An optimized protocol for routine development of cell culture from adult oyster, Crassostrea madrasensis.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

Propagation of IHHNV (Infectious hypodermal and haematopoietic necrosis virus)/PstDVI and MBV (Monodon baculovirus)/PmNV in shrimp primary haemocyte culture.

In vitro propagation of shrimp viruses IHHNV (Infectious hematopoietic hypodermal necrosis virus)/PstDV1 and MBV (Monodon baculovirus)/PmNV in the primary haemocyte culture was successfully be accomplished in the present study. The haemocytes were maintained in double-strength L15 medium with growth supplements and antibiotics. Cytopathic changes observed in the cells were detachment, slendering of cells, vacuolation, and rounding of the cells. Replication of viruses in the culture was confirmed through the expression of DNA polymerase, polyhedrin and lef 4 of MBV and capsid gene of IHHNV. Ultrathin sections of haemocytes observed through electron microscopy revealed scattered chromatin, virogenic stroma and presence of viruses and occlusion bodies (MBV). The study revealed that primary haemocyte culture could be used as a platform for the propagation of a variety of shrimp viruses.

Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

Two-Year Performance and Safety Results of the MINIject Supraciliary Implant in Patients With Primary Open-Angle Glaucoma: Meta-Analysis of the STAR-I, II, III Trials.

PURPOSE: To evaluate the performance and safety of minimally invasive glaucoma surgery with a supraciliary drainage device (MINIject; iSTAR Medical, Wavre, Belgium) in primary open-angle glaucoma (POAG) as a stand-alone procedure. DESIGN: Meta-analysis. METHODS: At 11 sites in Colombia, France, Germany, India, Panama, and Spain, 82 patients were treated in 3 prospective, multicenter, interventional, nonrandomized trials (STAR-I, II, III). Data were pooled in a meta-analysis of up to 2 years of follow-up postimplantation. The main outcome measures were mean relative and absolute reduction in diurnal intraocular pressure (IOP) compared to baseline. Secondary outcomes included patients with IOP ≤18 mmHg, patients with IOP reduction ≥20%, number of IOP-lowering medications, adverse events, and endothelial cell density loss. RESULTS: At the 2-year follow-up (n = 66), mean IOP was reduced from 23.8 ± 3.3 mmHg at baseline to 14.4 ± 4.5 mmHg (-39.3%; P < 0.0001). An IOP reduction of ≥20% was achieved in 89.4% of patients, with 84.8% having an IOP ≤18 mmHg. IOP-lowering medications were reduced from a mean of 2.4 ± 1.1 to 1.4 ± 1.4 (P < 0.0001), with 37.9% of patients being medication-free at 2 years. Mean endothelial cell density loss at 2 years was 6.2 ± 9.1% compared to baseline and no patient had a loss >30%. CONCLUSIONS: This meta-analysis demonstrates the favorable safety and efficacy profile of a supraciliary device implanted in a stand-alone, ab-interno procedure in patients with mild-to-moderate POAG. The data demonstrate that MINIject is a safe and effective, bleb-free treatment option for patients requiring low target IOP up to 2 years.

Residual Pattern of Chlorantraniliprole, Thiamethoxam, Flubendiamide and Deltamethrin in Tomato Fruit and Soil.

