RESUMO
Purpose: The aim of the present study was to evaluate the use of touchdown polymerase chain reaction (TD-PCR) for the detection of Entamoeba histolytica in liver pus samples obtained from patients with a clinical diagnosis of amoebic liver abscess (ALA) using small-subunit rRNA (SSU rRNA) as the target gene. Materials and Methods: Microscopic examination in vitro culture and serological test for the detection of E. histolytica in 67 pus samples obtained from ALA patients was performed. Molecular studies were carried out by both conventional PCR and TD-PCR targeting the SSU rRNA gene using the same sets of primers and the results were compared. Results: TD-PCR detected the presence of E. histolytica in 86.5% of the liver pus samples within 2.5 h as compared with 82.08% by conventional PCR within 3.5-4 h. Conclusion: TD-PCR assay may serve as a relatively better detection method for E. histolytica over conventional PCR with respect to the turnaround time, increased sensitivity, specificity and yield.
Assuntos
Técnicas de Laboratório Clínico/métodos , Primers do DNA/genética , Entamoeba histolytica/genética , Entamoeba histolytica/isolamento & purificação , Humanos , Abscesso Hepático Amebiano/diagnóstico , Abscesso Hepático Amebiano/parasitologia , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Supuração/parasitologia , Fatores de TempoRESUMO
A local strain of Entamoeba histolytica, the HTH-56: MUTM from a human liver abscess was successfully axenized. The culture was initially established monoxenically in Diamond's TYI-S-33 medium in the presence of Crithidia luciliae and maintained at 34 +/- 0.5 degrees C. After 5 passages it was adapted to axenic cultivation by addition of 0.02% Bacto agar in Diamond's TYI-S-33 medium in place of Crithidia. Subcultures or replacement with fresh complete media were done twice or thrice for 7 days, after which the agar was omitted and a stable culture was obtained. Isoenzyme analysis showed that this strain of E. histolytica belonged to the zymodeme II pattern, which is one out of 10 pathogenic zymodemes of E. histolytica most commonly found among the virulent strains.