks and, in a post hoc analysis, evaluated VL trajectories of individuals with viraemic episodes.Virologic suppression was 86%  74-93), 74% (95% CI 61-84) and 75% (95% CI 63-86) <50 copies/mL, and 95%, 84% and 77% <400 copies/mL at week 24, 48 and 72 respectively, with 89% (50/56) resistant (Stanford score ≥15) to tenofovir and/or lamivudine pre-switch. No participants developed integrase-inhibitor resistance. Of the 20 participants not suppressed at week 24 and/or 48, two developed virologic failure, one switched regimen (adverse event), two were lost to follow-up, one missed the visit, one transferred out, nine resuppressed <50 copies/mL with enhanced adherence counselling and four remained viraemic (three with <200 copies/mL) at week 72.Recycling NRTIs with dolutegravir was effective for most participants to 72 weeks. Most with viraemia did not develop virologic failure and subsequently suppressed with enhanced adherence counselling or continued to have low-level viraemia. No integrase-inhibitor resistance was detected despite low-level viraemia in a minority of participants. In both arms, the backbone was dual nucleoside reverse transcriptase inhibitors (NRTI) with at least one NRTI fully active on resistance testing1, which led the World Health Organization to recommend a second-line regimen of zidovudine, lamivudine and dolutegravir (ALD) after failing a first-line NNRTI-based regimen with tenofovir plus lamivudine or emtricitabine (XTC) in settings with limited access to antiretroviral resistance testing2. This recommendation for the zidovudine and lamivudine NRTI backbone is based on the rationale that the resistance mutation selected for by tenofovir (K65R), does not compromise the activity of zidovudine3.The DAWNING study showed that dolutegravir was superior to ritonavir-boosted lopinavir after failing first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART)5. Such a strategy would be beneficial to both patients and the health system as tenofovir is better tolerated than zidovudine, and TLD is available as a single fixed dose tablet taken once daily while ALD requires twice daily dosing of zidovudine and lamivudine 6.Recent evidence has emerged that recycling tenofovir and XTC with dolutegravir (TLD) in second-line ART, after failure of first-line NNRTI-based ART, is efficacious despite high rates of resistance to both tenofovir and XTCAntiRetroviral Therapy In Second-line: investigating Tenofovir-lamivudine-dolutegravir [ARTIST] trial), with 85% (51/60) achieving the primary end point of VL <50 copies/mL despite substantial baseline resistance to one or both of tenofovir and XTC . No participants developed study-defined virologic failure and no integrase resistance was detected in the one participant meeting criteria for resistance testing4.We have previously reported 24-week results of a single-arm interventional study of TLD as second-line ART , dolutegravir resistance developed in two patients who had virologic failure on second-line TLD14.The efficacy of second-line TLD in the absence of a fully active NRTI is likely due to dolutegravir’s high genetic barrier to resistance coupled with a cost in fitness from NRTI resistance mutations15. Here we report follow-up results of the ARTIST trial participants to 72 weeks to evaluate the maintenance of the virologic suppression on second-line TLD. Additionally, we conducted a post hoc descriptive analysis of the VL trajectories of those who had episodes of viraemia.Emergence of dolutegravir resistance in patients on second-line TLD could jeopardise the public health benefits that the combination potentially offers in lower and middle income country programmes. It is unclear whether the efficacy of TLD in those who have failed first-line NNRTI-based therapy is durable, particularly in those who develop viraemia, making longer follow-up crucial to inform treatment policy decisionsWe conducted a single arm, prospective, interventional study in two primary care clinics in Khayelitsha, South Africa. This study was designed to evaluate the virologic efficacy of TLD in second-line ART.4. The protocol was approved by the University of Cape Town’s Human Research Ethics Committee (039/2019) and is available with the statistical analysis plan on ClinicalTrials.gov (NCT03991013). Adults who had virologic failure  on a first-line regimen consisting of tenofovir, XTC and efavirenz or nevirapine were invited to participate. Exclusion criteria were CD4 cell count less than 100 cells/ml, active or suspected tuberculosis, active AIDS-defining conditions, an estimated glomerular filtration rate less than 50 ml/min per 1.73m2, haemoglobin less than 7.5 g/dl, alanine aminotransferase greater than 100 IU/l, a previous or current diagnosis of malignancy or any condition judged to put the patient at increased risk if participating, a condition judged likely to impact adherence (active psychiatric disease or substance abuse), pregnancy, breastfeeding, intention to fall pregnant or unable to take the study medication . Women of child-bearing potential were required to be on effective contraception.A full description of the study design was published with the week 24 primary endpoint resultsParticipants were enrolled and switched to TLD with additional 50 mg dolutegravir for two weeks to overcome efavirenz induction effects on dolutegravir metabolism and transport. Study visits with clinicians occurred every four weeks to week 24, then at week 36, 48 and 72. VL was assessed at baseline and every subsequent visit, with a repeat VL after two weeks of enhanced adherence counselling if VL was >50 copies/mL after week 12. If the repeat VL was >500 copies/mL, a genotypic resistance test was performed per protocol. Baseline genotypic resistance testing was performed retrospectively for all participants and was not available to inform treatment decisions.16. We used a ± two-week visit window for the first 20 weeks, then a ± six-week visit window from week 24 onwards. The extended window was introduced to accommodate COVID-19 restrictions in accessing health facilities. We conducted a modified intention-to-treat analysis (mITT), excluding those switching study drug due to contraception cessation, wishing to become pregnant, pregnancy, non-clinical transfer out or death from non-HIV and non-drug causes (assessed by the study doctor). Failure included: VL ≥50 copies/mL, missing VL within the visit window or loss to follow up, intolerance or adverse event due to any drug in the regimen requiring switch.The primary outcome was VL suppression (defined as VL <50 copies/mL), evaluated according to the FDA snapshot algorithm17.Virological failure was defined as having two consecutive VL >1000 copies/mL after week 12. Genotypic resistance was classified using the Stanford algorithm (version 8.9-1), with a score ≥15 indicating at least low-level resistance. Results were categorised as two fully active NRTIs (both with a Stanford score <15), resistance to one NRTI (one with a Stanford score <15 and one ≥15) and resistance to both NRTIs (both with a Stanford score ≥15)Virologic outcomes at week 24, 48 and 72 are presented as proportions with the corresponding exact binomial 95% confidence interval (CI). A pre-specified secondary analysis described VL suppression defined as <400 copies/mL. A sensitivity analysis of VL suppression excluded certain participants included in the mITT analysis: those lost to follow-up, missing a VL in the window and those who were changed from the study drug for reasons other than treatment failure. The pre-specified sensitivity analysis for the primary end-point of 24 weeks also excluded those with evidence of poor adherence at the visit (TFV-DP <350 fmol/punch), however these results were not available for the 48 and 72 week visits.In a post hoc descriptive analysis, the VL trajectories of participants who were on TLD and had a VL ≥50 copies/mL at weeks 24 and/or 48 were graphed along with a narrative description