Quality control in the diagnosis of Trichuris trichiura and Ascaris lumbricoides using the Kato-Katz technique: experience from three randomised controlled trials
© Speich et al.; licensee BioMed Central. 2015
Received: 17 December 2014
Accepted: 27 January 2015
Published: 5 February 2015
An accurate diagnosis of soil-transmitted helminthiasis is important for individual patient management, for drug efficacy evaluation and for monitoring control programmes. The Kato-Katz technique is the most widely used method detecting soil-transmitted helminth eggs in faecal samples. However, detailed analyses of quality control, including false-positive and faecal egg count (FEC) estimates, have received little attention.
Over a 3-year period, within the frame of a series of randomised controlled trials conducted in Pemba, United Republic of Tanzania, 10% of randomly selected Kato-Katz thick smears were re-read for Trichuris trichiura and Ascaris lumbricoides eggs. In case of discordant result (i.e. positive versus negative) the slides were re-examined a third time. A result was assumed to be false-positive or false-negative if the result from the initial reading did not agree with the quality control as well as the third reading. We also evaluated the general agreement in FECs between the first and second reading, according to internal and World Health Organization (WHO) guidelines.
From the 1,445 Kato-Katz thick smears subjected to quality control, 1,181 (81.7%) were positive for T. trichiura and 290 (20.1%) were positive for A. lumbricoides. During quality control, very low rates of false-positive results were observed; 0.35% (n = 5) for T. trichiura and 0.28% (n = 4) for A. lumbricoides. False-negative readings of Kato-Katz thick smears were obtained in 28 (1.94%) and 6 (0.42%) instances for T. trichiura and A. lumbricoides, respectively. A high frequency of discordant results in FECs was observed (i.e. 10.0-23.9% for T. trichiura, and 9.0-11.4% for A. lumbricoides).
Our analyses show that the rate of false-positive diagnoses of soil-transmitted helminths is low. As the probability of false-positive results increases after examination of multiple stool samples from a single individual, the potential influence of false-positive results on epidemiological studies and anthelminthic drug efficacy studies should be determined. Existing WHO guidelines for quality control might be overambitious and might have to be revised, specifically with regard to handling disagreements in FECs.
The common soil-transmitted helminths (i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura) affect approximately 1.5 billion people, cause considerable morbidity and account for an estimated 5.2 million disability adjusted life years (DALYs) [1,2]. An accurate diagnosis is important for the identification of infected individuals, for assessing anthelminthic drug efficacy, and for monitoring the control and elimination of soil-transmitted helminthiasis [3,4]. Currently, the most common diagnostic approach for soil-transmitted helminth infections in epidemiological studies is the copro-microscopic detection of helminth eggs using the Kato-Katz technique [5,6]. However, the Kato-Katz technique has limitations in terms of sensitivity, especially in low-intensity settings . The sensitivity of the Kato-Katz technique is increased by analysing multiple thick smears from a single or, ideally, from multiple stool samples [7-15]. The specificity of the Kato-Katz technique is rarely investigated as studies examining the effect of multiple diagnostics usually merge all results to create a composite ‘gold’ standard, considering all positive results as “true positives” . Henceforth, the possibility of false-positive results has been largely neglected and the frequency of false-positive is unknown . However, assuming 100% specificity is unrealistic as false-positive results can arise, for example, when debris is confused as helminth eggs or by mistakes recording the data . Tarafder et al. (2010) and Knopp et al. (2014) used Bayesian statistical methods to assess the specificity and sensitivity of the Kato-Katz technique for the diagnosis of soil-transmitted helminth infections [16,17].
Two guidelines how to judge differences in faecal egg counts between the initial reading and the quality control reading of Kato-Katz thick smears
Guideline from the World Health Organization (WHO)
Internal guideline developed at the Swiss Tropical and Public Health Institute
“If the expert identifies a difference in the egg count per gram of stoolb of more than 10% and more than four eggs, he or she should re-read the slide with the microscopist and discuss the reasons for the discrepancy”. a 
Results are considered as inconsistent if there is a difference in presence/absence of a specific helminth species, or if differences in egg counts exceed (i) 10 eggs for Kato-Katz thick smears with ≤100 eggs, or (ii) exceed 20% for Kato-Katz thick smears with more than 100 eggs.
