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Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

  • Lucas J. Cunningham (a1), J. Russell Stothard (a1), Mike Osei-Atweneboana (a2), Samuel Armoo (a2), Jaco J. Verweij (a3) and Emily R. Adams (a1)...


With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.


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Author for correspondence: Lucas J. Cunningham, E-mail:


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Al-Shehri, H, Koukounari, A, Stanton, MC, Adriko, M, Arinaitwe, M, Atuhaire, A, Kabatereine, NB and Stothard, RJ (2018) Surveillance of intestinal schistosomiasis during control: a comparison of four diagnostic tests across five Ugandan primary schools in the Lake Albert region. Parasitology.
Barenbold, O, Raso, G, Coulibaly, JT, N'Goran, EK, Utzinger, J and Vounatsou, P (2017) Estimating sensitivity of the Kato-Katz technique for the diagnosis of Schistosoma mansoni and hookworm in relation to infection intensity. PLoS Neglected Tropical Diseases 11.
Cunningham, LJ, Odoom, J, Pratt, D, Boatemaa, L, Asante-Ntim, N, Attiku, K, Banahene, B, Osei-Atweneboana, M, Verweij, JJ, Molyneux, D, Stothard, RJ and Adams, ER (2018) Expanding molecular diagnostics of helminthiasis: piloting use of the GPLN platform for surveillance of soil transmitted helminthiasis and schistosomiasis in Ghana. PLoS Neglected Tropical Diseases 12.
Fernandez-Soto, P, Arahuetes, JG, Hernandez, AS, Aban, JL, Santiago, BV and Muro, A (2014) A loop-mediated isothermal amplification (LAMP) assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model. PLoS Neglected Tropical Diseases 8.
Hotez, P, Ottesen, E, Fenwick, A and Molyneux, D (2006) The neglected tropical diseases: the ancient afflictions of stigma and poverty and the prospects for their control and elimination. Hot Topics in Infection and Immunity in Children III 582, 2333.
Katz, N, Chaves, A and Pellegrino, J (1972) A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Revista do Instituto Medicina Tropical Sao Paulo 14, 397400.
Kongs, A, Marks, G, Verle, P and Van der Stuyft, P (2001) The unreliability of the Kato-Katz technique limits its usefulness for evaluating S-mansoni infections. Tropical Medicine & International Health 6, 163169.
Liu, J, Gratz, J, Amour, C, Kibiki, G, Becker, S, Janaki, L, Verweij, JJ, Taniuchi, M, Sobuz, SU, Haque, R, Haverstick, DM and Houpt, ER (2013) A laboratory-developed TaqMan array card for simultaneous detection of 19 enteropathogens. Journal of Clinical Microbiology 51, 472480.
MEGA Molecular Evolutionary Genetics Analysis Home Page. Vol. 2013.
Meurs, L, Brienen, E, Mbow, M, Ochola, EA, Mboup, S, Karanja, DMS, Secor, WE, Polman, K and van Lieshout, L (2015) Is PCR the next reference standard for the diagnosis of schistosoma in stool? A comparison with microscopy in Senegal and Kenya. PLoS Neglected Tropical Diseases 9.
Minetti, C, LaCourse, J, Reimer, L and Stothard, R (2016) Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy. Parasitology Open 2.
Montresor, A, Crompton, DWT, Hall, A, Bundy, DAP and Savioli, L (1998) Guidelines for the evaluation of soil-transmitted helminthiasis and schistosomiasis at community level. (ed. WHO). Geneva: WHO.
Obeng, BB, Aryeetey, YA, de Dood, CJ, Amoah, AS, Larbi, IA, Deelder, AM, Yazdanbakhsh, M, Hartgers, FC, Boakye, DA, Verweij, JJ, van Dam, GJ and van Lieshout, L (2008) Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana. Annals of Tropical Medicine and Parasitology 102, 625633.
Rosser, A, Rollinson, D, Forrest, M and Webster, BL (2015) Isothermal recombinase polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection. Parasites & Vectors 8.
Savioli, L, Fenwick, A, Rollinson, D, Albonico, M and Ame, SM (2015) An achievable goal: control and elimination of schistosomiasis. The Lancet 386, 739.
Stothard, JR (2009) Improving control of African schistosomiasis: towards effective use of rapid diagnostic tests within an appropriate disease surveillance model. Transactions of the Royal Society of Tropical Medicine and Hygiene 103, 325332.
Stothard, JR, Stanton, MC, Bustinduy, AL, Sousa-Figueiredo, JC, Van Dam, GJ, Betson, M, Waterhouse, D, Ward, S, Allan, F, Hassan, AA, Al-Helal, MA, Memish, ZA and Rollinson, D (2014) Diagnostics for schistosomiasis in Africa and Arabia: a review of present options in control and future needs for elimination. Parasitology 141, 19471961.
Turner, HC, Bettis, AA, Dunn, JC, Whitton, JM, Hollingsworth, D, Fleming, FM and Anderson, RM (2017) Economic considerations for moving beyond the Kato-Katz technique for diagnosing intestinal parasites as we move towards elimination. Trends in Parasitology 33, 435443.
Verweij, JJ, Brienen, EAT, Ziem, J, Yelifari, L, Polderman, AM and Van Lieshout, L (2007) Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. American Journal of Tropical Medicine and Hygiene 77, 685690.
Verweij, JJ, Canales, M, Polman, K, Ziem, J, Brienen, EAT, Polderman, AM and van Lieshout, L (2010) Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR (vol 103, pg 342, 2009). Transactions of the Royal Society of Tropical Medicine and Hygiene 104, 378378.
Weatherhead, JE, Hotez, PJ and Mejia, R (2017) The global state of helminth control and elimination in children. Pediatric Clinics of North America 64, 867877.
WHO (2018) Fact Sheets Relating to NTDs. Vol. 2018.
Wiria, AE, Prasetyani, MA, Hamid, F, Wammes, LJ, Lell, B, Ariawan, I, Uh, HW, Wibowo, H, Djuardi, Y, Wahyuni, S, Sutanto, I, May, L, Luty, AJF, Verweij, JJ, Sartono, E, Yazdanbakhsh, M and Supali, T (2010) Does treatment of intestinal helminth infections influence malaria? Background and methodology of a longitudinal study of clinical, parasitological and immunological parameters in Nangapanda, Flores, Indonesia (ImmunoSPIN Study). BMC Infectious Diseases 10.


Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

  • Lucas J. Cunningham (a1), J. Russell Stothard (a1), Mike Osei-Atweneboana (a2), Samuel Armoo (a2), Jaco J. Verweij (a3) and Emily R. Adams (a1)...


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