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Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

ABSTRACT: BACKGROUND:Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs). METHODOLOGY:We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs. PRINCIPAL FINDINGS:All diagnostic methods showed a clinical sensitivity of ?90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration. CONCLUSIONS/SIGNIFICANCE:Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program. TRIAL REGISTRATION:ClinicalTrials.gov NCT03465488.

SUBMITTER: Cools P

PROVIDER: S-EPMC6675048 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

Cools Piet P Vlaminck Johnny J Albonico Marco M Ame Shaali S Ayana Mio M José Antonio Barrios Perez BP Cringoli Giuseppe G Dana Daniel D Keiser Jennifer J Maurelli Maria P MP Maya Catalina C Matoso Leonardo F LF Montresor Antonio A Mekonnen Zeleke Z Mirams Greg G Corrêa-Oliveira Rodrigo R Pinto Simone A SA Rinaldi Laura L Sayasone Somphou S Thomas Eurion E Verweij Jaco J JJ Vercruysse Jozef J Levecke Bruno B

PLoS neglected tropical diseases 20190801 8

<h4>Background</h4>Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs).<h4>Methodology</h4>We compared the diagnost ...[more]

PMID: 31369558

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Project description:BackgroundCopro-parasitological diagnosis is still a challenge in management of helminth infections at individual and community levels in resource-limited settings.The aim of our study was to compare the performance of three quantitative techniques: Kato-Katz, McMaster and Mini-FLOTAC methids. The study was carried out in Oran, Northern Argentina.Methods200 schoolchildren were enrolled to provide a single stool sample, which was tested for helminth infections with Kato-Katz, McMaster and Mini-FLOTAC methods. The Mini-FLOTAC was performed with two flotation solutions (FS2 saturated saline and FS7 zinc sulphate). Preparation and reading time for each of the three methods was calculated both when processing single and multiple samples.ResultsOut of 193 schoolchildren examined, 40% were positive for any helminth infection by any method; the most prevalent was Hymenolepis nana (23%) followed by Ascaris lumbricoides (17%) and a third group of less prevalent helminths: Enterobius vermicularis, Trichuris trichiura and hookworms (11% all together). Mini-FLOTAC FS2 was more sensitive than FS7 for H. nana (93% vs 78%) and for other helminths (85% vs 80%), whereas FS7 was more sensitive for A. lumbricoides (87% vs 61%). Kato-Katz method was more sensitive than McMaster method for A. lumbricoides (84% vs 48%) and for other helminths (48% vs 43%) except for H. nana (49% vs 61%). As for egg counts, Mini-FLOTAC FS2 reported 904 eggs per gram of faeces (EPG) for H. nana (vs 457 with McMaster and 111 with Kato-Katz) and 1177 EPG for A. lumbricoides (vs 1315 with Kato-Katz and 995 with McMaster); FS2 detected the highest EPG for both H.nana and A.lumbricoides (904 vs 568 and 1177 vs 643 respectively), the differences were not statistically significant. The technique feasibility was calculated: Kato-Katz mean time was 48 minutes/sample, Mini-FLOTAC 13 minutes/sample and McMaster 7 minutes/sample. However, especially for Kato-Katz and Mini-FLOTAC, the mean time (min/sample) decreased significantly when processing multiple samples.ConclusionsMini-FLOTAC is a promising technique for helminth diagnosis, it is more sensitive than Kato-Katz and McMaster for H. nana and as sensitive as Kato-Katz and more sensitive than McMaster for A. lumbricoides identification. Egg counts differences although relevant, did not reach statistical significance.

Project description:BackgroundSensitive 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.Methodology/principal findingsStool 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 Trichuris trichiura (95% vs. 88%, p = 0.012) and Ascaris lumbricoides (88% vs. 68%, p = 0.098), but a lower sensitivity for hookworm diagnosis (54% vs. 81%, p = 0.006). Considering the combined results from both methods as 'gold' standard, the prevalences of T. trichiura, hookworm and A. lumbricoides were 71% (95% confidence interval (CI): 66-75%), 22% (95% CI: 17-26%) and 12% (95% CI: 8-15%), respectively. At follow-up, 3-5 weeks after 174 among the 269 re-examined children were administered anthelmintic drugs, we observed cure rates (CRs) against A. lumbricoides, hookworm and T. trichiura of 91% (95% CI: 80-100%), 61% (95% CI: 48-75%) and 41% (95% CI: 34-49%), respectively, when using the Kato-Katz method. FLOTAC revealed lower CRs against A. lumbricoides (83%, 95% CI: 67-98%) and T. trichiura (36%, 95% CI: 29-43%), but a higher CR against hookworm (69%, 95% CI: 57-82%). These differences, however, lacked statistical significance. Considerable differences were observed in the geometric mean fecal egg counts between the two methods with lower egg reduction rates (ERRs) determined by FLOTAC.Conclusion/significanceOur 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.

Project description:The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings.

Project description:BackgroundThe Kato-Katz thick smear (Kato-Katz) is the diagnostic method recommended for monitoring large-scale treatment programs implemented for the control of soil-transmitted helminths (STH) in public health, yet it is difficult to standardize. A promising alternative is the McMaster egg counting method (McMaster), commonly used in veterinary parasitology, but rarely so for the detection of STH in human stool.Methodology/principal findingsThe Kato-Katz and McMaster methods were compared for the detection of STH in 1,543 subjects resident in five countries across Africa, Asia and South America. The consistency of the performance of both methods in different trials, the validity of the fixed multiplication factor employed in the Kato-Katz method and the accuracy of these methods for estimating 'true' drug efficacies were assessed. The Kato-Katz method detected significantly more Ascaris lumbricoides infections (88.1% vs. 75.6%, p<0.001), whereas the difference in sensitivity between the two methods was non-significant for hookworm (78.3% vs. 72.4%) and Trichuris trichiura (82.6% vs. 80.3%). The sensitivity of the methods varied significantly across trials and magnitude of fecal egg counts (FEC). Quantitative comparison revealed a significant correlation (Rs >0.32) in FEC between both methods, and indicated no significant difference in FEC, except for A. lumbricoides, where the Kato-Katz resulted in significantly higher FEC (14,197 eggs per gram of stool (EPG) vs. 5,982 EPG). For the Kato-Katz, the fixed multiplication factor resulted in significantly higher FEC than the multiplication factor adjusted for mass of feces examined for A. lumbricoides (16,538 EPG vs. 15,396 EPG) and T. trichiura (1,490 EPG vs. 1,363 EPG), but not for hookworm. The McMaster provided more accurate efficacy results (absolute difference to 'true' drug efficacy: 1.7% vs. 4.5%).Conclusions/significanceThe McMaster is an alternative method for monitoring large-scale treatment programs. It is a robust (accurate multiplication factor) and accurate (reliable efficacy results) method, which can be easily standardized.

Project description:BackgroundSoil-transmitted helminths infect about one fifth of the world's population and have a negative impact on health. The Kato-Katz technique is the recommended method to detect soil-transmitted helminth eggs in stool samples, particularly in programmatic settings. However, some questions in its procedure remain. Our study aimed to investigate the effect of storage time, storage temperature and stirring of stool samples on fecal egg counts (FECs).Methodology/principal findingsIn the framework of a clinical trial on Pemba Island, United Republic of Tanzania, 488 stool samples were collected from schoolchildren. These samples were evaluated in three experiments. In the first experiment (n = 92), two Kato-Katz slides were prepared from the same stool sample, one was stored at room temperature, the other in a refrigerator for 50 hours, and each slide was analyzed at nine time points (20, 50, 80, 110, 140 minutes, 18, 26, 42 and 50 hours). In the second experiment (n = 340), whole stool samples were split into two, one part was stored at room temperature, and the other part was put in a refrigerator for 48 hours. From each part one Kato-Katz slide was prepared and analyzed at three time points over two days (0, 24 and 48 hours). In the third experiment (n = 56), whole stool samples where stirred for 15 seconds six times and at each time point a Kato-Katz slide was prepared and analyzed. Mean hookworm FECs of Kato-Katz slides stored at room temperature steadily decreased following slide preparation. After two hours, mean hookworm FECs decreased from 22 to 16, whereas no reduction was observed if Kato-Katz slides were stored in the refrigerator (19 vs 21). The time x storage interaction effect was statistically significant (coefficient 0.26, 95% CI: 0.17 to 0.35, p < 0.0001). After 24 hours mean hookworm FECs dropped close to zero, irrespective of the storage condition. Whole stool samples stored at room temperature for one day resulted in a mean hookworm FEC decrease of 23% (p < 0.0001), compared to a 13% reduction (p < 0.0001) if samples were stored in the refrigerator. Fecal egg counts of A. lumbricoides and T. trichiura remained stable over time regardless of storage temperature of whole stool samples. Finally, we found a significant reduction of the variation of hookworm and T. trichiura eggs with increasing rounds of stirring the sample, but not for A. lumbricoides. For hookworm we observed a simultaneous decrease in mean FECs, making it difficult to draw recommendations on stirring samples.Conclusions/significanceOur findings suggest that stool samples (i) should be analyzed on the day of collection and (ii) should be analyzed between 20-30 minutes after slide preparation; if that is not possible, Kato-Katz slides can be stored in a refrigerator for a maximum of 110 minutes.

