Trends in neurologically intact survival after paediatric out-of-hospital cardiac arrest (OHCA) remain unclear. In the present study, we aimed to determine trends in 1-month neurologically intact survival after paediatric OHCA over time.We reviewed the data of 5461 children (aged < 18 years) who experienced bystander-witnessed OHCA and were included in the nationwide Japanese registry from 2005 to 2017. Patients were divided into three groups according to study period: 2005–2010, 2011–2015, and 2016–2017. We analysed the trends in 1-month neurologically intact survival rates over time.P for trend < 0.05 and < 0.001, respectively). In the subgroup analyses by aetiology, the 1-month neurologically intact survival rate in patients with OHCA of non-traumatic origin significantly increased from 11.8%–15.1% to 19.7% (P for trend < 0.001) but not in those with OHCA of traumatic origin .The risk-adjusted odds of 1-month neurologically intact survival  were significantly improved by 2016–2017 compared with baseline. Similar improvements in 1-month neurologically intact survival rates were observed with both standard bystander cardiopulmonary resuscitation (CPR) with rescue breaths and chest compression-only bystander CPR (The 1-month neurologically intact survival rate significantly increased from 2005 to 2017 in Japanese children with bystander-witnessed OHCA, regardless of bystander CPR type; This increase was noted in patients with OHCA of non-traumatic origin but not in those with OHCA of traumatic origin. Early bystander cardiopulmonary resuscitation (CPR) is a critical component in the chain of survival after out-of-hospital cardiac arrest (OHCA).In this study, we aimed to determine the change in outcomes after paediatric bystander-witnessed OHCA over time according to the CPR type and OHCA cause using the Japanese nationwide registry data.This nationwide, population-based observational study included all paediatric patients aged < 18 years) who experienced bystander-witnessed OHCA and were resuscitated by EMS personnel in Japan between January 1, 2005 and December 31, 2017. Patients were excluded if they (1) were aged ≥ 18 years, (2) did not receive resuscitation from an EMS personnel, (3) had no witnesses to their cardiac arrest, (4) had only EMS personnel as witnesses, (5) received rescue breathing-only bystander CPR or AED-only bystander intervention, or (6) had unknown outcomes or age. The study was approved by the institutional review board of Kanazawa University (No. 1263-8). The requirement for written informed consent was waived because the study used anonymised data. The Fire and Disaster Management Agency (FDMA) in Japan supervises the nationwide EMS system, while local fire stations operate the local EMS systems. As of 2017, the country has 732 fire departments and 5140 ambulance teams. years whIn 2005, the FDMA launched an ongoing, prospective, population-based observational study involving patients with OHCA who had received resuscitation from EMS personnel in Japan.The primary outcome measure was 1-month neurologically intact survival, defined as a CPC score of 1 or 2. The secondary outcome measure was 1-month survival after OHCA.P-value of < 0.05 was considered significant.Continuous variables are expressed as medians and interquartile ranges, or as means and standard deviations, whereas categorical variables are expressed as percentages. To reduce the bias derived from differences in CPR strategies used over time, the study periods were divided into three groups based on the year that the major CPR guidelines were released: 2005–2010, 2011–2015, and 2016–2017. To investigate the trends of outcomes in some subgroups, the patients were divided into subgroups according to the type of CPR , aetiology  and initial rhythm (shockable or non-shockable rhythm). We used the Kruskal–Wallis test followed by Dunn’s post hoc test to analyse continuous variables. The chi-square test and univariate logistic regression analyses were performed to compare the characteristics and outcomes of categorical variables. The Cochran–Armitage trend test was used to evaluate the trends in baseline characteristics and outcomes in the three study periods. To perform a rigorous adjustment of selection bias and thus determine the differences in the patients’ baseline characteristics, multivariate logistic regression analyses were used when comparing outcomes between groups and estimating the key prehospital variables associated with the outcomes. The potential prehospital confounders for the analytical model were selected based on biological plausibility and data from previous studies. In multivariate logistic regression analyses, eight prehospital variables were included: calendar year, age, sex, initial cardiac rhythm, cause of arrest, public-access defibrillation (PAD), bystander CPR status, and EMS response time . All statistical analyses were performed using the JMP statistical package, version 15-Pro . All reported tests were two-tailed, and a Details of attempted resuscitations performed for 1,550,356 patients with OHCA between 2005 and 2017 are documented in the database. The crude 1-month survival and CPC 1–2 rates significantly increased by year  of 1.89 (1.54–2.32) and 1.86 (1.41–2.44), respectively. A similar trend was observed in the 1-month outcomes of some subgroups: those who received CPR (both standard CPR and chest-compression-only CPR) and those with OHCA of non-traumatic origin (both cardiac and non-cardiac). Even after adjusting for confounding factors, both 1-month survival and neurologically intact survival in the 2016–2017 period were twice of those in the 2005–2010 period. To the best of our knowledge, this is the first and largest cohort study to show that 1-month survival and neurologically intact survival increased over time in Japanese paediatric patients with OHCA.There are many possible reasons for the improvement in outcomes during the study period. In accordance with the 2005 CPR guidelines,Regarding the CPR type, 1-month outcomes of both the standard CPR and chest-compression-only CPR cohorts improved over time. Although there may be a number of things contributing to this improvement that are clearly not explained by CPR, the present study supports the 2017 International Liaison Committee on Resuscitation summary statementA previous study showed that survival rates in paediatric patients who experienced in-hospital cardiac arrest in US significantly improved between 2000 and 2018.The increase in the rate of dispatcher-provided CPR instructions in cases of paediatric patients with OHCA may have improved overall outcomes because this rate significantly increased from 35% to 46% over time in this study. The proportion of patients who received CPR following dispatcher’s instructions also increased—from 74% to 83%. However, 17% of bystanders did not perform CPR despite the dispatcher’s offer. Accordingly, to improve neurological outcomes, every effort should be made to reduce the proportion of non-responders. Furthermore, to ensure that dispatcher-assisted CPR is administered, it is essential that dispatchers recognise cardiac arrest during an emergency call.This study has some limitations. First, we stratified the study periods into three unequal groups according to the release of major CPR guidelines to reduce the bias derived from differences in CPR strategies used over time. However, this may have led to the inclusion of unexpected confounders. Second, the actual aetiology of cardiac arrest was not fully verified. Therefore, some patients may have had sudden infant death syndrome, a common aetiology of cardiac arrest, followed by trauma and respiratory disease.Between 2005 and 2017 in Japan, the 1-month neurologically intact survival rate of paediatric OHCAs significantly increased in patients with bystander-witnessed OHCA of non-traumatic origin, regardless of the CPR type. No such improvement was seen in paediatric patients with bystander-witnessed OHCA of traumatic origin.apan Society for the Promotion of Science(Grant-in-Aid for Scientific Research: Grant Nos. 20H202271 and18K09999).This work was supported by the JYoshikazu Goto and Akira Funada designed the study. Yoshikazu Goto, Akira Funada, Tetsuo Maeda, and Yumiko Goto sorted the data. Yoshikazu Goto and Yumiko Goto analysed the data. Yoshikazu Goto drafted the manuscript, and Yumiko Goto and Akira Funada substantially contributed to its revision. Yoshikazu Goto takes responsibility for the paper as a whole. All authors read and approved the final manuscript.None.The datasets generated during and/or analysed during the current study are not publicly available because of the FDMA regulations but are available from the corresponding author on reasonable request.


