Studies in Mediterranean leishmaniasis. 3. The leishmanin skin test in kala-azar.

Leishmanin skin testing was carried out in the Emilia-Romagna region of Northern Italy, the site of an outbreak of kala-azar in 1971-72, and in Catania, Eastern Sicily an old endemic focus of Mediterranean kala-azar. Nearly all the people who had recovered from kala-azar in the past gave positive skin tests. Active cases of kala-azar gave negative tests. There was a higher proportion of positive reactors amongst the household contacts and neighbours of cases of kala-azar than among the general population. Age specific leishmanin rates showed an increasing positive rate with age in Catania, comparable to those found in endemic areas in Kenya, but in the Emilia-Romagna area all age groups showed a high positivity rate suggesting a simultaneous exposure to infection. The age specific rates from Catania suggest an interruption in transmission 20-30 years ago. The leishmanin skin test is a useful tool for the study of the epidemiology of Mediterranean kala-azar.     

Direct agglutination test for diagnosis and sero-epidemiological survey of kala-azar in the Sudan.

132 patients with suspected kala-azar (visceral leishmaniasis) were included in a prospective study to compare the performance of the direct agglutination test (DAT) with smears of lymph node, bone marrow and splenic aspiration. A titre greater than 1:3200 was considered positive. 67 patients provided positive smears and 65 were smear-negative. Compared with the results obtained from smears, the sensitivity of the DAT was 94% and its specificity 72%. Of 18 patients who were negative by smears but positive by DAT, 3 were treated on strong clinical suspicion of kala-azar and responded well to therapy. One other patient had post-kala-azar dermal leishmaniasis. Of the remaining 14, 8 were tested with the leishmanin skin test; 6 gave a positive result, suggesting past or sub-clinical infection. In 4 smear-positive patients, the DAT was negative; in 2 of these, the test remained negative during 6 months' follow-up. In a sero-epidemiological survey in a camp for displaced people, 30 of 600 sampled individuals gave a positive DAT. Of these, 11 had been treated for kala-azar; 4 others were leishmanin positive. The DAT is a useful screening test (sensitivity 94%; predictive value of a negative test 92%), but it does not differentiate between past kala-azar, sub-clinical infection and active disease.     

Cutaneous leishmaniasis in the Sudan

The epidemiology, clinical features, pathology, immunology and treatment of cutaneous leishmaniasis in the Sudan are described.The majority of patients came from kala-azar endemic areas of the central Sudan. The aetiology of the disease is discussed.Clinically the lesions are divided into 3 types but with rare exceptions the pathological changes in all types are essentially the same.Immunoglobulins IgG and IgM are elevated in cutaneous, mucosal and visceral leishmaniasis, but IgA is only significantly increased in the mucosal type. Compared with the other forms of leishmaniasis the cutaneous disease shows the lowest levels of fluorescent antibodies.In the Sudan cutaneous leishmaniasis responds well to sodium stibogluconate (Pentostam).The results of leishmanin skin test surveys carried out in 3 different regions of the country are reported and discussed.     

Studies on the immunology and serology of leishmaniasis. IX. Serological investigation of the parasites of Indian kala-azar and Indian post-kala-azar dermal leishmaniasis

The antigenic constitution of 4 strains of L. donovani from kala-azar and of 4 strains of the parasite from patients with post-kala-azar dermal leishmaniasis from West Bengal, India, was examined by indirect haemagglutination test performed on antigen cross-absorbed antisera. Antigenically all the strains of L. donovani from West Bengal, India, were essentially similar but differed from a strain of L. donovani from Ethiopia.     

