1. Introduction
Blastocystis sp. is the most prevalent enteric protist reported in human fecal samples [
1,
2]. Over one billion people are estimated to be infected with
Blastocystis sp. worldwide, especially in developing countries, where infection frequencies exceeding 50% are commonly reported, and even up to 100% in some tropical countries [
3,
4,
5]. Conversely, the prevalence is significantly lower in developed countries (10–15%) [
6]. The variations in prevalence between studies could be attributed to different factors, including the use of different diagnostic approaches in clinical microbiology laboratories, with many largely underestimating the prevalence in the context of enteric parasite diagnosis [
7,
8]. Nevertheless, it is well known that the high prevalence of this protist in developing countries is related to socioeconomic factors that lead to poor sanitation and higher potential sources of infection, including human-to-human, zoonotic, and waterborne transmission [
9].
Although this parasite was discovered more than a century ago, its pathogenic mechanisms still remain under discussion. Several studies support the pathogenic potential of
Blastocystis sp. in humans by reporting gastrointestinal symptoms, such as abdominal pain, diarrhea or vomiting, as well as a diverse spectrum of cutaneous symptoms, mainly urticaria, in the absence of any other cause of sickness identified in patients [
10,
11]. In contrast, some other studies did not report conclusive evidence for the pathogenic role of
Blastocystis sp. and consider this protist a common microorganism of healthy intestinal microbiota [
12,
13]. In the last decade, an increasing number of studies have explored the genetic diversity of
Blastocystis sp. in humans. A total of 33 different subtypes have been reported to date in humans and animals based on variations in the small subunit ribosomal RNA (SSU-rDNA) gene [
14]. However, not all strains of a specific subtype are clinically relevant and a possible correlation between the different subtypes and their pathogenic potential is still strongly debated [
15,
16,
17].
Multiple risk factors have been reported for
Blastocystis sp. human infections, including poor hygiene conditions, not washing hands after using toilets, drinking non-tap water and contact with animals [
18,
19]. The prevalence of the parasite was also correlated with a low socioeconomic status, low education and poor health conditions [
20]. Age, nutritional status and some clinical conditions (anemia, irritable bowel syndrome) have also been linked to
Blastocystis sp. infections [
19,
21]. In European countries, travelling abroad or infections with other enteric parasites significantly increase the risk of having a positive result for
Blastocystis sp [
17,
22,
23].
In Spain, studies on the prevalence and risk factors of
Blastocystis sp. infection in humans are limited and mostly focused on specific groups of the population. Studies in asymptomatic and symptomatic children have revealed prevalence rates of 5.3–19.4% using microscopy and/or PCR-based techniques, and up to 27.8% of adult patients tested positive for
Blastocystis sp. using both diagnostic methods [
24,
25,
26,
27]. Adult age, working with the public, being African and travelling to other countries have been reported to significantly increase the risk of having a positive diagnosis of
Blastocystis sp., while good hygiene practices, such as hand or vegetable washing, have been recommended to minimize the risk [
23,
26,
28,
29]. Few studies in Spain have analyzed the relationships of this parasite with the presence of digestive symptoms, which was not found as a significant factor in patients older than 18 years [
23]. The current case–control study was aimed to analyze the prevalence of
Blastocystis sp. in patients of different age groups with gastrointestinal manifestations in northern Spain. In addition, co-infection of
Blastocystis sp. and other species of pathogenic bacteria, helminths and protozoa were analyzed. Multiple variables, including anthropometric, socio-demographic and clinical data, were analyzed as potential associated factors with
Blastocystis sp. infection.
3. Results
Fecal specimens from 338 of 3682 patients (9.18%) tested positive for
Blastocystis sp. infection and were included as cases.
Figure 1 shows a microscope field obtained in a
Blastocystis positive sample.
The control group consisted of 3344 patients negative for this parasite. The case–control ratio group was 1: 9.89 (≈10).
Table 1 shows the distribution of the demographic/anthropometric parameters and treatment with immunosuppressive drugs in positive and negative
Blastocystis sp.-infected patients. Most of the variables showed statistically significant differences between the cases and controls. In relation to the geographical origin, the percentage of patients from Spain was significantly lower in the cases than in the controls, while the situation was reversed in the patients from other countries, with significant differences in those from Africa, America (Central and South America, plus one
Blastocystis negative patient from USA). When age was categorized based on a 16 years cut-off (child vs. adult), no significant difference was detected between cases and controls; in contrast, the cases were significantly younger than controls when age was considered as a quantitative variable. Underweight individuals were significantly more common in the cases than in controls; in contrast, both normal weight and overweight persons were significantly more frequent in the controls. The BMI as a quantitative variable also differed significantly between cases and controls, with lower values in the cases. Immunosuppressive treatment with chemotherapy, monoclonal antibodies or antiretroviral drugs were significantly more common in the cases than in controls, but treatment with corticosteroids was more frequent in the controls. Sex and proportion of patients undergoing treatment with nucleoside analogs did not differ between cases and controls.
The clinical symptoms, underlying diseases, co-infections with pathogenic bacteria or other enteric parasites and laboratory test results in the cases and controls are summarized in
Table 2. Most symptoms were significantly more common in the cases than in controls, including abdominal pain, nausea, anorexia, aerophagia, halitosis, urticaria, anal itching and dyspepsia. In contrast, the presence of diarrhea, and fever was more common in the controls than in cases, with statistically significant differences in the presence of fever. No significant differences were detected between the groups for the co-morbidities, except for type 2 diabetes, which was more frequent in the cases, while irritable bowel syndrome (IBS) was significantly more common in negative patients. There was no difference in biochemical parameters between
Blastocystis sp.-infected and uninfected patients. However, highly significant differences were found for relative eosinophilia, showing greater values in the cases.
Several variables related to the absence/presence of pathogens were not included in the PCA, whose results are described in
Table 3. Namely, a very low number of individuals (<2) showed presence of
Arcobacter butzleri,
Shigella,
Schistosoma intercalatum,
Taenia saginata and
Encephalitozoon hellem; therefore, these variables were not considered for PCA. Several variables had a KMO index <0.5, breaching the basic assumption for PCA (
Aeromonas sp.,
Campylobacter sp.,
E. coli,
Salmonella sp.,
Yersinia sp.,
Cryptosporidium sp.,
Hymenolepis nana and
Trichuris trichiura). Hence, only
Isospora belli,
Strongyloides stercoralis,
Giardia lamblia,
Dientamoeba, Entamoeba sp.,
Ascaris lumbricoides,
Enterobius vermicularis and
Blastocystis sp. were considered for PCA. Based on the criteria explained in the statistical methodology, three principal components were retained. The cumulative percentage of variance explained by them was 46.996%.
Table 3 shows the rotated component matrix (the key output of PCA); it shows the estimates of the correlation coefficients between each of the original variables and the estimated components. Hence, PCA summarizes the information about co-infection from the original 21 variables in only 3 variables (three principal components; 1, 2 and 3), including the original variables shown in
Table 3. The coefficients below 0.3 (absolute value) have been suppressed for easier interpretation. Positive loadings indicate that a variable and a principal component are positively correlated; an increase in one results in an increase in the other. Negative loadings indicate a negative correlation. Large (either positive or negative) loadings indicate that a variable has a strong effect on that principal component. Principal component 1 loads very strongly on
Isospora belli,
Strongyloides stercoralis and
Giardia lamblia but less strongly on
Dientamoeba sp.; this principal component mainly summarizes the information about the presence of these four agents. Similarly, principal component 3 loads very strongly on
Enterobius vermicularis and less strongly on
Blastocystis sp. Finally, principal component 2 loads strongly on
Entamoeba spp and
Ascaris lumbricoides, but lightly on
Blastocystis sp. and
Strongyloides stercoralis (negative load); this principal component summarizes the information about the presence of
Entamoeba spp and
Ascaris lumbricoides, and
Blastocystis and the absence of
Strongyloides stercoralis.
Once the original variables set was reduced by PCA, cluster grouping based on the three retained principal components must be applied in order to study co-infections. These clusters are groups of individuals that share similar characteristics, as it refers to the variables included in the three principal components; hence, this provides a simplified view of the most relevant co-infections in the considered sample of individuals.
