Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

Abstract

Loop-mediated isothermal amplification (LAMP) is ideal for the detection of DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of spp., and an ARR of between 1 × 10 and 1 × 10 equivalent parasites/mL was determined. An LoD of 1 × 10 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect DNA, which showed good diagnostic potential from sandflies and direct smear samples.

Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

“…being shown to have a detection limit of at least 0.01 parasites for LAMP [15] or 10-100 parasites/mL for RT-LAMP [116]. Testing the protocol described by Nzelu et al [15], León et al [100], found that LAMP sensitivity was greater (80%: 60.3-90.0 95% CI) than that by qPCR (78.0%: 64.0-88.5% 95% CI). Adams et al [102] tested a set of primers for amplifying the kDNA sequence.…”

Test accuracy of polymerase chain reaction methods against conventional diagnostic techniques for Cutaneous Leishmaniasis (CL) in patients with clinical or epidemiological suspicion of CL: Systematic review and meta-analysis

“…Articles were excluded at this stage due to (i) articles not available, (ii) not in the field of interest, (iii) use of an inappropriate reference standard, and (iv) inability to complete a 2-by-2 contingency table. A total of 13 articles were included in the systematic review [5,[12][13][14][15][16][17][18][19][20][21][22][23] while data of 12 articles were included in the meta-analysis. Data of 1 article were not considered in the meta-analysis because they did not fit the respective subgroups for molecular method and/or sample type.…”

Loop-mediated isothermal amplification (LAMP): An advanced molecular point-of-care technique for the detection of Leishmania infection

“…martiniquensis DNA in sand flies in Thailand; however, no Leishmania DNA was detected by LAMP and PCR when applied to 380 field-caught flies, probably due to low infection rate in the study area [79]. Additionally, the performance of the MG-LAMP was further validated using purified DNA from sand fly pools and was found to be of potential use for the entomological surveillance of CL in Colombia with 100% sensitivity and 96.8% specificity [80]. Indeed, these reported studies have shown the potential usefulness of LAMP for mass-screening of sand flies in the Old and New World.…”

“…HNB dye changes from violet to a sky blue color [82] and requires the operator to distinguish between positive and negative color results, which can be ambiguous. In contrast, MG signal recognition is highly sensitive, the system gives a clear-cut difference between positives (light blue color) and negatives (colorless) based on naked eye visualization, and interpretation by an independent observer is not required [9, 27, 41, 80]. MG-LAMP products are stable and thus can be kept for record purposes.…”

“…Several studies have shown the reliability, robust sensitivity, and wide applicability of MG-based-LAMP closed detection system for Leishmania and other purposes. MG has been used for detection of Leishmania DNA in crude Lutzomyia sand fly extract in Ecuador [27]; patient’s tissue spotted on FTA-card in Peru [9]; blood, saliva, and tissue biopsies in Thailand [41]; and direct smears and sand flies in Colombia [80]. Taken together, the incorporation of a closed-LAMP assay offer new prospects for improved detection of infections, as well as positioning LAMP as a quick, one-step, POC molecular diagnostic and xenomonitoring tool for leishmaniasis and other diseases.…”