of their baseline and follow-up resistance test results, reported adherence (taken from clinician notes) and missed visits (no visit within the window).The baseline characteristics of the 62 participants enrolled between August 2019 and July 2020, are described in The proportion of participants with virologic suppression (VL <50 copies/mL) decreased from week 24 to week 72 but was maintained above 70% across the mITT and sensitivity analyses. In the mITT analysis 75.4% of participants had a VL <50 copies/mL at 72 weeks. A sensitivity analysis examined virologic suppression in those with a VL result who remained on TLD throughout the study; at 72 weeks 83.6% had a VL <50 copies/mL in this analysis .The outcomes of those not suppressed at weeks 24, 48 and 72 are described in Two participants met criteria for virologic failure (two consecutive VL >1000 copies/mL after week 12) during the follow up period: one at 36 weeks and one at 52 weeks. Three participants were eligible for resistance testing and all three had resistance testing conducted , but none were found to have integrase resistance mutations.3) compared to those with two active NRTIs at baseline was 0.84 (95% CI 0.74-0.95) for week 24, 0.91 (95% CI 0.61-1.34) for week 48 and 0.91 (85% CI 0.61-1.34) for week 72  at week 24 . FollowiWe plotted the VL trajectories for participants who were on TLD, had a VL conducted and had a result ≥50 copies/mL at week 24 and/or week 48. Of the nine not suppressed at week 24, four were also not suppressed at week 48. Sixteen participants in total were not suppressed at week 48 . By weekOverall, of the 20 participants who had not been suppressed at week 24 and/or week 48 , six had a VL ≥50 copies/mL, three were missing VL results, one had switched due to an adverse event and ten were suppressed <50 copies/mL at week 72 .Our study demonstrated that virologic suppression is maintained above 70% to 72 weeks, despite almost 90% of participants having resistance to one or more NRTI drugs at baseline. The COVID-19 pandemic restricted access to facilities for many participants over the follow-up period: a sensitivity analysis that accounted for this and included only those on TLD with a VL result, also demonstrated high proportions with sustained virologic suppression: 88%, 79% and 84% with VL <50 copies/mL and 98%, 89% and 85% with VL <400 copies/mL at weeks 24, 48 and 72 respectively. Three quarters of the participants with episodes of viraemia re-suppressed to <50 copies/mL after enhanced adherence counselling, while two participants remained viraemic at low levels and only two went on to meet criteria for virological failure.7. Differences in suppression rates could be due to differences in patterns of adherence between the study populations. NADIA found suppression rates over 90% in those with no predicted NRTI activity5. We found a similar proportion suppressed in those with baseline NRTI resistance, and the Malawian observational study found no evidence for increased risk of viraemia or virological failure for participants with baseline NRTI resistance 14. This supports the conclusion from the NADIA trial that dolutegravir in combination with a recycled NRTI backbone is an effective second-line regimen. Most of our participants who were not suppressed had low-level viraemia (VL <200 copies/mL), and most of these resuppressed at later time points with enhanced adherence counselling. While there was little self-reported poor adherence, this may be due to a social desirability bias. Those with missed visits reflect gaps in medication on hand, leading to poor adherence. The NADIA trial also postulated that poor adherence was prevalent in their population5. We hypothesise that suboptimal adherence resulted in low-level viraemia and subsequent re-suppression with improved adherence.The outcomes we report are similar to the NADIA trial, where 92% were suppressed <400 copies/mL on a second-line regimen of dolutegravir or darunavir with a recycled tenofovir/lamivudine backbone at 48 and 96 weeks, and tenofovir/lamivudine was found to be superior to zidovudine/lamivudine at 96 weeks as a second-line backbone 7 and the Malawian observational study (two cases)14. The relatively low median baseline VL at switching and the small sample size in ARTIST may have contributed to our finding that no patients developed resistance to dolutegravir over 72 weeks. However, as resistance to dolutegravir has been shown in dolutegravir monotherapy trials to generally develop between 24 and 48 weeks11, these results are reassuring. This finding in participants using recycled tenofovir in the NRTI backbone, is important considering the high prevalence of tenofovir resistance in patients failing first-line ART in sub-Saharan Africa18. Cycling in and out of care with periods of viraemia is to be expected over a lifetime of ART 20. Thus, sustaining virologic suppression over time requires a regimen that is robust to the development of resistance despite fluctuating adherence. Dolutegravir has a particularly high barrier to resistance, comparable to protease inhibitors and much higher than efavirenz21. Low level viraemia has also been shown not to be associated with subsequent virologic failure in patients on a dolutegravir regimen, nearly half of whom had previous virologic failure22.Despite the substantial proportion with low-level viraemia, only two participants fulfilled the study definition of virological failure over 72 weeks and neither of these participants  had integrase resistance mutations. Resistance has been identified in NADIA (three cases in the TLD group)23. Low-level viraemia at the primary end-point did not predict later virologic failure in participants on first-line TLD in the ADVANCE study, resulting in a recommendation that the goal of treatment on dolutegravir regimens could be shifted to less stringent targets than a VL <50 copies/mL24. The low proportion of participants meeting criteria for virologic failure on TLD despite ongoing low-level viraemia in our study seems to support this recommendation.Dolutegravir with recycled tenofovir and lamivudine seems to fulfil this need for a tolerable, robust second-line regimen and could help in achieving the third UNAIDS target of 95% of those on treatment being virologically suppressed7 addresses two key concerns facing HIV services in this phase of the HIV pandemic: first, patients on second-line ART are in need of more tolerable regimens than the currently-recommended zidovudine/lamivudine/dolutegravir combination, and the fixed-dose, once-daily TLD provides a safe alternative to make treatment more acceptable and effective for individual patients.The demonstrated efficacy of recycling tenofovir and lamivudine with dolutegravir in patients failing first-line NNRTI regimens in our study and the NADIA trial26. The growing evidence from this study, the NADIA trial, and other studies, suggests that switching all patients on NNRTI-based first line to TLD regardless of VL is effective and safe, with the caveat that monitoring and interventions to reduce the development of dolutegravir resistance should be implemented alongside widespread switching. This approach could facilitate less expensive and simpler strategies for wider TLD use 21.Second, scale up of ART is complicated by resource constraints and many programmes are opting for universal TLD regimens, frequently without VL testing before switching patients due to high cost and low availability27. Effective monitoring and management of adherence issues is required to sustain the population health benefits that TLD promises.Suboptimal adherence in a number of participants in this study did not result in virological failure with the development of integrase-inhibitor resistance mutations. This is compatible with dolutegravir’s high resistance barrier, though larger numbers and longer follow-up are needed to verify this. However, adherence challenges remain an issue regardless of