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.
KeywordsGiardiasis Diagnosis Polymerase chain reaction Parasite load Water purification
We are grateful to Programa de Apoio à Pós Graduação—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROAP–CAPES) for financial support, and R.C. Bezagio has benefited from CAPES by the study scholarship, and we acknowledge the Parasitology Laboratory of UEM for support.
The experiments were designed by RCB, CRdA and MLG. Material preparation, data collection and analysis were performed by RCB, CMC, LILR, ÉCF, SM and MLG. The first draft of the manuscript was written by RCB and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Human Ethics Committee of the Faculdade Integrado of Campo Mourão (Paraná/Brasil) under registration number 1.594.078 and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
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