Devulcanization represents the recycling of choice for a homogenous rubber waste stream because it allows revulcanization of samples previously devulcanized, making the life of the rubber virtually endless, according to the principles of circular economy. Among the many devulcanization processes, the thermo-mechanical one is the most appealing because it is a continuous process, easy to be industrialized. In this paper a comprehensive set of analyses  were carried out on a post-industrial ethylene propylene diene monomer (EPDM), thermo-mechanical devulcanized in a co-rotating twin-screw extruder with different process parameters . Results of the swelling test according to the Flory–Rehner theory and Horikx analyses show that the higher the thermal profile and the higher the rpm, the higher is the percentage of devulcanization. The quality of the devulcanized sample in terms of sol fraction and percentage of random scissions depends on the process conditions. The screw profile concurs to the efficiency of the devulcanization: the different number of kneading elements and more in general the screw profile composition affects the percentage of devulcanization, making the results in some tests more dependent on the screw speed. Elastomers deform instantly under load application and soon return to their original size by load remotion. The structural reason for elastomeric behavior is the presence of flexible chains connected in a network with chemical unreversible crosslinks in the thermosetting elastomers or physical reversible crosslinks in the thermoplastic elastomers. Due to the permanent crosslinking, thermosetting elastomers are difficult to be recycled.Ethylene propylene diene monomer (EPDM) is a synthetic thermosetting elastomer. It is a copolymer of propylene and ethylene combined with pendant diene to crosslink the polymer. It shows strong resistance to many external agents such as heat, ozone, mild acids, synthetic brake fluids, water, ethylene glycol, and other liquids. Despite being an inert material with limited environmental impact, EPDM is used in a chemically cross-linked form in a variety of applications and this feature makes its recycling difficult. EPDM rubber is mainly vulcanized by two methods: (a) with sulfur, creating sulfuric crosslinks; or (b) with peroxides, linking polymer chains through C–C covalent bonds . The marIt should be emphasized that the EPDM parts in most goods  can be relatively easily dismantled and collected separately, constituting an important homogeneous waste stream which can be valorized according to the concepts of the circular economy . TherefoGenerally, rubber recycling can be carried out in three strategies: fragmentation, pyrolysis, devulcanization or reclaiming.Fragmentation, the most widely traditional approach for rubber recycling, is carried out by grinding the rubber to powdered particles that can be blended with thermoplastics or mixed with virgin EPDM compounds as a filler, to extend their life. A variety of products can be obtained in this way . This is an example of linear economy where the scraps progressively decrease their value; in addition, the market for these products is limited, hence the quality of recycled rubber products must be improved especially in the perspective of the circular economy .Pyrolysis is a sort of tertiary feedstock recycling in which feeding is converted merely by thermal energy into products such as monomers or pyrolysis oil depending on the polymer fed to the reactor. It does not require preliminary separation of the different polymers and is particularly suitable for mixed heterogeneous flow of waste or complex multicomponent items .Conventional reclamation processes a involveDevulcanization processes b cleave Among the four methods mentioned, devulcanization is the most appropriate process for a homogeneous waste stream because it better fits the circular recycling concepts, rubber scraps being converted into a material that can be revulcanized ,10.A few technologies of rubber devulcanization can be performed; for instance, chemical techniques use a devulcanizing agent to breakdown the crosslinked network. Among them, organic disulfides, such as 2,2′-dibenzothiazole disulfide, and dixylyl disulfide are the most commonly used to break down the crosslinked network. The devulcanization is carried out as a batch process by refluxing a mixture of rubber with the appropriate solvent and reclaiming agents ,12.On the contrary, physical techniques use physical agents such as heat, mechanical stress, microwaves, or ultrasound, alone or in combination, to break down the crosslinking bonds ,14,15,16Because of the weak penetration of devulcanization agents in the rubber matrix, mechanical devulcanization is a more promising route than chemical devulcanization .Thermo-mechanical devulcanization by extrusion seems to be even more promising, being a continuous process, easy to be industrialized. Usually, several parameters are needed to fully describe this reactive extrusion process and possibly to model it considering the experimental input data . In the One of the first mechanisms for thermo-mechanical devulcanization under shear was formulated by Mouri et al. ,21.The strength of the carbon bonds –C–C– of the rubber chain segments, monosulfidic bonds –S–, disulfidic bonds –S–S– and polysulfidic bonds –S–S–S─ are 352, 285, 268, and 251 kJ/mol . In the Another model for selective network scission by thermo-mechanical devulcanization is based on different elastic constants and binding energies of S–S, C–S, and C–C bonds. The differences between the binding energies are minor, and a purely thermal process leads to unselective cleavage. Since the elastic constant of S–S bonds is much smaller than that of C–C bonds, S–S bonds are the most extended under shear. As a result, the elastic energy generated by shear stress is most significant at S–S bonds leading to selective cleavage of crosslinks. However, this cleavage occurs only at high shear stress, otherwise, the entropic effect predominates .As far as thermo-mechanical devulcanization is concerned, apart from the undoubted advantage of being a continuous process easy to be industrialized, life cycle analysis (LCA) evidenced that the environmental impacts associated with this recycling process are limited, with the only concern being related to energy consumption . TherefoPost-industrial EPDM seals came from the portholes of washing machines. The EPDM seals were ground by F.lli Maris S.p.A. obtaining a free-flowing form material ready for feeding into the extruder. The devulcanized rubber, for each test, was collected and cut into pieces (approximatively 0.5 × 0.5 × 0.5 cm) for sample characterization and swelling tests.Devulcanization process: the devulcanization process using a co-rotating twin-screw extruder without the use of solvents/devulcanizing agents was studied, designed, and patented by F.lli Maris S.p.A.—an extruder manufacturer—in 2011.The co-rotating laboratory twin-screw extruder used to carry out the current study has the following technical characteristics: screw diameter = 20 mm, extruder length 64 L/D, which corresponds to 16 barrels (each barrel length is four times the diameter of the screw). Each barrel is equipped with a heating and a water-cooling system to guarantee precise control on the barrel temperature of the most suitable process temperature. The extruder was also equipped with water injection, a side degassing unit, and a sheet extrusion die. To devulcanize the EPDM rubber and subsequently to optimize the process parameters to increase the quality of the devulcanized product, the following parameters were changed: thermal profile , screw speed , screw profile configuration (three different screw profiles), and water injection .The screw profiles were designed to heat and to process the rubber in order to transform it—inside the extruder—from a vulcanized solid state to a plastomeric devulcanized state. Depending on the process parameters and the combination of them, the rubber is subjected to thermal energy and mechanical energy in different ratios, but only thanks to an appropriate balancing of all the parameters is it possible to reach the right temperature of the rubber and make devulcanization possible. If these parameters are not balanced correctly, however, the rubber exiting the extruder will be pulverized and degraded. The barrel temperatures were set between 200 and 300 °C.The injection of water in the final phase of the extrusion (devulcanization) process aims to rapidly cool the devulcanized rubber inside the extruder preserving it from possible degradation and it is also an excellent stripping agent for the reduction of gases developed during the process. In fact, the water injected into the extruder, after its mixing with the devulcanized rubber, is removed by means of the degassing unit.The same output was used for all tests (3.5 kg/h); Infrared analysis. The FTIR spectra were registered with a Perkin Elmer FTIR Spectrum 100, in ATR modality . The internal surface of samples was analyzed. The qualitative composition of the samples was determined.Pyrolysis–GC/MS. A Frontier Lab pyrolizer connected to a GC/MS Agilent 6890 Series and Agilent 5973 Network was used for analysis. The pyrolysis temperature was set to 600 °C, for 12 s.TGA. A TA Q500  equipment was used to ascertain the quantitative sample composition in terms of volatiles, polymer, CaCO3, carbon black, and inorganic residue. The following heating program was used: nitrogen flow 60 cc/min: ramp 10 °C/min from 50 °C to 300 °C, isothermal for 10 min; ramp 10 °C/min to 550 °C. Switched to air 60 cc/min: isothermal for 10 min, ramp 20 °C/min to 650 °C, isothermal for 15 min; ramp 20 °C/min to 800 °C, isothermal for 2 min.2 in the plateau at 550 °C in air and CaCO3 decomposes to CO2 (volatile) and CaO (in the residue) in the following ramp.Under these conditions plasticizing oils evaporate along the isothermal segment at 300 °C, rubber degrades in the first ramp in nitrogen up to 550 °C, carbon black, if present, is oxidized to CODSC. A DSC Q200  equipment was used to ascertain the glass transition temperature of the samples either in the original, or acetone extracted, or after-SW series as described below (swelling test paragraph). The following heating program was used: Heat cool heat method: equilibrate at −85 °C, ramp 20 °C/min to 80 °C (first heating). Ramp 20 °C/min to 85 °C (cooling). Ramp 20 °C/min to 80 °C (second heating). Tg was taken at the inflection point of the second heating. Duplicate analyses were carried out, the error was ±0.5 °C.Elemental analysis. Elemental analyses were performed with a CHNS-O Analyzer Flash EA, 1112 Series equipment on 2.5 mg of sample using V2O5 as a catalyzer, in duplicate. Error was ±0.05%Swelling test. The original rubber samples (ORIGINAL), directly obtained from Maris S.p.A., were weighed and reduced to smaller pieces. They were extracted in hot acetone for 16 h using a Soxhlet apparatus to eliminate the acetone-soluble species. The solids were dried at room temperature under a laboratory fume hood until constant weight. This operation generates a second series of samples (EXTRACTED) in which plasticizing oils are removed.bW) and immersed in 100 mL of toluene at room temperature for 24 h. After this time, the samples were quickly dried with absorbent paper, to remove the excess liquid from its surface, and immediately weighed (aW) in a cap-closed, tared, and weighted jar. Then the cap was removed from the jar and both the cap and the sample in the open jar were put into a forced-ventilating oven at 55 °C for 24 h.The Extracted samples were weighed (dW). The amount of the absorbed solvent is a − WdW, the amount of solubilized polymer is b − WdW [The jar containing the dried sample and the cap were removed from the oven; the jar was recapped and allowed to cool at room temperature. Finally, the closed jar was weighed and the weight of dried sample calculated ( Wb − Wd .