regimen. While TLD represents a tolerable and robust second-line that may be helpful in improving adherence and outcomes in those who have previously failed treatment, individuals on this regimen will still benefit from adherence supportLimitations of this study include the higher proportion of missing VL results at week 48 and 72. COVID-19 reduced access to the primary care facilities where the study took place, and while we widened the visit window and used telephonic follow-up to mitigate this, the pandemic may still be responsible for missing VL data. Interpretation of our study is also limited by the small sample size, not having a control arm and lack of long-term therapeutic drug monitoring. While the frequent VL monitoring is not pragmatic for clinical practice, it allowed exploration of virologic trajectories following episodes of viraemia. A strength of our study is that it was embedded within a primary care ART clinical service, increasing generalisabilityMost of our participants on a regimen of dolutegravir with a recycled tenofovir and lamivudine NRTI backbone were virologically suppressed at 72 weeks, despite substantial baseline NRTI resistance. Most of those who did not suppress had low-level viraemia or missed visits. Only two participants with raised VL developed virologic failure, and no participants were found to have developed dolutegravir resistance. With enhanced adherence counselling most participants re-suppressed or continued to have low-level viraemia. These findings, together with those of the NADIA trial and other studies, support the routine switching of all patients on tenofovir and NNRTI-based first-line therapy to TLD regardless of viral load, alongside measures to mitigate and detect the development of dolutegravir resistance. This would simplify rollout and make this effective, tolerable, and inexpensive regimen available to millions more patients.Supplementary material


Takotsubo syndrome (TTS) represents a form of acute heart failure featured by reversible left ventricular systolic dysfunction. The management during the acute phase is mainly performed with supportive pharmacological , anticoagulants, antiarrhythmics, non-catecholamine inotropics (levosimendan), and non-pharmacological  therapy, due to the wide clinical presentation and course of the disease. However, there is a gap in evidence and there are no randomized and adequately powered studies on clinical effectiveness of therapeutic approaches. Some evidence supports the use ACE-inhibitors/ARBs at long-term. A tailored approach based on cardiovascular and non-cardiovascular risk factors is strongly suggested for long-term management. The urgent need for evidence-based treatment approaches is also reflected by the prognosis following TTS. The acute phase of the disease can be accompanied by various cardiovascular complications. In addition, long term outcome of TTS patients is also related to non-cardiovascular comorbidities. Physical triggers such as hypoxia and acute neurological disorders in TTS are associated with a poor outcome. Although Takotsubo syndrome (TTS) has been classically described as a benign condition, life-threatening acute complications have been described in 52% of patients, while 11% have more than two complications that contribute to higher mortality rate . In-hospIn the acute phase, patients are generally treated with several supportive measures, pharmacological ), and nonpharmacological , depending on the grade of heart failure and the existence of concomitant complications.According to current literature “mild” form of TTS without the existence of acute heart failure require only therapy for comorbidities. However, no dedicated randomized trial has been published so far and few data come from adequately powered studies .Beta-blockers were commonly used in the acute phase of TTS due to the possibility of blocking adrenergic surge effects. Actually, no benefit for beta-blockers at short- and long-term has been proven in TTS ,6. In a p = 0.01) and systemic blood pressure [Esmolol, a β-1 cardio-selective blocker with a short half-life 9 min), may be an easily manageable tool for the treatment of patients with TTS and LVOTO. Santoro et al. found in a case series of 10 patients with TTS and LVOTO that Esmolol infusion was associated with a reduction of the intraventricular gradient  in TTS ranges from 6% to 20% ,11,12. AHowever, considering the pathophysiology of the syndrome, the Heart Failure Association of the European Society of Cardiology (ESC) has recommended to avoid or withdraw exogenous catecholamines as they could probably prolong or exacerbate the acute phase of the syndrome by activating catecholamine receptors or their downstream molecular pathways .Levosimendan is a non-catecholaminergic inotropic drug, commonly used in patients with acute heart failure; the drug may represent an option for TTS patients, but its use, is limited to patients with impaired systolic function, without LVOTO and with systolic arterial pressure ≥ 90 mm Hg. Clinical data from case series showed the safety and feasibility of this treatment in TTS patients ; no adveMechanical circulatory support (MCS) should be evaluated early in patients with TTS and cardiogenic shock. Intra-aortic balloon pump (IABP), TandemHeart, extracorporeal membrane oxygenation (ECMO), and microaxial pumps  are used in a bridge-to-recovery strategy .IABP use has been evaluated in a European registry of 2250 TTS patients, where 42 patients with CS were treated with this approach. IABP use did not provide any benefit in term of in-hospital mortality, length of hospitalization, and need of invasive ventilation .Recent data showed that there is a growing use of V-A ECMO and Impella in TTS patients with CS . HoweverIn TTS with impaired EF and cardiogenic shock a practical diagnostic and therapeutic flow chart can be suggested. In case of systolic blood pressure >90 mm Hg and left ventricular outflow tract obstruction, infusion of β1-selective beta-blockers  could be considered; if systolic blood pressure is > 90 mmHg in the absence of left ventricular outflow tract obstruction, levosimendan infusion may be considered; finally, in case of cardiogenic shock with blood pressure < 90 mmHg, without left ventricular outflow tract obstruction mechanical circulating support could be considered .Life-threatening arrhythmias may occur during the acute phase of TTS in up to 8% of cases . Data frAcute management of ventricular tachycardia includes a pharmacological approach with magnesium sulfate and/or short-acting β1-blocker or may require direct current cardioversion in case of sustained pulseless ventricular tachycardia. Of note, due to the high prevalence of prolonged QT interval in TTS patients during the acute phase , the usePersistent high-grade AV block should be managed with permanent pacemaker implantation. All TTS patients with complete AV block who received a pacemaker showed a permanent pacing over follow-up suggesting that TTS probably occurred secondarily due to the rhythm disorder ; patient3 cells/μL are independent predictors of LV thrombosis [LV thrombus formation has been reported in about 2.5% of all TTS patients and cardioembolic complications occur in 0.8% of this population . Time ofrombosis ,29,30.A simplified approach regarding OAC therapy in patients with TTS through echocardiographic evaluation combined with laboratory data (troponin levels) should be used for the acute management . In caseDespite data on long-term outcome from several registries, no randomized trial on pharmacological treatment has been published so far. Therefore, limited information is available and is mainly based on observational studies .Templin et al., in one of the largest registries on TTS patients, found that the use of angiotensin-converting–enzyme inhibitors (ACEi) or angiotensin-receptor blockers (ARBs) is associated with an improved survival at 1 year; in contrast, according to the same