Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

Abstract

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after “Boil & Spin” rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.

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

“…Evaluation of the analytical performance is vital to determine the relative analytical specificity and sensitivity of the various molecular methods. Such evaluations have been performed for qPCR and LAMP in Chagas disease 22,55,56 and conventional PCR and qPCR in toxoplasmosis, 57 but not for the LAMP assay in leishmaniasis. Herein, we evaluated the analytical sensitivity and specificity of a LAMP test targeting the 18S rRNA gene in six Leishmania species circulating in Colombia and determined the operational capabilities of the technique in direct smears from patients with presumptive CL and in sandflies.…”

“…[8][9][10][12][13][14] To date, LAMP has been used for the diagnosis of bacterial, 15 fungal, 16,17 and viral 18,19 infections, as well as parasitic infections such as those caused by Trypanosoma brucei, 20 Plasmodium falciparum, 21 and Trypanosoma cruzi. 22 For the diagnosis of leishmaniasis, LAMP has been used with the implementation of primers targeting the small ribosomal subunit (18S rRNA gene), 10,11,23 kinetoplast DNA (kDNA) for the detection of Old World Leishmania species, [24][25][26] and the Internal Transcribed Spacer 1 (ITS-1). 12 Loop-mediated isothermal amplification has also been used in the detection of parasites in their insect vectors, as in the case of Dirofilaria immitis detected in Aedes aegypti, 27 and T. cruzi and Trypanosoma rangeli.…”

“…28 Importantly, there has only been one report of the analytical validation of a LAMP assay for T. cruzi. 22 Although LAMP platform-based tests targeting different markers (most frequently 18S rRNA gene) have shown to be efficient for the detection of Leishmania in biological samples, a comprehensive assessment of the sensitivity and analytical specificity of these tests to elucidate their diagnostic potential is lacking, especially in endemic regions and where several species coexist. 29 Therefore, the objective of this study was to determine, for the first time in Leishmania, the analytical specificity (exclusivity and inclusivity) and sensitivity (anticipated reportable range [ARR], limit of detection [LoD], and accuracy) of the LAMP assay for detecting 18S rRNA gene to evaluate its performance against sandflies and direct smears of CL lesions from a recent outbreak of CL in Colombia.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Positive LAMP reaction was indicated with green colour, whereas negative LAMP reaction indicated with orange colour. To name a few, calcein has been used in detection of Trypanosoma cruzi (Besuschio et al 2017), avian influenza H5 virus (Liu et al 2013), hepatitis C virus (Zhao et al 2017), human papillomavirus , human coronavirus (Geng et al 2016) and Flavobacterium columnare in tilapia fish (Suebsing et al 2015). Calcein-based end-point detection is most commonly used due to its high sensitivity and notable colour change for naked eye observation (Fischbach et al 2015).…”

“…As been discussed in this paper, LAMP could be one of the diagnostic method alternative in relation to its rapidity, sensitivity (Ishiguro et al 2015;Kumvongpin et al 2016;Besuschio et al 2017) and specificity. The most significant advantage of LAMP would be its rapidity where LAMP allows immediate diagnosis.…”