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Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children
R Tolentino-Ruiz1, D Montoya-Varela, M García-Espitia
1Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, Escuela Nacional de Medicina y Homeopatía-IPN, Instituto Politécnico Nacional Guillermo Massieu Helguera No. 239, Col. Fracc La Escalera-Ticomán, 07320 México, DF, México.
Insights
A new multiplex PCR test can simultaneously detect multiple pathogens causing childhood acute gastroenteritis (AGE). This method aids in diagnosing viral, bacterial, and parasitic infections in children.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Acute gastroenteritis (AGE) is a significant global health concern in children, leading to substantial morbidity and mortality.
- The diverse etiology of AGE involves viruses, bacteria, and parasites, complicating diagnosis.
- Accurate and rapid diagnostic tools are crucial for effective management of pediatric AGE.
Purpose of the Study:
- To develop and evaluate a multiplex PCR assay for the simultaneous detection of common AGE pathogens.
- To compare the performance of the multiplex PCR assay with traditional methods like ELISA and single-target PCR/RT-PCR.
- To assess the utility of this novel assay in identifying multiple infectious agents in a single stool sample.
Main Methods:
- A multiplex PCR assay was designed to detect human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC).
- A total of 103 stool samples were analyzed using the multiplex PCR assay, ELISA, and RT-PCR/PCR.
- PCR products were sequenced to confirm the identity of detected pathogens.
Main Results:
- The multiplex PCR assay successfully detected HAstV, E. histolytica, and EIEC simultaneously in some samples.
- HAstV and HuCVs were detected in 3.8% of samples by RT-PCR, while ELISA identified only 2.5% for HuCVs.
- E. histolytica was found in 10.5% and EIEC in 70% of tested samples, with confirmed identities via sequencing. The HuCVs signal was consistently weak.
Conclusions:
- The developed multiplex PCR protocol is a novel and effective tool for identifying distinct pathogens causing AGE.
- This assay allows for the simultaneous detection of multiple enteric pathogens, offering a potential advantage over single-target methods.
- The multiplex PCR assay can support the diagnosis of acute gastroenteritis in children, improving etiological identification.
Abstract:
Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children.
