Biogenic silver nanoparticles reduce adherence, infection, and proliferation of toxoplasma gondii RH strain in HeLa cells without inflammatory mediators induction.

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity - depending on size and concentration - and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 µM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

Patterns of Transmission and Sources of Infection in Outbreaks of Human Toxoplasmosis.

We report on apparent temporal progression of probable sources of infection and transmission routes for global human toxoplasmosis outbreaks as described in published articles. We searched the Scientific Electronic Library Online, Web of Science, PubMed, and Scopus databases for articles on Toxoplasma, toxoplasmosis, and outbreaks. We found that transmission routes for Toxoplasma gondii varied by decade. In the 1960s and 1990s, toxoplasmosis outbreaks mainly occurred through ingestion of cysts in meat and meat derivatives; in the 1980s, through milk contaminated with tachyzoites; in 2000, due to the presence of oocysts in water, sand, and soil; and in 2010, due to oocysts in raw fruits and vegetables. Our study suggests a possible change in the epidemiology of reported toxoplasmosis outbreaks. Because of this change, we suggest that greater attention be paid to the disinfection of vegetables, as well as to the quality of water used for drinking and irrigation.

Molecular docking to Toxoplasma gondii thymidylate synthase-dihydrofolate reductase and efficacy of raltitrexed in infected mice.

Toxoplasmosis is a zoonosis of worldwide distribution. Currently, two drugs, pyrimethamine and sulfadiazine, are used as a reference in the treatment of toxoplasmosis, but the resistance of Toxoplasma gondii appears as a relevant public health problem. In order to identify new drugs to toxoplasmosis treatment, we performed a molecular docking of raltitrexed to T. gondii thymidylate synthase-dihydrofolate reductase (TS-DHFR) and also evaluated its efficacy in infected mice. Initially, raltitrexed was docked on the crystallographic structures of TS-DHFR from T. gondii and Mus musculus. Then, 48 h after infection with the T. gondii RH strain, different groups of mice received an oral dose of raltitrexed (0.15, 0.75, and 1.5 mg kg-1). Two days after treatments, raltitrexed was able to prevent mortality and reduce the number of tachyzoites in the peritoneal fluid and liver imprints from infected mice. The results showed that raltitrexed has important protective activities against the T. gondii RH strain. Molecular docking still suggests that the effects against the parasite may be dependent on the inhibition of T. gondii thymidylate synthase. This study opens new perspectives for the use of raltitrexed in patients infected with T. gondii, especially when conventional treatments do not exhibit the expected efficacy.

Caprine toxoplasmosis in Southern Brazil: a comparative seroepidemiological study between the indirect immunofluorescence assay, the enzyme-linked immunosorbent assay, and the modified agglutination test.

This study investigated the prevalence of caprine toxoplasmosis in goat herds from Southern Brazil by the indirect immunofluorescence assay (IFA), and compared these results with the enzyme-linked immunosorbent assay (ELISA) and the modified agglutination test (MAT). In addition, possible risk factors associated with infection due to Toxoplasma gondii were determined. The serum samples evaluated were from 1,058 goats derived from 94 goat herds within the state of Paraná, Southern Brazil. Seropositivity by IFA was 30.0%, 33.3% by ELISA, and 25.3% by MAT. The risk factors associated with infection by T. gondii in goats were pasture rental, female goats, the presence of cats, and pastures shared with several goat herds. Using IFA as a standard, ELISA and MAT showed substantial concordance (kappa = 0.74 and 0.61), with sensitivities of 87 and 66.2% and specificities of 89.7 and 92.2%, respectively. These results demonstrate that caprine toxoplasmosis is endemic within the state of Paraná, Southern Brazil. In addition, the results from the three assays were relevant, without any significant differences as demonstrated due to the substantial concordance based on the kappa index.

Activity of rosuvastatin in tachyzoites of Toxoplasma gondii (RH strain) in HeLa cells.

Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this study was to assess the activity of rosuvastatin in HeLa cells infected with the RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significant to promote further studies and the search for alternative treatment for toxoplasmosis.

Effects of extracts from Echinacea purpurea (L) MOENCH on mice infected with different strains of Toxoplasma gondii.

In recent years, due to the growing concern about recurrent epidemics by Toxoplasma gondii and other pathogens in Brazil, there has been an increase in the use of different preparations obtained from Echinacea purpurea in order to test their effectiveness against these infections. Although studies have suggested the beneficial effects of this species against the influenza virus, no data are available on the use of E. purpurea aqueous extract in T. gondii infections. Thus, the aim of this study was to analyze the effect of its administration in Swiss mice submitted to acute and prolonged infection with different T. gondii strains. This study showed that E. purpurea extract induced a significant reduction in the number of tachyzoites in the peritoneal fluid and liver imprints from mice infected by the RH strain. Moreover, prolonged treatment significantly increased the number of brain cysts of animals infected with ME 49 strain. The results obtained in this study suggest that the crude extract obtained from E. purpurea has important protective activities against infection with different T. gondii strains.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Occurrence of zoonotic enteric parasites in fecal samples from dogs in shelters, parks, squares and public roads, and the dog guardians' perception of zoonoses as for the risk to public health in the city of Guarapuava, Paraná, Brazil.

The aim of this study was to determine the occurrence of zoonotic enteroparasites in the feces of dogs from public shelters, squares, parks, and public roads in the city of Guarapuava, Paraná, Brazil, and to evaluate the perception of dog guardians regarding zoonoses and their risk to public health. Fecal samples were collected, coproparasitological examinations were performed to detect zoonotic enteroparasites, and polymerase chain reaction (PCR) was used to identify Giardia spp. and Cryptosporidium spp. Questionnaires were given to guardians who walked their dogs in parks, squares, and public roads, as to assess their perception of zoonoses. A total of 333 samples were collected, of these 75, 123, and 135 of them were from public shelters, squares and parks, and public roads, respectively. One or more parasites were identified in 166 (50 %) samples, of which 58/75 (77 %) were from public shelters, 50/123 (41 %) from squares and parks, and 58/135 (43 %) from public roads. The parasites detected included Ancylostoma spp., Giardia spp., Trichuris spp., Toxocara spp., and Cystoisospora spp., with Ancylostoma spp. having the highest occurrence. PCR was performed on 161 samples for convenience due to financial limitations, because only a portion of the study was funded by the municipal government, of which 15.6 % were positive for Giardia spp., and all were negative for Cryptosporidium spp. In total, 246 guardians were interviewed, of which 36 % said they did not collect their animals' feces during walks, 20 % did not use anti-helminthics on their dogs, and 23 % did not know which diseases could be transmitted by feces. Therefore, we conclude that there is a high infection rate of parasites with zoonotic potential in public places, showing the need to raise awareness among guardians about the diseases transmitted by dog feces, correct vermifugation and the importance of collecting feces in public places.

Zoonoses in humans from small rural properties in Jataizinho, Parana, Brazil.

The aim of this study was to conduct a serological survey for Lyme diseases, brucellosis, leptospirosis and toxoplasmosis and identify the risk variables related to these zoonoses in humans living in the rural area of Jataizinho, state of Parana, Brazil. A total of 63 rural properties were surveyed. Additionally, 207 serum samples collected from these rural area inhabitants were tested for indirect immunofluorescence (IFI) and western blots (WB) were performed to detect Borrelia burgdorferi (sensu lato); a tamponated acidified antigen test (AAT) and 2-mercaptoethanol (2-ME) were used to detect antibodies of Brucella abortus; the microscopic agglutination test (MAT) was carried out to detect antibodies anti-Leptospira spp. and IFI was used to find antibodies of Toxoplasma gondii. Two of the samples (0.96%) were reactive for Lyme borreliosis, three (1.4%) for brucellosis, 25 (12.1%) for leptospirosis and 143 (69.1%) for toxoplasmosis. Although the town of Jataizinho has a human development index (IDH) that was considered to be average (0.733) in the state of Parana, the low social, economic and cultural conditions of the population from small rural properties have resulted in lack of basic information on animal health and direct or indirect contact with the various species of domestic animals, wildlife and ticks have probably contributed to the prevalence levels found. These results show the need for additional regional studies in order to determine the epidemiological characteristics of these diseases as well as their respective vectors and reservoirs so that effective prophylaxis can be administered in the human population.

High titers of anti-Leishmania spp. antibodies in apparently healthy dogs in the North Pioneer Mesoregion of the state of Paraná, Brazil.