registry, there is no evidence of any survival benefit from the use of beta-blockers (BBs) .Chronic use of BBs has been commonly used on the base that BBs could prevent recurrence of TTs as countering the effect of increased catecholamine levels. However, no benefit in term of recurrence reduction or better survival have been proven . RecentlLong term use of antiplatelet drug still requires investigation. Recent studies showed that aspirin at hospital discharge did not relate to short- or long-term prognosis in a large population of TTS patients. The incidence of MACE in TTS patients discharged with aspirin was not significantly different when compared to patients without aspirin . The proOral anticoagulation should be prescribed long term among those patients with evidence of LV thrombi during hospitalization. Indeed, LV thrombi can be successfully managed with 3 months of OAC therapy with complete resolution of thrombosis . ObservaRecurrence of TTS is not infrequent; the recurrence rate is approximately 1.5% per year . SecondaAccording to current data, other pathophysiological pathways such as inflammatory or energetic–metabolic need to be further explored to identify a suitable therapy for TTS. A first-line approach remains the management of stressors and comorbidities that can trigger the onset of TTS. Cancer, pulmonary, and neuropsychiatric diseases are the most frequent comorbidities in these patients and should be carefully evaluated during hospitalization and at follow-up. Psychotherapy, counseling, and stress reduction approaches are reasonable and viable options for patients that experienced TTS due to emotional triggers. Therefore, among patients with several comorbidities, especially those with a psychical one, a multidisciplinary long-term approach is needed ,38.Recent evidence contradicts the assumption that TTS is a benign condition. Indeed, the initial presentation can be accompanied by fatal complications, including cardiogenic shock, congestive heart failure, and lethal arrhythmias, leading to an in-hospital mortality of 2–8.7% ,4.Among patients enrolled in the InterTak registry, during the first 30 days after admission, the rate of major adverse cardiac and cerebrovascular events was 7.1%, which included death and stroke or transient ischemic attack [Cardiogenic shock (CS) during TTS hospitalization is one of the stronger predictors of short and long-term outcome. Patients with CS have 5-fold higher risk of all-cause mortality at long-term. Clinical factors significantly related to CS included male sex, lower LVEF at presentation, longer QTc intervals, the presence of an LVOT gradient, or a physical trigger . AdditioMore strict surveillance during follow-up is mandatory for TTS patients with these risk factors.At long-term follow-up, the rate of death from any cause was 5.6% per patient-year and rate of major adverse cardiac and cerebrovascular events was 9.9% per patient-year .In an observational study by Stiermaier et al., TTS patients, when compared to ST elevation (STEMI) patients matched for age and gender, presented longer in-hospital stay and significantly higher all-cause mortality during the follow up period . Even the short-term mortality in patients presenting with TTS was higher than generally expected and comparable to a matched STEMI population . This study, moreover, highlighted that male sex, Killip class and diabetes mellitus were predictive factors of long-term mortality, allowing the definition of a prognostic risk score with a point for the presence of each factor. Mortality rates in patients with risk scores from 0 to 3 points were 13.7%, 37.3%, 64.7%, and 100%, respectively .One of the major predictors of long-term prognosis is represented by triggering stressor. Ghadri et al. proposed a patient’s classification according to the type of trigger to predict and stratify short- and long-term outcomes. Patients were stratified into three classes: Class I = triggered by emotional stress, Class II: triggered by physical stress , Class III = no identifiable triggering factor. TTS related to neurologic disorders had the highest mortality, while TTS patients with an emotional stressor showed a favorable short- and long-term outcome compared with those with a physical stressor . UribarrThe incidence of TTS recurrence is estimated to range from 4.7 to 5% at long term follow-up ,48. MostPrognosis of patients with TTS is featured by a high risk of major adverse cardiac and non-cardiac events during short and long-term follow-up, due to mainly non-cardiovascular comorbidities. Therefore, careful short- and long-term management is needed in these patients. During the acute phase, TTS is mainly managed with supportive therapy, while, in the chronic phase, a tailored approach based on the cardiovascular and non-cardiovascular risk factors is needed. However, randomized trials are warranted to define the optimal therapeutic approach for TTS patients.


The field of genome editing started with the discovery of meganucleases  in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy. In the last ten years, major breakthroughs have been made in the field of gene editing, which is the process where DNA is modified, deleted, inserted or replaced. The most recent discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened up many novel opportunities as well as applications for gene editing both in vitro and in vivo. In this review, we provide an overview of the different gene editing techniques including the CRISPR/Cas system, which was discovered in 2012. After this finding, different Cas proteins were developed with unique features that allow for distinct gene editing approaches. Here, we summarize these different Cas proteins and detail their specific features. Besides the development of different Cas proteins, endonuclease-deficient Cas proteins (dCas) allow for additional applications of the CRISPR/Cas system. These endonuclease-deficient Cas proteins (dCas) can be fused to effector domains which exert additional functions such as transcriptional activation or repression, but also tracking and base-editing. These dCas effectors as well as their functions and in vivo applications will be the main focus of this perspective.Gene editing started with the discovery of meganucleases in yeast as well as in other small organisms. There are hundreds of meganucleases which each contain a recognition sequence (homing site) between 14 and 40 base pairs  [1]. ThiIn 1994, the zinc finger nucleases (ZFN) were discovered as the first modulated gene editing tools . ZFN conIn 2009, the transcription activator-like effector nucleases (TALEN) were developed, which function in a similar way but have increased specificity when compared with ZFN ,8. TransStreptococcus pyogenes Cas9 (SpCas9) by Kleinstiver and his colleagues. These four amino acid substitutions alter the energetics of the binding of SpCas9 to the DNA, and thereby the specificity, which diminished off-target effects . Afre a PFS  28]. Ps. PsLeptoRuminococcus flavefaciens Cas13d (RfxCas13d) and is also not dependent on a PFS (The most recently discovered Cas13 protein is on a PFS  29]. Th. ThRuminCas9 and Cas13 can both serve as gene silencing tools for protein coding genes. The Cas9 system utilizes non-homologous end joining-based DNA damage repair to create a DNA insertion/deletion (indel) mutation to generate a frame shift or a point mutation  . In thisThe most recently discovered Cas14 family consists of the smallest RNA-guided nucleases, which depend on a tracRNA and, in contrast to the other Cas proteins, targets single-stranded DNA  32]. Th. Th32]. The CRISPR/Cas system can be repurposed in several ways by modification into an endonuclease-deficient Cas protein (dCas). dCas9 is generated via the introduction of two mutations in the cleavage domains of Cas9 (D10A and H840A). dCas9 is still capable to bind to the DNA in a sgRNA-mediated manner but does no longer cleave the DNA. Different effector domains with distinct gene regulatory functions, such as transcriptional activators, repressors or base editing domains, can be fused to dCas9. The subsequent introduction into cells results in sgRNA-guided gene activation, gene repression or base editing of the targeted locus. dCas9 can also be used to perform fluorescent imaging. These Cas9 derivatives will be discussed in more detail below.MUC4 locus . Th. Th33].  