Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Abstract

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…For example, some papers mention specifically that additional mechanical disruption is needed for the efficient isolation of DNA from Trichuris eggs or Entamoeba cysts, while others use a standard isolation protocol without mention of additional rigorous steps to break down the egg shell or cyst wall (15,(17)(18)(19). Another example is the negative effect of preserving feces in formalin or sodium acetate-acetic acid-formalin (SAF) on the specific amplification of Entamoeba, Giardia, and Cryptosporidium DNAs; this effect increases with the duration of fixation (20)(21)(22)(23). Species-specific DNA extraction using magnetic beads which capture oligonucleotides specific for Cryptosporidium and Giardia improved (lowered) real-time PCR C T values by averages of 10.7 and 9.7 cycles, respectively (24).…”

“…In developed countries, such sequelae may have a vast impact on quality of life; in developing countries, particularly in children, they add yet another burden to already disadvantaged populations (77). Several conventional and real-time PCRs for the primary diagnosis of giardiasis that target the SSU ribosomal DNA (rDNA), ␤-giardin, triosephosphate isomerase (TPI), and intergenic spacer (IGS) regions have been reported (21,(78)(79)(80)(81). Many targets are being used for further genotyping directly on DNA from fecal samples or from Giardia isolates.…”

Evaluation of a Real-Time Polymerase Chain Reaction for the Laboratory Diagnosis of Giardia intestinalis in Stool Samples from Schoolchildren from the Centre-Ouest and Plateau Central Regions of Burkina Faso

“…The lack of PCR detection of the 22 microscopy-positive samples suggests an absence of target DNA and could be explained by lysis of cysts followed by a possible degradation of the parasite DNA by the DNase contained in faeces during sample storage [33]. Indeed, no preservatives have been added to the samples during the storage period while keeping the samples at -20°C in contrast to other study protocols [33,34].…”

“…Indeed, no preservatives have been added to the samples during the storage period while keeping the samples at -20°C in contrast to other study protocols [33,34]. In addition, PCR-based DNA amplification can be inhibited by the decomposition products of haemoglobin in stool such as bilirubin, bile acids and inorganic ions [33,35].…”

Evaluation of Three Protocols of DNA Extraction for Detection of Giardia duodenalis in Human Fecal Specimens

“…Several laboratory techniques with different sensitivities are commonly used for detection of giardiasis, such as traditional microscopy method, enzyme-linked immunosorbent assay (ELISA), and direct fluorescent antibody test (DFA) (4,5,10,(14)(15)(16). However, microscopic examination is characterized as a definitive method for the diagnosis of giardiasis, however, it has some drawbacks such as expertise requirement in identifying the micro-organism and intermittent cyst shedding of parasite in stool (3,8,10).…”