Seven immunocompetent patients aged > 50 years old presented with herpes zoster (HZ) infection in a median of 9 days (range 7–20) after vaccination against SARS-CoV-2. The occurrence of HZ within the time window 1–21 days after vaccination defined for increased risk and the reported T cell-mediated immunity involvement suggest that COVID-19 vaccination is a probable cause of HZ. These cases support the importance of continuing assessment of vaccine safety during the ongoing massive vaccination for the COVID-19 pandemic and encourage reporting and communication of any vaccination-associated adverse event. As the pandemic of SARS-CoV-2 enters its second year and new variants of the virus emerge, vaccines are needed to protect individuals at high risk for complications and to potentially control disease outbreaks through the establishment of herd immunity ,2. The ESurveillance of rare safety issues related to the vaccines is progressing as more data accumulate during post-marketing surveillance ,5. AdverIn Greece, the vaccination program started in January 2021. Health care workers (HCW) accounted for a significant proportion of COVID-19 infections worldwide , and aloVaricella zoster virus (VZV) is a human neurotropic virus that causes varicella. This is followed by the establishment of latency in sensory ganglia, notably, the dorsal root ganglia, trigeminal ganglia, and enteric ganglia. The latent phase usually lasts for several decades before reactivation occurs. VZV reactivation normally presents as herpes zoster (HZ), which is characterized by a painful, unilateral vesicular eruption in the dermatome innervated by the ganglion where reactivation occurred and probably appears when the immune system fails to contain the latent VZV replication. Effective treatment is available ,9,10,11.VZV reactivation in COVID-19 cases has been already reported. COVID-19-associated lymphopenia, especially CD3+CD8+ lymphocyte and functional impairment of CD4+ T cells, can render a patient more susceptible to developing HZ by reactivating VZV. It is also reported that HZ could be a sign of undiagnosed COVID-19 infection in younger age groups ,13,14,15Here, we report on a series of patients with VZV reactivation after vaccination against SARS-CoV-2.Seven patients (3 of 7 were women) with a mean age of 77 years  had been initially diagnosed with HZ in primary care and they were referred to the Infectious Diseases Clinic of First Department of Internal Medicine, Laiko General Hospital, for further evaluation. All of them had been vaccinated with the Pfizer-BNT162b2 vaccine against SARS-CoV-2 and they developed HZ close to vaccination. Each case was reviewed for demographic information , vaccine-related symptoms, HZ-related symptoms, and management; as well, a brief medical history (comorbidities/drugs) was obtained for everyone. Participants were followed up prospectively and a serum serology test 7–14 days after vaccination was taken and tested for anti-SARS CoV-2 receptor binding domain (RBD) spike protein. Patients’ characteristics are shown in We present a series of 7 cases of HZ in immunocompetent individuals that occurred in a mean (range) of 9 days (7–20 days) after COVID-19 vaccination. The major risk factor for reactivation of VZV is increasing age, partly because of age-related decline in specific cell-mediated immune responses to VZV, while other risk factors include disease-related immunocompromise such as HIV infection, iatrogenic immunocompromission, physical trauma, or comorbid conditions such as malignancy or chronic kidney or liver disease ,18,19. TThe safety profile of BNT162b2 mRNA vaccine was characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache, while the incidence of serious adverse events was low and similar in vaccine (0.6%) and placebo (0.5%) groups [The incidence rate of HZ increases with age from 3.1 per 1000 person-years for persons 45–54 years old, to 5.7 for persons 55–64 years old, to 11.8 per 1000 person-years for persons older than 65 years. The incidence rate of HZ among the unvaccinated-for-VZV and immunocompetent population > 50 years old is 9.92  per 1000 person-years or 0.83  per 1000 person months, according to the Kaiser Permanente Southern California study ,21. n = 3763), the expected HZ cases is 3, based on the Kaiser Permanente Study [Concerning the mRNA -1273 SARS-CoV-2 vaccine, safety assessments included adverse events for 28 days after each injection . So, forte Study . Howeverte Study . HZ is ote Study . Moreovete Study . The incidence of HZ among individuals with HIV infection increases with decreasing CD4 cell counts, highlighting the importance of T-cell immunity in maintaining latency of VZV ,24. Post-vaccination HZ is rarely reported in the literature. Walter et al.  reportedRegarding COVID-19 vaccination, we could only retrieve a publication by Bostan et al. , who repDespite the rarity of publications, HZ is not a rare event. In the VAERS, when the COVID-19 and the varicella vaccine were excluded, there were 1653 reports of HZ-related complications from July 1990 to March 2021. The HZ-related adverse events reported for the COVID-19 vaccines were 232 . In the Reactivation of VZV is a failure of the T cell compartment to maintain control of the infection. This is supposed to occur more frequently with increasing age due to adaptive immunosenescence . On the Vaccine-induced reactivation of HZ may have similarities with immune reconstitution inflammatory syndrome (IRIS), which is a paradoxical worsening of preexisting infection unmasked by the host’s regained capacity to mount an inflammatory response following the initiation of ART . We postAEFI may be coincidental events that have occurred by chance or the vaccine may have increased the risk of the specific adverse event ,37. StanGiven that the decline in immune responses in the older population has been well documented and the severity of viral and bacterial infections is increased among older individuals, vaccination serves as the main strategy for preventing such infections or their severity by inducing efficient cellular and humoral responses. On the other hand, immunosenescence is often responsible for failing to induce long-term protective immunity after vaccination and offer limited duration of protection and so further studies are warranted to improve understanding of the immunological mechanisms that govern durable protection against SARS-CoV-2 and VZV. Even though the case fatality of HZ is extremely low , HZ is fHuge vaccination programs are ongoing. Post-marketing surveillance systems must be in place and continuing assessment of vaccine safety is important for the detection of any event that could attenuate the expected benefits, and thus to take any necessary action to minimize risks to vaccinated individuals. HZ seems to be a “probable’’ although rare AEFI, based on the criteria of temporal association with vaccination and a plausible biological association. In view of the hundreds of millions of individuals to be vaccinated for SARS-COV-2, a potential causal association may result in a large number of cases with potential severe complications among the elderly. Fortunately, HZ is a treatable situation and clinical review committees should decide whether VZV treatment is temporarily recommended before initiation of vaccination. In addition, mRNA platform is a promising new technology and demonstration of its safety is meaningful for the development of further vaccines against viruses and tumors .


We assessed whether famotidine improved inflammation and symptomatic recovery in outpatients with mild to moderate COVID-19.Randomised, double-blind, placebo-controlled, fully remote, phase 2 clinical trial (NCT04724720) enrolling symptomatic unvaccinated adult outpatients with confirmed COVID-19 between January 2021 and April 2021 from two US centres. Patients self-administered 80 mg famotidine (n=28) or placebo (n=27) orally three times a day for 14 consecutive days. Endpoints were time to (primary) or rate of (secondary) symptom resolution, and resolution of inflammation (exploratory).Of 55 patients in the intention-to-treat group (median age 35 years (IQR: 20); 35 women (64%); 18 African American (33%); 14 Hispanic (26%)), 52 (95%) completed the trial, submitting 1358 electronic symptom surveys. Time to symptom resolution was not statistically improved (p=0.4). Rate of symptom resolution was improved for patients taking famotidine (p<0.0001). Estimated 50% reduction of overall baseline symptom scores were achieved at 8.2 days (95% CI: 7 to 9.8 days) for famotidine and 11.4 days (95% CI: 10.3 to 12.6 days) for placebo treated patients. Differences were independent of patient sex, race or ethnicity. Five self-limiting adverse events occurred ; placebo, n=3 (60%)). On day 7, fewer patients on famotidine had detectable interferon alpha plasma levels (p=0.04). Plasma immunoglobulin type G levels to SARS-CoV-2 nucleocapsid core protein were similar between both arms.Famotidine was safe and well tolerated in outpatients with mild to moderate COVID-19. Famotidine led to earlier resolution of symptoms and inflammation without reducing anti-SARS-CoV-2 immunity. Additional randomised trials are required. COVID-19 is caused by the SARS-CoV-2.Cytokine release drives inflammation and poor outcome in patients with COVID-19.Famotidine is a histamine 2 receptor antagonist that is globally used to reduce gastric reflux symptoms and treat gastric ulcers.In laboratory studies, famotidine reduced type-I interferon release from virally infected epithelial cells.Famotidine improved the outcome of patients with COVID-19 in some retrospective studies and a case series, but evidence from a clinical trial is lacking.In this randomised, double-blind and placebo-controlled clinical trial, oral famotidine was safe and well tolerated.Patient with mild to moderate symptoms from COVID-19 on famotidine experienced more rapid symptom resolution.Famotidine induced earlier resolution of type-I interferon levels in patients with COVID-19.High-density data studies with fewer patients may improve access to clinical trial participation from more institutions globally and require less resource.In the absence of strong alternatives, famotidine might be considered as a treatment for symptomatic outpatients with COVID-19.Additional clinical trials are needed and may leverage the knowledge generated by this study.The search for safe, effective and affordable treatments for COVID-19 remains a global health priority. COVID-19 is pandemic, with an estimated 319 M cases and 5.5 M deaths worldwide to date.COVID-19 is caused by the SARS-CoV-2.COVID-19 preventions and treatments can be classified into the categories vaccination, anti-viral medication and host response modulator. Vaccinations prime the immune system, reduce disease severity and mortality and prevent spread of the disease.Famotidine is a widely available, safe, low-cost candidate medication for COVID-19. This selective histamine H2-receptor (H2R) antagonist reduced type-I interferon release from SARS-CoV-2-infected epithelial cells in a TLR3-dependent manner.We performed a randomised, double-blind, placebo-controlled, phase 2 clinical trial of oral famotidine (80 mg three times a day) and deeply profiled the enrolled diverse, non-hospitalised patients with mild to moderate symptoms from COVID-19. We devised a fully remote clinical trial strategy to reduce patient burden and exposure of the public and healthcare personnel. We aimed to assess the benefit of famotidine on resolution of symptoms and inflammation in patients with COVID-19.Extended information on methodology is provided in 10.1136/gutjnl-2022-326952.supp1Supplementary dataThis randomised, double-blind, placebo-controlled, fully remote phase 2 clinical trial  was regiFrom the day after consent (day 1), a period of 14 days of study medication intake was followed by 14 days of continued daily monitoring and a final telephone safety consultation and symptom review on day 60.Blood samples and nasopharyngeal swabs were collected by a mobile phlebotomy service at the patient’s residence on days 1, 7, 14 and 28. Remote blood draws on day 1 were timed prior to the patient’s first dose of study medication. These blood draws were used to isolate peripheral blood mononuclear cells (PBMCs), to analyse the complete blood counts and to isolate plasma for all of the biochemical, famotidine and interferon α analyses.2, suspected dysphagia and famotidine intake at the time of screening. Pregnant patients were eligible for enrollment.We screened and enrolled patients from two US sites: Northwell Health (NH) and New York City Health and Hospitals Corporation . Candidates were identified from laboratory lists, emergency department discharge lists (NH) or from COVID-19 isolation hotel admissions (NYCHHC) and contacted by screening team members by telephone or in person at the hotels by the nursing staff . EnrolleCentral randomisation, data storage, trial coordination, pharmacovigilance, patient follow-up and shipment of trial monitoring kits were performed by the Northwell Health Office of Clinical Research. All aspects of the study were conducted in accordance with the Declaration of Helsinki, including all of its relevant amendments, the guidelines for Good Clinical Practice of the International Conference on Harmonization, and all relevant New York state and US laws and directives. An independent monitor reviewed all study data. An independent Data Safety and Monitoring Board (DSMB) monitored recruitment, subject safety and outcome.All patients received a unique study ID, IRB001 to IRB056, and patient-reported data were submitted using the study ID by secure data link, in a Health Insurance Portability and Accountability Act of 1996 (HIPAA)-compliant manner, to a secure data