Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid, Spain

Abstract

BackgroundThe flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country.MethodsIn this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013–January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed.Principal findingsOf 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study.ConclusionsHuman giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.

Molecular diversity and frequency of the diarrheagenic enteric protozoan Giardia duodenalis and Cryptosporidium spp. in a hospital setting in Northern Spain

“…infections reported here very likely represent autochthonous cases, as most of the patients investigated were nationals with no recent history of travelling abroad. The same conclusion was reached in similar molecular epidemiological surveys carried out in the Madrid area [19,23]. As expected, acute diarrhoea, abdominal pain and nausea/vomit were the clinical manifestations more frequently identified in the symptomatic population under study.…”

“…duodenalis -positive isolates previously identified by qPCR. The performances of the GDH - and BG -PCRs carried out here were comparatively lower than those previously achieved by our research group using identical DNA extraction, purification, and amplification protocols in isolates from clinical (61% vs 47%) and general (34% vs 25%) mostly paediatric populations in Spain [19] and Ethiopia [50]. Although the amplification efficiency of genomic nucleic acids isolated from faecal material are known to be influenced by poor quality or low starting concentrations of DNA, the copy number of the targeted gene, and the primer set design [50,51], at present we cannot provide a clear explanation for this phenomenon.…”

“…duodenalis sub-assemblage more prevalent in the surveyed human population, being identified in 44% of the genotyped isolates. In contrast, molecular data from other Spanish regions found BIV as the most frequent sub-assemblage in the Madrid area [19] and Zaragossa and Leon [22]. Assemblage B is also responsible of two thirds of the human cases of giardiasis documented in Europe [8].…”

Giardia: a pathogen or commensal for children in high-prevalence settings?

“…Within assemblage B there is a consistently greater degree of genetic diversity than assemblage A [51, 52]. Additionally, there is ongoing population expansion of Giardia haplotype diversity across all continents [53].…”

Evaluation of Three Protocols of DNA Extraction for Detection of Giardia duodenalis in Human Fecal Specimens

“…Annually, about 200 million people are affected with symptomatic giardiasis in Asia, Africa, and Latin America (6,7). Giardia infections cause an economic burden in public health and veterinary (3,(8)(9)(10). Nowadays this protozoan parasite is considered as one of the most common causes of diarrhea in humans.…”