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      Association between Response to Albendazole Treatment and β-Tubulin Genotype Frequencies in Soil-transmitted Helminths

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          Abstract

          Background

          Albendazole (ABZ), a benzimidazole (BZ) anthelmintic (AH), is commonly used for treatment of soil-transmitted helminths (STHs). Its regular use increases the possibility that BZ resistance may develop, which, in veterinary nematodes is caused by single nucleotide polymorphisms (SNPs) in the β-tubulin gene at positions 200, 167 or 198. The relative importance of these SNPs varies among the different parasitic nematodes of animals studied to date, and it is currently unknown whether any of these are influencing BZ efficacy against STHs in humans. We assessed ABZ efficacy and SNP frequencies before and after treatment of Ascaris lumbricoides, Trichuris trichiura and hookworm infections.

          Methods

          Studies were performed in Haiti, Kenya, and Panama. Stool samples were examined prior to ABZ treatment and two weeks (Haiti), one week (Kenya) and three weeks (Panama) after treatment to determine egg reduction rate (ERR). Eggs were genotyped and frequencies of each SNP assessed.

          Findings

          In T. trichiura, polymorphism was detected at codon 200. Following treatment, there was a significant increase, from 3.1% to 55.3%, of homozygous resistance-type in Haiti, and from 51.3% to 67.8% in Kenya (ERRs were 49.7% and 10.1%, respectively). In A. lumbricoides, a SNP at position 167 was identified at high frequency, both before and after treatment, but ABZ efficacy remained high. In hookworms from Kenya we identified the resistance-associated SNP at position 200 at low frequency before and after treatment while ERR values indicated good drug efficacy.

          Conclusion

          Albendazole was effective for A. lumbricoides and hookworms. However, ABZ exerts a selection pressure on the β-tubulin gene at position 200 in T. trichiura, possibly explaining only moderate ABZ efficacy against this parasite. In A. lumbricoides, the codon 167 polymorphism seemed not to affect drug efficacy whilst the polymorphism at codon 200 in hookworms was at such low frequency that conclusions cannot be drawn.

          Author Summary

          The soil-transmitted helminths (STH) Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus are endemic in many tropical countries. Regular treatment with albendazole or mebendazole is the major means for controlling STHs. However, repeated treatment with the same class of benzimidazole anthelmintics has caused resistance in veterinary parasites, characterized by mutations at either codon 200, 167 or 198 in the β-tubulin gene. There is a concern that resistance may develop in human STH. Drug efficacy and mutation frequencies were assessed in T. trichiura, A. lumbricoides and hookworms collected in Haiti, Kenya and Panama prior to and after albendazole treatment. In T. trichiura from Haiti and Kenya, a significant increase of the frequency of the mutation at codon 200 was identified after treatment and drug efficacy was mediocre. Against A. lumbricoides, albendazole efficacy was good, even though the frequency of a mutation at codon 167 was relatively high, suggesting that, in this nematode, the codon 167 polymorphism does not impact efficacy. In hookworms, the mutation at codon 200 was identified, but at low frequencies and the response to albendazole was good. We conclude that monitoring for possible resistance in control programmes should be undertaken.

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          Arlequin (version 3.0): An integrated software package for population genetics data analysis

          Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies, tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several data types like DNA sequences, microsatellite data, or standard multi-locus genotypes. A Windows version of the software is freely available on http://cmpg.unibe.ch/software/arlequin3.
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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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              Assessment of the Anthelmintic Efficacy of Albendazole in School Children in Seven Countries Where Soil-Transmitted Helminths Are Endemic

