Avaliação de três esquemas terapêuticos com o antimoniato de N-metil-glucamina no tratamento da leishmaniose visceral no estado do Pará, Brasil

Avaliaram-se, de forma retrospectiva, três esquemas terapêuticos à base do antimoniato de N-metil-glucamina (Glucantime) usados no tratamento de 43 casos autóctones de leishmaniose visceral (Estado do Pará), observados em crianças de 1 a 12 anos de idade, no período de 1985 a 1990. Dos 43 casos, 28 (grupo A) foram tratados com 40 mg/SbV/kg administrados IV a intervalos de 48 hs, em séries de 15 doses (esquema I); 8 (grupo B) receberam 40mg/SbV/kg administrados IV diariamente, durante 15 dias (esquema II), e 7 (grupo C) receberam 20 mg/SbV/kg administrados IV diariamente, durante 15 dias (esquema III). Considerando que o controle de cura da doença foi essencialmente clínico, admitiu-se que o esquema III representaria a melhor opção terapêutica, em razão de: a) ter promovido taxa de cura equivalente aos esquemas que usaram o dobro dessa dose, b) a relação custo-benefício desse esquema torna-o menos dispendioso, c) pode ser usado durante período mais prolongado, com menor risco de produzir efeitos de toxicidade, e d) não existem, a nível local (Pará), relatos de casos de resistência da doença associados ao uso desse esquema.

Clinical and laboratorial features of visceral toxocariasis in infancy

Forty children with a diagnosis of Visceral Toxocariasis were evaluated prospectively from February 1982 to June 1989. Diagnosis was established by clinical, laboratorial and serological (ELISA - ES Toxocara canis antigen) evaluations. A great clinical polymorphism was found in our patients, ranging from unspecific or absent manifestations to an exhuberant symptomatology. The laboratorial findings were: leukocytosis,eosinophilia and elevation of serum gammaglobulin and isohemagglutinin levels. No significant relationship between clinical findings and laboratorial parameters was found. Serology (ELISA) was a method of great diagnostic support but did not show a correlation with clinical and laboratorial findings in this study. There was a significant relationship between pulmonary manifestations and the presence of signs and/or symptoms, when the patients were sent to us. Our findings, especially the high incidence of pulmonary manifestations, suggest that Visceral Toxocariasis has to be included in the differential diagnostic of children with pulmonary manifestations, characteristic epidemiological data and associated eosinophilia.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Leishmania (Viannia) panamensis - induced cutaneous leishmaniasis in susceptible and resistant mouse strains

We studied the susceptibility to Leishmania (Viannia) panamensis in strains of mice. The C57BL/6 strain was resistant and showed self-controlled lesion at the injected foot pad. The BALB/c and DBA/2J strains were susceptible and showed a foot swelling that started day 20 post-infection and progressed to a tumour-like lesion in later period of observation. The CBA/HJ strain was found to be of intermediary resistance. In contrast to other known cutaneous leishmaniasis in mice, the lesion in L. (V.) panamensis-infected mice was restricted to the inoculation site in the skin. In addition, we studied the development of cellular response and antibodies against Leishmania antigen in BALB/c and C57BL/6 strains. The proliferative response of lymph node cells against L. (V.) panamensis antigen was biphasic in both strains. An initial response was seen on day 20, followed by a refractory period between 40 and 80 days and a second response around fourth month post-infection. The response in the latter period was higher in C57BL/6 strain than in BALB/c strain. BALB/c strain presented much higher anti-Leishmania antibody level than C57BL/6 strain. The model and the correlation of immunological variables and the course of the infection are discussed.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

An Evaluation of clinical, serologic, anatomopathologic and immunohistochemical findings for fifteen patients with mucosal leishmaniasis before and after treatment

Treatment of mucosal leishmaniasis (ML) can be controlled by clinical examination and by serologic titers by the indirect immunofluorescence serologic reaction (IISR). We studied the correlation between the presence of antigen in tissue determined by immunohistochemistry, the IISR titers and the anatomopathologic findings in fifteen patients with ML before and after healing of the lesions as determined by otorhinolaryngologic evaluation, and evaluated these parameters to determine which of them could be useful during follow-up. Tissue antigens became negative in four patients (group A) after treatment, with a statistically significant reduction or negativity of IISR titers (p<0.05). This did not occur in patients in whom the antigen persisted after treatment (group B), suggesting that serologic follow-up should be performed together with the search for tissue antigen, a combination which, to our knowledge, has not been used in previous studies. The negativity of tissue antigens and the behavior of IIRS titers in group A patients probably indicate a lower possibility of recurrence. Upon anatomopathologic examination the inflammatory process was found to persist after treatment even in group A, suggesting that the permanence of inflammatory activity even in clinically healed lesions is possibly correlated with the presence of the antigen or of some unknown factor.

