Gabriela Motoie

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 parasites 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.

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.

Amphibian Secretions for Drug Discovery Studies: A Search for New Antiparasitic and Antifungal Compounds

The antiparasitic and antifungal activities of nine amphibian skin secretions were studied in different in vitro models. Seven secretions presented a considerable antiprotozoan activity and one showed promising results against Candida sp . These results can be the basis for the development of new drugs, especially for neglected parasitic diseases.

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.

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.

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.

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.

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 parasites 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.

WITHDRAWN: Selection of reference genes in five types of human tissues for normalization of gene expression studies in infectious diseases

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