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      Desempeño de McMaster y Mini-Flotac en el diagnóstico de Paramphistomum spp. en bovinos Translated title: Performance of McMaster and Mini-Flotac Techniques in the Diagnostic of Paramphistomum spp. in Bovines


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          Resumen Antecedentes: La Paramphistomosis es una enfermedad parasitaria emergente que causa cuantiosas pérdidas económicas, su diagnóstico es muy difícil. El objetivo del presente estudio fue evaluar el desempeño de las técnicas coprológicas McMaster y Mini- FLOTAC en el diagnóstico de Paramphistomum spp. de muestras provenientes de bovinos. Métodos Se analizó la recuperación de huevos de Paramphistomum spp. de McMaster y Mini-Flotac en muestras de heces de bovinos con cantidades conocidas de hpg. Se emplearon diferentes soluciones de flotación de ZnCl2 y ZnSO4, ambas con densidades de 1,45 y 1,50 mg/mL. Posteriormente se determinó el desempeño de McMaster y Mini- Flotac en muestras de heces de bovinos destinados al sacrificio (n=40); y otras provenientes de rebaños lecheros (n=155). Resultados Se obtuvieron similares exactitudes en la recuperación de huevos en ambas técnicas para hpg conocidos en diferentes soluciones de flotación (59-77 %). La precisión fue similar tanto en McMaster (16,93-25,83 %) y Mini-Flotac (17,83-25,05 %). Se observó linealidad de los conteos de hpg entre McMaster y Mini-Flotac tanto en muestras de heces de bovinos al sacrificio (R2=0,93), como de muestras de rebaños lecheros (R2=0,71). Las prevalencias de Paramphistomum spp. en rebaños lecheros fueron 42,9 % con McMaster y 43,5 % con Mini- FLOTAC. Conclusiones Ambas técnicas coprológicas tienen alta precisión y recuperación en la detección de huevos de Paramphistomum spp. en heces de bovinos, lo que indica la aplicabilidad para el diagnóstico parasitológico.

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          Abstract Background: Paramphistomosis is an emerging, hard to diagnose, parasitic disease, which causes enormous economic losses. The aim of this study was to evaluate the performance of two coprological techniques: McMaster and Mini- Flotac to diagnose Paramphistomum spp. from bovine samples. Methods: Paramphistomum spp. egg recovery through McMaster and Mini-Flotac was analyzed in bovine fecal samples, with known quantities of epg. Different floating solutions of ZnCl2 and ZnSO4 were used, with densities of 1.45 and 1.50 mg/mL. Then, the performances of McMaster and Mini-Flotac were determined in fecal samples from sacrifice bovines (n=40), as well as samples from dairy herds (n=155). Results: Both techniques yielded similar egg recovery accuracies, for known epg in different floating solutions (59-77 %). Accuracy was similar both in McMaster (16.93-25.83%) and Mini-Flotac (17.83-25.05%). Linearity was observed in epg counts between McMaster and Mini-Flotac, in both fecal samples from sacrifice bovines (R2=0.93) and from dairy herds (R=2=0.71). The prevalences of Paramphistomum spp. in dairy herds were 42.9% (McMaster), and 43.5% (Mini-Flotac). Conclusions: The two coprological techniques are highly accurate, with significant recovery at detecting Paramphistomum spp. eggs in bovine feces, thus indicating the feasibility of these parasitological diagnostic techniques.

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          FLOTAC: new multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans.

          Accurate diagnosis of parasitic infections is of pivotal importance for both individual patient management and population-based studies, such as drug efficacy trials and surveillance of parasitic disease control and elimination programs, in both human and veterinary public health. In this study, we present protocols for the FLOTAC basic, dual and double techniques, which are promising new multivalent, sensitive, accurate and precise methods for qualitative and quantitative copromicroscopic analysis. These various methods make use of the FLOTAC apparatus, a cylindrical device with two 5-ml flotation chambers, which allows up to 1 g of stool to be prepared for microscopic analysis. Compared with currently more widely used diagnostic methods for parasite detection in animals (e.g., McMaster and Wisconsin techniques) and humans (e.g., Kato-Katz and ether-based concentration techniques), the FLOTAC techniques show higher sensitivity and accuracy. All FLOTAC techniques can be performed on fresh fecal material as well as preserved stool samples, and require approximately 12-15 min of preparation time before microscopic analysis.
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            Comparing Diagnostic Accuracy of Kato-Katz, Koga Agar Plate, Ether-Concentration, and FLOTAC for Schistosoma mansoni and Soil-Transmitted Helminths

            Introduction Schistosomiasis and soil-transmitted helminthiasis are widespread in many parts of the developing world where they negatively impact on human health and wellbeing, and thus exacerbate poverty [1]–[4]. Schistosomiasis is caused by blood flukes of the genus Schistosoma; in Africa both S. mansoni and S. haematobium are endemic [2], [5]–[7]. Soil-transmitted helminthiasis is caused by intestinal nematodes; hookworms (Ancylostoma duodenale and Necator americanus), the roundworm (Ascaris lumbricoides), the whipworm (Trichuris trichiura), and the threadworm (Strongyloides stercoralis) [1], [3], [8], [9]. A detailed understanding of the epidemiology of these parasitic worm infections is important for the design, implementation, monitoring, and evaluation of helminth control programs [10], [11]. Commonly used diagnostic methods for these parasites rely on the detection of helminth eggs or larvae in human stool. These copromicroscopic approaches have drawbacks, such as low sensitivity for the detection of light-intensity infections [12], [13]. At present, the Kato-Katz technique is the most widely used copromicroscopic method in epidemiological surveys pertaining to human intestinal helminth infections because of its simplicity [14], low cost, and the established system to stratify infection intensity into different classes based on cut-offs of egg-counts [15]. The small amount of stool analyzed (usually 41.7 mg) explains why the Kato-Katz technique has a low sensitivity to detect eggs whenever they are present at low frequency or appear highly clustered (theoretical analytic sensitivity = 24 eggs per gram of stool (EPG)) [16], [17]. The sensitivity can be increased by examining multiple Kato-Katz thick smears prepared from the same stool sample or, better yet, from multiple stool samples [12],[18]–[22]. This point highlights the appeal of parasitological diagnostic methods capable of screening a larger amount of stool, e.g., 0.5 g or even 1 g in the case of the ether-concentration method [23] or the FLOTAC technique [24]. The ether-concentration method is often used for the diagnosis of helminth infections, particularly in specialized laboratories [23], [25]–[27]. Importantly, it allows the concurrent diagnosis of intestinal protozoa, and is sometimes used in combination with the Kato-Katz method to enhance diagnostic sensitivity for helminths, and hence to deepen our understanding of polyparasitism [28]–[30]. An important feature of the ether-concentration method is that it uses preserved stool samples, fixed in either sodium acetate-acetic acid-formalin (SAF) [23], or diluted formalin [24], thus allowing sample storage and analysis at later time points. However, considerable inter-laboratory discrepancies have been noted for helminth and particularly intestinal protozoa diagnosis [26], . The Koga agar plate technique allows direct observation of S. stercoralis and hookworm larvae hatched from fresh stool samples incubated on nutrient agar in a humid chamber, usually after 48 hours [31]. Recent studies suggest that the FLOTAC technique [24] holds promise for the diagnosis of soil-transmitted helminth infections in humans [32]–[34]. Its potential for the diagnosis of S. mansoni has yet to be investigated. The FLOTAC technique takes advantage of the fact that during flotation, parasitic elements such as helminth eggs gather in the apical portion of the flotation column and can be readily translated, i.e., cut transversally for subsequent viewing under a microscope. Thus, parasitic elements are separated from fecal debris, facilitating their identification and enumeration. Protocols have been developed for the FLOTAC basic technique (theoretical analytic sensitivity = 1 EPG), the FLOTAC dual technique, the FLOTAC double technique, and the FLOTAC pellet technique (all: theoretical analytic sensitivity = 2 EPG) [24]. Importantly, in a single FLOTAC