Draft genomes of Blastocystis subtypes from human samples of Colombia.

BACKGROUND: Blastocystis is one of the most common eukaryotic microorganisms colonizing the intestines of both humans and animals, but the conditions under which it may be a pathogen are unclear. METHODS: To study the genomic characteristics of circulating subtypes (ST) in Colombia, we established nine xenic cultures from Blastocystis isolated from human fecal samples, we identified 10 different subtypes, since one sample had a mixed infection. Thus, the genomes of the subtypes ST1 (n = 3), ST2 (n = 1), ST3 (n = 2), ST6 (n = 1), ST7 (n = 1), and ST8 (n = 2) were sequenced using Illumina and Oxford Nanopore Technologies (ONT). RESULTS: Analyses of these draft nuclear genomes indicated remarkable diversity in terms of genome size and guanine-cytosine (GC) content among the compared STs. Illumina sequencing-only draft genomes contained 824 to 2077 scaffolds, with total genome size ranging from 12 to 13.2 Mb and N50 values ranging from 10,585 to 29,404 base pairs (bp). The genome of one ST1 isolate was sequenced using ONT. This assembly was more contiguous, with a size of 20 million base pairs (Mb) spread over 116 scaffolds, and an N50 of 248,997 bp. CONCLUSION: This work represents one of the few large-scale comparative genomic analyses of Blastocystis isolates, providing an additional glimpse into its genomic diversity.

Identification of Multiple Blastocystis Subtypes in Domestic Animals From Colombia Using Amplicon-Based Next Generation Sequencing.

Blastocystis is frequently reported in fecal samples from animals and humans worldwide, and a variety of subtypes (STs) have been observed in wild and domestic animals. In Colombia, few studies have focused on the transmission dynamics and epidemiological importance of Blastocystis in animals. In this study, we characterized the frequency and subtypes of Blastocystis in fecal samples of domestic animals including pigs, minipigs, cows, dogs, horses, goats, sheep, and llama from three departments of Colombia. Of the 118 fecal samples included in this study 81.4% (n = 96) were positive for Blastocystis using a PCR that amplifies a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. PCR positive samples were sequenced by next generation amplicon sequencing (NGS) to determine subtypes. Eleven subtypes were detected, ten previously reported, ST5 (50.7%), ST10 (47.8%), ST25 (34.3%), ST26 (29.8%), ST21 (22.4%), ST23 (22.4%), ST1 (17.9%), ST14 (16.4%), ST24 (14.9%), ST3 (7.5%), and a novel subtype, named ST32 (3.0%). Mixed infection and/or intra -subtype variations were identified in most of the samples. Novel ST32 was observed in two samples from a goat and a cow. To support novel subtype designation, a MinION based sequencing strategy was used to generate the full-length of the SSU rRNA gene. Comparison of full-length nucleotide sequences with those from current valid subtypes supported the designation of ST32. This is the first study in Colombia using NGS to molecularly characterize subtypes of Blastocystis in farm animals. A great diversity of subtypes was observed in domestic animals including subtypes previously identified in humans. Additionally, subtype overlap between the different hosts examined in this study were observed. These findings highlight the presence of Blastocystis subtypes with zoonotic potential in farm animals indicating that farm animals could play a role in transmission to humans.

Epidemiological and Molecular Characterization of Blastocystis Infection in Children Attending Daycare Centers in Medellín, Colombia.

