Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP)

Abstract

BackgroundConfirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.Methodology/Principal findingsThe Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.Conclusions/SignificanceDue to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.

Clinical, Molecular and Serological Diagnosis of Canine Leishmaniosis: An Integrated Approach

“…Moreover, as also reported by other authors, it is important to point out that information provided by molecular analysis should not be separated from the data obtained from clinical signs and serological evaluations, because a positive PCR indicates a Leishmania infection, but not necessarily the disease development [5]. In fact, comparisons performed for the diagnosis of visceral leishmaniosis (VL) in humans between a commercial LAMP kit (EikenChemical Co., Tokyo, Japan) and other molecular and/or serological techniques, showed that LAMP could be included in the algorithm for VL, and could be used to support other laboratory findings [29,30].…”

“…These authors showed that LAMP is easy-to-use, highly sensitive (90-98%) and specific (80-100%) and allows to perform in-situ analysis, but no standardized protocols are available in the veterinary field. Contrariwise, to detect human leishmaniosis, the Loopamp™ Leishmania kit has been developed by the Eiken Chemical Co. (Tokyo, Japan) and successfully validated [29][30][31][32].…”

Applying Loop-mediated Isothermal Amplification (LAMP) in the Diagnosis of Malaria, Leishmaniasis and Trypanosomiasis as Point-of-Care Tests (POCTs)

“…The rK28 RDT presented higher sensitivity and specificity (98.81% and 100 % respectively) than DAT. The LAMP kit had high specificity regardless of the type of DNA extraction method used or sample analysed, (whole blood or buffy coat), avoiding the need for invasive lymph node aspiration [96]. Ibarra-Meneses et al, 2018, demonstrated that by combining this kit with a portable and robust real-time fluorimetry, a versatile assay able to diagnose VL in situations in which the serological diagnosis is useless such as in VL relapses and testof-cure, or VL / HIV co-infection and even in canine leishmaniasis was possible [97].…”

Evaluation of rapid extraction methods coupled with Recombinase polymerase amplification assay for point-of-need diagnosis of Post-kala-azar-dermal leishmaniasis

“…The SE method has not, however, been evaluated using skin samples. Alternatively, we found a relatively simple DNA extraction method based on an inhouse lysis buffer to be suitable for Loop-mediated isothermal assays (27,28). In an effort to develop a field-friendly diagnostic algorithm for detecting L. donovani DNA in skin samples from PKDL patients we therefore assessed various nucleic acid extraction techniques in combination with an RPA assay.…”