Acute ischemic stroke (AIS) is a common devastating disease that has increased yearly in absolute number of cases since 1990. While mechanical thrombectomy and tissue plasminogen activator (tPA) have proven to be effective treatments, their window-of-efficacy time is very short, leaving many patients with no viable treatment option. Over recent years there has been a growing interest in stimulating the facial nerves or ganglions to treat AIS. Pre-clinical studies have consistently demonstrated an increase in collateral blood flow (CBF) following ganglion stimulation, with positive indications in infarct size and neurological scores. Extensive human trials have focused on trans-oral electrical stimulation of the sphenopalatine ganglion, but have suffered from operational limitations and non-significant clinical findings. Regardless, the potential of ganglion stimulation to treat AIS or elongate the window-of-efficacy for current stroke treatments remains extremely promising. This review aims to summarize results from recent trial publications, highlight current innovations, and discuss future directions for the field. Importantly, this review comes after the release of four important clinical trials that were published in mid 2019. Stroke is the leading cause of disability and the fifth leading cause of death in the United States (US). Approximately 795,000 people experience a new or recurrent stroke each year . Acute iManagement for AIS relies on rapid treatment times to avoid penumbra evolution. The goal of modern stroke treatment facilities is to reperfuse the ischemic area via endovascular thrombectomy, mechanically removing the blood clot with catheter-based devices . The fasBoth endovascular thrombectomy and IV-tPA suffer from a limited window-of-efficacy. Currently, stroke guidelines list the acceptable window of treatment for mechanical thrombectomy at 24 h , 16, andThrombectomy is a well-established treatment , 16, 22,Researchers have recently sought to find new approaches to arrest the evolution of the ischemic penumbra and keep this susceptible region from becoming irreversibly damaged. Current approaches aim to either enhance oxygen delivery to the penumbra or reduce tissue oxygen demand . In addiFacial nerve-induced vasodilation of the cerebral arteries is an emerging therapeutic target that seeks to increase collateral blood flow in ischemic brain tissue, improve oxygen availability, and improve patient functional outcomes after stroke. Collateral circulation refers to alternative, pre-existing vascular pathways that deliver blood to target tissue when the primary vessel is occluded . ImagingThe sphenopalatine ganglion (SPG), also known as the pterygopalatine ganglion, is the largest and most superior ganglion of the sympathetic and parasympathetic nervous system and contains the largest collection of neurons in the calvarium outside of the brain . Humans The geniculate ganglion is a small collection of somatosensory and gustatory ganglion cells  located *,” “core,” and “penumbra” with intervention approach terms such as “parasympathetic,” “ganglion,” “facial nerve,” “stimulation,” “neuromodulate*,” “electrical acupuncture,” and “magnet*.”The literature search was conducted in three electronic databases MEDLINE, EMBASE, and SCOPUS, to obtain pre-clinical and clinical studies dating from 1986 to 2019. Search terms included “isch(a)emic,” “brain,” “cerebrStudies selected for final review included those assessing the effects of stimulation in the ischemic stroke indication, as well as the effects on blood flow in the brain from ganglion stimulation. The inclusion criteria for pre-clinical studies had at least one of the following outcome measures: (1) healthy and ischemic stroke-induced animal models, (2) invasive or non-invasive ganglia stimulation, and/or (3) studies providing at least one outcome measurement of cerebral or collateral blood flow, brain infarct size, neuronal survival, and neurological outcomes. Respectively, the inclusion criteria for clinical studies had at least one of the following outcome measures: (1) healthy human volunteers or patients with ischemic stroke, (2) invasive or non-invasive ganglia stimulation, (3) sham stimulation control group, (4) safety and tolerability of intervention, and/or (5) studies providing at least one outcome measurement of cerebral or collateral blood flow, brain infarct size, neuronal survival, functional and neurological outcomes including but not limited to mRS at 90 days post-stroke.Pre-clinical and clinical studies were excluded if they evaluated facial nerve stimulation for other indications such as hemorrhagic stroke, chronic headache, migraine, rhinitis, or stroke rehabilitation. Reviews, meta-analyses, observational studies, and commentaries were also excluded.The final emerging pre-clinical studies were subjected to the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias tool . The SYRThe initial search yielded 1,106 studies: 263 duplicates were removed, 843 studies were screened by title and abstract, and 717 studies were deemed irrelevant articles. A total of 126 studies were assessed for eligibility and were subjected to the predefined inclusion and exclusion criteria via full-text review, resulting in the removal of 100 studies. The final emerging 26 studies combined in this review assessed 22 pre-clinical studies and 5 human studies. One publication contained both a human and a pre-clinical trial. The methodological quality assessments of included pre-clinical and clinical studies are provided in Of the 22 pre-clinical studies included in this review, a majority , 57–59 wKey parameters of stimulation protocols included intensity, frequency, pulse, and duration , 4. SeveIntervention groups undergoing stimulation routinely demonstrated increased CBF. Three studies did not report an increase in CBF, with one not recording CBF , one repFour clinical studies using invasive electrical stimulation to treat AIS were identified  60–63).–63.60–63Khurana et al.  reportedPost-hoc analysis demonstrated significant improvements within a subsection of the population that had confirmed cortical involvement (CCI), defined as patients with NIHSS score ≥10 and signs of hypodensity or tissue swelling in at least one cortical region on initial imaging  to determine if 6 h of SPG stimulation using the same procedure in the ImpACT studies will “freeze” the ischemic penumbra in patients with acute ischemic stroke and reduce brain tissue death. The primary outcome measure is volume of core expansion determined by CT scan post SPG stimulation. This trial is expected to complete in spring 2021.Six of the 26 publications identified assessed non-invasive interventions to stimulate facial nerves or ganglions  43–45, , 52, 56.Magnetic stimulation interventions were primarily deployed through tesla coils located on either side of the subject's head. Magnetic intensities were reported in Tesla, and ranged from 0.5 to 1.9 T in the three studies , 44, 56.All non-invasive stimulation studies found an increase in CBF compared to controls , 52, 56,Sanchez et al.  was the There is a substantial amount of pre-clinical and clinical data to suggest that stimulation of the parasympathetic fibers of the facial nerve system is a promising option for rapid AIS treatment. Data from early trials with both invasive and non-invasive approaches have proven safe with very minimal reports of treatment-associated adverse events. Stimulation of the SPG or geniculate ganglion clearly results in increased CBF, and the technological means required to rapidly induce ganglion stimulation are certainly feasible. While the SPG has been the most well-studied ganglion in clinical trials to date, pre-clinical evidence suggests a variety of targets for the potential therapeutic development.Despite the tremendous upside it poses for treating AIS, ganglion stimulation has yet to demonstrate clinically meaningful outcomes. To date the clinical trials for SPG stimulation via a trans-oral device have suffered from various methodological shortcomings that obscure the reasons behind the lack of positive results. A recurring issue with incorrect device placement resulted in the study protocols for both the ImpACT-24A and ImpACT-24B trials to be repeatedly modified during the trial course. The mid-trial change of the neurostimulator, implantation technique and electrical transmitter-control unit in the ImpACT-24B trial is especially disconcerting and was not addressed in the outcome analysis. Additionally, while the trials claim to be double or triple blinded, it is unclear how successful the blinding can be for the subject and physician when a key element of the intervention's deployment is mild facial discomfort. It is also not clear why the ImpACT-1 and ImpACT-24A were published almost 10 years after completion, and simultaneously with ImpACT-24B and ImpACT-24M.Current strategies for employing ganglion stimulation to treat AIS suffer from three core limitations: (1) current trial design, (2), time-to-deployment, and (3) accessibility to trained physicians.The ImpACT trials have focused on independently improving AIS-related disability, but their negative results suggest that tPA and mechanical thrombectomy will remain the gold standard for AIS treatment. It is therefore optimal for facial nerve stimulation to augment and improve the applicability of these existing treatments. There is a clear need for interventions that elongate the current window-of-efficacy , with exHowever, if the goal of future trials is to stop the evolution of the penumbra prior to recanalization, it is key that ganglion stimulation protocols add minimal delay to the already extensive door-to-treatment time. The design of the current clinical stimulation devices and associated procedures are limited in their ability to accomplish this. The ImpACT-24B trial saw a 1.2-h difference in time from “last known well” on NIHSS to the first stimulation session between sham and control groups; this is likely due to the procedure associated with finding the ideal stimulation intensity, a step inherently missing from the sham procedure. Even without this optimization step, the implantation of the invasive device takes a reported average of 20 min, not including time for setting a sterile environment. This substantially eats away at thrombectomy's window-of-efficacy or the even shorter 60-min window for tPA induction , 68. TraThe requirement of highly trained physicians, specialized imaging, and sterile fields further limit the currently studied innovations. Current invasive techniques for ganglion stimulation can be employed only by specialized physicians, demonstrated by 58% of implantations in the ImpACT-24B trial being performed by surgeons and anesthesiologists, with the remaining performed by neurologists . ImplantThe research to date on ganglion stimulation is extremely promising, but further innovation is required to find a workable integration of ganglion stimulation into current clinical procedures. An inherent focus must be on limiting the evolution of the penumbra and minimizing the size of the ischemic core, with the primary goal of elongating the window-of-efficacy for mechanical thrombectomy or tPA. Solutions should look to benefit these current treatment options instead of supplanting them, and find a way to be used primarily at peripheral non-stroke centers.