Project description:BACKGROUND:Understanding and quantifying the sources and implications of error in the measurement of helminth egg intensity using Kato-Katz (KK) and the newly emerging "gold standard" quantitative polymerase chain reaction (qPCR) technique is necessary for the appropriate design of epidemiological studies, including impact assessments for deworming programs. METHODS:Repeated measurements of Ascaris lumbricoides infection intensity were made from samples collected in western Kenya using the qPCR and KK techniques. These data were combined with data on post-treatment worm expulsions. Random effects regression models were used to quantify the variability associated with different technical and biological factors for qPCR and KK diagnosis. The relative precision of these methods was compared, as was the precision of multiple qPCR replicates. RESULTS:For both KK and qPCR, intensity measurements were largely determined by the identity of the stool donor. Stool donor explained 92.4% of variability in qPCR measurements and 54.5% of observed measurement variance for KK. An additional 39.1% of variance in KK measurements was attributable to having expelled adult A. lumbricoides worms following anthelmintic treatment. For qPCR, the remaining 7.6% of variability was explained by the efficiency of the DNA extraction (2.4%), plate-to-plate variability (0.2%) and other residual factors (5%). Differences in replicate measurements by qPCR were comparatively small. In addition to KK variability based on stool donor infection levels, the slide reader was highly statistically significant, although it only explained 1.4% of the total variation. In a comparison of qPCR and KK variance to mean ratios under ideal conditions, the coefficient of variation was on average 3.6 times larger for KK highlighting increased precision of qPCR. CONCLUSIONS:Person-to-person differences explain the majority of variability in egg intensity measurements by qPCR and KK, with very little additional variability explained by the technical factors associated with the practical implementation of these techniques. qPCR provides approximately 3.6 times more precision in estimating A. lumbricoides egg intensity than KK, and could potentially be made more cost-effective by testing each sample only once without diminishing the power of a study to assess population-level intensity and prevalence.

Project description:BackgroundOver one billion people are infected with soil-transmitted helminths (STH), i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura. For estimating drug efficacy and monitoring anthelminthic drug resistance, accurate diagnostic methods are critical. FECPAKG2 is a new remote-diagnostic tool used in veterinary medicine, which produces an image of the stool sample that can be stored on an internet cloud. We compared for the first time FECPAKG2 with the recommended Kato-Katz method.Methodology/principal findingsTwo stool samples were collected from adolescent participants (age 15-18 years) at baseline and 14 to 21 days after treatment in the framework of a randomized clinical trial on Pemba Island, Tanzania. Stool samples were analyzed with different diagnostic efforts: i) one or ii) two Kato-Katz thick smears from the first sample, iii) two Kato-Katz thick smears from two samples and iv) FECPAKG2 from the first sample. Parameters were calculated based on a hierarchical Bayesian egg count model. Complete data for all diagnostic efforts were available from 615 participants at baseline and 231 hookworm-positive participants at follow-up. At baseline FECPAKG2 revealed a sensitivity of 75.6% (72.0-77.7) for detecting A. lumbricoides, 71.5% (67.4-95.3) for hookworm and 65.8% (64.9-66.2) for T. trichiura, which was significantly lower (all p<0.05) than any of the Kato-Katz methods and highly dependent on infection intensity. Despite that the egg counts based on FECPAKG2 were relatively lower compared to Kato-Katz by a ratio of 0.38 (0.32-0.43) for A. lumbricoides, 0.36 (0.33-0.40) for hookworm and 0.08 (0.07-0.09) for T. trichiura, the egg reduction rates (ERR) were correctly estimated with FECPAKG2.Conclusions/significanceThe sensitivity to identify any STH infection was considerably lower for FECPAKG2 compared to Kato-Katz. Following rigorous development, FECPAKG2 might be an interesting tool with unique features for epidemiological and clinical studies.

Dataset Information

Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

Publications

Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries.

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