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.

Induction of type 2 responses by schistosome worms during prepatent infection.

During natural schistosome infection, the induction of T helper type 2 (Th2) responses has been ascribed to parasite eggs, because exposure of the host to this life-cycle stage elicits a polarized Th2 response to egg antigens. In the present study, we show that schistosome worms also elicit systemic, antigen-specific type 2 responses during prepatent infection, before egg deposition begins. CD4(+) T cells producing interleukin (IL)-4 were induced by both male and female worms during single-sex infections, demonstrating that this response is independent of exposure to eggs. The Th2 response was accompanied by production of immunoglobulin E and the sensitization of circulating basophils to produce additional IL-4 in response to schistosome antigens. Together, our data show that schistosome worms establish an immunologic milieu where CD4(+) T cells and basophils are both primed to produce IL-4 before eggs are laid, suggesting that worms play a role in establishment of the Th2 response that is critical for host survival and parasite transmission.

Antiproliferative activity of Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum in peripheral blood mononuclear cells.

Several studies indicate Actinobacillus (Haemophilus) actinomycetemcomitans and Fusobacterium nucleatum as etiologic agents of periodontal disease. Immunosuppressive factors produced by microorganisms probably contribute to the initiation and evolution of this disease. This study evaluated the antiproliferative activity of ammonium precipitate fractions of A. (H.) actinomycetemcomitans and F. nucleatum isolates from humans and marmosets both with and without periodontal disease. All A. (H.) actinomycetemcomitans and most F. nucleatum strains inhibited PBMC proliferation in a dose-dependent manner. The degree of cell proliferative inhibition of each bacterial species differed among the strains and was independent of host clinical status. The in vitro inhibition of stimulated lymphocyte proliferation induced by different A. (H.) actinomycetemcomitans and F. nucleatum isolates demonstrated the importance of this phenomenon in bacterial virulence, playing a possible suppressor role in host defense mechanisms in vivo. Moreover, our findings pointed out a marked difference between A. (H.) actinomycetemcomitans and F. nucleatum cytoplasmic extracts in their antiproliferative activity, regarding the antigen concentration required for maximum inhibition and their vulnerability to heating and proteolytic treatment.