GRAPHICAL ABSTRACT

Key Message

This study provides prospective evidence that consumption of raw halibut infected with Kudoa septempunctata is associated with gastrointestinal symptoms, with the severity of symptoms increasing with the intensity of infection and the amount consumed. These results address a gap in epidemiological knowledge and highlight the need for strengthened surveillance systems and prevention-focused policies.

INTRODUCTION

Kudoa septempunctata was first reported during a mass food poisoning incident in Japan in 2010 and was subsequently identified as the causative pathogen responsible for symptoms following consumption of raw farmed Paralichthys olivaceus [1-4]. In Korea, exposure to K. septempunctata is relatively common because P. olivaceus is widely consumed [5,6]. Nevertheless, whether K. septempunctata definitively causes food poisoning symptoms remains a matter of debate [2,4,7-10].

P. olivaceus is the most commonly consumed marine species within the order Pleuronectiformes and accounted for 51% of all farmed marine fish production in 2013 [6,11]. As a result, several Korean studies have investigated the prevalence of K. septempunctata infection in P. olivaceus farms and have conducted related case-control studies [12].

Most existing studies are case reports or case-control investigations involving patients who developed gastrointestinal symptoms and, as such, cannot adequately substantiate a causal association between K. septempunctata and gastrointestinal symptoms [8,12,13]. Importantly, in many studies, consumed food specimens were unavailable, which precluded confirmation of infection in the raw fish [5,14,15]. In addition, biological studies have yielded inconsistent results due to variability in study populations and experimental methods, highlighting the need for epidemiological studies capable of more definitively clarifying this association [1,2,8,9,16-19].

The present study aimed to investigate the association between consumption of K. septempunctata–infected P. olivaceus (referred to in this manuscript as positive olive flounder) and the onset of gastrointestinal symptoms, as well as to evaluate the pathogenicity of K. septempunctata. The specific objectives were: (1) to analyze the prevalence of K. septempunctata infection among raw farmed P. olivaceus; and (2) to analyze the causal relationship between consumption of raw positive olive flounder and the onset of gastrointestinal symptoms.

MATERIALS AND METHODS

Study design

This prospective cohort study examined the incidence of gastrointestinal symptoms among individuals who purchased and consumed farmed P. olivaceus between 2020 and 2021 and investigated the association between gastrointestinal symptoms and K. septempunctata (Figure 1). Among the 1,755 individuals initially enrolled, participants who did not complete the second questionnaire, individuals younger than 19 years of age, and others (including participants who may have consumed multiple types of fish or who provided incorrect contact information) were excluded. As a result, a total of 1,708 participants were included in the final analysis.

To minimize potential bias, several measures were implemented: (1) researchers and investigators received standardized training to ensure consistency in study procedures and survey administration; (2) samples of raw fish were collected and tested for K. septempunctata infection before consumption by study participants; (3) participants were required to complete a second questionnaire within 24 hours after consuming raw fish, regardless of K. septempunctata infection status (Supplementary Material 1); and (4) for participants who developed symptoms, human biological specimens were examined to exclude potential causes other than K. septempunctata (Supplementary Material 2).

Study participants and sample size

Individuals who purchased farmed P. olivaceus at seafood markets or seafood sections of retail markets in Seoul, Gyeonggi, and Incheon between 2020 and 2021 and who provided informed consent were enrolled in this study. The authors or trained surveyors explained the purpose of the study to individuals purchasing farmed P. olivaceus. Participants completed a pre-consumption survey (Survey 1) and provided a specimen of the P. olivaceus they had purchased. In addition, individuals who consumed farmed P. olivaceus purchased together were asked to complete a post-consumption survey (Survey 2) if they agreed to participate.

For sample size estimation, we assumed a type I error of 0.05 and a type II error of 0.2. Based on previous studies, the prevalence of P. olivaceus infected with K. septempunctata has been reported to range from 3% to 5%; therefore, we conservatively estimated a prevalence of 2% [3,10]. We further assumed a 50% attack rate and a 10% incidence of gastrointestinal symptoms among individuals consuming infected P. olivaceus. Under these assumptions, a total sample size of 735 participants was required.