The role of Phlebotomus alexandri Sinton, 1928 in the transmission of kala-azar

Since 1968, kala-azar has been occurring sporadically in Meiyaogou, Turfan County, Xinjiang-Uygur Autonomous Region, where four species of sandflies are known to exist. The present study of sandflies collected in this area from May to August 1983 shows that Phlebotomus alexandri is the only anthropophilic and predominant species, accounting for 81.1% (7716/8843) of the sandfly population. After having been fed on cotton rats or hamsters infected with Leishmania donovani, 93.9% (230/245) of P. alexandri were infected, the promastigotes not only developing in the stomach but also invading the pharynx, buccal cavity and proboscis. Of the 643 female P. alexandri collected in the wild and in house, 13 (2.0%) were found to be naturally infected, with a distribution of promastigotes similar to that in the experimental infection. All the Chinese hamsters and golden hamsters that had been inoculated with 7 isolates of promastigotes from naturally infected sandflies developed visceral leishmaniasis. In addition, golden hamsters could be infected experimentally by the bite of P. alexandri which had an infection of promastigotes of L. donovani. This is the first demonstration of a species of Paraphlebotomus playing a role in the transmission of kala-azar in the Old World; studies for confirmation by biochemical identification of parasite isolates from naturally infected P. alexandri are now in progress.     

Leishmaniasis in Brazil: X. Some observations of intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and mucocutaneous leishmaniasis.

In spite of the difficulty of antigen standardization, paired intradermal skin tests demonstrated qualitative differences between leishmanin and in vitro exo-antigen. Some of these differences may be due to the destruction of certain antigens by phenol, while others seem to reflect basic antigenic differences. Leishmania mexicana amazonensis in vitro exo-antigen produced immediate anaphylactic reponses in 74.4% of the patients with parasitologically proven cutaneous leishmaniasis. Although more individuals responded to leishmanin, 82.4% as compared to 73.9%, the leishmanial in vitro exo-antigen gave significantly larger delayed responses, Trypanosoma cruzi trypamosomin elicited delayed reactions, while trypanosomal in vitro exo-antigen only produced immediate anaphylactic reactions in persons with active cutaneous leishmaniasis.     

The epidemiology of cutaneous leishmaniasis in Ethiopia

Parasites indistinguishable from those causing cutaneous leishmaniasis of man in Ethiopia were found in 3 widely separated localities in rock hyrax and phlebotomid sandflies. A close association between hyrax and the sandflies was demonstrated both in cliff and tree habitats. Procavia habessinica and Heterohyrax brucei were both shown to be reservoirs of the disease, and are thought to be the only significant ones. Phlebotomus longipes and P. pedifer are the vectors. Man intrudes on the hyrax/sandfly cycle, but in no way sustains it, and is an incidental host. This distinctive epidemiological system is thought to occur throughout the range of cutaneous leishmaniasis in Ethiopia.     

Post kala-azar dermal leishmaniasis in the Sudan: clinical features, pathology and treatment.

The clinical features, pathology, immune responses, diagnosis and treatment of post kala-azar dermal leishmaniasis (PKDL) in the Sudan are described and discussed. The disease is characterized by maculopapular or nodular lesions on the face, limbs or trunk. Lesions appear during or within months after the treatment of visceral leishmaniasis, but in 2 of 19 patients there was no previous history of kala-azar. PKDL may be confused with leprosy both clinically and pathologically. Similarities and differences between the 2 diseases are discussed. Unlike visceral leishmaniasis, the peripheral lymphoid cells of patients with PKDL respond to Leishmania antigen and some are leishmanin positive. The response to intravenous sodium stibogluconate (20 mg/kg for 30 d) was reasonably good but some patients required repeated or more prolonged treatment. Ketoconazole in a dose of 10 mg/kg daily for 4 weeks had no effect on PKDL.     

Knowledge, attitudes, and practices about kala-azar and its sandfly vector in rural communities of Nepal.