Table 4 shows the result of the clustering based on the three retained principal components. The first cluster included most of the individuals that were negative for
Blastocystis sp. (3277/3344 = 98%), while the sixth cluster contained the majority of the
Blastocystis sp. positive patients (301/336 = 89.50%), with no presence of other parasites in either cluster. Hence, most of the studied individuals, either cases or controls, did not show any co-infection. Each cluster of individuals concerned a particular microorganism, since most of the individuals that showed this microorganism are included. Cluster 2 concerned
Giardia lamblia (45/52 individuals infected with
Giardia = 86.53%) and included only 9 of the 336 patients infected with
Blastocystis sp. (2.68%). Cluster 3 included all patients infected with
Enterobius vermicularis (n: 16) and included eight patients positive for
Blastocystis sp. (2.38%). Cluster 4 contained all patients infected with
Entamoeba sp. (n: 19) and included twelve patients positive for
Blastocystis sp. (3.57%). Finally, all patients infected with
S. stercoralis were allocated to Cluster 5 (n: 6), which also included six
Blastocystis sp.-infected patients (1.78%). The
Table 4 shows the results obtained for all the kinds of co-infections. Since this study focuses on
Blastocystis,
Table 5 provides a more accurate description of co-infections, including more than one microorganism, in addition to
Blastocystis.
Table 6 shows the final model for binomial logistic regression; it was statistically significant (χ
2 (11) = 1955.696;
p < 0.001) and explained 94.6% of the total variance (Nagelkerke R
2). The probability of being infected with
Blastocystis sp. was similar for individuals from both Spain and the rest of Europe, but significantly increased for individuals from America and especially from Africa. No significant association was detected for individuals from Asia, which may be due to their scarcity. Abdominal pain, anorexia and type 2 diabetes were significantly associated with
Blastocystis sp. detection, but corticosteroid treatment was associated with the absence of this microorganism. Infection was strongly associated with the presence of pathogenic bacteria and halitosis; namely, individuals with bacterial co-infection were 132.960 times more likely to be infected with
Blastocystis sp. and this likelihood increased to 315.220 times more when halitosis was detected. However, the strongest association corresponded to relative eosinophilia; as relative eosinophilia increased by 1 unit, the likelihood for
Blastocystis sp. infection increased 1105.260 times. The accuracy parameters for the binomial logistic regression model were high and were as follows: Sn = 0.967 (95% CI: 0.969–0.998); Sb = 0.998 (95% CI: 0.996–0.998); PPV = 0.983 (95% CI: 0.968–0.997); NPV = 0.997 (95% CI: 0.995–0.999). The area under the ROC curve was 0.997 (95% CI: 0.993–1.000;
p > 0.001).
4. Discussion
Blastocystis sp. is among the most prevalent parasites found in human fecal specimens in diagnostic microbiology laboratories, although its clinical significance is still uncertain in contrast to common protozoa, which are responsible for a significant proportion of diarrheal morbidity globally, such as
Giardia duodenalis and
Cryptosporidium sp. [
40,
41,
42]. In Spain, previous studies in patients that reported digestive disorders and attended medical services have shown that the percentage of patients infected with
Blastocystis sp. is twice or even ten times higher than those infected with
G. duodenalis and
Cryptosporidium sp., respectively [
28]; however, studies in asymptomatic children have reported similar infection rates for
Blastocystis sp. and
G. duodenalis [
26].
The prevalence of
Blastocystis sp. infection in patients with gastrointestinal symptoms in this study was 9.18%. This result is consistent with previous studies in central Spain using conventional microscopy, which have documented prevalence rates of 9.6% in HIV-positive children and 5.3–19.4% in children attending daycare centers and primary schools [
24,
25], while 13% was found in asymptomatic school children using PCR-based methods [
26]. Higher prevalence rates have been reported in adult patients in Catalonia (northeastern Spain) through microscopic examination and PCR (27.8%) [
27], and up to 35.2% of PCR-
Blastocystis positive samples were found in humans sharing households with dogs and cats in northern Spain [
43]. In Europe, the prevalence of this protist has been reported to range from 3% to 7% in France, Italy and the United Kingdom using direct-light microscopy, but higher rates (14.5−24.2%) were found using PCR-based methods in France, the Netherlands and Denmark [
22,
28,
44,
45,
46,
47].
It is significant to mention that the infection rates detected in this study may be an underestimation, since molecular analyses are known to be much more sensitive than microscopic techniques and xenic in vitro culture for the detection of
Blastocystis sp. in fecal specimens from both humans and animals [
1,
45]. Culture from stool samples was significantly more sensitive than direct microscopic examination, but it is time consuming and not practical for diagnosis when a quick turnaround is required [
45]. The sensitivity of microscopy has been suggested to increase when increasing the number of investigated samples. Microscopy on two SAF (sodium acetate-acetic acid-formalin) preserved samples in the test called “triple faeces test”, which combines multiple fecal sampling (on 3 consecutive days) with a concentration method, has been reported to provide a similar sensitivity to sequence confirmed-PCR [
47,
48]. In contrast, other studies have shown that microscopic diagnosis through a concentration technique did not increase when two or three consecutive stool samples were investigated, compared with one simple stool investigation [
49].
Geographical location has been reported to influence the prevalence of parasitic infections [
50]. In this study, a significant effect of geographic origin of patients on the prevalence of
Blastoscystis sp. was found, with over 25% of infected patients originating from outside Europe, with the percentage of patients from Central and South America, and especially from Africa, being significantly higher in the cases than in controls. Poor sanitary and hygiene conditions have been proposed to explain the higher prevalence of
Blastocystis sp. in low-income countries, since it is mainly transmitted through the fecal-oral route via consumption of contaminated food or water [
3]. Infections rates ranging from 80.4% to 100% have been documented in school children in Senegal, 64% in Moroccan children, 71.1% in immigrant workers in Qatar, 78% in the Guinean population, and 63% in children in Lebanon [
4,
51,
52,
53,
54,
55]. In contrast, much lower rates are documented from European countries, including Denmark (5.6%), France (13.7–23.1%), Czech Republic (24%) and Spain (13%) [
22,
29,
56,
57].
In this study, the presence of
Blastocystis sp. was not associated with either of the two age classes (children or adults) or with sex, although the cases were significantly younger than controls when age was considered as a quantitative variable. Many studies in different parts of the world have investigated the effect of host sex on
Blastocystis sp. infection rates and most of them have shown that prevalence is not significantly related to sex [
57,
58,
59], although some studies have shown a higher prevalence in either females [
60] or males [
61]. Similarly, there are contradictory results regarding the association with host age. Some studies found no significant relationship [
21,
59,
62], but others reported higher
Blastocystis sp. infection rates among younger adult age groups [
22,
23], adults aged more than 18 years [
61] or even older than 60 years [
63]; other studies have documented patients younger than 30 years or children in the group of age 5–15 years to have a higher risk of being infected with
Blastocystis sp. [
43,
55].
Our results for BMI showed a highly significant difference between the groups of patients, with underweight individuals being significantly more common among the cases than in controls. This finding is consistent with previous studies that claimed a positive correlation between low BMI and
Blastocystis sp. infection, an observation which has been related to a potential negative effect of the protist on dietary intakes and energy metabolism [
42,
64,
65]. This potential negative impact on host weight could be of relevance when considering data that suggest that
Blastocystis sp. is capable of long-term host colonization, with some individuals testing positive for up to 10 years [
12].
Another potential factor that determines the transmission of pathogens is host immunity, since it is well known that patients with decreased immunity are particularly susceptible to opportunistic infections [
66].
Blastocystis sp. has been widely reported in both immunocompetent and immunocompromised individuals but its pathogenic, or rather opportunistic, role has yet not been clearly elucidated. Recent systematic reviews and meta-analyses have estimated that the global pooled prevalence rate of this protist in immunocompromised patients is around 10% [
67], and case–control studies have shown that immunosuppressive conditions, such as cancer, organ transplantation and hemodialysis, were associated with higher odds of infection [
68]. In contrast, similar or even lower prevalence was reported in some studies on immunodeficient patients compared to controls [
23]. In this study, immunosuppressive treatments with chemotherapy, monoclonal antibodies or antiretroviral drugs were more common in cases than in controls, which could support the role of
Blastocystis sp. as an opportunistic organism. However, it is worth mentioning that treatment with corticosteroids was much more frequent in controls than in cases (28.9% compared to 0.9%), and the use of these immunomodulatory drugs was also significant as a potential protective factor in the model for binary logistic regression. The reason for this observation is not known; in fact, a recent study on patients with inflammatory bowel disease showed no clear association between the presence of
Blastocystis sp. or the use of corticosteroids [
69].
In this study, type 2 diabetes was significantly more frequent in cases and this metabolic disease was also a significant factor in the binary logistic regression. Most studies have also reported patients with diabetes mellitus to be at higher risk of infection, which has been related to a weakened immune system [
70]. The presence of diabetes mellitus increased the risk of
Blastocystis sp. infection more than 9 times in patients with irritable bowel syndrome in Egypt [
71], although previous studies in Spain found no correlation among these factors [
23]. In contrast, our results do not support
Blastocystis infection to be a risk factor for irritable bowel syndrome, which was even more common among controls than in cases. The implication of
Blastocystis sp. in the development of IBS is controversial, although some studies have indicated a positive association and suggested that accurate diagnosis of this protist should be included in the clinical protocol of IBS patients [
72].
