Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil.

OBJECTIVE: To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS: Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS: Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION: Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil

BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil

ABSTRACT OBJECTIVE To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil.

American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Cytokine profile and parasite load in lymph nodes of dogs naturally infected with Leishmania infantum from distinct epidemiological scenarios in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil / Leishmaniose cutânea canina persistente causada por Leishmania (Viannia) braziliensis em uma área com predomínio de Nyssomyia neivai no estado de São Paulo, Brasil

Abstract American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Resumo A leishmaniose tegumentar Americana (LTA) é uma doença zoonótica negligenciada, causada principalmente por Leishmania (Viannia) braziliensis, sendo endêmica em todo o Brasil. Foram investigados casos de LTA canina em uma área rural da cidade de Monte Mor, São Paulo, onde foi confirmado um caso humano de LTA. Os cães foram avaliados por diagnóstico clínico e laboratorial, incluindo sorologia, esfregaços microscópicos e PCR de amostras em lesões de pele, linfonodos e medula óssea. Também foram realizadas investigações entomológicas durante 14 meses, usando-se armadilhas luminosas para flebotomíneos nas proximidades da área de estudo. Nyssomyia neivai foi a espécie de flebotomíneo predominante com 94,65% dos espécimes capturados (832 de 879). Essa espécie foi a mais abundante em todos os locais de captura, incluindo-se abrigos para humanos e cães. Foram também capturadas as espécies Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri e Expapilata firmatoi. Dos três cães examinados, dois apresentaram IgG anti-Leishmania positivo no ELISA, usando-se o antígeno "Fucose mannose ligand", PCR da lesão de pele positivo para L. (V.) braziliensis e negativo em todas amostras para L. (L.) infantum. Um dos cães apresentou infecção persistente por mais de um ano.

Gastrointestinal, skin and blood parasites in Didelphis spp. from urban and sylvatic areas in São Paulo state, Brazil.

Didelphis (Marsupialia, Didelphimorphia) are synanthropic mammals, whose omnivorous diet predisposes them to infections caused by endoparasites. Their higher frequency in urban areas makes them potential carriers of zoonotic protozoans and helminths, enhancing potential transmission to humans. Our purpose was to study two common species, Didelphis albiventris (54 individuals) and D. aurita (2 individuals), which were screened for blood, skin and intestinal parasites in animals captured in urban areas and in riparian forest regions associated with the Capivari River Basin, in Monte Mor's municipality, São Paulo state (SP), Brazil. Blood and tissue samples were collected for DNA extraction and PCR. Fecal samples were collected and submitted to two sedimentation and two flotation methods. 77.6% of fecal samples were positive for nematode eggs, 34.5% for trematode eggs and 32.7% for protozoans. Two D. aurita specimens were naturally infected by Trypanosoma cruzi. Molecular analysis in a D. albiventris captured on a forested rural area was positive for Leishmania sp. DNA. Several parasites were found infecting Didelphis sp., demonstrating that this group of animals can harbor important zoonotic parasites, potentially playing a role as sylvatic reservoirs for human and domestic animal pathogens.

Antibodies and Molecular Detection of Leishmania (Leishmania) infantum in Samples of Free-Ranging Marmosets (Primates: Callitrichidae: Callithrix spp.) in an Area of Canine Visceral Leishmaniasis in Southeastern Brazil.

Leishmaniasis is a vector-borne parasitic protozoan infection that affects mammals and involves a complex epidemiology. Although dogs are considered the main reservoir in zoonotic visceral leishmaniasis (VL), the possible presence of other mammalian species acting as reservoirs has been associated as a possible cause of lack of success in the control of human VL in many endemic areas. The knowledge about natural infections of some species is still scarce, such as nonhuman primates (NHP), especially from the genus Callithrix (marmosets). We investigated the infection by Leishmania (Leishmania) infantum, the agent of VL in the Americas, in 26 marmosets captured monthly, from April 2014 to March 2015, in an environmentally protected area (EPA) in Southeastern Brazil. The EPA has undergone significant environmental changes and has a transmission focus of canine VL since 2009. Serology was performed through the direct agglutination test, which detected low antibody titers in seven marmosets (7/26; 26.9%, 95% confidence interval 9.9-44.0), being five Callithrix penicillata (black-tufted-ear marmoset) and two Callithrix jacchus (white-tufted-ear marmoset). The presence of the DNA of Leishmania was investigated in blood and skin samples by PCR and genetic sequencing. This is the first report of the detection of L. (L.) infantum in the skin of a marmoset, which was verified in a sample from one C. penicillata. The results demonstrate the natural infection of marmosets by L. (L.) infantum and may suggest the participation of these animals as hosts in the parasite's transmission cycle in the EPA. However, more comprehensive studies are needed to elucidate their role on the VL epidemiology in this area and also in different endemic areas, especially because these NHP are increasingly in contact with humans and domestic animals, particularly due to environmental changes.

Molecular detection of Leishmania (Leishmania) infantum in phlebotomine sandflies from a visceral leishmaniasis endemic area in northwestern of São Paulo State, Brazil.

This study identified the natural infection rate of Leishmania (Leishmania) infantum in Lutzomyia longipalpis sandflies collected in a neighborhood around a kennel, in Dracena, northwestern of São Paulo state. This region is highly endemic for visceral leishmaniasis in Brazil. Insects were captured during 2-3 nights monthly for 11 months (January-November 2012) using 10 automatic light traps around a kennel in a transition between periurban and urban neighborhood. Capture aimed the determination of the minimal infection rate (MIR) on the area. A total of 1690 Lu. longipalpis were captured during the studied period. Out of them, 292 (17.25%) were females and were grouped in 165 pools containing 1 to five insects for DNA extraction and PCR analysis. Positive results for L. (L) infantum in conventional PCR and real time PCR were shown in 7.28% (12/165) and 4.85% (8/165) of the analysis respectively. These data confirm that Lu. longipalpis captured in the study area were infected by L. (L.) infantum. The MIR of sandflies during the 11 months of captures was 4.10% for female the total of 292 female sandflies collected. A high DNA concentration of L. (L.) infantum was detected on sandflies especially in kennel, chicken coop and neighboring houses, where higher abundance of hosts for blood source were present.

Visceral leishmaniasis in an environmentally protected area in southeastern Brazil: Epidemiological and laboratory cross-sectional investigation of phlebotomine fauna, wild hosts and canine cases.

BACKGROUND: Leishmaniasis is a rapidly expanding zoonosis that shows increasing urbanization. Concern exists regarding the role of wildlife in visceral leishmaniasis (VL) transmission, due to frequent natural or anthropogenic environmental changes that facilitate contact between wildlife, humans and their pets. The municipality of Campinas, in southeastern Brazil, initially recorded VL in 2009, when the first autochthonous case was confirmed in a dog living in an upscale residential condominium, located inside an environmentally protected area (EPA). Since then, disease transmission remains restricted to dogs inhabiting two geographically contiguous condominiums within the EPA. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional study of the VL focus to investigate Leishmania spp. infection in domestic dogs, wild mammals and sand flies using molecular tools and recommended serological techniques. Canine seroprevalences of 1.5% and 1.2% were observed in 2013 and 2015, respectively. Six insect species, confirmed or suspected vectors or potential transmitters of Leishmania, were identified. Two specimens of the main L. (L.) infantum vector in Brazil, Lutzomyia longipalpis, were captured in the EPA. Natural infection by L. (L.) infantum was recorded in one Expapillata firmatoi specimen and two Pintomyia monticola. Natural infection by L. (L.) infantum and Leishmania subgenus Viannia was also detected in two white-eared opossums (Didelphis albiventris), a known reservoir of VL. Geographical coordinates of each sampling of infected animals were plotted on a map of the EPA, demonstrating proximity between these animals, human residences, including the dogs positive for VL, and forest areas. CONCLUSIONS/SIGNIFICANCE: The EPA, which is inhabited by humans, has an active VL focus. The risk of establishing and maintaining disease transmission foci in similar scenarios, i.e. wild areas that undergo environmental modifications, is evident. Moreover, different epidemiological profiles of VL must be included to elaborate prevention and control measures that consider the particularities of each transmission area.