examination usually ∼1 g of stool is analyzed, and hence a single FLOTAC allows a 24-fold higher amount of stool to be examined than a single Kato-Katz thick smear, which is an important factor explaining the higher sensitivity of the FLOTAC technique. The aim of this study was to compare the diagnostic accuracy of different techniques and sampling efforts, i.e., single and multiple Kato-Katz thick smears, ether-concentration and the FLOTAC method, for the detection and quantification of helminth eggs. The performance of the Koga agar plate technique for the detection of helminth larvae was also assessed. Particular emphasis was placed on the diagnosis of S. mansoni. Methods Ethical considerations and treatment The study was cleared by the institutional research commissions of the Swiss Tropical and Public Health Institute (Basel, Switzerland) and the Centre Suisse de Recherches Scientifiques (CSRS; Abidjan, Côte d'Ivoire), and was approved by local and national health authorities of Côte d'Ivoire. The school director and teachers were informed about the objectives and procedures of the study. Parents, legal guardians, and children were informed about the study and sufficient time was given to ask questions. Written informed consent was obtained from the parents or legal guardians of all participating children. Participation was voluntary and children could withdraw from the study at any time without further obligations. At the end of the study, all children attending the school of Azaguié-IRFA (“Institut de Recherches sur les Fruits et Agrumes”) were treated free of charge with praziquantel (single 40 mg/kg oral dose using a ‘dose-pole’) and mebendazole (single 500 mg oral dose) according to WHO recommendations [35]. Additionally, children infected with S. stercoralis were treated with ivermectin (single 200 µg/kg oral dose). Study area and participants The study was carried out in June 2008 in the primary school of Azaguié-IRFA, located in a rural setting of Azaguié in the region of Agboville, south Côte d'Ivoire (geographical coordinates: 05°36′10.5″ N latitude, 04°00′58.5″ W longitude). The village is located 56 km north of Abidjan, the economic capital of Côte d'Ivoire. In the school year 2007/2008, 200 children attended grades 1–6. We aimed for a sample size of 120 school children, similar to our preceding FLOTAC research carried out elsewhere in Côte d'Ivoire [32]. Allowing for a drop out of ∼10%, we randomly selected 133 school children from all grades and invited them to submit a single fresh morning stool. Field and laboratory procedures School children were given plastic containers (125 ml) for stool collection. Upon submission, each container was labeled with a unique identification number and transferred within 1–2 hours to the CSRS in Abidjan. Stool samples were processed at the day of collection, usually within 2–3 hours after reaching the laboratory. Stool specimens were collected over six consecutive days (one sample from each of 12 children on day 1 and one sample from each of 20 children on the following 5 days). As depicted in Figure 1, each fresh stool sample was subjected to the Kato-Katz method (3 thick smears), the Koga agar plate method (1 examination), and the FLOTAC dual technique (1 examination, fresh stool sample homogenized in SAF). Additionally, ∼5 g of the fresh stool were preserved in 25 ml SAF. After 9 days of preservation, these samples were filtered in order to remove large fibers (wire mesh aperture: 250 µm) and split into 5 sub-samples, each weighing ∼1 g. Among them, 3 sub-samples were examined with the FLOTAC dual technique 10, 30, and 83 days post-stool collection, 1 sub-sample was subjected to the ether-concentration method 40 days post-stool collection, and the fifth sub-sample was used as back-up and was finally discarded. 10.1371/journal.pntd.0000754.g001 Figure 1 Diagnostic methods used to detect S. mansoni and soil-transmitted helminth infections. The flowchart details the diagnostic approaches and their temporal sequence, as well as the amount of stool examined for the detection of helminth eggs and larvae, and the comparison of the different diagnostic tools applied to 112 stool samples from school children in Azaguié-IRFA, Côte d'Ivoire, in June 2008. Processing of stool samples was as follows. First, triplicate Kato-Katz thick smears (41.7 mg each) were prepared. Slides were read twice; first within 30–60 min for detection of hookworm eggs and again after ∼2 hours for diagnosis of S. mansoni, A. lumbricoides, T. trichiura, and other helminths. For each helminth species, the number of eggs was counted under a microscope by one of four experienced laboratory technicians and recorded separately for both readings. Second, a hazelnut-sized stool sample (average weight = 2.29 g) was placed in the middle of an agar plate. The plates were incubated in a humid chamber at 28°C for 48 hours. Plates were inspected under a microscope for the characteristic traces of hookworm and S. stercoralis larvae. Subsequently, the plates were rinsed with SAF, the solutions centrifuged and the sediment examined for larvae under a microscope by one experienced laboratory technician. The technician was blinded to the preceding Kato-Katz test results. Third, the samples for FLOTAC were prepared as follows: the filtered stool suspensions were filled up to 10 ml with SAF and equally split into two 15-ml Falcon tubes. Three ml of ether were added to each tube to facilitate the removal of fatty compounds that could interfere with egg detection under a microscope. The Falcon tubes were shaken rigorously for at least 1 min and then centrifuged for 2 min at 170 g. The supernatant was discarded and each pellet suspended in 6 ml of either flotation solution FS4 (sodium nitrate: 315 g NaNO3 suspended in 685 ml H2O; specific gravity (s.g.) = 1.20) or FS7 (zinc sulphate: 685 g ZnSO4 H2O suspended in 685 ml H2O; s.g. = 1.35) [24]. In the present study, the FLOTAC dual technique was employed (i.e., one of two different FS in each of the two chambers of the same FLOTAC apparatus). From a panel of 14 different FS [36], we selected FS4 because it had proved to be useful for soil-transmitted helminth diagnosis in previous studies carried out in Côte d'Ivoire and Zanzibar [32], [33]. FS7 was chosen because it was particularly suitable for the detection of S. mansoni eggs in fecal samples obtained from infected mice and hamster (J. Keiser, L. Rinaldi, and J. Utzinger; unpublished data). Each suspension was transferred into one of the two 5-ml chambers of the FLOTAC apparatus. The apparatus was centrifuged (using a Hettich Universal 320 centrifuge; Tuttlingen, Germany) at 160 g for 5 min. Subsequently, the upper portion in the FLOTAC apparatus was translated, and the observation grid examined under a microscope at 100× magnification by one experienced laboratory technician. The technician was blinded to the preceding Kato-Katz and ether-concentration test results. Helminth eggs were counted and reported for each species in each chamber separately. Fourth, the ether-concentration method was performed as described in detail elsewhere [27]. Data management and statistical analysis Data were double-entered in Microsoft Office Access 2007, cross-checked using EpiInfo (TM 3.4.1), and analyzed with STATA version 9.1 (StataCorp LP; College Station, TX). The different diagnostic methods were compared using 2-way contingency tables of frequencies, and agreement between 2 diagnostic techniques was determined (e.g., Kato-Katz versus ether-concentration, and Kato-Katz versus FLOTAC for S. mansoni diagnosis). Kappa (ĸ) statistic was employed to determine the strength of agreement using the following cut-offs: (i) ĸ<0.00 indicating no agreement; (ii) ĸ = 0.00–0.20 indicating poor agreement; (iii) ĸ = 0.21–0.40 indicating fair agreement; (iv) ĸ = 0.41–0.60 indicating moderate agreement; ĸ = 0.61–0.80 indicating substantial agreement; and (v) ĸ = 0.81–1.00 indicating almost perfect agreement [37], [38]. It is important to note that ĸ values depend on the marginal distributions of contingency tables. We employed a test of marginal homogeneity and, since significant values were obtained, raked ĸ values were calculated throughout [39], [40]. The sensitivity and negative predictive value (NPV) were calculated for each method, considering the combined results from all different methods, FS, and at all time points investigated, as diagnostic ‘gold’ standard. Hence, any positive test result, regardless of the technique employed, was considered a true-positive result. Following this approach specificity was set to be 100%, justified by the direct observation of parasite eggs [21], [26], [32], [33]. Helminth-specific egg counts were expressed