title: Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.
abstract: Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD.
  
  
  
  A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh.
  
  
  
  The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent.
  
  
  
  The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.
authors.affiliation: Center for Nutrition and Food Security, International Center for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Mondal
authors.affiliation: Center for Nutrition and Food Security, International Center for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh.
authors.forename: Prakash
authors.initials: P
authors.lastname: Ghosh
authors.affiliation: Center for Nutrition and Food Security, International Center for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh.
authors.forename: Md Anik Ashfaq
authors.initials: MA
authors.lastname: Khan
authors.affiliation: Center for Nutrition and Food Security, International Center for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh.
authors.forename: Faria
authors.initials: F
authors.lastname: Hossain
authors.affiliation: Division of Microbiology and Animal Hygiene, Georg-August-University, Goettingen, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Böhlken-Fascher
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Matlashewski
authors.affiliation: University Medical Centre Freiburg, Centre for Medicine and Society, Freiburg, Germany.|UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland.
authors.forename: Axel
authors.initials: A
authors.lastname: Kroeger
authors.affiliation: UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland.|Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, UK.
authors.forename: Piero
authors.initials: P
authors.lastname: Olliaro
authors.affiliation: Division of Microbiology and Animal Hygiene, Georg-August-University, Goettingen, Germany. abdelwahed@gwdg.de.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abd El Wahed
mesh_terms: D000818:Animals; D001459:Bangladesh; D016054:DNA, Protozoan / Q000235:genetics; D005240:Feasibility Studies; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000469:parasitology; D008952:Mobile Health Units; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D045522:Recombinases; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan; D045522:Recombinases
keywords: Bangladesh; Leishmania donovani; Recombinase polymerase amplification assay; Suitcase laboratory; Visceral leishmaniasis