What antiviral agents for treating seasonal influenza are associated with the most safety and best outcomes among healthy adults and children?This network meta-analysis of 26 randomized clinical trials, including 11 897 patients, found that antiviral agents were associated with significantly greater efficacy than placebo in shortening the duration of influenza symptoms; zanamivir was associated with the shortest time to alleviation of influenza symptoms. Baloxavir was associated with a lower occurrence of influenza-related complications than other treatments.These findings suggest that zanamivir may be initiated as soon as possible for patients with influenza-like illness; in those who may be at high risk of developing influenza-related complications, baloxavir should be considered. This systematic review and network meta-analysis assesses neuraminidase inhibitors and an endonuclease inhibitor for safety and efficacy against seasonal influenza in children and adults. Antiviral treatment of influenza is recommended for patients with influenza-like illness during periods of community cocirculation of influenza viruses and SARS-CoV-2; however, questions remain about which treatment is associated with the best outcomes and fewest adverse events.To compare the efficacy and safety of neuraminidase inhibitors and the endonuclease inhibitor for the treatment of seasonal influenza among healthy adults and children.Medline, Embase, and the Cochrane Register of Clinical Trials were searched from inception to January 2020 (the last search was updated in October 2020).Included studies were randomized clinical trials conducted among patients of all ages with influenza treated with neuraminidase inhibitors  or an endonuclease inhibitor  compared with other active agents or placebo.Two investigators identified studies and independently abstracted data. Frequentist network meta-analyses were performed; relative ranking of agents was conducted using P-score probabilities. Quality of evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations criteria. Data were analyzed in October 2020.The time to alleviation of influenza symptoms (TTAS), complications of influenza, and adverse events .A total of 26 trials were identified that investigated antiviral drugs at high or low doses; these trials included 11 897 participants, among whom 6294 (52.9%) were men and the mean (SD) age was 32.5 (16.9) years. Of all treatments comparing with placebo in efficacy outcomes, high-quality evidence indicated that zanamivir was associated with the shortest TTAS , while baloxavir was associated with the lowest risk of influenza-related complications  based on moderate-quality evidence. In safety outcomes, baloxavir was associated with the lowest risk of total adverse events  compared with placebo based on moderate-quality evidence. There was no strong evidence of associations with risk of nausea or vomiting among all comparisons, except for 75 mg oseltamivir, which was associated with greater occurrence of nausea  and vomiting .In this systematic review and network meta-analysis, all 4 antiviral agents assessed were associated with shortening TTAS; zanamivir was associated with the shortest TTAS, and baloxavir was associated with reduced rate of influenza-related complications. During the SARS-CoV-2 pandemic, coinfection with influenza A or B viruses and SARS-CoV-2 can occur and should be considered. Influenza and COVID-19 have overlapping signs and symptoms.3 Another novel oral antiviral drug, baloxavir marboxil , is a selective inhibitor of influenza cap-dependent endonuclease, and blocks proliferation of the influenza virus by inhibiting the initiation of mRNA synthesis.4Antiviral medications can help to shorten the duration of illness and prevent serious complications. Neuraminidase inhibitors selectively block an enzyme conserved within all influenza viruses to inhibit virus particles from exiting host cells and thus interrupt person-to-person viral spread. Neuraminidase inhibitors also shorten the duration of symptoms, likely by decreasing the viral load, preventing the spread and release of cytokines, and reducing the risk of complications.6 When indicated, treatment should be initiated as soon as possible within the first 48 hours of symptoms developing. The adverse effects of each medication vary but generally are gastrointestinal. The most common adverse effects of oseltamivir are nausea and vomiting; zanamivir may cause bronchospasm; and peramivir may cause diarrhea.6 However, owing to a lack of high-quality evidence, questions about which treatment is associated with the best outcomes and fewest adverse events remain. In this study, we aimed to compare the efficacy and safety outcomes associated with different antiviral agents for adults and children with seasonal influenza.The Centers for Disease Control and Prevention has recommended 4 Food and Drug Administration–approved antiviral drugs for the treatment of influenza: oseltamivir, baloxavir , zanamivir , and peramivir (intravenous).PRISMA-NMA). The study protocol was published on the PROSPERO international prospective register of systematic reviews (CRD42020186433).This systematic review and network meta-analysis was performed in accordance with the PRISMA Extension Statement for Reporting of Systematic Reviews Incorporating Network Meta-analyses of Health Care Interventions  or endonuclease inhibitor  with each other or with a placebo for the treatment of seasonal influenza. Studies assessing previously healthy people of all ages (children and adults) were included. Influenza-like illness was defined as a fever (≥38.0 °C), and at least 2 of the following 4 symptoms: headache, sore throat, myalgia, and cough, with a duration of symptoms of 48 hours or less.7 Many clinical trials only report treatment effects as median or mean values, which were insufficient for our analysis. We used DigitizeIt software version 2.2 (DigitizeIt) to reconstruct the Kaplan-Meier curves and obtain the HRs and 95% CIs.8 The reliability and validity of the data obtained using this tool were confirmed by comparing the results with those of other data extraction programs.9 Most studies reported Kaplan-Meier curves in laboratory-confirmed population. We made the decision about using the intention-to-treat-influenza-infected (ITTI) population to enable a more comprehensive comparison of the effectiveness of all included antiviral agents.The primary efficacy outcome was time to symptom alleviation (TTAS), defined as time from the start of treatment to patient-reported improvement in all influenza-associated symptoms and measured using hazard ratios (HRs). When expressing the effect size of an intervention for time-to-event data, HRs were the most appropriate. Using median or mean time-to-event for continuous outcomes may introduce bias, given that censored participants must be excluded.The secondary efficacy outcome was complications of influenza, also based on the ITTI population. Complications of particular interest included pneumonia, bronchitis, otitis media, sinusitis, and other secondary illness, whether treated with antibiotics or not.11 Both outcomes of complications and adverse events were binary, reported as risk ratios (RRs) and 95% CIs. These outcomes were defined according to the authors of each study and were not exactly the same in all trials  independently extracted data from included trials, including published year, participants , interventions, results , and study design. Disagreements in extracted data were resolved by discussion.43 We also presented information related to potential issues of clinical heterogeneity for each study. For the network meta-analysis, the quality of direct and network evidence was graded using Grading of Recommendations Assessment, Development, and Evaluation.46 For each comparison, 2 of us (J.-W.L. and S.-H.L.) independently rated the quality of direct and network evidence for each outcome; discrepancies were resolved by consensus, and if necessary, with arbitration by a third author (J.-A. L.).We summarized the potential biases for each individual study using the Cochrane risk of bias tool.netmeta in R statistical software version 4.0.2 .47 For sparse binary outcome data , a continuity correction of 0.5 was added to each cell for each effect measure.48 We also pooled odds ratio outcomes using the Mantel-Haenszel method, which offers a reliable approach to assess binary outcomes, especially in the case of sparse data.49Network meta-analyses were fitted to a frequentist framework using a multivariate random-effects network meta-analyses model with the statistical package 50 We estimated the ranking probabilities of the different antiviral agents based on their P-scores. The P-score is measured on a scale from 0 (worst) to 1 (best), with a higher score indicating better overall performance of the competing treatment. The numerical P-score values are nearly identical to the surface under the cumulative ranking curve.51 It is also important to consider the relative risk and corresponding 95% CI for each comparison when interpreting the ranking results.52We produced network plots for each outcome to visualize network geometry and node connectivity.7 We assessed network heterogeneity across all treatment contrasts using τ2 and I2 statistics. We applied the Q statistic to test for global inconsistency using a design-by-treatment interaction random effects model.53 We evaluated local inconsistency via a node split method by splitting the network estimates into direct and indirect evidence using a back-calculation method.56P values were 2-sided, and P values less than .05 were considered statistically significant.We explored the transitivity assumption by assessing whether the distribution of patients and study characteristics, which could potentially modify the treatment effects across treatment comparisons, were significantly different.57 We hypothesized that studies that find a new form of treatment is superior to an existing treatment may have a higher chance of getting published. Symmetry around the point estimate line in the scatter plot may indicate the absence of publication bias, or small study effects. Data were analyzed in October 2020.We performed sensitivity analyses for the network meta-analysis outcomes of TTAS, nausea, and vomiting (self-report outcomes) to assess the robustness of the model by excluding trials with a high risk of bias in all domains overall and in the domain of blinding. We explored the potential for publication bias by visual inspection of the comparison-adjusted funnel plots and Egger test.42 after excluding 13 studies.70 Two studies (6%) published in Chinese13 and 1 study (3%) in Japanese20 were translated and included. A large-scale randomized clinical trial (CAPSTONE-2),11 published after our last electronic search, was also included.As shown in 42 (36%) assessing adult populations (age ≥18 years) and 5 trials41 (15%) including only children. Most trials recruited patients attending outpatient clinics ; 4 trials (12%) included hospitalized patients with rapid antigen test (RAT) results positive for influenza.39Participants included children and adults of all ages, with 12 trials42 Finally, 26 trials with 11 897 participants were included in the quantitative synthesis   were men, and the mean (SD) age was 32.5 (16.9) years.We focused on 4 Food and Drug Administration–approved antiviral agents at high or low doses: 3 neuraminidase inhibitors  and 1 endonuclease inhibitor . Laninamivir was not included in this meta-analysis because no available primary efficacy outcome data were available, and the drug was only investigated in a few Japanese trials.68 (6.1%) were judged to have a low risk of bias across all domains.The risk of bias judgments for the studies contributing to the analysis of each outcome are presented in eFigure 1 in the 35 No trial reported relevant TTAS data for laninamivir. Placebo was the most well-connected intervention and was directly linked to all other interventions. A total of 5 trials32 (33.3%) had 3 eligible groups, and the remaining 10 trials35 (66.7%) had 2 eligible groups. For the comparison of all treatments with placebo, 10 mg zanamivir was associated with the shortest TTAS , followed by 600 mg peramivir , 75 mg oseltamivir , 150 mg oseltamivir , 300 mg peramivir , and 40 mg or 80 mg baloxavir   .