Conceived and designed the experiments: SK BS LR KAM MA DR HM GC JU. Performed the experiments: SK BS LR ISK ASM. Analyzed the data: SK JH. Contributed reagents/materials/analysis tools: JH KAM ISK DR HM GC JU. Wrote the paper: SK BS JH LR KAM ISK ASM MA DR HM GC JU.
GC is the inventor and current patent holder of the FLOTAC apparatus. In case the current broad-scale validation of the FLOTAC technique will be successful, the method will be licensed free of charge to the WHO and interested public non-commercial research centers. None of the other authors has any conflict of interest concerning the work reported in this paper.
Sensitive diagnostic tools are required for an accurate assessment of prevalence and intensity of helminth infections in areas undergoing regular deworming, and for monitoring anthelmintic drug efficacy. We compared the diagnostic accuracy of the Kato-Katz and FLOTAC techniques in the frame of a drug efficacy trial.
Stool samples from 343 Zanzibari children were subjected to duplicate Kato-Katz thick smears and the FLOTAC basic technique in a baseline screening in early 2009. The FLOTAC showed a higher sensitivity than the Kato-Katz method for the diagnosis of
Our results suggest that the FLOTAC technique, following further optimization, might become a viable alternative to the Kato-Katz method for anthelmintic drug efficacy studies and for monitoring and evaluation of deworming programs. The lower CRs and ERRs determined by FLOTAC warrant consideration and could strategically impact future helminth control programs.
In areas where parasitic worm infections have been successfully reduced as a result of deworming campaigns, the level of infections and drug efficacy must be carefully monitored. For this purpose, diagnostic methods with a high sensitivity are needed. We compared the accuracy of the widely used Kato-Katz method with the more recently developed FLOTAC technique for the diagnosis of parasitic worms. Our study was done with children on Zanzibar island, Tanzania, within the frame of an anthelmintic drug efficacy study. We collected stool samples from 343 children in two primary schools before and after treatment and examined the stool samples with both methods. FLOTAC showed a higher sensitivity than Kato-Katz for the diagnosis of roundworm and whipworm, but a lower sensitivity for hookworm diagnosis. The cure rates determined by FLOTAC were lower for roundworm and whipworm when compared with Kato-Katz. The opposite was found for hookworm. Egg reduction rates were generally lower when the FLOTAC technique was used. Our results suggest that the FLOTAC method, after additional optimization, can become a viable alternative to the Kato-Katz method for anthelmintic drug efficacy studies and for monitoring and evaluation of deworming programs.
Current estimates suggest that soil-transmitted helminths might still affect over a quarter of the world's population and inflict a huge public health burden, particularly on rural and deprived urban communities in the developing world
The diagnosis of soil-transmitted helminth infections with direct parasitological methods based on egg detection in stool is unreliable among infected individuals who harbor only a few intestinal worms, since egg output is much lower than among heavily infected individuals. Hence, in settings where helminth control programs have been implemented and infection intensities dropped as a result of regular deworming, diagnostic methods with a high sensitivity are needed for an accurate assessment of the actual prevalence and intensity of soil-transmitted helminth infections
With the recently developed FLOTAC method, up to 1 g of stool can be examined – 24 times more than with a single Kato-Katz thick smear – and standard protocols are now available
The objective of this study was to compare the accuracy of the Kato-Katz method with the FLOTAC basic technique for the detection of soil-transmitted helminth infections within a randomized controlled trial on anthelmintic drug efficacy and safety carried out in Zanzibar in early 2009
The study presented here was embedded in a randomized controlled trial with the protocol being reviewed by the institutional research commission of the Swiss Tropical and Public Health Institute (Basel, Switzerland). Ethical approval was given by the ethics committee of Basel (EKBB, reference no. 13/09) and the Ministry of Health and Social Welfare of Zanzibar (MoHSW, reference no. ZAMEC/0001/09). The study is registered at controlled-trials.com, identifier ISRCTN08336605.
The directors and teachers of the primary schools in Kinyasini and Kilombero were informed about the purpose and procedures of the study. The study was explained in lay terms to the school children in their local language (Kiswahili). An informed consent sheet, including study information and the fact that participation was voluntary, was distributed to each child. Written informed consent was obtained from parents or guardians prior to stool sampling. Additionally, oral assent was sought from children.
