acid-phosphatase and Leishmaniasis--Visceral

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-μm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran.

Topics: Acid Phosphatase; Animals; Antibodies, Protozoan; Antigens, Protozoan; Case-Control Studies; Disease Reservoirs; Dog Diseases; Dogs; Electrophoresis, Polyacrylamide Gel; Endemic Diseases; Humans; Immunoblotting; Iran; Latex Fixation Tests; Leishmania infantum; Leishmaniasis, Visceral; Mass Screening; Reproducibility of Results; Rural Population; Sensitivity and Specificity

Leishmania donovani is the major causative agent of Old World human visceral leishmaniasis (VL). In vitro, both promastigotes and axenic amastigotes of L. donovani constitutively secrete soluble acid phosphatases (SAcPs), which contain conserved antigenic epitopes. These SAcPs are the most abundant and best characterized secretory proteins of this parasite. The aim of this study was to determine whether this enzyme was produced by intracellular amastigotes during the course of human infection. To that end, sera from acutely infected leishmaniasis patients were tested for anti-SAcP antibodies using L. donovani promastigote culture supernatants. Our results showed that VL patient sera from different endemic foci immunoprecipitated parasite SAcP enzyme activity. Further, these VL patient sera recognized the 110- and 130-kDa SAcPs in both Western blots and radioimmunoprecipitation assays. Results of tunicamycin experiments demonstrated that VL patient anti-SAcP antibodies were directed against the polypeptide backbone of the parasite SAcPs. In addition, both radiolabeled L. donovani SAcPs and native enzyme activities were immunoprecipitated by sera from patients with various forms of cutaneous leishmaniasis. Together, these studies demonstrate that Leishmania amastigotes produce SAcPs during the course of human infections.

Topics: Acid Phosphatase; Animals; Antibodies, Protozoan; Blotting, Western; Humans; Immune Sera; Leishmania donovani; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Mice; Molecular Weight; Precipitin Tests; Rabbits

A sensitive diagnostic assay for parasitic infections based on the detection of anti-enzyme antibodies is presented. All serum antibodies produced in response to parasite antigens are immobilized via their Fc domain on matrix-bound protein G. Incubation of the immobilized antibodies with saturating amounts of parasite extract results in the binding of all recognized antigens, including those directed against a specific and readily measurable enzyme. The amount of bound enzyme is proportional to the anti-enzyme antibody concentration in the serum. The application of this principle is demonstrated for the diagnosis of both human African trypanosomiasis and visceral leishmaniasis by the detection of antibodies against parasite acid phosphatases.

Topics: Acid Phosphatase; Animals; Antibodies, Protozoan; Humans; Leishmania mexicana; Leishmaniasis, Visceral; Trypanosoma brucei brucei; Trypanosomiasis, African

Analysis of lysosomes through acid phosphatase cytochemistry at the electron microscopy level has been performed in spleen and foot lesions from Leishmania-infected hamsters. The results showed that there is lysosomal depletion in macrophages from Leishmania donovani chagasi-infected hamster spleen and similar findings were obtained from foot lesions of Leishmania mexicana amazonensis-infected hamsters. The distribution of acid phosphatase and thiamine pyrophosphatase was also examined in the Golgi apparatus. It was possible to demonstrate that the activity of ACP is absent in infected macrophages from spleen and foot lesions of Leishmania-infected hamster while the distribution of TPP was very similar in control and infected macrophages from both systems. These results provide evidence that the lysosomal depletion can occur at the ACP synthesis and/or glycosylation level.

Topics: Acid Phosphatase; Animals; Cells, Cultured; Cricetinae; Golgi Apparatus; Histocytochemistry; Leishmania donovani; Leishmania mexicana; Leishmaniasis; Leishmaniasis, Visceral; Lysosomes; Macrophages; Mesocricetus; Microscopy, Electron; Spleen; Thiamine Pyrophosphatase

Topics: Acid Phosphatase; Alkaline Phosphatase; DNA; Histocytochemistry; Humans; Leishmaniasis, Visceral; Peroxidases; Reticulin; RNA; Skin