Infections in the era of immunobiologicals.

Immunobiologicals represent an innovative therapeutic option in dermatology. They are indicated in severe and refractory cases of different diseases when there is contraindication, intolerance, or failure of conventional systemic therapy and in cases with significant impairment of patient quality of life. The main immunobiologicals used in dermatology basically include inhibitors of tumor necrosis factor-alpha (anti-TNF), inhibitors of interleukin-12 and -23 (anti-IL12/23), inhibitors of interleukin-17 and its receptor (anti-IL17), inhibitors of interleukin-23 (anti-IL23), rituximab (anti-CD20 antibody), dupilumab (anti-IL4/IL13) and intravenous immunoglobulin. Their immunomodulatory action may be associated with an increase in the risk of infections in the short and long term, and each case must be assessed individually, according to the risk inherent to the drug, the patient general condition, and the need for precautions. This article will discuss the main risks of infection associated with the use of immunobiologicals, addressing the risk in immunocompetent and immunosuppressed patients, vaccination, fungal infections, tuberculosis, leprosy, and viral hepatitis, and how to manage the patient in the most diverse scenarios.

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.

Lucius phenomenon: the importance of a primary dermatological care

Abstract Leprosy presents a varied clinical spectrum. Lucius phenomenon is a rare leprosy reaction characterized by erythematous, painful, slightly infiltrated macules and hemorrhagic bullae that progress to ulceration. This case report describes a patient whose diagnosis of leprosy occurred in the presence of Lucius phenomenon. Late diagnosis and delay in the implementation of specific therapy contributed to an unfavorable outcome, highlighting the importance of early identification and treatment of this disease, as well as its complications.

Lucius phenomenon: the importance of a primary dermatological care.

Leprosy presents a varied clinical spectrum. Lucius phenomenon is a rare leprosy reaction characterized by erythematous, painful, slightly infiltrated macules and hemorrhagic bullae that progress to ulceration. This case report describes a patient whose diagnosis of leprosy occurred in the presence of Lucius phenomenon. Late diagnosis and delay in the implementation of specific therapy contributed to an unfavorable outcome, highlighting the importance of early identification and treatment of this disease, as well as its complications.

Human Genetic Susceptibility of Leprosy Recurrence.

Host genetic susceptibility to leprosy has been intensively investigated over the last decades; however, there are no studies on the role of genetic variants in disease recurrence. A previous initiative identified three recurrent cases of leprosy for which none of the M. leprae strains, as obtained in the first and the second diagnosis, had any known genomic variants associated to resistance to Multidrug therapy; in addition, whole genome sequencing indicated that the same M. leprae was causing two out of the three recurrences. Thus, these individuals were suspected of being particularly susceptible to M. leprae infection, either as relapse or reinfection. To verify this hypothesis, 19 genetic markers distributed across 11 loci (14 genes) classically associated with leprosy were genotyped in the recurrent and in three matching non-recurrent leprosy cases. An enrichment of risk alleles was observed in the recurrent cases, suggesting the existence of a particularly high susceptibility genetic profile among leprosy patients predisposing to disease recurrence.

Clinical trial for uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): adverse effects approach.

BACKGROUND: The Clinical Trial for Uniform Multidrug Therapy for Leprosy Patients in Brazil (U-MDT/CT-BR), designed to evaluate the effectiveness of a six-months regimen, assessed the adverse effects caused by the drugs. OBJECTIVE: Describe adverse effects due to MDT in U-MDT/CT-BR, comparing the uniform regimen (U-MDT) to the current WHO regimen (R-MDT). PATIENTS AND METHODS: After operational classification, patients were randomly allocated to the study groups. U-MDT PB and U-MDT MB groups, received the U-MDT regimen, six doses of MB-MDT (rifampicin, dapsone and clofazimine). R-MDT PB and R-MDT MB groups, received the WHO regimens: six doses (rifampicin and dapsone) for PB and 12 doses (rifampicin, dapsone and clofazimine) for MB. During treatment, patients returned monthly for clinical and laboratorial evaluation. Patients with single lesion were not included in this trial. RESULTS: Skin pigmentation (21.7%) and xerosis (16.9%) were the most frequent complaints among 753 patients. Laboratory exams showed hemoglobin concentration lower than 10g/dL in 23.3% of the patients, glutamic oxaloacetic transaminase (GOT) above 40U/L in 29.5% and glutamic pyruvic transaminase (GPT) above 40U/L in 28.5%. Twenty-four patients (3.2%) stopped dapsone intake due to adverse effects, of whom 16.6% due to severe anemia. One case of sulfone syndrome was reported. STUDY LIMITATIONS: Loss of some monthly laboratory sample collection. CONCLUSIONS: There was no statistical difference regarding adverse effects in the R-MDT and U-MDT groups but anemia was greater in patients from R-MDT/MB group, therefore adverse effects do not represent a constraint to recommend the six-month uniform regimen of treatment for all leprosy patients.

Mycobacterium leprae-Specific Antibodies in Multibacillary Leprosy Patients Decrease During and After Treatment With Either the Regular 12 Doses Multidrug Therapy (MDT) or the Uniform 6 Doses MDT.

Leprosy serology reflects the bacillary load of patients and multidrug therapy (MDT) reduces Mycobacterium leprae-specific antibody titers of multibacillary (MB) patients. The Clinical Trial for Uniform Multidrug Therapy Regimen for Leprosy Patients in Brazil (U-MDT/CT-BR) compared outcomes of regular 12 doses MDT/R-MDT and the uniform 6 doses MDT/U-MDT for MB leprosy, both of regimens including rifampicin, clofazimine, and dapsone. This study investigated the impact of R-MDT and U-MDT and the kinetic of antibody responses to M. leprae-specific antigens in MB patients from the U-MDT/CT-BR. We tested 3,400 serum samples from 263 MB patients (R-MDT:121; U-MDT:142) recruited at two Brazilian reference centers (Dona Libânia, Fortaleza, Ceará; Alfredo da Matta Foundation, Manaus, Amazonas). Enzyme-linked immunosorbent assays with three M. leprae antigens [NT-P-BSA: trisaccharide-phenyl of phenollic glycolipid-I antigen (PGL-I); LID-1: Leprosy Infectious Disease Research Institute Diagnostic 1 di-fusion recombinant protein; and ND-O-LID: fusion complex of disaccharide-octyl of PGL-I and LID-1] were performed using around 13 samples per patient. Samples were collected at baseline/M0, during MDT (R-MDT:M1-M12 months, U-MDT:M1-M6 months) and after MDT discontinuation (first, second year). Statistical significance was assessed by the Mann-Whitney U test for comparison between groups (p values < 0.05). Mixed effect multilevel regression analyses were used to investigate intraindividual serological changes overtime. In R-MDT and U-MDT groups, males predominated, median age was 41 and 40.5 years, most patients were borderline lepromatous and lepromatous leprosy (R-MDT:88%, U-MDT: 90%). The bacilloscopic index at diagnosis was similar (medians: 3.6 in the R-MDT and 3.8 in the U-MDT group). In R-MDT and U-MDT groups, a significant decline in anti-PGL-I positivity was observed from M0 to M5 (p = 0.035, p = 0.04, respectively), from M6 to M12 and at the first and second year posttreatment (p < 0.05). Anti-LID-1 antibodies declined from M0 to M6 (p = 0.024), M7 to M12 in the R-MDT; from M0 to M4 (p = 0.003), M5 to M12 in the U-MDT and posttreatment in both groups (p > 0.0001). Anti-ND-O-LID antibodies decreased during and after treatment in both groups, similarly to anti-PGL-I antibodies. Intraindividual serology results in R-MDT and U-MDT patients showed that the difference in serology decay to all three antigens was dependent upon time only. Our serology findings in MB leprosy show that regardless of the duration of the U-MDT and R-MDT, both of them reduce M. leprae-specific antibodies during and after treatment. In leprosy, antibody levels are considered a surrogate marker of the bacillary load; therefore, our serological results suggest that shorter U-MDT is also effective in reducing the patients' bacillary burden similarly to R-MDT. Clinical Trial Registration: ClinicalTrials.gov, NCT00669643.