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AC is the guarantor. TB conceptualized the study. TB and JR developed the search strategy and drafted the manuscript. JR extracted the data. All authors contributed to the development of the selection criteria, risk of bias assessment strategy, and data extraction criteria and also read, provided feedback, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clostridium botulinum, Bacillus cereus and Halomonas sp. are anaerobes found abundantly across the samples. Our findings highlight the significance of metagenomics based diagnosis and detection of SARS-CoV-2 and other respiratory co-infections in the current pandemic to enable efficient treatment administration and better clinical management. To our knowledge this is the first study from India with a focus on the role of co-infections in SARS-CoV-2 clinical sub-phenotype.Co-infection with ancillary pathogens is a significant modulator of morbidity and mortality in infectious diseases. There have been limited reports of co-infections accompanying SARS-CoV-2 infections, albeit lacking India specific study. The present study has made an effort toward elucidating the prevalence, diversity and characterization of co-infecting respiratory pathogens in the nasopharyngeal tract of SARS-CoV-2 positive patients. Two complementary metagenomics based sequencing approaches, Respiratory Virus Oligo Panel (RVOP) and Holo-seq, were utilized for unbiased detection of co-infecting viruses and bacteria. The limited SARS-CoV-2 clade diversity along with differential clinical phenotype seems to be partially explained by the observed spectrum of co-infections. We found a total of 43 bacteria and 29 viruses amongst the patients, with 18 viruses commonly captured by both the approaches. In addition to SARS-CoV-2, Human Mastadenovirus, known to cause respiratory distress, was present in a majority of the samples. We also found significant differences of bacterial reads based on clinical phenotype. Of all the bacterial species identified, ∼60% have been known to be involved in respiratory distress. Among the co-pathogens present in our sample cohort, anaerobic bacteria accounted for a preponderance of bacterial diversity with possible role in respiratory distress.  The COVID-19 disease that emerged in Wuhan, China has spread across the globe in the past 1 year and assumed pandemic proportions. The infection is caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is a betacoronavirus of the Coronaviridae family . SymptomHaemophilus influenzae, Pseudomonas aeruginosa, Klebsiella spp. and Mycoplasma spp.  has aided the characterization and analysis of the genomic profile of not just the primary pathogen, i.e., SARS-CoV-2, but also of the associated microbiome, using the nucleic acids extracted from the respiratory specimens of patients . The curThe study was conducted by the CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB) in collaboration with the MAX Hospital, Delhi, India. Ethical clearance for the study was obtained from the Institutional Ethics Committee at the IGIB and the Max Hospital, respectively. A total of 100 patients with confirmed COVID-19 positive status, based on RT-PCR results, hospitalised in MAX Hospital were enrolled in the study. The nasopharyngeal and/or throat swabs along with a sputum sample were collected by the paramedical staff at the MAX Hospital on the day of reporting to the hospital. The tip of the swab was put into a vial containing 3 ml of Viral Transport Media (VTM) , by breaking the applicator’s stick and sealing the tube tightly. The tube was then vortexed for 2 min to allow the dissolution of sample into the VTM solution followed by centrifugation and allowed to settle for some time before processing. For sputum samples, 200 μl of sputum was added to 200 μl of Sputum Liquefaction reagent, thoroughly mixed and incubated at 37°C for 10 min.Viral RNA from VTM solution or liquified sputum samples was extracted using commercially available RNA extraction kit . 200 μl of VTM solution or liquified sputum was processed for lysing and viral enrichment, in accordance with the kit protocol . After washing with the wash buffers, viral RNA was eluted in RNase-free water. qRT-PCR for SARS-CoV-2 detection was performed using the TRUPCR SARS-CoV-2 kit . In brief, 10 μl of RNA was added to 15 μl of reaction mix in accordance with the kit protocol. The qRT-PCR was run on Rotor-Gene Q (Qiagen) using the recommended cycling conditions. The cycle threshold (Ct) value of 35 was considered for interpretation of the results.Double-stranded cDNA (ds cDNA) was synthesised from 100 ng of total RNA for all SARS-CoV-2 positive samples. The first strand of cDNA was synthesised using Superscript IV First strand synthesis system  followed by single-stranded RNA (ssRNA) digestion with RNase H for second strand synthesis using DNA Polymerase I Large (Klenow) Fragment . The cDNA was purified using AMPure XP beads  and quantified using NanoDrop . 100 ng of purified ds cDNA was used for library prep using the Illumina DNA Prep with Enrichment kit . The process involves tagmentation followed by cleanup and amplification leading to indexed DNA fragments. Following tagmentation and indexing, enrichment was performed using the Illumina RVOP , wherein 500 ng of each sample were pooled by mass in accordance with the reference guide  for the 12-plex hybridisation with biotinylated adjacent oligo-probes of the RVOP. The hybridisation was performed overnight after which the probes were captured by streptavidin-biotin based interactions. The final library was PCR amplified and purified before sequencing. The quality and quantity of the sequencing library was checked using Agilent 2100 Bioanalyzer with high sensitivity DNA chip and the Qubit dsDNA HS Assay kit, respectively. A loading concentration of 10 pM was prepared by denaturing and diluting the libraries in accordance with the MiSeq System Denature and Dilute Libraries Guide . Sequencing was performed on the MiSeq system, using the MiSeq Reagent Kit v3 (150 cycles) at 2 × 75 bps read length.Whole transcriptome sequencing was performed using the Illumina TrueSeq Stranded Total RNA Library Prep Gold . A total RNA input of 500 ng of each sample was depleted of cytoplasmic and mitochondrial rRNA by Ribo-Zero Gold, followed by fragmentation, first and second strand cDNA synthesis and purification with AMPure XP beads  in accordance with the reference guide . The purified ds cDNA was adenylated at 3′ends, ligated with index adapters, which were further enriched by PCR based amplification. The final cDNA libraries were purified using AMPure XP beads and quantified using Qubit dsDNA HS Assay kit . The quality of cDNA libraries was checked by Agilent High Sensitivity DNA Kit using the Agilent 2100 Bioanalyzer. A final loading concentration of 400 pM was prepared by denaturing and diluting the libraries. Sequencing was performed on the NovaSeq 6000 system, using the NovaSeq SP reagents v1 at 2× 101 read length.In this study, the total sample set was divided into two subsets with some overlapping samples across the two subsets. Fifty samples consisting of one subset were used for detection of viruses other than the SARS-CoV-2, using the Illumina Respiratory Virus Oligo Panel (RVOP). The second subset consisted of 48 clinical phenotype defined samples that were studied to explore the presence of microbes using Holo-Seq. Of the 48 samples, 21 samples were common with RVOP based study. The downstream analysis was automated using the Nextflow script. The methodology includes steps for base calling, quality check, removal of adapters (trimming), alignment, generation of consensus genome FASTA, and evaluation of species diversity, as well as coexistence and visualisation.FastQC was used to check the Phred quality score for all sequences . HencefoThe Heatplus package from R was used to plot the heatmap . CytoscaU test and Chi-square tests to compare the differences, wherever appropriate. The Kruskal Wallis test was used to compare the distribution of bacterial presence across our patient subgroups. The Shannon Diversity index (H) was calculated to characterise the bacterial species diversity as aerobes and anaerobes, in 48 patient samples used for the Holo-transcriptomics study, to account for the abundance and evenness of bacterial species in each patient sample  and whole transcriptome RNA-Seq (Holo-seq). RNA samples from 50 patients with COVID-19 were used for Illumina RVOP analysis followed by Holo-Seq of 48 samples with an overlap of a subset of 21 samples which were used for both the analyses . The selThe spider web plot represents the distribution of patient samples included in the RVOP (represented in red) and Holo-seq (represented in purple) analysis with respect to their associated clinical symptoms and comorbidities . The comp-value < 0.05   . Lower O= 0.009) . We foune < 0.05 . While te < 0.05 .2 levels and the requirement of respiratory support after hospitalisation.To understand the correlation between different clinical parameters, we plotted the data using scatter plot and correlation matrix . Also, RWe performed the genome sequencing of SARS-CoV-2 isolates from all 100 patients to discover the viral clades as well as the phylogeny. We identified two clades according to the Nextclade classification, i.e., 19A and 20B . Clade 1missing link, which might help fill in the gap. Given the fact, that the presence of co-infections during SARS-CoV-2 is still being understood with only a limited number of studies till date, we thought that it would be important to explore and elucidate the role and presence of co-infections in SARS-CoV-2 positive patients in the Indian context.The above observations seem to indicate that there is a limited viral genomic diversity in terms of the SARS-CoV-2 clades. However, a larger diversity of symptoms and severity in SARS-CoV-2 positive individuals suggests a possibility of a We conducted a target enrichment based sequencing of RNA isolates from 50 SARS-CoV-2 positive individuals using Illumina RVOP panel. Alluvial plot depicts the reads that mapped to 10 predominant viral strains identified by the HISAT aligner (left panel) along with the NCBI ID for reference genome (right panel) . Human CThough we detected 10 different viral strains, the coverage of the aligned genome from the identified viruses was below 50%. To delve further, we used taxonomic sequence classifier, Kraken, to identify the overall diversity of co-infecting viral strains. Pavian was used to visualize the output from Kraken. For analysis, we included only those viruses whose relative abundance across at least one sample exceeded more than 1% of the total read count. Applying this cutoff value, we identified 26 viruses whose presence was detected across 50 samples . Of all Following the identification of viral species, we tried to correlate the differential presence of respiratory viruses in patients with their clinical parameters. A heatmap for differential presence of viruses across patients was plotted using the Heatplus package. We investigated whether levels of SpO2, condition of breathlessness and requirement of respiratory support across our samples are correlated with viral diversity . It was Clostridium botulinum, Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Neisseria gonorrhoeae were discovered in almost all samples. We, thus, looked into sub-clinical classification of the SARS-CoV-2 positive individuals and checked if the differential abundance of bacteria would help explain the observed variation in clinical symptoms. The patients who had respiratory distress symptoms were divided into three subgroups based on the report of shortness of breath (SOB) at admission and/or requirement of respiratory support during the hospital stay (RS). The other group of patients who didn’t have any respiratory symptoms or requirements of respiratory support were classified into two control groups with differing age . The bacterial presence in the control+old group (31%) differed significantly from that in the RS group (31.62%), though both lie within the higher age bracket . We also found a significant difference in the bacterial abundance between control+young (35.81%) and SOB (40.01%) groups despite both falling in the lower age bracket . Also, the bacterial presence within the RS and SOB groups differed significantly, with the latter showing a higher bacterial presence . Interestingly, the average bacterial read count in the SOB group at 45.85% was much higher than the other four groups including the SOB+RS group, which had an average bacterial read count of 35.12%, although not statistically significant.For individual patients in all the five groups, we calculated the percentage of cumulative bacterial reads (%) as well as viral reads (%) . The perIn a subset of 21 patients, microbial diversity was explored through both Holo-seq and RVOP, in correlation with clinical sub-phenotype diversity. We set up a cutoff value of >5% relative abundance for bacteria and >1% relative abundance of viral species for inclusion in our analysis. The threshold value was selected based on the required confidence for species identification and to exclude minimal sequence similarity with related organisms. In this subset, we identified 42 bacterial species (red nodes) and 21 viral species (blue and yellow nodes) using the Holo-seq . When weTo further understand the role of viruses and bacteria in modulating the disease severity, we looked into the literature for the functional role of the viral and bacterial species that were identified in our study. Respiratory pathology being the hallmark of COVID-19 disease severity, we especially looked for their etiological relevance to respiratory infections and distress . ImportaClostridium botulinum, Bacillus cereus and Halomonas spp. were found in all the samples  [Finally, we also visualised the taxonomic distribution of identified bacterial and viral species in each individual patient through Sankey plots . The lefThe present study utilized two different metagenomic approaches to identify the diversity of co-infecting species present within the upper respiratory tract of COVID-19 patients from the Delhi-NCR  during the initial phase of the epidemic in India (April–May 2020). Metagenomic sequencing offers advantages over other targeted methods of pathogen detection by factoring out the limitations associated with ascribing a particular disease state to a single causative agent. It allows autarkic verification of the primary pathogen along with the underlying microbiome which may be playing an important role in the disease progression and prognosis. Out of the three potential core elements that determine the course and end-point of an infection, our study tried to capture the possible role of two, i.e., pathogenic variation and the airway microbiome of the host . To our knowledge, this is the first study from India aiming to elucidate the role of co-inhabiting species (virus and bacteria) in modulating the severity and trajectory of SARS-CoV-2 infection .To appreciate the role of nasopharyngeal microbiome in affecting the SARS-CoV-2 disease course, we compared the demographic and associated clinical parameters in two sub-groups of patients who required or who did not require respiratory support during their stay in the hospital. The dichotomy in the requirement of respiratory support showed a correlation with older age. This is in sync with the previous studies which have reported older age as a risk factor for COVID-19 severity owing to weakened immune responses . The preWithin our samples, we identified two clades of SARS-CoV-2 virus, viz. 19A and 20B. 19A is the ancestral haplotype with presumed origin in China. A majority of the viral isolates from our cohort belonged to clade 19A, which has been previously reported to be mainly present in Northern India. Clade 20B with origin from European nations, along with other A2a haplotypes, became dominant in India after June 2020  and bloodstream infections  in mechanically ventilated patients  where most pathogenic species survive as commensals and become virulent only when they invade the lower respiratory tract.In view of the magnitude of the problem and excessive burden on the healthcare workers, there are certain limitations with respect to sampling that limited the scope of the present study. First, the nasopharyngeal samples were obtained from the patients only at the time of admission and there was no longitudinal follow-up, which could have allowed a better evaluation of the dynamic change in nasopharyngeal microbiome during the course of SARS-CoV-2 infection, including, of hospital-acquired infections. Second, all the samples were restricted to Delhi-NCR region and, as there was a nationwide lockdown imposed in the country at the time, hence much variation in terms of viral genome and phylogeny cannot be expected in viral isolates. Finally, the dataset lacked any mortality cases.The findings presented herewith have tried to correlate the role played by co-infecting respiratory pathogens in modulating the SARS-CoV-2 disease trajectory. The findings do not suggest a pan-study causal role for a particular co-infecting pathogen, but it does indicate possibilities of disease modulation in patient sub-groups. The findings also assume significance in the context of widespread usage of antibiotics during the current pandemic which may have long term impact in terms of increased antimicrobial resistance and emergence of multi-drug resistant strains. A further investigation into the role of co-infections in SARS-CoV-2 positive individuals in an extended dataset with matched SARS-CoV-2 negative controls is warranted to enable better understanding and management of the current COVID-19 disease pandemic.PRJNA676016 and PRJNA678831, the consensus fasta are available at the GISAID-EpiCoV (https://www.gisaid.org/) under the submission IDs: EPI_ISL_459911-EPI_ISL_459944 and EPI_ISL_641320-EPI_ISL_641376, and the classification reports of RVOP and Holo-transcriptomics for all patient samples are available for visualization at https://doi.org/10.6084/m9.figshare.13560506.The datasets presented in this study can be found online at the NCBI-SRA under the accession numbers The studies involving human participants were reviewed and approved by CSIR-IGIB’s Human Ethics Committee Clearance (Ref No: CSIR-IGIB/IHEC/2020-21/01). The patients/participants provided their written informed consent to participate in this study.PM, SSH, and SSA performed analysis, made figures, and wrote the manuscript. NM performed analysis and made figures. VA and BU performed analysis. PC made figures. SSI, AT, SWW, SB, VN, PPH, AD, BT, POD, and SJ contributed to the clinicians who diagnosed patients and shared samples with IGIB. PrD and AK contributed to the manuscript related literature survey. RM contributed to the data upload and helped in analysis. PS, US, SAW, NT, and MF performed genomic experiments. AA initiated collaboration and facilitated work. RP designed, conceptualized, implemented and coordinated the study, along with inferences of the results and wrote the manuscript. All 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.


Among patients with prostate cancer who have been operated on, a subset harboring high-risk features will present with a biochemical recurrence (BCR). Adjuvant radiotherapy (aRT) was proven to significantly reduce the risk of BCR when compared to salvage radiotherapy (SRT) but suffered from several limitations: a lack of patient selection criteria, a higher treatment-related morbidity and an uncertain benefit for long-term clinical endpoints. In the same clinical setting, early SRT (eSRT) appears as non-inferior to aRT with a lower morbidity, replacing aRT as the preferred option. In this review, we insist on the need for multidisciplinary discussions to fully comprehend the individual characteristics of each patient and propose the best treatment strategy for every patient.Despite three randomized trials indicating a significant reduction in biochemical recurrence (BCR) in high-risk patients, adjuvant radiotherapy (aRT) was rarely performed, even in patients harboring high-risk features. aRT is associated with a higher risk of urinary incontinence and is often criticized for the lack of patient selection criteria. With a BCR rate reaching 30–70% in high-risk patients, a consensus between urologists and radiation oncologists was needed, leading to three different randomized trials challenging aRT with early salvage radiotherapy (eSRT). In these three different randomized trials with event-free survival as the primary outcome and a planned meta-analysis, eSRT appeared as non-inferior to aRT, answering, for some, this never-ending question. For many, however, the debate persists; these results raised several questions among urologists and radiation oncologists. BCR is thought to be a surrogate for clinically meaningful endpoints such as overall survival and cancer-specific survival but may be poorly efficient in comparison with metastasis-free survival. Imaging of rising prostate-specific antigen (PSA), post-operative persistent PSA and BCR was revolutionized by the broader use of MRI and nuclear imaging such as PET-PSMA; these imaging modalities were not analyzed in the previous