WITHDRAWN: Selection of reference genes in five types of human tissues for normalization of gene expression studies in infectious diseases.

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Infection by Leishmania spp. in Free-Ranging Opossums (Didelphis albiventris) in an Environmentally Protected Area Inhabited by Humans in Southeastern Brazil.

There is a growing concern about the participation of wild hosts and reservoirs in the epidemiology of leishmaniasis, particularly within the context of increasingly frequent environmental changes and the expansion of the One Health concept. This work is a molecular research of infection by Leishmania spp. among the wildlife of an environmentally protected area located in the municipality of Campinas, São Paulo, Brazil. The studied area has a history of intense environmental changes, with notifications of human cases of cutaneous leishmaniasis in the 1990s, and a focus of canine visceral leishmaniasis since 2009. Eighty-two wild mammals were sampled by monthly captures in this region over a 1-year period. Blood samples were collected from each animal and subjected to DNA extraction and PCR using primers for the region of the internal transcribed spacer-1. The results of gene sequencing for the first time revealed the infection of opossums (Didelphis albiventris) by Leishmania spp., subgenera Leishmania and Viannia, in Campinas. These findings, in addition to environmental and historical characteristics of the studied area, indicate a possible role of wildlife in the introduction and/or maintenance of natural foci of leishmaniasis transmission.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Evolution of cytokine profile during the treatment of cerebral toxoplasmosis in HIV-infected patients.

This study was to follow IFN-γ, TNF-α and IL-10 modulation of peripheral blood mononuclear cells (PBMC) from HIV/cerebral toxoplasmosis patients (CT) during specific treatment. The results were compared with two other groups: HIV patients that had CT at least one year before (P/CT) and individuals with chronic toxoplasmosis (CHR). Blood samples (63) collected from three groups were analyzed. CT, 15 patients (3 blood samples collected one day before Toxoplasma gondii treatment; 7 and 15days during the treatment). P/CT, 5 patients (one blood sample collected at least, one year after the treatment). CHR, 13 individuals with chronic toxoplasmosis (one blood sample). Cytokine levels were assessed by ELISA after PBMC stimulation with T. gondii antigen. CT patients had low IFN-γ; discrete increase at 7th and 15th days; and the levels were recovered in cured patients (P/CT). CT patients had high TNF-α in the beginning of the treatment. TNF-α levels decrease during the treatment (7th and 15th) and in those patients who were treated (P/CT). IL-10 levels were almost similar in CT and P/CT groups but low when compared with CHR individuals. The evolution of the infection was correlated to restoration of IFN-γ response and a decrease of the inflammation. The evaluation of the immune response can provide valuable information and better monitoring of patients during specific treatment.

Cerebral and ocular toxoplasmosis related with IFN-γ, TNF-α, and IL-10 levels.

This study analyzed the synthesis of Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), and Interleukin 10 (IL-10) in chronically infected patients which developed the symptomatic disease as cerebral or ocular toxoplasmosis. Blood from 61 individuals were divided into four groups: Cerebral toxoplasmosis/AIDS patients (CT/AIDS group) (n = 15), ocular toxoplasmosis patients (OT group) (n = 23), chronic toxoplasmosis individuals (CHR group) (n = 13) and healthy individuals (HI group) (n = 10). OT, CHR, and HI groups were human immunodeficiency virus (HIV) seronegative. The diagnosis was made by laboratorial (PCR and ELISA) and clinical subjects. For cytokine determination, peripheral blood mononuclear cells (PBMC) of each patient were isolated and stimulated in vitro with T. gondii antigen. IFN-γ, TNF-α, and IL-10 activities were determined by ELISA. Patients from CT/AIDS and OT groups had low levels of IFN-γ when were compared with those from CHR group. These data suggest the low resistance to develop ocular lesions by the low ability to produce IFN-γ against the parasite. The same patients, which developed ocular or cerebral toxoplasmosis had higher TNF-α levels than CHR individuals. High TNF-α synthesis contribute to the inflammatory response and damage of the choroid and retina in OT patients and in AIDS patients caused a high inflammatory response as the TNF-α synthesis is not affected since monocytes are the major source this cytokine in response to soluble T. gondii antigens. IL-10 levels were almost similar in CT/AIDS and OT patients but low when compared with CHR individuals. The deviation to Th2 immune response including the production of anti-inflammatory cytokines, such as IL-10 may promote the parasite's survival causing the tissue immune destruction. IL-10 production in T. gondii-infected brains may support the persistence of parasites as down-regulating the intracerebral immune response. All these indicate that OT and CT/AIDS patients produced low levels of IL-10 (Th2 response) and IFN-γ (Th1 response). They produced high TNF-α suggesting a high inflammatory response triggered by the parasite.

