Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine.

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis.

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.

Knowledge about emergency dental trauma management among school teachers in Colombia: A baseline study to develop an education strategy.

BACKGROUND/AIM: Dental trauma in school populations has a high frequency and school teachers are often close to the place where such injuries occur. However, many studies have confirmed the lack of knowledge as to how to act in these cases. The aim of this study was to determine the knowledge in regard to dental trauma of school teachers in Colombia. MATERIALS AND METHODS: A multicentre, descriptive cross-sectional study was performed in 251 schools using a stratified randomized sampling. A structured hard copy survey was personally delivered to 2390 school teachers in the selected schools. The survey contained questions related to work experience, teaching level and demographic data, as well as questions related to their knowledge of and attitude towards emergency dental trauma management. RESULTS: The response rate was 96%. Most of the school teachers (95%) had never received training related to dental trauma, although 35% had witnessed at least 1 case. Of 2296 school teachers surveyed, only 5.8% would have replanted an avulsed tooth. An association was found between work experience and appropriate management of an avulsed tooth. No significant differences were found in regard to school location (city), school type (private/public), gender and school teachers' education level. CONCLUSIONS: The knowledge of school teachers in Colombia about emergency dental trauma management is inadequate. The findings strongly suggest a need for an education strategy with the involvement of the government, dentistry schools and private organizations.

Immunogencity of antigens from Mycobacterium tuberculosis self-assembled as particulate vaccines.

Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p<0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB.

In vivo polyester immobilized sortase for tagless protein purification.

BACKGROUND: Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcus aureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method. RESULTS: The generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacterium tuberculosis vaccine candidate Rv1626. CONCLUSIONS: We have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required.

Metals and nonsteroidal anti-inflammatory pharmaceuticals drugs present in water from Madín Reservoir (Mexico) induce oxidative stress in gill, blood, and muscle of common carp (Cyprinus carpio).

Many toxic xenobiotics that enter the aquatic environment exert their effects through redox cycling. Oxidative stress, which incorporates both oxidative damage and antioxidant defenses, is a common effect induced in organisms exposed to xenobiotics in their environment. The results of the present study aimed to determine the oxidative stress induced in the common carp Cyprinus carpio by contaminants [metals and nonsteroidal anti-inflammatory drugs (NSAIDs)] present in Madín Reservoir. Five sampling stations (SSs), considered to have the most problems due to discharges, were selected. Carp were exposed to water from each SS for 96 h, and the following biomarkers were evaluated in gill, blood, and muscle: hydroperoxide content, lipid peroxidation, protein carbonyl content, and the activity of antioxidant enzymes superoxide dismutase and catalase. Results show that contaminants (metals and NSAIDs) present in water from the different SSs induce oxidative stress. Thus, water in this reservoir is contaminated with xenobiotics that are hazardous to C. carpio, a species consumed by the local human population.

Tuning CAR T-cell therapies for efficacy and reduced toxicity.

Chimeric antigen receptor (CAR) T-cell therapies are a standard of care for certain relapsed or refractory B-cell cancers. However, many patients do not respond to CAR T-cell therapy or relapse later, short- and long-term toxicities are common, and current CAR T-cell therapies have limited efficacy for solid cancers. The gene engineering inherent in CAR T-cell manufacture offers an unprecedented opportunity to control cellular characteristics and design products that may overcome these limitations. This review summarises available methods to "tune" CAR T-cells for optimal efficacy and safety. The components of a typical CAR, and the modifications that can influence CAR T-cell function are discussed. Methods of engineering passive, inducible or autonomous control mechanisms into CAR T-cells, allowing selective limitation or enhancement of CAR T-cell activity are reviewed. The impact of manufacturing processes on CAR T-cell function are considered, including methods of limiting CAR T-cell terminal differentiation and exhaustion, and the use of specific T-cell subsets as the CAR T starting material. We discuss the use of multicistronic transgenes and multiplexed gene editing. Finally, we highlight the need for innovative clinical trial designs if we are to make the most of the opportunities offered by CAR T-cell therapies.

