*2.5. Co-Occurrence of Enteric Protozoa*

The null model analysis showed that the observed C-score (265) was lower than expected by chance (267.45), indicating the existence of a random, noncompetitively structured protist community. No evidence of a statistically significant protist combination could be demonstrated in the surveyed population (SES = −0.33, *p*-value = 0.414).

#### **3. Discussion**

Our study confirmed the single prevalence rates of enteric protist species, including *G. duodenalis*, *Blastocystis* sp., and *E. histolytica* commonly reported in previous studies in similar settings characterized by adequate access to water sanitation and personal hygiene practices (WASH) [1,17,27]. In the present PCR-based report, *Blastocystis* sp. was the predominant intestinal protist species identified, followed by *G. duodenalis* and *E. histolytica*, whereas *Cryptosporidium* spp. was not detected at all. These data were also consistent with those documented in previous studies conducted in human populations in China [28].

Regarding the genetic diversity of the protist species investigated, out of the 11 *G. duodenalis* isolates successfully genotyped at the *tpi* locus, 10 (90.9%) were classified as assemblage A and the remaining one (9.1%) as assemblage B. All assemblage A sequences were identified as sub-assemblage AI. This finding is interesting as sub-assemblage AI is typically reported at much lower frequencies than sub-assemblage AII in European [29–31] and Asian [32] countries. This discrepancy may be due to differences in infection sources or transmission pathways (e.g., a variable proportion of infections of zoonotic nature). No mixed coinfections involving assemblages A and B were found. Similarly, no infections caused by animal-specific assemblages C–D (dogs), E (domestic and wild ungulates) and F (cats) were detected, suggesting that these host species play a limited role as a source of human giardiasis in this geographical area.

It has been suggested that the presence and proportion of *G. duodenalis* assemblages may present spatiotemporal variations [33,34]. Additionally, socioeconomic factors have also been suggested as potential drivers for *G. duodenalis* assemblage distribution [35]. Interestingly, some molecular epidemiological investigations have reported that assemblage's segregation may be involved in infection outcome and clinical presentation, with assemblage B resulting in more frequently symptomatic infection in endemic settings [21,36]. Other surveys have indicated that sub-assemblage AII may represent a more virulent genetic variant of *G. duodenalis* in humans [17,37,38]. The data mentioned above may explain, at least partially, the lack of evidence in support of a potential association between the presence of *G. duodenalis* and the occurrence of clinical symptomatology in the present study. It should be noted, however, that a recent case–control study assessing the frequency and genetic diversity of *G. duodenalis* infections in children younger than five years of age with and without diarrhea in southern Mozambique has demonstrated that the occurrence of gastrointestinal illness was not associated with a given genotype of the parasite [39].

Five distinct *Blastocystis* STs were identified in the hospital inpatient population under study, including ST1–ST4 and ST7. This is well in agreement with the available molecular data from China, where ST1–ST7 and ST12 have been described at variable frequency rates in different human populations [40]. Interestingly, clinical studies on patients presenting with diarrhea have shown that ST1 was related to clinical manifestations, including diarrhea and may have, therefore, potential pathogenicity. In contrast, ST3 was the *Blastocystis* genetic variant more frequently identified in asymptomatic infections [41–43]. This potential link between a given *Blastocystis* ST and the presence/absence of clinical manifestations could not be demonstrated in the present survey. Of note, a relatively high proportion of individuals carried *Blastocystis* ST7, a subtype mostly identified in birds and rarely found in mammals, including humans [44]. This finding clearly indicates that contact with poultry or captive avian species (or with their fecal material) was the most likely source of infection of these inpatients.

Remarkably, *Cryptosporidium* spp. was not detected in any of the recruited patients in this survey. In China, an overall *Cryptosporidium* infection rate of 3% has been estimated for 1987-2018 [45]. In hospital settings, documented infection rates vary greatly depending on the clinical population under study. Cryptosporidiosis cases have been reported with low frequencies in children hospitalized primarily for non-gastrointestinal illnesses (1.6%, 102/6284) in Shanghai [46] and in diarrheic children (10/500) in Wuhan, Hubei province [47]. In contrast, comparatively much higher (6-40%) infection rates have been identified in gastrointestinal (including esophageal, small intestine, colorectal, and liver) cancer patients [48]. These discrepancies may be associated with differences in the age and immunological status of the patients, the diagnostic methods used or even the geographical area considered.

As we have shown, it seems that, at the enteric protist level, there is not any specific community assemblage in the study population considering the four protist species considered. Our work showed that the occurrence of those pathogens was purely random and asymptomatic in the study population. Furthermore, not specific interactions were detected between them, and there was no evidence suggesting that their presence, jointly or separately, was associated with developing clinical symptomatology classically associated with infections by these species. Although all of them have been reported as causative agents of gastrointestinal disorders, especially in children in LMIC [20], asymptomatic carriage has also been commonly reported elsewhere. In the case of *Blastocystis* sp., its pathogenic relevance is still controversial [10,11] since this species is one of the most common enteric protists detected in humans [12,49]. Therefore, it is not surprising that their presence was not associated with clinical symptomatology in immunocompetent individuals.

Given the results obtained, different factors might provide an explanation that helps to understand the pathogenesis of enteric protozoan infections and what are the conditions that lead to disease. Some of them are related to the characteristics of the pathogen, such as different pathogenicity due to virulence variability of strains or the need of, at least, a second infection with another pathogen to cause clinical symptomatology. Moreover, host factors can play a key role, too, such as individual susceptibility or the presence of healthy functional barriers that protect the human intestine: the mucus layer, the intestinal epithelial layer and the intestinal microbiota [5,15,18,26]. However, further research needs to be performed to establish the ecology of enteric communities in healthy and unhealthy individuals.

We are aware that our research may have some limitations. First, this was a hospitalbased cross-sectional study, so the surveyed population may not represent the general population in Tengchong City, and the generalizability to other populations may be rather limited. Although the occurrence rates of protist enteroparasites were consistent with those published in previous studies, low prevalences might have negatively influenced the reliability of the estimates in the PLS analyses. Second, the molecular methods (direct and nested PCRs) used here did not allow the quantification of parasites' DNA, so the direct association between parasites' burdens and occurrence of clinical manifestations could not be determined. In addition, acute bacterial or viral infections of the gastrointestinal tract were not found in this study. Additionally, molecular data on the diversity and frequency of species/genotypes were based on single-locus sequence analysis. Adopting multi-locus genotyping schemes (particularly for *G. duodenalis*) would likely improve the genetic data provided here. This is a task to be conducted soon.

Therefore, further research with a larger sample size, which considers a broader enteropathogen community, is needed to explore community assemblages present and their relationships with pathogenicity and clinical manifestations in rural and urban regions in China.

Undoubtedly, with the increasing awareness of the microbiota role in developing host immunity [5] and their potential relationship with many communicable and noncommunicable diseases, such as irritable bowel syndrome [50], there is a growing need to understand the complex relationships between different pathogens and as well as between the intestinal microbiota and pathogens.
