Coronavirus disease 2019 (COVID-19) can be a life-threatening lung disease or a trivial upper respiratory infection depending on whether the alveoli are involved. Emergency department (ED) evaluation of symptomatic patients with normal vital signs is frequently limited to chest auscultation and oro-nasopharyngeal swabs. We tested the null hypothesis that patients being screened for COVID-19 in the ED with normal vital signs and without hypoxia would have a point-of-care lung ultrasound (LUS) consistent with COVID-19 less than 2% of the time.We performed a retrospective, structured, blinded ultrasound review and chart review in patients 14 years or older with symptoms prompting ED evaluation for COVID-19. We excluded those with known congestive heart failure or other chronic lung conditions likely to cause excessive B-lines on LUS. We used a two-sided exact hypothesis test for binomial random variables. We measured LUS diagnostic performance using computed tomography as the gold standard.We reviewed 77 charts; 49 met inclusion criteria. Vital signs were normal in 30/49 patients; 10 (33%) of these patients had LUS consistent with viral pneumonitis. We rejected the null hypothesis . The treating physicians’ interpretations of their own point-of-care LUS had a sensitivity of 100% , 74%, 100%), specificity 88% , likelihood ratio (LR) positive of 5.8 , and LR negative of 0.05  when compared to CT findings.LUS had a meaningful detection rate for pneumonitis in symptomatic ED patients with normal vital signs who were being evaluated for COVID-19. We recommend at least LUS be used in addition to polymerase chain reaction testing when evaluating symptomatic ED patients for COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a variety of respiratory symptoms ranging from pharyngitis or rhinitis, through bronchitis to multifocal peripheral pneumonitis extending to the alveoli.Testing for SARS-CoV-2, therefore, frequently but not always includes both viral swabs from the oro-nasopharynx and imaging of the lower respiratory tract. This has included CXR, computed tomography (CT) imaging, and sometimes point-of-care lung ultrasound (LUS). Chest CT in the presence of lower respiratory tract involvement has a characteristic appearance and has been shown to be useful for diagnosing patients with COVID-19 pneumonia, including in the presence of negative nucleic acid testing. Some experienced centers even advocate CT imaging as a primary testing modality. However, CT imaging is slow, exposes the patient to ionizing radiation, and exposes additional staff to SARS-CoV-2.9Point-of-care LUS can detect SARS-CoV-2-induced lung disease, is readily available in most emergency departments (ED), does not expose the patient to ionizing radiation, and does not require the staff, expertise, and time necessary for traditional CT imaging.Our null hypothesis was that among symptomatic patients being screened for COVID-19 in the emergency department (ED) that the LUS would be consistent with COVID-19 less than 2% of the time if vital signs were normal. We also measured the diagnostic performance of LUS compared with CXR and CT chest. For comparative purposes we also measured the diagnostic performance of CXR and crackles or rales on auscultation with CT chest.The institutional review and privacy boards for Sutter Health approved this study and granted a waiver of informed consent .What do we already know about this issue?Auscultation and chest radiograph mostly fail to detect lung involvement in coronavirus disease 2019 (COVID-19).What was the research question?Do normal vital signs mean lung imaging is unnecessary when evaluating patients for COVID-19 in the ED?What was the major finding of the study?In symptomatic patients with normal vital signs 33% had lung ultrasound (LUS) evidence of alveolar involvement.How does this improve population health?Point-of-care LUS can aid in risk stratifying symptomatic ED patients in whom COVID-19 is suspected.This was a cross-sectional study with structured chart and ultrasound imaging review.Subjects were a consecutive sample of patients, 14 years of age and older, who received LUS and were evaluated for COVID-19 in an adult ED and a pediatric ED between March 4, 2020–May 19, 2020. We identified subjects from the imaging archive of the ED ultrasound machine. Patients had LUS performed if the treating physician was facile in point-of care LUS, presumably believed that lung imaging should form part of the COVID-19 evaluation, and did not send the patient for immediate CT of the chest.The physicians performing the LUS typically imaged the posterior acoustic windows by running the ultrasound probe down the patient’s back midway between the scapula and vertebral column. Axillary and anterior windows were typically interrogated with single views of each. Physicians sometimes chose to not interrogate all possible windows if they had already reached their diagnosis on the windows already imaged. Images were captured with a Zonare Z One ULTRA portable ultrasound machine . The probes available for use were linear 10-5 megahertz (MHz), linear 4-1 MHz, and curvilinear 9-3 MHz. For our primary analysis we used the interpretation of the LUS as documented in the chart.We also performed a second interpretation of the stored ultrasound images blinded to any clinical information and the original bedside interpretation. For this interpretation of the ultrasound images we considered the following findings to be consistent with viral pneumonitis: more than three simultaneous long coalescent B-lines per intercostal space occurring in more than one intercostal space; moth-eaten or irregular pleura in two or more interspaces or in one interspace with adjacent pleura showing excessive short B lines (comet tails). We considered A-lines, isolated short B-lines (comet tails) without adjacent moth-eaten pleura, and Z-lines  to be normal. Focal consolidations or effusions were taken as evidence against viral pneumonitis.We included subjects if they met the following criteria: they were 14 years of age or older; they had had ultrasound images archived with adequate identifiers; and they were being evaluated for SARS-CoV-2 infection causing a COVID-19 illness.Patients were excluded for a prior medical history of congestive heart failure, based on chart review, or other chronic lung disease likely to affect LUS interpretation  and if the point-of-care LUS was performed for a reason other than evaluating for COVID-19. We did not exclude patients with a history of asthma or chronic obstructive pulmonary disease. Patients were also excluded if we could not pair the written record of their ED visit with the ultrasound images. This happened when ultrasound images were saved without identifiers.We defined “symptomatic” as the documentation of any of the following in the electronic health record (EHR): cough; subjective fever; fatigue; weakness; sore throat or shortness of breath; nausea or vomiting; diarrhea; sore throat; fatigue; or headache. We defined “abnormal” vital signs as pulse or respiratory rate at or above the 98th percentile for age for children.We accepted the interpretation of the ultrasound by the performing physician as consistent with COVID-19 or viral pneumonitis for our primary analysis. On three occasions when the performing physician did not document an interpretation we substituted the blinded reading.One investigator (PW) performed a blinded reading of all LUS images using a structured template prior to performing chart review. Another (AH) extracted data from the EPIC/Clarity EHR  using SQL Server Management Studio . Vital signs for each visit and polymerase chain reaction (PCR) results of swabs were extracted from their respective fields in the EHR. Only the first set of vital signs was retained. Vital signs and lab results were directly extracted from the EHR. The full text of the ED visit was downloaded into a text file. EPIC EHR periodically automatically saves even incomplete notes as they are entered. The time of each (even incomplete) note is recorded. This allowed us to ensure the ultrasound note was entered before the CT resulted.The ultrasound note was typically entered either in free form or using personalized, physician-created templates. These were in various locations in the chart. Some were typed into distinct, stand-alone progress notes and others were included in the main chart, while still others were included in progress notes that included another patient’s information. We used a simplified sentiment analysis (sentimentr) in R  to locate the bedside ultrasound report in the chart.CT and CXR results have standardized headers and were located using regexm functions in Stata  and then manually reviewed and data abstracted using a standardized template by an author (PW). Because there was only one chart reviewer, inter-rater reliability was not a concern. We did not attempt intrarater reliability measurement of the chart abstraction process.We tested the null hypothesis using the bitest command in Stata. This performs exact hypothesis tests for binomial random variables. The null hypothesis was that the probability of a positive ultrasound was 2%. Our sample size calculations are shown in We identified 77 point-of-care LUS with associated medical records of which 49 met our inclusion and exclusion criteria. All 77 scans were used to measure inter-rater reliability and diagnostic performance characteristics. All the point-of-care LUS were performed before the CTs. The treating physician interpreted 18/49 (37%) point-of-care LUS as being consistent with COVID-19. Vital signs were normal in 30 patients, and 10 (33%) of these patients had LUS consistent with COVID-19. We therefore reject the null hypothesis that among symptomatic patients being screened for COVID-19 in the ED that the point-of-care LUS would be consistent with COVID-19 less than 2% of the time if vital signs were normal . We accept our alternative hypothesis that point-of-care LUS would be consistent with COVID-19 more than 2% of the time even if the vital signs were normal.When compared with the subsequent CT, the treating physicians’ interpretation of their own point-of-care LUS had a sensitivity of 100%  and specificity of 88% . For the over-reading physician relying only on archived images the sensitivity and specificity were 92%  and 37% , respectively. All but one of the CTs that were interpreted as positive reported multiple, ground-glass opacities. One CT report that did not explicitly report ground-glass opacities did report “bilateral interstitial changes” and an explicit radiology opinion that the CT lung appearance was consistent with COVID-19. The performance characteristics of point-of-care LUS using CT chest as the gold standard are detailed in Inter-rater agreement measured using Gwet’s AC1 between the bedside physician who performed the point-of-care LUS and the over-reading physician using only archives was 68%. Most characteristics showed acceptable inter-rater reliability between the bedside read and images that were over-read . Excess PCR testing for SARS-CoV-2 was not always available, but when it was a variety of tests performed at different sites were used. The results are shown in LUS detected lesions consistent with alveolar involvement in 33% of symptomatic patients with normal vital signs who were being screened for COVID-19. A key underlying assumption of our work was that a negative nasopharyngeal swab does not exclude COVID-19. This assumption has been repeatedly shown to be valid with studies finding negative nasopharyngeal swabs but positive bronchoalveolar lavage for SARS-CoV-2, SARS-CoV-1, and Middle East respiratory syndrome.21Our findings are consistent with published case series and social media reports of the utility of LUS in the diagnosis of COVID-19.28LUS has emerged as a clinical tool in human and veterinary medicine and in animal research with some advocates calling for it to replace the stethoscope.We believe that knowing whether a patient has alveolar involvement with COVID-19 is clinically important. Patients’ initially mild lung disease has been shown to progress, sometimes rapidly, on serial CTs as the disease progresses.This was a single-center study and was not a random sample. Whether a patient was seen by a physician who both believed that the COVID-19 evaluation should include lung imaging and was facile with ultrasound was a matter of luck rather than randomization. This adds uncertainty to estimates of the prevalence of pneumonitis that point-of-care LUS can detect among patients being screened for COVID-19. Other limitations of our work include its small sample size, and a single chart reviewer. Patients with mild disease, and especially those with normal vital signs, did not always have CT imaging performed. PCR testing for SARS-CoV-2 was not always available; and even when PCR testing was available, the gold standard of bronchoalveolar lavage to obtain a specimen was not performed.Our use of CT as a gold standard is imperfect as CT diagnosis of COVID-19 has its own limitations.38Our assessment of the performance characteristics of ultrasound is limited by our sample size. The relative subjectivity of LUS is also a limitation. We observed much less agreement between the blinded reviewer looking only at ultrasound images and the treating physician performing the LUS. We speculate that pleural findings were more subjective and the decision that pleural findings were abnormal might have been influenced by the clinical picture. However, describing the performance characteristics of LUS was not the primary aim of this study. Although falling out of favor, null hypothesis testing is well suited to answering our primary question when the sample size is small – after all, a single “red” (brown) Holstein cow demolishes the hypothesis that all cows are black and white, and careful planning minimizes the number of cows that need to be seen.Despite these limitations, we can be assured that the prevalence of pneumonitis in these patients was more than the 2% “acceptable miss rate” for high morbidity conditions, and this may be sufficient to adjust practice accordingly.In this small, single-center study, point-of-care lung ultrasound had a meaningful detection rate for pneumonitis in symptomatic ED patients with normal vital signs who were being evaluated for COVID-19. Test characteristics were as follows: sensitivity 100%; specificity 88%; PPV 92%; NPV 100%; LR+ 5.8; and LR− 0.1 with broad confidence intervals when compared to CT. We recommend at least point-of- care lung ultrasound be used in addition to PCR testing to identify lower airway disease when evaluating symptomatic patients in whom SARS-CoV-2 infection is suspected.


There is evidence to suggest that motor execution and motor imagery both involve planning and execution of the same motor plan, however, in the latter the output is inhibited. Currently, little is known about the underlying neural mechanisms of motor output inhibition during motor imagery. Uncovering the distinctive characteristics of motor imagery may help us better understand how we abstract complex thoughts and acquire new motor skills. The current study aimed to dissociate the cognitive processes involved in two distinct inhibitory mechanisms of motor inhibition and motor imagery restraint. Eleven healthy participants engaged in an imagined GO/NO-GO task during a 7 Tesla fMRI experiment. Participants planned a specific type of motor imagery, then, imagined the movements during the GO condition and restrained from making a response during the NO-GO condition. The results revealed that specific sub-regions of the supplementary motor cortex (SMC) and the primary motor cortex (M1) were recruited during the imagination of specific movements and information flowed from the SMC to the M1. Such condition-specific recruitment was not observed when motor imagery was restrained. Instead, general recruitment of the posterior parietal cortex (PPC) was observed, while the BOLD activity in the SMC and the M1 decreased below the baseline at the same time. Information flowed from the PPC to the SMC, and recurrently between the M1 and the SMC, and the M1 and the PPC. These results suggest that motor imagery involves task-specific motor output inhibition partly imposed by the SMC to the M1, while the PPC globally inhibits motor plans before they are passed on for execution during the restraint of responses. Participants engaged in an imagined GO/NO-GO motor task during a 7-tesla functional magnetic resonance imaging (7T-fMRI) experiment. The neural correlates of motor inhibition and motor imagery restraint were dissociated by contrasting the blood-oxygenation-level-dependent (BOLD) states yielded between the GO and NO-GO conditions against each other. The preferentiality and the spatiotemporal dynamics of significant BOLD activation were quantified to investigate the neural mechanisms underlying the inhibitory processes.Considering the above, we attempted to delineate the cortical networks of two distinct inhibitory mechanisms, Eleven healthy volunteers  participated in a single-session fMRI experiment after giving informed consent .The participants engaged in an imagined GO/NO-GO task by following the instructions presented on the screen . An expeON-trials refer to the time-points when the corresponding movements were imagined. Within the same sub-region, Off-trials refer to the time-points when all other movements were imagined .All imaging was performed on a 7T research scanner  with a 32-channel head-coil . A T3; in-plane field of view (FOV) = 222 × 222 mm2; flip angle = 34°, where T1 = 2,000 ms; partial Fourier = 6/8; phase encoding direction = A-P; multiband factor = 3; GRAPPA factor = 3; number of slices = 27; slice FOV = 40.5 mm]. Two additional sets of GE-EPI images with opposing phase encoding directions were acquired to perform B0-distortion correction.All fMRI images were acquired using 2D gradient echo-echo planar imaging (GE-EPI) with multiband and parallel imaging acceleration . In turn, three ROI masks were created for each participant.The individual-level activation maps were masked with the participant-specific region of interest (ROI) masks to ensure fair comparisons of metrics within the dorsal motor network regions across participants. The ROIs were manually defined in a study-specific template brain space, then were transformed into each participant’s functional space. The ROI masks were created for the posterior parietal cortex (PPC), the supplementary motor cortex (SMC), and the primary motor cortex (M1) using the following steps. First, a study-specific template brain was created using all T1-weighted anatomical images using the Advanced Normalization Tools .A group-level analysis was also performed. The contrast of parameter estimates and the variance maps from the individual-level analysis above were linearly registered into each subject’s Tp-values was tested against the False-Discovery Rate (FDR) adjusted threshold using the Benjamini–Hochberg procedure at Q = 0.05. For the tests that have survived the FDR-adjusted threshold, the original p-values were reported in the text.In any case of multiple comparisons, the significance of the S) was calculated by normalizing the data by subtracting the temporal mean signal then dividing it by the temporal mean signal of the voxel at each time point.We quantified the preferentiality of BOLD activity during specific motor imagery conditions  to investigate the neural correlates of motor inhibition and restraint. First, the BOLD percent signal change . To provide further support of the preferentiality of BOLD activity, random combinations of 6 ON and OFF-trials were sampled across all participants’ voxel averaged data for each region and condition. Then, all the ON and OFF-trial data were compared against each other without averaging across the samples within each region and condition using Wilcoxon rank-sum tests. This process was permuted 10,000 times with no repeating combinations of trials. The average percentage of significant predictive activity  was calculated across permutations for each region and condition.To investigate the neural correlates of motor inhibition, the %ΔS time-courses were extracted from voxels showing a significant activation during all NO-GO blocks at the individual level per ROI. The average %ΔS values between 4 s and 9 s after the start of the ON-trial GO blocks were compared against each of the three OFF-trials across the participants within each region using Wilcoxon rank-sum tests .To investigate the neural correlates of motor imagery restraint, the lack of condition-specific BOLD predictability was tested, because the group-level significant activation was observed in general across conditions and not for specific conditions. The %ΔS was compared against each other across participants using Wilcoxon rank-sum tests. In regions of significant deactivation, the latency to reach the trough %ΔS was compared.Then, we investigated the spatiotemporal dynamics of BOLD activation and deactivation onset. We controlled for any region-specific hemodynamic response function (HRF) driving the potential differences in the spatiotemporal dynamics across the dorsal motor network. We estimated the HRF of each voxel using a blind approach, then deconvoluted the BOLD signal with the estimated HRF . Significance was defined at p < 0.05 with FDR multiple comparisons correction.We further investigated how the information flow changed across the different ROIs during motor inhibition and motor imagery restraint. We subjected the voxel and trial averaged %ΔS time-courses from each region to a Multivariate Granger Causality (MVGC) analysis  and