Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba.

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.

Molecular analysis of Giardia duodenalis isolates from symptomatic and asymptomatic children from La Habana, Cuba.

Giardiasis is considered the most common intestinal parasitic disease in humans worldwide. In Cuba, this infection has particularly a strong clinical impact on the child population. Giardia duodenalis is a highly diverse protozoan, which comprises a complex of eight morphologically identical genetic assemblages, further divided into sub-assemblages. The present study used triose phosphate isomerase (tpi) and small-subunit ribosomal RNA (SSU rRNA) genes as genetic markers for the identification of G. duodenalis assemblages and sub-assemblages in correlation with clinical and epidemiological data in children attended at the Paediatric Hospital "William Soler" and at Pedro Kouri Institute, between 2015 and 2016. A prevalence of 8% of G. duodenalis infection was recorded in stool samples after concentration techniques from 68 children out of 847 analysed. A 100% detection of Giardia DNA was achieved by a SSU-rRNA PCR, whereas DNA from 63 of 68 (92.6%) was successfully amplified by tpi-PCR. By this assemblage-specific tpi-PCR 32 (50.8%) assemblage B, 17 (27.0%) assemblage A and 14 (22.2%) mixed infection (A + B) were identified. Assemblage B was significantly (P < 0.02) more frequently found in children with diarrhoea. Sequence analysis of the tpi gene of Giardia isolates from symptomatic children showed that assemblage A belonged to the sub-assemblage AII, and 4 sub assemblages BIV and 1 sub assemblage BIII were also recorded. Only 2 discordant genotyping results were observed by phylogenetic comparison of SSU-rRNA and tpi sequences. Further studies with novel molecular tools for a better discrimination at the sub-assemblage level are needed to identify the dynamics of spread of giardiasis and to verify possible correlations between Giardia genetic diversity and clinical manifestation.

Correlation of Giardia duodenalis assemblages with clinical and epidemiological data in Cuban children.

Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. In Cuba, this parasite has most clinical impact on children. The aim of this investigation was genetic characterization of G. duodenalis isolated from children with giardiasis diagnosed at the Paediatric Hospital "William Soler" between 2010 and 2011, and to compare the genetic results with clinical and epidemiological data. A total of 103 stool samples from 452 children were positive for G. duodenalis and co-infections with other parasites were noted in 5 cases. Assemblage identification was carried out by the amplification of a fragment of the triosephosphate isomerase (tpi) gene. Sub-assemblages of assemblage A (AI and AII) were identified by a nested PCR using the intergenic spacer (IGS) region of ribosomal deoxyribonucleic acid gene as a target. DNA from 90 of 103 (87.4%) samples was successfully amplified by PCR-tpi. The prevalence of assemblages A and B was 40% and 42%, respectively. Infections with both assemblages were reported in 16 cases. No associations between epidemiological information and assemblage was detected, but assemblage B was significantly (P<0.01) more frequently found in children with diarrhea, flatulence or abdominal pain than assemblage A. Sub-assemblage AII accounted for the majority of cases (86.5%).