Leishmaniasis is an anthropozoonosis with vector transmission, and knowledge regarding the occurrence of this parasitosis in sentinels can contribute to infection and disease control measures in humans. The objectives of this study were to evaluate the occurrence of Leishmania exposure and infection in dogs from urban and rural areas in the North Pioneer Mesoregion of the state of Paraná, to evaluate possible risk factors, and to analyze the statistical agreement between the serological techniques that were used. Using a convenience sampling, serum and whole blood samples were collected to perform serological and molecular assays, respectively. The enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) identified 29/204 (14.2%) and 20/204 (9.8%) seropositive dogs, respectively. Five dogs (2.4%) were seropositive for both serological tests, and four dogs presented high titers in the IFAT. None of the samples tested positive for Leishmania spp. DNA according to polymerase chain reaction analysis. No factors were significantly associated with infection. Leishmania parasites circulate in urban and rural dogs in the North Pioneer Mesoregion of the state of Paraná. Despite the absence of clinical cases, seropositive animals with high antibody titers should serve as a warning to the local population that should be properly informed regarding the prevention.

Oocyst shedding in cats vaccinated by the nasal and rectal routes with crude rhoptry proteins of Toxoplasma gondii.

During this study, cats were immunized by the intranasal and rectal routes with crude rhoptry proteins of Toxoplasma gondii admixed with Quil-A. Twenty-five domestic short hair cats divided into five groups (n=5) were used during this evaluation: G1 and G3 cats received 200 µg of the rhoptry proteins with Quil-A (20 µg) by the intranasal and rectal routes, respectively; G2 and G4 cats received bovine serum albumin (BSA, 200 µg/dose) with Quil-A (20 µg); and G5 animals served as unvaccinated controls. All treatments were performed at days 0, 21, 42, and 63. The challenge was done with 800 cysts of the ME49 of T. gondii strain at day 70 (challenge day). The serum IgG, IgM, IgA, and fecal IgA antibody levels were evaluated by using the indirect enzyme-linked immunosorbent assay (ELISA). Some animals produced antibody levels beyond cut-off; however, two animals from G1 (OD(mean)=0.308, OD(cut-off)=0.200) and three from G3 (OD(mean)=0.254) demonstrated IgG levels on being challenged, with similar results occurring in two cats from G1 to IgM (OD(mean)=0.279, OD(cut-off)=0.200). Fecal IgA levels were detected in all G1 cats (OD(mean)=0.330, OD(cut-off)=0.065), and in one cat from G3 (OD(mean)=0.167). The serum and fecal humoral immune responses did not correlate with oocyst shedding. Oocyst shedding varied from 98.4% (G1), 87.5% (G2), 53.0% (G3), to 58% (G4), and was lower than that of G5 cats. The prepatent period of cats vaccinated intranasally (G1) was reduced from 6-9.6 to 2.8 days, suggesting protection of environmental contamination, considering cats as the primary source of contamination. The intranasally and rectally administered rhoptry vaccines were able to partially protect cats against T. gondii cysts on being challenged; however, the intranasal method of vaccination yielded better results relative to the rectal route.

Socioeconomic factors associated with infection by Toxoplasma gondii and Toxocara canis in children.

The intense contact of children with domestic animals or environments contaminated with faeces of these animals, together with habits related to lack of hygiene, can facilitate infection by zoonoses. The study evaluated the seroprevalence and risk factors associated with toxoplasmosis and toxocariasis in schoolchildren in the city of Jataizinho, Paraná. Of the 412 children aged 4-15 years, 56.8% (234/412) presented antibodies reactive to Toxoplasma gondii, 42.5% (175/412) presented antibodies reactive to Toxocara canis, and 27.4% (113/412) were reactive for the two species. The analysis of risk factors showed that prevalence of toxoplasmosis and toxocariasis was associated with the level of education of the child's mother (less than eight years of schooling), age range (10-15 years) and the presence of cats in the residence. In addition, family income (up to a minimum wage), presence of a dog, the habit of playing in soil/sand and eosinophilia were associated with Toxocara canis infection. There was an association between the two zoonoses (p < .01), indicating the existence of coinfection. The results show high prevalence of these two important zoonoses, alerting to the need of implementing control measures in order to reduce the incidence and risks of sequelae in children.

Epidemiological study of toxoplasmosis outbreaks in Brazil.

In Brazil, notification of toxoplasmosis outbreaks and epidemiological investigation is a mandatory activity of health surveillance. We investigated the risk factors for toxoplasmosis during outbreaks, notifications of outbreaks by the health secretary and reports in the literature. Other factors related to the municipalities were determined through the Institute of Geography and Statistics portal. We found that fruits and vegetables were the most described transmission routes in outbreaks, and oocysts were the most common parasitic form; in recent years; there has been an increase in outbreak notifications. We also found that municipalities with high municipal human development index have a higher number of toxoplasmosis infections during outbreaks. There is a need to raise awareness among the population and producers regarding good water management and quality practices and to facilitate the acquisition of complex data to improve preventive strategies.

