Prevalence of Blastocystis sp. and other gastrointestinal pathogens among diarrheic COVID-19 patients in Italy

Background Gastrointestinal pathogens (GPs) contribute significantly to the burden of illness worldwide with diarrhoea being the most common among gastrointestinal symptoms (GSs). In the COVID-19 disease, diarrhoea, could be one of the initial presenting symptoms. However, no data on the potential correlation between diarrhoea-causing pathogens and SARS-CoV-2 infection are available. Therefore, we carried out a 2-years retrospective study aimed to evaluate the prevalence of “classic” GPs among SARS-CoV-2 infected and non-infected patients with diarrhoea in Italy. Methods Results of SARS-CoV-2 research from nasopharyngeal and detection of GPs from stool swab samples by Allplex™ SARS-CoV-2 and GI Virus, Bacteria and Parasite Assay were analysed for all patients with diarrhoea referring to Policlinico Ospedaliero Universitario, Foggia, (Italy) from February 2022 to October 2023. Results Out of the 833 involved patients, 81 (3.9%) were COVID-19 positive, while 752 (90.3%) were COVID-19 negative. Among COVID-19-positive patients, 37% (n = 30/81) were found positive for one or more GPs with a higher prevalence of protozoan parasites (18.5%) (Blastocystis ST1-ST4 subtypes, Dientamoeba fragilis genotype I), followed by bacteria (7.4%) (Campylobacter sp., Salmonella sp.). Viral pathogens were more frequent among COVID-19 negative patients (Adenovirus, Norovirus). Among GPs, Blastocystis ST3 subtype was the most prevalent registered in the 16% of patients (p = 0.0001). Conclusions Based on obtained results, a likely interaction between the classic GPs and SARS-CoV-2 infection can be speculated, driven by protozoan parasites. Moreover, these results also provide baseline data to understand more deeply Blastocystis sp. role in this scenario of dysbiosis, particularly in those cases of SARS-CoV-2 co-infection.


Introduction
Gastrointestinal infections (GIs) contribute significantly to the burden of infectious diseases illness worldwide [1,2].Centre for Disease Control and Prevention (CDC) estimates that each year about one billion people worldwide, especially children and elderly, are infected with at least one of the most prevalent species of intestinal pathogens, including virus, bacteria and protozoan parasites, with diarrhoea being the most frequent among gastrointestinal symptomology [2].While GIs are common in both high-income and medium/low-income countries, they are associated with different risk factors depending on the background.Indeed, within medium/low-income countries, illness is often linked to the lack of clean water and sanitation-related factors, whereas within high-income countries GIs are more often associated with foodborne transmission, seasonal prevalence as well as traveling [1].
The 2019 coronavirus disease (COVID- 19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic with cases spreading rapidly throughout the world [3].While fever and cough were the most frequently symptoms associated with COVID-19, the gastrointestinal manifestations have been reported for 32-61% of individuals with SARS-CoV-2 infection [4].In particular, diarrhoea has been reported from 4 to 34% of individuals in cohorts from China [5], from 12 to 34% in cohorts from USA [5] and approximately from 10 to 20% within hospitalized patients from Europe [6].A likely mechanism underlying the occurrence of gastrointestinal symptoms in COVID-19-positive patients is the presence of Angiotensin Converting Enzyme 2 (ACE2) receptors on gut epithelial cells, a well-described target used by SARS-CoV-2 for host cells binding and entry [7,8].Moreover, the diarrhoea and malabsorption caused by SARS-CoV-2 infection may be due to the dysregulation of intestinal ion transporters [9], leading to inflammation and gastrointestinal symptoms [10].The entry of inflammatory cells, including neutrophils and lymphocytes, into the intestinal mucosa alters the gut microbiota composition [11].Thus, this imbalance gut microbiome by SARS-CoV-2 could contribute or exacerbate the gastrointestinal symptoms related to infection by other "classic" gastrointestinal pathogens.
In the last years, polymerase chain reaction-based assays have improved the ability to diagnose GIs due to their high sensitivity and specificity, and are hence routinely used in many clinical laboratories and hospitals [12].
Until now, no data on the potential correlation between diarrhoeacausing pathogens and SARS-CoV-2 infection are available.Therefore, this 2-years retrospective study aimed to evaluate the prevalence of "classic" gastrointestinal pathogens among SARS-CoV-2 infected and non-infected patients with diarrhoea referring to a University Hospital in Italy.
Each patient was subjected (either for screening or for suspected infection) to nasopharyngeal swab for Real Time reverse-transcriptionpolymerase chain reaction (RT-PCR) detection of SARS-CoV-2 and to stool swab for the research of the most common gastrointestinal pathogens by Real Time PCR test.Patients tested positive for SARS-CoV-2 were defined as "COVID-19 positive".
The "Policlinico Riuniti" is a public, academic hospital that cares for a combined urban and suburban population of more than 594,000 citizens.Increasing immigrations rates, mainly from African countries and Eastern Europe, have been reported within this sanitary area in the last decade [14].
Demographic data (gender, geographical origin and age group) were obtained from the Hospital Information Management System.Clinical data (i.e., presence of gastrointestinal symptoms; eosinophilia; immunodeficiency and antibiotic treatments) were obtained from medical records.
Although data regarding duration of gastrointestinal symptoms in patients with COVID-19 are limited, a study found that COVID-19positive patients with gastrointestinal symptoms alone had on average 16 days from initial symptom onset to hospital admission and 31 days from symptom onset to viral clearance [15].Therefore, stool tests performed either 2 weeks before or 2 weeks after SARS-CoV-2 positivity were included in order to obtain an adequate time period during which patients may have had symptoms related to COVID-19.

