title: Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institute of Tropical Medicine and Infectious Diseases, Jomo Kenyatta University of Agriculture and Technology (JKUAT), P.O. Box 54840, 00200, Nairobi, Kenya. jrobert.joseph65@gmail.com.|Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O Box 54840, 00200, Nairobi, Kenya. jrobert.joseph65@gmail.com.
authors.forename: Robert Muriuki
authors.initials: RM
authors.lastname: Mugambi
authors.affiliation: Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O Box 54840, 00200, Nairobi, Kenya. Elelo@kemri.org.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Agola
authors.affiliation: Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O Box 54840, 00200, Nairobi, Kenya. INdungu@kemri.org.
authors.forename: Ibrahim N
authors.initials: IN
authors.lastname: Mwangi
authors.affiliation: Department of Biochemistry, Jomo Kenyatta University of Agriculture and technology (JKUAT), P.O. Box 62000, 00200, Nairobi, Kenya. johnsonkinyua@jkuat.ac.ke.
authors.forename: Johnson
authors.initials: J
authors.lastname: Kinyua
authors.affiliation: Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O Box 54840, 00200, Nairobi, Kenya. astar812@gmail.com.
authors.forename: Esther Andia
authors.initials: EA
authors.lastname: Shiraho
authors.affiliation: Center for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), P.O Box 54840, 00200, Nairobi, Kenya. GMkoji@kemri.org.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Mkoji
mesh_terms: D017954:Africa South of the Sahara; D000818:Animals; D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005243:Feces / Q000469:parasitology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D017221:Necator americanus / Q000235:genetics / Q000302:isolation & purification*; D009332:Necatoriasis / Q000175:diagnosis* / Q000469:parasitology; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer