Evaluation of Cytopathological Techniques for the Diagnosis of Canine Visceral Leishmaniosis with Lymph Node Samples.

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE-IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB-ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB-ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE-IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB-ICC and SC + CB-ICC was identical (18.46 amastigotes/mm2). Our study suggests that CB-ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.

Clinical analysis and parasite genetic diversity in human immunodeficiency virus/Chagas' disease coinfections in Brazil.

To evaluate the possible role of parasitemia on Chagas' disease reactivation in Chagas' disease/human immunodeficiency virus (HIV) coinfection cases and the impact of HIV coinfection on Trypanosoma cruzi genetic diversity, 71 patients with Chagas' disease (34 HIV+ and 37 HIV-) were surveyed. Moreover, 92 T. cruzi stocks from 47 chronic chagasic patients (29 HIV+ and 18 HIV-) were isolated and analyzed by multilocus enzyme electrophoresis and a random amplified polymorphic DNA procedure. High parasitemia appeared to play a major role in cases of Chagas' disease reactivation. In HIV+ patients, the genetic diversity and population structure (clonality) of T. cruzi was similar to that previously observed in HIV- patients, which indicates that immunodepression does not modify drastically genotype repartition of the parasite. There was no apparent association between given T. cruzi genotypes and specific clinical forms of Chagas' disease/HIV associations.

Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors.

Determination of the rate of Trypanosoma cruzi infection in its triatomine vectors is an element in control programmes directed at reducing transmission of the organism to humans. Traditionally, T. cruzi has been detected in these insects by microscopical examination of intestinal contents or excreta. The sensitivity of this laborious process has not been defined because of the lack of a bench-mark method against which microscopical examination could be compared. The purpose of this study was to compare the sensitivity of a polymerase chain reaction (PCR) assay with that of microscopical examination for detecting T. cruzi in Triatoma infestans nymphs that had fed on patients with chronic Chagas disease. To this end, we analysed 54 pairs of samples, each containing 2 groups of 10 insects, obtained by feedings on 19 patients with chronic T. cruzi infection, 17 of whom were fed upon 3 times. One group of insects in each pair was analysed by PCR and the other by microscopical examination of excreta. Overall, the PCR assay gave positive results in 32 of 54 groups of insects examined (59%), whereas only 7 of 54 groups (13%) were positive by microscopical examination (P = 0.038). These results demonstrate that the PCR assay is significantly more sensitive for the detection of T. cruzi in triatomine vectors than is microscopical examination, and suggest that the PCR assay could be a useful tool in epizootiological studies.

Regional variation in toxoplasmosis seronegativity in the São Paulo metropolitan region.

Toxoplasmosis is a highly prevalent zoonotic human infection caused by the Apicomplexa protozoon Toxoplasma gondii. The acute disease is usually mild or asymptomatic, except for foetal infection transmitted by acutely infected pregnant women, which courses as a devastating disease. In order to determine possible regional variations in risk factors, we studied the frequency of seronegativity in areas of the São Paulo Metropolitan Region, comparing titers and age groups. The prevalence of seronegativity was determined retrospectively in 1286 pregnant women receiving prenatal care at public health services in four selected areas of the São Paulo Metropolitan Region of similar socioeconomic background. The São Paulo City area had the higher frequency of seronegativity (41.1%), followed by the Northwest (31.5%) and Southwest (29.9%) areas, with similar intermediate levels, and by the Northeast (22.5%) area with the lowest frequency (p < 0.001). A rough estimate disclosed about 280 infected infants/year in the São Paulo Metropolitan Region. Serological titers analyzed by age group suggested a decline in antibody levels with age, as shown by a lower frequency of higher titers in older groups. Our study emphasizes the importance of determining the regional prevalence of toxoplasmosis for proper planning of public health prenatal care.

[Evaluation of the therapeutic activity of itraconazole in chronic infections, experimental and human, by Trypanosoma cruzi]. / Avaliação da atividade terapêutica do itraconazol nas infecções crônicas, experimental e humana, pelo Trypanosoma cruzi.

Other authors have demonstrated that itraconazole has antiparasitic activity against Trypanosoma cruzi both in vitro and in animal acute infection. Because of these observations, we decided to evaluate the chronic phase of this protozoal disease, since it is the most important form under a clinical and assistential point of view. We studied an infected mouse model as well as human cases of Chagas' disease. One hundred mg/kg/day by gastric tube, and 100 or 200 mg/day orally were given, respectively, during three months, without showing any beneficial effect, at least with the adopted methods.

Possible oral transmission of acute Chagas' disease in Brazil.

