Abstract
Objective. The aim of this study was to evaluate the operational characteristics (sensitivity and negative predictive value (NPV)) of wet mount, formol-ether concentration (FEC), and Kato-Katz techniques for the determination of intestinal parasitic infections. Method. A total of 354 faecal specimens were collected from students in Northwest Ethiopia and screened with Kato-Katz, wet mount, and FEC for the presence of intestinal parasitic infection. Since a gold standard test is not available for detection of intestinal parasites, the combined results from the three methods were used as diagnostic gold standard. Result. The prevalences of intestinal parasites using the single wet mount, FEC, and Kato-Katz thick smear techniques were 38.4%, 57.1%, and 59%, respectively. Taking the combined results of three techniques as a standard test for intestinal parasitic infection, the sensitivity and negative predictive value of Kato-Katz is 81.0% (confidence interval (CI) = 0.793–0.810) and 66.2% (CI = 0.63–0.622), respectively. The FEC detected 56 negative samples that were positive by the gold standard, indicating 78.3% (CI = 0.766–0.783) and 63.2% (CI = 0.603–63) sensitivity and NPV, respectively. Furthermore, Kato-Katz detects 113 cases that were negative by a single wet mount. The κ agreement between the wet mount and Kato-Katz methods for the diagnosis of Ascaris lumbricoides and hookworm was substantial (κ = 0.61 for Ascaris lumbricoides, κ = 0.65 for hookworm).
1. Background
Intestinal parasitic infections are among the most common infections worldwide. It is estimated that 3.5 billion people are affected, and 450 million are ill as a result of these infections, the majority being children [1]. In Ethiopia, intestinal parasitic infection is the second most predominant cause of outpatient morbidity. However, there are difficulties in estimating the exact burden of parasitic infections in the country [2]. Although there are several factors that make estimating the number and burden of intestinal parasitic infections difficult, lack of accurate diagnostic tools is the major one [3–9].
Although several diagnostic methods such as Kato-Katz and Formol-Ether Concentration (FEC) techniques are available, direct wet mount is the commonly used as a reliable diagnosis method for the diagnosis of intestinal parasitic infections generally in Africa and particularly in Ethiopia [10–13]. However, low sensitivity of the direct wet mount technique has been reported in the detection of low-intensity infection elsewhere [14]. This shows that the use of direct wet mount as a confirmatory test will significantly increase misdiagnosis of intestinal helminth infections. The reliable diagnosis of intestinal parasitic infections requires a more rapid, easy, and sensitive method. Therefore, this study aimed to evaluate the operational characteristics (sensitivity and negative predictive value (NPV)) of wet mount, FEC, and Kato-Katz techniques in intestinal parasitic infection endemic locality of Ethiopia.
2. Methods
2.1. Study Design and Area
A cross-sectional study was conducted in Atse Fasil Elementary School from March 10 to June 30, 2008 in Northwest Ethiopia, latitude of 12.55 (12°33′0 N), a longitude of 37.43 (37°25′32 E), and an elevation of 1,994 meters above sea level [15].
2.2. Study Population
A total of 354 students were included in the study. Students who had no history of antihelmenthic drug administration in the two weeks prior to screening, absence of any other serious chronic infection, and had ability to give stool samples were included in the study.
2.3. Stool Collection and Analysis
Students were provided with plastic stool cups and asked to bring approximately 3 gm of fresh stool of their own. Approximately 41.7 mg and 20 mg of the stool specimen were used to prepare a single Kato-Katz thick smear and wet mount preparation, respectively. One gram of stool sample was analyzed by FEC technique [16]. All the samples were processed in the temporally established laboratory except the Kato-Katz smear count of the common helminthes, which was done at the laboratory of the University of Gondar. The egg per gram of faeces (EPGs) counts for hookworm were done immediately in the temporary laboratory. The Kato-thick smear counts for all the common helminthes except for hookworm were done at the laboratory of University of Gondar.
2.4. Quality Control
Before starting the actual work, quality of reagents and instruments was checked by experienced laboratory technicians. The specimens were also checked for serial number, quantity, and procedures of collection.
To eliminate observer bias, each stool sample was examined immediately in the temporary laboratory by two experienced laboratory technicians. The technicians were not told about the health and other status of the study participants. In cases where the results were discordant, a third expert reader was used. The results of the third expert reader were considered the final result.