Tomato, Lycopersicon esculentum L. is grown widely as an important day-to-day demand vegetable. The crop is attacked by various polyphagous insect pests like tomato fruit borer, stink bug, cabbage looper, flea beetle, aphids, whitefly, two-spotted spider mite, etc., and oligophagous insects like leaf-miner, five-spotted hawkmoth, etc. To combat the damage and yield loss, various chemical insecticides were sprayed on tomatoes under field conditions. The residual pattern of insecticides like chlorantraniliprole, thiamethoxam, flubendiamide, and deltamethrin residues was studied following applications of chlorantraniliprole 18.5% SC (Coragen) @ 30 g a.i./ha, thiamethoxam 25% WG (Actara) @ 50 g a.i./ha, flubendiamide 39.35 M/M SC (Fame) @ 48 g a.i./ha and deltamethrin 2.8% EC (Decis 100) @ 12.5 g a.i./ha using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). Fruit samples were collected at 0 (1 h after application), 1, 2, 3, 5, 7 days and at harvest time. All the residues of insecticides such as chlorantraniliprole (0.09 mg kg- 1), thiamethoxam (0.03 mg kg- 1), flubendiamide (0.02 mg kg- 1), and deltamethrin (0.01 mg kg- 1) were persisted up to 5th day. There were no residues found at harvest time. The residues of chlorantraniliprole and deltamethrin persisted up to 3rd day of spraying whereas the residues of flubendiamide and thiamethoxam were not detected on the same day in the soil.

Differences in radiopacity among CT contrast agents and concentrations: A quantitative study.

BACKGROUND AND PURPOSE: Several studies in the literature have attempted to subjectively assess the degree of visualization of different neurovascular structures using different contrast agents and concentrations. Given the recent contrast shortages, we aim to objectively compare the radiopacity achieved with four angiographic contrast agents used in clinical practice. METHODS: Isovue 370, Visipaque 320, Omnipaque 300, and Isovue 300 were each drawn up at 25%, 50%, 75%, and 100% concentrations and compared against normal saline and air syringes. CT scans were obtained, and regions of interest were analyzed for radiopacity using Hounsfield unit (HU) measurements. An aneurysm model with different contrast concentrations was also scanned and dimensions compared. Two-tailed t-tests and Cohen's d coefficients were applied to assess for differences in mean HU measurements. RESULTS: Isovue 370 and Isovue 300 had the highest and lowest mean HU, respectively (p < .001). Visipaque 320 at 25% concentration had the lowest mean HU at -.76. Statistically similar agents (p < .05) were Visipaque 320 and Omnipaque 300 at a 100% concentration (p = .30), and Omnipaque 300 and Isovue 300 at a 25% concentration (p = .73). Aneurysm dimensions among Isovue 370, Visipaque 320, and Omnipaque 300 were all similar, whereas with Isovue 300, the dimensions were significantly smaller (p < .05). CONCLUSION: Isovue 370 provides the highest HU radiopacity and the most accurate aneurysm measurements. Angiographic measurements obtained with Isovue 300 may underestimate the actual aneurysmal dimensions. Visipaque 320 and Omnipaque 300 at 100% concentration have similar mean HUs and are beneficial for patients with chronic kidney or cardiac disease.

Evaluation of Deoxyribonucleic Acid Damage Using Neutral Comet Assay for High Radiation Doses: A Feasibility Study.

Purpose: This study aims to investigate the use of the neutral comet assay to assess deoxyribonucleic acid (DNA) damage in lymphocytes exposed to high doses of radiation. Materials and Methods: The research was conducted by obtaining informed consent, after which blood samples were taken from seven healthy individuals and this study was approved by the institutional ethics committee. At first, for the determination of dose-effect curves, samples obtained from the first five individuals were irradiated for doses ranging from 0 to 35 Gy after which they were processed under neutral comet assay. In order to verify the determined dose-effect curves, a test dose of 15 Gy was delivered to the samples obtained from the sixth and seventh individuals. The amount of DNA damage from the obtained comet assay images was analyzed using four comet assay parameters namely % tail DNA, tail length, tail moment (TM), and Olive TM (OTM). The most suitable comet assay parameter was evaluated based on the obtained dose-effect curves. Furthermore, the distribution of individual cells for each dose point was evaluated for all the four comet assay parameters to find the optimal parameter. Results: From our results, it was found that from 0 to 25 Gy all the four comet assay parameters fit well into a linear quadratic curve and above 25 Gy saturation was observed. Based on the individual cell distribution data, it was found that % tail DNA could be an optimal choice to evaluate DNA damage while using neutral comet assay for high-dose ionizing radiation. Conclusion: The neutral comet assay could be a potential tool to assess DNA damage from high doses of ionizing radiation greater than 5 Gy.

