C-ring compression tests in Ringer solution were used to cause a fracture. Crack initiation and propagation is influenced by twin boundaries and LPSO lamellae. The blocky LPSO phases also clearly hinder crack growth, by increasing the energy to pass either through the phase or along its interface. The microstructural features were characterized by micro- and nanohardness as well as the amount and location of LPSO phases in dependence on the heat treatment condition. By applying nanoindentation, blocky LPSO phases show a higher hardness than the grains with or without lamellar LPSO phases and their hardness decreases with heat treatment time. On the other hand, the matrix increases in hardness by solid solution strengthening. The microstructure consisting of a good balance of grain size, matrix and blocky LPSO phases and twins shows the highest fracture energy.The investigation of the crack propagation in as-extruded and heat-treated Mg-Dy-Nd-Zn-Zr alloy with a focus on the interaction of long-period stacking-ordered (LPSO) structures is the aim of this study. Solution heat treatment on a hot extruded Mg-Dy-Nd-Zn-Zr (RESOLOY Research on Mg-RE alloys covers many aspects: strength and ductility , creep , fatigue®, an Mg-Dy-Nd-Zn-Zr alloy high in Dy, was developed specifically for absorbable implants by Meko Laser Materials Processing in Sarstedt and the Helmholtz-Zentrum Geesthacht in Germany, is internationally patented [Resoloypatented  and showpatented ,10,11. Dpatented ,13. Mg-Rpatented . The LPSpatented , and apppatented . Studiespatented ,18, blocpatented ,20,21,22patented  and highpatented .®, in previous studies, shows a strong grain size increase by solution heat treatment at 500 °C and a change in the amount of blocky and lamellar LPSO structures with increasing solution heat treatment time [Resoloyent time ,26. LameWhile some microstructural features cause crack initiation under mechanical loading or stress corrosion , these fTo evaluate the hardness of individual microstructure features, tensile and compression tests and hardness measurements of higher loads reaching their limits were performed. Nanoindentation measurements with a small indent size show the higher hardness of precipitates in Mg alloys , the infThe motivation of this study is to explain the crack propagation in different microstructures by their microstructural features, especially by lamellar and blocky LPSO structures and their micro- and nanohardness. With this knowledge, the microstructure can be optimized and mechanical properties can be tailored to fit their application.The Mg-Dy-Nd-Zn-Zr alloy was cast using permanent mould direct chill casting  at the HC-ring samples with a width of 10 mm were machined from the extruded tubes with an outer diameter of 34 mm and a wall thickness of 2 mm. Their position in the initial extruded tubes can be seen in . C-ring i of each individual multi-indent was calculated by the hardness values in the range of the lowest dependence on the load applied, and its standard deviation was kept below 0.1 GPa. Their mean, minimum and maximum values are presented. The Vickers hardness HV1 and HV0.2 were tested with a universal hardness testing machine ZHU2.5 by Zwick , with up to 30 indents per condition applying HV1 and up to 15 indents using HV0.2. Individual values for HV0.005 were tested with a Zeiss Neophot 2 . In addition to the mean values, minimum and maximum values are reported and used for discussion. Due to the small indent size, only the microhardness by Vickers by HV0.005 could be used to determine the hardness of the blocky LPSO phases. Nanoindentation measurements were performed using the electrostatic transducer of the UBI 1 Hysitron TriboScope  with a diamond 90° cube corner tip. The detailed methodology is described in ,48. ProgThe Mg-Dy-Nd-Zn-Zr alloy consists of 12.63 wt.% Dy, 1.05 wt.% Nd, 0.94 wt.% Zn and 0.075 wt.% Zr. Fe, Cu and Ni do not exceed 0.001 wt.%. Whereas the fine-grained microstructure shows the highest number of grains at grain sizes around 10 µm and with no grains larger than 30 µm, the coarse-grained microstructure shows the highest number of grains around 24 µm at 500 °C for 24 h, at around 28 µm for 48 h and around 34 µm for 72 h. The blocky LPSO phases can reach a size of 10 µm; however, there are also phases in the sub-µm range. According to the mean value, the hardness HV1 decreases slightly by the heat treatment of 200 °C for 4 h (acting similar to stress relief), and further by solution heat treatment of 500 °C for 48 h, but increases slightly towards the 72 h heat treatment time g. The frThe C-ring compression tests were stopped when the significant force reduction during crack growth was exceeded (see force-displacement curves in ), so theMicrographs with higher magnifications present the interaction of the propagation of the sub-cracks branching off the main crack with the microstructural features. LPSO lamellae appear as thin, sometimes single lines, within a single grain in parallel formation and stops and starts at different locations. The contrast in between these lamellae does not differ from the rest of the grain. Twin boundaries show as thin parallel lines with a different contrast in between compared to the contrast outside the twin. The twins start at gb or other interfaces and run to the next interface or twin boundaries run towards each other within the grain, forming elliptical shapes. By examining the microstructure along the crack path of the solution heat-treated conditions at 500 °C, where the grains have grown, it can be seen that the grains are twinned and the amount of twins increases towards the compression side of the C-ring sample. During C-ring deformation the crack opens at the tensile side while the grains at the compression side are exposed to larger strain before the crack “arrives” and twins to a larger extent. The cross-sectional micrographs in The circle in To characterize the microstructural features regarding their hardness, individual regions were tested with the Vickers hardness HV0.2. The Vickers hardness indents of HV1 of this Mg-Dy-Nd-Zn-Zr alloy c have diThe graphs in It is worth mentioning that there is no obvious correlation of the number and orientation of LPSO lamellae, or of their spacing in between and the hardness value. The shape of some indents is not exactly symmetrical, but to determine any influence of the direction of the LPSO lamellae, further and detailed investigations are needed.As seen with the HV0.2 indents see ., they aThe bar chart in Since the imprints are of a size of around 10 µm, the Mg-matrix in between the LPSO lamellae could be measured individually; its mean value is smaller than the grains of the Mg-matrix with or without LPSO lamellae. Even so, its scattering is rather high. However, the scattering  of the hardness value of the blocky LPSO phases is very high (Δ hardness of 90 HV0.005), followed by the Mg-matrix without LPSO lamellae (Δ hardness of 48 HV0.005).Representative force-displacement curves of indents in blocky LPSO and of the Mg-matrix in Mg-Dy-Nd-Zn-Zr, in both the as-extruded condition and the solution heat-treated conditions at 500 °C for 24 h, 48 h, and 72 h, are shown in The depth-dependent hardness of the blocky LPSO phases has the highest value in the as-extruded material a. With hThe load-displacement curves of the solution heat-treated condition at 500 °C for 48 h in The highest dependence of the hardness on the load applied has been found for the regions of narrow lamellar LPSO structures within the Mg-matrix, as seen in i of the multi-indents from the stable range of hardness values, which it was aimed to keep below 0.1 GPa, could not be obtained in the region of LPSO lamellae with narrow distances in the solution heat-treated conditions at 500 °C for 72 h. Although the mean values mi were supposed to be calculated by the hardness values within a range of the lowest dependence on the load applied, in two cases the range of values resulted in a higher standard deviation, 0.2 GPa for lamella 1 and 0.13 GPa for lamella 3 , the hardness values are the lowest. The softer Mg-matrix consists of fewer solute atoms, which are instead in the LPSO phases in these grains.The nanohardness values clarify the higher hardness of the blocky LPSO phases: in the extruded condition, blocky LPSO phases reach 160% based on the hardness of the Mg-matrix and, in the solution heat-treated condition at 500 °C, the values exceed 130%. Due to the dissolution process of the blocky LPSO phases, where the phases can also fragment, meaning that their Dy-, Nd- and Zn-enriched layers widen, the decreasing density of rare earth elements in combination with Zn lowers the hardness. On the other hand, the hardness of the Mg-matrix increases with the increasing ageing time from 24 h to 72 h. The Mg-matrix in between the LPSO lamellae shows, in agreement with the HV0.005 hardness values, the lowest hardness. The size of the nanohardness indents even makes it possible to measure the hardness of the LPSO lamellae (or at least very close to them) and a higher mean value compared to LPSO lamellae free of Mg-matrix as well as LPSO lamellae containing matrix can be found. This proves that the Dy-, Nd- and Zn-enriched layers are the hardest features in the microstructure and, in case of indenting them in the right atomic layer, the hardness is even higher than that of the blocky LPSO phases. The significant load dependence of the hardness values of the LPSO lamellae region with narrow distances can be explained by the layered structure. The layers seen in the micrographs will not be cut at an exact 90-degree angle, which means that deeper measured areas will contain more or less Dy-, Nd- and Zn-enriched layers, leading to lower and higher values of one single multi-cycle indent, so-called depth-sensing nanoindentation, which is often found in multilayered coatings or functionally graded materials ,56.The evaluation of the hardness of each individual microstructural feature helps to understand and explain the crack propagation and its interaction with the microstructure. Micrographs of the fine-grained microstructure present sub-cracks, which branch off the main crack following the direction of the LPSO lamellae. The Mg-matrix in between the LPSO lamellae is