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Comparison of parasitological methods for the identification of soil-transmitted helminths, including Strongyloides stercoralis, in a regional reference laboratory in northwestern Argentina: An observational study
research-article
Author(s):
Elvia Nieves
a
,
b ,
Pedro Fleitas
c ,
Marisa Juárez
a
,
e ,
Cristina Almazán
a
,
b ,
Gabriela Flores
a ,
Jimena Alani
a ,
Ramón Diaz
a ,
Jorge Martos
a ,
Pamela Cajal
a
,
f
,
g ,
Rubén Cimino
a
,
b
,
d ,
Alejandro Krolewiecki
a
,
b
,
*
Publication date (Electronic):
20 July 2024
Journal:
Parasite Epidemiology and Control
Publisher:
Elsevier
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Abstract
Soil-transmitted helminths (STH) are a significant public health problem in impoverished communities of tropical and subtropical areas. Improved diagnostic methods are crucial for Neglected Tropical Diseases programs, particularly for S. stercoralis, as traditional methods are inadequate. Thus, it is important to identify the most accurate and efficient methods for the diagnosis of STH. We performed a retrospective study analyzing laboratory data at the Instituto de Investigaciones de Enfermedades Tropicales from 2010 to 2019. The study included data from outpatients referred for stool analysis and public health interventions from urban and rural communities in northern Salta province, Argentina. Samples were included in this analysis if processed through sedimentation/concentration, Baermann, Harada-Mori and McMaster's, with a subgroup that also included Agar plate culture method (APC). Sensitivity was calculated against a composite reference standard. Of the 5625 samples collected, 944 qualified for this analysis, with a prevalence of 11.14% for A. lumbricoides, 8.16% for hookworm, 1.38% for T. trichiura, and 6.36% for S. stercoralis. The sedimentation/concentration method was the most sensitive for A. lumbricoides (96%), compared to the McMaster method, with a sensitivity of 62%. Similarly, for hookworms, sedimentation/concentration was more sensitive than McMaster's, Harada-Mori, and Baermann with sensitivities of 87%, 70%, 43%, and 13%, respectively. Most of these infections were of light intensity. For S. stercoralis, Baermann and sedimentation/concentration methods were the most sensitive, with 70% and 62% respectively, while Harada-Mori was the least sensitive. In a subset of 389 samples also analyzed by the APC, Baermann was more sensitive than APC for detecting S. stercoralis, and both methods were superior to Harada-Mori. Parasitological methods, mostly when used combined, offer adequate opportunities for the diagnosis of STH in clinical and public health laboratories. The incorporation of S. stercoralis into the control strategies of the World Health Organization requires rethinking the current diagnostic approach used for surveys. With sedimentation/concentration and Baermann appearing as the most sensitive methods for this species. Further studies, including implementation assessments, should help in identifying the most adequate and feasible all-STH diagnostic approach.
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Most cited references32
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2017 Infectious Diseases Society of America Clinical Practice Guidelines for the Diagnosis and Management of Infectious Diarrhea
Andi L Shane, Rajal K. Mody, John Crump … (2017)
These guidelines are intended for use by healthcare professionals who care for children and adults with suspected or confirmed infectious diarrhea. They are not intended to replace physician judgement regarding specific patients or clinical or public health situations. This document does not provide detailed recommendations on infection prevention and control aspects related to infectious diarrhea.
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Diagnostic Accuracy of Five Serologic Tests for Strongyloides stercoralis Infection
Zeno Bisoffi, Dora Buonfrate, Marco Sequi … (2014)
Introduction Strongyloides stercoralis (S. stercoralis) is a nematode widely distributed all over the world, in areas where poor hygienic conditions permit the maintenance of its transmission. In the human host the infection is characterized by an autoinfective cycle, that can lead to life-long carriage of the parasite if left untreated [1]. For this reason, chronically infected patients are often found even in areas where transmission no longer occurs [2]. Chronic infection is often clinically silent. It is crucial, however, to detect and treat the infection in order to avoid the risk of the life-threatening complications (hyperinfection and dissemination) that can develop in the face of immunosuppression (e.g. underlying medical conditions and/or iatrogenic [steroids, other immunosuppressive agents]) [3]. Proper diagnostic testing is crucial both to identify S. stercoralis-infected individuals and to evaluate the prevalence of the infection among populations. One of the main problems with S. stercoralis is that its overall prevalence is probably underestimated [4], mostly due to the lack of sensitivity of fecal – based tests that are the most commonly used assessments for S. stercoralis infection. Serologic tests are also very useful, but their specificity is variable [5] and more difficult to assess because of the unreliability of the used reference test, i.e. microscopy. Discordant (fecal negative – serological positive) samples cannot be clearly defined. Furthermore, specificity is likely to be variable in different population groups and to be better in environments where other intestinal parasites are rare or absent, while sensitivity may be sub optimal in immunosuppressed patients [6]. An ideal diagnostic tool for S. stercoralis should have a very high sensitivity when used for screening (i.e. candidates for transplantation, chemotherapy, systemic corticosteroids) as well as to detect persistence of infection after treatment (therapeutic failure). Ideally the test should become negative or consistently show a marked decrease in titer in a predictable time after