Luciana Regina Meireles

Toxoplasma gondii spreading in an urban area evaluated by seroprevalence in free‐living cats and dogs

Infection by the protozoan parasite, Toxoplasma gondii is widely prevalent in humans and animals throughout the world. Transmission takes place mainly by ingestion of raw or undercooked meat that contains parasite cysts or by ingestion of oocysts excreted in cat faeces, which can contaminate water and raw vegetables. The incidence of toxoplasmosis in urban areas can thus be also related to environmental contamination with oocysts. A direct measure of this environmental contamination by oocyst counting is unfeasible for technical reasons. An interesting alternative for measuring T. gondii urban spreading is the seroprevalence in free‐living urban animals, used as sentinels, once they are exposed to similar risks of Toxoplasma infection‐like humans. With this aim, we tested serum samples from stray cats and dogs for antibodies to T. gondii by indirect haemagglutination assay (IHA) and enzyme‐linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 40% (40 of 100) of the cats, less than the 50.5% (101 of 200) found in dogs by ELISA (P < 0.05). Haemagglutination showed low resolution and concordance, precluding their use for diagnosis of T. gondii infection compared with ELISA. The prevalence of T. gondii was lower among stray cats probably due to their selective alimentary habits and lower water and food intake. Toxoplasma gondii seroprevalence in stray dogs and cats could be an indirect indicator of the parasite spreading in urban areas.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese

OBJECTIVE Analyze the infectivity and storage resistance of cysts of the ME-49 strain of Toxoplasma gondii in artificially infected bovine milk and homemade fresh cheese. METHODS Pasteurized bovine milk was infected with 10 cysts/ml of the ME-49 strain of T.gondii and inoculated in different groups of mice, immediately or after storage at 4 degrees C for 5, 10 and 20 days. Homemade fresh cheese was prepared with artificially infected milk, and also tested in groups of mice, using the same storage process. Infection was identified by the presence of cysts in the brain or serological testing in challenged mice after 5 weeks, confirmed by Western Blot and histology. RESULTS The infectivity of cysts of the ME-49 strain of T.gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions. CONCLUSIONS These data demonstrated that milk and dairy products could be an important source of T.gondii in human contamination, reinforcing the importance of milk pasteurization before any processing or ingestion.

Serological survey of antibodies to Toxoplasma gondii in food animals from São Paulo state, Brazil

Toxoplasmosis is one of the most prevalent parasitic infections of man and livestock, and its transmission has usually been attributed to ingestion of undercooked or raw meat from infected livestock, with the infection rate in those animals being an important risk predictor of human disease, high in Brazil and Sao Paulo State. Looking for this public health problem, we tested serum samples from cattle, goat, sheep and chicken from the State of Sao Paulo, Brazil, for IgG antibodies to Toxoplasma gondii by enzyme-linked immunosorbent assay (ELISA). Antibodies to Toxoplasma gondii were found in 31.00% (62/200) of sheep, 17.00% (34/200) of goat and 11.00% (22/200) of cattle, without positive sample in chicken. Despite differences in feeding habits of each species, the rate of infection of tested animals could be better attributed to livestock management methods, which improvement could reduce infection.

Seroprevalence of Toxoplasma gondii Antibodies in Humans From Rural Western Amazon, Brazil

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rondônia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (≥1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.

Preliminary report of HIV and Toxoplasma gondii occurrence in pregnant women from Mozambique

Toxoplasmosis, a protozoan disease, causes severe disease in fetuses during pregnancy and deadly encephalitis in HIV patients. There are several studies on its seroprevalence around the world, but studies focusing on African countries are limited in number and mostly anecdotal. We studied two groups of samples from Mozambique by ELISA, using serum samples from 150 pregnant women and six Cerebrospinal fluid (CSF) samples from AIDS patients with encephalitis. HIV status was confirmed, and CD4 blood counts were obtained from HIV-positive pregnant women. IgG seroprevalence of the group as a whole was 18.7% (28/150), with a higher prevalence in HIV-positive individuals compared to those who were HIV-negative (31.3%, [18/58] vs. 10.9%, [10/92]) patients. These data may be biased due to cumulative effects of exposition affecting disease prevalence. If corrected, this data may indicate an interaction of HIV and T. gondii. Prevalence of both diseases increases with age, but this is more clearly seen for toxoplasmosis (p < 0.005) than HIV infection, possibly explained by higher transmission of HIV after childhood. In HIV patients suffering from encephalitis, CSF serology showed that 33% of specific IgG CSF had a high avidity, which was in accordance with the data from the group of pregnant women. Lower prevalence rates of both infections in older groups could be explained by more deaths in the infected groups, resulting in an artificially lower prevalence. Using CD4 counts as a marker of time of HIV infection, and correcting for age, patients with contact with T. gondii had fewer CD4 cells, suggesting prolonged HIV disease or other causes. Toxoplasma IgG prevalence is higher in HIV+ groups, which could be ascribed to HIV- and T. gondii-associated risk factors, such as exposure to higher and more diverse social contacts. The low incidence of Toxoplasma IgG in younger age groups shows that transmission could be related to better access to cyst-containing meat in adulthood, as environmental transmission due to oocysts is usually blamed for higher incidence in children. Taken together, these data support the urgent need of research in toxoplasmosis in Africa, especially in the presence of HIV epidemics.

Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

Evidência sorológica da infecção aguda pelo Toxoplasma gondii em gestantes de Cascavel, Paraná

In order to investigate the incidence of toxoplasmosis in Cascavel, Paraná, a city near the region where the largest reported epidemic outbreak in the world occurred, 334 serum samples from pregnant women were screened using a commercial IgG immunoenzymatic assay at the Municipal Laboratory in Cascavel and were confirmed at the Institute of Tropical Medicine in São Paulo, by means of IgG immunofluorescence, immunoenzymatic assaying and the in-house IgG avidity test. The IgG seropositivity from the commercial test was 54.2%, from immunofluorescence 54.8% and from the in-house IgG 53.9%, with good agreement between immunofluorescence and the commercial IgG test (kappa = 0.963781; co-positivity = 97.8%; co-negativity = 98,7%) and between immunofluorescence and the in-house IgG (kappa = 0.975857; co-positivity = 97.8%; co-negativity = 100%). The evidence of acute infection among the pregnant women was similar, as estimated both by IgG avidity (2.4%/year) and by statistical trend analysis (chi(2) test) according to age group (2%/year). This suggests that prenatal serological screening and epidemiological surveillance are essential for reducing the risk of toxoplasmosis in the region, although without evidence of an epidemic outbreak.

Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISA test and its use for detection in rabbit meat cuts

Toxoplasma gondii causes severe disease both to man and livestock and its detection in meat after slaughtering requires PCR or biological tests. Meat packages contain retained exudate that could be used for serology due to its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or meat juice, after blood content correction. These assays were adequate regardless of the storage time up to 120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat juice may be useful for quality control for toxoplasmosis monitoring.

Canine visceral leishmaniasis and Chagas disease among dogs in Araguaína, Tocantins

The present study analyzed serum samples from 111 male and female dogs of various ages from the municipality of Araguaína in the State of Tocantins, Brazil. Serological diagnosis of canine visceral leishmaniasis (CVL) was initially performed at the Central Laboratory (Laboratório Central - LACEN) of Araguaína, resulting in 61 positive samples by an indirect immunofluorescence assay (IIFA) (≥1:40) and 50 non-reactive samples. The same samples were analyzed at the São Paulo Institute of Tropical Medicine (Instituto de Medicina Tropical de São Paulo - IMTSP) by an enzyme-linked-immunosorbent assay (ELISA), resulting in 57 positive samples (51.35%) and 54 negative samples (48.64%). The Kappa coefficient of agreement between the tests was 0.74. The serum samples were also subjected to a diagnostic assay for Trypanosoma cruzi (Trypomastigote Excreted/Secreted Antigens -TESA-blot) that detected five suspect animals; three of those animals were positive for leishmaniasis by ELISA but negative by IIFA. These findings suggest that the canine population of Araguaína may be simultaneously infected with Leishmania chagasi and T. cruzi. The results obtained demonstrate the difficulty of using serology to detect CVL, thus emphasizing the necessity for a reference test to diagnose CVL, particularly in regions where the infection is endemic.

A simple immune complex dissociation ELISA for leishmaniasis: Standardization of the assay in experimental models and preliminary results in canine and human samples

Visceral leishmaniasis, caused by Leishmania (Leishmania) chagasi, is a chronic parasitic disease of humans and dogs. Confirmation of the protozoal agent in bone marrow, lymph node or spleen aspirate is diagnostic, while specific-IgG serology is used mainly for epidemiology despite the general presence of high levels of serum immunoglobulin. Anecdotal reports of false-negative serology in active disease cases are known and are ascribed to the formation of immune complexes. Because dissociation of immune complexes can be accomplished by acid treatment, we devised a simple, routine enzyme immunoassay (ELISA) for the dissociation of immune complexes in serum samples using acid treatment in wells adsorbed with Leishmania antigen (dELISA). Confirmatory acid dot-blot was also developed for antigen detection by anti-Leishmania rabbit antiserum. In experimental L. chagasi hamster models, immune complexes interfered with ELISA mostly in the 30 and 60 days postinfection, according to both dELISA and antigen dot-blot results. In larger samples from endemic areas, dELISA was positive in 10% of seronegative dog samples (7/70) and 3.5% in negative human samples (3/88), showing that dELISA could be used in the serodiagnosis of visceral leishmaniasis. Moreover, dELISA could be used as an alternative approach to screening asymptomatic visceral leishmaniasis patients, instead of invasive confirmatory testing.