**1. Introduction**

Diarrhoea has long been recognized as a major cause of child morbidity and mortality globally [1]. The main diarrhoea-causing agents include viral (adenovirus, rotavirus), bacterial (*Campylobacter*, enterotoxigenic *Escherichia coli*, *Shigella*) and parasitic (*Giardia duodenalis*, *Cryptosporidium* spp.) pathogens. Recent data by the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) revealed that diarrhoea was the fifth leading cause of death among children younger than 5 years (446,000 deaths) only in 2016 [2]. In this group of age, diarrhoea causes more deaths than acquired immunodeficiency syndrome (AIDS), malaria, and measles combined [3]. Additionally, near 90% of diarrhoea-associated deaths are attributable to unsafe water, inadequate sanitation, and insufficient hygiene [4]. There is, therefore, a clear link between diarrhoea and poverty, particularly in deprived areas of low-income countries [5].

Dashti, A.; Bailo, B.; Hernández-de-Mingo, M.; Balasegaram, S.; Carmena, D. Molecular Diversity of *Giardia duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. in Symptomatic and Asymptomatic Schoolchildren in Zambézia Province (Mozambique). *Pathogens* **2021**, *10*, 255. https:// doi.org/10.3390/pathogens10030255

**Citation:** Muadica, A.S.; Köster, P.C.;

Academic Editor: Siddhartha Das

Received: 4 February 2021 Accepted: 22 February 2021 Published: 24 February 2021

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**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/).

The protozoa *G. duodenalis* and *Cryptosporidium* spp. and, to a lesser extent, the Stramenopile *Blastocystis* sp. are among the most important diarrheal pathogens of parasitic nature affecting humans [6]. Indeed, there are more than 200 million cases of symptomatic giardiosis worldwide each year only in developing countries [7]. The Global Enteric Multicenter Study (GEMS) has demonstrated that *Cryptosporidium* is second only to rotavirus in causing morbidity and mortality in young children living in South East Asian and sub-Saharan African countries [8]. *Blastocystis* sp. is the most prevalent intestinal microbial eukaryote colonising/infecting the human gut. Although its clinical significance remains controversial, *Blastocystis* carriage has been linked with intestinal (diarrhoea, irritable bowel syndrome) and extra-intestinal (urticarial) disorders [9]. In addition, childhood cryptosporidiosis and giardiosis frequently lead to stunted growth and cognitive impairment, particularly in poor-resource areas [10,11].

As with other enteric protists, *G. duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. are transmitted via the faecal-oral route through diverse pathways including person-toperson or animal-to-person direct contact and consumption of contaminated water or food. Indeed, waterborne and foodborne illnesses constitute a major area of concern [12,13]. Molecular epidemiological studies involving human, animal, and environmental samples are essential to determine the genetic diversity of these pathogens in a given host or geographical area, and to ascertain the exact contribution of the above-mentioned transmission routes to human infections.

*Giardia duodenalis* (the only *Giardia* species able to infect humans) is currently regarded as a multi-species complex comprising eight (A to H) distinct assemblages, of which zoonotic assemblages A and B (particularly the latter) are the most prevalent in humans [14]. The genus *Cryptosporidium* comprises at least 40 valid species and a similar number of genotypes of unclear taxonomic status, of which more than 20 have been reported to cause human infections. Only two species, *Cryptosporidium hominis* and *C. parvum*, account for up to 90% of the human cases of cryptosporidiosis documented globally [15]. Similarly, 22 distinct subtypes (ST) have been identified within *Blastocystis* sp. to date (ST1-17, ST21, ST23-26), of which ST1–9 and ST12 have been reported in humans [16].

The epidemiology of giardiosis, cryptosporidiosis, and blastocystosis in African countries is poorly understood [17–20]. Prevalence estimations have been frequently based on low-sensitive (e.g., microscopy) methods, were restricted to certain populations and geographical areas or are in need of update. When compared, polymerase chain reaction (PCR) methods outperformed microscopy in terms of sensitivity and range of parasite species detected [21]. Although recent large-scale epidemiological surveys such as GEMS or the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health (MAL-ED) Study have considerably improved our knowledge of certain species (particularly *Cryptosporidium* spp.), they did not consider others (e.g., *Blastocystis*) and did not conduct genotyping analyses on positive samples [8,22]. In Mozambique, GEMS-derived samples from young children positive for *G. duodenalis* and *Cryptosporidium* spp. in the Manhiça District (Maputo province) have been recently genotyped and sub-genotyped [23,24]. Earlier studies have molecularly characterised much smaller numbers of *Giardia*- and *Cryptosporidium*-positive isolates in diarrhoeal individuals and patients with human immunodeficiency virus (HIV) and/or tuberculosis in Maputo and Gaza provinces, respectively [25,26]. However, the current epidemiological situation of human blastocystosis in the country is completely unknown. This study aims to investigate the frequency and molecular diversity of *G. duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. in asymptomatic and symptomatic children in the Zambézia province in the central coastal region of Mozambique, an area where no studies on the presence of intestinal protist parasites have been previously conducted in human or animal populations.