successful treatment. Although some studies document a decline of antibody titer after effective treatment, a clear cut-off value has yet to be defined [7], [8], [9], [10]. For a clinical trial, however, a very high specificity is needed in order to avoid inclusion of false positive subjects. The main objective of the present study was to assess the accuracy of five serologic methods for the diagnosis of S. stercoralis infection in different patient populations. The serologic tools are intended for use both in highly endemic settings (screening of subjects at risk for complications, prevalence studies, clinical diagnosis in adequately equipped laboratories) and in areas of low or no endemicity (screening and diagnosis of immigrants, travelers, and autochthonous infection in elderly patients in countries previously endemic such as in Southern Europe). Methods Conduct of the study The study was carried out in two reference laboratories for parasitic diseases (CTD Negrar - Verona, Italy and NIAID-NIH, Bethesda, US) by well-trained staff members. Samples were selected from a composite study population that is described in detail below. As fecal based methods are virtually 100% specific but lack sensitivity [10], [11], [12], a composite reference standard was also used (see below) as a suggested procedure for the evaluation of diagnostic tests when there is no gold standard [13], [14]. Study design The study was designed as a retrospective comparative diagnostic study on archived, anonymized serum samples. Sensitivity, specificity and positive and negative predictive values (PPV, NPV) of the index tests calculated against the primary reference standard (direct demonstration of Strongyloides larvae in stools by microscopy or culture) was used as the primary endpoint. A secondary endpoint was a test's sensitivity, specificity and predictive values when compared to a composite reference standard (as defined below). Study samples The study was carried out on fully anonymized, coded serum samples already available at CTD that were selected randomly, within each study group outlined below. The archived specimens were kept frozen at −80°C from the day of the sample collection and tests were executed within 24 hours of unfreezing. Inclusion criteria Serum specimens were selected from a composite patient population including: Group I - Subjects of all ages with S. stercoralis larvae in fecal specimens, identified by microscopy and/or culture (primary reference standard) Group II - Subjects with no previous exposure to S. stercoralis: healthy blood donors and patients of all ages, born and resident in non-endemic areas of Europe and with no travel history to endemic countries. Group III - Subjects with potential, previous exposure to S. stercoralis but with negative fecal tests for strongyloidiasis: a) subjects routinely screened for parasites, with no known parasitic infections. b) patients with other parasitic infections (see below for details). Exclusion criteria Group I - Hyperinfection syndrome (HS) or disseminated strongyloidiasis (DS). HIV patients with CD4+ cells 50 years; previous residence in areas where Strongyloides transmission was known to occur in past decades Group III - HIV patients with CD4+ cells 70% sensitivity. Such standard and available tests could be used both in clinical and public health practices. It must be mentioned, however, that tests based on crude antigen may be difficult to ensure optimal reproducibility among different batches. We strongly recommend laboratories using these tests to put into place clear quality control methods. Study limitations This study has the potential limitations inherent to a retrospective study design. Some quite relevant data were missing for some of the control subjects (i.e. the continent of exposure when/if it did not coincide with the continent of origin). Moreover, as parasitological methods are not 100% sensitive, also for other parasitic infections, it may well be that some infections were missed in control subjects exposed, which may have caused cross reactivity. While we believe that subjects were better classified using the composite reference standard, we cannot exclude a possible misclassification of some of them. Conclusion and further research needs The issue of serology as a marker of cure remains an open question. If we were to rely on fecal-based diagnosis alone, we may wrongly consider cured a patient whose parasite load after treatment is too low to be detected. Thus, an evaluation of serologic tests to assess cure is currently underway. A prospective study that will include PCR on fecal samples is also planned. The ultimate aim is to identify the optimal diagnostic strategy for S. stercoralis for clinical and epidemiological purposes. Supporting Information Figure S1 STARD flow chart. (DOC) Click here for additional data file. Figure S2 ROC curve for IVD ELISA (primary reference standard). (JPG) Click here for additional data file. Figure S3 ROC curve for Bordier ELISA (primary reference standard). (JPG) Click here for additional data file. Figure S4 ROC curve for NIE-LIPS (primary reference standard). (JPG) Click here for additional data file. Figure S5 ROC curve for IFAT (primary reference standard) (numbers correspond to titers, 3 = 1/20 to 9 = 1/1280). (JPG) Click here for additional data file. Figure S6 ROC curve for NIE-ELISA (primary reference standard). (JPG) Click here for additional data file. Table S1 STARD checklist for reporting of studies of diagnostic accuracy. (DOC) Click here for additional data file. Table S2 Test accuracy (composite reference standard) at different cut-off levels of the index tests. (DOC) Click here for additional data file. Table S3 Positive and negative predictive values (PPV, NPV) for different theoretical prevalence levels. (DOC) Click here for additional data file. Table S4 Positive and negative predictive values (PPV, NPV) for different theoretical prevalence levels. (DOC) Click here for additional data file. Table S5 Concordance between pairs of index tests (Kappa test). (DOC) Click here for additional data file.
