Due to the recent increased use of the McMaster (MM) fecal egg counting method for assessing benzimidazole drug efficacy for treating soil-transmitted helminth (STH) infections, the aim of the current study was to determine the operational value of including the MM method alongside the Kato-Katz (KK) fecal thick smear to increase the diagnostic sensitivity when STHs are co-endemic with trematode helminths (e. g. , Schistosoma mansoni). A cross-sectional study was conducted in school-aged children aged 4-18 years in the northeastern region of the State of Minas Gerais (Brazil), where Necator americanus, Ascaris lumbricoides, Trichuris trichiura, and S. mansoni are co-endemic. One fecal sample from each participant was collected and transported to the field laboratory for analysis. Coprological diagnosis was performed on each fecal sample by three different methods: Formalin-Ether Sedimentation (FES), KK and the MM technique. The diagnostic sensitivity and negative predictive value (NPV) of each technique was calculated using the combination of all three techniques as the composite standard. In order to determine the agreement between the three techniques Fleiss´ kappa was used. Both the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) were calculated using the two quantification techniques (i. e. , the MM and KK). Fecal samples from 1260 children were analyzed. The KK had higher diagnostic sensitivity than the MM for the detection of both A. lumbricoides (KK 97. 3%, MM 69. 5%) and hookworm (KK 95. 1%, MM 80. 8%). The CR of a single dose of mebendazole varied significantly between the KK and MM for both A. lumbricoides (p = 0. 016) and hookworm (p = 0. 000), with lower rates obtained with the KK. On the other hand, the FECR was very similar between both techniques for both A. lumbricoides and hookworm. The MM did not add any diagnostic value over the KK in areas where both STHs and trematodes were co-endemic. The lower sensitivity of the MM would have an important impact on the administration of selective school-based treatment in this area since if only the MM were used, 36 (13. 9%) children diagnosed with A. lumbricoides would have gone untreated.
Despite numerous technological advances in the diagnosis of soil-transmitted helminth (STH) infections, such as the development of multiplex and multi-parallel fecal DNA assays [1,2], coprological microscopy techniques remain the standard for diagnosing these infections in humans [3], including the McMaster (MM) counting method [4] and the Kato-Katz (KK) fecal thick smear [5]. This is due to their simplicity, ease-of-use in the field, and low cost. Moreover, neither method requires expensive or highly calibrated instrumentation such as a real-time PCR instrument (multiplex and multi-parallel fecal DNA assay), so both can be performed on site in resource-limited endemic areas. The KK method has been the standard technique for the detection and quantification of STH and intestinal trematode infections globally for nearly forty years as reviewed in [6–12] and is recommended by the World Health Organization (WHO) for the detection of these infections [13]. However, as pointed out by Levecke et al. (2011) [14], the KK method has limitations with respect to its qualitative and quantitative diagnostic performance when used to assess STHs in areas endemic for multiple helminth species. These limitations are most apparent when hookworm (e. g. , Necator americanus) is one of the co-endemic STHs. With the use of the malachite green-glycerol solution in the KK method, helminth eggs are visualized at different time intervals or “clearing times” after the preparation of a slide. The clearing time for hookworm on a KK slide is limited to 30 minutes after preparation, whereas it is one hour for A. lumbricoides or S. mansoni. However, after 60 minutes, the glycerol solution begins to desiccates the hyaline shell of the hookworm eggs, distorting and eventually collapsing the eggs, so that they are mistaken as something else (e. g. , a vegetable spore) or are reported as entirely absent, hence affecting the specificity and sensitivity of the KK for the detection hookworm infection. In contrast, the eggs from A. lumbricoides or S. mansoni can be visualized after hours or even days after the KK slide has been prepared [15]. Moreover, the quantification of egg excretion (or the intensity of infection) by the KK method as determined by fecal egg counts (FEC) is based on a small fixed volume of feces (41. 7 mg) and not on the mass of the feces submitted [5]. Hence, the quantitative performance of the KK method is controversial as the density of the feces can vary while the intensity of the eggs excreted is expressed the same, as the number of eggs per gram (EPG) of stool [16]. This potential error in FECs obtained by the KK is considered critical in programs monitoring drug efficacy, where it is thought to “introduce variation in the results and broaden the confidence levels of the resulting statistical parameters” [14]. These properties have also impeded standardization of the KK method in large-scale studies conducted at different study sites. For these reasons, the MM was used as the diagnostic method in several recent large-scale multi-site trials [17,18] of efficacy of the anthelminthics albendazole and mebendazole in treating humans for STH infections. While the MM is the standard coprological method to assess STHs in veterinary parasitology, its estimates of FEC, ease of use, and suitability for poorly equipped laboratories, also make it a good choice for public health monitoring of human STHs. The aim of the current study was to determine the operational value of including the MM method in addition to the KK for routine diagnosis in areas co-endemic for several species of helminths (Fig 1). To this end, the MM and the KK methods were performed on the same fecal sample in a cross-sectional study of over 1,200 children aged 4–18 years of age in the northeastern region of the state of Minas Gerais, Brazil, where N. americanus, A. lumbricoides, T. trichiura, and S. mansoni are co-endemic, as shown in our previous studies [19,20]. More specifically, our objectives were to assess: (i) the qualitative diagnostic performance of the KK and MM (e. g. , diagnostic sensitivity and negative predictive value); (ii) the quantitative diagnostic performance of the KK and MM (fecal egg counts); and (iii) the accuracy of both methods for estimating drug efficacy. While recent multinational studies have focused on the relative performances of the MM and KK for monitoring drug efficacy for treatment of STH infections [17,18], to our knowledge, this is the first study to describe the operational value of combining these two techniques for routine public health monitoring of helminth endemicity.
