B lymphocytes deficiency results in altered immune response and increased susceptibility to Mycobacterium leprae in a murine leprosy model.

Leprosy is a chronic and infectious disease that primarily affects the skin and peripheral nervous system, presenting a wide spectrum of clinical forms with different degrees of severity. The distinct host immune response patters developed in the response to the bacillus Mycobacterium leprae, the leprosy etiologic agent, are associated with the spectral clinical forms and outcome of the disease. In this context, B cells are allegedly involved in the disease immunopathogenesis, usually as antibody-producing cells, but also as potential effector or regulatory elements. In order to determine the regulatory B cells role in experimental leprosy, this study evaluated the outcome of M. leprae infection in B cell deficient mice (BKO) and WT C57Bl/6 control, by means of microbiological/bacilloscopic, immunohistochemical and molecular analysis, performed 8 months after M. leprae inoculation. The results demonstrated that infected BKO showed a higher bacilli number when compared with WT animals, demonstrating the importance of these cells in experimental leprosy. The molecular analysis demonstrates that the expression of IL-4, IL-10 and TGF-ß was significantly higher in the BKO footpads when compared to WT group. Conversely, there was no difference in IFN-γ, TNF-α and IL-17 expression levels in BKO and WT groups. IL-17 expression was significantly higher in the lymph nodes of WT group. The immunohistochemical analysis revealed that M1 (CD80+) cells counts were significantly lower in the BKO group, while no significant difference was observed to M2 (CD206+) counts, resulting a skewed M1/M2 balance. These results demonstrated that the absence of B lymphocytes contribute to the persistence and multiplication of M. leprae, probably due to the increased expression of the IL-4, IL-10 and TGF-ß cytokines, as well as a decrease in the number of M1 macrophages in the inflammatory site.

Association of CD209 (DC-SIGN) rs735240 SNV with paucibacillary leprosy in the Brazilian population and its functional effects.

BACKGROUND: Leprosy, caused by Mycobacterium leprae, is a public health problem in Brazil that affects peripheral nerves, resulting in physical disabilities. During host-pathogen interactions, the immune response determines leprosy outcomes from a localised (paucibacillary) form to a disseminated (multibacillary) form. The recognition of M. leprae involves the DC-SIGN receptor, which is present on the dendritic cells (DCs) and participates in immune activation. OBJECTIVES: To evaluate the association of polymorphisms in the promoter region of the gene encoding DC-SIGN (CD209) and the clinical form of leprosy, and to investigate its functional effects. METHODS: The study population included 406 leprosy patients from an endemic area in Brazil [310 multibacillary (MB); 96 paucibacillary (PB)]. A functional evaluation based on the effects of the single nucleotide variant (SNV) associated with PB leprosy on the specific immune response was also performed. RESULTS: The GA genotype and the presence of the A allele of rs735240 (-939G>A) were associated with PB leprosy [OR: 2.09 (1.18-3.69) and 1.84 (1.07-3.14), respectively]. Carriers of the A allele showed reduced expression of CD209 and TGF-ß1 in leprosy lesions in comparison with individuals with GG genotype, in addition to a higher response to the Mitsuda test. CONCLUSION: These data suggest that rs735240 influences the immune response against M. leprae and clinical presentation of leprosy.

Emergence and Transmission of Drug-/Multidrug-resistant Mycobacterium leprae in a Former Leprosy Colony in the Brazilian Amazon.

BACKGROUND: 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. 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). RESULTS: 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. CONCLUSIONS: 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.

A genome wide association study identifies a lncRna as risk factor for pathological inflammatory responses in leprosy.

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.

Murine experimental leprosy: Evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology.

This study evaluated the immune response of nude and BALB/c mice inoculated in the footpads (FP) with Mycobacterium leprae after 3, 5 and 8 months. At each timepoint peritoneal cells, peripheral blood, FP and popliteal lymph nodes (PLN) were collected. Peritoneal cell cultures were performed to measure the H2 O2 , O2- , NO, IL-2, IL-4, IL-10, IL-12, IFN-γ and TNF levels. Serum levels of anti-PGL-I antibodies were also quantified. The results showed that the infection was progressive in nude mice with bacterial multiplication, development of macroscopic lesions in the FP and presence of bacilli in the PLN at 8 months. In BALB/c mice, the infection reached a plateau of bacillary multiplication at 5 months and regressed at 8 months. Histopathological analysis of FP revealed a mononuclear inflammatory infiltrate with a large number of neutrophils at 5 months, with a higher number in nude mice. At 8 months, the number of neutrophils decreased and the infiltrate was predominantly mononuclear in both mouse strains. There was no H2 O2, O2- , IL-2, IL-4, IL-10 and IFN-γ production in the course of infection in nude mice; however, in BALB/c, O2- and IL-12 production was higher at 5 months and NO, IFN-γ and TNF production was higher at 8 months when there was a decrease in the number of bacilli. The level of anti-PGL-I antibodies was higher in BALB/c mice. Thus, nude and BALB/c mice can be used as experimental models for the study of various aspects of leprosy.

Underlying mechanisms of leprosy recurrence in the Western Amazon: a retrospective cohort study.

BACKGROUND: The multidrug therapy (MDT) for leprosy treatment adopted by Brazil in the 1990s was important for reducing leprosy in the country; however, recurrent cases remained problematic. Mechanisms involved in leprosy recurrence are heterogeneous and can be sorted into three groups: insufficient therapy, bacillary persistence and new infections. This study aimed to analyse the time interval of leprosy recurrence in relation to the therapeutic scheme in the state of Acre. The hypotheses were as follows: 1) treatments (a) rifampicin, ofloxacin and minocycline (ROM) and (b) dapsone (DDS) have a short leprosy recurrence time, 2) treatments based on MDT have a long leprosy recurrence time, 3) there is a dose-response relationship between MDT and the time interval between leprosy episodes. METHODS: This retrospective cohort study included 201 patients with a second episode of clinical leprosy at the reference centers for leprosy control in the state of Acre. Exposure was the type of therapeutic scheme as follows: 1) ROM, 2) DDS, 3) MDT0-9 doses, 4) MDT10-19 doses, 5) MDT20-29 doses, and 6) MDT30+ doses. Outcome was the time interval between release from treatment and a diagnosis of a recurrent leprosy case. Incidence rate ratios and relative risk Poisson regressions adjusted by age and sex were calculated with 95% confidence intervals. RESULTS: The 201 patients studied during this retrospective follow-up resulted in a total of 224 cases of recurrent leprosy. Incidence rate ratios within this therapeutic scheme were as follows: 3.3 (2.39, 4.2; ROM/MDT30+), 1.12 (0.33, 1.92; DDS/MDT30+), 2.17 (1.39, 2.94; MDT0-9/MDT30+), 1.94 (1.13, 2.75; MDT10-19/MDT30+) and 1.26 (0.47, 2.05; MDT20-29/MDT30+). Relative risk Poisson regressions showed a protective effect of MDT30+ in comparison with ROM (0.22; 0.07, 0.72), MDT0-9 (0.42; 0.21, 0.85), and MDT10-19 (0.44; 0.21, 0.92). No differences among MDT30+ and DDS (0.71; 0.36, 1.41) and MDT20-29 (0.76; 0.38, 1.49) were observed. CONCLUSIONS: New infection is an important-yet neglected-mechanism in leprosy recurrence in the state of Acre and can challenge the leprosy elimination plan in Brazil. MDT with few doses might be associated with leprosy recurrence due to insufficient therapy or bacillary persistence.

Polymorphisms in the TGFB1 and IL2RA genes are associated with clinical forms of leprosy in Brazilian population.

BACKGROUND Leprosy is a chronic infectious disease caused by Mycobacterium leprae, and compromises the skin and peripheral nerves. This disease has been classified as multibacillary (MB) or paucibacillary (PB) depending on the host immune response. Genetic epidemiology studies in leprosy have shown the influence of human genetic components on the disease outcomes. OBJECTIVES We conducted an association study for IL2RA and TGFB1 genes with clinical forms of leprosy based on two case-control samples. These genes encode important molecules for the immunosuppressive activity of Treg cells and present differential expressions according to the clinical forms of leprosy. Furthermore, IL2RA is a positional candidate gene because it is located near the 10p13 chromosome region, presenting a linkage peak for PB leprosy. METHODS A total of 885 leprosy cases were included in the study; 406 cases from Rondonópolis County (start population), a hyperendemic region for leprosy in Brazil, and 479 cases from São Paulo state (replication population), which has lower epidemiological indexes for the disease. We tested 11 polymorphisms in the IL2RA gene and the missense variant rs1800470 in the TGFB1 gene. FINDINGS The AA genotype of rs2386841 in IL2RA was associated with the PB form in the start population. The AA genotype of rs1800470 in TGFB1 was associated with the MB form in the start population, and this association was confirmed for the replication population. MAIN CONCLUSIONS We demonstrated, for the first time, an association data with the PB form for a gene located on chromosome 10. In addition, we reported the association of TGFB1 gene with the MB form. Our results place these genes as candidates for validation and replication studies in leprosy polarisation.

