Foodborne concerns of Blastocystis spp. in marine animals (fish, bivalves, and sponges): A systematic review and meta-analysis of global prevalence and subtypes distribution

Blastocystis spp. is a common intestinal parasite found in humans and various animals, including marine species like fish, bivalves, and sponges. While traditionally considered non-pathogenic, emerging evidence suggests potential foodborne concerns, especially for vulnerable populations. The present systematic review and meta-analysis reviewed four electronic databases (PubMed, Scopus, Google Scholar, and Web of Science) until June 13, 2024, for studies reporting the prevalence and subtypes (STs) distribution of Blastocystis spp. in marine animals, including fish, bivalves, and sponges, to assess foodborne concern and zoonotic importance. In the analysis of 11 studies involving 1329 marine animals from nine countries, five studies/datasets (742 samples) focused on fish, five studies/datasets (567 samples) on bivalves, and one (20 samples) on sponges. This review found that 12.4 % (95 % CI: 4.3–31 %) of marine animals globally were infected by Blastocystis spp., with bivalves showing the highest infection rate at 32 % (95 % CI: 13–59.7 %), exceeding sponges with a single study at 10 % (95 % CI: 2.5–32.4 %), and fish at 4.4 % (95 % CI: 2–9.3 %). Sensitivity analysis assessed changes in weighted prevalence after excluding certain studies. A subgroup analysis of Blastocystis spp. prevalence was conducted based on publication years, countries, continents, WHO regions, and sample sizes. The data collected indicated that marine animals serve as suitable reservoirs for various Blastocystis spp. STs (ST1-ST4, ST7, ST8, ST10, ST14, ST23, ST26, and ST44), with most (except for ST26 and ST44) having the potential for zoonotic transmission. Overall, the findings emphasize the potential for foodborne risk posed by Blastocystis spp. in marine animals and highlight the need for improved monitoring and control measures to ensure food safety.


Introduction
Blastocystis spp. is a prevalent protozoan parasite found in the digestive tracts of animals and humans globally (Asghari et al., 2024c;Guilavogui et al., 2022).The frequency of Blastocystis spp.differs between developed and developing countries, with higher rates usually observed in regions with inadequate sanitation and restricted access to safe water (Asghari et al., 2024b;Mohammad et al., 2018).Risk factors for contracting Blastocystis spp.include poor hygiene practices, close contact with infected individuals, and consuming contaminated food or water (Asghari et al., 2024a;Mohammad et al., 2017).Clinical symptoms of infection can vary from mild gastrointestinal discomfort to more severe symptoms like diarrhea, nausea, and abdominal pain (Salvador et al., 2016).The pathogenesis of Blastocystis spp.infection is not fully understood, but Blastocystis spp.has the ability to regulate the gut microbiome and immune responses to maintain the homoeostasis as well (Roberts et al., 2014;Shams et al., 2024;Vassalos et al., 2008).
Given the zoonotic significance and genetic variety of this parasitic protozoan, numerous original and/or review studies have been carried out on humans and various animal categories, particularly zoo animals, domestic animals, and pets (Abedi et al., 2022;Asghari et al., 2021b;Asghari et al., 2021a;Badparva et al., 2017;Barati et al., 2022;Fusaro et al., 2024;Kumarasamy et al., 2023;Rostami et al., 2017;Shams et al., 2021Shams et al., , 2022b;;Shams et al., 2022a;dos Zanetti et al., 2020).However, there are few studies on the frequency and STs distribution of Blastocystis spp. in aquatic animals, possibly because of the challenge in sampling these animals.As many individuals worldwide are beginning to consume aquatic foods like fish and bivalves, the examination for parasitic infections such as Blastocystis spp. in these animals is crucial.Thus, this study was conducted to determine the prevalence and STs distribution of Blastocystis spp. in marine animals (fish, bivalves, and sponges) using existing information in this area.

Ethics approval and study type
The study received approval from the Ethics Committee of Qazvin University of Medical Sciences, Qazvin, Iran (approval no.IR.QUMS.REC.1403.176).It was a global systematic review and meta-analysis for the prevalence and subtype distribution of Blastocystis spp. in marine animals (fish, bivalves, and sponges).The research adhered to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines (Moher et al., 2015).