IgG4 specific to Toxoplasma gondii excretory/secretory antigens in serum and/or cerebrospinal fluid support the cerebral toxoplasmosis diagnosis in HIV-infected patients.

Cerebral toxoplasmosis is the most common neurological opportunistic disease manifested in HIV infected patients. Excretory/secretory antigens (ESA) are serological markers for the diagnosis of reactivation of the infection in HIV-infected patients with cerebral toxoplasmosis. Immunosuppressed patients develop high antibody titers for ESA. However, little is known about the humoral response for these antigens. The present study analyzed the profile of antibody recognition against ESA in comparison with tachyzoite lysate antigen (TLA) in 265 sera and 270 cerebrospinal fluid (CSF) samples from infected patients with Toxoplasma gondii and or HIV and in sera of 50 healthy individuals. The samples of sera and CSF were organized in 8 groups. The sera sample groups were: Group I - Se/CT/AIDS (patients with cerebral toxoplasmosis/AIDS) with 58 samples; Group II - Se/ONinf/AIDS/PosT (patients with AIDS/other neuroinfections/positive toxoplasmosis) with 49 samples; Group III - Se/ONinf/AIDS/NegT (patients with AIDS/other neuroinfections/negative toxoplasmosis) with 58 samples; Group IV - Se/PosT/NegHIV (individuals with asymptomatic toxoplasmosis/negative HIV) with 50 samples and Group V - Se/NegT/NegHIV (healthy individuals/negative toxoplasmosis and HIV) with 50 samples. The CSF sample groups were: Group VI - CSF/CT/AIDS (patients with cerebral toxoplasmosis/AIDS) with 99 samples; Group VII - CSF/ONinf/AIDS/PosT (patients with AIDS/other neuroinfections/positive toxoplasmosis) with 112 samples, and Group VIII - CSF/ONinf/AIDS/NegT (patients with AIDS/other neuroinfections/negative toxoplasmosis) with 59 samples. Levels of IgM, IgA, IgE, IgG and subclasses were determined by ELISA against TLA and ESA antigens. IgM, IgA or IgE antibodies against ESA or TLA were not detected in sera from patients with toxoplasmosis suggesting that all patients were in chronic phase of the infection. High levels of IgG1 against TLA were found in sera samples from groups I, II and IV and in CSF samples from groups VI and VII; whereas IgG2, IgG3 and IgG4 levels were not detected in the same sera or CSF sample groups. However, patients from groups I and VI, that had tachyzoites circulating in blood and CSF respectively, produced a mix of IgG1 and IgG4 antibodies against ESA. IgG2 against ESA were predominant in serum from patients with the latent (non-active) T. gondii infection/HIV negative and in CSF samples from patients with other neuroinfections and positive toxoplasmosis (groups IV and VII, respectively). IgG4 levels against ESA were found to be significantly (P<0.05 and P<0.005) higher in patients with cerebral toxoplasmosis (groups I and VI, respectively) in comparison with groups II, IV and VII. This data suggest that IgG4 can be valuable for supporting the diagnosis of focal brain lesions, caused by T. gondii infection, in HIV-infected patients. This approach might be useful, mainly when molecular investigation to detect parasites is not available.

Spatial distribution and population genetics of Leishmania infantum genotypes in São Paulo State, Brazil, employing multilocus microsatellite typing directly in dog infected tissues.

