• 1.

    WHO, 2015. Investing to Overcome the Global Impact of Neglected Tropical Diseases: Third WHO Report on Neglected Tropical Diseases 2015. Holmes P, ed. Geneva, Switzerland: World Health Organization, 161167.

    • Search Google Scholar
    • Export Citation
  • 2.

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

  • 3.

    Gill GV, Welch E, Bailey JW, Bell DR, Beeching NJ, 2004. Chronic Strongyloides stercoralis infection in former British Far East prisoners of war. QJM 97: 789795.

    • Search Google Scholar
    • Export Citation
  • 4.

    Schulte C, Krebs B, Jelinek T, Nothdurft HD, von Sonnenburg F, Loscher T, 2002. Diagnostic significance of blood eosinophilia in returning travelers. Clin Infect Dis 34: 407411.

    • Search Google Scholar
    • Export Citation
  • 5.

    Naidu P, Yanow SK, Kowalewska-Grochowska KT, 2013. Eosinophilia: a poor predictor of Strongyloides infection in refugees. Can J Infect Dis Med Microbiol 24: 9396.

    • Search Google Scholar
    • Export Citation
  • 6.

    Calderaro A, Montecchini S, Rossi S, Gorrini C, De Conto F, Medici MC, Chezzi C, Arcangeletti MC, 2014. Intestinal parasitoses in a tertiary-care hospital located in a non-endemic setting during 2006–2010. BMC Infect Dis 14: 264.

    • Search Google Scholar
    • Export Citation
  • 7.

    Becker SL, Sieto B, Silue KD, Adjossan L, Kone S, Hatz C, Kern WV, N'Goran EK, Utzinger J, 2011. Diagnosis, clinical features, and self-reported morbidity of Strongyloides stercoralis and hookworm infection in a co-endemic setting. PLoS Negl Trop Dis 5: e1292.

    • Search Google Scholar
    • Export Citation
  • 8.

    O'Brien DP, Leder K, Matchett E, Brown GV, Torresi J, 2006. Illness in returned travelers and immigrants/refugees: the 6-year experience of two Australian infectious diseases units. J Travel Med 13: 145152.

    • Search Google Scholar
    • Export Citation
  • 9.

    Schar F, Hattendorf J, Khieu V, Muth S, Char MC, Marti HP, Odermatt P, 2014. Strongyloides stercoralis larvae excretion patterns before and after treatment. Parasitology 141: 892897.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ramanathan R, Burbelo P, Groot S, Iadarola M, Neva F, Nutman T, 2008. A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection. J Infect Dis 198: 444451.

    • Search Google Scholar
    • Export Citation
  • 11.

    Soonawala D, van Lieshout L, den Boer MA, Claas EC, Verweij JJ, Godkewitsch A, Ratering M, Visser LG, 2014. Post-travel screening of asymptomatic long-term travelers to the tropics for intestinal parasites using molecular diagnostics. Am J Trop Med Hyg 90: 835839.

    • Search Google Scholar
    • Export Citation
  • 12.

    ten Hove RJ, van Esbroeck M, Vervoort T, van den Ende J, van Lieshout L, Verweij JJ, 2009. Molecular diagnostics of intestinal parasites in returning travellers. Eur J Clin Microbiol Infect Dis 28: 10451053.

    • Search Google Scholar
    • Export Citation
  • 13.

    Buss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK, Rogatcheva M, Kanack KJ, Bourzac KM, 2015. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol 53: 915925.

    • Search Google Scholar
    • Export Citation
  • 14.

    Reddington K, Tuite N, Minogue E, Barry T, 2014. A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens. Biomol Detect Quantif 1: 37.

    • Search Google Scholar
    • Export Citation
  • 15.

    Sleigh A, Hoff R, Mott K, Barreto M, de Paiva TM, Pedrosa J de S, Sherlock I, 1982. Comparison of filtration staining (Bell) and thick smear (Kato) for the detection and quantitation of Schistosoma mansoni eggs in faeces. Trans R Soc Trop Med Hyg 76: 403405.

    • Search Google Scholar
    • Export Citation
  • 16.

    WHO, 2015. Assessing the Epidemiology of Soil-Transmitted Helminths During a Transmission Assessment Survey in the Global Programme for the Elimination of Lymphatic Filariasis. Geneva, Switzerland: WHO Press.

    • Search Google Scholar
    • Export Citation
  • 17.

    van Mens SP, Aryeetey Y, Yazdanbakhsh M, van Lieshout L, Boakye D, Verweij JJ, 2013. Comparison of real-time PCR and Kato smear microscopy for the detection of hookworm infections in three consecutive faecal samples from schoolchildren in Ghana. Trans R Soc Trop Med Hyg 107: 269271.

    • Search Google Scholar
    • Export Citation
  • 18.

    Odongo-Aginya EI, Kabatereine N, Ludwig S, Wabinga H, Fenwick A, Montresor A, 2007. Substitution of malachite green with nigrosin–eosin yellow stain in the Kato-Katz method: microscopical appearance of the helminth eggs. Afr J Health Sci 7: 3336.

    • Search Google Scholar
    • Export Citation
  • 19.

    Mejia R, Vicuna Y, Broncano N, Sandoval C, Vaca M, Chico M, Cooper PJ, Nutman TB, 2013. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg 88: 10411047.

    • Search Google Scholar
    • Export Citation
  • 20.

    Arndt MB, John-Stewart G, Richardson BA, Singa B, van Lieshout L, Verweij JJ, Sangare LR, Mbogo LW, Naulikha JM, Walson JL, 2013. Impact of helminth diagnostic test performance on estimation of risk factors and outcomes in HIV-positive adults. PLoS One 8: e81915.

    • Search Google Scholar
    • Export Citation
  • 21.

    Easton AV, Oliveira RG, O'Connell EM, Kepha S, Mwandawiro CS, Njenga SM, Kihara JH, Mwatele C, Odiere MR, Brooker SJ, Webster JP, Anderson RM, Nutman TB, 2016. Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors 9: 38.

    • Search Google Scholar
    • Export Citation
  • 22.

    Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA, 2016. Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design. PLoS Negl Trop Dis 10: e0004578.

    • Search Google Scholar
    • Export Citation
  • 23.

    Knopp S, Salim N, Schindler T, Karagiannis Voules DA, Rothen J, Lweno O, Mohammed AS, Singo R, Benninghoff M, Nsojo AA, Genton B, Daubenberger C, 2014. Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania. Am J Trop Med Hyg 90: 535545.

    • Search Google Scholar
    • Export Citation
  • 24.

    Verweij JJ, Brienen EA, Ziem J, Yelifari L, Polderman AM, Van Lieshout L, 2007. Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77: 685690.

    • Search Google Scholar
    • Export Citation
  • 25.

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

    • Search Google Scholar
    • Export Citation
  • 26.

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

    • Search Google Scholar
    • Export Citation
  • 27.

    Sharifdini M, Mirhendi H, Ashrafi K, Hosseini M, Mohebali M, Khodadadi H, Kia EB, 2015. Comparison of nested polymerase chain reaction and real-time polymerase chain reaction with parasitological methods for detection of Strongyloides stercoralis in human fecal samples. Am J Trop Med Hyg 93: 12851291.

