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.

Prevalence of intestinal parasites and molecular characterization of Giardia duodenalis from dogs in La Habana, Cuba.

Giardia duodenalis is a worldwide protozoan parasite that infects humans and other mammals including dogs. Due to the risk of zoonotic transmission between dogs and humans, we aimed in this study to determine the prevalence of the intestinal parasites and the distribution of assemblages of G. duodenalis among dogs analysed. A descriptive cross-sectional study was carried out in La Habana from June 2014 to March 2015 in the Zoonosis Unit of La Lisa municipality. A total of 98 dogs were analysed by three different techniques (microscopy with faecal concentration, Enzyme-Linked ImmunoSorbent Assay, and Polymerase Chain Reaction) in order to detect Giardia in stool samples. Out of 98 dogs studied, 43 (43.9%) were infected with intestinal parasites. The zoonotic parasites Ancylostoma caninum (21.4%), Trichuris vulpis (16.3%) and the protozoan Giardia duodenalis (11.2%) were the most prevalent parasites. In regards to the G. duodenalis, seven dogs were positive by microscopy after faecal concentration, nine by NOVITEC® Giardia Microplate Assay, and ten and eleven samples were amplified by the ß-giardin and SSU-RNA PCRs, respectively. After PCR sequence analysis of both genes only zoonotic assemblages (A and B) were detected. The SSU-RNA sequence results revealed a distribution of 8 assemblage A and 4 assemblage B, whereas only assemblage A were identified by the ß-giardin analysis. Among subassemblage classification by ß-giardin phylogenetic tree, four isolates showed an AI pattern and one isolate displayed an AII distribution. Mixed infections were detected in three isolates. These findings highlight the risk of zoonotic transmission of Giardia duodenalis between dogs and humans.

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.

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B.

Molecular Characterization and Risk Factors of Giardia duodenalis among School Children from La Habana, Cuba.

Giardia duodenalis is considered the most common protozoan infecting humans worldwide. Molecular characterization of G. duodenalis isolates has revealed the existence of eight groups (assemblages A to H) which differ in their host distribution. A cross-sectional study was conducted in 639 children from La Habana between January and December 2013. Two assemblage-specific PCRs were carried out for the molecular characterization. The overall prevalence of Giardia infection was 11.9%. DNA from 63 of 76 (82.9%) samples was successfully amplified by PCR-tpi, while 58 from 76 (76.3%) were detected by PCRE1-HF. Similar results by both PCRs were obtained in 54 from 76 samples (71%). According to these analyses, assemblage B and mixed assemblages A + B account for most of the Giardia infections in the cohort of children tested. Our current study identified assemblage B as predominant genotype in children infected with Giardia. Univariate analysis indicated that omission of washing hands before eating and keeping dogs at home were significant risk factors for a Giardia infection. In the future, novel molecular tools for a better discrimination of assemblages at the subassemblages level are needed to verify possible correlations between Giardia genotypes and symptomatology of giardiasis.

14-3-3 gene characterization and description of a second 14-3-3 isoform in both Echinococcus granulosus and E. multilocularis.

Members of the 14-3-3 protein family have been identified as regulatory molecules in intracellular signaling pathways and cell cycle control. Previously, the first Echinococcus 14-3-3 isoform (E14-3-3.1) was isolated from E. granulosus and E. multilocularis metacestode stages. Hyperexpression of this isoform was claimed to be associated with non-restricted tumor-like growth of the E. multilocularis metacestode. In this report, we describe the characterization of a 14-3-3 cDNA from E. granulosus and E. multilocularis corresponding to a second isoform of this family, E14-3-3.2. The characterized 14-3-3 gene was interrupted by two introns whose sequence and positions were conserved in both Echinococcus species. The deduced amino acid sequence of E14-3-3.2 showed 88% identity to the E14-3-3.1 isoform and 52% identity to a third Echinococcus isoform (E14-3-3.3) described by other authors. These findings, coupled to Southern blot analysis, suggest the presence of more than one 14-3-3 gene in Echinococcus. Phylogenenetically, the Echinococcus 14-3-3.1 and 14-3-3.2 isoforms appeared to cluster with zeta-type ("pro-tumorigenic") 14-3-3 isoforms from closely related organisms, whereas the E14-3-3.3 isoform grouped with 14-3-3 epsilon isoforms. The presence of more than one 14-3-3 isoform might indicate isoform-specific roles in the different parasite stages of Echinococcus.