Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples

Abstract

BackgroundHookworm infection is a major concern in sub-Saharan Africa, particularly in children and pregnant women. Necator americanus and Ancylostoma duodenale are responsible for this condition. Hookworm disease is one of the Neglected tropical diseases (NTDs) that are targeted for elimination through global mass chemotherapy. To support this there is a need for reliable diagnostic tools. The conventional diagnostic test, Kato-Katz that is based on microscopic detection of parasite ova in faecal samples, is not effective due to its low sensitivity that is brought about mainly by non-random distribution of eggs in stool and day to day variation in egg output. It is tedious, cumbersome to perform and requires experience for correct diagnosis. LAMP-based tests are simple, relatively cheap, offer greater sensitivity, specificity than existing tests, have high throughput capability, and are ideal for use at the point of care.MethodsWe have developed a LAMP diagnostic test for detection of hookworm infection in faecal samples. LAMP relies on auto cycling strand displacement DNA synthesis performed at isothermal temperature by Bst polymerase and a set of 4 specific primers. The primers used in the LAMP assay were based on the second Internal Transcribed Spacer (ITS-2) region and designed using Primer Explorer version 4 Software. The ITS-2 region of the ribosomal gene (rDNA) was identified as a suitable target due to its low mutation rates and substantial differences between species. DNA was extracted directly from human faecal samples, followed by LAMP amplification at isothermal temperature of 63 °C for 1 h. Amplicons were visualized using gel electrophoresis and SYBR green dye. Both specificity and sensitivity of the assay were determined.ResultsThe LAMP based technique developed was able to detect N. americanus DNA in faecal samples. The assay showed 100 % specificity and no cross-reaction was observed with other helminth parasites (S. mansoni, A. lumbricoides or T. trichiura). The developed LAMP assay was 97 % sensitive and DNA at concentrations as low as 0.4 fg were amplified.ConclusionThe LAMP assay developed is an appropriate diagnostic method for the detection of N. americanus DNA in human stool samples because of its simplicity, low cost, sensitivity, and specificity. It holds great promise as a useful diagnostic tool for use in disease control where infection intensities have been significantly reduced.

Development of Visually Improved Loop Mediated Isothermal Amplification for the Diagnosis of Plasmodium vivax Malaria in a Tertiary Hospital in Chandigarh, North India

“…Of the many dyes, such as Malachite green, hydroxy napthol blue, and Calcein; SYBR green has been used for the diagnosis of a large number of parasitic diseases. [53][54][55][56] In the present study, the detection limit of P. vivax-specific LAMP was found to be 0.8 copies/μL which is in concordance with the previous report. 11 The detection limit of nPCR and RT-PCR have been reported up to 1-5 P/μL of blood which is much better than the sensitivity of microscopy and RDTs.…”

Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

“…In contrast to qPCR the use of isothermal amplification methods for STH stool diagnosis has been overlooked so far, with the exception of two LAMP assays targeting S. stercoralis (Watts et al 2014) and N. americanus (Mugambi et al 2015), respectively. The former assay, amplifying a region of the S. stercoralis 28S ribosomal DNA sequence, showed excellent performance (100% sensitivity and specificity) when tested on 28 stool specimens from patients with S. stercoralis infection confirmed by a combination of microscopy and real-time PCR (Watts et al 2014).…”

“…The former assay, amplifying a region of the S. stercoralis 28S ribosomal DNA sequence, showed excellent performance (100% sensitivity and specificity) when tested on 28 stool specimens from patients with S. stercoralis infection confirmed by a combination of microscopy and real-time PCR (Watts et al 2014). The latter assay, targeting the internal transcribed spacer region 2 of the N. americanus ribosomal DNA region, was used on 86 positive and 20 negative specimens (confirmed by Kato-Katz microscopy) detecting the parasite DNA with a 97% sensitivity and 100% specificity (Mugambi et al 2015). The N. americanus ITS2 LAMP was not tested on DNA from the other two hookworms infecting humans (A. duodenale and A. ceylanicum), so its applicability as a general hookworm diagnostic tool from feces has yet to be verified.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…They concluded that the newly developed LAMP was helpful when leishmania parasite density is very low, especially during the early infection stage (Nzelu et al 2014(Nzelu et al , 2016. There was a number of other reported significant practices of LAMP including Trichomonas viganalis (Reyes et al 2014), Necator americanus (Mugambi et al 2015) and Strongyloides stercoralis (Watts et al 2014), and it was shown that LAMP is at least 1000 times more sensitive than the conventional PCR.…”