Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

Romano Ngui,Kek Heng Chua,Yvonne A. L. Lim,David Joseph Diemert

doi:10.1371/journal.pone.0041996

Abstract

BackgroundHookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species.MethodsReal-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples.ConclusionThe HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

Highlights

Hookworms are blood feeding intestinal nematodes that infect almost 600 million people worldwide, resulting in up to 135,000 deaths annually [1]
We describe a new application of high resolution melting-curve (HRM) employing the ITS-2 of nuclear ribosomal DNA as the genetic marker for the rapid detection, quantification and speciation of hookworm species in human samples
DNA was obtained from faecal samples in which Necator americanus (n = 10), A. ceylanicum (n = 10) and A. caninum (n = 10) were confirmed by the polymerase chain reaction (PCR) coprodiagnostic technique, as described previously [2]

Summary

Introduction

Hookworms are blood feeding intestinal nematodes that infect almost 600 million people worldwide, resulting in up to 135,000 deaths annually [1]. Necator americanus and Ancylostoma duodenale are two most common species causing infection in humans. Mixed infections of these hookworms are common in many endemic areas especially among people in tropical and subtropical countries with low socioeconomic status. Besides the two human species, canine and/or feline hookworms such as Ancylostoma ceylanicum, Ancylostoma caninum and Ancylostoma braziliense can cause infections to human. Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world’s poorest people. A real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species

Methods

Results

Conclusion