Abstract
BackgroundEntamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available.MethodsIn this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples.ResultsThe stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10−7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10−5 (~3 ng/ml) and 10−4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide.ConclusionThe stem LAMP test developed in this study indicates potential towards detection of E. histolytica.
Highlights
Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections
We report an improved loop-mediated isothermal amplification (LAMP) test for E. histolytica with inclusion of stem primers
E. histolytica LAMP optimum reaction conditions The Taguchi method determined the optimal concentrations for the four reaction components in stem 18S LAMP test as 35 pmol for FIP/BIP, 18 pmol for loop primers, 23 pmol for stem primers and 2 mM deoxyribonucleotide triphosphates (dNTPs)
Summary
The causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. Amoebiasis caused by protozoan Entamoeba histolytica is an important human gastrointestinal infection responsible for over 50 million amoebic infection cases with over 100,000 deaths annually [1]. The E. histolytica antigen detection in the stool using ELISA tests [14,15,16] has proved more sensitive than microscopy, cross-reactivity with E. dispar limit their application [17, 18]. This far the PCR method has been the most sensitive method for discriminating between E. histolytica and E. dispar. Several PCR tests have been developed [19,20,21] but despite the reported advantage of PCR tests in diagnosis of E. histolytica, the method has limited use in routine diagnosis of amoebiasis in Kenya due to associated cost
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