title: Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection.
abstract: BACKGROUND
  Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.
  
  
  METHODS
  LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.
  
  
  RESULTS
  The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.
  
  
  CONCLUSIONS
  The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.
authors.affiliation: Bangkok Christian College, Bangkok, 10500, Thailand. chaichontat.s@gmail.com.
authors.forename: Chaichontat
authors.initials: C
authors.lastname: Sriworarat
authors.affiliation: Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand. amphumee@gmail.com.
authors.forename: Atchara
authors.initials: A
authors.lastname: Phumee
authors.affiliation: Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, 10400, Thailand. mathirut@hotmail.com.
authors.forename: Mathirut
authors.initials: M
authors.lastname: Mungthin
authors.affiliation: Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, 10400, Thailand. s_leelayoova@scientist.com.
authors.forename: Saovanee
authors.initials: S
authors.lastname: Leelayoova
authors.affiliation: Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand. padet.s@chula.ac.th.|Excellence Center for Emerging Infectious Diseases, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand. padet.s@chula.ac.th.
authors.forename: Padet
authors.initials: P
authors.lastname: Siriyasatien
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D013785:Thailand
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S