Indian journal of medical microbiology · 2026
Evaluation of duplex TaqMan real-time PCR assay for the simultaneous detection of Ancylostoma duodenale and Necator americanus.
СтатьяОценочное исследованиеПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Thakur Mamta, Mewara Abhishek, Lakshmi P V M, Guleria Sucheta, Khurana Sumeeta
Аннотация
PURPOSE: Hookworm infection affects approximately 438 million individuals globally and is a major cause of gastrointestinal distress and iron-deficiency anemia, particularly in tropical and subtropical regions. This study aimed to standardize and evaluate a duplex real-time PCR assay for the simultaneous detection of Ancylostoma duodenale and Necator americanus to improve diagnostic sensitivity and specificity.
METHODS: A total of 350 stool samples (50 for lab-based evaluation and 300 for clinical validation) were collected from the Department of Medical Parasitology, PGIMER, Chandigarh. Genomic DNA was extracted, and primers targeting the ITS-1 region were selected for PCR. Assay conditions were optimized using 13 microscopy-positive samples, negative samples and other parasite controls. In addition, 300 stool samples were analysed separately in a clinical setting to validate the applicability of the duplex real-time PCR assay under routine conditions.
RESULTS: The duplex real-time PCR assay demonstrated 100% sensitivity and 96.7% specificity in the lab-based evaluation. While only 13 samples were positive by microscopy, 20 were positive by conventional PCR and 21 by duplex real-time PCR. In the clinical setting, out of 300 samples analysed, 2 were positive by microscopy, whereas 15 were detected by duplex real-time PCR.
CONCLUSIONS: The duplex real-time PCR assay provides a sensitive alternative to conventional microscopy for hookworm detection. This is the first validated duplex assay for hookworm species in northern India, offering improved diagnostic efficiency and supporting enhanced epidemiological surveillance and disease management.
METHODS: A total of 350 stool samples (50 for lab-based evaluation and 300 for clinical validation) were collected from the Department of Medical Parasitology, PGIMER, Chandigarh. Genomic DNA was extracted, and primers targeting the ITS-1 region were selected for PCR. Assay conditions were optimized using 13 microscopy-positive samples, negative samples and other parasite controls. In addition, 300 stool samples were analysed separately in a clinical setting to validate the applicability of the duplex real-time PCR assay under routine conditions.
RESULTS: The duplex real-time PCR assay demonstrated 100% sensitivity and 96.7% specificity in the lab-based evaluation. While only 13 samples were positive by microscopy, 20 were positive by conventional PCR and 21 by duplex real-time PCR. In the clinical setting, out of 300 samples analysed, 2 were positive by microscopy, whereas 15 were detected by duplex real-time PCR.
CONCLUSIONS: The duplex real-time PCR assay provides a sensitive alternative to conventional microscopy for hookworm detection. This is the first validated duplex assay for hookworm species in northern India, offering improved diagnostic efficiency and supporting enhanced epidemiological surveillance and disease management.