softer and provides the weakest possibility for crack growth. The crack propagation is sidetracked by grain boundaries, due to the different orientation of LPSO lamellae in the neighboring grain, and by blocky LPSO phases due to their higher hardness, where the crack can find its way along the weaker interface of the blocky LPSO phase and Mg-matrix. Since the blocky LPSO phases build a network at the gb, the weaker interfaces have been found to carry the crack from grain to grain.The grain growth during solution heat-treated conditions at 500 °C allows twinning during plastic deformation. These twins also interact with the crack propagation: the crack surface appears jagged and uneven; a zig-zag crack pattern is the result of the crack following the twin boundaries, as seen for LPSO lamellae. These twin boundaries cause an accumulation of dislocation on new slip traces, finally leading to regions of secondary micro-cracking . Slip-coThe knowledge about the nanohardness helps to understand why the crack follows the lamellar LPSO phases within the Mg-matrix: due to a larger distance between the LPSO lamellae, a higher amount of dislocation gathers in the softer Mg-matrix at the interface of the LPSO lamellae, and micro-cracks initiate the crack propagation along the lamellae. The sub-cracks interact with LPSO lamellae, which disagree with the growth direction, and the LPSO lamellae redirect the crack in their own direction, until the main loading direction, given by the geometrical specimen shape and size during C-ring compression, will force it towards the next LPSO lamellae, also leading to a zig-zag pattern.The micrographs studied show that some blocky LPSO are crossed and some are passed sideways. Crossing the blocky LPSO phases is possible when the orientation of the lamellar structure agrees with the crack propagation or is not far off it. However, according to the higher hardness of the blocky LPSO phases, stresses pile up and more energy will be needed. Disagreement leads either to crack blunting or passing the blocky LPSO phase sideways along the interface . Any morThis study clearly helps to understand the role of blocky and lamellar LPSO phases on crack propagation and, thus, on the fracture toughness. The blocky LPSO phases are of a certain high hardness, providing some strength in general, but also serving as obstructions to crack growth; inner grain blocky LPSO phases are here very effective. In the fine-grained microstructure, the crack, which mainly follows the LPSO lamellae, can easier carry on growing along the LPSO lamellae in the neighboring grains, instead of being hindered by perpendicular lamellas in other grains. Current research is focusing on the detailed explanation of the high fracture toughness in the solution heat-treated condition at 500 °C for 48 h, so more nanohardness data are to come.The effect of blocky and lamellar LPSO phases and their hardness on the crack propagation in different microstructures of Mg-Dy-Nd-Zn-Zr alloy (RESOLOY) has been investigated. The fine-grained microstructure consists of lamellar LPSO structures within the matrix and in the coarser grains there are fewer lamellae, but blocky LPSO phases, whose amount decreases with heat treatment time, especially the inner grain blocky phases. The amount and location of LPSO phases were evaluated by optical microscopy.C-ring compression tests in Ringer solution were done to cause sample failure. Crack initiation and propagation were demonstrated with the focus on the interaction of the microstructural features and the crack growth. Twins and LPSO lamellae are responsible for crack initiation. The blocky LPSO phases clearly hinder crack growth, by increasing the energy needed to pass either through the phase or along its interface. When the crack growth direction is off the orientation of the matrix LPSO lamellae, they also redirect the crack. The crack runs in the softer Mg-matrix in between the LPSO lamellae. This softer Mg-matrix layer provides the possibility for slip band cracking. In the coarser-grained microstructure, the crack propagation is also influenced by twin boundaries, especially on the compression side of the C-ring sample.The microstructure, with a focus on microstructural features, was characterized by micro- and nanohardness. Hardness testing with a lower load was needed to measure the hardness of the blocky LPSO phases and LPSO lamellae individually . Blocky LPSO phases show a higher hardness than the grains with or without lamellar LPSO phases. The matrix in between matrix LPSO lamellae shows the smallest values. Whereas the blocky LPSO phases decrease with heat treatment time, the grains free of lamellar LPSO phases increase in hardness. The dissolution of blocky LPSO phases enriches the matrix by solid solution.The highest hardness and strength was found in the fine-grained extruded condition with grains completely filled by lamellar LPSO phases. However, the highest fracture toughness is seen in the solution heat-treated condition at 500 °C for 48 h, where the hardness shows the lowest value and the microstructure has a good mix of grain sizes, lamellar and blocky LPSO and twins formed under the mechanical loading. This study shows that the properties of the alloy system Mg-Dy-Nd-Zn-Zr can be easily tailored by post heat treatment. Since other studies have provided good corrosion resistance, it makes this alloy system an interesting candidate among the Mg-RE alloys.


Staphylococcus) and Staphylococcus aureus. Amongst the urinary isolates, most common pathogens were Escherichia coli and Staphylococcus aureus. A total of 60% MRSA was observed in urine and blood isolates. Up to 40% increase in AMR was observed amongst these isolates obtained during COVID-19 period compared to pre-COVID-19 times. The overuse of antibiotics gave abundant opportunity for the bacterial pathogens to gradually develop mechanisms and to acquire resistance. Since the dynamics of SARS-COV-2 are unpredictable, a compromise on hospital antibiotic policy may ultimately escalate the burden of drug resistant pathogens in hospitals. A shortage of trained staff during COVID-19 pandemic renders it impossible to maintain these records in places where the entire hospital staff is struggling to save lives. This study highlights the extensive rise in the use of antibiotics for respiratory illness due to COVID-19 compared to antibiotic use prior to COVID-19 in ICUs. The regular prescription audit followed by a constant surveillance of hospital infection control practices by the dedicated teams and training of clinicians can improve the quality of medications in the long run and help to fight the menace of AMR.Antimicrobial resistance (AMR) is an emerging public health problem in modern times and the current COVID-19 pandemic has further exaggerated this problem. Due to bacterial co-infection in COVID-19 cases, an irrational consumption of antibiotics has occurred during the pandemic. This study aimed to observe the COVID-19 patients hospitalized from 1 March 2019 to 31 December 2020 and to evaluate the AMR pattern of bacterial agents isolated. This was a single-center study comprising 494 bacterial isolates (blood and urine) that were obtained from patients with SARS-CoV-2 admitted to the ICU and investigated in the Department of Microbiology of a tertiary care hospital in Delhi, India. Out of the total bacterial isolates, 55.46% were gram negative and 44.53% were gram positive pathogens. Of the blood samples processed, the most common isolates were CoNS (Coagulase Negative  Coronavirus disease 2019 (COVID-19) had an immense worldwide impact on health care systems, economy and financial resources. The dynamics of COVID-19 disease and its treatment has been changing throughout the course of pandemic with new research guidelines constantly being added. Steroids, undeniably, are the mainstay in the management of COVID-19 illness, particularly in hypoxemic patients. There has been an irrational use of steroids in mild to moderate symptoms, thus, providing a niche for impending secondary infections. The COVID-19 illness has a wide spectrum of presentation, with a predominance of respiratory distress or fever and sore throat or may even remain asymptomatic. The development of sepsis with raised CRP is generally considered to be a domain of bacteria associated infection but, during this pandemic, viral sepsis emerged as a new entity that is relatively less suspected and undiagnosed, with a remarkable high risk of multiple organ failure . FurtherIt is yet to be ascertained whether the majority of severe COVID-19 hospitalized patients received any major benefits with the administration of antibiotics. In order to intensify the conflict, clinical evidence suggest that inadequate broad-spectrum empiric antibiotic use could be associated with higher mortality, especially in case of sepsis . Such diIn this study, we aimed to evaluate the AMR pattern of bacterial agents isolated from clinical samples (blood and urine) submitted to Microbiology laboratory from the patients with COVID-19 illness, to evaluate those admitted to a tertiary care center during the pandemic and compared the anti-microbial susceptibility profile of bacterial agents isolated in the previous year (pre-COVID-19) for strengthening the antimicrobial stewardship protocols during the disaster.The data were analyzed in two time periods: pre-COVID-19 (March 2019 to December 2019) and the COVID-19 period (March 2020 to December 2020).The total number of samples received during pre-pandemic-period (7309) was compared to the (4968) samples received during the pandemic phase in 2020. A decline in bacterial growth was observed during the pandemic (9.94 vs. 11.54%). A total of 494 bacterial isolates (blood and urine) were obtained from patients with SARS-CoV-2 admitted to the ICU of which 55.46% were gram negative and 44.53% were gram positive pathogens. The distribution of isolates among age groups did not show any significant variation during both time frames except in less than 18 years of age, including infants. A male preponderance was observed amongst the clinical isolates obtained during the COVID times compared to pre-COVID-19 period. There was a statistically significant increase in demand for culture requisition from the ICU