This study estimated the incidence of major amputation for people in North Queensland, Australia, examined changes in amputation rates over time and investigated survival after major amputation.This was a retrospective study of patients who underwent a major amputation above the ankle between 2000 and 2015. Major amputation rates and incidence rate ratios (IRR) were calculated using census data to define the at-risk population. Associations between risk factors and calendar year with major amputation were assessed using quasipoisson regression. Kaplan-Meier survival and Cox-proportional hazard analyses estimated the incidence of and risk factors for all-cause mortality.The annual incidence of major amputation was estimated to be greater in Aboriginal and Torres Strait Islanders than non-Indigenous people . After adjusting for population growth, the annual incidence of major amputations did not change significantly over time for either groups. Aboriginal and Torres Strait Islander people were at greater risk of all-cause mortality after major amputation compared to non-Indigenous people, although this association was not significant after adjusting for other risk factors .The incidence of major amputation in North Queensland has not reduced over time, indicating the need for better preventative treatments, particularly in Aboriginal and Torres Strait Islander people.The online version contains supplementary material available at 10.1186/s12902-021-00764-z. Lower extremity amputations, including excision of toes  and amputations above the ankle , are significant complications of diabetes-related peripheral neuropathy and peripheral artery disease (PAD) . IndividRecent studies have suggested a decline in major amputation rates despite a rise in the prevalence of diabetes , 3. A reThis was a retrospective study performed by assessing hospital records on all patients who underwent a non-traumatic major amputation at The Townsville Hospital and Health Service (THHS) between 2000 and 2015. The THHS is the main tertiary centre and health service for Northern Queensland. It has a geographical coverage of approximately 148,000 square kilometres and serves approximately 4.8% of the total Queensland population . A 5-memClinical characteristics collected for each patient included: age at the time of amputation; sex; history of diabetes, hypertension, PAD, ischaemic heart disease (IHD) and end-stage renal disease (ESRD); and operation type. Hypertension and diabetes were defined by history or medical treatment for these conditions. IHD was defined by a history of myocardial infarction, angina or coronary revascularization. ESRD was defined by requirement for dialysis. PAD was defined by an ankle-brachial index (ABI)<0.9 and/or imaging demonstrating stenosis or occlusion of the lower limb arteries as previously described , 15. OpeThe annual, population-standardised incidence of major amputation in relation to Aboriginal and Torres Strait Islander status, age and diabetes status was estimated with the following assumptions about the population serviced by the THHS:i)It included 217,893 individuals located in Townsville, Charters Towers, Ingham, Cardwell, Ayr, Magnetic and Palm Islands, Home Hill, Hughenden and Richmond  based onii)n=12,800) were re-assigned to the Aboriginal and Torres Strait Islander people or non-Indigenous categories in proportion to the numbers recorded for those who did report Aboriginal or Torres Strait Islander status .15,409 individuals were Aboriginal or Torres Strait Islander people and 189,684 were non-Indigenous based on the 2011 Australian Bureau of Statistics Census . Individiii)The numbers of at-risk individuals  in different age groups  were estimated using state-wide diabetes prevalence rates reported by the Australian Bureau of Statistics . The ageiv)Since an estimate of the at-risk population size was only available for the calendar year 2011, the at-risk population for the remaining years in the study period was estimated assuming constant population growth. In particular, estimates of the at-risk population sizes backward in time to the year 2000 and forward in time to the year 2015 were generated assuming a 2% (multiplicative) annual decrease or increase in the baseline 2011 value  across A reported weakness of the Australian census is an under-estimate of the number of Aboriginal and Torres Strait Islander people  for Queensland . Sensitiwww.r-project.org/). P values<0.01 were considered statistically significant.Clinical characteristics were compared between Aboriginal and Torres Strait Islander and non-Indigenous people. Histograms, skewness and kurtosis tests suggested that continuous data were not normally distributed. Thus, continuous data were summarized using median values and inter-quartile ranges and compared between groups using the Mann-Whitney U test. Nominal data were presented as count and percent (unless otherwise stated) and compared using chi-squared and Fishers exact tests. Kaplan-Meier analysis was used to examine patterns of all-cause mortality in Aboriginal and Torres Strait Islander and non-Indigenous people, with differences compared using the log rank test. Multivariable Cox proportional hazards analysis was used to assess the association between ethnicity and all-cause mortality, adjusting for relevant confounding variables . These variables were included in the Cox proportional hazard analysis as they are established risk factors for mortality . All Coxp=0.002), have diabetes  and ESRD  than non-Indigenous patients. There were no statistically significant differences in the type of major amputation performed in Aboriginal and Torres Strait Islander and non-Indigenous patients.During the study period, 374 patients underwent a non-traumatic lower limb major amputation. Of these, 70 18.7%) patients identified as Aboriginal and Torres Strait Islander people and 304 patients identified as non-Indigenous . Kaplan-Meier survival curves illustrating the cumulative proportion of patients with a major amputation who survived are shown in Fig.p=0.005). In the unadjusted Cox proportional analysis, Aboriginal and Torres Strait Islander patients who underwent a major amputation were at greater risk of all-cause mortality during follow-up compared to non-Indigenous patients . The association remained significant when adjusted for age and sex  but no longer remained significant when adjusted for age, sex, IHD, ESRD and hypertension  (Tablep=0.001). No significant difference in the incidence of contralateral major amputation was observed between Aboriginal and Torres Strait Islander or non-Indigenous patients  follow-up of Aboriginal and Torres Strait Islander and non-Indigenous patients were 2.3 (0.4 to 5.7) and 3.3 1.3 to 8.1) years, respectively (.3 to 8.1Recent systematic reviews have reported a decline in the incidence of major amputation in various countries and attributed this to improved multi-disciplinary care of the high-risk foot , 3 This Direct comparisons of major amputation rates between other studies are difficult since there is considerable variation in the methods used in previous investigations , 20. A nThe clinical characteristics of people undergoing major amputation in this study were comparable to other Australian reports , 11. AboThis study had several limitations including its retrospective design and relatively small sample size. The study used participants self-identifying as Aboriginal people and Torres Strait Islanders at the time of admission. This was not cross-referenced with any other records and it is therefore possible that the number of Aboriginal people and Torres Strait Islanders may have been under or over estimated. Patient data were obtained retrospectively from hospital records and data on smoking were not recorded due to inconsistent reporting of smoking behaviour in hospital charts. This is a recognised limitation of data from hospital records and has been highlighted in previous datasets . Higher In conclusion, this study suggests that the annual incidence of major amputation in North Queensland is high and is not decreasing over time, which is in contrast with reports of reducing amputation rates in other regions within Australia , 22, 23 Additional file 1: Supplementary Table 1. Quasipoisson regression analysis examiningtheassociation between Aboriginal and Torres Strait Islander status, sex, diabetes status, age and time on major amputation rates for varying assumptions aboutthe classification of Aboriginal and Torres-Strait Islander and non-Indigenous peoplein the Townsville census data. SupplementaryTable 2. Quasipoissonregressionanalysis examiningthe associationbetween time and major amputation rates for varying assumptions about thegrowth rate. Supplementary Figure 1. Kaplan-Meiercurve illustrating the cumulative proportion of Aboriginal and Torres Strait Islander andnon-Indigenous patients who had a contralateral major amputation.


Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors . PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers  during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata  was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs  The pancreatic epithelium gives rise to functionally distinct endocrine and exocrine cells through a well-orchestrated multi-step process. In the developing human pancreas, the 1st trimester is characterized by the proliferation of branched tubules into primitive acini, islets and ducts . By the PaSCs are non-endocrine, mesenchymal-like cells that reside in the periacinar, periductal, perivascular, and peri/intra-islet regions of human and rodent pancreas . The embin vitro co-culture systems. Here, we showed, using transmission electron microscopy and double immunofluorescence staining, that PaSCs can be identified based on their stellate-like morphology and noticeable foot-like processes, and that a high population of PaSCs in the developing human pancreas are located near ductal cells and newly formed islet cells during the 1st trimester of pregnancy. Co-culture of freshly isolated hIECCs and active PaSCs resulted in a significant decrease in the number of beta-cells and increased exocrine cell number. Suppressing the activation of PaSCs or Notch signaling resulted in enhanced beta-cell differentiation and a decreased number of exocrine cells in hIECCs, suggesting that Notch signaling may be one mechanism by which PaSCs regulate human fetal pancreatic cell differentiation.In the present study, the spatial and temporal localization patterns of PaSCs were examined in the human fetal pancreas from 1st to 2nd trimester of pregnancy . Using isolated human fetal PaSCs, the possible effects of PaSCs on human fetal islet-epithelial cell cluster (hIECC) differentiation were investigated in Human fetal pancreases  were collected according to protocols approved by the Health Sciences Research Ethics Board at Western University , in accordance with the Canadian Council on Health Sciences Research Involving Human Subjects guidelines. Tissues were immediately processed for electron microscopy, immunohistology, RNA or protein extraction or culture, with a minimum of three pancreases per age group .Human fetal pancreases at 14–16 weeks of fetal age, a stage at which there is an enriched PaSC population, were carefully dissected from surrounding tissues and immediately digested using dissociation buffer containing collagenase V  followed by filtration through a 250 μm nylon mesh to yield fine cell-clusters. PaSCs were isolated from human fetal pancreases using an outgrowth method and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (1:1 v/v) containing 10% fetal bovine serum , as described previously . The ver® Spectrum spectrophotometer , as previously described (Cultured PaSCs at passage 3–5 were plated in 96- or 24-well culture plates with triplicate wells per group . PaSCs wescribed . Each ex+/CK19+) and endocrine cells, as described previously (Direct co-cultures: sub-confluent (80–90%) PaSCs in a 12-well plate were pretreated with or without 100 nM ATRA for 24 h before hIECCs were plated on top. Indirect co-cultures: hIECCs were plated on Millicell® Inserts and co-cultured with sub-confluent PaSCs pretreated with or without ATRA (Conditioned media cultures: hIECCs were treated with or without media from PaSCs (PaSC-conditioned media) diluted to 20% (v/v) with CMRL1066.Pancreatic tissues  were pooled at the time of collection and digested to yield hIECCs, which contained epithelial progenitors  or DLL4 plus gamma secretase inhibitor DAPT , which prevents Notch receptor signal transduction . The conFor scanning electron microscopy (SEM), human fetal pancreas samples were cut into 2 × 2 mm fragments and fixed with 2.5% glutaraldehyde, as described previously (eviously . SamplesFor transmission electron microscopy (TEM), pancreata were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and post-fixed with 1% osmium tetroxide and embedded in araldite medium (2).e medium . Semi-the medium . Cells w+ and αSMA+ cell populations. To assess the effect of PaSCs on pancreatic cell differentiation, the hIECCs cultured with PaSCs were stained for endocrine, exocrine, and transcription factor markers. The percentage of immunoreactive cells was obtained by counting 500–1,000 cells per section per experimental group, with a minimum of three repeat experiments per group (in situ stained for ECM markers (Whole pancreases or cultured hIECCs wrapped in 2% (wt/vol.) agarose were fixed and embedded in paraffin . Sectioner group . To veri markers .RNA from pancreatic tissue and cultured hIECCs was extracted using either TRIZOL reagent or the RNAqueous-4PCR kit (Invitrogen) . Real-tiProteins from human fetal pancreases and cultured PaSCs were extracted in Non-idet-P40 lysis buffer. Equal amounts of protein were separated in 12% SDS-PAGE gels, transferred to a nitrocellulose membrane (Bio-Rad), and incubated with appropriate dilutions of primary antibodies  followedt-test, difference among more than two groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Differences were considered to be statistically significant when p < 0.05.Data are expressed as mean ± SEM. Statistical significance was analyzed using GraphPad Prism  with 95% of confidence interval. The difference between two groups were analyzed by paired Student’s +, SOX9+ and NKX6-1+ cells were found to have positive labeling for desmin or αSMA, but the majority of the cells showed no co-staining  cells with no co-localization  were found to co-label with desmin or αSMA, suggesting that PaSCs may not be a primary progenitor cell source for either endocrine or exocrine cells in the developing human pancreas. The concept that PaSCs represent a progenitor or stem cell pool that can differentiate to other pancreatic lineages is controversial, as demonstrated in multiple publications  resulted in a loss of ECM production, leading to decreased islet-acinar cell contact, acinar cell function and survival  and tumor cells of mice with advanced pancreatic ductal adenocarcinoma . The patients/participants provided their written informed consent to participate in this study.JL and BC contributed to the acquisition of data, interpretation of results, preparation of the manuscript, revisions, and final approval for the revision to be published. GF and CG contributed to collect all human fetal pancreas used in this study, critical comments for manuscript and revision preparation, and final approval for the revision to be published. RW contributed to the experimental design, interpretation of data, the manuscript preparation, revisions, final approval for the revision to be published, and is the guarantor of this work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


TTR gene that predominantly involve the heart include Val122Ile, Leu111Met, and Ile68Leu. The wild-type form of ATTR is also predominantly cardiac. Phenotypic heterogeneity is linked to differences among specific pathogenic TTR variants, geography, and the subtype of endemic versus nonendemic disease. Factors contributing to wild-type ATTR are largely unknown, but similar factors likely influence the penetrance of hereditary ATTR. Recognition of ATTR-CM is improving due to the increased use of cardiac scintigraphy as a noninvasive diagnostic tool, and early recognition of cardiac infiltration is crucial to optimize long-term prognosis.Transthyretin amyloid cardiomyopathy (ATTR-CM) is an underdiagnosed disease and an underestimated cause of both heart failure and conduction abnormalities. It is characterized by pathologic accumulation of extracellular protein arising from unstable transthyretin (TTR) tetramers, which dissociate into monomers that misfold, aggregate, and form insoluble fibrils that are resistant to proteolysis. Cardiac amyloidosis appears in two distinct forms: hereditary and wild-type. There is considerable heterogeneity in the clinical presentation of ATTR, ranging from primarily cardiac, primarily neuropathic, or mixed cardiac and neuropathic disease. Pathogenic variants in the  In a cohort of 109 individuals with an antemortem diagnosis of HFpEF without known amyloid who underwent autopsy, 5% had robust ATTR deposition in their hearts, while 12% had more mild cardiac deposition.20 This suggests that at least 155,000 people  have ATTR, but milder deposition is probably playing a role in many others with HFpEF. Additionally, several studies indicate disparity in the racial prevalence of HF,21 suggesting that the prevalence of ATTR among non-Whites with HFpEF may be higher than among Whites.The US Congress, through the Rare Disease Act of 2002, defines rare disease as fewer than 200,000 affected individuals in the United States, whereas the Public Health section of the European Commission defines rare disease as affecting fewer than 1 in 2,000 people in Europe. The prevalence of ATTR-CM may be substantially higher than these numbers,TTR gene on chromosome 18q12.1, and it spans 4 exons.22 Traditional numbering of TTR codons came from early amino acid sequence analysis of the circulating protein, which lacks an N-terminal signal peptide of 20 residues.23 Later analysis of the DNA sequence helped to determine the full-length proprotein.24 Based on proper nomenclature by the Human Gene Organization (HUGO), codon numbering should begin with methionine—which adds 20 residues to the traditional numbering that arose from early serum protein analysis—as well as three-letter abbreviations for amino acids. This manuscript follows the majority of published literature on ATTR, using the traditional numbering and three-letter abbreviations for amino acids. However, clinical genetic laboratories typically use HUGO-based nomenclature. For example, a common pathogenic variant in TTR may be designated as V30M or Val30Met by traditional annotation, or p.Val50Met by HUGO-based nomenclature.Transthyretin is a tetrameric serum transport protein encoded by the TTR related to familial ATTR have been reported.25 Heterozygosity is enough to prompt disease manifestations, although reduced penetrance and variable expression make family history an unreliable factor in recognizing ATTRv except among individuals and families with early-onset ATTRh.26 These mutations tend to cluster into geographic or ethnic groups with an autosomal-dominant pattern of inheritance.10 In the US, the most common TTR mutations are Val122Ile, Thr60Ala, and Val30Met, whereas the most common TTR mutations worldwide are Val30Met, Val122Ile, and Glu89Gln.27 Val30Met may be the most commonly recognized mutation worldwide, mostly found in regions of Portugal, Spain, France, Japan, Sweden, South America, and some nonendemic parts of Africa.6 On the other hand, Val122Ile originated in West Africa and is found in approximately 3% to 4% of African Americans, with cardiac manifestations occurring in later stages of life.28 Although the worldwide prevalence of the Val122Ile variant may exceed that of the Val30Met variant, the penetrance of Val30Met appears higher than for Val122Ile, and the incidence of ATTRv related to Val30Met may be highest.29 Leu111Met and Ile68Leu are mutations mostly reported in Denmark and Italy, respectively, and cause severe cardiomyopathy in relatively early ages.30 The Thr60Ala variant is the second most-common TTR variant in the US and the most common in the UK and Ireland, affecting approximately 1% of the population of north-western Ireland.32 Val122Ile is known to be the most common cause of late-onset ATTR-CM in the US.33 Other common causes of ATTRv-CM include Thr60Ala (early-onset phenotype), Ile68Leu (late-onset phenotype) in Italy, and Leu111Met (early-onset phenotype) in Denmark.34 Worldwide distribution of most common ATTRv genotypes by country/region is shown in Figure 1.More than 130 pathogenic variants in 4 In addition, an oculoleptomeningeal type with symptoms predominantly involving the central nervous system has been reported with some rare mutations.35 According to an international registry, Transthyretin Amyloidosis Outcomes Survey (THAOS), most patients enrolled from the US are elderly men with wtATTR-CM and a cardiac-predominant phenotype compared with patients enrolled in this registry from Europe.27 ATTRwt tends to produce a predominantly cardiac phenotype. The predominant phenotype in patients with hereditary ATTR varies based on the underlying mutation. Both Val122Ile and Leu111Met typically cause cardiomyopathy. However, there also can be a substantial component of neuropathy in patients with Val122Ile, with almost 60% of these individuals in the THAOS survey reporting sensory neuropathy.27 In another study with a mixed population of self-reported White and Black individuals with Val122Ile, 20% to 27% reported neuropathy.36 Early-onset Val30Met predominantly causes polyneuropathy, but late onset often has cardiac involvement.6 Glu89Gln has a mixed phenotype of polyneuropathy and cardiomyopathy.36There is heterogeneity in disease phenotypes, ranging from predominantly neuropathic , predominantly cardiac , or a mixed phenotype.37 In one study evaluating a potential parent-of-origin effect, the authors compared age of onset of TTR-FAP among French and Portuguese families with the Val30Met pathogenic variant. Out of 33 Portuguese families, the transmitting parent was found to be the mother in 153 individuals and the father in 138.38 In comparison, out of 48 French families, the transmitting parent was the mother in 216 individuals and the father in 219. In the Portuguese families, phenotypic manifestations were earlier when the pathogenic TTR variant was inherited from the mother instead of the father (P < .002). The age of onset of TTR-FAP was not significantly different in the French families when comparing maternal and paternal transmission of the TTR mutation.Heterogeneity in disease penetrance also occurs across different geographic regions. For example, age of disease onset related to Val30Met is much later in Sweden than in Portugal and Japan, and the Portuguese population has 80% higher penetrance of the Val30Met mutation during mid-age (50 years) compared with the French population.39 Traditionally, TTR-FAP and ATTRv-CM were considered two distinct disorders, although ATTR is now considered to be a single disease with a spectrum of manifestations, including nearly exclusively neuropathy or cardiomyopathy, or (more commonly) a combination of these and other manifestations. In prototypical TTR-FAP, signs and symptoms develop most commonly in the third to fifth decade of life