- Record: found
- Abstract: found
- Article: found
Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming
research-article
Author(s):
Alice V. Easton
,
Rita G. Oliveira ,
Elise M. O’Connell ,
Stella Kepha ,
Charles S. Mwandawiro ,
Sammy M. Njenga ,
Jimmy H. Kihara ,
Cassian Mwatele ,
Maurice R. Odiere ,
Simon J. Brooker ,
Joanne P. Webster ,
Roy M. Anderson ,
Thomas B. Nutman
Publication date (Electronic):
27 January 2016
Journal:
Parasites & Vectors
Publisher:
BioMed Central
Read this article at
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Background
Although chronic morbidity in humans from soil transmitted helminth (STH) infections can be reduced by anthelmintic treatment, inconsistent diagnostic tools make it difficult to reliably measure the impact of deworming programs and often miss light helminth infections.
Methods
Cryopreserved stool samples from 796 people (aged 2–81 years) in four villages in Bungoma County, western Kenya, were assessed using multi-parallel qPCR for 8 parasites and compared to point-of-contact assessments of the same stools by the 2-stool 2-slide Kato-Katz (KK) method. All subjects were treated with albendazole and all Ascaris lumbricoides expelled post-treatment were collected. Three months later, samples from 633 of these people were re-assessed by both qPCR and KK, re-treated with albendazole and the expelled worms collected.
Results
Baseline prevalence by qPCR ( n = 796) was 17 % for A. lumbricoides, 18 % for Necator americanus, 41 % for Giardia lamblia and 15 % for Entamoeba histolytica. The prevalence was <1 % for Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis and Cryptosporidium parvum. The sensitivity of qPCR was 98 % for A. lumbricoides and N. americanus, whereas KK sensitivity was 70 % and 32 %, respectively. Furthermore, qPCR detected infections with T. trichiura and S. stercoralis that were missed by KK, and infections with G. lamblia and E. histolytica that cannot be detected by KK. Infection intensities measured by qPCR and by KK were correlated for A. lumbricoides (r = 0.83, p < 0.0001) and N. americanus (r = 0.55, p < 0.0001). The number of A. lumbricoides worms expelled was correlated ( p < 0.0001) with both the KK (r = 0.63) and qPCR intensity measurements (r = 0.60).
Conclusions
KK may be an inadequate tool for stool-based surveillance in areas where hookworm or Strongyloides are common or where intensity of helminth infection is low after repeated rounds of chemotherapy. Because deworming programs need to distinguish between populations where parasitic infection is controlled and those where further treatment is required, multi-parallel qPCR (or similar high throughput molecular diagnostics) may provide new and important diagnostic information.
Related collections
Most cited references44
- Record: found
- Abstract: found
- Article: not found
Sensitivity of diagnostic tests for human soil-transmitted helminth infections: a meta-analysis in the absence of a true gold standard
1 Introduction Reliable, sensitive and practical diagnostic tests are an essential tool in disease control programmes, including those for neglected tropical diseases. The requirements and expectations for a diagnostic tool in terms of technical performance, feasibility and costs change as control programmes progress through different phases, from initially high levels of infections to the confirmation of absence of infections. More precisely, during initial mapping to identify priority areas for control, when infection levels are typically highest, a diagnostic test with moderate sensitivity is acceptable, although the chosen tool needs to be easy to use, cost-effective and allow for the high-throughput screening of large populations (McCarthy et al., 2012; Solomon et al., 2012). Since mapping data can also serve as a baseline for the monitoring and evaluation of programme impact, diagnostic tests must have sufficient performance to detect changes in the prevalence and intensity of infection (Solomon et al., 2012). In later stages of programmes, when infection prevalence and intensity have decreased significantly, more sensitive diagnostic tools are needed to establish an endpoint of treatment programmes. If test sensitivity is insufficient at this point, light infections might be missed and this runs the risk of stopping control programmes too early, before programme endpoints have been achieved. Highly sensitive tests are also required for surveillance once treatment has been stopped to detect the potential re-occurrence of infections (McCarthy et al., 2012; Solomon et al., 2012). Finally, diagnostic tests play an important role in the assessment of treatment efficacy (Albonico et al., 2012) and in patient