A Loop-Mediated Isothermal Amplification (LAMP) Assay for Strongyloides stercoralis in Stool That Uses a Visual Detection Method with SYTO-82 Fluorescent Dye

Matthew R. Watts Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Matthew R. Watts in
Current site
Google Scholar
PubMed
Close
,
Gregory James Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Gregory James in
Current site
Google Scholar
PubMed
Close
,
Yasmin Sultana Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Yasmin Sultana in
Current site
Google Scholar
PubMed
Close
,
Andrew N. Ginn Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Andrew N. Ginn in
Current site
Google Scholar
PubMed
Close
,
Alexander C. Outhred Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Alexander C. Outhred in
Current site
Google Scholar
PubMed
Close
,
Fanrong Kong Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Fanrong Kong in
Current site
Google Scholar
PubMed
Close
,
Jaco J. Verweij Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Jaco J. Verweij in
Current site
Google Scholar
PubMed
Close
,
Jonathan R. Iredell Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Jonathan R. Iredell in
Current site
Google Scholar
PubMed
Close
,
Sharon C-A. Chen Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Sharon C-A. Chen in
Current site
Google Scholar
PubMed
Close
, and
Rogan Lee Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands

Search for other papers by Rogan Lee in
Current site
Google Scholar
PubMed
Close
Restricted access

An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10−2 dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

Author Notes

* Address correspondence to Matthew R. Watts, Centre for Infectious Diseases and Microbiology, Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, Darcy Road, Westmead, New South Wales, 2145 Australia. E-mail: watts.idmicro@gmail.com

Financial support: Matthew R. Watts received financial assistance through an Australian NHMRC Postgraduate Scholarship. This project was partially funded through the Centre for Infectious Diseases and Microbiology Public Health, Institute of Clinical Pathology and Medical Research, Westmead Hospital.

Authors' addresses: Matthew R. Watts, Gregory James, Andrew N. Ginn, Alexander C. Outhred, Fanrong Kong, Jonathan R. Iredell, Sharon C-A. Chen, and Rogan Lee, Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research Westmead, Westmead Hospital, Westmead, New South Wales, Australia, E-mails: watts.idmicro@gmail.com, greg.james@swahs.health.nsw.gov.au, andrew.ginn@sydney.edu.au, aouthred@gmail.com, fanrong.kong@swahs.health.nsw.gov.au, jonathan.iredell@sydney.edu.au, sharon.chen@swahs.health.nsw.gov.au, and rogan.lee@swahs.health.nsw.gov.au. Yasmin Sultana, Department of Zoology, University of Dhaka, Dhaka, Bangladesh, E-mail: ysultana@du.ac.bd. Jaco J. Verweij, Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands, E-mail: j.verweij@elisabeth.nl.

  • 1.

    Siddiqui AA, Berk SL, 2001. Diagnosis of Strongyloides stercoralis infection. Clin Infect Dis 33: 10401047.

  • 2.

    Keiser PB, Nutman TB, 2004. Strongyloides stercoralis in the immunocompromised population. Clin Microbiol Rev 17: 208217.

  • 3.

    Mangklabruks A, Rerkasem A, Wongthanee A, Rerkasem K, Chiowanich P, Sritara P, Pruenglampoo S, Yipintsoi T, Tongsong T, Marshall T, Tantiprabha W, 2012. The risk factors of low birth weight infants in the northern part of Thailand. J Med Assoc Thai 95: 358365.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Dreyfuss ML, Msamanga GI, Spiegelman D, Hunter DJ, Urassa EJ, Hertzmark E, Fawzi WW, 2001. Determinants of low birth weight among HIV-infected pregnant women in Tanzania. Am J Clin Nutr 74: 814826.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Schaffel R, Nucci M, Carvalho E, Braga M, Almeida L, Portugal R, Pulcheri W, 2001. The value of an immunoenzymatic test (enzyme-linked immunosorbent assay) for the diagnosis of strongyloidiasis in patients immunosuppressed by hematologic malignancies. Am J Trop Med Hyg 65: 346350.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Sato Y, Kobayashi J, Toma H, Shiroma Y, 1995. Efficacy of stool examination for detection of Strongyloides infection. Am J Trop Med Hyg 53: 248250.

  • 7.

    Steinmann P, Zhou X-N, Du Z-W, Jiang J-Y, Wang L-B, Li L-H, Marti H, Utzinger J, 2007. Occurrence of Strongyloides stercoralis in Yunnan Province, China, and comparison of diagnostic methods. PLoS Negl Trop Dis 1: e75.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    Verweij JJ, Canales M, Polman K, Ziem J, Brienen EA, Polderman AM, van Lieshout L, 2009. Molecular diagnosis of Strongyloides stercoralis in fecal specimens using real-time PCR. Trans R Soc Trop Med Hyg 103: 342346.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Nilforoushan MR, Mirhendi H, Rezaian M, Meamar AR, Kia EB, 2007. A DNA-based identification of Strongyloides stercoralis isolates from Iran. Iran J Public Health 36: 1620.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Kramme S, Nissen N, Soblik H, Erttmann K, Tannich E, Fleischer B, Panning M, Brattig N, 2011. Novel real-time PCR for the universal detection of Strongyloides species. J Med Microbiol 60: 454458.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Herwaldt BL, 2001. Laboratory-acquired parasitic infections from accidental exposures. Clin Microbiol Rev 14: 659688.