Database search
The study analysed four global databases: Medline/PubMed, ProQuest, Scopus, and the Web of Knowledge, for articles published until June 13, 2024.Google Scholar was consulted for grey literature.The search was performed using Medical Subject Heading (MeSH) terms alone or in combination: ("Intestinal Parasites" OR "Parasitic Infections" OR "Blastocystis spp.")AND ("Prevalence" OR "Epidemiology" OR "Frequency" OR "Occurrence") AND ("Subtype" OR "Subtyping") AND ("Aquatic animals" OR "Marine animals" OR "Fish" OR "Molluscs" OR "Bivalves" OR "Sponges").To include more pertinent papers, extra keywords were utilized, and the references of relevant papers were examined.The data collected was input into EndNote X9 software, and duplicate articles were automatically removed.Two researchers independently reviewed the articles.

Inclusion/exclusion criteria
Cross-sectional studies from all languages, regions, and time periods that reported Blastocystis spp.prevalence in marine animals using microscopy, molecular, and serological methods were evaluated in this review.Studies on non-marine animals, humans and plants, case reports, commentaries, reviews, and studies that lacked total sample size or Blastocystis spp.prevalence rate were excluded from this review.

Data selection criteria
Papers were evaluated for inclusion or exclusion based on the Joanna Briggs Critical Appraisal Checklist for Studies Reporting Prevalence Data (Munn et al., 2014).Studies scoring 4-6 points were considered as moderate-quality, while those scoring 7 points or more were classified as high-quality.Articles with scores ≤3 were excluded.Two researchers extracted key data from the chosen papers, which were then validated by other researchers.The extracted information included the primary author's last name, animal type, quality assessment score, publication year, implementation year, continent, country, World Health Organization (WHO) classification, total sample size, and number of infected samples.

Statistical analysis
Statistical analyses utilized the Comprehensive Meta-Analysis (CMA) v3 software.P-values below 0.05 were deemed statistically significant.The random-effects model was employed to evaluate Blastocystis spp.prevalence by estimating pooled prevalence and 95 % CIs.Subgroup analysis was conducted to assess the weighted prevalence of infection by animal types, WHO regions, countries, publication years, continents, sample size, and diagnostic methods.A forest plot diagram was created to display the pooled prevalence with 95 % CIs.The funnel plot was used to assess publication bias in the analysis.Heterogeneity across studies was evaluated using the I2 index.Values below 25 %, 25-50 %, and over 50 % were categorized as low, moderate, and high heterogeneity.Furthermore, a sensitivity analysis was conducted to examine changes in the final weighted prevalence of Blastocystis spp.by excluding specific studies.

Article selection
Rigorous searches of four international databases found a total of 5792 initial records.After removing duplicates and reviewing the remaining 3547 records, a total of 13 articles were ultimately selected.Additionally, a quality assessment based on JBI criteria led to the exclusion of two more studies.Finally, a total of 11 relevant papers (11 datasets) met the inclusion criteria for this study (Fig. 1).

Qualitative and quantitative characteristics of the papers included
The analysis included 11 articles/datasets (five related to fish, five to bivalves, and one to sponges) published between 1997 and 2024.Out of the 1329 marine hosts, 742 were fish, 567 were bivalves, and 20 were sponges.France and Mexico published the most studies with two papers each, followed by single papers from each of Chile, China, Germany, Iran, Malaysia, Poland, and Qatar.Sample sizes ranged from 18 to 374 marine animals in the 11 studies.Seven papers detailed the subtype distribution of Blastocystis spp. in marine animals.Molecular method was the prevalent diagnostic technique in eight studies, with microscopy and culture techniques utilized in one and two studies, respectively (Table 1).The evaluation with the JBI checklist indicated that 6 papers were classified as high quality (>6 points), while the other 5 articles were deemed to be of moderate quality (4-6 points) (Supplementary Table 1).

Overall prevalence of Blastocystis spp. in marine animals
The global occurrence of Blastocystis spp. in marine animals across different countries is illustrated in Fig. 2.This study revealed  g This subtype, identified as ST10, exhibits a sequence similarity of 92 % with sequences found in GenBank, suggesting that it may be a new subtype.that 12.4 % (95 % CI: 4.3-31 %) of global marine animals were infected by Blastocystis spp.(Fig. 3).Statistical analysis revealed a considerable degree of heterogeneity among the included studies in the current systematic review and meta-analysis (Q = 328.1,I 2 = 96.9%, P = 0.000).

Weighted prevalence of Blastocystis spp. in marine animals based on examined subgroups
The subgroup-based prevalence of Blastocystis spp. in marine animals is displayed in Table 2 and Supplementary Figs.2-7.

Sensitivity analysis
Based on the sensitivity analysis, excluding particular datasets on Blastocystis spp. in marine animals did not notably alter the overall frequency (Supplementary Fig. 8).