but can be noticed from the second decade of life. Penetrance is variable and disease is usually present as a progressive sensory motor neuropathy with autonomic manifestations.40 Without treatment, the disease is usually progressive and fatal about 10 to 13 years after the onset of symptoms. Men and women are equally affected.41 On the other hand, prototypical hATTR-CM usually has a later onset than TTR-FAP, with symptoms occurring mostly after age 60.42 It manifests as unexplained left ventricular hypertrophy leading to restrictive cardiomyopathy, HF, atrial fibrillation, and conduction abnormalities.45Variability among patients in terms of age of disease onset also may depend on the phenotype.46 Family history is usually positive in endemic areas because inheritance is autosomal dominant.10 Val30Met can manifest as early- or late-onset disease and, as a result of this heterogeneity, may be difficult to diagnose.46 Although cardiac involvement is late among those with endemic disease, it tends to be more prominent in nonendemic areas and can lead to conduction abnormalities.7 Late-onset Val30Met manifests as ATTRv-CM as the disease progresses.47 The Val30Met variant is the most common mutation in several regions around the world; the largest group of individuals carrying this TTR variant may be found in northern Sweden with a prevalence of 4%,49 with a gene carrier frequency of 1:538 in northern Portugal.50 The usual age of disease onset in Portugal, Brazil, and Japan is in the third and fourth decade of life. In Portugal and Japan, more than 90% of TTR Val30Met gene carriers are symptomatic. In endemic areas of Portugal, the penetrance is high, with approximately 80% developing disease by age 50 years.52 Despite the high frequency of the Val30Met mutation in the endemic areas of Sweden (4%), there is relatively low penetrance: 11% by age 50, with older age of onset and a slower disease progression than in Portugal.37 Female gender seems to provide some protection from myocardial involvement, at least before the onset of menopause.53Discovered in the 1950s, Val30Met was the first TTR variant, initially identified in Portugal and later in Sweden and Japan. It typically causes FAP, resulting from a point mutation causing substitution of methionine for valine at position 30 of the mature protein.TTR mutation in the US, resulting from a valine-to-isoleucine substitution at position 122 of the mature protein.28 It has a prevalence of approximately 3.4% among African Americans, with a low penetrance that is as yet undefined. It presents with a cardiac phenotype, usually earlier than ATTRwt, typically during or after the sixth decade of life, and this variant is associated with more neurologic symptoms than ATTRwt. The Val122Ile mutation has been found in 10% of African Americans over the age of 65 years who have severe HF.28 The mutation ultimately leads to misfolding of free TTR monomers, with an age-dependent phenotypic penetrance. After age 70, carriers show a higher frequency of HF and increased mortality.28Val122Ile is the most common 32 Neurologic manifestations are more prevalent at an earlier stage, while cardiac involvement is usually present on diagnosis and is a major determinant of its poor prognosis. In the US, carriers of this pathogenic variant are more commonly in the Appalachian region with Irish ancestry, likely due to a common founder effect.Thr60Ala is the most common variant in the British and Irish populations, presenting with a mixed phenotype of HF and polyneuropathy, including severe autonomic neuropathy.5 This rare coding sequence is kinetically very stable and has been reported to be associated with increased longevity in a large Danish population study.55 When it occurs in trans with the Val30Met mutation, it counteracts the destabilizing effect and delays or prevents ATTR.55Thr119Met is a variant known to stabilize the transthyretin tetramer and has been associated with more favorable outcomes.With advancements in diagnostic imaging and the emergence of novel TTR-targeted therapies, the prognosis of ATTR-CM is improving. However, for those who remain undiagnosed or cannot access novel treatments, there remains significant morbidity and mortality.56 It should be pointed out, however, that these were developed prior to the more widespread use of disease-modifying therapies, which are associated with notable improvements in survival.To help prognosticate at the time of diagnosis, two staging systems are widely used for patients with ATTR-CM, one from Mayo Clinic and one from the UK National Amyloidosis Centre.14 a cohort of ATTRwt patients were classified into three prognostic stages based on elevations of troponin T (> 0.05 ng/mL) and N-terminal proB-type natriuretic peptide (NT-proBNP)  biomarkers. Stage I included patients with no elevated biomarkers, Stage II with elevation in one biomarker, and Stage III with elevations in both biomarkers. Patients classified in Stage III had significantly worse median survival compared with those classified in Stage I. Median survival for patients classified in Stages I, II, and III was 66, 40, and 20 months, respectively.For the Mayo risk model,56 the study cohort included both wild-type and hereditary ATTR variants, including the more common Val122Ile variant. Again, three prognostic stages were defined based on renal dysfunction (eGFR threshold of < 45 mL/min/1.73 m2) and elevations in NT-proBNP . Stage I included patients with no elevation in NT-proBNP and eGFR above or equal to the threshold, Stage III with elevation in NT-proBNP and eGFR below the threshold, and Stage II included the remainder of the cohort . Outcomes were significantly worse with higher stages. Median survival for patients classified in Stage I, II, and III was 69.2, 46.7, and 24.1 months, respectively. This finding remained significant even after controlling for different genotypes and was further validated in a separate cohort of ATTR-CM patients.For the UK risk model,57 Daily diuretic dosing was categorized into furosemide equivalents and assigned to a point system, with 0 points for 0 mg/kg, 1 point for > 0 to 0.5 mg/kg, 2 points for > 0.5 to 1 mg/kg, and 3 points for > 1 mg/kg. A similar point system was used for NYHA functional class with 1 point per class ranging from 1 to 4 points. The addition of these variables significantly improved the accuracy of the preexisting risk staging systems, increasing the area under the curve for the Mayo risk model from 0.693 to 0.798 and the UK risk model from 0.711 to 0.813 for all-cause mortality.More recently, a group from Columbia University evaluated the additive prognostic value of diuretic dose and New York Heart Association (NYHA) functional class to these preexisting risk staging systems.58 As such, high-sensitivity troponin and BNP concentrations are not interchangeable biomarkers for the previously described staging systems.It should be noted that while these biomarkers tend to be readily available and should be considered during the initial evaluation for all patients with ATTR-CM, analogous blood tests—including high-sensitivity troponin assays and B-type natriuretic peptide (BNP)—circulate at different concentrations in plasma. A separate biologically active hormone from NT-proBNP, BNP has a shorter half-life and can be affected by use of the angiotensin receptor-neprilysin inhibitor (ARNI) class of HF therapeutics.59 Additionally, NYHA functional class III or IV was identified as an independent risk factor for adverse cardiovascular outcomes that include the development of conduction disease, HF hospitalization, and stroke.61 Results from the Transthyretin Amyloidosis Cardiomyopathy Clinical Trial (ATTR-ACT) indicated diminished benefit from the TTR stabilizing agent, tafamidis, in patients with NYHA functional class III compared to those with NYHA class I or II disease, highlighting the need for early detection of ATTR-CM.62Clinical features in patients with ATTR-CM, including functional status, certain comorbid conditions, and specific genotypes, have been independently associated with increased mortality. Worsening survival among those with ATTRwt-CM has been observed with declining NYHA functional classes.63 The presence of atrial fibrillation, atrial flutter, or atrioventricular block is associated with worse prognosis. Although not independently associated with worse mortality compared to those with sinus rhythm, ATTR-CM patients with atrial fibrillation were more likely to have severe HF symptoms (NYHA class III or IV) and a higher frequency of renal dysfunction.65 Furthermore, ATTR-CM patients are at increased risk for thromboembolism,66 with one study examining cardioversion outcomes for atrial arrhythmias, noting a significantly high procedural cancellation rate mainly due to the presence of intracardiac thrombus (up to 30%) despite adequate anticoagulation.67 Another study of patients with ATTR-CM and atrial fibrillation noted no association between CHA2DS2-VASc score and the presence of left atrial appendage thrombus.68Atrial fibrillation is more common in ATTRwt compared with ATTRv and is present in up to 70% of ATTRwt patients.59 In a recent study examining ATTR-CM patients with implantable cardiac devices, those with a higher RV pacing burden (> 40%) experienced worsening HF, left ventricular ejection fraction (LVEF), and mitral regurgitation severity.69 In contrast, those with biventricular pacing noted improvements in LVEF, NYHA class, and severity of mitral regurgitation. These findings suggest that biventricular pacing should be used in patients with ATTR-CM and an indication for pacing.70 Ventricular arrhythmias are common in ATTR-CM, most commonly nonsustained ventricular tachycardia (up to 74%). However, placement of an implantable cardioverter defibrillator (ICD) for primary or secondary prevention has not been shown to improve survival.71 Currently, ICD implantation is of unclear benefit in this population and will need prospective studies to determine their utility as medical therapies for ATTR-CM that may improve survival and alter disease course.Conduction system disease often develops in patients with ATTR-CM, and requirement of a pacemaker has been associated with worse survival.72Finally, specific genotypes have been identified as carrying a worse prognosis compared with other familial variants and ATTRwt. In a large ATTR-CM cohort, patients with the Val122Ile variant had significantly worse median survival compared to those with non-Val122Ile variants or wild-type disease.73 Furthermore, results from the ATTR-ACT trial noted significant improvement in several key secondary end points, including distance walked during the 6-minute walk test and KCCQ-OS score, a measure of quality of life, with differences first observed at 6 months.62 Results from the APOLLO study—a phase 3 trial for patients with familial amyloid polyneuropathy randomized to placebo versus patisiran, a small-interfering RNA-based drug targeting hepatic synthesis of TTR—noted significant improvements in neuropathy impairment scores, 10-meter walk test, and quality of life.74 Treatment with another TTR “silencer,” inotersen, led to similar outcomes, although inotersen use was complicated by serious adverse events with glomerulonephritis (3%) and severe thrombocytopenia (3%) in this trial.75 Both patisiran and inotersen are approved by the US Food and Drug Administration for TTR-FAP but not indicated for ATTRwt or ATTRv-CM without neuropathy. The CARDIO-TTRansform trial is currently randomizing participants with ATTR-CM to receive placebo versus eplontersen, a ligand-conjugated investigational antisense medicine designed to reduce the production of transthyretin. The HELIOS-B study is a phase 3 trial of vutrisiran, an investigational therapeutic for patients with ATTR-CM similarly designed to reduce production of transthyretin with a longer acting effect.While not associated with survival, neuropathic involvement and functional decline in patients with ATTR-CM can add significant morbidity. This is best illustrated by studies of TTR-targeted therapies and their respective improvements in neuropathic impairment scores, functional assessments, and quality of life questionnaires. The early studies on the clinical applications of tafamidis indicated its ability to stabilize the TTR tetramer and delay peripheral neurologic impairment.In summary, long-term prognostic data are limited in the era of TTR-targeted therapeutics for patients with ATTR-CM. Current staging systems were published prior to the development and more widespread use of these novel agents. As such, studies indicate that in patients with ATTR-CM, those receiving treatment have significantly improved morbidity and mortality, especially if diagnosed at an early stage of disease.Two online databases were explored, Google Scholar and PubMed, to retrieve relevant articles and published studies, and different keywords and search terms were used to find those most relevant. These keywords were “amyloidosis,” “transthyretin amyloidosis,” “epidemiology of ATTR amyloidosis,” “genetics of ATTR amyloidosis,” “prognosis of ATTR amyloidosis,” “TTR variants,” and “ATTR amyloidosis AND treatments.”Exclusion and inclusion criteria were applied to the complete contents of searched articles to identify the most relevant studies. Only randomized controlled trials, meta-analyses, observational studies with controls, case series, review articles, and reports were included. Cross references of retrieved articles were also explored to find the relevant articles. Articles from non-peer review journals, editorials, conference papers, abstracts, and letters were excluded.Our recognition of the epidemiology of ATTR-CM, a disease that was once considered rare, is significantly changing due to increased awareness among healthcare providers and broader use of cardiac scintigraphy. There is considerable heterogeneity in the clinical presentation, and phenotypic heterogeneity is dependent on both genetic and environmental factors. Early diagnoses, recognition of the early stages of cardiac infiltration, and initiation of treatment are essential to optimize long-term prognosis.Phenotypic heterogeneity is linked to specific pathogenic variants in transthyretin, geography, and type of disease (endemic versus nonendemic).Early diagnosis is essential for effective treatment and favorable prognosis.Patients receiving treatment for transthyretin amyloid cardiomyopathy have significantly improved morbidity and mortality compared with historical data on patients without disease-modifying therapies.