τ2 = 0 and I2 = 0%; 95% CI, 0%-50.2%). There was no measurable global inconsistency based on a random effects design-by-treatment model  . There was no measurable global inconsistency based on a random effects design-by-treatment model  . There was no measurable global inconsistency based on a random effects design-by-treatment model  . There was no measurable global inconsistency based on a random effects design-by-treatment model  . There was no measurable global inconsistency based on a random effects design-by-treatment model   indicated trials of innovative treatments with favorable trial effects were more likely to get published .11 Additionally, we assessed HRs as our treatment outcome in TTAS, while Taieb et al71 assessed median values. We also conducted a test of assumption and evaluation of the quality of evidence in this study.To our knowledge, this is the first study to simultaneously compare the efficacy of baloxavir and 3 neuraminidase inhibitors, the 4 drugs most commonly used for patients with influenza in current clinical practice, using HRs. Compared with a 2019 network meta-analysis by Taieb et al,72 We also used the common-effect Mantel-Haenszel network meta-analysis, which is helpful to ensure the robustness of evidence when dealing with sparse data.49 We included participants of all ages with influenza-like illness; for the primary outcome, TTAS, we only focused on patients with laboratory-confirmed influenza based on a reverse transcription–polymerase chain reaction assay or viral culture with high specificity. The ITT population included patients without confirmed influenza and individuals who may have had poor medication adherence; thus, the analyses may underestimate the efficacy of the antiviral agents.73 Therefore, the efficacy analyses of the ITTI population may be closer to the truth than the analyses of the ITT population. Besides, most randomized clinical trials included in this study reported the Kaplan-Meier curves for their ITTI populations, which enabled a more comprehensive comparison of the effectiveness of the drugs.We applied a frequentist framework to perform the analysis. In theory, similar results should be reported under a frequentist framework, as well as a Bayesian framework with noninformative priors.75 Since this network meta-analysis was based on study-level data, the definition of influenza-related complications may be limited to original included trials. Additionally, we investigated all published reports of randomized clinical trials. Although nonrandomized clinical studies were not included, previous nonrandomized clinical studies reported minimal differences in the point estimates compared with our network meta-analysis. Given that nonrandomized clinical studies may be limited by the risk of confounding and selection, reporting, and publication bias, we have reservations about the credibility of their results.77 Since we did not include regulator reports, the possibility of publication bias cannot be completely ruled out.This network meta-analysis has several limitations. First, because HRs were not fully reported in most studies, our calculations based on reconstruction of the Kaplan-Meier curves may have subtle differences from the actual HRs. Second, there are a lack of studies on some antiviral agents, and the limited number of studies for some treatment comparisons may potentially dominate our network estimates. Third, the definition of complications varied among the studies, which could influence our interpretation of the effectiveness of the drugs for reducing the risk of complications. However, the network meta-analysis mostly identified influenza-related complications, which has a degree of credibility. Due to the lack of standardized definitions of influenza-related complications, meta‐analyses of these outcomes should be interpreted conservatively.The findings of this network meta-analysis suggest that the examined antiviral agents assessed were associated with shortening TTAS; zanamivir was associated with the lowest TTAS, and baloxavir was associated with the lowest rate of influenza-related complications. Moreover, baloxavir had the lowest risk of total adverse events. Compared with oseltamivir, zanamivir and baloxavir were associated with lower frequencies of nausea and 300 mg peramivir was associated with lower occurrences of vomiting. Network meta-analysis studies such as this may have utility in informing treatment guidelines for viral conditions, like influenza, when few direct comparisons between individual therapies are available.


Objective: Re-hospitalization after sepsis can lead to impaired quality of life. Predictors of re-hospitalization could help identify sepsis survivors who may benefit from targeted interventions. Our goal was to determine whether low heart rate variability (HRV), a measure of autonomic nervous system dysfunction, is associated with re-hospitalization in pediatric septic shock survivors.Materials and Methods: This was a retrospective, observational cohort study of patients admitted between 6/2012 and 10/2020 at a single institution. Patients admitted to the pediatric intensive care unit with septic shock who had continuous heart rate data available from the bedside monitors and survived their hospitalization were included. HRV was measured using age-normalized z-scores of the integer HRV (HRVi), which is the standard deviation of the heart rate sampled every 1 s over 5 consecutive minutes. The 24-h median HRVi was assessed on two different days: the last 24 h of PICU admission (“last HRVi”) and the 24-h period with the lowest median HRVi (“lowest HRVi”). The change between the lowest and last HRVi was termed “delta HRVi.” The primary outcome was re-hospitalization within 1 year of discharge, including both emergency department encounters and hospital readmission, with sensitivity analyses at 30 and 90 days. Kruskal-Wallis, logistic regression, and Poisson regression evaluated the association between HRVi and re-hospitalizations and adjusted for potential confounders.Results: Of the 463 patients who met inclusion criteria, 306 (66%) were re-hospitalized, including 270 readmissions (58%). The last HRVi was significantly lower among re-hospitalized patients compared to those who were not (p = 0.02). There was no difference in the lowest HRVi, but patients who were re-hospitalized showed a smaller recovery in their delta HRVi compared to those who were not re-hospitalized (p = 0.02). This association remained significant after adjusting for potential confounders. In the sensitivity analysis, a smaller recovery in delta HRVi was consistently associated with a higher likelihood of re-hospitalization.Conclusion: In pediatric septic shock survivors, a smaller recovery in HRV during the index admission is significantly associated with re-hospitalization. This continuous physiologic measure could potentially be used as a predictor of patients at risk for re-hospitalization and lower health-related quality of life. Sepsis is a leading cause of morbidity and mortality worldwide –3. Up toThe ANS is a unique system as it regulates functions in all organ systems, maintaining homeostasis when confronted with stressors, including sepsis . ANS dysThis was a retrospective, observational cohort study from 6/2012 to 10/2020 at a single institution. Patients admitted to the pediatric intensive care unit (PICU) with septic shock who had heart rate data available from bedside monitors were included in the analysis. Septic shock was defined as the need for vasoactive medications in children with confirmed or suspected infection within 72 h of PICU admission. The study was approved with a waiver of consent by Ann & Robert H. Lurie Children's Hospital of Chicago institutional review board (IRB #2020-3868).Continuous heart rate data from the entire PICU stay were extracted from bedside monitors using the BedMaster system  and stored locally in an archiving system within hospital servers. HRV was estimated using the integer HRV (HRVi), which was calculated as the standard deviation of the heart rate sampled every 1 s over 5 consecutive minutes and normalized for age as previously described . A minimThe primary outcome was all cause re-hospitalization to the same institution within 1 year from the index admission, including emergency department visits and hospital readmissions (both inpatient and PICU). The secondary outcome was the total number of re-hospitalizations (both inpatient and ICUs) within 1 year from the index admission to the same institution.a priori based on common risk factors associated with poor outcomes in children with septic shock that may also influence HRV.To address possible confounders of re-hospitalization, we used a multivariable regression analysis to adjust for age, severity of illness on admission using the Pediatric Risk of Mortality (PRISM) III , immunocTo determine if there were differences in the associations based on the timeframe of re-hospitalization, we performed a sensitivity analysis of patients re-hospitalized within 30 days and within 90 days from the index admission.Data were analyzed using R version 3.6.1  . KruskalThere were 463 patients who met inclusion criteria during the study period. Of those patients, 306 (66%) were re-hospitalized within 1 year, and 270 (58%) were readmitted to an inpatient ward or an ICU. The demographics and clinical characteristics of these patients during their index admission are presented in p = 0.02, p = 0.67), but patients who were re-hospitalized showed a smaller improvement in their delta HRVi  using logistic regression, the