High-Resolution Plasma Metabolomics Identifies Alterations in Fatty Acid, Energy, and Micronutrient Metabolism in Adults Across the Leprosy Spectrum.

BACKGROUND: High-resolution metabolomics (HRM) is an innovative tool to study challenging infectious diseases like leprosy, where the pathogen cannot be grown with standard methods. Here, we use HRM to better understand associations between disease manifestations, nutrition, and host metabolism. METHODS: From 2018 to 2019, adults with leprosy and controls were recruited in Minas Gerais, Brazil. Plasma metabolites were detected using an established HRM workflow and characterized by accurate mass, mass to charge ratio m/z and retention time. The mummichog informatics package compared metabolic pathways between cases and controls and between multibacillary (MB) and paucibacillary (PB) leprosy. Additionally, select individual metabolites were quantified and compared. RESULTS: Thirty-nine cases (62% MB and 38% PB) and 25 controls were enrolled. We found differences (P < .05) in several metabolic pathways, including fatty acid metabolism, carnitine shuttle, retinol, vitamin D3, and C-21 steroid metabolism, between cases and controls with lower retinol and associated metabolites in cases. Between MB and PB, leukotrienes, prostaglandins, tryptophan, and cortisol were all found to be lower in MB (P < .05). DISCUSSION: Metabolites associated with several nutrient-related metabolic pathways appeared differentially regulated in leprosy, especially MB versus PB. This pilot study demonstrates the metabolic interdependency of these pathways, which may play a role in the pathophysiology of disease.

Models for predicting the risk of illness in leprosy contacts in Brazil: Leprosy prediction models in Brazilian contacts.

OBJECTIVE: This study aims to develop and validate predictive models that assess the risk of leprosy development among contacts, contributing to an enhanced understanding of disease occurrence in this population. METHODS: A cohort of 600 contacts of people with leprosy treated at the National Reference Center for Leprosy and Health Dermatology at the Federal University of Uberlândia (CREDESH/HC-UFU) was followed up between 2002 and 2022. The database was divided into two parts: two-third to construct the disease risk score and one-third to validate this score. Multivariate logistic regression models were used to construct the disease score. RESULTS: Of the four models constructed, model 3, which included the variables anti-phenolic glycolipid I immunoglobulin M positive, absence of Bacillus Calmette-Guérin vaccine scar and age ≥60 years, was considered the best for identifying a higher risk of illness, with a specificity of 89.2%, a positive predictive value of 60% and an accuracy of 78%. CONCLUSIONS: Risk prediction models can contribute to the management of leprosy contacts and the systematisation of contact surveillance protocols.

Clinical factors associated with systemic sporotrichosis in Brazil.

BACKGROUND: Systemic sporotrichosis occurs when organs, other than subcutaneous tissues and lymph nodes, are infected. Interestingly, systemic sporotrichosis in apparently immunocompetent individuals is increasing in Brazil, but data on clinical manifestations and risk factors are scarce. Most of the existing data on such condition relate to people living with HIV. We aimed to study the risk factors associated with systemic sporotrichosis among HIV-negative and HIV-positive patients. METHODS: We performed a retrospective cross-sectional study with 80 patients from Brazil, diagnosed between 2014 and 2021. The association between disease classification, clinical presentation and risk factors were analysed by logistic regression. RESULTS: Of the 80 patients, 29 (36.3%) presented with systemic sporotrichosis. All HIV-positive patients developed the systemic form, with increased mortality (p = .002). Alcohol ingestion (p = .009) and diabetes (p = .010) were associated with systemic disease. Alcohol ingestion seemed to favour pulmonary infection (p = .013) and, diabetes favoured osteoarticular (p = .009) and ocular involvement (p = .033). The occurrence of fever (p = .001) and weight loss (p = .006) were significantly associated with systemic sporotrichosis, while meningeal involvement (p = .001) increased mortality risk. Nine (11.3%) patients died from sporotrichosis. The presence of fungal structures in the mycological examination of the patients' lesions were associated with the systemic form (p = .017) and death (p = .002). CONCLUSION: Our study points to the factors that drive systemic sporotrichosis other than HIV, such as alcohol ingestion and diabetes. Considering the high number of patients presenting severe sporotrichosis, the recognising these factors may contribute to timely diagnosis and proper treatment.

Hypovitaminosis D and reduced cathelicidin are strongly correlated during the multidrug therapy against leprosy.

