RESUMO
Little is known about the existence of drug-resistant Toxoplasma gondii strains and their possible impact on clinic outcomes. To expand our knowledge about the existence of natural variations on drug susceptibility of T. gondii strains in Brazil, we evaluated the in vitro and in vivo susceptibility to sulfadiazine (SDZ) and pyrimethamine (PYR) of three atypical strains (Wild2, Wild3, and Wild4) isolated from free-living wild birds. In vitro susceptibility assay showed that the three strains were equally susceptible to SDZ and PYR but variations in the susceptibility were observed to SDZ plus PYR treatment. Variations in the proliferation rates in vitro and spontaneous conversion to bradyzoites were also accessed for all strains. Wild2 showed a lower cystogenesis capacity compared to Wild3 and Wild4. The in vivo analysis showed that while Wild3 was highly susceptible to all SDZ and PYR doses, and their combination, Wild2 and Wild4 showed low susceptibility to the lower doses of SDZ or PYR. Interestingly, Wild2 presented low susceptibility to the higher doses of SDZ, PYR and their combination. Our results suggest that the variability in treatment response by T. gondii isolates could possibly be related not only to drug resistance but also to the strain cystogenesis capacity.
Assuntos
Antiprotozoários , Toxoplasma , Sulfadiazina/farmacologia , Sulfadiazina/uso terapêutico , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Antiprotozoários/uso terapêutico , BrasilRESUMO
BACKGROUND: Resistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used. METHODS: The presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether-lumefantrine conducted in 2018-2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72-76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed. RESULTS: All isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified. CONCLUSIONS: Overall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Sulfadoxina/farmacologia , Sulfadoxina/uso terapêutico , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Colômbia , Malária Falciparum/epidemiologia , Artemeter/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Combinação de Medicamentos , Resistência a Medicamentos/genética , Polimorfismo Genético , CódonRESUMO
Toxoplasma gondii infects one-third of the world population. For decades, it has been considered a silent lifelong infection. However, chronically T. gondii-infected persons may present psychiatric and neurocognitive changes as anxiety, depression, and memory loss. In a model of long-term chronic infection, behavioral alterations parallel neuroinflammation and systemic high cytokine levels, and may reflect brain cyst load. Recent findings support that in chronic infection an active parasite-host interplay involves an immune-mediated control of tissue cysts. Here, we tested the idea that etiological treatment in chronic phase may add advantage to intrinsic immune-mediated cyst control and impact behavioral changes. Thus, we combined sulfadiazine-plus-pyrimethamine (S+P), the first-choice therapy for toxoplasmosis, to study the association of brain cyst load and biological processes related to the immune response (neuroinflammation, blood-brain barrier -BBB- disruption and serum cytokine levels), with behavioral and neurocognitive changes of long-term chronic infection. Female C57BL/6 mice (H-2b) were infected (5 cysts, ME-49 strain) and treated with S+P from 30 to 60 days postinfection (dpi), compared with vehicle (Veh)-treated and noninfected controls. At endpoints (pre-therapy, 30 dpi; S+P therapy, 60 dpi; after ceased therapy, 90 dpi), independent groups were subjected to behavioral tests, and brain tissues and sera were collected. Multiple behavioral and neurocognitive changes were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic infection. S+P therapy resolved locomotor alterations, anxiety, and depressive-like behavior, partially or transiently ameliorated hyperactivity and habituation memory loss. Analysis after therapy cessation showed that S+P therapy reduced the number of stimuli required for aversive memory consolidation. S+P therapy resulted in reduced brain cyst load, neuroinflammation and BBB disruption, and lowered systemic Th1-cytokine levels. Correlation analysis revealed association between IFNγ, TNF and MCP-1/CCL2 serum levels, brain cyst load and behavioral and neurocognitive alterations. Moreover, principal-component analysis (PCA-2D and 3D projections) highlighted distinction between clusters (noninfected; Veh-treated and S+P-treated infected). Thus, our data suggest that S+P therapy added gain to intrinsic brain cyst control and, direct or indirectly, ameliorated inflammation-related alterations, traits associated with behavioral and neurocognitive alterations.
