Stool sample storage conditions for the preservation of Giardia intestinalis DNA

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.

Dust-mites: effect on lipid peroxidation.

BACKGROUND: Dust-mites are present in our homes, feed on dead exfoliated skin and other organic material. It is also known that oxidative stress may lead to cellular damage that can be confirmed by markers of cellular disruption. Oxidative stress in various infective processes has been documented. We investigated whether house dust-mites cause oxidative stress in patients. METHODS: Products of lipid peroxidation in erythrocytes and lymphocytes were assessed by measuring malondialdehyde concentration. RESULTS: Our results showed that patients who had a positive skin test for dust-mite antigens and had dust-mites present in their houses (dust-mite positive) had increased erythrocyte malondialdehyde levels (62.39 [18.56] nmol/g-Hb) compared with those who were skin test positive, dust-mite negative (45.45 [10.82]) or skin test negative, dust-mite negative (42.20 [5.68]). They also had significantly higher levels of lymphocyte malondialdehyde (4.22 [0.55] nmol/g-protein) compared with those who were skin test positive, dust-mite negative (3.46 [0.29]) or skin test negative, dust-mite negative (1.25 [0.31]; p <0.05). However, there was no statistically significant difference between the malondialdehyde levels of dust-mite negative/skin test positive and dust-mite negative/skin test negative patients. CONCLUSION: Increased malondialdehyde activity in lymphocytes and erythrocytes in the dust-mite positive/skin test positive group shows the presence of the oxidative stress in patients with dust-mite infestation.