Ecological aspects of American cutaneous leishmaniasis: 6. Man-biting sandfly fauna of a patch of residual forest in the middle and north-eastern region of the São Paulo State, Brazil

This report shows the phlebotomine vectorial role in the endemic cutaneous leishmaniasis situated in the middle and north-eastern region of the São Paulo, Brazil. Analysis of information encloses a sandfly man-biting fauna of four different patches of residual forest. So, using the human-bait and Shannon trap for a period of one year we have caught 16,869 sandflies. The predominance of Lutzomyia intermedia (85,6%) was clear. Moreover, the low density of Lutzomyia whitmani and Lutzomyia pessoai and their epidemiological implications at present and in the past are discussed. Information about the daily activity of some species is given and we emphasize for the first time, the diurnal activity of Lutzomyia firmatoi in the São Paulo State.

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.

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.

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.

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-g (IFN-g) 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-g 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.

Study of a cutaneous leishmaniasis outbreak in General Vedia, Province of Chaco, 1996

Between March and July 1996, a focalized epidemic outbreak of cutaneous leishmaniasis in General Vedia, province of Chaco, associated to the gallery forest of the Oro river was verified. The incidence rate in the area, which was 0-2/000 cases in preceding years, reached 8/000 cases in 1996. The risk of symptomatic infection was similar between sexes, but was different when analizing the different age groups by sex, suggesting a greater relative importance of the peridomestic transmission for the females and of the transmission in the subtropical forest for the males. Specimens of Lutzomyia intermedia, a species already incriminated as a vector of Leishmaniasis in other provinces of northern Argentina, were captured and identified in the focus locality in May 1996. The possible causes of the outbreak related to the climatic variables and the vector abundance are analyzed and the results in the framework of possible preventive and control activities are discussed.

American cutaneous leishmaniasis outbreak, Tartagal city, province of Salta, Argentina, 1993

An American cutaneous leishmaniasis outbreak, with cases clustering during 1993 in Tartagal city, Salta, was reported. The outbreak involved 102 individuals, 43.1% of them with multiple ulcers. Age (mean: 33 years old) and sex distribution of cases (74.5% males), as well as working activity (70 forest-related), support the hypothesis of classical forest transmission leishmaniasis, despite the fact that the place of permanent residence was in periurban Tartagal. Moreover, during July, sandflies were only collected from one of the 'deforestation areas'. Lutzomyia intermedia was the single species of the 491 phlebotomines captured, reinforcing the vector incrimination of this species. Most infections must have been acquired during the fall (April to June), a pattern consistent with previous sandfly population dynamics data. Based on the epidemiological and entomological results, it was advised not to do any vector-targeted periurban control measures during July. Further studies should be done to assess if the high rate of multiple lesions was due to parasite factors or to infective vector density factors.

The histopathology of cutaneous leishmaniasis due to Leishmania (Leishmania) mexicana in the Yucatan peninsula, Mexico

Localized Cutaneous Leishmaniasis (LCL) known as "chiclero's ulcer" in southeast Mexico, was described by SEIDELIN in 1912. Since then the sylvatic region of the Yucatan peninsula has been documented as an endemic focus of LCL. This study of 73 biopsies from parasitological confirmed lesions of LCL cases of Leishmania (Leishmania) mexicana infection was undertaken: 1) to examine host response at tissue level; and 2) to relate manifestations of this response to some characteristics of clinical presentation. Based on Magalhães' classification we found that the most common pattern in our LCL cases caused by L. (L.) mexicana was predominantly characterized by the presence of unorganized granuloma without necrosis, (43.8%). Another important finding to be highlighted is the fact that in 50/73 (68.5%) parasite identification was positive. There was direct relation between the size of the lesion and time of evolution (r s = 0.3079, p = 0.03), and inverse correlation between size of the lesion and abundance of amastigotes (r s = -0.2467, p = 0.03). In view of the complexity of clinical and histopathological findings, cell-mediated immune response of the disease related to clinical and histopathological features, as so genetic background should be studied.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Low efficacy of azithromycin to treat cutaneous leishmaniasis in Manaus, AM, Brazil

An open trial to evaluate the azithromycin efficacy in cutaneous leishmaniasis patients was carried out in Manaus (AM), where Leishmania (Viannia) guyanensis is the main etiologic agent. Forty-one patients with skin lesions of less than 12 weeks duration, without specific treatment for the last three months and a positive imprint to Leishmania sp. were included. From these, 31 (75.6%) were male with median age of 30.2. All of them received a daily-single oral dose of 500 mg of azithromycin for ten days. At 25th day, 16 (39%) presented therapeutic failure and received intramuscular pentavalent antimonial, four were considered lost, 21, that had improved or were inaltered received another ten-day series of azithromycin and were monthly followed, but nine (21.9%) of them presented a poor clinical response and switched to intramuscular pentavalent antimonial on day 55. Of the 12 remaining cases evaluated on day 55, despite of clinical improvement, three asked for antimony therapy and 9 (21.9%) continued the follow-up but, only three were cured on 55th, 85th and 115th days, and six did not come back for final evaluation. The intention-treatment overall response rate was 22% and whole cure was seen in three (7.3%) of cases. Thus, azithromycin showed a low efficacy to treat cutaneous leishmaniasis in Manaus.