Pathogen-Specific Epitopes as Epidemiological Tools for Defining the Magnitude of Mycobacterium leprae Transmission in Areas Endemic for Leprosy

During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population

Low rate of relapse after twelve-dose multidrug therapy for hansen's disease: A 20-year cohort study in a brazilian reference center.

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 model for modulation of IFN-γ production during asymptomatic <i>M. leprae</i> infection and active disease.

<p>A model for modulation of IFN-γ production during asymptomatic <i>M. leprae</i> infection and active disease.</p

Leprosy detection rates and responsiveness to <i>M. leprae</i> specific peptides.

<p>Peripheral blood mononuclear cells (PBMCs) from individuals with different levels of exposure to <i>M. leprae</i> were stimulated with 17 <i>M.leprae</i>-specific peptides, and the concentration of IFN-γ measured in culture supernatants. The boxes include response rates of 75% of the sample, and the horizontal bars in bold identify the medians. Points outside the deviation correspond to outliers. ○, Values between 1.5 and 3 box lengths from the upper or lower edge of the box. *, Values more than 3 box lengths from the upper or lower edge of the box. <b>A</b>: A Dutch group of healthy non-endemic controls (NEC_Netherlands); <b>B</b>: healthy controls from Porto Alegre, Brazil (NEC_Brazi); <b>C</b>: healthy endemic controls from an area with medium annual new case detection rate for leprosy (Meireles, Fortaleza, CE, Brazil; EC_low); <b>D</b>: another area with hyperendemic leprosy annual new case detection rate (Bom Jardim, Fortaleza, CE, Brazil; EC_high); <b>E</b>: a Dutch group of tuberculosis patients (TB).</p

Selection of <i>M. leprae</i>-specific peptides.

<p>Fifty-eight <i>M. leprae</i>-specific peptides were previously tested for induction of IFN-γ release by PBMC from leprosy patients and contacts, endemic and non-endemic controls <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001616#pntd.0001616-Spencer1" target="_blank">[13]</a>. The IFN-γ levels induced by the peptides in non-exposed (EC_low) and <i>M. leprae</i>-exposed individuals (HCMB) were used for selecting the best set of peptides allowing discrimination of the exposed group by applying an ANN algorithm. The final 12- peptide ANN made the right choice in 96% of the tests, identifying <i>M.leprae</i>-exposed or non-exposed individuals. *, individuals recruited from the city of Rio de Janeiro.</p

Responsiveness to <i>M. leprae</i>-specific peptides in PB and MB leprosy patients.

<p>The ex vivo stimulation of PBMC was done as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001616#pntd-0001616-g002" target="_blank">Fig. 2</a> legend. <b>A</b>: Paucibacillary (PB); and <b>B</b>: multibacillary (MB) leprosy patients.</p

IFN-γ production in response to pools of <i>M. leprae</i>-specific peptides.

<p>PBMCs from PB (<b>A</b>), HCMB (<b>B</b>), EC_high (<b>C</b>) and EC_low (<b>D</b>) groups stimulated with the 9 mer peptide pool (p52, p61, p68, p69 – 0.1, 1.0 and 10 µg/mL) and the 15 mer peptide pool(p38, p51, p56, p59, p65, p67, p70, p71, p88, p91, p92 - 0.1, 1.0 and 10 µg/ml). After 5 days culture, supernatants were harvested and assessed for levels of IFN-γ by ELISA. Each circle indicates an individual and the dash the median.</p

Baseline characteristics of Brazilian patients and healthy controls.

<p>Baseline characteristics of Brazilian patients and healthy controls.</p