median last and delta HRVi measurements remained significantly associated with re-hospitalization, with higher HRVi measurements associated with a lower adjusted odds ratio of re-hospitalization. The lowest HRVi measurement was not associated with re-hospitalization in the unadjusted or adjusted models .The total number of re-hospitalizations in the year after the index admission were analyzed as count data. The median last HRVi and delta HRVi measurements were significantly associated with the total number of re-hospitalizations after adjusting for age, PRISM III, immunocompromised state, and multi-comorbidity using Poisson regression, with higher HRVi measurements associated with a lower adjusted incidence rate ratio of a re-hospitalization. The lowest HRVi measurement was also associated with the number of re-hospitalizations with a higher value associated with increased likelihood of more re-hospitalizations .p < 0.001). The median last HRVi was not significantly associated with re-hospitalization at 30 days, while the median lowest HRVi had positive, statistically significant association with re-hospitalization at 30 and 90 days  measured using age-normalized integer heart rate variability (HRVi) is independently associated with re-hospitalization in pediatric septic shock survivors. Specifically, the delta HRVi between the day with the lowest HRVi and the last day of PICU during the index hospitalization was significantly associated with re-hospitalization within 1 year, as well as within 30 and 90 days in sensitivity analysis, with a smaller recovery in the HRVi trajectory associated with higher likelihood of re-hospitalization. This association remained significant after controlling for several potential confounders, including age, PRISM III score, immunocompromised state, and multi-comorbidity. The absolute HRVi mean values on the lowest and last day of PICU stay were inconsistently associated with the outcome across the primary endpoint of readmissions at 1 year and the sensitivity analyses at 30 and 90 days. This suggests that the relative trajectory of recovery of the HRVi at the patient level was more predictive of re-hospitalization than the absolute values at a population level. Furthermore, although commonly considered confounders of poor outcomes, the severity of illness on admission based on PRISM III, patient age, duration of mechanical ventilation and vasoactive-inotrope days did not differ between patients requiring re-hospitalization and those who did not. This lack of association between common physiologic and clinical variables during the index admission and our outcome of interest  makes the independent association between the recovery trajectory of HRVi during PICU admission and re-hospitalization all the more notable.Readmissions after sepsis are common in children. Our rates of re-hospitalization of 66% and hospital readmission of 58% within a year after septic shock are consistent with prior studies. In a state-wide analysis of pediatric severe sepsis survivors, 47% of children were readmitted within 3 months and 85% of these readmissions were considered urgent or emergent . In anotHealth-related quality of life (HRQL) among pediatric sepsis survivors frequently declines, with 17–35% of sepsis survivors demonstrating more disability at discharge when compared to admission , 6. ThisWhile pre-existing comorbidities have been shown to be a driver of readmission following sepsis, our data support that other measures, such as HRVi, are worth considering. In our study, delta HRVi remained significantly associated with re-hospitalization even after controlling for multi-comorbidity and across sensitivity analyses. HRVi is a continuous, non-invasive physiologic marker of ANS function and can be measured in virtually all critically ill patients with high-frequency data collected from the bedside monitor. HRV has been shown to be predictive of in-hospital mortality in adults with sepsis  and in cOur study has several strengths and limitations. First, this was a single center study and therefore may have limited generalizability. However, we present data on a relatively large cohort of septic shock survivors with available high-frequency physiologic bedside monitor data. Second, our definition of re-hospitalization only included patients who were re-hospitalized to our institution. It is possible that some patients were readmitted to other institutions. Third, we calculated the HRVi using high-frequency integer heart rate data, which is a lightweight proxy of HRV. While this method may be more generalizable than the derivation of HRV from R-R intervals in the electrocardiogram waveform, which is more computationally intense, HRVi may not fully capture the physiologic complexity of the ANS. Of note, we were unable to examine medications or specific conditions that may directly affect the autonomic nervous system. Finally, there may have been bedside monitor data losses during the study period, most commonly due to server downtimes without back-ups. We do not believe these data losses resulted in systematic bias in the study population since they occurred at random.In conclusion, our data demonstrate a significant association between HRVi recovery and re-hospitalization among survivors of pediatric septic shock. This continuous, non-invasive physiologic marker of ANS function may be useful in the derivation of prediction models to stratify and mitigate the risk of re-hospitalization in survivors of pediatric septic shock.The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.The studies involving human participants were reviewed and approved by Ann & Robert H. Lurie Children's Hospital of Chicago Institutional Review Board. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.CB and LS-P designed the study and performed the data analysis. All authors made substantial contributions to drafting and final approval of the manuscript and agree to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In March 2020, an influx of admissions in COVID-19 positive patients threatened to overwhelm healthcare facilities in East Baton Rouge Parish, Louisiana. Exacerbating this problem was an overall shortage of diagnostic testing capability at that time, resulting in a delay in time-to-result return. An improvement in diagnostic testing availability and timeliness was necessary to improve the allocation of resources and ultimate throughput of patients. The management of a COVID-19 positive patient or patient under investigation requires infection control measures that can quickly consume personal protective equipment (PPE) stores and personnel available to treat these patients. Critical shortages of both PPE and personnel also negatively impact care in patients admitted with non-COVID-19 illnesses.A multisectoral partnership of healthcare providers, facilities and academicians created a molecular diagnostic lab within an academic research facility dedicated to testing inpatients and healthcare personnel for SARS-CoV-2. The purpose of the laboratory was to provide a temporary solution to the East Baton Rouge Parish healthcare community until individual facilities were self-sustaining in testing capabilities. We describe the partnership and the impacts of this endeavor by developing a model derived from a combination of data sources, including electronic health records, hospital operations, and state and local resources.Our model demonstrates two important principles: the impact of reduced turnaround times (TAT) on potential differences in inpatient population numbers for COVID-19 and savings in PPE attributed to the more rapid TAT. Betacoronavirus genus in the Coronaviridae family [SARS-CoV-2, the etiologic agent of the disease known as COVID-19, is a member of the e family . Followie family  and, on Outbreaks place extraordinary stress on healthcare systems, and a primary goal of public health response is to avoid accelerated utilization and depletion of resources , 6. DataClinicians at the two hospitals with emergency departments admitted PUI’s to cohorted units until confirmation of SARS-CoV-2 nucleic acid amplification. Because these hospitals maintained PUI’s in COVID-specific units, and because there was no specific treatment for COVID-19, a positive result for SARS-CoV-2 did not impact individual patient care; however, clinicians acted on the presumptive negative results with admission to a non-COVID-19 unit. This action often resulted in unencumbered pursual of the differential diagnosis, relief of PPE use, and bed turnover for subsequent COVID-19 patients , 11.During the first weeks of the outbreak, there was a shortage of diagnostic testing ability in the EBRP area. The Louisiana Office of Public Health Laboratory became overwhelmed with specimens from across the state, and commercial laboratories were overwhelmed with specimens from around the country. Furthermore, the local hospitals did not have in-house capability for SARS-CoV-2 testing. However, the Louisiana State University School of Veterinary Medicine (LSU SVM), housed faculty with expertise in molecular diagnostics, including real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) and human respiratory pathobiology and viral pathogens. Recognizing the unique capabilities of these faculty and their laboratory, in an effort to improve local diagnostic testing capacity and turnaround time (TAT), a multisectoral partnership of healthcare providers, facilities, and academicians repurposed academic research facilities as a dedicated COVID-19 inpatient clinical testing entity, named River Road Testing Lab (RRTL).The following analysis of the RRTL demonstrates how research laboratories can complement existing diagnostic infrastructure in times of crisis due to an emerging infection/ pandemic, until commercial and hospital-based laboratories are able to bear the burden of testing. Further, a simple data-driven model demonstrates the differences in COVID-19 inpatient population numbers and PPE usage in scenarios where a RRTL-like, dedicated laboratory with accelerated TAT compared to the scenario where no RRTL-like laboratory exists.The study of the impact of the RRTL on the Baton Rouge area was approved by the LSU Health Sciences Center–New Orleans Institutional Review Board (LSU HSC IRB #20–043).The creation of RRTL began in early March 2020, prior to the first confirmed case in the COVID-19-units, by exploring supply chain options for testing reagents and supplies. On 16 March 2020, physician-scientists from the area hospitals approached the LSU SVM faculty about both supplying viral transport media (VTM) and the potential for diagnostic testing. Also, on this date, the FDA issued a “guidance to provide a policy to help accelerate the availability of novel coronavirus (COVID-19) diagnostic tests developed by laboratories and commercial manufacturers during the public health emergency .” With aTo estimate the impact of faster TAT due to the formation of RRTL, a model was