(%sol) is:ϕ is the percent of polymer in the acetone-extracted sample as derived from TGA.Therefore, the percentage of extracted polymer Swelling tests generated a third series of samples (after-SW) in which the sol fraction of the polymer was removed.The higher the amount of absorbed solvent, the lower is the crosslinking degree. From this consideration Flory–Rehner (FR) equated the free mixing energy and the elastic recovery force at the equilibrium:By substituting the appropriate thermodynamic equations, the classical FR equation is recovered .Their model was later extended considering the more realistic phantom model instead of the classical one of affine deformation .SW)n is:The final equation for density of crosslinking (SWn (density of crosslinking) corresponds to sub,1/M with subM the average number molecular weight of the chains between two adjacent crosslinking;sV is the molar volume of the solvent, taken from literature (here 106.52 cm3/mol);f is the functionality of crosslinking (here 4 was considered);χ is the interaction parameter polymer-solvent; values for χ ranging from 0.45 to 0.51 are reported in the literature [subM;terature  and in trϕ is the volume fraction of the expander rubber and is calculated from the swelling test:bW, a,W and dW have been previously described and rρ and sρ are the rubber and the solvent densities, taken from literature and respectively 0.860 and 0.865 gr/cm3;Φ is the percentage of the polymer in the sample undergoing swelling, evaluated from TGA of the after-extraction sample series;α is the fraction of sample extracted during the swelling test.i(% DVZ) is given by the following equation, which takes into account the density of crosslinking of RM (feedstock) and of each devulcanized sample:The percentage of devulcanization of each sample Horikx analysis. The fraction of soluble material in the degraded network is a measure of the number of scissions occurring in the network, provided that the original molecular weight of the chain before crosslinking and the average number of crosslinking/chains are known.M is the molecular weight of the original chains, iγ, the number of crosslinking/original chains, is:iS is, according to Charlesby [Under the assumption that harlesby :(7)Si=(2randomX) is:Chain scission at random: The required number of chain scissions is randomly applied to the not vulcanized polymer, after which the crosslinks are brought into the same positions that they would have occupied if no scission had occurred. In this case the fraction of broken links ;Horikx recovered a simple relation between the soluble fraction of a network that has undergone scission and the effective number of chains in the gel fraction (as determined by swelling measurements) for two limit cases ,32:Chain2.crossX) is:Severance of the crosslinks: there is no chain scission, but only opening of the crosslinks. In this case the fraction of broken links  and the shoulder at 2953 cm−1 (stretching -CH3). The -CH2- bending is overlapped by the band at 1455 cm−1 due to -O-(C=O)-O- group (CaCO3). The -CH3- bending appears at 1377 cm−1. The band at 720 cm−1 is due to the amorphous phase -CH2- rocking; the analogous in the crystalline phase at 730 cm−1 is not evident here. The insulated propylene structural units in EPDM (amorphous phase) absorb at 970 cm−1 and at 1158 cm−1 (weak bands); here the second band is hidden [Polymer and plasticizing oils are recognizable by the peaks at 2920–2852 cms hidden .−1 (-Si-O-H), 1092–1088 and 799 cm−1 (-Si-O-Si-) are attributed to silicates [−1 are attributed to calcium carbonate .Signals at 3643 cmilicates . Signals−1 bands. The band at 1450 cm−1 appears less affected because of the overlapping with the strong absorption of CaCO3. Similarly, these absorptions are further reduced by remotion of the sol fraction after SW. Therefore, FTIR accounts for the plasticizing oil extraction in acetone and the polymer sol fraction in swelling (in toluene).By comparing the FTIR spectra in The pyrogram of the extracted EPDM RM is displayed in PY-GC/MS highlights the nature of the diene in EPDM through several specific degradation products . In thisSeveral benzothiazole derivatives appear in the pyrogram such as benzothiazole , methylbenzothiazole,  and 2-(methyl mercapto) benzothiazole (MMBT r.t 16.182 min), indicating that the accelerator in the vulcanization package was benzothiazole-based. All MS spectra are reported in the m/z 94) in comparison to BT (m/z 135) or MeBT (m/z 149) is higher in the extracted than in the after-SW series. This supports the idea that unvulcanized EBN units are mostly confined to the sol fraction and the monosulfide crosslinkings in the gel phase.In m/z 181) is only found in the RMs and not in devulcanized samples. This support the hypothesis that polysulfides were destroyed in the devulcanization process.In addition, MMBT  slightly decreases mainly because of the decreasing CaCO3 content. On the contrary the organic fraction slightly increases mainly due to increased content of rubber, especially in the tests carried out with screw 1 and 2 (test 2–17). It appears that devulcanization affects the amount of CaCO3 (from 15.41% in RM to 14–14.6% in devulcanized samples) possibly by its thermal decomposition and on the amount of rubber (from 32.20% in RM up to 33.19% in devulcanized samples) because the volatilization of plasticizing oils in the devulcanizing equipment  compared to those of the devulcanized samples (220–226 K). This supports the loss of connecting ties in the network during devulcanization. Eventually the EPDM samples after-SW exhibit an even large Tg compared to those of the other series, and this supports the view that a (mobile) sol fraction is removed from the network during swelling. However, RM exhibits similar Tgs either after extraction (229.9 K) or after-SW (231.0 K) meaning that in this case the sol fraction is very low while it increases in the devulcanized samples due to the action of mechanical and thermal forces.The results of elemental analyses for extracted and after-SW series of EPDM are reported in 3 and silicates). Their amount is in line with the inorganic fraction in the original EPDMs, subtracting the plasticizing oils contribution when calculating the percentages of the various components.Other elements come from the inorganic fillers (CaCO3. From data in The content of C and H in extracted samples is higher than that in after-SW samples. The polymer is insoluble in acetone (extraction) but soluble in toluene (swelling test). Therefore, these data clearly highlight that during swelling the sol fraction initially present in the network or formed in the devulcanization process is removed from the sample, decreasing the percentage of the polymer in the sample and consequently the % of C and H;In the after-SW series the content of C and H of RM is higher than that of the devulcanized samples, confirming that in the devulcanization process the sol fraction increases;3 during the devulcanization process.In the extracted series the content of C and H of RM is slightly higher than that of the devulcanized samples. This points contextually with the slight decrease of the other elements percentages, and agrees with the partial decomposition of CaCOC and H arise from the polymer. In addition, part of the C is due to the presence of CaCOThe amount of N is similar and remains nearly constant along both the series. It means that the benzothiazole-containing units are neither soluble in acetone nor in toluene. Consequently, it can be argued that these groups are mainly connected, as terminal units, to the crosslinked network, as expected from the vulcanization mechanism;In the after-SW series the amount of S increases in comparison to that in the extracted ones. Considering that the elemental sulfur possibly formed in the devulcanization process is nearly insoluble in toluene, its increase can be due to the remotion of the sol phase, which decreases the amount of polymer in the sample under analysis;In the extracted series the N/S mol/mol ratio is lower for RM (1.20) than in devulcanized EPDM. Considering the insolubility of elemental sulfur and of benzothiazole-units in acetone, this means that possibly part of the elemental S can be removed in the devulcanization process.N and S arise from the vulcanization package based on accelerators  and elemental sulfur, used in the vulcanization process. Even if their amount is close to the experimental error several tendencies can be envisaged:%DVZ and %sol are reported in %random derived from the Horikx (Equation (10) approach is reported also, which will be discussed later.Results of the swelling tests in term of %DVZ and that when using low and very low rpm the %DVZ slightly decreases in comparison to the test carried out at high and medium rpm. As for the screw profiles, comparing the screw profiles 1 and 2, the %DVZ obtained with screw profile 2 (characterized by a different number of kneading elements), is slightly lower. The screw profile 3 gives %DVZ quite similar to that of screw profile 1 but, if the amount of injected water is high enough (15%) the %DVZ slightly decreases, probably due to the temperature drop of the rubber inside the extruder.There is clear evidence that the higher the thermal profile, the higher %DVZ is always greater when the thermal profile is high and in this case, the influence of the screw speed is less important. The influence of the screw speed, however, is greater at medium/low thermal profiles.The extruder provides mechanical energy to the rubber through correct construction of the screw profile and the screw speed, while the thermal energy is supplied through the extruder heating apparatus. As can be seen from %sol; likewise, at lower rpm, generally, the %sol is lower. However, when rpm are very low, %sol seems to increase in comparison to low rpm tests. This can be due to the longer residence time that samples experience in these conditions. Again, %sol obtained with screw profile n°2, characterized by a different number of kneading elements, seems to be less affected by the rpm, at least at higher thermal profiles. In tests with screw profile n°3, if the amount of injected water is high enough (15%), mostly, %sol slightly decreases at medium, low and very low rpm.Similarly, the higher the thermal profile, the higher is In f (=%sol/100) lay in between the crosslinking model and the random scission model curves; this means that both crosslink scission and random scission occur. However, it should be noted that all the experimental points are close to the crosslink scission model curve and this is indication of a preferential crosslink-scission mechanism during the devulcanization process.All experimental sol fractions values, S%random was calculated through Equation (10) : against the %sol expected from pure random scission , at the same level of devulcanization.To investigate the relevance of main chain scissions and crosslinking severance in each sample the ion (10) . %random%sol and %random for all samples.%sol and the %random since their relative trends are interdependent with the process parameters  and this does not allow a general trend to be provided.There is no global correlation between the %sol and the %random for the tests carried out with a high thermal profile (tests 2–4) is opposite to that obtained at medium (tests 5–7) and low thermal profiles  at different screw speeds. For instance, at high thermal profile %sol and %random decrease as rpm decreases except in test 10, carried out at very low rpm, in which both show a remarkable rise.However, it should be noted that in general, for the screw profile n°1, the trend of the %random by reducing the effect of mechanical shear given by the reduction in rpm. Conversely, at medium temperature, it is mainly the residence time of the material in the extruder, which decreases as rpm increases, that generates a lower %random at higher rpm.It is therefore to be observed that at high temperatures there is a greater selectivity in the breaking of the crosslinks and therefore a lower %random and %sol in tests performed with screw profile n°2 are less affected by the process conditions  in comparison to tests performed with screw profile n°1.