Emerging Subspecialties in Neurology: Interventional Neurology: A Starter Guide for Neurology Residents.

Interventional neurology is a subspecialty at the cross-section of neurology, neurosurgery, and neuroradiology that uses image-guided endovascular catheter techniques to diagnose and treat neurovascular disorders. Although interventional neurology techniques have existed for decades, recent landmark trials in stroke thrombectomy have catalyzed dramatic changes to the interventional neurology field, and there is rising neurology interest and representation. Unlike other neurology subspecialties, however, interventional neurology training and fellowship applications are not standardized. Thus, it can be difficult for neurology trainees to navigate this rapidly changing and multidisciplinary field. This article seeks to provide neurology residents and residency program directors with a brief overview of the modern interventional neurology field, the current fellowship application process, and tips on how neurologists can best prepare for a career in interventional neurology. To this end, we conducted a nationwide survey of interventional neurology fellowship program directors regarding training neurologists. We distill survey responses into concrete, actionable items for neurology trainees.

Identifying and addressing common contributors to nonadherence with ophthalmic medical therapy.

PURPOSE OF REVIEW: To discuss common reasons for nonadherence and review existing and emerging options to reduce nonadherence with ocular medical therapy and optimize therapeutic outcomes. RECENT FINDINGS: Nonadherence can arise from patient-related issues (e.g. physical, cognitive) and healthcare-related issues (e.g. cost, access to care). Multiple strategies have been developed and evaluated to overcome these barriers to adherence. Identifying nonadherence and its cause(s) facilitates the development of strategies to overcome it. SUMMARY: Many common causes of nonadherence can be mitigated through a variety of strategies presented.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Procedural and Clinical Outcome Analysis of Monoplane versus Biplane Angiography Suites in Stroke Thrombectomies.

BACKGROUND: Thrombectomy is now the standard of care in the treatment of acute ischemic stroke caused by emergent large vessel occlusion. Therefore thrombectomy services have expanded from Comprehensive Stroke Centers to Thrombectomy-Capable Stroke Centers. Stroke interventions at these sites are performed in both biplane and monoplane angiography suites. It has been hypothesized that differences in these systems may affect time to successful reperfusion, with a potentially significant effect on neurologic outcomes. With an increase in TSCs, this study aims to evaluate the safety and efficacy of monoplane thrombectomy versus biplane thrombectomy. METHODS: Patients who presented with isolated proximal middle cerebral artery M1 occlusions and underwent endovascular thrombectomy from March 2015 to August 2018 at 5 different centers within a single health system were included. Thrombectomy was performed by the same group of experienced neurointerventionalists. The primary endpoint was functional outcome as measured by the modified Rankin scale at 90 days. Secondary endpoints included recanalization grade as measured by the Thrombolysis in Cerebral Infarction score, time to final reperfusion, and incidence of hemorrhagic conversion. RESULTS: A total of 197 patients were included in this study. Of them, 80.7% underwent thrombectomy on biplane systems. Time to final reperfusion was 10.2 minutes longer in the monoplane group but was not statistically significant (P = 0.252). There was no significant difference in the rates of favorable reperfusion (P = 0.755), hemorrhagic conversion (P = 0.580), or functional outcome at 90 days (favorable modified Rankin Scale 0-2, P = 0.210; favorable modified Rankin Scale 0-3, P = 0.697). CONCLUSION: Despite perceived advantages of biplane systems in reducing procedural time, our study demonstrates no significant differences between systems. These data support the safety and efficacy of performing thrombectomy on monoplane systems and may also carry implications for reducing patient transfer times and potentially increasing thrombectomy access to areas of the world where biplane suites may not be available. The next step would be a prospective randomized trial comparing both systems in different settings.