developed to assess the time of March 23 –April 22, 2020 when RRTL was the primary local testing laboratory for the hospitals. We focused on the largest of the three hospitals to demonstrate the impact on a large acute care facility, with daily admissions of PUI’s shown in The model updates the population of the COVID-19-unit daily to include daily admissions of PUI’s based on data from a hospital partner . Daily Pi, the number of individuals comprising the COVID-19-unit (C19U) population was calculated as:i-1 is the C19U yesterday (i-1), Ai is the number of new admissions today, NTi is the number of ND test results at time i, and Ei is the number of patients that egress from the COVID-19-unit at time i. NTi was estimated based on the % positivity associated with the testing date  was used to inform TAT in the model for non-RRTL lab entities  when we did not operate and samples were submitted to the state reference lab or commercial laboratories. The TAT for these individuals (TATties see . Using tties see  to assigt would be resulted at day (t + TATX) where X corresponds to either RRTL or OTHER.Individuals admitted at day Data was also provided from partner hospitals to estimate the egress from COVID-19-unit based on length of stay (LoS) data provided  used daily per each individual in the COVID-19-unit as 23 per patient per day (Source: onsite counts performed by COVID-19-unit team). Cumulative savings in PPE was calculated by subtracting PPE use for scenarios 1–3 from scenario 4 to get the daily difference, which was cumulatively summed over all days of the simulation.Three scenarios were considered: 1–2) RRTL capacity of only 25% or 50% of inpatient, 3) the actual RRTL capacity for testing 90% of COVID-19 inpatients and 4) no RRTL and a TATRRTL served two major purposes during this first wave of COVID-19 in EBRP: supply of viral testing kits and testing of inpatients. First, RRTL made and distributed nearly 8,000 viral testing kits consisting of tubes, swabs, and VTM to local healthcare, with assistance from volunteers from hospital and other academic partners. These kits were critically important, as they enabled testing despite compromised supply chains amid the growing epidemic in Europe, Washington State, New York City (NYC), and elsewhere , 16. RRTThe typical daily testing capacity was 95 unique samples, with a maximum capacity of 190 samples. This capacity resulted in RRTL ability to run samples from additional healthcare facilities, congregate living institutions, as well as healthcare workers and first responders. RRTL was the primary testing site for the area’s two largest hospitals with emergency departments and one specialized care hospital from March 23 through April 22, 2020. The period between April 23 and May 15 served as a transition period, when local hospitals were increasing in-house testing capacity. By May 15, RRTL had run 3,857 samples since opening on March 23. The overall positivity rate was 33%, including 23.6% of hospitalized inpatients, meaning that RRTL was able to quickly provide operationally actionable results to move over 76.4% of tested patients out of COVID-19 inpatient units, provided there was no clinical or operational indication to keep the patient in the unit. Patient ages ranged from a premature infant to a 99-year-old patient. The TAT (defined as the time of collection to the time of result reporting) for the period of March 23, 2020 to May 15, 2020 was median of 1.67 days from the time of collection .OTHER was found to be distributed as follows: 4% of daily C19U inpatients was 4 days, 27% of daily C19U inpatients was 5 days, 30% of daily C19U inpatients was 6 days, and 14% of daily C19U inpatients was 7 days .This is in comparison to the TAT from other entities during that period, which we determined from patient records. TATLonger TATs for COVID-19 tests resulted in higher C19U population numbers  comparedeach type of PPE  at the peak day  significantly impact hospital resource caches and bed capacity. The model is based on estimated parameter values aggregated from patient data, with the assumption that these data are representative of that period. Since the authors were all directly involved in RRTL, we have confidence that the data and model are representative. Further, in addition to testing in general acute care hospitals, RRTL provided testing for local specialty healthcare facilities as well as congregate living settings and front-line emergency and healthcare workers. These results were not included in this modeling effort, and so the impacts of RRTL herein are strictly limited to demonstration of the general acute care hospital operational support. In addition, as lab capacity grew and as clinical diagnostic criteria changed, hospital operations evolved and so the model–while appropriate for this particular scenario–would likely need to be updated for contextual relevance.Understanding this impact proved critical as the expected second wave of COVID-19 proved to be bigger than the first . HospitaThe repurposing of an academic virology lab for diagnostic testing–especially within the timeframe that was accomplished here–was not without challenges, including the cross-translation of regulatory lexicon and scientific communication in the setting of an academic partnership with clinical laboratory, hospital administration, finance, and operations . Of the Fourth, reasonable relaxation of regulatory requirements was a major determinant in the speed at which RRTL came online. As more and more academic labs partnered with clinical testing entities to support pandemic response, it has become apparent that the capabilities exist for rapid response due to these partnerships . We suggIn summary, the formation of a targeted testing lab focused primarily on COVID-19 patients with faster-than-average TAT was critical for maintaining control over hospital capacity and resources until in-hospital and/or reference clinical laboratories were able to take over testing requirements. Given this dedicated system, RRTL was able to alleviate stress on state and hospital laboratories for the region serviced and prevent unnecessary stress on healthcare associated with testing backlogs. Academic biomedical laboratories are resourced with the capabilities to have significant impacts for their communities during public health emergencies. Similar to the reserve forces of the United States Armed Forces and National Guard, public health emergency responses should be enabled to draw upon a reserve of healthcare and biomedical research professionals embedded within communities who have the expertise, experience, and knowledge to fill critical gaps in capacity when necessary.S1 File(DOCX)Click here for additional data file.


The COVID-19 pandemic has caused widespread disruptions including to health services. In the early response to the pandemic many countries restricted population movements and some health services were suspended or limited. In late 2020 and early 2021 some countries re-imposed restrictions. Health authorities need to balance the potential harms of additional SARS-CoV-2 transmission due to contacts associated with health services against the benefits of those services, including fewer new HIV infections and deaths. This paper examines these trade-offs for select HIV services.We used four HIV simulation models  to estimate the benefits of continuing HIV services in terms of fewer new HIV infections and deaths. We used three COVID-19 transmission models  to estimate the additional deaths due to SARS-CoV-2 transmission among health workers and clients. We examined four HIV services: voluntary medical male circumcision, HIV diagnostic testing, viral load testing and programs to prevent mother-to-child transmission. We compared COVID-19 deaths in 2020 and 2021 with HIV deaths occurring now and over the next 50 years discounted to present value. The models were applied to countries with a range of HIV and COVID-19 epidemics.Maintaining these HIV services could lead to additional COVID-19 deaths of 0.002 to 0.15 per 10,000 clients. HIV-related deaths averted are estimated to be much larger, 19–146 discounted deaths per 10,000 clients.While there is some additional short-term risk of SARS-CoV-2 transmission associated with providing HIV services, the risk of additional COVID-19 deaths is at least 100 times less than the HIV deaths averted by those services. Ministries of Health need to take into account many factors in deciding when and how to offer essential health services during the COVID-19 pandemic. This work shows that the benefits of continuing key HIV services are far larger than the risks of additional SARS-CoV-2 transmission. Many health services, including those providing HIV prevention, testing and care, have been restricted during the COVID-19 pandemic because services have been closed for some periods of time, capacity has been limited, or people were reluctant to access health services because of the risk of becoming infected. WHO released guidelines for essential health services that prioritized maintaining services for antiretroviral therapy (ART) . These dFor example, consider the situation in Zimbabwe. At the end of March 2020, Zimbabwe released guidance for several HIV services . For VMMRestricting some services makes sense if the benefits of those services are small compared to the potential harm of additional COVID-19 transmission. If the benefits are large, then there will be advantages to continuing those services even if it means some additional COVID-19 transmission and deaths. The purpose of this paper is to examine the benefits and disadvantages of providing HIV services during the COVID-19 pandemic.We used four existing HIV simulation models to estimate the HIV-related deaths that would be averted through delivery of HIV services had they been available and three existing COVID-19 simulation models to estimate the additional COVID-related deaths that could happen as a result of keeping the HIV services open. The use of existing models, rather than constructing a new model with both HIV and COVID-19 allowed us to involve several modeling groups and use models that had already been calibrated to several different settings.We used multiple models for both COVID-19 and HIV to remove some of the uncertainty about model assumptions and fits to country data. The results from different models will vary because of differences in model structure and assumptions as well as different modeling teams. If there is consensus on the main results in spite of these differences, it indicates that the conclusions are robust and relieves the reader of the need to understand the details of each individual model.The COVID models looked at the possibilities of additional transmission from infected patients to health care workers and vice versa as well as potential transmission during patient travel to and from health clinics and thereafter within the household and beyond to the general population. COVID-19 