Absence of Trichinella spp. larvae in carcasses of road-killed wild animals in Paraná state, Brazil.

Trichinella spp. are zoonotic parasites that are widely distributed in warm-blooded carnivores and omnivores, including humans. Until the present moment, Brazil has been considered by World Animal Health Organization free from the domestic cycle of trichinellosis, whereas the parasite's sylvatic cycle has the status of infection in limited zones. However, neighboring countries such as Argentina have reports of parasite larvae in the wild fauna. The present study aimed to determine the occurrence of Trichinella spp. in road-killed wild animals in Paraná, Brazil. Biological samples from 71 wild animals-29 Didelphis albiventris, 11 Nasua nasua, ten Cerdocyon thous, seven Dasypus novemcinctus, six Leopardus guttulus, six Sphiggurus spinosus and two Puma concolor-collected from November 2016 to November 2021 were subjected to artificial digestion, following the methodology described in the REGULATION (EC) No. 2075/2005. No Trichinella spp. larvae were detected in the carcasses of the road-killed wild animals. However, considering the wide spectrum of possible reservoirs that could act as a link between the sylvatic and domestic cycles and considering the current Brazilian status of sylvatic trichinellosis in limited zones, frequent monitoring of wild fauna remains necessary.

Urban capuchin monkeys Sapajus nigritus (Goldfuss, 1809) (Primates, Cebidae) as environmental bioindicators of leishmaniasis.

The aim of this study was to investigate the presence of Leishmania sp. DNA and anti-Leishmania spp. antibodies in free-ranging Sapajus nigritus from an urban forest located in a city in the North Central region of the state of Paraná. For the indirect diagnosis, the direct agglutination test was used with promastigote forms of Leishmania (V.) braziliensis, where it was possible to detect the agglutination reaction in 53.33% of the S. nigritus blood samples. For direct diagnosis, the samples were submitted to real-time quantitative polymerase chain reaction, which confirmed the presence of Leishmania spp. DNA in 26.66% of the tested samples. It reinforces the importance of considering the concept of One Health in the face of diseases with high prevalence, such as leishmaniasis and the need for health education measures. This result shows that the animals in the present study have a role as environmental bioindicators for leishmaniasis.

Evaluation of implementation of the primary, secondary and tertiary prevention measures of the Surveillance Program of Gestational and Congenital Toxoplasmosis in the city of Londrina-PR.

Toxoplasmosis acquired during pregnancy is one that can lead to death or malformations of the foetus, and it is a complex disease to diagnose. The objective of the study was to evaluate the Surveillance Program of Gestational and Congenital Toxoplasmosis. To assess primary prevention, 424 pregnant women were interviewed regarding their knowledge of prevention measures in 2019. Secondary prevention measures were assessed, and the results of anti-Toxoplasma gondii serological tests were collected from pregnant women, from 2015 to 2018. In tertiary prevention measures, babies of mothers with a recent suspicion of T. gondii infection were screened to verify forwarding to the reference service. As a result, 45.5% (192/424) reported that they had received guidance from health professionals; 35.4% (68/192) changed their risk habits. The variables of schooling and age, having received prior guidance from health professionals and feline possession, proved to be significant when associated with the notions of preventive measures. 90.2% (17,423/19,319) of pregnant women had undergone serological tests to detect anti-T. gondii antibodies, but there was an excess in requests for tests and medication and only 40.6% (26/64) of the children were referred to the reference hospital. The Program presents positive results about the performance of serological screening in prenatal care; however, the dissemination of knowledge as for the prevention of toxoplasmosis and the request for tests need to be improved.

Evaluation of an active and early surveillance methodology for visceral leishmaniasis by molecular detection in road-killed wild fauna.

The present study aimed to evaluate a methodology for active surveillance of visceral leishmaniasis by detecting Leishmania DNA in organs of wild road-killed animals from November 2016 to October 2018 in the North of Paraná, Brazil. The collection points of road-killed wild animals were georeferenced. The animals were autopsied and samples of bone marrow, lymph node, liver, spleen, and ear skin were collected. Genomic DNA was extracted and subjected to PCR for amplification of Leishmania spp. 18S, kinetoplastic DNA (kDNA), HSP70, and ITS1 genes, and DNA sequencing was performed. The primers used for the amplification of kDNA, ITS1, and HSP70 genes presented non-specific results. Of the 66 mammals collected from 24 different municipalities, one Southern Tamandua (Tamandua tetradactyla) presented DNA of Leishmania spp. in lymph nodes by 18S PCR. DNA sequencing confirmed the results of the subgenus, Viannia, identification. We suggest using the methodology showed in the present study in the active and early surveillance of visceral leishmaniasis in a non-endemic area.