At the end of the study in May 2009, all children attending the two schools were offered free anthelmintic drugs, as part of the regular deworming done by the Helminth Control Laboratory Unguja (HCLU). Single oral doses of albendazole (400 mg) and praziquantel (40 mg/kg) were administered to school-aged children for preventing morbidity due to soil-transmitted helminthiasis and urinary schistosomiasis, respectively.
The study was carried out on Unguja island, Zanzibar, in the first half of 2009 within the frame of a randomized controlled trial to assess the efficacy and safety of different anthelmintic drugs against
The baseline screening was carried out as follows: in early March 2009, over a period of 3 weeks, every morning between 08:00 and 09:30 hours, ∼120 children were called from class and given a stool collection container labeled with unique identification numbers (IDs). Children were asked to return the container filled with a lime-sized own fresh morning stool sample the following day. Upon collection, filled stool containers were ordered by increasing IDs in a wooden transport-shelf and promptly transferred to the HCLU in Zanzibar Town.
At HCLU, duplicate Kato-Katz thick smears were prepared from each stool sample, using standard 41.7 mg templates
With regard to the FLOTAC method, ∼1 g of stool, systematically obtained from each third stool sample in the transport-shelf, was weighed in a plastic tube, filled with 10 ml of 5% formaldehyde. Stool samples were suspended with a wooden spatula and stored at room temperature until further use.
Three to 9 weeks after the collection of the first stool sample, children who had their stool samples examined both by the Kato-Katz and FLOTAC techniques were invited to submit a second stool sample. Samples were again processed with duplicate Kato-Katz, and ∼1 g of stool was preserved in 5% formaldehyde and stored at room temperature. Of note, children with a
The administration of albendazole and praziquantel to all school children in Kinyasini and Kilombero (and other schools) regardless of whether or not children participated in our study was carried out by members of the HCLU in late May 2009, when all available follow-up stool samples had been collected.
After completion of the trial in late May 2009, in the last 2 weeks of the study, all formaldehyde-preserved stool samples were examined with the FLOTAC basic technique
We used flotation solution no. 4 (FS4; sodium nitrate: NaNO3 315 g plus 685 ml H2O; specific gravity (s.g.) = 1.20) in light of our preceding results for the diagnosis of soil-transmitted helminth infections
Fecal egg counts (FECs) for each helminth species were recorded separately for each Kato-Katz thick smear and each of the two FLOTAC observation grids. For quality control, 10% of the slides and observation grids were re-examined by a senior laboratory technician. In case the senior technician detected one or several eggs of a helminth species that had not been recorded in the original reading, the former result was considered as false-negative and replaced by the result of the senior technician. Moreover, in case of deviations in FECs of more than 10%, the original egg count was replaced by the result of the senior technician. In both cases, the microscopist was advised to read more carefully the following days. All Kato-Katz thick smears would have been re-read if there were discrepancies in the FECs in more than 20% of the re-examined slides, but this was never the case over the course of our study. The microscopists reading the FLOTAC observation grids were blinded to the results derived from the Kato-Katz method.
Data were entered twice in Microsoft Excel version 10.0 (2002, Microsoft Corporation; Redmond, WA, USA) and checked for consistency with EpiData version 3.1 (EpiData Association; Odense, Denmark). Discrepancies were removed by consulting original data records. Data sets for Kato-Katz and FLOTAC results from the baseline screening and follow-up were merged by ID. Statistical analyses were carried out with STATA version 10 (StataCorp.; College Station, TX, USA).
For method comparisons, only individuals whose stool samples were subjected to duplicate Kato-Katz and FLOTAC at baseline
Helminth-specific FECs of each individual were expressed as eggs per gram of stool (EPG), calculated by multiplying the sum of the two FECs from duplicate Kato-Katz thick smears by a factor 12. For FLOTAC, the FECs obtained from the two observation grids were added and multiplied by a factor (1/weight of stool sample) and expressed as EPG. The geometric mean (GM) EPG of the study cohort was calculated using the normal logarithm of the EPG plus 1 (GM = exp ((∑ log (EPG+1))/n)−1), where log (EPG+1) is the sum of the logarithm of each individual EPG, and one egg is added to each count to permit the calculation of the logarithm in case of EPG = 0
Participants with complete data from the baseline
Consent to participate in our trial was given by the parents and guardians of 1240 children, among whom 1066 children provided a stool sample at baseline. For FLOTAC examinations, 385 (36%) stool samples were preserved in 5% formaldehyde at baseline (
Flow chart detailing the data loss during stool preservation for FLOTAC, examination, data recording and matching FLOTAC results with duplicate Kato-Katz thick smear readings within the frame of a randomized controlled trial on anthelmintic drug efficacy and safety carried out in Zanzibar in early 2009.