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A Comparison of the Sensitivity and Fecal Egg Counts of the McMaster Egg Counting and Kato-Katz Thick Smear Methods for Soil-Transmitted Helminths
Bruno Levecke, Jerzy Behnke, Sitara S. R. Ajjampur … (2011)
Introduction Infection with soil-transmitted helminths (STH), including Ascaris lumbricoides, Trichuris trichiura and hookworm (Ancylostoma duodenale and Necator americanus) are of major importance for public health in tropical and subtropical countries [1], [2]. Current approaches proposed for controlling STH infections entail periodic large-scale administration of anthelmintic drugs, particularly targeting school-aged children [3], [4]. Since such large-scale interventions are likely to intensify as more attention is given to these neglected tropical diseases [5], monitoring drug efficacy will assume increasing importance for assessment of drug efficacy [6] and for detection of the emergence of resistance [7], [8]. A weakness of published studies reporting anthelmintic efficacy in human trials has been the focus on qualitative diagnosis of infections (presence/absence of STH eggs in stool) after treatment, that is, on the cure rate. Quantitative studies, reporting the reductions in the number of eggs excreted are published more rarely (fecal egg count reduction (FECR)) [9], yet are likely to provide the best summary measure for assessment of anthelmintic efficacy in large-scale treatment programs [10]. Although this implies the need for methods to accurately quantify egg excretion levels, studies where more than one coprological method based on fecal egg counts (FEC) has been used, are scarce. In addition, little is known about the variability in qualitative and quantitative diagnosis by these methods between different laboratories [11] or about the accuracy of the methods for estimating drug efficacies in monitoring programs. To date, the Kato-Katz thick smear method (Kato-Katz) is the diagnostic method recommended by the World Health Organization (WHO) for the quantification of STH eggs in human stool [12], because of its simple format and ease-of-use in the field. The chief limitation of the Kato-Katz method, however, arises when it is used with the objective of simultaneous assessment of STH in fecal samples from subjects with multiple species infections. This is because helminth eggs of different species of helminths appear at different time intervals (clearing times). In addition, hookworm eggs rapidly disappear in cleared slides, resulting in false negative test results if the interval between preparation and examination of the slides is too long (>30 min). These properties have impeded standardization of the Kato-Katz method in large-scale studies at different study sites [13]–[15]. Moreover, quantification of the intensity of egg excretion is based on a fixed volume of feces, rather than the mass of feces examined. Its quantitative performance is, therefore, questionable, as the intensity of eggs excreted is expressed as the number of eggs per gram of stool (EPG) [16], and the density of feces can vary. This potential bias in the value of FEC is likely to be important in programs monitoring drug efficacy by the Kato-Katz, where it may introduce additional variation in the results of FECR and broaden the confidence levels of the resulting statistical parameters. A recent study in non-human primates, demonstrated that the McMaster egg counting method (McMaster) holds promise for the assessment of the efficacy of anthelmintics by FECR [17], as it provided accurate estimates of FEC, and was very easy to use, making it particularly suitable for use in poorly equipped and often short-staffed laboratories. However, despite the fact that McMaster is the method of choice for efficacy monitoring programs in veterinary medicine [18], its performance for the detection and enumeration of STH eggs in human public health remains unknown. Therefore, a multinational study was conducted to evaluate the relative performance of the McMaster and Kato-Katz methods for monitoring drug efficacy in STH in humans. To this end, these methods were compared for both qualitative and quantitative detection of STH in human populations in Brazil, Cameroon, India, Tanzania and Vietnam. The three specific objectives of the current study were (i) to assess the consistency of the performance of these two methods in trials conducted in these different countries located in three continents; (ii) to validate the fixed multiplication factor employed in the Kato-Katz method; and (iii) to assess the accuracy of both methods for estimating drug efficacies based on FECR. Methods Ethics statement The overall protocol of the study was approved by the ethics committee of the Faculty of Medicine, Ghent University (Nr B67020084254), followed by a separate local ethical approval for each study site. For Brazil, approval was obtained from the institutional review board from Centro de Pesquisas René Rachou (Nr 21/2008), for Cameroon from the national ethics committee (Nr 