management. For the detection of the human soil-transmitted helminth (STH) species, Ascaris lumbricoides, Trichuris trichiura and the hookworms (Necator americanus and Ancylostoma duodenale), The World Health Organization (WHO) currently recommends the use of the Kato-Katz method, based on duplicate slides (WHO, 2002). Other commonly used methods include direct smear microscopy, formol-ether concentration (FEC), McMaster, FLOTAC and Mini-FLOTAC. All of these techniques rely on visual examination of a small sample of stool to determine the presence and number of STH eggs (WHO, 1994). Due to intra- and inter-sample variation in egg counts (Booth et al., 2003; Krauth et al., 2012), microscopy-based techniques can have differing sensitivities, especially in low transmission settings. Moreover, diagnostic methods vary considerably in the quantification of egg counts, which is necessary to establish intensity of infection and to evaluate treatment effects (Knopp et al., 2011; Albonico et al., 2012; Levecke et al., 2014). In order to better understand the suitability of diagnostic tools for various transmission settings and stages of disease control programmes, we performed a meta-analysis of the most commonly used copro-microscopic STH diagnostic tests. Our main study objective was an independent and global assessment of the relative performance of commonly used diagnostic methods for STH, as well as factors associated with heterogeneity in test sensitivity. Previous evaluations of STH diagnostics have generally relied on comparisons with a combined reference standard (generated by adding the results of several compared tests or consecutively obtained samples), an approach which has been widely criticised (Enoe et al., 2000; Ihorst et al., 2007). Moreover, the absence of a common reference standard has been a major obstacle for combined evaluations of diagnostic tests in the form of a meta-analysis. We have addressed this problem by using Bayesian latent class analysis (LCA), which allows simultaneous estimation of the unknown true prevalence of infection and the sensitivities and specificities of compared diagnostic tests. This approach has been previously applied to the evaluation of imperfect diagnostic tests for Chagas disease, leishmaniasis and malaria (Menten et al., 2008; de Araujo Pereira et al., 2012; Goncalves et al., 2012), as well as specific studies evaluating STH diagnostic methods (Booth et al., 2003; Tarafder et al., 2010; Assefa et al., 2014; Knopp et al., 2014). The approach has also been used for the meta-analyses of diagnostic test performance (Ochola et al., 2006; Menten et al., 2008; Limmathurotsakul et al., 2012). The current paper presents a Bayesian meta-analysis of different diagnostic tests for the detection of STH species. 2 Materials and methods 2.1 Literature search A systematic literature search was performed to identify publications presenting the evaluation of diagnostic techniques for the human STH species, A. lumbricoides, T. trichiura and hookworms (N. americanus and A. duodenale). Systematic searches were performed (date of search 25th February 2014) using the electronic databases PubMed (http://www.ncbi.nlm.nih.gov/), MEDLINE and EMBASE (via OvidSP) (http://ovidsp.uk.ovid.com/) and the medical subject headings and search terms as detailed in Supplementary Data S1. Articles were considered if written in English, German, French or Spanish. The search was validated by verifying that a number of previously identified key readings were included in the retrieved search results. The titles of initially obtained search results were screened for suitable content and all abstracts mentioning studies on helminths were retrieved. The abstracts were subsequently screened for studies using more than one diagnostic test for the determination of infections, even if not directly mentioning a comparison of test performances. Full texts were read and information on test outcomes, egg counts, age-groups, countries of the studies and years of publication was extracted where results were presented in a suitable format as explained below. Reference lists were screened for additional publications. The literature selection process is outlined in Fig. 1. Data were collected separately for A. lumbricoides, T. trichiura and hookworms, and restricted to the most commonly used diagnostic methods for STH, namely Kato-Katz (Katz et al., 1972), direct microscopy (WHO, 1994), formol-ether concentration (FEC) (Ritchie, 1948), McMaster (Ministry of Agriculture Fisheries and Food, 1986), FLOTAC (Cringoli et al., 2010) and Mini-FLOTAC (Barda et al., 2013a). Other techniques such as midi-Parasep, Koga Agar Plate, Willis technique and Spontaneous tube sedimentation technique (SSTT) were not included due to a lack of suitable data. As performance during field surveys was the main interest, evaluations of diagnostic tests on samples from diagnostic laboratories of hospitals were excluded. Only data provided in the form of 2 × 2 comparisons (T1+T2+, T1+T2−, T1−T2+, T1−T2−, where T1 and T2 are the two diagnostic methods and + and − indicate the observed positive or negative results) were retained. This also included data for which these 2 × 2 comparisons could be created by transforming the original data provided, e.g. where comparisons were made against a combined ‘gold standard’ of two diagnostic methods. Additionally, data on egg counts obtained by the various techniques were retrieved, including those studies that did not provide data in a suitable format for the LCA. Arithmetic mean egg counts were the most commonly reported measures and therefore used for the analysis. For articles where data could not be directly extracted, corresponding authors were invited to contribute additional study results. Three authors replied and provided four datasets for the analysis; we were also able to contribute a further two datasets to the analysis. 2.2 Bayesian LCA A Bayesian latent class model was used to estimate the sensitivity of different diagnostic tests as described elsewhere (Dendukuri and Joseph, 2001; Branscum et al., 2005). LCA allows estimation of the sensitivity and specificity of imperfect diagnostic tests by assuming a probabilistic model for the relationship between five unobserved, or latent, parameters: true disease prevalence π k and the sensitivities S i , S j and specificities C i , C j of diagnostic methods i and j (Pepe and Janes, 2007). The model additionally incorporates the covariance terms covD ij + , covD ij - to account for conditional dependency between compared diagnostic tests amongst infected and non-infected individuals, which is necessary as the included diagnostic tests are based on the same biological principle (detection of eggs under a microscope) and therefore factors other than the true infection status are likely to influence both test outcomes simultaneously (Dendukuri and Joseph, 2001). Thus, the joint distribution of the results of a 2 × 2 table follows a multinomial distribution, ( X k + + , X k + - , X k - + , X k - - ) ∼ Multi ( p k + + , p k + - , p k - + , p k - - , N k ) with the multinomial probabilities calculated as follows: p k + + = P ( T i + , T j + | k th population ) = [ S i S j + covD ij + ] π k + [ ( 1 - C i ) ( 1 - C j ) + covD ij - ] ( 1 - π k ) p k + - = P ( T i + , T j - | k th population ) = [ S i ( S j - 1 ) - covD ij + ] π k + [ ( 1 - C i ) C j - covD ij - ] ( 1 - π k ) p k - + = P ( T i - , T j + | k th population ) = [ ( S i - 1 ) S j - covD ij + ] π k + [ C i ( 1 - C j ) - covD ij - ] ( 1 - π k ) p k - - = P ( T i - , T j - | k th population ) = [ ( S i - 1 ) ( S j - 1 ) + covD ij + ] π k + [ C i C j + covD ij - ] ( 1 - π k ) The conditional correlations between two test outcomes for infected and non-infected individuals were calculated as ρ D + = covD + S i ( 1 - S i ) S j ( 1 - S j ) and ρ D - = covD - C i ( 1 - C i ) C j ( 1 - C j ) , respectively. Uninformative prior information was provided for the sensitivity and underlying true prevalence (using a beta distribution with the shape parameters alpha and beta equal to 1). For the covariance terms, a uniform prior distribution was assumed with limits as described in Dendukuri and Joseph (2001) and Branscum et al. (2005) to ensure that probabilities are confined to values between 0 and 1. Specificity was included as a fixed term based on the most parsimonious, best-fitting model (i.e. that with the lowest deviance information criterion (DIC) value) and was assumed to be the same for all compared methods. This was justified on the dual assumption that false positives are rarely obtained by any type of copro-microscopic technique (Knopp et al., 2011; Levecke et al., 2011) and the necessity to restrict the number of estimated parameters for the identifiability of the model. The models, built separately for A. lumbricoides, T. trichiura and hookworms, were computed using WinBUGS software version 14 (Spiegelhalter, D., Thomas, A., Best, N., Gilks, W., 1996. BUGS: Bayesian Inference Using Gibbs Sampling. MRC Biostatistics Unit, Cambridge). Models were also developed separately for low and high intensity settings. Stratification was based on reported arithmetic mean egg counts (in eggs per gram of faeces, epg). Empirical cut-offs of 2500 epg, 400 epg and 165 epg average infection intensity were used for A. lumbricoides, T. trichiura and hookworms, respectively. These cut-offs were established based on the overall average infection intensity of studies included in the meta-analysis. Data with only geometric means reported were excluded from this analysis unless the geometric mean, which is lower than the average egg count, exceeded the cut-off value. Further details of model parameterisation, including handling of multiple slides, are provided in Supplementary Data S2. 