Simultaneous analysis of multiple T helper subsets in leprosy reveals distinct patterns of Th1, Th2, Th17 and Tregs markers expression in clinical forms and reactional events.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Previous studies have demonstrated that the difference among clinical forms of leprosy can be associated with the immune response of patients, mainly by T helper (Th) and regulatory T cells (Tregs). Then, aiming at clarifying the immune response, the expression of cytokines related to Th1, Th2, Th17 and Tregs profiles were evaluated by qPCR in 87 skin biopsies from leprosy patients. Additionally, cytokines and anti-PGL-1 antibodies were determined in serum by ELISA. The results showed that the expression of various targets (mRNA) related to Th1, Th2, Th17 and Tregs were significantly modulated in leprosy when compared with healthy individuals, suggesting the presence of a mixed profile. In addition, the targets related to Th1 predominated in the tuberculoid pole and side and Th2 and Tregs predominated in the lepromatous pole and side; however, Th17 targets showed a mixed profile. Concerning reactional events, Tregs markers were decreased and IL-15 was increased in reversal reaction and IL-17F, CCL20 and IL-8 in erythema nodosum leprosum, when compared with the respective non-reactional leprosy patients. Additionally, ELISA analysis demonstrated that IL-22, IL-6, IL-10 and anti-PGL-1 antibody levels were significantly higher in the serum of patients when compared with healthy individuals, and IL-10 and anti-PGL-1 antibodies were also increased in the lepromatous pole and side. Together, these results indicate that Th1, Th2 and Th17 are involved in the determination of clinical forms of leprosy and suggest that decreased Tregs activity may be involved in the pathogenesis of reactional events.

Association of TNFSF8 regulatory variants with excessive inflammatory responses but not leprosy per se.

BACKGROUND: Type 1 reactions (T1R) affect a considerable proportion of patients with leprosy. In those with T1R, the host immune response pathologically overcompensates for the actual infectious threat, resulting in nerve damage and permanent disability. Based on the results of a genome-wide association study of leprosy per se, we investigated the TNFSF15 chromosomal region for a possible contribution to susceptibility to T1R. METHODS: We performed a high-resolution association scan of the TNFSF15 locus to evaluate the association with T1R in 2 geographically and ethnically distinct populations: a family-based sample from Vietnam and a case-control sample from Brazil, comprising a total of 1768 subjects. RESULTS: In the Vietnamese sample, 47 single-nucleotide polymorphisms (SNPs) overlapping TNFSF15 and the adjacent TNFSF8 gene were associated with T1R but not with leprosy. Of the 47 SNPs, 39 were cis-expression quantitative trait loci (cis-eQTL) for TNFSF8 including SNPs located within the TNFSF15 gene. In the Brazilian sample, 18 of these cis-eQTL SNPs overlapping the TNFSF8 gene were validated for association with T1R. CONCLUSIONS: Taken together, these results indicate TNFSF8 and not TNFSF15 as an important T1R susceptibility gene. Our data support the need for infection genetics to go beyond genes for pathogen control to explore genes involved in a commensurate host response.

Synthesis and evaluation of novel dapsone-thalidomide hybrids for the treatment of type 2 leprosy reactions.

We synthesized a series of novel dapsone-thalidomide hybrids (3a-i) by molecular hybridization and evaluated their potential for the treatment of type 2 leprosy reactions. All of the compounds had analgesic properties. Compounds 3c and 3h were the most active antinociceptive compounds and reduced acetic acid-induced abdominal constrictions by 49.8% and 39.1%, respectively. The hybrid compounds also reduced tumor necrosis factor-α levels in lipopolysaccharide-stimulated L929 cells. Compound 3i was the most active compound; at concentrations of 15.62 and 125 µM, compound 3i decreased tumor necrosis factor-α levels by 86.33% and 87.80%, respectively. In nude mice infected with Mycobacterium leprae in vivo, compound 3i did not reduce the number of bacilli compared with controls. Compound 3i did not have mutagenic effects in Salmonella typhimurium strains TA100 and TA102, with or without metabolic activation (S9 mixture). Our results indicate that compound 3i is a novel lead compound for the treatment of type 2 leprosy reactions.