Mycobacterium leprae infection depends on the competence of the host immune defense to induce effective protection against this intracellular pathogen. The present study investigated the serum levels of vitamin D and the antimicrobial peptide cathelicidin, to determine the statistical correlation between them in leprosy patients before and post-six months of multidrug therapy (MDT), household contacts, and healthy individuals. Previous studies associated these molecules with high risks to develop mycobacterial diseases, such as tuberculosis and leprosy. A total of 34 leprosy patients [paucibacillary (n = 14), multibacillary (n = 20)], and 25 household contacts were recruited. Eighteen healthy adults were selected as a control group. Serum concentrations of vitamin D (25(OH)VD3) and cathelicidin were measured using high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit, respectively. There were no significant differences in serum levels of 25(OH)VD3 between all groups, and the overall prevalence rate of vitamin D deficiency was 67.1%. Cathelicidin levels were significantly lower in both untreated and treated patients when compared to controls and household contacts (p < 0.05). Strong correlations between hypovitaminosis D and reduced cathelicidin in untreated (r = 0.86) and post-six months of MDT (r = 0.79) leprosy patients were observed. These results suggest that vitamin D status and cathelicidin levels are strongly correlated during multidrug therapy for leprosy and nutritional supplementation from the beginning of treatment could strengthen the immune response against leprosy.

Biotechnological applications from a Leishmania amastigote-specific hypothetical protein in the canine and human visceral leishmaniasis.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a rapid and precise diagnosis of the disease should be performed, mainly to treat patients as soon as possible, aiming to reduce the treatment time and the toxicity of the therapeutics. In the present study, the diagnostic role of an amastigote-specific Leishmania protein was evaluated in the canine and human VL. Results showed that the recombinant protein (called rLiHyJ) and one specific B cell epitope (called PeptJ) predicted from protein sequence presented high sensitivity and specificity values to diagnose canine and human disease, showing also a low reactivity against cross-reactive samples. The rA2 protein and a parasite antigenic extract showed variable sensitivity and/or specificity values in the ELISA experiments. A prognostic evaluation of protein and peptide in the human VL indicated that specific IgG antibodies significantly decreased after treatment, when compared to be values obtained before therapy. The in vitro immunogenicity using rLiHyJ in peripheral blood mononuclear cell (PBMC) cultures collected of such patients and healthy subjects suggested that the protein induced lymphoproliferation and high IFN-γ production in the stimulated cells. In conclusion, although preliminary, results suggest that rLiHyJ and PeptJ could present distinct biotechnological applications in the canine and human VL.

A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease.

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.

Recombinant Leishmania eukaryotic elongation factor-1 beta protein: A potential diagnostic antigen to detect tegumentary and visceral leishmaniasis in dogs and humans.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

Cytokines as biomarkers to monitoring the impact of multidrug therapy in immune response of leprosy patients.

Leprosy or Hansen's disease is a chronic infectious disease of the skin and nerves, caused by the intracellular bacilli Mycobacterium leprae. It is characterized by a spectrum of clinical forms depending on the host's immune response to M. leprae. Patients with tuberculoid (TT) leprosy have strong cell-mediated immunity (CMI) with elimination of the bacilli, whereas patients with lepromatous (LL) leprosy exhibit defective CMI to M. leprae. Despite advances in the understanding of the pathogenesis of leprosy and the development of new therapeutic strategies, there is a need for the identification of biomarkers which be used for early diagnosis and to discrimination between different forms of the disease, as prognostic markers. Here, we analyzed the serum levels of IL-1ß, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-γ and TNF in order to address the contribution of these cytokines in late phase of M. leprae infection, and the impact of multidrug therapy (MDT). Our results demonstrated that patients of LL group presented higher expression of serum levels of inflammatory cytokines before MDT, while TT patients presented a balance between inflammatory and regulatory cytokines. MDT changes the profile of serum cytokines in M. leprae infected patients, as evidenced by the cytokine network, especially in TT patients. LL patients displayed a multifaceted cytokine system characterized by strong connecting axes involving inflammatory/regulatory molecules, while TT patients showed low involvement of regulatory cytokines in network overall. Cytokines can be identified as good biomarkers of the impact of MDT on the immune system and the effectiveness of treatment.

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment.

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

rMELEISH: A Novel Recombinant Multiepitope-Based Protein Applied to the Serodiagnosis of Both Canine and Human Visceral Leishmaniasis.

BACKGROUND: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem. METHODS: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of L. infantum infection using canine (n = 140) and human (n = 145) sera samples. RESULTS: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA). CONCLUSIONS: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL.

Factors associated with the development of leprosy in Brazilian contacts: a systematic review.