as EPGs for three among the four techniques. For the Kato-Katz method, the number of helminth eggs in each of the three thick smears prepared from a single fresh stool sample collected from each participant were added and then multiplied by a factor 8 to obtain EPGs. For the ether-concentration method, EPGs were estimated by dividing the number of helminth eggs with the amount of stool examined after 40 days of preservation (∼1 g; see Figure 1), that is one-fifth of the total amount of weighed stool that was preserved and used for four different tests with the remaining fifth part of the preserved stool sample finally discarded. For the examination of stool samples with the FLOTAC dual technique, FS-specific EPGs were estimated as follows. For the fresh stool sample (∼1 g; see Figure 1), the number of counted helminth eggs was divided by the exact amount of stool and multiplied with a factor 2 (stool sample was equally aliquoted to one of the two FLOTAC chambers) and, additionally, with a factor 1.2 (stool sample was solved in 6 ml FS, but only 5 ml was filled into the FLOTAC apparatus). For the SAF-preserved stool, the number of counted helminth eggs was divided by one-fifth of the total amount of weighed stool (∼1 g; see Figure 1), and then multiplied by factors 2 and 1.2 as described above. Since EPGs were not normally distributed (as assessed by quantile normal plots), the geometric mean (GM), including 95% confidence intervals (CI), was calculated and graphically displayed. Our assumption was that non-overlapping 95% CIs indicate statistical significance (p<0.05). Post-calibration for FLOTAC A post-calibration was performed 4 months post-stool collection to determine whether FS4 and FS7 are indeed among the most suitable FS for the FLOTAC technique for differential helminth diagnosis. For this purpose, we employed a composite human stool sample (∼100 g), pooling stool from 8 selected children with high-intensity helminth infections who provided a second stool sample just before administering anthelmintic drugs. This composite stool sample was preserved in SAF and transferred to Naples, Italy. From the panel of 14 different available FS [36], a total of 9 were selected, including FS4 and FS7. At least 3 replicates were examined for each of the 9 FS with and without a prior ether washing step, to allow an appraisal of the effect of ether on helminth diagnosis. Results Study cohort We obtained one sufficiently large stool sample to perform triplicate Kato-Katz, multiple FLOTAC, a single ether-concentration, and a single Koga agar plate test from 112 school children. Our study cohort comprised 61 (54.5%) boys. The median age was 10 years (range: 6–15 years). Prevalence of S. mansoni and soil-transmitted helminths The combined results from the different copromicroscopic techniques were considered as diagnostic ‘gold’ standard. As shown in Figure 2, eggs of S. mansoni were detected in 93 children (83.0%). The overall prevalences of hookworm, T. trichiura and A. lumbricoides were 55.4%, 40.2% and 28.6%, respectively. S. stercoralis larvae were detected in the stools of 38 children (33.9%). 10.1371/journal.pntd.0000754.g002 Figure 2 Prevalence of S. mansoni and soil-transmitted helminth infections. Bar charts indicate the prevalence of S. mansoni (A) and soil-transmitted helminth infections, i.e., hookworm (B), T. trichiura (C), and A. lumbricoides (D) among 112 school children from Azaguié-IRFA, Côte d'Ivoire, in June 2008. Results are stratified by diagnostic methods. The combined results from the different methods were considered as diagnostic ‘gold’ standard. Fresh stool examinations were subjected to triplicate Kato-Katz thick smears, a single Koga agar plate test, and a single FLOTAC examination. The fresh stool sample for FLOTAC (0 days) was homogenized in SAF. The SAF-preserved stool samples were examined once with the ether-concentration method (after 40 days) and 3 times with the FLOTAC method (at days 10, 30, and 83 post-stool collection). Prevalence estimates for S. mansoni using the FLOTAC method only considered the results of FS7. With regard to soil-transmitted helminth infections, the combined results of FS4 and FS7 were considered. Methods comparison for the diagnosis of S. mansoni Table 1 summarizes the characteristics of 3 different techniques for S. mansoni diagnosis. Whilst a single Kato-Katz thick smear revealed S. mansoni at a prevalence of 56.3%, the cumulative prevalence after examination of 3 Kato-Katz thick smears was 64.3%; an increase of 14.2%. The observed S. mansoni prevalence based on a single ether-concentration test was 70.5%. Subjecting a fresh stool sample homogenized in SAF to the FLOTAC dual technique, but considering only results from FS7, revealed a S. mansoni prevalence of 53.6%. Preservation of stool samples in SAF for 10, 30, and 83 days, and examination with FLOTAC FS7 revealed point prevalence estimates of 72.3–75.9%. 10.1371/journal.pntd.0000754.t001 Table 1 Prevalence, sensitivity, and negative predicted value (NPV) derived by different diagnostic methods. Parasite Technique Number of infected school children (%) Sensitivity in % (95% CI) NPV (95% CI) S. mansoni ‘Gold’ standard 93 (83.0) 100 100 Kato-Katz (single) 63 (56.3) 67.7 (59.1–76.4) 38.8 (29.8–47.8) Kato-Katz (triplicate) 72 (64.3) 77.4 (69.7–85.2) 47.5 (38.3–56.7) Ether-concentration 79 (70.5) 85.0 (78.3–91.6) 57.6 (48.4–66.7) FLOTAC (fresh) 60 (53.6) 64.5 (55.7–73.4) 36.5 (27.6–45.5) FLOTAC (10 days) 81 (72.3) 87.1 (80.9–93.3) 61.3 (52.3–70.3) FLOTAC (30 days) 85 (75.9) 91.4 (86.2–96.6) 70.4 (61.9–78.8) FLOTAC (83 days) 85 (75.9) 91.4 (86.2–96.6) 70.4 (61.9–78.8) Hookworm ‘Gold’ standard 62 (55.4) 100 100 Kato-Katz (single) 14 (12.5) 22.6 (14.8–30.3) 51.0 (41.8–60.3) Kato-Katz (triplicate) 24 (21.4) 38.7 (29.7–47.7) 56.8 (47.6–66.0) Ether-concentration 22 (19.6) 35.5 (26.6–44.3) 55.6 (46.4–64.8) Koga agar plate 28 (25.0) 45.2 (35.9–54.4) 59.5 (50.4–68.6) FLOTAC (fresh) 49 (43.8) 79.0 (71.5–86.6) 79.4 (71.9–86.9) FLOTAC (10 days) 35 (31.3) 56.5 (47.3–65.6) 64.9 (56.1–73.8) FLOTAC (30 days) 29 (25.9) 46.8 (37.5–56.0) 60.2 (51.2–69.3) FLOTAC (83 days) 19 (17.0) 30.6 (22.1–39.2) 53.8 (44.5–63.0) T. trichiura ‘Gold’ standard 45 (40.2) 100 100 Kato-Katz (single) 9 (8.0) 20.0 (12.6–27.4) 65.0 (56.2–73.9) Kato-Katz (triplicate) 14 (12.5) 31.1 (22.5–39.7) 68.4 (59.8–77.0) Ether-concentration 23 (20.5) 51.1 (41.9–60.4) 75.3 (67.3–83.3) FLOTAC (fresh) 35 (31.3) 77.8 (70.1–85.5) 87.0 (80.8–93.2) FLOTAC (10 days) 31 (27.7) 68.9 (60.3–77.5) 82.7 (75.7–89.7) FLOTAC (30 days) 36 (32.1) 80.0 (72.6–87.4) 88.2 (82.2–94.1) FLOTAC (83 days) 34 (30.4) 75.6 (67.6–83.5) 85.9 (79.5–92.3) A. lumbricoides ‘Gold’ standard 32 (28.6) 100 100 Kato-Katz (single) 22 (19.6) 68.8 (60.2–77.3) 88.9 (83.1–94.7) Kato-Katz (triplicate) 22 (19.6) 68.8 (60.2–77.3) 88.9 (83.1–94.7) Ether-concentration 14 (12.5) 43.8 (34.6–52.9) 81.6 (74.5–88.8) FLOTAC (fresh) 23 (20.5) 71.9 (63.5–80.2) 89.9 (84.3–95.5) FLOTAC (10 days) 15 (13.4) 46.9 (37.6–56.1) 82.5 (75.4–89.5) FLOTAC (30 days) 13 (11.6) 40.6 (31.5–49.7) 80.8 (73.5–88.1) FLOTAC (83 days) 12 (10.7) 37.5 (28.5–46.5) 80.0 (72.6–87.4) Number of school children (total: 112 individuals from Azaguié-IRFA, Côte d'Ivoire) diagnosed with S. mansoni, hookworm, T. trichiura, and A. lumbricoides in a single stool sample using different methods (Kato-Katz, ether-concentration, FLOTAC, and Koga agar plate, where appropriate), and sensitivity, and NPV of the respective technique. Fresh stool samples for FLOTAC were homogenized in SAF. Stool samples for ether-concentration and FLOTAC at day 10, 30, and 83 were preserved in SAF. The highest sensitivity for S. mansoni diagnosis (91.4%) was found for the FLOTAC dual technique after 30 and 83 days of preservation in SAF. The sensitivity of a single ether-concentration test was 85.0%, whereas triplicate or only a single Kato-Katz revealed sensitivities of 77.4% and 67.7%, respectively. Table 2 shows 2-way contingency tables comparing the results of a single FLOTAC (fresh stool, SAF preservation for 10, 30, or 83 days) and the single ether-concentration test (SAF preservation for 40 days) with the combined results of the triplicate Kato-Katz thick smear readings. Using fresh stool samples for FLOTAC (homogenized in SAF) and Kato-Katz, 51 S. mansoni infections were concurrently detected by both techniques, whereas 21 additional infections were diagnosed by triplicate Kato-Katz only, and 9 additional infections were detected by FLOTAC only. The agreement between these 2 methods was moderate (raked ĸ = 0.49). Comparing the results of FLOTAC performed with stool samples preserved in SAF for 10, 30, or 83 days with triplicate Kato-Katz from fresh stool, both methods concurrently detected between 69 and 71 S. mansoni infections, whereas 12–16 additional infections were only found by the FLOTAC technique and 1–3 infections were only detected by triplicate Kato-Katz thick smears. Substantial agreement was found for the stool samples preserved in SAF for 10 days (raked ĸ = 0.76) or 83 days (raked ĸ = 0.71), and an almost perfect agreement after 30 days of SAF preservation (raked ĸ = 0.84). 10.1371/journal.pntd.0000754.t002 Table 2 Agreement between different diagnostic techniques for the detection of S. mansoni. Triplicate Kato-Katz Raked kappa Marginal homogeneity Positive Negative Total P-values FLOTAC Fresh stool homogenized in SAF Positive 51 9 60 Negative 21 31 52 Total 72 40 112 0.49 0.029 Preserved stool in SAF (10 days) Positive 69 12 81 Negative 3 28 31 Total 72 40 112 0.76 0.020 Preserved stool in SAF (30 days) Positive 71 14 85 Negative 1 26 27 Total 72 40 112 0.84 <0.001 Preserved stool in SAF (83 days) Positive 69 16 85 Negative 3 24 27 Total 72 40 112 0.71 0.003 Ether-concentration method Preserved stool in SAF (40 days) Positive 69 10 79 Negative 3 30 33 Total 72 40 112 0.79 0.052 SAF: sodium acetate-acetic acid-formalin. The 2-way contingency tables show the agreement between triplicate Kato-Katz thick smears, FLOTAC, and the ether-concentration for the diagnosis of S. mansoni in stool samples from 112 school children from Azaguié-IRFA, Côte d'Ivoire, in June 2008. The ether-concentration method and triplicate Kato-Katz diagnosed 69 S. mansoni infections concurrently, whereas 10 infections were only found by the ether-concentration method and 3 were detected by triplicate Kato-Katz thick smears only, owing to a substantial agreement (raked ĸ = 0.79). Triplicate Kato-Katz thick smears revealed a mean infection intensity of 121.2 EPG (95% CI: 86.8–169.2 EPG). The mean infection intensity based on a single ether-concentration examination was 110.7 EPG (95% CI: 76.0–161.1 EPG). These mean egg counts were significantly higher than those obtained with the FLOTAC dual technique, regardless of whether FS4 or FS7 were employed (Figure 3A). There was one exception: a single FLOTAC performed on stool samples preserved in SAF for 83 days and using FS7 revealed similar EPGs as triplicate Kato-Katz thick smears and a single ether-concentration test. 10.1371/journal.pntd.0000754.g003 Figure 3 Geometric mean (GM) fecal egg counts according to different diagnostic techniques. Bar charts indicate the GM of fecal egg counts (as expressed in eggs per gram of stool (EPG) according to different techniques for the diagnosis of S. mansoni (A), hookworm (B), A. lumbricoides (C), and T. trichiura (D) in stool samples from 112 school children from Azaguié-IRFA, Côte d'Ivoire, in June 2008. The results for the FLOTAC method are presented separately for FS4 and FS7. Error bars indicate 95% confidence intervals (CIs) of the GM. Using FS4 with stool preserved in SAF for 10 days resulted in the detection of only 10 (8.9%), and after 83 days of preservation of no S. mansoni-positive individuals. The use of FS7, on the other hand, revealed 81 (72.3%) and 85 (75.9%) infections after 10 and 83 days of preservation, respectively. While the observed S. mansoni fecal egg counts in FS4 decreased over time to zero, an increase was observed in FS7 from a mean of 32.3 EPG (95% CI: 24.7–42.3 EPG) at day 10 to 57.7 EPG (95% CI: 41.5–80.2 EPG) at day 83. Figure 4 shows that the shape of S. mansoni eggs was somewhat altered when using the FLOTAC dual technique and employing FS7. However, the characteristic lateral spine remained, and hence unambiguous diagnosis was ascertained. 10.1371/journal.pntd.0000754.g004 Figure 4 S. mansoni eggs detected by the Kato-Katz or FLOTAC method. The pictures show a S. mansoni egg as seen under a light microscope using 100× magnification. S. mansoni egg without deformation as seen in a Kato-Katz thick smear (A), and egg deformed through the influence of zinc sulphate in FS7 and centrifugation as seen under the FLOTAC reading disc (B). Methods comparison for the diagnosis of soil-transmitted helminths For hookworm, the observed prevalence increased from 12.5% after a single to 21.4% after triplicate Kato-Katz examination, an increase of 71.2%. For T. trichiura there was an increase from 8.0% to 12.5% (+56.3%). No increase was observed for A. lumbricoides; a prevalence of 19.6% was obtained already after the first Kato-Katz thick smear reading (Figure 2B–D and Table 1). Analyses with the Koga agar plate method revealed 28 (25.0%) hookworm infections. The highest observed prevalence of hookworm infection was obtained with the FLOTAC dual technique using fresh stool samples homogenized in SAF (43.8%). The observed prevalence at days 10 and 83 post-conservation decreased to 31.3% and 17.0%, respectively. A single Kato-Katz revealed a hookworm infection intensity of 165.7 EPG (95% CI: 97.0–282.4 EPG), whereas triplicate Kato-Katz thick smears suggested an intensity of less than half of this value (64.6 EPG; 95% CI: 32.8–127.2 EPG). The difference in fecal egg counts determined by the Kato-Katz and the FLOTAC techniques in both FS (fresh stool: FS4 = 18.1 EPG; 95% CI: 11.3–29.2 EPG, and FS7 = 18.7 EPG; 95% CI: 11.3–30.9 EPG) was significant. In the preserved stool samples, the observed EPGs for hookworm decreased significantly in FS4 from day 10 (13.3 EPG; 95% CI: 7.4–23.8 EPG) to day 83 (2.7 EPG; 95% CI: 1.2–5.9 EPG) (Figure 3B). In FS7, the fecal egg counts also decreased, but the difference showed no statistical significance after 10 and 83 days of SAF conservation (from 18.1 EPG (95% CI: 11.5–28.6 EPG) to 11.1 EPG (95% CI: 6.1–20.2 EPG)). The highest prevalence of A. lumbricoides was estimated by a single FLOTAC from fresh stool homogenized in SAF (20.5%), but triplicate Kato-Katz showed only a marginally lower prevalence (19.6%). The examination of stool samples preserved in SAF for 83 days using FLOTAC or an ether-concentration test at day 40 after stool collection resulted in observed prevalences of 10.7% and 12.5%, respectively. The A. lumbricoides fecal egg counts determined with Kato-Katz were significantly higher than those obtained with FLOTAC, except for the results generated after 83 days of stool preservation. There was a significant increase for A. lumbricoides egg counts both with FS4 (from 148.4 EPG (95% CI: 39.9–551.5 EPG) to 1159.3 EPG (95% CI: 596.6–2252.7 EPG); a 7.8-fold increase) and with FS7 (from 181.2 EPG (95% CI: 60.0–546.9 EPG) to 1688.5 EPG (95% CI: 889.4–3205.8 EPG); a 9.3–fold increase) when comparing the 10 and 83 post-stool collection preservation time points (Figure 3C). The highest observed T. trichiura prevalence (32.1%) was obtained with a single FLOTAC examined after 30 days of stool preservation in SAF. Considerably lower prevalences were obtained with a single ether-concentration test (20.5%) and triplicate Kato-Katz thick smears (12.5%). The T. trichiura fecal egg count for a single Kato-Katz thick smear was 56.3 EPG (95% CI: 22.4–141.4 EPG). For triplicate Kato-Katz, the respective egg count was 24.0 EPG (95% CI: 11.4–50.6 EPG). Consistently more T. trichiura eggs were found in FS4 than in FS7, but the difference was only significant at day 30 post-preservation (FS4 = 35.1 EPG, 95% CI: 24.4–50.4 EPG; FS7 = 15.8 EPG, 95% CI: 10.2–24.4 EPG). There was an apparent increase of egg counts over the course of stool preservation in SAF, i.e., in FS4 from 17.5 EPG (95% CI: 11.2–27.2 EPG) to 39.6 EPG (95% CI: 25.8–60.8 EPG), a 2.3-fold increase, and in FS7 from 11.8 EPG (95% CI: 7.9–17.5 EPG) to 22.2 EPG (95% CI: 14.3–34.7 EPG), a 1.9-fold increase (Figure 3D). S. stercoralis larvae were found on Koga agar plates prepared with stool samples from 38 children but only 1 of these 38 infection was diagnosed after triplicate Kato-Katz examinations and 2 of these 38 infections were detected with FLOTAC in the fresh stool samples processed in SAF. No S. stercoralis larvae were found in the preserved samples, neither by FLOTAC nor by ether-concentration. Post-calibration of FLOTAC The use of FS7 resulted in the highest S. mansoni fecal egg count (average: 38.3 EPG, SD: 2.7 EPG; 6 replications), but stool samples had to be washed with ether as otherwise, due to the darkening effect of organic debris, accurate reading was not feasible. For hookworm diagnosis, FS4 produced the highest fecal egg count (average: 103.2 EPG, SD: 23.6 EPG; 6 replications). Of note, hookworm eggs were readily detected only in the absence of ether for sample preparation. With regard to A. lumbricoides and T. trichiura, the results from the post-calibration were less clear-cut than those for S. mansoni and hookworm, as all tested FS resulted in relatively high egg count averages for A. lumbricoides (306.0–515.0 EPG, SD: 22.2–176.8 EPG; 3–4 replications) and T. trichiura (6.0–26.0 EPG, SD: 2.8–15.0 EPG; 3–4 replications), whenever an ether washing step was included. Discussion Accurate diagnosis is key for adequate patient management and for guiding the design, implementation, and monitoring of community-based infectious disease control programs [13], [41]. We compared the diagnostic accuracy of two widely used techniques for detection and quantification of helminth eggs in fecal samples – the Kato-Katz thick smear using fresh stool, and the ether-concentration method using SAF-preserved samples – with the recently developed FLOTAC technique. Particular emphasis was placed on the diagnosis of S. mansoni because of the public health importance of intestinal schistosomiasis [2], [5]–[7], and because the FLOTAC method had not previously been investigated for this parasite. Additionally, the Koga agar plate method was employed, mainly for the diagnosis of S. stercoralis, but also for the detection of hookworm larvae. Best results, i.e., high sensitivities (87.1–91.4%) for detecting S. mansoni eggs (prevalence: 72.3–75.9%), were achieved after stool samples were homogenized, preserved in SAF, and examined after 10–83 days with the FLOTAC dual technique. Almost as sensitive was a single ether-concentration (85.0%), revealing a S. mansoni prevalence of 70.5%. Triplicate Kato-Katz examinations resulted in a prevalence estimate of 64.3%. A single Kato-Katz and FLOTAC using fresh stool revealed considerably lower S. mansoni point prevalences of 56.3% and 53.6%, respectively. It should be noted, however that neither FLOTAC, nor the ether-concentration test, nor multiple Kato-Katz readings detected ‘all’ S. mansoni or ‘all’ soil-transmitted helminth infections. There was moderate to almost perfect agreement between the FLOTAC or ether-concentration method and triplicate Kato-Katz thick smears according to raked ĸ values. With regard to soil-transmitted helminth diagnosis, our results confirm that a single FLOTAC is more sensitive than multiple Kato-Katz thick smears for the detection of hookworm, A. lumbricoides, and T. trichiura eggs in fecal samples [32]–[34]. A single FLOTAC using fresh stool processed with SAF was also more sensitive than a single ether-concentration test for the diagnosis of hookworm, A. lumbricoides, and T. trichiura infections. Finally, for hookworm diagnosis, a single FLOTAC using fresh stool was more sensitive than a single Koga agar plate test. The design of our study allowed investigating the effect of the duration of stool preservation on helminth species-specific diagnosis. The duration of stool fixation had a considerable effect on the diagnostic performance of copromicroscopic techniques. While the number of S. mansoni infections detected after 10, 30, and 83 days of stool preservation in SAF remained constant (81–85 infections), there was an apparent increase in fecal egg counts from day 10 to day 83, from 32.3 EPG to 57.7 EPG (considering only FS7). The observed prevalence of hookworm and A. lumbricoides decreased with increasing duration of SAF conservation before FLOTAC analysis. For example, while the point prevalence of hookworm was 43.8% for FLOTAC using fresh stool, it decreased to 17.0% after stool samples had been preserved in SAF for 83 days. On the other hand, there was no apparent decline in the prevalence of T. trichiura over the 83-day SAF preservation period. Higher fecal egg counts were observed for A. lumbricoides and T. trichiura as a function of stool preservation duration. Regarding hookworm diagnosis, there was a sharp decrease in fecal egg counts as a function of preservation time using SAF, suggesting a negative impact of this preservation medium on hookworm eggs. However, during the post-calibration investigation and subsequent studies, it was found that the introduction of an ether washing step resulted in lower hookworm egg counts. Indeed, considerably higher hookworm egg counts were revealed in SAF-preserved stool samples in the absence of ether, whereas destroyed hookworm eggs could be observed after exposure of the sample to ether. These observations indicate that the prolonged preservation of stool in SAF in combination with ether used for sample preparation might destroy the fragile hookworm eggs. To test this hypothesis, it will be interesting to investigate the exact influence of the preservation media alone, i.e., we still lack data on the influence of SAF and/or ether on helminth egg counts. The underlying mechanisms resulting in increasing S. mansoni, A. lumbricoides, and T. trichiura fecal egg counts estimates with time of preservation, and for the higher diagnostic sensitivity, yet lower egg counts when using FLOTAC as opposed to the Kato-Katz thick smear method, remain elusive and are the subject of ongoing deliberations and studies. We speculate that the helminth larvae are able to further develop and gain weight in these environmentally resistant eggs. This might lead to a change in density, and hence altered floating behavior of the eggs. However, these apparent fluctuations give rise to fears regarding the consistency of the diagnostic performance of FLOTAC when performed after non-standardized preservation time, on different populations, and by different laboratories. The somewhat higher fecal egg counts of S. mansoni, A. lumbricoides, and T. trichiura using FLOTAC at later time points of stool conservation, and the consistently higher fecal egg counts using Kato-Katz might be explained by the following additional reasons. First, helminth eggs should not be considered “inert elements”. Instead, interactions occur between the different compartments within a floating fecal suspension (e.g., FS components, parasitic elements, fixative, ether, and residues of the host alimentation), and these might be complex [24]. New research is therefore needed to elucidate potential interactions between these compartments. Second, the high fecal egg counts derived from the Kato-Katz thick smear readings shown in Fig. 3 might be misleading. EPG values obtained from Kato-Katz thick smear readings are not continuous due to the multiplication factor used (i.e., a factor 24 for a single, and a factor 8 for triplicate Kato-Katz thick smear readings). Hence, the minimum positive value for a single measurement is 24 EPG. Third, there are additional reasons why the Kato-Katz technique might overestimate fecal egg counts. For example, when scraping the plastic spatula of the Kato-Katz kit across the upper surface of the fine-meshed screen placed on top of the stool sample, the feces is sieved, and helminth eggs are concentrated [42], [43], an issue we are currently investigating. The available results pose considerable challenges for articulating recommendations regarding the optimal deployment of diagnostic tools for patient diagnosis, drug efficacy evaluations, and surveillance in areas where soil-transmitted helminths and S. mansoni are co-endemic. While the results pertaining to S. mansoni clearly argue for SAF-conservation of stool samples and their analysis with FS7 at a later time point, the data regarding common soil-transmitted helminth infections suggest that immediate diagnosis with FS4 should be pursued. Important trade-offs between time and fecal egg counts also exist for different soil-transmitted helminth species. Sound conclusions can probably only be drawn once more results and experience from the field are available, but it is clear that a method which needs different times and solutions to reliably diagnose distinct helminth species infections in a poly-parasitized patient is not ideal. The current study is part of a broad attempt at validating the FLOTAC technique for human helminth diagnosis. Although we knew from previous investigations that FS4 is particularly suitable for the detection of soil-transmitted helminth eggs [32], , and prior investigations with fecal pellets obtained from S. mansoni-infected mice and hamster revealed that FS7 is suitable for detection of S. mansoni eggs, this issue has never been addressed in a systematic manner, using a single pool of stool of uniform characteristics. In a post-calibration approach, we employed a composite human stool sample of ∼100 g, pooling stool from a few selected children with high-intensity helminth infections. The results of the post-calibration underscore that the ether washing step potentially destroys hookworm eggs after a certain conservation period in SAF. No S. mansoni eggs were found when using FS4, and only few hookworm eggs were found in FS7. With regard to A. lumbricoides and T. trichiura, the results from the post-calibration were less clear-cut than those for S. mansoni and hookworm, as a number of FS resulted in high fecal egg counts for A. lumbricoides and T. trichiura. Regarding parasitological S. stercoralis diagnosis, it is conventionally either done with the Koga agar plate method – as in the present study – or the Baermann method [12], [21], [31], [44]–[46]. Our study offered an opportunity to also obtain preliminary results with FLOTAC for S. stercoralis diagnosis. Only 2 individuals were found positive for S. stercoralis when fresh stool samples were subjected to FLOTAC, whereas the Koga agar plate method revealed 38 infections. One of the 2 samples determined as S. stercoralis-positive using the FLOTAC method contained so many larvae that they were even observed in the Kato-Katz thick smears. Of note, the tegument of the S. stercoralis larvae detected by FLOTAC showed signs of degeneration, which might be due to the SAF preservation, the prior washing step with ether, the FS, or a combination of these chemicals. More research is needed to determine whether the FLOTAC technique might be further adapted to allow S. stercoralis diagnosis. In conclusion, the high sensitivity of a single FLOTAC examination for diagnosing common soil-transmitted helminth infections has been confirmed, but fecal egg counts are consistently lower when compared to the Kato-Katz method. This is an important issue and warrants additional studies. Importantly, we have shown that the FLOTAC method holds promise for the detection of S. mansoni eggs, particularly in well-homogenized stool samples after preservation in SAF for at least 10 days. Further validation of the FLOTAC technique is under way in different parts of the world, as this technique might become an indispensable tool for patient management and rigorous monitoring of anthelmintic drug efficacy studies and community-based helminth control programs. Supporting Information Checklist S1 STARD checklist (0.89 MB PDF) Click here for additional data file.
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              Mini-FLOTAC, an Innovative Direct Diagnostic Technique for Intestinal Parasitic Infections: Experience from the Field

              Introduction Soil-transmitted helminths (STH) (Ascaris lumbricoides, Trichuris trichiura, hookworm, Strongyloides stercoralis) and intestinal protozoa (e.g. Giardia intestinalis, Entamoeba histolytica/dispar) are widespread in developing countries, especially where poor hygienic conditions facilitate infection with eggs, larvae and cysts through contact with contaminated soil, food or water. Soil-transmitted helminths affect more than 1 billion people worldwide and are part of the neglected tropical diseases (NTDs), which are linked to poverty and underdevelopment [1]. Soil-transmitted helminth infections in endemic countries are a major cause of malnutrition, anemia and growth delay. Often they are linked to minor symptoms such as sub-acute diarrhea or can occur in a subtle and asymptomatic way and are often underreported [2]. Intestinal protozoa infections (especially G.intestinalis and Entamoeba spp.) are of considerable public health importance [3], [4]. For example, Giardia prevalence is 2–7% in developed countries, whereas it is 20–30% in developing countries due to water and food contamination [5]. Around 200 million people are infected around the world with 50,000 new cases occurring every year. E. histolytica infects hundreds of millions of people per year; while most individuals are asymptomatic, perpetuating the natural cycle of the organism through fecal excretion of infective cysts, a minority suffers from the severe morbidity associated with invasive disease (approximately 50 million) with an estimated 100,000 dying every year from severe and invasive amebiasis [6]. The technique recommended for the qualitative diagnosis of intestinal parasites (both helminths and intestinal protozoa) is the formol-ether concentration method (FECM) [7], [8], [9] performed on three different samples, but the direct fecal smear on a single sample is used as diagnostic method more often in resource-constrained settings. The recommended quantitative technique for the diagnosis of soil-transmitted helminths is the Kato-Katz method, except for S. stercoralis, which requires the Bearmann, the Koga agar plate or the Harada Mori method as recommended direct diagnostic techniques [8]. Recently, fecal egg count (FEC) techniques used for the diagnosis of helminths in veterinary parasitology, such as the FLOTAC [10], [11] and the McMaster [12] techniques have been successfully used for diagnosis of soil-transmitted helminths infections in humans [13], [14], [15], [16]. Molecular tools such as polymerase chain reaction (PCR) and multiplex PCR are now increasingly common as diagnostic methods for parasitic infection in laboratories where the equipment is available, although this is often not the case in rural and district laboratories in developing countries [17], [18], [19], [20]. Despite their high sensitivity, a main limitation of the FLOTAC techniques is the complexity of the method which involve centrifugation of the sample with a specific device, equipment that is often not available in laboratories in developing countries [14]. To overcome this bottleneck, under the “FLOTAC strategy” of improving the quality of copromicroscopic diagnosis, a new simplified device has been developed, namely the mini-FLOTAC. One of the main advantages of this new method is that it can be more easily transferred and carried out in laboratories with limited facilities due to the lack of a centrifugation step. The aim of this study was to evaluate and compare widely used, standard qualitative diagnostic techniques with the mini-FLOTAC in different settings. The current report focuses on the diagnostic accuracy and feasibility of the different methods, placing particular emphasis on the application of the mini-FLOTAC method in rural/peripheral laboratories with basic facilities. Materials and Methods Ethic Statement The overall protocol of the study on the evaluation of the mini-FLOTAC was reviewed and approved by the Ethics Committee of the Faculty of Medicine, San Raffaele Hospital, Milan, Italy. For each proposed study site a separate ethical clearance was obtained from the local review board. All children were given an informed consent form to be read and approved by their parents/guardian before being enrolled into the study. All children diagnosed positive for intestinal parasitic infections by any of the methods performed were treated according to standard protocol in use within the country. Data were kept anonymous and patients were identified by code; the study data were safely filed and stored in a cabinet within the data management unit of each research site and remained confidential. Study Sites The study sites have been chosen among places where hospitals and health facilities are currently collaborating with an Italian non-governmental organization (Italian Association for Solidarity among People) and San Raffaele hospital. In particular, collaboration focused on facilities that were in need of technical support with laboratory diagnosis for intestinal parasitic infections. The first part of the study was carried out in February and March 2012 in a school in Dharamsala, Himachal Pradesh, India. Available data from Tibetan Delek hospital show that the prevalence of intestinal parasitic infection is around 10% in patients seen in the outpatient department, but this number is likely to be underestimated as stool diagnosis is made by direct smear. Published data show that Giardia infection is estimated to be around 20%, and the most common infection among soil-transmitted helminths is A. lumbricoides (11%) [21], [22]. All pupils (n = 80) of a school under the authority of the Department of Education of the Central Tibetan Administration in exile were analyzed for intestinal parasites. The second part of the study was conducted in May and June 2012 in Bukumbi, Mwanza district, Tanzania. In this region the commonest intestinal parasites are hookworm, Schistosoma mansoni, S. stercoralis, G. intestinalis and Entoamoeba spp. as determined from hospital records based on direct fecal smear. One hundred (n = 100) children were randomly selected from the only primary school in Bukumbi. Sample Size and Randomization The sample size has been calculated on the basis of mainly historical and unpublished data on the prevalence of intestinal parasitic infections in the study areas, conservatively estimated to be around 20%. In order to have an ideal number of positive/negatives (50%) to determine comparison among the techniques, the adequate sample size to have a significant difference with 95% confidence interval (CI) and with 80% of power was 88 in each site. The selection between the two primary schools in Dharamsala was made at random and all 80 children were examined in the Tibetan school selected. Three classes of children (grades 2, 3 and 4) from the only primary school in Bukumbi were selected at random and all children in those classes were examined for intestinal parasites. Parasitological Methods Stool containers were distributed to the children together with the consent forms, and the next day one fecal sample (minimum 12 g) was collected from each child and analysed on the same day. Samples were examined in parallel by direct smear, FECM and mini-FLOTAC in the hospital laboratory, and were processed and blindly read by two experienced parasitologists (BB and DI among the authors). In brief, approximately 2 mg of stool were used to perform a direct fecal smear [7]. With regard to the mini-FLOTAC, the technique evolved from FLOTAC techniques [10], [11], adapted in order to perform the techniques without the necessity of a centrifugation step. The mini-FLOTAC comprises two physical components, the base and the reading disc. There are two 1-ml flotation chambers, which are designed for optimal examination of fecal sample suspensions in each flotation chamber (total volume = 2 ml) and which permits a maximum magnification of 400×. Fill-FLOTAC are disposable sampling devices, which are part of the FLOTAC and mini-FLOTAC kits [10], [11]. They consist of a container, a collector and a filter (Figure 1). These kits facilitate the performance of the first four consecutive steps of the mini-FLOTAC techniques, i.e. collection (including weighing), homogenization, filtration and filling. The process of the mini-FLOTAC is illustrated in Figure 2. 10.1371/journal.pntd.0002344.g001 Figure 1 The fill-FLOTAC and the mini-FLOTAC kit. 10.1371/journal.pntd.0002344.g002 Figure 2 The steps of the mini-FLOTAC technique. The stools were processed as follows for the mini-FLOTAC basic technique (analytic sensitivity = 10 eggs or cysts per gram of feces). Eight grams of stool were placed in the fill-FLOTAC, diluted with 8 ml of formalin 5%, and thoroughly homogenized and filtered. Two ml of the suspension (1 g of stool+1 ml of formalin) were directly added to 18 ml of each of the two floatation solutions (FS), namely FS2 (saturated sodium chloride; specific gravity (s.g.) = 1.20) and FS7 (zinc sulphate; s.g. = 1.35). The flotation solutions are the same described in the FLOTAC protocols. The FS2 solution is recommended for the diagnosis of soil-transmitted helminths, the FS7 solution is recommended for S. mansoni and for intestinal protozoa [10]. Two mini-FLOTAC were performed for each sample, one filled with the fecal suspension in FS2 and the other with the fecal suspension in FS7. Before reading the slide and translating the reading dish, an average time of 10 min was needed for the eggs and cysts to float. Two ml of the initial 1∶1 solution (1 g of faeces plus 1 ml of 5% formalin solution) in the fill-FLOTAC were used to perform the FECM according to WHO recommendations [7]. Eggs of STHs were detected and counted. In addition, parasitic elements of other helminth genera (e.g. Strongyloides, Enterobius, Hymenolepis, Taenia) and intestinal protozoa were detected. The comparison between the three techniques was made on qualitative diagnosis as direct smear and FECM are not quantitative methods. Statistical Analysis Results were entered in an Excel file. Analysis was performed using the EpiData program (version 3.1; Area of Health Analysis and Information Systems Pan American Health Organization (PAHO/WHO) January 2006). The results were analysed by 2×2 contingency tables and Cohen's kappa statistics was calculated to assess the agreement among all the three diagnostic techniques. Kappa (k) statistic was employed to determine to strength of agreement using follows criteria: ≤0 = poor, 0.01–0.20 = slight, 0.21–0.40 = fair, 0.41–0.60 = moderate, 0.61–0.80 = substantial and 0.81–1 = almost perfect. We checked for any significant difference by calculating interference about proportions (p) in two independent populations with the level of significance set at p value<0.05, and a 95% CI was calculated. Sensitivity (probability to detect a “true” positive case) and negative predictive value (NPV; probability for a negative result to be a “true” negative case) were calculated for each method. The total number of positive samples detected by any of the methods was taken as diagnostic “gold” standard for each parasite species. Results Out of 200 students that were enrolled, 180, 47% (85/180) girls and 53% (95/180) boys, aged between 6 and 18 years (mean age 12 years) returned the sample and had their stool analysed for intestinal parasites. The results of the study are shown in Table 1, Table 2, and in Figure 3. Overall, 72% (129/180) of the samples were positive for any intestinal parasitic infection, 24% (43/180) carried double infections, and 11% (20/180) three infections or more. 10.1371/journal.pntd.0002344.g003 Figure 3 A) Prevalence of intestinal helminth and protozoa infections detected by each of the three methods. B) Prevalence focus on protozoa, Entamoeba coli, Entamoeba histolytica/dispar and Giardia intestinalis results. 10.1371/journal.pntd.0002344.t001 Table 1 Sensitivity and negative predictive value (NPV) of each diagnostic method. Tot (n = 180) Direct smear FECM Mini-FLOTAC FS2 Mini-FLOTAC FS7 N. positives N. positives sensitivity NPV N. positives sensitivity NPV N. positives sensitivity NPV N. positives sensitivity NPV Helminth 72 19 0.27 0.68 40 0.56 0.78 45 0.63 0.81 67 0.94 0.96 Hookworm 36 10 0.27 0.84 22 0.60 0.90 36 0.97 0.99 33 0.89 0.97 S. mansoni 22 4 0.18 0.89 12 0.36 0.92 0 N/A N/A 18 0.82 0.98 Protozoa 97 66 0.70 0.75 86 0.88 0.88 0 N/A N/A 64 0.68 0.74 E. coli 59 37 0.63 0.84 51 0.87 0.94 0 N/A N/A 42 0.71 0.88 E. histolytica / dispar 48 32 0.67 0.89 33 0.68 0.89 0 N/A N/A 22 0.46 0.84 G. intestinalis 33 26 0.82 0.95 23 0.70 0.94 0 N/A N/A 12 0.40 0.87 10.1371/journal.pntd.0002344.t002 Table 2 Qualitative diagnosis of intestinal helminth and protozoan infections by direct smear, FECM and mini-FLOTAC method. Combined results Direct smear FECM mini - FLOTAC N pos (%) K Cohen (CI 95%)1 N pos (%) N pos (%) N pos (%) K Cohen(CL 95%)2 Helminth 72 (40) 0.41 (0.29–0.52) 19 (11) 40 (22) 68 (38) 0.54 (0.41–0.66) Hookworm 36 (20) 0.53 (0.39–0.67) 10 (6) 22 (12) 36 (20) 0.72 (0.58–0.85) S. mansoni 18 (10) 0.40 (0.19–0.61) 4 (2) 12 (7) 18 (10) 0.49 (0.26–0.72) Protozoa 97 (54) 0.57 (0.48–0.67) 66 (37) 86 (48) 64 (36) 0.70 (0.60–0.80) E. coli 59 (33) 0.64 (0.55–0.74) 37 (21) 51 (28) 42 (23) 0.81 (0.71–0.90) E. histolytica/dispar 48 (27) 0.48 (0.35–0.60) 32 (18) 33 (18) 22 (12) 0.60 (0.43–0.76) G. intestinalis 33 (18) 0.57 (0.42–0.73) 26 (14) 23 (13) 12 (7) 0.66 (0.47–0.84) 1 K Cohen among all methods. 2 K Cohen between two methods. We found a difference in helminth distribution according to the geographical area: hookworm (20%), S. mansoni (10%), S. stercoralis and E. vermicularis (2%) were found in the African school, whereas A. lumbricoides (5%), T. trichiura (2%), H. nana and Taenia spp. (1%) were found in the Indian school. Intestinal protozoa were more homogeneously found in the two study sites. Mini-FLOTAC detected a higher number of helminth infections than the FECM or direct smear (38%, 22% and 11%, respectively) and the differences were significant (p<0.002 and p<0.001, respectively). Mini-FLOTAC had a sensitivity of around 90%, followed by FECM and direct fecal smear (60% and 30% respectively). The FECM detected the highest number of intestinal protozoa infection followed by the direct fecal smear and mini-FLOTAC technique (48%, 37% and 36%, respectively). The differences between FECM and mini-FLOTAC/direct fecal smear were significant (p<0.04 and p<0.02, respectively). Results are shown in Figure 3. The most sensitive method for intestinal protozoa diagnosis was FECM (88%), followed by direct fecal smear and mini-FLOTAC (70% and 68% respectively). The NPV were above 70% with all the three methods for both helminth and intestinal protozoa and they did not differ significantly (Table 1). The agreement between techniques is shown in Table 2. The agreement among the three techniques was only moderate (k = 0.40; p<0.001) and the best match was between FECM and mini-FLOTAC (k = 0.49–0.81; p<0.001). Eighteen children were found positive for other parasitic infections such as A. lumbricoides, E. vermicularis, Hymenolepis nana, S. stercoralis, Taenia spp. and T. trichiura, but they were too few for a meaningful statistic and were not included in the analysis. Considering the practical feasibility of the techniques, mini-FLOTAC took approximately 12 min to process a sample: 2 min to prepare the sample, 10 min to wait for the eggs/cysts to float, and 5–7 min to read the reading grid. The FECM on average took 2 min to prepare the sample, 10 min of centrifugation and 3 min to read the slide (a total of 15 min). The simplest and quickest method to perform was the direct smear as it did not require any further step beyond approximately 3 min for reading of the slide. Discussion For the first time the mini-FLOTAC apparatus was used for detecting soil-transmitted helminths and other helminths and intestinal protozoa and compared with two diagnostic techniques that are widely used in human parasitology. In the present study, adhering to standard protocols, all techniques revealed the same intestinal protozoa species (E. coli, E. histolytica/dispar, G. intestinalis), whereas the mini–FLOTAC revealed more helminth species than the two other techniques (E. vermicularis was found only with mini–FLOTAC and T. trichiura was not detected by the direct fecal smear). The number of individuals identified as positive by each technique varied considerably from one species to another. Our results show, similar to Levecke et al [23] for FLOTAC, that the mini–FLOTAC turned out to be the most sensitive approach (95% of the positive samples) for helminth diagnosis. An important observation of our study is that the diagnostic accuracy of the mini-FLOTAC changes according to the FS used: hookworm and E. vermicularis were more accurately diagnosed by FS2, whilst S. mansoni was diagnosed with a higher sensitivity by the FS7 solution, thus supporting the application of the FLOTAC method [11], [24]. When considering intestinal protozoa infections, the diagnostic pattern changes, and our results demonstrated relatively poor performance of mini-FLOTAC. These data differ substantially from the ones reported by Becker et al. [13], where FLOTAC was found to be more sensitive for E. coli and for G. intestinalis detection, whilst FECM was more sensitive for E.histolytica/dispar. It must be considered however, that the centrifugation step makes the FLOTAC different and not easily comparable to the mini-FLOTAC, especially for the diagnosis of intestinal protozoa. In the present papers we did not compare the FLOTAC with the mini-FLOTAC as the latter is not a replacement of the former, but the two methods should be used in different laboratory settings according to the facilities and equipment available. We realised, however, that a standard direct comparison of the mini-FLOTAC versus the FLOTAC method could be important both to compare the sensitivity of the methods and to evaluate their feasibility and cost-benefits. For the diagnosis of the protozoa in area with heavy parasite load and limited resources, the direct smear could be considered a sufficient method, as in this study it does not lose too much sensitivity compared to the FECM. It is worth noticing that in our study the sensitivity of the direct fecal smear for the diagnosis of G.intestinalis was even greater than the FECM, which could be explained by the fact that most of the samples were positive for trophozoites that were killed by formol-preservation, and therefore resulted negative with the other two techniques. Our study suffers from some limitations, which could partially explain the discordance between the three methods in parasitic diagnosis. The agreement between the three methods was variable (k values ranged between 0.4 and 0.8), but generally moderate (k = 0.6). Low agreement in protozoa diagnosis has been observed in other studies in which the same set of samples was analysed in different referral laboratories [25]. Due to low prevalence of other helminth infections, the findings of this study are mainly based on hookworm and S. mansoni diagnosis and further studies are needed in different settings to formulate more reliable and conclusive recommendations for use of mini-FLOTAC for other soil-transmitted helminths diagnosis. It is important to consider that the mini- FLOTAC technique is still under refinement and two main aspects need to be addressed in more details, especially for intestinal protozoa diagnosis. Firstly, stool consistency plays an important role, especially in light infections, as loose stools contain a lower amount of cysts. Mucous stool could be treated adding a mucosolvant or, where the laboratory facilities can afford it, a centrifugation step with ether could be performed in order to eliminate the debris. The visibility of internal structures is often impaired because of debris, thus rendering complicated the differentiation among Entamoeba spp. Moreover, the reading at high power of magnitude (400×) was not clear as the reading disk of the mini-FLOTAC does not yet allow perfect visibility. Secondly, mini–FLOTAC has been tested only using two different flotation solutions (saturated NaCl and Zinc sulphate), but other FS could be tested and the visibility of the samples compared. Furthermore, the flotation solution interacts with and may alter the external membrane of parasites (such as E. coli, G. intestinalis, S. mansoni) somehow distorting their images from the classical pattern. For this reason, well-trained laboratory technicians are needed for reading mini-FLOTAC and a teaching Atlas that could help reading the sample might be a useful tool to add to the mini-FLOTAC kit. An advantage of the mini-FLOTAC technique is that it permits work with fixed fecal sample, but can also be performed on fresh samples. This allows the possibility of examining the samples in different days and also improves the quality control process; in addition, the combined use of the fill-FLOTAC device prevents any hazard of contamination of the operator. A series of studies is further warranted to compare the quantitative performance of the mini –FLOTAC with the other recommended standard techniques for helminth diagnosis such as the Kato Katz and the McMaster. This is an important aspect in helminth control as intensity of infections measured as eggs per gram is directly related to morbidity, and is an important indicator to monitor the impact of control programs as well as drug efficacy [23], [26]. The preliminary qualitative results of the mini-FLOTAC performance from this study should foster the planning of a robust independent multicentric trial that compares the mini-FLOTAC with standard protocols both on qualitative and quantitative diagnosis of helminth infections. Considering the cost-benefit and the feasibility of the techniques, we demonstrated that mini-FLOTAC takes approximately the same time to process the sample as the FECM. As for the cost of the equipment, the only cost for the mini-FLOTAC (apart the cost of the kit which is presently given free of charge for research purposes) is the purchase of sodium chloride (NaCl) and zinc sulphate (ZnSO4) for the flotation solutions, whereas the FECM requires a centrifuge, formol and ether that are not always easy to purchase, especially in laboratories with limited resources. All mini-FLOTAC reading disks and the fill-FLOTAC containers are reusable after careful washing. However, the fact that zinc sulphate is a quite expensive salt and not easily available in peripheral laboratories should be taken into consideration. To conclude, this study demonstrates that mini-FLOTAC is definitely a sensitive and relatively simple technique for the qualitative diagnosis of helminth infections, whilst it still could be improved for the diagnosis of intestinal protozoa. We believe that this work is a step forward in the fight against NTDs, because neglected diagnostics sustain the vicious cycle of hidden diseases and poor access to clinical management and treatment. Innovative direct diagnostic tools that match modern technology and sensitivity with affordability and feasibility in resource-limited settings are most needed, and the development of the mini-FLOTAC kit is an advance towards this direction. Supporting Information Figure S1 STARD checklist. (DOC) Click here for additional data file. Figure S2 Flow diagram. (DOCX) Click here for additional data file.

                Author and article information

                Revista de Producción Animal
                Rev. prod. anim.
                Ediciones Universidad de Camagüey (Camagüey, , Cuba )
                April 2020
                : 32
                : 1
                : 100-112
                [1] Camagüey orgnameUniversidad de Camagüey Ignacio Agramonte Loynaz orgdiv1Facultad de Ciencias Agropecuarias Cuba
                [2] Camagüey orgnameUniversidad de Camagüey Ignacio Agramonte Loynaz orgdiv1Vicerrectoría de Informatización Cuba
                S2224-79202020000100100 S2224-7920(20)03200100100

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