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Abstract
Giardia duodenalis is an enteric parasite commonly detected in children. Exposure to this organism may lead to asymptomatic or symptomatic infection. Additionally, early-life infections by this protozoan have been associated with impaired growth and cognitive function in poor resource settings. The Global Enteric Multicenter Study (GEMS) in Mozambique demonstrated that G. duodenalis was more frequent among controls than in diarrhoeal cases (≥3 loosing stools in the previous 24 hours). However, no molecular investigation was conducted to ascertain the molecular variability of the parasite. Therefore, we describe here the frequency and genetic diversity of G. duodenalis infections in children younger than five years of age with and without diarrhoea from the Manhiça district in southern Mozambique enrolled in the context of GEMS. Genomic DNA from 757 G. duodenalis-positive stool samples by immunoassay collected between 2007-2012, were reanalysed by multiplex PCR targeting the E1-HP and C1-P21 genes for the differentiation of assemblages A and B. Overall, 47% (353) of the samples were successfully amplified in at least one locus. Assemblage B accounted for 90% (319/353) of all positives, followed by assemblage A (8%, 29/353) and mixed A+B infections (1%, 5/353). No association between the presence of a given assemblage and the occurrence of diarrhoea could be demonstrated. A total of 351 samples were further analysed by a multi-locus sequence genotyping (MLSG) approach at the glutamate dehydrogenase (gdh), ß-giardin (bg) and triose phosphate isomerase (tpi) genes. Overall, 63% (222/351) of samples were genotyped and/or sub-genotyped in at least one of the three markers. Sequence analysis revealed the presence of assemblages A (10%; 23/222) and B (90%; 199/222) with high molecular diversity at the nucleotide level within the latter; no mixed infections were identified under the MLSG scheme. Assemblage A sequences were assigned to sub-assemblages AI (0.5%, 1/222), AII (7%, 15/222) or ambiguous AII/AIII (3%, 7/222). Within assemblage B, sequences were assigned to sub-assemblages BIII (13%, 28/222), BIV (14%, 31/222) and ambiguous BIII/BIV (59%, 132/222). BIII/BIV sequences accumulated the majority of the single nucleotide polymorphisms detected, particularly in the form of double peaks at chromatogram inspection. This study demonstrated that the occurrence of gastrointestinal illness (diarrhoea) was not associated to a given genotype of G. duodenalis in Mozambican children younger than five years of age. The assemblage B of the parasite was responsible for nine out of ten infections detected in this paediatric population. The extremely high genetic diversity observed within assemblage B isolates was compatible with an hyperendemic epidemiological scenario where infections and reinfections were common. The obtained molecular data may be indicative of high coinfection rates by different G. duodenalis assemblages/sub-assemblages and/or genetic recombination events, although the exact contribution of both mechanisms to the genetic diversity of the parasite remains unknown.
Highlights
Diarrheal diseases remain the second leading cause of mortality in children under 5 years of age worldwide, accounting for 9% of the approximately 5.8 million deaths in 2015 [1,2]
By using targeted-molecular genotyping methods based on PCR and sequencing, we demonstrated here that i) the occurrence of diarrhoea in Mozambican young children infected with G. duodenalis was not linked to a given assemblage of the parasite, ii) assemblage B was the most prevalent genetic variant of G. duodenalis circulating in the surveyed paediatric population, being responsible for 90% of the infections, and iii) G. duodenalis assemblage B was characterised by an extremely high genetic diversity impairing the correct allocation of isolates to sub-assemblages BIII or BIV, a fact that may be due to elevated coinfection rates or the occurrence of genetic recombination
The recent update on diarrheal burden from the Global Enteric Multicenter Study (GEMS) [4,5] demonstrated that Rotavirus, Cryptosporidium spp., enterotoxigenic Escherichia coli producing heat stable toxin (ST_ETEC), and Shigella were the leading pathogens associated with moderate-to-severe diarrhoea (MSD) in African and Asian children, in contrast to Giardia duodenalis that was inversely associated with MSD [4]
Summary
Diarrheal diseases remain the second leading cause of mortality in children under 5 years of age worldwide, accounting for 9% of the approximately 5.8 million deaths in 2015 [1,2]. Most of these deaths occurred in sub-Saharan Africa and South Asia, where poor hygiene conditions and insufficient sanitation facilities prevail [3]. Asymptomatic infections in African children have been commonly reported in several sub-Saharan countries [11,12,13,14,15]. Progression from infection to disease is a multifactorial process involving both host (age, immune and nutritional status, microbiota composition and abundance) and pathogen (burden, genotype, virulence, immunomodulatory potential) determinants whose interactions and exact contribution to the tilting of the health-disease balance are not completely understood [16,17]
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