BACKGROUND: The present study aims to perform an epidemiological and molecular characterization of Blastocystis infection in a child population attending daycare centers of Medellín, Colombia. METHODS: A total of 265 children aged 0-5 years were enrolled in five children's centers in urban sectors of Medellín, northwestern Colombia. Stool samples were taken to identify intestinal parasites by direct examination, Ritchie-Frick concentration, and molecular identification of Blastocystis by conventional PCR and subtype (ST) identification by PCR barcoding with subsequent phylogenetic reconstruction. Kappa index was calculated to evaluate the agreement between microscopy and PCR for the diagnosis of Blastocystis. RESULTS: The prevalence of intestinal protozoa was 36.6% (97/265), with Blastocystis as the most frequent parasitic protozoan at 15.8% (42/265), followed by Giardia intestinalis at 15.5% (41/265) and Endolimax nana at 15.1% (40/265). The prevalence of Blastocystis by PCR was 53.2% (141/265), the subtypes identified were ST3 at 30.5% (18/59), ST2 at 23.7% (14/59), ST1 at 20.3% (12/59), and with less frequency, ST4 at 5.1% (3/59), ST6 at 1.7% (1/59) and ST16 at 15.3% (9/59) allele 162. CONCLUSION: This study provides the first genetic characterization of Blastocystis subtypes circulating in a population of Medellín, Colombia, and also updates the epidemiology of Blastocystis subtypes in the world with the first identification of ST16 in humans.

Succinate dehydrogenase gene as a marker for studying Blastocystis genetic diversity.

Blastocystis has been reported as the most common eukaryotic microorganism residing in the intestines of both humans and animals, with a prevalence of up to 100% in some populations. Since this is a cryptic species, sequence polymorphism are the single strategy to analyses its genetic diversity, being traditionally used the analysis of ssu rRNA gene sequence to determine alleles and subtypes (STs) for this species. This multicopy gene has shown high diversity among different STs, making necessary to explore other genes to assess intraspecific diversity. This study evaluated the use of a novel genetic marker, succinate dehydrogenase (SDHA), for the typing and evaluation of the genetic diversity and genetic population structure of Blastocystis. In total, 375 human fecal samples were collected and subjected to PCR, subtyped using the ssu rRNA marker, and then the SDHA gene was amplified via PCR for 117 samples. We found some incongruences between tree topologies for both molecular markers. However, the clustering by ST previously established for Blastocystis was congruent in the concatenated sequence. SDHA showed lower reticulation (The origination of a lineage through the partial merging of two ancestor lineages) signals and better intra ST clustering ability. Clusters with geographical associations were observed intra ST. The genetic diversity was lower in the marker evaluated compared to that of the ssu rRNA gene (nucleotide diversity = 0.03344 and 0.16986, respectively) and the sequences analyzed showed population expansion with genetic differentiation principally among STs. The ssu rRNA gene was useful to explore interspecific diversity but together with the SDHA gene the resolution power to evaluate intra ST diversity was higher. These results showed the potential of the SDHA marker for studying the intra ST genetic diversity of Blastocystis related with geographical location and the inter ST diversity using the concatenated sequences.

Development of a Multilocus Sequence Typing Scheme for Giardia intestinalis.

Giardia intestinalis is an intestinal protozoan most commonly found in humans. It has been grouped into 8 assemblages (A-H). Markers such as the glutamate dehydrogenase gene, triose phosphate isomerase and beta-giardin (ß-giardin) have been widely used for genotyping. In addition, different genetic targets have been proposed as a valuable alternative to assess diversity and genetics of this microorganism. Thus, our objective was to evaluate new markers for the study of the diversity and intra-taxa genetic structure of G. intestinalis in silico and in DNA obtained from stool samples. We analysed nine constitutive genes in 80 complete genome sequences and in a group of 24 stool samples from Colombia. Allelic diversity was evaluated by locus and for the concatenated sequence of nine loci that could discriminate up to 53 alleles. Phylogenetic reconstructions allowed us to identify AI, AII and B assemblages. We found evidence of intra- and inter-assemblage recombination events. Population structure analysis showed genetic differentiation among the assemblages analysed.

Molecular detection and genotyping of intestinal protozoa from different biogeographical regions of Colombia.

