**Molecular Characterisation of** *Cryptosporidium* **spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease**

**Augusto Messa Jr. 1, Pamela C. Köster 2, Marcelino Garrine 1,3, Tacilta Nhampossa 1,4, Sérgio Massora 1, Anélsio Cossa 1, Quique Bassat 1,5,6,7,8, Karen Kotloff 9, Myron M. Levine 9, Pedro L. Alonso 1,5,10, David Carmena 2,\* and Inácio Mandomando 1,4,\***


**Abstract:** *Cryptosporidium* is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. *Cryptosporidium*-positive faecal samples (*n* = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the *gp60* or *ssu* rRNA loci for species determination and genotyping. Three *Cryptosporidium* species including *C. hominis* (72.6%, 138/190), *C. parvum* (22.6%, 43/190), and *C. meleagridis* (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to *Cryptosporidium* spp. infections than older children. Both *C. hominis* and *C. parvum* were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, *p* = 0.007 and 23.7% vs. 11.8%, *p* = 0.014, respectively). A high intra-species genetic variability was observed within *C. hominis* (subtype families Ia, Ib, Id, Ie, and If) and *C. parvum* (subtype families IIb, IIc, IIe, and IIi) but not within *C. meleagridis* (subtype family IIIb). No association between *Cryptosporidium* species/genotypes and child's age was demonstrated. The predominance of *C. hominis* and *C. parvum* IIc suggests that most of the *Cryptosporidium* infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.

**Keywords:** *Cryptosporidium*; *gp60*; *ssu* rRNA; genotyping; children; diarrhoea; prevalence; molecular epidemiology; Mozambique; GEMS

**Citation:** Messa, A., Jr.; Köster, P.C.; Garrine, M.; Nhampossa, T.; Massora, S.; Cossa, A.; Bassat, Q.; Kotloff, K.; Levine, M.M.; Alonso, P.L.; et al. Molecular Characterisation of *Cryptosporidium* spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease. *Pathogens* **2021**, *10*, 452. https://doi.org/10.3390/ pathogens10040452

Academic Editor: Luiz Shozo Ozaki

Received: 22 March 2021 Accepted: 7 April 2021 Published: 9 April 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/).

#### **1. Introduction**

Diarrhoeal diseases remain the second leading cause of mortality after pneumonia in children under 5 years worldwide, accounting for approximately 9% of the 5.8 million deaths associated to this condition reported in 2015 [1,2]. Most of these fatalities disproportionately occur in poor-resource settings where suboptimal hygiene conditions and sanitation prevail [3]. A recent update on diarrhoeal burden from the Global Enteric Multicenter Study (GEMS and GEMS1A) demonstrated that Rotavirus, *Cryptosporidium*, enterotoxigenic *Escherichia coli* producing heat stable toxin (ST\_ETEC), and *Shigella* were the main pathogens associated with moderate-to-severe diarrhoea (MSD) and less severe diarrhoea (LSD) in African and Asian children [4,5]. Cryptosporidiosis in children under 5 years presents with watery diarrhoea, abdominal pain, nausea, and vomiting, being often associated to growth faltering and cognitive development impairment [6,7]. In severe cases, the disease can lead to life-threatening sequelae among malnourished and immunocompromised children [8]. *Cryptosporidium* spp. is also a major contributor to the burden of diarrhoeal disease in HIV-positive patients [9].

As other diarrhoea-causing pathogens, *Cryptosporidium* spp. are transmitted through the faecal–oral route. Humans acquire the infection through direct contact with infected hosts (person-to-person or zoonotic transmission) or by ingestion of contaminated food or water (foodborne and waterborne transmission), but the relative importance of these routes is still unclear [10]. At least 40 known *Cryptosporidium* species are currently recognised, and among these, more than 20 species and genotypes have been reported to cause human infections. *Cryptosporidium hominis* and *C. parvum* cause more than 90% of the human cases documented globally [11–13].

Molecular tools for the differentiation of *Cryptosporidium* species and genotypes are currently available, mostly using PCR followed by either restriction length fragment polymorphisms (RFLP) analysis or Sanger sequencing of the small subunit ribosomal ribonucleic acid (*ssu* rRNA) and the 60 kDa glycoprotein (*gp60*) genes of the parasite [10,13]. The *ssu* rRNA gene is largely used for the differential diagnosis of *Cryptosporidium* species due to its multicopy nature and associated high sensitivity. Subtype identification is primarily achieved through DNA sequence analysis of the highly polymorphic *gp60* gene. Subtype assignment is based on the number of TCA, TCG, and TCT repeats in addition to other repetitive sequences, such as the ACATCA, within the *gp60* tandem repeat motif region. Subtype families are named as Ia, Ib, Ic, Id, Ie, If, etc. for *C. hominis* and IIa, IIb, IIc, IId, etc. for *C. parvum*, with further species families named in ascending order [12,14].

In Africa, the epidemiology and genetic diversity of *Cryptosporidium* spp. remains relatively unknown. However, as noted by a recent literature review, at least 13 species and genotypes have been identified in humans, with *C. hominis* followed by *C. parvum* once again dominating the epidemiological landscape [11]. Subtyping studies support the dominance of anthroponotic over zoonotic transmission in African countries, regardless of the close contact with farm and domestic animals. Another interesting observation in Africa is the high level of subtype diversity, where at least six subtype families for *C. hominis* (Ia, Ib, Id, Ie, If, and Ih) have been described. For *C. parvum* the predominant subtypes identified in humans belong to the IIc family, in addition to IIa, IIb, IId, IIg, IIi, IIh, IIm and the rarer anthroponotically transmitted IIe subtype family [11].

In Mozambique, diarrhoea is ranked as the third cause of death in children under 14 years from the capital city Maputo [15] and fourth in children under 5 years from the Manhiça district (Maputo province) [16], being responsible for 20% of hospital paediatric admissions in this district [17]. The GEMS data support that prevention strategies targeting Rotavirus, *Cryptosporidium*, ST\_ETEC and *Shigella* could contribute to reduce diarrhoeal cases by approximately 50% in infants, and hence diarrhoeal-associated mortality [18]. However, the genetic diversity of *Cryptosporidium* spp. in GEMS was not investigated. Two previous hospital-based studies carried out in the southern part of the country have identified IaA23R3, IIcA5G3, and IIeA12G1 subtypes among nine isolates from patients with diarrhoea in the capital city Maputo [19], and IbA10G2 and IdA22 subtypes among eight

isolates in patients with HIV and tuberculosis in the Chokwe district of Gaza province [20]. However, no extensive molecular epidemiological studies have been conducted to evaluate the genetic diversity within *Cryptosporidium* spp. Herein, we aimed to analyse the diversity and frequency of *Cryptosporidium* species and subtypes detected in stools from children younger than 5 years from the Manhiça district, Mozambique, enrolled in the context of GEMS between 2007 and 2012.