%random and water injection at different percentages. However, it should be noted that an increase in the %random occur for the tests carried out at high thermal profile and high rpm. On the other hand, it should be noted that in particular for the tests carried out at medium thermal profile and medium rpm with 15% of water (test 26) the %random is the lowest for this screw profile.For screw profile n°3, there is not a global correlation between the Devulcanization represents the ideal recycling process for a homogenous rubber waste stream because it allows revulcanization of devulcanized samples, enabling virtually endless life of the rubber in accordance with the principles of circular economy. Among several devulcanization processes, the thermomechanical devulcanization—using a co-rotating twin-screw extruder—is the more appealing because it is a continuous process, easy to be industrialized, and furthermore the patented Maris process does not require the use of solvents/devulcanizing agents, making it more environmentally sustainable. However, if thermomechanical devulcanization is carried out in not-optimal conditions a still too crosslinked rubber or a too devulcanized rubber is obtained and in both of these cases an unsuccessful revulcanization is expected.A comprehensive set of analyses were carried out on a post-industrial EPDM seal from the porthole of a washing machine  devulcanized in a co-rotating twin-screw extruder using different screw profiles, different thermal profiles, different screw speeds, and water injection.3 was decomposed and a small part of the plasticizing oils volatized . The original EPDM was an ENB-EPDM, vulcanized with a benzothiazole vulcanizing package. Benzothiazole residues remained in the network as end groups connected to the rubber by monosulfide and polysulfide bridges. There is some evidence that polysulfide bridges are converted to S plus monosulfide bridges during devulcanization (Py-GC/MS).Characterization of devulcanized samples showed that during the devulcanization process, no matter the condition adopted, a small part of the CaCOMoreover, during devulcanization part of the rubber network is converted to a toluene-soluble sol phase (FTIR and swelling test).Devulcanization was ascertained by the decreasing Tg of the network after devulcanization (DSC) and, quantitatively, by the results of the swelling test according to the Flory–Rehner theory. Eventually the occurrence of random scission in the rubber chains, which is mostly an undesired phenomenon because it decreases the mechanical performance and the economic value of the rubber, was put to test by comparing the sol fraction experimentally obtained with the expected in the case of only-random or only-crosslinking scissions, according to the Horikx analyses. In all investigated cases the devulcanized samples lay between the two theoretical Horikx models, showing that both random scissions and severance of crosslinking occurred. However, it should be noted that all the experimental points are close to the crosslink scission model curve, and this is an index of the preferential crosslink scission mechanism during the devulcanization process.As for the devulcanization condition effects, it can be argued that the higher the thermal profile and the higher the rpm, the higher is the percentage of devulcanization. However, the quality of the devulcanized sample in terms of sol fraction and percentage of random scissions depends on the process parameters and the screw profile which affect the rheological properties and the residence time of the rubber in the extruder. Therefore, it is difficult to provide a general behavior for all the performed tests, because the different process parameters affect the process in different ways.%sol and %random obtained.Eventually the screw profile concurs to the efficiency of the devulcanization. The different number of kneading elements along the screw and more in general the screw profile composition affects the percentage of devulcanization, making in some tests the results more dependent on the screw speed. The water injection along the screw affects devulcanization by decreasing the rubber temperature. The water injection also affects the %sol and %random.This work enabled the evaluation of the characteristics of the devulcanized material obtained from the devulcanization process in a co-rotating twin-screw extruder, patented by Maris. This evaluation is only the first step related to the concept of circular economy and was useful to obtain information on the % of devulcanization and on the quality of the devulcanized material in terms of The final goal is, therefore, to obtain a new vulcanized compound produced from virgin material and devulcanized material in the highest possible percentage, so as to guarantee chemical–physical properties similar to virgin material, with the aim of replacing it for the same application which allows the reuse of waste material within the production chain. For this reason, there will be significant advantages in terms of environmental impact and cost effectiveness of the entire production process.However, it must be underlined that the devulcanized product could also be used as such (not vulcanized or vulcanized) or used in very high percentages with the virgin material, in all those applications that do not require specific chemical–physical properties.


In low-resource settings, antimicrobial resistance (AMR) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. The AMR Scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures.To evaluate the performance of the AMR Scorecard in 11 pilot laboratory evaluations in three countries also assessed with the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist.Pilot laboratory evaluations were conducted in Cameroon, Ethiopia and Kenya between February 2019 and March 2019. Assessors with previous SLIPTA and microbiology experience were trained. Assessors performed the laboratory assessments using the SLIPTA and AMR Scorecard tools.Weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. Safety had the highest mean performance score  while management review had the lowest  across all laboratories. The AMR Scorecard scores were generally consistent with SLIPTA scores. The AMR Scorecard identified technical weaknesses in AMR testing, and SLIPTA identified weaknesses in the quality management systems in the laboratories.Since the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA, it is recommended that microbiology laboratories use SLIPTA and the AMR Scorecard in parallel when preparing for accreditation. Expanding the use of the AMR Scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and AMR surveillance. Ensuring the quality of such services is a challenge because, in addition to the pre-analytical, analytical and post-analytical phases that take place within the laboratory, there are numerous other key drivers of overall diagnostic quality,10 including clinical question formulation and test selection, test ordering, sample collection and transportation to the laboratory, testing and results reporting, test results interpretation, and patient follow-up for clinical management or referral for further testing. Laboratories with weak systems have higher levels of errors, which can affect patient care and undermine the confidence that clinicians have in laboratory services.12 To address the coronavirus disease 2019 pandemic, laboratories are receiving new molecular and point-of-care technologies, thus increasing the number of samples processed and reinforcing the need for high-performing and high-quality laboratories and systems, particularly in LMICs.While recent advances in molecular methods to detect AMR are being increasingly implemented in high-income settings,15 However, only a few clinical microbiology laboratories in LMICs have achieved any form of accreditation, that is, a formal recognition that their quality management system (QMS) complies with international standards.18Significant advances have been made towards improving laboratory capacity and quality in disease areas such as HIV and tuberculosis. One example is the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) initiative developed by the United States Centers for Disease Control and Prevention in collaboration with the American Society for Clinical Pathology, the Clinton Health Access Initiative, and the World Health Organization Regional Office for Africa to promote the uptake of quality improvement initiatives in LMIC laboratories.Although the SLIPTA initiative has, to some extent, facilitated laboratory improvement in Africa, it may not specifically address the quality of processes in AMR laboratories, including sample culture, species identification, and susceptibility testing. While implementing QMS elements is critical, improving compliance with a technical standard of testing is equally important.19 which aims to improve the appropriate use of diagnostics to identify pathogens and guide patient treatment and management, and to optimise the surveillance and early detection of AMR. The AMR Scorecard focuses on priority specimen types such as blood, urine, and faecal samples, and includes the culture, pathogen detection, species identification, and antimicrobial susceptibility testing (AST) processes. The AMR Scorecard is designed to assess these technical processes for the priority pathogens reported to the Global Antimicrobial Resistance Surveillance System. This includes pathogens associated with hospital and community-acquired infections in which AMR is reportedly increasing, threatening the use of key drugs.5 In this article, we describe the performance of the developed AMR Scorecard during pilot laboratory evaluations in three countries.In recognition of the potential gap in the quality of AMR-related testing, we developed the AMR Laboratory Scorecard (AMR Scorecard),This article describes the performance of the AMR Scorecard during pilot evaluations and does not require ethical clearance. This article followed all ethical standards for research without direct contact with human or animal subjects.Pilot evaluations of the AMR Scorecard were conducted in Cameroon, Ethiopia, and Kenya between February and March 2019. The countries were selected based on their enrolment in the Global Antimicrobial Resistance Surveillance System and engagement with Becton Dickinson or Foundation for Innovative New Diagnostics (FIND), the global alliance for diagnostics, in ongoing laboratory strengthening activities. The laboratories in these five countries are representative of microbiology services across the diagnostic network from central level laboratories to district-level laboratories. Assessors were selected and trained on the use of the AMR Scorecard before performing the pilot laboratory assessments.20 and the United States Centers for Disease Control and Prevention.21The AMR Scorecard was developed based on the latest guidance and requirements for AMR testing obtained from a review of existing tools, checklists, and guidelines, including those of the Healthcare-Associated Infection Surveillance IndiaThe AMR Scorecard is based on the World Health Organization Regional Office for Africa SLIPTA Checklist version 2:2015 and incorporates clinical microbiology laboratory-specific requirements linked to sub-clauses in the SLIPTA checklist. It consists of three scorecard modules that are used to assess the technical procedures for processing blood, urine, and faecal samples. Assessment of technical procedures includes questions to determine if isolation procedures (e.g. ‘Are media used for primary culture of faeces incubated at 35 °C – 37 °C for at least 18 hours?’), identification procedures  and AST procedures  are being performed according to microbiology best practices.22 standard, the AMR Scorecard assessment compares technical laboratory practices against best practices for microbiology and AMR.The AMR Scorecard is designed to be used as a stand-alone internal assessment tool or as part of a comprehensive SLIPTA assessment to ensure the application of SLIPTA requirements to these test methods. The AMR Scorecard uses the same scoring convention and the same 12-section structure as the SLIPTA checklist. Individual AMR testing modules are scored according to the percentage of requirements met in each modular checklist. However, unlike SLIPTA, whose laboratory assessment is based on the International Standards Organization (ISO) 15 18922 The eTool consists of a general AMR testing spreadsheet for recording responses to questions common to all the technical modules, spreadsheets for information specific to faeces, urine and blood, a spreadsheet for previous audit information, and a summary spreadsheet with automated analysis and visualisation of the technical assessment scores. A spreadsheet corresponding to the 12 sections of SLIPTA is also provided to allow simultaneous SLIPTA evaluations. The summary spreadsheet with automated analysis provides a detailed overview and visualisation of the SLIPTA assessment scores.An AMR Scorecard eTool was developed to supplement the hard-copy technical modules and SLIPTA checklist and to allow automated analyses and reporting of the laboratory assessments.Trainee assessors who had microbiology experience and had participated in SLIPTA assessments in their laboratories (but were not necessarily African Society for Laboratory Medicine SLIPTA certified) were chosen to attend the training workshops. Training on the AMR Scorecard was conducted in Ethiopia from 11 to 13 February 2019, in Kenya from 25 to 26 February 2019, in Uganda on 18 February 2020, and in Ghana on 26 February 2020. Facilitators trained two assessors from Cameroon, eight assessors from Ethiopia, and six assessors from Kenya on the interpretation of AMR Scorecard questions, the use of the eTool, and the procedures