2.5. Data Analysis
Since a “gold” standard test is not available for detection of intestinal parasites, the operational characteristics (sensitivity and NPV) were estimated using the combined results from the three methods as diagnostic “gold” standard [17, 18].
Data were analyzed using SPSS version 16 and JavaStat software. Sensitivities, NPV, and kappa were determined for the various tests to evaluate their operational characteristics. P values <0.05 were considered statistically significant.
2.6. Ethical Considerations
The study protocol was reviewed and approved by the Ethical Review Committee of the Department of Medical Laboratory Technology, University of Gondar. Written informed consent was obtained from all study participants and mothers/caretakers of students under 18 who participated in the study after explaining the purpose and objective of the study. Students who were positive for intestinal parasites were treated based on the recommended drug regimen at Azezo Clinic, Gondar.
3. Result
A total of 354 students aged 5–19 years participated in the study; 146 (53.9%) and 125 (46.1%) were males and females, respectively.
3.1. Prevalence
The prevalences of intestinal parasites using single wet mount, FEC, and Kato-Katz thick smear techniques were 38.4%, 57.1%, and 59%, respectively (Table 1). The detection rate when two techniques used at a time was 69.8% (for Kato-thick and FEC), 61% (for wet mount and FEC), and 68% (for wet mount and Kato-thick smear technique). The detection rate was 72.9% (258/354) when all the three techniques were used together. The overall prevalence of Schistosoma mansoni, Ascaris lumbricoides, Trichuris trichiura, and hookworm infections was 43.5%, 28.8%, 18.1%, and 8.2%, respectively (Table 2).
Table 1.
Wet mount, Kato-Katz, and FEC techniques results compared to the gold standard from Atse Fasil General Elementary School, Northwest Ethiopia, March 10–June 30, 2008.
| Method | Result | Gold standard | Total (%) | NPV | 95% CI | Sensitivity | 95% CI | |
|---|---|---|---|---|---|---|---|---|
| Positive (%) | Negative (%) | |||||||
| Wet mount | Positive | 136 (38.4) | 0 | 136 (38.4) | 44.0% | 0.42–0.44 | 52.7% | 0.51–0.53 |
| Negative | 122 (34.5) | 96 (27.1) | 218 (61.6) | |||||
| FEC | Positive | 202 (57.1) | 0 | 202 (57.1) | 63.2% | 0.60–0.63 | 78.3% | 0.76–0.78 |
| Negative | 56 (15.8) | 96 (27.1) | 152 (42.9) | |||||
| Kato-Katz | Positive | 209 (59.0) | 0 | 209 (59.0) | 66.2% | 0.63–0.62 | 81.0% | 0.79–0.81 |
| Negative | 49 (13.8) | 96 (27.1) | 145 (41.0) | |||||
FEC: formol-ether concentration, NPV: negative predictive value.
Table 2.
Wet mount, Kato-Katz, and FEC techniques results compared to the gold standard by species of common helminthes from students of Atse Fasil General Elementary School, Northwest Ethiopia, March 10–June 30, 2008.
| Parasite | Technique | Infected students no. (%) | NPV (95% CI) | Sensitivity (95% CI) |
|---|---|---|---|---|
| S. mansoni | Gold standard | 154 (43.5) | ||
| Kato katz | 148 (41.8) | 97.1% (0.95–0.97) | 96.1% (0.94–0.96) | |
| FEC | 90 (25.4) | 75.8% (0.74–0.75) | 58.4% (0.55–0.58) | |
| Wet mount | 34 (9.6) | 62.5% (0.61–0.62) | 22.1% (0.194–0.221) | |
|
| ||||
| A. lumbricoides | Gold standard | 102 (28.8) | ||
| Kato-Katz | 95 (26.8) | 97.3% (0.95–0.97) | 93.1% (0.89–0.93) | |
| FEC | 83 (23.4) | 93.0% (0.92–0.93) | 81.4% (0.77–0.81) | |
| Wet mount | 53 (15) | 83.7% (0.82–0.83) | 52.0% (0.48–0.52) | |
|
| ||||
| T. trichiura | Gold standard | 64 (18.1) | ||
| Kato-Katz | 58 (16.4) | 98.0% (0.97–0.98) | 90.6% (0.85–0.91) | |
| FEC | 37 (10.5) | 91.5% (0.90–0.92) | 57.8% (0.52–0.58) | |
| Wet mount | 8 (2.3) | 83.8% (0.83–0.84) | 12.5% (0.07–0.12) | |
|
| ||||
| Hook worm | Gold standard | 29 (8.2) | ||
| Kato-Katz | 20 (5.6) | 97.3% (0.96–0.97) | 69.0% (0.57–0.69) | |
| FEC | 21 (5.9) | 97.6% (0.97–0.98) | 72.4% (0.60–0.72) | |
| Wet mount | 11 (3.1) | 94.8% (0.94–0.95) | 37.9% (0.27–0.38) | |
FEC: formol-ether concentration, NPV: negative predictive value.
3.2. Sensitivity and NPV
Among the 354 study participants diagnosed 209 were found to be positive while 145 were negative by Kato-Katz. The sensitivity and NPV of Kato-Katz are 81.0% (confidence interval (CI) = 0.793–0.810) and 66.2% (CI = 0.63–0.622), respectively (Table 1). The FEC detected 56 negative samples that were positive by the gold standard, indicating 78.3% (CI = 0.766–0.783) and 63.2% (CI = 0.603–63) sensitivity and NPV, respectively (Table 1).
3.3. Agreement in Test Results
As compared to single FEC, single Kato-Katz detected 53, 83, 56, and 95 cases of Ascaris lumbricoides, Schistosoma mansoni, Trichuris trichiura, and hook worm, respectively (Table 3), indicating substantial (κ = 0.80), moderate (κ = 0.58), and moderate (κ = 0.59) κ agreement between the FEC and Kato-Katz for the diagnosis of Ascaris lumbricoides, Schistosoma mansoni and Trichuris trichiura, respectively (Table 3). Furthermore, Kato-Katz detects 113 cases that were negative by a single wet mount (Table 4). The κ agreement between the wet mount and Kato-Katz methods for the diagnosis of Ascaris lumbricoides and hook worm was substantial (κ = 0.61 for Ascaris lumbricoides, κ = 0.65 for hookworm) (Table 4).
Table 3.
Agreement between a single Kato-Katz and FEC technique for the diagnosis of each intestinal parasite in students from Atse Fasil General Elementary School, Northwest Ethiopia, March 10–June 30, 2008.
| Parasite | FEC | Kato-Katz | Total | κ agreement (P) | |
|---|---|---|---|---|---|
| Positive | Negative | ||||
| S. mansoni | Positive | 85 | 5 | 85 | 0.58 (P < 0.001) |
| Negative | 62 | 202 | 264 | ||
|
| |||||
| Total | 147 | 207 | 354 | ||
|
| |||||
| A. lumbricoides | Positive | 75 | 8 | 83 | 0.80 (P < 0.001) |
| Negative | 18 | 253 | 271 | ||
|
| |||||
| Total | 93 | 261 | 354 | ||
|
| |||||
| T. trichiura | Positive | 31 | 6 | 37 | 0.59 (P < 0.001) |
| Negative | 28 | 289 | 317 | ||
|
| |||||
| Total | 59 | 295 | 354 | ||
|
| |||||
| Hook worm | Positive | 12 | 9 | 21 | 0.57 (P < 0.001) |
| Negative | 7 | 326 | 333 | ||
|
| |||||
| Total | 19 | 335 | 354 | ||
FEC: formol-ether concentration.
Table 4.
Agreement between a single Kato-Katz and wet mount for the diagnosis of each intestinal parasite in students from Atse Fasil General Elementary School, Northwest Ethiopia, March 10–June 30, 2008.
| Parasite | Wet mount | Kato-Katz | Total | κ agreement (P) | |
|---|---|---|---|---|---|
| Positive | Negative | ||||
| S. mansoni | Positive | 33 | 1 | 34 | 0.25 (P < 0.001) |
| Negative | 114 | 206 | 320 | ||
|
| |||||
| Total | 147 | 207 | 354 | ||
|
| |||||
| A. lumbricoides | Positive | 50 | 3 | 53 | 0.61 (P < 0.001) |
| Negative | 43 | 258 | 301 | ||
|
| |||||
| Total | 93 | 261 | 354 | ||
|
| |||||
| T. trichiura | Positive | 8 | 0 | 8 | 0.21 (P < 0.001) |
| Negative | 51 | 295 | 346 | ||
|
| |||||
| Total | 59 | 295 | 354 | ||
|
| |||||
| Hook worm | Positive | 10 | 1 | 11 | 0.65 (P < 0.001) |
| Negative | 9 | 334 | 343 | ||
|
| |||||
| Total | 19 | 335 | 354 | ||
4. Discussion
A single Kato-Katz had significantly lower detection capacity than the FEC method in diagnosing hookworm. This poor performance of the Kato-Katz in detecting hookworm infection is explained by the following facts. First, hookworm has lower egg laying capacity, more likely to be missed by Kato-Katz. Second, hookworm eggs disappear due to glycerin when long time delays occur between Kato-Katz smear preparation and microscopic examination [19]. Furthermore, unlike FEC, small amount of fecal material is processed in Kato-Katz technique. The chance of detecting hookworm infection by Kato-Katz from small amount of faecal material is suggested to be lower [20, 21]. Therefore, small amount of fecal material used in Kato-Katz technique may be the reason for lower detection capacity of Kato-Katz.