Enterobacterales (CRE) is a global threat. Enterobacterales develops carbapenem resistance through several mechanisms, including the production of carbapenemases. We aim to describe the prevalence of Carbapenem-resistant Enterobacterales (CRE) with and without carbapenemase production and distribution of carbapenemase-producing (CP) genes in Thailand using 2016–2018 data from a national antimicrobial resistance surveillance system developed by the Thailand National Institute of Health (NIH).Carbapenem-resistant Enterobacterales resistant to ertapenem, imipenem, or meropenem. Starting in 2016, 25 tertiary care hospitals from the five regions of Thailand submitted the first CRE isolate from each specimen type and patient admission to Thailand NIH, accompanied by a case report form with patient information. NIH performed confirmatory identification and antimicrobial susceptibility testing and performed multiplex polymerase chain reaction testing to detect CP-genes. Using 2016–2018 data, we calculated proportions of CP-CRE, stratified by specimen type, organism, and CP-gene using SAS 9.4.CRE was defined as any Klebsiella pneumoniae , followed by Escherichia coli . The proportion of CP-CRE was high for all organism types (range: 85–98%). Of all CRE isolates, 2909 (80%) had one CP-gene and 629 (17%) had > 1 CP-gene. New Delhi metallo-beta-lactamase (NDM) was the most common CP-gene, present in 2392 (65%) CRE isolates. K. pneumoniae carbapenemase (KPC) and Verona integron-encoded metallo-β-lactamase (VIM) genes were not detected among any isolates.Overall, 4,296 presumed CRE isolates were submitted to Thailand NIH; 3,946 (93%) were confirmed CRE. Urine  and sputum  were the most common specimen types, while blood only accounted for 323 (8%) CRE isolates. The most common organism was CP genes were found in a high proportion (97%) of CRE isolates from hospitals across Thailand. The prevalence of NDM and OXA-48-like genes in Thailand is consistent with pattern seen in Southeast Asia, but different from that in the United States and other regions. As carbapenemase testing is not routinely performed in Thailand, hospital staff should consider treating all patients with CRE with enhanced infection control measures; in line with CDC recommendation for enhanced infection control measures for CP-CRE because of their high propensity to spread.The online version contains supplementary material available at 10.1186/s13756-021-00950-7. Antimicrobial resistance (AMR) is a global health priority considering its association with high morbidity and mortality and increased healthcare costs –3. The bEnterobacterales (CRE) are an especially concerning AMR threat because they are resistant to many last-resort antibiotics, making it difficult to treat and leading to high mortality rates. CRE are often the result of healthcare transmission and associated with risk factors such as previous antibiotic use, prolonged hospitalization, and medical device use [Carbapenem-resistant vice use –11. CRE vice use –14. For vice use .Klebsiella pneumoniae carbapenemase (KPC) which is the most common carbapenemase in the United States and New Delhi metallo-beta-lactamase (NDM) which was first identified in a traveler returning from India but has now spread worldwide. Others include Verona integrin-encoded metallo-β-lactamase (VIM) and imipenemase (IMP) which are most prevalent in Southern Europe and Asia and oxacillinase-48 (OXA-48) which is most prevalent in the Mediterranean region, Europe, and India [Carbapenemase enzymes are encoded by genes on mobile genetic elements, such as plasmids, which are highly transmissible between organisms and increase the potential spread of resistance. There are several genes encoding different carbapenemases, including nd India .K. pneumoniae isolates resistant to meropenem increased from 0.4% in 2000 to 6.5% in 2016 and the overall percentage of Escherichia coli isolates resistant to meropenem increased from 0.6 to 1.6% during the same period [The National Antimicrobial Resistance Surveillance center, Thailand (NARST) which is managed by Thailand’s National Institute of Health (NIH), was established in 1998 and includes 92 hospital laboratories from all five regions of Thailand. NARST data show the overall percentage of NARST contains limited information on resistance genes because resistance mechanism testing is not routinely performed at participating hospital laboratories. NIH established the Enhanced Incorporation of Global and National AMR (EIGNA) Surveillance System to understand the distribution of resistance genes and identify novel resistance genes among CRE in Thailand. We analyzed 2016–2018 EIGNA data to better understand the distribution of resistance genes and identify novel resistance genes among CP-CRE in Thailand.EIGNA includes 25 of 92 hospital laboratories participating in NARST surveillance from all five regions of Thailand; 21 (84%) of the labs are regional, tertiary care hospitals Table . These lEnterobacterales isolate resistant to ertapenem, imipenem, or meropenem according to CLSI M100-S26 standards [Hospital laboratories performed Gram stains, cultures, biochemical tests, and antimicrobial susceptibility testing (AST) using disk diffusion and/or E-test on routine clinical specimens from hospitalized patients. CRE was defined as any tandards . ParticiblaKPC, blaNDM, blaOXA-48-like, blaVIM, and blaIMP [blaMCR-1 . Since hyperproduction of AmpC β-lactamase combined with altered membrane permeability can result in carbapenem resistance [blaAmpC was started for CRE isolates in the middle of this surveillance period and included testing for some isolates from 2017 and all isolates from 2018. Isolates negative by PCR for the six targeted genes were tested using modified Carbapenem Inactivation Method (mCIM) [NIH repeated AST using disk diffusion. E-test was done to confirm CRE for isolates that had discordant results. CRE isolates confirmed by NIH underwent multiplex polymerase chain reaction (PCR) to detect five carbapenemase-producing (CP) genes, including d blaIMP . As colisistance , 19, 20,d (mCIM)  for carbd (mCIM)  to confiFor isolates with evidence of carbapenemase production but no CP gene identified, whole-genome sequencing was conducted by isolating DNA from overnight cultures with a DNeasy Blood and Tissue kit  and quantifying the extracted DNA using the Qubit dsDNA HS Assay Kit (Invitrogen), both according to the manufacturer’s protocols. Genomic libraries were generated with the QIAGEN® QIAseq FX DNA Library Kit  following to the manufacturer’s protocol. The Whole-genome sequencing (WGS) was carried out using the Illumina® MiSeq platforms to obtain 250-bp paired-end reads chemistry  according to manufacturer’s instructions. The 250-bp paired-end reads was de novo assembled using the CLC Genomics Workbench 12.0.2  using defaults settings except that the minimum contig size threshold was set to 500 bp in length.EIGNA data from September 2016 through August 2018 were analyzed using SAS 9.4 . Proportions of CP-CRE isolates among CRE isolates were calculated and stratified by specimen type, organism, and carbapenemase-producing gene. For the analysis of patient characteristics, isolate data was deduplicated by including CRF information from the first isolate submitted. However, for one patient whose first isolate was CP-CRE negative and subsequent isolates were CP-CRE positive, the first CP-CRE isolate was included and prior non-CP-CRE isolates were excluded. Clinical characteristics were calculated for CP-CRE and non-CP-CRE patients and compared using the Wilcoxon-Mann–Whitney test for continuous variables and chi-square and Fisher’s exact tests for categorical variables.K. pneumoniae (73%) and E. coli (21%) were the most common organisms among all confirmed CRE isolates [Table A total of 4296 presumed CRE isolates were submitted to NIH by participating hospital laboratories and 3946 (92%) isolates from 3748 patients were confirmed by NIH as CRE Fig. , with 22E. coli 2% were thOf 3748 patients with confirmed CRE, 2368 (63%) were collected more than two calendar days after admission , 2502 (67%) had antibiotic exposures during 14 days prior to specimen collection, and 885 (24%) died during hospitalization. For the 3724 (99%) with information on age available, median age was 66 years (IQR: 52–77). Of the 3031 (80%) with information on length of stay (LOS) available, median LOS was 20 days (IQR: 9–39). Of the 3318 (80%) with information on duration from admission to specimen collection date available, median duration was 10 days (IQR: 2–23) [Table Enterobacterales species ranged from 89 to 100% [Table Of 3,946 CRE isolates, 3,844 (97%) tested positive for at least one CP gene and the proportion of CP-CRE for all 0% Table .Table 3CblaNDM and 1892 (48%) were positive for blaOXA-48-like (48%). There were 634 (16%) isolates with coexistence of both blaNDM and blaOXA-48-like genes, seven (< 1%) isolates with coexistence of both blaIMP and blaOXA-48-like genes, and five (< 1%) isolates with coexistence of blaIMP and blaNDM. When stratified by organism, blaNDM remained the most common CP gene across all organisms, except in K. pneumoniae, which had a similar percentage of isolates with blaNDM (55%) and blaOXA-48-like (59%) genes. The highest proportion of blaNDM-positive isolates were among Citrobacter freundii  and E. coli . E. cloacae had the highest proportion of isolates positive for blaIMP . blaKPC and blaVIM genes were not detected among any of the isolates. Of the 102 isolates with no CP gene identified, the most common pathogen was K. pneumoniae . The distribution of CP genes by region is presented in Additional file blaAmpC, four (8%) of which were positive. Of the six mCIM positive isolates, one was positive for both mCIM positive and blaAmpC and was later found to have blaIMI by whole-genome sequencing. One mCIM positive isolate was also later found to have Non-Metalloenzyme Carbapenemase (blaNMC) and blaIMI genes by AMR Direct Flow Chip kit. The MCR-1 gene was identified in ten isolates, including six isolates that also had blaNDM and three isolates that also had blaOXA-48-like.The distribution of carbapenemase genes among these isolates can be found in Table Enterobacterales isolates from Bangkok were CP-CRE [The EIGNA surveillance system provides new information about carbapenemase distribution across Thailand. While previous studies show that there is a high burden of CRE in Thailand , 7, 23, e CP-CRE . While te CP-CRE , we did The distribution of CP genes in Thailand also differs from that found in other parts of the world. The most common CP genes in Thailand were NDM (69%) and OXA-48-like (38%), regardless of organism. These results support other studies showing that NDM is the most common gene in South and Southeast Asia –30. HoweThe U.S. Centers for Disease Control and Prevention recommends enhanced infection control measures specifically for CP-CRE compared to non-CP-CRE because of their high propensity to spread. However, testing for the presence of a CP gene is not routinely done in Thailand’s clinical laboratories and reference laboratory testing results are often delayed, hindering their use in guiding infection control measures. Given the high proportion of CP- CRE, clinicians in Thailand that do not have access to genotyping or carbapenemase production testing should consider that any CRE isolate is likely to have a CP gene and use appropriate infection control precautions for all patients with CRE. Testing for specific CP genes is more difficult and less available than testing for the presence of any carbapenemase activity. While there is currently no evidence or recommendation about special infection control measures associated with specific CP genes, information about CP genes can be useful to guide antimicrobial therapy. NDM is associated with limited treatment options. Newer combination drugs such as meropenem-vaborbactam and imipenem-relebactam are effective against KPC, but not NDM or OXA-48-like carbapenemases. Ceftazadime-avibactam is effective against both KPC and OXA-48-like carbapenemases, but not against NDM . HoweverThere are several limitations to this study that restrict its generalizability. Participating sites were chosen based on microbiology capacity and surveillance performance to optimize data accuracy but may not reflect a nationally representative sample and gaps in reporting may have resulted in an underestimate of total CRE cases. Different hospital types and locations were included, but regional hospitals (82%), which provide tertiary care and likely have more severely ill patients, were disproportionately represented. Results may also have been biased by inconsistent culturing practices or laboratory testing at participating sites. For example, specimen collection and testing at hospital sites was left to the clinical discretion of each hospital or clinician and therefore not standardized. As a result, some clinicians may have collected samples only from patients with more severe conditions or those who did not respond to initial therapy with first-line antibiotics. Additionally, ertapenem susceptibility testing was not performed at all sites; only 14 (17%) of 84 sites participating in NARST do ertapenem susceptibility testing. As a result, isolates resistant to only ertapenem, which are more likely to be non-CP-CRE, may not have been identified or included in the analysis , 35. WhoClinical data collected as part of the EIGNA surveillance system was limited and did not include information about some risk factors or exposures  which may be associated with CP-CRE. Outcome data was only reported for the hospitalization and did not include death after discharge.We found that carbapenemase production is predominant among CRE across Thailand. The distribution of carbapenemase-producing genes and the prevalence of NDM and OXA-48-like genes in Thailand differs from that in the United States and other regions of the world but seems to be consistent with patterns demonstrated by the limited data from Southeast Asia. Our analysis demonstrated a high frequency of established healthcare risk factors and poor outcomes among patients with CP-CRE. Efforts to strengthen clinical laboratory capacity for carbapenemase testing is important to improve early identification and appropriate response by guiding clinical treatment and control measures. Given that current availability of carbapenemase testing results is limited, all patients with CRE should be considered to have CP-CRE and treated with appropriate use of infection control measures. Ongoing CRE surveillance is necessary to detect new, emerging resistance mechanisms, monitor CRE trends, and determine the effectiveness of control measures in preventing transmission.Additional file 1: Table 1. Presence of carbapenemase* and MCR-1 genes in isolates by region, Thailand, 2016–2018.


Withania somnifera (L.) Dunal, WS), may be beneficial in the management of COVID-19. WS is a widely prescribed Ayurvedic botanical known as an immunomodulatory, antiviral, anti-inflammatory, and adaptogenic agent. The chemical profile and pharmacological activities of WS have been extensively reported. Several clinical studies have reported its safety for use in humans. This review presents a research synthesis of in silico, in vitro, in vivo, and clinical studies on Withania somnifera (L.) Dunal (WS) and discusses its potential for prophylaxis and management of COVID-19. We have collated the data from studies on WS that focused on viral infections  and noncommunicable diseases . The experimental literature indicates that WS has the potential for 1) maintaining immune homeostasis, 2) regulating inflammation, 3) suppressing pro-inflammatory cytokines, 4) organ protection , and 5) anti-stress, antihypertensive, and antidiabetic activities. Using these trends, the review presents a triangulation of Ayurveda wisdom, pharmacological properties, and COVID-19 pathophysiology ranging from viral entry to end-stage acute respiratory distress syndrome (ARDS). The review proposes WS as a potential therapeutic adjuvant for various stages of COVID-19 management. WS may also have beneficial effects on comorbidities associated with the COVID-19. However, systematic studies are needed to realize the potential of WS for improving clinical outcome of patients with COVID-19.As the COVID-19 pandemic is progressing, the therapeutic gaps in conventional management have highlighted the need for the integration of traditional knowledge systems with modern medicine. Ayurvedic medicines, especially Ashwagandha ( The COVID-19 or coronavirus disease 2019 is a contagious disease caused by SARS-CoV-2. The rapidly spreading disease is considered as one of the causes of mortality globally  “WHO An “WHO Co.Understanding the pathophysiology of this disease is rapidly advancing with the availability of new research data. Current evidence suggests that most individuals are asymptomatic or are suffering from mild symptoms. The patients who progress to severity develop pneumonia and ARDS and require hospitalization. The SARS-CoV-2 virus uses the angiotensin-converting enzyme 2 (ACE2) receptor for cell entry and the transmembrane serine protease 2 (TMPRSS2) for spike protein priming . Upon hoThe extent of COVID-19 has highlighted challenges for healthcare systems. The advancing clinical observations are rapidly changing the management protocols. The present empirical pharmacotherapeutics involves antivirals , antimalarials , antibiotics , and in few cases immunomodulators   “Revise “Clinic. HoweverWithania somnifera (L.) Dunal (Ashwagandha/WS) is one of the extensively prescribed botanicals in Ayurveda practice for its multimodal effects , the main phytochemical of WS, play a central role in exhibiting multimodal effects synergistically. These are a group of Cic agent   as was indicated by a significant activation of T cells and NK cells after 4 days of twice daily consumption  and lowered cell-mediated immunity (B and T cells) . The T-c control  .The property of WS in optimizing immune response is being explored in the management of COVID-19. The Ministry of AYUSH, Govt. of India, has initiated multicentric clinical studies in India. These studies have been aimed to understand WS applications in the management of COVID-19  in rats  expressed by alveolar macrophages are immune factors capable of inducing acute inflammation in the lungs  Kinnula Diet et. Prx1 ac in rats .in vitro and in silico analysis of aqueous and hydroethanolic extracts of WS with 5-lipoxygenase (isolated from human polymorphonuclear leukocytes) showed a significant inhibition better than conventional anti-inflammatory agents (Inflammasomes (intracellular multiprotein complexes) and macrophage polarization enables the regulation of inflammation in chronic diseases  Guo et . AIM2 any agents . WS aquey agents .An activation of the NF-κB pathway is a decisive mechanism in inducing inflammation . It poteThe role of WS in inflammatory diseases is explored by several researchers. Mazzio et al. explored several natural products for their anti-inflammatory activity in the context of the acute systemic inflammatory response to prevent sepsis. The study revealed explicitly that WS has better anti-inflammatory potential than all other natural products accompanied with negligible toxicity . SimilarThe collected mechanistic studies suggest that WS and withanolides restrict inflammatory response through regulating cytokine expression, modulating inflammatory receptor proteins, and inhibiting the NF-κB pathway. This encourages the investigation of WS in COVID-19 hyperinflammation.The effect of inflammatory mediators on blood vessels that are carrying them is obvious. Patients with COVID-19 showed that immune cells, inflammatory cytokines, and vasoactive molecules increase gaps between endothelial cells that line blood vessels. This leads to vascular leakage that causes infiltration of inflammatory cells . As a rein vitro and in vivo. Withaferin A significantly inhibited HMGB1-mediated barrier disruption, acetic acid-induced hyperpermeability, and restricted transendothelial migration of leukocytes  to explore bevacizumab, an anti-VEGF, in patients with COVID-19 has been registered with clear pathological rationale , a vascular permeability inducing factor, in the blood  Huang e. A cliniationale . Moleculationale . Withafeationale  and lungationale .These studies support the protective and preventive effects of WS in vascular permeability-induced alveolar consolidation. Thereby, WS may help reduce breathlessness in patients with COVID-19. Therefore, systematic clinical studies are required to validate this effect of WS.The clinical complications of COVID-19 have been demonstrated to influence multiple vital organs . The sysThe clinically evident holistic approach of Ayurveda designates health as the central point of concern. Ayurveda interventions are aimed at strengthening homeostatic mechanisms and optimizing adaptation limits. Unlike the organ-specific approach of modern biomedicine, Ayurveda focuses on systemic revitalization to protect organs thereby avoiding failure . WS has The neurological deformations are associated with COVID-19 pathophysiology. The major clinical observations of patients incorporate encephalitis, necrotic hemorrhage, and epileptic seizures . A systeNeuroinflammation triggered by immune cells is a key mechanism to induce neurological complications  Vito et. WS leafThe COVID-19 patients also showed heart-related complications such as cardiomyopathy and myocarditis. The pathological observations revealed elevated levels of cardiac troponin I, IL-6, and ACE2 receptors .Myocarditis and further heart dysfunction are coupled with systemic inflammation. The alcoholic extract of WS leaf normalized troponin I release in the blood and thereby preserved structural and functional integrity of contractile myocardium in a rat model . WS rootPatients with COVID-19 with severe categories showed liver damage, which was evident by abnormal ranges of liver enzymes and other markers. There was a significant rate of mortality due to liver failure on SARS-CoV-2 infection . HepaticWithanolide-rich fraction isolated from WS root methanolic extract found to restore the marker enzyme levels in drug-induced hepatic cytotoxicity in rat models of cytokines . Pre-admThe nasal tract, nasopharyngeal cavity, and lungs are the main sites for SARS-CoV-2 infection. Pulmonary inflammation, anosmia, respiratory distress, and lung endothelial dysfunction with endothelitis are the major symptoms associated with COVID-19  Varga e. ReportsEndothelial dysfunction due to inflammation results in pulmonary hypertension. WS root powder attenuated endothelitis in a rat model by limiting expression of proliferating cell nuclear antigen (PCNA). It also suppressed the level of inflammatory markers IL-10 and TNF-α by regulating the NF-κB transcription factor , while pAnosmia is neuroinflammatory dysfunction of olfactory cells due to entry of COVID-19 