randomized trials. A sub-group of very high-risk patients could possibly benefit from an adjuvant radiotherapy; but their usual risk factors such as high Gleason score or invaded surgical margins mean they are unable to be selected. More precise biomarkers of early BCR or even metastatic-relapse were developed in this setting and could be useful for the patients’ stratification. In this review, we insist on the need for multidisciplinary discussions to fully comprehend the individual characteristics of each patient and propose the best treatment strategy for every patient. Approximately 30% of operated on prostate cancer (PCa) patients will experience biochemical recurrence (BCR), this rising to 50–70% in very high-risk patients ,2,3. SinGiven the possible lack of benefit on OS and its higher toxicity profile, aRT was challenged by early SRT (eSRT) in three recently published RCTs ,7,8 poolSeveral reserves must be raised before closing the debate so rapidly. The number of events was quite low in the three trials with a relatively short follow-up. Despite a non-significant benefit in the ARTISTIC meta-analysis, the EFS favored aRT over eSRT in the GETUG-AFU17 and the RAVES trials. Inclusion criteria and study design substantially differed from one trial to another, especially regarding the inclusion/exclusion of patients with a lymph node invasion (LNI). Contemporary imaging modalities such as PET-PSMA were not used, and the performances of recent prognostic tools such as genomic tests  were notIn this review, we aim to revisit the place of aRT and eSRT in patients operated on with high-risk PCa and give an overview of the tools available for personalizing the best treatment selection for each patient.The prostate-specific antigen (PSA) level at the time of post-operative RT was proved to be critical, with a high correlation between the PSA value and the risk of bRFS . Pre-SRTAmong the three RCTs, only the SWOG S8794 trial used metastasis recurrence-free survival as the primary endpoint, while the EORTC 22911 used bRFS and ARO 96-02/AUO AP 09/95 a progression-free survival (PFS), but defined as the non-occurrence of BCR, local or distant clinical recurrence or death from any cause. The three RCTs met their respective primary endpoints, but both EORTC 22911 and ARO 96-02/AUO AP 09/95 failed to prove the benefit of aRT on MFS and OS (secondary endpoints). Results from these three RCTs were pooled in a meta-analysis conducted by the Cochrane Library . DespiteSince these three RCTs, a fourth RCT has also been presented confirming the bRFS benefit of aRT vs. observation but with a mean follow-up of 8.6 years in the observation group, with no benefit on MFS and OS .In spite of this level A data, aRT remained widely underused. For instance, in the United States of America, between 2004 and 2015, only 11.7% of the 189,240 eligible patients (adverse events on pathology reports) received aRT [Genitourinary morbidity after post-operative radiotherapy is correlated to the delay between surgery and start of RT and the patient’s complete functional recovery . Two con-When given (RADICALS-RT and GETUG-AFU-17), ADT artificially prolongs bRFS. Indeed, BCR under ADT at this stage of the disease almost never happens. On the aRT arm, ADT is administered shortly after the randomization and probably 1 to 2 months before the start of aRT. On the eSRT arm, ADT and eSRT could start concomitantly. This design adds a systematic bias that probably negatively impacts the aRT’s results-Because of the definition of BCR, patients with the same biochemical control can be classified differently only because of their affected treatment group. For instance, in the GETUG-AFU-17 trial: BCR will be reached as soon as the PSA rises to 0.4 ng/mL at least 6 months after RT completion for the aRT arm, whereas in the SRT arm, BCR will be reached at the time of the follow-up meetings several months after RT completion. Similarly, defining the bRFS from the date of randomization contributes to this statistical bias. This difference adds a systematic extension of the bRFS in the SRT arm that is problematic with such a short follow-up. This directly induces a possible bias as observed in the RAVES trial with a 5-year BCR-free rate of 86% and 87% for the aRT and eSRT arm, while the 8-year BCR-free rate fell to 80% and 75% for the aRT and eSRT arms, respectively [-The GETUG-AFU-17 trial was closed prematurely because of the low rate of events.Early salvage radiotherapy (eSRT) is defined as the delivery of SRT to patients with low level rising PSA values (PSA > 0.1–0.2 ng/mL). Use of eSRT is supported by solid retrospective data such as multi-institutional retrospective studies . With 51The results from the three RCTs SRT has focused specifically on patients with a pN1 status .LNI was an exclusion criteria for the three RCTs comparing aRT and observation ,4,5. RetAs previously stated, only two of the three RCTs pooled in the ARTISTIC meta-analysis allowed patients with LNI to be included. Patients with LNI represent a very small subset of the overall cohort (3.0%). No conclusions regarding the impact of LNI on the effect of RT timing could thus be proposed, leaving clinicians wondering about the generalizability of the aRT vs. eSRT results to the pN1 patients.A major possible confounding factor was the possibility (or non-possibility) of concomitant ADT, depending on the RCT. The addition of 24 months of ADT to SRT was significantly associated with longer MFS and OS in the RTOG 9601 trial . The valA note must be made regarding the design of the six RCTs focusing on eSRT, SRT and aRT. Apart from the RADICALS trial, none of the RCTs were designed or powered enough to evaluate the impact of ADT in the post-operative setting.All the data seem to converge towards a single challenge, namely the selection of patients for either aRT or (e)SRT. Numerous clinical features are taken into account, with a consensus being adopted on the definition of high-risk and very high-risk patients. Data on high-risk patients are temporary awaiting a longer follow-up, while data on very high-risk patients are scarce or unavailable. The ARTISTIC meta-analysis used the CAPRA-S risk score to combine risk features and stratify the included patients but did not find a significant benefit of aRT over eSRT, even in patients with a high CAPRA-S score. Being an aggregate study-level analysis and not an individual patient-level analysis, all subset analyses in the ARTISTIC meta-analysis were probably underpowered. Before detailing other selection modalities, the importance of a central pathology review must be stressed ,29. PathCharacterization of the disease with genomic or radiomics approaches also appear as an efficient tools for patients’ selection. In this setting and on a sub-analysis of 486 patients, a genomic-classifier was significantly correlated with MFS and OS . The DecAs presented, several key clinical issues were defined to summarize the abundance of literature on the aRT vs. (e)SRT debate. Data regarding aRT vs. SRT and aRT vs. eSRT were limited to published RCTs, while data regarding the impact of LNI and ADT were limited to RCTs or large and/or multicentric retrospective studies. This methodology has the advantage of a clear and concise report but with a risk of selection bias. However, the goal was mainly to discuss current literature data and offer practical insights on present and future perspectives in this clinical setting.With a 5-years follow-up, adjuvant radiotherapy does not appear to significantly impact MFS and OS over eSRT in high-risk PCa patients. These aRT vs. eSRT RCTs confirmed the low value of bRFS as an intermediate surrogate of MFS and OS  and prob


Pseudomonas aeruginosa isolates for their antimicrobial susceptibility profiles under biofilm growth conditions towards the antibiotics ciprofloxacin, tobramycin, and colistin. We discovered characteristic patterns of drug-specific killing activity and detected conditional tolerance levels far lower ), but also far higher  than generally believed. This extremely broad distribution of biofilm-induced tolerance phenotypes across the clinical isolates was greatly influenced by the choice of the antibiotic. We furthermore describe cross-tolerance against ciprofloxacin and tobramycin, but not colistin, and observed an additive activity between biofilm-induced tolerance and genetically determined resistance. This became less evident when the biofilm-grown cells were exposed to very high antibiotic concentrations. Although much more remains to be learned on the molecular mechanisms underlying biofilm-induced tolerance, our data on intra-species variations in tolerance profiles provide valuable new insights. Furthermore, our observation that colistin appears to act independently of the tolerance mechanisms of individual clinical strains could make colistin a valuable therapeutic option in chronic biofilm-associated infections characterized by the presence of particularly tolerant strains.Biofilm-associated bacteria exhibit profound changes in bacterial physiology. They thrive in the environment but also in the human host in protected sessile communities. Antimicrobial therapy usually fails, despite the absence of genotypic resistance, and it is commonly accepted that biofilm-grown bacteria are up to 1,000-fold more resistant than planktonic cells. We are only at the beginning to understand the reasons for biofilm recalcitrance, and systematic approaches to describe biofilm-induced tolerance phenotypes are lacking. In this study, we investigated a large and highly diverse collection of 352 clinical  Frequent use of antibiotics favors bacterial strains that have acquired mechanisms to overcome drug inhibition and lethality. Various genetically inherited mechanisms have been identified in bacteria that reduce the efficacy of a drug. These include, for example, mutations of the target structures, enzymatic inactivation of the antibiotic, or the reduction of its intracellular concentrations through reduced influx and/or increased efflux. The phenotypic consequences of genetically conferred resistance are usually monitored by the use of standard antimicrobial susceptibility testing (AST), which largely relies on the determination of the minimal inhibitory concentration (MIC) values of the antibiotic. While the assessment of MICs has a significant impact on the choice of a therapeutic strategy, it has been argued that the predictive power of MIC measurements is less suitable for the implementation of successful treatment regimens in patients with chronic, biofilm-associated infections . The abiThe biofilm mode of growth enables bacteria to evade the attacks from the immune system and to survive exposure to high concentrations of antimicrobial agents. It is commonly accepted that bacteria exhibit an up to 1,000-fold increased tolerance towards a broad range of different classes of antibiotics