In this study, we report new insights from detailed analysis of quality control data obtained over a 3-year period in a series of randomised controlled trials conducted in Pemba, United Republic of Tanzania. We assessed the frequency of false-positive results when using the Kato-Katz technique for the diagnosis of T. trichiura and A. lumbricoides. We also analysed the frequency of false-negative results and differences in FECs according to guidelines put forth by the WHO  and Swiss TPH.
Studies, subjects and quality control samples
In the years 2011, 2012 and 2013, we conducted three randomised controlled trials which evaluated new anthelminthic drugs or drug combinations against soil-transmitted helminths on Pemba Island, United Republic of Tanzania [21-23]. Children attending the schools in Wawi and Al-Sadik (both in 2011), Mchangamdogo and Shungi (both in 2012 and 2013) were invited to participate in the clinical trials. Within the frame of these randomised controlled trials, a total of 14,855 Kato-Katz thick smears were prepared and examined under a microscope at the Public Health Laboratory-Ivo de Carneri (PHL-IdC) by experienced laboratory technicians. Kato-Katz thick smears were prepared according to standard protocols. In brief, we used 41.7 mg templates, and the slides were read within 60 min to avoid over clearing of hookworm eggs [5,24,25]. Soil-transmitted helminth eggs were counted for each species separately (i.e. A. lumbricoides, hookworm and T. trichiura).
The results from the initial reading and from the subsequent quality control were compared. In case of discordant results (i.e. positive versus negative for a specific soil-transmitted helminth species and if the investigator judged the difference in FECs subjectively as too large) between the first two readers, a third microscopist was asked to re-examine the respective slide. Quality control (i.e. the second reading) was performed by a senior laboratory technician or by an investigator of the clinical trial. If a third reading was necessary, a third technician who did not previously examine the Kato-Katz thick smear was randomly chosen to re-examine the slide. The only exception was that in cases where a false-positive result was suspected, the first reader was asked to re-read the slide and show the observed egg to the investigator. All microscopists were blinded to previous results.
For each of the three trials, ethical clearances from the cantonal ethics commission of Basel, Switzerland (EKBB) and from the Ministry of Health and Social Welfare of Zanzibar, United Republic of Tanzania were obtained [21-23]. The trials are registered at Current Controlled Trials (identifiers: ISRCTN08336605, ISRCTN54577342 and ISRCTN80245406). It was emphasised that study participation was voluntary and withdrawal possible at any time without further obligation. At the end of each study, all school-going children were offered albendazole (at a dose of 400 mg) according to national guidelines [26,27].
Results were classified as false-positive if the original result was positive for a specific soil-transmitted helminth, but the results from the quality control (second reading), as well as from the third reading, were negative. A result was judged as false-negative if the original result was negative, but the quality control as well as the result from the third reading, were positive. We retrospectively calculated (i) the proportion of false-positive results on the overall number of samples; (ii) the proportion of false-positive results among the negative samples; (iii) the proportion of false-negative results among the overall number of samples; and (iv) the proportion of false-negative results among the positive samples. Species-specific differences among (ii) and (iv) were calculated with a two-sample test of proportion. FECs of confirmed false-positive and false-negative readings were descriptively analysed.
