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Development and evaluation of a loop-mediated isothermal amplification (LAMP) diagnostic test for detection of whipworm, Trichuris trichiura, in faecal samples

Published online by Cambridge University Press:  02 April 2020

M.G. Ngari ,
I.N. Mwangi ,
M.P. Njoroge ,
J. Kinyua ,
F.A. Osuna ,
B.M. Kimeu ,
P.W. Okanya  and
E.L. Agola Open the ORCID record for E.L. Agola [Opens in a new window]
M.G. Ngari
Affiliation:
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, PO Box 62000, 00200Nairobi, Kenya
I.N. Mwangi
Affiliation:
Centre for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), PO Box 54840, 00200Nairobi, Kenya
M.P. Njoroge
Affiliation:
Department of Health, PO Box 24, Kirinyaga County, Kerugoya, Kenya
J. Kinyua
Affiliation:
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, PO Box 62000, 00200Nairobi, Kenya
F.A. Osuna
Affiliation:
Centre for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), PO Box 54840, 00200Nairobi, Kenya
B.M. Kimeu
Affiliation:
Department of Biochemistry and Biotechnology, Technical University of Kenya, PO Box 52428, 00200Nairobi, Kenya
P.W. Okanya
Affiliation:
Department of Biochemistry and Biotechnology, Technical University of Kenya, PO Box 52428, 00200Nairobi, Kenya
E.L. Agola*
Affiliation:
Centre for Biotechnology Research and Development, Kenya Medical Research Institute (KEMRI), PO Box 54840, 00200Nairobi, Kenya Department of Biochemistry and Biotechnology, Technical University of Kenya, PO Box 52428, 00200Nairobi, Kenya
*
Author for correspondence: E.L. Agola, E-mail: elelo@kemri.org
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Abstract

Whipworm infection or trichuriasis caused by Trichuris trichiura is of major public health concern in sub-Saharan Africa, particularly among pre-school and school-going children. It is among the neglected tropical diseases targeted for elimination through mass drug administration (MDA). One of the outcomes of MDA is a rapid decline in levels of infection intensity, making it difficult to monitor effectiveness of control measures using the conventional Kato–Katz procedure, which relies on the microscopic detection of parasite ova in faecal samples. In the present study, a loop-mediated isothermal amplification (LAMP) test was developed for the detection of T. trichiura infection in faecal samples. LAMP technology offers greater sensitivity and specificity than the microscopy-based tests. A set of four specific primers targeting the internal transcribed spacer 2 region of the ribosomal DNA were designed using Primer Explorer software. DNA was extracted from faecal samples using the alkaline lysis method (HotSHOT) and the LAMP reaction performed at 63°C for 1 h. The amplicons were visualized by both gel electrophoresis and with the naked eye following staining with SYBR green dye. Sensitivity and specificity tests were determined using the standard Kato–Katz diagnostic procedure as a reference test. The developed LAMP assay reliably detected T. trichiura DNA in faecal samples, with a specificity and sensitivity of 88% and 77%, respectively. No cross-reactivity was observed with several common helminth parasites. The developed LAMP assay is an appropriate diagnostic method for the detection of T. trichiura DNA in human faecal samples due to its simplicity, low cost, high sensitivity and specificity.

Keywords

Kato–KatzLAMP assayTrichuris trichiuratrichuriasisdiagnosisITS-2Kenya
Type
Research Paper
Information
Journal of Helminthology , Volume 94 , 2020 , e142
Copyright
Copyright © The Author(s), 2020. Published by Cambridge University Press