the SMC  for all participants. Significant group-level activation was observed bilaterally for each condition in the PPC  and the SMC . In the 9/11 participants, significant activation was also observed in the SMC (776 ± 123 mm3) and the PPC . Significant group-level activation was observed in the SMC bilaterally and in the M1 contralaterally for conditions LA (932 mm3), RA (240 mm3), and LB (84 mm3) but not for WF  at the individual-level in the M1 . Significant BOLD activation was observed at the group-level across all conditions in general in the right PPC .We investigated the cortical regions involved in motor inhibition and motor imagery restraint by quantifying the preferentiality of the BOLD activation during the GO and NO-GO blocks. The results revealed that during motor imagery, and thus motor inhibition, different sub-regions of the SMC and the M1 showed preferential BOLD activation to specific imagery conditions. Averaging across significant voxels of activation revealed greater %ΔS between the ON-trials and OFF-trials during motor imagery restraint .In contrast, the results revealed that during motor imagery restraint , there was no significant group-level BOLD activation in the SMC or the M1. Instead, the BOLD activation was observed primarily in the PPC and was not preferential for specific motor imagery conditions . AveragiS from the start of the prompt blocks  and the M1 (8.5 ± 0.6 s), and the latency to reach the peak %ΔS in the PPC  and from the PPC to the SMC during motor inhibition , from the M1 to the PPC (p = 0.0388) and from the M1 to the SMC (p = 0.0071) and that from the SMC to the M1 (p < 0.0001) and from the PPC to the SMC remained (p = 0.0133; Furthermore, the %ΔWe distinguished the neural correlates of motor inhibition and motor imagery restraint using an imagined GO/NO-GO task. During motor inhibition, different spatial patterns of preferential BOLD activity were observed primarily in the SMC and the M1 for each type of imagined movement , 3. BOLDThe current inhibitory network overlaps well with previous motor execution studies (Coxon et al., The current study extends upon previous findings and shows that the inhibitory mechanism of the SMC on the M1 is also task-specific. Despite a degree of overlap, spatially distinct patterns of BOLD activity were observed during the imagination of specific movements. Furthermore, the BOLD activation within each distinct pattern was preferential for each movement. Previous decision-making and executive-function studies have shown that SMC is involved in complex motor response tasks that require inhibition of distracting stimuli (Curtis et al., A task-specific inhibitory mechanism is self-evident, as one can still perform overt actions while simultaneously imagining alternate tasks or planning for the next move—a behavior that requires inhibition of specific movements while performing others. Current results are also consistent with our previous work, where it was shown that specific sub-regions of the SMC orchestrates the flow of cortical information across the dorsal motor network to encode and execute specific motor plans (Yoo et al., The current study provides further support for the task-specific inhibitory mechanism of the SMC during motor imagery by dissociating between motor inhibition and motor imagery restraint. Our results show that motor imagery restraint specifically involves PPC. Previously, the PPC has been shown to activate when stopping from performing pre-planned movements (Garavan et al., The ratio of the GO and NO-GO trials was kept in favor of the former to induce an environment that strategically benefits the GO response Wessel, . The uneThe current study focused on the cortical inhibitory mechanism coordinated from the SMC and the PPC, which resulted in a limited field-of-view. Previous studies have shown that other regions that could not be imaged in this study may play a role during motor inhibition (Coxon et al., We distinguished the neural correlates underlying two inhibitory mechanisms, motor inhibition and motor imagery restraint. Specific sub-regions of the SMC and the M1 showed BOLD activation during the imagination of specific movements and the G-causal relationship was found from the SMC to the M1. General BOLD activation in the PPC was observed during motor imagery restraint, which coincided with a BOLD deactivation in the same regions of the SMC and the M1 that were activating during motor imagery. Furthermore, G-causal relationships were observed from the PPC to the SMC and the M1. These results suggest that the SMC inhibits motor outputs in a task-specific manner, while the PPC inhibits motor plans to restrain responses in general, providing evidence that motor imagery involves inhibition at the level of motor output in a task-specific manner.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by The University of Melbourne Human Ethics Committee; Ethics ID: 1340926.1. The patients/participants provided their written informed consent to participate in this study.PY designed the experiments, performed the experiments, performed the analyses and wrote the manuscript. MH, SJ, NO, TO, SR, GR, AB, BM, and YW provided comments and made edits to the manuscript. BM and YW are joint last authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diarrhea is a leading cause of death among children under five (U5) in Lao People’s Democratic Republic (PDR). This study assessed the association between the presence of household hand-washing facilities with water and soap and diarrhea episodes among children U5 in Lao PDR. Data from the Lao Social Indicator Survey II were used. The outcome variable was diarrhea episodes in the two weeks preceding the survey. The main predictor variable was the presence of household hand-washing facilities with or without water and/or soap. Mixed-effect logistic regression analysis was used to assess the association, controlling for clustering, and other predictor variables. Of the 8640 households surveyed with 11,404 children, 49.1% possessed hand-washing facilities with both water and soap and 34.7% possessed hand-washing facilities with water alone. Children whose households possessed hand-washing facilities with water alone were significantly more likely to have a diarrhea episode compared to children whose households possessed hand-washing facilities with both water and soap . The association remained significant even after adjusting for other predictors. The absence of soap in hand-washing facilities was associated with higher odds of having a diarrhea episode among children U5 in Lao PDR. Diarrhea is a leading cause of death among children under five years of age U5), especially in low- and lower middle-income countries ,2. Accor, especiaHand-washing in everyday life is effective for reducing the risk of diarrhea. A systematic review examined the impact of hand-washing with soap on the risk of diarrheal disease and reported that hand-washing with soap in community settings can reduce the risk of diarrheal disease by 42–47%; therefore, interventions to promote hand-washing can save millions of lives a year . AccordiRapid observations are widely used as a proxy measure of hand-washing practice, assuming that household members practice hand-washing with soap if a specific place for hand-washing is observed in a household with available water and soap . In a naIn Lao PDR, expanding the coverage of water, sanitation, and hygiene (WASH) facilities has been prioritized especially in rural areas and many WASH projects including open defecation free projects have been implemented . HoweverThe association between hand-washing facilities with soap and diarrhea incidence among children U5 remains poorly understood in Lao PDR. To the best of our knowledge, two studies have assessed this association in Lao PDR. A cross-sectional study with 297 households in 50 villages in Saravan province examined associations between the presence of household hand-washing facilities and the infection status of diarrhea-causing pathogens among household members, including children U5 . The stuTherefore, the present study aimed to assess the association between the presence of household hand-washing facilities with water and soap and diarrhea episodes among children U5 in Lao PDR using data from the LSIS II. We have long conducted research concerning community health in rural Lao PDR. The present study was conducted as a part of a larger study that aims to inform strategies to promote hand-washing with soap in rural Lao PDR.https://mics.unicef.org/surveys). Out of 11,812 children U5 eligible for the LSIS II, data were available for 11,720 children whose primary caretakers responded to the survey. From the 11,720 children, the present study excluded 316 children because of missing values or “don’t know” responses to question items that were relevant to the present study. Overall, the present study included 11,404 children U5 with 9038 primary caretakers in 8640 households (spreadsheet S1: Dataset). The present study used data from the LSIS II that were obtained from UNICEF  had diarrhea?”The predictor variable of interest was the presence of hand-washing facilities with water and soap. This variable had four categories: (1) hand-washing facilities with both water and soap, (2) hand-washing facilities with water alone, (3) hand-washing facilities without water and with/without soap, and (4) no hand-washing facilities. The variable was developed on the basis of three original variables of the LSIS II: “hand-washing facility,” “water availability,” and “soap availability.” “Hand-washing facility” was measured by the following question item: “Can you please show me where members of your household most often wash their hands?” “Water availability” was measured by the following observation item: observe the presence of water at the place for hand-washing. “Soap availability” was measured by the following observation item: is soap, detergent, or ash/mud/sand present at the place for hand-washing?Based on the LSIS II report and similar studies that examined the factors associated with childhood diarrhea, the present study included the 12 predictor variables below, which were categorized into four levels: the individual, caretaker, household, and village levels ,18,19,20Escherichia coli (E. coli) contamination in the three household-based samples per village (<11 colony-forming units (CFUs) in all three samples/≥11 CFUs in one or two samples/≥11 CFUs in all three samples). The individual-level variables included the sex and age of the child U5 and the supervision by primary caretaker (adequate/inadequate). The caretaker-level variables included the age and educational attainment of the primary caretaker . The household-level variables included the number of household members (six or fewer/over six), sanitation facilities (improved/unimproved/no facilities), source of drinking water (improved/unimproved), ownership of domestic animals (yes/no), and household wealth (quartiles). To assess household wealth, an asset-based index was originally built using principal component analysis . The maip < 0.05 for all tests. The presence of multicollinearity was assessed using variance inflation factor (VIF) and a VIF > 5 was considered to indicate multicollinearity. These analyses were performed using Stata 14.2 .Bivariate analyses were conducted to assess the association between the outcome variable and each of the predictor variables using Fisher’s exact test or the chi-square test. Multivariate analyses were conducted using mixed-effect logistic regression. In the multivariate analyses, multi-level modeling was used to account for the hierarchical structure of the data: individuals (level 1) were nested within caretakers (level 2), caretakers were nested within households (level 3), and households were nested within villages (level 4). In the multivariate analyses, three models were examined: in Model 1, only the predictor of interest was included. In Model 2, the predictor of interest and the household-level variables were included. In Model 3, all of the predictor variables were included. The likelihood ratio test was used to assess the fitness of these models. The significance level was set at n = 5805, 50.9%) of the children U5 were male. The number of children in each age group did not differ greatly, ranging from 2187 (19.2%) in 1 year to 2413 (21.2%) in 3 years. The majority  of the children U5 were supervised adequately by their primary caretaker.Of the 11,404 children U5, 803 (7.0%) children had experienced a diarrhea episode in the two weeks preceding the survey . Approxin = 4241, 49.1%) possessed hand-washing facilities with both water and soap available, whereas 2994 (34.7%) households possessed hand-washing facilities with water alone. Meanwhile, 580 (6.7%) households possessed hand-washing facilities without water and with/without soap, and 825 (9.5%) households did not possess any hand-washing facilities. The majority  of the households possessed improved sanitation facilities, while 2350 (27.2%) households did not have any sanitation facilities. Most households had an improved source of drinking water  and owned domestic animals . The number of households in each household wealth quintile was almost the same across quintiles, ranging from 2141 (24.8%) in the fourth group (richest group) to 2176 (25.2%) in the third group (second richest group).The median age of the primary caretakers (interquartile range) was 28 years (range = 23–33). Of the 9038 primary caretakers, 7148 (79.1%) had completed at least primary education . The medE. coli contamination, the quality of source water was considered to be safe in 159 villages (13.7%), whereas it was considered to be unsafe in 419 villages (36.2%).Of the 1159 villages included in the LSIS II, 792 (68.3%) were rural villages . In termThe factors significantly associated with diarrhea episodes among children U5 were the sex of the child, the age of the child, the supervision by the primary caretaker, the age of the primary caretaker, the educational attainment of the primary caretaker, the number of household members, soap availability, hand-washing facilities with water and soap, sanitation facilities, source of drinking water, and household wealth . No signIn Model 1, the children whose households possess hand-washing facilities with water alone were significantly more likely to have a diarrhea episode compared to the reference group  . This diAdditionally, in Model 3 there was a significant difference between the reference group and comparison group in the following characteristics; sex of child, age of child, supervision by primary caretaker, sanitation facilities, and household wealth.The main finding of the present study was that children whose households possess hand-washing facilities with both water and soap were significantly less likely to experience diarrhea episodes compared to children whose households possess hand-washing facilities with water alone. This finding suggests that in the Lao setting, hand-washing with soap is more effective for preventing childhood diarrhea episodes compared to hand-washing without soap. This finding is important because hand-washing facilities with water are available in most households in Lao PDR. If soap use becomes more common, the mortality and morbidity due to diarrhea could be widely reduced.Escherichia coli, and Salmonella spp. [Clostridium perfringens compared to hand-washing with water alone [This main finding is biologically plausible. Analysis of the samples collected at Lao healthcare facilities showed that the major etiologic agents of acute childhood diarrhea are rotavirus, lla spp. , which alla spp. . A randolla spp. . A commuer alone . Additioer alone .The main finding is also consistent with those reported from similar observational studies. A cross-sectional study involving 347 households in rural Bangladesh showed that children U5 whose family members washed their hands with soap after defecation were significantly less likely to experience a diarrhea episode in the 48 h preceding the survey compared to children whose family members washed their hands with water only . A crossAlthough the effect of soap being present in hand-washing facilities on diarrhea incidence was not large , placing soap in hand-washing facilities could widely impact the health of Lao children, as more than one-third of Lao households do not have soap in their hand-washing facilities. Based on the assumption that a household has one child U5, 283,000 out of the total 786,000 children U5 in Lao could benefit from placing soap in hand-washing facilities. Additionally, the use of soap could contribute to preventing not only diarrhea, but also other illnesses including pneumonia, which is also a leading cause of death among Lao children [The reasons for the absence of soap in hand-washing facilities in many households remain poorly understood in Lao PDR, as no study has been conducted in the country to explore these reasons. The LSIS II report showed, however, that there are some household trends for the absence of soap in handwashing facilities: rural households, households whose heads have lower educational attainment, households of lower wealth quintiles, and households of minority language groups are less likely to have soap in their hand-washing facilities compared to their counterparts . A studyThe results of the present study also showed that there was no significant difference in the incidence of diarrhea between households with hand-washing facilities where soap and water are available and households without hand-washing facilities. A possible explanation for the lack of a difference is that the households without hand-washing facilities include a substantial proportion of households that live near a community well, and thus household members use the community well as a hand-washing facility. A community-shared well is commonly seen in rural villages of Lao PDR. In fact, wells are a major source of water for housework, including hand-washing, in rural Lao PDR . AdditioLikewise, in the present study there was no significant difference in the incidence of diarrhea between households with improved sanitation facility and households with no sanitation facility. Currently, we are unable to provide a possible reason for the lack of a difference. A multi-country case control study, which assessed sanitation and hygiene-specific risk factors for moderate-to-severe childhood diarrhea, also showed that there was no significant difference in the risk of diarrhea between households with private sanitation facilities and households with no sanitation facility in, for example, Bangladesh . HoweverA major limitation of the present study is the absence of information about actual hand-washing practices. It is of concern whether the study participants of the households where soap is available in hand-washing facilities actually use soap, as studies have shown that in settings where soap is available, people do not necessarily use soap when washing their hands before/after critical events such as fecal contact, food preparation, eating, and feeding a child. A school-based study in Lao PDR observed that of the pupils who used the school toilet, only 23.9% washed their hands with soap afterward . A multiAnother limitation is that the present study was not able to incorporate all the factors which are reported to be associated with childhood diarrhea episodes in similar studies. Such factors include food preparation practices and child feces disposal practices. For example, a cross-sectional study in Viet Nam showed that the risk of childhood diarrhea was significantly higher among children whose mothers prepared food for cooking somewhere other than the table, compared to children whose mothers prepared food on the table . A crossIn order to maximize the effect of hand-washing on preventing communicable diseases, merely recommending hand-washing with soap before/after critical events is not enough. The Centers for Disease Control and Prevention recommends the five steps for domestic hand-washing: wetting hands with clean, running water; lathering hands by rubbing hands together with soap; scrubbing hands for at least 20 s; rinsing hands well under clean, running water; and drying hands using a clean towel or air dry hands . A commuThe results of the present study showed that most of the households with children U5 already had improved sanitation facilities (70.2%) and improved source of drinking water (82.2%), whereas only 49.1% of the households with children U5 had hand-washing facilities with water and soap. The results suggest that hygiene education does not keep up with the increased coverage of sanitation and water supply. Therefore, more efforts should be made in promoting hygiene education in Lao PDR. The proportion of the population that use hand-washing facilities with water and soap is one of the indicators for Target 6.2 of Sustainable Development Goal 6: “By 2030, achieve access to adequate and equitable sanitation and hygiene for all and end open defecation, paying special attention to the needs of women and girls and those in vulnerable situations” . The preThe absence of soap in hand-washing facilities was associated with higher odds of having a diarrhea episode among children under five years of age. This suggests that hand-washing with soap is effective for preventing childhood diarrhea in Lao household settings. Households can reduce the risk of diarrhea among their children by making soap available in hand-washing facilities. Further study is necessary to inform strategies for increasing the availability of soap in hand-washing facilities in every household of Lao PDR.