during the COVID-19 period (63.36%) compared to previous years (5.68%).There was a significant increase in the proportion of blood culture samples (67.87%) received as compared to urine culture (32.12%) during the pandemic. However, blood culture positivity was lower (7.53%) during the pandemic period compared to the previous year (15.45%). The percentage of urinary bacterial isolates (15.03%) was much higher in samples received from Covid-19 patients in 2020 compared to positivity (8.30%) in the previous year .Staphylococcus aureus, Coagulase Negative Staphylococcus (CoNS) and Enterococcus spp. during both periods. Among the Gram negative bacteria, even though the positivity rate was similar, there was an increase in the isolation of Acinetobacter baumannii and Escherichia coli during the pandemic. There was significant increase in urinary isolates detected during the COVID period (82.08% vs. 69.27%).In Klebsiella pneumoniae was less susceptible to aminoglycosides, fluoroquinolones and carbapenems. On the other hand, Pseudomonas aeruginosa isolates showed alarming resistance to imipenem along with third generation cephalosporins.In the antimicrobial susceptibility pattern of blood culture isolates, ciprofloxacin and cotrimoxazole showed reduced susceptibility among the Gram negative bacteria . KlebsieStaphylococcus aureus depicted reduced susceptibility to all classes of drug during the pandemic period. Significant increases in MRSA during the COVID-19 period with decreases in susceptibility to commonly prescribed drugs such as clindamycin, erythromycin and fluroquinolones were observed as shown in Escherichia coli and Acinetobacter baumannii showed increased resistance to most of the drugs, while the susceptibility pattern for Klebsiella pneumoniae did not vary in both periods. Although Pseudomonas aeruginosa isolates were less in number, the resistance patterns for various antibiotics were not significantly different except for piperacillin-tazobactum and cefepime  data, which reported a high rate of resistance to ciprofloxacin, gentamicin, cotrimoxazole, erythromycin and clindamycin. On the contrary, among Gram negative bacteria, Enterobacteriaceae as a group accounted for maximum number of cases (20–21%) similar to findings reported in other studies [The  studies .Acinetobacter baumannii was the predominant bacteria isolated during COVID-19 compared to the pre–COVID-19 period with reduced susceptibility to gentamicin, amikacin and ciprofloxacin but an alarming decline in susceptibility was observed for Cotrimoxazole and piperacillin-tazobactam. Before the pandemic, Klebsiella pnemoniae, followed by Acinetobacter spp., Escherichia coli and Pseudomonas aeruginosa were the common isolates in blood culture. The COVID-19 period witnessed a shift towards Acinetobacter baumannii emerging as the most predominant pathogen followed by Escherichia coli and Klebsiella pneumonia. Acinetobacter baumannii associated VAP (ventilator-associated pneumonia) and associated secondary bacteraemia among patients in ICUs contribute to high mortality rates up to 50% [Escherichia coli is also responsible for community-acquired pneumonia with 11–21% mortality and a disproportionally high rate of bacteraemia [Amongst the Gram negative bacteria, p to 50% ,38. Eschteraemia .Several factors such as the overuse of cephalosporins and fluroquinolones for respiratory illness in admitted patients as well as for outpatients accounted for the development of a high degree of resistance amongst this class of antimicrobials. Additionally, the rise of Carbapenem resistant Enterobacteriaceae (CRE), CRPA and CRAB pose serious challenges in the management of critically ill patients in all health care settings. Therefore, utmost caution should be advocated, especially in the use of reserve drugs such as amikacin, polymyxin and Tigecycline and combinations for managing bacterial infections in COVID-19 patients ,41,42. AEscherichia coli remained the most common pathogen followed by Enterococcus, Staphylococcus and Klebsiella pneumoniae in the urine samples of the COVID patients. The distribution shifted in 2020 with an increase in the proportion of Gram negative isolates from 69 percent to 82 percent. Similar to blood culture isolates, a low susceptibility was observed for cephalosporins, fluoroquinolones and cotrimoxazolein. Higher susceptibility was observed for aminoglycosides, carbapenems, nitrofurantoin and Fosfomycin; resistance exhibited for fluoroquinolones.Amongst the urinary isolates, the culture positivity increased to almost double (15% versus 8.3%) during the pandemic in comparison to previous year. A study by Can O et al. showed that urine tract infections were significantly increased in elderly patients after COVID-19 infection ,48. EschThe critical observation of our study was the excessive use of intravenous antimicrobial agents such as fluoroquinolones, cephalosporins, carbapenems, azithromycin, vancomycin and linezolid, which belong to the watch and reserve group of drugs of AWARE classification by the WHO. The trend of increasing antimicrobial resistance observed during the pandemic compared to the preceding pre-COVID-19 year was comparable to observations from Clancy et al. , who empOur study emphasizes that the threat of AMR is large, thus, when faced with difficultly to demarcate whether a COVID-19 patient is progressing to a bacterial infection, a strong clinical suspicion aided with serum biomarkers assays such as PCT, CRP and bacterial cultures should be adopted as potential laboratory tools to guide the clinicians to administer antibiotics rationally ,51.The association between AMR and COVID pandemic is slowly emerging with many studies (mostly from Asian countries) showing an unbalanced approach toward antimicrobial consumption . AlthougPerhaps more stringent antimicrobial stewardship protocols in COVID-19 management plans may prevent the AMR emergence. Descriptive data of antimicrobial susceptibility should be available to the clinicians for timely de-escalation and interventions in order to reduce inappropriate prescription of antibiotics for COVID-19 patients. Further studies are required to explore the risk factors in secondary bacterial co-infections in COVID-19 patients and severity should be monitored with serum biomarkers before administering empirical antimicrobials. The regular prescription audit followed by a constant surveillance of hospital infection control practices by the clinical hospital infection control team and the training of clinicians can improve the quality of medications in the long run and to help to fight the menace of AMR.This was a single-center study conducted at the Department of Microbiology of a tertiary care hospital in Delhi, India. The hospital catered to COVID-19 suspects/ confirmed patients and was declared as a dedicated COVID-19 hospital by the government of Delhi, India. The study was conducted from 1 March 2019 to 31 December 2020. Bacterial isolates from blood and urine samples submitted for microbiological analysis for admitted patients were included in the study. A total of 844 pre-COVID-19 (Mar 2019 to December 2019) and 494 COVID-19 period (March 2020 to December 2020) bacterial isolates were identified and the antimicrobial susceptibility pattern was studied.Clinical samples (blood and urine) from patients admitted to ICU and wards of our tertiary care COVID-19 hospital were included in the study. COVID-19 period samples were transported and processed as per standardized laboratory protocols set up for COVID-19 patients.Patients admitted in the hospital were either suspects, clinically diagnosed COVID-19 or confirmed COVID-19 patients. The decision to request for cultures was based on the individual clinical judgment of the treating physician and no systematic protocol was used. Blood and urine samples from these patients were transported to the department as per COVID-19 related precautions. All samples were handled by trained personal while wearing N95 mask and suitable personal protective equipment in a class-II A2 biosafety cabinet. The amount of 10–20 mL venous blood sample was collected and sent in automated BACTEC aerobic blood culture bottles . Both adult and pediatric bottles were incubated up to 7 days in automated BACTEC machine. Bottles that were flagged positive by the machine were withdrawn and gram staining was performed. Subcultures were performed on blood agar and MacConkey agar plates were incubated overnight at 37 °C for the isolation of the pathogen. Growth was subjected to the microscopic examination of gram-stained smears prepared from the colonies.For urine cultures, the samples included midstream clean catch and sample from catheterized patients. Urine routine microscopy and cultures were performed. Cysteine lactose electrolyte deficient media were used for semi quantitative methods by using calibrated loops. This method provides information regarding the number of CFU/mL. All cultures were read after 18 to 24 h of inoculation for bacterial growth. Bacterial isolates obtained were processed by the automated identification method using Microscan WalkAway  .Corynebacteria, Bacillus spp., Propionibacterium spp., etc.) [BSI was defined as the presence of at least one positive blood culture from a patient. At least two consecutive positive blood cultures were needed to define BSI due to CoNS or other common skin colonizers  .Staphylococcus aureus 25923 and Escherichia coli 25922). Vancomycin and Daptomycin for S. aureus could not be included as MIC could not be performed due to the lack of resources.The antimicrobial susceptibility test (AST) was performed by the Kirby Bauer disk diffusion method as per CLSI guidelines . QualityBiomedical waste management guidelines for COVID-19 were followed for all laboratory work including double bag use and onsite inactivation of samples. Single isolate was reported from all the samples. Duplicate isolates from the same patient on subsequent testing were excluded from the study.Statistical analysis was performed using the SAS 9.1 software . Categorical variables were described by using frequencies and 95% confidence intervals (CIs). Skewed distribution was described using medians, interquartile ranges and maximums and minimums. The significance threshold was 0.05.