The finding of the present study showed that, as compared to the Kato-Katz and FEC techniques, direct wet mount exhibited very low sensitivity for the detection of Ascaris lumbricoides, Schistosoma mansoni, Trichuris trichiura and hookworm. The use of direct wet mount alone as an indicator of intestinal parasitic infections is also suggested to be insufficient by other studies [22]. However, in most laboratories in Ethiopia, the direct wet mount is the preferred stool parasitological detection technique. This shows that, since the use of direct wet mount as a confirmatory test will significantly increase misdiagnosis of intestinal helminth infections, the use of another diagnosing method is mandatory to decrease the consequences caused in the community due to intestinal helminth infections.
As compared to FEC and direct wet mount techniques, the Kato-Katz exhibited high sensitivity for the detection of Schistosoma mansoni, Trichuris trichiura, and Ascaris lumbricoides. This shows that the use of the Kato-Katz as a confirmatory test for Schistosoma mansoni, Trichuris trichiura, and Ascaris lumbricoides will reduce the morbidity and mortality caused by these parasites by reducing misdiagnosis. However, lack of previous similar study makes difficulty in making rigorous discussion on this finding.
In conclusion, the present study revealed that Kato-Katz technique and FEC methods showed a better sensitivity than the traditional direct wet mount method. Therefore, the employment of FEC technique as a confirmatory test in routine laboratory examination of stool and Kato-Katz in epidemiological studies will significantly aid in accurate determination and management of parasitic infections in the community.
Acknowledgments
The study was financially supported by College of Medicine and Health Sciences, University of Gondar. The authors thank the study participants for their cooperation and the health management and working staff of Gondar District Health Office and University of Gondar.
Conflict of Interests
The authors declare that they have no conflict of interests.
Authors' Contribution
M. Endris was involved in all aspects of the project, study design, data collection, analysis, and in writing of the paper. Z. Tekeste was involved in data analysis, interpretation, and in writing of the paper. A. Kassu and W. Lemma have made a contribution in supervision and in critically revising the paper. All authors read and approved the manuscript.
References
- 1.World Health Organization (WHO) Control of Tropical Diseases. Geneva, Switzerland: WHO; 1998. [Google Scholar]
- 2.Tesfa-Yohannes T. M., Kloos H. Intestinal parasitism. In: Zein A. Z., Kloos H., editors. Ecology of Health and Disease in Ethiopia. Addis Ababa, Ethiopia: Ministry of Health; 1988. pp. 214–230. [Google Scholar]
- 3.Booth M., Vounatsou P., N'goran E. K., Tanner M., Utzinger J. The influence of sampling effort and the performance of the Kato-Katz technique in diagnosing Schistosoma mansoni and hookworm co-infections in rural Côte d'Ivoire. Parasitology. 2003;127(6):525–531. doi: 10.1017/S0031182003004128. [DOI] [PubMed] [Google Scholar]
- 4.Utzinger J., N'Goran E. K., Caffrey C. R., Keiser J. From innovation to application: social-ecological context, diagnostics, drugs and integrated control of schistosomiasis. Acta Tropica. 2010;120(1):121–137. doi: 10.1016/j.actatropica.2010.08.020. [DOI] [PubMed] [Google Scholar]
- 5.Montresor A., Gyorkos T. W., Crompton D. W., Bundy D., Savioli : L. Monitoring Helminth Control Programmes: Guidelines for Monitoring the Impact of Control Programmes Aimed at Reducing Morbidity Caused by Soil-Transmitted Helminthes and Schistosomes, with Particular Reference to School-age Children. Geneva, Switzerland: WHO; 1998. [Google Scholar]
- 6.WHO. Prevention and control of schistosomiasis and soil-transmitted helminthiasis; report of a WHO expert committee. WHO Technical Report Series. 2002;(912) [PubMed]
