A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples.

BACKGROUND: Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. METHODS: Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. RESULTS: The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. CONCLUSION: The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.

Bartonella henselae as a putative trigger for chronic type 2 leprosy reactions.

Leprosy reactions are an acute inflammatory phenomenon that can arise before diagnosis, during treatment, or after cure of leprosy. These reactions are considered one of the main diseases that cause physical disabilities. Immunosuppressive treatment for these immune responses makes these patients susceptible to coinfections, which can trigger new leprosy reactions. The main objective of this study was to evaluate the occurrence of infection by Bartonella sp. in blood samples from 47 patients who had untreatable episodes of type 2 leprosy reactions for more than six months, comparing them with a control group. Cultures and molecular methods (PCR) were used. Amplicons from species-specific reactions and sequencing showed a higher prevalence of Bartonella henselae infection in patients, 19/47 (40.4 %), compared to control, 9/50 (18.0 %), p = 0.0149. Five patients accepted treatment for coinfection, and all showed improvement in leprosy reactions with treatment for B. henselae infection. We conclude that these bacteria can trigger chronic reactions of type 2 leprosy and should be investigated in these patients. SUMMARY LINE: Patients who have chronic type 2 leprosy reactions are more susceptible to Bartonella henselae infection than controls: 19/47 (40.4 %) compared 9/50 (18.0 %), p = 0.0149.

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques.

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index.

INTRODUCTION: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. METHODS: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. RESULTS: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. CONCLUSIONS: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools.

The diagnosis of leprosy poses several challenges. The bacillary load, serology, and tissue response are determined by the host immune status, which make individual tests unsuitable across the spectrum. The sensitivity of tests for identifying paucibacillary cases remains limited, on the other hand, many tests lack specificity in differentiating contacts from diseased cases. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy. In the current review, we critically analyze the tests available for diagnosis, prognostication, and prediction of treatment response in leprosy. We discuss in brief the conventional tests available and detail the newer serologic and molecular tests added over the past few years with an attempt to suggest the pros and cons of each, and the tests best fit for each clinical scenario. Slit skin smears and skin or nerve biopsies are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. Antibody titres of phenolic glycolipid-1 and its synthetic variants can be measured in serum and saliva and provide noninvasive means to detect leprosy with good specificity. Conventional, quantitative, real-time, and other variants of PCR can detect M. leprae DNA and have been used to effect in blood, tissue, and urine samples. T helper I and II cytokine signatures can be used to differentiate the subtypes of leprosy. Newer machine learning algorithms use combinations of these tests to predict the development of leprosy in contacts. Tests to detect treatment response, antimicrobial drug resistance, and predict the onset of reactions in leprosy can be used to advantage. We compare the characteristics of these tests and suggest an algorithm for leprosy diagnosis optimally utilizing them in various clinical settings.

Utility of Multi-target Nested PCR and ELISPOT Assays for the Detection of Paucibacillary Leprosy: A Possible Conclusion of Clinical Laboratory Misdiagnosis.

The diagnosis of paucibacillary (PB) leprosy often possesses a diagnostic challenge, especially for pure neuritic and lesser skin lesions with the zero bacillary load, requiring a sensitive and accurate diagnostic tool. We have included 300 clinically diagnosed new leprosy cases (comprising 98 PB cases) and analyzed the sensitivity and specificity of PB leprosy cases by nested PCR with folP, gyrA, rpoB, RLEP, and 16SrRNA and Enzyme-linked Immunospot Assay test (ELISPOT) with MMPII, NDO-BSA, and LID-1 antigens by detecting interferon gamma (IFN-γ) release. The overall positivity rates of genes tested in 300 clinical specimens were identified as 55% of 16SrRNA, 59% of RLEP, 59.3% of folP, 57.3% of rpoB, 61% of gyrA while 90% of nested folP, 92.6% of nested rpoB, and 95% of nested gyrA, and 285 (95%) of at least one gene positive cases. For PB specimens, 95% PCR positivity was achieved by three tested genes in nested PCR. The data obtained from ELISPOT for three antigens were analyzed for IFN-γ expression with 600 subjects. Among 98 PB leprosy cases, the sensitivity of MMP II, LID-1, and NDO-BSA was 90%, 87%, and 83%, respectively, and the specificity was 90%, 91%, and 86%, respectively. The total number of cases positive for at least one antigen was 90 (91.8%) in PB, which is significantly higher than that in multibacillary (MB) leprosy (56.7%). The combination of multi-targets nested PCR and ELISPOT assay provides a specific tool to early clinical laboratory diagnosis of PB leprosy cases. The two assays are complementary to each other and beneficial for screening PB patients.