through the nasal tract. WS has a neuroprotective activity by regulating Sema3a factor in olfactory cells in a mouse model .GI tract dysfunction is also found to be associated with COVID-19. A population study of COVID-19 patients reported common symptoms as nausea, diarrhea, abdominal pain, anorexia, and vomiting . PatholoThe immune response-induced inflammation against COVID-19 is the main cause of GI tract dysfunction. WS root aqueous extract expressed antidiarrheal activity and maintained gastrointestinal mobility in the rat model by inhibiting cyclooxygenase and regulating inflammatory markers induced by NF-κB transcription factor . WithafeThe renal system is also found to be a target of COVID-19. Pathological findings reported direct entry of COVID-19 in kidney cells through increased ACE-2 receptor, while the inflammatory response to infection shows proteinuria and increased levels of serum creatinine and urea nitrogen .The inflammatory mechanism is responsible for renal dysfunction. WS root powder was found to show a nephroprotective effect by stabilizing urea nitrogen and creatine level and restoring antioxidant enzyme expression in animal models  Vedi et. This isRhabdomyolysis, myalgia, and muscle weakness are symptoms found in COVID-19 patients . They peSeveral clinical trials involving the administration of WS aqueous extract found an increase in the overall muscle strength  Ziegenf. RhabdomThe complications of COVID-19 have also found to induce acute pancreatitis . The bioWithaferin A  and aqueCOVID-19 is associated with gonadal inflammation. Primary pathological symptoms include damage to spermatozoan, abnormal secretion of sex hormones, and increased level of ACE-2 in Leydig cells in testes .Inflammation-induced cytokines are the main molecules for the dysfunction of the testes. Administration of methanolic extract of WS root has shown regulation of gonadotropic hormone by controlling GABA neurons in a mouse model . This saAn intervention provided in addition to mainstream administration is considered as an adjuvant. The primary aim of the adjuvant is to enhance the beneficial effects and reduce the adverse effects of mainstream administration. This section of the review discusses the potential of WS as an adjuvant to current pharmacotherapeutics and vaccine development in COVID-19.The current therapeutic approaches in the management of COVID-19 involves the use of hydroxychloroquine, chloroquine, azithromycin, and emergency antiviral drug remdesivir  “Clinic. The benThe major concern with the use of hydroxychloroquine and chloroquine is cardiac arrhythmia  Blignau. A combiA few cases reported hydroxychloroquine-associated hepatic failure leading to death. The histopathological observations showed necrosis of liver parenchymal cells and moderately percolated inflammatory cells . WS is aAn approach of convalescent plasma therapy is also showing promising results in COVID-19 management. The beneficial effects of convalescent plasma therapy have been demonstrated by increased neutralizing antibody titer accompanied by reduced SARS-CoV-2 RNA in patients . WS is aOverall, the preceding studies suggest that WS may show benefits to decrease adverse effects of drugs and adds up to antibody response in COVID-19 management. The multimodal activities of WS described in the previous section may exhibit the probable synergistic effects. Therefore, WS can be a promising candidate to be considered as a therapeutic adjuvant in COVID-19 management.B. pertussis cells. This was accompanied by improved overall health status and reduced mortality  maintaining immune homeostasis, 2) regulating inflammation, 3) suppressing pro-inflammatory cytokines, 4) organ protection , and 5) anti-stress, antihypertensive, and antidiabetic activities. The dose of WS extracts ranged from 10–300 mg/kg in various preclinical experimental models have been discussed in this review. However, it was found that the concentration range of 50–200 mg/kg of different extracts of WS and 2 mg/kg of withaferin A tested in animal models significantly showed anti-inflammatory, cytokine regulatory, and immunomodulatory effects. Moreover, 10–50 μg/ml of withaferin A stimulated cytokine expression in monocyte and macrophage cell lines  would throw light on the fundamental benefits of WS.The post-COVID-19 complications also deserve a serious attention to achieve quality of life . Severalin silico observations followed by clinical studies. The focus on evaluating pharmacokinetic–pharmacodynamic profile and drug interactions along with safety and efficacy in healthy volunteers and patients with COVID-19 for prophylactic and therapeutic use is required. This review attempts mapping of phytochemical diversity and reported pharmacological activities of WS with COVID-19 pathophysiology. The review proposes potential pharmacological activities of WS extracts and metabolites in the context of COVID-19, which are based on rational extrapolations from available literature leading to testable hypotheses. However, there is a need for systematic studies with appropriate controls to validate these hypotheses in suitable experimental models. Moreover, the activity of any botanical depends on a number of variables including geographical variation of species, cultivation and collection methods, extraction process, and administration . The extract should be prepared from the authentic and standardized raw material with a robust protocol to ensure batch-to-batch reproducibility, potency, safety, and efficacy. These sources of variability need to be accounted during experimental designs for exploring WS effects.The study of literature suggests that WS has important molecular and pharmacological characteristics to act as a therapeutic adjuvant for prophylaxis and treatment of COVID-19. Therefore, WS may synergistically improve the clinical outcomes of COVID-19 pharmacotherapeutics in current use. WS may be explored in healthcare workers undergoing COVID-19 vaccination for modulation in antibody titer to understand the real-world adjuvant potential. Besides, WS may have a salutary effect on other comorbidities associated with the COVID-19 disease. Our recently published review has addressed this possibility in cancer . To realRasayana botanicals.The multidimensional research on WS with a deeper understanding of biological mechanisms is a promising area for future exploration as a therapeutic adjuvant. Given the recent observations that COVID-19 pandemic will have a long-term trajectory, there will be a significant need for research and development of COVID-19 therapeutics and adjuvants . In this


Scattering-type scanningnear-field optical microscopy (s-SNOM)has emerged over the past years as a powerful characterization toolthat can probe important properties of advanced materials and biologicalsamples in a label-free manner, with spatial resolutions lying inthe nanoscale realm. In this work, we explore such usefulness in relationshipwith an interesting class of materials: polymer-coated gold nanoparticles(NPs). As thoroughly discussed in recent works, the interplay betweenthe Au core and the polymeric shell has been found to be importantin many applications devoted to biomedicine. We investigate bare AuNPs next to polystyrenesulfonate (PSS) and poly (PDDA) coated ones under 532 nm laser excitation, an wavelengthmatching the surface plasmon band of the custom-synthesized nanoparticles.We observe consistent s-SNOM phase signals in the case of bare andshallow-coated Au NPs, whereas for thicker shell instances, thesesignals fade. For all investigated samples, the s-SNOM amplitude signalswere found to be very weak, which may be related to reduced scatteringefficiency due to absorption of the incident beam. We consider theseobservations important, as they may facilitate studies and applicationsin nanomedicine and nanotechnology where the precise positioning ofpolymer-coated Au NPs with nanoscale resolution is needed besidestheir dielectric function and related intrinsic optical properties,which are also quantitatively available with s-SNOM. For example, while in generalgold nanomaterials are considered safe, studies suggest that suchstructures can be toxic in some configurations, being prone to beuptaken by kidneys, causing nephrotoxicity.6 Given that size, surface charge, chemical composition, and shapeare key factors related to the potential toxicity and stability ofAu nanomaterials, coating them with polymers in order to tune theseproperties to values that raise no health hazards represents an interestingsolution.7 Moreover, given that the elasticityof NPs dictates how these are endocytosed by cells,8 controlling this property by coating the Au NPs with softor rigid polymers9 can facilitate variousapplications that rely on Au NPs uptake or can prevent their internalizationwhen this is not wanted. Other surface properties of the polymer shellhave also been found to be important in the context of Au NPs’cell trafficking.9 Exploiting the propertiesof gold nanostructures to tune the properties of polymer-based materialsis also possible. For example, in a past study,10 the interrelationship between Au NPs cores and the yieldof a fluorescent polymer shell was discussed. Additional aspects onrelated topics are provided in refs (15Gold nanoparticles(Au NPs) represent one of the most popular typesof nanomaterials as they are highly stable and easy to synthesizein various shapes and sizes with reproducible procedures in refs . We also19 However, conventional optical microscopies are limited in resolvingthe properties of Au NPs, as their resolution is limited by the diffractionphenomena to ∼200 nm, depending on the wavelength being used,which is insufficient for assessing nanoscale features of interest.Scattering-type scanning near-field optical microscopy (s-SNOM) representsan emerging optical characterization technique that can help in thisregard, offering possibilities for both imaging and spectroscopicassays.20 Its working principles rely ona sharp tip that is scanned across the sample’s surface whileit is being excited with a focused laser beam. The tip converts theincident radiation into a highly localized and enhanced near fieldat the tip apex, which modifies both the amplitude and the phase ofthe scattered light via the near-field interaction with the sampleunderneath. This process depends on the local dielectric propertiesof the sample,22 which can thus be probed withthis technique at a resolution dictated only by the dimension of thesharp tip used for scanning and the sensitivity of the detector. Suchcapabilities are very important for exploring the optical propertiesof various nanostructures, including various Au-based nanomaterials.28 Additionally, in previous studies, it was shown that besides usings-SNOM to collect raw near-field optical signals, which are extremelyuseful, but also quite difficult to interpret, this technique canalso be used to quantitatively map with nanoscale spatial resolutionthe dielectric function and intrinsic optical properties, e.g., refractiveindex, reflectance, etc.30 Such earlier efforts are currently being extended by means of modernmachine learning methods.32 The attainable resolutionin s-SNOM depends on the size and geometry of the probe, with manyimportant applications having been reported for tip sizes rangingbetween 5 and 150 nm.34 In our experiment, we use a Co–Crtip with a 60 nm radius of curvature in its apex. Also worthy to mentionare s-SNOM’s capabilities to lithographically modify materialswith