              Introduction The three major Soil-Transmitted Helminths (STH), Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) and Necator americanus/Ancylostoma duodenale (the hookworms) are amongst the most widespread parasites worldwide. An estimated 4.5 billion individuals are at risk of STH infection and more than one billion individuals are thought to be infected, of whom 450 million suffer morbidity from their infection, the majority of who are children. An additional 44 million infected pregnant women suffer significant morbidity and mortality due to hookworm-associated anemia. Approximately 135,000 deaths occur per year, mainly due to infections with hookworms or A. lumbricoides [1]. The most widely implemented method of controlling STH infections is through periodic administration of anthelmintics. Rather than aiming to achieve eradication, current control programs are focused on reducing infection intensity and transmission potential, primarily to reduce morbidity and avoid mortality associated with the disease [2]. The benzimidazole (BZ) drugs, i.e. albendazole (ALB) and mebendazole, are the most widely used drugs for the control of STH. While both show broad-spectrum anthelmintic activity, for hookworms a single dose of ALB is more effective than mebendazole [3]. The scale up of chemotherapy programs that is underway in various parts of Africa, Asia and South America, particularly targeting school children, is likely to exert increasing drug pressure on parasite populations, a circumstance that is likely to favor parasite genotypes that can resist anthelmintic drugs. Given the paucity of suitable alternative anthelmintics it is imperative that monitoring programs are introduced, both to assess progress and to detect any changes in therapeutic efficacy that may arise from the selection of worms carrying genes responsible for drug resistance. The well documented occurrence of resistance to anthelmintics in nematode populations of livestock [4], highlights the potential for frequent treatments used in chemotherapy programs to select drug resistant worms. Such an eventuality threatens the success of treatment programs in humans, both at individual and community levels [5]. Although some small scale studies [6], [7], have suggested emerging drug resistance in human STH, these studies should be interpreted with some caution, since suboptimal efficacy could have been due to factors other than drug resistance. Moreover, although for the BZ drugs there are many published studies reporting the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR), the two most widely used indicators for assessing the efficacy of an anthelmintic in human medicine, comparison of such studies is difficult, largely because there is no widely accepted standard operating procedure for undertaking such trials [8]. Published studies are confounded by methodological variations including treatment regimens, poor quality of drugs, differing statistical analyses used to calculate therapeutic efficacy, as well as a range of other problems in study design, such as small sample size, diagnostic methods, variation in pre-intervention infection intensities and confounding factors related to geographical locations. Such variation among studies greatly complicates direct comparison [3]. A World Health Organization-World Bank (WHO-WB) meeting on “Monitoring of Drug Efficacy in Large Scale Treatment Programs for Human Helminthiasis”, held in Washington DC at the end of 2007, highlighted the need to closely monitor anthelmintic drug efficacy and to develop standard operating procedures for this purpose. In a systematic meta-analysis of published single-dose studies, Keiser and Utzinger [8], confirmed that there was a paucity of high quality trials, and that the majority of trials were carried out more than 20 years ago. They recommended that well-designed, adequately powered, and rigorously implemented trials should be undertaken to provide current data regarding the efficacy of anthelmintics against the main species of STH. These should be designed to establish benchmarks (including standard operating procedures) for subsequent monitoring of drug resistance. The objective of the present work was to validate a standard protocol that has been developed for monitoring efficacy of anthelmintics against STH. To give the study wide relevance, we conducted the trial in seven populations in different geographic locations in Brazil, Cameroon, Cambodia, Ethiopia, India, Tanzania and Vietnam. In each of the study sites, different epidemiologic patterns of infection prevail, including different combinations of STH. We assessed the efficacy of a single dose (400 mg) of ALB in terms of the CR and the FECR in school children between 14 and 30 days following treatment. The McMaster egg counting technique was used in a standardized fashion, with rigorous quality control. Levecke et al. [9] reported that the McMaster holds promise as a standardized method on account of its applicability for quantitative screening of large numbers of subjects. This method is the recommended method for measuring fecal egg counts (FEC) when performing FECR for the detection of anthelmintic resistance in veterinary medicine [10], [11]. Methods Study sites This study was carried out in seven different countries covering Africa (Cameroon, Ethiopia and Tanzania), Asia (Cambodia, India and Vietnam) and South-America (Brazil). However, it is important to note, that while we refer to individual countries to identify results from particular trials, we do not make any conclusions about any country as such. Here, names of countries are used only to distinguish between 7 separate trials that were conducted in 7 geographically disparate regions of the world. In total ten study sites with varying STH and treatment history were included. These seven STH endemic countries were selected because of the presence of investigator groups with previous extensive experience in the diagnosis and control of STH. Table 1 provides their specific locations (district/province/state) and treatment history. Both species of hookworms (N. americanus and A. duodenale) were present in all study sites in varying degree with the exception of Brazil where only N. americanus was present. 10.1371/journal.pntd.0000948.t001 Table 1 The location and treatment history of the ten study sites. Country District/Province/State Treatment history Brazil Minas Gerais LSAT since 2007 (ALB) Cambodia Kratie LSAT since 1997, last in 2007 (MBD) Cameroon Loum LSAT (MBD/ALB) since 1999, last in 2008 (MBD) Yoyo No LSAT Ethiopia Jimma No LSAT India Vellore LSAT, since 2001, last in 2008 (ALB) Thiruvanamalai No LSAT Tanzania (Zanzibar) Pemba Island LSAT since 1994, last in 2006 (PZQ, IVM, ALB) Vietnam Thái Nguyên LSAT since 2005 Tuyên Quang No LSAT LSAT: large scale anthelmintic treatment, MBD: mebendazole, PZQ: praziquantel, IVM: ivermectine, ALB: albendazole. Trial design During the pre-intervention survey, school children aged 4 to 18 years at the different study sites were asked to provide a stool sample. For the initial sampling the aim was to enroll at least 250 infected children with a minimum of 150 eggs per gram of feces (EPG) for at least one of the STH. This sample size was selected based on statistical analysis of study power, using random simulations of correlated over-dispersed FEC data reflecting the variance-covariance structure in a selection of real FEC data sets. This analysis suggested that a sample size of up to 200 individuals (α = 0.05, power = 80%) was required to detect a 10 percentage point drop from a null efficacy of ∼ 80% (mean percentage FEC Δ per individual) over a wide range of infection scenarios. Standard power analyses for proportions also indicated that the detection of a ∼10 percentage point drop from a null cure rate required sample sizes up to 200 (the largest samples being required to detect departures from null efficacies of around 50%). Given an anticipated non-compliance rate of 25%, a sample of 250 individuals with >150 EPG pre-treatment was therefore considered necessary at each study site. Fecal samples were processed using the McMaster technique (analytic sensitivity of 50 EPG) for the detection and the enumeration of infections with A. lumbricoides, T. trichiura and hookworms [9]. None of the samples were preserved. Samples which could not be processed within 24 hours were kept at 4°C. A single dose of 400 mg ALB (Zentel) from the same manufacturer (GlaxoSmithKline Pharmaceuticals Limited, India) and same lot (batch number: B.N°: L298) was used at all trial sites. No placebo control subjects were included in the trial for ethical and operational reasons. Between 14 to 30 days after the pre-intervention survey, stool samples were collected from the treated subjects and processed by the McMaster technique. All of the trials were carried out in a single calendar year (2009). Subjects who were unable to provide a stool sample at follow-up, or who were experiencing a severe concurrent medical condition or had diarrhea at time of the first sampling, were excluded from the study. The participation, the occurrence of STH and sample submission compliance for pre- and post-intervention surveys are summarized in Figure 1. 