Parasites, Hosts and Diseases

"feces"

Original Articles

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation: Yousry Hawash, M. M. Ghonaim, Ayman S. Al-Hazmi; Korean J Parasitol 2015;53(2):147-154.
Published online April 22, 2015; DOI: https://doi.org/10.3347/kjp.2015.53.2.147

Abstract
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Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (?375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ?550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ?2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

Citations

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An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting
Yoursry Hawash, M. M. Ghonaim, S. S. Al-Shehri
The Korean Journal of Parasitology.2016; 54(1): 1. CrossRef
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Lucas H Rijsman, Jan F Monkelbaan, Johannes G Kusters
Journal of Gastroenterology and Hepatology.2016; 31(11): 1808. CrossRef

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DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR: Yousry Hawash; Korean J Parasitol 2014;52(3):263-271.
Published online June 26, 2014; DOI: https://doi.org/10.3347/kjp.2014.52.3.263

Abstract
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PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 ?l) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ? 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

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Brief Communications

Modified Formalin-Ether Concentration Technique for Diagnosis of Human Strongyloidiasis: Witthaya Anamnart, Pewpan M. Intapan, Wanchai Maleewong; Korean J Parasitol 2013;51(6):743-745.
Published online December 31, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.6.743

Abstract
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We compared the efficacy and applicability of a modified formalin-ether concentration technique (M-FECT) to the conventional FECT (C-FECT) and the agar plate culture (APC) method for the detection of Strongyloides stercoralis larvae. For this purpose, we used 600 human fecal specimens collected in an endemic area of southern Thailand. In the M-FECT, we used 2 layers of wire meshes, instead of gauze, to avoid the loss by absorption/adhesion of larvae to the gauze during filtration, and we reduced the exposure time of S. stercoralis larvae in stool samples to formalin. By such simple modifications, the efficacy of M-FECT has become comparable to APC and was much better than that of C-FECT for the diagnosis of strongyloidiasis.

Citations

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Egg Laying Capacity of Haplorchis taichui (Digenea: Heterophyidae) in Humans: Megumi Sato, Surapol Sanguankiat, Somchit Pubampen, Teera Kusolsuk, Wanna Maipanich, Jitra Waikagul; Korean J Parasitol 2009;47(3):315-318.
Published online August 28, 2009; DOI: https://doi.org/10.3347/kjp.2009.47.3.315

Abstract
PDF

Quantitative fecal egg counts represented as the number of eggs per gram of feces (EPG) are generally a reliable parameter to estimate the worm burden of intestinal and hepatic parasitoses. Although Haplorchis taichui (Digenea: Heterophyidae) is one of the most common minute human intestinal flukes, little is known about the relationship between EPG and the actual worm burden in patients or the severity of the disease. In the present study, fecal samples were collected from 25 villagers in northern Thailand before and after praziquantel treatment. The EPG values of each participant were determined by the modified cellophane thick smear method, and adult worms were collected from the whole stool after the treatment. Eggs per day per worm (EPDPW) of H. taichui were estimated 82 from egg counts and expelled worms. The EPG was not well correlated with the worm burden, and a reverse correlation was observed between the EPDPW and the worm burden.

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