Clinical trial for uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): adverse effects approach

Abstract: BACKGROUND: The Clinical Trial for Uniform Multidrug Therapy for Leprosy Patients in Brazil (U-MDT/CT-BR), designed to evaluate the effectiveness of a six-months regimen, assessed the adverse effects caused by the drugs. OBJECTIVE: Describe adverse effects due to MDT in U-MDT/CT-BR, comparing the uniform regimen (U-MDT) to the current WHO regimen (R-MDT). Patients and methods: After operational classification, patients were randomly allocated to the study groups. U-MDT PB and U-MDT MB groups, received the U-MDT regimen, six doses of MB-MDT (rifampicin, dapsone and clofazimine). R-MDT PB and R-MDT MB groups, received the WHO regimens: six doses (rifampicin and dapsone) for PB and 12 doses (rifampicin, dapsone and clofazimine) for MB. During treatment, patients returned monthly for clinical and laboratorial evaluation. Patients with single lesion were not included in this trial. RESULTS: Skin pigmentation (21.7%) and xerosis (16.9%) were the most frequent complaints among 753 patients. Laboratory exams showed hemoglobin concentration lower than 10g/dL in 23.3% of the patients, glutamic oxaloacetic transaminase (GOT) above 40U/L in 29.5% and glutamic pyruvic transaminase (GPT) above 40U/L in 28.5%. Twenty-four patients (3.2%) stopped dapsone intake due to adverse effects, of whom 16.6% due to severe anemia. One case of sulfone syndrome was reported. STUDY LIMITATIONS: Loss of some monthly laboratory sample collection. CONCLUSIONS: There was no statistical difference regarding adverse effects in the R-MDT and U-MDT groups but anemia was greater in patients from R-MDT/MB group, therefore adverse effects do not represent a constraint to recommend the six-month uniform regimen of treatment for all leprosy patients.

Genotyping comparison of Mycobacterium leprae isolates by VNTR analysis from nasal samples in a Brazilian endemic region.

This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Ceará. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS.

Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (Tm = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 103-8.02 × 105, and in SB samples from MB patients were 1.87 × 103-1.50 × 106. Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Genotyping of Mycobacterium leprae for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil.

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1-4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.

Uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): Results of an open label, randomized and controlled clinical trial, among multibacillary patients.

BACKGROUND: Leprosy control is based on early diagnosis and multidrug therapy. For treatment purposes, leprosy patients can be classified as paucibacillary (PB) or multibacillary (MB), according to the number of skin lesions. Studies regarding a uniform treatment regimen (U-MDT) for all leprosy patients have been encouraged by the WHO, rendering disease classification unnecessary. METHODOLOGY AND FINDINGS: An independent, randomized, controlled clinical trial conducted from 2007 to 2015 in Brazil, compared main outcomes (frequency of reactions, bacilloscopic index trend, disability progression and relapse rates) among MB patients treated with a uniform regimen/U-MDT (dapsone+rifampicin+clofazimine for six months) versus WHO regular-MDT/R-MDT (dapsone+rifampicin+clofazimine for 12 months). A total of 613 newly diagnosed, untreated MB patients with high bacterial load were included. There was no statistically significant difference in Kaplan-Meyer survival function regarding reaction or disability progression among patients in the U-MDT and R-MDT groups, with more than 25% disability progression in both groups. The full mixed effects model adjusted for the bacilloscopic index average trend in time showed no statistically significant difference for the regression coefficient in both groups and for interaction variables that included treatment group. During active follow up, four patients in U-MDT group relapsed representing a relapse rate of 2.6 per 1000 patients per year of active follow up (95% CI [0·81, 6·2] per 1000). During passive follow up three patients relapsed in U-MDT and one in R-MTD. As this period corresponds to passive follow up, sensitivity analysis estimated the relapse rate for the entire follow up period between 2·9- and 4·5 per 1000 people per year. CONCLUSION: Our results on the first randomized and controlled study on U-MDT together with the results from three previous studies performed in China, India and Bangladesh, support the hypothesis that UMDT is an acceptable option to be adopted in endemic countries to treat leprosy patients in the field worldwide. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00669643.

Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases.

BACKGROUND: Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. METHODOLOGY: DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. PRINCIPAL FINDINGS: In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. CONCLUSIONS/SIGNIFICANCE: This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

Leprosy reactions: The predictive value of Mycobacterium leprae-specific serology evaluated in a Brazilian cohort of leprosy patients (U-MDT/CT-BR).

