Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification.

BACKGROUND: Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. RESULTS: In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. CONCLUSION: The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

Characteristics of children with leprosy: Factors associated with delay in disease diagnosis.

OBJECTIVES: The objectives of the study were to characterize the clinical profile of childhood leprosy presenting at tertiary leprosy care hospitals in the states of Bihar, West Bengal and Uttar Pradesh in India, and to determine the possible risk factors associated with disabilities at presentation. METHODS: Subjects were children with newly diagnosed leprosy registered for treatment at tertiary Leprosy Mission Hospitals in Muzaffarpur (Bihar), Purulia (West Bengal) and Faizabad (Uttar Pradesh), India, between June and December 2019. Demographic and leprosy characteristics were collected at the time of diagnosis. Parents/guardians were interviewed on reasons for delay in presenting at the hospital. Associations between various factors and delay in diagnosis were assessed. RESULTS: Among the 84 children, the mean (SD) age was 10 (3) years with a range of 4-14 years. There were more boys (58%) and most children were currently in school (93%), resident in rural areas (90%) and belonged to a lower socioeconomic status (68%). More children were diagnosed with multibacillary leprosy (69%), one-third of them being skin smear positive for Mycobacterium leprae. On presentation, 17% had deformity (5% grade 1 deformity and 12% grade 2), 29% had nerve involvement and skin lesions were spread across the body in half of the children. Mean (SD) duration of delay was 10.5 (9.8) months. Delayed presentation was more in boys (43% vs. 17%; P = 0.01), those without a history of migration for work compared to those who had a history of migration (40% vs. 9%; P = 0.008) and in those children who were from a poor economic status compared with those that came from a better economic status (44% vs. 7%; P = 0.001) Limitations: Because our study was conducted at tertiary care hospitals, the findings are not representative of the situation in the field. Furthermore, a comparison group of newly diagnosed adult leprosy patients with disability could have been included in the study. CONCLUSION: Childhood leprosy continues to occur in endemic pockets in India and a substantial number present with skin smear positivity and deformity. Guardians of these children cite many reasons for the delay in presentation.

Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

Potential Role of CXCL10 in Monitoring Response to Treatment in Leprosy Patients.

The treatment of multibacillary cases of leprosy with multidrug therapy (MDT) comprises 12 doses of a combination of rifampicin, dapsone and clofazimine. Previous studies have described the immunological phenotypic pattern in skin lesions in multibacillary patients. Here, we evaluated the effect of MDT on skin cell phenotype and on the Mycobacterium leprae-specific immune response. An analysis of skin cell phenotype demonstrated a significant decrease in MRS1 (SR-A), CXCL10 (IP-10) and IFNG (IFN-γ) gene and protein expression after MDT release. Patients were randomized according to whether they experienced a reduction in bacillary load after MDT. A reduction in CXCL10 (IP-10) in sera was associated with the absence of a reduction in the bacillary load at release. Although IFN-γ production in response to M. leprae was not affected by MDT, CXCL10 (IP-10) levels in response to M. leprae increased in cells from patients who experienced a reduction in bacillary load after treatment. Together, our results suggest that CXCL10 (IP-10) may be a good marker for monitoring treatment efficacy in multibacillary patients.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Low rate of relapse after twelve-dose multidrug therapy for hansen's disease: A 20-year cohort study in a brazilian reference center.

The World Health Organization has raised concerns about the increasing number of Hansen disease (HD) relapses worldwide, especially in Brazil, India, and Indonesia that report the highest number of recurrent cases. Relapses are an indicator of MDT effectiveness and can reflect Mycobacterium leprae persistence or re-infection. Relapse is also a potential marker for the development or progression of disability. In this research, we studied a large cohort of persons affected by HD treated with full fixed-dose multibacillary (MB) multidrug therapy (MDT) followed for up to 20 years and observed that relapses are a rare event. We estimated the incidence density of relapse in a cohort of patients classified to receive MB regime (bacillary index (BI) > 0), diagnosed between September 1997 and June 2017, and treated with twelve-dose MB-MDT at a HD reference center in Rio de Janeiro, Brazil. We obtained the data from the data management system of the clinic routine service. We linked the selected cases to the dataset of relapses of the national HD data to confirm possible relapse cases diagnosed elsewhere. We diagnosed ten cases of relapse in a cohort of 713 patients followed-up for a mean of 12.1 years. This resulted in an incidence rate of 1.16 relapse cases per 1000 person-year (95% CI = 0.5915-2.076). The accumulated risk was 0.025 in 20 years. The very low risk observed in this cohort of twelve-dose-treated MB patients reinforces the success of the current MDT scheme.

IL-1R1-Dependent Signals Improve Control of Cytosolic Virulent Mycobacteria In Vivo.

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals.IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Chronic Unilateral Uveitis as a Manifestation of Leprosy: A Case Report and Literature Review.