In October 1986, 7 to 22 days after a meeting at a farm in Paraíba state, 26 individuals presented with a febrile illness associated with bilateral eyelid and lower limb edema, mild hepatosplenomegaly, lymphadenopathy and, occasionally a skin rash. A 11-year-old boy exhibited atrial premature complexes and a 74-year-old patient developed acute heart failure. In two patients hospitalized in São Paulo city, acute Chagas' disease was diagnosed by the demonstration of circulating Trypanosoma cruzi. At autopsy in a fatal case, acute Chagas' cardiomyopathy was demonstrated. Xenodiagnosis were positive in 9 out of 14 tested patients. A specific IgG immune response was found in all patients and specific IgM antibodies were identified in 20 out of 22 tested patients. A epidemiological survey showed the existence of Triatoma brasiliensis in the outbuildings of this farm, but none in the house where most of the guests stayed. A high rate of infection with Trypanosoma cruzi was found in opossums. These observations together with those related to the food consumed by the patients, lead the authors to suggest that the human infections resulted from oral contamination probably originating from naturally infected marsupials in the area or crushed infected bugs.

[Acute Chagas' disease: transmission routes, clinical aspects and response to specific therapy in diagnosed cases in an urban center]. / Doença de Chagas aguda: vias de transmissão, aspectos clínicos e resposta à terapêutica específica em casos diagnosticados em um centro urbano.

The authors report clinical features and therapeutic response of 24 outpatients with acute Chagas' disease, and 3 in the initial chronic phase, referred to the Clinic for Infectious and Parasitic Diseases of the FMUSP "Clínicas" Hospital between 1974 and 1987. The following transmission routes were involved: triatominae in 7 cases, blood transfusion in 9, kidney transplantation and/or blood transfusion in 4, accidental in 1, oral route in 3, probably breast feeding in 1, congenital or breast feeding in 1, and congenital or blood transfusion in 1. Six patients infected by triatominac acquired the disease between 1974 and 1980 and one in 1987. The blood transfusion infected patients acquired the disease in Greater São Paulo, seven of whom after 1983. The acute phase Chagas' disease was oligosymptomatic in 4 patients: three of such patients being immunocompromised by drugs or other diseases. Another two adult immunocompromised patients developed myocarditis and congestive heart failure. Clinical features were severe in 5 from 6 children under two years, irrespective of the transmission route. Evaluation of the acute phase patients treated with benznidazol (4-10 mg/kg/day) showed: therapeutic failure in 4/16 (25.0%); possible cure in 9/16 (53.2%) and inconclusive results in 3/16 (18.8%). The antibody and complement-mediated lysis reaction was in keeping with the xenodiagnosis in 18/22 cases, having shown negative results after treatment earlier than classical serological reactions. One aplastic anaemia patient receiving corticosteroid presented lymphoproliferative disease 6 years after being treated with benznidazol for acute Chagas' disease.

[Xenodiagnosis in Chagas disease: influence of the triatominae sex]. / O xenodiagnóstico na doença de chagas: influência do sexo dos triatomíneos.

We evaluated, comparatively, the performance of female and male Triatoma infestans in the 5th instar of development, in xenodiagnosis for Chagas' disease. Xenodiagnosis were done in 40 patients with chronic infectans. For each diagnosis 20 nymphs female and 20 nymphs males were used, which were weighed immediately before and after the meal. Intestinal contents were examined about 20, 30, 60 and 90 days after the xenodiagnosis application. The females and males ingested the mean of 230 and 210 mg of blood, respectively. The results revealed positivity of 15 (37.5%) out of 40 xenodiagnosis, but any of the positive tests show 100% of the nymphs, female and/or male infected by T. cruzi. The females ingested significantly more blood than the males; this is coincidental with the higher female positivation to T. cruzi here observed. Our results suggest that the female nymphs seem able to increase the sensibility of the xenodiagnosis for chronic patients with Chagas' disease being necessary further studies are necessary to confirm this hypothesis.

Ecoepidemiological aspects of American cutaneous leishmaniasis in the state of São Paulo, Brazil.

The aim of this study is to review some of the ecoepidemiological aspects of american cutaneous leishmaniasis (ACL) in the State of São Paulo, Brazil. During the first half of this century ACL occurred in São Paulo, predominantly on the bank of the Tietê River, where there were railroad constructions and there was inevitable contact between workers and forested areas. Man's activities resulted in a drastic reduction of the forested regions of the State and molded the present landscape found in São Paulo, which brought a gradual change in the epidemiology of ACL during this century. Currently ACL can be considered as an endemic disease. Nowadays, ACL is found in different regions of São Paulo State, and is no longer limited to the bank of the Tietê River. The disease occurs in all age groups and sexes. Lack of knowledge about wild reservoir hosts of Leishmania (V.) braziliensis has simulated speculation about the possible role played by domestic animals (dogs and equines). Man's activities also favoured Lutzomyia intermedia a sandfly species which can clearly thrive in changed environments. L.(V.) braziliensis continues to be transmitted, even after decades of forest destruction in São Paulo.

