ABSTRACT
Leprosy and post-kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2-100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1-99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.
Subject(s)
Leishmania donovani/genetics , Leishmaniasis, Cutaneous/diagnosis , Leprosy/diagnosis , Molecular Diagnostic Techniques/methods , Mycobacterium leprae/genetics , Nucleic Acid Amplification Techniques/methods , Adolescent , Adult , Aged , Antigens, Protozoan/genetics , Diagnosis, Differential , Female , Humans , Leishmaniasis, Cutaneous/parasitology , Leprosy/microbiology , Male , Middle Aged , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/standards , Multiplex Polymerase Chain Reaction/economics , Multiplex Polymerase Chain Reaction/methods , Multiplex Polymerase Chain Reaction/standards , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/standards , Young AdultABSTRACT
An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.
Subject(s)
Antigens, Protozoan , Chagas Disease/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Serologic Tests/methods , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/genetics , Chagas Disease/immunology , Chagas Disease/parasitology , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Evaluation Studies as Topic , False Positive Reactions , Humans , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/genetics , Peptides/immunology , Sensitivity and Specificity , Serologic Tests/statistics & numerical data , Trypanosoma cruzi/genetics , Trypanosoma cruzi/immunologyABSTRACT
We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.