Functional characterization of a histone H2A derived antimicrobial peptide HARRIOTTIN-1 from sicklefin chimaera, Neoharriotta pinnata.

Antimicrobial peptides (AMPs) are gene encoded short peptides which play an important role in the innate immunity of almost all living organisms ranging from bacteria to mammals. Histones play a very important role in defense as precursors to bioactive peptides. The present study is an attempt to decipher the antimicrobial activity of a histone H2A derived peptide, Harriottin-1 from sicklefin chimaera, Neoharriotta pinnata. Analysis in silico predicted the molecule with potent antibacterial and anticancer property. The Harriottin-1 was recombinantly produced and the recombinant peptide rHar-1 demonstrated potent antibacterial activity at 25 µM besides anticancer activity. The study strongly suggests the importance of histone H2A derived peptides as a model for the design and synthesis of potent peptide drugs.

On Comparison of Heat Treated and Non-Heat-Treated LOM Manufactured Sample for Poly(lactic)acid: Mechanical and Morphological View Point.

This work reports the comparison of heat-treated and non-heat-treated laminated object-manufactured (LOM) 3D-printed specimens from mechanical and morphological viewpoints. The study suggests that heat treatment of the FDM-printed specimen may have a significant impact on the material characteristics of the polymer. The work has been performed at two stages for the characterization of (a) non-heat-treated samples and (b) heat-treated samples. The results for stage 1 (non-heat-treated samples) suggest that the infill density: 70%, infill pattern: honeycomb, and six number of discs in a single LOM-manufactured sample is the optimized condition with a compression strength of 42.47 MPa. The heat treatment analysis at stage 2 suggests that a high temperature: 65 °C, low time interval: 10 min, works equally well as the low temperature: 55 °C, high time interval: 30 min. The post-heat treatment near Tg (65 °C) for a time interval of 10 min improved the compressive strength by 105.42%.

On Laminated Object Manufactured FDM-Printed ABS/TPU Multimaterial Specimens: An Insight into Mechanical and Morphological Characteristics.

Fused deposition modeling (FDM) printing of commercial and reinforced filaments is a proven and well-explored method for the enhancement of mechanical properties. However, little has hitherto been reported on the multi-material components, fused or laminated together into a single specimen by using the laminated object manufacturing (LOM) technique for sustainable/renewable polymers. TPU is one such durable and flexible, sustainable material exhibiting renewable and biocompatible properties that have been explored very less often in combination with the ABS polymer matrix in a single specimen, such as the LOM specimen. The current research work presents the LOM manufacturing of 3D-printed flexural specimens of two different, widely used polymers available viz. ABS and TPU and tested as per ASTM D790 standards. The specimens were made and laminated in three layers. They were grouped into two categories, namely ABS: TPU: ABS (ATA) and TPU: ABS: TPU (TAT), which are functionally graded, sandwiched structures of polymeric material. The investigation of the flexural properties, microscopic imaging, and porosity characteristics of the specimens was made for the above categories. The results of the study suggest that ATA-based samples held larger flexural strength than TAT laminated manufactured samples. A significant improvement in the peak elongation and break elongation of the samples was achieved and has shown a 187% increase in the break elongation. Similarly, for the TAT-based specimen, flexural strength was improved significantly from approximately 6.8 MPa to 13 MPa, which represents a nearly 92% increase in the flexural strength. The morphological testing using Tool Maker's microscopic analysis and porosity analysis has supported the observed trends of mechanical behavior of ATA and TAT samples.

Status in molluscan cell line development in last one decade (2010-2020): impediments and way forward.

