Direct smears are commonly used to detect the trophozoites of Entaemeba histolytica and Giardia lamblia from the diarrheic stools of acute-phase patients. This method is not useful for examining helminthic infections because the egg detection rate is very low, except for diphyllobothiid tapeworms.

The cellophane thick smear (Kato’s method) is performed using a cellophane (No. 600) strip pre-soaked in Kato’s solution instead of a cover slip. Kato’s solution is a mixture of glycerin (500 mL), distilled water (DW, 500 mL), and 3% malachite green (recently, methylene blue has been highly recommended) solution (5 mL). This method is discussed in detail in the quantitative technique.

The Faust’s zinc sulfate flotation technique is commonly used to detect cysts and/or oocysts of intestinal protozoa, such as E. histolytica, G. lamblia, and Cryptosporidium parvum, in the stool of patients in the chronic phase. It is not suitable for the diagnosis of helminthic infections because the specific gravity of helminth eggs is relatively high.

FEST is somewhat complicated and troublesome; however, it is better than the Kato's method for the exact identification of helminth eggs, particularly the STE of opisthorchiid and heterophyid flukes.

FEST procedure (Supplementary Fig. 1 in the online-only Data Supplement)

1. Sample a thumb head-size amount of feces into a beaker with DW.

2. Stir with a wooden applicator to form a stool suspension.

3. Filter the stool suspension through two layers of wet gauze.

4. Centrifuge the conical tip tube with the filtered stool suspension at 500 × g for 3 min.

5. Discard the supernatant in a tube.

6. Add 10% formalin (10 mL) and ether (3 mL) to the tube.

7. Shake the tube vigorously for 15 s after placing a rubber stopper or cap on it.

8. Centrifuge the tube at 500 × g for 3 min.

9. Gently remove the plug of debris and discard the supernatant.

10. Drop the sediment onto the slide glass.

11. Cover the glass slip (cover slip) and examine it under a light microscope.

In stool examinations using FEST in FBH-endemic riverside areas of Korea (2011–2023), the prevalence of clonorchiasis was the highest (4.8%), followed by heterophyid fluke infection (1.5%), trichuriasis (0.2%), and Gymnophalloides seoi infection (0.06%) (Table 1) [12,13]. Results of stool examinations with FEST in health checkups in the KAHP revealed a similar pattern, with a higher prevalence of FBH, such as clonorchiasis (1.5%) and heterophyid fluke infections (0.34%), including metagonimiasis, compared to STH, that is, ascariasis and trichuriasis (0.13%) (Table 2) [14]. These findings suggest that FEST is the gold standard for the diagnosis of helminthic infections, including clonorchiasis and metagonimiasis, in Korea.

1. Fill a 0.1 N NaOH or KOH solution into the 56 mL scale of a Stoll’s flask (Supplementary Fig. 2 in the online-only Data Supplement).

2. Add the fecal sample in the Stoll’s flask to a volume of 60 mL (1 g of feces is dissolved in 15 mL of fecal solution).

3. Sample the fecal solution (0.15 or 0.075 mL) with a Stoll pipette after shaking the flask, and drop it onto the slide glass when the feces are completely dissolved in the solution.

4. Cover the glass slip, examine the fecal specimen under a light microscope, and count the number of eggs.

5. EPG (No. eggs per gram of feces), n (correction factor for the condition of the stool) × No. of eggs × 100 (200 for the 0.075 mL sample)

6. EPD (No. eggs per day): EPG × whole-day stool (200 g in adults)

7. EPDPF (EPDPW), No. of eggs per day per female (worm) Ascaris lumbricoides: 200,000, Trichuris trichiura: 5,000, hookworms: 10,000, Clonorchis sinensis: 3,000.

8. No. of worms: EPD/EPDPF (EPDPW)

Materials (Supplementary Fig. 3 in the online-only Data Supplement)

1. Plastic template: 50 mg (hole: 9 mm diameter × 1 mm thick). 41.7 mg (hole: 6 mm in diameter × 1.5 mm thick), 20 mg (hole: 6.5 mm in diameter × 0.5 mm thick).

