Interplay among differential exposure to Mycobacterium leprae and TLR4 polymorphism impacts the immune response in household contacts of leprosy patients.

Introduction: The aim of the present study was to investigate the association between the single nucleotide polymorphism (SNP) rs1927914 A/G in TLR4 gene and the immunological profile of household contacts (HHC) of leprosy patients. Leprosy classification is usually complex and requires the assessment of several clinical and laboratorial features. Methods: Herein, we have applied distinct models of descriptive analysis to explore qualitative/quantitative changes in chemokine and cytokine production in HHC further categorized according to operational classification [HHC(PB) and HHC(MB)] and according to TLR4SNP. Results and discussion: Our results showed that M. leprae stimuli induced an outstanding production of chemokines (CXCL8;CCL2; CXCL9; CXCL10) by HHC(PB), while increase levels of pro-inflammatory cytokines (IL-6; TNF; IFN-γ; IL-17) were observed for HHC(MB). Moreover, the analysis of chemokine and cytokine signatures demonstrated that A allele was associated with a prominent soluble mediator secretion (CXCL8; CXCL9; IL-6; TNF; IFN-γ). Data analysis according to TLR4 SNP genotypes further demonstrated that AA and AG were associated with a more prominent secretion of soluble mediators as compared to GG, supporting the clustering of AA and AG genotypes into dominant genetic model. CXCL8, IL-6, TNF and IL-17 displayed distinct profiles in HHC(PB) vs HHC(MB) or AA+AG vs GG genotype. In general, chemokine/cytokine networks analysis showed an overall profile of AA+GA-selective (CXCL9-CXCL10) and GG-selective (CXCL10-IL-6) axis regardless of the operational classification. However, mirrored inverted CCL2-IL-10 axis and a (IFN-γ-IL-2)-selective axis were identified in HHC(MB). CXCL8 presented outstanding performance to classify AA+AG from GG genotypes and HHC(PB) from HHC(MB). TNF and IL-17 presented elevated accuracy to classify AA+AG from GG genotypes and HHC(PB) (low levels) from HHC(MB) (high levels), respectively. Our results highlighted that both factors: i) differential exposure to M. leprae and ii) TLR4 rs1927914 genetic background impact the immune response of HHC. Our main results reinforce the relevance of integrated studies of immunological and genetic biomarkers that may have implications to improve the classification and monitoring of HHC in future studies.

Algorithm Design for a Cytokine Release Assay of Antigen-Specific In Vitro Stimuli of Circulating Leukocytes to Classify Leprosy Patients and Household Contacts.

BACKGROUND: Immunological biomarkers have often been used as a complementary approach to support clinical diagnosis in several infectious diseases. The lack of commercially available laboratory tests for conclusive early diagnosis of leprosy has motivated the search for novel methods for accurate diagnosis. In the present study, we describe an integrated analysis of a cytokine release assay using a machine learning approach to create a decision tree algorithm. This algorithm was used to classify leprosy clinical forms and monitor household contacts. METHODS: A model of Mycobacterium leprae antigen-specific in vitro assay with subsequent cytokine measurements by enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor (TNF), interferon-γ, interleukin 4, and interleukin 10 (IL-10) in culture supernatants of peripheral blood mononuclear cells from patients with leprosy, healthy controls, and household contacts. Receiver operating characteristic curve analysis was carried out to define each cytokine's global accuracy and performance indices to identify clinical subgroups. RESULTS: Data demonstrated that TNF (control culture [CC]: AUC = 0.72; antigen-stimulated culture [Ml]: AUC = 0.80) and IL-10 (CC: AUC = 0.77; Ml: AUC = 0.71) were the most accurate biomarkers to classify subgroups of household contacts and patients with leprosy, respectively. Decision tree classifier algorithms for TNF analysis categorized subgroups of household contacts according to the operational classification with moderate accuracy (CC: 79% [48/61]; Ml: 84% [51/61]). Additionally, IL-10 analysis categorized leprosy patients' subgroups with moderate accuracy (CC: 73% [22/30] and Ml: 70% [21/30]). CONCLUSIONS: Together, our findings demonstrated that a cytokine release assay is a promising method to complement clinical diagnosis, ultimately contributing to effective control of the disease.

Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts.

BACKGROUND: Leprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes: paucibacillary (PB) and multibacillary (MB), closely related to the host immune responses. In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts. METHODS: The cross-sectional study enrolled 49 participants, including 19 patients and 30 healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated and incubated in the presence of Mycobacterium leprae bacilli. The cells were prepared for surface (CD4+ and CD8+) and intracytoplasmic cytokine staining (IFN-γ, IL-4 and IL-10). Multiple comparisons amongst groups were carried out by ANOVA, Kruskal-Wallis, Student T or Mann-Whitney test. Comparative analysis of categorical variables was performed by Chi-square. Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels. RESULTS: The cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets, with increased frequency of IFN-γ+ T-cell subsets along with IL-10+ and IL-4+ from CD4+ T-cells, as compared to health Controls (Venn diagram report). Moreover, statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons, according to the data normality distribution. L(PB) displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+, IL-10+ and IL-4+ produced by most T-cell subsets, as compared to L(MB) that presented a more restricted cytokine functional profile mediated by IL-10+ and IL-4+ T-cells with minor contribution of IFN-γ produced by CD4+ T-cells. Noteworthy was that HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB) that presented a cytokine profile limited to IL-10 and IL-4. CONCLUSIONS: Our data demonstrated that L(PB) displayed enhanced percentage of IFN-γ+, IL-10+ and IL-4+ as compared to L(MB) that presented functional profile mediated by IL-10+ and IL-4+ T-cells and HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB). Together, our findings provide additional immunological features associated with leprosy and household contacts. These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.

High frequency of M. leprae DNA detection in asymptomatic household contacts.

BACKGROUND: Characterization of the Mycobacterium leprae genome has made possible the development of Polymerase Chain Reaction (PCR) systems that can amplify different genomic regions. Increased reliability and technical efficiency of quantitative PCR (qPCR) makes it a promising tool for early diagnosis of leprosy. Index cases that are multibacillary spread the bacillus silently, even before they are clinically diagnosed. Early detection and treatment could prevent transmission in endemic areas. METHODS: In this study, the qPCR technique is used to detect DNA of M. leprae in samples of slit skin smears (SSS) of the ear lobe and blood of leprosy patients and their asymptomatic household contacts residing in Governador Valadares, MG, Brazil, a hyperendemic area for leprosy. A total of 164 subjects participated in the study: 43 index cases, 113 household contacts, and, as negative controls, 8 individuals who reported no contact with patients nor history of leprosy in the family. The qPCR was performed to amplify 16S rRNA fragments and was specifically designed for M. leprae. RESULTS: Of asymptomatic household contacts, 23.89% showed bacillary DNA by qPCR in samples of SSS and blood. Also, 48.84% of patients diagnosed with leprosy were positive for qPCR while the bacillary load was positive in only 30.23% of patients. It is important to note that most patients were already receiving treatment when the collection of biological material for qPCR was performed. The level of bacillary DNA from household contacts was similar to the DNA levels detected in the group of paucibacillary patients. CONCLUSION: Considering that household contacts comprise a recognizable group of individuals with a high risk of disease, as they live in close proximity to a source of infection, qPCR can be used to estimate the risk of progress towards leprosy among household contacts and as a routine screening method for a chemoprophylactic protocol.

Utility of immunoglobulin isotypes against LID-1 and NDO-LID for, particularly IgG1, confirming the diagnosis of multibacillary leprosy.

BACKGROUND Leprosy remains a health problem in many countries, with difficulties in diagnosis resulting in delayed treatment and more severe disabilities. Antibodies against several Mycobacterium leprae antigens have, however, shown value as diagnostic and/or prognostic markers. OBJECTIVES The objective of this study was to evaluate serum immunoglobulin (Ig) IgM and IgG subclass reactivity against three M. leprae specific antigens: NDO-HSA, a conjugate formed by natural octyl disaccharide bound to human serum albumin; LID-1, the fusion protein product of the ml0405 and ml2331 genes; and NDO-LID, a combination of LID-1 and NDO. METHODS Sera from healthy controls, paucibacillary (PB) and multibacillary (MB) leprosy patients, and their respective household contacts, were evaluated for the presence of antigen-specific IgM, IgG, and IgG subclass antibodies by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of each ELISA were evaluated by receiver operating characteristic (ROC) curve analysis. FINDINGS Our data confirm that serum IgM antibodies against NDO-HSA and IgG antibodies against LID-1, as well as IgG/M antibodies against NDO-LID, are markedly increased in MB patients. For the first time, our data reveal a selective increase in IgG1 and IgG3 antibodies against LID-1 and NDO-LID in MB patients, demonstrating that these antibody isotypes are suitable for differentiation between MB and PB patients. ROC curve analysis indicates an improved capacity for diagnosing MB leprosy patients using the detection of IgG antibodies, particularly the IgG1 isotype, specific to LID-1 and NDO-LID over the performance levels attained with NDO-HSA. CONCLUSIONS Our findings indicate that serological tests based on the detection of antigen-specific IgG1 antibodies are a useful tool to differentiate MB from PB patients, and indicate the enhanced performance of the LID-1 and NDO-LID antigens in the serodiagnosis of leprosy.

Utility of immunoglobulin isotypes against LID-1 and NDO-LID for, particularly IgG1, confirming the diagnosis of multibacillary leprosy

BACKGROUND Leprosy remains a health problem in many countries, with difficulties in diagnosis resulting in delayed treatment and more severe disabilities. Antibodies against several Mycobacterium leprae antigens have, however, shown value as diagnostic and/or prognostic markers. OBJECTIVES The objective of this study was to evaluate serum immunoglobulin (Ig) IgM and IgG subclass reactivity against three M. leprae specific antigens: NDO-HSA, a conjugate formed by natural octyl disaccharide bound to human serum albumin; LID-1, the fusion protein product of the ml0405 and ml2331 genes; and NDO-LID, a combination of LID-1 and NDO. METHODS Sera from healthy controls, paucibacillary (PB) and multibacillary (MB) leprosy patients, and their respective household contacts, were evaluated for the presence of antigen-specific IgM, IgG, and IgG subclass antibodies by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of each ELISA were evaluated by receiver operating characteristic (ROC) curve analysis. FINDINGS Our data confirm that serum IgM antibodies against NDO-HSA and IgG antibodies against LID-1, as well as IgG/M antibodies against NDO-LID, are markedly increased in MB patients. For the first time, our data reveal a selective increase in IgG1 and IgG3 antibodies against LID-1 and NDO-LID in MB patients, demonstrating that these antibody isotypes are suitable for differentiation between MB and PB patients. ROC curve analysis indicates an improved capacity for diagnosing MB leprosy patients using the detection of IgG antibodies, particularly the IgG1 isotype, specific to LID-1 and NDO-LID over the performance levels attained with NDO-HSA. CONCLUSIONS Our findings indicate that serological tests based on the detection of antigen-specific IgG1 antibodies are a useful tool to differentiate MB from PB patients, and indicate the enhanced performance of the LID-1 and NDO-LID antigens in the serodiagnosis of leprosy.

Dexamethasone abrogates the antimicrobial and antibiofilm activities of different drugs against clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa.

Staphylococcus aureus and Pseudomonas aeruginosa are part of the human microbiota and are also important bacterial pathogens, for which therapeutic options are lacking nowadays. The combined administration of corticosteroids and antimicrobials is commonly used in the treatment of infectious diseases to control inflammatory processes and to minimize potential toxicity of antimicrobials, avoiding sequelae. Although different pharmaceutical dosage forms of antimicrobials combined to corticosteroids are available, studies on the interference of corticosteroids on the pharmacological activity of antimicrobials are scarce and controversial. Here, we provide evidence of the interference of dexamethasone on the pharmacological activity of clinically important antimicrobial drugs against biofilms and planktonic cells of S. aureus and P. aeruginosa. Broth microdilution assays of minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) of gentamicin, chloramphenicol, oxacillin, ceftriaxone and meropenem were conducted with and without the addition of dexamethasone. The effect of all drugs was abrogated by dexamethasone in their MIC, MBC, and MBEC, except gentamicin and meropenem, for which the MBC was not affected in some strains. The present study opens doors for more investigations on in vitro and in vivo effects and safety of the combination of antimicrobials and glucocorticoids.