The COVID-19 pandemic has hit Northern Italy’s regions hard in terms of deaths since February 2020. Containment measures have been applied to avoid contagion and reduce the patient infection rate. In this manuscript, we report the experience of the Pathology Department of the Fondazione IRCCS Istituto Nazionale Tumori in Milan, during the period of the first lockdown that occurred in Lombardy from March to May 2020, focusing on the variation in terms of exams between the pre-COVID-19 and COVID-19 periods and describing the measures applied to guarantee the safeguarding of workers. Moreover, we calculated if changes introduced within the workflow affected the average diagnosis time using Turn-Around-Time (TAT) metrics released by the Lombardy Region. We showed a sharp slowdown in exams during the first wave of COVID-19 and that the measures applied for the safeguarding of the personnel turned out to be feasible and did not affect the overall performance of the Pathology Department.Background: On December 2019, an outbreak of atypical pneumonia, known as COVID-19, was identified in Wuhan, China. This disease, characterized by the rapid human-to-human transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly in more than 200 countries. Northern Italy’s regions have been hit hard in terms of deaths. Here, we report the experience of the Pathology Department of the Fondazione IRCCS Istituto Nazionale Tumori (INT) in Milan, the first Italian public cancer center, in the period of the lockdown that took place in Lombardy from March to May 2020. Method: The variation in terms of exams was calculated in two different timeframes: December 2019–February 2020 (pre-COVID-19) and March–May 2020 (COVID-19). During these periods, Turn-Around-Time (TAT) metrics released by the Lombardy Region were calculated to assess if changes applied to guarantee the safeguarding of workers affected the average diagnosis time. Results: In the COVID-19 period, there was a decrease for all the performed exams. The most considerable decrease was observed for PAP tests (−81.6%), followed by biopsies (−48.8%), second opinions (−41.7%), and surgical (−31.5%), molecular (−29.4%) and cytological (−18.1%) tests. Measures applied within the Pathology Department, such as digital pathology, remote working, rotations and changes in operating procedures, improved the diagnostic performance as required by the guidelines of the Lombardy Region in terms of TAT. At the same time, the measures applied for the safeguarding of the personnel turned out to be feasible and did not affect the overall performance of the Pathology Department. Conclusions: The sharp slowdown in cancer screening during the first wave of COVID-19 could seriously endanger cancer prevention in the near future. With over 165 million cases reported and 2 million new cases per day  has hit The main objective of this study was to quantify the negative impact of the first SARS-CoV-2 outbreak on cancer patients of our Institution by focusing on the number of exams performed by the INT Department of Pathology. For this reason, we evaluated the number of exams in two different timeframes, i.e., December 2019–February 2020 (pre-COVID-19 period) and March–May 2020 (COVID-19 period). The exams were grouped into different classes—“surgical”, “biopsy”, “second opinion”, “molecular”, “cytology” and “PAP test”—and differentiated by the origins of the exams, namely, (i) day hospital, (ii) inpatients and (iii) outpatients.During the lockdown period, several to-hospital and within-hospital filters were applied to protect our Institution, as described by Valenza et al. . AccordiThe main modifications to the operating procedures and working environment are listed below. (a)Gross examinationAll the samples were already treated in a safe environment using personal protective equipment. Before the SARS-CoV-2 outbreak, sampling procedures were performed on fresh surgery tissues. The first measure adopted was to submit all the specimens to a 24–72 h fixation with 10% buffered formalin. All the fresh surgical materials referred to our department were properly handled by a pathologist, wearing a FFP2 safety mask and single-use sterile gown under a chemical fume cabinet, to improve formalin penetration, and then fixed for 24 h before routine sampling. All the intraoperative examinations were performed wearing FFP2 safe masks and single-use sterile gowns. The cryostat was decontaminated after every procedure using a 95% alcoholic solution.(b) CytologyAll the instruments dedicated to cytology were secured under a class I biosafety cabinet. Fresh samples, such as bronchoalveolar washes, urine and expectorates were processed by operators wearing FFP2 safe masks and disposable gowns. Fluids  posing a risk of aerosol generation were processed under a laminar flow hood using additional disposables such as FFP2/FFP3 masks. Steps outside the flow hood were always carried out wearing an FFP2/FFP3 mask, glasses or visors, using sealed tubes. As during the pre-COVID-19 period, alcoholic or different fixatives were avoided in order to preserve the integrity of samples and prevent coarctation or flocculation.(c) Flow cytometrySample processing and immunofluorescence labeling were carried out under a laminar flow hood using individual disposables. For the steps performed outside the hood, such as centrifugation, tubes were hermetically sealed. All the instruments were properly sanitized .(d) CytogeneticsCell cultures from peripheral blood and bone marrow, cell cultures for the analysis of B lymphocyte aspirates, and chromosomal preparations from blood and/or bone marrow aspirates were handled under a sterile laminar flow hood. The operators always wore double gloves, protective glasses or visors and appropriate clothing with additional disposable gowns. Surfaces and centrifuges were sanitized using hydroalcoholic solution.(e) DNA extractionDNA extraction from blood samples was always performed using an automated Maxwell extractor . Cartridges were loaded under a laminar flow hood with closed glass. According to the regulations published by the Center for Disease Control and Prevention CDC , the ope(f) Digital Pathology®  at 40× and 400× magnifications and loaded on a Network Attached Storage (NAS) server in order for them to be accessible and viewable by remotely working pathologists. Software for digital slide viewing and analysis  was installed on the pathologist’s personal computer, as well as them being provided with access to the institutional virtual private network (VPN) to work remotely.Slides were digitalized for diagnostic use. Images were acquired on an Aperio ScanscopeXTIn order to prevent the possible transmission of COVID-19 within the Laboratory Department, several security measures were undertaken. On 8 March 2020, a presidential decree (Law no. 34/2020) established that up to 50% of the staff personnel could be placed in remote working. The decree was then implemented during the second peak of SARS-CoV-2 infections in October 2020, in order to further balance the need to face the pandemic and the continuity of services through flexibility in working hours. We prioritized remote working for the personnel who were actually able to work from home. In particular, pathologists who shared offices with other colleagues were equipped with software for digital slide viewing and VPN access. Accordingly, biologists were also connected to the VPN in order to enable them to use the lab software remotely. These procedures allowed obtaining a 50% reduction in onsite pathologists and biologists, respectively. By contrast, only 15% of the laboratory technician staff were able to carry out remote working, due to the need to manually perform working procedures on site. Rotations were established to guarantee a safe alternation between onsite and remote working. The access of personnel external to the pathology department was restricted to operators carrying samples from surgical rooms and the day hospital. All the offices of the department were already equipped with sensors unlocked through electronic badges, a measure further helping to reduce contacts among inter- and intra-department operators. The reception of patients for pathology report collection or consultation sample delivery was restricted to offices equipped by glass separators, and the opening time was reduced.p-values < 0.05 were considered statistically significant.All the statistical analyses were performed using the R environment for statistical computing and graphics . Comparisons of exams among groups were performed by Fisher’s exact test or the Chi square test for categorical variables. All the tests were two-sided, and The number and distribution of the exams carried out in the department in the pre-COVID-19 and COVID-19 periods are outlined in During the COVID-19 period, the distribution of the exam sources was modified. Overall, the most important reduction was observed for outpatient exams (−1989 or −40.9%), followed by day hospital exams (−589 or −83.9%) and inpatient exams (−311 or −12.0%). Specifically, the relative frequency of surgical samples and biopsies from the day hospital dropped . Cytological exams also decreased, though to a lesser extent (4.7% vs. 3.5%), while molecular analyses and second opinions were unaltered . A similar trend was observed for the outpatients: the surgical (35.1% vs. 32.3%), cytological (46.6% vs. 35.9%), molecular (73.0% vs. 68.9%) and second opinion (61.7% vs. 55.4%) workloads decreased during the COVID-19 period, while biopsies were unchanged (80.8% vs. 80.4%). Finally, we registered an increase in relative frequency in all the exams within the inpatient subgroup during the COVID-19 period.p < 0.0001) and molecular analyses  and second opinions  in the COVID-19 period compared to the pre-COVID-19 period. On the other hand, no significant TAT changes were observed for biopsies , cytological exams  or pap-test exams .The pre-specified TAT was matched for all the test typologies, in both the pre-COVID-19 and COVID-19 quarters under evaluation . In partThe COVID-19 pandemic keeps challenging health systems worldwide, with repeated infection peaks urging deeply modifying approaches and procedures for safeguarding patients and personnel. During the first lockdown in Lombardy, lasting from March to May 2020, several structures were converted into COVID-19-hospitals, and cancer patients were centralized to a few hubs, including INT Milan. Cancer screening programs including gynecology and mammographic exams were suspended, first and follow-up visits were often postponed, and surgery was delayed except for cases defined as urgent according to specific guidelines released by the regional department of health. In this scenario, cancer centers had the twofold goal of maintaining their proficiency within a COVID-19-low environment through careful screening for COVID-19 infections and new procedures for patient triage as well as internal reorganization .Here, we describe the impact of COVID-19-related procedures  applied upon the first peak of the SARS-CoV-2 outbreak in Lombardy on the TAT of the exams carried out at the units of surgical and molecular pathology in INT Milan. The TAT measures the proficiency of a pathology lab in providing timely diagnoses, a prerequisite for properly applying personalized treatments, especially in cancer patients. We found that the TAT of all the diagnostic procedures evaluated during the COVID-19 period was always >90%; specifically, 93.5–100% of the exams were completed within the prespecified timeframe. This result in spite of a 50% reduction in onsite personnel (mainly pathologists and biologists) and the implementation of time-consuming procedures including 24–72 h formalin fixation for all the samples admitted to the department likely stems from a generalized decrease in the overall workload due to lockdown restrictions. Actually, the volume of histological, cytological and molecular procedures showed a 14.1–36% reduction during the COVID-19 period. The reduction was particularly evident for day-hospital histological and cytological exams, while samples from inpatients subjected to surgery slightly increased, a trend attributable to social confinement with the centralization of cancer patients from general hospitals. PAP tests showed the greatest decrease −81.6%), further confirming that cancer screening had an abrupt slowdown during the first wave of COVID-19, a trend endangering cancer prevention in the near future. These data are in keeping with a recent report of London J.W. et al. [1.6%, furWe used digital pathology to conjugate personnel safeguarding with diagnostic performance: among the procedural changes made upon the COVID-19 crisis, telemedicine is likely to be maintained and further implemented in the future. Digital pathology allows effectively making diagnoses remotely based on cytological, histological, immunohistochemical and in situ hybridization slides. When properly integrated with the institutional software and the electronical clinical record, this model permits handling all the diagnostic steps following macroscopic examination, eventually enhancing the flexibility of the personnel work schedule.Collectively, our data provide evidence that implementing measures for the safeguarding of the personnel is feasible and does not affect the overall performance of the pathology laboratory. Although the model was implemented upon the first lockdown, when a significant reduction in the overall workload was observed, its efficacy was maintained in the second wave of the infection, is likely to be maintained beyond, and may be useful for other pathology laboratories included in a hub and spoke model. Finally, confirming the principle that every crisis represents an opportunity for progress, our data demonstrate that digital pathology should be implemented for a new and more efficient diagnostic approach.