BACKGROUND: Leprosy reactions, reversal reactions/RR and erythema nodosum leprosum/ENL, can cause irreversible nerve damage, handicaps and deformities. The study of Mycobacterium leprae-specific serologic responses at diagnosis in the cohort of patients enrolled at the Clinical Trial for Uniform Multidrug Therapy Regimen for Leprosy Patients in Brazil/U-MDT/CT-BR is suitable to evaluate its prognostic value for the development of reactions. METHODOLOGY: IgM and IgG antibody responses to PGL-I, LID-1, ND-O-LID were evaluated by ELISA in 452 reaction-free leprosy patients at diagnosis, enrolled and monitored for the development of leprosy reactions during a total person-time of 780,930 person-days, i.e. 2139.5 person-years, with a maximum of 6.66 years follow-up time. PRINCIPAL FINDINGS: Among these patients, 36% (160/452) developed reactions during follow-up: 26% (119/452) RR and 10% (41/452) had ENL. At baseline higher anti-PGL-I, anti-LID-1 and anti-ND-O-LID seropositivity rates were seen in patients who developed ENL and RR compared to reaction-free patients (p<0.0001). Seroreactivity in reactional and reaction-free patients was stratified by bacilloscopic index/BI categories. Among BI negative patients, higher anti-PGL-I levels were seen in RR compared to reaction-free patients (p = 0.014). In patients with 0

Antigen-specific secretion of IFNγ and CXCL10 in whole blood assay detects Mycobacterium leprae infection but does not discriminate asymptomatic infection from symptomatic leprosy.

To advance toward a whole blood assay (WBA)-based test capable of facilitating the diagnosis of paucibacillary (PB) leprosy, we evaluated a prototype in-tube WBA using combinations of Mycobacterium leprae antigens. Blood was collected from newly diagnosed untreated PB (n=38), multibacillary (MB) (n=30), healthy household contacts (HHC) of MB (n=27), and endemic controls (n=61) residing in Goiânia and Fortaleza, Brazil. Blood was incubated with M. leprae cell sonicate, recombinant proteins (46f+LID-1; ML0276+LID-1), or controls (phosphate-buffered saline, phytohemagglutinin, M. tuberculosis purified protein derivative). Antigen-specific IFNγ production was observed in 71-84% and 55% of PB and HHC, respectively. Antigen-specific CXCL10 levels were similarly assessed to determine if, unlike IFNγ, CXCL10 could differentiate PB from HHC with repeated exposure/asymptomatic M. leprae infection. The CXCL10 levels induced in response to M. leprae antigens could not, however, differentiate PB from HHC. Despite these limitations, the WBAs reported here still represent important tools for assessing M. leprae infection rates and evaluating the impact of control measures.

Uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): results of an open label, randomized and controlled clinical trial, among multibacillary patients

BACKGROUND: Leprosy control is based on early diagnosis and multidrug therapy. For treatment purposes, leprosy patients can be classified as paucibacillary (PB) or multibacillary (MB), according to the number of skin lesions. Studies regarding a uniform treatment regimen (U-MDT) for all leprosy patients have been encouraged by the WHO, rendering disease classification unnecessary. METHODOLOGY AND FINDINGS: An independent, randomized, controlled clinical trial conducted from 2007 to 2015 in Brazil, compared main outcomes (frequency of reactions, bacilloscopic index trend, disability progression and relapse rates) among MB patients treated with a uniform regimen/U-MDT (dapsone+rifampicin+clofazimine for six months) versus WHO regular-MDT/R-MDT (dapsone+rifampicin+clofazimine for 12 months). A total of 613 newly diagnosed, untreated MB patients with high bacterial load were included. There was no statistically significant difference in Kaplan-Meyer survival function regarding reaction or disability progression among patients in the U-MDT and R-MDT groups, with more than 25% disability progression in both groups. The full mixed effects model adjusted for the bacilloscopic index average trend in time showed no statistically significant difference for the regression coefficient in both groups and for interaction variables that included treatment group. During active follow up, four patients in U-MDT group relapsed representing a relapse rate of 2.6 per 1000 patients per year of active follow up (95% CI [0·81, 6·2] per 1000). During passive follow up three patients relapsed in U-MDT and one in R-MTD. As this period corresponds to passive follow up, sensitivity analysis estimated the relapse rate for the entire follow up period between 2·9- and 4·5 per 1000 people per year. CONCLUSION: Our results on the first randomized and controlled study on U-MDT together with the results from three previous studies performed in China, India and Bangladesh, support the hypothesis that UMDT is an acceptable option to be adopted in endemic countries to treat leprosy patients in the field worldwide.