Molecular Characterization Of Pathogen And Molecular Testing

E. histolytica and E. dispar genome shotgun sequencings are underway as a joint effort from TIGR[11] and the Sanger Centre,[12] funded by the National Institute for Allergy and Infectious Diseases and the Wellcome Trust, respectively. The genome is around 18-20 Mb in size in 14 chromosomes.[11-13] However, partial genomic data from these organisms are available since the end of the 1980s.[14,15] These data allowed designing molecular assays that are sensitive to detection and, more interestingly, that can differentiate between both.[16,17] Following these pioneering works, other early molecular techniques were designed as well.[18-21] To be readily used in the clinic, molecular detection and differentiation techniques have to be usable on feces, and as any infectious disease diagnostics technique, to be both sensitive and specific (or in fact, have good positive and negative predictive values). As these values depend on prevalence, it is quite clear that a ''good'' technique in a Western country, where E. histolytica is not endemic and quite rare, will not be as good as in a developing area, where it is much more prevalent. Moreover, there is a cost issue, regarding techniques such as PCR.

Nowadays, to distinguish between E. histolytica and E. dispar, there are mainly two alternate means: protein differentiation using monoclonal antibodies[22-25] or genetic differentiation using probes and/or PCR. As a commercial kit, the only available is the Techlab ELISA

on stool samples. Numerous PCR techniques have been developed since 1989[18-21,26,27,29-33] (see Table 1 for a comparison). So far, only one real-time quantitative technique has been published, using the Roche Light-Cycler, and thus no internal probe.[26] The authors claim a sensitivity of 0.1 parasite per gram of feces, which is high, with a 100% specificity. Samples were from Vietnam and South Africa, and data are provided allowing for predictive value calculations.

On top of the requirements cited above, a ''good'' PCR technique should be usable on clinical feces such as sodium acetate-acetic acid-formalin (SAF) fixative or frozen samples. As SAF-fixed samples are the easiest to obtain and ship (because they are used for other examinations, and because they have no temperature requirements for shipping), they would be the best. Several authors have tried to design such techniques and investigated the effect of SAF fixative on PCR subsequent detection. It was shown that even if its effect on DNA is indirect, concentrations of formalin higher than 1% seemed to inhibit PCR amplification from 4 days of fixation.[34] This was confirmed by the work of Troll et al.[29] who showed that sensitivity of PCR usually decreased within 2 days in feces stored in SAF fixative. Both teams concluded that the effects of formalin are time-dependent. However, provided that PCR assay is carried out swiftly after fixation, SAF fixative-prepared samples are suitable and have been successfully used in the field by several teams.[35,36]

Several works are underway to design a vaccine, and the completion of E. histolytica and E. dispar genomes will open new avenues for diagnostics and vaccination.

Significant data have also now been accumulated on the molecular pathways of E. histolytica infection (reviewed in Refs. [6] and [4]). Briefly, the amoeba

Table 1 Comparison of several published PCR techniques for Entamoeba histolytica/dispar detection/differentiation

Gene target

PCR type

Amplicon sizea

Specimen

Reference

30 kDa antigen

Classic

100/101

Stools

[16]

SSU rDNA

Classic

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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