072/CNE/DNM08), for India from the institutional review board of the Christian Medical College (Nr 6541), for Tanzania (Nr 20) from the Zanzibar Health Research Council and the Ministry of Health and Social Welfare, for Vietnam by the Ministry of Health of Vietnam. All subjects included in the study, or the parents in the case of school-aged children, signed an informed consent form. The clinical trial in this study was registered under the ClinicalTrials.gov identifier NCT01087099. Study sites and population The study was undertaken in five countries across Africa (Cameroon, Tanzania), Asia (India, Vietnam) and South America (Brazil). For Brazil, Cameroon, Tanzania, and Vietnam, the subjects involved also participated in a multinational trial of the efficacy of a single-dose albendazole (400 mg) against STH infections, which has been presented elsewhere [10]. It is important to note that here we do not make comparison between countries as such, but rather between five distinct trials conducted in five countries in geographically contrasting regions of the world, and reference to country is only for the purpose of distinguishing between specific trials. For this multinational efficacy trial, only subjects meeting the required criteria were included: attending school, aged of 4–18 years, not experiencing a severe concurrent medical condition or diarrhea at time of first sampling. For the trial conducted in India, stool samples of patients presented at the Christian Medical College hospital in August 2009 were included. A subset of at least 100 subjects (first screened) from each site was included in the analysis. This sample size was based on available prevalence data [19]–[23], and was sufficient in size to enable analysis by logistic regression modeling (10 infected subjects per predictor included in the model) [24]. Parasitological methods All stool samples were processed by the McMaster and the Kato-Katz methods as described below. For each stool sample, both methods were applied on the same day by experienced laboratory technicians blinded to any preceding test results. McMaster The McMaster method was based on the modified McMaster described by the Ministry of Agriculture, Fisheries, and Food (1986) [25]. Two grams of stool were suspended in 30 ml of saturated salt solution at room temperature (density ∼1.2, prepared by adding NaCl to 5 l of warm distilled water (40–50°C) until no more salt went into solution and the excess settled on the bottom of the container). The fecal suspension was poured three times through a wire mesh (aperture of 250 µm) to remove large debris. Then, 0.5 ml aliquots were added to each of the two chambers of a McMaster slide (http://www.mcmaster.co.za). Both chambers were examined under a light microscope using a 100x magnification and the FEC, expressed as EPG for each helminth species, were obtained by multiplying the total number of eggs by 50. A tutorial for performing the McMaster is made available at http://www.vetparasitology.ugent.be/page30/page30.html. Kato-Katz The Kato-Katz thick smears were prepared as described by WHO (1991) [12] on microscope slides using a square template with a hole diameter of 6 mm and depth of 1.5 mm, which is assumed to sample 41.7 mg of feces. All samples were examined within 30–60 min for the presence of hookworm and re-examined after ∼2 hours for the remaining STH eggs. The number of helminth eggs was counted on a per species basis and multiplied by 24 to obtain the FEC in units of EPG. In addition, in Tanzania and Cameroon, the validity of the multiplication factor was investigated by weighing the mass of feces examined, and then by comparing FEC based on the fixed multiplication factor of 24 with those based on a multiplication factor adjusted for the actual weight of the amount of feces examined. To this end, microscope slides were weighed (scale precision of 0.01 g) individually, without and with their aliquot of stool. The multiplication factor adjusted for the mass of feces examined was therefore 1 over the mass of the feces examined in grams (mass slide with feces – mass slide without feces). Statistical analysis As described below both diagnostic methods were compared qualitatively (sensitivity and negative predictive value (NPV)) and quantitatively (FEC) for each of the three STH species. In addition, the validity of the fixed multiplication factor for the Kato-Katz was examined. Finally, the accuracy of both methods for estimating drug efficacy by means of FECR was assessed. Both the qualitative and quantitative comparisons for each of the three STH separately were based only on subjects meeting the following inclusion criteria: (i) excreting STH eggs and (ii) originating from a trial were a minimal of 30 infected subjects were detected at the initial survey. The number of subjects enrolled, the occurrence of STH and the number of subjects included for this qualitative and quantitative comparison are shown in Figure 1. 