Detection of Group 1 Trypanosoma brucei gambiense by Loop-Mediated Isothermal Amplification

Abstract

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3 end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ϳ1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10 3 trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

“…More promising is the ability of the LAMP method to overcome potential inhibitors in crude sand fly templates, an issue of particular concern for PCR assays, and still not compromise its detection sensitivity down to levels of 0.1 and 0.01 parasites. It is suggested that a significant amount of parasite DNA is lost during the extraction process, resulting in a lower detection limit when purified DNA is used as a template (Njiru et al, 2011). Other studies have shown superior tolerance of LAMP tests for biological substances (Kaneko et al, 2007;Poon et al, 2006).…”

Molecular Diagnostics in the Diagnosis of Parasitic Infection

“…One approach for simplifying testing has been to incorporate an oligochromatographic dipstick device for amplicon detection after conventional PCR (Deborggraeve et al, 2006). However, amplification can be further simplified through use of isothermal amplification methods such as LAMP (Kuboki et al, 2003;Njiru, Mikosza, Armstrong, et al, 2008;Njiru, Mikosza, Matovu, et al, 2008;Njiru, Traub, Ouma, Enyaru, & Matovu, 2011) and NASBA (Matovu et al, 2010;Mugasa et al, 2009). These isothermal techniques have demonstrated high analytical sensitivity and specificity, comparable or superior to PCR.…”

Recent progress in molecular diagnosis of sleeping sickness

“…gambiense-and T.b. rhodesiense-specific LAMP assays have been developed by Njiru et al [41,45]. LAMP targeting the SRA gene for diagnosis of rhodesiense sleeping sickness showed a detection limit of one trypanosome per ml blood using heat-treated buffy coat, which is 1000-fold more sensitive than conventional SRA PCR [45].…”

“…A similar low detection level was reached for the T.b. gambiense LAMP using the TgsGP gene [41]. LAMP product visualization with hydroxynaphtol blue was scored favorable over turbidimetry and colorimetry with Quant-iT™ PicoGreen ® and calcein-MnCl 2 in a comparative study performed in 2010 [50].…”