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References

Abbasi, I, King, CH, Muchiri, EM and Hamburger, J (2010) Detection of Schistosoma mansoni and Schistosoma haematobium DNA by loop-mediated isothermal amplification: identification of infected snails from early prepatency. The American Journal of Tropical Medicine and Hygiene 83(2), 427–432.CrossRefGoogle ScholarPubMed
Acosta Soto, L, Santisima-Trinidad, AB, Bornay-Llinares, FJ, Martin Gonzalez, M, Pascual Valero, JA and Ros Munoz, M (2017) Quantitative PCR and digital PCR for detection of Ascaris lumbricoides eggs in reclaimed water. Biomedical Research International 2017, 7515409.CrossRefGoogle ScholarPubMed
Amoah, ID, Singh, G, Stenstrom, TA and Reddy, P (2017) Detection and quantification of soil-transmitted helminths in environmental samples: a review of current state-of-the-art and future perspectives. Acta Tropica 169, 187–201.CrossRefGoogle ScholarPubMed
Allen, AV and Ridley, DS (1970) Further observations on the formol-ether concentration technique for faecal parasites. Journal of Clinical Pathology 23(6), 545–546.CrossRefGoogle ScholarPubMed
Boehme, CC, Nabeta, P, Henostroza, G, Raqib, R, Rahim, Z, Gerhardt, M and Perkins, MD (2007) Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. Journal of Clinical Microbiology 45(6), 1936–1940.CrossRefGoogle ScholarPubMed
Booth, M, Vounatsou, P, N'goran, EK, Tanner, M and Utzinger, J (2003) The influence of sampling effort and the performance of the Kato-Katz technique in diagnosing Schistosoma mansoni and hookworm co-infections in rural Côte d'Ivoire. Parasitology 127(6), 525–531.CrossRefGoogle ScholarPubMed
J, Cohen (1960) A coefficient of agreement for nominal scale. Educational and Psychological Measurement 20, 37–46.Google Scholar
Cook, J, Aydin-Schmidt, B, González, IJ, Bell, D, Edlund, E, Nassor, MH and Björkman, A (2015) Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar. Malaria Journal 14, 43.CrossRefGoogle ScholarPubMed
George, EI and McCulloch, RE (2011) Variable selection via gibbs sampling. Journal of the American Statistical Association 88(423), 881–889.CrossRefGoogle Scholar
G/hiwot, Y, Degarege, A and Erko, B (2014) Prevalence of intestinal parasitic infections among children under five years of age with emphasis on Schistosoma mansoni in Wonji Shoa sugar estate, Ethiopia. PLoS ONE 9(10), e109793.CrossRefGoogle ScholarPubMed
Goodman, D, Haji, HJ, Bickle, QD, Stoltzfus, RJ, Tielsch, JM, Ramsan, M, Savioli, L and Albonico, M (2007) A comparison of methods for detecting the eggs of Ascaris, Trichuris, and hookworm in infant stool, and the epidemiology of infection in Zanzibari infants. American Journal of Tropical Medicine and Hygiene 74(4), 725–731.CrossRefGoogle Scholar
Han, ET, Watanabe, R, Sattabongkot, J, Khuntirat, B, Sirichaisinthop, J, Iriko, H and Tsuboi, T (2007) Detection of four plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. Journal of Clinical Microbiology 45(8), 2521–2528.CrossRefGoogle ScholarPubMed
Harris, JR, Worrell, CM, Davis, SM, Odero, K, Mogeni, OD, Deming, MS and Addiss, DG (2015) Unprogrammed deworming in the Kibera slum, Nairobi: implications for control of soil-transmitted helminthiases. PLoS Neglected Tropical Diseases 9(3), e0003590.CrossRefGoogle ScholarPubMed
Kaisar, MMM, Brienen, EAT, Djuardi, Y, Sartono, E, Yazdanbakhsh, M, Verweij, JJ and Van Lieshout, L (2017) Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites. Parasitology 144(7), 965–974.CrossRefGoogle ScholarPubMed
Katz, N, Chaves, A and Pellegrino, J (1972) A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Revista Do Instituto De Medicina Tropical De Sao Paulo 14(6), 397–400.Google ScholarPubMed
Knopp, S, Rinaldi, L, Khamis, IS, Stothard, JR, Rollinson, D, Maurelli, MP and Utzinger, J (2009) A single FLOTAC is more sensitive than triplicate Kato–Katz for the diagnosis of low-intensity soil-transmitted helminth infections. Transactions of the Royal Society of Tropical Medicine and Hygiene 103(4), 347–354.CrossRefGoogle Scholar
Kuboki, N, Inoue, N, Sakurai, T, Di Cello, F, Grab, DJ, Suzuki, H, Sugimoto, C and Igarashi, I (2003) Loop-mediated isothermal amplification for detection of African trypanosomes. Journal of Clinical Microbiology 41(12), 5517–5524.CrossRefGoogle ScholarPubMed
Leal, I, Green, M, Allen, E, Humble, L and Rott, M (2005) An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine. Nematology 7(6), 833–842.Google Scholar
Mugambi, RM, Agola, EL, Mwangi, IN, Kinyua, J, Shiraho, EA and Mkoji, GM (2015) Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in faecal samples. Parasites & Vectors 8, 574.CrossRefGoogle Scholar
Mwangi, IN, Agola, EL, Mugambi, RM, Shiraho, EA and Mkoji, GM (2018) Development and evaluation of a loop mediated isothermal amplification assay for diagnosis of Schistosoma mansoni infection in faecal samples. Journal of Parasitology Research 2018, 1267826.CrossRefGoogle ScholarPubMed
Njiru, ZK, Mikosza, ASJ, Matovu, E, Enyaru, JCK, Ouma, JO, Kibona, SN and Ndung'u, JM (2008) African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. International Journal for Parasitology 38(5), 589–599.CrossRefGoogle ScholarPubMed
Njiru, J, Muhoho, N, Simbauni, J and Kabiru, E (2016) Effects of soil- transmitted helminths and Schistosoma species on nutritional status of children in Mwea irrigation scheme, Kenya. Journal of Applied Life Sciences International 5(1), 1–8.CrossRefGoogle Scholar
Notomi, T, Okayama, H, Masubuchi, H, Yonekawa, T, Watanabe, K, Amino, N and Hase, T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28(12), e63.CrossRefGoogle ScholarPubMed
Phuphisut, O, Yoonuan, T, Sanguankiat, S, Chaisiri, K, Maipanich, W, Pubampen, S and Adisakwattana, P (2014) Triplex polymerase chain reaction assay for detection of major soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, in faecal samples. The Southeast Asian Journal of Tropical Medicine and Public Health 45(2), 267–275.Google Scholar
Pontes, LA, Oliveira, MC, Katz, N, Dias-Neto, E and Rabello, A (2003) Comparison of a polymerase chain reaction and the Kato-Katz technique for diagnosing infection with Schistosoma mansoni. The American Journal of Tropical Medicine and Hygiene 68(6), 652–656.CrossRefGoogle ScholarPubMed
Shiraho, EA, Agola, LE, Mwangi, IN, Maina, GM, Kinuthia, JM, Mutuku, MW and Mkoji, GM (2016) Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Faecal Samples. Journal of Parasitology Research 2016, 7376207. Epub 1 November 2016.CrossRefGoogle Scholar
Truett, GE, Heeger, P, Mynatt, RL, Truett, AA, Walker, JA and Warman, ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). BioTechniques 29(1), 52–54.CrossRefGoogle Scholar
Webster, JP, Molyneux, DH, Hotez, PJ and Fenwick, A (2014) The contribution of mass drug administration to global health: past, present and future. Philosophical Transactions of the Royal Society B: Biological Sciences 369(1645), 20130434.CrossRefGoogle ScholarPubMed
World Health Organization (2010) Working to Overcome the Global Impact of Neglected Tropical Diseases. First WHO Report on Neglected Tropical Diseases. Geneva, Switzerland: World Health Organization.Google Scholar
World Health Organization (2017) Preventive chemotherapy to control soil-transmitted helminth infections in at-risk population groups. Geneva: World Health Organization.Google Scholar