Multiple-subgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach

To evaluate the geographic distribution of Giardia intestinalis genotypes in Nablus, West Bank, Palestine, a genotyping study was performed using clinical fecal samples. Microscopic examination confirmed that 8 of 69 (11.6%) samples were G. intestinalis positive, and subsequent genotyping analyses targeting the small-subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase (GDH) genes revealed the G. intestinalis genotypes within the 8 samples. Of these 8 samples, 6 were clustered with assemblage A-II and the remaining 2 samples were clustered with assemblage B by 18S rRNA gene analysis; however, direct sequencing of the GDH gene segments from the latter 2 samples showed a mixed infection profile. To assess those samples, we employed a subcloning approach and successfully isolated 6 independent assemblage B subgenotypes. These partial GDH gene sequences (393 bp) had 15 single-nucleotide polymorphisms, all of which were synonymous transition substitutions at the third nucleotide position of codons. From the results, we concluded that the highly polymorphic gene loci such as GDH gene locus might provide us an opportunity to obtain a detailed molecular data even from the samples with multiple-subgenotype mixed infections. Therefore, subcloning approach is recommended in genotyping studies, especially in those conducted in giardiasis-endemic areas, where the repeated and cumulative infections could be commonly expected.