Rattus norvegicus and/or Mus musculus, suggesting that rodents are the intermediate reservoir of SARS-CoV-2 and SARS-CoV. To evaluate the allergenicity values, the binding affinities of human leukocyte antigen (HLA) class I or II molecules with the spike proteins were calculated, and the results showed that both SARS-CoV-2 and SARS-CoV are predicted to bind to fourteen HLA class I and II molecules with super-high HLA allele-peptide affinities. The infection rate of individuals who have HLA alleles with very high binding affinities who might become infected and develop into refractory patients if there were no medical or non-medical interventions is about 7.36% and 4.78% of Chinese and Americans, respectively. Extremely high temperature and exceptionally low precipitation, the common climate factors between the outbreak sites of COVID-19 in Wuhan in 2019 and SARS in Guangdong in 2002, might have promoted coronavirus evolution into more virulent forms. Our hypothesis suggests that early immunization with an allergenically-engineered virus, in combination with continued surveillance of meteorological factors and viral mutations, may be one of the most powerful prophylactic modalities to fight this virus.The goal of this study is to investigate the probable intermediate hosts and the allergenicity of the notorious virus SARS-CoV-2 to understand how this virus emerged. The phylogenetic analysis of the virus spike proteins indicates that SARS-CoV-2 falls into various small subclades that include a bat coronavirus RaTG13, suggesting bats as a likely natural origin. Refined alignment of the spike protein in NCBI found several fragments that are specific to SARS-CoV-2 and/or SARS-CoV are specific to  Betacoronavirus sarbecovirus) originally found in bats in China [The recent outbreak of COVID-19 across the whole world was caused by a novel beta coronavirus isoform which was designated as SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV) based on the sequence of the viral RNA genome. The World Health Organization (WHO) claimed an international public health emergency for the outbreak in January and, later, a global pandemic in March 2020. As of 22 March 2021, more than 25,794,100 infected cases were reported in over 200 countries and regions. This prompted scientists to identify how the epidemic affected such a substantial amount of people in the world. Coronaviruses are known to circulate in birds and mammals, including bats [in China . Guo et Manis javanica) should be considered as a possible intermediate host for the novel coronavirus based on the 85.5% to 92.4% similarity of the viruses found in pangolins to a partial length (~86.3%) of the SARS-CoV-2 genome sequence [It is critical to identify the immediate reservoirs of SARS-CoV-2 and how the virus is passed onto humans. Recently reported results have been controversial. The results from Wei et al. suggested that snakes are the most probable wild animal reservoir for the virus, based on their relatively synonymous codon usage bias compared to other animal species . Work frlength ~8.3% of thsequence ,8. SARS-sequence ,11. How α-β-α layers, a similar structure component element shared by different allergens [Allergenicity is referred to as the ability of an antigen to induce an aberrant or detrimental immune response in the host, which is an overreaction and different from a normal immune response in that it does not result in a protective/prophylactic effect but instead causes physiological dysfunction and/or tissue damage . In the llergens . It can llergens ,22. TherThis study aims to: (1) Determine the potential virus intermediate reservoir by carrying out comprehensive amino acid sequence analysis and comparison of sliding sequence fragments of the novel virus with all sequences from mammals available in the NCBI database; (2) Analyze the allergenicity of the spike protein in SARS-CoV-2 and compare it with that of SARS-CoV to explain the mechanism of the COVID-19 pandemic from a new perspective. Furthermore, we systematically compare climate data in the past 50 years to predict any relationship between the meteorological conditions and the survival/development of the virus.The amino acid sequences of the SARS-CoV spike glycoprotein derived from humans, civets and bats were downloaded from NCBI. Sequence alignment was performed to identify three functional subunits: receptor binding domain, N-terminal domain and coronavirus S2 glycoprotein. A phylogenetic tree was constructed using the maximum likelihood method with best protein models for different sequence groups using the MEGA7.0 program ,24.First, protein sequences of SARS-CoV-2 and SARS-CoV derived from humans, civets and bats were analyzed through multiple sequence alignment to locate all potential homologous sites. Then, a homology site screening program was constructed to classify these sites and to screen out the homologous key sites in accord with consistent sites among human SARS-CoV-2, SARS-CoV, and civet SARS-CoV, but 50–100% different from bat SARS-CoV.The fixed-length sliding window method was used to split the spike glycoprotein sequence into equal length, non-overlapping peptide segments. Based on the latest version of the NCBI Reference protein library of whole organisms, a peptide source scanner was constructed to analyze the source of each peptide of the spike glycoprotein. The proportion of mouse-derived peptide was calculated, and the possible mouse-derived peptides were retained. By scanning in the NCBI Reference protein library and the non-redundant protein sequence library, Protein BLAST was performed to verify the exclusive origin of the peptides retained by the scanner.http://www.allelefrequencies.net/, accessed on 27 August 2021) [The allergenicity of SARS-CoV-2 and SARS-CoV was assessed by using the software NetMHC-4.0 ,26 and Nst 2021) , the numhttp://www.weather.com.cn/, accessed on 27 August 2021). Different meteorological factors, including 6 climate factors and 12 months from 1959 to 2019, were analyzed to obtain any clues regarding the outbreak of SARS-CoV-2. Using 12 months and 6 climatic features as primary data, 257,985 different combinations between month and climatic feature were produced. The correlation coefficients of different years under the combination of month and climatic features were calculated using R language. To construct a screening program aimed at 2019 and 2002, we screened out the climate combination features that exhibited a special strong correlation between the two years; that is, the corresponding climate combination features of Wuhan 2019 are very similar to those of Guangdong in 2002 (Pearson correlation coefficient > 0.8), but are less similar to those in most other years (Pearson correlation coefficient < 0.5). The monthly climate data of Wuhan and Guangdong from 1951 to 2019 were downloaded from the China Meteorological Network, including six climate characteristics, such as average temperature, average maximum temperature, average minimum temperature, precipitation, sunshine hours, and relative humidity  but no other mammal databases  in bat SARS-CoV . The eigA variety of pathogenic viruses can cause hypersensitivity reactions and are threats to human health. Therefore, it is of interest to investigate the allergenicity between virus proteins and host immune defenses. To assess the allergenicity and the infection potential of the virus to humans, we analyzed and classified HLA molecules that can tightly bind to the spike proteins of SARS-CoV-2 and SARS-CoV. Through the binding affinity analysis of human SARS-CoV-2 and SARS-CoV with HLA class I and II molecules, we found those HLA alleles that are predicted to bind to the spike protein from SARS-CoV-2 are very similar to those from SARS-CoV. The spike proteins from SARS-CoV-2, as well as SARS-CoV, are predicted to bind with high affinity to five human HLA class I alleles  and seveAs listed in http://www.allelefrequencies.net, accessed on 27 August 2021). This allele has a low binding affinity to SARS-CoV-2. In addition, SARS-CoV-2 harbors far fewer peptides with high binding affinity to HLA class I alleles than class II alleles . Currently, it is unknown how the virus can be transferred directly from animal species to humans in the seafood market and how the virus could be spread among diverse foods in this market. This suggested that there may be animals that freely contact all kinds of foods and spread the virus everywhere in the market. With the number of confirmed COVID-19 cases reaching 5,267,452 as of 24 May 2020, which is far more than the number of cases of severe acute respiratory syndrome (SARS), it is clear that the world is in the midst of a global pandemic. It is of the utmost importance to quickly discover the intermediate hosts of this virus and eradicate the source in order to prevent future outbreaks. Several research groups have recently attempted to address this issue . Bats, md market , which id market . HoweverMus musculus) and Norway rats  are the most widely distributed and most successful mammals, except for humans, on the planet and have been commensal with humans for thousands of years [https://tech.sina.com.cn/roll/2020-01-23/doc-iihnzhha4251798.shtml, accessed on 27 August 2021), thereby providing a food source for rats and mice. . The viruses harbored by wild animals would be therefore taken away by the foodies, thus rendering the viruses scattered everywhere in or even outside the Market and then transferred to humans.The house mouse , respectively. Two fragments (EAEVQID/NHTSPDV) shared by both viruses are more conserved than other proteins encoded by the viruses. This explains why the two viruses cross-react with the antibodies generated against the other . It alsoWith regard to SARS-CoV-2, even though the Huanan Seafood Wholesale Market was shut down on 1 January 2020, animal carcasses and viscera were observed and living rats and mice were still present through 17–19 January when the High-level Experts Group of the National Health Commission arrived to investigate the outbreak. Similar to the SARS-CoV outbreak 18 years ago, it is possible that rats and/or mice acquired the SARS-CoV-2 virus from the viscera of butchered animals, including, for example, bats, minks, pangolins that served as natural reservoirs for the virus, when these animals were traded as delicacies in the Market. Two studies showed that SARS-CoV-2 has infected cat populations in Wuhan during the outbreak and argued that the virus was transmitted in cats ,8. This Mice and humans have large-scale synteny across over 90% of their genomes but have a much lower extent of sequence orthology covering less than half of the two genomes . TherefoRhinolophus affinis (but not Rhinolophus sinicus), with 96.2% identity at the whole-genome level to SARS-CoV-2 [R. sinicus and R. affinis, have a similar appearance, and R. affinis is the main variety of bat in the Hubei Province. Nevertheless, no SARS-CoV-like virus has been identified from the Hubei R. affinis bats, but from the bats R. macrotis and R. ferrumequinum, in which no viruses had been isolated by culture with Vero E6 cells from fecal swabs of the PCR-positive samples [R. pearsoni bats are indigenous across the Yunnan province, China and Southeast Asia, and were suggested to harbor coronaviruses closely related to SARS that infected the human population [R. sinicus, not R. affinis), civets, pigs, and mouses concluded that SARS-CoV-2 could use all but mouse ACE2 as an entry receptor in the ACE2-expressing cells; that is to say, the