Identification of cases

Cases were defined based on the severity of gastrointestinal symptoms that developed within 24 hours after consumption of raw farmed P. olivaceus, as follows: (1) Case definition 1: Individuals who experienced at least 1 gastrointestinal symptom, such as diarrhea, vomiting, nausea, or stomach pain; (2) Case definition 2: Individuals who had 1 episode of diarrhea or vomiting along with other gastrointestinal symptoms, such as nausea or stomach pain.

Collection of raw flounder specimens and examination for K. septempunctata infection

The Kudoa Rapido A rapid diagnostic test kit was used to detect Kudoa antibodies in raw P. olivaceus. In addition, to exclude false-negative results, approximately 10 g of tissue was collected from 2 separate sites in each fish, and K. septempunctata infection was confirmed through visual inspection. No other infectious agents were investigated.

Human specimen testing from symptomatic individuals

More than 5 g of human specimens (vomitus or feces) were collected from participants who developed gastrointestinal symptoms after consuming fish infected with K. septempunctata. Specimens were stored at 4°C and examined using standard laboratory procedures, including protozoan analysis. Detailed information on test items for human specimens is provided in Supplementary Material 2.

Statistical analysis

All statistical analyses were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and statistical significance was defined as p-value<0.05. Data distributions and mean values between groups exposed and not exposed to K. septempunctata were compared using chi-square tests for categorical variables and independent t-tests for continuous variables. Relative risks (RRs) and corresponding 95% confidence intervals (CIs) are reported.

Ethics statement

The study protocol was approved by the Institutional Review Boards of Gacheon University Gil Medical Center (GAIRB-2020-158) and Seoul National University Bundang Hospital (B-2107-696-301).

RESULTS

General characteristics

Participants were divided into 2 groups based on whether they consumed raw fish infected with K. septempunctata. The general characteristics of the study participants are summarized in Table 1. A total of 1,708 participants were included in the analysis, of whom 27 (1.6%) were exposed to K. septempunctata and 1,681 (98.4%) were classified as unexposed.

Among the participants, 754 (44.1%) were men and 954 (55.9%) were women. The most common age group among individuals who consumed raw fish was 50–59 years (n=458, 26.8%), followed by those aged ≥60 years (n=373, 21.8%) and those aged 40–49 years (n=314, 18.4%).

Methods of storing raw flounder before ingestion differed between groups. In the exposure group, storage methods included immediate ingestion (n=9, 33.3%), storage at room temperature (n=3, 11.1%), and refrigeration (n=15, 55.6%). In the non-exposure group, storage methods included immediate ingestion (n=174, 10.4%), room temperature storage (n=110, 6.5%), refrigeration (n=1,370, 81.5%), and freezing (n=27, 1.6%). The time from purchase of raw farmed P. olivaceus to consumption was within 1 hour (n=7, 25.9%), 1–2 hours (n=16, 59.3%), and 2–3 hours (n=4, 14.8%) in the exposure group, and within 1 hour (n=494, 29.4%), 1–2 hours (n=371, 22.1%), 2–3 hours (n=243, 14.5%), and ≥3 hours (n=573, 34.1%) in the non-exposure group.

The number of raw slices of farmed P. olivaceus consumed in the exposure group was 3–7 (n=6, 22.2%), 8–12 (n=6, 22.2%), 13–17 (n=6, 22.2%), 18–22 (n=4, 14.8%), and ≥23 slices (n=5, 18.5%). In the non-exposure group, the corresponding consumption categories were 3–7 (n=369, 22.0%), 8–12 (n=632, 37.6%), 13–17 (n=390, 23.2%), 18–22 (n=185, 11.0%), and ≥23 slices (n=105, 6.3%).

In the exposure group, 2 participants (7.4%) reported having food allergies, while 25 (92.6%) reported no food allergies. In the non-exposure group, 21 participants (1.3%) reported food allergies, 1,605 (95.5%) reported no food allergies, and 55 (3.3%) reported that they did not know their allergy status.