People who interact with leprosy patients in their environment, neighborhood, family, or social relationships are at risk to develop the disease. This systematic review investigated the risk and protective factors associated with the development of leprosy in Brazilian contacts. The studies were found in Cochrane Library, PubMed (MEDLINE), Embase, Virtual Health Library, grey literature and hand search until July 2021. The study selection, data extraction and quality assessment were independently performed by two investigators. The quality assessment was performed using the Newcastle-Ottawa Scale (NOS). This review was registered in PROSPERO (CRD42020160680). Seventeen articles fulfilled the inclusion criteria (n=544). The immunological and molecular factors, such as Anti-phenolic Glycolipid Antibodies (Anti-PGL-1) seropositivity, negative Mitsuda test, absence of Bacillus Calmette-Guérin (BCG) scar, positive Polymerase Chain Reaction (PCR) in blood; age and race; conviviality, education, contact time and type of contact, as well as elements related to the index case (bacilloscopic index; genetic conditions, family relationships), and some combined factors were shown to be relevant risk factors associated with the development of the disease in Brazilian leprosy contacts. The protective factors reported were the presence of one or more BCG scars, positive Mitsuda test, and education level. All selected studies were considered of high quality according to NOS. The knowledge of disease-related risk and protective factors provides the scientific basis for decision-making in the management of the disease in leprosy contacts.

Associations between occupation, leprosy disability and other sociodemographic factors in an endemic area of Brazil.

BACKGROUND: In Brazil, new leprosy cases with grade-2 disability (G2D) have been increasing. Physical disability has been associated with experienced stigmatization, psychological distress, and social restriction. OBJECTIVES: To identify factors associated with leprosy disability in an endemic area of Brazil focusing on occupational and other sociodemographic factors. METHODS: Between July and December 2015, adult patients with multibacillary leprosy who attended a clinic in Belo Horizonte, Brazil were enrolled. Social, clinical, and demographic factors were collected from an administered questionnaire and medical charts. Occupations were categorized as manual vs non-manual. Descriptive statistics and multivariable logistic regression were performed to study associated factors with disability (Grade 1 disability (G1D) and G2D combined). FINDINGS: Seventy-three patients were enrolled with 48 (65.8%) presenting with either G1D or G2D at the time of enrollment. Twenty-nine (39.7%) had G2D. About half of the patients (n = 36, 49%) reported a manual labor occupation and reactions were common (n = 53, 73%). On univariate analyses, older age (p = 0.048) and low education (p = 0.007) were associated with disability. On multivariable analyses, only low education (primary or less) was associated with disability (OR = 6.34, 95% CI 1.37, 29.26). Greater distance from clinic, income, smoking, marital status, and occupation were not associated. MAIN CONCLUSIONS: Low education was associated with leprosy disability, consistent with prior studies, and therefore should be a focus for disability reduction programs. While the sample size of this study may have limited detection of associations between disability and social determinants tested, half of the patients reported a manual job, highlighting the need for more extensive studies on associations between occupation, disability, and related injuries.

Reduced vitamin D receptor (VDR) and cathelicidin antimicrobial peptide (CAMP) gene expression contribute to the maintenance of inflammatory immune response in leprosy patients.

Leprosy is an infectious disease influenced by genetic, immunological, and environmental factors. Reduced gene expressions may be associated with the immunological response pattern and leprosy susceptibility. We investigated the direct and indirect effects of Vitamin D Receptor (VDR) and Cathelicidin Antimicrobial Peptide (CAMP) gene expressions on the serum levels of vitamin D, Cathelicidin, and cytokines in newly-diagnosed leprosy patients and post-six-months of multidrug therapy (MDT). Thirty-four leprosy patients were assessed, paucibacillary (PB; n = 14) and multibacillary (MB; n = 20) cases, untreated or having received six months of MDT, 18 healthy controls, and 25 household contacts. VDR and CAMP gene expression levels were strongly correlated to some important cytokines in both, untreated leprosy patients (PB, r = 0.9319; MB, r = 0.9569) and patients who had undergone MDT (PB, r = 0.9667; MB, r = 0.9569). We observed that both gene expressions directly influenced IL-2, IFN-γ, and IL-17F serum levels in leprosy patients compared to the household contacts and healthy individuals. VDR and CAMP gene expressions induced a persistent inflammatory response in PB and MB leprosy patients, even after six months of MDT, to fight the Mycobacterium leprae infection. Due to the persistent inflammatory profile, multidrug therapy is suggested to be maintained for more than six months, especially for MB patients. Vitamin D supplementation is recommended from the onset as a transcription factor to improve VDR and CAMP gene expression in leprosy patients.

