RESUMO
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.
Assuntos
Doença de Chagas/sangue , DNA de Protozoário/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Doença de Chagas/diagnóstico , Doença de Chagas/genética , Doença de Chagas/parasitologia , DNA de Protozoário/isolamento & purificação , Humanos , Cooperação Internacional , Ensaio de Proficiência Laboratorial , Tipagem Molecular , Parasitemia/sangue , Parasitemia/diagnóstico , Parasitemia/genética , Sensibilidade e Especificidade , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Aiming at contributing to the knowledge of the relationships between bovines and rumen ciliates that naturally occur in Brazilian beef cattle, this paper presents a survey of these ciliate populations in bovines killed in Juiz de Fora Municipal Slaughterhouse, Minas Gerais State, Brazil. Material samplings were carried out monthly from August 1996 to May 1997, totalizing 100 bovines. All rumen contents samples were obtained from recentlyslaughtered animals, cross-bred holstein friesianzebu, kept in Brachiaria decumbens grass. The ciliates were quantified in a Sedgewick-Rafter chamber. The ciliate genus found and the corresponding total and average numbers, relative composition in the population and prevalence were the following: Entodinium (5.163.200; 51.632; 50,48; 100), Diplodinium (572.000; 5.837; 5,59; 98), Eudiplodinium (706.400; 7.283; 6,91; 97), Ostracodinium (989.599; 10.417; 9,68; 95), Isotricha (424.400; 4.563; 4,15; 93), Dasytricha (338.800; 3.643; 3,31; 93), Metadinium (312.800; 3.476; 3,06; 90), Eremoplastron (756.000; 8.690; 7,39; 87), Epidinium (543.200; 7441; 5,31; 73),Charonina (136.400; 2.099; 1,33; 65), Eodinium (152.400 2.583; 1,49; 59), Diploplastron (90.000; 3.750; 0,88; 24), Elytroplastron (36.400; 2.275; 0,36; 16), Polyplastron (3.600 1.200; 0,04; 03) e Buetschlia (2.000; 2.000; 0,02; 01). The populations are characterized as type B in 94% of samples.
RESUMO
Aiming at contributing to the knowledge of the relationships between bovines and rumen ciliates that naturally occur in Brazilian beef cattle, this paper presents a survey of these ciliate populations in bovines killed in Juiz de Fora Municipal Slaughterhouse, Minas Gerais State, Brazil. Material samplings were carried out monthly from August 1996 to May 1997, totalizing 100 bovines. All rumen contents samples were obtained from recentlyslaughtered animals, cross-bred holstein friesianzebu, kept in Brachiaria decumbens grass. The ciliates were quantified in a Sedgewick-Rafter chamber. The ciliate genus found and the corresponding total and average numbers, relative composition in the population and prevalence were the following: Entodinium (5.163.200; 51.632; 50,48; 100), Diplodinium (572.000; 5.837; 5,59; 98), Eudiplodinium (706.400; 7.283; 6,91; 97), Ostracodinium (989.599; 10.417; 9,68; 95), Isotricha (424.400; 4.563; 4,15; 93), Dasytricha (338.800; 3.643; 3,31; 93), Metadinium (312.800; 3.476; 3,06; 90), Eremoplastron (756.000; 8.690; 7,39; 87), Epidinium (543.200; 7441; 5,31; 73),Charonina (136.400; 2.099; 1,33; 65), Eodinium (152.400 2.583; 1,49; 59), Diploplastron (90.000; 3.750; 0,88; 24), Elytroplastron (36.400; 2.275; 0,36; 16), Polyplastron (3.600 1.200; 0,04; 03) e Buetschlia (2.000; 2.000; 0,02; 01). The populations are characterized as type B in 94% of samples.