Despite the attempts that have started since the 1960s, not even a single cell line of marine molluscs is available. Considering the vast contribution of marine bivalve aquaculture to the world economy, the prevailing viral threats, and the dismaying lack of advancements in molluscan virology, the requirement of a marine molluscan cell line is indispensable. This synthetic review discusses the obstacles in developing a marine molluscan cell line concerning the choice of species, the selection of tissue and decontamination, and cell culture media, with emphasis given on the current decade 2010-2020. Detailed accounts on the experiments on the virus cultivation in vitro and molluscan cell immortalization, with a brief note on the history and applications of the molluscan cell culture, are elucidated to give a holistic picture of the current status and future trends in molluscan cell line development. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00539-x.

First Real-World, Multicenter, Post-Marketing, Retrospective Study of Dexamethasone Intraocular Suspension for Inflammation After Cataract Surgery.

Purpose: To evaluate dexamethasone intraocular suspension 9% (intraocular DXM) in real-world clinical use to manage inflammation associated with cataract surgery. Setting: Patients who underwent cataract surgery and received intraocular DXM at 22 outpatient eye surgery centers in the US. Design: Retrospective, observational chart review. Methods: Records of all patients who received intraocular DXM from March to December 2019 at participating centers were reviewed. Main Outcome Measures: Outcomes included anterior chamber cell (ACC) grades, anterior chamber flare (ACF) grades, and visual acuity, as well as intraocular pressure (IOP) and adverse events (AEs) at postoperative days (PODs) 1, 8, 14, 30. Descriptive statistics were generated. Results: The study population included 527 patients (641 eyes), with glaucoma history in 66 patients (80 eyes). Among eyes with recorded ACC grades, the percentage with grade 0 increased from 40% at POD 1 to 89.7% at POD 30, with similar results in eyes with glaucoma history. Among eyes with recorded ACF grades, the percentage with grade 0 increased from 78.4% at POD 1 to 97.1% at POD 30. At POD 30, 96.6% eyes with recorded results achieved target acuity. Mean IOP was 18.6 mmHg at POD 1 but declined to ≤15.2 mmHg thereafter. Investigators reported 22 AEs in 20 patients, all reported mild or moderate, the most common: IOP increase (7 events). Conclusion: Patients undergoing cataract surgery and treated with intraocular DXM showed favorable inflammatory and visual outcomes, without unanticipated safety problems, consistent with results of previous controlled clinical trials.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Equal rates of drug resistance in leprosy cases with relapse and recurrent/chronic Type 2 reaction: time to revise the guidelines for drug-resistance testing in leprosy?

BACKGROUND: Leprosy relapse/recurrence is a serious concern particularly in a leprosy-endemic nation such as India. It is believed that bacilli persisting even after multidrug therapy can cause relapse; recently, however, drug resistance as a cause for recurrences and chronic erythema nodosum leprosum (ENL) has been speculated. AIM: To study drug-resistance patterns in cases of leprosy relapse and chronic/recurrent (c/r)ENL. METHODS: This cross-sectional study conducted over a period of 1 year included patients diagnosed as having leprosy relapse and those with c/rENL. Skin biopsy specimens were examined by conventional PCR for resistance testing for rifampicin, dapsone and ofloxacin, respectively targeting the rpoB, folP and gyrA genes of Mycobacterium leprae. RESULTS: In total, 61 patients (25 smear-negative) were included in the study. Of these, 37 were diagnosed as having leprosy relapse and 24 as having c/rENL. Drug resistance to at least one drug was identified in 10 cases (16.4%). Rates of drug resistance were 5.4% (2 of 37) for dapsone, 10.8% (4 of 37) for rifampicin and 2.7% (1 of 37) for ofloxacin among cases of relapse, whereas it was 12.5% (3 of 24) and 8.3% (2 of 24) for dapsone and rifampicin respectively among those with c/rENL. Multidrug resistance was seen in 3.3% patients (2 of 61). CONCLUSION: Drug-resistance rate among those with c/rENL was almost equalled that of relapse. Smear-negative leprosy relapse cases also had resistance to bactericidal drugs. These findings call for modifications in criteria for testing under leprosy drug-resistance surveillance and all cases of relapse and those with recalcitrant c/rENL should be tested.