10.1371/journal.pntd.0000948.g001 Figure 1 The participation, occurrence of STH and sample submission compliance for pre- and post-intervention surveys. Subjects who were not able to provide a sample for the follow-up, or who were experiencing a severe current medical condition or had diarrhea at the time of the first sampling were excluded from the trial. The McMaster counting technique The McMaster counting technique (McMaster) was based on the modified McMaster described by the Ministry of Agriculture, Fisheries and Food (UK; 1986) [12]. Two grams of fresh stool samples were suspended in 30 ml of saturated salt solution (density = 1.2). The suspension was poured three times through a wire mesh to remove large debris. Then 0.15 ml aliquots were added to each of the 2 chambers of a McMaster slide. Both chambers were examined under a light microscope using a 100x magnification and the FEC for each helminth species was obtained by multiplying the total number of eggs by 50. Statistical analysis The efficacy of the treatment for each of the three STH was evaluated qualitatively based on the reduction in infected children (CR) and quantitatively based on the reduction in fecal egg counts (FECR). The outcome of the FECR was calculated using three formulae. The first two formulae were based on the mean (arithmetic/geometric) of the pre- and post-intervention fecal egg count (FEC) ignoring the individual variability, whereas the third formula represented the mean of the reduction in the FEC per subject. The latter is the only quantitative indicator of efficacy for which the importance of confounding factors can be assessed by statistical analysis. The CR and the FECR (1-3) outputs were calculated for the different trials, both sexes, age classes (A: 4–8 years; B: 9–13 years and C: 14–18 years) and for the level of egg excretion intensity at the pre-intervention survey. These levels corresponded to the low, moderate and high intensities of infection as described Montresor et al. [13] For A. lumbricoides these were 1–4,999 EPG, 5,000–49,999 EPG and >49,999 EPG; for T. trichiura these levels were 1–999 EPG, 1000–9,999 EPG and >9,999 EPG; and for hookworms these were 1–1,999 EPG, 2,000–3,999 EPG and >3,999 EPG, respectively. In addition, the robustness of the three FECR formulae was explored by comparing the FEC reduction rate obtained from all samples containing STH and those obtained from samples containing more than 150 EPG as recommended in the anthelmintic resistance guidelines of the World Association for the Advancement of Veterinary Parasitology [9]. Finally, putative factors affecting the CR and the FECR (3) were evaluated. For the CR, generalized linear models (binomial error) were built with the test result (infected /uninfected) as the outcome, ‘trial’ (7 levels: trials in Brazil, Cambodia, Cameroon, Ethiopia, India, Tanzania and Vietnam) and ‘sex’ (2 levels: female and male) as factors, and ‘age’ and the log transformed pre-intervention FEC as covariates. Full factorial models were evaluated by the backward selection procedure using the likelihood ratio test of χ2. Finally, the CR for each of the observed values of the covariate and factor was calculated based on these models (The R Foundation for Statistical Computing, version 2.10.0 [14]). For analysis of the data from FECR (3), non-parametric methods were used, because models based on parametric statistics, even with negative binomial error structures, or based on transformed data would not converge satisfactorily as a consequence of the high proportion of FEC with zero EPG. Hence, the impact of the factors ‘trial’ and ‘sex’ were assessed by the Kruskal-Wallis test (for more than 2 group comparisons) and the Mann-Whitney U test, respectively. The correlation between the outputs of FECR (3) and the covariates (age and pre-intervention FEC) was estimated by the Spearman rank order correlation coefficient (SAS 9.1.3, SAS Institute Inc.; Cary, NC, USA). Ethics statement The overall protocol of the study was approved by the Ethics committee of the Faculty of Medicine, Ghent University (Nr B67020084254) and was followed by a separate local ethical approval for each study site. For Brazil, approval was obtained from the Institutional Review Board from Centro de Pesquisas René Rachou (Nr 21/2008), for Cambodia from the National Ethic Commitee for Health Research, for Cameroon from the National Ethics Committee (Nr 072/CNE/DNM08), for Ethiopia from the Ethical Review Board of Jimma University, for India from the Institutional Review Board of the Christian Medical College (Nr 6541), for Tanzania (Nr 20) from the Zanzibar Health Research Council and the Ministry of Health and Social Welfare, for Vietnam by the Ministry of Health of Vietnam. An informed consent form was signed by the parents of all subjects included in the study. This clinical trial was registered under the ClinicalTrials.gov Identifier NCT01087099. Results The cure rate (CR) Overall, the highest CRs were observed for A. lumbricoides (98.2%), followed by hookworm (87.8%) and T. trichiura (46.6%). However, as shown in Table 2, the CRs varied