Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.
ABSTRACT: We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.
Project description:Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.
Project description:BACKGROUND:Kinesin-related gene diversity among strains and species of Leishmania may impact the sensitivity and specificity of serodiagnostic tests for visceral leishmaniasis (VL). METHODS:In this study, we report on the recombinant expression of this novel Iranian Leishmania infantum (MCAN14/47) homologue of rK39 (Li-rK39), in L. tarentolae. The diagnostic potential of the Li-rK39 antigen was evaluated in an ELISA, using sera from 100 VL patients, 190 healthy endemic controls, 46 non-endemic healthy controls and 47 patients with other infections. RESULTS:The results showed a sensitivity of 96% and a specificity of 93.8%. A commercial rK39 immunochromatographic test (ICT) was 90% sensitive and 100% specific on the same cohort. CONCLUSIONS:Here, we show that the K39 gene from an Iranian L. infantum isolate is heterozygous as compared to the sequence of the Brazilian L. infantum (former L. chagasi), whose antigen is incorporated in most rK39-based immunochromatographic tests. Therefore, Li-rK39 has the potential to be used as an alternative for VL diagnosis in Iran.
Project description:BACKGROUND: For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan. METHODOLOGY AND PRINCIPLE FINDINGS: We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity. CONCLUSION: The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.
Project description:Canine leishmaniosis (CanL) prevention in the Mediterranean basin is considered essential to stop human zoonotic visceral leishmaniasis. In this context, vaccination of dogs is expected to have a significant impact in disease control. CaniLeish® (Virbac Animal Health) is one of a few CanL vaccines that are at this moment licensed in Europe. This vaccine contains purified excreted-secreted proteins of Leishmania having several antigens/immunogens with potential to influence serological response. Therefore, it is important to know if CaniLeish vaccination increased the diagnostic challenges associated with conventional serology, limiting the value of some antigens. To address this 20 dogs from a cohort of 35 healthy dogs that were vaccinated, maintained indoor for 1 month and then returned to their natural domiciles for 2 years. After this period, they were re-called to evaluate their clinical/parasitological condition and assess the evolution of seroreactivity against different antigens: soluble promastigote Leishmania antigens (SPLA), recombinant protein Leishmania infantum cytosolic peroxiredoxin, recombinant protein K39 (rK39), recombinant protein K28 and recombinant kinesin degenerated derived repeat using ELISA. Two years after vaccination all vaccinated non-infected animals were seropositive for SPLA. For the other antigens the serological profile was indistinguishable from non-infected animals. Moreover, vaccinated animals presented a characteristic relative serological profile, with higher normalized serological response to SPLA than rK39. This fact enabled to distinguish with sensitivity 92.3% and specificity 95.4%, vaccinated non-infected dogs from infected and non-infected dogs. Ultimately, relative serological profile enabled the detection of healthy vaccinated animals enabling more accurate serological surveys.
Project description:Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis. Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host. To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library. One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L. chagasi infection. The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene. Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa. Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L. chagasi infection. Immunization with Lcr1 partially protected BALB/c mice against challenge with L. chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens.
Project description:Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96-100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64-88%) and France (73.1-88.5% and 63.6-81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7-100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests.
Project description:The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.
Project description:Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.
Project description:The sensitivity of a K39 ELISA (Leishmania IgG, Virion/Serion) for the detection of antibodies in patients with imported leishmaniasis was compared with an immunofluorescence assay (IFA), which was applied as "golden standard". The retrospective study comprised 93 IFA-positive or borderline sera from 42 patients with visceral (n?=?16) or cutaneous (n?=?26) leishmaniasis. Patients had acquired infection predominately in the Mediterranean area or the Middle East. The Leishmania species (Leishmania donovani/infantum, Leishmania tropica, Leishmania major) were identified by real-time PCR. The majority (94%) of first samples from patients with visceral leishmaniasis (VL) tested positive by K39 ELISA. Antibody levels ranged from low to very high (33.19-1990.00 U/ml; median 596.66 U/ml) but did not correlate with the respective IFA titers. High K39 ELISA values correlated with acute infection in immunocompetent individuals. K39 antibodies declined in all individuals after clinically successful therapy, but time to seronegativity varied considerably (51 weeks to >6 years). In patients with cutaneous leishmaniasis (CL), the sensitivity of the K39 ELISA was low (23%) compared to IFA (92% positive). Antibody levels ranged from low to medium (10.85-524.77 U/ml; median 19.77 U/ml). The highest antibody concentrations were seen in L. infantum-infected individuals. Summarizing, a high K39 ELISA value indicates active VL. The assay is, like IFA, not a measure for effective therapy but may support post-treatment monitoring. Low level positivity can indicate subclinical, previous or clinically cured VL or even CL. The K39 ELISA can supplement highly sensitive screening tests in the diagnosis and follow-up of imported leishmaniasis.
Project description:Leishmania infantum chagasi is the causative agent of visceral leishmaniasis in Brazil. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of kinetoplast DNA (kDNA) minicircles was used to evaluate genetic profiles of 48 Leishmania infantum chagasi strains from dog and human parasite cultures, fresh collected dog bone marrow aspirates, and from infected sand flies. Results revealed that heterogeneity in kDNA minicircles depends mostly on the source of the samples, with cultured parasites showing a high degree of homogeneity.