Diagnostic utility of PCR in detection of clinical cases and carriers of leprosy: A cross sectional study at a tertiary care teaching hospital in central India.

PURPOSE: Since ancient era leprosy is existing across the world. India, Indonesia and Brazil still harbour major proportion of global cases. Child leprosy and Grade II disability indicate delayed diagnosis and persistence of transmission in community. So, this study was conducted with aim to evaluate the diagnostic efficacy of PCR in comparison to SSS (Slit Skin Smear) microscopy for detection of leprosy in early stages in both cases and carriers (contacts). METHODS: A cross sectional observational study was conducted on 100 subjects including 50 clinically diagnosed new cases of leprosy and their 50 contacts. Each group was subjected to SSS (Slit Skin Smear) microscopy and PCR using RLEP gene as target. RESULTS: The overall male: female ratio was 2.44. The Slit Skin smear (SSS) microscopy positivity was 34% (n = 17/50) among cases while it was 0% (n = 0/50) among contacts. The overall positivity for PCR was 42% (n = 42/100) being 66% (n = 33/50) in cases and 18% (n = 9/50) in contacts. About 30% (n = 25/83) of all the microscopically negative subjects were found to be positive by PCR. CONCLUSIONS: PCR was found to be a better diagnostic tool both among cases and their contacts. It should be used for screening contacts for early diagnosis and treatment and thus preventing transmission in community. KEY MESSAGE: To diagnose case and contacts of leprosy in early stages even in very low bacterial density using PCR.

A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Simultaneous detection and differentiation between Mycobacterium leprae and Mycobacterium lepromatosis using novel polymerase chain reaction primers.

We have developed a polymerase chain reaction-based method to detect and distinguish Mycobacterium leprae and Mycobacterium lepromatosis using a single set of primers based on a 45-bp difference in the amplicon size of their rpoT gene. This method can also help in detecting the cases of co-infection in a single experiment.

Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable, making it difficult to diagnose and elucidate details of its transmission chain. Thus, this study aimed to analyze the dynamics of environmental transmission of M. leprae in a case-control study in the city of Mossoró, Brazil. METHODS AND RESULTS: Data of clinical, epidemiological, bacilloscopic, and serological evaluation of 22 newly diagnosed patients were compared, with molecular results of detection of specific genome regions RLEP and 16S rRNA of M. leprae in samples of the nasal swab, saliva, and house dust of these individuals and their controls (44 household contacts and 44 peridomiciliar contacts). The rapid serological tests evaluated, ML flow (IgM ND-O-BSA) and OrangeLife® (IgM and IgG anti NDO-LID 1) showed similar results, with greater positivity among paucibacillaries by OrangeLife® (54.5%). Positivity for nasal swab and saliva in multibacillary patients with RLEP primer was 16.7% and 33.3%, respectively. There was no detection of bacterial DNA in house dust or among paucibacillaries. The OrangeLife® test indicated that the lower the amount of windows, the more transmission in the house (3.79 more chances). Having a history of leprosy cases in the family increased the risk by 2.89 times, and being over 60 years of age gave 3.6 times more chances of acquiring the disease. PCR positivity was higher among all clinical samples using the M. leprae RLEP region than 16S rRNA. CONCLUSIONS: In this study, the serological and PCR analysis were capable of detecting M. leprae DNA in clinical samples but not in the environmental samples. Close monitoring of patients and household contacts appears an effective measure to reduce the transmission of leprosy in endemic areas.

Case Report: Diagnostic Pitfalls in an Atypical Case of Primary Neuritic Leprosy.

Primary neuritic leprosy is a form of leprosy clinically limited to the peripheral nerves without obvious skin lesions. Diagnosing leprosy in the absence of typical dermatological features is challenging and often causes a delay in diagnosis. We describe a case of primary neuritic leprosy with atypical features and the roles that histological confirmation using nerve biopsy of an unenlarged nerve and newer techniques, such as polymerase chain reaction and high-resolution ultrasonography, play in improving the diagnosis.

The unprecedented epidemic-like scenario of dermatophytosis in India: II. Diagnostic methods and taxonomical aspects.

Trichophyton (T.) mentagrophytes now accounts for an overwhelming majority of clinical cases in India, a new "Indian genotype" (T. mentagrophytes ITS genotype VIII) having been isolated from skin samples obtained from cases across a wide geographical distribution in this country. The conventional diagnostic methods, like fungal culture, are, however, inadequate for diagnosing this agent. Thus, molecular methods of diagnosis are necessary for proper characterization of the causative agent. The shift in the predominant agent of dermatophytosis from T. rubrum to T. mentagrophytes, within a relatively short span of time, is without historic parallel. The apparent ease of transmission of a zoophilic fungus among human hosts can also be explained by means of mycological phenomena, like anthropization.

