Abstract
This protocol is an extension to: Nat. Protoc. 5, 503–515 (2010); doi: 10.1038/nprot.2009.235; published online 25 February 2010
The FLOTAC is a sensitive, accurate, and precise technique for the diagnosis of protozoan and helminth infections in humans and animals. However, it requires centrifugation, and hence might be out of reach in resource-constrained settings. As an extension of the original FLOTAC protocol, this protocol describes the Mini-FLOTAC technique, a logical evolution of FLOTAC conceived to perform multivalent, qualitative, and quantitative diagnosis of helminth and protozoan infections in human and animal feces, and urine. This has been found to be of most use in the processing of large numbers of samples with rapid laboratory workup, and for veterinary applications directly on-farm. In addition to the Mini-FLOTAC apparatus, we describe the use of the Fill-FLOTAC, a closed system used to facilitate the performance of the first four consecutive steps of the Mini-FLOTAC technique: fecal sample collection and weighing, homogenization, filtration, and filling of the Mini-FLOTAC chambers. Processing of an individual sample using this protocol requires ∼12 min.
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References
Pedrique, B. et al. The drug and vaccine landscape for neglected diseases (2010-2011): a systematic assessment. Lancet Glob. Health 1, e371–e379 (2013).
Moran, M. et al. Neglected disease research and development: how much are we really spending? PLoS Med. 6, e1000030 (2009).
Bergquist, R., Johansen, M.V. & Utzinger, J. Diagnostic dilemmas in helminthology: what tools to use and when? Trends Parasitol. 25, 151–156 (2009).
Cringoli, G., Rinaldi, L., Maurelli, M.P. & Utzinger, J. FLOTAC: new multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat. Protoc. 5, 503–515 (2010).
Utzinger, J., Becker, S.L., van Lieshout, L., van Dam, G.J. & Knopp, S. New diagnostic tools in schistosomiasis. Clin. Microbiol. Infect. 21, 529–542 (2015).
Knopp, S. et al. A single FLOTAC is more sensitive than triplicate Kato-Katz for the diagnosis of low-intensity soil-transmitted helminth infections. Trans. R. Soc. Trop. Med. Hyg. 103, 347–354 (2009).
El-Abdellati, A. et al. The use of a simplified faecal egg count reduction test for assessing anthelmintic efficacy on Belgian and German cattle farms. Vet. Parasitol. 169, 352–357 (2010).
Becker, S.L. et al. Comparison of the Flotac-400 dual technique and the formalin-ether concentration technique for diagnosis of human intestinal protozoon infection. J. Clin. Microbiol. 49, 2183–2190 (2011).
Levecke, B. et al. Monitoring drug efficacy against gastrointestinal nematodes when faecal egg counts are low: do the analytic sensitivity and the formula matter? Parasitol. Res. 109, 953–957 (2011).
Albonico, M. et al. Comparison of three copromicroscopic methods to assess albendazole efficacy against soil-transmitted helminth infections in school-aged children on Pemba Island. Trans. R. Soc. Trop. Med. Hyg. 107, 493–501 (2013).
Bogoch, I.I., Raso, G., N'Goran, E.K., Marti, H.P. & Utzinger, J. Differences in microscopic diagnosis of helminths and intestinal protozoa among diagnostic centres. Eur. J. Clin. Microbiol. Infect. Dis. 25, 344–347 (2006).
Levecke, B. et al. A comparison of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz thick smear methods for soil-transmitted helminths. PLoS Negl. Trop. Dis. 5, e1201 (2011).
Becker, S.L. et al. Experiences and lessons from a multi-country NIDIAG study on persistent digestive disorders in the tropics. PLoS Negl. Trop.Dis 10, e0004818 (2016).
Fisheries and Food Reference Book: Manual of Veterinary Parasitological Laboratory Techniques, Vol. 418, Ministry of Agriculture, Fisheries and Food, HMSO, London (1986).
Katz, N., Chaves, A. & Pellegrino, J. A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Rev. Inst. Med. Trop. São Paulo 14, 397–400 (1972).
Yap, P. et al. Determining soil-transmitted helminth infection status and physical fitness of school-aged children. J. Vis. Exp. 66, e3966 (2012).
Allen, A.V.H. & Ridley, D.S. Further observations on the formol-ether concentration technique for faecal parasites. J. Clin. Pathol. 23, 545–546 (1970).
Utzinger, J. et al. Microscopic diagnosis of sodium acetate-acetic acid-formalin-fixed stool samples for helminths and intestinal protozoa: a comparison among European reference laboratories. Clin. Microbiol. Infect. 16, 267–273 (2010).
Cringoli, G. Coprological diagnosis: what's new? Parassitologia 46, 137–139 (2004).
Cringoli, G., Rinaldi, L., Albonico, M., Bergquist, R. & Utzinger, J. Geospatial (s)tools: integration of advanced epidemiological sampling and novel diagnostics. Geospat. Health 7, 399–404 (2013).