10.1371/journal.pntd.0001201.g001 Figure 1 The number of subjects involved in the statistical analysis for agreement in test results. Qualitative agreement Sensitivity was calculated for each method, using the combined results of both methods as the diagnostic ‘gold’ standard. Therefore, the specificity of both methods was set at 100%, as indicated by the morphology of the eggs. Differences in sensitivity between methods was assessed by the Z-test. The variation in sensitivity within each method was explored by a logistic regression model, which was fitted for each of the two methods with their test result (positive/negative) as the outcome, the mean FEC of both methods as covariate, and trial as a factor (five levels: Brazil, Cameroon, India, Tanzania, and Vietnam). The final models were evaluated from the full factorial model (including interactions) by a backward selection procedure (least significant predictor was step wise omitted from the model) using the χ 2 likelihood ratio statistic. The level of significance was set at p 0.5 was set as a positive test result, and negative if different. Finally, the sensitivity for each of the observed values of the covariate and factor, was calculated based on these models (R Foundation for Statistical Computing, version 2.10.0). The NPV was calculated according the theorem of Bayes. The 95% confidence intervals (CIs) for NPV were obtained by statistical simulation (R Foundation for Statistical Computing, version 2.10.0). Quantitative agreement The agreement in quantitative test results was estimated by the Spearman rank correlation coefficient (Rs) (SAS 9.1.3, SAS Institute Inc.; Cary, NC, USA). The Wilcoxon signed rank test was used to test for differences in FEC between the methods. Furthermore, samples were subdivided into low, moderate, and high egg excretion intensities according to thresholds proposed by WHO [9]; for A. lumbricoides these were 1–4,999 EPG, 5,000–49,999 EPG, and >49,999 EPG; for T. trichiura these were 1–999 EPG, 1000–9,999 EPG, and >9,999 EPG; and for hookworm these were 1–1,999 EPG, 2,000–3,999 EPG, and >3,999 EPG, respectively. Finally, the agreement in the assignment to these three levels of egg excretion intensity by the McMaster and Kato-Katz methods was evaluated by the Cohen’s kappa statistic (κ). The value of this statistic indicates a slight (κ 80%) for each of the three STH in all trials (Table 1), except for the detection of T. trichiura (McMaster: 65.2%; Kato-Katz: 63.5%) and hookworm (McMaster: 73.4%; Kato-Katz: 72.9%) in Tanzania. In the majority of the cases, there was a large overlap in the 95% CI of both diagnostic methods, except in the Brazilian trial for the detection of A. lumbricoides (McMaster: [87.8–94.2%] vs. Kato-Katz: [100–100%]) and hookworm ([88.3–94.7%] vs. [96.9–99.7%]) and in Cameroon for T. trichiura ([70.4–90.5%] vs. [89.4–100%]). In each of these trials, the overlap was either small or absent. Agreement in quantitative test results Overall there was a significant correlation between the FEC of the McMaster and those obtained by Kato-Katz (A. lumbricoides: Rs = 0.70, n = 312, p<0.001; T. trichiura: Rs = 0.49, n = 345, p<0.001; hookworm: Rs = 0.32, n = 290, p<0.001) (Table 2). Assessment of egg excretion intensity by the Kato-Katz resulted in significantly more eggs of A. lumbricoides (14,197 EPG vs. 5,982, n = 312, p<0.001), but not for hookworm (468 EPG vs. 409, n = 290, p = 0.10) and T. trichiura (784 EPG vs. 604, n = 345, p = 1.00). However, these findings were not consistent across the different trials. A significant positive correlation between both methods was found for each of the three STH in all countries (Rs = 0.28–0.88, p<0.05), except for trials in Tanzania and Vietnam. In Tanzania, no significant correlation was found between the two methods for the quantification of hookworm eggs (Rs = −0.05, n = 116, p = 0.56), while in the trial in Vietnam, a significant negative correlation was found for T. trichiura (Rs = −0.24, n = 107, p = 0.01) and hookworm (Rs = −0.49, n = 51, p<0.001). A significant difference in the enumeration of STH eggs between the Kato-Katz and McMaster methods was found for Brazil, Cameroon, and Vietnam. In both the Brazilian and Cameroonian trials, the Kato-Katz method yielded higher FEC compared to the McMaster method. In the Vietnamese trial, the McMaster method resulted in detection of more T. trichiura and hookworm eggs. In trials in India and Tanzania, no significant differences between the methods were found. 10.1371/journal.pntd.0001201.t002 Table 2 The quantitative agreement in fecal egg counts (FEC) between McMaster and Kato-Katz. Country n Mean FEC (EPG) Rs (p-value) p- value for Δ FEC McMaster Kato-Katz A. lumbricoides 312 5,982 14,197 0.70 (<0.001) <0.001 Brazil 81 6,490 25,079 0.88 (<0.001) <0.001 Cameroon 61 10,643 2,0531 0.82 (<0.001) <0.001 Tanzania 74 4,460 6,876 0.58 (<0.001) 0.08 Vietnam 96 3,559 6,560 0.28 (0.015) 0.20 T. trichiura 345 604 784 0.49 (<0.001) 1.00 Cameroon 67 1,168 1,938 0.76 (<0.001) 0.001 Tanzania 171 671 769 0.38 (<0.001) 0.60 Vietnam 107 143 84 −0.24 (0.01) 0.006 Hookworm 290 409 468 0.32 (<0.001) 0.10 Brazil 84 422 796 0.66 (<0.001) <0.001 India 39 1,031 1,630 0.67 (<0.001) 0.57 Tanzania 116 300 783 −0.05 (0.56) 0.09 Vietnam 51 162 32 −0.49 (<0.001) <0.001 Rs: Spearman correlation coefficient; ΔFEC: FECKato-Katz – FECMcMaster. Overall, there was a fair agreement (0.2≤κ<0.4) between the methods in the assignment of the samples to the three levels of egg excretion intensity as recommended by WHO (A. lumbricoides: κ = 0.37 (n = 199, p<0.001); T. trichiura: κ = 0.39 (n = 217, p<0.001); hookworm: κ = 0.34 (n = 147, p<0.001). As shown in the Figure 4, the McMaster method often assigned the samples to a lower level of egg excretion intensity compared to the Kato-Katz method. 10.1371/journal.pntd.0001201.g004 Figure 4 The agreement in the assignment to egg excretion intensity obtained by McMaster and Kato-Katz. The distribution of egg excretion intensity obtained by the McMaster method (low [white], moderate [grey], and high [black] over the egg excretion intensity observed by the Kato-Katz method for A. lumbricoides (A) (n = 199), T. trichiura (B) (n = 217), and hookworm (C) (n = 147). The validity of the multiplication factor employed in the Kato-Katz The mass of feces was measured in 207 Kato-Katz thick smears (Cameroon, n = 107; Tanzania, n = 100) in order to assess the validity of the multiplication factor used. Overall, the adjusted multiplication factor was 23.7, but it was subject to considerable variation (95% CI: [14.3–66.7]). This variation was observed in both trials (Cameroon 23.3 [13.4–83.3], and Tanzania 23.7 [15.3–54.3]) (p = 0.82). Table 3 summarizes the quantitative agreement between the FEC based on the fixed and adjusted multiplication factor, respectively. There was a high correlation between both approaches (Rs = 0.98, n = 39–146, p<0.001), regardless of in which country the trial was based. However, FEC obtained on the fixed multiplication factor were significantly higher compared to those adjusted for the mass of feces examined for A. lumbricoides (16,538 EPG vs. 15,396 EPG, n = 99, p<0.001), T. trichiura (1,490 EPG vs. 1,363 EPG, n = 146, p<0.001), but not for hookworm (351 EPG vs. 301 EPG, n = 39, p = 0.05). These findings were confirmed in both countries, though not significant in the case of A. lumbricoides in Tanzania. Despite the differences in FEC, there was a substantial to almost perfect agreement in the assignment to the different levels of egg excretion intensity between both approaches (κ A. lumbricoides = 0.93, n = 99, p<0.001; κ T. trichiura = 0.89, n = 146, p<0.001; κhookworm = 0.93, n = 39, p<0.001). 10.1371/journal.pntd.0001201.t003 Table 3 The quantitative agreement in fecal egg counts (FEC) between Kato-Katz using different multiplication factors. n Mean FEC (EPG) Rs (p-value) p- value for Δ FEC Fixed Adjusted A. lumbricoides 99 16,538 15,396 0.98 (<0.001) <0.001 Cameroon 54 12,307 11,702 0.98 (<0.001) <0.001 Tanzania 45 4,527 3,953 0.98 (<0.001) 0.11 T. trichuria 146 1,490 1,363 0.98 (<0.001) <0.001 Cameroon 62 2,268 2,023 0.98 (<0.001) 0.001 Tanzania 84 904 865 0.98 (<0.001) 0.02 Hookworm 39 351 301 0.98 (<0.001) 0.05 Tanzania 39 351 301 0.98 (<0.001) 0.05 Rs: Spearman correlation coefficient; ΔFEC: FECfixed – FECadjusted. Accuracy of estimating drug efficacy Overall, the mean bias (departure from the TDE in either direction) was 1.7% for McMaster and 4.5% for Kato-Katz. The bias for each of the two methods by trials (different countries), by pre-DA FEC and by TDE are illustrated in Figure 5. The bias for McMaster did not exceed 5%. Differences in bias across trials were small (Cameroon: 0.3–4.6%; Tanzania: 0.1–3.6%; Vietnam: 0.3–4.7%), but there was a decrease in bias across both pre-DA FEC (100 EPG: 0.3–4.6%; 250 EPG: 0.3–3.8%; 500 EPG: 0.2–4.7%; 750 EPG: 0.1–2.1%; 1,000 EPG: 0.1–2.6%) and TDE (90%: 0.1–4.7%; 95%: 0.7–2.4%; 99%: 0.1–0.5%). The bias for Kato-Katz ranged from 0.01% to 25.7%, and decreased when pre-DA FEC increased (100 EPG: 5.3–25.7%; 250 EPG: 0.2–8.0%; 500 EPG: 0.5–4.4%; 750 EPG: 0.3–4.0%; 1,000 EPG: 0.1–4.0%). Across trials (Cameroon: 0.3–14.8%; Tanzania: 0.4–20.9%; Vietnam: 0.1–25.7%) and TDE (90%: 0.5–25.7%; 95%: 0.2–17.9%; 99%: 0.1–20.9%), the bias remained largely unchanged. McMaster was significantly more accurate in estimating FECR compared to Kato-Katz (p = 0.006). Yet, these differences in accuracy of FECR between the methods became non-significant when only pre-DA FEC above 100 EPG were considered (p = 0.40, McMaster: 1.6% (range: 0.01–4.7%), Kato-Katz: 2.0% (range: 0.01–8.0%)). A detailed overview of the calculations made is available in Table S1. 