    • Search Google Scholar
    • Export Citation
  • 28.

    Moghaddassani H, Mirhendi H, Hosseini M, Rokni MB, Mowlavi GH, Kia EB, 2011. Molecular diagnosis of Strongyloides stercoralis infection by PCR detection of specific DNA in human stool samples. Iran J Parasitol 6: 2330.

    • Search Google Scholar
    • Export Citation
  • 29.

    de Gruijter JM, van Lieshout L, Gasser RB, Verweij JJ, Brienen EA, Ziem JB, Yelifari L, Polderman AM, 2005. Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana. Trop Med Int Health 10: 574580.

    • Search Google Scholar
    • Export Citation
  • 30.

    Traub RJ, Inpankaew T, Sutthikornchai C, Sukthana Y, Thompson RC, 2008. PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok. Vet Parasitol 155: 6773.

    • Search Google Scholar
    • Export Citation
  • 31.

    Gasser RB, Stewart LE, Speare R, 1996. Genetic markers in ribosomal DNA for hookworm identification. Acta Trop 62: 1521.

  • 32.

    Zhan B, Li T, Xiao S, Zheng F, Hawdon JM, 2001. Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene. J Parasitol 87: 12271229.

    • Search Google Scholar
    • Export Citation
  • 33.

    Pecson BM, Barrios JA, Johnson DR, Nelson KL, 2006. A real-time PCR method for quantifying viable Ascaris eggs using the first internally transcribed spacer region of ribosomal DNA. Appl Environ Microbiol 72: 78647872.

    • Search Google Scholar
    • Export Citation
  • 34.

    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 AJ, Verweij JJ, Sartono E, Yazdanbakhsh M, 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 Infect Dis 10: 77.

    • Search Google Scholar
    • Export Citation
  • 35.

    Loreille O, Roumat E, Verneau O, Bouchet F, Hanni C, 2001. Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites. Int J Parasitol 31: 11011106.

    • Search Google Scholar
    • Export Citation
  • 36.

    Wang JX, Pan CS, Cui LW, 2012. Application of a real-time PCR method for detecting and monitoring hookworm Necator americanus infections in southern China. Asian Pac J Trop Biomed 2: 925929.

    • Search Google Scholar
    • Export Citation
  • 37.

    Verweij JJ, Pit DS, van Lieshout L, Baeta SM, Dery GD, Gasser RB, Polderman AM, 2001. Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Trop Med Int Health 6: 726731.

    • Search Google Scholar
    • Export Citation
  • 38.

    Romstad A, Gasser RB, Monti JR, Polderman AM, Nansen P, Pit DS, Chilton NB, 1997. Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA. Mol Cell Probes 11: 169176.

    • Search Google Scholar
    • Export Citation
  • 39.

    Romstad A, Gasser RB, Nansen P, Polderman AM, Monti JR, Chilton NB, 1997. Characterization of Oesophagostomum bifurcum and Necator americanus by PCR-RFLP of rDNA. J Parasitol 83: 963966.

    • Search Google Scholar
    • Export Citation
  • 40.

    Ramachandran S, Gam AA, Neva FA, 1997. Molecular differences between several species of Strongyloides and comparison of selected isolates of S. stercoralis using a polymerase chain reaction-linked restriction fragment length polymorphism approach. Am J Trop Med Hyg 56: 6165.

    • Search Google Scholar
    • Export Citation
  • 41.

    Nissen S, Al-Jubury A, Hansen TV, Olsen A, Christensen H, Thamsborg SM, Nejsum P, 2012. Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a sympatric setting in Uganda. Vet Parasitol 188: 6877.

    • Search Google Scholar
    • Export Citation
  • 42.

    Phuphisut O, Yoonuan T, Sanguankiat S, Chaisiri K, Maipanich W, Pubampen S, Komalamisra C, Adisakwattana P, 2014. Triplex polymerase chain reaction assay for detection of major soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, in fecal samples. Southeast Asian J Trop Med Public Health 45: 267275.

    • Search Google Scholar
    • Export Citation
  • 43.

    Llewellyn S, Inpankaew T, Nery SV, Gray DJ, Verweij JJ, Clements AC, Gomes SJ, Traub R, McCarthy JS, 2016. Application of a multiplex quantitative PCR to assess prevalence and intensity of intestinal parasite infections in a controlled clinical trial. PLoS Negl Trop Dis 10: e0004380.

    • Search Google Scholar
    • Export Citation
  • 44.

    Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R, 2012. Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR. Trop Biomed 29: 434442.

    • Search Google Scholar
    • Export Citation
  • 45.

    Basuni M, Muhi J, Othman N, Verweij JJ, Ahmad M, Miswan N, Rahumatullah A, Aziz FA, Zainudin NS, Noordin R, 2011. A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. Am J Trop Med Hyg 84: 338343.

    • Search Google Scholar
    • Export Citation
  • 46.

    Yooseph S, Kirkness EF, Tran TM, Harkins DM, Jones MB, Torralba MG, O'Connell E, Nutman TB, Doumbo S, Doumbo OK, Traore B, Crompton PD, Nelson KE, 2015. Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection. BMC Genomics 16: 631.

    • Search Google Scholar
    • Export Citation
  • 47.

    Al-Soud WA, Ouis IS, Li DQ, Ljungh S, Wadstrom T, 2005. Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species. FEMS Immunol Med Microbiol 44: 177182.

    • Search Google Scholar
    • Export Citation
  • 48.

    Sato M, Sanguankiat S, Yoonuan T, Pongvongsa T, Keomoungkhoun M, Phimmayoi I, Boupa B, Moji K, Waikagul J, 2010. Copro-molecular identification of infections with hookworm eggs in rural Lao PDR. Trans R Soc Trop Med Hyg 104: 617622.

    • Search Google Scholar
    • Export Citation
  • 49.

    Repetto SA, Alba Soto CD, Cazorla SI, Tayeldin ML, Cuello S, Lasala MB, Tekiel VS, Gonzalez Cappa SM, 2013. An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples. Acta Trop 126: 110114.

    • Search Google Scholar
    • Export Citation
  • 50.

    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.

    • Search Google Scholar
    • Export Citation
  • 51.

    Saugar JM, Merino FJ, Martin-Rabadan P, Fernandez-Soto P, Ortega S, Garate T, Rodriguez E, 2015. Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain. Acta Trop 142: 2025.

    • Search Google Scholar
    • Export Citation
  • 52.

    Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, Verweij JJ, Taniuchi M, Sobuz SU, Haque R, Haverstick DM, Houpt ER, 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472480.

    • Search Google Scholar
    • Export Citation
  • 53.

    Taniuchi M, Verweij JJ, Noor Z, Sobuz SU, Lieshout L, Petri WA Jr, Haque R, Houpt ER, 2011. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. Am J Trop Med Hyg 84: 332337.

    • Search Google Scholar
    • Export Citation
  • 54.

    Harmon AF, Williams ZB, Holler LD, Hildreth MB, 2007. Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs. Vet Parasitol 145: 361365.

    • Search Google Scholar
    • Export Citation
  • 55.

    Williams RB, Thebo P, Marshall RN, Marshall JA, 2010. Coccidian oocysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA. Syst Parasitol 76: 6976.

    • Search Google Scholar
    • Export Citation
  • 56.

    Wilke H, Robertson LJ, 2009. Preservation of Giardia cysts in stool samples for subsequent PCR analysis. J Microbiol Methods 78: 292296.

    • Search Google Scholar
    • Export Citation
  • 57.

    Kuk S, Yazar S, Cetinkaya U, 2012. Stool sample storage conditions for the preservation of Giardia intestinalis DNA. Mem Inst Oswaldo Cruz 107: 965968.

    • Search Google Scholar
    • Export Citation
  • 58.

    Halstead FD, Lee AV, Couto-Parada X, Polley SD, Ling C, Jenkins C, Chalmers RM, Elwin K, Gray JJ, Iturriza-Gomara M, Wain J, Clark DA, Bolton FJ, Manuel RJ, Olympics GI Group, 2013. Universal extraction method for gastrointestinal pathogens. J Med Microbiol 62: 15351539.

    • Search Google Scholar
    • Export Citation
  • 59.

    Leles D, Araujo A, Vicente AC, Iniguez AM, 2009. Molecular diagnosis of ascariasis from human feces and description of a new Ascaris sp. genotype in Brazil. Vet Parasitol 163: 167170.

    • Search Google Scholar
    • Export Citation
  • 60.

    Harmon AF, Zarlenga DS, Hildreth MB, 2006. Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces. Vet Parasitol 135: 297302.

    • Search Google Scholar
    • Export Citation
  • 61.

    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.

    • Search Google Scholar
    • Export Citation
  • 62.

    Becker SL, Piraisoody N, Kramme S, Marti H, Silue KD, Panning M, Nickel B, Kern WV, Herrmann M, Hatz CF, N'Goran EK, Utzinger J, von Muller L, 2015. Real-time PCR for detection of Strongyloides stercoralis in human stool samples from Cote d'Ivoire: diagnostic accuracy, inter-laboratory comparison and patterns of hookworm co-infection. Acta Trop 150: 210217.

    • Search Google Scholar
    • Export Citation
  • 63.

    Cimino RO, Jeun R, Juarez M, Cajal PS, Vargas P, Echazu A, Bryan PE, Nasser J, Krolewiecki A, Mejia R, 2015. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction. Parasit Vectors 8: 380.

    • Search Google Scholar
    • Export Citation
  • 64.

    Gordon CA, McManus DP, Acosta LP, Olveda RM, Williams GM, Ross AG, Gray DJ, Gobert GN, 2015. Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines. Int J Parasitol 45: 477483.

    • Search Google Scholar
    • Export Citation
  • 65.

    Hu W, Wu S, Yu X, Abullahi AY, Song M, Tan L, Wang Z, Jiang B, Li G, 2015. A multiplex PCR for simultaneous detection of three zoonotic parasites Ancylostoma ceylanicum, A. caninum, and Giardia lamblia assemblage A. BioMed Res Int 2015: 406168.

    • Search Google Scholar
    • Export Citation
  • 66.

    Pullan RL, Smith JL, Jasrasaria R, Brooker SJ, 2014. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit Vectors 7: 37.

    • Search Google Scholar
    • Export Citation
  • 67.

    Jonker FA, Calis JC, Phiri K, Brienen EA, Khoffi H, Brabin BJ, Verweij JJ, van Hensbroek MB, van Lieshout L, 2012. Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children. PLoS Negl Trop Dis 6: e1555.

    • Search Google Scholar
    • Export Citation
  • 68.

    Getachew M, Yewhalaw D, Tafess K, Getachew Y, Zeynudin A, 2012. Anaemia and associated risk factors among pregnant women in Gilgel Gibe dam area, southwest Ethiopia. Parasit Vectors 5: 296.

    • Search Google Scholar
    • Export Citation
  • 69.

    Gyorkos TW, Gilbert NL, Larocque R, Casapia M, Montresor A, 2012. Re-visiting Trichuris trichiura intensity thresholds based on anemia during pregnancy. PLoS Negl Trop Dis 6: e1783.

    • Search Google Scholar
    • Export Citation
  • 70.

    Ngui R, Lim YA, Chong Kin L, Sek Chuen C, Jaffar S, 2012. Association between anaemia, iron deficiency anaemia, neglected parasitic infections and socioeconomic factors in rural children of west Malaysia. PLoS Negl Trop Dis 6: e1550.

    • Search Google Scholar
    • Export Citation
  • 71.

    Demeler J, Kruger N, Krucken J, von der Heyden VC, Ramunke S, Kuttler U, Miltsch S, Lopez Cepeda M, Knox M, Vercruysse J, Geldhof P, Harder A, von Samson-Himmelstjerna G, 2013. Phylogenetic characterization of β-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes. PLoS One 8: e70212.

    • Search Google Scholar
    • Export Citation
  • 72.

    Areekul P, Putaporntip C, Pattanawong U, Sitthicharoenchai P, Jongwutiwes S, 2010. Trichuris vulpis and T. trichiura infections among schoolchildren of a rural community in northwestern Thailand: the possible role of dogs in disease transmission. Asian Biomed 4: 4960.

    • Search Google Scholar
    • Export Citation
  • 73.

    Ahmad AF, Hadip F, Ngui R, Lim YA, Mahmud R, 2013. Serological and molecular detection of Strongyloides stercoralis infection among an Orang Asli community in Malaysia. Parasitol Res 112: 28112816.

    • Search Google Scholar
    • Export Citation
  • 74.

    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic local alignment search tool. J Mol Biol 215: 403410.

Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 1084 430 46
PDF Downloads 720 249 25
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Molecular Diagnostics for Soil-Transmitted Helminths

Elise M. O'ConnellLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

Search for other papers by Elise M. O'Connell in
Current site
Google Scholar
PubMed
Close
and
Thomas B. NutmanLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

Search for other papers by Thomas B. Nutman in
Current site
Google Scholar
PubMed
Close

Historically, the diagnosis of soil-transmitted helminths (STHs) (e.g., Strongyloides stercoralis, Trichuris trichiura, Ancylostoma duodenale, Necator americanus, and Ascaris lumbricoides) has relied on often-insensitive microscopy techniques. Over the past several years, there has been an effort to use molecular diagnostics, particularly quantitative polymerase chain reaction (qPCR), to detect intestinal pathogens. While some platforms have been approved by regulatory bodies (e.g., Food and Drug Administration) to detect intestinal bacteria, viruses, and protozoa, there are no approved tests currently available for STH. Although studies comparing qPCR to microscopy methods for STH are imperfect, due in large part to a lack of a sufficient gold standard, they do show a significant increase in sensitivity and specificity of qPCR compared with microscopic techniques. These studies, as well as the advantages and disadvantages of using qPCR for STH diagnosis, are discussed. Guidelines for those designing future studies utilizing qPCR are proposed for optimizing results, as is the proposition for using standardized molecular diagnostics routinely for STH in clinical laboratories and for field-based studies when possible.

Introduction

Soil-transmitted helminths (STHs) encompass a number of intestinal parasitic nematodes that are acquired either by larvae burrowing through intact skin (Strongyloides stercoralis, the hookworms Ancylostoma duodenale and related spp., and Necator americanus) or by the fecal oral route (Ascaris lumbricoides and Trichuris trichiura). As a group, STH are on the World Health Organization's (WHO's) list of 17 neglected tropical diseases1 because of the significant morbidity they cause and their propensity to be poverty promoting. Although WHO does not include S. stercoralis on its formal list of STH, we include it here because it is highly prevalent and can be a significant cause of morbidity and mortality2 in low-, middle- and high-income countries alike.