Vaccines, invited experts contribute a series of articles to the current understanding of antibody-based HIV-1 vaccine development.Despite the great success of antiretroviral therapy, both in the treatment and prevention of HIV-1 infection, a vaccine is still urgently needed to end the epidemic. According to UNAIDS, in 2018, about 35% of HIV-1 infected persons did not receive antiretroviral therapy (ART), resulting in 1.7 million new infections in that year. One major reason for actual HIV-1 transmissions is the fact that about 20% of HIV-1 infected persons do not know about their HIV-positive status and therefore are not on ART despite the potential high viral load linked to high risk of virus transmission. Therefore, in particular in countries with high HIV incidence, a preventive vaccine is required to reduce HIV transmissions in the general population. In this special issue of HIV vaccine development has a long history of negative results, which nevertheless helped to accumulate detailed insights into the underlying reasons for their failure. Due to the tremendous intrinsic variability of HIV-1, the epidemic is dominated by multiple subtypes and recombinant forms, which circulate worldwide. Within an infected person, viral variants continously evolve from the transmitted/founder virus into a divergent “quasispecies” allowing their escape from the antiviral pressure imposed by the immune response or antiretroviral therapy (ART). In the paper by Stefic et al., the authors stress the importance of considering transmitted/founder viruses in vaccine development and in neutralization evaluation studies, as these are primarily the viruses a preventive vaccine has to target [Intensive collaborative work in the last decade resulted in the isolation of antibodies from a subset of HIV-positive patients, that are able to potently neutralize a broad spectrum of primary  HIV-1 isolates in vitro and to protect from infection in animal models, underlining the importance of such broadly neutralizing antibodies (bnAbs) as correlates of protection. However, despite the fact that many different HIV-1 Env immunogens have been studied in non-human primates and in clinical trials, no bnAbs could be induced so far upon vaccination. Nevertheless, recent advances in single B cell cloning in conjunction with next generation sequencing have allowed the study of the gradual development of bnAbs in patients over time. The review by Kreer et al. from the group of Florian Klein, one of the pioneers of this work, excellently summarizes how B cell receptor variability is generated and, most importantly, how B cell receptor analysis in patients developing bnAbs can guide vaccination strategies to induce such antibodies . It is cAlthough this sophisticated type of bnAbs against HIV-1 has not been induced thus far upon vaccination in humans or non-human primates, they could be induced in camelids. Camelids, besides conventional antibodies, encode heavy-chain-only antibodies, the variabel domain of which is called VHH or nanobody. Weiss and Verrips summarize here the promising work on the selection and characterization of broadly neutralizing nanobodies against HIV-1 that has been performed in recent years . Due to Numerous preclinical vaccination approaches have been performed with the aim to induce bnAbs against HIV-1 using various soluble or vector-expressed Env antigens. However, so far, at best, neutralizing antibodies were induced against easy to neutralize HIV-1 strains  or against autologous HIV-1 strains. One reason for the lack of induction of Abs with broad neutralizing activity against more difficult to neutralize patient-derived HIV-1 strains (Tiers 2 and 3) is certainly due to the complex in vivo maturation pathways of bnAbs described above, which are generated by the evolving HIV-1 quasispecies in patients. Nevertheless, promising vaccine vectors have been developed in these studies, and their pros and cons have been studied. The contribution of Wilmschen et al. in this issue nicely elaborates on the different vector systems and their optimization to achieve long-lasting antiviral immune responses, while minimizing antivector immunity and side effects . Once suBesides viral vectors, nanoparticles have been shown to be suitable carriers of viral immunogens for the induction of protective antibodies, i.e., against the human Hepatitis B Virus (HBV) and the human Papilloma Virus (HPV). Likewise, the highly repetitive expression of HIV-1 Env antigens on nanoparticles allows strong B cell activation via BCR crosslinking in the B cell follicles, which is particularly important for the very rare B cell precursors of bnAbs. Brinkkemper and Sliepen excellently summarize the different studies of nanoparticles as carriers of HIV-1 Env immunogens in vaccination studies, some of which are very promising and have advanced to phase 1 clinical trials .Advances in reverse vaccinology based on bioinformatic tools and the phage display technology have also led to the identification of short peptides mimicking linear or conformational epitopes as targets of bnAbs against HIV-1. As such, these peptides represent the minimal antigenic Env components relevant for vaccine development, which, however, have to be coupled to carriers to increase their antigenicity. Combadière et al. report on the actual status of HIV-1 peptide vaccines, most of which are derived from the transmembrane protein gp41 . One of So far, the only clinical trial in the HIV-1 vaccine field showing a moderate reduction in HIV-1 acquisition was the RV144 prime-boost study performed in Thailand about 10 years ago. The detailed analysis of non-infected and infected individuals from this trial in the following years identified antibodies against the second variable region in Env (V2) to correlate with protection in conjunction with low IgA/IgG serum levels. The paper by Duerr and Gorny nicely summarizes the complexity of V2-specific antibodies mounted during natural infection or after vaccination . Four diInterestingly, the RV144 vaccine trial mentioned above identified Fc-mediated effector functions of antibodies that correlate with protection. By attracting components of the innate immune system, such Fc-mediated effector functions can also target infected cells, thus contributing to a reduction in viremia. Anand and Finzi further explore in their excellent review the viral and cellular mechanisms affecting the different Fc-mediated effector functions, and their contribution to the control of viremia . In thisAltogether, the articles in this issue highlight different, important aspects of antibody-based HIV-1 vaccine development. Besides antibody-mediated neutralization, which is usually the protective mechanism against viral infections, antibody-mediated effector functions also play an essential role in controlling HIV-1 infection. Although the individual components necessary to develop an HIV-1 vaccine, like bnAbs, structural knowledge of their epitopes, knowledge of the antibody effector functions and a series of vaccine vectors, are known, bnAbs against primary HIV-1 strains have not yet been induced by the current Env immunogens. However, increasing knowledge on the complex maturation pathways of bnAbs in infected persons in recent years has set the basis to derive a series of recombinant Env immunogens able to engage the germline B-cell receptors of bnAbs and subsequent B cell receptors on the affinity maturation pathway towards the generation of bnAbs. Thus, learning from nature in conjunction with the development of suited techniques has rapidly advanced antibody-based HIV-1 vaccine development in the last years, providing hope that we might be a big step closer to an HIV-1 vaccine.


The latest chapter of the historic battle of humans against pathogenic microbes is the severe acute respiratory syndrome (SARS)-like coronavirus-2 (SARS-CoV-2), responsible for COVID-19, a respiratory disease declared a global pandemic by the WHO on March 11, 2020. Less than one year later, at the beginning of February 2021, nearly 104 million cases and 2.3 million deaths have been reported worldwide [The progressive approval and utilization of effective vaccines, including two unprecedented mRNA-based ones, is very good news that was largely unexpected only a few months ago, and that could allow us to watch with a cautious optimism to the months to come. However, the full control of COVID-19 cannot rely only on vaccines; it will also require efficacious therapeutics, which could possibly be used to face future related infections more efficiently. Although several molecules are currently undergoing clinical trials, the single FDA-approved drug is remdesivir , alone ocytokine storm) resulting from the SARS-CoV-2 infection.Repurposing existing drugs has been a very popular strategy among scientists searching for possible COVID-19 treatments, especially those trying to block the recognition between the SARS-CoV-2 spike glycoprotein and the cellular angiotensin-converting enzyme 2 (ACE2). Additionally, many anti-inflammatory and anticancer drugs have been repurposed to inhibit the massive inflammatory response , a depsipeptide originally isolated from the tunicate Aplidium albicans and marketed with the name Aplidin® by Pharmamar, largely outperformed remdesivir against SARS-CoV-2. Plitidepsin target is the human protein eEF1A, whose expression is related to cancer insurgence, but which is also involved in the interaction with the coronavirus nucleocapsid protein during the viral infection. The researchers tested the drug in two different mouse models: in mice that were administered plitidepsin shortly before being infected with SARS-CoV-2, the drug significantly reduced viral load (similarly to remdesivir) and lung inflammation (much better than remdesivir) compared with controls. Plitidepsin is approved for use against multiple myeloma, and its repurposing could allow the compound to directly enter phase III clinical trials against COVID-19.However, probably the most exciting discovery was published at the end of January 2021 in Science . White, These results once more certify that sea organisms/micro-organisms are incredibly prolific sources of bioactive secondary metabolites, and they are a strong encouragement for the marine natural product scientific community to continue in the efforts to exploit this unique resource.


COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements. CoVs sp surface . The emeAs of now, over 24.9 million confirmed cases and approximately 0.84 million deaths have been reported from over 215 countries across the world . SeveralSeeing the emergence of SARS-CoV-2, an understanding of the zoonotic transfer of viruses may help in preventing such events in future. Three major factors including behavioral changes in human beings, environmental change and change in diversity of microorganisms can be attributed to the emergence/re-emergence of infectious diseases. Such changes are considered to have a synergistic effect in increasing the risks for the emergence of pathogens and its transmission to the susceptible hosts, apart from creating a favorable environment for the pathogen to sustain itself in a reservoir host and/or an intermediate host before reaching humans and causing a major devastating effect .Betacoronavirus genus of the Coronaviridae family (SARS-CoV-2 is a positive sense single-stranded RNA (ssRNA) virus that is highly contagious among human beings. The virus belongs to the e family . Other ce family  and encoe family . Its gene family . The S, e family .The transmission of SARS-CoV-2 to susceptible hosts occurs primarily through aerosols originating from coughing and sneezing by an infected individual; however, indirect contact through contaminated surfaces has also been shown to be responsible for its transmission . The incSARS-CoV-2 causes flu-like symptoms, and hence, a specific diagnosis of the infection is very important to reduce the mortality rate. Currently, SARS-CoV-2 is detected using real time RT-PCR which is validated by the WHO. However, being an RNA virus, it mutates at a significant rate and may render the present diagnostic tools inefficient in due course of time. A recent study using submitted sequences of SARS-CoV-2 has revealed the mutations in viral genome that have occurred independently multiple times, and almost 80% of such recurrent mutations produce non-synonymous changes that enable the evolution and adaptation of the virus .Accurate diagnosis is very important for the management of COVID-19 as no standard therapy or vaccines are currently available for the disease; currently, only supportive and/or management treatment procedures are being followed worldwide. Therefore, research and development are on fast-track mode for the development of appropriate diagnostics, vaccines, and/or suitable therapeutics .The devastating effect of SARS-CoV-2 across the world invites a better management strategy that may include both diagnostic as well as therapeutic tools. The use of CRISPR -Cas technology could be an effective, novel, and holistic approach to target viral RNA for its degradation and thus, limit the replication of the virus in host cells, resulting in control of its transmission. CRISPR-Cas system has been identified as a part of adaptive immune system in archaea and bacteria against the virus infections  and its In addition, the CRISPR-Cas system is also used for the treatment of cancer and blindness . The tecCRISPR-Cas technology is a highly flexible RNA-guided endonuclease (RGEN)-based nucleic acid editing tool that has transformed the field of genomics, gene editing, gene therapy, and genome imaging. The broad range application of this technology provides immense scope to understand and manipulate genetic or epigenetic elements. CRISPR and Cas proteins are the part of natural adaptive immune systems against invading viruses in archae and bacteria . CRISPR-The adaptation stage begins with the expression of a complex of Cas proteins by the CRISPR-Cas loci and binding of these Cas proteins to the target DNA sequence, followed by two double-strand breaks in the target DNA based on the Protospacer Adjacent Motif (PAM) which is a distinct short motif of 2-4 bases. The released segment of the target DNA, called as protospacer, is inserted between two repeats of the CRISPR array and then acts as a new spacer . The traThere are two classes of CRISPR-Cas systems viz. class I and class II. Each of the two classes is further classified into three different subtypes, i.e., Type I, III, and IV in class I and type II, V, and VI in class II . The ribThe binding of complementary crRNA to the target RNA activates the Cas13 protein, which then degrades the collateral ssRNA . This prThe gene-editing application of the CRISPR-Cas system was recognized initially and exploited for developing anti-viral therapies. Later, its application as a gene-detection system was recognized and it has brought about a revolution in diagnostics. Point-of-care CRISPR diagnostics enable the accurate and rapid identification of any pathogen in clinical settings. The CRISPR-Cas system has been used for the development of novel therapeutics against many viruses and it may serve as an advanced-stage therapeutic option for treating HIV infections .The CRISPR-Cas technology has been utilized by molecular biologists for epigenetic regulation in eukaryotic systems. It has been used as a laboratory tool to edit DNA/RNA for fixing genetic defects and enhancing genetic traits Figure 2The group also demonstrated that multiplexing in the form of CRISPR array or multiple crRNAs could be a powerful advantage of using Cas13-based therapeutics. Cas13 could process the CRISPR array to yield individual crRNA for targeting multiple viral RNAs. There have been reports on the induction of mutations in target sites by CRISPR-based antivirals using Cas9 , while nAs of now, any SARS-CoV-2 specific therapy is not available to cure COVID-19 and also, the vaccine development is estimated to take 12–18 months. Recently, CRISPR-Cas13 has emerged as a potent system having the abilityto protect host bacterial cells against bacteriophage infection by using sequence specific crRNAs . A similvia recombinant adeno-associated virus (AAV) in patients suffering from COVID-19. The small size of the Cas13d effector is expected to facilitate the AAV to carry the CRISPR array containing more than two crRNAs, ensuring efficient virus clearance and reducing the probability of the generation of resistant strains. Moreover, the availability of an AAV serotype having lung tissue specificity can facilitate the targeted delivery of CRISPR.Recently, in vivo delivery of PAC-MAN to the target organ is a big challenge and needs to be addressed to achieve the best result. Viral vectors including AAV, adenovirus and lentiviral vectors may be opted as vehicles for delivery of such CRISPR based antivirals  has been proposed by Abbott et al. (2020) to inhibit SARS-CoV-2. Effective degradation of genomic sequences of SARS-CoV-2 and influenza A virus (IAV) has been demonstrated in human lung epithelial cells . The PACtivirals . Althougtivirals . The CRICRISPR-Cas systems have shown their potential to be utilized as molecular diagnostics for detecting nucleic acids  and may trans-cleavage. Developers have used this property and created fluorescently labeled ssDNA/RNA reporter probes to detect visible bands through the lateral flow assay in a paper strip, making it possible to develop a novel nucleic acid-based diagnostic tool . Recently developed CRISPR-based diagnosis tools using Cas12a or Cas13 nuclease have been following a principle of “collateral cleavage activity”. Cas12a/Cas13 nuclease, a component of the CRISPR tool, gets activated after the CRISPR-RNA (crRNA) targeted cleavage, and once activated, it cleaves all the nearby ssDNA/RNA molecules non-specifically, a feature called collateral cleavage or tic tool . The crRtic tool .The recently invented Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK) technology utilizes the activity of the crRNA-Cas13a protein complex to accurately identify RNA molecules and cleave collateral RNA along with target RNAs. This technology uses non-targeted reporter RNA tagged to a fluorescent dye for the identification of specific RNA molecules . The CRItargeted CRISPR trans-reporter (DETECTR) is a similar technique that amplifies the pathogenic DNA through isothermal recombinase polymerase amplification (RPA), and similar to the SHERLOCK system, reverse transcription is combined in the process for detecting RNA viruses. Target recognition by Cas12a-crRNA complex activates the Cas12a nuclease which further exhibits indiscriminate cleavage of fluorescence tagged reporter ssDNA. DETECTR has successfully been used to differentiate between human papillomavirus 16 (HPV16) and human papillomavirus 18 (HPV18) within an hour in the crude DNA isolated from cultured human cells as well as from clinical samples  in combination with reverse transcription to diagnose COVID-19 in just 30 min .Francisella novicida (FnCas9) that is very sensitive to nucleotide mismatches and developed the FnCas9 Editor Linked Uniform Detection Assay (FELUDA) for the diagnosis of SARS-CoV-2 infection in clinical settings as a point-of-care low-cost test  assay method which facilitates highly specific, ultrasensitive and faster visual detection of viral nucleic acids. In this assay, all the reagents required for the detection of viral nucleic acids are incubated at 37°C in one pot, which simplifies the detection procedure and reduces the chances of carryover contamination. The AIOD-CRISPR assay method has been utilized to detect the genomic RNA of HIV and SARS-CoV-2 with high sensitivity within an hour . The assost test .https://www.fda.gov/media/136151/download) . Therefore, a diagnostic kit having rapid and early detection ability, as well as sensitivity for very low viral titre, is necessarily required. SHERLOCK system is sensitive for around 2 attomolar [2 aM] of DNA and RNA, i.e., around 1cp/µl while the average sensitivity for an existing qRT-PCR method is 6.25 cp/µl ; LabCorpownload) . AIOD-CRAs CRISPR-Cas tools are efficient; simple to use, and need low cost infrastructure making it accessible by healthcare workers even in resource limited areas. The advantages of these tools are helping in their adoption in basic and translational research including diagnostic and therapeutic development. Though these tools have several merits, there are drawbacks which are being addressed herein Figure 4Staphylococcus aureus (SaCas9) having novel mutation (Mut268) could reduce the off-target effects while maintaining the functionality of the protein  by the CRISPR system has been a matter of concern, especially when it is intended for therapeutic and diagnostic usage . Efficie protein . A modif protein . It is uThe safety and efficacy assessment need to be monitored closely during the ongoing studies. A potential risk of using of CRISPR is the introduction of off-target changes to the genome sequence, and thus improving methods for detection of rare mutations and quantifying their potential risks will be important for future clinical advancementin vivo condition. Viral vectors are the most preferred vehicle for delivery of these tools and adeno-associated virus (AAV) have been a vector of choice by several researchers but these recombinant viruses show limitations in the size of foreign genes to be carried by them. Most of the Cas proteins have high molecular weight and therefore, it’s a challenge to generate recombinant AAV carrying the gene encoding Cas proteins along with crRNA/gRNA. To overcome this challenge, several low molecular weight Cas proteins viz. Cas14 cjCas9, ScCas9 etc. have been discovered which can also be used for the intended use and can also be delivered by rAAV (in vivo delivery using viral vectors suffer from limitations like immunogenicity and duration of Cas expression. However, non-viral vectors which are based on lipids and inorganic particles are devoid of such limitations and could be investigated as alternative for delivery of CRISPR-cas systems for therapeutic purpose (Therapeutic applications of CRISPR technology include the delivery of various tools inside the target cells under  by rAAV . Though  purpose .in vivo delivery of these proteins may result in toxicity in the human cells harboring these proteins and also in immune activation leading to production of Cas protein specific antibodies (Cas proteins are derived from prokaryotic sources and therefore, tibodies . These atibodies . Furthertibodies  and remoSeveral Cas proteins including Cas9 and Cas12a need PAM sequence for targeting a specific region in the target genome. The PAM sequences are highly specific for the type of CRISPR-Cas system and unavailability of specific PAM sequence near the target region greatly hinders the use of this technology for therapeutic purpose . FlexibiRNA generally acquires secondary structure inside the cells and this can affect the efficiency of RNA specific Cas proteins like Cas13. Elucidation of mechanisms behind the sensitivity of Cas13 proteins could help in engineering these proteins so as to make them less sensitive to RNA secondary structure .Fragile nature of RNA due to ubiquitous presence of RNase may significantly affect the efficiency of the CRISPR based diagnostic system. Other limitations in the use of CRISPR system for therapeutic applications include occurrence of mosaicism which happens when transduced cells divide before the editing or cleavage of target nucleic acids .In the modern era of science and technology, although significant advancements have been made in the field of infectious diseases, human beings become helpless whenever novel virulent viruses such as SARS-CoV-2 emerge in an unsuspecting population. Now, as per the current prevailing situations where the major hurdles are the requirements of specific equipment, the time-consuming processes associated with disease diagnosis and limited availability of diagnostic centers, there is an urgent need for such low-cost, portable, rapid, and easy-to-use diagnostic techniques that could be applicable in resource limited remote areas which are at higher risk of SARS-CoV-2 transmission, including airports and local community hospitals in low- to middle-income countries. Since SARS-CoV-2 infection causes flu-like symptoms, the accurate diagnosis of such infections is essential to control the dissemination of the virus within a population and beyond the affected people. Moreover, SARS-CoV-2 causes symptomatic as well as asymptomatic infection, and hence, it has been able to cross the territories without getting noticed in the infected but asymptomatic individuals. Such individuals are not screened, and they keep shedding the virus in the environment, infecting several other individuals around them. The availability of an affordable and portable diagnostic system may aid in extensive scale screening of affected populations to stop the chain of transmission of highly infectious viral infection.CRISPR-Cas system is a next-generation technology helping in to develop novel highly specific anti-viral therapeutics and molecular diagnostics with rapid, accurate, point-of-care use without any need for technical expertise and expensive equipment in resource poor setting. CRISPR based diagnosis could fulfil this requirement being easy to use, portable, and take less time than existing real-time PCR based standard diagnosis method for the diagnosis of COVID-19. Though CRISPR based therapies are still not approved, the CRISPR based diagnostic has obtained the US FDA emergency use authorization licence. Currently, the screening and diagnosis of COVID-19 are being done by real time RT-PCR, which detects viral RNA in respiratory samples. Though the diagnosis is specific and sensitive, but infrastructure requirements, need of trained manpower, and cost are significant limitations. CRISPR-Cas technology proved highly effective in developing diagnostics against some viruses like dengue, Zika, etc. Diagnostics based on CRISPR-Cas technology like DETECTR and AIOD-CRISPR has been shown to be fast, reliable, and cost-effective. Although not yet commercialized, the paper-based diagnostics can be a very easy to use tool to diagnose the virus infection at the point-of-care and in resource-limited remote areas. Large scale screening of affected populations can be done using such diagnostic tools, which will help in routine surveillance and restricting the community spread of this virus. The development of therapeutic drugs against RNA viruses is a great challenge as favourable mutations keep on accumulating in their genome, leading to the development of antiviral resistance. To face the challenges, strategies have been explored for the development of therapeutics targeting CRISPR-Cas technology, which targets not only the viral genome but also the host machinery involved in virus replication. As CRISPR mediated abrogation of CCR5 co-receptor gene conferred resistance to CD4 T cells for HIV infection, similar approach can be adopted to make the host cell resistant to SARS-CoV-2 by delivering the CRISPR-Cas13d into the susceptible host cells. The proposed strategy of CRISPR based therapeutics for COVID-19 are next generation technologies with significant challenges in terms of delivery, safety, efficacy and regulatory approval for use in human subjects. Delivery remains the biggest obstacle in the use of CRISPR based therapeutics. Several methods have been explored for the delivery of CRISPR with each method having some merits as well as demerits. Among the various approaches available for targeting nucleic acids, delivery of ribonucleoproteins, rather than plasmid DNA or mRNA, is associated with minimal off-target effects . TherefoAll authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current literature has documented numerous different presentations of SARS-COV-2 (COVID-19). Common symptoms include fever, cough and shortness of breath, however, lack of these symptoms does not exclude COVID-19. Given the incomplete understanding of the virus at this time, healthcare professionals must continue to remain informed of the vast number of clinical presentations of the virus to ensure early supportive treatment, ideally leading to improved outcomes. The typical presentation of SARS-COV-2 COVID-19) is fever, cough, tachypnea and shortness of breath [9 is feveThe primary objective of this case report is to present an atypical presentation of the SARS-COV-2 virus.A 90-year-old African American female with no documented past medical history presented to the emergency department following a welfare check from Adult Protective Services. The patient was noted to have altered mentation by emergency medical services (EMS). In the emergency room, vitals demonstrated a temperature of 97.9, blood pressure of 141/78, heart rate of 140, and a respiratory rate of 24 not requiring oxygen supplementation. Physical exam was notable for tachycardia with irregularly irregular rhythm and mild bibasilar crackles with no jugular venous distention or lower extremity edema. The patient was found to be in atrial fibrillation with a rapid ventricular response (afib with RVR) on initial EKG Figure . Repeat Given the patient’s imaging, COVID-19 polymerase chain reaction (PCR) testing was performed with a positive result on day three of hospitalization. That same day, the patient rapidly deteriorated and became hypoxic prompting intubation and vasopressor support. The patient was treated with supportive care in the intensive care unit (ICU) and was started on hydroxychloroquine and azithromycin. She was extubated on day nine of admission.Since the onset of COVID-19, a number of presentations have been documented. The typical presentation includes symptoms of fever, cough, and shortness of breath. However, these symptoms are not always seen on the initial presentation. As a result, suspicion for this viral infection may erroneously become lower on the differential diagnosis. As noted in our case presentation, the patient presented with altered mental status and afib with RVR. A study published in “Heart Rhythm” suggested that atrial fibrillation can be induced by a systemic inflammatory response and increased sympathetic tone . The stuSinus tachycardia (the most common arrhythmia in COVID-19 patients to date), myocardial injury, hypotension, bradycardia, and transient cardiomegaly have been documented as complications of SARS, particularly in patients with underlying cardiovascular disease -5. CurreNew manifestations of COVID-19 are being documented on a daily basis. Our patient's initial presentation resulted in decreased initial suspicion for COVID-19 infection given the lack of typical signs and symptoms. As the literature grows, earlier testing and diagnosis will follow ultimately leading to better outcomes. Further research and data collection will be necessary to develop a better understanding of the underlying mechanisms and clinical outcomes involved with the manifestations of COVID-19.


As of 8 January 2021, there were 86,749,940 confirmed coronavirus disease 2019 (COVID-19) cases and 1,890,342 COVID-19-related deaths worldwide, as reported by the World Health Organization (WHO). In order to address the COVID-19 pandemic by limiting transmission, an intense global effort is underway to develop a vaccine against SARS-CoV-2. The development of a safe and effective vaccine usually requires several years of pre-clinical and clinical stages of evaluation and requires strict regulatory approvals before it can be manufactured in bulk and distributed. Since the global impact of COVID-19 is unprecedented in the modern era, the development and testing of a new vaccine are being expedited. Given the high-level of attrition during vaccine development, simultaneous testing of multiple candidates increases the probability of finding one that is effective. Over 200 vaccines are currently in development, with over 60 candidate vaccines being tested in clinical trials. These make use of various platforms and are at different stages of development. This review discusses the different phases of vaccine development and the various platforms in use for candidate COVID-19 vaccines, including their progress to date. The potential challenges once a vaccine becomes available are also addressed. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) was responsible for 86,749,940 reported infections and 1,890,342 reported deaths as of 8 January 2021, as indicated by the World Health Organization (WHO) . This leThe purpose of vaccines is to initiate a primary immune response by introducing altered or weakened antigens (or parts thereof) that usually cause disease, in order to develop immune memory without the host becoming infected naturally . VaccineThe initial assessment of vaccines relied on observing the response of the recipient, but this has been replaced by advanced technologies that have allowed vaccines to be more specific and safer . VaccinePurified inactivated (or killed) viruses have been used traditionally for vaccine development and consist of chemically inactivated versions that are incapable of causing disease . InactivThe development of a safe and effective vaccine usually takes several years as it needs to proceed through clinical trials and requires strict regulatory approvals before it can be manufactured and distributed . Since tThe first phase involves testing candidate vaccines on a small number (20–80) of healthy individuals to ensure that the vaccine is not harmful and to determine efficacy. Tolerance and expected adverse effects due to the vaccine or vaccination process are evaluated in phase I trials . To ensuIf favorable results are achieved in phase I, candidate vaccines proceed to phase II. The second phase involves testing the vaccine on a larger sample size (100–300) and is expected to provide clinically meaningful information on safety, immunogenicity and efficacy of the candidate vaccine . This phPhase III is the most pivotal part of a study on which licensing is based. In this phase the vaccine is given to many people (1000–3000) in the target population and is tested for efficacy and safety. A single study may not be sufficient to address all questions and it may therefore be necessary to test the vaccine under different conditions including disease patterns and populations . If the As of 8 January 2021, there were 233 candidate vaccines in development for COVID-19, with 170 being evaluated in the pre-clinical and 63 in the clinical phases of testing a [9]. ThCOVID-19 vaccines in phase III were initially evaluated in young healthy volunteers. Older participants and individuals with comorbidities at higher risk of developing severe disease were included once the vaccines were proven to be safe in healthy volunteers . Some caThe first vaccine entered human trials on 16 March 2020 and was an RNA-based vaccine, mRNA-1273, developed by Moderna and the National Institute of Allergy and Infectious Diseases (NIAID). Phase I results indicated that protective antibodies were produced in a group of 45 healthy volunteers. The levels of protective antibodies in participants were similar to those detected in recovered COVID-19 patients. Effectiveness and optimal dose were also determined during phase I . A separBioNTech and Pfizer developed an RNA vaccine, BNT162b2. Initial phase I/II results involving 60 healthy volunteers aged between 18 and 55 years were encouraging. Broadly neutralizing antibodies were detected and CD4+ and CD8+ T-cell responses against the SARS-CoV-2 receptor binding domain (RBD) were observed at low doses. Phase III testing involved 30,000 healthy participants and showed 95% efficacy . The vacBoth mRNA vaccines use a lipid-based nanoparticle (LNP) carrier system, which also acts as an adjuvant. The LNPs are stabilized with polyethylene glycol (PEG), prolonging their lifespan. Scientists speculate that these allergic reactions might be related to either the lipid or the PEG component of these vaccines .PicoVacc/CoronaVac, an inactivated COVID-19 vaccine developed by Sinovac, received approval on 3 July 2020 to proceed to phase III after promising pre-clinical results and the absence of serious adverse events in 743 volunteers in phase I/II testing .Bharat Biotech in collaboration with the Indian Council of Medical Research (ICMR) recently registered a final phase testing protocol of candidate vaccine BBV152, which will take place at 25 centers in India. The plan is to launch this vaccine in the second quarter of 2021 once regulatory approval has been obtained.10 viral particles) vaccine showed better safety and immunogenicity [Four non-replicating viral vector vaccines have entered phase III . Phase Igenicity . It was genicity .A non-replicating viral vector vaccine developed by the Gamaleya Research Institute of Epidemiology and Microbiology in Moscow was approved in Russia. At the time of registration, the vaccine had not yet entered the final testing phase in humans, and no results had been published on earlier clinical phase testing . ScientiJanssen Pharmaceutica is using Ad26 to develop a COVID-19 vaccine. This single dose vaccine provided protection in monkeys and showed a promising safety profile and immunogenicity in phase I human trials . A phaseThe COVID-19 Oxford vaccine (AZD1222) is a collaborative effort between the University of Oxford, the Oxford Jenner Institute and AstraZeneca. This vaccine makes use of a non-replicating chimpanzee adenovirus  that contains the genetic sequence of the SARS-CoV-2 surface spike (S) protein . More thThe first protein subunit vaccine is being developed by Novavax and has entered phase III testing. Phase I results, which included 131 healthy adults, were recently published. This vaccine includes an adjuvant (Matrix-M1) in some groups that resulted in an enhanced immune response with acceptable safety. A second vaccination in the same individuals resulted in increased neutralizing antibody and T-helper-cell responses when the Matrix-M1 adjuvant was included. The levels observed in participants were equal to those seen in hospitalized patients with COVID-19 .The first protein-based vaccine from China  recentlyDespite efforts to develop a vaccine against SARS-CoV-2, experts warn that a vaccine might not completely eradicate this disease as levels of efficacy may vary or be incomplete, and not everyone will have access or wish to be vaccinated. As discussed below, there are several challenges that need to be addressed for a vaccine to achieve maximum effect.Historical studies on the coronavirus family including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) as well as an early study on SARS-CoV-2 suggested that infection does not provide long-term immunity ,64. HoweAntibody-mediated protection is well described, and antibody detection is used to determine effectiveness of many human vaccines. However, past experience with other viruses suggests that antibodies might enhance inflammatory responses. This is termed antibody-dependent enhancement (ADE) of the disease, and is due to the presence of poorly neutralizing cross-reactive antibodies that bind to the virus and enhance viral entry into cells . This isIf and when a vaccine against SARS-CoV-2 becomes available, there will be a need to manufacture and distribute sufficient quantities of the vaccine in order to immunize the global population. Many countries have pre-emptively secured millions of vaccine doses from pharmaceutical companies without the knowledge that these candidate vaccines will in fact work . At thisThe African continent has limited capacity for vaccine manufacturing, even in countries such as South Africa. COVID-19 has demonstrated the need for such capacity, and it is hoped that this will be the catalyst for more countries, including LMICs, to develop vaccine manufacturing capability. To ensure access to sufficient quantities, South Africa is part of Covax, an initiative supported by the WHO that will pool resources for vaccine development and global distribution. South Africa has entered into three COVID-19 vaccine trials and is investing in its own capacity to produce and distribute a vaccine locally . The SouA major obstacle to the success of a vaccine is vaccine hesitancy. Individuals opposed to vaccines do not consider them to be necessary and believe that the response of the immune system to the virus would be stronger if a person were to contract the illness naturally, and recover. Others feel that vaccines may not have been adequately tested and regulated before licensing. A significant increase in vaccine hesitancy was triggered following a 1998 Lancet publication, which suggested that the measles, mumps, and rubella (MMR) vaccine could lead to a higher incidence of developmental disorders in children, even though the research was discredited and the article retracted . Those aMorbidity, mortality and the enormous socioeconomic consequences that have resulted from the COVID-19 pandemic necessitate the urgent development of a SARS-CoV-2 vaccine. Safety and efficacy at both pre-clinical and clinical stages are essential and are the reason for extended timelines normally required for vaccine development. The pandemic has seen the establishment of novel paradigms to fast-track candidate vaccine development in order to curb the spread of the virus. Although it is important to find a vaccine quickly, evidence-based decisions should remain the priority, and this takes time. Knowledge about SARS-CoV-2 and COVID-19 is accumulating rapidly; it is however unclear whether a vaccine will be able to completely eradicate this disease. Irrespective of the outcome, we will, at least in the short- to medium-term, need to find ways to manage life with the virus, and vaccination is a key requirement if we are to do so successfully.


Displaced populations living in overcrowded settlements present an emerging and severeCOVID-19 public health risk in conflict-affected countries across the globe. InAfghanistan, the scale of the risk is sobering: over 8 million people have beendisplaced since 2012, including 4.2 million internally displaced people (IDPs) and 4million returnees from Iran and Pakistan . In thisTo date, there have been few empirical investigations of COVID-19 vulnerability in urbanIDP and returnee settlements. However informal settlements around the world are becomingincreasingly important sites in the fight against the pandemic. A recent case inMumbai’s Dharvi – the world’s largest informal settlement – drewa swift containment response from Indian authorities . PreventThe case of the settlement of Surkh Dewall in the Afghan city of Jalalabad is useful tohighlight the public health and socioeconomic aspects COVID-19. The settlement consistsof 643 households, who were displaced from the place of origin because of conflictbetween ISIS-K and government forces; 638 households were surveyed in 2019 as partof a UN-Habitat vulnerability assessment . ThecasLike many informal settlements around the world, overcrowding and multiple occupancyhousing presents a high risk of COVID-19 transmission for the population of SurkhDewall. On average, there are over 15 people per dwelling, with the majority ofhouseholds (92 per cent) residing in single roomed mud-brick dwellings. There is also ahighly vulnerable households residing in tents, accounting for a total of 30 familiesand over 150 people. The high number of people living in close proximity is alarmingbecause it has been known since January 2020 that the main spread of COVID-19 hasoccurred within households , driven Overcrowding synergises with low capacity WASH services to present additionaltransmission risks for residents. Limited access to running water, open defecation, andthe price of hygiene items all impact on inhabitants’ ability to protectthemselves from the spread of SARS-CoV-2. In this context, 9 per cent of householdspractice open defecation, with female-headed and lower-income households overrepresented in this bracket. The remainder of households use only basic open latrines,which also present high contamination risks, particularly in the context of overcrowdedhousing. These practices can lead to physical and psychological stress, and result inadverse health impacts that could complicate COVID-19 and result in worse healthoutcomes.The precarious livelihood characteristics of residents of Surkh Dewall render themvulnerable to COVID-19 transmission, and the secondary socioeconomic impacts of thedisease. Many of the residents are living in extreme poverty with just under a thirdhaving a family income of less than 1000AFG a month (US$13), equating to around US$ 0.08per person per day; 50% of female-headed households were in this bracket. Residentsreported informal sector livelihood activities, with the majority unskilled labourers(around 80 per cent), and the remainder as street vendors: such activities are at-riskof disruption due to government lockdowns, which are currently being implemented acrossAfghan cities. In this context, low and insecure incomes constrain residents’ability to take measures to prevent COVID-19 transmission, such as purchasing personalhygiene products or staying at home and self-isolating when COVID-19 symptomatic.Households also have limited resources to meet the costs associated with health care andlost income if a family member becomes ill.In the context of reduced income, food security has been highlighted by the World FoodProgramme as a key source of COVID-19 associated vulnerability . In SurkLancet call to action stressed the need for vulnerabledisplaced populations to be included in the health sector’s COVID-19 response[A recent