Epidemiology and Molecular Characterization of Giardia lamblia in Children

Abstract

Giardia lamblia is the most common protozoan infecting the small intestine of human beings and a major cause of enteric infection especially in children throughout the world. This is the first comprehensive study on giardiasis in children of Punjab, Pakistan. The aim of the present study was to detect the prevalence of Giárdiá lámbliá infection and its associated risk factors in children. A cross sectional study was designed and total 800 samples were collected from children of different district healthcare hospitals of Punjab, Pakistan. The stool samples were subjected to conventional microscopy and enzyme linked immunosorbent assay (ELISA). Polymerase chain reaction (PCR) was employed for molecular detection of Giárdiá lámbliá and differentiation of its assemblages. Different DNA extraction protocols were also evaluated for G. lámbliá. All the information regarding personal data, demographic data and supposed risk factors was collected through a structured questionnaire. The results indicated the prevalence of Giárdiá lámbliá as 2.75% with microscopy alone, 8.88% with ELISA alone and overall prevalence on the basis of both the test was 9.5% (76/800). On the basis of spatial trend, highest prevalence was recorded in district Khanewal (12.5%) while the least was observed in district Faisalabad (6.5%). The demographic factors positively associated with giardiasis were age (P = 0.035; odds ratio, OR = 1.96), mother’s educational level (P = 0.031; OR = 2.67) and father’s educational level (P = 0.004; OR = 3.56) while amongst risk factors, rural residency (P = 0.032; OR = 1.76), absence of proper sewerage system (P = 0.000; OR = 6.60) and unavailability of safe drinking water (P = 0.000; OR = 4.08) were strongly connected with giardiasis. All the 76 positive samples were subjected to PCR by using small-subunit ribosomal RNA (SSU rRNA) and assemblage specific triose phosphate isomerase (tpi) gene primers. The results showed 100% sensitivity by SSU rRNA-PCR while tpi-PCR successfully amplified 69 (90.80%) isolates. Among these tpi gene positive samples, 38 were successfully typed into assemblage B (55.07%) followed by 20 (28.98%) into assemblage A and 11 (15.94%) in mixed type assemblages (A&B). All the DNA extraction protocols produced 100% sensitivity but the FavorPrep Stool DNA kit was found effective when other parameters (DNA quantity, quality, sample cost and time) were tested. The clinical presentation prominently found in Assemblage B subjects was abdominal pain (57.89%) and Assemblage A was vomiting (40%). X It was concluded that prevalence of Giárdiá lámbliá is high among children of Punjab and an issue of public health concern. The prominent contributing factors were unsafe drinking water, hygiene status, rural residency and parent’s education level of children. Molecular detection is very helpful tool for estimation of true prevalence of G. lámbliá and its assemblages. On the basis of conclusions, it was recommended that creating awareness about risk factors, improving education level of parents and providing a better hygiene level to children can be helpful to reduce the burden of giardiasis in children. The use of advanced molecular detection tools for giardiasis should be adapted to get true prevalence, better discrimination of assemblages and their correlation with clinical signs.