Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy ...susceptibility locus to chromosome 6 region q25-q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5′ regulatory region shared by the Parkinson's disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.
Canine Visceral Leishmaniasis (CVL) shares many aspects with the human disease and dogs are considered the main urban reservoir of L. infantum in zoonotic VL. Infected dogs develop progressive ...disease with a large clinical spectrum. A complex balance between the parasite and the genetic/immunological background of the host are decisive for infection evolution and clinical outcome. This study comprised 92 Leishmania infected mongrel dogs of various ages from Mato Grosso, Brazil. Spleen samples were collected for determining parasite load, humoral response, cytokine mRNA expression and histopathology alterations. By real-time PCR for the ssrRNA Leishmania gene, two groups were defined; a low (lowP, n = 46) and a high parasite load groups (highP, n = 42). When comparing these groups, results show variable individual humoral immune response with higher specific IgG production in infected animals but with a notable difference in CVL rapid test optical densities (DPP) between highP and lowP groups. Splenic architecture disruption was characterized by disorganization of white pulp, more evident in animals with high parasitism. All cytokine transcripts in spleen were less expressed in highP than lowP groups with a large heterogeneous variation in response. Individual correlation analysis between cytokine expression and parasite load revealed a negative correlation for both pro-inflammatory cytokines: IFNγ, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and TGFβ. TNF showed the best negative correlation (r2 = 0.231; p<0.001). Herein we describe impairment on mRNA cytokine expression in leishmania infected dogs with high parasite load associated with a structural modification in the splenic lymphoid micro-architecture. We also discuss the possible mechanism responsible for the uncontrolled parasite growth and clinical outcome.
Leprosy Type-1 Reactions (T1Rs) are pathological inflammatory responses that afflict a sub-group of leprosy patients and result in peripheral nerve damage. Here, we employed a family-based GWAS in ...221 families with 229 T1R-affect offspring with stepwise replication to identify risk factors for T1R. We discovered, replicated and validated T1R-specific associations with SNPs located in chromosome region 10p21.2. Combined analysis across the three independent samples resulted in strong evidence of association of rs1875147 with T1R (p = 4.5x10-8; OR = 1.54, 95% CI = 1.32-1.80). The T1R-risk locus was restricted to a lncRNA-encoding genomic interval with rs1875147 being an eQTL for the lncRNA. Since a genetic overlap between leprosy and inflammatory bowel disease (IBD) has been detected, we evaluated if the shared genetic control could be traced to the T1R endophenotype. Employing the results of a recent IBD GWAS meta-analysis we found that 10.6% of IBD SNPs available in our dataset shared a common risk-allele with T1R (p = 2.4x10-4). This finding points to a substantial overlap in the genetic control of clinically diverse inflammatory disorders.
The diagnosis of paucibacillary (PB) leprosy cases remains a challenge because of the absence of a confirmatory laboratory method. While quantitative polymerase chain reaction (qPCR) has been shown ...to provide reliable sensitivity and specificity in PB diagnoses, a thorough investigation of its efficacy in clinical practice has not yet been published. The present study evaluated patients with suspected leprosy skin lesions by using qPCR to identify PB individuals in the Leprosy Outpatient clinic at the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil.
One hundred seventy-two suspected PB cases were included in the study. The patients were evaluated by a dermatologist at three different times. The clinical dermato-neurological examination and collected samples were performed on the first visit. On the second visit, the results of the histopathological analysis and PCR assay (DNA-based Mycobacterium leprae qPCR-targeting 16S gene) results were analyzed, and a decision regarding multi-drug therapy was made. A year later, the patients were re-examined, and the consensus diagnosis was established.
In 58% (100/172) of cases, a conclusive diagnosis via histopathological analysis was not possible; however, 30% (30/100) of these cases had a positive PCR. One hundred ten patients (110/172) attended the third visit. The analysis showed that while the sensitivity of the histopathological test was very low (35%), a qPCR alone was more effective for identifying leprosy, with 57% sensitivity.
The use of qPCR in suspected PB cases with an inconclusive histology improved the sensitivity of leprosy diagnoses.
Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies ...(TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid‐liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25‐mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP90‐231) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP90‐231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP90‐231 phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration‐dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.
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.