Impact of K. septempunctata-infected P. olivaceus on risk

Table 2 presents the RRs for gastrointestinal symptoms in the exposure group according to case definition. Due to the small number of events, exact RRs and 95% CIs were calculated using the Koopman asymptotic score method for 2×2 contingency tables. For case definition 1, 8 participants in the exposure group and 7 participants in the non-exposure group developed symptoms, yielding an RR of 71.2 (95% CI, 27.0 to 178.6) for gastrointestinal symptoms in the exposure group compared with the non-exposure group. For case definition 2, 6 participants in the exposure group and 3 participants in the non-exposure group developed symptoms, yielding an RR of 124.5 (95% CI, 43.5 to 355.0) for gastrointestinal symptoms in the exposure group compared with the non-exposure group.

Human specimen test results for symptomatic individuals

As shown in Supplementary Material 3, gastrointestinal symptoms such as diarrhea and nausea occurred more frequently and with greater severity among individuals who consumed larger amounts of raw positive olive flounder, particularly when the infection intensity exceeded the minimum infectious dose threshold reported in previous studies [4,12].

Standard testing of human specimens, including protozoan assays, yielded negative results for all tested agents except K. septempunctata [8,16-19] (Supplementary Material 4).

DISCUSSION

Various myxosporean infections have been reported in farmed P. olivaceus in Korea, raising concerns regarding their persistence and potential impacts on aquaculture production [3,6,11]. K. septempunctata is a muscle parasite of P. olivaceus; however, it is not readily detected by microscopic examination during the early stages of infection [3,4,12,15,20,21].

In Japan, an epidemiological investigation of a mass food poisoning outbreak affecting 113 individuals in Ehime Prefecture and 8 other prefectures in October 2010 examined 60 specimens of P. olivaceus (farmed P. olivaceus in Korea and Japan) and identified a new species belonging to the order Multivalvulid, later named K. septempunctata, in the muscle tissue of farmed P. olivaceus imported from Korea. This strain was classified as the ST3 genotype, which differs from the K. septempunctata strains found in P. olivaceus inhabiting waters surrounding Japan (ST1 and ST2) [1,8,16]. The Japanese Ministry of Health, Labor, and Welfare subsequently recognized this protozoan species as a cause of food poisoning and designated the condition as Kudoa food poisoning [2-4,22].

To date, the association between K. septempunctata and gastrointestinal symptoms has remained unclear [5,7-9,14-17,19]. The present prospective cohort study aimed to analyze the prevalence of K. septempunctata infection in the raw flesh of farmed P. olivaceus and to evaluate the causal relationship between consumption of raw positive olive flounder and the onset of gastrointestinal symptoms. The exposure group exhibited a higher RR of gastrointestinal symptoms than the non-exposure group, and analyses stratified by case definition demonstrated that consumption of raw positive olive flounder was associated with the occurrence of gastrointestinal symptoms. Notably, higher infection intensity in positive olive flounder and greater amounts consumed were associated with more severe gastrointestinal symptoms. Among participants who consumed P. olivaceus infected with K. septempunctata, symptom presence, frequency, and incubation period are summarized in Supplementary Material 3. The raw positive olive flounder sample numbers presented in Supplementary Materials 3 and 4, as well as the exposure numbers of symptomatic participants according to raw positive olive flounder sample number, were arbitrarily assigned by the researchers. In a previous study, the minimum ingestion threshold for the development of gastrointestinal symptoms was reported as 7.2×10⁷ spores/g, and in the present study, gastrointestinal symptoms were observed in exposed participants as the infection intensity of raw positive olive flounder approached this minimum intake threshold [4,12]. A previous study also reported relatively consistent findings regarding the incubation period and clinical manifestations of gastrointestinal symptoms following consumption of raw positive olive flounder [2,4].

Some studies have argued that the association between K. septempunctata and gastrointestinal symptoms cannot be conclusively established because other bacterial pathogens with similar epidemiological characteristics, such as Staphylococcus aureus and Bacillus cereus, may also be involved [8,15,19].