BACKGROUND: Intestinal parasitic protozoa represent a serious problem of public health particularly in developing countries. Protozoa such as Blastocystis, Giardia intestinalis, Entamoeba histolytica and Cryptosporidium spp. are associated with diarrheal symptoms. In Colombia, there is little region-specific data on the frequency and circulating genotypes/species of these microorganisms. Therefore, the main objective of our study was to employ molecular detection and genotyping of G. intestinalis and Blastocystis, Cryptosporidium and Entamoeba spp. in samples from different biogeographical regions of Colombia. METHODS: We collected 649 human fecal samples from five biogeographical regions of Colombia: the Amazon, Andean, Caribbean, Orinoco and Pacific regions. Blastocystis, G. intestinalis, Cryptosporidium spp. and Entamoeba complex were detected by microscopy and conventional PCR. Molecular genotyping was conducted to identify Blastocystis subtypes (STs) (18s), G. intestinalis assemblages (triose phosphate isomerase and glutamate dehydrogenase) and Cryptosporidium species (18s). Genetic diversity indices were determined using dnasp.5. RESULTS: We detected G. intestinalis in 45.4% (n = 280) of samples, Blastocystis in 54.5% (n = 336) of samples, Cryptosporidium spp. in 7.3% (n = 45) of samples, Entamoeba dispar in 1.5% (n = 9) of samples, and Entamoeba moshkovskii in 0.32% (n = 2) of samples. Blastocystis STs 1-4, 8 and 9 and G. intestinalis assemblages AII, BIII, BIV, D and G were identified. The following Cryptosporidium species were identified: C. hominis, C. parvum, C. bovis, C. andersoni, C. muris, C. ubiquitum and C. felis. The Caribbean region had the highest frequency for each of the microorganisms evaluated (91.9% for G. duodenalis, 97.3% for Blastocystis, 10.8% for Cryptosporidium spp., 13.5% for E. dispar and 2.7% for E. moshkovskii). The Orinoco region had a high frequency of Blastocystis (97.2%) and the Andean region had a high frequency of G. intestinalis (69.4%). High and active transmission was apparent in several regions of the country, implying that mechanisms for prevention and control of intestinal parasitosis in different parts of the country must be improved.

Trypanosoma cruzi infection, discrete typing units and feeding sources among Psammolestes arthuri (Reduviidae: Triatominae) collected in eastern Colombia.

BACKGROUND: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi, and is transmitted by hematophagous insects of the family Reduviidae. Psammolestes arthuri is a sylvatic triatomine distributed in Colombia and Venezuela which feeds on birds and there are a few studies that have reported Ps. arthuri naturally infected with T. cruzi. In Colombia, Ps. arthuri has been found in dwellings, making it important to evaluate its possible role in the T. cruzi transmission cycle. We aimed to evaluate the presence of T. cruzi and feeding sources of Ps. arthuri to elucidate new possible scenarios of T. cruzi transmission in the country. METHODS: A total of 60 Ps. arthuri were collected in Arauca and Casanare, Colombia. We detected and genotyped T. cruzi and identified feeding sources. The frequency of the presence of T. cruzi was obtained and compared with different eco-epidemiological variables. Multiple correspondence analysis was conducted to explore associations between eco-epidemiological variables and the presence of T. cruzi; with these results, a logistic regression was used to determine statistical associations. RESULTS: The infection rate of T. cruzi was 70.7% and was mostly associated with insect stage, sex, bird nest and feeding source. Regarding discrete typing units (DTUs), TcI was found in 54.7% samples, of which 21.7% (5/23) were TcIDom, 52.1% (12/23) had mixed infection (TcIDom-TcISylv), and single infection with TcISylv was not detected. Mixed infections (TcI/TcII-TcVI) were found in 9.52% (4/42) of the samples; of these, 14.2% (6/42) were TcII-TcVI. A total of 15 feeding sources were identified and the most frequent were: Cranioleuca baroni (35.85%), Homo sapiens (26.42%), Thraupis episcopus (11.32%) and Serinus albogularis (3.77%). CONCLUSIONS: Although Ps. arthuri is mainly ornithophilic, this species may be feeding on other animals that can be infected with T. cruzi, possibly playing a role maintaining the zoonotic cycle of the parasite. Further studies with molecular techniques and wider sampling are needed to improve information regarding infection rates, ecotopes and habits with the aim of evaluating whether Ps. arthuri could be a potential T. cruzi vector.

Molecular and descriptive epidemiology of intestinal protozoan parasites of children and their pets in Cauca, Colombia: a cross-sectional study.

BACKGROUND: Parasitic infections, particularly those caused by protozoa, represent a considerable public health problem in developing countries. Blastocystis, Giardia duodenalis, Cryptosporidium spp. and the Entamoeba complex (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii) are the most common etiological causes of intestinal parasitic infections. METHODS: We carried out a descriptive cross-sectional study in school-age children attending a daycare institution in commune eight of Popayán, Cauca (Southwest Colombia). A total of 266 fecal samples were collected (258 from children and eight from pets). Blastocystis, G. duodenalis, Cryptosporidium spp. and the Entamoeba complex were identified by microscopy, quantitative real-time PCR (qPCR) and conventional PCR. The concordance of qPCR and microscopy was assessed using the Kappa index. Molecular characterization was conducted to identify Blastocystis subtypes (18S), G. duodenalis assemblages (tpi and gdh) and Cryptosporidium species/subtypes (18S and GP60). Potential associations between intestinal parasitism and sociodemographic factors were examined using bivariate analyses. RESULTS: A total of 258 fecal samples from children were analyzed by microscopy and 255 samples were analyzed by qPCR. The prevalence of Blastocystis was between 25.19% (microscopy) and 39.22% (qPCR), that of G. duodenalis was between 8.14% (microscopy) and 10.59% (qPCR), that of Cryptosporidium spp. was estimated at 9.8% (qPCR), and that of the Entamoeba complex was between 0.39% (conventional PCR) and 0.78% (microscopy). The concordance between microscopy and qPCR was very low. Blastocystis ST1 (alleles 4, 8, and 80), ST2 (alleles 11, 12, and 15), ST3 (alleles 31, 34, 36, 38,57, and 151), and ST4 (alleles 42 and 91), G. duodenalis assemblages AII, BIII, BIV and D, C. parvum subtype IIa and C. hominis subtype IbA9G3R2 were identified. The only identified member of the Entamoeba complex corresponded to E. histolytica. No statistically significant association was identified between parasitic infection and any sociodemographic variable. CONCLUSION: This study revealed the usefulness of molecular methods to depict the transmission dynamics of parasitic protozoa in southwest Colombia. The presence of some of these protozoa in domestic animals may be involved in their transmission.

Molecular epidemiology of dengue, yellow fever, Zika and Chikungunya arboviruses: An update.

Arboviruses are a group of viruses transmitted by arthropods. They are characterized by a wide geographic distribution, which is associated with the presence of the vector, and cause asymptomatic infections or febrile diseases in humans in both enzootic and urban cycles. Recent reports of human infections caused by viruses such as dengue, Zika, and chikungunya have raised concern regarding public health, and have led to the re-evaluation of surveillance mechanisms and measures to control the transmission of these arboviruses. Viruses such as Mayaro and Usutu are not currently responsible for a high number of symptomatic infections in humans, but should remain under epidemiological surveillance to avoid the emergence of new epidemics, as happened with Zika virus, that are associated with new or more severe symptoms. Additionally, significant variation has been observed in these viruses, giving rise to different lineages. Until recently, the emergence of new lineages has primarily been related to geographical distribution and dispersion, allowing us to ascertain the possible origins and direction of expansion of each virus type, and to make predictions regarding regions where active infections in humans are likely to occur. Therefore, this review is focused on untangling the molecular epidemiology of Dengue, Yellow fever, Zika and Chikungunya due to their recent epidemics in Latinamerica but provides an update on the geographical distribution globally of these viral variants, and outlines the need for further understanding of the genotypes/lineages assignment.

Description of Leishmania species among dogs and humans in Colombian Visceral Leishmaniasis outbreaks.

