Development and Evaluation of a Modified Loop-Mediated Isothermal Amplification (mLAMP) Test for the Detection of Entamoeba histolytica

Abstract

Entamoeba histolytica, the causative agent for amoebiasis is of considerable burden to populations 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. This study aimed at developing a rapid modified LAMP test that can be applied in the detection of E. histolytica. Three hundred and thirty four samples were collected and microscopically examined for the presence of E. histolytica, from children who presented with diarrhea, abdominal pain and/or discomfort at three participating outpatient clinics at Mukuru informal settlements and one inpatient paediatric ward of Mbagathi District Hospital. Microscopic examination scored 126 of the 334 samples as positive for Entamoeba. A loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with a pair of extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of Entamoeba histolytica DNA in clinical samples. The 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 ten-fold serial dilution of DNA. In the analysis of microscopy Entamoeba spp trophozites and cysts positive clinical samples, stem LAMP detected Entamoeba histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 18/126 respectively. There was 100% agreement in detection of the stem LAMP 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. A new stem 18S LAMP test which is a modification of the standard LAMP test through inclusion of stem primers was developed. It is recommended that this new stem 18S LAMP test be further evaluated using a larger sample size and be part of diagnostic algorithms for amoebiasis.