2. Plastic spatula.

3. 60–105 nylon mesh.

4. Slide glass.

5, Newspaper (Fig. 1A).

6. Cellophane: 40–50 µm thick (25 × 30 mm), flat-bottom jar with lid, and forceps (Fig. 1B).

1. Place a small amount of fecal sample on a newspaper and press the nylon mesh on top so that some of the feces are sieved through the mesh and accumulate on top (Fig. 1C).

2. Scrape the flat-sided spatula across the upper surface of the mesh to collect the sieved feces (Fig. 1D).

3. Place the template at the center of a glass slide and add feces from the spatula to completely fill the hole (Fig. 1E).

4. Remove the template carefully so that the mound of feces remained on the slide.

5. Cover the fecal mound with the presoaked cellophane strip (Fig. 1F).

6. Invert the slide and firmly press the fecal sample against cellophane strips to evenly spread the feces (Fig. 1G).

7. Maintain CTS specimens at room temperature for more than 1 h for clearance (Supplementary Fig. 3 in the online-only Data Supplement).

8. Examine the fecal specimen under a light microscope and count the number of eggs.

9. EPG: No. of eggs × 20 (50 mg template), No. eggs × 24 (41.7 mg template).

10. EPD = EPG × whole-day stool amount (200 g in adults)

11. No. of the infected worms: EPD/EPDPF (EPDPW)

Control and elimination programs for helminthiases have been performed using the Kato-Katz thick smear technique by NGOs including KAHP, the Korea International Cooperation Agency, and the Korea Foundation for International Healthcare in endemic countries (Myamar, Cambodia, and Lao PDR) in Southeast Asia [15–18]. The individual prevalence of helminthiasis differed between the survey regions and subjects. However, in these programs, STHs, such as ascariasis (17.7%), trichuriasis (14.0%), and hookworm infections (14.9%), were the main target diseases, and FBHs, that is opisthorchiid and heterophyid fluke infections (12.1%), were additional health problems (Table 3). Eom et al. [17] conducted a Korea-Laos collaborative project for the control of foodborne trematode infections in Lao PDR (2007–2011). They examined 6,178 fecal samples using a Kato-Katz thick smear and Stoll’s egg counting technique to determine the infection status of helminths. The prevalence of STE was the highest (55.6%), followed by hookworm infection (27.8%), trichuriasis (6.5%), and ascariasis (3.7%).

In contrast, McCarthy et al. [23] reviewed the diagnostic technologies relevant to the control of helminth infections, either available or under development. They also discussed the critical gaps to be identified and the opportunities to improve diagnostic technologies for control and elimination programs as a research agenda for helminthic diseases in humans. Some research groups have adopted new devices and kits to efficiently detect helminth eggs. Boonyong et al. [24] compared the detection performance of a fully automatic digital feces analyzer (Orienter Model FA280) with that of formalin-ethyl acetate concentration technique. Lefaure et al. [25] evaluated the detection efficacy of the Parasep® device, which was adapted to the Faust’s flotation technique, with conventional techniques. Sarirah et al. [26] evaluated the detection efficacy of the MiniFLOTAC and compared it with that of KKECT, a quantitative technique recommended by the World Health Organization (WHO). Khanna et al. [27] also evaluated the detection rate of Mini Parasep® solvent-free method in the rapid diagnosis of intestinal helminthiases through comparing it with that of formalin-ethyl acetate sedimentation technique (FEAST, has been recently recommended instead of FEST by WHO) [28]. Recently, the Korea DCPA developed the ParaEgg kit and adopted it for stool examinations in the FBHendemic riverside areas of Korea (personal communication). The detection efficiency of new devices or kits is promising compared to that of conventional techniques in stool examinations; however, some limitations, such as sensitivity, specificity, practical difficulty, cost, and infrastructure demand, need to be solved preemptively for their widespread use.

The cellotape anal swab is used for the diagnosis of pinworm, Enterobius vermicularis, and sometimes beef tapeworm, Taenia saginata, infections.

The eggs of the opisthorchiid fluke (C. sinensis) and heterophyid flukes including Metagonimus spp. are relatively small and include an operculum and an embryo (miracidium).

Clonorchis sinensis: Ovoid and sesame seed-shaped with a miracidium inside, 27–35 × 15–20 µm in size, have a dominantly developed (conspicuous) operculum and shoulder rim, a shell surface with muskmelonlike wrinkles, and maximum width in the posterior 1/3 level (Fig. 2A, 2 B).

Heterophyids: Ovoid in shape with a miracidium inside, commonly having an inconspicuous operculum and shoulder rim, smooth shell surface without muskmelon wrinkle pattern, and maximum width in the median (Fig. 2C–2F). More than 11 species, including Metagonimus spp., Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Pygidiopsis summa, Centrocestus armatus, Stictodora fuscata, Stictodora lari, and Acanthotrema felis, have been reported in Korea. They are divided into 3 groups according to their lengths: < 25 µm, 26–30 µm, and > 31µm.

In the CTS specimens, the eggs of opisthorchiid flukes are indistinguishable from those of heterophyid f lukes including Metagonimus spp. (Supplementary Fig. 5 in the online-only Data Supplement). However, they can be easily differentiated based on the dominantly developed operculum and muskmelon surface appearance in FEST fecal specimens. Unlike opisthorchiid flukes, the eggs of heterophyid flukes had a smooth shell surface.

Stictodora fuscata: Ovoid with a miracidium inside, larger (34–38 × 20–23 µm) than other heterophyid f luke eggs, with thick shells, which are sometimes dark brown in color (Fig. 2F).

Plagiorchis muris: Elliptical with a miracidium inside, 32–38 × 20–24 µm in size, thick shell, conspicuous operculum, and sometimes an abopercular knob (Fig. 2G).

Eurytrema pancreaticum: Oval with a miracidium inside, 48–53 × 25–33 µm in size, thick shell, inconspicuous operculum, dark brown in color (Fig. 2H).

Gymnophalloides seoi: Very small with a miracidium inside, 20–25 × 11–15 µm in size, very thin shell, and large operculum (Fig. 2I).

The eggs of Diphyllobothrium spp., echinostomatid flukes, Neodiplostomum seoulense, Paragonimus westermani, Fasciola hepatica, and Fasciolopsis buski are relatively larger in size and have an operculum, but do not have an embryo. They consist of germ cells and yolk inside.

Echinostomatids: The echinostomes infecting humans belong to 4 species, Echinostoma cinetorchis (99–116 × 66–76 µm), Isthmiophora hortensis (127–139 × 71–81 µm), Echinochasmus japonicus (76–87 × 52–63 µm), and Acanthoparyphium tyosenense (78–93 × 63–73 µm), have been reported in Korea. Echinostomatid eggs differentiate from other fluke eggs in this group by the presence of abopercular wrinkles, and are roughly divided into 2 groups based on their average length: < 100 µm and > 101 µm. They are commonly oval in shape, with a thin shell, a small operculum, and abopercular wrinkles (Fig. 3A–3D).

Paragonimus westermani: Elliptical and asymmetrical in shape, 80–100 × 45–65 µm in size, have a wide operculum, shoulder rim, and abopercular thickening (Fig. 3E).

Neodiplostomum seoulense: Elliptical and asymmetrical in shape, 86–99 × 55–63 µm in size, have thin shell, and an inapparent operculum (Fig. 3F).

Fasciola hepatica: Very big, 145 × 80 µm in average size, with thin shell, and inconspicuous smaller operculum (Fig. 3G).

Fasciolopsis buski: Very big, 147 × 75 µm in average size, with thin shell, and inconspicuous smaller operculum.

Diphyllobothrium spp.: Ovoid and symmetrical in shape, 58–76 × 40–51 µm in size, with a thick shell, and smaller operculum, and sometimes with an abopercular knob (Fig. 3H).

The eggs of cyclophyllidian tapeworms, such as Taenia spp. and Hymenolepis spp., and blood flukes, Schistosoma spp., are embryonated, but they do not have an operculum.

Taenia spp.: Round, 30–45 µm in diameter size, with thick and striated embryophore, and have a hexacanth embryo. Three species, Taenia asiatica, T. saginata, and T. solium, are not microscopically differentiable at the species level (Fig. 4A).

Hymenolepis nana: Round, 30–47 µm in diameter size, with transparent shell, and have a hexacanth embryo with polar filaments (Fig. 4B, 4C).

Hymenolepis diminuta: Round, 70–80 µm in diameter size, with thick shell, and have a hexacanth embryo with polar thickenings (Fig. 4D).

Schistosoma japonicum: Ovoid, 70–100 × 50–65 µm in size, with thin shell, a small lateral spine and a miracidium inside.

Schistosoma mansoni: Long, elliptical, 114–175 × 45–68 µm in size, with thin shell, a stout lateral spine, and a miracidium inside.

Schistosoma haematobium: Long, elliptical, 112–170 × 40–70 µm in size, with thin shell, a terminal spine, and a miracidium inside.

The nematode eggs have no operculum and embryo, but those of hookworms and pinworms are sometimes observed in embryonated stages.

Ascaris lumbricoides (F): Subspherical or round, 50–75 × 35–50 µm in size, with 3 layered shells: outer mammilated protein coat, middle chitinous, and inner lipoid layers. In fresh stool, they retain a single blastomere (Fig. 5A, 5 B). They sometimes develop in CTS specimens as 2-, 4-, 8-, 16-, 32-cells, and morular stages in a warm environment and rarely have a decorticated (no protein coat) appearance. Fertilized eggs of A. lumbricoides are clearly differentiated from toxocarid (Toxocara spp.) eggs from cats and dogs by their morphological shape, being nearly round with a thinner shell, but nearly identical to those of Ascaris suum from pigs.

Ascaris lumbricoides (U): Long, elliptical, 80–90 × 40–45 µm in size, thin shell with poorly developed protein coat and granules inside (Fig. 5C, 5D). A variety of shapes are frequently observed in the CTS specimens (Supplementary Fig. 6 in the online-only Data Supplement). Characteristic broken shell fragments with a protein coat and internal granules are helpful in identifying unfertilized A. lumbricoides eggs.

Trichuris trichiura: Barrel shape, 50–55 × 22–23 µm in size, with thick shell and polar mucoid plugs on both sides and a single blastomere in fresh stool specimens (Fig. 5E, 5F).

Recently, two types (standard- and large-sized) of whipworm (T. trichiura) eggs from school children in Yangon, Myanmar were molecularly confirmed to be the same species. The size of standard-eggs was 52–58 × 21–28 μm and that of large- eggs was 65–76 × 30–35 μm in the study by Ryoo et al. [29]. The large eggs were slightly smaller than the known size of Trichuris vulpis (dog whipworm). Some parasitologists strongly suspect T. vulpis eggs in CTS specimens from prevalent countries, such as Lao PDR, Cambodia, and Myanmar.

Enterobius vermicularis: Persimmon seed shape, 50–60 × 20–30 µm in size, with double-layered shell and larva. Pinworm eggs are detected in the cellotape anal swab specimens but are sometimes found in the fecal specimens of CTS (Fig. 5G, 5H).

Hookworms: Ovoid, 55–75 × 35–45 µm in size, have hyaline thin shells with blunting of both ends, and 4–8 cell stage in the fresh fecal specimen (Fig. 5I). They rapidly develop and are embryonated in warm environments, and sometimes show an air-bubble-like appearance (Fig. 5J, 5 K). In this case, they are difficult to recognize. To solve this problem, the microscope should be finely adjusted. The definite and continuous demarcation of egg shells may be helpful in the diagnosis of hookworm eggs. As humaninfecting hookworms, 3 species, i.e., Ancylostoma duodenale, A. cylanicum, and Necator americanus, have been reported.

Trichostrongylus orientalis: Long, elliptical, 75–95 × 45–50 µm in size, with a thin hyaline shell and a morular stage inside (Fig. 5L). These nematode eggs are easily differentiated from hookworm eggs by their large size, one pointed end (both blunted ends in hookworms), and the internal morular stage.

The morphological characteristics of helminth eggs are summarized in Tables 4 and 5.