A New Multiplex PCR Assay Reveals the Occurrence of E. bangladeshi alongside E. histolytica and E. moshkovskii in Eastern India

• Published in: Acta parasitologica 2024-12, Vol.69 (4), p.1886-1895
• Main Authors: Sardar, Sanjib Kumar, Mal, Sweety, Ghosal, Ajanta, Haldar, Tapas, Prasad, Akash, Roy, Chayanika, Ghosh, Arjun, Saito-Nakano, Yumiko, Kobayashi, Seiki, Dutta, Shanta, Nozaki, Tomoysohi, Ganguly, Sandipan
• Format: Article
• Language: English
• Subjects: Animal Systematics/Taxonomy/Biogeography; Assaying; Biomedical and Life Sciences; Biomedicine; Deoxyribonucleic acid; Diarrhea; Diarrhea - parasitology; DNA; DNA Primers - genetics; DNA sequencing; DNA, Protozoan - genetics; E coli; Ecology; Entamoeba; Entamoeba - classification; Entamoeba - genetics; Entamoeba - isolation & purification; Entamoeba histolytica; Entamoeba histolytica - classification; Entamoeba histolytica - genetics; Entamoeba histolytica - isolation & purification; Entamoeba moshkovskii; Entamoebiasis - diagnosis; Entamoebiasis - epidemiology; Entamoebiasis - parasitology; Epidemiology; Feces - parasitology; Gene sequencing; Humans; India - epidemiology; Indigenous species; Medical Microbiology; Microbiology; Multiplex Polymerase Chain Reaction - methods; Multiplexing; Nucleotide sequence; Original Paper; Parasitology; Phylogenetics; Phylogeny; Polymerase chain reaction; RNA, Ribosomal, 18S - genetics; rRNA; Sensitivity analysis; Sensitivity and Specificity; Sequence Analysis, DNA; Species Specificity; Statistical analysis
• ISSN: 1230-2821; 1896-1851; 1896-1851
• DOI: 10.1007/s11686-024-00921-z

Purpose Epidemiological studies on amoebic infections are complicated due to morphologically identical and clinically important Entamoeba species. Therefore, newer, simpler, and more economical diagnostic techniques are required for differentiating clinically important Entamoeba species. Methods We developed a single-round multiplex PCR assay to identify E. histolytica , E. moshkovskii , E. dispar , E. bangladeshi , and E. coli . Primers were designed based on variations in 18 S rRNA sequences. Sensitivity and specificity were assessed using known positive and negative samples. Furthermore, we screened 472 diarrheal samples using this technique alongside the reference PCR method to evaluate its suitability for epidemiological studies and clinical diagnosis. DNA sequencing and phylogenetic analysis of the isolates were conducted. All statistical analyses of the data were performed using GraphPad Prism. Results The designed primers successfully yielded species-specific PCR products of different sizes as expected. We did not observe any non-specific amplifications of the primer set. The diagnostic performance was also convincing. After screening clinical samples using the method, we observed that 2.33% ( n  = 11) tested positive for E. moshkovskii , 1.06% ( n  = 5) tested positive for E. histolytica , and 0.85% ( n  = 4) tested positive for E. bangladeshi in the studied area. DNA sequencing further confirmed the identified species. The constructed phylogenetic tree also demonstrated clear separation of the detected species lineages. Conclusion The study suggests the multiplex PCR assay could be a reliable diagnostic tool for amoebic infections. This study is particularly significant as it marks the first reported occurrence of E. bangladeshi since its documentation in South Africa and its native Bangladesh.