storage portal. Data were then combined with the full REDCap trial database prior to analysis.We randomly assigned patients in a 1:1 ratio to either placebo or famotidine treatment groups using central block randomisation generated by the Northwell Office of Clinical Research. Stratification was by patient sex and study site. Trial participants, investigators and trial staff remained blinded for the entirety of the study duration and clinical data analysis.Size #000 opaque white capsules (Gelatin-free Capsugel) either filled with microcrystalline cellulose  or famotidine 80 mg  were manufactured by Alchem Laboratories in concordance with current Good Manufacturing Practice regulations . PatientTo assess toxicity, patients were reviewed by telehealth consultation, and complete blood count, serum liver chemistries and serum creatinine with estimated glomerular filtration rate were monitored. All results and adverse events were reviewed and acted on by the blinded principal investigator and blinded clinical team. The blinded DSMB reviewed all adverse events, and no events that required unblinding were encountered.Data were collected from the REDCap database downloaded on 30 August 2021 and VitalCare Symptom Surveys on 12 May 2021. All symptom-related and general inflammatory endpoints were analysed using both intention-to-treat (ITT) analysis and per-protocol (PP) analysis of the subgroup of patients who had confirmed famotidine intake by plasma analysis. Statistical analysis was performed using SAS V.9.4 (SAS Institute), and the significance level was set at 0.05. We believed data was missing at random, and did not impute missing data.The endpoints were developed in close collaboration with the Food and Drug Administration. The strength and weaknesses of using fewer data points and comparing a single endpoint that captures sustained clinical recovery or of using all survey supplied daily total symptom scores and comparing modelled change of symptoms over time were considered. A shared decision was made to capture both these symptom resolution endpoints as primary and first secondary endpoint, reflecting that this phase 2 trial was in part designed to identify the strongest endpoint for future studies.Time to symptom resolution was defined as days from treatment start to either the first-time achieving symptom resolution or the last follow-up, up to day 28, whichever occurred first. Symptom resolution was defined as the first time that the total symptom score was ≤3, and no individual symptom score was >1 for two consecutive days. The famotidine and placebo arm were compared using stratified log-rank test.The decreasing rate of symptom resolution from day 0 to day 28 was assessed using a mixed random effect model assuming a linear change over time in ln(score+1) which was applied to overall symptom score to meet the normality model assumption. Factors adjusted in the model included time (Day in Study), treatment arm and sex. The covariance structure considered to model the correlation among longitudinal measurements from the same patient was Toeplitz. The selection was based on convergence status and Arkaike Information Criteria. Other structure considered included compound symmetry (CS), heterogeneous CS, first-order autoregressive (AR(1)), heterogeneous AR(1) and unstructured. Two-way interaction term between treatment arm and time was used to model the different changing rate in each treatment arm. To explore if difference in the changing rate between two study arms was similar between sex, race and ethnicity groups, corresponding three-way interaction terms were further examined in the linear mixed effect model.Cumulative incidence of resolution (symptom score decreased to ≤1 for two consecutive days) of each individual symptom that are >1 at baseline (day 0) was estimated for days 7, 14, 21 and 28. The symptom diarrhoea was not included in this analysis because it had low baseline prevalence and all patients were asymptomatic before day 7. Analysis method was correspondent to the analysis of primary endpoint.CRP and ferritin levels were measured on days 1, 7, 14 and 28. Values under detection limit were treated as missing. Linear mixed effect models were used to compare the levels at different time points for each treatment arm. Log-transformation was applied, that is, ln(level), to meet the normality assumption. Factors adjusted in models included time (day in study), treatment arm and gender. Time was treated as a discrete variable. The interaction term between treatment arm and time was used to estimate ferritin relative change over time within each treatment group. Based on the similar strategy, the selected covariance structure in the models was CS.The power calculation was based on an assumption of cumulative incidences of symptom resolution at day 28 in the treatment arm and placebo arm of 80% and 50%, respectively. Based on this assumption, 84 patients would have achieved 92.34% power to detect such a difference using a two-sided log-rank test with type-I error controlled at 0.05.Liquid chromatography-mass spectrometry with isotope-labelled internal standardisation for famotidine was used to determine calculated plasma famotidine concentrations.RNA was isolated from PBMCs using the PureLink RNA Mini Kit .RNA sequencing libraries were generated by poly(A) capture prior to reverse transcription into reverse strand-specific cDNA libraries. Sequencing  was performed on a NextSeq 2000 machine with a P3 kit . Read alignment and quantification was implemented using the nf-core/rnaseq pipeline V3.0.The funders and sponsor did not participate in the trial design, data accrual, analysis or manuscript preparation. The equipment provider  only participated in patient onboarding and in HIPAA-compliant data submission of deidentified primary data. Access to the raw data was available to the statisticians and study investigators. The corresponding author had full access to all data and the final responsibility for manuscript submission.Patients or the public were not directly involved in this study, including but not limited to the trial design, patient recruitment, conduct of the trial, analysis of data and manuscript preparation.Cumulative longitudinal symptom data are available on reasonable request. The gene list for assessing type-I interferon response is provided in Between January 2021 and April 2021, 56 patients were enrolled , and 55 A total of 1358 electronic symptom surveys were submitted by the 55 patients in the ITT group (n=1215 for the PP group). Symptom frequency (counting symptoms with a score of at least 1) at baseline was equally distributed between the study arms . Lack ofThe time to symptom resolution by study day 28 (primary endpoint) was not significantly different between patients in the famotidine and placebo arm in either the ITT  were −0.085 (95% CI: −0.099 to −0.071) in the famotidine group, and −0.061 (95% CI: −0.067 to −0.055) in the placebo group , and those on placebo for 2 out of 16 symptoms (12.5%) . The PP We next assessed the safety of famotidine. No severe adverse effects were observed. Five possibly medication-related adverse events were experienced during the 28-day study period, one each by five patients. Two patients, one from each arm, had mild elevations of serum markers of liver function. One patient in the famotidine arm had nausea and vomiting for 1 day, which settled after a 24-hour medication break and did not reoccur on restarting medication. In the placebo arm, one patient developed mild thrombocytopenia, and another patient had non-itching hives for 8 days. All of these events resolved spontaneously and were managed with interruption of study medication. We observed neutropenia (neutrophil count <1500/µL) in 11 patients. In the famotidine arm, all three patients with neutropenia on day 1 had normal neutrophil counts on day 7; in the placebo arm, 3 out of 8 patients with neutropenia on day 1 remained neutropenic at later timepoints.With regard to clinical outcomes and their surrogates, no deaths, no hospitalisations and, therefore, no admissions to intensive care units occurred in either study arm. One emergency department assessment for symptomatic hypoxia during the study occurred in the placebo arm. Most laboratory measurements were in the normal range. No changes were detected for CRP . HoweverTo examine the biological effect of famotidine in the experimental medicine group of the study, we identified patients for inclusion by determining plasma famotidine levels during the treatment period using mass spectrometry and RNA availability from PBMCs . Five paThis randomised, double-blind and placebo-controlled phase 2 clinical trial examined the effect of oral famotidine (80 mg three times a day) taken for 14 days by a diverse patient population with mild to moderate COVID-19. In concordance with pre-clinical mechanistic work,Our study has limitations. Although the chosen primary endpoint of symptom resolution was not met at statistical threshold, this analysis showed that twice as many patients stayed symptomatic on the placebo arm from day 14 onwards. In addition, most patients of our study, by physiological and standard laboratory measures, had mild disease only. Follow-up studies should perhaps focus on patients with moderate disease severity or patients with symptomatic deterioration. At the time of our trial, patients over the age of 65 or over 55 years with comorbidities received antibody therapy, which in turn led to exclusion from this study. Also, our patients had not received vaccinations to SARS-CoV-2 prior to or during the study and were not infected by the variants of SARS-CoV-2 that currently cause most global cases. Nevertheless, our results are likely to be less affected by the emergence of viral variants and more relevant to patients with breakthrough infections than findings from studies related to vaccinations or anti-viral therapiesThe power of a dataset in clinical trials, an essential consideration in the study design, is not simply based on the number of enrolled patients. Studies that use rare events in patients as endpoints, for example hospitalisation or death in patients with mild to moderately severe COVID-19, require large patient numbers to generate a small number of endpoint-related events.Our study has additional strengths. Famotidine is a safe, low-cost and widely available medication with excellent tolerability and minimal known drug–drug interactionsIn accordance with mechanistic laboratory studies,Important information in this study was gathered by determining plasma famotidine levels. Measured treatment concordance was 76% in keeping with other outpatient trials.The type-I interferon response has a dual role in COVID-19, and the chronicity of type-I interferon release needs to be carefully considered.Famotidine is likely to remain a drug used for COVID-19, either prescribed by physicians or self-administered by patients. Our finding may support this use of famotidine, given that we show that famotidine is well tolerated and that it accelerates the resolution of symptoms and inflammation without compromising immunity. We acknowledge that additional research studies and clinical trials remain a priority.


Farnoosh et al. aimed to investigate about “the different pathologic aspects of lung injury caused by mustard gas and also the relationship between this damage and the increased susceptibility of Iranian mustard gas exposed survivors to COVID-19.”The authors first discussed pulmonary complications of sulfur mustard (SM) in clinic and radiologic studies and then pathogenesis of mustard lung, pathogenesis of COVID-19, and the risk of COVID-19 for SM injured cases was discussed. Finally, in conclusion, they predicted that the respiratory and pulmonary clinical manifestations of COVID-19 will be more severe in SM-exposed patients; therefore, COVID-19 is more lethal in SM-exposed individuals.2 Evidence for both hypotheses is interesting and acceptable, with most cases presenting a suitable response to anti-inflammatory or anti-oxidative medications,3 but it seems that antioxidant imbalance may be the main basic pathogenesis that triggers the long-term chronic inflammatory process in the lung.4This review presented compact data from chemically injured lung secondary to SM, which previously presented as mustard lung, and it can be a research model for other industrial and inhalation chemical injuries. There are 2 main pathogenic hypotheses in survivors who inhaled SM: chronic inflammatory process with fluctuation in severity, and oxidative stress-antioxidant imbalance.5 Therefore, the conclusions reached by Farnoosh et al. can be closer to the fact. Here, CT scans of 4 SM pulmonary injury survivors who were affected by COVID-19 will be presented. A CT scan provides the most useful and reliable evidence for the diagnosis of pulmonary COVID-19. Bilateral ground glass opacities and patchy consolidation as the most frequent findings in pulmonary SARS-CoV-2 were observed in these 4 cases and pneumomediastinum was present in 1 case. Two of 4 presented cases were expired due to severity of the diseases  infections. Although there is no original research on COVID-19 in SM-injured cases, due to the similarity with chronic obstructive pulmonary disease (COPD) and bronchiolitis obliterans (BO), as the main possible lung pathology in SM injury, we can temporally rely on the COPD case findings. A recent meta-analysis declared that “underlying respiratory diseases, specifically COPD, and smoking were associated with severe COVID-19 outcomes.”This feature can accelerate the deterioration and severe phase of the illness and impair the oxygenation cycle. A cohort study on SM-lung-injured survivors, along with healthy controls, is needed to finally prove this vulnerability.


