Ana C. González

Isolation and characterization of Acanthamoeba strains from soil samples in Gran Canaria, Canary Islands, Spain.

Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1–T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains in soil samples collected between 2012 and 2013 in Gran Canaria Island, Canary Islands, Spain. Samples were inoculated onto non-nutrient agar (NNA) plates and were checked for the presence of Acanthamoeba. Identification of Acanthamoeba strains was based on the morphology of the cyst and trophozoite forms. Subsequently, positive samples were cloned for their molecular characterization at the genotype level by sequencing the DF3 region located in the 18S rDNA gene of Acanthamoeba as previously described. Sequencing results revealed the presence of T2, T5 and T4 genotypes within the studied samples. To the best of our knowledge, this is the first report demonstrating the presence of Acanthamoeba in Gran Canaria Island and the first study at the genotype level in the Canary Islands.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Genotyping of potentially pathogenic Acanthamoeba strains isolated from nasal swabs of healthy individuals in Peru.

Free Living Amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can be found in the air, soil and water; and have also been isolated from air-conditioning units. In humans, they are causative agents of a sight-threating infection of the cornea, Acanthamoeba keratitis (AK) and a fatal infection of the central nervous system known as Granulomatous Amoebic Encephalitis (GAE). In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in nasal swabs from individuals in two regions of Peru. Identification of isolates was based on cyst morphology and PCR-sequencing of the Diagnostic Fragment 3 to identify strains at the genotype level. The pathogenic potential of the isolates was also assayed using temperature and osmotolerance assays and extracellular proteases zymograms. The obtained results revealed that all isolated strains exhibited pathogenic potential. After sequencing the highly variable DF3 (Diagnostic Fragment 3) region in the 18S rRNA gene as previously described, genotype T4 was found to be the most common one in the samples included in this study but also genotype T15 was identified. To the best of our knowledge, this is the first study on the characterization of Acanthamoeba strains at the genotype level and the first report of genotype T4 and T15 in Peru.

Analysis of NLS and rRNA binding motifs in the L25 ribosomal protein from Leishmania (viannia) braziliensis: investigation of its diagnostic capabilities.

A cDNA clone codifying ribosomal protein L25 was isolated from a Leishmania braziliensis cDNA gene library. The alignment of the amino acid sequence deduced from this gene with other proteins revealed that this protein is related to the L23/25 rihosomal protein family. This is so because this protein shows, in its C-terminal end, the rRNA binding domains characteristic of these proteins and at the N-terminal end the NLS sequence necessary for its entry into the nucleus. Southern blot analysis showed 2 copies of gene L25 per genome arranged in tandem position and pointing in the same direction. Northern blot analysis showed that this gene is transcribed in 2 mRNAs when parasite promastigotes are in the logarithmic phase. In order to analyse the antigenic properties of L. braziliensis RPL25, it was purified as a recombinant protein and ELISA-tested against cutaneous, mucocutaneous and Chagasic sera. The results indicate that the recombinant RPL25 from L. braziliensis presents a non-specific reaction that disqualifies it for the diagnosis of cutaneous leishmaniasis. In contrast, some of the synthetic peptides derived from its sequence may serve as promising tools for the diagnosis of this disease.

First larval record of Mesocestoides in carnivora of Tenerife (Canary Islands).

Larvae of Mesocestoides sp. were recovered in Tenerife (Canary Islands) in 2004 from the peritoneal cavities of 2 domestic dogs and a domestic cat. Morphological and molecular identification were carried out. Mesocestoides litteratus from Vulpes vulpes was sequenced for the first time using the ITS-2 region (18S rDNA), and was included in the phylogenetic analysis to compare the sequence variability among these and other Mesocestoides spp. belonging to different carnivores. Phylogenetic studies were carried out based on maximum parsimony and neighbor-joining analysis. The results showed the relationships between these and other previously published Mesocestoides species. Moreover, it is demonstrated that Mesocestoides sp. from Tenerife comprises a previously unreported sequence. This is the first larval record of Mesocestoides sp. in domestic animals from Tenerife, Canary Islands, Spain.

RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis

The technique of Random Amplification Polymorphic DNA allows fragments of the genome to be amplified by means of polymerase chain reaction (PCR) without previous knowledge of their sequences. The protozoa of the genus Leishmania present great genetic variability, making it difficult to characterize the different species. A method is developed with a single 10-mers long primer, which allows the species L. braziliensis, L. mexicana, L. infantum, L. tropica, L. chagasi, L. amazonensis and L. major to be differentiated. These products amplified by RAPD have also facilitated the design of some primers that amplify L. braziliensis DNA exclusively.

Cloning and Molecular Characterization of the cDNA Encoding Histone H1 From Leishmania braziliensis

The isolation and molecular characterization of the histone H1-encoding gene from Leishmania braziliensis was carried out. The gene is present in the genome as a single copy and transcribed as a polyadenylated transcript of 830 nucleotides. The deduced amino acid sequence has in its central region the DNA binding K-[K/R]-A-A-[A/P] motif, which is repeated in tandem 9 times.

Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru.

In March 2010, a 35 year-old HIV/AIDS female patient was admitted to hospital to start treatment with Highly Active Antiretroviral Therapy (HAART) since during a routine control a dramatic decrease in the CD4(+) levels was detected. At this stage, a nasal swab from each nostril was collected from the patient to include it in the samples for the case study mentioned above. Moreover, it is important to mention that the patient was diagnosed in 2009 with invasive pneumococcal disease, acute cholecystitis, pancreatitis and pulmonary tuberculosis. The collected nasal swabs from both nostrils were positive for Vermamoeba vermiformis species which was identified using morphological and PCR/DNA sequencing approaches. Basic Local Alignment Search Tool (BLAST) homology and phylogenetic analysis confirmed the amoebic strain to belong to V.vermiformis species. Molecular identification of the Mycobacterium strain was carried out using a bacterial universal primer pair for the 16S rDNA gene at the genus level and the rpoB gene was amplified and sequenced as previously described to identify the Mycobacterium species (Shin et al., 2008; Sheen et al., 2013). Homology and phylogenetic analyses of the rpoB gene confirmed the species as Mycobacterium chelonae. In parallel, collected swabs were tested by PCR and were positive for the presence of V.vermiformis and M.chelonae. This work describes the identification of an emerging bacterial pathogen,M.chelonae from a Free-Living Amoebae (FLA) strain belonging to the species V.vermiformis that colonized the nasal cavities of an HIV/AIDS patient, previously diagnosed with TB. Awareness within clinicians and public health professionals should be raised, as pathogenic agents such as M.chelonae may be using FLA to propagate and survive in the environment.

MC1R gene variants and sporadic malignant melanoma susceptibility in the Canary Islands population

Several MC1R variants are associated with increased risk of malignant melanoma (MM) in a variety of populations. We aim to examine the influence of the MC1R variants (RHC: D84E, R151C, R160W; NRHC: V60L, R163Q and the synonymous polymorphism T314T) on the MM risk in a population from the Canary Islands. Overall, 1,046 Caucasian individuals were included in the study. A thousand of them were genotyped for MC1R variants: 509 were sporadic MM patients and 491 were healthy control subjects from general population. The analysis was adjusted for age, sex, hair colour, eye colour, skin phototype and ancestry. We found that carriers of the R151C and R163Q variants were at an increased risk for melanoma OR 2.76 (1.59–4.78) and OR 5.62 (2.54–12.42), respectively. The risk of carrying RHC variants was 3.04 (1.90–4.86). Current study confirms the increased MM risk for R151C carriers. It also supports the association between R163Q variant and MM risk in the population on the Canary Islands, as opposed to reported on northern populations. These results highlight the importance of the sample population selection in this kind of studies.

Acanthamoeba Belonging to T3, T4, and T11: Genotypes Isolated from Air-Conditioning Units in Santiago, Chile

Free‐living amoebae (FLA) of the genus Acanthamoeba are widely distributed in the environment, in the air, soil, and water, and have also been isolated from air‐conditioning units. The objective of this work was to investigate the presence of this genus of FLA in the air‐conditioning equipment at the Institute of Public Health of Chile in Santiago, Chile. Water and air samples were collected from air‐conditioning systems and were checked for the presence of Acanthamoeba spp. Positive samples were further classified at the genotype level after sequencing the highly variable diagnostic fragment 3 (DF3) region of the 18S rRNA gene. This is the first report of the T3, T4, and T11 genotypes of Acanthamoeba in air‐conditioning units from Chile. Overall, the widespread distribution of potentially pathogenic Acanthamoeba strains in the studied source demands more awareness within the public and health professionals in Chile as this pathogen is emerging as a risk for human health worldwide.