FECs from the primary reading were compared to the data from quality control according to two different guidelines put forward by WHO and Swiss TPH. Of note, at the time the clinical trials were implemented, no guidelines on judging FEC differences were available. In contrast to our in-house guideline, it is not explicitly stated in the WHO guideline how to address differences between the observed presence or absence of helminth eggs. Hence, we assumed that, according to WHO guideline, differences in presence/absence of helminth eggs do not require re-reading as long as the difference does not exceed the given range of 4 eggs (see Table 1). By comparing the initial readings to the results of the quality control, we retrospectively assessed the proportion of Kato-Katz thick smears which would have required a third re-reading according to the two different guidelines and which were therefore judged as discordant results. In addition, the proportion of agreeing Kato-Katz thick smears among the non-negative slides (i.e. FEC ≥ 1 either in the initial reading or the quality control) was calculated. These analyses were performed separately for A. lumbricoides and T. trichiura. All data were double entered into an Excel spreadsheet (Microsoft 2010) and cross-checked. Statistical analysis was conducted with Stata version 10.1 software (StataCorp.; College Station, TX, USA).
Proportion of false-positive and false-negative results after a second quality control reading of Kato-Katz thick smears (in case of discordant results, slides were read a third time)
Total Kato-Katz thick smears read for quality control
Positive Kato-Katz thick smears
Negative Kato-Katz thick smears
Percentage of positive Kato-Katz thick smears
Percentage of false-positive results
Percentage of false-positive results among negative Kato-Katz thick smears
Faecal egg counts of the false-positive Kato-Katz thick smearsb
1, 3, 4, 7, 10
2, 2, 2, 12
Percentage of false-negative results
Percentage of false-negative results among positive Kato-Katz thick smears
Faecal egg counts from the quality control of the false-negative Kato-Katz thick smearsb
1, 1, 1, 1, 1, 1, 1, 1, 1, 2, 2, 2, 2, 3, 3, 3, 3, 3, 4, 4, 5, 6, 9, 10, 11, 12, 15, 16
1, 1, 1, 1, 6, 8
Among the 1,445 re-examined slides, a total of 28 (1.94%) and 6 (0.42%) slides were detected as false-negative for T. trichiura and A. lumbricoides, respectively. The proportion of false-negative results among the positive Kato-Katz thick smears was 2.37% for T. trichiura and 2.07% for A. lumbricoides. No significant difference in proportion of false-negative results was detected between T. trichiura and A. lumbricoides (p = 0.76). FECs of the false-positive and false-negative results are listed in Table 2.
Agreement in faecal egg counts (FECs) between initial reading and second quality control reading of Kato-Katz thick smears according to two different guidelines
WHO guideline a
Swiss TPH guideline a
Total number of Kato-Katz thick smears (positive/negative)
1. No. of Kato-Katz thick smears with difference in FECs (%)
2. No. of Kato-Katz thick smears with differences in presence/absence of helminth eggs (%)
3. No. of Kato-Katz thick smears with difference in FECs (%) among samples with egg counts
4. No. of Kato-Katz thick smears with differences in presence/absence of helminth eggs (%) among samples with egg counts
1. and/or 2. (%)
No. of Kato-Katz thick smears with discrepancies according to respective guidelineb (%)
3. and/or 4. and proportion as percentage (%) among Kato-Katz thick smears with positive egg counts
No. of Kato-Katz thick smears with discrepancies according to respective guidelineb (%) among Kato-Katz thick smears with positive egg counts
Even though the proportion of false-positive was relatively low (i.e. 1.89% for T. trichiura and 0.35% for A. lumbricoides) its impact should not be neglected. While false-negative results can be corrected to some extent by examining multiple Kato-Katz thick smears, the probability of false-positive results increases as a function of examining multiple Kato-Katz thick smears . Hence, in studies where multiple Kato-Katz thick smears are examined from each participant, the false-positive rate per individual might not be negligible. This hypothesis is confirmed by a recent study by Tarafder and colleagues (2010) who used Bayesian statistical methods to calculate the specificity of a single and multiple Kato-Katz thick smears. While they reported high specificity for a single Kato-Katz thick smear, specificity decreased by examining multiple Kato-Katz slides .
