Comparison of the Kato-Katz, Wet Mount, and Formol-Ether Concentration Diagnostic Techniques for Intestinal Helminth Infections in Ethiopia

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).


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][4][5][6][7][8][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][11][12][13]. However, low sensitivity of the direct wet mount technique has been reported in the detection of lowintensity infection elsewhere [14]. is shows that the use of direct wet mount as a con�rmatory test will signi�cantly increase misdiagnosis of intestinal helminth infections. e reliable diagnosis of intestinal parasitic infections requires a more rapid, easy, and sensitive method. erefore, 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.

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.

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. e egg per gram of faeces (EPGs) counts for hookworm were done immediately in the temporary laboratory. e Kato-thick smear counts for all the common helminthes except for hookworm were done at the laboratory of University of Gondar.

Quality Control.
Before starting the actual work, quality of reagents and instruments was checked by experienced laboratory technicians. e 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. e 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. e results of the third expert reader were considered the �nal result.

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  ( ), moderate ( ), and moderate ( 9) 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). e agreement between the wet mount and Kato-Katz methods for the diagnosis of Ascaris lumbricoides and hook worm was substantial ( for Ascaris lumbricoides, for hookworm) ( Table 4).

Discussion
A single Kato-Katz had signi�cantly lower detection capacity than the FEC method in diagnosing hookworm. is 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. e chance of detecting hookworm infection by Kato-Katz from small amount of faecal material is suggested to be lower [20,21]. exhibited very low sensitivity for the detection of Ascaris lumbricoides, Schistosoma mansoni, Trichuris trichiura and hookworm. e use of direct wet mount alone as an indicator of intestinal parasitic infections is also suggested to be insuf-�cient by other studies [22]. However, in most laboratories in Ethiopia, the direct wet mount is the preferred stool parasitological detection technique. is shows that, since the use of direct wet mount as a con�rmatory test will signi�cantly 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. is shows that the use of the Kato-Katz as a con�rmatory 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 �nding.
In conclusion, the present study revealed that Kato-Katz technique and FEC methods showed a better sensitivity than the traditional direct wet mount method. erefore, the employment of FEC technique as a con�rmatory test in routine laboratory examination of stool and Kato-Katz in epidemiological studies will signi�cantly aid in accurate determination and management of parasitic infections in the community.

Con�ict of �nterests
e authors declare that they have no con�ict 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.