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1.
Braz. j. biol ; 83: e247422, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1285631

ABSTRACT

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.


Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.


Subject(s)
Plasmodium falciparum/genetics , Antimalarials/pharmacology , Pakistan , Membrane Transport Proteins/genetics , Drug Resistance/genetics , Protozoan Proteins/genetics , Chloroquine/pharmacology , Multidrug Resistance-Associated Proteins/genetics , Alleles
2.
Braz. j. biol ; 82: e241110, 2022. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1278500

ABSTRACT

Abstract Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp-3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.


Resumo O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp-3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados de parasitas circulantes na área de estudo. Os resultados desse estudo contribuirão em estudos futuros sobre a estrutura genética do parasita e o desenvolvimento de vacinas contra a malária.


Subject(s)
Humans , Plasmodium vivax/genetics , Protozoan Proteins/genetics , Pakistan , Genetic Variation , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , Genotype
3.
Mem. Inst. Oswaldo Cruz ; 116: e200584, 2021. graf
Article in English | LILACS | ID: biblio-1250360

ABSTRACT

In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.


Subject(s)
Humans , Plasmodium vivax/genetics , Malaria, Vivax , Genetic Variation/genetics , Brazil , Protozoan Proteins/genetics , Catalytic Domain
4.
Rev. Soc. Bras. Med. Trop ; 52: e20180225, 2019. tab, graf
Article in English | LILACS | ID: biblio-1041579

ABSTRACT

Abstract INTRODUCTION Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.


Subject(s)
Humans , Plasmodium falciparum/genetics , Drug Resistance/genetics , Protozoan Proteins/genetics , Malaria, Falciparum/parasitology , Artemisinins/pharmacology , Mutation/genetics , Phenotype , Plasmodium falciparum/drug effects , Genotype
5.
Mem. Inst. Oswaldo Cruz ; 113(8): e180086, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040598

ABSTRACT

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.


Subject(s)
Palmitates/metabolism , Trypanosoma cruzi/metabolism , Protozoan Proteins/metabolism , Protein S/metabolism , Lipoylation/genetics , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/genetics , Protozoan Proteins/genetics , Gene Expression Regulation , Protein S/genetics
7.
Rev. bras. parasitol. vet ; 26(2): 185-204, Apr.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-899279

ABSTRACT

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.


Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.


Subject(s)
Animals , Female , Protozoan Proteins/metabolism , Gene Expression Profiling/veterinary , Ornithodoros/metabolism , Intestinal Mucosa/metabolism , Phylogeny , Protozoan Proteins/genetics , Gene Expression Profiling/methods , Ornithodoros/classification
8.
Mem. Inst. Oswaldo Cruz ; 111(7): 460-468, tab, graf
Article in English | LILACS | ID: lil-787557

ABSTRACT

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.


Subject(s)
Animals , Female , HSP70 Heat-Shock Proteins/physiology , Leishmania mexicana/physiology , Leishmaniasis, Cutaneous/parasitology , Protozoan Proteins/physiology , Stress, Physiological , HSP70 Heat-Shock Proteins/genetics , Leishmania mexicana/genetics , Leishmania mexicana/ultrastructure , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Mitochondria/physiology , Oxidative Stress , Protozoan Proteins/genetics , Transfection/methods
9.
Rev. bras. parasitol. vet ; 25(2): 187-195, graf
Article in English | LILACS | ID: lil-785166

ABSTRACT

Abstract Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.


Resumo A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.


Subject(s)
Animals , Protozoan Proteins/genetics , Giardia lamblia/genetics , Cysts/genetics , Cytoskeletal Proteins/genetics , Alleles , Genetic Carrier Screening/veterinary , Giardia lamblia/classification , Genotype
10.
Recife; s.n; 2016. 62 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-871421