Discussion
The present review with 12.4 % (95 % CI: 4.3-31 %) found a moderate occurrence of Blastocystis spp. in marine animals, with bivalves showing a higher occurrence (32 %; 95 % CI: 13-59.7 %) compared to sponges (10 %; 95 % CI: 2.5-32.4%) and fish (4.4 %; 95 % CI: 2-9.3 %).Of note, due to limited research, the true prevalence of Blastocystis spp. in these hosts remains unclear.Therefore, caution is advised when interpreting the prevalence rates from this study's analysis.Of note, in areas with high marine animal consumption, the risk of parasitic infections such as Blastocystis spp.should be taken seriously.After conducting the sensitivity analysis, the exclusion of specific datasets on Blastocystis spp. in marine animals did not significantly change the overall frequency.Furthermore, following the elimination of these datasets, the estimated prevalence of Blastocystis spp. in marine animals was around 9.6-15.3%.
Subgroup analysis by year of publication revealed that more recent studies indicate a lower prevalence [7.8 % (95 % CI 1.8-28.8)] of Blastocystis spp. in marine animals.This shift could be attributed to the adoption of advanced molecular techniques and more accurate reporting of infection rates.Furthermore, research conducted across WHO regions, continents, and countries revealed a significantly high prevalence of Blastocystis spp.infection in marine animals in the AMR region [40 % (95 % CI: 12.2-76.2)]and on the continent of North America including Mexico [60.5 % (95 % CI: 44.5-74.5)].Adherence to health protocols and raising public awareness regarding the consumption of raw or undercooked food in these regions is strongly advised.Increasing the sample size from <100 to 100-400 resulted in nearly halving the reported prevalence of Blastocystis spp. in marine animals, underscoring the significance of a large sample size in epidemiological research to ascertain the accurate prevalence of the targeted infection.Molecular methods (18.7 %; 95 % CI: 6-45.2) showed notably better diagnostic accuracy for identifying Blastocystis spp. in samples from marine animals compared to microscopic (3.5 %; 95 % CI: 1.3-9) and culture (2.7 %; 95 % CI: 0.1-43.8)methods.However, the number of studies varied significantly across diagnostic methods, and with an equal number of studies, the results could change entirely.Overall, the analyses in this study are mainly derived from a small pool of research and necessitate thorough interpretation.
Parasitic infections like Blastocystis spp.can be transmitted from marine animals to humans through various means, including consumption of contaminated seafood, contact with infected water, environmental contamination, and food preparation practices.Preventive measures to reduce transmission include thoroughly cooking seafood, practising good hygiene, avoiding contaminated water, ensuring safe food preparation, and undergoing regular health check-ups.Following these practices can significantly lower the risk of transmission.To enhance the sampling of marine animals for studying parasitic infections like Blastocystis spp., future research can benefit from strategies such as collaborating with fisheries, employing non-invasive techniques like collecting water or fecal samples, and utilizing technologies like drones or underwater robots for preliminary surveys to locate and identify target species without disturbance.This review encountered certain constraints, including a scarcity of prevalence data in many global regions, imprecise diagnostics such as molecular tests with good sensitivity and specificity, the inclusion of only one study in certain marine animal categories (e.g.sponges), and reliance on studies with limited sample sizes.These limitations hindered arriving at a definitive conclusion, yet our findings provided an assessment of the present status of Blastocystis spp. in marine animals.

Conclusion
This systematic review and meta-analysis study presented a 12.4 % moderate prevalence of Blastocystis spp. in marine animals, indicating that various species like fish, bivalves, and sponges can be infected with this protozoan parasite.Our findings highlighted that marine animals serve as proper hosts for different Blastocystis spp.STs, including zoonotic ones.Therefore, the significance of Blastocystis spp.infection, particularly its zoonotic STs, in the transmission from marine animals to humans and marine predatory species, should not be underestimated.Overall, investigating the prevalence and STs distribution of Blastocystis spp. in marine animals is an emerging field that warrants further exploration.

Fig. 1 .
Fig. 1.Flowchart of the included eligible studies in the present study.

Fig. 2 .
Fig. 2. The global occurrence of Blastocystis spp. in marine animals across different countries (the numbers following the country names and the cases in parentheses indicate the prevalence rate of Blastocystis spp.)

Fig.
Fig. The worldwide prevalence of Blastocystis spp. in marine animals using a random-effects model and 95 % CIs.

Table 1
The main data from 11 studies in this review about the prevalence and subtype distribution of Blastocystis spp. in marine animals.
a Unclear.b Culture method.c Microscopic detection.d Molecular detection.e Subtypes.f Unidentified subtypes.

Table 2
Subgroup analysis of Blastocystis spp. in marine animals by publication year, continent, WHO region, country, sample size, and diagnostic method.