Introduction
Kudoa septempunctata, a myxosporean parasite originally identified in olive flounder (
Paralichthys olivaceus), has been implicated in foodborne gastroenteritis associated with the consumption of raw olive flounder. In 2012, the Ministry of Health, Labour and Welfare of Japan designated
K. septempunctata as a foodborne pathogen [
1–
3]. Previous epidemiological reports have described vomiting, diarrhea, and abdominal pain as the principal symptoms of foodborne illness caused by
K. septempunctata, with most patients recovering within 24 hours [
3]. Humans are considered accidental hosts and are exposed through the ingestion of infected fish tissue, particularly when fish is consumed raw or undercooked [
4,
5]. Although
K. septempunctata demonstrates high host specificity for olive flounder, sporadic infections have been reported in other species, including wild grass puffer (
Takifugu alboplumbeus), Japanese whiting (
Sillago japonica), and black scraper (
Thamnaconus modestus) [
6,
7]. Because the spore density of
K. septempunctata in these incidental hosts is relatively low, their potential to cause human illness appears limited. Nevertheless, olive flounder remains the primary vector implicated in human foodborne outbreaks associated with
K. septempunctata.
K. septempunctata belongs to the class Myxosporea and possesses 6 or 7 shell valves with polar capsules [
8]. Its natural life cycle has not been fully elucidated. Although many myxosporeans use annelids as alternate hosts, no definitive intermediate host has been identified for
K. septempunctata; therefore, general myxosporean life-cycle paradigms are referenced without proposing a specific annelid host [
9–
11]. Animal experiments involving
K. septempunctata have demonstrated transient gastrointestinal symptoms in some models, whereas others have shown little or no histological evidence of tissue injury [
3,
12–
14]. Therefore, polymerase chain reaction (PCR) positivity for
K. septempunctata in stool or vomit indicates recent exposure but does not, by itself, establish that the parasite caused the illness [
4].
Mitochondrial sequence types of
K. septempunctata, defined by
cox1/
rnl haplotypes, are classified as sequence type 1 (ST1;
cox1-1/
rnl-1), sequence type 2 (ST2;
cox1-2/
rnl-2), and sequence type 3 (ST3;
cox1-3/
rnl-2), with ST3 predominating in the Republic of Korea (ROK) [
15,
16]. Although genotype-phenotype relationships are well established for other enteric pathogens, such as
Cryptosporidium and norovirus [
17,
18], the clinical relevance of
K. septempunctata sequence types, particularly ST3, remains unclear. This uncertainty underscores the need for integrated epidemiological and molecular analyses.
In the ROK,
K. septempunctata has been monitored as a foodborne pathogen since 2015 and was incorporated into the National Water and Foodborne Disease Guidelines in 2017 [
19,
20]. From 2016 to 2019, surveillance focused on laboratory detection (presence or absence of
Kudoa) and lacked standardized clinical and epidemiological data. To address this gap, standardized investigation workflows for foodborne illness caused by
K. septempunctata were implemented in 2020.
Accordingly, this study aimed to elucidate the molecular and genetic characteristics of K. septempunctata outbreaks and to provide a scientific basis for effective public health interventions in the ROK. By analyzing nationwide surveillance data from 2016 to 2024, focusing on and conducting in-depth analyses of specific outbreaks from 2020 to 2024, we sought to determine the predominant K. septempunctata sequence types, evaluate seasonal outbreak trends, and assess the effect of the interval between consumption and specimen collection on pathogen detection rates.
Materials and Methods
Outbreak Criteria
We defined a
Kudoa-associated outbreak as the occurrence of 2 or more epidemiologically linked cases with compatible gastrointestinal symptoms after the consumption of raw fish, typically raw olive flounder. Confirmed outbreaks required at least 1 case to be PCR-positive for
K. septempunctata rRNA, with alternative enteric pathogens excluded through laboratory testing or deemed unlikely on the basis of incompatible incubation periods. Events that did not satisfy all of these criteria were classified as possible
Kudoa-associated outbreaks. Among the total reported cases, we analyzed 37 outbreaks occurring between 2020–2024 and evaluated standardized variables, including outbreak date, number of patients, and symptom types [
20]. Outbreaks were categorized geographically according to the location where cases were identified and field investigations were conducted, as determined by the investigating public health centers (PHCs) or health and environment research institutes (HERIs), rather than by the production origin of the implicated seafood.
Specimen Collection and Ethical Statement
A total of 1,200 clinical specimens (stool and/or vomitus) were tested to identify pathogens in patients presenting with diarrhea or vomiting after suspected seafood-borne illness outbreaks during 2016–2024. This study focused exclusively on the molecular detection and genetic typing of
K. septempunctata in clinical specimens because food samples, including leftover raw fish, were not included in the analysis. This study was conducted as a retrospective public-health surveillance investigation using specimens collected after symptom onset, and leftover fish from the exposure meal was not available for collection or testing. Clinical specimens were collected primarily at PHCs from suspected cases and were then referred to provincial HERIs under cold-chain conditions for testing [
21].
In this study, a retrospective analysis was performed using data collected during routine public health investigations for outbreak diagnosis. Institutional review board approval was waived because the data had been collected for diagnostic purposes during public health practice rather than for human participant research.
DNA Extraction and PCR Assays
Total DNA was extracted from approximately 250 mg of each clinical specimen (stool and/or vomitus) using the FastDNA SPIN Kit for Soil (MP Biomedicals) after mechanical disruption with a FastPrep-24 instrument (MP Biomedicals) at 6.0 m/s for 40 seconds. To detect
K. septempunctata, nested PCR targeting the 18S and 28S rRNA genes was performed using a thermal cycler (C1000 Touch Thermal Cycler; Bio-Rad Laboratories) (
Table 1) [
22]. The cycling conditions for the primary PCR were 95 °C for 5 minutes; 35 cycles of 95 °C for 30 seconds, 60 °C for 45 seconds, and 72 °C for 30 seconds; followed by 72 °C for 5 minutes. The cycling conditions for the secondary PCR were 95 °C for 5 minutes; 35 cycles of 95 °C for 30 seconds, 58 °C for 45 seconds, and 72 °C for 30 seconds; followed by 72 °C for 5 minutes. From 2021 onward, a multiplex real-time PCR assay (PowerChek
K. septempunctata 18S/28S rRNA Real-time PCR Kit II; Kogene Biotech) was used for screening.
Determination of Sequence Type
For mitochondrial typing of
K. septempunctata, the cytochrome c oxidase subunit I (
cox1) and large-subunit rRNA (
rnl) genes were amplified by nested PCR [
16]. The amplified DNA fragments were sequenced, and phylogenetic trees were constructed using the maximum-likelihood method with the Kimura 2-parameter model in MEGA 11 and 1,000 bootstrap replicates to determine the sequence type (ST1, ST2, or ST3).
Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics ver. 25.0 (IBM Corp.), with statistical significance set at α=0.05. Seasonality was assessed by comparing the proportion of Kudoa-positive outbreaks between May–October and November–April using Pearson’s χ² test. The effect of specimen collection timing on K. septempunctata detection was analyzed using Fisher’s exact test by comparing specimens collected ≤24 hours with those collected >24 hours after contaminated food consumption. Relative risk (RR) with 95% confidence intervals (CIs) was calculated for both analyses.
Results
Detection of K. septempunctata in Clinical Specimens
Of the 415 outbreaks linked to olive flounder sashimi, 237 events (57.1%) met the prespecified definition of
Kudoa-associated outbreaks. The proportion of outbreaks confirmed as
Kudoa-associated increased from 2016 and peaked at 77.8% in 2020. During 2021–2024, although the number of reported outbreaks decreased,
K. septempunctata was consistently identified in nearly all tested outbreaks (e.g., 5/7 in 2023 and 10/12 in 2024;
Figure 1A). By region, the number of outbreaks was highest in Gyeonggi-do, followed by Gyeongsangbuk-do and Seoul (
Table 2). Overall, 431 of 1,200 clinical specimens (35.9%) tested positive for
K. septempunctata. Except for 2 outbreaks in which enteropathogenic
Escherichia coli (EPEC) was identified and 1 outbreak in which norovirus was detected in 2020, no other pathogens were identified in specimens during the study period.
