Development and evaluation of a loop mediated isothermal amplification technique for the detection of hookworm infection in feacal samples

Abstract

Hookworm 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. Accurate diagnosis is essential for the formulation of effective control measures. Currently most research conducted on epidemiology of hookworms has relied on the use of conventional microscopy for identification of parasite ova in faeces. The benefits are mainly technical simplicity and low cost; however, these techniques are limited to the fact that most of nematode eggs are morphologically indistinguishable from those of other species, they also lack the sensitivity to detect low level infections and thus cannot be effective in areas where mass chemotherapy is employed as a control strategy against hookworms and other soil transmitted helminths. Over-reliance on this microscopy based techniques that is Kato-Katz that is prone to human errors and leads to in most cases misdiagnosis and poor implemented control strategies. With that in mind it is of great importance to develop efficient molecular diagnostic tools, which have proven to be more sensitive in detecting low level infections with reliability and reproducibility and specific enough to differentiate between infections by different species. In this study, I aimed to employ a Loop mediated isothermal amplification to diagnose hookworm infection, specifically N. americanus from faecal samples. This LAMP assay was based on internal transcribed spacer 2 (ITS-2) of nuclear ribosomal DNA (rDNA) because of its low mutation rates and ability to differentiate between species. The assay employed four primers; two inner primers and two outer primers designed by an online based program, Primer explorer version 4. Bst DNA polymerase containing the 5’-3’ polymerase and lacking 5’-3’ exonuclease activity with high displacement activity was used. This technique showed to be 100 % specific when used in detecting DNA from other commonly occurring soil transmitted helminths, that is, S. mansoni, A. lumbricoides and T. trichiura and 97% sensitive with a detection limit as low as 0.4FG. When compared to Kato-Katz, LAMP proved more robust by detecting three samples negatives, which were diagnosed as positive by Kato-Katz, highlighting the limitation that Kato-Katz readings are subject to human errors non-random distribution of eggs in stool and day to day variation in egg output. In terms of agreement, between the two techniques, there was an excellent agreement level with a kappa coefficient of 0.9. The LAMP assay has demonstrated that it can be utilized as an appropriate diagnostic method for the detection of N. americanus DNA in human faecal samples because of its high sensitivity, specificity. It holds great promise as a useful tool for use in disease control where infection intensities have been significantly reduced.