Risk factors for physical disability upon release from multidrug therapy in new cases of leprosy at a referral center in Brazil.

The present study sought to investigate the risk factors for physical disability upon release from multidrug therapy (MDT) in new cases of leprosy, registered at a referral center in Brazil. This is a longitudinal and retrospective study that evaluated 260 patients. Multivariate analyses, using both the ordinal logistic regression, as well as the classification and regression tree (CART) algorithm were performed to determine the factors associated with physical disability upon release from treatment. The prevalence of disability did not differ significantly between diagnosis and release from treatment. Number of affected nerves and sensory impairment upon diagnosis were risk factors for disability at the end of MDT. The analysis using the CART algorithm resulted in the development of a clinical score to predict the risk of disability upon release from MDT. The decision tree may have a direct applicability in clinical practice for professionals dealing with leprosy, as it allows them to identify patients with a higher risk of physical disability through the use of simple and widely available clinical tests. This study also shows that the disability grade upon admission is the main risk factor for disability upon release from MDT. This result draws attention to the importance of early diagnosis in disability prevention.

Reading of the Mitsuda test: comparison between diameter and total area by means of a computerized method.

The Mitsuda test is a skin test based on the individual's immune response through late and highly specific hypersensitivity reaction to the Mycobacterium leprae bacillus. A negative reaction identifies individuals who present a higher risk of becoming ill if exposed to M. leprae and, if they become ill, to develop the virchowian form of disease. The Mitsuda test reading is performed by means of a millimeter ruler. The dermatoscopy is a method that has not been used in the evaluation of cutaneous tests, although its use has increased in several areas. The study aimed to compare the results between the standardized reading and the total area of the Mitsuda test obtained by a computerized method which was structured by the association of digital dermatoscopy, the Dermatology Web system and the Image Tool 3.0 software. Data collection was performed at the Dermatology Outpatient Clinic of the Eduardo de Menezes Hospital, in Belo Horizonte, from November 2015 to August 2016. The sample consisted of 100 leprosy domiciliary contacts. There was an excellent agreement between the Mitsuda test (diameter and area), with a coefficient greater than 80%, and an excellent correlation with the Spearman's correlation coefficient (0.936). The intraclass correlation coefficient indicated a low (0.219) but significant agreement between the two measurements. In conclusion, there is a significant correlation between the standardized reading and the total area of the Mitsuda test. Digital dermoscopy can be an alternative instrument of evaluation, allowing the computerization and recording of the Mitsuda test.

Deep Venous Thrombosis in Patients with Erythema Nodosum Leprosum in the Use of Thalidomide and Systemic Corticosteroid in Reference Service in Belo Horizonte, Minas Gerais.

INTRODUCTION: Erythema nodosum leprosum (ENL) is a type of lepra reaction treated with corticosteroids and thalidomide, but this association increases the risk of deep venous thrombosis (DVT). OBJECTIVE: To report cases of ENL with DVT in the use of thalidomide/corticosteroid associated. METHODOLOGY: The study was conducted between December 2015 and December 2016 at the Eduardo de Menezes Hospital (HEM-FHEMIG). RESULTS: A clinical case series of 16 patients, eight from HEM-FHEMIG and eight from the literature. DVT occurred on 4 continents, mainly in adults and men. All patients were multibacillary; four people had pulmonary embolism (PE); there were 11 unilateral and five bilateral DVT cases; 12 cases were proximal, two distal, and two unspecified. Pharmacological thromboprophylaxis was used on two individuals. Outcome after DVT, 14 patients improved, one had sequelae, and one died. DISCUSSION: DVT increased in association with thalidomide/corticosteroid in multiple myeloma, but this complication is poorly described in ENL. In proximal DVT, there was a greater risk of PE and sequelae venous insufficiency. After DVT, start anticoagulation. ASA 100mg/day as prophylaxis for DVT in case of this drug association in ENL is recommended. CONCLUSION: The article illustrates the incidence increase of DVT because of the thalidomide/corticosteroid combination in ENL. When this association is necessary, use ASA 100mg/day as prophylaxis.

The Burden of Helminth Coinfections and Micronutrient Deficiencies in Patients with and without Leprosy Reactions: A Pilot Study in Minas Gerais, Brazil.