Whole blood transcriptomics reveals the enrichment of neutrophil activation pathways during erythema nodosum leprosum reaction.

Introduction: Patients with the multibacillary form of leprosy can develop reactional episodes of acute inflammation, known as erythema nodosum leprosum (ENL), which are characterized by the appearance of painful cutaneous nodules and systemic symptoms. Neutrophils have been recognized to play a role in the pathogenesis of ENL, and recent global transcriptomic analysis revealed neutrophil-related processes as a signature of ENL skin lesions. Methods: In this study, we expanded this analysis to the blood compartment, comparing whole blood transcriptomics of patients with non-reactional lepromatous leprosy at diagnosis (LL, n=7) and patients with ENL before administration of anti-reactional treatment (ENL, n=15). Furthermore, a follow-up study was performed with patients experiencing an ENL episode at the time of diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Validation in an independent cohort (ENL=8; LL=7) was performed by RT-qPCR. Results: An enrichment of neutrophil activation and degranulation-related genes was observed in the ENL group, with the gene for the neutrophil activation marker CD177 being the most enriched gene of ENL episode when compared to its expression in the LL group. A more pro-inflammatory transcriptome was also observed, with increased expression of genes related to innate immunity. Validation in an independent cohort indicated that S100A8 expression could discriminate ENL from LL. Supernatants of blood cells stimulated in vitro with Mycobacterium leprae sonicate showed higher levels of CD177 compared to the level of untreated cells, indicating that the leprosy bacillus can activate neutrophils expressing CD177. Of note, suggestive higher CD177 protein levels were found in the sera of patients with severe/moderate ENL episodes when compared with patients with mild episodes and LL patients, highlighting CD177 as a potential systemic marker of ENL severity that deserves future confirmation. Furthermore, a follow-up study was performed with patients at the time of ENL diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Enrichment of neutrophil pathways was sustained in the transcriptomic profile of patients undergoing treatment; however, important immune targets that might be relevant to the effect of thalidomide at a systemic level, particularly NLRP6 and IL5RA, were revealed. Discussion: In conclusion, our study reinforces the key role played by neutrophils in ENL pathogenesis and shed lights on potential diagnostic candidates and novel therapeutic targets that could benefit patients with leprosy.

Time between symptom and testing in relation to familial transmission of severe acute respiratory syndrome coronavirus 2.

Brazil has a huge number of cases and deaths due to coronavirus disease 2019 (COVID-19); however, few studies have dealt with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among familial contacts in Brazil. Here, we report our findings on transmission in a family-based study in Bauru, São Paulo, Brazil. The study, conducted from July to November 2020, comprised 974 individuals with 233 index patients and 741 familial contacts. Familial contacts were evaluated using the rapid COVID-19 Ag ECO and reverse transcription-polymerase chain reaction (RT-PCR) tests immediately after the index patient diagnosis. The antigen-based rapid test was validated in 121 individuals using RT-PCR as the gold standard. Additionally, 30 days later, familial contacts were evaluated for IgM and IgG antibodies against SARS-CoV-2. We found 333 cases of COVID-19 among familial contacts (44.9%). A positive correlation was observed between the time elapsed from the onset of symptoms until the index patient's COVID-19 testing and the number of family contacts infected by SARS-CoV-2. Early SARS-CoV-2 testing and familial contact evaluation are relevant strategies to contain transmission.

Prediction of the occurrence of leprosy reactions based on Bayesian networks.

Introduction: Leprosy reactions (LR) are severe episodes of intense activation of the host inflammatory response of uncertain etiology, today the leading cause of permanent nerve damage in leprosy patients. Several genetic and non-genetic risk factors for LR have been described; however, there are limited attempts to combine this information to estimate the risk of a leprosy patient developing LR. Here we present an artificial intelligence (AI)-based system that can assess LR risk using clinical, demographic, and genetic data. Methods: The study includes four datasets from different regions of Brazil, totalizing 1,450 leprosy patients followed prospectively for at least 2 years to assess the occurrence of LR. Data mining using WEKA software was performed following a two-step protocol to select the variables included in the AI system, based on Bayesian Networks, and developed using the NETICA software. Results: Analysis of the complete database resulted in a system able to estimate LR risk with 82.7% accuracy, 79.3% sensitivity, and 86.2% specificity. When using only databases for which host genetic information associated with LR was included, the performance increased to 87.7% accuracy, 85.7% sensitivity, and 89.4% specificity. Conclusion: We produced an easy-to-use, online, free-access system that identifies leprosy patients at risk of developing LR. Risk assessment of LR for individual patients may detect candidates for close monitoring, with a potentially positive impact on the prevention of permanent disabilities, the quality of life of the patients, and upon leprosy control programs.