and HIV were compared on the basis of additional COVID-19 deaths caused by continuing HIV services and HIV-related deaths averted. Since COVID-19 deaths would occur immediately and HIV-related deaths would occur later, possibly 30 years or more later if patients with new infections receive antiretroviral therapy (ART), we discounted all HIV-related deaths to the present year using a 3% annual discount rate as recommended for comparisons of this type . DiffereThe HIV services we examined included: the provision of voluntary medical male circumcision (VMMC) which reduces susceptibility to HIV transmission, HIV diagnostic testing which provides an entryway into treatment, viral load testing which determines whether ART is successfully controlling HIV, and programs to prevent mother-to-child transmission (PMTCT) which provides HIV testing and antiretroviral therapy to prevent transmission of HIV from mother to newborn. We did not include antiretroviral treatment in this analysis since previous work  has alreWe use modeling results from different countries to take advantage of country calibrations that have been done previously. Direct comparisons between COVID-19 deaths and HIV deaths averted in the same country are only possible for Malawi and South Africa, but we also compare the range of results across many countries to understand the variations in results.The four HIV simulation models are described below. In each case, the team responsible for developing the model applied it to one or more countries using the best available data on HIV prevalence, behaviors and treatment over time. Goals is a compartmental HIV simulation model developed by Avenir Health . It divist April 2020. We then consider 5 service resumption policies: (i) all three services remain unavailable until 1 July 2021; (ii) viral load monitoring and ART switching are resumed on 1 July 2020, HIV testing and VMMC remain unavailable until 1 July 2021; (iii) HIV testing is resumed on 1 July 2020; Viral load monitoring and ART switching and VMMC remain unavailable until 1 July 2021; (iv) HIV testing and Viral load monitoring and ART switching are resumed on 1 July 2020, VMMC remain unavailable until 1 July 2021; (v) all three services are resumed on 1 July 2020. We assume that people on ART are able to continue taking ART throughout this period , but the suspension of HIV testing impacts on ART initiations. The effect of resuming HIV testing on numbers of deaths per 10,000 people receiving the service is derived from the difference between policies (i) and (iii), the effect of resuming viral load monitoring from the difference between policies (i) and (ii), and the effect of resuming VMMC services from the difference between policies (iv) and (v).The HIV Synthesis model is an individual-based model of sexual behaviour, transmission and progression of HIV and the effect of ART in an adult population updating in 3 month time steps. For each model run we sample from distributions of parameters and generate a range of “setting scenarios” which we consider reflect the diversity of epidemic and programmatic situations in sub-settings within countries sub-Saharan Africa, as well as reflecting uncertainty over some parameter values. The effect of opening three services is modelled: HIV testing, viral load monitoring and ART switching, and VMMC. We assume that each of these stopped on 1The Optima HIV model is a compartmental model with populations disaggregated by sex, age, and risk. Models for 38 countries , Malawi, Mexico, Moldova, Mozambique, Nepal, Nigeria, Papua New Guinea, Peru, Russian Federation, Senegal, South Africa, Sudan, Tajikistan, Tanzania, Thailand, Togo, Uganda, Ukraine, Uzbekistan, Vietnam, and Zimbabwe) were used in this analysis. The effect of opening each HIV service was modelled in countries based on where HIV services were implemented in a comparable format. This included nine countries in sub-Saharan Africa with coverage disaggregated by VMMC and traditional circumcision, 19 countries with viral load monitoring coverage disaggregated from ART, and all 38 countries for HIV testing. ART and PMTCT coverage were held constant over the study period to reflect 2020 values for the proportion of people diagnosed with HIV, while coverage of other programs was held constant based on the latest reported spending. Where extended disruptions occurred to VMMC, it was assumed that VMMCs would be deferred to the 12-months following the resumption of service coverage rather than not occurring.th and 75th percentile for individual countries to capture the variation across countries representing local conditions.The same analysis approach described above for the HIV Synthesis model was used. The number of client visits averted by not re-opening services was based on the assumption that each HIV test, each viral load test, and each voluntary medical male circumcision consisted of one client visit, giving the deaths averted per 10,000 clients as EMOD-HIV is a network transmission model of HIV  built inThree COVID-19 simulation models were applied to estimate the potential COVID-related deaths that could occur due to transmission related to HIV services. IHW are estimated as:p is the prevalence of infectious SARS-CoV-2 in the population, e is the effectiveness of personal protective equipment used by health care workers (set at 90%), w is the number of health care workers that have contact with each client and R0 is the average number of people who will contract an infection from one infected person .We developed a simple contact model of COVID-19 transmission based on an approach developed by Abbas et al. to examine the risks related to childhood immunization . We estip’ is the prevalence of infections SARS-CoV-2 in health care workers. This is assumed to be lower than the general population due to the use of personal protective equipment.Client infections at health care settings are estimated as:r is the probability of infection given contact with an infectious person during travel or in the waiting area and n is the number of contacts during travel to and from the clinic or in the clinic waiting area.For client contact with the general public during travel to and from, and while awaiting, services the probability of infections is estimated as:The population prevalence of infectious SARS-CoV-2 is estimated as the average reported number of daily new cases of COVID-19 multiplied by the average duration of the infectious period (5–11 days ) dividedThe number of deaths due to COVID-19 is estimated as the number of new infections multiplied by the mortality rate and adjusted for the age. The majority of COVID-19 deaths occur to people over the age of 65. Health care workers and clients are likely to be much younger. We adjust the population mortality rate downward to account for these age effects. In the United States the age group 35–44 accounts for 1.8% of all COVID-19  deaths ast for one year. The model has a SEIR structure and estimates infections, hospitalization need, intensive care need, and deaths and includes daily estimates of cloth mask utilization and physical distancing. We adjust the risk of severe disease resulting in hospitalization, intensive care, and death according to age using age-specific rates from the literature. Finally, community spread of COVID-19 did not begin on the same date in each sub-district. In lieu of sufficiently granular testing data, we estimate each sub-district’s epidemic start date by leveraging estimates of connectedness between sub-districts from mobile phone data. These daily estimates of connectedness act as a likelihood of transmission based on the pool of susceptibles and infectious individuals in source and sink sub-districts, respectively.We developed a deterministic, compartmental, age-structured transmission model for COVID-19 down to the sub-district level in Malawi beginning on April 1Surveillance of infections, hospitalizations, and deaths are highly limited in Malawi, with one of the lowest testing rates in the world and very limited death registration. We triangulate limited seroprevalence estimates in Malawi, with seroprevalence estimates from other settings including South Africa in combination with their respective test rates. We estimate that actual infections of COVID-19 in Malawi may be undercounted by 500-times, while COVID deaths are undercounted by approximately 12-times. We adjusted model parameters (within sensible range of literature review estimates) including R0, generation time, and hospitalization risk to align the projection with the calibrated trajectory of infections and deaths.Over one year, we estimate approximately 3,500,000 COVID-19 infections and 6,600 deaths among adults. We then estimate what proportion of infections and deaths are attributable to HIV service delivery as those services are a proportion of total close contacts with similar transmission probability. We estimate under a range of HIV service delivery scenarios that those services contribute approximately 1.2%-2.5% of total contacts–and therefore that same proportion of infections and deaths. We express these COVID-19 infections and deaths as a rate per 10,000 HIV clients, and break results down by service type.Covasim is an agent-based, age-and sex-structured SEIR model for COVID-19 transmission. A model for KwaZulu-Natal, South Africa was informed using estimates for household size distribution and contact network matrices  and was calibrated to daily numbers of COVID-19 cases, deaths and tests using changes in viral transmission to reflect implementation of different COVID-19 policies over time. The model was also informed by global COVID-19 disease parameter estimates as described by Kerr et . To simuOur simulations estimate that the additional COVID-19 deaths per 10,000 in-person HIV service clients ranges from 0.002 to 0.155 across all countries and models while the additional HIV-related deaths averted by keeping those services open range from 19 to 146. The average ratio of discounted HIV-related deaths to COVID-19 deaths ranges from 140 for HIV testing to 740 for VMMC services. Our results clearly show that the potential increase in HIV transmission and subsequent death that would be associated with curtailing HIV services during the COVID-19 pandemic are many times larger than the expected COVID-19 deaths due to transmission during visits to access HIV services. COVID-19 deaths would have to be at least 1000 times higher than we estimated to result in an equivalent number of deaths.As indicated by the ranges for the HIV outcomes in This result derives mostly from the differential mortality rates of the two diseases. Untreated HIV generally leads to death although a person with drug sensitive virus who takes their regimen every day is unlikely to die from HIV, even after 30 or 40 years . If HIV The results presented here come from seven different models applied to different sets of countries and settings. The variation in the results