Leprosy reactions are immune-mediated complications occurring in up to 50% of patients. The immune consequences of helminth infections and micronutrient deficiencies suggest a potential role in type 1 reactions (T1R) or type 2 reactions (T2R). We conducted a case-control study in Minas Gerais, Brazil, to evaluate whether comorbidities and other factors are associated with reactions in patients with multibacillary leprosy. Stool and serum were tested for helminth infections. Deficiencies of vitamin A, D, and iron were measured using serum retinol, 25-hydroxyvitamin D, and ferritin, respectively. Logistic regression models identified associations between reactions and helminth infections, micronutrient deficiencies, and other variables. Seventy-three patients were enrolled, 24 (33%) with T1R, 21 (29%) with T2R, 8 (15%) with mixed T1R/T2R, and 20 (27%) without reactions. Evidence of helminth infections were found in 11 participants (15%) and included IgG4 reactivity against Schistosoma mansoni, Strongyloides, and Ascaris antigens. Thirty-eight (52%) had vitamin D deficiency, eight (11%) had vitamin A insufficiency, 21 (29%) had anemia, and one (1.4%) had iron deficiency. Multivariable logistic regression showed no statistically significant associations between helminth coinfections and total reactions (adjusted odds ratios [aOR]: 1.36, 95% CI: 0.22, 8.33), T1R (aOR: 0.85, 95% CI: 0.17, 4.17), or T2R (aOR: 2.41, 95% CI: 0.29, 20.0). Vitamin D deficiency and vitamin A insufficiency were also not statistically associated with reactions. However, vitamin deficiencies and helminth infections were prevalent in these patients, suggesting a potential role for additional treatment interventions. Studying reactions prospectively may further clarify the role of comorbidities in the clinical presentation of leprosy.

A biomarker for tegumentary and visceral leishmaniasis based on a recombinant Leishmania hypothetical protein.

The measures for leishmaniasis control include the precise diagnosis of disease. However, although several recombinant antigens have been tested with this biotechnological purpose, no effective product exists, which could detects patients with the active disease, as well as differentiates them from cured and treated patients. In this study, a conserved Leishmania hypothetical protein, which was identified in Leishmania infantum parasites, but evaluated to presents high homology in the amino acid sequences between distinct parasite species, was evaluated for the diagnosis of tegumentary and visceral leishmaniasis. In addition, PBMCs collected from treated and untreated mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) patients, as well as in healthy subjects living in endemic region of disease, were in vitro stimulated, when IFN-γ, IL-4 and IL-10 levels were evaluated in the cell supernatant. Regarding the serological analyses, ELISA experiments using the recombinant protein (rLiHyL) and a human serological panel revealed high sensitivity and specificity values to detect both diseases, while control antigens showed worst results. Regarding the cellular response, results showed that rLiHyL-stimulated cells produced higher IFN-γ and lower IL-4 and IL-10 levels in the supernatants. Also, the anti-protein antibody production was evaluated in these patients, and data showed higher IgG2 and lower IgG1 levels found in the treated patients and healthy controls, demonstrating the stimulation of a Th1-type response induced by the rLiHyL protein. In conclusion, this hypothetical protein can be considered as antigenic in TL and VL, as well as a vaccine candidate to be tested in future studies to protect against disease.

A comparison of ML Flow serology and slit skin smears to assess the bacterial load in newly diagnosed leprosy patients in Brazil.

INTRODUCTION: The ML Flow test is an immunochromatographic assay that detects IgM antibodies against M. leprae-specific anti-phenolic glycolipid I (PGL-I). In addition to slit skin smears stained by the Ziehl-Neelsen technique, it can be helpful in the operational classification of leprosy patients for treatment purposes. OBJECTIVE: This work studied the relationship between antibody levels as detected by semi-quantitative ML Flow serologic test and bacterial load as quantified by slit skin smear. PATIENTS AND METHODS: 135 patients with newly detected leprosy at the reference service in Sanitary Dermatology in Brazil had slit skin smears (registered as bacillary index - BI) and an ML Flow test (registered qualitatively and semi-quantitatively) performed at admission. A logistic regression and agreement measures (kappa index) were calculated. RESULTS: Slit skin smears were positive in 35.9% of patients and 57% of patients were seropositive for PGL-1 antibodies. Among the seropositive patients, 416% had five or fewer skin lesions, and 65.8% had more than one peripheral nerve involved. Slit skin smears were positive in only three seronegative patients (5.6%), and negative in 41.9% of seropositive patients. Patients with a BI of 4 + had an OR of 33 for being seropositive in comparison to those with a low BI. CONCLUSIONS: There is a correlation between serologic test and slit skin smear results. Therefore, an ML Flow test may become a useful tool in the clinical classification of leprosy, besides slit skin smears, which require a proper laboratory infrastructure and experienced personnel.