Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation Ying.

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Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation Ying Li, Nirbhay Kumar, Anusha Gopalakrishnan, Christine Ginocchio, Ryhana Manji, Maureen Bythrow, Bertrand Lemieux, Huimin Kong The Journal of Molecular Diagnostics Volume 15, Issue 5, Pages 634-641 (September 2013) DOI: 10.1016/j.jmoldx.2013.05.005 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Alignment of P. falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax 18S rRNA sequences. The sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/nucleotide; accession numbers AL010278, L07560, AB489196, AJ001527, and U93233) for each species, respectively. Only the region surrounding the tHDA amplification product is shown. The primers and probes used in the two tHDA tests are indicated on the alignment. The Journal of Molecular Diagnostics 2013 15, 634-641DOI: (10.1016/j.jmoldx.2013.05.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Schematic diagram of the tHDA-based malaria assay results. There are three antibody lines on the lateral-flow test strip: anti-DNP, anti-DIG, and anti-FAM (from top to bottom). In test 1, if only the DNP line is present, the test result is negative for Plasmodium. If the FAM line is present (the other two lines can be present or absent), the test result is positive for P. falciparum. If the FAM line is absent, but the DIG line is present (the DNP line can be either present or absent), the result is positive for Plasmodium but negative for P. falciparum. In test 2, if only the DNP line is present, the test result is negative for P. vivax; and if the DIG line is present (the DNP line can be either present or absent), the test is positive for P. vivax. The FAM line should always be absent for test 2. The Journal of Molecular Diagnostics 2013 15, 634-641DOI: (10.1016/j.jmoldx.2013.05.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Amplification and detection of all five Plasmodium species and identification of P. falciparum and P. vivax by tHDA and lateral-flow cassettes. A: Test 1. Genomic DNA of P. falciparum (lane F) or plasmids containing the 18S rRNA fragment of the other Plasmodium species (lanes V, O, M, and K) were added to 50 μL of tHDA test 1 amplification reaction to a final amount of 50 or 500 copies. B: Test 2. Plasmid DNA containing the 18S rRNA fragment of P. vivax was added to tHDA test 2 at 50 copies per reaction (lane V). Plasmid containing P. ovale, P. malariae, or P. knowlesi DNA fragment or genomic DNA of P. falciparum was added to tHDA test 2 at 1000× limit of detection (final 50,000 copies) per reaction (lanes F, O, M, and K). In both A and B, the reactions were incubated at 64°C for 90 minutes before the tubes were applied to BESt cassettes. Negative reactions without input template (NTC) were also performed (lane N). Each reaction was performed in triplicate. In A, only NTC and reactions containing 50 copies of the input DNA template were shown. Only one of the triplicates was shown for each reaction. Label C on cassettes indicates the DNP line for IC; M, DIG line for Plasmodium; and S, FAM line for P. falciparum. The Journal of Molecular Diagnostics 2013 15, 634-641DOI: (10.1016/j.jmoldx.2013.05.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Histogram of the parasitemia levels of the positive clinical samples. A: P. falciparum–positive samples. B: All other Plasmodium-positive samples. The Journal of Molecular Diagnostics 2013 15, 634-641DOI: (10.1016/j.jmoldx.2013.05.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions