Evaluation of Immunodiagnostic Tests for Leprosy in Brazil, China and Ethiopia.

Leprosy remains persistently endemic in several low- or middle income countries. Transmission is still ongoing as indicated by the unabated rate of leprosy new case detection, illustrating the insufficiency of current prevention methods. Therefore, low-complexity tools suitable for large scale screening efforts to specifically detect M. leprae infection and diagnose disease are required. Previously, we showed that combined detection of cellular and humoral markers, using field-friendly lateral flow assays (LFAs), increased diagnostic potential for detecting leprosy in Bangladesh compared to antibody serology alone. In the current study we assessed the diagnostic performance of similar LFAs in three other geographical settings in Asia, Africa and South-America with different leprosy endemicity. Levels of anti-PGL-I IgM antibody (humoral immunity), IP-10, CCL4 and CRP (cellular immunity) were measured in blood collected from leprosy patients, household contacts and healthy controls from each area. Combined detection of these biomarkers significantly improved the diagnostic potential, particularly for paucibacillary leprosy in all three regions, in line with data obtained in Bangladesh. These data hold promise for the use of low-complexity, multibiomarker LFAs as universal tools for more accurate detection of M. leprae infection and different phenotypes of clinical leprosy.

Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.

BACKGROUND: Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. METHODS: The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. RESULTS: Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. CONCLUSIONS: Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.

Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.

Comparison of three immunological tests for leprosy diagnosis and detection of subclinical infection.

OBJECTIVE: Our aim was to compare the performance of three serological assays in leprosy patients and their household contacts utilising two quantitative ELISA tests using native PGL-I (PGL-1 ELISA), synthetic ND-O-HSA (ND-O-HSA ELISA), and the semi-quantitative lateral flow test (ML Flow). METHODS: Comparisons among three immunological assays, PGL-I ELISA, ND-O-HSA ELISA, and ML Flow were performed in 154 leprosy patients, 191 household contacts and 52 health subjects. RESULTS: The sensitivity results of the PGL-1, ND-O-HSA, and ML Flow were 68.83%, 63.84%, and 60.65%, respectively, with specificity of 98% for both ELISA assays. The native and synthetic PGL-I ELISA assays detected antibodies in 22.73% and 31.82% of the paucibacillary (PB) patients, respectively and the ML Flow test did not detect antibodies in this group. The ML Flow test was able to discriminate patients into PB or multibacillary (MB) forms, while the native PGL-I and ND-O-HSA was correlated with the bacillary load and the Ridley-Jopling clinical forms. In household contacts, the native PGL-I, ND-O-HSA, and ML Flow assays detected seropositivity of 25%, 17%, and 10%, respectively. CONCLUSIONS: The use of ELISA and ML Flow tests are thus recommended as additional tools in the diagnosis and classification of the clinical forms, aiding in prescribing the correct treatment regimen to prevent subsequent nerve damage and disability, and besides, the PGL-I ELISA may be used to detect subclinical infection in leprosy.

Leprosy: diagnostic and control challenges for a worldwide disease.

Leprosy is a curable disease with well-defined etiology, but lacks better diagnostic tools, preventive and therapeutic strategies. The continued application of the Ridley-Jopling clinical classification that recognizes the natural diversity of the immune response has provided the basis for understanding leprosy, and this review proposes its implementation in all Reference Centers in order to standardize the diagnostic resources, aiming at the improvement of the disease control. Due to the broad bioepidemiological aspects of infection its eradication is difficult, and proper diagnosis of the disease and the correct clinical classification are required to ensure proper treatment. Tools and markers for diagnosis and prognosis, and the novel use of nanotechnology, as well as strategies for disease control and monitoring populations at higher risk are still continuous challenges, which will be specifically reviewed with additional insights. The use of the current diagnostic tools, such as ELISA and PCR has a very limited approach for leprosy that has been considered as a marginal disease; therefore, the current diagnostic tools must be applied extensively in the routine to accumulate clinical experience in order to improve their precise application, like what has been done in many other infectious diseases. Since a vaccine for leprosy presents an unpredictable future, the proposed chemoprophylaxis of contacts (healthy carriers and/or with subclinical infection) must also be employed in referral centers of endemic countries not only to evaluate its efficacy, but also because of the favorable cost-benefit ratio, given that there is no other available approach, besides the multi-drug therapy of patients. This strategy should readily be applied as a public health policy, and may lead to a substantial breakage of the transmission chain aiming a world without leprosy.

Towards an immunodiagnostic test for leprosy.

In addition to multidrug therapy, elimination of leprosy requires improved diagnostic methods. Using a comparative genomics approach, 17 potential protein antigens (MLP) that are restricted to Mycobacterium leprae, or of limited distribution, were produced and tested for antigen-specific immune responses on leprosy patients, healthy contacts of leprosy patients, and tuberculosis patients in Mali and Bangladesh, as well as on non-endemic controls. T-cell antigenicity of MLP was confirmed by IFN-gamma production in whole-blood assays with the highest responses observed in paucibacillary leprosy patients and healthy contacts. Four MLP behaved well in both countries and induced significantly different responses between the study groups. Peptides carrying T cell epitopes from one of the antigens gave promising results in restimulation assays in mice and immune responses were not influenced by prior exposure to BCG or environmental mycobacteria. This study provides the immunological framework for the development of a specific, peptide-based immunodiagnostic test for leprosy.

Evaluation of a rapid immunochromatographic test for diagnosis of kala-azar & post kala-azar dermal leishmaniasis at a tertiary care centre of north India.

BACKGROUND & OBJECTIVE: Definitive diagnosis of kala-azar requires demonstration of parasites by diagnostic protocols based on invasive organ aspirations. We evaluated in the present study the diagnostic utility of an immunochromatographic test (ICT) for detection of anti- rK-39 antibodies for the non-invasive diagnosis of kala-azar and post kala-azar dermal leishmaniasis (PKDL) at a tertiary care centre of north India. METHODS: The study was conducted in the Department of Microbiology, All India Institute of Medical Sciences, New Delhi, from July 2003 to October 2004. Of the 120 samples tested, 57 were found to be positive by ICT; of which, 51 were diagnosed as kala-azar and 6 as PKDL. The controls included individuals from endemic (50) and non endemic (19) areas with malignancies, haemolytic disorders, chronic liver diseases, hypersplenism, portal hypertension, metabolic disorders and sarcoidosis. In addition, 47 sera from confirmed cases of tuberculosis, malaria, typhoid, filariasis, leptospirosis, histoplasmosis, toxoplasmosis, invasive aspergillosis, amoebic liver abscess, AIDS, leprosy, cryptococcosis, strongyloidiasis, cyclosporosis, patients having collagen vascular diseases and hypergammaglobulinaemia were also tested to check the specificity of the test. RESULTS: Of the 51 cases with kala-azar 43 were males, children accounted for 25 per cent of these cases. All had fever of duration ranging from <1 month to 1.5 yr (median 4.5 months). All PKDL patients (n=6, 4 males) gave a history of having suffered from kala-azar in the past, and their slit skin test smears were microscopically positive for Leishman-Donovan (LD) bodies. The strip test was positive in all the cases of kala-azar and PKDL (estimated sensitivity 100%), all control sera were negative by the ICT (specificity 100%). INTERPRETATION & CONCLUSION: The rK-39 ICT is a highly sensitive and specific test, and may be suitable for a rapid, cost-effective and reliable field diagnosis of kala-azar and PKDL.