mitosis . This sy mitosis . This sy mitosis . This syRecruitment of transcriptional factors or activation domains to initiate gene expression can be achieved by fusion of various activation domains to dCas9 . dCas9-bBesides fusing transcriptional activators to dCas9, it is also possible to fuse dCas9 to epigenetic-modifying domains. Here, the gene is activated via their endogenous transcription system and is therefore expressed at physiological levels ,40. In gdCas9-based gene activation systems  showed great gene reactivation effects, but they mainly depend on using a pool of different guide RNAs, which makes it more difficult to use them effectively in genome-wide screens. Additionally, these ectopic transcription factors increase the gene reactivation level beyond physiological conditions, which is likely to bring unwanted side effects. Also, VP64 and Rta are derived from the herpes simplex viral protein 16 and Epstein–Barr virus Rta Protein, respectively, which might lead to cell type-specific efficiency and toxicity issues .TET protein-mediated DNA demethylation and histone modification enzyme-induced chromatin remodeling can also reactivate gene expression. These methods rely on increasing the chromatin accessibility of the gene promoter/enhancer regions to recruit endogenous transcription factors, thereby enhancing the transcription. These methods have the advantage over dCas9 fusion with activators such as VP64, p65, Rta or HSF1 since they maintain the reactivated gene expression within a physiological level ,40. HoweBesides transcriptional activation, effector domains which facilitate transcriptional repression can also be fused to dCas9 . ExampleThe advantage of dCas9-based gene repression modules  is that it can efficiently and specifically decrease gene expression by up to 99% . As compNotably, dCas9-fused DNMTs also hold a great ability in gene silencing, however, this is only applicable for those genes which have CpG-rich regions in the promoter . In addition, Galonska et al. have reported that dCas9-DNMT modules have global off-target effects by adding methylation footprints that are independent of the sgRNA and delivery methods .S. pyogenes Cas9-derived base editor) and SaBE4 (Staphylococcus aureus Cas9-derived BE4)) [The CRISPR/Cas9 system can also be repurposed to perform base editing in the DNA to correct mutations relevant to human disease . This ened BE4)) . Furthered BE4)) . Anothered BE4)) . Here, ded BE4)) . It is ied BE4)) . AltogetBase editing ingeniously avoids DNA breaks and was thought to minimize inaccuracy . However6A  reader proteins YTDHF1 and YTDHF2 results in enhanced translation and the induction of mRNA degradation, respectively [Where Cas9 derivatives can be used to induce transcriptional activation or repression at the DNA level, Cas13 derivatives can be used for RNA targeting. Similar to dCas9, dCas13 is able to bind to the target RNA but is unable to cleave it. Further, and also similar to dCas9, an EGFP-tagged dCas13 can be used to track transcripts in living cells . Moreoveectively . Taken tThe CRISPR/Cas derivatives have distinct effects in vitro, such as transcriptional activation or repression, but only some of them have been demonstrated to function in vivo. The first description of dCas9-mediated transcriptional activation in vivo was in drosophila . Here, dAs described before, dCas9-based transcriptional activation can not only occur via fusion of dCas9 to the transcriptional activators but also to the epigenetic modifiers, such as TETs, which induce DNA demethylation, and thus the re-activation of gene expression. The dCas9-SunTag (with recruiting TET1CDs) is used to perform targeted demethylation, and thus the upregulation of genes in the brain of mouse fetuses via in utero electroporation . dCas9-TBesides transcriptional activation, CRISPR-dCas9-based transcriptional repression has also been reported in a few in vivo studies. In general, CRISPR-dCas9 derivatives are delivered in vivo via lentiviral vectors  and elecThe dCas9-KRAB, but also dCas9 fused to the epigenetic repressor LSD1 (lysine-specific demethylase 1) are electroporated in the chick embryo to inactivate enhancers . dCas9-VAltogether, this shows the in vivo applications of different CRISPR/Cas derivatives  tissue-specificity, (2) target cell entry ability and (3) without/low immunogenicity. Initially, as proof-of-concept, many of the therapeutic applications of the dCas9 derivatives were performed on animal models via the electroporation of plasmid DNA ,76. ElecThe discovery of the CRISPR/Cas9 system has revolutionized the field of epigenetic and genetic editing in the pre-clinic. This is a fast-growing field where different Cas9 proteins have been discovered with each their distinct features . The discovery of endonuclease-deficient Cas9 has led to the development of different CRISPR/dCas9 derivatives, which can be used to perform distinct functions such as tracking, transcriptional activation and repression, and base editing. Some of these dCas9-based derivatives have also been applied in vivo, where they contribute to transcriptional activation or repression which can lead to changes in disease phenotypes. This holds great promise for genetic screens to identify novel genes which are associated with a disease phenotype, and of course for potential therapies. Even though the discovery of high-fidelity Cas9 has significantly improved off-target effects, future research needs to be directed to improve methods to detect off-target effects  can increase the specificity by several orders of magnitude) . In addi


Simultaneous occurrence of acute ischemic stroke and myocardial infarction is reported to have variable precipitating causes. This occurrence has been rarely reported in the literature and described only in very few case reports. During the surge of coronavirus disease (COVID-19) in our region, we noted an increase in the simultaneous occurrence of cardio-cerebral infarction. This led us to explore the possible mechanisms and pathophysiology that could contribute to this increase. The retrospective nature of the study limited us from drawing any conclusion about causation. Rather, we aimed to formulate a hypothesis for future, more rigorous studies.We present an overview of 5 cases of simultaneous cardio-cerebral infarction that we encountered in our emergency department within 1 month.In all cases, diagnosis was confirmed using an electrocardiogram, assessment of laboratory cardiac markers, and imaging.In all cases, dual antiplatelet therapy was started and thrombolysis was held, as the condition was considered high risk in most of the patients. Cardiac catheterization lab was not activated either because the patient was unstable or the risk of COVID-19 in staff outweighed the benefit added in patient treatment.Two out of 5 patients died because of early complications that lasted for few days. The remaining 3 were discharged from the hospital in moderate functionality for extensive therapy and rehabilitation.Early recognition and immediate treatment is important in different scenarios leading to thrombosis as the outcome. Additionally, addressing the unknown risks that could contribute to our traditional understanding of these causative mechanisms is important. The hypothesis of exacerbated damage caused by inflammatory and immunological endothelial systemic damage should further be explored to be able to delineate new possibilities in managing these conditions. During the coronavirus disease (COVID-19) pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a stronger association between COVID-19 and venous and large vessel arterial thromboses has been noted, with up to 25% in hospitalized patients.–9 This association is not clearly defined, and further studies are needed to determine whether this relationship exists. During the COVID-19 pandemic, in our region, we noted an increase in cases with rare, simultaneous cardio-cerebral events within a few weeks. While we did not confirm the direct association with COVID-19 in these cases, we hypothesize that COVID-19 increased the risk of this presentation. We herein discuss in detail the possible mechanisms and pathophysiology of thrombosis that can explain this increase in the occurrence. Due to the retrospective study design, which is a limitation of this case series, we hypothesize that only COVID-19 could be a major contributing factor for the increased occurrence. Any direct association remains yet open for further studies. All cases presented here had a high score suspicion for COVID-19 based on our national COVID-19 triage criteria  saturation, body temperature, and blood glucose level were 141/83 mmHg; 119 beats per minute; 48 breaths per minute; 76% at room air; 37 °C; and 252 mg/dL, respectively. He was immediately placed on 10 L oxygen which improved his saturation to 95%. At neurological examination, he was confused and uncooperative. He showed global aphasia, right sided weakness, right sided facial palsy, and left sided gaze. His National Institute of Health Stroke Scale (NIHSS) score was 22. An immediate stroke code activation was initiated as per our institutional protocol, and an initial unenhanced cranial computed tomography (CT) revealed left middle cerebral artery (MCA) ischemic stroke , heart rate (HR), respiratory rate (RR), oxygen , with both most likely related to her old ischemic insult .3 The term “cardio-cerebral infarction” was coined in 2010 by Omar et al, and it describes the simultaneous occurrence of acute ischemic stroke and acute myocardial infarction. According to the scientific statement of the American Heart Association/American Stroke Association (AHA/ASA), it is recommended to start intravenous alteplase at a dose appropriate for cerebral ischemia, followed by percutaneous coronary angioplasty when hyperacute simultaneous cardio-cerebral infarction occurs, and this approach is considered reasonable. Both conditions have a high risk of mortality and a narrow therapeutic window. Delaying treatment of one, over the other, may result in irreversible damage. There is no evidence-based guideline for the best treatment approach or clinical studies to guide the management. The incidence is reported to be 0.009%. Few case reports have been described in the literature with simultaneous cardio-cerebral infarction.,12,14,15 Multiple mechanisms can be proposed as possible causes for cardio-cerebral infarction. First, it could occur in the presence of intracardiac thrombus with poor ventricular function, which can simultaneously embolize the cerebral and coronary arteries. Second, arterial thromboembolism caused by atrial fibrillation is another cause for simultaneous cardio-cerebral emboli. Cardiac emboli caused by atrial fibrillation are usually large and can cause a massive infarct in the MCA. Third, type-I aortic dissection can cause simultaneous myocardial infarction and ischemic stroke. Fourth, right ventricular infarction with a patent foramen ovale can embolize to vascular territories and cause an ischemic stroke.The simultaneous occurrence of acute ischemic stroke and myocardial infarction is rare but life-threatening. It is challenging because both conditions require timely diagnosis and management.This case series describes 5 cases from the ongoing COVID-19 pandemic. We suggest that COVID-19 be considered a high-risk factor for ischemic stroke, myocardial infarction, and systemic thrombosis. Vascular endothelial dysfunction and coagulopathy are the likely etiologies. In a Dutch study, it was found that 31% of COVID-19 patients admitted to the intensive care unit had thrombotic complications. Of those, 3.7% were arterial thrombotic events. The elevated levels of D-dimer and C reactive protein indicate an increased inflammatory and an abnormal coagulopathy state. The ischemic manifestations of COVID-19 could be explained by this increase in the inflammatory state, abnormal coagulopathy, cytokine storm, viral-mediated disruption of the endothelium.–23 These all have been hypothesized to cause an increase in the risk of large vessel arterial thrombosis in addition to the postulated increase in venous thrombosis and COVID-19-related microangiopathy. The pathogenic mechanism of this relationship is still to be determined in further studies.The incidence of stroke in a retrospective study in Wuhan, China, showed an incidence of 5% among hospitalized patients infected with SARS-CoV-2. It is proposed that SARS-CoV-2 enters the central nervous system (CNS) through the hematogenous or retrograde neuronal route, similar to other respiratory viruses. In one case, SARS-CoV-2 was identified in the cerebrospinal fluid by PCR. Other reports were not able to identify the virus in the cerebrospinal fluid despite positive COVID-19 PCR in nasal swabs with concurrent neurological manifestations.In hospitalized patients in Wuhan, China, it was found that 36.7% of COVID-19 patients had neurological manifestations. In previous SARS viruses, it has been postulated that the SARS-CoV-1 can infect and invade the blood-brain barrier (BBB) endothelial cells, allowing direct passage of the virus across the BBB into the CNS. Binding of the virus to the angiotensin-converting enzyme 2 (ACE2) receptors expressed in the capillary endothelium of the BBB has been proposed to have a major role in the direct viral access into the CNS.,29 Depletion of ACE2 receptors may play a major role in endothelial damage and overexpression of angiotensin II because of the unopposed ACE1 receptors. Accumulation of angiotensin II can also activate the nuclear factor-kappa (NF-κB)-signaling pathway and the interleuking-6 (IL-6) pathway. The activation of the NF-κB pathway has an important role in the pathogenesis of inflammatory diseases -6, tumor necrosis factor (TNF)-α, interferons (IFNs), and chemokines  are likely related to the extensive systemic inflammation and more severe multi-organ involvement Table .  Even though the role of ROS has been implicated in different pathologies such as cancer, atherosclerosis, and neurodegenerative diseases; the brain is particularly sensitive to oxidative stress and excessive production of ROS. ROS imbalance is further exacerbated by the endothelial disruption and conformational changes that interrupts cell-to-cell adhesion inducing BBB breakdown, viral invasion, and inflammatory mediator infiltration.The inflammatory damage by the renin-angiotensin-aldosterone system (RAAS) is further exacerbated by the oxidative stress caused by increased reactive oxygen species (ROS). Oxidative stress, mainly Toll-like receptor 4 pathway, is thought to be triggered by viral pathogens such as SARS-CoV-2, amplifying host inflammatory response.2–) into hydrogen peroxide (H2O2) and the more reactive hydroxyl radical (OH). The different free radical species affect many processes within the brain parenchyma and can result in detrimental cellular effects such as lipid peroxidation, affecting cellular signaling and initiating apoptosis.,49 The activation of the NF-κB pathway have an important role in the pathogenesis of inflammatory diseases.,32 When stimulated by the release of TNFα, or other cell stressors, the BNF-κ pathway can have a neuroprotective or a proinflammatory role; and influence cell regulation and survival based on the tissue location and pathological state. The binding of the COVID-19 virus to the ACE2 receptors can also activate BNF-κ. Angiotensin II accumulation will lead to increased vasoconstriction, increased oxidative stress, and inflammation.The oxygen-depleted ischemic cells have increased acidosis, which will further exacerbate the conversion of superoxide radical (O,53The unique features of COVID-19 pathophysiology make it more challenging to tackle and make it an open area for further studies to delineate the best modality of treatment. Immune modulatory therapy is an area for further research because it is proposed to have a major role in the COVID-19 pathophysiology. The inflammatory dysregulation is further worsened with chronic conditions that have similar immunological disruption such as diabetes mellitus and dyslipidemia. This could explain the COVID-19 exacerbation of existing chronic conditions with increasing incidence of COVID-19-related thrombotic cardio-cerebral events. Despite this recommendation, it is challenging to treat simultaneous cardio-cerebral infarction because of different risks including time of presentation, different dosing requirements, availability of endovascular therapy, and the timing of fibrinolytic therapy required for the different conditions. Individualized treatment approaches are recommended to be considered according to the different operating factors.,55The proposed systemic viral endothelial disruption, inflammatory dysregulation, cytokine storm, ROS imbalance, and RAAS disturbance can all play a major role in macro-thrombosis formation. The relation of COVID-19 with arterial thrombosis should be further explored. The treatment approaches for simultaneous STEMI and acute ischemic stroke are extremely challenging with a high risk of morbidity and mortality even in the regular circumstances and much more so in the COVID-19-related settings. In the context of simultaneous cardio-cerebral infarction, the AHA/ASA in 2018 recommended that intravenous alteplase can be used at the same dose for cerebral ischemia, followed by percutaneous coronary intervention.4In the cases mentioned above, different treatment approaches were considered based on the individual presentation and risk of bleeding. Cardio-cerebral infarction is challenging concerning diagnosis, and treatment is not straightforward. If COVID-19 infection is to be considered, then this challenge is further complicated, as pathophysiology and treatment approaches are not yet fully determined. Despite the retrospective study design, which is a limitation in our cases, COVID-19 should always be considered and further testing strategies should be considered, if available, because the pathophysiology is different and requires different treatment modalities and strategies. To date, the testing modalities and immunological effects of COVID-19 are not fully understood. New treatment strategies such as antivirals, antioxidants, anti-inflammatory agents, targeted immunomodulatory therapies, antifibrotics, and anticoagulants could prove beneficial in these cases, and further research is warranted.The authors would like to acknowledge Dr. Areej Almweisheer and Dr. Fai Aldosari for their help in tracing back patients’ events upon admission.Conceptualization: Rawan Eskandarani, Seham Sahli.Data curation: Rawan Eskandarani, Seham Sahli, Asim Alsaeed.Investigation: Rawan Eskandarani, Seham Sahli, Shaima Sawan, Asim Alsaeed.Supervision: Rawan Eskandarani, Asim Alsaeed.Writing – original draft: Rawan Eskandarani, Seham Sahli, Shaima Sawan.Writing – review & editing: Rawan Eskandarani, Asim Alsaeed.


Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes.  Bladder cancer (BC) is the most common malignancy of the urinary tract . CystoscDue to their high throughput and low cost, microfluidic-based diagnostic tools hold the promise for improved patient care and outcomes considering the limitations of current screening and diagnostic techniques as well as the societal and economic impact of BC , 23. MicBuilt on our previous work regarding the development of microfluidic platforms for the capture of BC CTCs , 29, we 4F8, Sigma Aldrich, USA). The etching of the features was confirmed by SEM inspection. Photoresist residues were stripped by oxygen plasma  and the wafer was diced using a DAD 3350 Dicing Saw . Masters were then cleaned with isopropyl alcohol , rinsed with deionized water and dried at 150°C. Finally, masters were hydrophobized through treatment with trichlorosilane  and cured for 1 h at 65°C.Masters were designed in AutoCAD software  and consist of five rows of posts with increasingly narrower gap widths  , allowinDevices were fabricated in polydimethylsiloxane , which was prepared as a two-part system with mix ratio of 10:1 (w/w) base/curing agent, poured over the master, degassed and cured for 2 h at 65°C. Following that, the PDMS was unmolded and inlet and outlet made using a puncher. Irreversible bonding was achieved through surface activation of clean glass slides and PDMS replicas by low power oxygen plasma for 15 s . Immediately after fabrication and bonding , microfl2 in RPMI 1640 GlutaMAX  and DMEM high glucose , respectively. Media were supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin . Cells were continuously monitored by microscopy to ensure they maintained their original morphology. Where appropriate, BC cells were harvested by incubation in 0.25% Trypsin-EDTA , washed with PBS and labeled with 12.5 μM calcein-AM  according to the manufacturer's instructions.Human urinary BC cell lines HT1376 and MCR-STn [overexpressing the sialyl-Tn antigen ] were grPeripheral blood (3 mL) was collected from healthy blood donors after informed consent, layered over histopaque-1077  and centrifuged at room temperature for 10 min at 650 g, without active break. Peripheral blood mononuclear cells (PBMCs) were then gently collected from the gradient interface, washed twice and resuspended in PBS supplemented with 2% bovine serum albumin . Cell viability and concentration were determined using the Tuerk solution .n = 8) and Hospital Universitario 12 de Ocubre, Madrid, Spain (n = 6). Voided urine samples (30–50 mL) from 14 patients were collected prior to transurethral resection of bladder tumor (TURBT). Bladder wash samples (10–30 mL) were obtained after flushing the bladder with saline buffer immediately before TURBT. n = 6) from healthy subjects were obtained and subjected to the same protocol. Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were also included in the study. All procedures were performed after patient informed consent and approval by the Ethics Committee of both hospitals.BC patients were enrolled at the Urology department of Hospital da Senhora da Oliveira, Guimarães, Portugal  previously labeled with calcein-AM were injected into the UriChip microfluidic devices at two different inlet pressures (200 and 300 mbar) with the help of a pressure pump. Trapped cells were then fixed with 4% (w/v) formaldehyde solution  during 20 min and finally washed with PBS. Capture efficiency (CE) of HT1376 cells was determined by imaging and counting the number of calcein-AM-positive cells captured and comparing with the total input.CE (%) = (captured HT1376 cells)/ × 100To determine cancer cell capture purity in the presence of leucocytes (which may be found in the urine due to cancer associated inflammation), calcein-AM-stained HT1376 cells were next spiked in 500 μL of PBS containing unlabeled PBMCs at a 1:10 ratio, run at 200 mbar and fixed with 4% (w/v) formaldehyde solution as described above. Cell purity and PBMC retention were determined according the following formulas:Purity (%) = [(captured HT1376 cells)/(captured HT1376 cells + captured PBMCs)] × 100PBMC retention (%) = (captured PBMCs)/ × 100Similarly, to evaluate the efficiency of isolating cancer cells from voided urine, MCR-STn cells were pre-stained with calcein-AM for 30 min at 37°C, spiked in 500 μL of PBS-2%BSA containing urine sediment from healthy subjects and run at 200 mbar. Cells were then fixed with 4% (w/v) formaldehyde solution and finally washed with PBS. The number of