Reported are the results of a study of the knowledge, attitudes and practices (KAP) about kala-azar of the inhabitants of two villages (Titaria and Haraincha) situated in terai (plain) areas of Nepal. The villagers had poor knowledge about the transmission of kala-azar, with most villagers perceiving that mosquitos, instead of sandflies, were responsible for transmission of the infection. Most also failed to recognize the common symptoms of kala-azar. The majority of the respondents, 78.9% in Titaria and 48.4% in Haraincha, were aware that the condition can be treated, while fewer than 2% believed that it cannot be treated at all. More than 58% of villagers in Titaria and 36.8% in Haraincha used bednets. The residents of both villages were highly responsive to a programme to spray houses with insecticides. Fewer than 5% of respondents slept outdoors in farm outhouses and these individuals did not take any personal vector control measures. The results of this study show the importance of understanding the beliefs and practices of communities in the successful planning and implementation of kala-azar control activities in Nepal.     

Canine visceral leishmaniasis in villages West of Lanchow,China

Forty-four dogs naturally infected with visceral leishmaniasis were found among 1,430 examined in villages about 50 km. west of Lanchow. Epilation and seborrhoea were present in all but one of the dogs. Leishmania were found by skin biopsy in thirty-eight of the forty-four dogs and by ilium puncture in thirty-nine of forty-one dogs in which this operation was performed. The altitude of the urea under study ranges from 5,656 to 7,788 feet above sea level. On this high plateau kala-azar, as well as sandflies, was also found to be present.     

3. Visceral leishmaniasis

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent—in Western Upper Nile province in southern Sudan, detected in 1988 — claiming over 100000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70–93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for interleukin (IL)-10, interferon γ and IL-2. Treatment of peripheral blood mononuclear cells from VL patients with IL-12 was found to drive the immune response toward a Th1 type response with the production of interferon γ, indicating a potential therapeutic role for IL-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of 20 mg/kg intravenously or intramuscularly per day for 15–30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.     

Inorganic particles in the skin of inhabitants of volcanic areas of Central America: their possible immunomodulatory influence in leishmaniasis and leprosy

We have evaluated biopsies from patients with atypical nodular and typical ulcerated lesions of cutaneous leishmaniasis, from leishmanin reactions and skin from normal individuals from Nicaragua, Honduras and Guatemala for the presence of inorganic particles using confocal microscopy with a polarised light source and conventional histopathological techniques. Analysis by semiquantitative confocal microscopy permitted the demonstration of significantly larger numbers of particles in atypical lesions. Silica and aluminium, important components of these particles, were less abundant in particles from normal skin. The histology of these atypical lesions, characterised by 'naked' sarcoidal granulomas with epithelioid differentiation but very few lymphocytes, was very similar to the histological reaction observed after 14 days in persisting inflammation at leishmanin skin test sites. The presence of these unusual lesions in areas of Central American countries characterised by the presence of large amounts of volcanic ash, as well the unexpectedly low prevalence of leprosy in Central America, suggest that environmental factors may contribute significantly to the frequency and clinical manifestations of these infections. Among possible environmental features, the presence of inorganic particles with immunomodulatory properties in the skin may be a significant factor.     

Leishmaniasis in Brazil: IX. Considerations on the Leishmania braziliensis complex:—Importance of sandflies of the genus Psychodopygus (mangabeira) in the transmission of L. braziliensis braziliensis in North Brazil

At least 3 different parasites may produce cutaneous leishmaniasis in man in Brazil. Leishmania mexicana amazonensis is widely distributed and very common in wild animals, but rarely infects man because the vector is not anthropophilic. L. braziliensis guyanensis and L. braziliensis braziliensis have highly anthropophilic vectors and are thus the major causes of the disease in man.Infections with Leishmania b. braziliensis are described in 17 persons from an endemic area in the Serra dos Carajás, Pará, north Brazil. A nearby Indian tribe (Xikrín) was examined and leishmanin skin-tests carried out on 116 individuals. No evidence of past or present infection was found, but strong positive reactions were noted in 83·9% adult males, 38·8% adult females, 14·3% male children and 4·8% female children.A total of 2,701 man-biting and rodent-biting sandflies were dissected and the guts examined for flagellates. Promastigotes were found in 3 out of 1,656 Psychodopygus wellcomei, 2 out of 175 Ps. paraensis and 1 out of 127 Ps. amazonensis. Hamsters inoculated with promastigotes from one Ps. wellcomei developed a typical L. b. braziliensis infection. The importance of sandflies of the genus Psychodopygus is stressed: Ps. wellcomei is of particular importance due to its tendency to bite man during the day and the fact that it is attracted to both man and rodents in large numbers.Nearly all the sandflies were caught at ground level. The absence of a large, arboreal population of man-biters suggests that the reservoir-hosts of L. b. braziliensis are likely to be terrestrial or frequently coming to the ground.     