SARS-CoV-2 PCR Panel Assay
All nasopharyngeal swab samples were subjected to RNA extraction by using MagCore® Nucleic Acid Extraction Kit (RBC Bioscience, Taiwan) and immediately subjected to real time RT-PCR screening by the Allplex™ SARS-CoV-2 Assay (Seegene Inc.Seoul, Korea) to determine SARS-CoV-2 infection, following the manufacturer's protocol.

Gastrointestinal pathogen PCR Panel Assay
All the stool swab samples were subjected to total nucleic acid (DNA/ RNA) extraction by using MagCore® Nucleic Acid Extraction Kit (RBC Bioscience, Taiwan) and immediately subjected to multiplex real time PCR assay by the Allplex™ Platform (Seegene Inc.Seoul, Korea) in order to identify the causative pathogens of the gastrointestinal diseases.Allplex™ GI-Virus, Allplex™ GI-Bacteria and Allplex™ GI-Parasite (Seegene Inc.Seoul, Korea) assays were used to detect virus, bacteria and protozoan parasites, respectively (Table 1).

Statistical analysis
The statistical model was built to examine whether the gastrointestinal infections (overall and singular viral, bacteria and protozoan parasites infections) were associated with COVID-19 positivity.Gender (male vs female), origin (Western Europe, North Africa, Eastern Europe, East/Central Asia, South America) and age classes (0-18; 19-39; 40-59; 60-79; 80-99) were analysed as secondary variables.Statistical analysis was performed using the QuickCalcs GraphPad online tool (available at https://www.graphpad.com/quickcalcs/).The relationship between variables was examined by Chi-square test.A p value < 0.05 was considered statistically significant.

Association between SARS-CoV-2 and gastrointestinal pathogens infection
Out of 833 patients, 81 (9.7%) were positive for SARS-CoV-2 PCR Panel Assay, while 752 (90.3%) were negative.The distribution of patients positive and negative to COVID-19 according the gender, origin and age classes is showed in Table 2.The positivity to COVID-19 were statistically associated with North Africa origin (p = 0.0273) and with age classes 60-79 (p = 0.0054), while no statistically significant correlation were observed with gender.By contrast COVID-19 negative patients were more frequently in the age classes 0-18 (p = 0.0020).
Considering the single group of pathogens, a highly statistically significant correlation was observed between COVID-19 positivity and presence of protozoan parasites (p = 0.0001) and multiple pathogens (p = 0.0004) (Table 3).Conversely, a statistically significant correlation was observed between COVID-19 negativity and viral pathogens (p = 0.0149).The association between COVID-19 and presence of bacterial pathogens was not significant (Table 3).

Genetic characterization of protozoan parasites
Following the molecular characterization of isolated protozoan parasites, four subtypes (ST1-ST4) were identified for Blastocystis sp., Assemblages A and A1 for G. duodenalis, genotype IIa for C. parvum and genotype 1 for D. fragilis (Table 5).

Discussions
Nowadays gastrointestinal disease still remains a significant global health concern.Indeed, according to the World Health Organization, there are 1.7 billion total cases each year, causing approximately 750,000 deaths mainly among children younger than 5 years old [3].Gastrointestinal infections caused by bacteria (i.e., Clostridioides difficile, Escherichia coli, Shigella), viruses (i.e., norovirus, rotavirus), or protozoan parasites (i.e., Cryptosporidium, Giardia) are major causes of diarrheal illness worldwide, often resulting from contaminated food/water and poor sanitation [21].Disruption of normal mucosal defences by one enteric infection is recognized as a risk factor for other infections, and prior studies have found that up to 26% of patients undergoing stool pathogen testing carry multiple pathogens [5].
It is known that among patients positive to COVID-19, the most common manifestations include fever, dry cough, dyspnoea, weakness,  breathing difficulty, fatigue, and myalgia [22].However, some patients also present gastrointestinal symptoms including nausea, vomiting, diarrhoea, and abdominal pain.Within patients suspected for COVID-19 infection, gastrointestinal symptoms could be the initial presenting symptoms [23].Some patients may present only gastrointestinal symptoms during the disease, which can delay the diagnosis leading hence to potential complications for themselves and infection transmission to others [23].Previous studies have indicated that SARS-CoV-2 enters cells through the ACE2 receptors expressed within the human respiratory and gastrointestinal (oesophagus, ileum, and colon) tracts.
The ACE2 receptors in the gastrointestinal tract maintain a regulatory role in amino acid homeostasis, gut microbiome, and innate immunity [24].Consequently, the binding of SARS-CoV-2 to ACE2 receptors in the gastrointestinal tract may result in a dysbiosis with gastrointestinal symptoms such as abdominal pain and diarrhoea [23].In addition, it is possible that this intestinal dysbiosis and inflammation due to SARS-CoV-2 infection could predispose individuals to co-infection with other gastrointestinal pathogens that can be assumed by oral-faecal route.
In this retrospective study, a potential interaction between COVID-19 positivity and the presence of "classic" gastrointestinal pathogens in patients with diarrheal disease has been investigated.
Out of 833 diarrheic patients and subjected to SARS-CoV-2 test, 9.7% (81/833) resulted positive and 90.3% (752/833) resulted negative to COVID-19.Although the SARS-CoV-2 infection was more frequent in male patients, any statistically significant correlation has been found between COVID-19 positivity and gender.By contrast, a statistically significant correlation has been found between COVID-19 positivity and patients come from North Africa (unfortunately, we are not able to know if the infection was acquired in Italy or in their country of origin) and in elderly people (age group 60-79 years), as expected.
Amongst protozoan parasites, a higher prevalence of Blastocystis sp.(n = 13, 16%) was reported within COVID-19-positive subjects when compared to the COVID-19-negative patient prevalence (n = 8, 1.1%).This result (although with a higher prevalence) is in accordance with a recent study conducted in an Hospital in Teheran, Iran, that focused on studying the frequency of intestinal parasitic infections within a cohort of COVID-19 positive patients and reporting hence a Blastocystis sp.prevalence of 6% [25].In another study conducted in Arabia Saudia, a SARS-CoV-2 and Blastocystis sp.mixed infection was reported in a woman with a congenital chloride losing diarrhoea (CCLD) [26] leading the authors to hypothesize that the two microorganisms could have exacerbated the osmotic diarrhoea typical of CCLD [27].Although the pathogenic role of Blastocystis sp.remains still controversial since this protozoan parasite has been found in healthy and non -healthy patients [28,29], the last literature data showed the potential ability of Blastocystis sp. to alter the gut microbiota ecosystem, which may lead to beneficial or harmful functions in the digestive system [29].This gut microbiome alteration could predispose individuals to co-infection with other gastrointestinal pathogens and favour or exacerbate also the inflammation due to SARS-CoV-2 infection.Based on the results here obtained a potential interaction between SARS-CoV-2 and Blastocystis sp. could be then hypothesized.
By genotyping our Blastocystis sp.isolates, subtypes from ST1 to ST4 were identified, with ST3 the most prevalent subtype, as expected [16].Actually, among thirteen Blastocystis-COVID-19 positive patients, subtype ST3 was detected in six patients, following by ST1 in four patients, ST2 in two patients and ST1 in one patient.The higher number of ST3 subtypes were also reported in a similar study carried out in a hospital in Teheran, in which a high prevalence of Blastocystis sp. has been found in COVID-19-positive patients with ST3 being the most common detected subtype [30,31].Nevertheless, these results prone the basis for future investigations focusing on the association between Blastocystis and COVID-19-positive samples in order to establish potential risk factors for gastrointestinal symptoms.
In our work, D. fragilis was found in 2.5% of patients with COVID-19 when compared with those COVID-19 negative (0.5%).Similar to Blastocystis sp., the pathogenic role of D. fragilis is still controversial but several studies reported a cooperation between the two protozoan parasites [32].Indeed, here, a mixed infection Blastocystis sp./D.fragilis were reported in two patients COVID-19 positive patients.Interestingly, all our D. fragilis isolates have been genotyping as Genotype 1 variant that being the most reported and pathogenic genotype [33,34].Quite unexpected was the detection of C. parvum genotype IIa (n. 4, all Italian origin) and G. duodenalis Assemblages A and A1 (n. 4, all Italian origin) (the most frequently protozoan parasites causing diarrhoea and with a high zoonotic impact) only within our COVID19negative patients whereas the presence of C. parvum IIa and G. duodenalis was observed in COVID-19 positive patients from a study carried out in Teheran, although with a very low prevalence [25].In these patients group any correlation with origin, gender or age were found and mostly probably the diarrhoea was related just to parasite infection.
Among bacterial pathogens, Campylobacter sp. and Salmonella sp. were the only bacteria species found in COVID-19-positive patients with prevalence of 4.9% and 2.5%, respectively although without any statistically significant correlation.Interestingly, mixed infections Campylobacter/Blastocystis and Clostridioides/Blastocystis were reported in one and two COVID-19 positive patients, respectively, with a statistically significant association.In a recent work analysing the occurrence of Blastocystis sp. in patients with C. difficile in a cohort study from Colombia, the authors demonstrated a significant association between the presence of Blastocystis and C. difficile infection (CDI), with 61 cases of co-infection among CDI patients.This co-infection could support hypotheses that Blastocystis may adapt to dysbiosis and oxidative stress by C. difficile and together exacerbated the gastrointestinal symptoms [35].
Concerning viral pathogens, although Adenovirus and Norovirus were reported in the 3.7% and 1.2% of COVID-19-positive patients, we found a statistically significant association between COVID-19 negative patients and presence of viral pathogens (p = 0.0139).This result was quite unexpected since previous reports stating that the circulation and morbidity burden of other respiratory viruses (including adenoviruses) are also highly impacted by the COVID-19 pandemic, with wide temporal and geographic fluctuations [36].Conversely, a mixed infection Adenovirus/Blastocystis/Dientamoeba was registered in one COVID-19 positive patient.
In this study we found that patients with COVID-19 were more likely to test positive for co-infection with classic gastrointestinal pathogens compared to those without COVID-19, although any correlation with gender, origin or age group has been found.This difference was mainly driven by protozoan parasites (highly statistically significant) followed by mixed infection (statistically significant).Although the majority of gastrointestinal symptoms in COVID-19 patients may be attributable to viruses, our results show that the most commonly identified enteric infections were related to protozoan parasites species, which was unexpected given these are relatively neglected pathogens.Based on the literature reports, intestinal protozoa can potentially polarize the helper T cells towards type 1 (Th1) [25].Additionally, co-infections with intestinal protozoa and some intracellular pathogens, such as Mycobacterium tuberculosis and human immunodeficiency virus (HIV), may substantially cause imbalances in the host and lead hence to further pathological consequences [37].Since the emergence of the SARS-CoV-2, there have been some hypotheses on the likely interaction between the intestinal parasites and COVID-19 [38].Nevertheless in COVID-19 positive patients investigated in the present study, diarrhoea cannot be reliably attributed solely to the Blastocystis sp., because other infectious (bacterial and viral agents) and/or non-infectious diseases may have had a role in the initiation and progression of diarrhoea.For this reason, in order to establish the existence of a probable correlation between the Blastocystis sp. and SARS-CoV-2 infection, more deeply studies are needed.In particular, as our next goal, a prospective study on a large scale, regarding the analysis of the composition of gut microbiota and its correlations with different risks factors for each patient with gastrointestinal diseases will be designed.This will allow us to better understand the role of protozoan parasites and in particular for Blastocystis sp. in a scenario of intestinal dysbiosis and try to elucidate the modulating role of this eukaryote on the members of the bacterial microbiome, particularly in those cases of co-infection with SARS-CoV-2.

Study limitations
To the best of our knowledge, this is the first study, conducted over a period time of almost two years, reporting the prevalence of co-infection with gastrointestinal pathogens using PCR-based assays in patients under evaluation for COVID-19 infection.However, the present study has some limitations, mainly due to its retrospective design.In particular, we have had not the change to distinguish the severity of diarrhoea and therefore the differences between groups.In addition, we were not able to accurately retrieve the medical history for each patient (i.e.travel to other countries, the time between the arrival in Italy and clinical manifestation) and evaluate clinical features such as patients' immunological conditions and haematological parameters.

Table 2
Demographics data (gender, origin and age classes) of the enrolled patients and of COVID-19 positive and negative patients.
a p value: statistically significant.

Table 3
Viruses, bacteria and protozoan parasites (singular and mixed infection) detected among COVID-19 positive and negative patients.
a p value: statistically significant; b p value: highly statistically significant.

Table 5
Number of subtypes/assemblages/genotypes and sequence Accession Number of protozoan parasites isolated in COVID-19-positive and COVID-19 negative patients.