Understanding the impact of behaviors on COVID-19 severity can improve health promotion strategies. We investigated the association between health-related behaviors and odds of hospitalization for COVID-19 in a cohort of military personnel.This case-controlled study compared all active-duty US Air Force service members hospitalized for COVID-19 between March 5, 2020, and March 10, 2021 (cases), with their geographically matched peers who had COVID-19 and were treated as outpatients (controls). We used logistic regression to compare cases and controls according to self-reported sleep duration, physical activity, dietary factors, binge alcohol consumption, and tobacco use — with and without adjustment for sociodemographic factors, body mass index, physical fitness level, pertinent disease history, and psychological distress — resulting in crude and adjusted odds ratios (ORs) with 95% CIs. The trend between sugar-sweetened beverage (SSB) consumption and hospitalization odds was assessed by using the Cochran-Armitage test.P value for trend = .02).Ninety-three hospitalized cases were matched to 372 ambulatory controls. Adjusting for baseline characteristics and other health-related behaviors, cases were more likely than controls to report fewer than 7 hours of sleep, compared with 7 to 9 hours , and were more likely than controls to consume 3 or more SSBs per week, compared with fewer than 3 SSBs . In a dose–response relationship, higher SSB consumption was associated with greater odds of being hospitalized (Interventions that address short sleep duration and SSB consumption may reduce morbidity from COVID-19 among military service members and potentially in the broader US population. Certain lifestyle-mediated diseases and health-related behaviors are associated with significant morbidity from COVID-19.Among active-duty US Air Force service members, short sleep duration and sugar-sweetened beverage consumption were associated with higher odds of being hospitalized for COVID-19, even after controlling for sociodemographic factors, body mass index, physical fitness level, underlying disease history, and other health-related behaviors.To mitigate the health effects of COVID-19, individuals should be encouraged to obtain adequate sleep and minimize sugar-sweetened beverage consumption.Several chronic conditions have been implicated in the development of severe COVID-19 illness, including obesity , hyperteUnderstanding the relationship between health-related behaviors and COVID-19 severity is important for guiding population-wide preventive strategies. We designed this study to determine whether short or long sleep duration, insufficient aerobic or strength-training activity, unhealthy dietary habits, binge alcohol consumption, tobacco use, and secondhand smoke exposure were associated with COVID-19 hospitalization in a military population. We chose this target population based on the potential national security ramifications of rampant illness, and the study affords a valuable opportunity for investigation due to availability of exposure and outcome data.This is a case-control study of active-duty US Air Force service members diagnosed with COVID-19 with a reported onset date between March 5, 2020, and March 10, 2021. Service members who were hospitalized for COVID-19 during the surveillance period served as cases. We defined controls as active-duty service members with COVID-19 who did not require hospitalization.  We randomly selected 4 controls for each case, matching on Air Force installation to account for potential geographic heterogeneity in virus variants and hospital admission criteria. All Air Force installations worldwide were eligible. Hospitalized cases and ambulatory controls were retrieved from the Disease Reporting System internet, the passive surveillance system for reportable medical events used by the US Department of Defense. Hospitalization status is a required variable in the system and is defined as admission to an inpatient ward; emergency department visits are ineligible. We included both confirmed and probable cases, which were defined according to the Department of Defense COVID-19 reporting case definition, adapted from the national definition then in use. Confirmed cases required a positive molecular amplification test, such as polymerase chain reaction. Probable cases required clinical compatibility and an epidemiologic link, or a positive antigen test, or a death certificate listing COVID-19 as either the underlying cause of death or a significant condition contributing to death (2), overweight (25.0–29.9 kg/m2), and obese (≥30.0 kg/m2). The composite fitness score is calculated by summing the scores on each component: run time (up to 60 points), abdominal circumference (20 points), push-ups (10 points), and sit-ups (10 points). Per Air Force policy  as weight divided by height squared and stratified as normal . Active-duty service members complete the online questionnaire annually, and answers are reviewed and signed by an authorized military health care provider . We defiWe also obtained self-reported health-related behaviors from the PHAQ. These questions are modeled after the Behavioral Risk Factor Surveillance System . We defiWhenever possible, we used the fitness assessment and PHAQ immediately antecedent to the COVID-19 onset date, but not earlier than January 2018. For service members exempted from the antecedent fitness assessment, we used the last available assessment. In the absence of any prevenient values, we used the first values after the COVID-19 onset date. We considered variables missing if no records existed between January 1, 2018, and February 28, 2021. By using the Wilcoxon rank sum test, we compared cases and controls in terms of the time duration between their fitness assessment and PHAQ dates and their COVID-19 onset dates.2 test or Fisher exact test .We categorized race or ethnicity as Hispanic, non-Hispanic Black, non-Hispanic White, non-Hispanic Asian, and other/unknown. This variable was included given known differences in COVID-19 hospitalization rates by race and ethnicity in the US  and the 2 test or Fisher exact test . We calculated adjusted ORs with 95% CIs using multivariable logistic regression. Partially adjusted ORs incorporated age, sex, self-reported race or ethnicity, military rank, BMI, physical fitness level, disease history, and psychological distress, while fully adjusted ORs incorporated these baseline characteristics and all other health-related behaviors. This reflected a priori model considerations rather than a stepwise variable selection strategy.For each health-related behavior , we calculated descriptive statistics and a crude odds ratio (OR) with 95% CIs using the Wald χWe assessed the trend between dietary factors  and hospitalization odds by using the exact Cochran-Armitage test. We used the 6 categories of the PHAQ: rarely, 1–2 servings/week, 3–6 servings/week, 1 serving/d, 2–3 servings/d, and ≥4 servings/d.To assess the impact of missing race or ethnicity data and the use of the probable case definition, we conducted sensitivity analyses in which we calculated the fully adjusted ORs after excluding each set of individuals.We used SAS version 9.4  to match controls with cases and to conduct all analyses. This project was reviewed by the Air Force Research Laboratory Institutional Review Board and determined to be a public health, nonresearch activity.A total of 93 active-duty service members from 46 Air Force installations were hospitalized with COVID-19 during the surveillance period . These cases were matched to 372 ambulatory controls, service members from the same installations who were diagnosed with COVID-19 but did not require hospitalization .P = .03). Two fitness assessments (1 each for cases and controls) occurred after the onset date. Among cases and controls, PHAQs occurred by a respective median of 231 days and 203 days before the COVID-19 onset date (P = .32). Eleven PHAQs (1 case and 10 controls) occurred after the onset date.Some service members had missing data for race or ethnicity (n = 129), BMI (n = 23), and physical fitness level (n = 15). (The discrepancy between BMI and fitness level was due to 8 members having the latter without an associated weight and height.) Among cases and controls, fitness assessments occurred by a respective median of 337 days and 377 days before the COVID-19 onset date (P = .92) and similar median BMI of 25.9 and 26.3 (P = .92).Cases and controls were similar by age category, sex, race or ethnicity, military rank, BMI category, and disease history. Cases were more likely than controls to have a satisfactory physical fitness level, compared with an excellent level , and they were less likely to report a major life stressor within the prior month that caused significant concern or disturbed life, work, or relationships  . Cases aSelf-reported health-related behaviors were similar between cases and controls with 2 notable exceptions. After adjusting for baseline characteristics and all other health-related behaviors, cases were more likely to report <7 hours of sleep per night, compared with 7–9 hours . They were also more likely to consume 3 or more SSBs per week, compared with fewer than 3 SSBs  .P value for trend = .02) (P = .38) or fruits (P = .31). Compared with individuals who reported sleeping 7–9 hours nightly and consuming fewer than 3 SSBs weekly, those who reported sleeping less than 7 hours nightly and consuming 3 or more SSBs weekly had even greater odds of hospitalization .SSB consumption followed a dose–response relationship, with greater consumption associated with greater odds of being hospitalized (d = .02) . Dose–reThe exclusion of individuals with missing race or ethnicity data or who were classified as probable cases did not substantially alter the findings beyond decreasing the precision of the effect estimates .We analyzed the association of health-related behaviors and COVID-19 severity, assessed by hospitalization, in a population of US Air Force service members. By matching on military installation and controlling for sociodemographic factors, BMI, physical fitness level, underlying disease history, and psychological distress, this study isolates the impact of health-related behaviors. Regardless of these baseline attributes, the odds of service members reporting less sleep and greater SSB consumption were higher in hospitalized cases when compared with nonhospitalized controls.Obtaining 7 to 9 hours of sleep nightly is foundational for human health . Sleep rSSB consumption was associated with 74% increased odds of hospitalization when dichotomized at 3 or more servings per week versus fewer than 3 servings per week, and a dose–response relationship was evident when assessed as a trend of ordinal categories. These findings are striking given the low prevalence of SSB intake among cases and controls. Compared with the 63% of US adults who report consuming SSBs at least daily , only 18The reason why service members who reported a significant life stressor had lower crude odds of hospitalization for COVID-19 is unclear. We anticipated the opposite finding because acute stress has been linked to impaired immune function and higher risk of respiratory infections . A potenThe null findings in this study are also interesting. Contrary to our expectations, inadequate consumption of vegetables and fruits, physical inactivity, and tobacco use were not associated with hospitalization in crude and adjusted models, and trends for vegetable and fruit consumption were not significant. These findings may be due to the small sample size of hospitalized cases in this study. The null finding for physical inactivity may reflect its low prevalence in this cohort of active-duty service members. Whereas more than 75% of American adults fail to achieve the minimum recommended level of aerobic and strength-training activity , fewer tIn addition to the small sample size, this study has 6 important limitations. First, data were self-reported and some questions considered a condensed timeframe. Questions related to major life stressors and sleep duration, for example, were restricted to 30- and 14-day windows before the PHAQ, respectively. Self-reported surveys also raise the issues of recall and reporting biases. Exposure misclassification may be present due to perceived stigma and military career consequences, especially concerning alcohol and tobacco use, as these questionnaires are reviewed by military medical personnel. We suspect any misclassification would be nondifferential by outcome, thus biasing results toward the null. Second, missing baseline data were present for some variables, most notably for race or ethnicity, which has been associated with COVID-19 severity ,18; howeFinally, hospitalization is an imperfect surrogate for severity. Nondisease considerations such as improvised isolation and psychosocial issues can affect decisions to hospitalize a patient. However, we believe these factors would be rare for active-duty military service members. Hospitalization criteria should be similar across Air Force military treatment facilities, and potential heterogeneity was accounted for by geographical matching of cases and controls. Furthermore, the use of hospitalization as an indicator of COVID-19 severity is widespread in studies of civilian populations ,6–8.Because this study was conducted in a military context, external validity to civilian populations may be questioned. For example, the findings may not apply to children and older adults, or to young and middle-aged adults who are ineligible for military service due to medical or educational reasons. Conversely, the findings may not apply to all members of the US Armed Forces given heterogeneity in physical fitness and health requirements among the military services and among units within each service . The inclusion of cases detected outside the US may be considered another threat to external validity, but given the rapid international spread of variant strains, this may be a strength of the study, especially since controls were matched on military installation.With SARS-CoV-2 variants spreading, actionable public health messages are needed to empower individuals and communities. Adequate sleep and reduced consumption of SSBs, like vaccination and respiratory etiquette, are controllable behaviors that could mitigate hospitalization associated with COVID-19. Although causality cannot be presumed and generalizability beyond the military should proceed cautiously, these behaviors appeared to be protective irrespective of BMI, baseline physical fitness, and disease history. Prioritizing sleep and limiting SSB consumption are evidence-based messages that may reduce susceptibility to severe COVID-19 illness among active-duty military personnel.