Improving Healthcare Quality and Clinical Outcomes for Persons with Dementia in the Sub-Acute Hospital Through Person-Centered Care Practice.

Background: Person-centered care is considered beneficial for persons with dementia. Objective: To evaluate the impact of a person-centered knowledge translation intervention on the quality of healthcare and outcomes for persons with dementia. Methods: Over nine months, sub-acute hospital nursing, allied health, and medical staff (n = 90) participated in online and/or face-to-face person-centered education and were supported by senior nursing, allied health, and medical staff champions (n = 8) to implement person-centered healthcare. The quality of healthcare service, ward climate and care delivery were evaluated pre/post study intervention. In the week following hospital admission (Time 1) and week of discharge (Time 3), agitation incidence (co-primary outcome) was assessed in participants with dementia (n = 80). Participant delirium (co-primary outcome), accidents/injuries, psychotropic medicines, length of stay, readmission and discharge destination (secondary outcomes) were compared with a retrospective group (n = 77) matched on demographics, cognition and function in activities of daily living. Results: Improvements occurred post-intervention in service quality by 17.5% (p = 0.369, phi = 0.08), ward climate by 18.1% (p = 0.291, phi = 0.08), and care quality by 50% (p = 0.000, phi = 0.37). Participant agitation did not change from Time 1 to Time 3 (p = 0.223). Relative to the retrospective group, significant reductions occurred in participant delirium (p = 0.000, phi = 0.73), incidents/injuries (p = 0.000, phi = 0.99), psychotropic medicine use (p = 0.030, phi = 0.09), and hospital readmissions within 30 days (p = 0.002, phi = 0.25), but not in discharge to home (p = 0.171). Conclusions: When person-centered healthcare knowledge is translated through staff education and practice support, persons with dementia can experience improved healthcare services and clinical outcomes, while healthcare services can benefit through reductions in unplanned service use.

The impact of elder abuse training on subacute health providers and older adults: study protocol for a randomized control trial.

BACKGROUND: Elder abuse often goes unreported and undetected. Older people may be ashamed, fearful, or otherwise reticent to disclose abuse, and many health providers are not confident in asking about it. In the No More Shame study, we will evaluate a co-designed, multi-component intervention that aims to improve health providers' recognition, response, and referral of elder abuse. METHODS: This is a single-blinded, pragmatic, cluster randomised controlled trial. Ten subacute hospital sites (i.e. clusters) across Australia will be allocated 1:1, stratified by state to a multi-component intervention comprising a training programme for health providers, implementation of a screening tool and use of site champions, or no additional training or support. Outcomes will be collected at baseline, 4 and 9 months. Our co-primary outcomes are change in health providers' knowledge of responding to elder abuse and older people's sense of safety and quality of life. We will include all inpatients at participating sites, aged 65 + (or aged 50 + if Aboriginal or Torres Strait Islander), who are able to provide informed consent and all unit staff who provide direct care to older people; a sample size of at least 92 health providers and 612 older people will provide sufficient power for primary analyses. DISCUSSION: This will be one of the first trials in the world to evaluate a multi-component elder abuse intervention. If successful, it will provide the most robust evidence base to date for health providers to draw on to create a safe environment for reporting, response, and referral. TRIAL REGISTRATION: ANZCTR, ACTRN12623000676617p . Registered 22 June 2023.

A Comparative Analysis on the Potential Anticancer Properties of Tetrahydrocannabinol, Cannabidiol, and Tetrahydrocannabivarin Compounds Through In Silico Approach.