across the different trials, age classes and pre-intervention FEC levels. The differences in CRs between trials were most pronounced for T. trichiura, ranging from 21.0 (Tanzania) to 88.9% (India). The T. trichiura CRs of 100% for the trials in Brazil and Cambodia are not considered here as they were based on only 1 and 2 individuals, respectively. For hookworms and A. lumbricoides, the CRs varied from 74.7 (India) to 100% (Vietnam) and from 96.4 (Tanzania) to 99.3% (Ethiopia and Cameroon), respectively. The CRs for A. lumbricoides in Cambodia (100%) and India (95.2%) are not considered here as they were based on fewer than 50 individuals. The CRs increased over the three age classes (A. lumbricoides: 95.8 to 100%; T. trichiura: 44.7 to 54.1%), except for hookworms where the CRs ranged from 86.1 to 88.3, and then to 87.5%. For each of the three STH, there was a decline in the CR with increasing levels of infection intensities at the pre-intervention survey. The largest drop was observed for T. trichiura, which decreased from 53.9 to 12.5%. For the two other STH, the drop in the CR was less pronounced, ranging from 88.6 to 76.9% for hookworms and only from 98.3 to 95% for A. lumbricoides. The observed differences between sexes were negligible for all three STH. 10.1371/journal.pntd.0000948.t002 Table 2 The cure rate (CR) for treatment with a single dose of albendazole against soil-transmitted helminths. A. lumbricoides T. trichiura Hookworms n CR (%) N CR (%) n CR (%) Country Brazil 50 98.0 1* 100 52 88.5 Cambodia 5* 100 2* 100 127 87.4 Cameroon 298 99.3 386 47.4 140 87.1 Ethiopia 151 99.3 105 85.7 91 98.9 India 21* 95.2 18* 88.9 95 74.7 Tanzania 279 96.4 396 21.0 349 86.8 Vietnam 148 98.6 138 81.2 58 100 Age class A (4–8) 215 95.8 219 44.7 173 86.1 B (9–13) 669 98.8 753 46.3 643 88.3 C (14–18) 68 100 74 54.1 96 87.5 Sex Female 462 98.1 503 48.5 393 89.1 Male 490 98.4 543 44.8 519 86.9 Pre-intervention infection intensity Low 662 98.3 823 53.9 859 88.6 Moderate 270 98.1 215 19.5 40 75.0 High 20 95.0 8 12.5 13 76.9 Total 952 98.2 1046 46.6 912 87.8 *Due to the low number of infected subjects ( 75%). The pre-intervention FEC was probably the most important as it had a considerable effect on the CR of A. lumbricoides (χ2 1 = 4.14, p 99.3%). The results of FECR (3) mostly yielded comparable or lower values than those from FECR (1). The low values (sometimes negative) can be explained by subjects for whom the post-intervention FEC exceeded the pre-intervention FEC. These subjects contributed to a negative FEC reduction rate which had a significant impact on the final FEC reduction rate calculated with FECR (3). This became apparent in the FEC reduction rate for A. lumbricoides, where a Cameroonian male subject of 7 years with a pre-intervention FEC of 100 and a post-intervention FEC of 22,050 EPG, contributed markedly to lowering the overall values for the data-set from the trial in Cameroon (FECR (1): 99.2%; FECR (3): 26.0%). This lowering of FECR (3) compared to FECR (1) for A. lumbricoides also occurred with age class A (FECR (1): 98.9%; FECR (3): −2.7%) and the low pre-intervention infection intensity level (FECR (1): 97.8%; FECR (3): 66.6%), but not for the remaining variables. The number of negative individual FEC reduction rates, and the magnitude of the difference between pre- and post-intervention FEC, both contributed to the discrepancies found for T. trichiura (176 subjects) and hookworms (10 subjects). Robustness of FECR formulae Table 5 summarizes the FEC reduction rates restricted to samples of more than 150 EPG indicating that the results of FECR (1) and FECR (2) remained roughly unchanged. The values from FECR (3) increased and were mostly comparable with those obtained by FECR (1). This change in the results of FECR (3) is due to the exclusion of negative individual FEC reduction rates which mostly occurred among the subjects with low pre-intervention FEC (see also Table 4). Differences of more than 5% between the results of FECR (3) and FECR (1) were limited to T. trichiura (country: Cameroon, India, Tanzania and Vietnam; age class: A and C). 