The diverse effects of brain glucagon-like peptide 1 receptors on ingestive behaviour.

Glucagon-like peptide 1 (GLP-1) is well known as a gut hormone and also acts as a neuropeptide, produced in a discrete population of caudal brainstem neurons that project widely throughout the brain. GLP-1 receptors are expressed in many brain areas of relevance to energy balance, and stimulation of these receptors at many of these sites potently suppresses food intake. This review surveys the current evidence for effects mediated by GLP-1 receptors on feeding behaviour at a wide array of brain sites and discusses behavioural and neurophysiological mechanisms for the effects identified thus far. Taken together, it is clear that GLP-1 receptor activity in the brain can influence feeding by diverse means, including mediation of gastrointestinal satiation and/or satiety signalling, suppression of motivation for food reward, induction of nausea and mediation of restraint stress-induced hypophagia, but many questions about the organization of this system remain. LINKED ARTICLES: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc.

Mycobacterium leprae Transcriptome During In Vivo Growth and Ex Vivo Stationary Phases.

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen primarily residing within host macrophages and Schwann cells. Whole genome sequencing predicts a highly degraded genome with approximately one third of the coding capacity resulting in the loss of many catabolic pathways. Therefore, it can be assumed that M. leprae obtains many of the necessary metabolites for intracellular survival and growth from the host cells. In this study, global transcriptomic analyses were done on freshly harvested M. leprae growing in athymic mouse footpads for five months (MFP5) and compared to those held in axenic medium for 48 (ML48) and 96 (ML96) hours. Results show that all of the genes and pseudogenes were transcribed under both in vivo and in vitro conditions. 24% and 33% of gene transcript levels were significantly altered in ML48 and ML96 respectively, compared to MFP5. Approximately 45% (39/86) of lipid metabolism genes were significantly downregulated in ML96 compared to MFP5, majority of which are in the ß-oxidation pathway. Cholesterol oxidase, acyl-CoA dehydrogenase, and coenzyme F420-dependent oxidoreductase, were significantly upregulated in both ML48 and ML96 compared to MFP5. 30% of cell wall and cell processes functional category genes had altered gene transcription at 96hr compared to MFP5. 40% of 57 genes associated with mycobacterial virulence showed significantly altered transcript levels with 52% significantly downregulated in ML96, including most of the Pro-Glu/Pro-Pro-Glu genes. All 111 hypothetical protein genes with unknown function were expressed. Adenosine triphosphate (ATP) synthesis in M. leprae appears to be significantly downregulated under ex vivo conditions. This is the first study comparing M. leprae global gene expression during in vivo growth and ex vivo stationery phase in axenic medium confirming that during the growth phase in the footpads of experimentally infected mice, M. leprae is metabolically active and its primary source of energy production is probably lipids.

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae.

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen's obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.

Hindbrain orexin 1 receptors blunt intake suppression by gastrointestinal nutrients and cholecystokinin in male rats.

Hypothalamic orexin neurons project to many brain areas, including hindbrain structures such as the nucleus of the solitary tract (NTS) and area postrema (AP), where orexin 1 receptors (OX1Rs) are expressed. Hindbrain administration of orexin-A increases feeding and meal size, and blockade of hindbrain OX1Rs with the selective antagonist SB334867 has the opposite effect. Here we asked whether hindbrain OX1R stimulation or blockade alter rats' sensitivity to gastrointestinal satiety signals. Rats received 4th intracerebroventricular (icv) injections of vehicle or orexin-A, at a dose with no effect on its own, prior to an intragastric (IG) infusion of saline or a satiating volume of Ensure. IG Ensure suppressed subsequent chow intake, but orexin-A pretreatment significantly attenuated this IG nutrient-induced satiety at 2 h into the dark phase. In a second experiment, rats received NTS injections of vehicle or orexin-A before intraperitoneal (IP) injection of vehicle or the satiation hormone cholecystokinin (CCK). NTS orexin-A pretreatment completely blocked the intake-suppressive effect of CCK on dark-phase chow intake. Finally, we investigated the role of endogenous hindbrain OX1R activation by pretreating rats with 4th-icv injection of vehicle or SB334867 followed by IG infusion of saline or Ensure just before a chocolate Ensure licking test session. IG nutrient infusion suppressed Ensure intake, and blockade of hindbrain OX1Rs significantly prolonged that intake-suppressive effect. We conclude that hindbrain OX1Rs are a mechanism though which hypothalamic orexin neurons can reduce animals' sensitivity to gastrointestinal nutrient load, allowing them to consume more food.

Intermittent High-Fat Diet Intake Reduces Sensitivity to Intragastric Nutrient Infusion and Exogenous Amylin in Female Rats.

OBJECTIVE: Intermittent (INT) access to a high-fat diet (HFD) can induce excessive-intake phenotypes in rodents. This study hypothesized that impaired satiation responses contribute to elevated intake in an INT-HFD access model. METHODS: First, this study characterized the intake and meal patterns of female rats that were subjected to an INT HFD in which a 45% HFD was presented for 20 hours every fourth day. To examine nutrient-induced satiation, rats received intragastric infusions of saline or Ensure Plus prior to darkness-onset food access. A similar design was used to examine sensitivity to the satiating effect of amylin. This study then examined whether an INT HFD influences amylin-induced c-Fos in feeding-relevant brain areas. RESULTS: Upon INT HFD access, rats consumed meals of larger size. The anorexic response to intragastric Ensure infusion and exogenous amylin treatment was blunted in INT rats on both chow-only and INT-HFD days of the diet regimen, compared with chow-maintained and continuous-HFD rats. An INT HFD did not influence amylin-induced c-Fos in the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus. CONCLUSIONS: Impaired satiation responses, mediated in part by reduced sensitivity to amylin, may explain the elevated intake observed upon INT HFD access and may play a role in disorders of INT overconsumption, including binge eating.

Preliminary examination of insulin and amylin levels in women with purging disorder.

OBJECTIVE: This preliminary study explored whether differences in meal-stimulated insulin or amylin release are linked to altered ingestive behaviors in individuals with bulimia nervosa (BN) or purging disorder (PD). METHOD: Women with BN (n = 15), PD (n = 16), or no eating disorder (n = 18) underwent structured clinical interviews and assessments of gut hormone and subjective responses to a fixed test meal. Multilevel model analyses were used to explore whether gut hormone responses contribute to subjective responses to the test meal and whether these associations differed by group. RESULTS: Insulin and amylin levels significantly increased following the test meal. Women with PD showed greater insulin release compared to those with BN, but not controls. Multilevel models support significant group X insulin interactions predicting subjective ratings of nausea and urge to vomit, with a stronger association between higher insulin responses and higher nausea and urge to vomit in women with PD and BN. Amylin responses did not differ by group. CONCLUSION: Increased sensitivity to the effects of insulin on nausea and urge to vomit may be linked to purging in both PD and BN. Differences in postprandial insulin levels may be linked to purging behavior in the absence versus presence of binge eating.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Endogenous GLP-1 in lateral septum promotes satiety and suppresses motivation for food in mice.

