Detection and subtyping of Blastocystis sp. in human and animal stool samples using high-resolution melting analysis

This study aimed to investigate the prevalence and subtype distribution of the genus Blastocystis using high-resolution melting curve analysis (HRM), a novel molecular technique. Blastocystis sp., a unicellular, anaerobic, and zoonotic parasite, is commonly found in the digestive tracts of humans and animals. Transmission occurs via the fecal-oral route, with prevalence rates ranging from 20% in developed countries to over 60% in developing regions. Although the pathogenicity of Blastocystis remains debated, it has been detected in both symptomatic and asymptomatic individuals. Traditionally, direct microscopy with wet mounts is used for the rapid identification of Blastocystis in stool samples. However, molecular diagnostics have become essential for the accurate detection and differentiation of Blastocystis subtypes MATERIALS AND METHODS: We collected 730 stool samples from humans and domestic animals. Initial screening for Blastocystis sp. was performed using direct microscopy. Negative samples were cultured in a two-phase culture medium and re-examined after 2-3 days. HRM analysis was then employed to identify and differentiate Blastocystis subtypes using specific primers via real-time PCR.

Six subtypes were identified, with ST7 (30%) being the most prevalent, followed by ST3 (28%), ST2 (16%), ST1 (14%), ST5 (6%), and ST14 (6%). In humans, ST3 was the most common subtype, also found in poultry and sheep, whereas ST7 was mainly detected in domestic animals. Notably, ST1-ST3 were identified in domesticated animals, indicating a pattern of cross-species transmission.

ST3 (28%) and ST7 (30%) were the most prevalent subtypes across all samples, with distinct distributions in human and animal hosts. The HRM technique demonstrated efficiency and cost-effectiveness, providing a rapid and accurate method for Blastocystis subtype identification in developing countries, which can expedite diagnostic responses and reduce the need for sequencing. This supports the potential for HRM to improve epidemiological surveillance and understanding of cross-species transmission.