Epidemiological Analysis of Outbreaks
An epidemiological analysis was performed for 37
Kudoa-associated foodborne outbreaks identified between 2020 and 2024 (
Table 3). All outbreaks involved the consumption of raw fish, primarily olive flounder. The mean attack rate across outbreaks was 87.5% (range, 25%–100%). The mean symptom onset time was 5 hours (range, 1.2–11 hours) after consumption of the contaminated food. The most common symptom was diarrhea, reported in 88.8% of patients, followed by vomiting (61.2%) and abdominal pain (32.7%). Outbreaks tended to occur more frequently during warmer months (May–October;
Figure 2) than during colder months, with a notable peak in August (14.8%). However, this seasonal difference was not statistically significant (RR, 1.14; 95% CI, 0.93–1.39;
p=0.115). The clinical-specimen detection rate of
K. septempunctata averaged 53.6%, ranging from 16.7% to 100.0% across outbreaks.
Correlation Between Specimen Collection Interval and Pathogen Positivity
To assess the effect of the interval between contaminated food consumption and specimen collection on diagnostic yield, we analyzed 112 specimens for which complete timing data were available in the epidemiological investigation forms. Among specimens collected ≤24 hours after consumption, 69.8% (44/63) tested positive. In contrast, only 35.9% (14/39) of specimens collected >24 hours after consumption were positive. This difference was statistically significant (RR, 1.95; 95% CI, 1.24–3.05; Fisher’s exact test, p=0.0010), indicating that prompt sampling nearly doubled the diagnostic yield. Specimens with unknown intervals (n=10; 4 positive) were excluded from this comparison.
Discussion
National surveillance data indicate an increase in reported
Kudoa-associated outbreaks since 2020. Although previous investigations emphasized laboratory detection and suggested possible dose-response relationships [
23], PCR positivity alone indicates only the presence of
K. septempunctata DNA and cannot establish causation without clinical correlation [
1,
4,
12]. A recent prospective cohort study in the ROK reported a statistically significant association between consumption of
K. septempunctata-infected farmed olive flounder and the occurrence of acute gastrointestinal symptoms [
24]. In that cohort, symptoms occurred more frequently and were more severe among participants who consumed larger amounts of
K. septempunctata-positive olive flounder, particularly at higher infection intensities [
24]. Clinically, acute diarrhea and vomiting predominated among cases of foodborne illness associated with
K. septempunctata in the ROK [
19], consistent with reports from Japan [
2,
25].
Prompt specimen collection after contaminated food consumption is essential. Our results demonstrated a significant decline in
K. septempunctata detection rates after more than 24 hours had elapsed since contaminated food consumption. This time-dependent decline is consistent with previous outbreak investigations in Japan, in which the detection rate of
K. septempunctata in stool decreased significantly when specimens were collected more than 2.5 days after contaminated food consumption [
1]. In addition, experimental studies have shown that myxospores can be detected in feces within hours of exposure, supporting the narrow diagnostic window observed in our surveillance data [
13]. This short detection window is consistent with observations that spores are rapidly excreted and lack the capacity to colonize or proliferate in the mammalian gastrointestinal tract [
12]. In epidemiological investigations, specimen collection times are typically recorded in days (e.g., 0.5, 1, or 2 days); accordingly, we used 24 hours as the reference cutoff.
Regarding seasonality, our data showed a visual trend toward an increased number of
Kudoa-positive outbreaks from May to October, with a notable peak in August. However, this apparent increase was not statistically significant (
p=0.115). This seasonal pattern appears broader than that reported in Japan, where foodborne illness caused by
K. septempunctata occurs predominantly between August and November [
3]. The tendency toward more gastroenteritis cases caused by
K. septempunctata during warmer months in the ROK may be partly explained by seasonal variation in raw olive flounder consumption [
26]. Therefore, integrating seafood consumption data with epidemiological surveillance is essential for understanding the transmission dynamics of
K. septempunctata.
During the study period, the number of reported patients with
K. septempunctata infection decreased between 2020 and 2021, coinciding with the coronavirus disease 2019 pandemic. This decrease may have been attributable to social distancing measures, which likely reduced dining out and the consumption of raw fish, rather than to a biological decline in parasite prevalence. Although social distancing measures were relaxed between 2022 and 2024, patient numbers did not fully return to prepandemic levels, and the reasons for this pattern remain unclear. In addition, higher seawater temperatures have been associated with an increased prevalence of other
Kudoa spp. [
27–
29], raising the possibility that environmental factors influence the epidemiology of