Purpose: To describe a case of leprosy presenting chronic anterior uveitis associated with other systemic lesions.Methods: Case report and systematic literature review.Results: We describe the case of a 65-year-old patient presenting clinical features of chronic uveitis and poor response to topical and intravitreal steroid treatment. Upon ocular examination, diffuse iris atrophy and macular edema were observed and laboratory tests for autoimmune and infectious diseases were within normal range. Physical examination revealed the presence of skin lesions on trunk and extremities, which were biopsied and identified as positive for leprosy.Conclusion: The case reported herein presented atypical characteristics of uveitis due to the involvement of the posterior segment of the eye. Leprosy diagnosis could be a challenge, a systematic approach is mandatory to achieve adequate treatment.

Multidrug Therapy for Leprosy Can Cure Patients with Lobomycosis in Acre State, Brazil: A Proof of Therapy Study.

Lobomycosis, also referred to as lacaziosis, is an endemic cutaneous and subcutaneous fungal disease that mainly affects Amazonian forest dwellers in Brazil. There is no disease control program in place in Brazil, and antifungal therapy failures are common, and the therapy is inaccessible to most patients. We performed a randomized, unblinded clinical trial testing the cure rate of multiple drug therapy (MDT) for leprosy with surgical excision, with or without itraconazole. A control arm consisted of patients who did not adhere to either therapeutic regimens but continued to be followed up. Multiple drug therapy consisted of monthly supervised doses of 600 mg rifampicin, 300 mg clofazimine, and 100 mg dapsone, in addition to daily doses of 50 mg clofazimine and 100 mg dapsone. The patients in the MDT plus itraconazole arm also received itraconazole 100 mg twice daily. We followed up 54 patients from the MDT group and 26 patients from the MDT plus itraconazole group for an average of 4 years and 9 months. The 23 controls were followed up for 6 months on average. The following endpoints were observed: 1) unchanged (no apparent improvement), 2) improved (reduction in lesion size and/or pruritus), and 3) cured (complete remission of the lesions, no viable fungi, and no relapse for 2 years after the end of the drug treatment). The results indicated a significantly greater likelihood of cure associated with the use of multidrug therapy for leprosy with or without itraconazole when compared with the control group. The addition of itraconazole to MDT was not associated with improved outcomes, suggesting that MDT alone is effective.

The function of peroxisome proliferator-activated receptors PPAR-γ and PPAR-δ in Mycobacterium leprae-induced foam cell formation in host macrophages.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae). In lepromatous leprosy (LL), skin macrophages, harboring extensive bacterial multiplication, gain a distinctive foamy appearance due to increased intracellular lipid load. To determine the mechanism by which M. leprae modifies the lipid homeostasis in host cells, an in vitro M. leprae infection system, using human macrophage precursor THP-1 cells and M. leprae prepared from the footpads of nude mice, was employed. RNA extracted from skin smear samples of patients was used to investigate host gene expressions before and after multidrug therapy (MDT). We found that a cluster of peroxisome proliferator-activated receptor (PPAR) target genes associated with adipocyte differentiation were strongly induced in M. leprae-infected THP-1 cells, with increased intracellular lipid accumulation. PPAR-δ and PPAR-γ expressions were induced by M. leprae infection in a bacterial load-dependent manner, and their proteins underwent nuclear translocalization after infection, indicating activation of PPAR signaling in host cells. Either PPAR-δ or PPAR-γ antagonist abolished the effect of M. leprae to modify host gene expressions and inhibited intracellular lipid accumulation in host cells. M. leprae-specific gene expressions were detected in the skin smear samples both before and after MDT, whereas PPAR target gene expressions were dramatically diminished after MDT. These results suggest that M. leprae infection activates host PPAR signaling to induce an array of adipocyte differentiation-associated genes, leading to accumulation of intracellular lipids to accommodate M. leprae parasitization. Certain PPAR target genes in skin lesions may serve as biomarkers for monitoring treatment efficacy.

Delayed diagnosis and ongoing transmission of leprosy in the post-elimination era in Boru Meda hospital, Ethiopia.

