title: Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid, Spain.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  In 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.
  
  
  RESULTS
  Of 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.
  
  
  CONCLUSIONS
  Human 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.
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Majadahonda, Madrid, Spain.
authors.forename: Aida
authors.initials: A
authors.lastname: de Lucio
authors.affiliation: Microbiology and Clinical Parasitology Service, University Hospital Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: Martínez-Ruiz
authors.affiliation: Microbiology Service, University Hospital Severo Ochoa, Leganés, Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Merino
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Majadahonda, Madrid, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Bailo
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Majadahonda, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Aguilera
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Majadahonda, Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fuentes
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Majadahonda, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Carmena
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D005260:Female; D005838:Genotype; D016829:Giardia lamblia / Q000235:genetics*; D005873:Giardiasis / Q000469:parasitology*; D006779:Hospitals, Public; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D013030:Spain; D055815:Young Adult