We compared the diagnostic performance of the standard method (Kato-Katz) with two recently developed methods (Mini-FLOTAC and Flukefinder) for the detection and quantification of Fasciola hepatica eggs in human stool. Uninfected human stool samples were artificially spiked with F. hepatica eggs to reach final concentrations of 14, 28, 41, or 96 eggs per gram of stool (epg). Only Flukefinder showed 100% sensitivity in all but the samples with the lowest concentration of eggs (14 epg), in which it had a sensitivity of 60%. Each of the methods underestimated the true fecal egg counts (FECs), Flukefinder resulting in the most biased egg counts (egg counts 0.18 times lower than the expected FECs). Only the Flukefinder resulted in more precise results (coefficient of variance < 30%) from FECs of 96 epg onward. The outcome of this study indicates that the Flukefinder is a useful alternative diagnostic method for human fascioliasis in stool

Briones-Montero, Andrea

Geldhof, Peter

Levecke, Bruno

Vlaminck, Johnny

Zárate-Rendón, Daniel

English

Ghent University Academic Bibliography

Am. J. Trop. Med. Hyg., 101(1), 2019, pp. 59–61doi:10.4269/ajtmh.18-0988Copyright © 2019 by The American Society of Tropical Medicine and HygieneComparison of Kato-Katz Thick Smear, Mini-FLOTAC, and Flukeﬁnder for the Detection andQuantiﬁcation of Fasciola hepatica Eggs in Artiﬁcially Spiked Human StoolDaniel A. Za´rate-Rendo´n,1* Johnny Vlaminck,2 Bruno Levecke,2 Andrea Briones-Montero,1 and Peter Geldhof21Laboratorio de Parasitologı´a, Facultad de Zootecnia, Universiad Nacional Agraria La Molina, Lima, Peru; 2Laboratory of Parasitology, Faculty ofVeterinary Medicine, University of Ghent, Merelbeke, BelgiumAbstract. Wecompared the diagnostic performance of the standardmethod (Kato-Katz) with two recently developedmethods (Mini-FLOTAC and Flukeﬁnder) for the detection and quantiﬁcation of Fasciola hepatica eggs in human stool.Uninfected human stool samples were artiﬁcially spiked with F. hepatica eggs to reach ﬁnal concentrations of 14, 28, 41,or 96 eggs per gram of stool (epg). Only Flukeﬁnder showed 100% sensitivity in all but the samples with the lowestconcentration of eggs (14 epg), inwhich it had a sensitivity of 60%.Eachof themethods underestimated the true fecal eggcounts (FECs), Flukeﬁnder resulting in themost biased egg counts (egg counts 0.18 times lower than the expected FECs).Only the Flukeﬁnder resulted in more precise results (coefﬁcient of variance < 30%) from FECs of 96 epg onward. Theoutcome of this study indicates that the Flukeﬁnder is a useful alternative diagnostic method for human fascioliasis instool.Fascioliasis is caused by the ﬂatworms Fasciola hepaticaandF. giganticaandposesamajor problem toboth animal andpublic health.1,2 In humans, this parasitosis is an acknowl-edged (re-)emerging human disease in approximately 51countries, its spread being closely linked to climatic conditionsand affected by climatic change.3 The more important regionsfor human disease in the world are located in South America,4with highly endemic areas in the Andean highlands.2,3 In recentdecades, a variety of studies reported a hyperdendemic prev-alence (> 10%) of human fascioliasis in these areas,5,6 ac-counting for approximately 17 million infected people and 180million at risk of infection.1 Morbidity caused by fascioliasis isparticularly pronounced in children,7–9 contributing to anemiaand poor development.10,11 Despite the increased use of se-rological tests, the routine tests used to diagnose humanfascioliasis in endemic rural areas are based on the micro-scopical demonstration and quantiﬁcation of eggs in stoolusing coprological methods. One of the most widely usedcoprological methods is the Kato-Katz thick smear.9 Thismethod has been extensively used to determine both preva-lence and intensity of infection (based on the number of eggsper gram of stool; epg) in highly endemic areas.12,13 It is acheap and simple method that has been standardized andrecommended for a wider range of parasitic diseases in hu-mans.5 However, an important limitation of this method is thatit fails to detect infection of low intensity.14 Other coprologicalmethods that hold promise as an alternative for Kato-Katz arethe recently developed Mini-FLOTAC and Flukeﬁnder test.Both