10.1371/journal.pntd.0001201.g005 Figure 5 The absolute bias for McMaster and Kato-Katz in the assessment of drug efficacy. The bias (i.e., absolute value of the differences between the ‘true’ drug efficacy (TDE) and the observed fecal egg count reduction) for McMaster and Kato-Katz across the different trials (countries), pre-drug administration fecal egg counts (pre-DA FEC) and ‘true’ drug efficacies (TDE) based on predictions from statistical models. Discussion In the present study, the McMaster and Kato-Katz were compared for both qualitative and quantitative detection of STH infections in human populations on a scale that is unprecedented in the literature. Moreover, we assessed (i) the consistency of the performance of these two methods across five trials in different countries, (ii) the validity of a fixed multiplication factor for the Kato-Katz, and (iii) the ability of both methods to estimate a ‘true’ drug efficacy. The qualitative comparison revealed that Kato-Katz was more sensitive for the detection of A. lumbricoides, but not for hookworm and T. trichiura. These differences in sensitivity can be explained to some extent by the intrinsic properties of the methods. In the Kato-Katz method, a larger quantity of stool is examined (Kato-Katz: 41.7 mg, McMaster: 20 mg). Moreover, this quantity of stool is determined after the larger items in fecal debris have been removed by sieving, whereas the initial quantity of stool used in the McMaster method includes large items of debris. Finally, the McMaster method is based on the flotation of eggs, but it is clear that the buoyancy of eggs differs between the different STHs. For example, it was noticed that unfertilized eggs of A. lumbricoides (heavier than fertilized ones) were rarely detected in McMaster chambers, even when a high numbers of eggs was being excreted. For both methods there was a considerable variation in sensitivity between the different trials. This variation was largely explained by intensity of egg excretion (FEC) and factors inherent to the different laboratories involved in the trials and the countries where they were located. The probability of the diagnosis of STH infections increased as the number of eggs excreted increased. Although this finding is not unexpected, it highlights the importance of quantifying infection intensity in future studies comparing diagnostic methods. This will enable ready comparison of the sensitivity reported in different studies. The differences between countries/laboratories are not easily explained and are likely multi-factorial. An important factor, which may have contributed to this difference, is human error. Although we employed standardized methods throughout based on identical written protocols, small differences in processing samples and/or examination of the slides between laboratories/countries cannot be ruled out. This is particularly the case in the use of the Kato-Katz, for which the time between processing and examination is extremely difficult to standardize (in the present study ranging from 30 to 60 min), yet crucial for the detection of hookworm eggs [12]. Similar major inter-laboratory differences also became apparent when their performance of diagnostic testing for STH was compared between European and African laboratories [11]. Therefore in future, rigorous quality control for similar studies is recommended to minimize human error. A set of control samples from the same source could have been examined independently by the different laboratories involved (so-called ring test). However, this would have required preservation of the samples, which may itself have thwarted the interpretation of the quality control, and dispatch to the laboratories involved would have resulted in different time periods between collection of sample from the donor and fixation, and eventual assessment of FEC, adding yet more variables and uncertainties to the outcome. Preservation (e.g., formaldehyde) is known to alter the morphology/density of eggs, resulting in false negative test results and an underestimation of FEC [29]. Moreover, when preserved by the addition of a preservant in a liquid formulation, it would no longer be possible to process samples as fresh samples, as normally done under field conditions, because then centrifugation would have to be implemented to discard the preservant prior to assay. This additional step, therefore, is likely not only to generate extra variation in the test results, but also to concentrate the eggs, hence increasing the sensitivity and FEC [30]. Other factors which cannot be excluded are differences in fecundity of worms [31], the number of samples containing unfertilized eggs (A. lumbricoides), the diet of subjects or the proportion of N. americanus/A. duodenale. The diet varied considerably across the five participating countries, and thus differences in the quality of food consumed would have created differences in fat and roughage content, which may have influenced the buoyancy of helminth eggs, particularly for the McMaster method as it is based on flotation of the eggs. Our study did not distinguish between N. americanus and A. duodenale eggs, yet it was remarkable that the effect of magnitude of FEC on sensitivity differed markedly between countries only for hookworm (interaction term), suggesting that sensitivity may also vary between hookworms species. At present, it remains unclear which factor(s) is (are) causing the observed variation across laboratories/countries, however, differences in sensitivity between