Leishmaniasis in Sudan. Visceral leishmaniasis

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent--in Western Upper Nile province in southern Sudan, detected in 1988--claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70-93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for interleukin (IL)-10, interferon gamma and IL-2. Treatment of peripheral blood mononuclear cells from VL patients with IL-12 was found to drive the immune response toward a Th1 type response with the production of interferon gamma, indicating a potential therapeutic role for IL-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of 20 mg/kg intravenously or intramuscularly per day for 15-30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.     

Comparison of leishmanin skin test and direct smear for the diagnosis of cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is an endemic disease in some parts of Iran and it has high morbidity in some areas of the country. The disease is detected by parasitological examinations including direct microscopic and culture tests. This comparative study aimed to evaluate the relationship between positivity of the leishmanin skin test (LST), microscopically examination and clinical forms of CL for the diagnosis of human cutaneous leishmaniasis. This study was performed on 66 patients suspected to cutaneous leishmaniasis. CL cases evaluated by both microscopical examination and leishmanin skin test. In this study, 1 ml of leishmanin fluid (lot no 121/1, produced in Pasteur institute of Iran) was injected intradermally in forearms of all patients and indurations were measured after 72 hours. Induration of 5 mm and higher was considered as positive results. The collected data were statistically analyzed using the SPSS version 13.5. From 66 CL patients who were evaluated in this study, 30 (45.5%) of them had positive microscopically results while 28(42/4%) of them had showed positive leishmanin skin test (≥ 5 mm diameter). From 36 (54.5%) patients who had negative microscopical examination, only 6(16/6%) of them had positive leishmanin skin test. The agreement between two tests was 87.9 % by kappa analysis (p< 0.01). In attention to the results of this study, it seems the LST would be used as an alternative diagnosis method when there is a strong clinical doubt to cutaneous leishmaniasis even there is no parasite in direct smear.     

Circulating factor from a kala-azar patient suppresses in vitro antileishmanial T cell proliferation

In vitro lymphocyte blastogenesis stimulated by phytohaemagglutinin (PHA), streptococcal antigens (SKSD) and leishmanial antigens were used to assess T cell responsiveness in one patient with kala-azar before and after therapy. During the illness, responses to PHA and SKSD but not to leishmanial antigens could be detected. After treatment lymphocytes responded to all three stimuli when cells were cultured in convalescent plasma, but failed to respond to leishmanial antigens when cultured in plasma obtained before treatment. These observations suggest the presence of a circulating inhibitor of antileishmanial T cell responsiveness in kala-azar, and warrant further investigation.     

Phlebotomid sandflies

The article presents a synthesis of present knowledge concerning sandflies of the family Phlebotomidae in relation to leishmaniasis. Over 500 species of Phlebotominae are known, most of which belong to the genera Phlebotomus and Sergentomyia in the Old World and Lutzomyia in the New World. Phlebotomus is the dominant genus in the palaearctic region, extending also into the other regions of the Old World where Sergentomyia is the principal genus. Sandflies are of little importance in temperate North America but in tropical America Lutzomyia is the main genus and is found over large areas. The distribution of sandflies largely determines the occurrence of leishmaniasis. Certain species of Phlebotomus and Lutzomyia transmit dermal leishmaniasis in large areas of the palaearctic and neotropical regions. Visceral leishmaniasis is transmitted by some species of Phlebotomus in much of the palaearctic region, except desert areas of North Africa, and in limited areas of Africa and India, and by a species of Lutzomyia in north-eastern Brazil. Sandflies are quite likely to bite man in the open country of much of the Old World; they tend however to be localized in distribution on account of their need for a suitable microhabitat (e.g., the burrow of the Central Asian large gerbil, which has been extensively studied in relation to dermal leishmaniasis). It is noted that the distribution of sandflies and leishmaniasis appears to be changing.     