Infections with STHs are often clinically asymptomatic, but they can be associated with eosinophilia and/or prolonged gastrointestinal symptoms, which most often occur in returning travelers35 and immigrants.68 These infections are often underdiagnosed given the decreasing number of well-trained personnel with the competence in identifying eggs and/or larvae in traditional stool-based microscopic methods and the intermittent shedding of eggs and/or larvae by some of these parasites (e.g., Strongyloides6,9,10).

Over the past 20 years, there has been an increasing effort to use molecular diagnostics for STH in epidemiologic studies and for the diagnosis of individual patients in some high-income countries.11,12 With the recent Food and Drug Administration (FDA) approval of several molecular diagnostic tools for stool bacterial and a few protozoa pathogens,13,14 the use of twenty-first century technology for the detection of intestinal pathogens has finally begun. However, there is no such FDA-approved molecular platform for diagnosing gastrointestinal helminth infections. In this review, we summarize the work that has been done in this growing field with specific attention paid to the strengths and limitations of using molecular diagnostics (particularly quantitative polymerase chain reaction [qPCR] platforms) in detecting STH.

An Inadequate Gold Standard

Part of the difficulty in determining the sensitivity and specificity of qPCR and other molecular-based diagnostics is the lack of a sufficient gold standard against which to compare these newer techniques, given the general insensitivity of stool-based microscopic methods commonly in use. For example, the Kato-Katz (KK) technique was initially developed to detect Schistosoma spp. eggs,15 but is currently the most commonly used technique in STH surveys.16 It is particularly problematic in accurately detecting hookworm infections as the stool must be prepared immediately,17 and the clarification step (i.e., glycerin) can make the eggs unrecognizable.18 Interestingly, it is partly due to the lack of sensitivity of KK in identifying the larvae of S. stercoralis that has informed the decision by the WHO to not include this pathogen as an STH.16

Some studies have attempted to deal with this (lack of sufficient gold standard) problem by considering true positives to be the sum of the positives found by microscopy and/or PCR.1922 Others have used several different microscopic detection methods with or without the use of statistical modeling to project the true prevalence, sensitivity, and specificity for any given method.23 Regardless of the method used, with rare exception, studies comparing microscopy to molecular methods (primarily qPCR) to diagnose STH have found markedly increased sensitivities with molecular diagnostics (Table 1). Moreover, because many of the molecular diagnostic techniques are multiplexed24,42,43 or multi-parallel,19,21,22 studies have also shown an increased ability to detect multiple concurrent infections using qPCR when compared with microscopy.19,21,4345

Table 1

Targets and unique primer sequences reported in the literature for PCR for STH*

Organism Target region Citations of unique primer sequences
Strongyloides stercoralis ITS-1 25
18S 26
Cytochrome oxidase 1 26,27
Sequence repeats 22,26
ITS-1, 5.8S, ITS-2 28
Ancylostoma duodenale/ceylanicum ITS-2 24,29
ITS-1, 5.8S, ITS-2 30,31
A. duodenale Cytochrome oxidase 1 32
Sequence repeats 22
Ascaris lumbricoides ITS-1 22,33,34
Cytochrome oxidase 1 35
Necator americanus ITS-2 24,3639
ITS-1, 5.8S, ITS-2 30,31,39,40
Cytochrome oxidase 1 32
Sequence repeats 22
Trichuris trichiura ITS-1 19
ITS-2 41
Sequence repeats 22

ITS = internal transcribed spacer; qPCR = quantitative polymerase chain reaction; STH = soil-transmitted helminth.

Included are studies utilizing conventional, nested, qPCR.

Applications of qPCR as an STH Diagnostic

The WHO has set a worldwide goal to eliminate childhood morbidity caused by STH by 2020. Dictating which communities receive anthelmintic drugs, how often they receive them, and when community treatment is stopped is based on population prevalence rates determined by a small community sampling.16 Therefore, the very sensitive nature of qPCR (even post-treatment where eggs are often no longer visible), the improved ability to detect multiple helminths in a given sample and the ability to reproducibly quantitate egg burden, would be a huge asset to control programs.

Research studies are already utilizing qPCR for mass screening of stool samples for STH in malaria46 and in vaccine studies (currently underway) as stool can be stored relatively easily (see about preservatives in the section Technical Considerations in Using qPCR as an STH diagnostic) and sent offsite for qPCR not only for STH but also for protozoa and other gastrointestinal pathogens.

One of the most common criticisms of qPCR is the high cost compared with traditional methods. In one price comparison, a multiplex platform was estimated to cost about the same in consumables as the cost of microscopy,45 and consumable cost for multi-parallel singleplex qPCR of one group was estimated to be almost half the cost of microscopy19 on a per test basis. Thus, depending on the technique used, cost may not be a prohibitive factor.

Technical Considerations in Using qPCR as an STH Diagnostic

Despite the sensitive, rapid, and quantitative nature of qPCR for the diagnosis of STH, there are important methodological considerations in test design and results interpretation. Stool contains bile acids and other substances that inhibit the PCR product amplification.47 Early on in the use of molecular diagnostics for fecal pathogens, there were significant sensitivity problems when DNA extraction techniques that were not specifically designed to remove these inhibitors48 were used. PCR inhibitors are largely removed without problem using 1) more recent in-house developed protocols,27,49 2) tissue kits with additional inhibitor removal steps,24,50 and 3) stool and soil-engineered kits for DNA extraction.19,21,43,5053

A more clinically validated study will also amplify an internal control for each specimen to ensure efficient PCR inhibitor removal and to eliminate the possibility of false negatives in the molecular diagnostic results.21,24,43,49,50,52

Sensitivity can also be decreased by formalin fixation of the stool prior to DNA extraction.54 While specific stool preservation methods have not been directly compared in helminth detection through qPCR, in studies assessing the impact of different preservative measures on protozoal DNA amplification,5557 it has been shown that samples stored in potassium dichromate can be stored at room temperature for prolonged periods before qPCR without any significant loss in sensitivity.

Unlike bacteria, which have cell walls that are easy to lyse, some physical stress is required to optimize the release of the nucleic acids from helminth eggs or larvae (and even some protozoa58). While freeze-thaw cycles, heating, and/or sonication do appear to offer an advantage over standard lysis buffer methods,49,59 the use of a tissue homogenization (“bead beating”) step with beads resistant to degradation (i.e., not glass) likely offers the most thorough disruption of parasite ova, thereby increasing molecular-based assay sensitivities significantly.60 The amount of stool extracted, the type of physical disruption method used, as well as the relative efficiency of commercial kits in extracting DNA50,60 likely explain much of the variability in sensitivity in published studies (see Table 1).