largely reflect those differences. There are also differences in model structure relating to how age and risk behaviors are handled that affect the results. The modeling teams often made different assumptions about how to interpret available data and which dynamics were important to model explicitly. The models agree surprisingly well on some results and vary by an order of magnitude on others, which we hope to explore further in future collaborations. However, in all cases the variation in estimates across models is quite small compared to the differences in estimated COVID-19-related and HIV-related mortality. In all cases the lowest estimate of HIV-related deaths averted by re-opening HIV services was at least two orders of magnitude higher than the highest estimate of additional COVID-19 related deaths as a result of re-opening HIV services. Although the models were applied to different countries, we also present results for two countries  with both COVID-19 and HIV models. Those results reinforce the conclusion that HIV services will prevent many more HIV deaths than COVID-19 deaths that might results from increased COVID-19 transmission.The contact model does not account for potential immunity to SARS-CoV-2 that may have accrued among health care workers and patients prior to potential exposures related to HIV service delivery. Thus, this model is expected to produce much higher COVID-19 risk than one that accounts for accrual of immunity, and therefore should be interpreted as an upper bound to COVID-19 risk. However, this upper bound is worth considering given uncertainties about the cumulative number of SARS-CoV-2 infections in these settings, the duration and effectiveness of SARS-CoV-2 immunity in these settings, and the potential for new variants of SARS-CoV-2 to evade immune responses.There are limitations to this analysis. COVID-19 is a new disease, so models are also new and relatively untested in projecting into the future. We have modelled HIV services in isolation but they are imbedded within a larger health system and effects of shortages of health personnel to deal with COVID-19 hospitalizations, testing and vaccination campaigns may also affect HIV services. Decision to continue a specific HIV service, such as testing, may be determined more by decisions about how to provide all types of health services rather than just those related to HIV. Disruptions to supply chains may also affect the delivery of health services. In most cases we modeled each HIV service as a single visit. Some may require two or three visits which would increase the risk of COVID-19 transmission. However, doubling or tripling the potential COVID-19 deaths still leaves them well below potential HIV-related deaths of not using the services.Most programs have taken steps to ensure that essential HIV services such as ART re-supply can continue. Often this involves multi-month scripting to reduce the number of visits required for patients doing well on ART. Countries have learned from past epidemics, such as Ebola, how to maintain essential services under extraordinary circumstances .Our analysis only examines the relative mortality risks and benefits of providing HIV services during the COVID-19 pandemic. Some services might still be restricted because of a lack of adequate health care workers, lack of supplies or fears of patients. These are all important factors. Our analysis is intended to show that fear of causing greater harm through additional COVID-19 deaths should not be a concern since the HIV services will avert many more HIV deaths.Many health systems are under great strain trying to cope with the surge of hospitalizations due to COVID-19 and meanwhile maintain other essential health services, but many are finding ways to continue these important services in spite of the obstacles. These efforts are important not only for averting new HIV infections but also for averting other infectious diseases through continued childhood immunization, avoiding unintended pregnancies by continuing family planning services, avoiding increased TB transmission by continuing diagnoses and treatment programs, and for many other health challenges as well. Our efforts should focus on how to maintain these services so that we do not lose the benefits of the progress in HIV that has been made in recent years.


A 75-year-old man with a 23-year history of poorly controlled type 2 diabetes mellitus presented with chronic constipation. Colonoscopy showed heavily pigmented mucosa, resembling leopard skin, from the cecum through the ascending colon . The pat(1). Melanosis coli is associated with the chronic use of laxatives, particularly those containing anthraquinones, such as senna, rhubarb, and cascara. It can develop within a few months of using anthraquinone-containing laxatives. Anthraquinones cause direct injury to and apoptosis of the colonic epithelial cells, resulting in lipofuscin deposition in the macrophages of the lamina propria (2), which is visible as a dark pigment. Melanosis coli disappears with the discontinuation of the drug.A multicenter observational study with patients who had undergone colonoscopy suggested that the prevalence of melanosis coli is approximately 1.8% (4), melanosis coli may be associated with a higher incidence and number of colonic non-adenoma polyps and low-grade adenomas (5). Thus, follow-up colonoscopy should be considered in patients with melanosis coli.Although there is no known association between melanosis coli and colorectal cancer NoneAK took care of the patient, wrote the manuscript, and submitted the current article.Written informed consent was obtained from the patient to publish this case report including the accompanying images.


Direct metal printing is a promising technique for manufacturing injection molds with complex conformal cooling channels from maraging steel powder, which is widely applied in automotive or aerospace industries. However, two major disadvantages of direct metal printing are the narrow process window and length of time consumed. The fabrication of high-density injection molds is frequently applied to prevent coolant leakage during the cooling stage. In this study, we propose a simple method of reducing coolant leakage for a direct-metal-printed injection mold with conformal cooling channels by combining injection mold fabrication with general process parameters, as well as solution and aging treatment (SAT). This study comprehensively investigates the microstructural evolution of the injection mold after SAT using field-emission scanning electron microscopy and energy-dispersive X-ray spectroscopy. We found that the surface hardness of the injection mold was enhanced from HV 189 to HV 546 as the Ni-Mo precipitates increased from 12.8 to 18.5%. The size of the pores was reduced significantly due to iron oxide precipitates because the relative density of the injection mold increased from 99.18 to 99.72%. The total production time of the wax injection mold without coolant leakage during the cooling stage was only 62% that of the production time of the wax injection mold fabricated with high-density process parameters. A significant savings of up to 46% of the production cost of the injection mold was obtained. Additive manufacturing (AM) ,2 technoThe conformal cooling channel (CCC) is frequently employed in injection molding because of its more uniform cooling in comparison to conventional straight cooling channels during the cooling stage. An injection mold with the appropriate CCC is considered to be a piece of technology that can reduce cycle time. The manufacturing of an injection mold with CCC is affordable due to the recent developments in AM that provide increased design freedom, less wasted material, and minimized machining. In practice, coolant leakage will appear when the injection mold is not fabricated with high-density process parameters. The cooling time is affected by coolant leakage. This results in a defect named short shot: when the molded wax pattern is observed. To solve this major disadvantage, optimum process parameters are widely applied to manufacture high-density injection molds. A distinct disadvantage is that the process window is very narrow, which leads to a high failure rate in mold making when using direct metal printing technology; furthermore, it is a time-consuming process. Thus, a simple method of reducing coolant leakage is proposed in this study, integrating injection mold fabrication with general process parameters as well as solution and aging treatment (SAT). We evaluate the surface hardnesses of the test specimens before and after heat treatment ,11. A co® 18Ni-300 Inc. South Carolina, USA) was used to manufacture injection molds using an SLM machine with a building volume of 100 × 100 × 80 mm3 , equipped with an optical-path transmission system, a f-θ lens, a scanning galvanometer mirror, and a Q-switched ytterbium-doped yttrium aluminum garnet 50 W fiber laser with a wavelength of 1070 nm. The geometric models of the injection-molded part, CCC, and injection mold proposed in this work were designed using computer-aided design software . Selective laser melting (SLM) is a metal AM technology that uses a powder bed with a source of heat to create a metal mold or die. Stainless-steel powder (LaserFormTo evaluate the Vickers hardness of test specimens after heat treatment, a cylindrical test specimen was designed with a height of 20 mm and a diameter of 20 mm. Wax patterns can be fabricated through low-pressure wax injection molding. We carried out wax injection molding to study the difference in the cooling times of the wax pattern for injection molds with and without coolant leakage. 6Al4V, Al-Si alloy, Ni-Ti alloy, 304 stainless steel, 17-4 PH stainless steel, 316-L stainless steel, CoCrMo, or Al-Fe-V-Si can also be applied to manufacture functional components, molds, or dies for industrial applications. These tasks are currently being investigated and the results will be presented in further studies.According to the results described above, the remarkable findings of this study have great practical value and show excellent potential for application in the precision investment casting industry ,21. The This study shows great potential for application in the mold industry for its improved fabrication of an injection mold in terms of economics, efficiency, and speed. This simple method is applicable to any direct-metal-printed injection molds that incorporate CCC.We comprehensively investigated and described the mechanism of the prevention of coolant leakage in direct-metal-printed injection molds with CCC.The proposed simple approach solves the coolant leakage problem without any detrimental effects on the functions of either the CCC or the injection mold. The surface hardnesses of the injection mold were improved from HV 189 to HV 546 due to the Ni-Mo precipitates, increasing from 12.8 to 18.5%. The pore sizes were greatly reduced due to the iron oxide precipitates and the surface hardness of the injection mold was increased from 99.18 to 99.72%.The total production time of a wax injection mold without coolant leakage was only 62% that of a wax injection mold fabricated with high-density process parameters. In addition, up to 46% of production cost savings could be obtained when an injection mold is fabricated using general process parameters.Direct metal printing is a useful three-dimensional printing technology that can build any metal component with sophisticated geometry by melting metal powder layer by layer. In practice, two disadvantages of fabricating a high-density injection mold to prevent coolant leakage during the cooling stage are the narrow process window and the length of time consumed. The main findings and contributions of this study are summarized as follows:


After the initiation of the current outbreak, humans’ lives have been profoundly impacted by COVID-19. During the first months, no rapid and reliable detecting tool was readily available to sufficiently respond to the requirement of massive testing. In this situation, when the development of an effective vaccine requires at least a few months, it is crucial to be prepared by developing and commercializing affordable, accurate, rapid and adaptable biosensors not only to fight Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) but also to be armed to avoid the pandemic in the earliest stages in the future. The COVID-19 diagnostic tools are categorized into two main groups of Nucleic Acid (NA)-based and protein-based tests. To date, nucleic acid-based detection has been announced as the gold-standard strategy for coronavirus detection; however, protein-based tests are promising alternatives for rapid and large-scale screening of susceptible groups. In this review, we discuss the current protein-based biosensing tools, the research advances and the potential protein-detecting strategies for COVID-19 detection. This narrative review aims to highlight the importance of the diagnostic tests, encourage the academic research groups and the companies to eliminate the shortcomings of the current techniques and step forward to mass-producing reliable point-of-care (POC) and point-of-need (PON) adaptable diagnostic tools for large-scale screening in the future outbreaks. The spread of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a life-threatening novel respiratory disease worldwide . By 5 MaBased on FIND, as of March 5, at least 1130 diagnostic tests have been submitted to various regulatory organizations in different countries, about 1030 tests have received emergency authorization and are manufactured . Two maiThe genetic material of the SARS-CoV-2 is an RNA including six open reading frames (ORFs) responsible for the production of the nucleocapsid (N), spike (S), membrane (M) and a small envelope (E) structural protein subunits; the rest of the genes produce 16 functional proteins such as RNA dependent RNA polymerase (RdRP) and helicase . N proteFrom a diagnostic point of view, specific virus antigens or specific antibodies against these antigens are detectable in the specimens collected from COVID-19 positive patients e.g., respiratory swabs, saliva, blood, serum, stool and other types of samples . ConsideSerological tests have been serving as one of the main categories to detect infectious diseases over time by targeting the specific antibodies (Ab) or so-called immunoglobulins (Ig) secreted in the human body. These systemic immunoglobulin are secreted from white blood cells (WBCs) particularly B cells (lymphocytes) as protective proteins during the infection ,20. EachAll of the serological assays rely on capturing specific antibodies in a mixture using an antibody–antigen attachment . Based oBy the date, hundreds of serological tests have been developed and received FDA EUA approval to be performed for COVID-19 detection to target one specific Ab or a different combination of them in the blood or blood products of the suspected individuals either in a lab-based or in a POC setting . ConsideEnzyme-linked immunoassays (EIA) are NIIA assays that are suitable for lab-based detection or measurement of specific antibodies in the blood and serve as the main strategies which regularly assist laboratory scientists for COVID-19 screening. Generally, the main drawbacks limiting the clinical application of the immunoassays are high costs, their complexity and long duration making them not applicable as a simple and rapid POC and near-patient tool .EIA variants including particularly Chemiluminescence Immunoassays (CLIA) and Enzyme-linked immunosorbent assay (ELISA) are frequently being employed for COVID-19 detection; they are also the routine serological methods for convalescence prediction and quantifying materials including antibodies and hormones. All of the immunoassays in this category take advantage of the affinity between antigens and specific antibodies with enzyme-based labels usually immobilized on the microplate surface .CLIA is one of the most popular immunoassays which utilizes chemiluminescent or light-emitting labels for the detection of biomolecules present in blood, serum or plasma in a total duration of about 1–2 h . In thisMany researchers and manufacturers have developed diagnostic tests based on CLIA variants, electrochemiluminescence immunoassay (ECLIA) and chemiluminescent microparticle immunoassay (CMIA), to fight the current pandemic . The maiAnother group employed the same assay with fewer specimens and reported low sensitivity of 48% but 100% specificity for the IgM test, while IgG detection illustrated 89% sensitivity and 91% specificity, respectively. By combining IgM and IgG testing, they achieved the highest accuracy for COVID-19 detection . Ma et aELISA is one of the most frequently used methods for serological detection of the infection with about 1–5 h. ELISA, as the manual version of the automated CLIA, is a microwell/plate-based assay employing immobilized capture antigen, and a secondary/tracer antigen which targets specific antibodies in the serum, plasma and/or whole blood samples , resultiFor ELISA-based COVID-19 detection, the researchers represent the immune response of the human body to the SARS-CoV-2 infection, indicating prior or recent infection. Based on the literature, IgM and IgG are recognized to be more upregulated compared to IgA and act as better targets in COVID-19 investigations. However, studies have demonstrated that IgA is also increased in response to SARS-CoV-2 infection and some tests developed to target IgA in blood or serum samples as well .Although a few numbers of the assays employ full-length S antigen as capture molecules in the test, it is commonly referred to use specific shorter peptides from this protein such as RBD domain. A research group developed two different versions of ELISA assay for the detection of S-specific antibodies using full-length S protein and RBD domain. When evaluated the tests, the data revealed that the reactivity of both of the antigens was high, with a significantly higher reactivity for S antigen . Okba etCompanies such as Bio-Rad Laboratories, DRG Diagnostics GmbH, Euroimmun, IBL International and Epitope Diagnostics have developed manual ELISA tests for COVID-19 detection. Various FDA EUA approved, and CE marked ELISA kits, have been commercially available targeting IgM, IgG, IgA, IgG/IgM or total antibodies in the collected specimens, whose names and characteristics are listed in Lassaunière et al. validated three ELISAs with serum samples and demonstrated the best performance from Wantai SARS-CoV-2 Total Antibody ELISA with 100% specificity and 90% sensitivity, Euroimmun IgA ELISA showed 93% specificity and 90 sensitivity and Euroimmun IgG ELISA displayed the lowest sensitivity of 65% with 96% specificity . BeijingMicrosphere Immunoassay (MIA) is one of the ELISA variants using fluorescent material-labelled secondary antibody and magnetic carboxylated microspheres-virus antigen particle conjugates for detection of the antibodies in serum . This teFIA is a quantitative fluorescent-labelled immunoassay, a biochemical technique that detects the attachment of the capture antibody and the desired analyte . This te® (NxTAG®-enabled) system is 71.4% and 96.2% for the samples collected on days 8–14 and >14 from symptom onset, respectively, while the NPA is 100% .,77.76,77For POC detection of the infection, the potential monitoring strategy is relying on the colour change visible to the naked eyes. Hence, colloidal gold nanoparticle (AuNP) is usually the preferred reporter which is widely observed in the developed and commercialized rapid tests due to its long-term stability, easy operation, rapid onsite detection, low costs, no requirement for complex and expensive instruments and eye-reading results, being highly biocompatible and negligible biological toxicity. However, AuNP-based LFIAs are not capable of performing quantitative measurements. One other drawback of these strips is the lower sensitivity compared with reader-based reporters . The oveAlmost all of these tests require a small amount of 10–20 µL of the sample from suspected patients, and the results are reported in about less than 30 min  52]. Ce. Ce52]. Although reporter nanoparticles in most of the rapid tests are gold, some researchers and companies have developed strips using different detection techniques. The DPP COVID-19 IgM/IgG test introduced by Chembio Diagnostics reporting the results in 15 min requiring optical readout using MicroReader 1 and 2 analyzers . The FDAUntil now, rapid strips have been widely adapted for novel coronavirus detection; however, this strategy requires to be modified in the future to be more accurate and reliable. The investigations demonstrate that the accuracy of the LFIA tests is lower compared with qRT-PCR tests. As a critical shortcoming, many reports illustrate that many of the developed LF tests suffer from sensitivity lower than 70% ,102,103.Antibody microarrays, or so-called antigen microarrays, belong to the category of protein microarrays with the unique capabilities and taking advantage of a novel promising proteomic technology performing high throughput, multiplex and miniaturized tests to target low-abundant analytes in the samples . ELISA aE. coli BL21. Then, a total of 18 proteins including proteins extracted from the sequences of N gene, S gene or other ORFs of viral RNA including E gene and nsp genes were spotted on the PATH substrate slide and formed a 2 × 7 subarray format. The test was evaluated using 29 serum samples collected from recovered patients, and the results clearly illustrated that S1 and N protein are suitable for detection, S1 with higher sensitivity than N protein. The responses of the antibodies to ORF9b and NSP5 proteins were also significant. The data provide insights in the field of vaccine development as well as diagnostics and therapeutics . A . A 52]. On May 8, FDA authorized the first