2.3 Comparison of quantitative performances To compare the various diagnostic tests in terms of their quantitative performance, we compared the arithmetic mean egg count obtained by various techniques. Statistical significance of differences was assessed using the non-parametric paired Wilcoxon signed-ranks test and the linearity of the relationship between counts was assessed by scatter plots of log-transformed (natural logarithm) average egg counts. Moreover, we evaluated the percentage of studies reporting egg counts of other techniques that were lower/higher than the Kato-Katz method, which currently forms the basis of the WHO defined intensity thresholds. To allow for a small variation in counts, egg counts were considered as lower or higher than the Kato-Katz method if these were lower or higher than the Kato-Katz egg count plus or minus 10%. Due to the limited availability of data and the fact that faecal egg counts do not vary significantly by the sampling effort for Kato-Katz analysis, all versions of Kato-Katz were combined (Levecke et al., 2014). 3 Results 3.1 Identification of diagnostic test comparisons The initial literature search identified 56 articles which were retrieved for full-text review. Of these, 32 studies fulfilled the inclusion criteria and 2 × 2 comparison data could be obtained for 20 studies (Table 1) (see Fig. 1 for an outline of literature selection steps). The number of extracted 2 × 2 comparisons by species and diagnostic methods is shown in Fig. 2. The included studies were published between 2003 and 2014 and conducted in 12 countries, primarily among school-aged children. The inclusion of only recent studies was somewhat surprising. Even though the original literature search had retrieved studies published since 1967, the non-availability of 2 × 2 data, the type of compared techniques and the evaluation of methods in laboratory or hospital samples led to their exclusion. The evaluation of diagnostic tests was mainly based on comparison with a combined reference-standard (14 of 20 studies); few studies used predicted estimates as a reference (1/20), an LCA approach (1/20) or a combination of the two (1/20). Three studies did not provide sensitivity estimates. The most widely applied method was the Kato-Katz method in 18 of 20 studies (mostly 1-slide or 2-slides on a single sample). The main characteristics of included studies are summarised in Table 1. 3.2 LCA of diagnostic test sensitivities (presence of infection) For all STH species, the models allowing for dependency between compared diagnostic tests showed a better fit, indicated by a lower DIC (not shown). Significant positive correlation between diagnostic test outcomes for infected individuals was observed, especially for comparisons of a 1-slide 1-sample Kato-Katz test with other diagnostic tests (details are provided in Supplementary Data S2). Taking this dependency into account, the sensitivities of selected diagnostic methods were estimated separately for A. lumbricoides, T. trichiura and hookworm and are provided in Table 2 and Fig. 3. Generally, sensitivities of all compared tests were higher for T. trichiuria (Fig. 3B) than for hookworm (Fig. 3C) and A. lumbricoides (Fig. 3A). The obtained sensitivities were highest overall for the FLOTAC method with 79.7% (95% Bayesian credible interval (BCI): 72.8–86.0%), 91.0% (95% BCI: 88.8–93.5%), and 92.4% (95% BCI: 87.6–96.2%) for A. lumbricoides, T. trichiura and hookworm, respectively (Table 2). The lowest sensitivity was observed for the direct microscopy method with 52.1% (95% BCI: 46.6–57.7%), 62.8% (95% BCI: 56.9–68.9%), and 42.8% (95% BCI: 38.3–48.4%), respectively. The estimated sensitivity of the 2-slide 1-sample Kato-Katz test for A. lumbricoides was 64.6% (95% BCI: 59.7–69.8%), for T. trichiura was 84.8% (95% BCI: 82.5–87.1%) and for hookworm was 63.0% (95% BCI: 59.8–66.4%). These estimates were only a slight improvement upon the sensitivities of a 1-slide 1-sample Kato-Katz test. However, increased sensitivities could be observed for 1-slide Kato-Katz performed on two consecutive samples. The sensitivity for Kato-Katz tests performed on three consecutive samples was only slightly further improved. Test specificities were not the main outcome and were fixed at 99.6% for A. lumbricoides, 97.5% for T. trichiura and 98.0% for hookworm, based upon model fit. 3.3 Effect of infection intensity on diagnostic test sensitivity The obtained sensitivity estimates by intensity group are presented in Table 3 and Fig. 4. For all tests and STH species evaluated in both intensity groups, sensitivity varied markedly and most strongly for the Kato-Katz method. For example, for A. lumbricoides the 1-slide Kato-Katz method had a sensitivity of 48.8% (95% BCI: 37.6–58.2%) in the low intensity group compared with 95.8% (95% BCI: 91.8–98.5%) in the high intensity group. Interestingly, in the low intensity group the sensitivity of Kato-Katz was improved markedly by performance of a second slide on the same sample. The sensitivity of the FLOTAC method was highest at 81.8% (95% BCI: 65.5–90.3%) at low intensity compared with 97.1% (95% BCI: 93.1–99.7%) at high intensity. 3.4 Comparison of quantitative test performances A total of 17, 16 and 27 comparisons of average Kato-Katz A. lumbricoides, T. trichiura and hookworm egg counts with other diagnostic methods were obtained from 11 articles (Table 1, analysis 2). The majority of comparisons were between versions of Kato-Katz and FLOTAC or McMaster techniques. Only a few studies compared egg counts between Kato-Katz and FEC or Mini-FLOTAC methods; none with direct microscopy. Table 4 shows that the FLOTAC method generally underestimates the average egg counts compared with Kato-Katz, even though the difference is not statistically significant for T. trichiura. The McMaster technique, however, resulted in a higher egg count for six of 11 comparisons (55%) for T. trichiura and four of 12 comparisons (33%) for hookworm whilst A. lumbricoides egg counts were significantly lower. The relationships between the logarithmic average measurements of Kato-Katz and FLOTAC or McMaster techniques followed a linear trend as shown by the scatter plots presented in Fig. 5. 4 Discussion A global assessment of STH diagnostic test sensitivities and their extent of variation is required to investigate the suitability of diagnostic tools for different transmission settings or stages of STH control programmes. Here we present, to our knowledge, the first meta-analysis of STH diagnostic method performance using a Bayesian LCA framework to overcome the absence of a true gold standard (Dendukuri and Joseph, 2001; Branscum et al., 2005). Our results demonstrate that sensitivities of evaluated diagnostic tests are low overall and cannot be generalised over different transmission settings. Sensitivity, overall and in both intensity groups, was highest for the FLOTAC method, but was comparable for Mini-FLOTAC and Kato-Katz methods. Test sensitivities are strongly influenced by intensity of infection and this variation needs to be taken into account for the choice of a diagnostic test in a specific setting. Moreover, reduced test sensitivity at low infection intensities is of increasing importance as ongoing control programmes reduce the prevalence and intensity of STH infections within endemic communities. The Kato-Katz method is the most widely used and reported diagnostic method, due to its simplicity and low cost (Katz et al., 1972), and is recommended by the WHO for the quantification of STH eggs in the human stool (WHO, 2002). Even though the overall sensitivity of the Kato-Katz method was low, the results of the stratified analysis suggest a high sensitivity of 74–95% when infection intensity is high, which is likely the case for mapping and baseline assessment. However, the test sensitivity dropped dramatically in low transmission settings, making the method a less valuable option in later stages of control programmes. This is likely a reflection of methodological problems specific to the Kato-Katz method, especially when diagnosing multiple STH species infections, as different helminth eggs have different clearing times (Bergquist et al., 2009). In high intensity settings, little value was added by performing a 2-slide test on the same sample, even though this is the currently recommended protocol; whereas in low intensity settings sensitivity was improved by performing a second slide. Sensitivity increased significantly when performing the Kato-Katz method on multiple consecutive samples, which is most likely explained by daily variations of egg excretions and the non-equal distribution of eggs in the faeces leading to substantial variation in egg numbers between stool samples from the same person (Booth et al., 2003; Krauth et al., 2012). For all investigated STH species, sensitivity was highest for the FLOTAC method, even when evaluated in low intensity settings, a finding which is consistent with previous evaluations (Utzinger et al., 2008; Knopp et