When interpreting molecular diagnostic results, it is also important to know the DNA sequence being amplified (see Table 2) and the limitations inherent in testing for a widely conserved sequence compared with sequence(s) that are species specific. In terms of qPCR platforms, singleplex (often using a multi-parallel approach19,21,22,52,63) offers slightly more sensitivity compared with multiplexed assays24,42,44,53,64,65(in which reagents in a given tube/well may be limiting). However, it may be possible to optimize a multiplex format to be equally sensitive to a singleplex approach.43 Multiplexed assays do, however, require more sophisticated and expensive equipment and labeled probes that may be less universally available (and more costly).

Table 2

Reported sensitivities of qPCR for diagnosing STH compared with reported stool microscopy sensitivities*

Organism Sensitivities reported by qPCR Sensitivities by microscopy
Ascaris lumbricoides 85.7%,20 98%,**21 100%52 71.4%,20 70,** 21 88%52
Hookworms 96.9%,20 78.9%,61 75.7–83.3%,23 98%,21 100%36 31.3%,20 79.2–88.8%,23 32%**21
Strongyloides stercoralis 76%,62 83.3%,20 83.3–88.9%,§61 11.6%,23 93.8%,‡‡51 86.4%,§26 84.7%††27 16.7%,20 50%,62 28.3%23
Trichuris trichiura 100%52 88%52

qPCR = quantitative polymerase chain reaction; STH = soil-transmitted helminth.

Unless otherwise stated, gold standard in determining true positives was the sum of positives by qPCR and microscopy method used. Study specific microscopy methods (if described) are listed below.

Single stool sample prepared with a combination of Kato-Katz, wet preparation, and formol-ether concentration methods.

Single stool sample subjected to Baermann funnel concentration and Koga agar plate culture.

Sensitivity excludes those positive by Koga agar and negative by Baermann funnel. See text for details.

No internal controls used to rule out PCR inhibition in this study.

Mathematical modeling to determine gold standard taking into account results of single stool subjected to FLOTAC, Kato-Katz, Baermann, and qPCR.

Single stool sample prepared by Kato-Katz, with duplicate slides assessed by two technicians.

Gold standard a combination of nested PCR, Koga agar plate culture, and formalin ether concentration.

Single stool sample subjected to formalin-ethyl acetate concentration, agar plate culture, and Harada Mori technique.

qPCR in the Detection of Each of the Major STH

Ascaris lumbricoides.

At an estimated prevalence rate of 819 million infections worldwide, A. lumbricoides is by far the most common STH.66 Several large studies have shown a clear relationship between stool DNA concentration as determined by qPCR and egg counts.19,21,43 Interestingly, there is also a good correlation between DNA quantification in the stool (presumably reflecting egg DNA) and the number of expelled adult worms after albendazole treatment.21 The evaluation of the existing data on A. lumbricoides across multiple studies (Table 1) has been aided enormously by the fact that all of the studies have targeted the internal transcribed spacer 1 region (see Table 2). In addition, of all the STH, KK identifies A. lumbricoides the most easily. More reliable reproducibility likely explains the relatively small gap (still in favor of the molecular diagnostics) in sensitivities between KK and qPCR for Ascaris (Table 1).

Hookworms.

Necator americanus and A. duodenale are classically the two species of hookworm considered to be the most prevalent and clinically relevant worldwide. However, in certain regions of the world, Ancylostoma ceylanicum30 and Oesophagostomum bifurcum24 are very prevalent intestinal parasites, both of which are indistinguishable from N. americanus and A. duodenale using standard microscopic diagnostic methods. Thus, in areas where molecular testing has yet to characterize the exact species of infecting hookworms, the presumed predominant hookworm species may not always be present. This inability to accurately distinguish among the hookworms species morphologically has led to some confusion in that highly species-specific primer/probe combinations have led to some “false negatives” when comparing stool microscopy to qPCR.17,61 Indeed, egg-spiking experiments have determined that qPCR can detect a single hookworm egg in 200 mg of stool36 suggesting qPCR is extraordinarily sensitive, but the eggs seen by microscopy may not be the species that the primer/probe set was designed to detect.

Under optimal microscopic conditions, stool egg counts, stool larval counts, and clinical outcomes have been highly correlated with qPCR cycle times.21,23,24,43 For example, a study on hookworm infection in Malawian children found a significant interrelationship between the burden of hookworms as determined by qPCR, particularly A. duodenale, and the severity of iron deficiency and anemia.67

Trichuris trichiura.

Trichuris infections in some populations are associated with iron deficiency anemia.6870 The ova of Trichuris spp. are notorious for being more difficult to break open in the DNA extraction process than any other STH. Indeed, it is clear that a tissue homogenization step is imperative to achieve egg disruption.71 This parasite is the least well studied in the context of molecularly based diagnosis. However in the few studies where T. trichiura infection has been assessed,19,22,52 homogenization with or without a heating step has shown to yield highly sensitive qPCR results (Table 1), in one case predicting the limit of detection to be a single egg.22 Egg counts have also been shown to correlate highly when comparing microscopy to qPCR.19 Similar to hookworm, a number of Trichuris species besides T. trichiura are increasingly being recognized as having the potential to be a human pathogen.72 In cases where sequences highly specific for T. trichiura are targeted in qPCR, other related species (e.g., Trichuris ovis) may be missed.22

Strongyloides stercoralis.

Strongyloidiasis, caused by S. stercoralis, is problematic due to the prolonged period maintained by this infection, as well as the potential for accelerated autoinfection with immune suppression (most often associated with corticosteroid use or human T-lymphotropic virus type 1 infection). Of all the intestinal helminths, it is certainly the one most difficult to diagnose through stool microscopy. This is due to the intermittent nature of larval shedding in the stool, as well as the relatively low numbers of larvae found in the stool (with the exception during accelerated autoinfection).9

Of the DNA sequences targeted (see Table 2), amplification of the 18S small subunit (Genbank accession number AY029262) has been proven to be more sensitive than cytochrome c oxidase 1 or the dispersed repeat sequence,26 and has been one of the most (if not the most) used sequence in qPCR for S. stercoralis.

One of the easiest and most sensitive methods for detecting infection with S. stercoralis is through the Koga agar plate culture, and so is often the method to which qPCR is compared. However, this test also can give a positive result in the setting of hookworm infection and can contribute to perceived false negatives and an overall low sensitivity seen in some studies using qPCR.26,61,62 However, in many epidemiologic surveys, qPCR has typically increased overall diagnostic yield by 2- to 8-fold when compared with a variety of microscopic techniques.12,19,27,45,51,63,73 Unlike the other STH, however, the quantification of larvae in the stool has not been shown to predict the adult worm burden, and so qPCR would not be used as a reflection of adult worm burden in a clinical setting.

Proposed Guidelines in Using qPCR to Diagnose STH in Research Studies

Given the variability in sensitivity and specificity of qPCR found in previous studies, we propose some guidelines for use in designing trials comparing qPCR with microscopy results. First, DNA extraction should be performed using a commercial stool or soil DNA extraction kit, or published protocols validated for use in stool-based PCR. A physical disruption step early in DNA extraction (use of a tissue homogenizer with ceramic or zirconia/silica beads, particularly if attempting to detect Trichuris spp.) should be used. To exclude the possibility of false-negative results due to PCR inhibitors, samples should be spiked with an internal control and amplified along with target sequences of interest. Only validated primer/probe sequences should be used for a particular target. Alternatively, one can validate new sequences through Basic Local Alignment Search Tool74 searches. Once identified, the primers/probes derived from these new targets can be tested using genomic DNA from the species of interest and from potentially cross-reactive organisms to determine specificity. In performing stool PCR surveys, one should ideally know the endemic hookworm and Trichuris species in the population to be tested based on previous molecular testing/sequencing; if unavailable it might be important to include a pilot discovery phase to determine the actual species of infecting parasitic helminth found in the stool.

Conclusion

The highly sensitive, rapid, and scalable nature of qPCR makes its utilization in diagnosing STH extremely appealing over insensitive and labor-intensive traditional microscopic methods. We have suggested here its superiority over microscopy methods (particularly KK) in sensitivity while still providing a quantitative measure of infection intensity. There are, however, important technical lessons that have been learned that provide a framework to maximize the utility of this potentially valuable molecular approach, which we have highlighted here. Until now molecular detection of STH has been restricted to the research setting. Given the neglected nature of STH and the large cost of the regulatory processes, moving forward in developing a FDA- or European Union-approved platform would require significant political will and/or philanthropic efforts. Nevertheless, given that surveillance and monitoring for many other pathogens are being done using standardized (but not commercialized) molecular techniques, we would argue that use of qPCR in detecting STH would likely provide a more accurate and cost-effective approach to the WHO STH elimination strategy and should be considered seriously.

  • 1.

    WHO, 2015. Investing to Overcome the Global Impact of Neglected Tropical Diseases: Third WHO Report on Neglected Tropical Diseases 2015. Holmes P, ed. Geneva, Switzerland: World Health Organization, 161167.

    • Search Google Scholar
    • Export Citation
  • 2.

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

  • 3.

    Gill GV, Welch E, Bailey JW, Bell DR, Beeching NJ, 2004. Chronic Strongyloides stercoralis infection in former British Far East prisoners of war. QJM 97: 789795.

    • Search Google Scholar
    • Export Citation
  • 4.

    Schulte C, Krebs B, Jelinek T, Nothdurft HD, von Sonnenburg F, Loscher T, 2002. Diagnostic significance of blood eosinophilia in returning travelers. Clin Infect Dis 34: 407411.

    • Search Google Scholar
    • Export Citation
  • 5.

    Naidu P, Yanow SK, Kowalewska-Grochowska KT, 2013. Eosinophilia: a poor predictor of Strongyloides infection in refugees. Can J Infect Dis Med Microbiol 24: 9396.

    • Search Google Scholar
    • Export Citation
  • 6.

    Calderaro A, Montecchini S, Rossi S, Gorrini C, De Conto F, Medici MC, Chezzi C, Arcangeletti MC, 2014. Intestinal parasitoses in a tertiary-care hospital located in a non-endemic setting during 2006–2010. BMC Infect Dis 14: 264.

    • Search Google Scholar
    • Export Citation
  • 7.

    Becker SL, Sieto B, Silue KD, Adjossan L, Kone S, Hatz C, Kern WV, N'Goran EK, Utzinger J, 2011. Diagnosis, clinical features, and self-reported morbidity of Strongyloides stercoralis and hookworm infection in a co-endemic setting. PLoS Negl Trop Dis 5: e1292.

    • Search Google Scholar
    • Export Citation
  • 8.

    O'Brien DP, Leder K, Matchett E, Brown GV, Torresi J, 2006. Illness in returned travelers and immigrants/refugees: the 6-year experience of two Australian infectious diseases units. J Travel Med 13: 145152.

    • Search Google Scholar
    • Export Citation
  • 9.

    Schar F, Hattendorf J, Khieu V, Muth S, Char MC, Marti HP, Odermatt P, 2014. Strongyloides stercoralis larvae excretion patterns before and after treatment. Parasitology 141: 892897.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ramanathan R, Burbelo P, Groot S, Iadarola M, Neva F, Nutman T, 2008. A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection. J Infect Dis 198: 444451.

    • Search Google Scholar
    • Export Citation
  • 11.

    Soonawala D, van Lieshout L, den Boer MA, Claas EC, Verweij JJ, Godkewitsch A, Ratering M, Visser LG, 2014. Post-travel screening of asymptomatic long-term travelers to the tropics for intestinal parasites using molecular diagnostics. Am J Trop Med Hyg 90: 835839.

    • Search Google Scholar
    • Export Citation
  • 12.

    ten Hove RJ, van Esbroeck M, Vervoort T, van den Ende J, van Lieshout L, Verweij JJ, 2009. Molecular diagnostics of intestinal parasites in returning travellers. Eur J Clin Microbiol Infect Dis 28: 10451053.

    • Search Google Scholar
    • Export Citation
  • 13.

    Buss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK, Rogatcheva M, Kanack KJ, Bourzac KM, 2015. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol 53: 915925.

    • Search Google Scholar
    • Export Citation
  • 14.

    Reddington K, Tuite N, Minogue E, Barry T, 2014. A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens. Biomol Detect Quantif 1: 37.

    • Search Google Scholar
    • Export Citation
  • 15.

    Sleigh A, Hoff R, Mott K, Barreto M, de Paiva TM, Pedrosa J de S, Sherlock I, 1982. Comparison of filtration staining (Bell) and thick smear (Kato) for the detection and quantitation of Schistosoma mansoni eggs in faeces. Trans R Soc Trop Med Hyg 76: 403405.

    • Search Google Scholar
    • Export Citation
  • 16.

    WHO, 2015. Assessing the Epidemiology of Soil-Transmitted Helminths During a Transmission Assessment Survey in the Global Programme for the Elimination of Lymphatic Filariasis. Geneva, Switzerland: WHO Press.

    • Search Google Scholar
    • Export Citation
  • 17.

    van Mens SP, Aryeetey Y, Yazdanbakhsh M, van Lieshout L, Boakye D, Verweij JJ, 2013. Comparison of real-time PCR and Kato smear microscopy for the detection of hookworm infections in three consecutive faecal samples from schoolchildren in Ghana. Trans R Soc Trop Med Hyg 107: 269271.

    • Search Google Scholar
    • Export Citation
  • 18.

    Odongo-Aginya EI, Kabatereine N, Ludwig S, Wabinga H, Fenwick A, Montresor A, 2007. Substitution of malachite green with nigrosin–eosin yellow stain in the Kato-Katz method: microscopical appearance of the helminth eggs. Afr J Health Sci 7: 3336.

    • Search Google Scholar
    • Export Citation
  • 19.

    Mejia R, Vicuna Y, Broncano N, Sandoval C, Vaca M, Chico M, Cooper PJ, Nutman TB, 2013. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg 88: 10411047.

    • Search Google Scholar
    • Export Citation
  • 20.

    Arndt MB, John-Stewart G, Richardson BA, Singa B, van Lieshout L, Verweij JJ, Sangare LR, Mbogo LW, Naulikha JM, Walson JL, 2013. Impact of helminth diagnostic test performance on estimation of risk factors and outcomes in HIV-positive adults. PLoS One 8: e81915.

    • Search Google Scholar
    • Export Citation
  • 21.

    Easton AV, Oliveira RG, O'Connell EM, Kepha S, Mwandawiro CS, Njenga SM, Kihara JH, Mwatele C, Odiere MR, Brooker SJ, Webster JP, Anderson RM, Nutman TB, 2016. Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors 9: 38.