Differences in FECs between the initial and the quality control readings were assessed according to two different guidelines (Table 1). To our knowledge, we evaluated for the first time these guidelines with a large dataset from three randomised controlled trials. A relatively high frequency of discordant results was observed according to both guidelines. This observation indicates that an accurate counting of eggs in studies where high numbers of Kato-Katz thick smears are read in relatively short time frames is challenging. A particularly large number of disagreeing results was observed for the A. lumbricoides-positive slides. It is not entirely clear, whether A. lumbricoides eggs are generally more difficult to detect or if the high egg counts, which are more common for A. lumbricoides, were responsible for this result [30,31]. Given the fact that enumerating soil-transmitted helminth eggs is challenging, one might consider revising the current WHO guideline, which currently seem to be too firm and therefore might be overambitious. In our opinion another limitation of the WHO guideline is that a difference in presence versus absence of eggs is not considered a discordant result, as long as the FEC differed by not more than 4 eggs (see Table 1). However, verification of presence or absence of a helminth infection is crucial, as false-negative or false-positive diagnoses have important ramifications on patient management.
A limitation of our study is that we cannot evaluate the impact of further error sources of false-positive results, as for example sampling errors which can occur when children mix up or even share stool samples. Additionally, the aforementioned guidelines on how to judge and handle FEC differences in quality control had not yet been available at the time the clinical trials were conducted. Therefore, Kato-Katz thick smears with differing FECs were only re-read a third time if the investigator judged the difference in FECs as too large. This was done in a subjective way and not as strictly as might be suggested by the guidelines. Due to this reason, we did not have many third reading results for discordant FECs and could thus only compare the first and the quality control reading. It is important to note that discrepancies in those two readings could have equally arose due to reading errors by the initial microscopist as well as reading errors from the microscopist who conducted the quality control.
It should also be highlighted that our results are not directly transferable to other epidemiological settings. First of all, the technicians from the WHO Collaborating Centre PHL-IdC are highly skilled and they have examined tens of thousands of Kato-Katz thick smears over the past several years. Furthermore, the rigorous implementation of a quality control scheme might have increased the overall quality of the Kato-Katz thick smear readings leading to a low number of false-positive and false-negative readings. However, a high frequency of discordant FECs was observed, even though rigorous quality control was in place and technicians were highly experienced.
We observed low rates of false-positive results of A. lumbricoides and T. trichiura when using the Kato-Katz technique. Our results indicate that especially in a setting where soil-transmitted helminth infections are highly prevalent, false-positive results will only have a small effect in overall prevalence estimates. However, in settings with low prevalence where it is challenging to identify infected individuals, false-positive results might influence treatment allocation, results of epidemiological studies and monitoring of control programmes. Examining multiple stool samples from participating individuals will further increase the frequency of false-positive results. Additionally, we have shown that an accurate counting of helminth eggs is a formidable challenge, even for highly skilled technicians. Therefore, the WHO guideline might be too strict and one might additionally cast doubt on the reliability of FECs as well as egg reduction rate, which has recently been proposed as the single most important metrics for assessing anthelminthic drug efficacy. We recommend to validate the current WHO guideline within different settings and to adapt it if the frequency of discordant results remains equally high.
