ABSTRACT
Cutaneous leishmaniasis (CL) is one of the most frequent parasitic zoonoses in Panama. Currently, conventional, molecular and histopathological tests are performed to diagnose CL. Immunohistochemistry (IHC) has proven to be a valuable tool to facilitate the diagnosis of leishmaniasis and to study the cellular immune response developed during the infection. Therefore, considering the absence of IHC in the diagnostic routine in Panama, the objective of this study is to demonstrate the usefulness of this test as a complementary diagnostic tool for improving the sensitivity of histopathology (HP) and helping to study the cellular immune response of patients. Samples from patients with suspected CL were analysed by intradermal reaction of Montenegro (IDRM), smears, culture, PCR (Viannia, Hsp-70), HP and IHC. According to the diagnostic criteria, 95.8% of patients were positive for Leishmania sp., that was characterized as Leishmania (V.) panamensis by PCR-HSP70/RFLP. From positive samples, Leishmania was detected by the tested diagnostic methods in the following degrees: 100% by IDRM, 60% by smears, 93.3% by culture, 100% by kDNA PCR, 78.3% by PCR Hsp-70, 50% by HP and 73.9% by IHC. Although IHC had a poor correlation (kâ¯=â¯0.191) with the diagnostic criteria, the sensitivities of both HP (76.1%) and smears (89.1%) were improved by combining them with IHC. IHC considerably improved the detection of the Leishmania parasites in the histopathological sections, supporting the need to implement this diagnostic tool in Panama. In addition, immunohistochemistry allows evaluation of the patient's immune response and thus provides new guidelines for the treatment and control of CL in Panama.
Subject(s)
Immunohistochemistry/standards , Leishmania/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Skin/pathology , Adult , Aged , Antigens, Protozoan/immunology , Biopsy , DNA, Protozoan/genetics , Female , Histological Techniques , Humans , Immunity, Cellular , Leishmania/genetics , Male , Middle Aged , Panama , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity , Skin/parasitology , Young AdultABSTRACT
We investigated the performance of the DPP(®) canine visceral leishmaniasis (CVL) rapid test, a novel immunochromatographic assay launched by BioManguinhos (Brazil), which was recently included in the new Brazilian protocol for screening CVL in serological surveys. The present study compared the DPP(®) with the ELISA and IFA produced by BioManguinhos (Brazil) both with L. major-like antigens and with in-house tests using Leishmania infantum chagasi (in-house ELISA and in-house IFA). We analyzed the sera from clinically symptomatic (n=47) and asymptomatic (n=38) infected dogs from an endemic area of CVL, as well as from healthy (n=18) dogs, in addition to the sera of dogs (n=81) infected with other pathogens. The DPP(®) and the in-house ELISA showed a sensitivity of 90.6% and 94.1%, respectively, and specificity of 95.1% and 97.5%, respectively, and both presented cross-reactivity only with the sera of dogs with babesiosis, 44% for the DPP(®) and 22% for the in-house ELISA. The clinical groups were detected equally by the two assays. The ELISA BioManguinhos, IFA BioManguinhos, and in house-IFA showed a good sensitivity, 90.6%, 96.5% and 89.4%, respectively, but very low specificity, 77.8%, 69.1% and 65.8%, respectively, due to the high cross-reactivity with the sera from the animals harboring other pathogens. The in-house ELISA provided the highest accuracy (95.8%), followed by the DPP(®) (92.7%), ELISA BioManguinhos (84.3%), IFA BioManguinhos (83.1%), and in-house IFA (78.0%). The simultaneous use of the DPP(®) and ELISA BioManguinhos reached a sensitivity of 99.1% and 82.1% when used sequentially. In conclusion, the DPP(®) performed well as serological test for CVL, and detected both asymptomatic and symptomatic dogs in equal proportions. Although its sensitivity is not ideal yet, discarding the IFA and including the DPP(®) improved the accuracy of the new Brazilian CVL diagnostic protocol, particularly of detecting truly infected dogs. Moreover, considering the higher specificity of DPP(®) (95.1% vs 77.8%), positive predictive value (95.1% vs 81.1%) and positive likelihood value (18.3% vs 4.1%) in comparison with the ELISA BioManguinhos, the use of DPP(®) as a confirmatory test instead of a screening test is suggested.
Subject(s)
Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Dog Diseases/epidemiology , Leishmania infantum/immunology , Leishmaniasis, Visceral/diagnosis , Animals , Brazil , Chromatography, Affinity , Cross Reactions , Dogs , Enzyme-Linked Immunosorbent Assay/veterinary , Fluorescent Antibody Technique, Indirect , Leishmania infantum/isolation & purification , Rabbits , Sensitivity and Specificity , Serologic Tests/veterinary , Time FactorsABSTRACT
In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70+/-1.72 microg/ml) in comparison with that of Cdca venom (IC50 value of 9.41+/-1.21 microg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30+/-2.18 microg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00+/-9.50 microg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80+/-0.52 microg/ml and 19.95+/-4.21 microg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80+/-2.21 microg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.
Subject(s)
Antiprotozoal Agents/toxicity , Crotalid Venoms/toxicity , Leishmania/drug effects , Animals , Antiprotozoal Agents/chemistry , Cell Line , Crotalid Venoms/chemistry , Crotalid Venoms/isolation & purification , Crotoxin/isolation & purification , Crotoxin/toxicity , Inhibitory Concentration 50 , Lectins, C-Type/isolation & purification , Macrophages/drug effects , Mice , Mice, Inbred BALB C , PoisoningABSTRACT
The crude methanolic extract from leaves of Jacaranda puberula showed activity against Leishmania (Leishmania) amazonensis. The extract presented active against promastigote forms with an inhibitory concentration 50% (IC(50)) value of 88.0 mug/ml, but only moderated activity against amastigote forms; however in higher concentrations the extract showed cytotoxic effects. The bio-guided chromatographic fractionation the crude methanolic extract against amastigotes yielded a fraction with an IC(50) value of 14.0 mug/ml (without cytotoxic activity) in relation to the crude extract (IC(50) value, 359.0 microg/ml). These data indicate that J. puberula leaves contain active compounds, which should be further investigated for the development of new potential drugs against cutaneous leishmaniasis.
