ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Efficiency of rLb6H recombinant protein from Leishma-nia (Viannia) braziliensis for the detection of canine visceral leishmaniasis / Eficiência da proteína recombinante rLb6H de Leishmania (Viannia) brazi-liensis na detecção da leishmaniose visceral canina

Introduction: Visceral leishmaniasis (VL) is a serious endemic disease in many tropical and subtropical countries, with a strong incidence in Brazil. The disease is transmitted by the bite of infected female phlebotomine sandflies, with dogs being the main urban reservoirs of the parasite. The diverse clinical profile and the long incubation period are challenges for the diagnosis of canine visceral leishmaniasis (CVL). Recombinant proteins from Leishmania spp. have been studied as antigens that can increase the accuracy of serological tests. Objective: To evaluate the diagnostic performance of the recombinant protein rLb6H, from Leishmania braziliensis, in comparison to the reference antigens rK39 and rK28, from L. donovani, prioritizing the identification of subclinical infected dogs. Material and Methods: Serum IgG reactivity to rLb6H, rK28, and rK39 recombinant proteins was assessed in dogs with previously parasitological confirmation of CVL, subdivided according to their clinical status, using immunoenzymatic assay (ELISA). Diagnostic accuracy of each ELISA was evaluated by receiver operating characteristic (ROC) curve analysis. Results: While all antigens showed a better performance in detecting CVL in symptomatic dogs (SD), detection of CVL in the oligosymptomatic (OD) and asymptomatic (AD) groups was lower, but rLb6H achieved high sensitivity for asymptomatic CVL. Interestingly, the most reactive CVL samples to rK28 were barely detected by rLb6H, while the less reactive to rK28, mostly from the AD group, presented higher reactivity to rLb6H. Conclusion: The recombinant protein rLb6H showed utility in the detection of asymptomatic CVL, displaying a complementary reactivity to rK39 and rK28. Thus, these results suggest that rLb6H could be incorporated into multi-antigen strategies, to increase diagnostic accuracy of CVL.

Introdução: A leishmaniose visceral (LV) é uma doença endêmica grave em muitos países tropicais e subtropicais, tendo forte incidência no Brasil. A doença é transmitida pela picada de flebotomíneos fêmeas infectadas, sendo os cães os principais reservatórios urbanos do parasito. O perfil clínico diversificado e o longo período de incubação são desafios para o diagnóstico da leishmaniose visceral canina (LVC). Proteínas recombinantes de Leishmania spp. têm sido estudadas como antígenos que podem aumentar a precisão de testes sorológicos. Objetivo: Avaliar o desempenho diagnóstico da proteína recombinante rLb6H, de Leishmania braziliensis, em comparação com os antígenos de referência rK39 e rK28, de L. donovani, priorizando a identificação de cães com infecção subclínica. Material e Métodos: A reatividade de anticorpos IgG séricos às proteínas recombinantes rLb6H, rK28 e rK39 foi avaliada em cães com confirmação parasitológica prévia de LVC, subdivididos de acordo com seu quadro clínico, utilizando ensaio imunoenzimático (ELISA). A precisão diagnóstica de cada ELISA foi avaliada pela análise da curva ROC (receiver operating characteristic curve). Resultados: Enquanto todos os antígenos mostraram um melhor desempenho na detecção de CVL em cães sintomáticos (SD), a detecção de CVL nos grupos oligossintomáticos (OD) e assintomáticos (AD) foi menor, mas rLb6H alcançou alta sensibilidade para CVL assintomática. Curiosamente, as amostras de CVL mais reativas a rK28 foram pouco detectadas por rLb6H, enquanto as menos reativas a rK28, principalmente do grupo AD, apresentaram maior reatividade a rLb6H. Conclusão: A proteína recombinante rLb6H mostrou utilidade na detecção de CVL assintomática, apresentando uma reatividade complementar a rK39 e rK28. Assim, estes resultados sugerem que o rLb6H pode ser incorporado em estratégias multi-antígeno para aumentar a acurácia diagnóstica da leishmaniose visceral.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

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.

Integrative literature review of the reported uses of serological tests in leprosy management

Abstract: An integrative literature review was conducted to synthesize available publications regarding the potential use of serological tests in leprosy programs. We searched the databases Literatura Latino-Americana e do Caribe em Ciências da Saúde, Índice Bibliográfico Espanhol em Ciências da Saúde, Acervo da Biblioteca da Organização Pan-Americana da Saúde, Medical Literature Analysis and Retrieval System Online, Hanseníase, National Library of Medicine, Scopus, Ovid, Cinahl, and Web of Science for articles investigating the use of serological tests for antibodies against phenolic glycolipid-I (PGL-I), ML0405, ML2331, leprosy IDRI diagnostic-1 (LID-1), and natural disaccharide octyl-leprosy IDRI diagnostic-1 (NDO-LID). From an initial pool of 3.514 articles, 40 full-length articles fulfilled our inclusion criteria. Based on these papers, we concluded that these antibodies can be used to assist in diagnosing leprosy, detecting neuritis, monitoring therapeutic efficacy, and monitoring household contacts or at-risk populations in leprosy-endemic areas. Thus, available data suggest that serological tests could contribute substantially to leprosy management.

