title: Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  In 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.
  
  
  RESULTS
  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 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.
  
  
  CONCLUSIONS
  The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.
authors.affiliation: Institute of Tropical Medicine and Infectious Diseases, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-0200, Nairobi, Kenya. fridahmwendwa.fm@gmail.com.
authors.forename: Fridah
authors.identifier: http://orcid.org/0000-0002-8358-5037
authors.initials: F
authors.lastname: Mwendwa
authors.affiliation: Centre for Microbiology Research, Kenya Medical Research Institute, P.O Box 19464-00202, Nairobi, Kenya.
authors.forename: Cecilia K
authors.initials: CK
authors.lastname: Mbae
authors.affiliation: Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-0200, Nairobi, Kenya.
authors.forename: Johnson
authors.initials: J
authors.lastname: Kinyua
authors.affiliation: Centre for Microbiology Research, Kenya Medical Research Institute, P.O Box 19464-00202, Nairobi, Kenya.
authors.forename: Erastus
authors.initials: E
authors.lastname: Mulinge
authors.affiliation: Meru University of Science and Technology, P. O. Box 972-60200, Meru, Kenya.
authors.forename: Gitonga Nkanata
authors.initials: GN
authors.lastname: Mburugu
authors.affiliation: Meru University of Science and Technology, P. O. Box 972-60200, Meru, Kenya.|School of Health Professions, Mandurah Campus, Murdoch University, Murdoch, WA, 6210, Australia.
authors.forename: Zablon K
authors.initials: ZK
authors.lastname: Njiru
mesh_terms: D001483:Base Sequence; D002648:Child; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics* / Q000502:physiology; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D005243:Feces / Q000469:parasitology; D006790:Host-Parasite Interactions; D006801:Humans; D007630:Kenya; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S
keywords: Amoebiasis; Diagnosis; Entamoeba histolytica; Kenya; Loop-mediated isothermal amplification, LAMP; Stem-LAMP test