ABSTRACT

A leishmaniose visceral(LV) é uma doença grave que afeta a população de vários países, onde o Brasil apresenta a maior prevalência da infecção nas Américas. Com o estudo do gene codificante da proteína B de superfície (HASPB ou K26) de Leishmania infantum é possível identificar as variações polimórficas intraespecíficas e, assim, será possível consolidar a descrição de um perfil polimórfico presente no Estado de Pernambuco. O objetivo do trabalho foi analisar as regiões polimórficas do gene HASPB (K26) de Leishmania infantum em amostras clínicas positivas para leishmaniose visceral e coinfecção LV/HIV. O sistema K26 PCR foi otimizado utilizando concentrações variadas de DNA genômico de L. infantum. Foi realizado o screening de amostras clínicas de DNA através de dois sistemas de PCR simples, kDNA e ITS1/RFLP, para ensaios posteriores com a K26 PCR nas amostras positivas. A curva de dissociação de alta definição (qPCR-HRM) foi empregada na localização de temperaturas de melting específicas para L. infantum. Os amplicons do gene K26 foram sequenciados e alinhados as sequencias selecionadas em base de dados. A K26 PCR apresentou limiar de detecção de 1 pg para amplicon de 700 pb. A especificidade dos primers foi avaliada experimentalmente e in silico, apresentando anelamento inespecífico com DNA humano. Em paralelo, foram selecionadas 78 amostras de DNA através dos dois sistemas screening, sendo 17 caracterizadas como L. infantum. Os ensaios com DNA das amostras clínicas para o sistema K26 PCR revelaram bandas espúrias. A análise através qPCR-HRM em DNA genômico do parasita resultou em amplificação com Tm de 88,2 °C, já o ensaio com amostra clínica revelou duas amplificações com distintas temperaturas de melting, 84,6 e 88,2°C. Três amplicons do gene K26 foram sequenciados e alinhados a cinco sequencias da base de dados, indicando 38,2 por cento de similaridade. Pode-se concluir que o sistema K26 PCR é recomendável para análise dos polimorfismos genéticos, contanto que o DNA seja extraído diretamente de espécies isoladas em meio de cultura.


Subject(s)
Humans , Animals , HIV Infections/complications , Leishmania infantum/genetics , Leishmaniasis, Visceral/complications , Leishmaniasis, Visceral/diagnosis , Polymorphism, Genetic , Protozoan Proteins/genetics , Polymerase Chain Reaction/methods , Coinfection/parasitology , DNA, Protozoan/genetics , Molecular Sequence Data , Sensitivity and Specificity , Sequence Analysis, DNA
11.
Article in English | WPRIM | ID: wpr-36487

ABSTRACT

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.


Subject(s)
Animals , Cell Line , Immune Sera/genetics , Male , Mice , Protozoan Proteins/genetics , Rabbits , Recombinant Proteins/immunology , Sheep , Sodium-Hydrogen Exchangers/genetics , Toxoplasma/genetics , Toxoplasmosis/parasitology
12.
Mem. Inst. Oswaldo Cruz ; 110(7): 877-883, Nov. 2015. tab, graf
Article in English | LILACS | ID: lil-764587

ABSTRACT

Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalisisolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.


Subject(s)
Animals , Cattle , Female , Humans , Adenosine Deaminase/metabolism , Iron Chelating Agents/pharmacology , Trichomonas vaginalis/drug effects , Trichomonas vaginalis/enzymology , Adenosine Deaminase/drug effects , Gene Expression Regulation, Enzymologic , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trichomonas vaginalis/growth & development
13.
Rev. Soc. Bras. Med. Trop ; 48(5): 555-559, Sept.-Oct. 2015. graf
Article in English | LILACS | ID: lil-763336

ABSTRACT

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.


Subject(s)
Female , Humans , Male , Leishmania braziliensis/genetics , Leishmania guyanensis/genetics , Leishmaniasis, Mucocutaneous/parasitology , Mucous Membrane/parasitology , DNA, Protozoan/analysis , Leishmania braziliensis/isolation & purification , Leishmania guyanensis/isolation & purification , Paraffin , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Protozoan Proteins/genetics
14.
Mem. Inst. Oswaldo Cruz ; 110(6): 732-738, Sept. 2015. tab, graf
Article in English | LILACS | ID: lil-763098

ABSTRACT

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma 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.


Subject(s)
Female , Humans , Pregnancy , Antigens, Protozoan/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Membrane Proteins/immunology , Pregnancy Complications, Parasitic/diagnosis , Protozoan Proteins/immunology , Toxoplasmosis/diagnosis , Antigens, Protozoan/blood , Confidence Intervals , Enzyme-Linked Immunosorbent Assay/standards , Fluorescent Antibody Technique, Indirect , Immunoglobulin G/isolation & purification , Immunoglobulin M/blood , Immunoglobulin M/isolation & purification , Inventions/standards , Membrane Proteins/genetics , Predictive Value of Tests , Pregnancy Complications, Parasitic/blood , Pregnancy Complications, Parasitic/immunology , Protozoan Proteins/genetics , Recombinant Proteins , Reference Standards , Sensitivity and Specificity , Toxoplasma/immunology , Toxoplasmosis/blood , Toxoplasmosis/immunology
15.
Rev. Inst. Med. Trop. Säo Paulo ; 57(3): 205-209, May-Jun/2015. graf
Article in English | LILACS | ID: lil-752605

ABSTRACT

Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites. Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI. Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences. Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them. Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny. .


Contexto e objetivo. A caracterização molecular de isolados indianos de Toxoplasma gondii é importante para a investigação de variações genéticas existentes entre cepas do parasito em diferentes locos gênicos. Delineamento e disposição. A presente comunicação realizou a clonagem e o sequenciamento dos 1158 pares de base correspondendo à totalidade do quadro de leitura do antígeno de superfície 3 (SAG3) de Toxoplasma gondii em dois isolados indianos (Chennai e Izatnagar) mantidos em um biorrepositório localizado em IVRI. Método. As sequências do SAG3 dos dois isolados indianos foram clonadas, sequenciadas e posteriormente comparadas com sequências SAG3 de Toxoplasma gondii disponíveis em publicações. Resultados. A comparação das sequências revelou 99,9% de homologia com a cepa RH padrão; 99,3% de homologia com as cepas P-Br e CEP; e 98,4% de homologia com a cepa PRU. Os dois isolados indianos eram 100% idênticos no que diz respeito à sequência SAG3. Conclusão. Concluiu-se que os isolados indianos são filogeneticamente mais próximos da cepa RH em relação à cepa brasileira P-Br, ou às cepas CEP e PRU (USA). No entanto, a análise de outros genes de Toxoplasma gondii destes dois isolados indianos mostrou diferenças na composição de nucleotídeos, ao contrário do que foi encontrado para o locus SAG3. Estes resultados poderiam ser atribuídos ao fato do locus SAG3 ser altamente conservado, necessitando de estudos adicionais para determinar se SAG3 poderia ser utilizado no diagnóstico da toxoplasmose. No entanto, estes resultados são importantes do ponto de vista da filogenia molecular. .


Subject(s)
Animals , Male , Mice , DNA, Protozoan/genetics , Membrane Glycoproteins/genetics , Protozoan Proteins/genetics , Toxoplasma/genetics , Genotype , India , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Toxoplasma/classification
16.
Clinics ; 70(3): 157-161, 03/2015. tab
Article in English | LILACS | ID: lil-747105

ABSTRACT

OBJECTIVE: To determine whether Tai Chi Chuan or ballroom dancing promotes better performance with respect to postural balance, gait, and postural transfer among elderly people. METHODS: We evaluated 76 elderly individuals who were divided into two groups: the Tai Chi Chuan Group and the Dance Group. The subjects were tested using the NeuroCom Balance Master¯ force platform system with the following protocols: static balance tests (the Modified Clinical Tests of Sensory Interaction on Balance and Unilateral Stance) and dynamic balance tests (the Walk Across Test and Sit-to-stand Transfer Test). RESULTS: In the Modified Clinical Test of Sensory Interaction on Balance, the Tai Chi Chuan Group presented a lower sway velocity on a firm surface with open and closed eyes, as well as on a foam surface with closed eyes. In the Modified Clinical Test of Sensory Interaction on Unilateral Stance, the Tai Chi Chuan Group presented a lower sway velocity with open eyes, whereas the Dance Group presented a lower sway velocity with closed eyes. In the Walk Across Test, the Tai Chi Chuan Group presented faster walking speeds than those of the Dance Group. In the Sit-to-stand Transfer Test, the Tai Chi Chuan Group presented shorter transfer times from the sitting to the standing position, with less sway in the final standing position. CONCLUSION: The elderly individuals who practiced Tai Chi Chuan had better bilateral balance with eyes open on both types of surfaces compared with the Dance Group. The Dance Group had better unilateral postural balance with eyes closed. The Tai Chi Chuan Group had faster walking speeds, shorter transfer times, and better postural balance in the final standing position during the Sit-to-stand Test. .