for conducting the assessments. As all the trainee assessors were already familiar with the use of the SLIPTA checklist, training on SLIPTA was not provided.Theoretical training was supplemented by practical assessments of three facilities (two in Ethiopia and one in Kenya). The national or reference laboratories were chosen for the practical assessment to provide trainee assessors exposure to all the procedures evaluated using the scorecard . All thrBefore commencing the pilot assessments at the respective laboratories, assessors introduced the AMR Scorecard to the laboratory head and quality officer. All the assessments were conducted using the SLIPTA checklist and the AMR Scorecard and transferred to the eTool for further analysis.Assessors evaluated the laboratory operations based on the SLIPTA checklist and AMR Scorecard items, recording scores for each item and documenting findings in detail. During the assessment, assessors reviewed laboratory documentation to verify that policies, manuals, and standard operating procedures were complete, current, and accurate. They also reviewed records and observed laboratory procedures to verify that AMR policies were being followed and that laboratory procedures used were appropriate for the testing performed. In addition, the assessors determined the availability of functional and well-maintained equipment, reviewed data on the number of processed samples, isolates, contaminated cultures, and negative cultures for each sample type, and reviewed the internal quality control and external quality assessment results. These data provided assessors with an overview of the laboratories’ operations and allowed the identification of systemic technical issues not easily determined using SLIPTA alone.23 and non-conformities with microbiology best practices identified by the AMR Scorecard were tabulated and presented to the laboratory along with copies of the completed checklists.Following the assessments, the assessors provided feedback to the laboratory head, quality officer, and laboratory technologists. Non-conformities with the ISO 15189:2012 standard identified by SLIPTAThe results of the pilot laboratory assessments were transferred to the eTool, which then automatically calculated the scores and totals for each section and generated a bar graph of laboratory performance by section. The AMR Scorecard results for blood, faeces, and urine were analysed with the SLIPTA scores.In the pilot assessments conducted in this study, weaknesses in technical procedures and the QMS were identified in all areas and all laboratories . The meaBased on the AMR Scorecard assessments, all 11 laboratories performed best with all sample types in Section 12: Facilities and Safety . The weakest performance for all sample types in all laboratories was in Section 2: Management Reviews , followed by Section 11: Occurrence Management , and Section 6: Evaluations and Audits .Technical issues with the processing of all sample types  were identified in Section 8: Process Control and Internal and External Quality Assessment of the AMR Scorecard . In 10 of 11 laboratories, data on the number of isolated pathogens and cumulative AST patterns were not collected and reported to the relevant oversight committees such as the antimicrobial stewardship committee, or hospital surveillance or outbreak team.Technical issues with the processing of urine samples included failure to perform cell counts or wet preparations (6 of 11 laboratories), lack of rejection criteria (8 of 11 laboratories), lack of quality controls for media (4 of 11 laboratories), lack of antibiotic discs for AST (9 of 11 laboratories), and failure to use purity plates or standardised inocula for AST (7 of 11 laboratories).Salmonella or Shigella species (5 of 11 laboratories).Technical issues with the processing of faeces samples included failure to perform wet preparations for parasites (5 of 11 laboratories), shortage of Selenite F Broth and lack of sub-culture testing (8 of 11 laboratories), and failure to perform serological identification of either Technical issues with the processing of blood samples included failure to perform extended-spectrum beta-lactamase and carbapenemase detection tests (11 of 11 laboratories).As with the results of the AMR Scorecard assessments, the SLIPTA assessment also identified Section 12: Facilities and Safety to be the strongest area  in all the laboratories, followed by Section 3: Organization and Personnel  and Section 7: Purchasing and Inventory . The weakest area was Section 2: Management Reviews . Some reasons for low SLIPTA scores included failure to conduct regular management reviews or audits (6 of 11 laboratories), and failure to collect and analyse quality indicators (6 of 11 laboratories). Weaknesses specific to the QMS were identified using SLIPTA as these are not assessed by the AMR Scorecard. For example, the content of the quality manual (Documents and Records) was identified as a weakness in 7 of 11 laboratories.The assessment scores obtained using the AMR Scorecard and SLIPTA were disaggregated by laboratory area and laboratory level –4. Two lAt least one laboratory performed poorly at each level. At the central level, Laboratory A had the lowest performance, with a mean AMR Scorecard assessment score of 30% and a mean SLIPTA score of 35%. At the regional-level, Laboratory A had a mean AMR Scorecard assessment score of 6% and a mean SLIPTA score of 8%. At the district-level, Laboratory A had a mean AMR Scorecard assessment score of 15% and a mean SLIPTA score of 17%. As these results suggest, the overall AMR Scorecard scores were similar to the overall SLIPTA scores, irrespective of the laboratory performance. However, differences between the SLIPTA and AMR Scorecard assessment scores in the different areas of the laboratory were noted at all laboratory levels. At the central laboratories, the largest differences between the AMR Scorecard and SLIPTA scores occurred in Section 2: Management Reviews (0% vs 25%), Section 9: Information Management (42% vs 63%) and Section 10: Corrective Action (10% vs 39%). At the regional laboratories, the largest differences between the AMR Scorecard and SLIPTA scores occurred in Section 1: Documents and Records (33% vs 59%), Section 2: Management Reviews (10% vs 36%), Section 9: Information Management (22% vs 60%), and Section 11: Occurrence Management (7% vs 50%). Finally, at the district laboratories, the largest differences between the AMR Scorecard and SLIPTA scores occurred in Section 1: Documents and Records (26% vs 47%), Section 4: Client Management (17% vs 40%), and Section 7: Purchasing and Inventory (18% vs 48%). Where there were large differences (> 20%) in the scores, the SLIPTA score was always higher.24 While implementing QMS elements in the microbiology laboratory is critical, improving compliance to a technical standard of testing is equally important. The AMR Scorecard provides laboratories with a specific technical assessment tool for strengthening the quality of culture, identification, and AST laboratory procedures.Quality clinical microbiology services are an essential element of the AMR response that enable the appropriate use of antibiotics, improve AMR surveillance, and reduce the development of resistance.25 in both the AMR Scorecard and SLIPTA assessments, Section 12: Facilities and Safety was found to be the strongest area in the laboratories. This focus on safety, even in laboratories with weak systems , is encouraging.In the pilot assessments conducted in this study, the AMR Scorecard scores generally correlated well with the SLIPTA scores. Safety in the laboratory has been identified as an increasingly important subject because of the emergence of highly infectious diseases, including coronavirus disease 2019. Although safety has traditionally been regarded as a low-priority issue in developing countries,26 Overall, six laboratories (55%) received zero stars (< 55%) using the official SLIPTA system, with only two laboratories (18%) scoring two stars  and three stars . The SLIPTA scores in this study are consistent with the SLIPTA scores from 47 countries worldwide, including 23 countries in Africa, assessed using the Strengthening Laboratory Management Toward Accreditation methodology prior to the initiation of laboratory strengthening activities (i.e. at baseline). Yao et al.16 found that the mean score in these laboratories at baseline was 39% (median 37%), with 84% of the laboratories scoring zero stars (i.e. score < 55%). It has been suggested that microbiology laboratories are trailing other clinical laboratories in achieving accreditation.27The SLIPTA assessments identified weaknesses in the QMS of the microbiology laboratories assessed. In 6 of the 11 laboratories, weaknesses identified included failure to conduct regular management reviews or audits. These findings are consistent with previous reports that some of the weakest areas in the laboratory are internal auditing and the collection of quality indicator data.The overall poor performance recorded at each laboratory level suggests that providing quality microbiology services is a challenge across the tiered network. The exclusion of national or reference microbiology laboratories in the assessments likely resulted in the lower overall scores observed in the pilot. While national or reference microbiology laboratories were assessed using the AMR Scorecard during the training, SLIPTA assessments were not conducted due to time constraints and thus the AMR Scorecard results were not included in this report. In settings with limited resources, strengthening technical testing and QMS (including accreditation) is often initiated at the national level, suggesting that overall scores may have been higher had they been included.28By providing a specific technical focus, the AMR Scorecard identified important gaps in AMR technical testing not detected by SLIPTA alone. The AMR Scorecard assesses the step-by-step procedures for sample processing, bacterial isolation and identification, and AST. Approximately 44% of the AMR Scorecard focuses on these procedures in contrast to SLIPTA which has a limited focus on technical procedures. Topics covered by SLIPTA are also covered by the AMR Scorecard but the latter focuses on the specific details related to AMR. For example, SLIPTA assesses whether standard operating procedures for laboratory functions and technical and managerial procedures are available, while the AMR Scorecard assesses whether the laboratory has, for example, documented procedures for microscopic examination and urine cell count. As the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA alone, and SLIPTA identified specific weaknesses in the QMS that were beyond the scope of the AMR Scorecard, it is recommended that microbiology laboratories that require a comprehensive assessment or are developing their QMS through continuous improvement toward accreditation be assessed using both the SLIPTA and the AMR Scorecard in parallel. Thus, the AMR Scorecard can be used as an entry point into the QMS journey, with laboratories choosing to apply for SLIPTA certification (or ISO certification) after reaching a satisfactory level . It should be noted that the official star recognition system provided by African Society for Laboratory Medicine can only be obtained through the SLIPTA certification provided by the African Society for Laboratory Medicine Secretariat.5 cells/mL?’) and assessors are encouraged to assist laboratories to collect and analyse their data. The clinical laboratory is a major source of healthcare data that can be used to inform health system-wide actions meant to improve diagnostic test utilisation, service efficiency, and patient outcomes.29The importance of data collection and analysis is also highlighted in several questions in the AMR Scorecard (e.g. ‘Are the following performance indicators collected – Number and percentage of urine cultures with cell counts > 10In these evaluations, cumulative quality indicator data on isolated pathogens and AST were not collected by 10 of the 11 laboratories assessed. This was due to the lack of automated instruments or an electronic laboratory information system, meaning that staff were required to manually record and calculate isolation rates and AST patterns. In one laboratory, the compilation of data by the assessors exposed a very low isolation rate of enteric pathogens that required further investigation. Quality indicator data, if available, could have been used to identify the cause of the low isolation rate, thereby improving the quality of enteric bacterial culture.In addition to improving the quality of laboratory testing, laboratory data can also be used to influence clinical decisions. For example, cumulative data on pathogens and AST results can be used to inform treatment guidelines. However, for laboratory data to impact health systems in such a way, the laboratory needs to carefully consider how the data are collated, communicated, and disseminated. Data from the assessments in this pilot revealed that 10 of the 11 laboratories failed to collect data on cumulative AST patterns and report these data to oversight committees, thereby missing the opportunity to inform antimicrobial stewardship decisions with laboratory data.15 Only 2 of the 11 laboratories reported a previous SLIPTA assessment. It is expected that laboratories participating in quality improvement programmes are more likely to score higher on both assessment tools. Second, the AMR Scorecard is based on microbiology best practices and not on an ISO standard such as SLIPTA. Without a standard to guide preparations, it may not be clear to laboratories what requirements need to be in place to assure quality AMR testing. In this respect, the AMR Scorecard has a role to play in educating laboratories regarding the technical requirements for AMR testing.In the pilot assessments, where there were differences between the AMR Scorecard assessment and SLIPTA scores, the SLIPTA scores were consistently higher. In addition to the differences in the content of the two assessment tools, other factors may contribute to this finding. First, the laboratories included in the pilot were not part of any active and ongoing programmes to improve laboratory quality such as the Strengthening Laboratory Management Toward Accreditation programme.30 thus, it is important to ensure that enough resources in terms of funding and personnel are put in place to allow mentoring to take place. Ideally, laboratories should be mentored by reference laboratories within the AMR surveillance network.There were several challenges to implementing the AMR Scorecard in the initial cohort of laboratories, including procurement of funding support for quality improvement and provision of cover for the trainers and mentors during programme-related absences. Mentoring of laboratories has been reported to be an important component of successful and sustainable quality improvement initiatives;Based on feedback from facilitators and assessors, the AMR Scorecard needed to be revised to strengthen identified weaknesses. Suggested changes included revising the language of some questions and adding ‘Not Applicable’ options to others. Two additional questions to determine whether laboratories were performing extended-spectrum beta-lactamase and carbapenemase screening on faecal samples were added to the faeces module. This increased the score of the faeces module by four points. The total score of the blood and urine modules remained unaltered in the revised AMR Scorecard.In 2020, the structure of the AMR Scorecard was changed, and additional scorecards were added to allow for assessments of other sample types including pulmonary, genital, and wound samples. Evaluations of the revised AMR Scorecard were performed in three laboratories in Ghana and Uganda.The microbiology laboratories selected for the pilot of the AMR Scorecard do not represent the scope of microbiology technical abilities across Africa. While care was taken during the selection of laboratories for the study, the AMR Scorecard may be less useful in identifying and addressing gaps that impact the quality of testing in certain settings. In addition, all assessments, except those performed in Cameroon, included FIND and Becton Dickinson facilitators. As these facilitators were involved in the development of the AMR Scorecard, they may have influenced the outcomes of the assessments in favour of the AMR Scorecard.This study showed that a customised scorecard to guide the establishment and strengthening of AMR testing quality in resource-limited settings can assist in identifying and addressing quality gaps. The AMR Scorecard, used in conjunction with SLIPTA, found important gaps in the procedures for identification and AST of priority pathogens that were not identified by SLIPTA alone. Expanding the use of this scorecard will help address the need for quality clinical microbiology laboratory services to support optimal patient care and AMR surveillance.


Research on aggregation-induced emission (AIE) has attracted much attention since the concept of AIE was coined in 2001 . AggregaPengfei Zhang et al. explores a double-checked Logic Gate system comprised of both CRISPR/Cas12a and DNA-binding AIE molecule for the detection of pathogenic microorganisms. The presented AIE molecules are non-emissive in an aqueous solution but emit intensively in the presence of a large amount of DNA produced by recombinase polymerase amplification (RPA) within a short time period, with an extremely high targeting-specificity via a restriction of intramolecular motion mechanism. This finding is useful for detecting pathogenic microorganisms in environmental water samples.Biosensing and bioimaging represent important applications of molecular aggregates, in which enhanced emission can be achieved upon the formation of molecular aggregates or binding molecules with biomolecules in the surrounding bio-environment. The collaborative study led by Cheng et al. reviewed the 3D fibrous aerogels for solar vapor generation (SVG) based on building block synthesis, photothermal materials selection, porous structure construction, and device design. In this study building blocks based on natural cellulose nanofibers, petrochemical polymer nanofibers, and inorganic nanofibers were introduced to fabricate aerogels. Plasmonic nanometals, semiconductors, carbonaceous polymers, MXene, and AIE molecules are selected as photothermal materials. To improve the evaporation efficiency, the porous structures of these building blocks and photothermal materials are crucial factors. For one of the applications, water purification, the SVG-based technology shows advantages for both large-scale and personal use with inexhaustible solar energy, independent of electricity and complex infrastructure. At the end of the review, they propose three strategies to further promote SVG utility, such as tailoring the hydrophilicity of the fibrous building blocks to increase the interaction with bonded water, multifunctional SVG with anti-biofouling activity, photocatalysis, and energy transformation property, exploring simple fabrication methods and low-cost starting fiber materials.Zhu. Interestingly, unlike the conventional AIEgens with conjugated aromatic structure, they possess a non-aromatic chiral central ring (tetrahydropyrimidine), which is attached by three completely non-crowded and freely rotatable phenyl rings. Although non-successive conjugation is found in these AIEgens, the through-space conjugation between the isolated phenyl rings can induce a remarkable emission in the visible light region. Their molecular conformation is flexible and thus, the RIM can be explained by the AIE behavior. Moreover, the AIEgens can be readily obtained with different substituents which allow for tuning the AIE effect, emission wavelength, luminescent efficiency (up to 100%), packing model, and polymorph in the aggregate state. The collective properties enable them to be competent in the applications of critical micelle concentration (CMC) fluorescent detection, long-term cell imaging, fluorescence on-off thermometer, mechanofluorochromism, and copper (II) probe. This unusual structure with interesting properties will likely inspire more studies in the future.A new class of AIEgens of racemic C6-unsubstituted tetrahydropyrimidines (THPs) are reviewed by Zheng et al. review recent works investigating the structure-property relationships of organic molecular aggregates in different environments, including crystal, co-crystal, amorphous aggregate, and doped systems, by the multiscale modeling protocol. Different molecular models were summarized according to the relevant micro-environments: 1) the implicit solvation model was used to consider the solvent effect; 2) the hybrid quantum mechanics/molecular mechanics (QM/MM) model was chosen to consider the influence of molecular packing on photophysical properties; 3) molecular dynamics simulation was applied for the complicated conformations of amorphous aggregates. This work systematically discussed the influence of intermolecular noncovalent interactions on molecular packing and their photophysical properties. It could therefore provide powerful guidelines for the design, synthesis, and development of advanced organic luminescent materials.


A woman in her 20s with a history of systemic lupus erythematosus controlled with azathioprine and low-dose prednisone presented at ten weeks of pregnancy after developing pruritic, painful facial lesions over three weeks. She had no history of rosacea, topical corticosteroid use, or recent fevers, but was wearing masks frequently. On examination, she appeared non-toxic and had numerous coalescing erythematous papules and pustules with yellow-gray crusting around her nose, mouth, and glabella Fig. . The verA. Pyoderma facialeB. ImpetigoC. Acute lupus flareD. Pyostomatitis vegetansE. Amicrobial pustulosis associated with autoimmune disease (APAD)Correct answer: A. Pyoderma faciale. Disease is characterized by numerous erythematous nodules, papules, and pustules erupting on the central face.She was diagnosed with periorificial pyoderma faciale. With her obstetrician’s approval, we initiated a three-month course of cephalexin, in line with standard treatment durations for rosacea. At two months follow-up, there was near-resolution.1 Approximately 10% of cases occur during pregnancy, and around 40% can be triggered by menopause, medication changes, or emotional stressors.1 Patients present suddenly with tender coalescing inflamed pustules, nodules, draining sinuses, and cysts/abscesses on the central face.2 Our patient presented with numerous pustules, with the additional and unique feature of thick, confluent, yellow-gray crusting. To our knowledge, prior reports of patients with crusted papulopustules have not shown such exuberant facial crusting.3 The reasons for our patient’s dramatic presentation are unclear. Possibly, her underlying systemic lupus erythematosus (SLE), stress and altered immunity during pregnancy, and mask wearing promoted vascular hyperreactivity and inflammation predisposing to development of pyoderma faciale.2Pyoderma faciale, also known as rosacea fulminans, is a rare inflammatory facial dermatosis most commonly affecting women between 20 and 40 years old with a history of flushing or rosacea.Pyoderma faciale can mimic infectious and inflammatory processes. In our patient with SLE on immunosuppression, the differential diagnosis included infections such as impetigo, an acute lupus flare, pyostomatitis vegetans, or an atypical facial presentation of amicrobial pustulosis associated with autoimmune disease (APAD). Given the patient’s pregnancy status, morphology of lesions with centrofacial distribution and sparing of the vermilion border, non-toxic appearance, stable SLE and negative infectious work-up, pyoderma faciale was felt to be the best diagnosis. Notably, the perioral and perinasal distribution of the rash resembled periorificial dermatitis, which is on the rosacea spectrum, much like pyoderma faciale.1 Recently, erythromycin monotherapy has shown promising results in pregnant individuals.1 Our case provides evidence that a robust presentation of pyoderma faciale with a periorificial distribution can be treated safely and successfully with cephalexin monotherapy during pregnancy.Typically, pyoderma faciale is treated initially with systemic corticosteroids to rapidly reduce inflammation, followed by longer courses of isotretinoin or tetracyclines to maintain improvement. In our patient, the latter 2 treatment options were contraindicated due to pregnancy. Beta-lactam antibiotics can improve pyoderma faciale, although they often fail to achieve resolution as monotherapy and are typically given in conjunction with corticosteroids or isotretinoin.A. Atopic dermatitisB. AcneC. Rosacea/flushingD. ImpetigoE. FolliculitisCorrect answer: C. Rosacea/flushing. Walsh et al.1 demonstrated that history of rosacea/flushing is present in 80% of cases of pyoderma faciale.None.None.Raymond Zhao contributed to the drafting of the manuscript, figure design, and background research. Severine Cao and Frank Wang contributed to the conception and overall planning of the project, supervision of the project, and critical revision of the manuscript.N/A.


The therapeutic landscape for patients with non-small cell lung cancer (NSCLC) has dramatically evolved with the development and adoption of immune checkpoint inhibitors (ICI) as front-line therapy. These novel antibodies target the interactions in immunoregulatory pathways, between programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), or cytotoxic T-lymphocyte antigen 4 (CTLA-4) and B7, resulting in the activation of T cells and cytotoxic response to induce an immunologic response. ICIs have demonstrated significant survival benefits and sustained responses in the treatment of NSCLC leading to the long-term survival of up to 5 year. One unusual response to ICI is a phenomenon termed Hyperprogressive Disease (HYD), which occurs in a subset of patients for whom ICI therapy can induce rapid disease growth, which ultimately leads to poorer outcomes with an incidence rate ranging from 5 to 37% in NSCLC patients. Prior reviews demonstrated that HYD can be defined by rapid tumor progression, deterioration of patient’s symptoms or new onset of disease. The mechanism of HYD could be related to genomic and tumor microenvironment changes and altered immune response. It will be important to establish a common definition of HYD for future research and clinical care. The therapeutic landscape for patients with non-small cell lung cancer (NSCLC) has dramatically evolved in the last several years with the development and adoption of immune checkpoint inhibitors (ICI) as front-line therapy. These novel antibodies target the interactions in immunoregulatory pathways, such as those between programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), or cytotoxic T-lymphocyte antigen 4 (CTLA-4) and B7, resulting in the activation of T cells and cytotoxic response to induce an immunologic response in several solid tumor types –4.One unusual response to ICI is a phenomenon termed Hyperprogressive Disease (HYD), which occurs in a subset of patients for whom ICI therapy can induce rapid disease growth, which ultimately leads to poorer outcomes . Existin61-year-old white female presented on 3/17/2022 with several months of cognitive changes (including confusion) and gait instability. CT of the head done on 3/17/2022 showed a large frontal lobe mass measuring 3.2 x 2.6 cm with extensive adjacent edema of the left frontal and parietal lobes, midline shift of 7 mm, and marked compression/distortion of the left frontal horn of the left lateral ventricle. The patient was admitted to the neurosurgery service and CT chest/abdomen/pelvis on 3/18/2022 showed a 2.1 cm nodule in the medial azygos lobe of the right upper lobe, compatible with primary lung ca. There were several smaller irregular ground glass and nodular opacities in the left lower lobe . She also had mild mediastinal and right hilar lymphadenopathy, but no abdominal pelvic metastatic disease. The patient underwent endobronchial ultrasound biopsy and bronchioalveolar lavage on 3/21/2022. Fine Needle Aspiration of the right hilar mass showed poorly differentiated carcinoma pulmonary non-small cell carcinoma. PD-L1 TPS was 40%. MRI of the brain done on 3/22/2022 showed enhancing anteromedial left frontal cerebral cortical nodule, indicating solitary cerebral metastasis, marked associated left anterior cerebral vasogenic edema, and mild rightward frontal midline shift. The patient underwent a left frontal craniotomy on 3/23/2022 and pathology showed poorly differentiated carcinoma consistent with metastasis from lung adenocarcinoma primary. PD-L1 TPS was 1 to 2%. NGS testing showed mutation of TP53, BRAF L597Q (not V660E), STK-11, PDGFRA, KMT2D, ZNF217, and RNA testing was negative for an actionable mutation. She was discharged on 3/25/22 and underwent post-operative radiation to the surgical bed from 4/12/22 to 4/25/22. She tapered off Decadron and enrolled in a trial randomized to Pembrolizumab single agent which started on 5/13/22. After 2 cycles of therapy, the patient developed deterioration of her performance status, and required hospital admission 6/30/22. The CT scan after 2 cycles done on 6/23/22 showed significant progression of her disease Figure 1HYD is generally described as unexpected, accelerated tumor growth after treatment with ICI therapy . Early aDespite increased recognition of the hyperprogressive phenomenon, there is a lack of a unifying definition of this process. Several previous studies have sought to define HYD across a broad range of tumor types. Definitions thus far largely have been categorized into time-dependent criteria or size/clinically dependent criteria.Examples of time-dependent criteria employed in the literature include tumor growth rate (TGR) and tumor growth kinetics (TGK). TGR calculation involves the difference (or ratio) of 3-dimensional tumor volume per month, related to the sum of the target lesion(s) diameter(s) as well as the time between imaging evaluations . Tumor gChampiat et al. (2017) were the first to collectively describe and define HYD in solid-tumor patients who were treated with immunotherapy . This reA later study by Singavi et al. incorporated a similar definition to the criteria set out by Champiat, including a TGR increase of two-fold or higher per RECIST 1.1 criteria, with an additional requirement of RECIST 1.1 tumor size increase of 50% or higher . EventuaAdditional data from Kato et al. 2017) reviewed 155 patients with advanced solid tumors who received immunotherapies and had their tumors evaluated by next-generation sequencing. This study defined HYD as a TTF of fewer than 2 months, a 50% or higher increase in tumor burden compared to pretreatment imaging, and a 2-fold or higher increase in progression pace . Notably reviewedThe heterogeneity in definitions of HYD has real-world implications in the current diagnosis of HYD. A recent retrospective cohort study evaluated 406 patients with NSCLC, analyzing the incidence and outcomes in a single population of patients with HYD as defined by five different, established definitions per previous trials. The data revealed a variance in reported incidence (5.4%-18.5%) of HYD, with concordance between definitions ranging from 33.3% to 69.3% . Indeed,Beyond HYD, other patterns of progression on ICI have been described in the literature. Gandara et al. described fast progression (FP) and early death (ED) in a retrospective evaluation of the OAK study. FP was defined on size-based criteria (50% or greater increase in the sum of largest diameters of target lesions per RECIST 1.1 criteria) and did not require pre-baseline assessment. ED was defined as death due to disease progression within 12 weeks from baseline in patients without a response assessment . FurtherThe use of parameters such as the TGR or TGK allows for the evaluation of tumor kinetics as guided by tumor size. Of note, the TGK does not involve a three-dimensional evaluation of tumor size, which may lead to some overestimation of the incidence of HYD , 15, 18.Size or clinically dependent criteria require pre-baseline imaging but do require dynamic data regarding tumor momentum in growth, i.e., RECIST criteria measuring size  or reliaMatos et al. used RECIST and defined HYD as a progression of disease within the first 8 weeks after treatment with ICI, an increase of a minimum of 10mm and addition to increasing≥ 40% in the sum of target lesions compared with baseline (double of the RECIST 1.1 definition of progression) and/or increase of ≥ 20% in the sum of target lesions compared with baseline and the appearance of new lesions in at least 2 different organs. In this study, they analyzed 287 patients treated with ICI monotherapy or in combination. HYD by RECIST definition occurred in 10.7% of patients representing 27.1% of patients with disease progression. Their outcome was worse with median overall survival (mOS) of 5.23 months vs. 7.3 months without HYD .Furthermore, size- or clinical-dependent criteria may be easier to implement in the real-world setting and possibly in clinical trials. However, these evaluations cannot describe the rates or speed of tumor growth inherently associated with time-based evaluations, and thus distinguishing between natural disease progression and HYD remains difficult . A limitFuture implementation of early disease assessments and integrating time-based tumor kinetic evaluation will be crucial in identifying those with HYD. A proposed set of parameters as the definition of HYD based on the review of the literature is shown in via oncogenic driver mutations , may lead to IFN-γ-dependent recruitment of immunosuppressive myeloid-derived suppressor cells . Inflammvia proteasome  score greater than 1, Royal Marsden Hospital (RMH) score of two or higher, serum LDH greater than the upper limit of normal, more than two metastatic sites, and presence of liver metastasis , with another study by Kim et al. showing an mOS of 50 days in patients with HYD (vs. 205 days in those without) . In the Since the advent and adoption of ICI therapy in the treatment of advanced NSCLC, multiple studies have shown significant improvements in outcomes for these patients , 4, but CHH review and editing of manuscript, conceptualization of the review, tables and figures, and literature review. AB literature review, draft of manuscript and literature review. CH review and editing, creation of figures. All authors contributed to the article and approved the submitted version.Professional CME funds.CHH is an investigator in clinical trial funded by Genetech, BMS, Amgen, RRX, Pfizer, Exelixis, Incyte, Sanofi and Mirati.The remaining 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.


Antimicrobial resistance (AMR) is a crucial and emerging multifactorial “One Health” problem involving human and animal health, agriculture, aquaculture, and environment; and posing a potential public health hazard globally. The containment of AMR justifies effective surveillance programs to explicate the magnitude of the problem across the contributing sectors. Laboratory-based AMR testing and characterization is the key component of an AMR surveillance program. An AMR surveillance program should have a “top management” for fund mobilization, planning, formulating, and multilateral coordinating of the surveillance activities. The top management should identify competent participating laboratories to form a network comprising a reference laboratory and an adequate number of sentinel laboratories. The responsibilities of the reference laboratory include the development of standardized test methods for ensuring quality and homogeneity of surveillance activities, providing training to the laboratory personnel, and in-depth AMR characterization. The sentinel laboratories will take the responsibilities of receiving samples, isolation and identification of microbes, and initial AMR characterization. The sentinel laboratories will use simple antimicrobial susceptibility test (AST) methods such as disk diffusion tests, whereas the reference laboratories should use automated quantitative AST methods as well as advanced molecular methods to explicit AMR emergence mechanisms. Standard guidelines set by Clinical Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing, should be followed to bring about conformity and harmonization in the AST procedures. AMR surveillance program in animals is eventually similar to that in human health with the exception is that veterinary antibiotics and veterinary pathogens should be given preference here. Hence, the review study was envisaged to look deep into the structure of the AMR surveillance program with significance on laboratory-based AMR testing and characterization methods. Antimicrobials are small molecules that can inhibit or kill bacteria, but some bacteria can grow and survive despite antimicrobial pressures, a property known as antimicrobial resistance (AMR). Antibiotics are being used for several decades to tackle infections by pathogenic microorganisms in humans, animals, and plants . AlongsiGlobal and national concerned organizations have categorized the AMR issue as an imminent hazard and have unanimously agreed that tracking the emergence and prevalence of AMR is crucial to minimize the threat to public health ,6. RecogLaboratory-based AMR surveillance starts on the receipt of samples (considering acceptance/rejection criteria) in the laboratory and involves isolation of bacteria and performing antimicrobial susceptibility tests (ASTs). Among the ASTs, disk diffusion and broth dilution methods are usually used in most laboratories for phenotypic AMR characterization . Along wHence, the review study was envisaged to look deep into the structure of the AMR surveillance program with significance on laboratory-based AMR testing and characterization methods.Google Scholar, PubMed, ResearchGate, and Crossref databases. Our research question was “what are the methods used for AMR surveillance in animals?” and the databases were searched using the phrases “AMR surveillance in animals,” “Methods of AMR surveillance in animals,” “Animals and/AMR surveillance.” Searches were filtered for research or review articles published in the English language from January 2011 to June 2021. Grey materials/unpublished documents were searched using Google and retrieved from the relevant institutional websites. The articles describing sampling procedure, AST methods, and molecular methods for AMR characterization were considered for inclusion in the review, whereas articles on AMR surveillance methodologies in humans, agriculture, aquaculture, and environment were excluded from the study.We conducted literature searches in The selection of competent laboratories to form a network is crucial for the successful implementation of AMR surveillance programs in animals. This network should include regional or sentinel as well as one or more reference laboratories . There sAll possible animal species should be included in the AMR surveillance program to achieve the general objectives of establishing baseline data on the prevalence of drug-resistant bacteria among animals. The animal species usually targeted for inclusion in AMR surveillance programs are divided into companion animals, performance animals, food production animals, exotics, and wildlife . Among tEscherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Acinetobacter baumannii, Salmonella spp., Shigella spp., and Neisseria gonorrhoeae [Staphylococcus pseudintermedius is the major companion animals associated bacteria requiring monitoring for AMR. For AMR surveillance in cattle, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni are included traditionally in surveillance programs [Salmonella spp. and Campylobacter spp. For monitoring of AMR in healthy livestock and poultry, surveillance is usually conducted in indicator bacteria as these organisms are ubiquitously distributed in nature, food, animals, and humans and reflect AMR characteristics arising from selective pressure across these environments. Enterococcus spp. and E. coli are as such indicator bacteria commonly included in AMR surveillance programs in animals [WHO has prioritized a list of pathogens for antimicrobial susceptibility testing in the AMR surveillance program among humans which are orrhoeae . Howeverorrhoeae ,21,24-27 animals . The selEnterococcus spp. and E. coli; and nasopharyngeal swabs for M. haemolytica as well as other respiratory bacterial species [For surveillance programs in healthy livestock feces samples are collected to monitor AMR in  species ,30. AMR  species . Clinica species .A statistically valid number of farms, pens, or individual animals are randomly selected to enroll in the program depending on the AMR surveillance strategy. Alternatively, 30% of farms or pens in a defined area may be selected for sampling, and within each farm or pen 10% of all animals may be randomly sampled for AMR surveillance. Enrolled farms, pens, or individual animals are usually sampled once or more than once over the course of the study ,33.Fecal samples are collected per-rectum in the case of an individual animal. A minimum of 4 g of feces is placed in a vial containing modified Cary Blair transport media. Composite fecal samples are collected from floors of pens using a sterile plastic spoon by placing approximately 0.5-1 g of feces into a sterile plastic container. The composite sample is mixed thoroughly, and approximately 4 g of feces from each container is then transferred into a vial containing modified Cary Blair transport media. Nasopharyngeal samples are collected from the deep pharynx using commercially available sterile swabs . After cThe collected samples shall be properly labeled with the date of collection, farm or pen number, and the individual ID number for each sample . The samBased on the type of samples, different recovery methods can be used for the isolation and ID of bacteria. On arrival at the laboratory, the samples must be processed following a standard protocol for the recovery of the target bacteria . The refThe isolated bacterial strains are preserved in 20-30% glycerol at –80°C temperature until further phenotypic and genotypic AMR characterization is conducted . IsolateAntimicrobial drugs are selected in AMR surveillance programs depending on their present and history of usage in animals, clinical and epidemiological importance, target organisms, as well as the previous reports on the development of resistance against the drugs. Reports on the evolution of new mechanisms of resistance along with the development of resistance against new drugs also influence the selection of antimicrobials. The future concern of the drugs, specifically critical importance to human health, is also an inclusion criterion in surveillance programs ,32. The AMR surveillance in animal health currently relies largely on the isolation of indicator and clinical microorganisms from livestock, environmental, and food samples followed by the phenotypic AMR characterization of the isolates . This cut® , and various commercially available semi-automated and automated systems /mL for E. coli. Inoculum is then spread across MHA plates to form bacterial lawn, followed by placement of the antibiotic-impregnated disks on top of the bacteria. Plates are then incubated overnight, usually at 35°C [Disk diffusion methods measure bacterial growth inhibition zone around paper disks impregnated with a specific concentration of the target drug on agar plates. The Kirby–Bauer disk diffusion assay is widely used to characterize AMR for bacterial isolates in surveillance programs . Disk di at 35°C .The antibiotics diffuse from the disks and form a gradient with the target antibiotic compound into the agar. The inoculated bacteria will grow on the agar to an extent to which the drug is concentrated enough to inhibit the growth. The diameter of the inhibition zone is measured in millimeters either manually or using an automated machine and then is compared with standardized CLSI or EUCAST interpretive criteria to characterize the isolate as sensitive (S), intermediate (I), or resistant (R) to the antibiotic .Quantitative phenotypic AST methods are used to determine the MIC of antibiotics against bacterial isolates. MIC is the lowest concentration of an antibiotic that prevents the visible growth of bacteria and is often expressed in micrograms per milliliter or milligrams per liter .In broth dilution methods, a single bacterial isolate is incubated at 35±1°C for 18-24 h with sequential 2-fold dilutions of the target antibiotics . This te5 CFU/mL. For obtaining the desired bacterial concentration, a suspension of 0.5 McFarland is prepared first and diluted 100× to reach a density of 106 CFU/mL by adding 0.1 ml 0.5 McFarland suspensions to 9.9 mL broth. Finally, an equal volume of bacterial inoculum is added with an equal volume of a liquid medium with antibiotics in test tubes (macro-dilution) or wells of a microtiter plate (micro-dilution) [For bacterial inoculum preparation in broth dilution methods, morphologically similar colonies cultured overnight on a nonselective solid medium are suspended in MHB medium and adjusted to 5×10ilution) .4 CFU/spot by diluting the 0.5 McFarland suspension 10× in NACL or MHB and spotting 1 mL of such suspension on the MHA media with appropriate antibiotic dilutions [The agar dilution method is similar to broth dilution in principle with the exception that; two-fold serial dilutions of the target antibiotic are added to melted agar before solidification. The inoculum is prepared to obtain a final concentration of 1×10ilutions . The preilutions .In both broth and agar dilution methods, dissolutions of antibiotics are also required for MIC determination. Several types of solvents such as water for most beta-lactams, fluoroquinolones, and aminoglycosides; alcohol for macrolides, chloramphenicol, and rifampicin; dimethyl sulfoxide for carbapenems are used for preparing stock solutions of antibiotics .® is an assay for simultaneous determination of antimicrobial sensitivity as well as MIC value [® can be used to test multiple antibiotics per plate against only one organism and the time required to yield results is comparable to other agar diffusion or dilution methods [EtestIC value . This coIC value . Etest®  methods .® (bioMérieux), MicroScan® , and Phoenix™ . These systems are reliable, easy to use, and can be integrated with laboratory information management systems (LIMS) [Uncertainties in result interpretation, labor intensiveness, and long AST profiling time have driven the laboratories to use automated systems to determine antibiotic susceptibility profiles. There are several commercial automated MIC determination systems are currently available. Among these, the following four systems have been approved by FDA: Sensitire™ , Vitek 2s (LIMS) .Sensitire™ is based on the broth microdilution method and the actual detection of bacterial growth can be read through a fully automated approach using fluorescence technology. The specific enzymes produced by the organism over the overnight incubation period cleave the bond between the fluorophore and the quencher substrate; release the fluorophore to emit fluorescence . The amo® system utilizes the broth microdilution technique and is used simultaneously for bacterial ID and AST profiling [8 CFU/mL; henceforth, the vial is coupled with an AST card, scanned, and placed into the VITEK® system. The suspension is finally diluted automatically to 5×105 CFU/mL, loaded into the AST cards, sealed, and incubated within the VITEK® system. The bacteria are incubated for 18-24 h and yeast for 36 h with periodic growth monitoring. The MIC values are calculated from the OD values in individual wells arising from bacterial growth or no growth, and a MIC table for different antibiotics. A report, along with its interpretation, is generated automatically by the system [Vitek 2rofiling . This syrofiling . For thee system .BD Phoenix™ is a microdilution-based automated system used for simultaneous bacterial ID and antibiotic susceptibility testing . The sys® is also an automated system for bacterial ID and AST based on broth microdilution method and capable of medium and large-scale operations using 40 and 96-panel modules, respectively. Like other automated systems, test assays are prepared by rehydrating dried antibiotics and media in the wells by inoculating the bacterial suspension. Test assays are incubated for 4.5-18 h and growth or no growth of bacteria in individual wells are determined by colorimetric readings. The threshold concentration for bacterial detection in this system is 2×107 CFU/mL [Micro-scan walk away7 CFU/mL .Quality control should be carried out in each of the phenotypic AST methods following standardized SOPs, controlling medium sterility, using reference strains, and obtaining quality test results. The reference bacterial strains recommended by either CLSI or EUCAST must be used in each test  29,45,4,56,57. TPhenotypic methods for AMR determination are applicable only to the cultivable microbes and incapable of interpreting the mechanisms of emergence and spread of AMR in diverse and complex microbial communities where large fractions are uncultivable bacteria. Genotypic methods can be applied to overcome this limitation and are applicable to both cultivable and uncultivable bacteria. Bacterial AMR is usually genetically encoded and these genetic determinants can be identified and characterized by PCR and gene sequencing technologies .vanA (encoding vancomycin resistance), mecA (encoding methicillin resistance), ampC (encoding ampicillin resistance), mcr-1 and mcr-2 (encoding colistin resistance), ndm-1 (encoding carbapenems resistance), ESBL genes blaTEM, blaSHV, blaCTX-M, blaOXA, blaVIM, blaNDM, and blaKPC, etc. [PCR assay is being used to detect AMR genes in microorganisms through a target-based approach. This method enables the rapid detection of previously known and characterized antibiotic-resistant genes harbored in the microorganisms, thus having the potential to be an important tool for inclusion in AMR surveillance programs. For PCR, DNA from the culture-isolated microorganisms, or even directly from samples is extracted first and used as the template for PCR reaction. Predesigned primers are used to amplify the target region on template DNA . Along wPC, etc. .The introduction of high throughput sequencing technologies and bioinformatics tools has enabled WGS and WMS as potential methods for rapid species and AMR gene ID and characterization. Both WGS and WMS can be used reliably to predict the phenotypic AMR using readily-available online tools and is suitable to be incorporated in AMR surveillance programs in animals ,63.In a standard WGS protocol, DNA is extracted from the isolated target bacteria followed by library preparation by shearing DNA into a pool of fragments. Thereafter, the library is sequenced through a set of sequencing reactions and analyzed in a sequencing machine capable of determining the DNA sequence of each fragment in the library. These fragments are assembled into short contigs, ultimately to the complete genome for further analyses, including gene prediction and annotation, comparative genomics, and evolutionary analysis . WMS tecBoth WGS and WMS are based on high throughput next-generation sequencing techniques and have been developed by a number of commercial organizations and operated on different platforms such as the Illumina/Solexa,454/Roche, Ion PGM from Ion Torrent, AB SOLiD System, and Oxford Nanopore MinION. These systems use different chemistry for their operation . The tecde novo assembled draft genome against a reference database and mapping analysis of raw sequencing reads [de novo assembly of the bacterial genome from short-reads is performed by De Bruijn graph-based assemblers such as SPAdes, Velvet, ABySS, and SOAPdenovo. Sequencing reads are divided into shorter overlapping fragments (k-mers) to form a network graph. Henceforth, the assemblers reconstruct the genome sequence by finding an optimum path (Euler’s path) through the graph that visits each edge once. IDBA-UD, MEGAHIT, MetaSPAdes, and MetaVelvet are the assemblers used to assemble metagenomic sequences [Two common bioinformatic approaches for detecting AMR genes are Basic Local Alignment Search Tool (BLAST)-based analysis of ng reads . In asseequences . Followiequences . In readequences ,62.The reference databases used to identify AMR genes in the genome of an isolate or in metagenome are ResFinder, ABRicate, Search Engine for Antimicrobial Resistance, Comprehensive Antibiotic Resistance Database, Antibiotic Resistance Gene-ANNOTation, Antimicrobial Resistance Identification By Assembly, or Resistance Gene Identifier . AnotherAMR is a serious global problem of complex epidemiology as resistant organisms exist in multiple sectors like humans, animals, food, and the environment . The evoE. coli bacteria in feces samples or M. haemolytica in nasopharyngeal swabs are suggested to explicate an in-depth picture of the problem [AMR surveillance programs in the animal health sector should be coordinated by top management. The top management eventually acts in the role of centralized leadership and is responsible for fund mobilization and networking between disciplines and sectors through shared meetings, discussions, and reporting. The top management will analyze the needs of participating labs and respond accordingly ,23,69. S problem -30.The establishment of a laboratory network engaging a competent central reference laboratory and a sufficient number of sentinel laboratories is another important responsibility of the top management. The central reference laboratory should be identified from existing facilities or be established for AMR surveillance. If such scope does not exist, assistance from a competent laboratory in neighboring countries should be availed through collaborative agreement. The central reference AMR laboratory should be accredited in accordance with global standard ISO/IEC 17025, or be working toward laboratory accreditation for ensuring reliable test results ,70.Laboratory-based activities in AMR surveillance programs are eventually started at sentinel laboratories. However, the activities of the sentinel labs should be limited to the sample reception, isolation and ID of target bacteria, and AMR characterization by disk diffusion test. Disk diffusion test is easy to execute and suitable for testing multiple drugs on a single agar plate, and thus can be established in sentinel laboratories with limited-resource settings ,68.Central reference laboratory should determine MIC values of the antibiotics through broth or agar dilution methods. Although both broth macro-dilution and micro-dilution methods are easy to interpret and efficient in determining MIC of antibiotics, the broth macro-dilution method is cumbersome, time-consuming, and difficult to run multiple samples simultaneously. Whereas broth micro-dilution method is easier to perform with minimal expertise and enables multiple samples or antibiotics testing at a time; hence, it can be conveniently used in reference laboratories. In the agar dilution method, the preparation of agar plates is cumbersome, but this method is reproducible and enables the efficient testing of large numbers of bacterial isolates . HoweverAutomated MIC determination systems can be used in the reference laboratories if the sample size is too large. However, the scope of using automated systems has been limited by its high cost. Moreover, the systems require a pure isolated culture of bacteria for AST determination and interpretation, which is time-consuming. However, automated systems are user-friendly by their easy and smooth workflow . These sGenotypic AMR characterization is performed to explicit the genetic determinants of AMR emergence. PCR is used commonly for this purpose, but this method only enables the detection of previously known and characterized antibiotic-resistant genes harbored in the microorganisms. Even, a single nucleotide polymorphism in the AMR gene may mislead the false-negative result . AMR genBased on the above discussion, the following recommendations could be followed for setting and executing an AMR surveillance program in animals-The AMR surveillance program in animals should be well-structured, comprising top management and a competent laboratory networkThe top management of the AMR surveillance program will identify a competent laboratory network and define a catchment area for surveillance studyThe top management will formulate a pragmatic AMR surveillance programThe top management should provide support to improve the capacity and capability for AMR surveillance and monitoring at all levels involved, allocate sufficient funds to ensure the availability of consumables, diagnostics, and reagentsStandardized protocols should be developed by the reference laboratory for the harmonization of all activities at all levelsThe sentinel laboratories will be responsible for sample receiving, bacteria isolation and ID, and performing AST by disk diffusion testThe reference laboratory will go for in-depth AMR characterization through both phenotypic and genotypic methods using automated AST devices and WGS or WMS approach, respectivelyAppropriate reference bacterial strains must be used for antibiotic susceptibility testingAntibiotic susceptibility test reports should be given following internationally accepted CLSI or EUCAST breakpointsEvaluate the surveillance methods used and the data collected periodically to ensure that they are serving the purposes; make necessary adjustments to address emerging issues such as emerging pathogens and new commodities.AMR is considered one of the most dreadful public health hazards and its surveillance is crucial for the containment of the problem. AMR is eventually a “One Health” problem where the animal sector is a significant contributor to it. Therefore, AMR surveillance in animals is very important for understanding the emergence and magnitude of the burden and thereafter for mitigating the hazard. The surveillance system is also very important to gather data that will increase our knowledge and understanding of the complex mechanism and epidemiology of AMR. This information is crucial for treating infection, making policy recommendations, and developing strategies to reduce the magnitude of AMR burden. This review study explicit the current structure and methodology of an AMR surveillance program used in animals and recommends an efficient and pragmatic surveillance system comprising both phenotypic and genotypic ASTs. This comprehensive review tried to retrieve the maximum available relevant information. Nevertheless, due to the limited open data sharing policy by some of the journals and restricted access to some of the databases, the search may not be exhaustive and the review is limited by the inclusion of a small number of articles. Even though the study sufficiently illustrated the structure, objectives, and methodologies of an AMR surveillance program. Hence, this review article could be used as a reference work to formulate, adopt, and implement an effective and pragmatic AMR surveillance program in veterinary practices.MAA: Conceptualized and designed the review. MAA, MHP, and MNH: Collected the literature. MAA and MNH: Drafted the manuscript. MHP, AZS, SS, and MMK: Edited and finalized the manuscript. All authors have read and approved the final manuscript.