OBJECTIVE: The purpose of this study is to comparatively analyze the anticancer properties of Tetrahydrocannabinol (THC), Cannabidiol (CBD), and Tetrahydrocannabivarin (THCV) using In silico tools. METHODS: Using SwissADME and pkCSM, the physicochemical and pharmacokinetics properties of the cannabinoids were evaluated. Protox-II was utilized for the assessment of their cytotoxicity. The chemical-biological interactions of the cannabinoids were also predicted using the Way2Drug Predictive Server which comprises Acute Rat Toxicity, Adver-Pred, CLC-Pred, and Pass Target Prediction. RESULTS: Both physicochemical and drug-likeness analysis using SwissADME favored THCV due to high water solubility and lower MLOGP value. On the other hand, ADMET assessment demonstrated that THC and CBD have good skin permeability while both THC and THCV exhibited better BBB permeability and have low inhibitory activity on the CYP1A2 enzyme. Furthermore, toxicity predictions by Protox-II revealed that CBD has the lowest probability of hepatotoxicity, carcinogenicity, and immunotoxicity. Contrarily, it has the highest probability of being inactive in mutagenicity and cytotoxicity. Additionally, CLC results revealed that CBD has the highest probability against lung carcinoma. The rat toxicity prediction showed that among the cannabinoids, THCV had the lowest LD50 concentration in rat oral and IV. CONCLUSION: Overall, in silico predictions of the three cannabinoid compounds revealed that they are good candidates for oral drug formulation. Among the three cannabinoids, THCV is an excellent anticancer aspirant for future chemotherapy with the most favorable results in drug-likeness and ADMET analysis, pharmacological properties evaluation, and cytotoxicity assessment results. Further study on bioevaluation of compounds is needed to elucidate their potential pharmacological activities.

Enamel fluorosis related with fluoride-containing water ingestion and urinary excretion in schoolchildren.

Background: Natural water sources are considered as the major environmental exposure of fluoride, resulting in increased prevalence of enamel fluorosis. This type of natural exposure should be permanently monitored to avoid the interactions with other non-natural fluoride sources. We evaluated the prevalence of enamel fluorosis in Colombian schoolchildren and its relationship with fluoride-containing water ingestion exposure dose and urinary fluoride excretion. Material and Methods: We included 923 schoolchildren aged 7-12 years residing in eight municipalities in Colombia. Sampling of consumption water was performed in major aquifers used for daily supply. Samples were collected in 98 polyethylene containers and refrigerated until analysis. Water and urine fluoride concentrations were measured using the fluoride selective electrode method. Enamel fluorosis was evaluated using Thylstrup and Ferjerskov Index (TFI). Demographic and anthropometric characteristics were assessed. Besides, other exposures to non-natural fluoride were also evaluated. Logistic regression was applied for multiple analyses. Results: The median fluoride concentration in water and urine samples was 10.5 mg/L and 0.63 mg/L respectively, with the highest value found in Algarrobo-Magdalena, and the lowest value found in Manzanares-Caldas. The overall prevalence of enamel fluorosis was 86.1%, being more frequent the mild codes with TFI-1 to TFI-2. The highest prevalence was found in Margarita-Bolívar and Manzanares-Caldas, and the most severe codes (TFI-5 to TFI-9) were detected in Manzanares-Caldas. The multiple analysis revealed water ingestion exposure dose, urinary excretion, involuntary intake of toothpaste, amount of table salt consumption and sex as significant factors (p< 0.001). Conclusions: The fluoride ingestion exposure dose and its subsequent urinary excretion could be used as estimators of past fluoride exposure, explaining the current prevalence of enamel fluorosis in Colombian schoolchildren. Key words:Fluoride, groundwater ingestion, enamel fluorosis, prevalence, severity.

Evaluating Implementation and Outcomes of a Person-Centered Care Model for People with Dementia in the Rehabilitation In-Patient Setting: Project Protocol.

BACKGROUND: While Australian guidelines promote person-centered healthcare (PCC) for persons with dementia, healthcare systems, routines, rules, and workplace cultures can pose challenges in the provision of PCC. OBJECTIVE: To present a knowledge translation protocol of the PCC model in a sub-acute rehabilitation hospital. METHODS: The two-year pre/post/follow-up translation project will include (n = 80) persons with dementia, (n = 80) adult family/carers of patient participants, (n = 60) healthcare staff (medical, nursing, allied health), and (n = 8) PCC staff champions. Champions will complete six half-days' training in PCC. Medical, nursing, and allied health staff will be provided with PCC learning manuals, complete six hours of online PCC education and attend six face-to-face PCC education sessions. Champions will provide ongoing support to staff in PCC practice. The Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework will be used to evaluate: i) outcomes for prospective patients provided with PCC, compared with a matched sample of retrospective patients (primary outcomes agitation incidence and severity); 2) champion and staff PCC knowledge, confidence, engagement, and practice quality; 3) person, family/carer, champion, and staff satisfaction with PCC; 4) PCC costs and benefits; and 5) organizational structures, systems and policies required to implement and maintain PCC in sub-acute healthcare. RESULTS: We will identify if PCC benefits persons with dementia, staff, and healthcare services, and we will generate evidence on the educational and organizational resources required to embed PCC in practice. CONCLUSION: Project findings will inform tailored PCC education applications for dissemination in healthcare and produce evidence-based PCC practice guidelines to improve healthcare for persons with dementia.

Invasive Pneumococcal Disease Characterization in Adults and Subgroups aged < 60 years and ≥ 60 years in Bogota, Colombia.

Background: There is scarce information on the burden of invasive pneumococcal disease (IPD) among adults in low- and middle-income countries. This study aimed to describe the clinical outcomes and microbiological characteristics associated with IPD in adults and subgroups aged 18-59 years and ≥60 years in Colombia. Methods: A retrospective chart review study was conducted in five institutions of Bogotá from January 2011 to December 2017. Analyses were carried out for overall population and stratified by age group (18-59; ≥ 60 years). Results: There were 169 IPD cases; median age was 58 years, 51.5% were male, and 80.5% had at least one comorbidity. Bacteremic pneumonia was the most common presentation (63.9%). The median length of hospital stay was 12 days with high healthcare resource utilization (HCRU): 58.6% required ICU and 53.3% inotropic support. Overall case-fatality rate (CFR) was 41.4%. Clinical outcomes were worse in patients ≥60 years old with significantly higher CFR and HCRU (ICU admission, mechanical ventilation, and inotropic support) compared to those aged 18-59 years. The most frequent serotypes were 3, 6 A/C, 14, and 19A. The sensitivity to penicillin in meningitis and non-meningitis isolates were 75% and 89.1% respectively. Conclusions: IPD was associated with a substantial burden in adults and worse clinical outcomes and HCRU in older adults in Colombia. Surveillance data combined with clinical outcomes have the potential to inform age-based pneumococcal vaccination policies.

Translation, adaptation, and psychometrically validation of an instrument to assess disease-related knowledge in Spanish-speaking cardiac rehabilitation participants: The Spanish CADE-Q SV.

OBJECTIVE: To validate the Spanish Coronary Artery Disease Education Questionnaire Short Version(CADE-QSV). BACKGROUND: Knowledge assessment can tailor patient education strategies in CR. METHODS: Ten CR experts culturally-adapted the translated tool and 249 CR patients in three countries participated in the psychometrically validation. The internal consistency was assessed using KR-20 and Cronbach's alpha. Distribution- and anchor-based methods were used to estimate the MCID for interpretability analysis. Factor structure, acceptability, floor and ceiling effects and discriminant validity were also assessed. RESULTS: KR-20 was 0.75.Factor analysis revealed 5 factors, all internally consistent. Discriminant validity was supported by significant differences in educational level and family income at post-CR. Results showed that increases in knowledge can moderately increase mean steps per day, with MCID=3.00. Items were completed by 98% of participants. No evidence for ceiling/floor effects on total score. The area with the highest knowledge was nutrition and the lowest was medical condition. CONCLUSION: The Spanish CADE-QSV demonstrated preliminary validity and reliability.

Risk Factors for Mortality in Colombian Patients with Candidemia.

The aim of the study was to describe the microbiology and susceptibility profile of candidemia and to identify the risk factors associated with mortality in Colombia. A cohort of patients was followed for 30 days during 2008 to 2010. Microbiological identification and susceptibility assessments were performed in a reference centre. Demographic, clinical and treatment variables were evaluated for their associations with mortality. A parametric survival regression analysis was used to identify the risk factors associated with mortality. A total of 109 patients with candidemia in four hospitals in Colombia were identified, with a median age of 30 years old. C. parapsilosis was the most frequently identified microorganism (38.5%); the susceptibility of all isolates was high to fluconazole and anidulafungin, except for C. glabrata isolates. The overall mortality was 35.7%, and the risk factors associated with mortality included lack of antifungal treatment (HR 5.5, 95% CI 3.6-11.4), cancer (HR 3.9, 95% CI 2.3-8.0), diabetes (HR 2.5, 95% CI 1.03-6.4), and age (HR 1.13 per every 10 years, 95% CI 1.02-1.24). Catheter removal was associated with a low mortality rate (HR 0.06, 95% CI 0.00-0.49). Prompt antifungal treatment, better glycemic control and catheter removal should be prioritized in the management of candidemia.

Translation and evaluation of a comprehensive educational program for cardiac rehabilitation patients in Latin America: A multi-national, longitudinal study.

OBJECTIVES: To translate, cross-culturally adapt and validate a comprehensive evidence- and theoretically-based CR education intervention in Latin America. METHODS: First, best practices in translation and cross-cultural adaptation were applied through 6 steps. Then, the Spanish version was delivered to CR participants from programs in Colombia, Costa Rica and Peru for validation, such that the evaluation was pre-post, uncontrolled, pragmatic, observational, and prospective in design. Participants completed surveys assessing knowledge, health literacy, self-efficacy, and health behaviours. All outcomes were assessed pre-, and post-CR, as well as 6 months after CR completion. RESULTS: After translation of the patient guide from English to Spanish, 5 of the 9 booklets were culturally adapted. Two-hundred and forty-nine patients consented to participate, of which 184 (74 %) completed post-CR, and 121 (48 %) completed final assessments. There was a significant improvement in disease-related knowledge pre- to post-CR, as well as in health literacy, self-efficacy, and health behaviours (all p < 0.05). These gains were sustained 6 months post-program. With adjustment, CR attendance (i.e., exposure to the education) was associated with greater post-CR knowledge (ß = 0.026; p = 0.01). CONCLUSION: A patient education intervention for CR patients in Latin America has been validated, and wider implementation is warranted. PRACTICE IMPLICATIONS: Application of this first-ever validated CR education program for Spanish-speaking settings may result in secondary prevention.

Prescribing antibiotics by dentists in Colombia: Toward a conscientious prescription.

OBJECTIVES: Inappropriate prescription of antibiotics contributes to antibiotic resistance. Therefore, the objective of this study was to determine the awareness, attitudes, and intention to practice of dentists prescribing antibiotics in Colombia in order to design a virtual learning environment on this subject. METHODS: In a descriptive study across seven cities, 700 dentists from different Colombian cities were requested to complete a validated questionnaire containing five sections: general information, awareness on antibiotic effectiveness and antibiotic resistance, attitudes regarding prescription decision, intention to practice concerning clinical cases, and complementary information. The level of awareness, attitudes, and intention to practice was determined and Chi-square test was used to determine the existence of significant differences among cities. RESULTS: The majority of dentists showed a medium level regarding the number of correct answers on awareness (62.4 percent) and attitudes (88.7 percent) and a high level on intention to practice (91.7 percent). Common errors within the awareness section included the meaning of the term "antibiotic resistance" (35 percent) and most dentists were not convinced that such resistance could be derived from prescription of antibiotics (51.2 percent). In the attitudes section, only 45 percent declared that they prescribe antibiotics based mainly on symptoms, and the intention to practice section showed a significant percentage of unnecessary prescription (51 percent for pacemaker users) or absence of prescription (53.9 percent for ventricular septal defect) in antibiotic prophylaxis for infectious endocarditis (IE). CONCLUSION: The dentists interviewed should be trained and made aware of antibiotic resistance, microbiological and clinical foundations, and current antibiotic prophylaxis guidelines.

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.