Lobomycosis Epidemiology and Management: The Quest for a Cure for the Most Neglected of Neglected Tropical Diseases.

Lobomycosis is a chronic disease caused by Lacazia loboi, which is endemic to the Amazon rainforest, where it affects forest dwellers in Brazil. There is no disease control program and no official therapeutic protocol. This situation contributes to an unknown disease prevalence and unmet needs of people disabled by this disease who seek access to treatment. This review provides an update on the subject with an emphasis on therapeutic advances in humans. All relevant studies that addressed epidemiology, diagnosis, or therapeutics of lobomycosis were considered. Seventy-one articles published between 1931 and 2021 were included for a narrative literature review on the epidemiology and quest for a cure. An effective therapy for lobomycosis has been found following decades of research led by the State Dermatology Program of Acre in the Amazon rainforest, where the largest number of cases occur. This discovery opened new avenues for future studies. The main recommendations here, addressed to the Brazilian Ministry of Health, are for lobomycosis to become a reportable disease to ensure that disease prevalence is measured, and that it be prioritized such that affected individuals may access treatment free-of-charge.

Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America.

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

Detection of cytokines and nitric oxide synthase in skin lesions of Jorge Lobo's disease patients.

Studies investigating the immunopathological aspects of Jorge Lobo's disease have shown that the inflammatory infiltrate consists mainly of histiocytes and multinucleated giant cells involving numerous yeast-like cells of Lacazia loboi, with the T lymphocytes more common than B lymphocytes and plasma cells. The quantification of cytokines in peripheral blood mononuclear cells culture supernatant has revealed alterations in the cytokines profile, characterized by predominance of a Th2 profile. In view of these findings and of the role of cytokines in cell interactions, the objective of the present study was to investigate the presence of the cytokines IL-10, TGF-ß1 and TNF-α, as well as iNOS enzyme in granulomas induced by L. loboi. Histological sections obtained from skin lesions of 16 patients were analyzed by immunohistochemistry for the presence of these cytokines and iNOS. The results showed that TGF-ß1 was the cytokine most frequently expressed by cells present in the inflammatory infiltrate, followed by IL-10. There was a minimum to discrete positivity of cells expressing TNF-α and iNOS. The results suggest that the presence of immunosuppressive cytokines in skin lesions of patients with the mycosis might be responsible for the lack of containment of the pathogen as demonstrated by the presence of numerous fungi in the granuloma.

In vitro and skin lesion cytokine profile in Brazilian patients with borderline tuberculoid and borderline lepromatous leprosy.

OBJECTIVE: We investigated the in vitro and skin lesions production of cytokines in non-treated borderline tuberculoid (BT) and borderline lepromatous (BL) patients. PATIENTS AND METHODS: Seven untreated, non-reactional BT patients and 12 untreated, non-reactional BL patients were studied. Levels of the cytokines IFN-gamma, IL-10, TGF-beta1 and TNF-alpha were measured in supernantant of peripheral blood mononuclear cells (PBMC) cultures, stimulated with specific M. leprae antigen (sonicated and whole). The cytokines iNOS, IL-10 and TGF-beta1 were detected by immunohistochemistry in skin biopsies. RESULTS: BT patients produced higher levels of IFN-gamma than BL patients; iNOS expression in skin lesions was also higher in BT patients. TGF-beta1 was detected in more cells in BL patients; IL-10 expression was similar in both groups. There was a negative correlation between iNOS and TGF-beta1 expression in skin biopsies, positive correlation between TGF-beta1 in skin lesions and bacillary index, as well as positive correlation between iNOS detected in skin biopsies and PBMC IFN-gamma production. CONCLUSIONS: The BT patients had a mainly a Th1-profile of cytokines in their skin lesions and BL patients had a Th2 profile.

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.

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences.

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.