    • Search Google Scholar
    • Export Citation
  • 22.

    Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA, 2016. Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design. PLoS Negl Trop Dis 10: e0004578.

    • Search Google Scholar
    • Export Citation
  • 23.

    Knopp S, Salim N, Schindler T, Karagiannis Voules DA, Rothen J, Lweno O, Mohammed AS, Singo R, Benninghoff M, Nsojo AA, Genton B, Daubenberger C, 2014. Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania. Am J Trop Med Hyg 90: 535545.

    • Search Google Scholar
    • Export Citation
  • 24.

    Verweij JJ, Brienen EA, Ziem J, Yelifari L, Polderman AM, Van Lieshout L, 2007. Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77: 685690.

    • Search Google Scholar
    • Export Citation
  • 25.

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

    • Search Google Scholar
    • Export Citation
  • 26.

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

    • Search Google Scholar
    • Export Citation
  • 27.

    Sharifdini M, Mirhendi H, Ashrafi K, Hosseini M, Mohebali M, Khodadadi H, Kia EB, 2015. Comparison of nested polymerase chain reaction and real-time polymerase chain reaction with parasitological methods for detection of Strongyloides stercoralis in human fecal samples. Am J Trop Med Hyg 93: 12851291.

    • Search Google Scholar
    • Export Citation
  • 28.

    Moghaddassani H, Mirhendi H, Hosseini M, Rokni MB, Mowlavi GH, Kia EB, 2011. Molecular diagnosis of Strongyloides stercoralis infection by PCR detection of specific DNA in human stool samples. Iran J Parasitol 6: 2330.

    • Search Google Scholar
    • Export Citation
  • 29.

    de Gruijter JM, van Lieshout L, Gasser RB, Verweij JJ, Brienen EA, Ziem JB, Yelifari L, Polderman AM, 2005. Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana. Trop Med Int Health 10: 574580.

    • Search Google Scholar
    • Export Citation
  • 30.

    Traub RJ, Inpankaew T, Sutthikornchai C, Sukthana Y, Thompson RC, 2008. PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok. Vet Parasitol 155: 6773.

    • Search Google Scholar
    • Export Citation
  • 31.

    Gasser RB, Stewart LE, Speare R, 1996. Genetic markers in ribosomal DNA for hookworm identification. Acta Trop 62: 1521.

  • 32.

    Zhan B, Li T, Xiao S, Zheng F, Hawdon JM, 2001. Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene. J Parasitol 87: 12271229.

    • Search Google Scholar
    • Export Citation
  • 33.

    Pecson BM, Barrios JA, Johnson DR, Nelson KL, 2006. A real-time PCR method for quantifying viable Ascaris eggs using the first internally transcribed spacer region of ribosomal DNA. Appl Environ Microbiol 72: 78647872.

    • Search Google Scholar
    • Export Citation
  • 34.

    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 AJ, Verweij JJ, Sartono E, Yazdanbakhsh M, 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 Infect Dis 10: 77.

    • Search Google Scholar
    • Export Citation
  • 35.

    Loreille O, Roumat E, Verneau O, Bouchet F, Hanni C, 2001. Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites. Int J Parasitol 31: 11011106.

    • Search Google Scholar
    • Export Citation
  • 36.

    Wang JX, Pan CS, Cui LW, 2012. Application of a real-time PCR method for detecting and monitoring hookworm Necator americanus infections in southern China. Asian Pac J Trop Biomed 2: 925929.

    • Search Google Scholar
    • Export Citation
  • 37.

    Verweij JJ, Pit DS, van Lieshout L, Baeta SM, Dery GD, Gasser RB, Polderman AM, 2001. Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Trop Med Int Health 6: 726731.

    • Search Google Scholar
    • Export Citation
  • 38.

    Romstad A, Gasser RB, Monti JR, Polderman AM, Nansen P, Pit DS, Chilton NB, 1997. Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA. Mol Cell Probes 11: 169176.

    • Search Google Scholar
    • Export Citation
  • 39.

    Romstad A, Gasser RB, Nansen P, Polderman AM, Monti JR, Chilton NB, 1997. Characterization of Oesophagostomum bifurcum and Necator americanus by PCR-RFLP of rDNA. J Parasitol 83: 963966.

    • Search Google Scholar
    • Export Citation
  • 40.

    Ramachandran S, Gam AA, Neva FA, 1997. Molecular differences between several species of Strongyloides and comparison of selected isolates of S. stercoralis using a polymerase chain reaction-linked restriction fragment length polymorphism approach. Am J Trop Med Hyg 56: 6165.

    • Search Google Scholar
    • Export Citation
  • 41.

    Nissen S, Al-Jubury A, Hansen TV, Olsen A, Christensen H, Thamsborg SM, Nejsum P, 2012. Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a sympatric setting in Uganda. Vet Parasitol 188: 6877.

    • Search Google Scholar
    • Export Citation
  • 42.

    Phuphisut O, Yoonuan T, Sanguankiat S, Chaisiri K, Maipanich W, Pubampen S, Komalamisra C, Adisakwattana P, 2014. Triplex polymerase chain reaction assay for detection of major soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, in fecal samples. Southeast Asian J Trop Med Public Health 45: 267275.

    • Search Google Scholar
    • Export Citation
  • 43.

    Llewellyn S, Inpankaew T, Nery SV, Gray DJ, Verweij JJ, Clements AC, Gomes SJ, Traub R, McCarthy JS, 2016. Application of a multiplex quantitative PCR to assess prevalence and intensity of intestinal parasite infections in a controlled clinical trial. PLoS Negl Trop Dis 10: e0004380.

    • Search Google Scholar
    • Export Citation
  • 44.

    Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R, 2012. Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR. Trop Biomed 29: 434442.

    • Search Google Scholar
    • Export Citation
  • 45.

    Basuni M, Muhi J, Othman N, Verweij JJ, Ahmad M, Miswan N, Rahumatullah A, Aziz FA, Zainudin NS, Noordin R, 2011. A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. Am J Trop Med Hyg 84: 338343.

    • Search Google Scholar
    • Export Citation
  • 46.

    Yooseph S, Kirkness EF, Tran TM, Harkins DM, Jones MB, Torralba MG, O'Connell E, Nutman TB, Doumbo S, Doumbo OK, Traore B, Crompton PD, Nelson KE, 2015. Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection. BMC Genomics 16: 631.

    • Search Google Scholar
    • Export Citation
  • 47.

    Al-Soud WA, Ouis IS, Li DQ, Ljungh S, Wadstrom T, 2005. Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species. FEMS Immunol Med Microbiol 44: 177182.

    • Search Google Scholar
    • Export Citation
  • 48.

    Sato M, Sanguankiat S, Yoonuan T, Pongvongsa T, Keomoungkhoun M, Phimmayoi I, Boupa B, Moji K, Waikagul J, 2010. Copro-molecular identification of infections with hookworm eggs in rural Lao PDR. Trans R Soc Trop Med Hyg 104: 617622.

    • Search Google Scholar
    • Export Citation
  • 49.

    Repetto SA, Alba Soto CD, Cazorla SI, Tayeldin ML, Cuello S, Lasala MB, Tekiel VS, Gonzalez Cappa SM, 2013. An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples. Acta Trop 126: 110114.