al., 2009b; Glinz et al., 2010). However, despite its improved performance compared with other copro-microscopic methods, FLOTAC has several practical constraints including higher associated costs, necessity of a centrifuge and longer sample preparation time, decreasing its value as a universal diagnostic method (Knopp et al., 2009a). To enable its use in settings with limited facilities, the Mini-FLOTAC method, a simplified form of FLOTAC, was developed (Barda et al., 2013a). Our findings suggest that the sensitivity of Mini-FLOTAC is much lower than FLOTAC, and it does not outperform the less expensive Kato-Katz method according to a recent study in Kenya (Speich et al., 2010; Assefa et al., 2014). A recognised advantage of the Mini-FLOTAC method, however, is that it can be performed on fixed stools, enabling processing at a later date in a central laboratory. This can help to increase the quality control process and overcomes some of the logistical difficulties in examining fresh stool samples in the field on the day of collection (Barda et al., 2013a). The obtained Mini-FLOTAC sensitivity estimates have relatively high uncertainty, visible in the wide confidence intervals, probably due to the limited number of studies available for the analysis and their evaluation primarily in low transmission settings, where the number of positive individuals is very limited. The detection or failure of detection of a single individual therefore might have a large impact on the sensitivity estimate. In remote areas where microscopy is often unavailable, studies can also use FEC, which allows the fixation of stool samples for later examination (WHO, 1994); several authors have also suggested the use of the McMaster technique as it is easier to standardise than Kato-Katz (Levecke et al., 2011; Albonico et al., 2012). Overall, the observed relative performances of these diagnostic tests when compared with the Kato-Katz method are consistent with those presented in the literature: the performance of Kato-Katz and McMaster methods were comparable, although this did vary by setting (Levecke et al., 2011; Albonico et al., 2013). Similarly, even though FEC had predominantly lower sensitivity than Kato-Katz in included studies, the reported relative performance varies in the literature (Glinz et al., 2010; Speich et al., 2013). The sensitivity of direct microscopy was consistently lower than the Kato-Katz method. Other available methods which were not included in our meta-analysis due to limited data availability, such as the midi-Parasep, do not show any improved test performance in their previous evaluations (Funk et al., 2013). Although we present an improved approach for evaluating diagnostic test performances, accounting for the absence of a perfect gold standard by estimating the true unmeasured infection status and allowing for conditional dependency between the test outcomes, our analysis is subject to several limitations. The results presented here are limited by the low availability of comparable data for each diagnostic test, especially when performing the analysis stratified by intensity group. Direct microscopy was primarily evaluated in low intensity settings, which could have led to the lower observed sensitivity estimates, whereas the Kato-Katz method was evaluated in a full range of settings. The cut-off value to define high and low intensity groups of study populations was chosen based on the data included in the meta-analysis, but does not necessarily represent two main types of transmission settings. Nevertheless, the groupings demonstrate the substantial differences in test performance across varying infection intensities. As the investigated range of transmission settings was limited, further diagnostic test evaluations in specified transmission settings will be needed to provide concrete test performance estimates for each of the settings. To take into account the conditional dependency between compared diagnostic tests, we used a fixed effects model, assuming that conditional dependency is the same for all study settings. Different approaches allowing for varying correlations by using random effects to model sensitivities and specificities as a function of a latent subject-specific random variable could be explored further (Dendukuri and Joseph, 2001). Moreover, our findings might be biased towards results from studies comparing multiple diagnostic tests at the same time, as these are underpinned by a larger amount of data. Assumptions had to ensure identifiability of the model by limiting the number of parameters to be estimated. We focussed our analysis on the sensitivity of diagnostic tests, assuming that specificity of various methods do not differ largely, and therefore included the specificity of all single sample diagnostic tests as one fixed parameter. This assumption can be questioned, as for example Kato-Katz slides are more difficult to read than FLOTAC slides due to debris (Glinz et al., 2010); however, it is still an improvement on the assumption of 100% test specificity for all diagnostic tests as applied in previous publications (Booth et al., 2003; Knopp et al., 2011; Levecke et al., 2011). Using uninformative priors instead of fixed terms did not improve model fit and led to slightly wider BCIs. Importantly, the current model assumes that sensitivities are identical within all populations, which is not fulfilled if sensitivity varies by study setting (Toft et al., 2005). Indeed, the stratified analysis showed that sensitivity varied by infection intensity; however, there were not sufficient data to obtain good estimates for all tests in various transmission settings. Additionally, sensitivity in a specific study setting might be affected by other factors including stool consistency and diet, standardisation and adherence to protocols, equipment quality and human error (Bogoch et al., 2006; Bergquist et al., 2009; Levecke et al., 2011). To overcome the limited comparability of evaluations from different studies, purposeful evaluations of test sensitivity over a continuous range of infection intensities in comparable populations, for example before and after treatment rounds, are clearly necessary to better refine sensitivity estimates, and could be used to identify intensity categories within which sensitivity remains comparable. Results could then be transformed into recommendations for the use of diagnostic tests for different stages of disease control programmes. The performance of a diagnostic tool should not only be measured in terms of sensitivity, but also needs to consider the ability of the test to quantify faecal egg counts. Current infection and treatment effect indicators are based on the Kato-Katz method, and the question arises whether the increasing use of other methods will constitute a problem for standardised recommendations (WHO, 2002). The comparison of average egg counts obtained by Kato-Katz and FLOTAC methods shows a broad agreement with previous studies with generally higher Kato-Katz egg counts (Knopp et al., 2009b, 2011; Albonico et al., 2013). The quantitative performance of the McMaster technique, however, varied in comparison to the Kato-Katz method as higher McMaster average egg counts were observed in several studies, especially for T. trichiura and hookworms (Levecke et al., 2011; Albonico et al., 2012, 2013). The current analysis has focussed on copro-microscopic diagnostic tests, which are based on examination of stool samples. There is current interest in developing more sensitive assays that allow a high sample throughput for screening of large populations using other biological samples and the simultaneous detection of several parasite species in co-endemic settings (Bergquist et al., 2009; Knopp et al., 2014). Recently, assays based on PCR have been developed for the detection of STH (Verweij et al., 2007; Schar et al., 2013; Knopp et al., 2014); however, we did not include this method in our meta-analysis due to limited data availability from field settings. Nonetheless, a recent study showed that the sensitivity of PCR methods was comparable with the Kato-Katz method, especially in low endemicity settings (Knopp et al., 2014). In conclusion, we provide a first known meta-analysis of the sensitivity and quantitative performance of STH diagnostic methods most widely used in resource-limited settings. Our results show that the FLOTAC method had the highest sensitivity both overall and in low intensity settings; however this technique requires a centrifuge and has relatively low throughput. Our results further show that the sensitivities of the Kato-Katz and Mini-FLOTAC techniques were comparable and in high intensity settings both techniques provide a practical and reliable diagnostic method. A particular advantage of the Kato-Katz method is the ability to simultaneously detect STH and schistosome species at low cost; whereas the Mini-FLOTAC method has the advantage that it can be used on preserved samples. As control programmes reduce the intensity of infection, there is a need for diagnostic methods which are more sensitive than these currently used. In evaluating the performance of new diagnostic methods we recommend a standardised evaluation in multiple transmission settings, using the robust statistical methods presented here, as well as a consideration of the cost-effectiveness of alternative methods (Assefa et al., 2014).
- Record: found
- Abstract: found
- Article: not found
Estimation of diagnostic-test sensitivity and specificity through Bayesian modeling.
Adam J. Branscum, David Johnson, Richard Gardner (2005)
We review recent Bayesian approaches to estimation (based on cross-sectional sampling designs) of the sensitivity and specificity of one or more diagnostic tests. Our primary goal is to provide veterinary researchers with a concise presentation of the computational aspects involved in using the Bayesian framework for test evaluation. We consider estimation of diagnostic-test sensitivity and specificity in the following settings: (i) one test in one population, (ii) two conditionally independent tests in two or more populations, (iii) two correlated tests in two or more populations, and (iv) three tests in two or more populations, where two tests are correlated but jointly independent of the third test. For each scenario, we describe a Bayesian model that incorporates parameters of interest. The WinBUGS code used to fit each model, which is available at http://www.epi.ucdavis.edu/diagnos-tictests/, can be altered readily to conform to different data.
- Record: found
- Abstract: found
- Article: found
Comparing Diagnostic Accuracy of Kato-Katz, Koga Agar Plate, Ether-Concentration, and FLOTAC for Schistosoma mansoni and Soil-Transmitted Helminths
Dominik Glinz, Kigbafori D Silué, Stefanie Knopp … (2010)
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.
Author and article information
Contributors
Alice V. Easton: alice.easton@nih.gov
,
aeaston@alumni.princeton.edu
Rita G. Oliveira: r.oliveira11@imperial.ac.uk
Elise M. O’Connell: elise.o'connell@nih.gov
Stella Kepha: stellakepha2005@yahoo.com
Charles S. Mwandawiro: CMwandawiro@kemri.org
Sammy M. Njenga: SNjenga@kemri.org
Jimmy H. Kihara: jimmykihara09@gmail.com
Cassian Mwatele: cmwatele@gmail.com
Maurice R. Odiere: MOdiere@kemricdc.org
Simon J. Brooker: simon.brooker@gatesfoundation.org
Joanne P. Webster: jowebster@rvc.ac.uk
Roy M. Anderson: roy.anderson@imperial.ac.uk
Thomas B. Nutman: tnutman@niaid.nih.gov
Journal
Journal ID (nlm-ta): Parasit Vectors
Journal ID (iso-abbrev): Parasit Vectors
Title:
Parasites & Vectors
Publisher:
BioMed Central
(London
)
ISSN
(Electronic):
1756-3305
Publication date
(Electronic):
27
January
2016
Publication date PMC-release: 27
January
2016
Publication date Collection: 2016
Volume: 9
Electronic Location Identifier: 38
Affiliations
[
]Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College
London, London, UK
[
]Eastern and Southern Africa Centre of International Parasite Control, Kenya Medical
Research Institute, Nairobi, Kenya
[
]Neglected Tropical Diseases Research Unit, Center for Global Health Research, Kenya
Medical Research Institute, Kisumu, Kenya
Article
Publisher ID: 1314DOI: 10.1186/s13071-016-1314-y
PMC ID: 4729172
PubMed ID: 26813411
SO-VID: 857042cb-05d9-4513-9c31-d718a7e5b6bb
Copyright © © Easton et al. 2016
License:
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
History
Date
received
: 19
November
2015
Date
accepted
: 5
January
2016
Categories
Subject:
Research
Custom metadata
issue-copyright-statement © The Author(s) 2016
ScienceOpen disciplines: Parasitology
Data availability:
ScienceOpen disciplines: Parasitology