الفهرس 
Giardia duodenalisOnly 14 pages are availabe for public view
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Abstract
Giardia duodenalis (syn. G. lamblia, G. intestinalis) is considered the most common cause of parasitic diarrhoea worldwide with estimated prevalence rates of 20-30 % in developing countries and 2-5% in developed countries. Genetic studies revealed that at least eight assemblages (A–H) exist. Of these assemblages, A and B are found primarily in human beings and occasionally in animals. The association between clinical symptoms and G. duodenalis assemblages is controversial.
The aim of the present study was to use multilocus sequence typing (MLST) to identify genotypes among Egyptian isolates of G. duodenalis. In part this study aimed to evaluate possible relationship between these genotypes and clinical features of giardiasis. Additionally, optimized and standardized protocols for future genotyping of G. duodenalis isolates in Egypt were provided.
A descriptive study was done over the period from May 2013 to March 2014. DNA was extracted from 96 randomly selected light microscopy-Giardia positive stool samples. The extracted DNA samples were transferred to the Mycotic and Parasitic agents and Mycobacteria, Department of Infectious Diseases, Robert Koch-Institute, Berlin, Germany for further genotyping by MLST targeting triose phosphate isomerase (tpi), β-giardin (bg), and glutamate dehydrogenase (gdh) genes. Amplified polymerase chain reaction (PCR) products were then purified, sequenced, and aligned with reference strains to determine the assemblages of the Giardia isolates.
The Results have shown that:
• Of the 96 selected cases 43 (44.8%) and 53 (55.2%) were from Ain Shams and Cairo University Pediatrics’ Hospitals, respectively. The age of participants ranged from 8 months to 16 years old with with a median age of 5 years and interquartile range (IQR) 3–6 years old. Of these, 66 were males and 30 were females. Almost 93 % had soft or loose stool with moderate to many cysts numbers/HPF Abdominal pain and diarrhea were the most frequent symptoms.
• Out of the 96 Giardia microscopically positive samples, 77 samples (80 %) were successfully amplified at least at one of the three genetic loci. Nineteen (20 %) gave negative results. In total, among the 77 samples, nPCR gave positive amplification in 67 (70 %), 69 (72 %), and 73 (76 %) for tpi, bg, and gdh genetic loci, respectively. Sixty-two (65 %) were amplified at the three loci, 4 (4 %) gave positive results for both bg and gdh, 3 (3 %) gave positive results for both tpi and gdh, 1 (1 %) gave positive results for both tpi and bg, 4 (4 %) gave positive results for gdh only, 1 (1 %) gave positive results for bg only, while 1 (1 %) gave positive results for tpi only.
• Comparative analysis of the sequences of the 77 isolates, that gave positive results at one or more of the three molecular markers, with reference strains revealed assemblages A and B in 21 (27.3%) and 54 (70.1%) of the samples, respectively, while discordant sequence type results were noted in 2 (2.6%) isolates. At the individual loci, tpi, bg, and gdh, 19 (28.4%), 22 (31.8%) and 20 (27.4 %) were identified as assemblage A, while 47 (70.1%), 47 (68.2%) and 54 (70.1 %) were identified as assemblage B, respectively. Sixty isolates gave similar assemblage at the three loci, of these 18 (30%), 42 (70%) were of assemblage A and B, respectively. Two isolates gave discordant assemblages. All assemblage A sequences belonged to sub-assemblage AII. Generally, it was difficult to sub-classify assemblage B, thus no further sub-classification has been attempted.
• Comparative analysis of Giardia sequences by distance matrices and simple phylogeny tree builder by Unweighted Pair group Method with Arithmetic Mean (UPGMA) has shown the presence of unique sequences in 65 %, 43 % 69%, and 78 % at the tpi, bg, gdh and concatenated levels and that such uniqueness was mainly at the B assemblage level; 85 %, 56 % 94%, and 100 %, respectively for the aforementioned sequences.
• No significant statistical differences were found either between Giardia assemblages and gender, or with the type of drinking water. However, a statistical significant difference in the distribution of assemblages B over A at all age groups was found (P = 0.01). Additionally, no significant statistical difference (P = 0.92) was found between Giardia assemblages and the site of sample collection, i.e. Cairo and Ain Shams Pediatric Hospitals. Children infected with assemblage B had considerably soft to loose stool. Additionally, G. duodenalis cysts were roughly quantified in all the genotyped stool samples, children with assemblage B shed more G. duodenalis cysts than children infected with assemblage A. However, these differences did not reach statistical significance (P = 0.16 and P = 0.74) respectively. Though infections with assemblage B were more frequently associated with abdominal pain and diarrhea than with assemblage A, yet the difference was not statistically significant (P ˃ 0.05).
The present study, as with others, has shown that multilocus genotyping based on the commonly used set of housekeeping genes might be more useful for the typing of assemblage A. For assemblage B characterization at the subassemblage level, a more defining tool is required. The study has also shown that the discrimination of identical or near identical strains could be possible in order to trace a specific strain locally or longitudinally in one patient.