Genotyping of Giardia Intestinalis and Cryptosporidium from human and environmental samples

Abstract

This study was conducted to identify species and genotypes of Cryptosporidium and Giardia intestinalis in Orang Asli (aboriginal) communities and environmental samples in Pahang, Malaysia. Stool samples were collected from 321 individuals aged between 2 and 76 years old, of whom 160 were males and 161 were females. Water was collected from the river flowing through this community. Faecal samples were processed with Trichrome stain and Ziehl-Neelsen stain for the primary identification of G. intestinalis and Cryptosporidium, respectively. Water samples were processed and examined with epifluorescence microscopy. Molecular identification was carried out by the amplific tion of a partial SSU rRNA gene using nested PCR. Genotyping was done by DNA sequencing of the SSU rRNA in both directions followed with phylogenetic analysis. Biodata were collected via pre-tested standard questionnaire. The prevalence rate of G. intestinalis and Cryptosporidium based on faecal samples was 23.7% and 4.1%, respectively. The predictors of Giardia infection in this community which were confirmed with multivariate analysis using logistic regression were: age ≤ 12 years (Odd Ratio (OR) = 6.2, 95% Confidence Interval (CI) = 1.5-27.0, p < 0.005), drinking piped water (OR = 5.1, 95% CI = 0.06-0.7, p < 0.005) and eating raw vegetables (OR = 2.4, 95% CI = 0.2 -0.6, p < 0.005). Giardiasis was significantly associated with diarrhoea and symptoms of gastroenteritis (p < 0.05). Genotyping analysis of Cryptosporidium isolated from human faecal samples identified one isolate as Cryptosporidium hominis and four isolates as unidentified genotypes which may represent local strains. Two G. intestinalis assemblages were found; assemblage A and assemblage B with the predominance of assemblage B. Risk analysis based on the detected genotypes of Giardia using univariate analysis and logistic regression identified three significant risk factors of giardiasis caused by assemblage B. These factors included children ≤ 12 years (OR = 9.42, 95% CI = (1.3 – 67.5), p = 0.004), females (OR= 2.23, 95% CI = 1.04 – 4.79, p = 0.035) and eating fresh fruits (OR = 7.28, 95% CI = 0.96-54.99, p = 0.026). Assemblage B infection was also significantly correlated with clinical symptoms of giardiasis (OR = 2.4, 95% CI = 1.13 – 5.12, p = 0.019). All the river water samples were tested negative for Cryptosporidium and G. intestinalis (oo)cysts. In conclusion, cryptosporidiosis and giardiasis are still public health problems in Orang Asli communities. The predominance of G. intestinalis assemblage B and the identification of Cryptosporidium hominis which have been postulated commonly found in humans suggest anthroponotic transmission as the most possible way of G. intestinalis and Cryptosporidium transmission implicating humans as the main source of infection. It would seem that transmission occurs either directly or indirectly through contaminated water and food. In future, it is necessary that these two aspects be considered in control strategies. The high risk groups should receive greater attention from public health authorities.