Comparative analysis of routine parasitological methods for recovery of cysts, molecular detection, and genotyping of Giardia duodenalis.

In order to improve the diagnosis of giardiasis, fecal samples (high/medium/low concentration of cysts) were processed by the parasitological methods used in the routine: Faust, Lutz e Ritchie modified (replacement of formaldehyde by distilled water). The cysts were quantified; the DNA was extracted and amplified by semi-nested PCR (GDH gene). Fifteen clinical samples were analyzed to validate the study by PCR-RFLP. The results showed that the parasite was only detected and genotyped correctly when samples from children with high, medium, and low parasitic load, belonging to genotype AII, were processed by the modified Ritchie method, different from what was observed for the other methods used in laboratory routine (Faust and Lutz). The modified Ritchie method proved to be more suitable, recovering a greater number of cysts from samples, regardless of parasitic load, which reduces the chance of false negative results and has epidemiological repercussions since individuals with low parasite load are usually asymptomatic and the main disseminators of this infection.

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 ß-giardin (ßg) 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 ßg 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 ßg genes, even at low concentration of cysts.