African trypanosomiasis: Sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA

Abstract

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

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

“…This technique has been proven to be an accurate, rapid and simple method, which amplifies the target nucleic acid under isothermal conditions (Notomi et al, 2000). Recently, wide applicability of LAMP in the detection of parasitic protozoa such as Babesia, Plasmodium, Leishmania and Trypanosoma in clinical samples has been demonstrated (Ikadai et al, 2004;Poon et al, 2006;Njiru et al, 2008;Takagi et al, 2009;Thekisoe et al, 2007;Laohasinnarong et al, 2011). Studies have also shown the application of LAMP to survey vectors of infectious diseases (Aonuma et al, 2009;Thekisoe et al, 2010;Nakao et al, 2010); however, these studies used purified DNA as a template for the LAMP assays.…”

Comparative Detection of Trypanosomal DNA by Loop-Mediated Isothermal Amplification and PCR from Flinders Technology Associates Cards Spotted with Patient Blood

“…These tests amplify 18S ribosomal DNA or RNA, respectively, and as such cannot be used to distinguish between the two HAT types. Recently, loop-mediated isothermal amplification of DNA (LAMP) has been used for detection of members of the Trypanozoon subgenus as well as T. b. rhodesiense (11,12) subsequent to earlier studies targeting various trypanosomal genes (8,16). The reportedly high sen-sitivity, coupled with no need for specialized equipment, increases the likelihood of its adoption in resource-poor health centers where HAT is endemic.…”

Human African Trypanosomiasis

“…110 Present research eff orts focus on several areas: (i) recombinant or native trypanosome antigens that could be used to develop an improved serological test; (ii) methods to detect parasite antigens in blood or CSF; (iii) proteomic fi ngerprinting; (iv) low-tech PCR methods such as loop-mediated isothermal amplifi cation or oligochromatography; and (v) new blood or CSF markers of second-stage disease. [111][112][113][114][115] …”