Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based...

Area of Science:

• Medical Parasitology
• Molecular Diagnostics
• Microbiology

Background:

• The standard barcoding-PCR method for Blastocystis subtyping has limitations, including potential false positives and reduced sensitivity with direct stool samples.
• Current methods struggle with accurate Blastocystis subtyping directly from stool DNA, impacting diagnostic reliability.

Purpose of the Study:

• To develop and evaluate a Semi-Nested barcode PCR method for improved Blastocystis subtyping directly from stool samples.
• To enhance the sensitivity and specificity of Blastocystis subtyping compared to the classical barcoding-PCR.

Main Methods:

• Development of a Semi-Nested PCR assay amplifying the 18S rRNA gene's barcoding region in two steps.
• Comparison of the Semi-Nested PCR with classical barcoding-PCR using DNA from stool samples.
• Validation against a reference standard using Santin-PCR and Next-Generation Sequencing (NGS).

Main Results:

• The Semi-Nested barcode PCR demonstrated superior performance over classical barcoding-PCR for stool-derived DNA.
• This new method showed increased reliability, stronger gel signals, and no false positives.
• High concordance (68/70 samples) was observed with the NGS-based reference standard, including the identification of a rare subtype (ST35).

Conclusions:

• Semi-Nested barcoded PCR is a more robust and sensitive method for Blastocystis subtyping directly from stool.
• This improved technique enhances the reliable detection and characterization of Blastocystis subtypes in clinical samples.
• The study successfully identified a second global case of Blastocystis ST35 in Colombia.