antigenic test developed by Quidel Corporation, Sofia SARS Antigen FIA to perform tests in authorized laboratories and also POC settings. This test is a cassette-based LF immunofluorescent sandwich assay that detects viral N protein. Sofia2 or Sofia analyzer is required for qualitative detection. The results are reported in 15 min with 87.5% sensitivity. The test detects both SARS-CoV and SARS-CoV-2 but is not capable of differentiating them from each other . Sofia 2Lin et al. have developed a POC microfluidic immunoassay for detection of IgG/IgM/ SARS-CoV-2 Antigen simultaneously in 15 min and evaluated its clinical performance using 28 healthy and 26 COVID-19 samples . They coLumiraDx SARS-CoV-2 Ag Test takes advantage of microfluidic FIA, antibodies specific to SARS-CoV-2 N protein are applied in the FIA and target viral N antigen in the collected specimens. The test has demonstrated no cross-reaction with other respiratory pathogens such as SARS-CoV-2 and reports the results in 12 min as one of the fastest antigenic tests. However, like the BD Veritor System, the LumiraDx SARS-CoV-2 Ag Test does not present visual results and requires to be monitored using the LumiraDx Instrument. The sensitivity and specificity of the test are measured 97.6% and 96.6% with an LoD of 32 TCID50/mL . BinaxNOPCL Inc. has also introduced the new PCL COVID19 Ag Rapid FIA test. This test has received a certificate from different regulators including CE-IVD. This device is a POC rapid and cassette-based fluorescent immunoassay targeting SARS-CoV-2 N protein as an antigen is oropharyngeal, nasopharyngeal and sputum samples. The results are achieved in as fast as 10 min and FLA Analyzer is used as a fluorescent reader. Based on the manufacturing, the LoD of the test is 1000 PFU (active viruses), and its sensitivity and specificity are 100% and 97.78%, respectively . AnotherThe Simoa SARS-CoV-2 N Protein Antigen Test has recently been given FDA EUA for COVID-19 detection. Among all of the approved antigen tests, this test is the only high throughput kit with a higher TaT of about 80 min, although results are reported in 150 min for 96 tests. This test is a Paramagnetic Microbead-based Immunoassay requiring an analyzer for interpretation but does not differentiate SARS-CoV-2 and SARS-CoV from each other in the infected specimens . The CliAn important application of the rapid diagnostic tools is at home and near-patient testing. Fortunately, two rapid antigenic tests have recently been given FDA EUA to be applied as home tests with or without prescription, . The EllConsidering the increasing demand for rapid, cost-effective and accurate tests for COVID-19 detection, a wide range of technologies have been developed to overcome the shortages in the testing area . ResearcCOVID-19 has also been detected using field-deployable/portable plasmonic fibre-optic absorbance biosensors (P-FAB). This device is developed based on P-FAB with the LoD of detecting down to attomolar (10–18 M) protein concentrations. P-FAB technology monitors the changes in the intensity/absorbent or power loss in the light which is propagated in a multimode U-bent fibre-optic probe using a green LED and a photodetector. Two assays have been suggested for this aim, one using a labelled approach and another one is label-free. In the former, AuNPs are immobilized on the biosensor and then covalently conjugated with anti-N protein monoclonal antibodies (detector antibody) by thiol-PEG-NHS binding. Non-specific interactions are prevented by bovine serum albumin (BSA) treatment. These biofunctionalized probes will detect N protein of the SARS-CoV-2 in saliva samples in 15 min. On the other hand, the latter employs AuNP-labeled capture and detector antibodies in a sandwich immunoassay. The biosensor matrix is first coated with anti-N protein monoclonal antibodies on the U-bent fibre-optic probe and then treated with BSA. The sample should be mixed with anti-N protein antibodies-gold nanoparticles’ conjugates and introduced to the sensing area. The results will be achieved in 5 min. Among these two introduced technologies, the label-free assay is more promising due to its one-step response with no need for reagents, but its drawback is poor specificity. In general, the sensitivity and low LoD of the devices over LFAs are considerable, and P-FAB technology has the potential to be developed as a COVID-19 diagnostic test to fight the pandemic .Bioelectric recognition assay has been used in the development of a novel rapid and portable cell-based biosensor for COVID-19 detection. This method is based on Molecular Identification through Membrane Engineering, in which human chimeric spike S1-RBD specific antibody, recombinant human IgG1, was inserted into mammalian Vero cells via electroinserting. By the presence of viral S proteins is positive samples, they attach to their specific antibody on the surface of the cells and make a unique difference in biorecognition elements’ electric properties. This hyperpolarization was measured and recorded by a cell-biosensor, a customized multichannel potentiometer with a polydimethylsiloxane (PDMS) layer containing eight holes on its electrode’s polyester part. After the application, for the readout section, the potentiometer was connected to a tablet and recorded the measurements. The test did not require any sample-preparation steps, and the LoD of the test was 1 fg/mL and showed no cross-reaction with SARS-CoV-2 N protein. The range of responses was semi-linear from 10 fg to 1 µg/mL .PathSensors Inc. is another company developing its sensor, CANARY biosensor, for novel coronavirus detection using a cell-based technology in 5 min. The test is based on CANARY ™ technology. In summary, the test is made by genetically engineering B lymphocytes with bioluminescence from jellyfish and specific antibodies developed in mice. The engineered cells are designed to emit light when they are exposed and attached to a secondary pathogen . When SAConvat project is one of the funded projects by the H2020 European Union Framework program with the main goal of developing a POC nanophotonic sensor based on silicon photonics interferometric technology and microfluidics lab-on-chip integration. For this aim, three distinct assays are in development, the first one for viral genomic analysis and two others for direct virus detection and serological testing. For direct detection of the virus, SARS-CoV-2 specific monoclonal antibodies and nanobodies are produced, linked to the chip for capturing the complete virus and evaluated using deactivated SARS-CoV-2 virus and real samples from COVID-19 positive patients. The test quantifies the viral load in the sample. For direct RNA detection, WHO recommended sequences for SARS-CoV-2 PCR detection were evaluated and three highly specific candidate sequences were selected as targets with a similarity of 100% to SARS-CoV-2 and 0% to other genomes  with no need for PCR amplification. Different complementary probes are immobilized on the chip to hybridize with the target viral-specific sequences. The device is evaluated with synthetic RNA targets, viral SARS-CoV-2 RNA and RNA of other coronaviruses and the target sequence in the E gene and N1 gene have an LoD of 1 nM and 3 nM, respectively. Direct virus detection takes place by immobilizing anti-S1 antibodies on the surface of the chip and evaluating of its affinity and specificity. The LoD is measured at 19 ng/mL for this test. For the serological test, the targets are viral N, S1 and RBD antigens, and the intact virus is detected. The device is evaluated using S serum samples and COVID-19 positive samples. The LoD value of this chip is equal to 446 FFU/mL. The tests are performed in 30 min .Förster or fluorescence resonance energy transfer (FRET) is another method achieving high resolutions of detection in the range of 1–10 nm compared with optical techniques. Through FRET signals, protein–protein interactions, protein conformation changes and proteolytic cleaves can be studied even in living cells. For viral protein detection, FRET is capable of sensing protein–protein interactions such as antibody–antigen . FRET teIn this review, we discussed the current and potential protein-based strategies which are developed for COVID-19 detection and have the potential to be adapted for the detection of other pathogens in future pandemics. Having many unsuspected asymptomatic COVID-19 carriers interacting with other people in the society increases the risk of infecting healthy people and makes the virus spread much more quickly. This situation may lead to problems such as overloaded clinics and hospitals. This fact has raised concern regarding not only new coronavirus but also to avoid such probable outbreaks in the future. Early detection and isolation of the positive cases is pivotal for controlling any outbreak. For this aim, the development of rapid and adaptable diagnostic tools plays a critical role to limit the spread of the virus in the earliest stages in the future to avoid such pandemics. Although vaccination has the potential of improving the immune state of our bodies, the challenge is that, after the initiation of each outbreak, at least a few months are required to develop a safe and effective vaccine and large-scale vaccination itself required a long time. More importantly, natural mutations in virus RNA may immunize the virus against the vaccine. For these reasons, mass production of sensitive and low-cost POC diagnostic tools is critical to saving thousands of lives during the first months of the upcoming outbreaks.Although a large number of the COVID-19 tests have been developed over the last year, it is still difficult to recommend the advantage of each technology over the other ones due to the lack of sufficient clinical reports for most of these tests. However, the main goal of this review is to open the discussion and stimulate the questions for the Bioengineering community and also the designers who are interested in developing and contributing in the diagnostic technologies. For this reason, in each section, we have provided the performance, advantages and disadvantages of each strategy, and we hope this review opens new discussions in the development of the novel assays such as combining the current technologies with electronic biosensors or developing fully integrated devices. As an illustration, in the future, we expect to see the fully integrated lower-cost technologies adoptable for the detection of pathogens in the early stages of the spread to avoid any pandemics. Generally, the protein-based tests have been demonstrated to be very useful during the previous SARS and MERS pandemics, and, in the meantime, they are widely employed for COVID-19 diagnostics as attractive strategies especially for large-scale screening purposes. In conclusion, novel, reliable, accurate, prompt and adaptable protein-based strategies are urgently needed to assist us in the current pandemic and future hazards.