INTRODUCTION: Following the recommendation of the Global Leprosy Strategy, Ethiopia targeted to reduce the incidence of new leprosy cases, and the proportion with severe disability (grade 2) from 13.6% in 2016 to < 1% in 2020. This study assessed the clinical profile of new leprosy cases and the sequelae of previously treated ones 20 years after leprosy was eliminated as a public health problem in the country. METHODOLOGY: Hospital based cross sectional study was conducted  by reviewing the medical records of all leprosy patients seen at the dermatology clinic of Boru Meda Hospital from August to December 2018.The  data were captured using a standard data collection form. RESULTS: Over the study period, 57 (27.4%) new cases and 151 (72.6%) previously treated cases were seen.The median age was 44 years (interquartile range 32-57). Among the newly diagnosed cases, two were under the age of 15 years , 51 (89.5%) were multibacillary and 34 (59.6%) had grade 2 disability. This included visual impairment in 10 (17.5%) and neurological complications in 44 (77.2%). Of the 151 previously treated cases, 104 (68.9%) presented with disabilities, including 97 (64.2%) with grade 2. Amongst previously treated cases, 130 (86.1%) had neurological complications. In addition, 53 (35.1%) had vision impairment. CONCLUSIONS: This study showed evidence of ongoing leprosy transmission and delayed diagnosis in the country. This calls for operational research to determine the underlying reasons and provide ways forward. At the same time, the high burden of disabilities in previously treated cases should be addressed.

Zosteriform multidermatomal nodules and plaques in a case of lepromatous leprosy: an uncommon presentation.

Leprosy is caused by the obligate intracellular organism Mycobacterium leprae which mainly affects the skin and nervous system. The course of the disease is determined by host immunity, it is thus believed that in lepromatous leprosy (LL), all manifestations are bilaterally symmetrical. This is because of the inability of the host to mount an adequate cell-mediated immune response, resulting in widespread haematogenous dissemination of bacilli. Varied manifestations of LL have been reported; however, a multidermatomal pattern of nodules is hitherto unreported and we suggest a hypothesis for its presentation.

Dominant marker (inter-simple sequence repeat-polymerase chain reaction) versus codominant marker (RLEP-polymerase chain reaction) for laboratory diagnosis of leprosy: A comparative evaluation.

Background: Leprosy is a contagious disease and was eliminated globally in 2002. Since then, new cases were continuously detected from different parts of the world. Untreated leprosy cases shed millions of bacteria and are the main cause of dissemination of the disease. Currently, leprosy is detected by acid-fast bacilli (AFB) microscopy and has a low sensitivity ranging from 10% to 50%. The correlation between clinical findings and microscopy is unable to provide a conclusive case detection. Thus, in the present study, we compared to molecular methods, namely RLEP-polymerase chain reaction (RLEP-PCR) and inter-simple sequence repeat-PCR (ISSR-PCR) taking AFB microscopy as a gold standard for the detection of leprosy. Methods: A total of 168 clinically diagnosed leprosy patients were recruited in this study including 58 multibacillary and 110 paucibacillary patients. Slit-skin smear samples were taken for both microscopy and molecular study. Primers for RLEP-PCR were taken from the previous reports. The primers for ISSR-PCR were designed by screening the whole genome of Mycobacterium leprae TN strain (GenBank accession AL450380) for the presence of simple sequence repeats. One primer (TA)8CA3was synthesized and used for molecular amplification of ISSR-PCR. Results: We found that the efficacy of the AFB microscopy was 24.40%, whereas the efficacy of RLEP-PCR and ISSR-PCR was 63.09% and 73.21% (P = 0.000, 0.000, and 0.469), respectively. The area under the curve of receiver operating characteristic curve for the comparison of three diagnostic methods was 0.845. An enhancement of 48.81% in the case detection rate by ISSR-PCR over AFB microscopy and 10.12% over RLEP-PCR was also found. Our study clearly reveals that ISSR-PCR is a better tool for diagnosis of leprosy than AFB microscopy and RLEP-PCR. Interestingly, both the PCR techniques RLEP-PCR and ISSR-PCR are able to detect samples which were negative for AFB microscopy. Conclusion: Thus, the demonstration of ISSR-PCR in SSS samples can provide a better sensitive and confirmative tool for early diagnosis of leprosy.

Long-term ulcerations caused by Mycobacterium lepromatosis.

Patients with leprosy rarely present ulcerated lesions that can appear during reactional states like Lucio's phenomenon (LP), as in our case. LP is a rare complication of multibacillary leprosy due to massive bacilli invasion of endothelial cells causing a thrombotic syndrome. The initial macular lesion is purpuric followed by multiple infiltrated papules and nodules, some of them ulcerated, associated to loss of sensation on lower limbs. The importance of recognizing ulcers as a specific cutaneous manifestation of leprosy allows early diagnosis and treatment, and therefore avoiding the development of disabilities and persistence of illness. Infection by Mycobacterium lepromatosis is associated with LP and it should be especially sought in patients from endemic areas.

Hansen's disease with lepra reaction presenting with IgA dominant infection related glomerulonephritis.

Various renal abnormalities in leprosy have been described largely in literature but the occurrence of IgA dominant infection related glomerulonephritis in leprosy with type 2 lepra reaction has not been reported so far. We present here a 60-year-old man with a history of leprosy in the past admitted with type 2 lepra reaction, rapidly progressive glomerulonephritis with severe renal failure requiring dialysis and diagnosed to have IgA dominant infection related glomerulonephritis.