Mobile Suitcase Laboratory Rapid Detects Visceral Leishmaniasis

Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL) and currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms.

Molecular diagnosis would require real-time polymerase chain reaction (qPCR), which is not easy to implement at field settings; however a recombinase polymerase amplification (RPA) assay has been developed and tested for the detection of LD.

Scientists at the Georg-August-University (Gottingen, Germany) and their colleagues tested a total of 48 archived DNA samples from VL patients, asymptomatic individuals and post-kala-azar dermal leishmaniasis (PKDL) patients were tested by both RPA and real-time PCR assays. To determine the specificity of the assay, the archived DNA samples including 35 endemic healthy controls, five non-endemic healthy controls and disease controls including six malaria cases and two tuberculosis cases were also investigated.

All VL, PKDL and asymptomatic cases were positive for LD DNA by realtime PCR, while controls (healthy and disease) were negative for rK39, direct antiglobulin test (DAT) and LD DNA by real-time PCR. Quantitative detection of Leishmania DNA was performed on a CFX96 real time detection system (Bio-Rad, Hercules CA, USA). Two mobile suitcase laboratories were constructed to have separate workspaces for nucleic acid extraction and detection in order to avoid any possible contamination. Blood samples were collected from seven patients hospitalized at the Surya Kanta Kala-azar Research Center, and nucleic acid was extracted from whole blood and RPA test was performed in the field.

The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature of 42 °C in 15 minutes. 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, Qiagen, Hilden, Germany), in which a simple fast lysis protocol was applied. Moreover, all reagents were cold-chain independent.

The authors concluded that the use of a mobile suitcase laboratory is advantageous for rapid, sensitive and specific detection of LD using SpeedXtract and RPA assay, especially, at low resource settings such as Bangladesh and could contribute to VL control and elimination program. The study was published in the May 2016 issue of the journal Parasites &Vectors.

Related Links:
Georg-August-University
Bio-Rad
Qiagen