Phenotypical and genotypical differences among Leishmania (Leishmania) amazonensis isolates that caused different clinical frames in humans and dogs: A systematic review.

Leishmania (Leishmania) amazonensis is an important etiological agent of American cutaneous leishmaniasis (ACL) in Brazil. The species causes a large spectrum of clinical manifestations in humans and dogs, ranging from cutaneous, cutaneous diffuse, mucocutaneous, and visceral involvement, however, the factors that drive the development of different disease forms by the same species are not yet fully known. In the present work, it was systematically reviewed the studies addressing phenotypic and genotypic characteristics of Leishmania (L.) amazonensis isolates causing cutaneous and visceral clinical frames in humans and dogs, comparing the results observed. For this, four research databases were searched for the following keywords: (Leishmania amazonensis AND visceral leishmaniasis) AND (tropism OR virulence OR visceralization OR adaptations OR mutation OR clinical presentation OR resistance OR survival OR wide spectrum). The results revealed that the complexity disease seems to involve the combination of genetic factors of the parasite (as modifications in molecules related to the virulence and metabolism) and also of the host's immune background and status. Nonetheless, the exact mechanism that leads to different clinical manifestations between strains of the same species is still uncertain and future studies must be developed to better elucidate this phenomenon.

Techniques for inactivating Toxoplasma gondii oocysts: a systematic review.

The oocyst, a resistant form of Toxoplasma gondii, plays an important role in the transmission of this protozoan. The objective of this review was to report the methods capable of inactivating oocysts through a systematic review of the literature carried out in the Scientific Electronic Library Online, Web of Science, Science Direct, PubMed and Scopus databases. The keywords searched were (((effects OR infectivity OR resistance) AND Toxoplasma) AND oocyst). We selected 16 articles that described 309 different treatments. Among all the protocols evaluated, 35.60% (110/309) were effective in inactivating oocysts. Physical methods were more effective than other methods (p <0.05). Sporulated oocysts and the T. gondii VEG strain were more resistant (p <0.05) to treatments. Although it is effective against viruses and bacteria, the use of disinfectants in water has little or no effect on T. gondii oocysts. The use of radiation and pressure were effective in inactivating oocysts, as these treatments do not include changes in temperature, they can be used in foods for raw consumption, such as vegetables, as it will not cause substantially changes in their physical and chemical characteristics. Therefore, these methods can be viable alternatives for the control of T. gondii.

American cutaneous leishmaniasis associated with degradation of native forest, regardless of economic, social and infrastructure vulnerability.

American cutaneous leishmaniasis (ACL) is a notifiable dermatozoonosis with relevant morbidity. The present study aimed to evaluate the epidemiological aspects of cases of ACL reported in Northern Paraná (2007-2016), as well as to georeference and analyse the influence of economic, social and environmental variables. Data from ACL notification forms were obtained and the patients' houses were georeferenced. Descriptive statistics, calculations of disease incidence and proportion of vegetation cover by municipality, spatial analysis, multiple linear regression and vulnerability analysis by census sector (CS) were performed. One thousand four hundred fifty-one cases of ACL were reported in the 89 municipalities of four regional health (RH). The average incidence of cases per 10,000 inhabitants was as follows: 11.58, 8.79, 4.92 and 4.03 in 18thRH, 15thRH, 16thRH and 17thRH. Peaks of incident cases were observed in 2008, 2012 and 2015. There was a statistically significant difference between the HR when comparing the proportions of the variables gender, age, education level, area of residence, clinical form, diagnostic criteria, response to treatment and drug used in case of failure. It was observed that the lower the proportion of remaining Atlantic Forest, the greater the incidence of ACL in the municipality. With regard to clusters analysis, treatment abandonment clusters were observed in the 15thRH and mucous form clusters were observed in the 15thRH, 16thRH and 17thRH. The vulnerability analysis by CS allowed us to observe a statistically significant difference in all vulnerability indicators: economic and social in 15thRH and 17thRH; home infrastructure in 15thRH and 18thRH, urban infrastructure in 15thRH and 18thRH and compound vulnerability index in 15thRH and 18thRH. However, the most vulnerable areas did not always have the highest number of cases. The data presented demonstrate that preventive and health education measures must be mainly directed to areas of greater degradation of native forest; regardless of the vulnerability situation.