Genotype Characteristics of Giardia duodenalis in Patients Using High Resolution Melting Analysis Technique in Khorramabad, Iran.

BACKGROUND: We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a cost-effective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared. METHODS: Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software. RESULTS: The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea. CONCLUSION: The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.

Prevalence and associated risk factors of Cystoisospora belli and Cyclospora cayetanensis infection among Iranian patients with colorectal cancer.

From among intestinal parasites, coccidian intestinal parasites such as Cystoisospora belli (previously known as Isospora belli), and Cyclospora cayetanensis are well-known as opportunist parasites, particularly in patients with cancer. This study assessed the prevalence of C. belli and C. cayetanensis in patients with cancer in Lorestan Province, Southwest of Iran. This cross-sectional descriptive study was conducted on 87 patients with colorectal cancers, referred to the general hospitals of Lorestan from October 2017 to August 2018. A fresh stool specimen was collected from each subject in a sterile labeled container. The collected stool samples were concentrated through sucrose flotation method and then prepared for Ziehl-Neelsen staining for microscopic examination. Demographic and possible risk factors such as age, sex, education, residence, and unwashed vegetable/fruit consumption were collected by an applied questionnaire. Out of the 87 patients with colorectal cancer, eight (9.2%) were found positive for C. belli and C. cayetanensis infections, with five (5.74%) and three (3.44%) patients positive for C. belli and C. cayetanensis infections, respectively. Results also showed that sex and unwashed vegetable/fruit consumption were significantly associated with the prevalence of these parasites (p < 0.01). The findings revealed the considerable prevalence of C. belli and C. cayetanensis in patients with colorectal cancers. It is therefore essential for gastrointestinal specialists to pay special attention to the prevalence of coccidian parasites in patients with colorectal cancer.

Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for PCR targeting the tpi gene.

Giardia duodenalis is an intestinal flagellated protozoan and the common cause of gastrointestinal diseases in human. This parasite can be seen in two different forms in its life cycle including as cyst and trophozoite. Due to presence of resistant cyst wall, DNA extraction inhibitors along with artifact in stool specimens, this study was performed aiming to evaluate four methods for DNA extraction from G. duodenalis cysts. Seventy G. duodenalis positive stool specimens that were confirmed by light microscope were included in this study. All stool samples were concentrated using four layered discontinuous sucrose flotation technique (0.5, 0.75, 1, and 1.5 M) and single-layered sucrose solution (0.85 M). The isolated cysts were then subjected to DNA extraction by four methods. To remove the artifacts, the extracted DNA were evaluated by PCR. The results of the present study showed the high level of optical density (OD) in the method I (P < 0.01) with the following steps; Giardia cysts plus crushed cover glass were vortexed. Then, the samples were boiled and then followed by freeze-thaw cycles, yet this method yielded the lowest concentration. Furthermore, the highest concentration were observed in the method II (P <0.01) with the following steps; Giardia cysts plus crushed cover glass and TAE buffer were mixed and then shaken, followed by boiling. Based on the results of the present study, using crushed cover glass, boiling and freeze-thaw cycles can be effective in destruction of G. duodenalis cyst wall and have enough efficiency for extracting DNA from G. duodenalis cysts.

Diagnosis of Giardia duodenalis Infection using Dot Blot in Comparison with Microscopy.

BACKGROUND AND PURPOSE: Giardia duodenalis is an intestinal flagellate parasite which spreads all over the world and is considered as a health problem in the most rural and low sanitation areas. Many diagnostic tests have been developed for the detection of Giardia parasite in stool samples but all of them have some disadvantages such as lack of sensitivity and specificity. In search for a simple and accurate test, diagnosis of Giardia infection using dot blot method has been investigated in this work. MATERIALS AND METHODS: In this descriptive study, 30 stool samples which their infection with Giardia were confirmed by direct examination and formalin ether considered as case group. Thirty stool samples without Giardia infection according to formalin ether examination were also considered as a control group. Giardia cysts were isolated from the stool samples using sucrose method. In order to raise antiserum against Giardia cysts, the purified cysts were then sonicated and injected to a rabbit. A mono specific antiserum against the 66KDa band of Giardia cyst antigen was also prepared. The two antisera were used in the dot blot test. Finally, the sensitivity and specificity of the dot-blot method were estimated by considering formalin ether as the gold standard. RESULTS: When Poly specific antiserum was used, the sensitivity and specificity of the dot blot for detection of Giardia infection were 77% and 64% respectively. However the sensitivity and specificity of this assay were 97% and 64% respectively when monospecific antiserum was used. CONCLUSION: It seems that dot blot is an easy method for the diagnosis of Giardia especially in the rural areas. However more work is recommended for further development of this test.