Borrelli, L. et al. New diagnostic insights for Macrorhabdus ornithogaster infection. J. Clin. Microbiol. 3, 3448–3350 (2015).
Barda, B. et al. Mini-FLOTAC, an innovative direct diagnostic technique for intestinal parasitic infections: experience from the field. PLoS Negl. Trop. Dis. 7, e2344 (2013).
Barda, B. et al. Parasitic infections on the shore of Lake Victoria (East Africa) detected by Mini-FLOTAC and standard techniques. Acta Trop. 137, 140–146 (2014).
Kenyon, F. et al. Pooling sheep faecal samples for the assessment of gastrointestinal strongyle and Nematodirus infection intensity and anthelmintic drug efficacy using McMaster and Mini-FLOTAC. Vet. Parasitol. 225, 53–60 (2016).
King, J.D. et al. Intestinal parasite prevalence in an area of Ethiopia after implementing the SAFE strategy, enhanced outreach services, and health extension program. PLoS Negl. Trop. Dis. 7, e2223 (2013).
Maurelli, M.P. et al. FLOTAC and Mini-FLOTAC for uro-microscopic diagnosis of Capillaria plica (syn. Pearsonema plica) in dogs. BMC Res. Notes 7, 591 (2014).
Kochanowski, M., Karamon, J., Dąbrowska, J. & Cencek, T. Experimental estimation of the efficacy of the FLOTAC basic technique. J. Parasitol. 100, 633–639 (2014).
Ruzicova, M., Petrzelkova, K., Kalousova, B., Modry, D. & Pomajbìkovà, K. Validation of FLOTAC for the detection and quantification of Troglodytella abrassarti and Neobalantidium coli in chimpanzees and pigs. J. Parasitol. 100, 662–670 (2014).
Noel, M.L., Scare, J.A., Bellaw, J.L. & Nielsen, M.K. Accuracy and precision of Mini-FLOTAC and McMaster techniques for determining equine strongyle egg counts. J. Equin. Vet. Sci. 48, 182–187e1 (2017).
Bosco, A. et al. Mini-FLOTAC, an accurate method for the diagnosis of nematode infections in horses and sheep. Proceedings of the XXIX Congress of the Italian Society of Parasitology (SOIPA) Bari, Italy, June 21–24, p. 32 (2016).
Van den Putte, N., Claerebout, E. & Levecke, B. Evaluation of the Mini-FLOTAC technique for detection of gastro-intestinal parasites in large companion animals. Vlams Diergeneskd. Tijdschr. 85, 15–22 (2016).
Maurelli, M.P. et al. Mini-FLOTAC, a new tool for copromicroscopic diagnosis of common intestinal nematodes in dogs. Parasit. Vectors 7, 356 (2014).
Torgerson, P.R., Paul, M. & Lewis, F.I. The contribution of simple random sampling to observed variations in faecal egg counts. Vet. Parasitol. 188, 397–401 (2012).
Engels, D., Nahimana, S., de Vlas, S.J. & Gryseels, B. Variation in weight of stool samples prepared by the Kato-Katz method and its implications. Trop. Med. Int. Health. 2, 265–271 (1997).
Krauth, S.J. et al. An in-depth analysis of a piece of shit: distribution of Schistosoma mansoni and hookworm eggs in human stool. PLoS Negl. Trop. Dis. 6, e1969 (2012).
Levecke, B. et al. Mathematical inference on helminth egg counts in stool and its applications in mass drug administration programmes to control soil-transmitted helminthiasis in public health. Adv. Parasitol. 87, 193–247 (2015).
Rinaldi, L. et al. Comparison of individual and pooled faecal samples in sheep for the assessment of gastrointestinal strongyle infection intensity and anthelmintic drug efficacy using McMaster and Mini-FLOTAC. Vet. Parasitol. 205, 216–223 (2014).
Mekonnen, Z. et al. Comparison of individual and pooled stool samples for the assessment of soil-transmitted helminth infection intensity and drug efficacy. PLoS Negl. Trop. Dis. 7, e2189 (2013).
Kure, A. et al. Comparison of individual and pooled stool samples for the assessment of intensity of Schistosoma mansoni and soil-transmitted helminth infections using the Kato-Katz technique. Parasit. Vectors 8, 489 (2015).
Degarege, A. et al. Comparison of individual and pooled urine samples for estimating the presence and intensity of Schistosoma haematobium infections at the population level. Parasit. Vectors 8, 593 (2015).
Lo, N.C. et al. Evaluation of a urine pooling strategy for the rapid and cost-efficient prevalence classification of schistosomiasis. PLoS Negl. Trop. Dis. 10, e0004894 (2016).
Barda, B. et al. How long can stool samples be fixed for an accurate diagnosis of soil-transmitted helminth infection using Mini-FLOTAC? PLoS Negl. Trop. Dis. 9, e0003698 (2015).
Rinaldi, L., Coles, G.C., Maurelli, M.P., Musella,, V. & Cringoli, G. Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep. Vet. Parasitol. 177, 345–352 (2011).
Barda, B. et al. Mini-FLOTAC and Kato-Katz: helminth eggs watching on the shore of Lake Victoria. Parasit. Vectors 6, 220 (2013).
Barda, B. et al. “Freezing” parasites in pre-Himalayan region, Himachal Pradesh: experience with Mini-FLOTAC. Acta Trop. 130, 11–16 (2013).
Barda, B. et al. Mini-FLOTAC, Kato-Katz and McMaster: three methods, one goal; highlights from north Argentina. Parasit. Vectors 7, 271 (2014).
Lima, V.F. et al. A comparison of Mini-FLOTAC and FLOTAC with classic methods to diagnosing intestinal parasites of dogs from Brazil. Parasitol. Res. 114, 3529–3533 (2015).
Djokic, V. et al. Mini-FLOTAC for counting Toxoplasma gondii oocysts from cat faeces-comparison with cell counting plates. Exp. Parasitol. 14, 67–71 (2014).
Silva, L.M.R. et al. Mini-FLOTAC for the diagnosis of Eimeria infection in goats: an alternative to McMaster. Small Rum. Res. 114, 280–283 (2013).
Godber, O.F. et al. A comparison of the FECPAK and Mini-FLOTAC faecal egg counting techniques. Vet. Parasitol. 207, 342–345 (2015).
Assefa, L.M. et al. Diagnostic accuracy and cost-effectiveness of alternative methods for detection of soil-transmitted helminths in a post-treatment setting in western Kenya. PLoS Negl. Trop. Dis. 8, e2843 (2014).
Nikolay, B., Brooker, S.J. & Pullan, R.L. Sensitivity of diagnostic tests for human soil-transmitted helminth infections: a meta-analysis in the absence of a true gold standard. Int. J. Parasitol. 44, 765–774 (2014).
Smith, J.L. et al. Factors associated with the performance and cost-effectiveness of using lymphatic filariasis transmission assessment surveys for monitoring soil-transmitted helminths: a case study in Kenya. Am. J. Trop. Med. Hyg. 92, 342–353 (2015).
Benjamin-Chung, J. et al. The interaction of deworming, improved sanitation, and household flooring with soil-transmitted helminth infection in rural Bangladesh. PLoS Negl. Trop. Dis. 9, e0004256 (2015).
Malrait, K. et al. Novel insights into the pathogenic importance, diagnosis and treatment of the rumen fluke (Calicophoron daubneyi) in cattle. Vet. Parasitol. 207, 134–139 (2015).
Donoghue, E.M., Lyons, E.T., Bellaw, J.L. & Nielsen, M.K. Biphasic appearance of corticated and decorticated ascarid egg shedding in untreated horse foals. Vet. Parasitol. 214, 114–117 (2015).
Lamberton, P.H. & Jourdan, P.M. Human ascariasis: diagnostics update. Curr. Trop. Med. Rep. 2, 189–200 (2015).
Acknowledgements
G.C. acknowledges G. and M. Federico for their technical expertise and their enthusiasm in participating in the development of the Mini-FLOTAC and Fill-FLOTAC apparatuses. G.C. also acknowledges M.E. Morgoglione, M. Santaniello, P. Pepe, D. Ianniello, S. Pennacchio, A. Amadesi, L. Del Prete, R. Vascone, E. Noviello, V. Musella, and M. Parrilla for their participation in the application and validation of the Mini-FLOTAC technique.
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G.C. invented the Mini-FLOTAC and the Fill-FLOTAC devices and developed the Mini-FLOTAC technique; M.P.M., B.L., A.B., J.V., J.U., and L.R. participated in the application and validation of the technique in the medical and veterinary fields. All authors read, revised, and approved the final submitted paper.
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The Mini-FLOTAC and Fill-FLOTAC devices have been developed by and are patented by G. Cringoli, but the patent will be handed over to the University of Naples ‘Federico II’. At present, the Mini-FLOTAC technique is being further validated by different research groups focusing on human and veterinary parasitology. The fact that one of the authors is the current patent holder of the Mini-FLOTAC technique had no role in the preparation and submission of the protocols reported or the design and implementation of ongoing and future studies. To obtain Mini-FLOTAC or Fill-FLOTAC devices, a contribution is required that is used only to (i) cover costs of production and packaging, and (ii) contribute to the ongoing FLOTAC research. The remaining authors have no competing financial interests.
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Cringoli, G., Maurelli, M., Levecke, B. et al. The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals. Nat Protoc 12, 1723–1732 (2017). https://doi.org/10.1038/nprot.2017.067
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DOI: https://doi.org/10.1038/nprot.2017.067
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