Prevalence of Entamoeba histolytica in Phayao Lake, Phayao Province, Thailand

Abstract

Entamoeba histolytica is a parasitic protozoan that lives in the large intestine of humans and other primates causing amoebic dysentery or amoebiasis in infected people. In severe cases or immunocompromised people, Entamoeba histolytica leads to complications because this type of protozoa can cause intestinal perforation initiating liver or lung abscesses when protozoa spread to the brain, leading to death. The protozoa can contaminate the environment, water, natural water resources, and fresh vegetables throughout pandemic areas. This can spread due to consumption of contaminated food or drink and unhygienic behavior, especially in tropical regions with inadequate public health priorities. Phayao Lake is a large natural water reservoir in Phayao Province, Thailand, used as a water source and for agriculture, fishery, breeding of freshwater animals, and tourism. Farmers bring cattle to feed from the surrounding area. These activities support the growth of microorganisms in water sources, especially when temperatures increase during the summer. The microscope detection method is an important and standard technique for detection of protozoal parasites collected from clinical and environmental samples prior to the laboratory. This method is an easy and convenient technique but requires expertise to identify the type of protozoa, including rarely detectable mild infection. Molecular biology has been introduced to detect protozoal parasites. Real-time polymerase chain reaction (PCR) can detect Entamoeba histolytica with high sensitivity and specificity. However, molecular biology methods are costly and require several testing steps. The loop-mediated isothermal amplification (LAMP) technique was developed and used in the diagnosis of diseases by increasing the amount of DNA under a single temperature. It does not require a thermocycler and takes less time compared to other molecular biology techniques. Therefore, LAMP can be used in field detection of protozoal parasites. Previous studies have shown that the LAMP technique is more sensitive than PCR and nested PCR. The present study aimed to determine the prevalence of Entamoeba histolytica in 70 water samples from Phayao Lake and compared protozoal detection using a standard microscopic method and a loop-mediated isothermal amplification (LAMP) method. Prior to detection, water samples (5 liters per sampling point) were collected from surface water covering Phayao Lake.  The physical quality of water, including dissolved oxygen (DO), pH, conductivity and temperature, by multimeter. All sample collection points were marked by GPS on the Google Map application. Water samples were centrifuged to sediment and concentrated with ethyl acetate before iodine staining with glass slides under the microscope. For the LAMP method, the sediment of all samples was extracted by QIAamp DNA Mini Kit before LAMP reaction with reagents including 10X DNA polymerase buffer, primers (Ehd1-F3, Ehd1-B3, Eh1-FIP and Ehd1-BIP), MgSO₄, dNTPs mix, Bst DNA polymerase, RNase free water and templates. Data were analyzed by SPSS version 20.0 and the concordance of Entamoeba histolytica detection was analyzed by Cohen’s Kappa-coefficient. The proportion of positive results in detecting Entamoeba histolytica was 30.0% (21/70). 7.1% (5/70) and 30.0% (21/70) of Entamoeba histolytica detections were by microscope and LAMP methods, respectively. The LAMP method of identifying Entamoeba histolytica was more sensitive and specific than the microscopic method, at 100.0% and 75.4%, respectively. The concordance of Entamoeba histolytica detection was fair between the methods (Kappa coefficient = 0.30). The results of this study could be applied to detect protozoal contamination in water sources with suitable techniques in water-borne protozoal surveillance.