Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay

Abstract

BackgroundLeishmania 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.MethodsA 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1572-8) contains supplementary material, which is available to authorized users.

Recombinase polymerase amplification: Basics, applications and recent advances

“…However, this tolerance to mismatches can also lead to cross-reactivity as demonstrated by an RPA assay developed to detect the three different genotypes of Chikungunya virus that was [129] observed to have cross-reactivity with another related alphavirus, the O'nyong'nyong virus, based on 4 to 7 mismatches in the primers. A further example is an assay to determine Leishmania donovi that was observed to also amplify other Leishmania spp [62]. However, a method to detect EGFR mutations in lung cancer cells with specificity of just one base mismatch or single nucleotide polymorphism has been developed.…”

Chagas Disease Diagnostic Applications

“…Serological tests, however show substantial variations in their sensitivity in different geographical areas (Verani et al, 2009), and display sub-optimal performances (Sanchez-Camargo et al, 2014). Unfortunately, and despite being widely used in different parasitic diseases (Mondal et al, 2016; Sriworarat et al, 2015), PCR-based methods applicable in PoC settings such as those based on simple colorimetric loop-mediated isothermal amplification or Recombinase Polymerase Amplification (RPA) techniques are not yet available for T. cruzi detection.…”

Towards elimination of visceral leishmaniasis in the Indian subcontinent—Translating research to practice to public health

“…To identify active VL, it is used as one element of the diagnostic algorithm whereby it is applied to subjects with persistent fever and palpable spleen. By itself, it cannot differentiate active from past infection [41], and it has limited value as a marker for disease progression, cure, or relapse [18]. The search continues for a new diagnostic marker that can be used at the population level—a marker for asymptomatic infection, for progression to PKDL, that performs well even in VL–HIV-coinfected individuals [42].…”

“…The implications of HIV coinfection with VL for treatment failure and relapse, transmission dynamics, and development of parasite resistance to drugs need to be studied further, and strategies need to be developed and tested as appropriate [42]. The development of innovative approaches to impair infection through early case detection and treatment, particularly in remote or previously nonendemic areas, as well as vector surveillance systems, new methods to measure transmission, mathematical transmission modelling to measure progress post elimination, xenodiagnostic studies to measure reservoir potential, new noninvasive antigen-based diagnostic tools [41], better treatment of PKDL, and surveillance for drug resistance are some of the urgent research priorities for the immediate future [122, 128]. DWL as an option for vector control needs to be further explored, as well as other alternatives that can be applied by communities themselves, such as insecticidal paint or LLIN combined with other affordable “do-it-yourself” measures with appropriate support.…”