Glucagon-like peptide 1 receptors (GLP-1R) are expressed in the lateral septum (LS) of rats and mice, and we have published that endogenous LS GLP-1 affects feeding and motivation for food in rats. Here we asked if these effects are also observed in mice. In separate dose-response studies using male C57Bl6J mice, intra-LS GLP-1 or the GLP-1R antagonist Exendin 9 (Ex9) was delivered shortly before dark onset, at doses subthreshold for effect when injected intracerebroventricularly (icv). Intra-LS GLP-1 significantly suppressed chow intake early in the dark phase and tended to reduce overnight intake. However, blockade of LS GLP-1R with Ex9 had no effect on ad libitum dark onset chow intake. We then asked if LS GLP-1R blockade blunts nutrient preload-induced intake suppression. Mice were trained to consume Ensure immediately before dark onset, which suppressed subsequent chow intake, and intra-LS Ex9 attenuated that preload-induced intake suppression. We also found that restraint stress robustly activates hindbrain GLP-1-producing neurons, and that LS GLP-1R blockade attenuates 30-min restraint stress-induced hypophagia in mice. Furthermore, we have reported that in the rat, GLP-1R in the dorsal subregion of the LS (dLS) affect motivation for food. We examined this in food-restricted mice responding for sucrose pellets on a progressive ratio (PR) schedule. Intra-dLS GLP-1R stimulation significantly suppressed, and Ex9 significantly increased, operant responding, and the Ex9 effect remained after mice returned to ad libitum conditions. Similarly, we found that stimulation of dLS GLP-1 suppressed licking for sucrose and conversely, Ex9 increased licking under ad libitum feeding conditions. Together, our data suggest that endogenous activation of LS GLP-1R plays a role in feeding in mice under some but not all conditions, and that these receptors strongly influence motivation for food.

Dual RNA-Seq of Human Leprosy Lesions Identifies Bacterial Determinants Linked to Host Immune Response.

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. Dual RNA-seq on patient lesions identifies two independent molecular measures of M. leprae, each of which correlates with distinct aspects of the host immune response. The fraction of bacterial transcripts, reflecting bacterial burden, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial mRNA:rRNA ratio, reflecting bacterial viability, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for the interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Preproglucagon Neurons in the Nucleus of the Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress-Induced Hypophagia, and Limit Unusually Large Intakes of Food.

Centrally administered glucagon-like peptide 1 (GLP-1) supresses food intake. Here we demonstrate that GLP-1-producing (PPG) neurons in the nucleus tractus solitarii (NTS) are the predominant source of endogenous GLP-1 within the brain. Selective ablation of NTS PPG neurons by viral expression of diphtheria toxin subunit A substantially reduced active GLP-1 concentrations in brain and spinal cord. Contrary to expectations, this loss of central GLP-1 had no significant effect on the ad libitum feeding of mice, affecting neither daily chow intake nor body weight or glucose tolerance. Only after bigger challenges to homeostasis were PPG neurons necessary for food intake control. PPG-ablated mice increased food intake after a prolonged fast and after a liquid diet preload. Consistent with our ablation data, acute inhibition of hM4Di-expressing PPG neurons did not affect ad libitum feeding; however, it increased refeeding intake after fast and blocked stress-induced hypophagia. Additionally, chemogenetic PPG neuron activation through hM3Dq caused a strong acute anorectic effect. We conclude that PPG neurons are not involved in primary intake regulation but form part of a secondary satiation/satiety circuit, which is activated by both psychogenic stress and large meals. Given their hypophagic capacity, PPG neurons might be an attractive drug target in obesity treatment.

Dapsone Resistance in Leprosy Patients Originally from American Samoa, United States, 2010-2012.

Skin biopsies from US leprosy patients were tested for mutations associated with drug resistance. Dapsone resistance was found in 4 of 6 biopsies from American Samoa patients. No resistance was observed in patients from other origins. The high rate of dapsone resistance in patients from American Samoa warrants further investigation.

Intragastric nutrient infusion reduces motivation for food in male and female rats.

The idea that gut-derived satiation signals influence food reward has recently gained traction, but this hypothesis is largely based on studies focused on neural circuitry, not the peripherally released signals. Here, we directly tested the hypothesis that intragastric (IG) nutrient infusion can suppress motivation for food. In a series of experiments, IG sucrose infusion (15 kcal) significantly and reliably reduced operant responding for a sucrose reward on a progressive ratio (PR) schedule. Moreover, food deprivation for 24 h before the test session did not prevent the suppressive effect of nutrients. The suppressive effect of IG sucrose on fixed ratio 5 (FR5) operant responding was also assessed as a comparison. The effect of IG nutrients to reduce motivation was not limited to sucrose; IG Ensure infusion (9.3 kcal) also significantly reduced PR operant responding for sucrose pellets. To verify that these effects were not secondary to the osmotic challenge of concentrated nutrients, we tested IG infusion of noncaloric saline solutions equiosmolar to 40% sucrose or Ensure and found no effect. Finally, we focused on glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) as candidate mediators for the effect of IG nutrients. Pretreatment with exendin-9, a GLP-1 receptor antagonist, delivered intraperitoneally, significantly attenuated the ability of IG nutrients to suppress PR responding and breakpoint in males, but not in females, whereas pretreatment with devazepide, a CCKA receptor antagonist, failed to do so in both sexes. Together, these data support the idea that nutrient-induced satiation signals influence food reward and may implicate GLP-1 in this process.