  • 12.

    Mori Y, Kanda H, Notomi T, 2013. Loop-mediated isothermal amplification (LAMP): recent progress in research and development. J Infect Chemother 19: 404411.

  • 13.

    Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M, Sanga E, Hoelscher M, Notomi T, Hase T, Perkins MD, 2007. Operational feasibility of using loop-mediated isothermal amplification for the diagnosis of pulmonary tuberculosis in microscopy centres of developing countries. J Clin Microbiol 45: 19361940.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Mori Y, Notomi T, 2009. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-cost effective diagnostic method for infectious diseases. J Infect Chemother 15: 6269.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Theksioe OM, Bazie RS, Coronel-Servain AM, Sugimoto C, Kawazu S, Inoue N, 2009. Stability of loop-mediated isothermal amplification (LAMP) reagents and its amplification efficacy on crude trypanosome DNA templates. J Vet Med Sci 71: 471475.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Myers FB, Henrikson RH, Bone J, Lee LP, 2013. A handheld point-of-care genomic diagnostic system. PLoS ONE 8: e70266.

  • 17.

    Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, Matsushita Y, Okutani A, Bazartseren B, Inoue S, Sata T, Katano H, 2010. LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn J Infect Dis 63: 3640.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18.

    Garcia LS, 2007. Diagnostic Medical Parasitology. Washington, DC: ASM Press.

  • 19.

    Sultana Y, Jeoffreys N, Watts MR, Gilbert GL, Lee R, 2013. Real-time polymerase chain reaction for detection of Strongyloides stercoralis in stool. Am J Trop Med Hyg 88: 10481051.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20.

    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: e63.

  • 21.

    Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877882.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I, 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41: 55175524.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Xin Z, Velten JP, Oliver MJ, Burke JJ, 2003. High-throughput DNA extraction method suitable for PCR. Biotechniques 34: 820826.

  • 24.

    Goto M, Honda E, Ogura A, Nomoto A, Hanaki K, 2009. Colorimetric detection of loop-mediated isothermal amplification reaction using hydroxy naphthol blue. Biotechniques 46: 167172.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25.

    Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, Thompson RC, Ndung'u JM, 2008. African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 38: 589599.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26.

    Sun J, Najafzadeh MJ, Vicente V, Xi L, de Hoog GS, 2010. Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea agents of chromoblastomycosis. J Microbiol Methods 80: 1924.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27.

    Gudnason H, Dufva M, Bang DD, Wolff A, 2007. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res 35: e127.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28.

    Ihira M, Akimoto S, Miyake F, Fujita A, Sugata K, Suga S, Ohashi M, Nishimura N, Ozaki T, Asano Y, Yoshikawa T, 2007. Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method. J Clin Virol 39: 2226.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29.

    Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M, 2005. Loop-mediated isothermal amplification method targeting the lytA gene for the detection of Streptococcus pneumoniae. J Clin Microbiol 43: 15811586.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30.

    Kimura Y, de Hoon MJ, Aoki S, Ishizu Y, Kawai Y, Kogo Y, Daub CO, Lezhava A, Arner E, Hayashizaki Y, 2011. Optimization of turn-back primers in isothermal amplification. Nucleic Acids Res 39: e59.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31.

    Lee D, La Mura M, Allnutt TR, Powell W, 2009. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences. BMC Biotechnol 9: 7.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32.

    Nagamine K, Hase T, Notomi T, 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16: 223229.

  • 33.

    Ihira M, Ohta A, Sugata K, Suga S, Asano Y, Yoshikawa T, 2008. Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B. J Virol Methods 154: 223225.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34.

    Dreyer G, Fernandes-Silva E, Alves S, Rocha A, Albuquerque R, Addiss D, 1996. Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials. J Clin Microbiol 34: 25692571.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 35.

    McOrist AL, Jackson M, Bird AR, 2002. A comparison of five methods for extraction of bacterial DNA from human fecal samples. J Microbiol Methods 50: 131139.

  • 36.

    Samuelson J, Acuna-Soto R, Reed S, Biagi F, Wirth D, 1989. DNA hybridization probe for clinical diagnosis of Entamoeba histolytica. J Clin Microbiol 27: 671676.

  • 37.

    Schar F, Odermatt P, Khieu V, Panning M, Duong S, Muth S, Marti H, Kramme S, 2013. Evaluation of real-time PCR for Strongyloides stercoralis and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia. Acta Trop 126: 8992.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38.

    Ellison SL, English CA, Burns MJ, Keer JT, 2006. Routes to improving the reliability of low level DNA analysis using real-time PCR. BMC Biotechnol 6: 33.

Past two years Past Year Past 30 Days
Abstract Views 678 500 50
Full Text Views 857 22 5
PDF Downloads 468 15 4
 
 
 
 
Affiliate Membership Banner
 
 
Research for Health Information Banner
 
 
CLOCKSS
 
 
 
Society Publishers Coalition Banner
Save