In risk-stratified cancer screening, multiple risk factors are incorporated into the risk assessment. An individual’s estimated absolute cancer risk is linked to risk categories with tailored screening recommendations for each risk category. Absolute risk, expressed as either remaining lifetime risk or shorter-term (five- or ten-year) risk, is estimated from the age at assessment. These risk estimates vary by age; however, some clinical guidelines  and ongoing personalised breast screening trials, stratify women based on absolute risk thresholds that do not vary by age. We examine an alternative approach in which the risk thresholds used for risk stratification vary by age and consider the implications of using age-independent risk thresholds on risk stratification. We demonstrate that using an age-independent remaining lifetime risk threshold approach could identify high-risk younger women but would miss high-risk older women, whereas an age-independent 5-year or 10-year absolute risk threshold could miss high-risk younger women and classify lower-risk older women as high risk. With risk misclassification, women with an equivalent risk level would be offered a different screening plan. To mitigate these problems, age-dependent absolute risk thresholds should be used to inform risk stratification. Risk-stratified screening approaches have emerged as promising strategies to improve the efficiency of cancer screening programmes and to reduce their adverse consequences ,2,3. A rSeveral breast cancer risk assessment models are available. These models estimate a woman’s absolute risk of developing breast cancer, either over a fixed time horizon  or over her remaining lifetime  ,7,8,9.Clinical guidelines in the US ,11 and iFive-year absolute risk thresholds are also used by clinical guidelines  and by oIn each of these examples, the risk thresholds do not take into account a woman’s age at the time of risk assessment. However, since the absolute risk of breast cancer varies substantially by age, this will have a substantial influence on the proportion of women falling into each risk category. Here, we examine an alternative approach in which risk thresholds vary by age, consider the implications of using age-independent risk thresholds on risk stratification, and present age-dependent absolute risk thresholds for tailoring screening recommendations in Canada.relative risk of breast cancer, relative to the population average. We calculated age-dependent risk thresholds (thresholdrisk (t) at age t) that are equivalent to those for a 30-year-old woman having a specified absolute risk, with ‘equivalence’ defined as having the same relative risk (RR) ).We defined age-dependent risk thresholds based on poprisk), 5- and 10-year absolute risk and remaining lifetime risk to age 80 for women in Canada of varying ages 30 to 79 years  and lifestyle, hormonal, and reproductive risk factors is estimated to have an Area Under the Curve (AUC) of 0.65 . Assumin of 0.30 . We set to unity . Given tTen-year absolute risk thresholds of 1.1% and 3.7% at ages 30 and 40 would be equivalent to remaining lifetime risk of 25% at ages 30 and 40, respectively. An age-independent 10-year absolute risk threshold  could miss high-risk younger women and classify lower-risk older women as at high risk b. SimilaUsing an age-independent remaining lifetime risk threshold approach could identify high-risk younger women but would miss high-risk older women, whereas an age-independent 10-year or 5-year absolute risk threshold could miss high-risk younger women and classify lower-risk older women as high risk. Using an age-dependent absolute risk threshold for risk stratification, women with the same absolute risk level but at different ages could receive different screening recommendations. For example, a 40-year-old woman with a 10-year absolute risk of breast cancer of 3.7% would be recommended enhanced screening, whereas a 60-year-old woman with the same risk level would not.With risk misclassification, women with equivalent risk levels would be offered a different screening plan. An average-risk woman, when classified as high risk, would unnecessarily receive enhanced screening, annual mammograms, and adjunctive breast MRI. This potentially could increase the likelihood of experiencing adverse consequences of screening, false screening findings and overdiagnosis, as well as of inefficient use of healthcare resources, whereas a high-risk woman missing being classified as high risk would miss the benefits of enhanced screening.If risk is assessed from current age, and the age at assessment varies between individuals, then risk stratification with risk thresholds that are age-dependent should be used to mitigate the problems of misclassification and inequity in provision of the screening services. An alternative is to compute risk from a fixed index age or have a one-off risk assessment at a particular age only in order to use age-independent absolute risk thresholds. For example, the UK National Institute for Health and Clinical Care Excellence (NICE) guidelines define breast cancer risk categories based on remaining lifetime risk from a fixed index age of 20 to 80 years .In the Personalized Risk Assessment for the Prevention and Early Detection of Breast Cancer: Integration and Implementation (PERSPECTIVE I&I) study currently underway in Canada, women aged 40 to 69 years are invited for risk assessment, and a screening plan is proposed based on the women’s risk category, age at risk assessment, and provincial breast screening guidelines . PERSPECIn this analysis we have calculated age-dependent absolute risk thresholds equivalent to the thresholds recommended by the clinical guidelines. The relative risk threshold approach is a pragmatic approach to set age-dependent absolute risk thresholds. However, evidence from empirical data is still needed to determine the absolute risk thresholds to be used in risk-stratified breast screening programmes. In addition, evidence is needed on whether an age-dependent, compared to age-independent, absolute risk threshold would improve screening outcomes, such as reducing breast cancer death and overdiagnosis, and improve the cost-effectiveness of the screening programme. Ongoing studies, such as the PERSPECTIVE I&I, will offer evidence on the optimal risk thresholds to improve the benefit to harm ratio and the cost-effectiveness of risk-stratified breast cancer screening programmes, the clinical utility, and feasibility of implementing age-dependent absolute risk thresholds for risk-stratification in breast cancer screening.