countries for the McMaster were less pronounced compared to Kato-Katz, indicating that the McMaster is a more robust method under field conditions. The quantitative comparison revealed an overall positive correlation. Yet, the Kato-Katz method resulted in significantly higher FECs than the McMaster method for A. lumbricoides, but not for T. trichiura or hookworm. These findings partially confirm previous studies summarized by Knopp et al. (2009) [32], where differences in FEC between Kato-Katz and FLOTAC (a derivative of the McMaster method) were more pronounced for A. lumbricoides and hookworm, than for T. trichiura. It is clear that intrinsic aspects of both methods explaining the discrepancy in sensitivity for STH will also contribute to the discrepancy in FEC. In addition, it is important to bear in mind that the Kato-Katz method does not include the homogenization of a large mass of the stool sample (41.7 mg compared to 2 g for the McMaster) prior to examination, that in certain cases may result in higher counts, as eggs are not equally distributed among the sample [33], [34]. The level of quantitative agreement was not consistent across the different trials involved, but this can be explained mostly either by a small number of samples containing STH (type error II) or differences in sensitivity. The present study also confirms that the use of a fixed multiplication factor of 24 for the Kato-Katz should be revised to enable more accurate quantification of the eggs excreted [16]. Although the mean of the multiplication factor adjusted for the mass of feces examined (23.7) approached the conventially used 24, there was considerable variation in the multiplication factor across the different samples ranging from 11 to 100. Moreover, FECs based on the fixed multiplication factor resulted in significantly higher FECs compared to those based on a multiplication factor adjusted for the actual mass of feces examined, which may explain the above described difference in FEC between McMaster and Kato-Katz. The statistical simulation revealed that both methods provide reliable estimates of drug efficacies, supporting the use of both methods for monitoring large-scale treatment programs implemented for the control of STH in public health. However, the McMaster method has several advantages when a large number of samples need to be examined because the microscopy is readily performed, and all parasites can be examined simultaneously, in contrast to the Kato-Katz method where different clearing times for the different STH require re-examination at times optimal for different species [15]. These findings also confirms that FECR is preferred as a summary measure for assessment of drug efficacy, since it allows an accurate and realistic comparison of FECR across laboratories or the locations where the trials have been conducted, and this regardless of differences in sensitivity between trials. In conclusion, this multinational study highlights considerable variation in the performance of two methods used for the diagnosis of STH, particularly for the commonly used Kato-Katz. Both the McMaster and the Kato-Katz methods are valid methods for monitoring large-scale treatment administration programs. Yet, the McMaster method seems more suitable for further standardization because of its robust multiplication factor, and allowing for simultaneous detection of all species of STH. Supporting Information Checklist S1 STARD checklist (DOC) Click here for additional data file. Table S1 A detailed overview of the calculations made to assess the accuracy of estimating drug efficacy (XLS) Click here for additional data file.
Author and article information
Contributors
Alejandro Krolewiecki
Journal
Journal ID (nlm-ta): Parasite Epidemiol Control
Journal ID (iso-abbrev): Parasite Epidemiol Control
Title:
Parasite Epidemiology and Control
Publisher:
Elsevier
ISSN
(Electronic):
2405-6731
Publication date PMC-release: 20
July
2024
Publication date Collection:
August
2024
Publication date
(Electronic):
20
July
2024
Volume: 26
Electronic Location Identifier: e00370
Affiliations
[a
]Universidad Nacional de Salta, Sede Regional Orán, Instituto de Investigaciones de
Enfermedades Tropicales, Salta, Argentina (IIET)
[d
]Universidad Nacional de Salta, Facultad de Ciencias Naturales, Cátedra de Química
Biológica, Salta, Argentina
Author notes
[*
]Corresponding author at: Universidad Nacional de Salta. Instituto de Investigaciones
de Enfermedades Tropicales. Alvarado 751 (4530), Orán, Argentina.
alekrol@
123456mundosano.org
Article
Publisher Item ID: S2405-6731(24)00034-5 Publisher ID: e00370DOI: 10.1016/j.parepi.2024.e00370
PMC ID: 11321430
PubMed ID: 39139793
SO-VID: 021369b9-1e91-4557-84f7-91c43e1d13d6
Copyright © © 2024 Published by Elsevier Ltd on behalf of World Federation of Parasitologists.
License:
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
History
Date
received
: 13
June
2023
Date
revision received
: 3
June
2024
Date
accepted
: 18
July
2024
Categories
Subject:
Original Research article
Data availability: