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A New Multiplex PCR Assay Reveals the Occurrence of E. bangladeshi alongside E. histolytica and E. moshkovskii in Eastern India
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A New Multiplex PCR Assay Reveals the Occurrence of E. bangladeshi alongside E. histolytica and E. moshkovskii in Eastern India
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Journal Article
A New Multiplex PCR Assay Reveals the Occurrence of E. bangladeshi alongside E. histolytica and E. moshkovskii in Eastern India
2024
Overview
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.
Publisher
Springer International Publishing,Springer Nature B.V
Subject
Animal Systematics/Taxonomy/Biogeography
/ Assaying
/ Biomedical and Life Sciences
/ Cysts
/ Design
/ Diarrhea
/ DNA
/ E coli
/ Ecology
/ Entamoeba - isolation & purification
/ Entamoeba histolytica - classification
/ Entamoeba histolytica - genetics
/ Entamoeba histolytica - isolation & purification
/ Entamoebiasis - epidemiology
/ Entamoebiasis - parasitology
/ Humans
/ LDCs
/ Multiplex Polymerase Chain Reaction - methods
/ Plasmids
/ RNA, Ribosomal, 18S - genetics
/ rRNA
/ Species