Sensitivity and specificity of multibacillary and paucibacillary leprosy laboratory tests: A systematic review and meta-analysis.

This systematic review (number register: CRD42018112736) was performed to compare the sensitivity and specificity of leprosy diagnostic methods. The search was conducted in 3 electronic databases in January 2021. Studies evaluating leprosy diagnostic tests were included according the eligibility criteria. Meta-analysis was performed to calculate the sensibility and specificity of the groups. We included 36 studies. The test sensitivity for paucibacillary patients was 0.31 (95%CI: 0.29-0.33) and the specificity was 0.92 (95%CI: 0.92-0.93). In multibacillary patients, the sensitivity was 0.78 (95%CI: 0.77-0.80) and specificity was 0.92 (95%CI: 0.92-0.93). Comparing the sensitivity and specificity of the different techniques included, it should be noted that polymerase chain reaction (PCR) test presented the highest sensitivity for paucibacillary patients, while the western blot technique showed the highest sensitivity for multibacillary patients. However, further studies are needed to optimise the diagnosis of leprosy, requiring research with a larger number of samples and more uniform protocols.

Structural aspects of lesional and non-lesional skin microbiota reveal key community changes in leprosy patients from India.

Although skin is the primary affected organ in Leprosy, the role of the skin microbiome in its pathogenesis is not well understood. Recent reports have shown that skin of leprosy patients (LP) harbours perturbed microbiota which grants inflammation and disease progression. Herein, we present the results of nested Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) which was initially performed for investigating the diversity of bacterial communities from lesional skin (LS) and non-lesional skin (NLS) sites of LP (n = 11). Further, we performed comprehensive analysis of 16S rRNA profiles corresponding to skin samples from participants (n = 90) located in two geographical locations i.e. Hyderabad and Miraj in India. The genus Staphylococcus was observed to be one of the representative bacteria characterizing healthy controls (HC; n = 30), which in contrast was underrepresented in skin microbiota of LP. Taxa affiliated to phyla Firmicutes and Proteobacteria were found to be signatures of HC and LS, respectively. Observed diversity level changes, shifts in core microbiota, and community network structure support the evident dysbiosis in normal skin microbiota due to leprosy. Insights obtained indicate the need for exploring skin microbiota modulation as a potential therapeutic option for leprosy.

Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population.

BACKGROUND: A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. RESULTS: Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. CONCLUSION: A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

CLINICAL PROGRESSION OF LEPROSY IN EURASIAN RED SQUIRRELS (SCIURUS VULGARIS) IN A NATURALLY INFECTED WILD POPULATION.

Leprosy has been described in Eurasian red squirrel (Sciurus vulgaris; ERS) carcasses since 2014. Studies of ERS carcasses have not provided information about incubation or disease progression in this host but have provided important insights into pathogen presence and distribution throughout the United Kingdom. Here we present field study data on 31 live ERS from an island population naturally infected with Mycobacterium leprae that were assessed longitudinally over a 2-yr time period. Clinical assessment, serologic (anti-phenolic glycolipid-I antibody [αPGL-I] detection) and molecular methods (polymerase chain reaction) were used to diagnose and categorize ERS at each assessment as a leprosy case, a leprosy suspect, colonized by M. leprae, or a contact ERS. Eight ERS (25.8%) were identified as leprosy cases: four at initial assessment, two at 6 mon and two at 24 mon after initial assessment. One ERS was categorized a leprosy suspect when it developed typical lesions 12 mon after initial assessment, despite negative serologic and molecular test results at this time, though M. leprae DNA had been isolated during the initial assessment. Seven ERS (22.6%) were categorized as colonized and of these, six were reassessed but did not develop clinical signs of leprosy within 6 (n = 2), 12 (n = 3), and 18 (n = 1) mon. Most (48.4%, n = 15) were categorized as contact ERS. Progression of leprosy lesions varied between ERS, but always increased in severity over time and was paralleled with increased antibody response. Based on our dataset, we propose the hypotheses: 1) leprosy in ERS is a chronic, slowly progressing disease in this species, similar to that described for other hosts; 2) lesions can undergo repeated ulceration-healing cycles; and 3) in some instances M. leprae DNA and αPGL-I antibodies are detectable before the onset of clinical signs of disease. Future studies addressing the progression of leprosy in ERS should follow affected animals over a longer time period and include tissue samples to pair molecular diagnostics with serologic results.