mouse ACE2 would not facilitate SARS-CoV-2 entry to mouse cells. This conclusion may be doubtful based on the homology analysis performed on ACE2. The alignment result indicated that the identity of ACE2 amino acid sequences between human and mice (Mus musculus) or rats (Rattus norvegicus) are 81.05% to 82.49% . Therefore, the entry receptor ACE2 is not a problem for the coronavirus to attack humans from then on, no matter whether RaTK15 was isolated from R. pearsoni, R. sinicus, or R. affinis. According to sequence alignment with other SARS-CoVs, SARS-CoV-2 has an unusual insert of 10 amino acids (HVSGTNGTKR) in the N-terminal domain . This inRS-CoV-2 . It is n samples . In addipulation . The stuo 82.49% , respect−3 nucleotide substitutions per site per year for SARS-CoV [SARS-CoV-2 is a positive-sense, single-stranded RNA coronavirus. It possesses a large RNA genome and undergoes RNA recombination, as in other coronaviruses, at a high frequency of nearly 25% for the entire genome , thus drSARS-CoV . Further+CCR6+ Th17 in CD4+ T cells [https://arxiv.org/abs/1305.2659, accessed on 27 August 2021). Interestingly, both SARS-CoV and SARS-CoV-2 are inherently capable of reacting with different allelic forms of HLA molecules and tightly binding dozens of different HLA molecules. This means that these two viruses would have similar allergenicity and would trigger similar pathophysiological insults in humans. This is supported by autopsy and biopsies of cadavers of patients who died from SARS-CoV-2, because the pathological characteristics of COVID-19 strongly resemble those seen in SARS and Middle Eastern respiratory syndrome (MERS) coronavirus infections ,62,63. H T cells  would be T cells ,66. Ther T cells ,67. Therhttps://arxiv.org/abs/1305.2659, accessed on 27 August 2021). These existing SARS-CoVs have significant potential to evolve into highly virulent strains when favorable meteorological conditions occur, highlighting the potential risk for reemergence of SARS as well. Based on the mutation rate of coronavirus and meteorological extremes occurring because of climate change, we speculate that SARS could re-emerge in the near future in a new form. A SARS vaccine is therefore urgently needed. However, a SARS-like chimeric virus experiment demonstrated that both monoclonal antibody and vaccine approaches and prophylactic modalities failed to neutralize and protect from infection by those CoVs that possess a novel spike protein [It is thus likely that a SARS epidemic could recur when the meteorological conditions in the world are suitable for SARS-CoV-2 mutation. The virus would be maintained in general populations who have no high binding-affinity HLA alleles and be transferred between individuals. As described above, more than 92.64% of the population harbors the virus with no obvious symptoms, meaning that many people will be SARS-CoV-2 positive as detected by nucleic acid testing. We have demonstrated that weakly virulent SARS-CoVs might still exist in humans for years  group(s) of individuals with specific MHC genotype(s), these susceptible individuals either die or heal with the development of immunity. The vast majority of individuals who range from being paucisymptomatic to asymptomatic or having recovered from the disease still harbor the novel virus while it is spread within the population with no epidemic. When conditions favor mutations, the virus becomes more virulent and targets individuals harboring other type(s) of MHC genotype(s), leading to soaring infection numbers and another epidemic. A new cycle would therefore start within the population. When the virus has acquired mutations favoring a cross-species jump, increasing infection numbers would lead to an epidemic followed by asymptomatic transmission within the new species into which the virus has jumped. This phenomenon will occur in many different mammals including humans, bats, rodents, etc. Furthermore, when the virus accumulates the requisite mutations enabling interspecies transmission and binding of all MHC alleles among another species population with high affinity, a much deadlier super virus could emerge to eradicate the species. The only way to defend against such a super virus is to employ prophylactic modalities, such as early immunization with a hypoallergenic virus that has been gradually attenuated from the super virus, as exemplified by the incidence of smallpox versus cowpox.In summary, our results indicate that both SARS-CoV-2 and SARS-CoV are naturally originated from bats and might be transmitted to humans through rodents. This was demonstrated by carrying out comprehensive amino acid sequence analysis and comparison of sliding sequence fragments of the novel virus with all sequences from mammals available in the NCBI database. SARS-CoV-2 and SARS-CoV have similar binding affinities to the HLA antigen and would have similar potential to induce inflammation. Different populations have distinct allele distribution patterns and thus variable infection rates. It is predicted that the virus will severely infect 4.78% to 7.36% of the American and Chinese populations, respectively, and would make them suffer severe symptoms. Meteorological factor analysis indicates that Wuhan 2019 and Guangdong 2002–2003 have similar climate features, with extremely high temperatures and exceptionally low precipitation, which might imply some link between the climate environment and the survival and development of the coronaviruses. Early immunization with allergenically-engineered virus together with a continued surveillance of meteorological factors and viral mutations may serve as one of the most powerful prophylactic modalities to fight this virus.


Increasing evidence has demonstrated that pyroptosis, a type of inflammatory programmed cell death, plays an important role in the pathogenesis and progression of gastric cancer. However, it remains unclear whether pyroptosis-related long non-coding RNAs (lncRNAs) can be used to predict the diagnosis and prognosis of gastric adenocarcinoma. This study aimed to evaluate and test the role of the lncRNA signature associated with pyroptosis as a prognostic tool for stomach adenocarcinoma (STAD) and to ascertain their immune value. Relative RNA-sequencing data were extracted from The Cancer Genome Atlas database (TCGA), and data preprocessing was performed for STAD. Pearson correlation analysis was used to determine whether lncRNAs were significantly correlated with pyroptosis based on 23 genes related to pyroptosis. Univariate Cox regression and least absolute shrinkage and selection operator(LASSO) analyses were both adopted to select features and establish the pyroptosis-related lncRNA (PRL) prognostic signature. Kaplan–Meier(KM) survival analysis of the different risk groups was conducted according to the risk scores. We further examined the functional enrichment, tumor microenvironment, and landscape of mutation status among the different risk groups, and these analyses further explained the reasons for the differences in the prediction as well as survival value of the different risk groups. Four lncRNAs, including HAND2-AS1, LINC01354, RP11-276H19.1, and PGM5-AS1, were involved in the PRL signature and used to split STAD patients into two risk groups. Overall survival time(OS) was significantly higher in the low-risk group than in the high-risk group in both the training and validation groups. Functional enrichment analysis was further employed to analyze differentially expressed genes in high- and low-risk groups to identify potential molecular functions and pathways associated with pyroptosis in the gastric cancer microenvironment. Protein-protein interaction (PPI) and Friends analysis identified hub genes that may play a key role in differentially expressed genes in high- and low-risk groups. In addition, there were remarkable discrepancies between the different risk groups in the tumor stage (P < 0.01) and histologic grade (P < 0.05). Furthermore, drug-susceptibility testing indicated potential sensitive chemotherapeutic drugs for each risk group. This study is the first to establish and validate STAD-associated PRLs that can effectively guide the prognosis and the immune microenvironment in STAD patients and provide evidence for the development of molecularly targeted therapies related to pyroptosis. Gastric cancer (GC), an extremely heterogeneous disease, is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide , 2. Stomvia the activation of caspase-1/4/5/11 by inflammasomes leads to cell membrane rupture and the release of intracellular pro-inflammatory substances, which triggers a strong inflammatory response in the immune microenvironment (in vivo and in vitro (Pyroptosis is a novel mode of programmed cell death mediated by gasdermin proteins . The cleironment . Pyroptoin vitro .Long non-coding RNAs (lncRNAs), with a length of more than 200  nucleotides, are emerging as key regulators of multiple biological processes. Recently, the aberrant expression of lncRNA genes has been suggested to be critical in regulating tumor progression, which is involved in the regulation of tumor proliferation, immune evasion, resistance to cell death, and regional or distant metastasis in STAD .Therefore, lncRNAs are an important breakthrough in deciphering the molecular mechanism of the pyroptosis process, and lncRNA-based studies provide evidence for the development of lncRNAs as therapeutic targets for inducing pyroptosis in gastric cancer. Notably, it has been recently revealed that lncRNAs are crucial modulators of pyroptosis . Howeverhttps://xenabrowser.net/datapages/) was downloaded. Gene mRNA data, copy number variation (CNV) data, Single nucleotide polymorphism (SNP) mutation data, and clinical information for STAD were downloaded from UCSC Xena; the detailed clinical results are presented in the TCGA dataset of STAD with 373 samples and clinical information from 406 patients in the UCSC Xena , the lncRNA expression profiles of tumor samples , the mRNA expression profiles of all samples , and the lncRNA expression profiles of all samples .http://www.genecards.org/), GO BP(http://geneontology.org/), and Reactome databases(https://reactome.org/) with the keyword “pyroptosis”. Then, the jveen package  analysis was performed using the samples, as indicated. To acquire tumor-associated lncRNAs, differential expression analysis was performed using the limma package  on gastrSection 2.2 and differential lncRNAs acquired in Section 2.3 in the tumor samples. We adjusted p-values for multiple testing using the Benjamini–Hochberg (BH) correction and selected the differentially expressed lncRNAs with BH-adjusted P < 0.01 as significantly co-expressed lncRNAs.To derive the differentially expressed lncRNAs associated with the pyroptosis process, we performed Pearson co-expression analysis of the expression of the pyroptosis-related genes from To determine the potential pyroptosis-related prognostic lncRNAs, survival analysis was carried out on these candidate PRLs to determine their effects on STAD patient survival. The lncRNAs with P-values < 0.05 were considered as candidate prognostic lncRNAs and were analyzed further.The STAD group was randomly divided into a training set and a validation set . The LASSO method was implemented using the glmSparseNet package  in the tEach sample received a risk score according to the novel lncRNA risk score model. Finally, based on the optimal cutoff separation, we divided the TCGA-STAD samples into high- and low-risk groups. To evaluate the differences in the