Subject(s)
/metabolism , Cyclic AMP/metabolism , Dictyostelium/enzymology , Dictyostelium/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Protozoan Proteins/metabolism , /genetics , Dictyostelium/growth & development , Dictyostelium/metabolism , Down-Regulation/drug effects , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Folic Acid/pharmacology , /deficiency , /genetics , /metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/deficiency , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Mutation , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Protozoan Proteins/genetics , Signal Transduction , Spores, Protozoan/enzymology , Spores, Protozoan/genetics , Vitamin B Complex/pharmacology
17.
Indian J Biochem Biophys ; 2015 Feb; 52 (1): 14-22
Article in English | IMSEAR | ID: sea-157950

ABSTRACT

Proteases have been considered as an important group of targets for development of antiprotozoal drugs due to their essential roles in host-parasite interactions, parasite immune evasion, life cycle transition and pathogenesis of parasitic diseases. The development of potent and selective serine protease inhibitors targeting L. donovani secretory serine protease (pSP) could pave the way to the discovery of potential antileishmanial drugs. Here, we employed different classical serine protease inhibitors (SPIs), such as aprotinin, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N-tosyl-lysine chloromethyl ketone (TLCK), benzamidine (Bza) and pSP-antibody to determine the role of the protease in parasitic survival, growth and infectivity. Among the different classical SPIs, aprotinin appeared to be more potent in arresting L. donovani promastigotes growth with significant morphological alterations. Furthermore, aprotinin and anti-pSP treated parasites significantly decreased the intracellular parasites and percentage of infected macrophages. These results suggest that SPIs may reduce the infectivity by targeting the serine protease activity and may prove useful to elucidate defined molecular mechanisms of pSP, as well as for the development of novel antileishmanial drugs in future.


Subject(s)
Antiprotozoal Agents/therapeutic use , Leishmania donovani/drug effects , Leishmania donovani/genetics , Leishmaniasis/drug therapy , Leishmaniasis Vaccines/immunology , Protozoan Proteins/genetics , Serine Proteases/therapeutic use , Serine Proteinase Inhibitors/therapeutic use
18.
Exp. Parasitol ; 157: 156-162, 2015.
Article in English | LILACS, SES-SP, SESSP-IALPROD, SES-SP, SESSP-IALACERVO | ID: biblio-1022550

ABSTRACT

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.


Subject(s)
Animals , Reference Standards , Protozoan Proteins/genetics , DNA, Protozoan/chemistry , Sensitivity and Specificity , Leishmania infantum/genetics , Leishmania infantum/chemistry , Dog Diseases/diagnosis , Dog Diseases/parasitology , Dogs , Leishmania/classification , Leishmania/genetics , Leishmania/chemistry , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/veterinary , Animals
19.
Article in English | WPRIM | ID: wpr-51160

ABSTRACT

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.


Subject(s)
Antigens, Protozoan/genetics , Gene Frequency , Genetic Variation , Genotype , Humans , Malaria, Falciparum/epidemiology , Plasmodium falciparum/classification , Polymorphism, Genetic , Protozoan Proteins/genetics , Thailand/epidemiology
20.
Article in English | WPRIM | ID: wpr-19165

ABSTRACT

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.


Subject(s)
Animals , Base Sequence , Brazil , China , Deer , Genetic Variation , Genotype , Goats , Humans , Molecular Sequence Data , Phylogeny , Protozoan Proteins/genetics , Sheep , Swine , Toxoplasma/classification , Toxoplasmosis/parasitology , Toxoplasmosis, Animal/parasitology , United States
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