Molecular Diagnosis and Characterization of Two Intestinal Protozoa : Entamoeba histolytica & Giardia intestinalis

Entamoeba histolytica and Giardia intestinalis are two of the most important and most widespread diarrhea-related parasitic protozoa in the world. Approximately 1200–1500 cases of Giardia and 200–400 cases of Entamoeba are reported each year in Sweden, whereas the corresponding numbers are much higher in developing countries like Nicaragua. Traditionally, diagnosis of these parasites depends on microscopic detection of cysts or trophozoites, even though such methodology is neither sensitive nor species specific. Recently, more specific and sensitive molecular techniques have been introduced to identify these infections, but routine use of these tests in diagnostic laboratories is still limited. Amoebiasis, infection with E. histolytica, is a notifiable disease in Sweden. Despite that, diagnosis depends mainly on stool microscopy, which cannot distinguish between the invasive and pathogenic E. histolytica and the nonpathogenic E. dispar. We used a PCR technique to evaluate the proportions of these two species in patients who had amoebic infections and had initially been diagnosed by microscopy, and we found E. histolytica in less than 10% of those cases. Differentiation of E. histolytica and E. dispar is now a recommended analysis in Sweden, and this approach has greatly decreased the number of patients receiving unnecessary treatment. Giardia intestinalis consists of seven morphologically identical but genetically distinct genotypes or assemblages, which are designated A to G. Assemblages A and B can infect both humans and other mammals, whereas assemblages C to G are host specific. Sequence-based genotyping of Giardia isolates was applied to investigate the relationship between assemblages and symptoms, the zoonotic potential, sequence divergence, and transmission dynamics. Giardia samples obtained from humans and dogs living in the same area in Leόn, Nicaragua, as well as from humans and various animals in Sweden were analyzed using molecular methods. The investigation of the human samples indicated the following: assemblage B was the most common genotype in both countries, and this assemblage was largely polymorphic with ambiguous nucleotide positions in many sequences. In contrast, assemblage A isolates were genetically more homogeneous, and multilocus genotypes (MLGs) were easily determined for this assemblage. Most animals were infected with the host-specific assemblages C–G, but assemblages A and B were detected as well. A few animal and human isolates shared the same assemblage A MLGs, which suggests that zoonotic transmission of Giardia can occur in Sweden. Determination of MLGs may be a useful tool for source tracing in outbreak situations, although assignment of a specific MLG was hampered in many of the current isolates due to the large sequence polymorphism seen in assemblage B. The only correlation found between assemblages and symptoms concerned flatulence, which was noted to be significantly more common in young children infected with assemblage B. In conclusion, the combination of microscopy and molecular methods enabled us to differentiate between pathogenic and nonpathogenic Entamoeba species and thereby decrease the number of unnecessarily treated patients. Furthermore this methodological approach also gave us basic knowledge about Giardia genotypes in humans and animals, both in Nicaragua and Sweden, information that is useful for understanding the transmission and the clinical presentation of Giardia infection. LIST OF PUBLICATIONS This thesis is based on the following papers. They will be referred to by their Roman numerals in the text. I. Lebbad M, Svärd SG. PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy. Scand J Infect Dis. 2005;37(9):680-5. II. Lebbad M, Ankarklev J, Tellez A, Leiva B, Andersson JO, Svärd S. Dominance of Giardia assemblage B in León, Nicaragua. Acta Trop. 2008;106(1):44-53. III. Lebbad M, Mattsson JG, Christensson B, Ljungström B, Backhans A, Andersson JO, Svärd SG. From mouse to moose: multilocus genotyping of Giardia isolates from various animal species. Vet Parasitol. 2010 25;168(34):231-9. IV. Lebbad M, Petersson I, Karlsson L, Botero-Kleiven S, Andersson JO, Svenungsson B, Svärd SG. Multilocus sequence based genotyping of Giardia isolates from Swedish patients. In manuscript