Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples.

Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due to low cyst concentrations. In order to eliminate inhibitors, improve cyst recovery and molecular detection of G. duodenalis, different types of water, distillates (MDs), deionized (MDz), injection (MI) or Milli-Q (MM) were used instead of formaldehyde (F) in the laboratory routine method (Ritchie). Cysts were isolated from faecal samples with low cyst concentrations (< 1 cyst/field), medium (1-2 cysts/field) or high (> 2 cysts/field). Cyst recovery was improved using all water types (MDs, MDz, MI, MM) compared to formaldehyde. At all cyst concentrations, the use of MM consistently showed the greatest recovery of G. duodenalis cysts . DNA samples from recovered cysts were tested for the glutamate dehydrogenase (GDH) and β-giardin (βg) genes. The use of Milli-Q water allowed to detect both genes in all cyst concentrations, including low. The method processed with the other types of water amplified these genes at high and medium cyst concentrations. GDH and βg genes were not detected when the sample was processed with formaldehyde. These experimental results were confirmed in clinical samples. The results suggest that Milli-Q water provides the highest cyst recovery from stool samples and, correspondingly, the highest sensitivity for detecting G. duodenalis by microscopy or PCR for GDH and βg genes, even at low concentration of cysts.