Endogenous GLP-1 in lateral septum contributes to stress-induced hypophagia.

Glucagon-like peptide 1 (GLP-1) neurons of the caudal brainstem project to many brain areas, including the lateral septum (LS), which has a known role in stress responses. Previously, we showed that endogenous GLP-1 in the LS plays a physiologic role in the control of feeding under non-stressed conditions, however, central GLP-1 is also involved in behavioral and endocrine responses to stress. Here, we asked whether LS GLP-1 receptors (GLP-1R) contribute to stress-induced hypophagia. Male rats were implanted with bilateral cannulas targeting the dorsal subregion of the LS (dLS). In a within-subjects design, shortly before the onset of the dark phase, rats received dLS injections of saline or the GLP-1R antagonist Exendin (9-39) (Ex9) prior to 30 min restraint stress. Food intake was measured continuously for the next 20 h. The stress-induced hypophagia observed within the first 30 min of dark was not influenced by Ex9 pretreatment, but Ex9 tended to blunt the effect of stress as early as 1 and 2 h into the dark phase. By 4-6 h, there were significant stress X drug interactions, and Ex9 pretreatment blocked the stress-induced suppression of feeding. These effects were mediated entirely through changes in average meal size; stress suppressed meal size while dLS Ex9 attenuated this effect. Using a similar design, we examined the role of dLS GLP-1R in the neuroendocrine response to acute restraint stress. As expected, stress potently increased serum corticosterone, but blockade of dLS GLP-1Rs did not affect this response. Together, these data show that endogenous GLP-1 action in the dLS plays a role in some but not all of the physiologic responses to acute stress.

Lateral septum growth hormone secretagogue receptor affects food intake and motivation for sucrose reinforcement.

The hormone ghrelin promotes eating and is widely considered to be a hunger signal. Ghrelin receptors, growth hormone secretagogue receptors (GHSRs), are found in a number of specific regions throughout the brain, including the lateral septum (LS), an area not traditionally associated with the control of feeding. Here we investigated whether GHSRs in the LS play a role in the control of food intake. We examined the feeding effects of ghrelin and the GHSR antagonists ([d-Lys3]-growth hormone-releasing peptide-6 and JMV-2959) at doses subthreshold for effect when delivered to the lateral ventricle. Intra-LS ghrelin significantly increased chow intake during the midlight phase, suggesting that pharmacological activation of LS GHSRs promotes feeding. Conversely, GHSR antagonist delivered to the LS shortly before dark onset significantly reduced chow intake. These data support the hypothesis that exogenous and endogenous stimulation of GHSRs in the LS influence feeding. Ghrelin is known to affect motivation for food, and the dorsal subdivision of LS (dLS) has been shown to play a role in motivation. Thus, we investigated the role of dLS GHSRs in motivation for food reward by examining operant responding for sucrose on a progressive ratio (PR) schedule. Intra-dLS ghrelin increased PR responding for sucrose, whereas blockade of LS GHSRs did not affect responding in either a fed or fasted state. Together these findings for the first time substantiate the LS as a site of action for ghrelin signaling in the control of food intake.

GLP-1 action in the mouse bed nucleus of the stria terminalis.