methods have proven to have a high sensitivity for thediagnosis of fascioliasis in livestock,15–17 but they have not yetbeen fully validated for the diagnosis of human fascioliasis. Inthis study, we compared the diagnostic performance of thestandard method (Kato-Katz) with the recently developedmethods Mini-FLOTAC and Flukeﬁnder for the detection andquantiﬁcation of F. hepatica eggs in human stool samples.The study protocol was approved by the Ethics Committeeof the Universidad Peruana Cayetano Heredia (Code SIDISI:17010) and Ghent University (Belgium (Code OZ 2017/0869).Negative stool samples were collected from children attend-ing Institutional Day Care of the Universidad Nacional Agrariala Molina in Lima (Peru). This day care lies in an area which isnonendemic for fascioliasis. The collected samples were re-peatedly examined by a modiﬁed rapid sedimentation test5 toconﬁrm the absence of F. hepatica eggs. Conﬁrmed Fasciola-negative stool samples were subsequently pooled and ho-mogenized using wooden tongue depressors. The pooledsample was subsequently divided into four aliquots of 100 geach. This amount of stool allowed for repeated examinationby all diagnostic methods. The aliquots were subsequentlystored at 4C for approximately 2weeks until the experiments.Eggs of F. hepatica were obtained from bile and gall bladderscollected from livers of naturally infected cattle that wereslaughtered at an authorized abattoir in Lima. The eggs werepuriﬁed and collected using the commercial Flukeﬁndermethod (Flukeﬁnder) as described by the manufacturer’s in-structions. The puriﬁed eggs were suspended in distilled wa-ter, and their concentration was determined by counting thenumber of eggs present in 10 aliquots of 10 μL each andsubsequently calculating the arithmetic mean of these eggcounts. Different volumes of the egg suspension were sub-sequently pipetted to the four aliquots of the pooled stoolsample to obtain a ﬁnal concentration of approximately 14, 28,41, and 96 epg. A homogeneous distribution of the eggs wasobtained in the stool by vigorously mixing the pooled stoolusing a metal plaster spatula.The Kato-Katz thick smear (SterliTech, Kent, WA),17 Flu-keﬁnder (Flukeﬁnder, Soda Springs, ID),16 and Mini-FLOTAC(Zinc Chloride ﬂotation solution with a speciﬁc gravity of 1.30;University of Naples, Naples, Italy)14methodswere performedaccording to the manufacturer’s instructions. The fecal eggcounts (FECs; expressed in epg) were obtained bymultiplyingthe egg counts by 24 for Kato-Katz (0.417 g of stool is ex-amined), 10 for Mini-FLOTAC (0.1 g of stool is examined =2 mL of stool suspension which contains 2 g in a total volume40mL), and 0.5 for Flukeﬁnder (2 g of stool is examined). Eachmethod was performed 10 times on each of the four spikedsamples. To avoid any systematic error in the results (e.g., ﬁrstexamining all aliquots with one particular method), the se-quence of stool analysis was randomized. The sensitivity* Address correspondence to Daniel A. Za´rate-Rendo´n, Laboratoriode Parasitologı´a, Facultad de Zootecnia, Universidad NacionalAgraria La Molina, Lima 12, Peru. E-mail: dazre@lamolina.edu.pe59(proportion of samples in which eggs were found) was cal-culated across the three diagnostic methods and the four eggconcentrations. The accuracy and precision of the FECsmeasured by the methods were based on the ratio observedover the expected FECs and the coefﬁcient of variance (CV)(= SD/mean), respectively.Overall, Flukeﬁnder was the most sensitive method (90.0%[95%CI: 80.0; 97.5]), followedbyMini-FLOTAC (67.5 [95%CI:52.5; 80.0]) and Kato-Katz (32.5% [95% CI: 17.5; 47.5]). Asshown in Table 1, the sensitivity increased as a function of thenumber of eggs spiked, although this increase varied acrossthe methods. The Flukeﬁnder already reached a sensitivity of100% for FECs of at least 28 epg, whereas for Mini-FLOTAC,thiswasonly the case for the highest FECs (96 epg). Kato-Katznever reached a sensitivity of 100%, the sensitivity being 70%at most.The ratio of the observed epg over the expected FECs was< 1 for all the three methods, indicating that the methodsunderestimated FECs. The least biased FECs were obtainedby Kato-Katz and Mini-FLOTAC, both methods resulting inFECs that on averagewere 0.31 times lower than the expectedFECs. Fecal egg counts obtained by Flukeﬁnder were 0.19times lower. Table 1 illustrates the accuracy across the dif-ferent levels of seeded eggs for the different methods. ForMini-FLOTAC, the percentage of accuracy in FECs remainedrelatively unchanged, ranging from 24% to 35.7%. This was incontrast to the other methods for which a wider range wasobserved (Kato-Katz: 17.1–55%; Flukeﬁnder: 4.3–33.6%).Flukeﬁnderwas themost precisemethod of all, resulting in thelowest CV values across the different levels of seeded eggs. ACV not higher than 30%was obtained for FECs of 96 epg. Thetwo other methods did not achieve a CV < 30%, although theCV of Mini-FLOTAC was 35% for the highest FECs (98 epg).To the best of our knowledge, this is the ﬁrst work thatcompares Kato-Katz, Mini-FLOTAC, and Flukeﬁnder eggdetection and quantiﬁcation of F. hepatica eggs in artiﬁciallyTABLE 1The results of the spiking experiment using the Kato-Katz thick smear, Mini-FLOTAC, and Flukeﬁnder on the samples artiﬁcially spikedwith 14, 28,41, or 96 Fasciola hepatica eggs per gram (epg)Method ReplicateConcentration of F. hepatica eggs per gram of stool (epg)14 28 41 96Kato-Katz 1 0 0 0 722 0 24 0 1203 0 0 0 04 0 48 0 05 0 0 0 486 0 24 48 1687 0 0 0 728 24 24 0 249 0 0 0 010 0 0 0 24Sensitivity (%) 10.0 40.0 10.0 70.0Mean + SD 2.4 + 7.6 12 + 17.0 4.8 + 15.2 52.8 + 56.3Accuracy (%) 17.1 42.9 11.7 55.0CV (%) 44.3 39.6 129.7 102.4Mini-FLOTAC 1 0 20 0 302 10 20 0 303 0 20 10 304 20 10 0 205 0 0 20 106 10 0 20 307 10 10 20 308 0 10 10 109 0 0 30 2010 0 0 30 20Sensitivity (%) 40.0 60.0 70.0 100Mean + SD 5 ± 7.1 9 ± 8.8 14 ± 11.7 23 ± 8.2Accuracy (%) 35.7 32.1 34.1 24.9CV (%) 19.8 27.2 34.4 34.3Flukeﬁnder 1 1 4.5 2.5 29.52 0.5 9.5 1.5 283 0 7 5 234 1.5 6 5 325 0 4 5 42.56 1 11 2.5 30.57 0.5 7.5 0.5 468 0 6 3 24.59 1.5 6.5 6 3010 0 17.5 6 37Sensitivity (%) 60.0 100 100 100Mean + SD 0.6 ± 0.6 7.95 ± 4.0 3.7 ± 1.9 32.3 ± 7.4Accuracy (%) 4.3 28.4 9.0 33.6CV (%) 14.3 14.0 21.6 22.1CV = coefﬁcient of variance; FEC = fecal egg count. Ten FEC replicates were performed for each method and the results are shown in epg. The accuracy equals the ratio observed over theexpected FECs. CV = mean/SD.60 ZA´RATE-RENDO´N AND OTHERSspiked human stool samples. Our results indicate that Fluke-ﬁnder holds promise as an alternative to Kato-Katz for thediagnosis of human fascioliasis in stool. In contrast to theother methods, it shows a high sensitivity at low FECs (up toFECs of 28 epg) and it results in highly precise egg counts(CV < 30%) at FECs of 96 epg. The method underestimatesFECs, but this was observed by all methods. Moreover, theFlukeﬁnder could potentially also be used for the diagnosis ofother major trematodiasis, such as paragonimiasis or schis-tosomiasis. Further research is now required to assess theclinical sensitivity and the user-friendliness of the Flukeﬁnderunder ﬁeld conditions.Received December 14, 2018. Accepted for publication March 14,2019.Published online May 6, 2019.Financial support: This work was supported by the Consortium ofFlemish Universities of Belgium (VLIR) through the Project VLIR UOS-UNALM. Both J. V. and B. L. are ﬁnancially supported by an FWOgrant.Authors’ addresses: Daniel A. Za´rate-Rendo´n and Andrea Briones-Montero, Facultad de Zootecnia, Laboratorio de Parasitologı´a, Uni-versidad Nacional Agraria La Molina, Lima, Peru, E-mails: dazre@lamolina.edu.pe and abriones.montero@gmail.com. Johnny Vlaminck,Bruno Levecke, and Peter Geldhof, Faculty of Veterinary Medicine,Laboratory of Parasitology, GhentUniversity, Ghent, Belgium, E-mails:johnny.vlaminck@ugent.be, bruno.levecke@ugent.be, and peter.geldhof@ugent.be.This is an open-access article distributed under the terms of theCreative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided theoriginal author and source are credited.REFERENCES1. 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Comparison of Kato-Katz thick smear, Mini-FLOTAC, and Flukefinder for the detection and quantification of Fasciola hepatica eggs in artificially spiked human stool

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Comparison of Kato-Katz thick smear, Mini-FLOTAC, and Flukefinder for the detection and quantification of Fasciola hepatica eggs in artificially spiked human stool

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