EPIDEMIOLOGICAL SURVEY ON CANINE POPULATION WITH THE USE OF IMMUNOLEISH SKIN TEST IN ENDEMIC AREAS OF HUMAN AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO, BRAZIL

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V.) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p>0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.

Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

VISCERAL LARVA MIGRANS AND TROPICAL PYOMYOSITIS: A CASE REPORT

We report a case of tropical pyomyositis in a boy who presented with a severe febrile illness associated with diffuse erythema, and swelling in many areas of the body which revealed on operation extensive necrotic areas of various muscles that required repeated débridement. The patient gave a history of contact with dogs, and an ELISA test for Toxocara canis was positive. He also presented eosinophilia and high serum IgE levels. Staphylococcus aureus was the sole bacteria isolated from the muscles affected. We suggest that tropical pyomyositis may be caused by the presence of migrating larvae of this or other parasites in the muscles. The immunologic and structural alterations caused by the larvae, in the presence of concomitant bacteremia, would favour seeding of the bacteria and the development of pyomyositis.

NEPHROTOXICITY ATTRIBUTED TO MEGLUMINE ANTIMONIATE (GLUCANTIME) IN THE TREATMENT OF GENERALIZED CUTANEOUS LEISHMANIASIS

Background: Pentavalent antimonials have became of basic importance for the treatment of leishmaniasis. Their most severe side effects have been reported to be increased hepatic enzyme levels and electrocardiographic abnormalities. Nephrotoxicity has been rarely related. Observations: We report a case of generalized cutaneous leishmaniasis involving a 50-year old male patient who was submitted to treatment with meglumine antimoniate (Glucantime). He developed acute renal failure (ARF) due to acute tubular necrosis (ATN), followed by death after receiving a total of 53 ampoules of Glucantime. Conclusions: The treatment with Glucantime was responsible by ARF diagnosed in this patient. The previous urine osmolarity and serum creatinine levels were normal and the autopsy showed ATN. It should be pointed out if ARF may also be explained by massive deposits of immunocomplexes by leishmania antibodies and antigens due to the antigenic break by the antimonial compound, since our patient presented countless lesions covering the entire tegument, similar to the Hexheimer phenomenon, but at the autopsy no glomerular alterations were seen.

INFLUENCE OF MICROBIOTA IN EXPERIMENTAL CUTANEOUS LEISHMANIASIS IN SWISS MICE

Infection of Swiss/NIH mice with Leishmania major was compared with infection in isogenic resistant C57BL/6 and susceptible BALB/c mice. Swiss/NIH mice showed self-controlled lesions in the injected foot pad. The production of high levels of interferon-<FONT FACE="Symbol">g</font> (IFN-<FONT FACE="Symbol">g</font>) and low levels of interleukin-4 (IL-4) by cells from these animals suggests that they mount a Th1-type immune response. The importance of the indigenous microbiota on the development of murine leishmaniasis was investigated by infecting germfree Swiss/NIH in the hind footpad with L. major and conventionalizing after 3 weeks of infection. Lesions from conventionalized Swiss/NIH mice were significantly larger than conventional mice. Histopathological analysis of lesions from conventionalized animals showed abscesses of variable shapes and sizes and high numbers of parasitized macrophages. In the lesions from conventional mice, besides the absence of abscess formation, parasites were rarely observed. On the other hand, cells from conventional and conventionalized mice produced similar Th1-type response characterized by high levels of IFN-<FONT FACE="Symbol">g</font> and low levels of IL-4. In this study, we demonstrated that Swiss/NIH mice are resistant to L. major infection and that the absence of the normal microbiota at the beginning of infection significantly influenced the lesion size and the inflammatory response at the site of infection.