under biofilm- as opposed to planktonic growth conditions  e.g. . Of notePseudomonas aeruginosa isolates and determined their susceptibility profiles against the three commonly used antibiotics tobramycin, ciprofloxacin, and colistin under biofilm growth conditions. We describe an extremely broad distribution of conditional tolerance phenotypes across the various clinical isolates, which are dependent on the individual strain background and influenced by the choice of the antibiotic. We furthermore describe an additive activity between biofilm-induced tolerance and genetically determined resistance, which however becomes less apparent if the biofilm-grown cells are exposed to very high antibiotic concentrations.In this study, we investigated overall 352 clinical P. aeruginosa isolates, which have been previously collected across Europe . The plate was sealed with an air-permeable BREATHseal cover foil (Greiner Bio-One) and incubated without shaking at 37°C in humid atmosphere. After 24 h, 60 μl of antibiotic solution in diluted LB media were carefully added, resulting in a total volume of 160 µl per well. Antibiotic solutions were adjusted to final concentrations of 1; 4; 16; 64; 256; and 1,024 μg/ml. For the growth control, 60 µl diluted LB media (1:3 in deionized water) without antibiotics were added. The antibiotic exposed biofilms were incubated for 24 h and subsequently resuspended by the use of a multichannel pipette. Tenfold serial dilutions were spotted onto rectangular LB agar plates and incubated at 37°C. Growth was evaluated after 16 h of incubation and further monitored over a period of 48 h to ensure that all surviving bacteria were detected. Colony-forming units (CFU) per ml were calculated for surviving bacteria.We performed large-scale antimicrobial susceptibility testing of the clinical isolate collection for tobramycin, ciprofloxacin, and colistin under biofilm growth conditions as previously described . In brieAntibiotic concentration-dependent killing of biofilm-grown cells was determined based on the reduction of CFUs following the addition of different antibiotic concentrations as compared to an untreated control well. The number of surviving bacteria for each clinical isolate for overall six different concentrations of the three different antibiotics was recorded. From those data the minimum antibiotic concentrations of killing (MCK) that resulted in a CFU reduction of biofilm-grown cells of at least 1-log, 2-log, 3-log, 4-log, 5-log, 6-log or eradication (below the detection limit) could be deduced.To quantify biofilm-induced tolerance of the individual isolates, the biofilm tolerance factor (BTF) was introduced. The BTF results from the ratio of the concentration that is required to achieve a certain log-CFU reduction in biofilm-grown cells, and the respective MIC of the strain:BTF: Biofilm tolerance factor; MCK(CFU log-reduction): minimum antibiotic concentration of killing at which a certain log-reduction  in the CFUs of biofilm-grown cells was observed; MIC: minimal inhibitory concentrationAn integrated analysis of biofilm tolerance factors (BTFs) for the three antibiotics tested was used to uncover cross-tolerance and characteristic patterns in the tolerance behavior of biofilm-grown cells. Only discrete BTFs were used, where the highest antibiotic concentration tested  resulted in a measurable reduction of CFUs. BTFs were log2-transformed prior to further analyses.Correlation analyses were performed in order to access cross-tolerance. Correlation coefficients for log2-transformed BTFs for the different antibiotics were calculated pairwise using the Correlation analysis tool in Microsoft Excel. Simple linear regression was calculated and plotted in GraphPad Prism (v 8.3.0). A hierarchical clustering approach was performed using the web tool ClustVis  to groupP. aeruginosa strains isolated from various infection sites  under planktonic conditions. Some clinical Of note, several clinical isolates showed a remarkable biofilm recalcitrance, particularly towards tobramycin and ciprofloxacin. Even the highest concentration applied in this screening  was not sufficient to reduce the CFUs of biofilm-grown cells  required for a certain reduction in CFUs were very different across the individual clinical strains. To evaluate whether killing efficiency is dependent on the underlying MIC, we sorted the 352 clinical strains according to their MIC values and depicted the MCKs for those clinical strains that exhibited a MIC around the CLSI breakpoints Figure 2It is commonly claimed that biofilm-grown bacteria exhibit an up to 1,000-fold increased tolerance towards antibiotics as compared to their planktonically grown counterparts. In order to provide a better data basis, we determined the biofilm tolerance factor (BTF) of the clinical isolates against the three tested antibiotics. The BTF indicates which multiple of antibiotic concentration under biofilm-growth conditions as compared to the MIC (determined under planktonic growth conditions) must be used to achieve a CFU reduction of at least 1 log (BTF-1), 2 log (BTF-2), 3 log (BTF-3), 4 log (BTF-4), 5 log (BTF-5), 6 log (BTF-6) units or to observe eradication (BTF-E) below the detection limit  was sufficient to achieve a 2-log (99 %) CFU reduction of biofilm-grown bacteria (We found that for 1-log CFU reductions the median biofilm tolerance factor (BTF-1) across the clinical isolates was 4 for tobramycin, 2 for ciprofloxacin and 32 for colistin. This factor shifted to a median BTF-E (biofilm tolerance factor for eradication) of 256 for tobramycin, and 1,024 for ciprofloxacin, while it reached the maximum of 128 for colistin already at BTF-2 Figure 3Our data also illustrate that the distribution of the BTFs among the clinical isolates was different in the three antibiotics tested. The fraction of clinical isolates that were effectively reduced by 2-log already at a low BTF was highest for ciprofloxacin. However, the proportion of isolates that required a high BTF to achieve a substantial reduction in the CFU of biofilm-grown bacteria was also highest for ciprofloxacin  between the killing efficiency of ciprofloxacin and tobramycin (reduction of 99.99 % of the biofilm-grown cells (BTF-4), In order to characterize the conditional tolerance phenotype of the individual clinical isolates in more detail, we integrated all data on the BTFs of the three antibiotics and performed a hierarchical cluster analysis across all clinical isolates. We identified six clusters of clinical strains with characteristic patterns in their biofilm tolerance factors . Those have been isolated from various infection sites and from acute and chronic infections. As opposed to previous studies, which had introduced the minimum duration of killing (MDK) as a quantitative indicator for tolerance of planktonic cultures in time-resolved killing experiments , a concentration of 256 µg/ml or more was required to achieve the same biofilm killing in 31 % of the isolates.Variation in tobramycin MCKs was lower, while the clinical isolates responded most consistently to colistin. The great majority of the clinical isolates (90 %) were eradicated at a colistin concentration of 256 µg/ml. While this concentration was required to kill at least 90 % of the biofilm-grown bacterial population, the same colistin concentration was also sufficient to eradicate the biofilm-grown cells to levels below the detection limit.Our observation of a broad distribution of the MCK values across the clinical isolates is remarkable and suggests that while the biofilm growth mode in itself is sufficient to explain tolerance in clinical isolates, the respective tolerance levels seem to be largely modulated by the individual strain background. Interestingly, biofilm-induced tolerance against ciprofloxacin and tobramycin, but not colistin, were correlated in the individual clinical isolates. This result underlines that the conditional tolerance phenotype seems to be non-specific and is determined both, by the biofilm growth pattern and by additional isolate-specific, tolerance-promoting mechanisms that help bacteria survive transient exposure to high antibiotic concentrations. Despite extensive research on the identification of bacterial factors that determine tolerance-promoting phenotypes, we are only at the beginning to understand the molecular mechanisms that drive bacterial tolerance. It seems that changes in the activity of the tricarboxylic acid (TCA) cycle, cellular respiration, the proton motive force as well as shifts in the intracellular pH drive bacterial tolerance phenotypes . FurtherDespite the large variability in the MCKs, our extensive data on many clinical isolates allowed the detection of a positive correlation between the MIC values of the individual isolates and the concentration of antibiotics (MCK) that was required to kill biofilm-grown cells. These results suggest that genetically inherited resistance mechanisms affect the biofilm-induced tolerance phenotype. In general, strains found to be resistant as categorized by elevated MIC levels also required high concentrations of antibiotics to achieve biofilm reduction of 4-logs or more. A synergistic interaction between tolerance and resistance has been described previously  and it hStrikingly, at the same time we observed that the strains exhibiting high MIC values seemed to express lower biofilm-specific tolerance factors (BTFs) as defined by the multiple of the MIC concentration required to kill the biofilm-grown population. Thus, it seems that once a certain threshold concentration of an antibiotic is reached, biofilm-grown bacteria are efficiently killed, independent on their individual genetically determined resistance profile. A recent study demonstrated that antibiotic exposed strains developed either high-level multidrug tolerance or antibiotic resistance, but not both . It coulIt has already been shown several times that antibiotic susceptibility profiles of biofilm-grown cells cannot be predicted on the basis of MIC profiles. Thus MIC determinations cannot serve as an approximation on which is the best therapy to apply to treat patients suffering from chronic biofilm-associated diseases . HoweverThe original contributions presented in the study are included in the article/JT, conception and design, acquisition and curation of the data, analysis and interpretation of the data, drafting and revising the manuscript. SH, conception and design, analysis and interpretation of the data, drafting and revising the manuscript, funding acquisition. All authors have read and agreed to the published version of the manuscript.SH was funded by the European Union  and received funding as part of the excellence cluster RESIST . Furthermore, SH received funding from the German Research Foundation (DFG SPP 1879) and the Novo Nordisk Foundation (NNF 18OC0033946).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.