A second stool sample was preserved from 288 among the 385 individuals (75%) 3–9 weeks after the collection of the first stool sample (
Since 29 IDs from the first and second stool examination data set did not match, complete examination data from the baseline and follow-up survey were available from 240 among the 385 originally selected study participants (62%). Among them, 174 were given anthelmintic drugs.
Results presented in
Hookworm | ||||||||||
n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | ||
Kato-Katz | 213 | 62.1 | (56.9–67.3) | 60 | 17.5 | (13.5–21.5) | 27 | 7.9 | (5.0–10.7) | |
FLOTAC | 230 | 67.1 | (62.1–72.1) | 40 | 11.7 | (8.3–15.1) | 35 | 10.2 | (7.0–13.4) | |
‘Gold’ standard | 242 | 70.6 | (65.7–75.4) | 74 | 21.6 | (17.2–25.9) | 40 | 11.7 | (8.3–15.1) | |
Lower quartile (25%) | 0 | 0 | 0 | |||||||
Median | 36.0 | 0 | 0 | |||||||
Upper quartile (75%) | 156.0 | 0 | 0 | |||||||
Arithmetic mean | 235.2 | (153.9–316.5) | 43.6 | (20.2–67.0) | 970.4 | (257.0–1683.8) | ||||
Geometric mean | 18.9 | (14.2–25.2) | 1.2 | (0.8–1.7) | 0.9 | (0.5–1.3) | ||||
Lower quartile (25%) | 0 | 0 | 0 | |||||||
Median | 11.3 | 0 | 0 | |||||||
Upper quartile (75%) | 60.0 | 0 | 0 | |||||||
Arithmetic mean | 75.6 | (47.7–103.5) | 3.1 | (0.2–5.9) | 101.5 | (10.1–193.0) | ||||
Geometric mean | 9.7 | (7.6–12.3) | 0.3 | (0.2–0.4) | 0.6 | (0.3–0.9) | ||||
Kato-Katz | 88.0 | (84.6–91.5) |
81.1 | (76.9–85.2) |
67.5 | (62.5–72.5) |
||||
FLOTAC | 95.0 | (92.7–97.3) | 54.1 | (48.8–59.3) | 87.5 | (84.0–91.0) |
Prevalence, quartiles, arithmetic mean and geometric mean eggs per gram of stool (EPG), and sensitivity with 95% confidence intervals (CI), as determined from stool samples examined with duplicate Kato-Katz thick smears and the FLOTAC basic technique at baseline in 343 school children from Kinyasini and Kilombero primary schools, Zanzibar, in March 2009. The diagnostic ‘gold’ standard was derived by the combined results of duplicate Kato-Katz thick smears and the FLOTAC basic technique.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.74.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.44.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.68.
Hookworm | ||||||||||
n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | ||
Kato-Katz | 146 | 54.0 | (48.3–60.3) | 36 | 13.4 | (9.3–17.5) | 5 | 1.9 | (0.6–4.3) |
|
FLOTAC | 154 | 57.3 | (51.3–63.2) | 30 | 11.2 | (7.4–14.9) | 11 | 4.1 | (2.1–7.2) |
|
‘Gold’ standard | 165 | 61.3 | (55.5–67.2) | 49 | 18.2 | (13.6–22.9) | 12 | 4.5 | (2.3–7.7) |
|
Lower quartile (25%) | 0 | 0 | 0 | |||||||
Median | 12.0 | 0 | 0 | |||||||
Upper quartile (75%) | 90.0 | 0 | 0 | |||||||
Arithmetic mean | 138.3 | (90.6–186.0) | 31.1 | (4.0–58.1) | 168.0 | (0–341.5) | ||||
Geometric mean | 10.5 | (7.5–14.6) | 0.8 | (0.5–1.2) | 0.2 | (0.01–0.3) | ||||
Lower quartile (25%) | 0 | 0 | 0 | |||||||
Median | 2.7 | 0 | 0 | |||||||
Upper quartile (75%) | 18.0 | 0 | 0 | |||||||
Arithmetic mean | 35.6 | (22.1–49.1) | 1.4 | (0.1–2.7) | 78.2 | (0–201.8) | ||||
Geometric mean | 4.4 | (3.3–5.7) | 0.2 | (0.1–0.3) | 0.2 | (0.03–0.3) | ||||
Kato-Katz | 84.9 | (80.6–89.1) |
77.6 | (72.6–82.5) |
41.7 | (35.8–47.6) |
||||
FLOTAC | 93.3 | (90.4–96.3) | 61.2 | (55.4–67.1) | 97.4 | (95.4–99.3) |
Prevalence, quartiles, arithmetic mean and geometric mean eggs per gram of stool (EPG), and sensitivity with 95% confidence intervals (CI), as determined from stool samples examined with duplicate Kato-Katz thick smears and the FLOTAC basic technique at follow-up in 269 school children from Kinyasini and Kilombero primary schools, Zanzibar, in May 2009. The diagnostic ‘gold’ standard was derived by the combined results of duplicate Kato-Katz thick smears and the FLOTAC basic technique.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.73.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.50.