calcein-AM-positive MCR-STn cells captured in the device was compared to the total number of MCR-STn cells spiked, and CE determined.Patient samples (processed bladder washes or voided urine) were resuspended in PBS-2%BSA and injected into the chips at 200 mbar. After a 30 min incubation period with no flow to reduce unspecific binding, cells were fixed with 4% (w/v) formaldehyde for 20 min and further treated with 0.25% Triton X-100 in PBS for 5 min to induce cellular permeability. Subsequently, two washes with PBS were performed and mouse monoclonal anti-CK pan-FITC antibody (1:100 Clone C-11) and DAPI  diluted in PBS-2%BSA were loaded into the UriChips for 1 h in the dark. The immunostaining process ended by washing with PBS before imaging, with the flow rate set at 200 mbar during the entire procedure. Where appropriate, cells were also immunostained using the anti-STn mouse monoclonal antibody clone B72.3  for 1 h and 30 min at room temperature, and labeled with a secondary goat anti-mouse IgG-TRITC antibody  for one additional hour, prior to cell fixation and permeabilization. Following sample processing, a fluorescence microscopy analysis of the captured cells was performed under an inverted fluorescence microscope . A total of twenty-five fields (20x) per device were selected randomly and the number of captured cells counted. Only DAPI and STn-positive cells with identifiable cellular morphology and well-delimited cytoplasm were considered for cell enumeration, and count normalized to the total number of DAPI-positive cells.FFPE tissue sections from patients with BC were screened for STn staining using the streptavidin/biotin peroxidase method with the anti-STn antibody, as previously described .Clostridium perfringens neuraminidase  for 2 h at 37°C to cleave terminal sialic acid residues from glycoproteins on cell surfaces. The reaction was stopped with PBS washes.Samples were incubated with p < 0.05 .Statistical analysis was performed using GraphPad Prism, version 5 . Data is presented as mean ± SD. Deviation from normality was tested using the D'Agostino and Pearson normality test. The Mann-Whitney test was used for unpaired samples and differences were considered to be significant when The performance of the microfluidic device to capture BC cells was firstly investigated using HT1376 BC cells as a model, pre-stained with calcein-AM and spiked in PBS. As illustrated in To avoid the risk of device leaking and ensure the proper and intact morphology of urothelial exfoliated cells during the microfluidic capture, 300 mbar of inlet pressure was applied in the UriChip. On the other hand, to prevent squeezing of urothelial cells through the microposts under pressure and consequently their loss to the outlet, the minimum inlet pressure of 200 mbar was tested. By working on the range of 200–300 mbar, results revealed a higher CE of HT1376 cells at 200 mbar when compared to 300 mbar input pressure . This reGiven that the urine of BC patients is heterogeneous and usually contains leucocytes due to cancer associated inflammation, we next investigated the CE of HT1376 cells pre-stained with calcein-AM in the presence of non-labeled PBMCs isolated from healthy donors, spiked in PBS. PBMC population comprises lymphocytes and monocytes that range between 7 and 15 μm in diameter with a high deformability capacity . NotablyHaving confirmed the ability of the UriChip to isolate BC cells in single and mixed model samples, we further evaluated its potential for the capture and analysis of cells from clinical samples, known to be much more complex and heterogeneous. As such, we analyzed both bladder wash samples, which are highly cellular and contain well-preserved cells , as wellCytokeratins are highly expressed in intermediate filaments of normal and neoplastic epithelial cells . Hence, in situ immunofluorescence.These results highlight the potential of the UriChip to retain cells of different sizes and shapes from voided urine, using a label-free approach. Moreover, morphological analysis of captured cells can be performed, and phenotypic characteristics identified via In order to accurately identify urothelial ETCs and distinguish them from benign ones, we selected STn, a tumor associated antigen overexpressed in BC but absent in the healthy urothelium, as malignant biomarker . Indeed,STn expression enhances the migration and invasive capacity of MCR cells , which aWe next moved to the pre-clinical testing of the UriChip using both bladder washes and fresh voided urine from BC patients and a combination of two biomarkers, STn and CK to detect malignant cells. Of note, we extended this analysis and evaluated UriChip and STn/CK immunoassay feasibility to detect cells from cryopreserved samples, greatly relevant for retrospective studies. The working principle of UriChip device is based on size-exclusion and cell deformability, which allows for the capture and characterization of urothelial ETCs. In fact, urothelial ETCs, which are shed directly by the growing tumor in the bladder, are known to reflect disease progression  and for Optimization tests were conducted with two distinct BC cell models, prior to the analysis of clinical samples: bladder washes, which contain well-preserved cells when collected before TURBT , and voiSpiking experiments demonstrated that, using the UriChip, the CE of BC cells spiked in leukocytes and urine from healthy donors achieved 53%, typical for size-based filtration . Higher in situ, via a microfluidic immunoassay involving the epithelial marker, pan-CK, and the tumor-associated glycan, STn. On-chip immunostaining of CTCs isolated from blood samples by microfluidics with distinct biomarkers has been extensively performed  will enable to assess the detection accuracy of UriChip. Additionally, further studies by combining UriChip immunoassay with traditional cytology and comparing with commercial available FDA-approved markers are warranted to validate the present findings. Furthermore, the capture and enrichment of intact urothelial exfoliated cells within UriChip provide the opportunity for downstream on-chip proteomic and off-chip single-cell genomic analyses for a precise diagnosis of BC.Overall, UriChip microfluidic-based platform is able to efficiently capture and enrich urothelial exfoliated cells, from both fresh and frozen voided urine of BC patients, according to their size and deformability. Notably, STn expression in ETCs from patients from two independent data sets was demonstrated herein for the first time, which correlated with staging and grading of BC.Combination with high-throughput processing and automation may constitute a first step toward a fully integrated system for rapid label-free capture, on-chip phenotypic characterization and enumeration of BC cells. In addition, exploring glycosylation of tumor cells in body fluids, namely STn expression, will offer a more selective malignant cell isolation, paving the way to downstream molecular analysis and fostering precision medicine applications in bladder cancer and other malignancies.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Hospital Senhora da Oliveira, Guimarães, Portugal. The patients/participants provided their written informed consent to participate in this study.in vitro, data analysis, and manuscript writing. CA and LD were responsible for the design and fabrication of the UriChip microfluidic device and evaluation of its performance. DF performed tissue immunohistochemistry experiments. RR, VG, and MD selected and collected patient samples. LL, JF, LS, MM-F, MD, CS-C, JP, and PF contributed to the interpretation of the experimental data. MO was responsible for the project conception, study design, data interpretation, and manuscript writing. All authors discussed the results and commented on the manuscript.SC was responsible for the study design, patient samples processing, analytical measurements 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.