expression profiles of the high- and low-risk subgroups, after PCA, we performed differential analysis between high- and low-risk subgroups using the limma package  with a sSection 2.7 based on the clusterProfiler package , GO-Cellular Component (GO CC), and GO-Molecular Function (GO MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the differential genes acquired in  package  and visuhttp://www.gsea-msigdb.org/gsea/index.jsp) was carried out between high- and low-risk groups to evaluate their potential biological functional alterations. Absolute values of normalized enrichment score (NES) >1 and nominal P-values < 0.05 were selected as thresholds of significance.To obtain the expression distribution patterns of the optimal PRLs in the different risk groups, we performed a PCA using scatter plots. Furthermore, GSEA   at1-, 3‐ and 5‐years were calculated to assess the predictive performance of the top 10 hub genes using the timeROC package (version 0.4) (Section 2.12. Tumor mutation burden (TMB) was defined as the total number of somatic mutations per megabase in each tumor sample. Therefore, we calculated the number of gene mutations in each tumor sample to determine the TMB of each tumor sample. We then performed statistical analysis between groups to determine the TMB in the high- and low-risk subgroups.To investigate the difference in SNP expression between the high- and low-risk subgroups, we analyzed the downloaded SNP data of STAD using the maftools package , in whicWe processed the downloaded CNV segment files to obtain the marker files and uploaded the files to the GenePattern Gistic 2.0 module for CNV analysis. The database default was selected for the analysis parameters, and the maftools package  was usedCIBERSORT  is an onBased on the different high- and low-risk groups, we performed Chi-square tests on the relevant clinical features in different risk groups , pathologic stage, TNM staging, vital status, gender, and race) to analyze and filter the disease processes that may be influenced by high- and low-tumor risk and disease-related events that affect the high- and low-risk subgroups.® Premix Ex Taq™ (TaKaRa) for qRT-PCR. All primers used in the PCR are listed in A total of 15 human gastric cancer tissues and paired adjacent non-tumor tissues (more than 2.5 cm from the edge of the cancer tissue), were surgically resected and first diagnosed for primary gastric cancer in the Foshan First People’s Hospital between January 2018 and December 2020. Patients who did not receive preoperative chemoradiation treatment were selected for this study. Ethical approval was obtained from the subject review committee of Foshan First People’s Hospital, and all 15 patients informed consent and signed the consent form. Gastric cancer tissues and adjacent tissues were immediately stored in liquid nitrogen after resection for further RNA extraction. Total tissue RNA was isolated using Trizol reagent , followed by cDNA synthesis using Prime-Script RT Master Mix  for reverse transcription and TaKaRa SYBR50 of different anticancer drugs and different risk groups  website to download the anticancer drug dataset and used the oncoPredict package to calculate the relationship between the ICk groups . We screAll statistical analyses and plots were performed using R (version 4.0.2). All data are presented as the mean ± standard deviation (SD) derived from at least three separate experiments. Student’s t-test or one-way analysis of variance (ANOVA) was used to evaluate differences among different groups. Otherwise, the correlation was evaluated using the Pearson correlation analysis. The correlations between risk groups and clinical variables were determined using the Chi-square and Student’s t-tests. Survival-related lncRNAs were identified using univariate Cox regression analysis. OS was calculated using the KM method and estimated by the log-rank test. For all statistical methods, statistical significance was set at P < 0.05.). According to the cut-off criteria of | log2FC | > 1.5 and adjusted P-value < 0.01, there were 388 differentially expressed lncRNAs between cancer and adjacent normal tissues . Using jvenn, we found that 27 (9 + 5 + 13) mRNAs were correlated with pyroptosis  through using univariate Cox regression analysis for selection, then we applied the LASSO method in the training group to establish a PRL signature to evaluate the prognosis of patients with STAD . The high-risk group was found to be mainly enriched in malignant pathways, such as focal adhesion; ECM receptor interaction; TGF-beta signaling pathway; and metabolism-related pathways, such as glycosaminoglycan biosynthesis. These findings indicate a relatively high level of metabolism in individuals at high risk.via miRNA sponges  can be found in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Foshan First People’s Hospital. The patients/participants provided their written informed consent to participate in this study.ZW, RY, and YG conceived and designed this research, obtained the funding and supervised the study. ZW, LC, and SZ performed relevant experiments, the data analysis, and wrote the manuscript. J L collected clinical gastric cancer samples. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (82002913), Natural Science Foundation of Tianjin (Nos. 20JCQNJC01850), and Guangdong Basic and Applied Basic ResearchFoundation (2021B1515120036).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.


Patients diagnosed with breast cancer treated with chemotherapy before surgery were included in this study. The tumor was imaged using ultrasound before the chemotherapy was started and in the middle of chemotherapy treatment (one month after starting). After treatment completion, patients were followed up according to standard clinical practice and categorized into two groups based on disease recurrence until the last follow-up. The ultrasound imaging was analyzed using advanced computational techniques, and artificial intelligence was used to develop models to differentiate between the two outcomes. We demonstrated that using the ultrasound image data, the final outcomes can be predicted as early as within one month of the start of chemotherapy.1) were determined from parametric maps acquired using grey-level co-occurrence matrices (GLCM) for each patient, which were further processed to generate 64 texture derivatives (QUS-Tex1-Tex2), leading to a total of 95 features from each time point. Analysis was carried out on week 4 data and compared to baseline (week 0) data. ∆Week 4 data was obtained from the difference in QUS parameters, texture features (QUS-Tex1), and texture derivatives (QUS-Tex1-Tex2) of week 4 data and week 0 data. Patients were divided into two groups: recurrence and non-recurrence. Machine learning algorithms using k-nearest neighbor (k-NN) and support vector machines (SVMs) were used to generate radiomic models. Internal validation was undertaken using leave-one patient out cross-validation method. Results: With a median follow up of 69 months (range 7–118 months), 28 patients had disease recurrence. The k-NN classifier was the best performing algorithm at week 4 with sensitivity, specificity, accuracy, and area under curve (AUC) of 87%, 75%, 81%, and 0.83, respectively. The inclusion of texture derivatives (QUS-Tex1-Tex2) in week 4 QUS data analysis led to the improvement of the classifier performances. The AUC increased from 0.70  without texture derivatives to 0.83 (0.73 to 0.92) with texture derivatives. The most relevant features separating the two groups were higher-order texture derivatives obtained from scatterer diameter and acoustic concentration-related parametric images. Conclusions: This is the first study highlighting the utility of QUS radiomics in the prediction of recurrence during the treatment of LABC. It reflects that the ongoing treatment-related changes can predict clinical outcomes with higher accuracy as compared to pretreatment features alone.Background: This study was conducted to explore the use of quantitative ultrasound (QUS) in predicting recurrence for patients with locally advanced breast cancer (LABC) early during neoadjuvant chemotherapy (NAC). Methods: Eighty-three patients with LABC were scanned with 7 MHz ultrasound before starting NAC (week 0) and during treatment (week 4). Spectral parametric maps were generated corresponding to tumor volume. Twenty-four textural features (QUS-Tex Breast cancer is the most commonly diagnosed malignancy and is the leading cause of cancer-related death among women . LocallyUltrasound (US) is a commonly used imaging modality with widespread clinical applications in differentiating benign versus malignant primary lesions in the breast or characterizing lymph nodes and facilitating tissue acquisition for histological evaluation. Quantitative ultrasound (QUS) is similar to the clinically used ultrasound device, with the primary difference of acquisition and analyzing the raw radiofrequency (RF) data, which aids in more detailed biological characterization of the imaged tissues. The biophysical basis of QUS imaging is the differences in the elastic properties, leading to differential scattering reflected as backscatter ultrasound signals from tissues ,11. ApplThe study here explored the role of QUS features, including higher-order texture features obtained during the early course of NAC, in predicting the recurrence for patients with LABC. We have demonstrated the utility of QUS in predicting clinical outcomes with reasonable accuracy. Being a portable technique with rapid scan acquisition, QUS may be beneficial over other imaging modalities for response monitoring mid-treatment as well as in characterizing long term outcomes.The prospective study was approved by the Research Ethics Board at Sunnybrook Health Sciences Centre (SHSC), Toronto, Canada, and registered with Clinical Trials.gov (NCT00437879). All study participants signed a written consent form. Inclusion criteria were the presence of biopsy-proven LABC and scheduled to receive NAC as part of their standard care of treatment. Clinical information was obtained from a prospectively maintained database with additional information collected from patient electronic medical records. Patients diagnosed with inflammatory breast cancer or upfront metastasis were excluded from the analysis. The time to recurrence was defined from the start of NAC to the clinical or radiological evidence of disease recurrence. In order to avoid underestimating the risk of recurrence, the minimum follow-up time for patients without any recurrence was required to be at least four years.The majority of patients were treated with an anthracycline and taxane-based NAC, while some patients received other chemotherapy treatments. The chemotherapy regimen and the interval between two cycles were based on the decision of the treating medical oncologist. Patients with human epidermal growth factor receptor 2 (HER2) positive status received targeted therapy in combination with NAC. After receiving NAC, all patients underwent surgery (mastectomy or breast-conserving surgery), in conjunction with sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), based on the pretreatment disease stage and response. Patients received adjuvant locoregional radiation therapy and maintenance targeted therapy or endocrinal therapy according to standard institutional practice. Patients were followed up every 3–6 months during the first two years, and then after every 6–12 months, as indicated clinically. Oncologists confirmed all recurrences