2. Mucosal leishmaniasis

Sudanese mucosal leishmaniasis is a chronic infection of the upper respiratory tract and/or oral mucosa caused mainly by Leishmania donovani. The disease occurs in areas of the country endemic for visceral leishmaniasis, particularly among Masalit and other closely related tribes in western Sudan. The condition may develop during or after an attack of visceral leishmaniasis, but in most cases it is a primary mucosal disease. Unlike South American mucocutaneous leishmaniasis, mucosal leishmaniasis in Sudan is not preceded or accompanied by a cutaneous lesion. Pathologically, the lesions show a mixture of macrophages, plasma cells and lymphocytes. An epithelioid granuloma may also be found. Parasites are scanty. Diagnosis is established by demonstration of parasites in smears or biopsies, by culture or animal inoculation, or with the aid of the polymerase chain reaction. Most patients give positive results in the direct agglutination test and leishmanin skin test. Patients respond well to treatment with pentavalent antimony compounds.     

4. Post kala-azar dermal leishmaniasis

Post kala-azar dermal leishmaniasis (PKDL) is increasingly recognized in Sudan as a complication of visceral leishmaniasis (VL), occurring in c. 55% of patients after, or during treatment of, VL. The development of PKDL seems to be restricted to parasites of the Leishmania donovani sensu stricto cluster; no particular zymodeme has been found to be associated with it. In contrast to PKDL in India, PKDL in Sudan occurs within 0–6 months after treatment for VL. The rash may be macular, maculo-papular or nodular, and spreads from the perioral area to other parts of the body, depending on grade of severity. Young children are particularly at risk of developing more severe disease. In 16% of PKDL patients, parasites can be demonstrated by microscopy in lymph node or bone marrow aspirates and, with the aid of the polymerase chain reaction (PCR), in lymph nodes of 81% of patients, possibly indicating persistent visceralized infection. Diagnosis can be made by demonstration of parasites in skin smears or biopsies in 20–30% of cases; newer techniques, using PCR with skin smears, have higher sensitivity (83%). Monoclonal antibodies against L. donovani can detect parasites in 88% of biopsies. Serological tests are of limited value. The leishmanin skin test is positive in 50–60% of cases; there is an inverse relationship between the skin test result and severity of PKDL. In differential diagnosis, miliaria rubra is the most common problem; differentiation from leprosy is the most difficult. In biopsies, hyperkeratosis, parakeratosis, acanthosis, follicular plugging and liquefaction degeneration of the basal layer may be found in the epidermis; in the dermis there are varying intensities of inflammation with scanty parasites and mainly lymphocytes; macrophages and epithelioid cells may also be found. In 20% of cases discrete granulomas may be found. After VL, the immune response shifts from a Th₂-type to a mixed Th₁/Th₂-type. High levels of interleukin-10 in skin biopsies as well as in peripheral blood mononuclear cells and plasma in patients with VL predict the development of PKDL. Treatment is needed only for those who have severe and prolonged disease; sodium stibogluconate (20 mg/kg/d for 2 months) is usually sufficient. (Liposomal) amphotericin B is effective, whereas ketoconazole, terbinafine and itraconazole are not.     

The effect of repeated leishmanin skin testing on the immune responses to Leishmania antigen in healthy volunteers

The leishmanin skin test (LST) is used in immunogenicity studies. The effect of multiple LSTs on immune responses was assessed. None of the volunteers converted to LST positive. IFN-gamma and IL-10 levels remained unchanged. Repetition of LST does not modulate the in vivo or in vitro immune responses to Leishmania antigen.