Enterobacterales (MDRE) are an emerging threat to global health, leading to rising health care costs, morbidity and mortality. Multidrug-resistance is commonly caused by different β-lactamases , sometimes in combination with other resistance mechanisms . The continuous spread of MDRE among patients in hospital settings and the healthy population require adjustments in healthcare management and routine diagnostics. Rapid and reliable detection of MDRE infections as well as gastrointestinal colonization is key to guide therapy and infection control measures. However, proper implementation of these strategies requires diagnostic methods with short time-to-result, high sensitivity and specificity. Therefore, research on new techniques and improvement of already established protocols is inevitable. In this review, current methods for detection of MDRE are summarized with focus on culture based and molecular techniques, which are useful for the clinical microbiology laboratory.Multidrug-resistant  Enterobacterales (MDRE) are a major cause of hospital-acquired infections, which are associated with high morbidity and mortality as well as rising healthcare costs [Enterobacterales as well as MDRE are part of the normal intestinal microbiota of healthy individuals [Enterobacterales, which account for only 0.01% of 1013–1014 organisms, are those mainly associated with antibiotic resistance [The dissemination of multidrug-resistant (MDR) bacteria is one of the biggest threats to global health ,2. In pare costs ,4,5. On ividuals . The majsistance . Enterobacterales. Great variations are observed in different regions, with lower rates in some European countries like Hungary (2.6%) and Sweden (7%) and higher rates in countries like Nepal (9.8%), Mozambique (20%) and Taiwan (41.4%) [Enterobacterales (ESBLE) and 6.9% for carbapenemase—producing Enterobacterales (CPE). In North America, Europe and Oceania, prevalence is 9%, 12.9% and 6% for ESBLE and 5%, 0.2% and 0.4% for CPE, respectively [Colonization-rates with MDRE in healthy adults are mostly studied for extended spectrum β-lactamase (ESBL) and AmpC producing  Nepal 9.%, Mozamb (41.4%) ,11,12. T (41.4%) . Risk fa (41.4%) . Prevaleectively . The preectively . In Germectively , whereasectively ,19. Enterobacterales as the highest priority for the development of new antibiotics [E. coli clinical isolates resistant to 3rd generation cephalosporins has been increasing in European countries since 2015 and ranged from 6.2% in Norway to 38.6% in Bulgaria in 2019. Even higher rates have been observed in Klebsiella pneumoniae (4.3–75.7%). In parallel, the proportion of E. coli and K. pneumoniae with resistance to carbapenems increased sharply by 2019 . The prevalence of E. coli and K. pneumoniae isolates resistant to carbapenems or 3rd generation cephalosporins varies widely among EU/EEA countries, with the overall highest resistance rates being reported from Italy, Bulgaria, Greece and Romania [Among MDR pathogens, WHO has declared carbapenem- and 3rd generation cephalosporin-resistant ibiotics . Surveil Romania .Resistance to 3rd generation cephalosporins and carbapenems is mostly caused by different β-lactamases, with >7000 different β-lactamases known, and more being published almost daily, illustrating the high mutation and adaptation frequency in different bacteria . β-lactaEnterobacterales in each class are shown in Extended-spectrum β-lactamases (ESBLs) have been increasingly detected in hospitalized patients and the community since the 1980s and are of great clinical importance. These enzymes are capable of hydrolyzing penicillins, monobactams and 3rd generation cephalosporins. ESBLs belong to Ambler classes A and D in most cases, with those in Ambler class A being mostly sensitive to inhibitors such as clavulanic acid . RelevanKlebsiella pneumoniae carbapenemase (KPC-2) [Infections with ESBL-harboring bacteria have been successfully treated with carbapenems, but the massive use of this antibiotic class accelerated the dissemination of the second important group of β-lactamases, the carbapenemases. These enzymes belong to Ambler classes A, B and D and differ in the resistance pattern they induce . Class A (KPC-2) ,28, non- (KPC-2) , imipene (KPC-2)  and othe (KPC-2) . Well-kn (KPC-2) , VIM [32 (KPC-2) . Oxacill (KPC-2) . Enterobacterales seems inevitable. High consumption of antibiotics in health care and livestock as well as increased mobility represent only two accelerators in the process, which is accompanied by an ongoing evolution of resistance plasmids and mobile genetic elements [With the increasing use of antibiotics, the further emergence of MDR elements ,36. Neveelements . In addiEnterobacterales (CPE) [Every clinical laboratory must be able to reliably detect MDR bacteria and provide regular reports on the epidemiology of MDRE in order to guide infection control measures. This baseline epidemiological monitoring uses data from clinical samples in most institutions. For high-risk patients, in outbreak situations and in the epidemic setting, the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) as well as the Healthcare Infection Control Practices Advisory Committee and Center for Diseases Control and Prevention (HICPAC/CDC) guidelines recommend active screening cultures (ASC) for the detection of colonization with MDRE, especially for carbapenemase-producing es (CPE) ,39.An often-overlooked issue which impacts the sensitivity of microbiological cultures and in particular ASC are preanalytical factors including appropriate specimens, the collection site as well as the collection device. Stool is considered the gold standard specimen for studying gastrointestinal microbiota and detection of MDRE colonization . BecauseEnterobacterales. Frequently, agars are supplemented with chromogens which allow presumptive species identification using species-specific enzymes, namely, β-galactosidase, β-glucuronidase and deaminase [Selective media are suitable for screening of patient samples for ESBL- and carbapenemase-producing eaminase . TM ESBL , Chromatic ESBL agar , Brilliance ESBL agar , BLSE agar , and others , chromID ESBL agar , ESBL chromogenic agar , chromogenic ESBL agar , ESBL ChromoSelect Agar , CHROMagar d others . Most chEnterobacterales from clinical samples . After 24 h, incubation sensitivity was 88% and 85% for chromID and BLSE agar, respectively. Further 24 h incubation increased sensitivity of chromID to 94% but did not affect performance of BLSE agar. The reason for these differences was likely the choice of cefpodoxime in chromID ESBL but cefotaxime or ceftazidime in BLSE agar. False-positive results were obtained for isolates overproducing a chromosomal cephalosporinase or penicillinase [Many of these products have been evaluated in different studies. ChromID ESBL agar  and BLSE agar medium  were challenged with various illinase . Sensitiillinase . Compariillinase . Enterobacterales with low cephalosporin MICs. High specificity and sensitivity for CPE detection was recorded for Brilliance CRE and the in-house agars, whereas chromID CARBA failed to detect 15% of all CPE and 8/20 OXA-48-producing isolates. Because detection of OXA-48-producing Enterobacterales is difficult, the use of the specific chromID OXA-48 agar is beneficial according to the authors, especially in outbreaks of OXA-48 CPE [In additional to ESBL agars, chromogenic agars for the detection of CPE have become commercially available in the last decade. In a recent study, seven commercially available screening media and in-house agars were investigated . The autA-48 CPE . The resEnterobacterales by 21% to 32% [Pre-enrichment of screening cultures has been proposed as an additional means to increase the sensitivity of MDRE detection. An unselective or semi-selective broth  is inoculated with the screening sample and incubated overnight before an aliquot is spread on a selective agar. In several studies, pre-enrichment enhanced the detection rate of ESBL or 3rd generation cephalosporin resistant % to 32% ,102,103.Obviously, the performance of the different chromogenic screening media may vary depending on the type of β-lactamase and the analyzed organism. Therefore, the proper choice of chromogenic agar must be adapted to the specific objectives and the epidemiological landscape of each medical center. While the ease of use and experience in the microbiology laboratories are good arguments in favor of chromogenic agar, this technique has some drawbacks. These include a relatively high frequency of unspecific growth on the plates resulting in considerable subsequent work-up, problems in the detection of some β-lactamases  and lack of information on the type of β-lactamase present. Therefore, additional work-up is required to assess susceptibility and identify the ESBL or carbapenemase variant.Susceptibility can be assessed by various assays, e.g., disc diffusion according to EUCAST or CLSI, semi-automated susceptibility with commercial assays , Phoenix  or WalkAway ) or broth microdilution.The sensitivity and specificity of commercially available semi-automatic systems varies widely for detection of ESBLs and carbapenemases ,108,109.If the results of susceptibility testing show an ESBL- or CPE-phenotype, confirmation tests are necessary to rule out resistance by other mechanisms . A great variety of confirmation tests are available, differing in performance, costs and TAT .In disc diffusion assays, an antibiotics disc is placed on an agar plate inoculated with the test strains. If the test strain is susceptible to the antibiotic, a growth inhibition zone becomes visible after incubation A. For coSimilarly, inhibitor-based tests can be used for carbapenemase identification. Carbapenemase inhibitors include boronic acid derivates (KPC and Ambler class A carbapenemase and AmpC β-lactamase inhibition), cloxacillin (AmpC inhibition) and zinc chelators such as EDTA and dipicolinic acid (MBL inhibition) ,114,115.Enterobacterales often have a double inhibition zone, which is highly specific. The combination of temocillin and faropenem discs increases specificity for OXA-48-like carbapenemase-producing Enterobacterales [The faropenem disc test is a highly sensitive screening method for CPE ,118. Whicterales .Enterobacterales. Examples of ESBL tests are Liofilchem ESBL disc kits  and Mastdiscs ESBL detection set , while tests for carbapenemase detection are more numerous and include Mastdiscs Carbapenemase Detection Set, Combi Carba Plus (both MAST Group), KPC/MBL & OXA-48 Confirm Kits  and KPC&MBL&OXA-48 disc kit  [Both CDT and DDST assays are commercially available for screening ESBL- and carbapenemase-producing , Italy) ,50,51. TE. coli ATCC 25922) is inoculated onto an agar plate, and a carbapenem disc is placed in the center. The test strains as well as control strains are streaked in lines from the center to the periphery of the plate. If the test strain produces a carbapenemase, the carbapenem is inactivated and the susceptible E. coli strain grows alongside the test strain, which can be observed by cloverleaf-like indentations (The modified Hodge test (MHT) is an additional test for the detection of carbapenemases and based on the inactivation of a carbapenem. A susceptible strain , the test strain is suspended in tryptic soy broth instead of water, and incubation time of the antibiotic disc in the bacterial suspension was increased from 2 h to 4 h, which improves sensitivity. If the test strains are additionally incubated with the antibiotic disc and either EDTA (eCIM) or EDTA and phenylboronic acid (CIMPlus), the putative Ambler class of the isolate can be identified ,124. TheAll CIM assays perform well with high specificity and sensitivity , but false-positive results can occur in strains with reduced porin expression and overexpression of AmpC β-lactamase or ESBL. In addition, detection of strains with low carbapenemase-activity remains challenging.While CIM assays still have a time-to-result of 5 to 20 h, colorimetric assays have been developed for rapid detection of ESBL and carbapenemases. The ESBL-NDP test is employed for detection of ESBL activity. Lysed bacteria are incubated in a medium containing red phenol as pH indicator and supplemented with either cefotaxime, cefotaxime and tazobactam or without antibiotics. Hydrolysis of cefotaxime by ESBL leads to the formation of carboxylic acid which induces