    • Search Google Scholar
    • Export Citation
  • 50.

    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.

    • Search Google Scholar
    • Export Citation
  • 51.

    Saugar JM, Merino FJ, Martin-Rabadan P, Fernandez-Soto P, Ortega S, Garate T, Rodriguez E, 2015. Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain. Acta Trop 142: 2025.

    • Search Google Scholar
    • Export Citation
  • 52.

    Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, Verweij JJ, Taniuchi M, Sobuz SU, Haque R, Haverstick DM, Houpt ER, 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472480.

    • Search Google Scholar
    • Export Citation
  • 53.

    Taniuchi M, Verweij JJ, Noor Z, Sobuz SU, Lieshout L, Petri WA Jr, Haque R, Houpt ER, 2011. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. Am J Trop Med Hyg 84: 332337.

    • Search Google Scholar
    • Export Citation
  • 54.

    Harmon AF, Williams ZB, Holler LD, Hildreth MB, 2007. Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs. Vet Parasitol 145: 361365.

    • Search Google Scholar
    • Export Citation
  • 55.

    Williams RB, Thebo P, Marshall RN, Marshall JA, 2010. Coccidian oocysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA. Syst Parasitol 76: 6976.

    • Search Google Scholar
    • Export Citation
  • 56.

    Wilke H, Robertson LJ, 2009. Preservation of Giardia cysts in stool samples for subsequent PCR analysis. J Microbiol Methods 78: 292296.

    • Search Google Scholar
    • Export Citation
  • 57.

    Kuk S, Yazar S, Cetinkaya U, 2012. Stool sample storage conditions for the preservation of Giardia intestinalis DNA. Mem Inst Oswaldo Cruz 107: 965968.

    • Search Google Scholar
    • Export Citation
  • 58.

    Halstead FD, Lee AV, Couto-Parada X, Polley SD, Ling C, Jenkins C, Chalmers RM, Elwin K, Gray JJ, Iturriza-Gomara M, Wain J, Clark DA, Bolton FJ, Manuel RJ, Olympics GI Group, 2013. Universal extraction method for gastrointestinal pathogens. J Med Microbiol 62: 15351539.

    • Search Google Scholar
    • Export Citation
  • 59.

    Leles D, Araujo A, Vicente AC, Iniguez AM, 2009. Molecular diagnosis of ascariasis from human feces and description of a new Ascaris sp. genotype in Brazil. Vet Parasitol 163: 167170.

    • Search Google Scholar
    • Export Citation
  • 60.

    Harmon AF, Zarlenga DS, Hildreth MB, 2006. Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces. Vet Parasitol 135: 297302.

    • Search Google Scholar
    • Export Citation
  • 61.

    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.

    • Search Google Scholar
    • Export Citation
  • 62.

    Becker SL, Piraisoody N, Kramme S, Marti H, Silue KD, Panning M, Nickel B, Kern WV, Herrmann M, Hatz CF, N'Goran EK, Utzinger J, von Muller L, 2015. Real-time PCR for detection of Strongyloides stercoralis in human stool samples from Cote d'Ivoire: diagnostic accuracy, inter-laboratory comparison and patterns of hookworm co-infection. Acta Trop 150: 210217.

    • Search Google Scholar
    • Export Citation
  • 63.

    Cimino RO, Jeun R, Juarez M, Cajal PS, Vargas P, Echazu A, Bryan PE, Nasser J, Krolewiecki A, Mejia R, 2015. Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction. Parasit Vectors 8: 380.

    • Search Google Scholar
    • Export Citation
  • 64.

    Gordon CA, McManus DP, Acosta LP, Olveda RM, Williams GM, Ross AG, Gray DJ, Gobert GN, 2015. Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines. Int J Parasitol 45: 477483.

    • Search Google Scholar
    • Export Citation
  • 65.

    Hu W, Wu S, Yu X, Abullahi AY, Song M, Tan L, Wang Z, Jiang B, Li G, 2015. A multiplex PCR for simultaneous detection of three zoonotic parasites Ancylostoma ceylanicum, A. caninum, and Giardia lamblia assemblage A. BioMed Res Int 2015: 406168.

    • Search Google Scholar
    • Export Citation
  • 66.

    Pullan RL, Smith JL, Jasrasaria R, Brooker SJ, 2014. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit Vectors 7: 37.

    • Search Google Scholar
    • Export Citation
  • 67.

    Jonker FA, Calis JC, Phiri K, Brienen EA, Khoffi H, Brabin BJ, Verweij JJ, van Hensbroek MB, van Lieshout L, 2012. Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children. PLoS Negl Trop Dis 6: e1555.

    • Search Google Scholar
    • Export Citation
  • 68.

    Getachew M, Yewhalaw D, Tafess K, Getachew Y, Zeynudin A, 2012. Anaemia and associated risk factors among pregnant women in Gilgel Gibe dam area, southwest Ethiopia. Parasit Vectors 5: 296.

    • Search Google Scholar
    • Export Citation
  • 69.

    Gyorkos TW, Gilbert NL, Larocque R, Casapia M, Montresor A, 2012. Re-visiting Trichuris trichiura intensity thresholds based on anemia during pregnancy. PLoS Negl Trop Dis 6: e1783.

    • Search Google Scholar
    • Export Citation
  • 70.

    Ngui R, Lim YA, Chong Kin L, Sek Chuen C, Jaffar S, 2012. Association between anaemia, iron deficiency anaemia, neglected parasitic infections and socioeconomic factors in rural children of west Malaysia. PLoS Negl Trop Dis 6: e1550.

    • Search Google Scholar
    • Export Citation
  • 71.

    Demeler J, Kruger N, Krucken J, von der Heyden VC, Ramunke S, Kuttler U, Miltsch S, Lopez Cepeda M, Knox M, Vercruysse J, Geldhof P, Harder A, von Samson-Himmelstjerna G, 2013. Phylogenetic characterization of β-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes. PLoS One 8: e70212.

    • Search Google Scholar
    • Export Citation
  • 72.

    Areekul P, Putaporntip C, Pattanawong U, Sitthicharoenchai P, Jongwutiwes S, 2010. Trichuris vulpis and T. trichiura infections among schoolchildren of a rural community in northwestern Thailand: the possible role of dogs in disease transmission. Asian Biomed 4: 4960.

    • Search Google Scholar
    • Export Citation
  • 73.

    Ahmad AF, Hadip F, Ngui R, Lim YA, Mahmud R, 2013. Serological and molecular detection of Strongyloides stercoralis infection among an Orang Asli community in Malaysia. Parasitol Res 112: 28112816.

    • Search Google Scholar
    • Export Citation
  • 74.

    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic local alignment search tool. J Mol Biol 215: 403410.

Author Notes

* Address correspondence to Elise M. O'Connell, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4/Room B1-05, 4 Center Drive, Bethesda, MD 20892. E-mail: oconnellem@niaid.nih.gov

Financial support: This work was supported by the Division of Intramural Research (DIR) of the National Institute of Allergy and Infectious Diseases.

Authors' addresses: Elise M. O'Connell and Thomas B. Nutman Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, E-mails: oconnellem@niaid.nih.gov and tnutman@niaid.nih.gov.

Save