**1. Introduction**

Parasitic infections have been frequently reported as significant causes of gastrointestinal disorders and major contributors to the global burden of diarrheal disease globally [1–5].

**Citation:** Zhang, S.-X.; Carmena, D.; Ballesteros, C.; Yang, C.-L.; Chen, J.-X.; Chu, Y.-H.; Yu, Y.-F.; Wu, X.-P.; Tian, L.-G.; Serrano, E. Symptomatic and Asymptomatic Protist Infections in Hospital Inpatients in Southwestern China. *Pathogens* **2021**, *10*, 684. https://doi.org/10.3390/ pathogens10060684

Academic Editors: Lawrence S. Young and Siddhartha Das

Received: 28 March 2021 Accepted: 19 May 2021 Published: 31 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Several diarrhea-causing enteric parasite species have been described in humans. Among them, the most relevant are the protozoa *Cryptosporidium* spp., *Entamoeba histolytica*, and *Giardia duodenalis*, and the stramenopile *Blastocystis* sp. [5,6]. However, the impact that these agents exert on public health has not been fully characterized yet as a large amount of the epidemiological information currently available is mainly based on single protist infections, and the role of coinfections is often understated. It is estimated that around 20% of child diarrheal episodes are reported in low and middle-income countries, and 9% of those documented in high-income settings are caused by *Cryptosporidium* spp. [1]. Amebiasis, the acute disease caused by *E. histolytica*, affects near 50 million people and causes 100,000 deaths each year [7]. Both *Cryptosporidium* spp. and *E. histolytica* have been recognized as significant causes of morbidity and mortality associated with diarrhea by the 2017 Global Burden Disease Study [4]. On the other hand, *G. duodenalis* infection is estimated to result in 280 million cases annually worldwide [8] and was included, together with *Cryptosporidium* spp., in the "Neglected Disease Initiative" launched by the World Health Organization in 2004 [9]. *Blastocystis* sp. is regarded as the most prevalent enteric protist isolated from diarrheal patients in high-income countries [1]. However, its pathogenicity remains controversial partly because it is also the most frequent non-fungal eukaryotic organism detected in fecal samples from apparently healthy individuals [10–12]. It has been argued that this protist could be used as an indicator of potential exposure to other pathogenic enteric protozoa [13]. Besides *Blastocystis* sp., *Cryptosporidium* spp., *E. histolytica*, and *G. duodenalis* infections are common in apparently healthy individuals [1,14,15]. There is a lack of reliable data on the true burden of asymptomatic infections due to the absence of monitoring programs, underreporting and the fact that carriage of subclinical stages is often underdiagnosed [1,16,17].

Even though there have been notable advances in this field, the factors that determine the course of enteric protozoan infections and the development (or not) of gastrointestinal symptoms remain poorly understood [18]. Protist species/genotypes have been suggested as predictors of pathogenicity/virulence. It is expected that the growing development of new molecular diagnostic tools contributes to clarifying the distinction between pathogenic and nonpathogenic lineages, as well as pathophysiological interactions and other epidemiological features of interest [19].

Many previous studies have explored and reported the presence of more than one pathogen in cases with diarrheal disease [6,19,20] as well as in healthy individuals [21,22]. Comparatively, less effort has been devoted to exploring the impact of concomitant enteric infections involving viral, bacterial, and/or parasitic agents and characterizing the enteric communities and the existence of specific interactions among them [15,23,24]. These interactions, at least between 2 specific enteric pathogens, are well documented in the veterinary field, such as the association of enterotoxigenic *Escherichia coli* and rotavirus that lead to severe diarrhea in piglets as an example [25]. However, it has not been addressed in immunocompetent humans until recently [15,26].

To improve our current understanding of the existence and impact of intestinal protozoan coinfections in humans, we conducted a hospital-based cross-sectional study. The aims of the survey were: (i) to investigate the occurrence of the four protist species most commonly associated with gastrointestinal disorders and to determine their molecular profile, (ii) to assess whether these protists co-occur by chance or are community-structured in hospital-based patients, and (iii) to explore the potential impact of coinfections on the clinical symptomatology among immunocompetent patients attending the People's Hospital of Tengchong City and the Chinese Medicine Hospital in Tengchong City.

#### **2. Results**

#### *2.1. Characteristics of the Study Population*

A total of 507 subjects participated in the study. The male:female ratio was 1.1 (260/247). Han nationality was predominant (94.9%, 481/507), and the median age of recruited participants was 52 years (interquartile range (IQR): 38–63 years). Most of the subjects lived in rural areas (78.1%, 396/507). Around two-thirds of them had completed primary education (65.6%, 332/507), and 63.1% of the participants were farmers (320/507).

The most common clinical symptom was decreased appetite (17.9%, 91/507), followed by abdominal pain (16.6%, 84/507) and nausea (16.6%, 84/507); in addition, 10.4% (59/507) of recruited patients presenting with acute diarrhea. Other less frequently reported symptoms included abdominal distension (12.4%, 63/507), itchy skin 12.2%, 62/507), constipation (10.8%, 55/507) and perianal pruritus (5.5%, 28/507). Overall, 50.0% (233/507) of the patients did not have any gastrointestinal symptoms.

#### *2.2. Single Enteric Pathogen Infections and Coinfections*

*Blastocystis* sp. was the most prevalent protist species found (9.5%, 48/507; 95% CI: 7.1–12.4), followed by *G. duodenalis* (2.2%, 11/507; 95% CI: 1.1–3.8) and *E. histolytica* (2.0%, 10/507; 95% CI: 0.9–3.6), whereas *Cryptosporidium* spp. was not detected at all. *Entamoeba histolytica* was more frequently found in diarrheal (11.9%, 7/59) than in non-diarrheal (0.7%, 3/448) cases (OR = 19.9, 95% CI: 5.0–79.5). The same was true for *G. duodenalis* (6.8%, 4/59 versus 1.6%, 7/448; OR = 4.6, 95% CI: 1.3–16.2). In contrast, no significant differences were observed on the distribution of *Blastocystis* sp. between diarrheal and non-diarrheal cases (13.6%, 8/59 versus 8.9%, 40/448; OR = 1.6, 95% CI: 0.7–3.6).

Overall, 12.4% of patients (63/507, 95% CI: 9.7–15.6) were infected by at least one enteric protist. Infections caused by two different enteric protist species were detected in 1.2% of patients (6/507, 95% CI: 0.4–2.6). The most common coinfection found was *E. histolytica* and *Blastocystis* sp. (1.0%, 5/507; 95% CI: 0.3–2.2), and the coinfection of *E. histolytica* and *Blastocystis* sp. in diarrheal cases was more common than in non-diarrheal patients (5.1%, 3/59; 0.4%, 2/448, chi-squared = 10.8, *p*-value = 0.001). Coinfection by *G. duodenalis* and *Blastocystis* sp. was identified in a single case (0.2%, 1/59; 95% CI: 0.01–1.1), and no significant difference of the coinfection by *G. duodenalis* and *Blastocystis* sp. was found in subjects with and without diarrhea (0.2%, 1/59; 0.0%, 0/449, chi-squared = 2.81, *p*-value = 0.116). No other coinfection of these four enteric protozoa was found in diarrhea individuals and healthy controls.