Binomial exact 95% confidence intervals.
Differences in sensitivities determined by the McNemar test on positive individuals:
κ measure of agreement taking into account positive and negative individuals: κ = 0.49.
The inter-method sensitivity between FLOTAC and Kato-Katz differed significantly for the detection of
Moderate-to-substantial agreement between the two diagnostic techniques was observed for all helminths investigated, before and after treatment. The highest agreement (κ = 0.74) was observed for
In line with a higher sensitivity of the FLOTAC basic technique for the diagnosis of
At follow-up, after 193 out of the 269 children (72%) had received experimental treatment, observed prevalences of
Hookworm | ||||||||||
n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | ||
Kato-Katz | 174 | 100 | 41 | 83.7 | (73.0–94.4) | 19 | 82.6 | (65.9–99.4) | ||
FLOTAC | 163 | 93.7 | (90.0–97.3) | 25 | 51.0 | (36.5–65.5) | 22 | 95.7 | (86.6–100) | |
‘Gold’ standard | 174 | 100 | 49 | 100 | 23 | 100 | ||||
Kato-Katz | 121.4 | (98.3–149.7) | 42.6 | (22.4–80.5) | 1119.2 | (220.7–5659.6) | ||||
FLOTAC | 29.7 | (22.9–38.4) | 2.0 | (1.0–3.5) | 193.7 | (61.9–601.6) | ||||
Kato-Katz | 102 | 58.6 | (51.2–66.0) | 19 | 38.8 | (24.6–52.9) | 2 | 8.7 | (0–21.2) | |
FLOTAC | 111 | 63.8 | (56.6–71.0) | 15 | 30.6 | (17.2–44) | 4 | 17.4 | (6.3–34.2) | |
Kato-Katz | 15.1 | (10.0–22.5) | 4.3 | (1.8–9.3) | 1.2 | (0–5.8) | ||||
FLOTAC | 5.7 | (4.1–7.8) | 0.7 | (0.3–1.2) | 1.1 | (0–4.1) | ||||
Kato-Katz | 41.4 | (34.1–48.7) |
61.2 | (47.6–74.9) |
91.3 | (79.8–100) |
||||
FLOTAC | 36.2 | (29.1–43.3) | 69.4 | (56.5–82.3) | 82.6 | (67.1–98.1) | ||||
Kato-Katz | 87.6 | 89.9 | 99.9 | |||||||
FLOTAC | 80.7 | 65.5 | 99.4 |
Prevalence, geometric mean (GM) eggs per gram of stool (EPG), cure rate (CR) and egg reduction rate (ERR), as determined from stool samples examined with duplicate Kato-Katz thick smears and the FLOTAC basic technique in relation to the diagnostic ‘gold’ standard from school children treated with anthelmintic drugs. The diagnostic ‘gold’ standard was derived by the combined results of duplicate Kato-Katz thick smears and the FLOTAC basic technique.
Two-sample test of proportions:
Two-sample test of proportions:
Two-sample test of proportions:
A total of 66 children had a stool sample examined with duplicate Kato-Katz thick smears and the FLOTAC basic technique at baseline and follow-up, without treatment in between. Among them, eight, seven and four children were identified to be infected with
Hookworm | ||||||||||
n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | n pos/EPG | % | 95% CI | ||
Kato-Katz | 8 | 12.1 | 7 | 10.6 | 4 | 6.1 | ||||
FLOTAC | 22 | 33.3 | 5 | 7.6 | 4 | 6.1 | ||||
Kato-Katz | 0.8 | (0.2–1.7) | 0.6 | (0.1–1.2) | 0.4 | (0–0.9) | ||||
FLOTAC | 1.8 | (0.8–3.3) | 0.1 | (0–0.2) | 0.2 | (0–0.5) | ||||
Kato-Katz | 24 | 36.4 | 9 | 13.6 | 0 | 0 | ||||
FLOTAC | 26 | 39.4 | 10 | 15.2 | 1 | 1.5 | ||||
Kato-Katz | 4.2 | (2.0–8.2) | 0.8 | (0.2–1.7) | 0 | |||||
FLOTAC | 2.1 | (1.0–3.6) | 0.3 | (0.1–0.5) | 0.04 | (0–0.1) |
Prevalence and geometric mean (GM) eggs per gram of stool (EPG), as determined from stool samples examined with duplicate Kato-Katz thick smears and the FLOTAC basic technique at baseline and follow-up from 66 school children not treated with anthelmintic drugs.
We found a significantly higher sensitivity of the FLOTAC basic technique compared to the Kato-Katz method for the diagnosis of
The higher sensitivity of the FLOTAC basic technique for
Second, the stool samples in the previous studies were preserved in sodium acetate-acetic acid-formalin (SAF), whereas in the current study 5% formaldehyde was used. A potential negative impact of the stool preservation media and FS on fragile hookworm eggs have been discussed before
Third, the higher sensitivity of FLOTAC for hookworm diagnosis at follow-up compared to baseline is pointing to a negative impact of the duration of stool preservation on hookworm eggs (samples collected at follow-up had at least a 3-week shorter preservation period than samples preserved at the baseline survey), which is in line with findings from Côte d'Ivoire
Fourth, floated organic debris might have averted the accurate detection of transparent hookworm eggs in some of our stool samples, and hence negatively impacted on the sensitivity of FLOTAC for hookworm diagnosis. This latter problem was recently observed in stool samples collected from school children in Côte d'Ivoire and Pemba island, where it was overcome by including a washing step with ether or ethyl acetate to remove the organic debris or by a higher dilution of stool samples using tap water
The comparable sensitivities of either method at baseline and follow-up, despite a considerable decrease in FECs, suggest that a decrease in sensitivity only occurs if FECs fall under the lower detection limit of a method (i.e., 12 EPG for duplicate Kato-Katz thick smears, 24 EPG for a single Kato-Katz thick smear and 1 EPG for the FLOTAC basic technique). This suggestion is supported by the finding that those seven individuals found
The considerably lower numbers in the GM EPGs of our study cohort derived by FLOTAC in comparison to Kato-Katz are in line with previous studies
The somewhat lower observed CRs against
Before generalizing these results one must consider, however, that our study design suffers from the following shortcomings: (i) the study was not adequately powered for clinically important findings; (ii) CRs and ERRs were estimated only for individuals who were found
There are several causes for the high loss of samples from the baseline to the follow-up survey. First, 97 children did not submit a stool sample at follow-up. Among them 87 (89.7%) were from the
While it is still too early to generalize the results reported here, and the FLOTAC technique might need further optimization for reliable diagnosis of hookworm infections, this new copro-microscopic technique holds promise for simultaneous detection of the three common soil-transmitted helminths,
Diagnostische Genauigkeit der Kato-Katz und FLOTAC Methode bei der Bestimmung Anthelminthischer Medikamentenwirksamkeit - Translation of abstract into German by Stefanie Knopp.
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STARD checklist.
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Study protocol of the randomized controlled trial, which provided the base of our study on diagnostic accuracy of the Kato-Katz and FLOTAC techniques for assessing anthelmintic drug efficacy.
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We thank the children, school directors and teachers from Kinyasini and Kilombero schools in Unguja island, Zanzibar, for their support and commitment during the study. We are grateful to the whole team at the HCLU from the MoHSW Zanzibar for their help in the field and in performing hundreds of Kato-Katz thick smears and FLOTAC readings. We are indebted to the Department of Pathology and Animal Health of the University of Naples “Federico II” for the generous donation of a centrifuge to the HCLU in Zanzibar. We also thank several external referees for constructive and helpful comments and suggestions that further improved the quality of our manuscript.