We thank Dr. Peter Steinmann from the Swiss Tropical and Public Health Institute for developing the in-house guideline for handling discrepancies among faecal egg count readings. This work was financially supported by the Medicor Foundation and the Swiss National Science Foundation (Grant no. 320030_149310/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Murray CJL, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2197–223.View ArticlePubMedGoogle Scholar
- Pullan RL, Smith JL, Jasrasaria R, Brooker SJ. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit Vectors. 2014;7:37.View ArticlePubMed CentralPubMedGoogle Scholar
- Bergquist R, Johansen MV, Utzinger J. Diagnostic dilemmas in helminthology: what tools to use and when? Trends Parasitol. 2009;25:151–6.View ArticlePubMedGoogle Scholar
- McCarthy JS, Lustigman S, Yang GJ, Barakat RM, García HH, Sripa B, et al. A research agenda for helminth diseases of humans: diagnostics for control and elimination programmes. PLoS Negl Trop Dis. 2012;6:e1601.View ArticlePubMed CentralPubMedGoogle Scholar
- Katz N, Chaves A, Pellegrino J. A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Rev Inst Med Trop São Paulo. 1972;14:397–400.PubMedGoogle Scholar
- Speich B, Knopp S, Mohammed KA, Khamis IS, Rinaldi L, Cringoli G, et al. Comparative cost assessment of the Kato-Katz and FLOTAC techniques for soil-transmitted helminth diagnosis in epidemiological surveys. Parasit Vectors. 2010;3:71.View ArticlePubMed CentralPubMedGoogle Scholar
- Ebrahim A, El-Morshedy H, Omer E, El-Daly S, Barakat R. Evaluation of the Kato-Katz thick smear and formol ether sedimentation techniques for quantitative diagnosis of Schistosoma mansoni infection. Am J Trop Med Hyg. 1997;57:706–8.PubMedGoogle Scholar
- Utzinger J, Booth M, N’Goran EK, Müller I, Tanner M, Lengeler C. Relative contribution of day-to-day and intra-specimen variation in faecal egg counts of Schistosoma mansoni before and after treatment with praziquantel. Parasitology. 2001;122:537–44.View ArticlePubMedGoogle Scholar
- Booth M, Vounatsou P, N’Goran EK, Tanner M, Utzinger J. The influence of sampling effort on the performance of the Kato-Katz technique in diagnosing Schistosoma mansoni and hookworm co-infections in rural Côte d’Ivoire. Parasitology. 2003;127:525–31.View ArticlePubMedGoogle Scholar
- Steinmann P, Du ZW, Wang LB, Wang XZ, Jiang JY, Li LH, et al. Extensive multiparasitism in a village of Yunnan province, People’s Republic of China, revealed by a suite of diagnostic methods. Am J Trop Med Hyg. 2008;78:760–9.PubMedGoogle Scholar
- Knopp S, Rinaldi L, Khamis IS, Stothard JR, Rollinson D, Maurelli MP, et al. A single FLOTAC is more sensitive than triplicate Kato-Katz for the diagnosis of low-intensity soil-transmitted helminth infections. Trans R Soc Trop Med Hyg. 2009;103:347–54.View ArticlePubMedGoogle Scholar
- Glinz D, Silué KD, Knopp S, Lohourignon LK, Yao KP, Steinmann P, et al. Comparing diagnostic accuracy of Kato-Katz, Koga agar plate, ether-concentration, and FLOTAC for Schistosoma mansoni and soil-transmitted helminths. PLoS Negl Trop Dis. 2010;4:e754.View ArticlePubMed CentralPubMedGoogle Scholar
- Levallois P, Chevalier P, Gingras S, Déry P, Payment P, Michel P, et al. Risk of infectious gastroenteritis in young children living in Québec rural areas with intensive animal farming: results of a case–control study (2004–2007). Zoonoses Public Health. 2014;61:28–38.View ArticlePubMedGoogle Scholar
- Nikolay B, Brooker SJ, Pullan RL. Sensitivity of diagnostic tests for human soil-transmitted helminth infections: a meta-analysis in the absence of a true gold standard. Int J Parasitol. 2014;44:765–74.View ArticlePubMed CentralPubMedGoogle Scholar
- Speich B, Utzinger J, Marti H, Ame SM, Ali SM, Albonico M, et al. Comparison of the Kato-Katz method and ether-concentration technique for the diagnosis of soil-transmitted helminth infections in the framework of a randomised controlled trial. Eur J Clin Microbiol Infect Dis. 2014;33:815–22.View ArticlePubMedGoogle Scholar