a color change from red to yellow. In the case of ESBLs, this reaction can be inhibited by tazobactam . The tesEnterobacterales and Pseudomonas spp. with low hydrolytic activity, such as OXA-48 [Comparable to the detection of ESBL enzymes by pH decrease, the Carba NP test has been developed for detection of carbapenemases. The hydrolysis of imipenem by a carbapenemase leads to an acidification of a phenol red solution, resulting in color change from red to orange or yellow . The fols OXA-48 . In a vas OXA-48 . Improves OXA-48 .Due to the increasing number of CPE, a variety of detection kits are commercially available, which include the β-CARBA assay , NeoRapid CARB  and CARBA PAcE  . These tColorimetric tests provide a rapid indication if the test strain is an ESBL- or carbapenemase producer and tests with inhibitors can identify which Ambler class the enzymes belong to. However, precise identification of the type of ESBL or carbapenemase cannot be achieved using cultural methods and requires molecular techniques.Molecular methods have the highest sensitivity and specificity for the detection of resistance genes and can be applied to cultured isolates or directly to clinical specimens. They can provide accurate information on the type of ESBL enzyme or carbapenemase and have a relatively short time-to-result. Although molecular methods have the disadvantage of requiring qualified personnel and incur high costs for technical equipment and consumables, the number of laboratories using these techniques is growing. This is due in part to the continuously increasing number of different methods and commercially available assays . In partFor simultaneous detection of multiple target genes, multiplex PCR and microarrays are the methods of choice. These assays provide a more comprehensive picture of the resistance landscape of an isolate , since mn = 21). Specificity was slightly higher at 97.6% and modified cut-off values even increased specificity to 98.8% [Several in-house multiplex PCR assays have been published ,134,135.to 98.8% . Enterobacterales is even higher .E. coli isolates [Klebsiella spp. and Pseudomonas aeruginosa isolates [Enterobacterales and Pseudomonas isolates [LAMP can circumvent the disadvantages of PCR assays, namely the high costs and negative influence of compounds present in clinical samples which can inhibit DNA polymerase activity. Instead of repetitive temperature cycles like in PCR, only isothermal conditions are required. Good results were obtained with the eazyplex SuperBug CRE system , which can detect ESBLs of the CTX-M-1 and CTX-M-9 group, and the carbapenemases KPC, VIM, NDM, OXA-48 and OXA-181. The assay showed 100% concordant results compared to PCR based assays when using cultured isolates . Comparaisolates  and on cisolates ,144. Theisolates . A drawbMolecular methods are advantageous due to their high sensitivity and specificity, but not all diagnostic laboratories are equipped with the appropriate devices and trained personnel. In addition, molecular methods are expensive compared to phenotypic assays. Further developments in the field of molecular diagnostics could circumvent these drawbacks and lead to a more widespread implementation of these techniques in routine laboratories.Most ICTs in the microbiological laboratory are lateral flow double-antibody sandwich assays. After application of the analyte to the sample pad, the antigens  migrate along the nitrocellulose membrane by capillary flow. They form a complex with dye-labeled antibodies  on the conjugate pad and are captured by immobilized antibodies on a nitrocellulose membrane at the test line resulting in a visible band. The control line consists of immobilized anti-IgG antibodies which capture the excess dye-labeled antibodies, thereby serving as a control for the capillary flow . The maiEnterobacterales from bacterial colonies and positive blood cultures [The NG-Test CTX-MULTI  was developed to detect group CTX-M-1, -2, -8, -9, -25 producing cultures . The autProteus spp. and from positive blood cultures [For carbapenemases, different tests are commercially available and have been extensively studied. The NG-Test CARBA-5  detects the most prevalent carbapenemases KPC, OXA-48-like, VIM, IMP, and NDM from bacterial colonies and directly from positive blood cultures with high sensitivity and specificity ,  55,69,7,72. The cultures ,147,148.cultures ,149.Despite the numerous advantages of ICTs, they share the same problems with molecular methods, e.g., only the carbapenemases included in the test design can be detected, and rare carbapenemases are usually missed. To overcome this restriction, Baeza and colleagues proposed an algorithm applying immunochromatographic assays in combination with a subsequent zCIM test for the detection of common and rare carbapenemases . ReachinEnterobacterales from positive blood cultures combining the NG-Test CTX-M MULTI and the NG-Test Carba 5 assay after identification of Enterobacterales using the MBT Sepsityper IVD KIT . Analyzing a total of 236 episodes of Enterobacterales blood stream infections, a good agreement with conventional phenotypic results was recorded. Time-to-result (defined starting point was the processing of positive BC) for this fast-track workflow was 42 min compared to 38 h for the conventional workflow (identification and AST from overnight cultures) [Boattini and colleagues proposed a fast-track workflow for ultures) . In pracAnother way to detect resistance to carbapenems or 3rd generation cephalosporins with little equipment is by employing electrochemical assays. They use disposable sensors with screen printed carbon electrodes in combination with small electronical measuring devices.Enterobacterales and showed a good sensitivity and specificity for the confirmation of carbapenemases from bacterial colonies  [Bogaerts and colleagues developed the BYG Carba test for the rapid confirmation of carbapenemase activity. The test measures variations of conductivity on an electro-sensing polymer coated electrode conferred by imipenem hydrolysis. These variations are analyzed in real-time by a portable reader . The tesctively) .Rochelet and colleagues described a voltammetric assay for the detection of ESBLs. Bacterial isolates are incubated together with nitrocefin, and subsequently, the hydrolysis of nitrocefin into an electroactive product is detected by a voltammetric measurement on a disposable carbon screen-printed sensor .Enterobacterales spiked into blood cultures and reported a good detection of 3rd generation cephalosporin resistance conferred by Ambler class A β-lactamases (sensitivity 83.3% and specificity 99.1%), but the test failed to detect 3rd generation cephalosporin resistance conferred by Ambler classes B, C and D β-lactamases [The successor of this principle is the commercially available BL-RED test  which is designed for the detection 3rd generation cephalosporin hydrolysis from bacterial culture or directly from positive blood cultures. Durand and colleagues challenged the test with a set of 150 ctamases .Enterobacterales, there are three promising approaches for MALDI-TOF MS application available: The detection of β-lactamase activity, estimating the effect of antibiotics on the growth of bacteria, and the direct detection of biomarkers  associated with AMR , and this application will likely become an essential part of the routine laboratory in the near future ,154. Regwith AMR .The detection of β-lactamase activity using MALDI-TOF is already available for IVD use in Europe. The principle is to incubate the bacterial isolate in question with an antibiotic. Hydrolysis of the β-lactam antibiotic results in a mass shift of the corresponding antibiotic within the spectrum obtained by MALDI-TOF ,156. TheK. pneumoniae after 4 h incubation from culture plates [Enterobacterales [Enterobacterales as it uses an easy-to-follow protocol, an instrument that is available in many microbiological laboratories and has an acceptable TAT. Moreover, it is independent of the resistance mechanisms and can be applied to any combination of antibiotic and bacterial species.Another approach for MALDI-TOF MS is to use it as a sensitive instrument for growth detection in presence of defined antibiotic concentrations. For this purpose, Idelevich and colleagues developed the direct-on-target microdroplet growth assay (DOT-MGA) . In thise plates  as well e plates . The teccterales . While tcfiABacteroides fragilispos. [Klebsiella pneumoniae from cultured colonies as well as directly from positive blood cultures during the routine identification process  [Enterobacterales are not yet validated for routine laboratory use but are promising approaches for the near future. For now, biomarkers allow the detection of a few selected resistance determinants, and therefore, confirmation of negative results by phenotypic AST methods is still needed. With better and broader spectra libraries, further resistance mechanisms can likely be detected in the future. Accelerating bioinformatic tools like machine learning will play a crucial role in the construction of those libraries [Another technique is the direct detection of biomarkers of antimicrobial resistance in mass spectra. Using this approach, the same mass spectra are used for species identification and resistance detection. This could significantly shorten laboratory workflows and is already established for some resistance markers, e.g., for some MRSA strains and for carbapenem resistance in fiApos. ,159. ThefiApos.  and KPC-fiApos. ,162 was ty 100%) . The proibraries .Patients colonized or infected by MDRE can today be more rapidly and reliably identified using assays with shorter turn-around-time and improved sensitivity.Culture based approaches rely on the cultivation on selective or unselective agars, subsequent susceptibility testing and identification of resistance mechanisms. This workflow will likely remain the standard in the medical microbiology laboratory. This approach permits the subsequent characterization of isolates, e.g., by phenotypic methods or molecular methods including next generation sequencing (NGS). For patient screening using the classical culture approach, we recommend using an ESBL in combination with a CPE agar, as not all CPE can be detected by ESBL screening agars. The use of an enrichment broth is optional but will further improve the detection of MDRE.The traditional culture-based workflow requires about 2.5 days while using molecular techniques directly from screening specimens only requires a few hours. At the time of writing, PCR techniques are still costly when applied routinely for screening purposes from sample material . Since mMany tests presented in this review have primarily been designed for the confirmation of ESBL and carbapenemase production but have been evaluated for the use directly on clinical specimens, especially blood cultures. The more widespread use of these techniques will accelerate clinical decision making and ultimately improve patient outcomes. Different rapid diagnostic interventions  have shown to decrease the time for adequate therapy. For other parameters such as length of stay, overall mortality and cost effectiveness, the picture is less clear. Most studies combine the implementation of rapid diagnostics with antimicrobial stewardship programs making it difficult to assess the genuine impact of new technologies alone .Next generation sequencing (NGS) is increasingly used in microbiological laboratories, mainly in reference institutions and research. The DNA sequence of a bacterial genome can be obtained in a single sequencing run and is very successfully used in the determination of antibiotic resistance genes (ARG) and typing of pathogens in hospital outbreaks. NGS can be employed for in-depth analysis of single isolates but also allows culture-free identification of bacteria and ARG directly from complex samples like stool . New innAll these applications make NGS a valuable tool for identification, surveillance and most likely future clinical decision making regarding MDRE. With declining costs for the initial investment in an NGS platform, decreased costs per run and the development of accessible bioinformatics software, this technique will inevitably be incorporated into routine microbiological practice. However, standardization of protocols, quality control issues and bioinformatic capacities are serious obstacles that need to be addressed before a more widespread implementation in clinical microbiology laboratories seems realistic .Enterobacterales is a rapidly evolving field with a diverse armamentarium of in-house and commercially available tests as well as fascinating future perspectives. Overall rapid and reliable detection of MDRE in clinical specimens is a key capacity for every clinical microbiological laboratory. Multidrug resistance in


Rising demands for repetitive SARS-CoV-2 screens and mass testing necessitate additional test strategies. Saliva may serve as an alternative to nasopharyngeal swab (NPS) as its collection is simple, non-invasive and amenable for mass- and home testing, but its rigorous validation, particularly in children, is missing. We conducted a large-scale head-to-head comparison of SARS-CoV-2 detection by RT-PCR in saliva and NPS of 1270 adults and children reporting to outpatient test centers and an emergency unit. In total, 273 individuals were tested positive for SARS-CoV-2 in either NPS or saliva. SARS-CoV-2 RT-PCR results in the two specimens showed a high agreement . Despite lower viral loads in the saliva of both adults and children, detection of SARS-CoV-2 in saliva fared well compared to NPS (positive percent agreement = 92.5%). Importantly, in children, SARS-CoV-2 infections were more often detected in saliva than NPS , underlining that NPS sampling in children can be challenging. The comprehensive parallel analysis reported here establishes saliva as a generally reliable specimen for the detection of SARS-CoV-2, with particular advantages for testing children, that is readily applicable to increase and facilitate repetitive and mass testing in adults and children. The current gold standard for the diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection relies on the detection by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swabs. A range of RT-qPCRs methods have been developed and proven highly sensitive, accurate and reliable ,2. NasopSeveral recent studies have evaluated saliva as an alternate specimen ,27,28,29Adults (N = 1100) and children (N = 170) opting for a SARS-CoV-2 test at one of five participating test centers were included. Our study included five different test sites to ensure that data are not skewed due to specific procedures at one site. Four centers were dedicated outpatient test centers  and one was an emergency department. The study population comprised individuals with SARS-CoV-2-related symptoms based on Swiss testing criteria and asymptomatic individuals with relevant exposure to a SARS-CoV-2 index case. Hospitalized patients were not included. Individuals were included without further selection to avoid skewing. Information on symptomatic or asymptomatic status was acquired as part of the regular procedure for SARS-CoV-2 testing and reporting based on self-evaluation (asymptomatic/mild/strong) by the participants, as they did not see a physician in the test center setting.The Zurich Cantonal Ethics Commission waived the necessity for a formal ethical evaluation based on the Swiss Federal Human Research Act, as the collection of saliva in parallel to a scheduled nasopharyngeal swab induces no risk (Req-2020-00398). No additional personal data beyond the usual information on symptoms and duration required by the Swiss Federal Office of Public Health (FOPH) for all SARS-CoV-2 tests in Switzerland were retrieved. Due to the ethics waiver, no informed consent had to be requested.Test centers were advised to use their regular swab and virus transport medium (VTM)/universal transport medium (UTM) for nasopharyngeal sampling. Transport media used by the centers included Cobas PCR Medium , Liquid Amies preservation medium , Virus Preservative Medium  and in-house VTM (HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), antibiotics and antimycotics).Collection kits for saliva were supplied to the test centers: one tube for saliva collection (Sarsted 62.555.001) and a separate tube with 3 mL VTM (Axonlab AL0607). The procedure for saliva collection was described in an instruction leaflet . In StudNPS and saliva were processed identically using the procedures established for NPS in the diagnostics laboratory of the Institute of Medical Virology. Briefly, 500 uL of NPS or saliva in VTM was diluted in 500 uL of Nuclisens easyMAG Lysis Buffer , centrifuged  and analyzed with the Cobas SARS-CoV-2 IVD test (Roche) on a Cobas 6800. All testing for NPS and saliva was done in parallel on the same day. SARS-CoV-2 detection was further quantified using SARS-CoV-2 Frankfurt 1 RNA as a calibrator , allowing to report both Ct (cycle threshold) and genome equivalents.Discordant results of the Cobas SARS-CoV-2 test between NPS and saliva were re-analyzed using an in-house RT-qPCR targeting the E-gene based on Corman et al. . GAPDH wE-gene Ct values were used for comparison. If the E-gene reported negative but ORF1 reported positive by the Cobas SARS-CoV-2 IVD test, the ORF1 result was considered and the respective sample rated positive for SARS-CoV-2. This was the case for one saliva sample. Data were analyzed using R (version 4.0.2) . FurtherWe sought to design a saliva collection procedure that is safe, does not create infectious waste at the collection site and does not need trained medical personnel. In order to avoid having contaminated material that needs to be disposed at the collection sites, we refrained from using collection tubes with disposable funnels and opted for collection into commonly available, cheap, wide plastic tubes. Our protocol for saliva collection instructed participants to self-collect approx. 0.5 mL saliva into an empty, wide  tube. Initial attempts in a pilot experiment at the participating emergency department with smaller tubes  showed that spitting into narrower tubes is problematic for some participants, leading to a contamination of the outside of the tube with saliva in some cases. Sampling with the wider tubes was, in contrast, unproblematic and, thus, deemed safe. Saliva sampling in children was found equally unproblematic; children were collaborating and able to expectorate. We deliberately chose to add VTM after saliva collection and not to use VTM-filled tubes as some persons feel more comfortable with sampling into empty containers. This also circumvents that the VTM is mistakenly used to gurgle or swallowed, issues that have to be considered in home testing and with children.Saliva was mixed with VTM immediately after collection and shipped to the lab at room temperature the same day or stored at 4 °C until shipping the next day. The thus-diluted material was unproblematic for further processing in the laboratory, with no complications in pipetting, and only rarely, samples were rejected due to the viscosity of saliva or invalid results were observed.Adults and children that qualified for a regular SARS-CoV-2 test according to the FOPH and reported to one of the participating test centers or emergency units were enrolled from 20 October 2020 to 28 January 2021. In total, 1270 individuals  were included . The genThe SARS-CoV-2 positivity rate amongst study participants was 21.5%. Across the entire cohort and both study arms, NPS and saliva results showed a high overall percent agreement (OPA = 97.8%) and a good positive percent agreement  and saliva  produced false-negative results in cases where the other specimen showed a high viral load (Ct < 33), highlighting variability in collection for both specimens.Breakdown in adults and children  underlinCorrelation analysis of sample pairs that both tested positive (N = 245) confirmed that saliva and NPS results are in good agreement A. NotablOur study recorded the severity of symptoms (asymptomatic/mild/strong) at the sampling time point by self-evaluation A. We obsTo investigate if the intensity of saliva collection has an impact, we analyzed the two study arms of saliva collection separately. Participants were either asked to clear their throat thoroughly and collect about 0.5–1 mL of saliva  or, in an intensified protocol, to repeat throat clearing and spitting three times . To ensure appropriate statistical power, we investigated the influence of sampling solely in the full cohort. We found that intensified saliva collection appears favorable for samples with low viral load. With the enhanced sampling protocol, PPA with NPS of Ct >33 reached 66.7% (CI 35–90%), compared to 50.0% (CI 28–72%) with the basic protocol . The difBased on these findings, local health authorities in Zurich, Switzerland, have launched mass testing for outbreak management utilizing the enhanced saliva sampling procedure in schools. Thus far, we have conducted mass tests in six schools (N = 350–700 tests per school), and saliva sampling proved well accepted by children, parents and teachers and easily applicable for mass collection and laboratory processing. In the present study, we sought to devise and evaluate a saliva sampling strategy that provides (i) representative sampling of virus-containing material, (ii) easy and safe collection, (iii) comfort for repetitive testing in adults and children, (iv) possibility for home collection and (v) straightforward processing in the laboratory  in the corresponding NPS, a notably high PPA (97.8% for the full cohort), is reached. Of note, virus loads in an even lower range are considered to impose a marginal risk for transmission as suggested by contact tracing and in vitro culturing studies ,39,40. AIn view of the observed PPA in detection, saliva may safely be envisaged as a substitute for NPS detection in a range of settings. Possible scenarios include (i) sampling of children, (ii) home collection, (iii) test centers without trained medical personnel , (iv) a non-irritating alternative for persons that need frequent testing due to their occupation or health status and (v) repetitive mass testing. In situations where other respiratory viruses besides SARS-CoV-2, e.g., Influenza and Respiratory syncytial virus (RSV), need to be excluded, NPS should, however, remain the standard material of choice as it allows rapid detection with multiplex-PCR from a single specimen. In addition, if SARS-CoV-2 infection has to be ruled out with the highest possible sensitivity , NPS should equally continue to be used.The majority of SARS-CoV-2 in saliva represents likely virus secreted from infected cells in the nasopharynx and is not locally produced. Collecting material from the posterior oropharynx may, thus, be important. This is also highlighted in our study as the collection protocol with intensified throat clearing showed a trend to increase PPA at low viral loads.It remains possible that eating or drinking shortly before collection may decrease viral content in the oral cavity and throat. In the present study, neither eating, drinking nor smoking were controlled as study subjects came for an elective analysis by NPS and, thus, could only be informed about the saliva sampling on site immediately before the collection. Abstaining from food and beverage uptake shortly (1 h) before saliva collection could be considered in forthcoming applications of saliva as a test material, as it may increase the efficacy of SARS-CoV-2 detection in saliva even further.In summary, our analysis rates saliva as a valid alternate specimen for SARS-CoV-2 detection by RT-PCR, which is even superior in children when compared to NPS. Saliva collection is non-invasive, not strenuous for patients, does not need trained personnel, allows collection at any location and allows self-collection. Importantly, as we show here, saliva collection does not require any adjustments in the diagnostics tests; established RT-qPCR can be used. Of note, while using RT-PCR as a method for saliva testing causes only a minor loss in sensitivity compared to NPS, loss in sensitivity for other test systems must be carefully weighed and analyzed, as methods with generally lower sensitivity than RT-PCR, such as antigen tests and isothermal PCR methods, may depend on testing of NPS to sustain a high enough sensitivity. Future developments that allow sensitive detection of SARS-CoV-2 in saliva by rapid antigen tests and isothermal PCR would, however, have immense potential, as the ease in sample collection and rapid and inexpensive testing would provide important advantages for mass testing ,43,44,45