superb precision.35 Based on these,s-SNOM systems can be regarded as multipurpose platforms for nanoscalemanipulation and characterization of advanced functional bio(nano)materials.To improve existing applications of Au NPs and to enable novelones, a thorough understanding of these materials is required. Manycharacterization techniques have been used to date in this regard,with optical ones being very important to shed light on various aspectsof interest.43 the body of work performed to date to elucidate such aspects isstill limited. Our work aims at extending the current understandingof this problem, focusing on an application that, to the best of ourknowledge, has not been addressed before. Namely, we assess the attenuationof s-SNOM phase signals corresponding to a metallic nanoparticle corewith the thickness of the surrounding (polymer) shell, when excitingwith a laser beam with a wavelength matching the absorption band ofthe metallic core to promote phase contrast corresponding to the latter.This is a distinct problem compared to the case when the compositenanoparticle is imaged by s-SNOM phase contrast associated with theshell.44 Interestingly, we observe verydifferent results compared to previous experiments that evaluateds-SNOM signal attenuation with depth from the perspective of amplitudecontrast, instead of phase contrast, which is at the focus of ourwork. These differences, along with their significance, are discussedin the While s-SNOM is generally acknowledged as a surface characterizationtechnique, it exhibits nonetheless valuable capabilities for subsurfaceand 3D imaging. These are very important, considering that subsurfacenanostructures cannot be resolved by optical systems with diffractionlimited resolution and also cannot be observed by scanning probe orscanning electron microscopy. Although the topic of s-SNOM signals’attenuation with depth  has been discussedto date in several publications, in relationship with different problems,To be more specific,in this experiment, we explore s-SNOM’scharacterization potential with respect to a set of polymer-coatedAu NPs that we carefully synthesized. A custom-modified s-SNOM systemwas used to acquire amplitude and phase images in the visible frequencyrange, under illumination with 532 nm, a wavelength falling in theabsorption band of the investigated instances, which we demonstratein detail. In this configuration, with a Co-Cr tip, we obtain strongphase contrast for the bare and shallow-coated Au NPs, whereas thepolymer coating seems to attenuate the phase contrast in the caseof thick-shell instances. For all instances, the amplitude contrastis minimal, which may be ascribed to reduced scattering efficiencydue to absorption of the incident beam by the sample. We discuss aswell imaging results obtained with an Au-coated tip, and with theinitial Co–Cr one under illumination with an off-resonancewavelength, 1550 nm, none of these two configurations are capableof providing similar s-SNOM phase contrast as observed in the caseof the Co–Cr tip illuminated with 532 nm.46 viruses,49 prokaryotic and eukaryotic cells,52 both fixedand living tissues53 and various typesof nanostructured materials,34 includingnanoparticles of different kinds,55 providing valuable information based on the sampleintrinsic contrast. Such capabilities render s-SNOM as a superb toolfor supporting studies that focus on the interaction of advanced materialsand biological organisms and structures, at nanoscale. We speculatethat some of the important applications exploiting these capabilitiescould focus on resolving how nanoparticles of different shapes, sizes,and dielectric properties are specifically endocytosed by cells57 or how their positioning can be correlated with their interferencein important cell processes.60 The results presented here canpromote such applications by enabling a better understanding of theattainable s-SNOM contrast for a material class that is not only highlypopular in biomedicine61 but which can also be regardedas a relevant model for other composite nanoparticles comprised ofdifferent polymeric-shell metallic core combinations.62 Importantly, applications of this kind can also benefitof other opportunities offered by s-SNOM, such as quantitative dielectricfunction mapping,63 plasmonic behavior assessment,65 or the identification of vibrational signatures68 via spectroscopicvariants.We considerour observations to be important, as they may facilitatestudies and applications in nanomedicine and nanotechnology wherethe precise positioning of Au NPs with nanoscale resolution is needed.In this context, we find it worthy to mention that s-SNOM has beendemonstrated to date as a very useful tool to image proteins,4·3H2O, >99.9%), trisodium citrate(99%), polystyrenesulfonate, and poly are all purchased from Merck. Nanopurewater (≤15.0 MΩ) from a Millipore Milli-Q gradient systemfrom Merck was used as a solvent.Gold(III) chloride trihydrate was added to 50.0 mL of water,and the system was heated under magnetic stirring with the presenceof a condenser. Once the boiling was reached, 300 μL of HAuCl4 solution (25 mM) was quickly injected, and a sudden colorchange was observed. After 10 min, the seeds were formed, and 630μL of this colloidal solution was withdrawn for further characterization.The Au NPs were synthesized by meansof a multistep approach,4 solution (25 mM) was quickly injected, and thesystem was left under stirring for 20 min, after which 630 μLwas withdrawn for further characterization. This growth procedurewas repeated 13 times, which led to obtaining colloidal Au NPs withan absorption peak close to 532 nm, the wavelength of the visiblelaser line employed in this experiment for s-SNOM imaging. Throughthis article, we refer to these Au NPs as Au-citr NPs, consideringthem a type of Au NPs with particular properties as the interactionsof the citrate molecules with the gold surface can be different fromthose that occur in the case of other molecules (citrate establishesionic interactions that favor Au plasmon damping).71For the growth of the particles, in the same vessel, the temperatureof the system was lowered to 90 °C , and 330 μL of sodiumcitrate solution (60.0 mM) was added. After about 2 min, 300 μLof HAuCl72 wherein each polymeric layer was added sequentially. First, 5.0mL of freshly prepared Au-citr NPs was added dropwise to 5.0 mL ofan aqueous PSS solution (0.55 wt %). The system was kept under vigorousmagnetic stirring for 1 h. The particles were collected by means ofcentrifugation  and dissolvedin 5.0 mL of water (this sample was named Au-L1). Afterward, Au-L1solution was added dropwise to 5.0 mL of an aqueous PDDA solution(0.55 wt %), and the system was kept under vigorous magnetic stirringfor 30 min. The particles were collected by means of centrifugation and dissolved in 5.0 mL ofwater (this sample was named Au-L2). By repeating the layer depositionprocesses, by alternating PSS to PDDA, Au-citr NPs with various polymericshell thickness were synthesized. The investigations reported in thiswork were performed on the Au-L3, Au-L5, Au-L7, and Au-L9 samples,where the numerical value in the sample name denotes the number ofshell layers . For s-SNOMimaging, the Au-citr NPs and all synthesized Au-L# NPs were depositedon a glass coverslip by spin coating. The coverslip was initiallycleaned by immersion in an aqua regia solution (HCl/HNO3 3:1 v/v) for 1 h, followed by several immersions in clean Milli-Qwater and finally acetone. Then, 50 μL of Au-citr/Au-L# NPsaqueous suspension was deposited on the center of the coverslip andspin coated at 400 rpm for 10 s, immediately followed by 1500 rpmfor 30 s.Further, the Au-citr NPs synthesized as above-described werecoatedwith polymeric shells consisting of polystyrenesulfonate (PSS) andpoly (PDDA), of different thicknesses.The fabrication strategy was based on a layer-by-layer (LbL) process,73 inorder to have a statistically significant evaluation at least 120–150particles for each sample have been analyzed. The particle size analysiswas carried by the Nicomp Nano DLS/ZLS Systems , which allowed the determination of the hydrodynamicdiameters of the colloids. All the samples were equilibrated for 10min at 23 °C, and the runtime was set to 15 min to ensure a goodstatistic. The NPs were measured in water at 90°, using a reddiode laser (635 nm). The scattering data were then analyzed throughthe Nicomp algorithm.The optical properties of thesynthesized colloidal samples were first investigated through a Cary8454 UV–vis diode array spectrophotometer. Transmission electronmicroscope (TEM) investigations were performed using a Philips 208transmission electron microscope (TEM) with an 80 kV beam accelerationto assess the size and morphology of the synthesized materials, accordingto a previously described procedure;The s-SNOM experiments performed in thiswork were implemented on a custom-modified neaSNOM  system 1, workinFor s-SNOM imaging, a HQ:NSC18/Co-Cr/Al BS cantilever was used, with a radius of curvature < 60 nm. The cantileverhad a resonant frequency of 75 kHz and a force constant of 2.8 N/m.Imaging was also attempted with a sharper, Au-coated tip, HQ:NSC16/Cr-Au, but this configuration resulted in very weaknear-field signals which we hypothesize to be linked with the incidentlight being absorbed by the Au film coating the tip, resulting ina reduced scattering efficiency.74 and importantly, in the landmarkwork of Stiegler et al.75 it was shownthat the near-field phase spectra of small particles correlate wellwith their far-field absorption spectra. Later works77 had shed more light on the existing correlations occurring betweens-SNOM phase signals and the absorption properties of the investigatedsamples. These previous efforts showed that s-SNOM phase contrastthus provides specificity, as tuning the wavelength of excitationbeam to match the absorption properties of a particular element ofinterest found in an investigated sample leads to images where thiselement is highlighted. For example, in the recent work of Mészároset al.,78 s-SNOM imaging with illuminationat 1660 cm–1 (6024 nm) was employed to visualizethe protein content (amide I band) and distribution in a number ofrepresentative cells.The aim of this experiment was to assess the capabilities of s-SNOMto image polymer-coated Au NPs, based on phase contrast resultingfrom the absorption of the excitation beam by the probed sample. Asthoroughly discussed in previous works, the phase of near-field signalsis related to the complex optical constants using quasi-electrostatictheory,81 and for this class of materials, mid-IR illumination sources arerequired. Our interest addresses the distinct problem of probing thephase contrast arising from the Au-citr core, which has a two-foldreason. First, the findings can be generalized for additional compositecolloidal nanomaterials based on Au cores covered with shells composedof various other materials.82 Second, withcorrelative imaging approaches based on multimodal systems that combines-SNOM with other near-field and far-field modalities,83 the use of excitation sources in the visible or near-IR to servemultiple imaging modalities is more practical and more financiallyfeasible given that other complementary near-field modalities suchas tip-enhanced fluorescence, tip-enhanced Raman, tip-enhanced second-harmonicgeneration, as well as far-field modalities such as diffraction limitedand super-resolved techniques based on laser scanning, typically operateunder visible and