Complementarity stemming from the difference in properties and manifestations of the two mechanophores is essential. That is, the more labile Rh allows shifting the appreciable optical changes to a much lower force threshold; the transient nature and high dynamic range of mechanochemiluminescence from Ad map in real time where and when many of the covalently incorporated dioxetane bonds break; besides, the disrupted yet non-scissile structure of Rh acts as a fluorescent acceptor to effectively harvest chemiluminescence from ruptured Ad. The current strategy is thus empowering multi-functional mechano-responsive polymers with greatly improved sensitivity and resolution for multimodal stress reporting.Self-reporting polymers, which can indicate damage with perceptible optical signals in a tailored force range, are useful as stress-sensitive sensors. We demonstrate a simple approach to realize this function by embedding two distinct mechanophores — rhodamine (Rh) and bis(adamantyl)-1,2-dioxetane (Ad), in polyurethane/polylactic acid blends. The deformed blends generate red coloration and red chemiluminescence. Such a unique dual-responsive behavior was evaluated by solid-state UV-vis spectroscopy, macroscopic tensile tests with  Probing bond scission process in a broader force range was realized by embedding mechanochromic rhodamine and mechanochemiluminescent 1,2-dioxetane in polymer blends. However, exhibiting a more or less persistent mechanochromic response, the non-scissile, reversible Rh delivers limited temporal resolution in recording chain scission events.26,27 In this sense, real-time monitoring of bond breakage is possible when bis(adamantyl)-1,2-dioxetane is integrated as a chemiluminescent stress probe.17 Mechanical dissociation of the dioxetane ring leads to the excited adamantanone that relaxes to the ground state with blue auto-luminescence (ca. 420 nm). Unlike Rh, benefiting from the wide dynamic range of chemiluminescence without external excited sources required, Ad-derivatized polymers feature high spatial and temporal resolution of mechanochemiluminescence, which results in highly localized emission in front of a propagating crack tip.28 But due to the low quantum efficiency of the transient blue emission from broken Ad, its mechanical monitoring has to be performed in the dark. Another limitation lies in the high stability of Ad, so that its force threshold is fairly high.29Rhodamine (Rh) and bis(adamantyl)-1,2-dioxetane (Ad) have been demonstrated as versatile self-reporting mechanophores .17,19 Wh2 Firstly, orthogonal optical properties (absorption and luminescence) can be employed for multimodal analysis of damage and stress. Secondly, the force threshold that triggers discoloration and luminescence or the sensitivity of the rate to the applied force can be adjusted facilely.Our motivation was to create more sophisticated mechano-responsive polymers to self-report excessive stress with clearly perceptible signals, meanwhile with the rupture force tailored over a broad range. Rather than synthetic elaborations of mechanophores, combining mechanochromic and mechanochemiluminescent moieties is a straightforward and useful approach.26,29 we herein incorporated the two distinct mechanophores into polymer blends of polyurethane and polylactic acid that displayed successive occurrences of mechanochromism and mechanochemiluminescence. The rupture of the Rh ring was observed at a low strain, followed by the breakage of Ad, concomitant with intense red emission through effective fluorescence resonance energy transfer (FRET) from excited adamantone to the opened zwitterion of Rh  the amorphous parts facilitate the formation of hydrogen bonds with polyurethane and the ordering of the amorphous phase is responsive and tunable under tension, which are effective in toughening polyurethane.37,40 Translucent polymer blends PU/PLLAs with varied ratios of PLLA from 0 to 20 wt% were prepared via the solution casting method  mainly due to the restricted chain movement by hydrogen bonding between Rh-Ad-PU and PLLA; then, shifted to a lower temperature  with the emergence of the melting peak for PLLA at higher PLLA content, which could be ascribed to the increase of immiscibility between polyurethane and PLLA crystals.42–44For that, PLLA existing in the forms of crystalline and amorphous phase in blends was selected as a polymeric filler. The chemical and physical features of PLLA play an important role in the ultimate performance profile of the multiphase blends: (1) the crystalline domains of PLLA with high modulus and their orientation behaviors under applied force can reinforce the blends with increased tensile strength and toughness;44–46 Meanwhile, PU/PLLAs are mechanochromic, as can be readily distinguished by the naked eye .In darkness, transient intense light from the stretched blends was recorded by using a high-speed camera. The evolution of mechanoluminescence from a representative film demonstrated its high spatial and temporal resolution . BesidesTo gain a better understanding of the mechanochemical transduction from these self-reporting blends, the effects of PLLA content and strain rate on the mechanochromism and mechanoluminescence were studied. The total light intensity was found to correlate with mechanical stress. As shown in −1 to 0.2 s−1 increased the mechanical properties dramatically in terms of strength and toughness. Also, the slower strain rate on the sample led to a more significant color change and luminescence intensity ,26,29 their chemical conversion under tension is unpredictable. Revealing the competitive mechanochemical process of Ad and Rh is difficult in theory. This is because the mechanochemiluminescence of dioxetane follows the non-adiabatic dissociation mechanism that largely precludes the well-used quantum-chemical calculations of force-dependent kinetics.27 Pleasingly, herein experimentally, we found the successful FRET process under tension  were prepared for optomechanical studies  and ca. 357% (ca. 214 MPa), respectively. On the other hand, vs. strain for PU/PLLA-10% (Video S1 and S2ca. 260% (ca. 80 MPa); further elongation until a strain of ca. 310% (ca. 180 MPa) triggered the activation of Ad. Valuable information deduced from these data is as follows: (1) a distinct difference in the mechanical activation between Rh-PU/PLLA-10% and Ad-PU/PLLA-10% was indeed observed, which supported that many of the Rh units could be mechanically activated before Ad. (2) The existence of an overlapping stress region for chromism and luminescence implicated the simultaneous breakage of a fraction of the two mechanophores after many of the Rh rings were opened under low stress. (3) The force thresholds for the respective activation of Rh-PU/PLLA-10% and Ad-PU/PLLA-10% were similar to those of PU/PLLA-10%, except for the slight “shifting” of the chemiluminescent transition to the lower strain in our bis-mechanophore system mainly due to the additional FRET process to sensitize the luminescence signal. Therefore, our results indicated that the difference between Rh and Ad didn't translate into an observable impact on the activation of PU/PLLA-10% in the bulk state, or the mechanochemical coupling effect on the scission rate of adjacent Rh and Ad was not sufficient enough to be detected.52When different mechanophores were coupled closely, a reduction in their mechano-activation was very likely anticipated since they shared the overall stress at separate sites.via two different manifestations, but also the stress history can be captured, in that the distinctive change of chemiluminescence color from blue to red recorded a prior force-loading on the Rh moieties  into polymer main chains. The two mechanophores can be activated and exhibit red discoloration and red chemiluminescence, respectively, according to the different strengths of mechanical stimuli. Taking advantage of the two mechanophores, the yielded macroscopically detectable optical signals from these blends have been proved as stress-dependent, with high resolution to report the evolution and localization of damage at the molecular scale, which cannot be realized by using one mechanophore alone. Furthermore, the fact that mechanically generated discoloration takes place obviously earlier than that of luminescence, not only indicates an expanded responsive force range, but also in turn, confirms that the activation energy of Rh is less than that of Ad under mechanical conditions. Our strategy thus is applicable for the comparison of the bond energy of different mechanophores, even if it is difficult theoretically. Also, setting aside the enormous synthetic efforts on new multi-functional mechanophores, the present approach is versatile in unravelling and enhancing mechanochemical processes at the molecular level; thus, it can facilitate the full-day stress detection in a mutimodal analysis.There are no conflicts to declare.SC-012-D0SC06140A-s001SC-012-D0SC06140A-s002SC-012-D0SC06140A-s003


Coronavirus disease 2019 (COVID-19), which was named by the World Health Organization (WHO) in February 2020, has quickly spread to more than 200 countries around the world and was declared as a global pandemic in March 2020. The severity of the disease makes it more prone to severe symptoms and higher mortality rates in patients, especially those who are with comorbidities, including high blood pressure, cardiovascular disease, obesity, and diabetes, increases the concern over the consequences of this pandemic. However, initial reports do not clearly describe whether diabetes itself or associated comorbidities or treatment strategies contribute to the severe prognosis of COVID-19 infections. Various clinical trials are being conducted on glucose-lowering agents but to date, there is no standard treatment protocol approved for COVID-19 cases with pre-existing diabetes. This review is aimed to decipher the potential risk factors of COVID-19 involved from existing evidence. Identification of a novel therapeutic strategy could be beneficial for combating SARS-CoV-2, which might be dreadful to debilitating people who have diabetes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause for coronavirus disease 2019 (COVID-19), has infected more than 126 million people in the world and over 2.76 million deaths have been reported worldwide at the time this review was written . The COVStudies found that having one or more comorbidities is linked to the increased severity of COVID-19. In a systematic review and meta-analysis with a total number of 46248 confirmed cases, data showed that hypertension and diabetes are the most common comorbidities among COVID-19 patients , 8. StudHowever, whether diabetes per se or together with the concomitant comorbidities contribute to the worse prognosis remains to be fully uncovered. Therefore, this review aims to highlight potential risk factors in patients with COVID-19 comorbid with diabetes. We will also discuss the specific therapeutic strategies being used for people who have both COVID-19 and diabetes.COVID-19 morbidity and mortality are higher in older and male individuals. In a systematic literature review and meta-analysis of 13 studies including a total number of 3027 COVID-19 patients and in aged patients over 65, Zheng et al. reported a greater risk of mortality and more comorbidities such as hypertension, diabetes also greatly affecting the prognosis of the COVID-19 . In a laA preliminary study showed an overall even distribution of SARS-CoV-2 infections between men and women  . Howeverin vitro and animal studies have demonstrated that ACEI and ARBs increased the expression of ACE2, and those drugs could facilitate the infection of target organs and exacerbate COVID-19 disease progression (High blood pressure (hypertension) is the most common comorbidity in severe COVID-19 patients . Studiesgression , 29. Morgression . In recegression  and COVIgression  and alsogression .A high prevalence of the cardiovascular disease has been observed in patients with COVID-19  and clinRecently, several cases of stress-induced cardiomyopathy with COVID-19 have been reported, while the past medical histories of these patients were unremarkable –47. The In summary, these findings suggest that cardiovascular disease is not only a common symptom of COVID-19 but also a risk factor for poor prognosis. Although we do not understand the mechanisms underlying COVID-19-related cardiovascular disease, this could be largely attributable to systemic inflammation based on the available clinical findings.2) is one of the strongest hospitalization risks for COVID-19 positive patients. This finding was consistent with the results from the CORONADO study. It was found that in people with diabetes hospitalized for COVID-19, BMI (>40 kg/m2) was independently associated with the severity of COVID-19 but not long-term glycemic control assessed by HbA1c , an increased neutrophil-to-lymphocyte ratio and increased serum levels of inflammatory cytokines and chemokines have been associated with COVID-19 disease severity and death , 60. TheDiabetes is characterized by impaired glucose homeostasis resulting from insulin resistance or deficiency. As we know, diabetes is a well-established risk factor and predictor for elevated morbidity and mortality in various diseases such as cardiovascular diseases, cancer, and infection diseases –73.In a cohort of 1561 patients with COVID-19 in two hospitals from Wuhan, those with diabetes are more likely to require an intensive care unit (ICU) admission or to die . In a ceAs we know, the virulence of some pathogens is increased during the hyperglycemic environment. It was reported that phagocytosis and chemotaxis are impaired and the production of T cells and neutrophils in response to infection is also reduced in diabetes . Generalde novo diabetes development.A recent study reported that COVID-19 patients with diabetes and uncontrolled hyperglycemia  were associated with longer hospitalization and higher mortality . StudiesIn summary, uncontrolled glycemic at admission and hospitalization exacerbate poor outcomes of COVID-19 patients. In COVID-19 patients with hyperglycemia, therapeutic strategies combined with glycemic control should be considered to reduce the risk of severe outcomes and mortality.Medical teams should ensure sufficient glycemic control in COVID-19 patients with diabetes. This requires full consideration of all potential complications the therapies may generate which will be used for those patients.Insulin treatment is a general therapy for both types of diabetes. However, insulin therapy should be decided based on the severity of COVID-19 and those patients should be intensely monitored, although this treatment has been recommended in severe COVID-19 patients with diabetes . One stuSodium-glucose transporter 2 inhibitors should also be treated with caution due to their adverse effects such as ketoacidosis and impaired fat metabolism . BesidesAs we know, it is a multistep process for virus infection. Researchers have proposed several targets to treat COVID-19. The beneficial effects of ACEI and ARBs for kidney and heart in diabetes have already been proven . HoweverGlucocorticoids are known to cause hyperglycemia in patients with or without pre-existing diabetes. However, it has been used for the treatment of severely ill patients to suppress the very high levels of cytokines and c-reactive peptides which are often observed in those patients, although they can exacerbate insulin resistance, reduce insulin sensitivity and cause severe hyperglycemia. No studies have found that they could decrease mortality or slow viral clearance in clinical.Patients with COVID-19 and diabetes are at greater risk for more severe infections, poorer prognosis and much higher mortality compared to those patients without diabetes. The grave prognosis and the risk factors for patients with diabetes are well linked with older age, sex, high blood pressure, cardiovascular disease, obesity, inflammation and hyperglycemia. All those factors contribute to the increased risk of getting severely ill in those individuals. It is a great challenge for blood glucose management in COVID-19 patients because it requires more detailed strategies for medical team integration and fully considerations of all possible complications and death.de novo diabetes development.It is also clear that the relationship between diabetes and COVID-19 is tightly linked together and it requires more research to fully uncover the specific mechanisms of SARS-CoV-2 such as how SARS-CoV-2 impairs the pancreatic islets, deteriorates insulin homeostasis and induce SJ and WH wrote different sections of the manuscript. WH revised, wrote, and prepared the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Department of Science and Technology of Liaoning province (2019-ZD-0774).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.