Assuntos
Encéfalo , Pirimetamina , Sulfadiazina , Toxoplasmose , Animais , Encéfalo/parasitologia , Citocinas , Feminino , Inflamação/tratamento farmacológico , Transtornos da Memória/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Sulfadiazina/farmacologia , Sulfadiazina/uso terapêutico , Toxoplasmose/tratamento farmacológico , Toxoplasmose/patologiaRESUMO
Chloroquine remains the first-line treatment for uncomplicated malaria in Haiti, and until recently, sulfadoxine-pyrimethamine was the second-line treatment. A few studies have reported the presence of molecular markers for resistance in Plasmodium falciparum parasites, and in vivo therapeutic efficacy studies (TESs) have been limited. Recognizing the history of antimalarial resistance around the globe and the challenges of implementing TESs in low-endemic areas, the Ministry of Health established a surveillance program to detect molecular markers of antimalarial resistance in Haiti. Sentinel sites were purposefully selected in each of Haiti's 10 administrative departments; an 11th site was selected in Grand'Anse, the department with the highest number of reported cases. Factors considered for site selection included the number of malaria cases identified, observed skills of laboratory technicians conducting rapid diagnostic tests (RDTs), stock and storage conditions of RDTs, accuracy of data reporting to the national surveillance system, and motivation to participate. Epidemiologic data from 2,437 patients who tested positive for malaria from March 2016 to December 2018 and consented to provide samples for molecular sequencing are presented here. Of these, 936 (38.4%) patients reported self-treatment with any medication since the onset of their illness before diagnosis; overall, 69 (2.8%) patients reported taking an antimalarial. Ten patients (0.4%) reported travel away from their home for at least one night in the month before diagnosis. Establishing a molecular surveillance program for antimalarial drug resistance proved practical and feasible in a resource-limited setting and will provide the evidence needed to make informed treatment policy decisions at the national level.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos/genética , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Combinação de Medicamentos , Feminino , Haiti/epidemiologia , Humanos , Lactente , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/efeitos dos fármacos , Vigilância de Evento Sentinela , Adulto JovemRESUMO
Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.
Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.
Assuntos
Humanos , Plasmodium vivax/genética , Tetra-Hidrofolato Desidrogenase/genética , Variação Genética , Plasmodium vivax/enzimologia , Pirimetamina/farmacologia , América do Sul , Brasil , Resistência a Inseticidas/genética , Colômbia , Guiana Francesa , Honduras , México , Mutação , Nicarágua , Antiprotozoários/farmacologiaRESUMO
OBJECTIVE: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. MATERIALS AND METHODS: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. RESULTS: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. CONCLUSIONS: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.
OBJETIVO: Determinar mutaciones en la dihydrofolato reductasa de P. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. MATERIAL Y MÉTODOS: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. RESULTADOS: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. CONCLUSIONES: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.
Assuntos
Variação Genética , Plasmodium vivax/genética , Tetra-Hidrofolato Desidrogenase/genética , Antiprotozoários/farmacologia , Brasil , Colômbia , Guiana Francesa , Honduras , Humanos , Resistência a Inseticidas/genética , México , Mutação , Nicarágua , Plasmodium vivax/enzimologia , Pirimetamina/farmacologia , América do SulRESUMO
Haiti is striving for zero local malaria transmission by the year 2025. Chloroquine remains the first-line treatment, and sulfadoxine/pyrimethamine (SP) has been used for mass drug-administration pilot programs. In March 2016, nationwide molecular surveillance was initiated to assess molecular resistance signatures for chloroquine and SP. For 778 samples collected through December 2017, we used Sanger sequencing to investigate putative resistance markers to chloroquine (Pfcrt codons 72, 74, 75, and 76), sulfadoxine (Pfdhps codons 436, 437, 540, 581, 613), and pyrimethamine (Pfdhfr codons 50, 51, 59, 108, 164). No parasites harbored Pfcrt point mutations. Prevalence of the Pfdhfr S108N single mutation was 47%, and we found the triple mutant Pfdhfr haplotype (108N, 51I, and 59R) in a single isolate. We observed no Pfdhps variants except in 1 isolate (A437G mutation). These data confirm the lack of highly resistant chloroquine and SP alleles in Haiti and support the continued use of chloroquine and SP.
Assuntos
Antimaláricos , Malária Falciparum , Alelos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Haiti/epidemiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Mutação , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Sulfadoxina/farmacologia , Sulfadoxina/uso terapêuticoRESUMO
Toxoplasma gondii, the agent of toxoplasmosis, is an intracellular parasite that can infect a wide range of vertebrate hosts. Toxoplasmosis causes severe damage to immunocompromised hosts and its treatment is mainly based on the combination of pyrimethamine and sulfadiazine, which causes relevant side effects primarily observed in AIDS patients, including bone marrow suppression and hematological toxicity (pyrimethamine) and/or hypersensitivity and allergic skin reactions (sulfadiazine). Thus, it is important to investigate new compounds against T. gondii, particularly those that may act on bradyzoites, which are present in cysts during the chronic disease phase. We propose an in vitro model to simultaneously study new candidate compounds against the two main causative stages of Toxoplasma infection in humans, using the EGS-DC strain that was modified from a type I/III strain (EGS), isolated from a case of human congenital toxoplasmosis in Brazil and engineered to express markers for both stages of development. One feature of this strain is that it presents tachyzoite and bradyzoite in the same culture system and in the same host cell under normal culture conditions. Additionally, this strain presents stage-specific fluorescent protein expression, allowing for easy identification of both stages, thus making this strain useful in different studies. HFF cells were infected and after 4 and 7 days post infection the cells were treated with 10 µM of pyrimethamine or atovaquone, for 48 or 72 h. We used high-throughput screening to quantify the extent of parasite infection. Despite a reduction in tachyzoite infection caused by both treatments, the atovaquone treatment reduced the bradyzoite infection while the pyrimethamine one increased it. Ultrastructural analysis showed that after treatment with both drugs, parasites displayed altered mitochondria. Fluorescence microscopy of cells labeled with MitoTracker CMXRos showed that the cysts present inside the cells lost their mitochondrial membrane potential. Our results indicate that this experimental model is adequate to simultaneously analyze new active compounds against tachyzoite and bradyzoite forms.
Assuntos
Parasitologia/métodos , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/genética , Toxoplasmose Congênita/parasitologia , Antiprotozoários/farmacologia , Atovaquona/farmacologia , Brasil , Linhagem Celular , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Estágios do Ciclo de Vida , Pirimetamina/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose Congênita/diagnósticoRESUMO
PURPOSE: Toxoplasma gondii is a protozoan from phylum Apicomplexa, which causes the toxoplasmosis infection; this one exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. Our goal was to verify whether the Toxoplasma gondii (RH strain) tachyzoites form pretreated with pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations could affect the infection processes, suggesting direct action on protozoa intracellular proliferation through the inhibition of isoprenoids in the parasite's apicoplast. METHODS: To have the adhesion, infection, and parasite proliferation during experimental infection investigated, HeLa cells (105) were subjected to a 24-hour infection by T. gondii tachyzoites forms of RH strain (5 × 105) pretreated for 30 min with pravastatin and/or simvastatin combined or not with pyrimethamine and sulfadiazine. RESULTS: Combined with conventional drugs at low concentrations pravastatin and simvastatin inhibit the adhesion, invasion, and intracellular proliferation of T. gondii in HeLa cells which are similar to the positive control. CONCLUSION: Pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations can be regarded as a promising, effective alternative to toxoplasmosis treatment with reduced side effects.
Assuntos
Antiprotozoários/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Pravastatina/farmacologia , Pirimetamina/farmacologia , Sinvastatina/farmacologia , Sulfadiazina/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose/parasitologia , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células HeLa , Humanos , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/fisiologia , Toxoplasmose/tratamento farmacológicoRESUMO
Toxoplasmosis in South America presents great health impacts and is a topic of research interest not only because of the severity of native cases but also due to the predominant atypical genotypes of the parasite circulating in this continent. Typically, symptomatic toxoplasmosis is treated with a combination of sulfadiazine (SDZ) and pyrimethamine (PYR). However, some clinical cases present treatment failures due to an inability of the drugs to control the infection or their significant adverse effects, which can lead to treatment interruption. Although resistance/susceptibility to the aforementioned drugs has been well described for clonal strains of Toxoplasma gondii spread to the Northern Hemisphere, less is known about the South American atypical strains. In this study, the effectiveness of SDZ and PYR for the treatment of mice during acute infection with different atypical T. gondii strains was evaluated. Swiss mice were infected with seven T. gondii strains obtained from newborn patients with congenital toxoplasmosis in Brazil. The infected mice were treated with 10-640â¯mg/kg per day of SDZ, 3-200â¯mg/kg per day of PYR, or a combination of both drugs with a lower dosage. The mice were evaluated for parameters including mortality, anti-T. gondii IgG production by ELISA and the presence of brain cysts. In addition, the presence of polymorphisms in the dhps gene was verified by gene sequencing. A descriptive analysis was used to assess the association between susceptibility to SDZ and/or PYR and the genotype. The TgCTBr4 and TgCTBr17 strains (genotype 108) presented lower susceptibility to SDZ or PYR treatment. The TgCTBr1 and TgCTBr25 strains (genotype 206) presented similar susceptibility to PYR but not SDZ treatment. The TgCTBr9 strain (genotype 11) was the only strain with high susceptibility to treatment with both drugs. The TgCTBr13 strain (genotype 208) was not susceptible to treatment with the lower PYR or SDZ doses. The TgCTBR23 strain (genotype 41) was more susceptible to PYR than to SDZ treatment. However, the association of low SDZ and PYR doses showed good efficacy for the treatment of experimental toxoplasmosis with T. gondii atypical strains obtained from newborns in Brazil. A new mutation in the T. gondii dhps gene (I347M) was identified that might be associated with the SDZ low sensitivity profile observed for the TgCTBr4 and TgCTBr17 isolates.