- Tarafder MR, Carabin H, Joseph L, Balolong E, Olveda R, McGarvey ST. Estimating the sensitivity and specificity of Kato-Katz stool examination technique for detection of hookworms, Ascaris lumbricoides and Trichuris trichiura infections in humans in the absence of a “gold standard”. Int J Parasitol. 2010;40:399–404.View ArticlePubMed CentralPubMedGoogle Scholar
- Knopp S, Salim N, Schindler T, Karagiannis-Voules DA, Rothen J, Lweno O, et al. Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania. Am J Trop Med Hyg. 2014;90:535–45.View ArticlePubMed CentralPubMedGoogle Scholar
- WHO. Assessing the efficacy of anthelminthic drugs against schistosomiasis and soil-transmitted helminthiases. Geneva: World Health Organization; 2013.Google Scholar
- Ziegelbauer K, Speich B, Mäusezahl D, Bos R, Keiser J, Utzinger J. Effect of sanitation on soil-transmitted helminth infection: systematic review and meta-analysis. PLoS Med. 2012;9:e1001162.View ArticlePubMed CentralPubMedGoogle Scholar
- Strunz EC, Addiss DG, Stocks ME, Ogden S, Utzinger J, Freeman MC. Water, sanitation, hygiene, and soil-transmitted helminth infection: a systematic review and meta-analysis. PLoS Med. 2014;11:e1001620.View ArticlePubMed CentralPubMedGoogle Scholar
- Speich B, Ame SM, Ali SM, Alles R, Hattendorf J, Utzinger J, et al. Efficacy and safety of nitazoxanide, albendazole, and nitazoxanide-albendazole against Trichuris trichiura infection: a randomized controlled trial. PLoS Negl Trop Dis. 2012;6:e1685.View ArticlePubMed CentralPubMedGoogle Scholar
- Speich B, Ame SM, Ali SM, Alles R, Huwyler J, Hattendorf J, et al. Oxantel pamoate-albendazole for Trichuris trichiura infection. N Engl J Med. 2014;370:610–20.View ArticlePubMedGoogle Scholar
- Speich B, Ali SM, Ame SM, Bogoch II, Alles R, Huwyler J, et al. Efficacy and safety of albendazole-ivermectin, albendazole-mebendazole, albendazole-oxantel pamoate, and mebendazole against Trichuris trichiura and concomitant soil-transmitted helminth infections: a randomised controlled trial. Lancet Infect Dis. (in press); doi:10.1016/S1473-3099(14)71050-3.Google Scholar
- Martin LK, Beaver PC. Evaluation of Kato thick-smear technique for quantitative diagnosis of helminth infections. Am J Trop Med Hyg. 1968;17:382–91.PubMedGoogle Scholar
- Yap P, Fürst T, Müller I, Kriemler S, Utzinger J, Steinmann P. Determining soil-transmitted helminth infection status and physical fitness of school-aged children. J Vis Exp. 2012;66:e3966.PubMedGoogle Scholar
- Albonico M, Crompton DWT, Savioli L. Control strategies for human intestinal nematode infections. Adv Parasitol. 1999;42:277–341.View ArticlePubMedGoogle Scholar
- WHO. Model list of essential medicines for children (2nd list, March 2010 update). Geneva: World Health Organization; 2010.Google Scholar
- Steinmann P, Rinaldi L, Cringoli G, Du ZW, Marti H, Jiang JY, et al. Morphological diversity of Trichuris spp. eggs observed during an anthelminthic drug trial in Yunnan, China, and relative performance of parasitologic diagnostic tools. Acta Trop. 2015;141:184–8.View ArticlePubMedGoogle Scholar
- Knopp S, Mgeni AF, Khamis IS, Steinmann P, Stothard JR, Rollinson D, et al. Diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: effect of multiple stool sampling and use of different diagnostic techniques. PLoS Negl Trop Dis. 2008;2:e331.View ArticlePubMed CentralPubMedGoogle Scholar
- Bethony J, Brooker S, Albonico M, Geiger SM, Loukas A, Diemert D, et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet. 2006;367:1521–32.View ArticlePubMedGoogle Scholar
- Knopp S, Steinmann P, Keiser J, Utzinger J. Nematode infections: soil-transmitted helminths and Trichinella. Infect Dis Clin North Am. 2012;26:341–58.View ArticlePubMedGoogle Scholar
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