10.1371/journal.pntd.0000948.t005 Table 5 Fecal egg count reduction for samples with a pre-intervention FEC of more than 150 EPG. A. lumbricoides T. trichiura Hookworms n FECR(1)(%) FECR(2)(%) FECR(3)(%) n FECR(1)(%) FECR(2)(%) FECR(3)(%) n FECR(1)(%) FECR(2)(%) FECR(3)(%) Country Brazil 47* 100.0 100.0 100.0 0* _ _ _ 46* 97.5 99.6 97.5 Cambodia 1* 100.0 100.0 100.0 1* 100.0 99.7 100.0 100 97.7 99.6 96.7 Cameroon 266 99.8 100.0 100.0 233 39.9 93.4 50.4 71 93.6 99.5 95.1 Ethiopia 145 100.0 99.9 100.0 72 92.3 99.2 92.6 66 99.6 99.7 99.8 India 17* 98.9 99.9 99.6 11* 72.0 99.1 87.0 83 87.8 99.3 84.2 Tanzania 266 100.0 100.0 99.9 325 58.3 86.6 36.4 281 95.4 99.7 93.1 Vietnam 130 100.0 99.9 99.9 71 93.1 99.2 88.0 19* 100.0 99.7 100.0 Age class A (4–8) 196 99.9 100.0 99.8 153 65.1 94.8 57.2 130 94.7 99.6 94.4 B (9–12) 613 99.8 100.0 99.9 515 48.4 94.1 51.8 460 94.9 99.6 93.2 C (13–18) 63 100.0 100.0 100.0 45 60.2 94.4 46.4 76 96.4 99.6 97.1 Sex Female 428 100.0 100.0 99.9 343 54.0 94.7 57.7 286 95.2 99.6 93.8 Male 444 99.7 100.0 99.9 370 53.0 93.8 48.0 380 94.8 99.6 94.0 Pre-intervention infection intensity Low 582 99.9 99.9 99.9 490 49.0 95.1 50.2 613 94.1 99.6 93.6 Moderate 270 100.0 100.0 100.0 215 58.7 92.2 58.7 40 97.6 99.9 97.1 High 20 99.5 100.0 99.6 8 40.0 88.6 40.1 13 95.9 99.9 96.4 Total 872 99.9 100.0 99.9 713 53.5 94.3 52.7 666 95.0 99.6 93.9 FECR(1): group based and arithmetic mean; FECR(2): group based and geometric mean; FECR(3): individual based and arithmetic. *Due to the low number of infected subjects ( 95% for A. lumbricoides and >90% for hookworms are appropriate thresholds, and that efficacy levels below this should raise concern. The great variability of the FECR for T. trichiura and the relatively low efficacy of ALB, confirmed in this present study, indicate that it is not possible to propose an efficacy threshold for this parasite based on our data. In conclusion, the present study is the first to evaluate drug efficacy of a single-oral dose of ALB on such a scale and across three continents. The results confirm the therapeutic efficacy of this treatment against A. lumbricoides and hookworms, and the low efficacy against T. trichiura. Efficacy varied widely across the seven different trials, particularly in the case of T. trichiura and it remains unclear which factors were principally responsible for this variation, although pre-intervention FEC and age played clear roles in this respect. The FEC reduction rate based on arithmetic means is the best available indicator of drug efficacy, and should be adopted in future monitoring and evaluation studies of large scale anthelmintic treatment programs. Finally, our findings emphasize the need to revise the WHO recommended efficacy threshold for single dose ALB treatments. Supporting Information Checklist S1 CONSORT Checklist (0.22 MB DOC) Click here for additional data file. Protocol S1 Trial Protocol (1.17 MB PDF) Click here for additional data file.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                May 2013
                30 May 2013
                : 7
                : 5
                : e2247
                Affiliations
                [1 ]Institute of Parasitology, McGill University, Sainte-Anne-de-Bellevue, Quebéc, Canada
                [2 ]Department of Infectious Disease Epidemiology, School of Public Health, Faculty of Medicine, St Mary's Campus Imperial College London, London, United Kingdom
                [3 ]Eastern and Southern Africa Center of International Parasite Control, Kenya Medical Research Institute, Nairobi, Kenya
                [4 ]Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America
                [5 ]Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States of America
                [6 ]Sainte Justine Research Centre, Université de Montréal, Montréal, Québec, Canada
                Swiss Tropical and Public Health Institute, Switzerland
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AD RMK TSC MGB CMH MES RKP. Performed the experiments: AD CMH TSC CM JK. Analyzed the data: AD. Contributed reagents/materials/analysis tools: TSC CMH TGS YI. Wrote the paper: AD RKP. Contributed to improving the manuscript: RMK TSC MGB CMH MES JK.

                Article
                PNTD-D-13-00098
                10.1371/journal.pntd.0002247
                3667785
                23738029
                1239bb9e-c977-49b1-a3cc-ece9ebfcd9c4
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 November 2013
                : 16 April 2013
                Page count
                Pages: 11
                Funding
                This study received support from the Atlanta Research and Education Foundation, Inc. in collaboration with the Centers for Diseases Control and Prevention, United States of America; from the Centre for Host-Parasite Interactions, McGill University, and the Partnership for Child Development (PCD) at Imperial College London. AD was the recipient of a fellowship from FQRNT, Québec. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine
                Infectious Diseases
                Neglected Tropical Diseases
                Soil-Transmitted Helminths
                Parasitic Diseases
                Ascariasis
                Helminth Infection
                Hookworm Infection
                Trichuriasis
                Infectious Disease Control

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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