This study investigated the genetic characteristics of Leishmania infantum samples from São Paulo (SP) State, Brazil in order to collaborate with information about the possible origins of the parasites, as well as, the introduction and spread of visceral leishmaniasis in this Brazilian State. Multilocus microsatellite typing (MLMT) was performed using a set of 17 microsatellite markers. DNA was extracted from 250 samples collected from dogs diagnosed with visceral leishmaniasis and 112 (45%) were genotyped: 67 from the northwest region (NWSP), and 29 from the southeast region (SESP) of SP. The results were correlated with other 16 samples from Mato Grosso do Sul State (MS) (which borders NWSP). Although, a small portion of samples was genotyped, it was possible to genotype multiple loci using small amounts of Leishmania DNA extracted directly from dog tissues. Despite the fact that MLMT analysis defined 33 different genotypes, a low polymorphism was detected within the parasites studied with 10 polymorphic loci. There are two main genetic clusters circulating in SP with strong genetic differentiation, one (POP-A) is composed by samples from SESP and NWSP and presented a weak signal of geographical substructure. The other, belongs to the same cluster found in the state of MS (POP-B), which was the main one. The majority (93.75%) of MS parasite genotypes belonged to POP-B, with just one sample (6.25%) grouped in POP-A. POP-B also comprised 10.34% of SESP and 26.87% of NWSP samples. Besides one sample from MS, POP-A is composed by 73.13% of NWSP and 89.66% of SESP samples. The MLMT analysis supported the idea of canine visceral leishmaniasis being introduced in the Northwest region of SP State by the traffic of humans and dogs from MS. In the southeast region of SP occurred an introduction of a new L. infantum genetic cluster. Probably the transmission was spread by traffic of infected dogs from other Brazilian regions, or by introduction of imported dogs from other countries. All these data together contributed to the detection of the genetic profile of L. infantum population in SP State.

Caracterização genotípica de Leishmania (Leishmania) infantum proveniente de cães naturalmente infectados com leishmaniose visceral americana no Estado de São Paulo / Genotypic characterization of Leishmania (Leishmania) infantum from dogs naturally infected with visceral leishmaniasis americanano in the State of Sao Paulo

Mesmo sendo reconhecida a importância da leishmaniose visceral(LV) no Brasil, a diversidade genética de Leishmania (Leishmania) infantum ainda é pouco estudada. Desta forma, o uso de marcadores microssatélites torna-se uma ferramenta importante para o estudo de genótipos de Leishmania em regiões endêmicas. O objetivo do presente estudo foi determinar o perfil genotípico de isolados de L. infantum provenientes de duas regiões do Estado de São Paulo (SP) analisando regiões hipervariáveis do DNA com marcadores moleculares microssatélites dinucleotídeos. Este estudo visa contribuir com informações sobre as possíveis origens dos parasitas, bem como a introdução e propagação da LV neste Estado. A genotipagem foi realizada com um conjunto de 17 marcadores microssatélites e o DNA foi extraído a partir de 250 amostras coletadas de cães diagnosticados com LV.Do total de amostras, 112 (45%) foram genotipadas: 67 da região noroeste (NWSP) e 29 da região sudeste (SESP) de SP. Os resultados foram comparados com outras 16 amostras do Estadode Mato Grosso do Sul (MS) que faz fronteira com NWSP. Embora umapequena parte das amostras tenha sido genotipada, foi possível genotiparmúltiplos loci utilizando pequenas quantidades de DNA de Leishmaniaextraídos diretamente a partir de tecidos de cães. Apesar de 33 genótiposdiferentes terem sido detectados, um baixo polimorfismo foi encontrado nasamostras estudadas com somente 10 loci polimórficos. No presente estudoforam detectadas duas populações principais que circulam em SP comgrande diferenciação genética. A POP-A é composta por amostras de SESPe NWSP. A outra pertence à população principal encontrada em MS(POP-B). A maioria dos genótipos dos parasitas de MS (93,75%) pertence àPOP-B, com apenas uma amostra (6,25%) agrupada na POP-A. A POP-Btambém é composta por 10,34% das amostras de SESP e 26,87% deNWSP. Além de uma amostra de MS, a POP-A é composta por 73,13% deNWSP e 89,66% das amostras SESP...