On March 12, the World Health Organization declared a pandemic following the exponential increase of SARS-CoV-2 cases. The rapid spread of the virus is due to both its high infectivity and the free circulation of unrecognized infectious cases. Thus, diagnostic testing is a key element to prevent further dissemination of the virus. Urged by WHO's call, laboratories worldwide have been working on nucleic acid tests protocols and immunoassays that became available, albeit poorly validated, within a comparatively short time. Since then, external studies evaluating these diagnostic tests have been published. The present study is a review of the COVID-19 diagnostic approaches, discussing both direct and indirect microbiological diagnoses. A compendium of the literature on commercial assays kits available to date is provided together with the conclusions drawn as well as RT-PCR protocols published by the WHO. Briefly, diagnostic accuracy varies according to time elapsed since symptom onset and evolves together with understanding of the COVID-19 disease. Taking into account all these variables will allow determining the most adequate diagnostic test to use and how to optimize diagnostic testing for COVID-19. Both thDiagnostic tests must be specific enough to discriminate SARS-CoV-2 from other CoV with which it shares a high degree of homology . Thus, sThe present review aims to describe the current approaches to SARS-CoV-2 diagnosis including the different types of tests available and the current limitations and successful findings achieved during the previous 7 months of testing. Based on the current evidence and what has been learned to date about the infectivity and physiopathology of the virus, as well as the kinetics profile of the specific antibodies to SARS-CoV-2, we provide suggestions on how to optimize diagnostic testing for COVID-19 and the usefulness of antibody detection.Urged by the call from the WHO to develop reliable diagnostic tools, laboratories worldwide have developed several commercial assays that have been granted an Emergency Use Authorization (EUA) by the Food and Drug Administration . Among aSince the beginning of the pandemic, 7 different in-house developed molecular assays protocols (RT-PCR) have been posted on the WHO website . These pWith regard to immunoassays and Ag-RDTs, website of the Foundation for Innovative New Diagnostics (FIND) lists all the SARS-CoV-2 tests commercially currently available or under development , 38. DiaTo date, rRT-PCR is considered the gold standard technique for the diagnosis of SARS-CoV-2 infection since the symptomatology is non-specific and inconclusive, and other biological markers are non-exclusive of SARS-CoV-2 , 41. As rdRp gene assay for further discrimination of SARS-CoV-2 RNA from SARS-CoV RNA  is the first to have been published and is one of the protocols most commonly implemented by laboratories worldwide , 27, 43.-CoV RNA . Upon re-CoV RNA .Sarbecoviruses, since there was still not enough information of the virus genetic diversity. When aligned with the SARS-CoV-2 genome sequences, which were gradually being posted on GISAID, the primers were confirmed to match perfectly. Sequence variations among genome targets were translated into degenerate nucleotides on the primers. With the exception of the novel SARS-CoV-2 and SARS CoV, no other Sarbecoviruses have ever been detected in humans  using the ry assay . The US t result . Little t result ; Table 2t result .During the months that followed, WHO protocols were widely implemented in laboratories worldwide. Since then, some studies have assessed and compared the sensitivity and specificity of the different RT-PCR, reporting their limitations and assisting laboratories in their choice of protocol.2 viral RNA copies/μL concentrations while other assays were able to detect the RNA ]  was found to have the lowest sensitivity [it only partially detected SARS-CoV-2 RNA for all 10–10es <40)] . Etievanes <40)] .Vogels et al. reported that the E (Charité), the ORF1 (HKU Med), the N (HKU Med), the N (China CDC), the N1 (US CDC), and the N3 assays (US-CDC) were the most sensitive primer-probe sets, being able to detect SARS-CoV-2 at 1 and 10 viral RNA/μL . The stuThe main difference between the Vogels and Etievant studies is the HKU primer-probes assessment. While Vogels states that the ORF1 assay (HKU Med) is one of the most sensitive assays, the Etievant study places it below the N1 (US CDC) and the N assays (China CDC). The discrepancy in sensitivity in the case of the ORF1 assay (HKU Med) may be because Vogels standardized PCR conditions for primer-probes comparisons and did not reproduce the protocols characteristics, unlike Etievant, who did follow different PCR conditions. While it's not clear whether this influences the sensitivity, Vogels reproduced a more realistic scenario since not all laboratories will be able to work within protocols conditions. Another possible explanation could be the use of different RNA extraction kits: Etievant extracted nucleic acid using the EMAG platform  while Vogels used the MagMax Viral/Pathogen nucleic acid isolation kit (Thermofisher). Concerning specificity, background amplification was not observed in any of the nasopharyngeal swabs collected prior to the COVID-19 tested with primer-probe sets .E gene and RdRp gene tests cross-contaminated with synthetic controls.Although all the protocols have been considered reliable for achieving an accurate diagnosis, some irregularities have been noted. Etievant reported that the E (Charité) and the N2 assays (US CDC) were positive for all the dilutions tested including negative samples and controls. When analyzing amplicon size, both unspecific amplification and contamination were noted. It has been previously reported  that somRdRp_SARSr (Charité) contains a degenerate nucleotide (S) on the corresponding nucleotide (12th position of the primer) that is intended to pair with G or C (nucleotides found at this position on SARS-CoV and bat-SARS-related CoV genomes). The degenerated nucleotide was purposely added to help the primer anneal to SARS-CoV and bat-SARS-related CoV genomes (2019-nCoV_N3 (US CDC) forward primer annealing (CCDC-N (US China) primer target sequence, do not seem as critical since their placement upstream on the primer does not seem to compromise their capability to anneal and amplify (With regard to the RdRp assay (Charité), low performance , 36, and genomes . This sunnealing . This wannealing . The thr amplify .With the posting of the SARS-CoV-2 sequences from the beginning of the pandemics, GISAID has updated the information regarding the variability of the primer target sequences. The last update in March 2020 reported that the N (from China CDC and HKU Med) and the N3 assays (US CDC) had the highest rate of mutations in the 3′ end of the primer (defined as the last 5 nucleotides of the primer sequence). These mutations could partially compromise sensitivity .Even though RT-PCR is considered the gold standard technique to diagnose COVID-19, recurrent notifications of false negatives have cast doubts on this methodology. Although most false negatives can be associated with poor sample collection usually of nasopharyngeal or oropharyngeal swabs, false negatives can also be attributed to the technique itself, aside from personnel skill, and to a lack of knowledge of the characteristics of the virus , which is needed to address where and when to detect the virus .Sarbecoviruses family, and second, in using specific probes for the further discrimination of SARS-CoV-2. This was done on purpose since at the time the first molecular assays were developed, little was known about the genetic diversity of the virus. Targeting a broad range of specimens implies designing primers that recognize a range of variability in detriment of specificity, thereby resulting in a higher number of false negatives. New approaches have attempted to identify SARS-CoV-2 directly, by establishing globally conserved targets, such as the COVID-19-nsp2 or the COVID-19-RdRp/Hel assays  (8 copies per throat swab), the same time that it takes for symptomatology to appear. By the time patients are admitted to hospital and testing is performed, the shedding peak in the upper respiratory tract has already been reached, and possible contagions have already occurred   and by der swab) . Symptomoccurred . The virFinally, successful virus isolation from throat swabs, as well as identification of viral subgenomic messenger RNAs provided proof of virus replication in the upper respiratory tract . AnotherORF1ab and the E genes  has been working on the innovation of the molecular diagnosis, developing an automated molecular testing platform named GeneXpert System, which allows point-of-care testing . Followi E genes . DiaSori E genes , 65. The E genes . Lastly, E genes , 68.Indirect testing relies on the presence of host antibodies. SARS-CoV-2 antibody response has proven to be similar to that seen against many other acute viral infections , 69. IgMat the time and site of an encounter.” RDTs have proven to be effective for detecting other pathogens in the past (The development of rapid diagnostic tests (RDTs) began a short time after the outbreak of SARS-CoV-2, pursuing point-of-care (POC) diagnostic goals, which provide results “the past . The Ab-the past , 80. In the past , 39, 73.Besides Ab-RDTs, ELISAs, a well-established type of immunoassay technique, and CLIAs have also been developed and are included in the present review , 3. ELISThe heterogeneity of the testing conditions among the studies included such as the different number of days since symptom onset during sample collection and the different number of patients in which testing was performed, prevents the pooling of data to perform statistical analyses. In addition, some of the articles here reported data that have not yet been peer-reviewed, due to the recent onset of the pandemic. Therefore, this systematic search aims to be a compendium of the currently available literature on the commercial kits to test SARS-CoV-2 listed on the FIND website. Conclusions drawn by the different studies are collected and compared, with advice on which features provide better results. At present, FIND is evaluating some of the commercial immunoassays listed under the manufacturer's request, using a standardized independent protocol ; with thThe final selection of articles included in the present review comprised 18 articles testing 7 different commercial ELISAs, 2 different commercial CLIAs, 9 different commercial Ab-RDTs based on LFIA technology, and 1 Ag-RDT see –4. Some The tests differ, among other aspects, in the laboratory technique, the antigens used for antibody detection, and the type of antibody targeted. In addition, negative