Multibacillary leprosy patients with high and persistent serum antibodies to leprosy IDRI diagnostic-1/LID-1: higher susceptibility to develop type 2 reactions

Leprosy inflammatory episodes [type 1 (T1R) and type 2 (T2R) reactions] represent the major cause of irreversible nerve damage. Leprosy serology is known to be influenced by the patient’s bacterial index (BI) with higher positivity in multibacillary patients (MB) and specific multidrug therapy (MDT) reduces antibody production. This study evaluated by ELISA antibody responses to leprosy Infectious Disease Research Institute diagnostic-1 (LID-1) fusion protein and phenolic glycolipid I (PGL-I) in 100 paired serum samples of 50 MB patients collected in the presence/absence of reactions and in nonreactional patients before/after MDT. Patients who presented T2R had a median BI of 3+, while MB patients with T1R and nonreactional patients had median BI of 2.5+ (p > 0.05). Anti-LID-1 and anti-PGL-I antibodies declined in patients diagnosed during T1R (p < 0.05). Anti-LID-1 levels waned in MB with T2R at diagnosis and nonreactional MB patients (p < 0.05). Higher anti-LID-1 levels were seen in patients with T2R at diagnosis (vs. patients with T1R at diagnosis, p = 0.008; vs. nonreactional patients, p = 0.020) and in patients with T2R during MDT (vs. nonreactional MB, p = 0.020). In MB patients, high and persistent anti-LID-1 antibody levels might be a useful tool for clinicians to predict which patients are more susceptible to develop leprosy T2R.

Micronutrients influencing the immune response in leprosy / Micronutrientes que influyen en la respuesta immune en la lepra

La lepra es una enfermedad infecciosa crónica causada por el Mycobacterium leprae, un bacilo intracelular de transmisión aérea. La enfermedad afecta la piel y los nervios periféricos y causa secuelas neurológicas. El bacilo se multiplica lentamente en el hospedador y posiblemente la enfermedad ocurre por el mal funcionamiento de la respuesta inmunitaria del hospedador. Esta revisión aborda el papel de algunos micronutrientes específicos en la respuesta inmunitaria, tales como las vitaminas A, D, E, C, el cinc y el selenio, detallando sus mecanismos de acción en las enfermedades infecciosas y en la lepra. La respuesta inmunitaria a los patógenos libera sustancias nocivas que producen lesión tisular. Esta revisión también aborda cómo una menor cantidad de antioxidantes puede contribuir a un aumento del estrés oxidativo y a complicaciones de las enfermedades infecciosas y la lepra. Puesto que los micronutrientes poseen un efecto regulador de la respuesta inmunitaria innata y adaptativa, es importante un equilibrio perfecto de sus concentraciones para mejorar la respuesta inmunitaria frente a los patógenos (AU)

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an intracellular bacillus of airborne transmission. The disease affects the skin and peripheral nerves and can cause neurological sequelae. The bacillusmultiplies slowly in the host and the disease probably occurs due to malfunctioning in host immune response. This review addresses the role of some specific micronutrients in the immune response, such as Vitamins A, D, E, C, Zinc and Selenium, detailing their mechanisms of actions in infectious diseases, and in leprosy. The immune response to pathogens releases harmful substances, which lead to tissue damage. This review discusses how a decreased level of antioxidants may contribute to an increased oxidative stress and complications of infectious diseases and leprosy. As the nutrients have a regulatory effect in the innate and adaptative immune responses, a perfect balance in their concentrations is important to improve the immune response against the pathogens (AU)

Clinical variables associated with leprosy reactions and persistence of physical impairment

Introduction Leprosy is a chronic disease that affects skin and peripheral nerves. Disease complications include reactional episodes and physical impairment. One World Health Organization (WHO) goal of leprosy programs is to decrease the number of grade 2 impairment diagnoses by 2015. This study aims to evaluate clinical factors associated with the occurrence of leprosy reactions and physical impairment in leprosy patients. Methods We conducted a retrospective study of data from medical records of patients followed in two important centers for the treatment of leprosy in Aracaju, Sergipe, Brazil, from 2005 to 2011. We used the chi-square test to analyze associations between the following categorical variables: gender, age, operational classification, clinical forms, leprosy reactions, corticosteroid treatment, and physical impairment at the diagnosis and after cure. Clinical variables associated with multibacillary leprosy and/or reactional episodes and the presence of any grade of physical impairment after cure were evaluated using the logistic regression model. Results We found that men were more affected by multibacillary forms, reactional episodes, and grade 2 physical impairment at diagnosis. Leprosy reactions were detected in a total of 40% of patients and all were treated with corticosteroids. However, physical impairment was observed in 29.8% of the patients analyzed at the end of the treatment and our multivariate analysis associated a low dose and short period of corticosteroid treatment with persistence of physical impairments. Conclusions Physical impairment should receive an increased attention before and after treatment, and adequate treatment should be emphasized. .

The potential for vaccination in leprosy elimination: new tools for targeted interventions

Despite the huge effort and massive advances toward the elimination of leprosy over the last two decades, the disease has proven stubborn; new case detection rates have stabilised over the last few years and leprosy remains endemic in a number of localised regions. The American Leprosy Missions and Infectious Disease Research Institute have undertaken a large research effort aimed at developing new tools and a vaccine to continue the push for leprosy elimination. In this paper, we outline our strategy for the integration of rapid diagnostic tests and lab-based assays to facilitate the detection of early or asymptomatic leprosy cases, as well as the efficient and focused implementation of chemoprophylaxis and immunisation to intervene in leprosy development and transmission.

Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela

Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Pau-cibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additionally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.

Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil

New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil.

Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil

New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil