Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction

Abstract

An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4 degrees C. The present PCR was sensitive enough to detect ten cysts of either of the amebae.

Historia del protozoo Entamoeba histolytica

“…Acuña-Soto et al (1993) aplicaron PCR en muestras de materia fecal para diferenciar E. histolytica patógena de no patógena en una población rural de México 38 . En 1997 se utilizó este método para diferenciar los quistes de E. histolytica y E. dispar 3,39 . Con relación a la estructura molecular, Ghosh et al (2003) identificaron la estructura del ADN ribosomal de la E. histolytica 40 .…”

Laboratory Diagnostic Techniques for Entamoeba Species

“…However, methods of fixation of feces with fixatives or preservatives may result in a decreased sensitivity of PCR with time (143,192). A few groups have, however, shown good results using formalin-fixed samples for PCR (147,157). Ethanol is a simple transport medium that preserves amebic DNA.…”

Laboratory Diagnosis of Amebiasis

“…In reality, many of these organisms were probably genetically distinct from E. dispar (218). Currently, there are many molecular tools available to allow the differentiation E. histolytica from E. dispar, such as amoebic antigen and DNA detection enzyme immunoassay (EIA) and PCR (6,28,29,59,79,178,179,192,224,238). Reclassification of E. histolytica and E. dispar is of great importance because it allow the clinician to focus on early identification and treatment of E. histolytica infection in the minority of patients who are at highest personal risk and pose a major public health problem (175).…”

“…Nowadays, in addition to this technique, typing by using monoclonal antibodies to surface antigens (antigen-based enzyme-linked immunosorbent assay [ELISA]) (78,79,161), PCR-specific analysis (59,192,224), and restriction fragment length polymorphism (ribotyping) (41) have been of great value in understanding the epidemiology of these parasites and in investigating disease outbreaks. Epidemiological studies have shown that low socioeconomic status and unsanitary conditions are significant independent risk factors for infection.…”