The odds ratio for halitosis, pathogenic bacteria and relative eosinophilia shows very high values. When both the odds of achieving an outcome, if exposed (presence of Blastocystis, cases) and if not exposed (absence of Blastocystis, controls), are very low, but odds in controls are even lower than odds in cases, high values for OR are found. However, 95% confidence intervals are very wide, pointing to a low confidence in the obtained OR values. Therefore, these high OR values found for halitosis, pathogenic bacteria and relative eosinophilia suggest than these factors are associated with Blastocystosis, but one must take into account that these OR values may be overestimated, due to the low frequencies of these outcomes in the whole sample of cases and controls.
Relative eosinophilia has been associated with
Blastocystis sp. infection in this study. In parasitic diseases, blood eosinophilia is usually associated with helminth infections, especially coinciding with the larval migration through tissues, and travelers/immigrants from resource-limited countries, who are most likely to acquire these infections, have been reported to have a high likelihood of eosinophilia. However, it is usually considered uncommon for an eosinophilia to be produced by protozoan infections [
73]. Few studies have reported a high proportion of eosinophils in the peripheral blood of symptomatic patients infected with
Blastocystis sp. [
74], but our results indicate that patients with relative eosinophilia are more likely to be infected, suggesting that eosinophilia should be taken into consideration in the diagnosis of
Blastocystis sp. infection.
Human gut microbiota composition is considered a deciding agent in the pathogenicity and occurrence of
Blastocystis sp., and the alteration of the intestinal environment provoked by pathogens has been suggested to be involved in its pathogenicity [
15]. In healthy children, Kodio et al. (2019) [
75] concluded that
Blastocystis sp. colonization is associated with a higher diversity of the bacterial communities in the gut but is not associated with the presence of potentially pathogenic bacteria in the human gut. As higher bacterial diversity is commonly associated with health and lower incidence of inflammatory diseases, it has been suggested that
Blastocystis colonization is associated with a healthy gut microbiome [
76]. In this study, the cluster analysis showed that this protist was by far the most frequently detected intestinal parasite, and most patients infected had no presence of other helminths or protozoa included in the principal component analysis; however, co-infections with pathogenic bacteria or enteric parasites were much more common among cases than controls, which suggest that they are associated with
Blastocystis sp. presence. These findings contrast with the observations of Hidalgo et al., (2019) [
23] who suggested that the presence of other parasites significantly decreased the risk of positive detection of
Blastocystis sp., although the authors indicated that there could be evidence of bias in the study because all the participants were patients submitted for parasitological diagnosis.
Whether
Blastocystis sp. is a pathogen or a commensal of the human gut is still uncertain. Studies on healthy, randomly sampled individuals have shown a high presence of
Blastocystis sp. and a prolonged colonization of the gut, but others have implicated it in intestinal diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and even cutaneous disorders. The most common intestinal symptoms attributed to
Blastocystis infection are diarrhea and abdominal pain, as well as nonspecific symptoms, such as nausea, vomiting, fatigue and flatulence [
19,
22,
64,
67]. The findings of the current study support a potentially pathogenic role for this protist, since most symptoms reported by infected patients were significantly more common in cases than in controls, including nausea, aerophagia, urticaria, anal itching and dyspepsia; moreover, the likelihood of having a
Blastocystis infection increased with abdominal pain, anorexia and halitosis. In contrast, a higher proportion of patients in the control group presented episodes of diarrhea and fever, with the latter being even significantly more common in controls than in cases. However, our National Health Service only analyzes samples from patients attending consultations because of the presence of symptoms. For this reason, no controls without disease manifestations were available. Therefore, no conclusions on the pathogenic potential of
Blastocystis could be directly reached from the analysis of our data set.
Diagnosis based only by the microscopic examination of stool specimens, lacking information relating to subtypes, is also a limitation of this study. Remarkable genetic diversity has been revealed among
Blastocystis sp. from humans and animals, and differences in clinical significance have been suggested for different subtypes, an aspect which was not investigated in this study; however, at present, there is no widely accepted distinction between the pathogenic and non-pathogenic subtypes [
8,
16].