Experimental toxoplasmosis, and immunosuppression (parasite evaluation and histological alterations)

With the purpose of studying immunosuppressant drug action on parasites, the effect of Cyclosporine and cortisone in experimental toxoplasma evaluation, a highly virulent strain of Toxoplasma gondii (RH strain) was used. The animals were divided in two groups: control and immunosuppressed. Two animals from each group were sacrificed daily. Evaluation was based on parasitemia and parasitism of peritoneal exsudate and presence of the parasite in heart, lung, liver, spleen, small bowel, brain, cerebellum, spinal cord and eyes. Parasite infestation in peritoneal exsudate was 10 times greater than parasitemia. Red spots were observed after the second day; after the fourth day, focal necrosis with softening and fluidification of brain tissue spattered throughout the nervous tissue with enhanced vascularization. Capillaries obstructed by Toxoplasmas gondii were found, causing infarction and necrosis due to toxic or enzymatic substances. Images suggestive of cells an "owl-eye", Cytomegalovirus-like were found, with T. gondii, both in the brain, as well as in the tear glands and small bowel. Blood stream parasite infestation was observed, no histopathological lesions were found in the remaining tissue, possibly due to lack of time, for all animals died within a week's observation. We conclude that immunosuppression has worsened the outcome, anticipating the disease in 24 hours.

Regional variation in toxoplasmosis seronegativity in the Sao Paulo Metropolitan Region

Toxoplasmose e uma infeccao zoonotica humana de lata prevalencia, causada por um protozoario Apicomplexa, Toxoplasma gondii. a evolucao da doenca aguda e geralmente leve ou assintomatica, exceto nas infeccoes agudas das gestantes, quando a infeccao fetal causa uma doenca devastadora. Para determinar se haveriam fatores de risco regionais, foi analisada a frequencia de titulos de anticorpos T. gondii em areas na regiao Metropolitana de Sao Paulo, comparando grupos etarios....

Possible oral transmission of acute Chagas'disease in Brazil

Em outubro de 1986, 7 a 22 dias apos uma reuniao em uma fazenda no estado da Paraiba, 26 pessoas apresentaram doenca febril, associada a edema bipalpebral bilateral, e de membros inferiores, hepatoesplenomegalia leve, linfadenopatia e, mais raramente, a um exantema. Um menino de 11 anos apresentou arritmia atrial ao eletrocardiograma e um paciente de 74 anos desenvolveu insuficiencia cardiaca aguda. Em 2 pacientes hospitalizados em Sao Paulo, foi estabelecido o diagnostico de Doenca de Chagas aguda por observacao de T. cruzi em creme leucocitario. Em autopsia de um caso fatal foi demonstrada cradiomiopatia chagasica aguda. O xenodiagnostico foi positivo em 9 de 14 pacientes testados. Anticorpos especificos de classe IgG foram encontrados em todos os pacientes e da classe IgM em 20 de 22 doentes examinados. Estudo epidemiologico revelou Triatoma brasiliensis nas vizinhacas desta fazenda, porem tal vetor nao foi encontrado na casa onde a maioria dos hospedes pernoitou. Observou-se alta taxa de gambas infectados por Trypanosoma cruzi. Essas observacoes, associadas as informacoes relativas aos alimentos consumidos, sugerem que a contaminacao de alimentos tenha se originado de secrecoes de marsupiais naturalmente infectados ou de triatomineos infectados que poderiam ter sido esmagados durante o preparo do caldo de cana.

Doença de Chagas aguda: vias de transmissäo aspectos clínicos e resposta a terapêutica específica em casos diagnosticados em um centro urbano / Acute Chagas' disease: transmission routes, clinical aspects and response to specific therapy in diagnosed cases in an urban center

Relata-se o quadro clínico de 27 pacientes com doença de Chagas aguda, acompanhados no ambulatório da Clínica de Doenças Infecciosas e Parasitárias do Hospital das Clínicas da FM-USP no período de 1974 a 1987. As vias de transmissäo envolvidas foram: vetorial em 7 casos, transfusional em 9, transplante de rim e/ou transfusional em 4, acidental em 1, via oral em 3, provável alcitamento materno em 1, congênita ou aleitamento materno em 1, congênita ou transfusional em 1. Pacientes com infecçäo por via vetorial eram procedentes da Bahia e Minas Gerais, infectados por via transfusional adquiriram a doença na Grande Säo Paulo, 7 deles após 1983. O quadro clínico foi oligossintomático ou assintomático em 4 pacientes, sendo 3 deles imunodeprimidos por doença de base ou por medicamentos. Em outros 2 pacientes imunodeprimidos ocorreu miocardite grave com insuficiência cardíaca congetiva. O quadro clínico foi também mais grave em 5 de 6 crianças menores de dois anos de idade, qualquer dque fosse a via de transmissäo