A current major challenge in leprosy control is the prevention of permanent disabilities. Host pathological inflammatory responses termed type 1 reaction (T1R) are a leading cause of nerve damage for ...leprosy patients. The environmental or inherited factors that predispose leprosy cases to undergo T1R are not known. However, studies have shown an important contribution of host genetics for susceptibility to T1R. We have previously identified variants encompassing the
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genes as T1R risk factors in a Vietnamese sample and replicated this association in a Brazilian sample. However, we failed to validate in Brazilian patients the strong association of
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markers rs6478108 and rs7863183 with T1R that we had observed in Vietnamese patients. Here, we investigated if the lack of validation of these variants was due to age-dependent effects on association using four independent population samples, two from Brazil and two from Vietnam. In the combined analysis across the four samples, we observed a strong association of the
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variants rs6478108, rs7863183, and rs3181348 with T1R (
= 1.5E-05,
= 1.8E-05, and
= 6.5E-06, respectively). However, the association of rs6478108 with T1R was more pronounced in leprosy cases under 30 years of age compared to the global sample odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.54-2.46,
= 2.5E-08 versus OR = 1.46, 95% CI = 1.23-1.73,
= 1.5E-05. A multivariable analysis indicated that the association of rs6478108 with T1R was independent of either rs7863183 or rs3181348. These three variants are known regulators of the
gene transcription level in multiple tissues. The age dependency of association of rs6478108 and T1R suggests that the genetic control of gene expression varies across the human life span.
Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide ...new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (MΦ), or within immune-activated MΦ. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.
The enzyme IDO‐1 is involved in the first stage of tryptophan catabolism and has been described in both microbicidal and tolerogenic microenvironments. Previous data from our group have shown that ...IDO‐1 is differentially regulated in the distinctive clinical forms of leprosy. The present study aims to investigate the mechanisms associated with IDO‐1 expression and activity in human monocyte‐derived dendritic cells (mDCs) after stimulation with irradiated Mycobacterium leprae and its fractions. M. leprae and its fractions induced the expression and activity of IDO‐1 in human mDCs. Among the stimuli studied, irradiated M. leprae and its membrane fraction (MLMA) induced the production of proinflammatory cytokines TNF and IL‐6 whereas irradiated M. leprae and its cytosol fraction (MLSA) induced an increase in IL‐10. We investigated if TLR2 activation was necessary for IDO‐1 induction in mDCs. We observed that in cultures treated with a neutralizing anti‐TLR2 antibody, there was a decrease in IDO‐1 activity and expression induced by M. leprae and MLMA. The same effect was observed when we used a MyD88 inhibitor. Our data demonstrate that coculture of mDCs with autologous lymphocytes induced an increase in regulatory T (Treg) cell frequency in MLSA‐stimulated cultures, showing that M. leprae constituents may play opposite roles that may possibly be related to the dubious effect of IDO‐1 in the different clinical forms of disease. Our data show that M. leprae and its fractions are able to differentially modulate the activity and functionality of IDO‐1 in mDCs by a pathway that involves TLR2, suggesting that this enzyme may play an important role in leprosy immunopathogenesis.
Graphical
Mycobacterium leprae and its fractions are able to differentially modulate the activity and functionality of Indoleamine 2,3 dioxygenase in human monocyte‐derived dendritic cells via a pathway that involves TLR2.
Leprosy has been treated with multidrug therapy, which has been distributed for free across the globe and regarded as highly efficient. However, the impossibility of growing Mycobacterium leprae in ...axenic media has historically impaired assessments of M. leprae resistance, a parameter only recently detectable through molecular methods.
A systematic, population-based search for M. leprae resistance in suspected leprosy relapse cases and contacts was performed in Prata Village, an isolated, hyperendemic, former leprosy colony located in the Brazilian Amazon. Results led to an extended active search involving the entire Prata population. Confirmed leprosy cases were investigated for bacterial resistance using a combination of in vivo testing and direct sequencing of resistance genes folP1, rpoB, and gyrA. A molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR).
Mycobacterium leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new cases): 16 (43.24%) displayed drug-resistance variants. Multidrug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in 1 new case. Resistance to dapsone was present in 2 relapses and 1 new case. Combined molecular resistance and VNTR data revealed evidence of intra-familial primary transmission of resistant M. leprae.
A comprehensive, population-based systematic approach to investigate M. leprae resistance in a unique population revealed an alarming scenario of the emergence and transmission of resistant strains. These findings may be used for the development of new strategies for surveillance of drug resistance in other populations.
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