Although human studies investigating the biological mechanisms underlying gastrointestinal symptoms remain limited, several studies have suggested that K. septempunctata spores ingested through consumption of infected marine fish infiltrate intestinal epithelial cells and induce gastrointestinal symptoms [2,4,18,23]. In addition, toxicological studies have demonstrated that ingestion of 1.0×10⁷ spores/g of P. olivaceus induced vomiting in Suncus murinus [4].

We conducted a prospective epidemiological study to support previous findings regarding the onset of gastrointestinal symptoms following consumption of raw flesh from K. septempunctata–infected fish [2,4,7-10]. A portion of the raw fish consumed by participants was collected and tested for K. septempunctata infection. Through this approach, we identified the consumption histories of participants who consumed raw fish infected with K. septempunctata, as well as the presence or absence, frequency, and intensity of gastrointestinal symptoms. Our findings indicate that both the frequency and severity of gastrointestinal symptoms tended to be greater among participants who consumed larger amounts of raw fish with higher levels of K. septempunctata infection, as detailed in Table 2 and Supplementary Material 3. The primary significance of this study lies in demonstrating an association between consumption of raw positive olive flounder and gastrointestinal symptoms. Nevertheless, several limitations should be considered. First, farmed P. olivaceus samples were periodically tested for K. septempunctata at an external laboratory, which occasionally delayed the collection of human biological specimens, even when fish samples tested positive. As a result, evidence regarding the onset of gastrointestinal symptoms in relation to infection intensity may be limited. Second, Survey 2 was administered within 24 hours after consumption to assess symptom frequency, which may have introduced recall bias. Additional limitations include reliance on subjective reporting of symptom frequency, except for diarrhea and vomiting, and the use of symptom frequency alone as a proxy for symptom severity. Third, only 2 cases of gastrointestinal symptoms occurred following consumption of raw positive olive flounder. However, given that many participants exhibited gastrointestinal symptoms with clinical manifestations and incubation periods consistent with K. septempunctata–associated illness, and that no other causative pathogens were identified in human biological specimens, it is reasonable to infer that K. septempunctata was the causative agent in these cases. To address these limitations, further studies involving broader geographic regions and larger populations are warranted.

Despite these limitations, our findings demonstrate an association between consumption of raw positive olive flounder and the onset of gastrointestinal symptoms. These results underscore the need for policies and guidelines aimed at preventing gastrointestinal illness associated with consumption of raw infected fish.

Our findings further suggest an epidemiological association between ingestion of raw K. septempunctata–infected flounder and the occurrence of infectious gastrointestinal disease. Accordingly, policies to strengthen early management and continuous monitoring of raw farmed P. olivaceus prior to distribution are needed to prevent and reduce gastrointestinal symptoms caused by K. septempunctata infection.

Data availability

Supplementary materials

NOTES

Conflict of interest

The authors have no conflicts of interest to declare for this study.

Funding

This study was supported by the Korea Disease Control and Prevention Agency (No. 2019-P4402-00, No. 2021-07-002).

Acknowledgements

We appreciate Chung-Ang University for their support in observing K. septempunctata infection in raw fish in 2019, and the Division of Vectors and Parasitic Diseases, Korea Disease Control and Prevention Agency, for their assistance in 2020. We also acknowledge the Department of Diseases Research, Incheon Metropolitan City Research Institute of Public Health and Environment (the Disease Survey Division) for human specimen examinations in 2019-2020.

The study’s sponsor was not involved in the study design, analysis, and interpretation of data, writing of the report, or the decision to submit the study results for publication.

Author contributions

Conceptualization: An J, Ko KP. Data curation: An J, Ko KP, Lee SO, Hong SJ, Choi JH, Kim JH, Hong SH, Ju JW. Formal analysis: An J, Ko KP. Funding acquisition: Ko KP. Methodology: An J, Ko KP, Lee SO, Hong SJ, Choi JH, Kim JH, Hong SH, Ju JW. Project administration: Ko KP, An J, Kim H. Visualization: An J. Writing – original draft: An J, Jung EJ. Writing – review & editing: An J, Jung EJ, Ko KP, Lee SO, Choi JH, Kim JH, Hong SJ, Hong SH, Ju JW, Kim H.

Figure 1.

Flow chart of the cohort study design.

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Figure & Data

References

Citations

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