The study was approved by the National Ethics Committee of Brazil (Protocol 454/2009) and the Institutional Review Board of the Centro de Pesquisas René Rachou (Belo Horizonte, Brazil). Written informed consent was obtained from a parent or guardian of every child. This study was conducted in northeastern, Minas Gerais state, Brazil. Details of the study region have been extensively detailed elsewhere [21,22]. Prior studies have confirmed that N. americanus is the sole hookworm species endemic in this region [23,24]. The study was conducted as a school-based survey including children between the ages of 4 and 18 years old, inclusive. First, meetings were held with community members to explain the purpose of the study. For the parasitological survey, school personnel and students were informed 24 hours in advance of sample collection and labeled plastic containers were provided. Students were instructed to deposit one fecal sample of each child into the container and return it to school on the following day. Fresh fecal samples were transported at 4°C and those returned later than 48h after date of distribution were not accepted and new containers were issued. Refrigerated samples were transported to the nearby field laboratory located in Americaninhas, Minas Gerais state, Brazil were they were processed within 24 hours of receipt. Children positive for a STH by any of the three detection techniques described below were treated with a single oral dose of 500 mg mebendazole. Children were asked to provide a second fecal sample 7 to 14 days after treatment to monitor treatment efficacy; these samples were also analyzed by all three coprological techniques. The presence of A. lumbricoides, T. trichiura, hookworm and S. mansoni eggs in feces was determined by trained lab personnel in compliance with standard operating procedures for the 3 different techniques routinely used in the field laboratories of the Centro de Pesquisas René Rachou: a) formalin-ether sedimentation (FES) [25]; b) KK fecal thick smear [5]; and MM counting technique [4]. All data were double-entered in Microsoft Excel (2010 Microsoft Corporation). Statistical analyses were performed using SPSS Statistics for Windows, Version 19. 0 (Copyright IBM Corporation 1994,2013) and GraphPad Prism, Version 6. 0 (2015 GraphPad Software, Inc.) A total of 1,260 children from 23 schools were included in the study, of which 635 (50. 4%) were female and 625 (49. 6%) were male. The ages ranged from 4 to 18 years of age, with a mean age of 10. 1 years. All the children included in the study provided a sufficient volume of feces to perform all 3 diagnostic procedures. A total of 311 children were found to be positive for at least 1 of the 3 STHs and were therefore treated with a single dose of 500 mg mebendazole. Of these, 302 provided fecal samples at follow-up 7–14 days following treatment. A total of 312 children were infected with S. mansoni. Since the MM is a flotation technique and therefore does not detect eggs of S. mansoni, the overall prevalence for this helminth (24. 8%) was calculated using only the FES and KK techniques. Moreover, 46. 2% of the children diagnosed with S. mansoni were co-infected with at least 1 STH. By combining the results from the 3 different techniques into a composite standard outcome measure, the prevalence for each STH was: 22. 8% for hookworm, 20. 6% for A. lumbricoides and 1. 2% for T. trichiura. The overall prevalence and the individual prevalence obtained using each technique is detailed in Table 1. Table 1 summarizes the relative performance characteristics of the 3 different fecal techniques used for the diagnosis of STH infections. Although the technique with the best performance for the diagnosis of hookworm proved to be FES, the kappa statistic (Table 2) showed an almost perfect agreement between all 3 techniques, with a Fleiss’ κ of 0. 887. On the other hand, for A. lumbricoides, the technique with the best performance was the KK. Again, Fleiss’ kappa statistics comparing the techniques showed almost perfect agreement between them with a κ of 0. 843, although Cohen’s kappa statistics showed only substantial agreement between the KK and MM (κ = 0. 794) and between the FES and MM (κ = 0. 791). Regarding the diagnosis of T. trichiura, both FES and KK were equally useful, with almost perfect agreement between them (Cohen’s kappa of 0. 845). On the other hand, the agreement between the KK and MM techniques and between the MM and FES was only substantial (κ = 0. 629 and 0. 698, respectively). The overall agreement between all 3 techniques for T. trichiura as measured by Fleiss’ kappa was of substantial agreement (κ = 0. 694). The comparison of the 2 techniques used for the diagnosis of S. mansoni demonstrated that the KK performed better than the FES, although Cohen’s kappa statistic showed almost perfect agreement between them with a κ of 0. 841 (Table 2). The maximum EPG count, geometric and arithmetic mean of the EPG counts, standard deviation from the mean, and lower and upper 95% confidence intervals for STHs and S. mansoni obtained with the KK and MM counting techniques are summarized in Table 3. This table also summarizes the infection intensities of the positive samples, stratified into light, moderate or heavy infections. The mean EPG was significantly higher with the KK than the MM, as evaluated using the related-samples Wilcoxon Ranked Signed Test (p<0. 001), for hookworm (207. 39 vs. 86. 53 EPG) and A. lumbricoides (3940. 01 vs. 1329. 33 EPG) but not for T. trichiura (p = 0. 396). The maximum EPG value was lower with the KK for T. trichiura (252 vs. 400 EPG), although it was higher for both A. lumbricoides (129,120 vs. 51,250 EPG) and for hookworm (20,616 vs. 3850 EPG). The relationship between the EPG values obtained for each sample by each technique followed a linear trend as shown by scatter plots presented in Fig 2. The correlation between the KK and MM based on Spearman’s correlation coefficient was significant for all three STHs (0. 86 for A. lumbricoides, 0. 88 for hookworm and 0. 63 for T. trichiura). Moreover, when the sensitivity of the KK and MM was calculated according to the classification by infection intensity, the KK proved to be more sensitive than the MM for light, medium and heavy infections. This was true for all three STHs detected in the study (Table 4). In general, the KK technique detected more cases of moderate and heavy intensity infections when compared to the MM counting technique (Table 5). Using Cohen’s kappa statistics (κ), the agreement in the classification between the MM and KK for the STHs showed differences depending on the species being studied (Table 5). For hookworm, the agreement was substantial (κ = 0. 70) for light-intensity infections, slight (κ = 0. 11) for moderate-intensity infections and poor (κ = 0. 00) for heavy-intensity infections. For Ascaris, the agreement for both light- and heavy-intensity infections was slight (κ = 0. 20 and 0. 02, respectively) while for moderate-intensity infections it was fair (κ = 0. 33). The difference observed in A. lumbricoides infection intensities is probably due to the presence of infertile A. lumbricoides eggs since the MM does not detect them because they are too heavy to float. During this study, the presence of infertile A. lumbricoides eggs was annotated and it was observed that the MM does not detect them. In 41 of the 259 cases of A. lumbricoides that were detected (15. 8% of all cases detected); the presence of infertile eggs was observed, but only by the KK method. In 5 of these samples, the MM detected only fertile eggs while the KK detected both fertile and unfertile eggs; the remaining 36 cases that contained only infertile eggs were missed by the MM. For Trichuris, only light-intensity infections were diagnosed by both methods and the agreement between them was slight (κ = 0. 19). As shown in Table 6, the CR was significantly lower when assessed by the KK technique in comparison to the MM for both hookworm (p < 0. 001) and A. lumbricoides (p = 0. 016). Therefore, 22 of the 189 children (11. 6%) who were treated were determined to be cured from A. lumbricoides by the MM technique but were in fact still infected as determined by the KK. Also, after treatment, the MM failed to detect 38 light hookworm infections out of the 212 children treated (17. 9%) that were diagnosed using the KK. On the other hand, 4 children (1. 9%) with light hookworm infections after treatment went undetected by the KK method but were detected by the MM. Nonetheless, this difference in egg detection did not have an impact on the FECR since it was very similar between both techniques. The CR and FECR for T. trichiura were not calculated due to the small number of positive samples found.
Based on the diagnostic sensitivities of the MM and the KK as reported herein, we do not see the value of the extra time, materials, and labor required by the addition of the MM into a diagnostic strategy for STHs in areas where S. mansoni infection is also present, especially resource limited areas such as our study site in Brazil. Initially, we hypothesized that the MM method would improve the sensitivity for the detection of STHs over the use of the KK alone, especially for the diagnosis of hookworm infections. However, as shown here, the inclusion of the MM in our diagnostic workflow did not improve overall sensitivity. Similarly, the MM could not replace the KK in the study area given its lower sensitivity for diagnosing A. lumbricoides and its inability to detect S. mansoni eggs. Nonetheless, when conducting coprological studies in humans, determining which technique to use will depend on the research question, the expected species of interest, the expected prevalence and intensity of each species and finally, the field conditions for implementation—in other words, “finding the right tool for the job”.
Diagnosis of intestinal helminths and Schistosoma mansoni infections is based on the detection of eggs in feces. There are many techniques available for both detection and quantification of infection. For the quantification of helminth infections, the methods traditionally used are the Kato-Katz (KK) fecal think smear in humans, and the McMaster (MM) counting method in animals. Recently, the MM has been used for assessing the efficacy of benzimidazole drugs for treating soil-transmitted helminth (STH) infections in humans. In most parts of the world, however, STHs occur simultaneously with other helminth species, and the MM does not detect other helminth eggs. Therefore, in this study we sought to determine if the use of the MM in an area of Brazil were both STHs and S. mansoni are co-endemic, added any value to the current standard of diagnosis using the KK.
The Right Tool for the Job: Detection of Soil-Transmitted Helminths in Areas Co-endemic for Other Helminths