K. septempunctata infections. This hypothesis warrants longitudinal monitoring.
All identified specimens belonged to the ST3 genotype (
Figure 3), consistent with the known distribution in the ROK, whereas ST1 and ST2 predominate in Japan [
15,
16]. The clinical significance of each sequence type remains uncertain, and published data regarding differences in virulence are conflicting. Recently, Shamsi and Barton [
30] reviewed the global potential of
Kudoa spp. as emerging seafood-borne parasites and noted that, as the consumption of raw fish expands globally, these regional genotypes may have implications beyond East Asia. They also noted that
Kudoa-associated gastroenteric symptoms can be easily confused with viral or bacterial gastroenteritis, which may contribute to misdiagnosis or underdiagnosis, and that new hosts and expanded distribution patterns are increasingly reported [
30]. Comparative studies evaluating sequence type-specific pathogenicity are needed to refine risk assessments of gastroenteritis associated with
K. septempunctata.
The biological plausibility
that K. septempunctata exposure can cause transient gastrointestinal symptoms in humans is supported by animal-model studies. Some studies have shown that
K. septempunctata spores induce transient gastrointestinal symptoms, including diarrhea and fluid accumulation, in suckling mice, with resolution within 24 hours [
2]. Vomiting has also been observed in musk shrews [
2]. In contrast, other studies have reported detection of parasite DNA in mice without overt symptoms [
12,
13]. Taken together, these findings indicate that stool PCR positivity is evidence of recent exposure rather than definitive proof of pathogenic causation in an individual case. Moreover, the parasite appears unable to colonize or proliferate within the mammalian gastrointestinal tract, resulting in rapid excretion of
K. septempunctata and a short detection window that is much narrower than that of pathogens capable of replicating in the gut [
31–
33]. More recent
in vitro and
in vivo studies have suggested that
K. septempunctata may impair intestinal epithelial barrier integrity and stimulate serotonin release, providing a potential mechanistic basis for the acute gastrointestinal symptoms observed in affected individuals [
34].
Foodborne outbreaks linked to raw olive flounder continue to occur nationwide. Regional counts were highest in Gyeonggi-do, a pattern similar to the clustering of norovirus cases reported in metropolitan areas with high population density [
21,
23]. Because fish provenance has not been systematically recorded and seafood distribution networks move products from coastal production sites, such as Jeju-do, to inland markets, provincial counts likely reflect locations of consumption rather than production.
Despite the strengths of national surveillance, this study has several limitations. A major limitation is the absence of laboratory testing of food samples. Because of the retrospective nature of the surveillance data, matched leftover raw-fish specimens could not be obtained or analyzed. Consequently, although the epidemiological links were strong, we could not directly confirm the presence of the parasite in the implicated food items. This limitation complicates causal interpretation in mixed-infection scenarios. For example, in 3 outbreaks (outbreaks 1, 9, and 10) in which norovirus or EPEC was identified, alternative pathogens were carefully considered. Norovirus was considered less likely in outbreak 1 because symptom onset was short (<6 hours). Similarly, in outbreaks 9 and 10, symptoms appeared within 3–4 hours and resolved within 1 day, making EPEC an unlikely explanation. These findings are most consistent with Kudoa-associated illness, given the timing and test results.
More broadly, the absence of systematic dietary histories and the potential influence of unmeasured confounders, such as co-exposures and recall limitations, are important constraints. Future studies should incorporate standardized dietary surveys together with fish provenance and supply-chain information, including farmed versus wild origin, domestic versus imported source, and distribution pathways. These data should be integrated with experimental parasitology, such as host-parasite interaction assays and barrier-function models, to combine human, product, and environmental information.
Article information
Ethics Approval
Not applicable.
Conflicts of Interest
The authors have no conflicts of interest to declare.
Funding
This research was funded by the Korea Disease Control and Prevention Agency (KDCA; 6331-311-210-13) of the Republic of Korea.
Availability of Data
All data generated or analyzed during this study are included in this published article. Other data are available from the corresponding author upon request.
Authors’ Contributions
Conceptualization: JHC, SHH, HIL; Data curation: JHC, CYK, JYK, JYS, SJJ; Formal analysis: JHC, SHH; Funding acquisition: JWJ, HIL; Investigation: JHC, CYK, JYK, JYS, SJJ; Methodology: JHC, SHH, JYK, JYS, TYK; Project administration: SHH, TYK, JWJ, HIL; Resources: all authors; Software: JHC; Supervision: SHH, TYK, JWJ, HIL; Validation: JHC, CYK, JYK, JYS, SJJ; Visualization: JHC, SHH; Writing–original draft: JHC, SHH; Writing–review & editing: JHC, SHH, TYK, HIL. All authors read and approved the final manuscript.