with clinical examination, diagnostic imaging, and histological confirmation as indicated clinically.Ultrasound data were obtained using a Sonix RP clinical research system  coupled to a L14–5/60 transducer with a central frequency of 7 MHz and −6 dB bandwidth of 4–9 MHz. Data were digitally collected with a sampling frequency of 40 MHz with a 16-bit resolution. Patients were scanned before the initiation of systemic therapy (week 0) and at week 4 from the start date of NAC. The scan frames were collected at intervals of 1 cm, spanning the entire tumor, with the transducer focus towards the center of the tumor. On each ultrasound frame, a region of interest (ROI) was manually contoured corresponding to the tumor. Using a sliding window approach, each ROI was divided into analysis blocks of size 10λ × 10λ, with a 94% adjacent overlap in axial and lateral directions (2.2 mm × 2.2 mm approximately). The detailed description of standardization methods and calculation of quantitative ultrasound parameters have been explained in previous publications . In shorThe spatial distribution of QUS parameters was evaluated from parametric maps using grey-level co-occurrence matrices (GLCM) , represe1) were determined from the six spectral parametric maps (excluding ACE).In summary, the CON parameter measures the magnitude of intensity differences between two neighboring pixels. The COR parameter is a measure of how linearly a pixel is correlated to its adjacent pixel. The ENE parameter measures textural uniformity, and the HOM parameter estimates the incidence of pixel pairs of different intensities. A total of 24 textural features (QUS-Tex1-Tex2). The steps were repeated for all ultrasound scans obtained (at weeks 0 and 4). The ∆week 4 data was obtained from the difference in mean QUS parameters, texture features (QUS-Tex1), and texture derivatives (QUS-Tex1-Tex2) of week 4 data from the corresponding week 0 data.In order to determine third-order imaging features (texture derivatives), the GLCM method  was repeated on the 16 QUS-texture maps  described above. As a result, a total of 64 texture derivatives were determined . The list of all the features extracted is presented in Therefore we had a set of 95 features from each time point , which was a combination of week 0 data and ∆Week 4 data, were used for the model development. A p-value of < 0.05 was considered statistically significant.A Shapiro–Wilk normality test was applied to the feature sets to confirm data distribution for the two groups (patients with and without recurrence). For normally distributed faculty, a parametric unpaired student Two machine learning classifiers, consisting of k-nearest neighbor (k-NN), and support vector machine-radial based function (SVM), were independently tested for comparison using MATLAB 2016a to evaluate diagnostic performance. The maximum feature number was set to 3 in the classification model to avoid model overfitting due to the high dimensionality of the data set . Since tSurvival analysis was undertaken using the Kaplan–Meier product-limit method. Univariate analysis was done using log-rank tests to compare the survival estimates for the two predicted groups using the radiomics model (predicted recurrence group versus predicted non-recurrence group).The analysis included a sample size of 83 women with LABC as per inclusion criteria. The patient’s ages ranged from 29 to 79 years (median 50 years). Clinical characteristics, histopathologic features, and treatment details are mentioned in p = 0.033), and ∆MBF-HOM-CON (p = 0.038) parameters exhibited a statistically significant difference between the two groups of patients (Representative ultrasound B-mode images and parametric maps of QUS features and selected texture and texture-derivative maps for a patient with (1a) and without recurrence (1b) are presented in patients . The cor1 + QUS-Tex1-Tex2) performed better as compared to the week 0 data (QUS + QUS-Tex1 + QUS-Tex1-Tex2). The AUC improved from 0.78 to 0.83 in the k-NN classifier model, whereas it only enhanced marginally in the SVM model, from 0.76 to 0.78 . The AUC improved significantly from 0.70 to 0.83 , and presented in The median follow up for the entire cohort was 69 months (interquartile range 49–84 months). Out of eighty-three patients included in the analysis, 28 patients had developed disease recurrence, with the median time to recurrence being 24 months. More than 80% of the recurrences were seen in the first four years. The site of the first recurrence was isolated local disease in five, local and regional in one, regional and distant in four, isolated distant relapse in 17, and combined local, regional, and distant in one patient. For the entire group, five-year recurrence-free survival (RFS) and five-year overall survival (OS) were 68% and 79%, respectively. The predicted RFS using the k-NN and SVM classifier is shown in Statement of principal findings: In the study, 83 patients with LABC were imaged using QUS before starting NAC and at week 4 of treatment. The changes in spectral parameters, texture and higher-order texture-derivate features from chemotherapy were computed and radiomics models were developed with disease recurrence as the endpoint. A k-NN classifier was able to predict recurrence with sensitivity, specificity, accuracy, and AUC of 87%, 75%, 81%, and 0.83, respectively.Strengths and weakness: This is the first study demonstrating the ability of QUS obtained during the course of NAC in predicting disease recurrence in patients with breast cancer. The study included patients with diverse clinical and molecular characteristics suggesting the applicability of QUS radiomics model across different populations of patients with breast cancer. We had a reasonable follow up (median follow up > 5 years), with clinical outcomes recorded prospectively adding to the strengths of the study. One of the limitations of the study here was the relatively small number of patients who presented with recurrence. The inclusion of more patients will likely improve the performance and robustness of the classifiers, and also extending the study at other centers will help in undertaking external validation techniques.Relation to other studies: Several studies have identified various factors that can predict the recurrence of breast cancer ranging from clinical characteristics, immunohistochemical assays to gene expression levels. Clinically, the most reliable prognostic markers include nodal status and tumor size. Various other clinical factors such as tumor grade, patient age, and treatment type have been added to build a Clinical Treatment Score (CTS), which provides a recurrence risk estimate for ER+ breast cancer [t cancer . In the t cancer ,27,28. St cancer . Similart cancer .Meaning of the study: The role of ultrasound is widely recognized in the screening and diagnosis of breast masses. Applications include assessing morphological tumor details , anatomic relationships of masses to their surrounding tissues, and regional lymph nodal involvement. However, recent clinical applications of ultrasound have expanded in the management of breast cancer. For example, ultrasound elastography techniques can be used to differentiate benign from malignant breast lesions, contrast-enhanced ultrasound can characterize tissues with different vascularity, and three-dimensional ultrasound improves the characterization of breast lesions [ lesions . QUS Tec lesions ,32.In the past, it has been demonstrated in preclinical studies that QUS can evaluate cell death in response to different treatment modalities ,34. The W0, AAC-CON-CONW0, and ∆ASD-CON-CON, at week 4 (Week 0 + ∆Week 4). Amongst the three features, the two parameters (ACEW0 & AAC-CON-CONW0) are related to tissue composition and microstructural organization of cells. The selection of texture derivative ‘AAC-CON-CONW0′ technically represents the summation of other sub-variables and possibly depicts heterogeneity within a tumor at a more advanced level. Other parameters were selected from the pretreatment (week 0) data. This suggests that the spatial organization of the tissue as reflected in QUS parameters relates to tumor biology and may have a role in recurrence prediction to some extent before initiation of treatment. In previous studies, ACE W0 was found to be a significant predictive parameter to distinguish tissues of different types, supporting the findings in this study [The work presented in this study aimed to investigate the effectiveness of QUS texture derivatives in predicting recurrence risk rather than the local response in breast cancer within weeks of initiation of systemic therapy. The improvement in AUC performance with the addition of texture derivatives, as well as from week 0 to week 4 in the k-NN classifier , signifiis study ,38.The other texture derivative selected was ∆ASD-CON-CON, where ASD signifies microstructural size and may be related to lobular diameter, and the contrast derivatives (contrast of contrast), which measure the intensity differences. This was found to be significant after the initiation of treatment. As mentioned previously, changes occurring at the molecular level in tumor cells in response to the treatment can be captured by the QUS technique. Hence, it is postulated that, as treatment initiates, ASD and its derivatives have a potential role in predicting recurrence risk and differentiating the two groups.We estimated the survival outcomes based on a prediction by the two machine learning classifiers. Out of the two, the k-NN classifier using texture derivatives (week 4) was able to closely approximate the curve obtained from the patient’s clinical details on recurrence, thus displaying the efficacy of the classifier in recurrence prediction. Magnetic resonance imaging and mammography are used more commonly in clinical practice for imaging of breast malignancies aiding in diagnosis, staging, and response evaluation to treatment. Ultrasound forms an attractive strategy due to its portable nature, ability of rapid scan acquisition, no radiation hazards, and better patient compliance. Quantitative analysis of the ultrasound imaging also provides an opportunity to capture the changes in the tumor in real-time during treatment, much earlier than other imaging modalities which rely on the morphological changes.Unanswered question and future research: In the future, with a larger cohort, it may also be worthwhile to combine clinical features and molecular subgroups with a QUS-radiomics model. The promising results obtained in the study here emphasize the importance of QUS parameters as a valuable tool for the timely identification of patients whose tumors have a strong tendency towards recurrence. An early prediction of recurrence risk can potentially assist oncologists in making decisions in regards to selecting systemic agents for treatment or changing a less effective treatment to more effective therapy or maintenance therapies. It may provide an insight into an earlier shift to surgery or towards an intensification of systemic therapy before missing the ‘therapeutic window’ for benefit.To summarize, this study demonstrates the effectiveness of QUS-radiomics in predicting recurrence in patients with LABC early in the course of treatment. The work presented here highlights the role of higher-order imaging features (texture derivatives) in improving the performance of the classifiers at four weeks into neoadjuvant chemotherapy. Further studies externally validating these initial, single-center findings are warranted.