Glucagon-like peptide-1 (GLP-1) injected into the brain reduces food intake. Similarly, activation of preproglucagon (PPG) cells in the hindbrain which synthesize GLP-1, reduces food intake. However, it is far from clear whether this happens because of satiety, nausea, reduced reward, or even stress. Here we explore the role of the bed nucleus of the stria terminalis (BNST), an area involved in feeding control as well as stress responses, in GLP-1 responses. Using cre-expressing mice we visualized projections of NTS PPG neurons and GLP-1R-expressing BNST cells with AAV-driven Channelrhodopsin-YFP expression. The BNST displayed many varicose YFP+ PPG axons in the ventral and less in the dorsal regions. Mice which express RFP in GLP-1R neurons had RFP+ cells throughout the BNST with the highest density in the dorsal part, suggesting that PPG neuron-derived GLP-1 acts in the BNST. Indeed, injection of GLP-1 into the BNST reduced chow intake during the dark phase, whereas injection of the GLP-1 receptor antagonist Ex9 increased feeding. BNST-specific GLP-1-induced food suppression was less effective in mice on high fat (HF, 60%) diet, and Ex9 had no effect. Restraint stress-induced hypophagia was attenuated by BNST Ex9 treatment, further supporting a role for endogenous brain GLP-1. Finally, whole-cell patch clamp recordings of RFP+ BNST neurons demonstrated that GLP-1 elicited either a depolarizing or hyperpolarizing reversible response that was of opposite polarity to that under dopamine. Our data support a physiological role for BNST GLP-1R in feeding, and suggest complex cellular responses to GLP-1 in this nucleus.

Weight suppression and bulimic syndrome maintenance: Preliminary findings for the mediating role of leptin.

OBJECTIVE: Longitudinal studies support a prospective relationship between weight suppression (WS) and bulimic syndrome (BN-S) maintenance. Although biobehavioral mechanisms have been proposed to explain this link, such mechanisms have yet to be identified. Given that weight loss would reduce leptin levels which may influence eating, this study examined whether reduced leptin levels mediate the link between greater WS and longer illness duration. METHOD: Women (N = 53), ages 18-45 years, were recruited from the community if they met criteria for a BN-S, including either DSM-5 bulimia nervosa (BN; n = 33) or purging disorder (PD: n = 20), and fell within a healthy weight range (18.5-26.5 kg/m2 ). Participants completed clinical assessments and provided blood samples to measure circulating leptin. RESULTS: Significant associations were found among greater WS, lower leptin concentrations, and longer duration of illness. Mediation analyses using bootstrapping procedures indicated all paths were significant and that leptin mediated the link between WS and illness duration. An alternative model in which longer illness duration contributed to leptin, via greater WS, was not supported. DISCUSSION: Longitudinal research is needed to support temporal associations and explore behavioral mechanisms linking leptin to illness trajectory.

Estradiol modulates the anorexic response to central glucagon-like peptide 1.

Estrogens suppress feeding in part by enhancing the response to satiation signals. Glucagon-like peptide 1 (GLP-1) acts on receptor populations both peripherally and centrally to affect food intake. We hypothesized that modulation of the central GLP-1 system is one of the mechanisms underlying the effects of estrogens on feeding. We assessed the anorexic effect of 0, 1, and 10µg doses of GLP-1 administered into the lateral ventricle of bilaterally ovariectomized (OVX) female rats on a cyclic regimen of either 2µg ß-estradiol-3-benzoate (EB) or oil vehicle 30min prior to dark onset on the day following hormone treatment. Central GLP-1 treatment significantly suppressed food intake in EB-treated rats at both doses compared to vehicle, whereas only the 10µg GLP-1 dose was effective in oil-treated rats. To follow up, we examined whether physiologic-dose cyclic estradiol treatment influences GLP-1-induced c-Fos in feeding-relevant brain areas of OVX females. GLP-1 significantly increased c-Fos expression in the area postrema (AP) and nucleus of the solitary tract (NTS), and the presence of estrogens may be required for this effect in the paraventricular nucleus of the hypothalamus (PVN). Together, these data suggest that modulation of the central GLP-1 system may be one of the mechanisms by which estrogens suppress food intake, and highlight the PVN as a region of interest for future investigation.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.