Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples.

Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.

Multilocus sequence analysis of Giardia spp. isolated from patients with diarrhea in Austria.

Giardia duodenalis is a protozoan parasite causing intestinal infections in a wide range of mammals. Two distinct assemblages, A and B, infect humans predominantly; however, both are believed to be generally zoonotic. Giardia strains associated with infections in Austria have not been investigated at the molecular level. In this study, 65 human stool samples microscopically positive for Giardia spp. were subjected to DNA isolation and nested PCR targeting fragments of the glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), and beta-gardin (bg) genes. A total of 52 samples were successfully analyzed using PCR and DNA sequencing. Assemblage B was detected most frequently and accounted for 65.4% (34/52) of infections, while Assemblage A accounted for 34.6% (18/52). There was a high level of genetic diversity among the isolates with 46.2% designated as sub-assemblage BIV (24/52), 25% sub-assemblage AII (13/52), 19.2% sub-assemblage BIII (10/52), and 9.6% sub-assemblage AI (5/52). No mixed infections were detected. The results suggest that the majority of infections were imported and that endemic anthroponotic transmission plays a minor role in Austria.

Molecular epidemiology and multilocus sequence analysis of potentially zoonotic Giardia spp. from humans and dogs in Jamaica.

Giardia spp. are the causative agents of intestinal infections in a wide variety of mammals including humans and companion animals. Dogs may be reservoirs of zoonotic Giardia spp.; however, the potential for transmission between dogs and humans in Jamaica has not been studied. Conventional PCR was used to screen 285 human and 225 dog stool samples for Giardia targeting the SSU rDNA gene followed by multilocus sequencing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and ß-giardin (bg) genes. Prevalence of human infections based on PCR was 6.7 % (19/285) and canine infections 19.6 % (44/225). Nested PCR conducted on all 63 positive samples revealed the exclusive presence of assemblage A in both humans and dogs. Sub-assemblage A-II was responsible for 79.0 % (15/19) and 70.5 % (31/44) of the infections in humans and dogs, respectively, while sub-assemblage A-I was identified at a rate of 15.8 % (3/19) and 29.5 % (13/44) in humans and dogs, respectively. The predominance of a single circulating assemblage among both humans and dogs in Jamaica suggests possible zoonotic transmission of Giardia infections.