COVID-19 specimens used to test kit specificity across studies have a variable origin. Some studies assessed specificity testing in samples collected from healthy individuals prior to the SARS-CoV-2 outbreak, while others used samples from subjects with a negative COVID-19 result. It is important to note that almost none of the Ab-RDTs manufacturers provide information on which antigen was used for antibody detection. Finally, all the studies, with the exception of one, agree with the use of RT-PCR as the gold standard method for comparing test sensitivity.The specificity of all the commercial immunoassays was generally very satisfactory, while the sensitivity was far from adequate. The tests showing the best performance, according to sensitivity values, were ELISAs, followed by CLIAs and finally Ab-RDTs , althougMost of the studies evaluated sensitivity and specificity separately for IgM and IgG, with IgG mainly performing better than IgM. In scenarios in which this was not the case, the results are attributed to the difference in time of sample collection from symptom onset. A possible explanation would be that IgM detection covers a narrower phase of the infection time course than IgG. IgM appears earlier but also fades first, while IgG persists . AnotherFinally, the ELISA immunoassay studies reporting the best performance used a double sandwich assay instead of a capture or an indirect ELISA , 71.Whether test performance is affected when tested in milder COVID-19 cases remains unknown . Most ofOverall, Ab-RDTs are far from reliable in terms of sensitivity and specificity. Regardless of how attractive point-of-care diagnosis is, at present it cannot compensate for its poor performance. However, future improvements in these aspects, will make Ab-RDTs a promising solution for large-scale screening.Serological assays rely on the recombinant antigen with which they are coated. The antigen chosen must not only be immunogenic to ensure a high sensitivity but must also comprise specific epitopes to avoid cross-reactivity. In SARS-CoV, the N and the S proteins were found to be the dominant immunogenic antigens . This prThe N protein, is one of the major structural viral proteins and is involved in the transcription and replication of the genetic information of the virus and further encapsulation and packaging of the virions . It is aA) domain and a receptor binding domain (RBD) [ [, 88; Fign (RBD) [.In the years following the SARS-CoV outbreak, there was controversial literature as to whether the majority of neutralizing antibodies were directed against the N or S protein. Buchholz et al. reported that neutralizing antibodies against S proteins conferred protection and ultimately prevented host cell infection , 90. On The SARS-CoV-2 pandemic has revived the same doubts concerning which antigen protein is the most adequate for detecting antibodies. To date, the kits available include the use of the RBD, and the N, or the S protein .The Alphacoronavirus, another CoV genus, is known for being responsible for a number of seasonal common cold cases every year. Consequently, a large proportion of the population possesses antibodies against one of the four human endemic CoVs . PhylogeOkba et al. tested several in-house and commercial assays that used different recombinant antigens showing that the S1 subdomain is more appropriate for SARS-CoV-2 detection than the S2 , as the latter is more conserved in CoV  percent. The autA study comparing commercial test performance between a rN-based ELISA  and a rS(RBD)-based ELISA  was conducted by Liu et al.  Supplem. The resAmanat et al. developed two in-house ELISA versions coated with the S protein antigen: the first with the full-length protein, the second with only the RBD. The full-length version showed stronger reactivity possibly due to the larger number of epitopes that encodes the larger version of the antigen . StrongeRBD has demonstrated to be especially variable, varying more than the S2 subdomain or even than the N protein, being the major differentiator between the SARS-CoV-2 and the remaining CoVs, and is thus, becoming a promising antigen , 22, 88.The heterogeneity of the sensitivities reported across immunoassays is too high to be attributed only to the type of antibody detected or the antigen used in the assay. It should be noted that the number of days since symptom onset at which samples were collected to test commercial immunoassays vary across studies –4. IndeeConsidering that RT-PCR sensitivity is a dynamic value across time, it raises the question as to whether to use this technique as the gold standard method to compare test sensitivity. If RNA testing is performed in the second week after illness onset or later, the sensitivity falls, missing false-negatives, and thereby providing misleading immunoassays sensitivity values.It is essential to understand the difference between a diagnostic test and a test designed to study immunization status. The goal of the latter, which will not be discussed below, is the search for past infections, while the former searches for active infections . DiagnosLikewise, it is important to define what is meant by a screening test. Due to their easy-to-use and low turnaround condition, screening tests can cover a wide range of the population, which explains why they are so appealing for tracking infectious diseases and can be used to either diagnose unrecognized SARS-CoV-2 infectious cases or to determine seroprevalence levels among the population . If usedOptimizing diagnostics entails improving the choice of the diagnostic test based on the time since illness onset and understanding of COVID-19 disease, which in turn determine whether to look for antibodies or antigens . FurtherViral antigens and genome are specific markers of the virus that precede both symptomatology and immunoglobulin response . In the This data should not be misinterpreted, as they exist other Ag-RDT authorized by other sources besides the one just mentioned. As stated at the beginning, the present review only includes commercial kits fulfilling the inclusion criterion of diagnostic kits supported by published literature, tested independently from the manufacturer, and providing both sensitivity and specificity values. Following such a strict criterion resulted in the inclusion of only one Ag-RTDs following FIND data search. However, since July 2020, the Food and Drug Administration has conceded 10 Emergency Use Authorizations for SARS-CoV-2 Ag-RDT. Approved Ag-RDT, listed in the Food and Drug Administration webpage, have been developed by manufacturers such as Quidel Corporation, Becton Dickinson and Company (BD), LumiraDx UK Ltd., among others . BesidesCurrently, the World Health Organization  recommenIf it were not for the poor accuracy reported, Ag-RDTs would have been a suitable option for large-scale detection of infectious cases before the appearance of symptoms, since despite being highly sensitive, RT-PCR molecular tests have a slower turnaround time, and are therefore less suitable for population screening. Consequently, RT-PCR molecular tests have been the standard technique to diagnose and screen contacts among reported infectious cases.Active infections can also be diagnosed by detecting antibodies against SARS-CoV-2. A negative nucleic acid test result does not necessarily exclude the possibility of being infectious. Likewise, a positive immunoassay test result does not necessarily translate into antigen clearance. It has been demonstrated that RNA levels persist despite the appearance of antibodies . This raImmunoassays are considered as a complement to RNA testing, especially after the second week after symptoms onset [Besides being a complement to RNA testing in the diagnosis of COVID-19, antibody detection using immunoassay tests has many other applications. In the present situation, immunoassays are being used within the context of epidemiological studies to determine which are the seroprevalence levels among the population . FurtherThe studies conducted so far have revealed a significant correlation between antibody titers and that the clinical severity of the disease remains beyond the second week after illness onset—the higher the antibody titers, the worse the prognosis . MoreoveMoreover, Wu et al. measured SARS-CoV-2 specific neutralizing antibodies (NAbs) among recovered patients and observed that about 30% of subjects developed very low NAbs levels, suggesting the presence of alternatives pathways besides NAbs production against the virus .Despite the recent concern of the WHO in regard to the lack of data demonstrating immunization, and whether immunization protects against SARS-CoV-2 reinfection, a study conducted in China demonstrated that reinfection did not occur in Rhesus macaques after recovering from SARS-CoV-2 infection . FinallyThis review was aimed at analyzing the current COVID-19 diagnostic approaches available, with the added difficulties of the recent onset of the pandemic and the huge amount of incoming information. The constant development of new commercial diagnostic tests will subject any conclusion drawn here to obligatorily be revised. In the meantime, even though it is too soon to derive definitive results, the present work intends to be a helpful guide in terms of optimizing diagnosis.The high degree of homology shared with other human CoVs and the high number of mild COVID-19 cases demonstrate the need for both sensitive and specific diagnostic tests. RT-PCR is currently the most accurate test. Two automatized platforms can currently be used: (1) Integrated platforms which provide a result in 1 h or 1 h and a half, although they cannot process many samples at once, and (2) Integrated platforms which process more than 90 samples at once but the turnaround time is of around 3.5–4 h. Meanwhile, immunoassays, for which time-dependent accuracy is their major inconvenience, are considered as a complement to nucleic acid tests, especially after 14 days since illness onset. Moreover, these tests have many other applications besides diagnosis, as noted along the text. Finally, the primary goal of RDTs is to obtain point-of-care diagnosis, but they lack sensitivity and specificity and need further diagnostic confirmation.There is a need for sharing findings as well as providing transparent results when testing different diagnostic kits. As mentioned previously, at the time of writing FIND is conducting a generalized evaluation of the commercial kits available, using a standardized independent protocol, in order to provide practical advice based on robust evidence-based results.AM-V, CB-D, and JV contributed to the design of the text and writing. AM-V design the tables. JV design figures. All 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.