We report the species detected in dogs and humans from outbreaks of visceral leishmaniasis in Colombia. In this study, 91 sera from patients (n = 38) and dogs (n = 53) diagnosed with visceral leishmaniasis using IFAT were analyzed to determine the causative species. DNA extraction, PCR amplification, DNA sequencing and species identification was performed. Results were obtained with 13 of the sera. A phylogenetic tree and a network of haplotypes were constructed. Leishmania infantum chagasi (11/13), Leishmania braziliensis (1/13) and Leishmania amazonensis (1/13) were identified as the circulating species and genetic variability in one of the L. infantum chagasi strains was demonstrated. This is the first study describing Leishmania species in outbreaks of visceral leishmaniasis in Colombia.

Ecology of Trypanosoma cruzi I genotypes across Rhodnius prolixus captured in Attalea butyracea palms.

Trypanosoma cruzi, the agent of Chagas disease exhibits significant genetic diversity. This parasite is divided into six discrete typing units (DTUs) where T. cruzi I (TcI) is the most widespread in the Americas. TcI genotypes have been associated to domestic and sylvatic cycles of transmission (TcIDom and sylvatic TcI). Due to the importance of the enzootic transmission, we determined the frequency of TcI genotypes present in Rhodnius prolixus captured in different regions of the palm A. butyracea to understand the ecology of the disease and the importance of A. butyracea palms as ecotopes of R. prolixus. Forty A. butyracea palms were sampled (base crown, mid-point and crown) capturing 105 individuals identified as R. prolixus by morphological and molecular barcoding. We conducted molecular detection and typing of T. cruzi across 59 individuals. The results showed that all the insects were infected with TcI; 28.57% were sylvatic TcI, 12.38% TcIDom and 15,24% mixed infections (TcIDom/sylvatic TcI). Statistical analysis showed a similar behavior between TcIDom and mixed infections in the mid-point and at the crown of the palm, being more frequent in the crown, while sylvatic TcI does not seem to have a specific association with any of the sampled areas. These findings are consistent with other studies showing high mobility of the insect vector between different ecotopes, increasing the need to develop improvements in the programs of disease control.

Cerebral embolization in asymptomatic versus symptomatic patients after carotid stenting.

BACKGROUND: Previous studies have investigated the development of new ischemic brain lesions on diffusion-weighted magnetic resonance imaging (DW-MRI) after carotid artery stenting (CAS). The rate of ischemic brain injury after CAS for asymptomatic stenosis has not been extensively studied but is presumed to be less likely than in symptomatic patients. This study assessed the occurrence of cerebral embolization after CAS for asymptomatic vs symptomatic carotid stenosis. METHODS: During an 18-month period, 40 patients undergoing CAS under filter embolic protection were prospectively evaluated. Transcranial Doppler (TCD) during CAS and preprocedural and 24-hour postprocedural DW-MRI were used to assess cerebral embolization. Univariate and nonparametric analyses were used to compare differences in cerebral embolization after CAS in asymptomatic and symptomatic patients. RESULTS: CAS was performed for 23 asymptomatic (58%) and 17 symptomatic (42%) carotid stenoses. The median microembolic counts detected by TCD were 285 (interquartile range [IQR], 182-376) for asymptomatic and 313 (IQR, 170-426) for symptomatic carotid stenosis (P=.6). DW-MRI was available for assessment in 20 asymptomatic and 14 symptomatic patients. New acute cerebral emboli detected with DW-MRI occurred in 10 asymptomatic (50%) and 7 symptomatic patients (50%) undergoing CAS (P=.9). The ipsilateral and total median number of DW-MRI lesions between groups were not statistically significantly different at, respectively, 1 (IQR, 0-2.5) and 1.5 (IQR, 0-3) for asymptomatic vs 0.5 (IQR, 0-2) and 0.5 (IQR, 0-3) for symptomatic carotid stenosis (P>.5). One asymptomatic patient sustained a minor stroke after CAS. No new neurologic events occurred in symptomatic patients. The 30-day stroke-death rate was 2.5% in this series. CONCLUSIONS: Cerebral embolization, as detected by TCD and DW-MRI, occurs with a similar frequency after CAS for asymptomatic and symptomatic carotid stenosis. Because postprocedural ischemic brain injury occurs in approximately half of asymptomatic patients, the safety of CAS under filter embolic protection for asymptomatic carotid stenosis is uncertain and warrants further study.

Randomized clinical trial of open-cell vs closed-cell stents for carotid stenting and effects of stent design on cerebral embolization.

OBJECTIVE: The effect of stent design on cerebral embolization has not been established. The purpose of this trial was to contrast the incidence of subclinical cerebral embolization in high-risk patients undergoing carotid artery stenting (CAS) with open-cell vs closed-cell stents. METHODS: During an 18-month period, 40 patients were randomized (1:1) to undergo CAS with open-cell (Acculink, n = 20) or closed-cell stents (Xact, n = 20). A single filter device for embolic protection (Accunet filter) was used. Transcranial Doppler (TCD)-detected microembolic signals (MES) during CAS and preprocedural and 24-hour postprocedural diffusion-weighted magnetic resonance imaging (DW-MRI) were used to determine cerebral embolization. Univariate and nonparametric analyses were used to assess associations between stent design and cerebral embolization. RESULTS: CAS was performed in 17 symptomatic patients (43%) and 23 asymptomatic patients (57%) with a similar number of open-cell and closed-cell stents (9/8 and 11/12, respectively). The total and poststenting median ipsilateral MES counts detected by TCD were 264 (interquartile range [IQR], 222-343) and 48 (IQR, 41-66) for open-cell stents and 339 (IQR, 163-408) and 53 (IQR, 23-88) for closed-cell stents, respectively (P > .56). New acute cerebral emboli detected with DW-MRI occurred in 53% and 47% of patients undergoing CAS with open-cell and closed-cell stents, respectively (P = 1.0). The total and ipsilateral median numbers of DW-MRI lesions between groups were not statistically significantly different (ie, 2 [IQR, 0-4] and 1 [IQR, 0-3] for open-cell stents and 1 [IQR, 0-3] and 1 [IQR, 0-2] for closed cell-stents, respectively; P > .4). One asymptomatic patient undergoing CAS with an open-cell stent sustained a minor stroke; the 30-day stroke-death rate in this series was 2.5%. CONCLUSION: Cerebral embolization, as detected by TCD and DW-MRI, occurs with similar frequency after CAS with open-cell and closed-cell stents. This randomized trial does not support the superiority of any stent design with respect to cerebral embolization.

Diseño, construcción y validación de un instrumento para evaluar el riesgo psicolaboral en empresas colombianas / Design, construction and validation of an instrument to evaluate psicolaboral risk in colombian companies

La investigación tuvo como propósito el diseño, la construcción y la validación de un Instrumento de Medición de Riesgo Psicosocial (IMP), conformado por 52 ítems que evalúan el riesgo psicosocial a través de siete dimensiones: carga de trabajo, definición del rol, identificación con la tarea, nivel de responsabilidad del cargo, características de la gestión, características del grupo social de trabajo y características de la organización propiamente dicha. Para tal fin, se establecieron como fases: a) el abordaje teórico y conceptual del riesgo psicosocial; b) diseño del instrumento; c) evaluación de jueces; d) aplicación del instrumento; y e) análisis y discusión de resultados. Este instrumento se aplicó a una población de 442 personas de siete empresas bogotanas de los sectores privado y público, con el cual se obtuvo un coeficiente de confiabilidad mediante Alpha de Cronbach de 0,843.

The purpose of the study was to design, construct and validate a Psychosocial Risk Measure (IMP) Instrument. The IMP is conformed by 52 items which evaluate the psychosocial risk through seven dimensions: job loads, role definition, task identification, job level responsibility, management characteristics, social group of work characteristics and organizational characteristics. Five phases was carried out: a) theoretical and conceptual boarding of the psychosocial risk; b) test design; c) judges evaluation; d) instrument implementation; and e) results analysis and discussion. IMP was applied to 442 workers, from seven companies, some private and some public, in Bogotá. Reliability coefficient of Cronbach's Alpha was 0.843.