- 7.Berhe N., Medhin G., Erko B., et al. Variations in helminth faecal egg counts in Kato-Katz thick smears and their implications in assessing infection status with Schistosoma mansoni . Acta Tropica. 2004;92(3):205–212. doi: 10.1016/j.actatropica.2004.06.011. [DOI] [PubMed] [Google Scholar]
- 8.Speich B., Knopp S., Mohammed K. A., et al. Comparative cost assessment of the Kato-Katz and FLOTAC techniques for soil-transmitted helminth diagnosis in epidemiological surveys. Parasites and Vectors. 2010;3(1, article 71) doi: 10.1186/1756-3305-3-71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Knopp S., Mgeni A. F., Khamis I. S., 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 Neglected Tropical Diseases. 2008;2(11, article e331) doi: 10.1371/journal.pntd.0000331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Utzinger J., Rinaldi L., Lohourignon L. K., et al. FLOTAC: a new sensitive technique for the diagnosis of hookworm infections in humans. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2008;102(1):84–90. doi: 10.1016/j.trstmh.2007.09.009. [DOI] [PubMed] [Google Scholar]
- 11.Kassahun H., Abrham D., Yemane Y., Birhanu E. Comparision of the Kato katz and FLOTAC techniques for the diagnosis of soil transmitted helminth infections. Parasitology International. 2011;60(4):398–402. doi: 10.1016/j.parint.2011.06.020. [DOI] [PubMed] [Google Scholar]
- 12.Knopp S., Rinaldi L., Khamis I. S., et al. A single FLOTAC is more sensitive than triplicate Kato-Katz for the diagnosis of low-intensity soil-transmitted helminth infections. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2009;103(4):347–354. doi: 10.1016/j.trstmh.2008.11.013. [DOI] [PubMed] [Google Scholar]
- 13.Tarafder M. R., Carabin H., Joseph L., Balolong E., Olveda R., McGarvey S. T. 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’. International Journal for Parasitology. 2010;40(4):399–404. doi: 10.1016/j.ijpara.2009.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Levecke B., De Wilde N., Vandenhoute E., Vercruysse J. Field validity and feasibility of four techniques for the detection of Trichuris in Simians: a model for monitoring drug efficacy in public health? PLoS Neglected Tropical Diseases. 2009;3(1, article e366) doi: 10.1371/journal.pntd.0000366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Gondar, Ethiopia, 2012, http://en.wikipedia.org/wiki/Gondar.
- 16.WHO. Bench Aids for the Diagnosis of Intestinal Parasites. Geneva, Switzerland: WHO; 1994. [Google Scholar]
- 17.Joseph L., Gyorkos T. W., Coupal L. Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard. American Journal of Epidemiology. 1995;141(3):263–272. doi: 10.1093/oxfordjournals.aje.a117428. [DOI] [PubMed] [Google Scholar]
- 18.Dendukuri N., Rahme E., Bélisle P., Joseph L. Bayesian sample size determination for prevalence and diagnostic test studies in the absence of a gold standard test. Biometrics. 2004;60(2):388–397. doi: 10.1111/j.0006-341X.2004.00183.x. [DOI] [PubMed] [Google Scholar]
- 19.Dacombe R. J., Crampin A. C., Floyd S., et al. Time delays between patient and laboratory selectively affect accuracy of helminth diagnosis. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2007;101(2):140–145. doi: 10.1016/j.trstmh.2006.04.008. [DOI] [PubMed] [Google Scholar]
- 20.Hall A. Intestinal helminths of man: the interpretation of egg counts. Parasitology. 1982;85(3):605–613. doi: 10.1017/s0031182000056389. [DOI] [PubMed] [Google Scholar]
- 21.Lin D. D., Liu J. X., Liu Y. M., et al. Routine Kato-Katz technique underestimates the prevalence of Schistosoma japonicum: a case study in an endemic area of the People's Republic of China. Parasitology International. 2008;57(3):281–286. doi: 10.1016/j.parint.2008.04.005. [DOI] [PubMed] [Google Scholar]
- 22.Paraemeshwarappa K. D., Chandrakanth C., Sunil B. The prevalence of intestinal parasitic infections and the evaluation of different concentration techniques of the stool examination. Journal of Clinical and Diagnostic Research. 2012;6(7):1188–1191. [Google Scholar]