Diarrheal disease is a worldwide problem causing significant morbidity and mortality, especially in developing countries [1]. It is common practice to request stool specimens for culture and/or parasitological examination in patients with diarrhea. Although it is common to think that parasitic diseases only occur in tropical regions, most of the intestinal infections occur in temperate regions of the world [1]. In addition to common parasitic organisms, laboratories should identify some of the less common intestinal parasites often observed in individuals that have traveled abroad. In general, the diagnosis of parasites depends on microscopic identification; thus, it is crucial to maintain the inspection ability of each laboratory. Therefore, exact identification of intestinal parasites should be based on the quality control (QC) of microscopic examinations. The Clinical and Laboratory Standards Institute (CLSI) has established guidelines for stool examination re-garding collection, processing, and examination [2]. However, data on the status of QC systems in clinical laboratories performing stool examinations are not sufficient [3]. Thus, this study assessed the status of QC systems in Korean clinical laboratories performing stool examinations.

The study cohort consisted of clinical laboratories that perform stool examinations for parasitic infections within medical institutions (hospitals and medical centers) and in referral clinical laboratories accredited by the Korean Laboratory Accreditation Program [4]. A brief questionnaire was sent by electronic mail to the directors and clinical pathologists in charge of the laboratories in order to survey the clinical laboratory practices related to stool examinations. The questionnaire included the use/method of stool concentration, use of additional stains, reactivity testing of the dye solution, and internal and external QC systems.

In total, 39 clinical laboratories (24.2%, 39/161) responded to the survey. Most of the responses were from laboratories in medical institutions with 500–1,000 beds (53.8%, 21/39), followed by seven institutions with less than 200 beds (including three referral medical laboratories), seven institutions with 200–500 beds, and four institutions with greater than 1,000 beds (Table 1). Fecal concentration is recommended to increase the chance of detecting parasitic ova, cysts, and larvae, particularly in specimens where they are present in insufficient numbers to be seen using direct microscopy [5]. Although more than a half of the laboratories (51.3%) performed stool concentration using formalin-ether or Tween 80, a third of the laboratories (30.8%) performed direct smears only. As the prevalence of intestinal parasitic infections in Korea decreases, fecal concentration should be used to increase sensitivity. Notably, more than 70% of responders (28 institutions) did not utilize additional stains, and there were no cases diagnosed as Cryptosporidium parvum by stool examination during a 1-year period. It is difficult to identify cysts or trophozoites without the aid of special stains or molecular modalities, especially for protozoa such as Cryptosporidium species [6-8]. The diagnosis rate for protozoa may be underestimated because most laboratories typically perform only wet-mount preparations without additional special staining [9].

Table 1 . Current status of quality control systems for stool examination in Korea

Items	No. (%)
No. of respondents	39
No. of beds in the health care institute
>1,000	4 (10.3)
500–1,000	21 (53.8)
200–500	7 (17.9)
<200	7 (17.9)
Stool examination method
Concentration using formalin-ether or Tween 80	20 (51.3)
Direct smear only	12 (30.8)
Cellophane thick smear only	4 (10.3)
Direct smear and cellophane thick smear	3 (7.7)
Stain used for stool examinations
Unnecessary (in the case of cellophane thick smear only)	4 (10.3)
Saline only	28 (71.8)
Iodine or trichrome stain	7 (17.9)
Internal quality control system
Test patient samples without checking positive/negative controls	29 (74.4)
Test patient samples after examining negative and positive controls	7 (17.9)
Test patient samples after examining the positive control	3 (7.7)
Patient samples are tested without control materials due to:
Difficulty in obtaining adequate positive/negative control materials	22 (75.9)
Lack of necessity of internal quality control materials for stool examinations	4 (13.8)
No answer	3 (10.3)
Reactivity check of the dye solution
Do not check	35 (89.7)
Mix stool and fixation solution	4 (10.3)
Inspection cycle for the reactivity check of the dye solution
Do not check/no response	35 (89.7)
At every test	2 (5.1)
At least once per month	1 (2.6)
At least once per week	1 (2.6)
Management of the dye solution
Lot-to-lot variation check, Yes	3 (7.7)
Use of a positive control, Yes	3 (7.7)
Preference of external quality control system for stool examination
Quality control slides for stool examination	17 (43.6)
Positive/negative control materials	12 (30.8)
Virtual slide photo	7 (17.9)
Other (combination of the above options)	3 (7.7)
Diagnostic methods used for protozoa
Direct smear, cellophane thick smear or formalin-ether concentration without special stains	29 (74.4)
Special stain after direct smear or formalin-ether concentration	10 (25.6)
Opinion regarding special staining for the diagnosis of protozoan infection
Special staining should be performed only if a protozoan infection is suspected	20 (62.5)
Special staining should be performed for diarrhea specimens, even if a protozoan infection is not suspected	6 (18.8)
Special staining should be performed for stool examinations in general	3 (9.4)
Special staining is not necessary for the diagnosis of a protozoan infection	3 (9.4)
Opinion regarding the usefulness of enzyme-linked immunosorbent assays for the diagnosis of Cryptosporidium parvum, Giardia lamblia, and Entamoeba histolytica infections
Useful for more sensitive and economical diagnosis	30 (81.1)
Unnecessary	7 (18.9)

The current study showed that the majority of laboratories (74.4%) did not perform internal QC testing for stool examinations. When asked why positive and negative control materials were not included before testing the patient samples, 75.9% of the laboratories indicated it was difficult to secure adequate positive and negative control materials. According to CLSI guidelines [2], stool samples used for QC can be fixed stool specimens that contain protozoa or polyvinyl alcohol (PVA)-preserved negative stool samples to which buffy coat cells have been added. The CLSI recommends that a QC slide should be included in each run of stained slides; however, this step is not mandatory, and the exact QC assessment can be adjusted at the laboratory’s discretion [2]. A few laboratories (10.3%) checked the reactivity of the dye solution at least once every month. We found a gap between the laboratory protocols and the CLSI recommendation that fixative should be checked weekly or when using a new lot number [2]. Only three laboratories compared the reactivity of the staining reagent lot by lot and included positive control materials. A QC smear prepared with a PVA-preserved stool or buffy coat cells should be used when a new stain is prepared or at least once every month according to CLSI guidelines [2]. For external QC systems, the use of a QC slide (43.6%) was preferred, followed by QC materials (30.8%), virtual slides (17.9%), and a combination of the above options (7.7%). Generally, for external quality assessment programs, manufactured stool materials or slides have been used [10]. Liebman et al. suggested that pooling pairs of stool specimens for microscopy is likely to be more cost effective than commercial QC slides [10]. In order to obtain an adequate supply of pooling materials representing common and educationally important parasites, however, it might be necessary to survey endemic regions of parasitic disease around the world in addition to domestic multicenters. Moreover, the recently introduced Web Microscope for Parasitology could be an alternative tool [11].

In this study, 74.4% of respondents diagnosed protozoan infections without the aid of a special stain; however, 90.6% of respondents stated that special stains were necessary for the diagnosis of a protozoan infection. Furthermore, 81.1% of respondents indicated that additional enzyme-linked immunosorbent assays (ELISAs) could be necessary for the diagnoses of C. parvum, Giardia lamblia, and Entamoeba histolytica infections. These protozoan infections are monitored by the government, as they are pathogens relevant to public health. Over the last few years, several alternative diagnostic methods such as direct immunofluorescence staining or ELISAs have been developed and commercialized [12]. Previous researchers found that ELISAs were superior to conven-tional parasitological microscopy for the detection of protozoa, and they suggested that ELISAs should be used more routinely for diagnosis [13]. Special tests should be requested for patients who are suspected of having giardia, ameba, or cryptosporidium infections instead of conventional microscopic examinations for ova and parasites. An expanded parasite-screening repertoire that allows for more patient-specific options should be provided to clinicians. Although there are no available ELISA kits authorized by the Korea Centers for Disease Control and Prevention yet, for testing for protozoa, the introduction of ELISAs in the future could be useful to meet expanding clinical demands [13].

The current study demonstrates that Clonorchis sinensis, Enterobius vermicularis, and Endolimax nana are the most frequently observed parasite ova or protozoan cysts in stool samples (Fig. 1). This finding is consistent with a recent nationwide survey that showed large increases in the egg-positive rates of C. sinensis [14]. Importantly, the rate of positive stool tests differed markedly according to the laboratory performing the testing (ranging from 0.0% to 6.7%; data not shown). Previously, Manser et al. also demonstrated that variations in the procedures for stool examinations could reduce the recovery of parasites at different stages, particularly if present in small numbers [5]. We suggest that the standardization of stool examinations in regards to the overall methodology and QC is needed.

Figure 1. The most commonly observed (A) and the second-most commonly observed (B) parasites during stool examinations in Korean clinical laboratories participating in this study.

To our knowledge, this is the first report to assess the current status of QC systems in Korean clinical laboratories performing stool examinations. We have found that many laboratories have inadequate internal QC systems, mostly due to limitations in obtaining appropriate positive control materials. This study highlights that it is crucial to support the development of adequate QC materials for the establishment of a QC system in the field of stool examination.

No potential conflicts of interest relevant to this article were reported.

We thank the participating clinical laboratories for their valuable answers in this study. This study was supported by funding from the Laboratory Medicine Foundation (2016) and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2016R1C1B1009746).