near-infrared excitation. Correlative imaging approachesbased on multimodal systems that incorporate various combinationsof these aforementioned techniques could be particularly useful inassessing how polymer-coated Au NPs position inside cells, by providingboth a well understood biological context, based on fluorescence imagingof the cells at microscale, together with the possibilities for nanoscalelocalization of Au NPs via label-free s-SNOM phase contrast. Finally,illumination sources in the visible are more widespread and more affordablethan mid-IR and IR ones, which also motivates our interest to explorerelated s-SNOM applications.For the polymer-coated Au NPs discussedin this study, s-SNOM phasecontrast could thus arise either from the polymer shell or from theAu-citr NP core. s-SNOM phase contrast arising from polymers has beenthoroughly discussed in past works,Figure S1. As can be observed,the absorption/surface plasmon resonance maximum of both Au-citr andAu-L# instances lies close to 532 nm, the wavelength used in thisstudy for s-SNOM illumination. The TEM images of the Au-L3 NPs, asan example  between goldand silicon under a PMMA layer with a thickness > 100 nm, whereasin our experiment the s-SNOM phase contrast seems to attenuate morerapidly when imaging polymer-coated Au NPs under 532 nm illuminationmatching the absorption band of the Au-citr core. This suggests thatthe problem of s-SNOM signal attenuation with depth is specific tothe application at hand and to the considered s-SNOM signal type , aspects that should be carefully consideredwhen designing and implementing an experiment focused on the investigationof subsurface features/elements.Further, we discuss additionalaspects concerning the intensityof the phase signals observed in the case of all NPs. Given the lowintensity of the s-SNOM phase signals observed in the case of Au-L93, we ass85 For each structure of interest, a corresponding doughnut-shapedmask was drawn to cover the surrounding substrate. The width of thisirregular shape was manually defined in the range of 1–3 pixelsso that the NP structure mask and the surrounding substrate mask havesimilar areas. The phase of each Au NP was computed as the differencebetween the average phase in the Au NP mask and the average phasein the substrate mask. This approach is intended to compensate forthe phase drift/tilt that usually occurs across the y axis when collecting an s-SNOM phase image. Because of this effect,the phase signal values of two identical structures positioned inthe upper and lower part of an image frequently differ. This was thusaddressed by referring to the phase difference instead of the nominalphase value.To shed more light on this,we performed a statistical analysison the images collected from each sample (two images per sample),testing how the mean phase of the segmented structures varies dependingon the polymer shell thickness. In P valuesless than 0.05 were considered statistically significant. The 95%confidence intervals (CIs) of the mean differences between the fivegroups were also calculated.Statistical analysis of s-SNOM phase contrast was performed withPrism 9.1 . The one-way ANOVA test was usedto determine whether there is a statistical difference between themeans of the five independent groups . We conducted a Dunnett’s test formultiple comparisons to determine exactly which groups are different.Dunnett’s test performed pairwise comparisons between the controlAu-citr NPs and four polymer-coated instances. 86 indicating a statistically significant decrease in the s-SNOM phasefrom the control group (Au-citr NPs) to the Au-L7 and Au-L9 instances,which suggests that for both these samples the phase signals are significantlylower compared to the case of the bare Au-citr NPs. The results obtainedfor the Au-citr NPs versus Au-L3 and Au-citr versus Au-L5 are statisticallynonsignificant and thus should be considered with care. The 95% CIsof the differences in each of the two cases  nanomaterials, to resolve important pending issuessuch as nanoparticle trafficking by cells.87 Hopefully, our experiment will encourage additional work promotingthe use of s-SNOM for implementing advanced characterization assaysthat can resolve properties of polymer-coated Au NPs and also theirinteraction with biological species, with a specificity and levelof detail unavailable to other imaging techniques. Our further effortswill focus on characterizing such polymer-coated Au NPs structuresat the single nanoparticle level with ultrasharp tips. Besides experimentalwork, future efforts based on complex simulations with emerging s-SNOMmodeling tools88 can also contribute toa better understanding of subsurface s-SNOM imaging.We consider these observations to be important in light of s-SNOM’scapabilities for nanoscale spatial resolution, which represent animportant advantage when studying Au NP-related aspects, such as theirdistribution in doped materials or inside cells. Such latter studiesare greatly facilitated by recent efforts that have been focused ons-SNOM imaging of living


Coronavirus disease 2019 (COVID-19) is a pandemic disease that spread rapidly throughout the world and became a major public health concern. Approximately 5–12% of COVID-19 patients require admission to the intensive-care unit (ICU), where they often require oxygen therapy and prolonged intubation. Post-intubation laryngotracheal stenosis (PILS) is a complication that occurs in 10–22% of non-COVID-19 patients after prolonged intubation, while the rate of COVID-19 related PILS remains unknown. Additionally, there is still no consensus in the literature regarding the management modalities for PILS following COVID-19.Here we report two cases of tracheal stenosis after prolonged intubation due to severe COVID-19 infection. The first patient was admitted to the ICU and intubated for 21 days; 3 months after discharge, he developed a 3 cm long tracheal stenosis that narrowed 70% of the lumen. The second patient was intubated for 2 months and, 4 months after discharge, developed a 2.5 cm long tracheal stenosis that narrowed 80% of the lumen.In both cases, the diagnosis was confirmed by CT scan and Rigid bronchoscopy; then, they were managed successfully with tracheal resection and reconstruction by end-to-end anastomosis.In conclusion, we would like to highlight the importance of suspecting PILS in recovered COVID-19 patients re-presenting with breathing difficulties following weaning from mechanical ventilation; therefore, careful follow-up in such patients is required. Moreover, we would like to point out that the management of tracheal stenosis after COVID-19 appears to be similar to that of tracheal stenosis in general. •Tracheal stenosis (TS) is a rare complication that follows prolonged intubation.•Prolonged intubation causes tracheal wall damage, which leads to TS.•Suspect TS in recovered ICU-admitted COVID-19 patient re-presenting with dyspnea.•Stenosis length, severity, and location are essential in the management plan.•Consider non-surgical procedures in patients with “thin and young” stenoses. The disease has spread rapidly across every country and has become a major public health concern .Only 5–12% of COVID-19 patients require admission to the intensive-care unit (ICU); such patients often require prolonged mechanical ventilation with high positive end-expiratory pressure (PEEP) through an endotracheal tube [While tracheal intubation is a life-saving procedure, it may cause mucosal damage and tracheal wall necrosis, which leads to laryngotracheal stenosis . The ratIn this paper, we report two cases of patients with post-intubation tracheal stenosis diagnosed during their recovery phase of COVID-19 and treated by surgical resection and reconstruction by end-to-end anastomosis. This work has been reported in line with the SCARE criteria .22.12) was 96% on room air. CT scan showed signs of tracheal stenosis. He underwent 3 attempts of tracheal dilatation without a satisfactory outcome. Rigid bronchoscopy revealed a 3 cm long tracheal stenosis that narrowed 70% of the lumen; the stenosis was 5 mm below the cricoid cartilage. The stenosis was managed successfully with tracheal resection and reconstruction. The postoperative period was uneventful, and the patient was reassessed after 2 months: his general condition was good without complaints, and bronchoscopy revealed a non-narrowing anastomotic line.A 35-year-old man presented to our hospital complaining of dyspnea with stridor that started a month before and exacerbated during the last week. The patient was a non-smoker and had no significant surgical or family history. 4 months before, he was admitted to the intensive care unit (ICU) and intubated for 21 days due to acute respiratory failure that occurred as a complication of COVID-19. On admission, his peripheral oxygen saturation (SpO2.22 was 95% on room air. CT scan showed signs of tracheal stenosis (A 62-year-old man presented to our hospital complaining of dyspnea and wheezing that started 2 months before and exacerbated during the last 10 days. The patient was a moderate smoker with a history of poorly controlled hypertension diagnosed 23 years ago. 6 months before, he was diagnosed with COVID-19 and admitted to the ICU due to the rapid deterioration of his respiratory condition. He was put on a mechanical ventilator for 2 months. On admission, his SpOstenosis . Rigid b3PILS is a rare condition with an estimated incidence of 4.9 cases per million per year in the general population . PILS beThere are still no data about the occurrence of PILS in recovered COVID-19 patients. However, it is thought that those patients could be at increased risk of developing PILS; perhaps most notably due to their hyperinflammatory state, which causes altered fibrosis, but also because those patients are subjected to prolonged intubations and usually already have comorbidities, such as diabetes and hypertension .Early tracheostomy is recommended for patients who require prolonged tracheal intubation , which iAlthough Mr. Francesco Mattioli et al.  suggesteAs an approach for tracheal stenosis, the location, length, and severity of the stenosis are essential factors in the management plan. If the stenosis is suitable for resection and reconstruction by end-to-end anastomosis and the patient can withstand the operation, surgery is recommended; otherwise, other therapeutic procedures are performed. Thus, the management strategies for tracheal stenosis after COVID-19 are similar to those for tracheal stenosis in general.4We presented two cases of post-intubation tracheal stenosis that occurred after COVID-19. It should highlight the importance of suspecting tracheal stenosis in recovered COVID-19 patients re-presenting with breathing difficulties following weaning from mechanical ventilation; therefore, careful follow-up in such patients is required. Moreover, we would like to point out that the management of tracheal stenosis after COVID-19 appears to be similar to that of tracheal stenosis in general.Written informed consent was obtained from our two patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not commissioned, externally peer-reviewed.None.None.None.AB: reviewed the literature, wrote the abstract, and the introduction.AA: reviewed the literature, wrote the case presentation, and the discussion.BD: lead the surgical team, checked the spelling and grammar, revised the manuscript and helped in writing the discussion.All of the authors declared that they have no conflict of interest.