Prevalence of giardiasis and multi locus sequence typing of giardia lamblia isolates from human diarrheal patients
Student name: Ms Krittika Jana
Guide: Dr Anandita Singh, Dr Ramakrishnan Sitaraman & Dr Pallavi Somvanshi
Year of completion: 2014
Host Organisation: National Institute of Cholera and Enteric Diseases (Indian Council of Medical Research)
Supervisor (Host Organisation): Dr Sandipan Ganguly
Abstract: Prevalence of Giardiasis was studied with the detection of pathogen positive stool
samples from patients admitted in IDBG & BCH hospitals in the Kolkata region
through molecular assays. ELISA based detection ofantigen in Giardiasis patient
fecal samples was done to analyse the prevalence of the disease in the region. It was
found through monoclonal ELISA based detection techniques that Giardia is more
prevalent in this study region than other parasites like Cryptosporidium , with
Entamoeba being the least frequent. Percentage of prevalence of Giardia lamblia was
found to be 7.5%, the most frequent when compaired to other enteric parasites such
as Cryptosporidium sp( 3.7 %) and Entamoeba histolytica ( 1.8% ). Giardia lamblia
cysts isolated from human stool samples from the patients with diarrheal symptoms
admitted in the IDBG hospital were studied to characterize the genotype of the
parasite using PCR methods. DNA extracted from the patient fecal samples were
amplified through polymerase chain reactions specific for the 218 bp of the
Betagiardin( bg ) gene . Molecular assays such as nested PCR was used to study the
genotypic characterization of the parasite , based on three different loci. Nested PCR
based amplification of Betagiardin (ßg) 511 bp ,Triosephophate Isomerase (tpi) 530
bp and Glutamate Dehydrogenase (gdh) 434 bp genetic elements were done for future
DNA sequencing assay . Among 25 fecal samples from ELISA based diagnosis of
Giardiasis patients, the bg, tpi ,gdh genes were amplified in 20 ( 80%) , 15 (60%) ,19
(76%) cases respectively with a nested PCR assay and purified for future sequencing
assays. Of the 25 samples, 14 samples ( 56% ) were commonly amplified for all the
three loci. Purified nested PCR products of those 14 samples were amplified with the
individual respective ( bg, tpi, gdh ) nested primers for sequencing analysis.
Sequencing PCR products , done with purified nested PCR products amplified with
their respective nested primers, would be sequenced and then validated in database
search. Data indicated the effect of multilocus genetic elements for the pathogenic
property of the parasite. The study also revealed that Giardia is more frequent over
other enteric parasites in this region. Further study based on sequencing methods
would help to understand the genotypic features of this disease in different
populations.
Key Words: Giardia lamblia, Subculturing, Genotyping, Nested PCR, Sequencing,
Glutamate Dehydrogenase, Triosephosphate Isomerase, Betagiardin
