Pre-multidrug-resistant Mycobacterium tuberculosis Beijing strain associated with disseminated tuberculosis in a pet dog.

Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.

The use of Bowman's layer vertical topographic thickness map in the diagnosis of keratoconus.

PURPOSE: To evaluate the use of Bowman's layer (BL) vertical topographic thickness maps in diagnosing keratoconus (KC). DESIGN: Prospective, case control, interventional case series. PARTICIPANTS: A total of 42 eyes: 22 eyes of 15 normal subjects and 20 eyes of 15 patients with KC. INTERVENTION: Bowman's layer 2-dimensional 9-mm vertical topographic thickness maps were created using custom-made ultra high-resolution optical coherence tomography. MAIN OUTCOME MEASURES: Bowman's layer average and minimum thicknesses of the inferior half of the cornea, Bowman's ectasia index (BEI; defined as BL minimum thickness of the inferior half of the cornea divided by BL average thickness of the superior half of the cornea multiplied by 100), BEI-Max (defined as BL minimum thickness of the inferior half of the cornea divided by BL maximum thickness of the superior half of the cornea multiplied by 100), keratometric astigmatism (Ast-K) of patients with KC, and average keratometric (Avg-K) readings. RESULTS: In patients with KC, BL vertical thickness maps disclosed localized relative inferior thinning of the BL. Inferior BL average thickness (normal = 15±2, KC = 12±3 µm), inferior BL minimum thickness (normal = 13±2, KC = 7±3 µm), BEI (normal = 91±7, KC = 48±14), and BEI-Max (normal = 75±8; KC = 40±13) all showed highly significant differences in KC compared with normal subjects (P< 0.001). Receiver operating characteristic (ROC) curve analysis showed excellent predictive accuracy for BEI and BEI-Max with 100% sensitivity and specificity (area under the curve [AUC] of 1) with cutoff values of 80 and 60, respectively. The AUC of inferior BL average thickness and minimum thickness were 0.87 and 0.96 with a sensitivity of 80% and 93%, respectively, and a specificity of 93% and 93%, respectively. Inferior BL average thickness, inferior BL minimum thickness, BEI, and BEI-Max correlated highly to Ast-K (R = -0.72, -0.82, -0.84, and -0.82, respectively; P< 0.001) and to Avg-K (R = -0.62, P< 0.001; R = -0.59, P = 0.001; R = -0.60, P< 0.001; and R = -0.59, P = 0.001, respectively). CONCLUSIONS: Bowman's layer vertical topographic thickness maps of patients with KC disclose characteristic localized relative inferior thinning. Inferior BL average thickness, inferior BL minimum thickness, BEI, and BEI-Max are qualitative and quantitative indices for the diagnosis of KC that accurately correlate with the severity of KC. In our pilot study, BEI and BEI-Max showed excellent accuracy, sensitivity, and specificity in the diagnosis of KC.

Diagnosis of ocular surface lesions using ultra-high-resolution optical coherence tomography.

PURPOSE: To assess the use of ultra-high-resolution (UHR) optical coherence tomography (OCT) in the diagnosis of ocular surface lesions. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Fifty-four eyes of 53 consecutive patients with biopsy-proven ocular surface lesions: 8 primary acquired melanosis lesions, 5 amelanotic melanoma lesions, 2 nevi, 19 ocular surface squamous neoplasia lesions, 1 histiocytosis lesion, 6 conjunctival lymphoma lesions, 2 conjunctival amyloidosis lesions, and 11 pterygia lesions. INTERVENTION: Ultra-high-resolution OCT imaging of the ocular surface lesions. MAIN OUTCOME MEASURES: Clinical course and photographs, UHR OCT image, and histopathologic findings. RESULTS: Ultra-high-resolution OCT images of all examined ocular surface lesions showed close correlation with the obtained histopathologic specimens. When clinical differential diagnosis of ocular surface lesions was broad, UHR OCT images provided optical signs indicating a more specific diagnosis and management. In cases of amelanotic melanoma, conjunctival amyloidosis, and primary histiocytosis and in 1 case of ocular surface squamous neoplasia, UHR OCT was instrumental in guiding the diagnosis. In those cases, UHR OCT suggested that the presumed clinical diagnosis was incorrect and favored a diagnosis that later was confirmed by histopathologic examination. CONCLUSIONS: Correlations between UHR OCT and histopathologic findings confirm that UHR OCT is an adjunctive diagnostic method that can provide a noninvasive means to help guide diagnosis and management of ocular surface lesions. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Complications of femtosecond laser-assisted re-treatment for residual refractive errors after LASIK.

PURPOSE: To report complications of femtosecond laser-assisted re-treatment by the creation of side cuts within the old flaps for residual refractive error after primary LASIK in two patients. METHODS: Case report. RESULTS: Three eyes of two patients had complications with a circumferential sliver of stromal tissue displaced during surgery due to overlap of old and new side cuts. The displaced tissue was repositioned and corneal anatomy was restored. Two of three eyes demonstrated improvement in the uncorrected visual acuity, whereas one eye lost two lines of corrected visual acuity due to loss of tissue at side cut resulting from flap manipulation, which was done at 1 week. CONCLUSIONS: These cases demonstrate a complication of femtosecond laser-enabled side-cut for LASIK enhancement and factors that may lead to this complication and precautions to avoid it.

Comparison of IOL power calculation methods and intraoperative wavefront aberrometer in eyes after refractive surgery.

PURPOSE: To compare preoperative methods for calculating intraocular lens (IOL) power versus the intraoperative wavefront aberrometer in eyes with a history of refractive surgery. METHODS: A retrospective study of 46 eyes (33 patients) with previous refractive surgery that underwent subsequent cataract surgery was conducted. Suggested IOL power predicted by ORange intraoperative wavefront aberrometer (WaveTec Vision Systems, Inc., Aliso Viejo, CA) was compared to power predicted by the (1) SRK-T formula using keratometry and axial length measurements from the IOLMaster (Carl Zeiss Meditec, Dublin, CA), (2) average central keratometry (Avg K) from corneal topography, and (3) average IOL power predicted by the American Society of Cataract and Refractive Surgery (ASCRS) web site. No historical information was used for the calculations. IOL power required for emmetropia was back-calculated using manifest refraction and implanted IOL power after cataract surgery. RESULTS: Mean age was 60 ± 7.9 years. Fifteen percent had a history of myopic photorefractive keratectomy (n = 7), 57% myopic LASIK (n = 26), 13% hyperopic LASIK (n = 6), and 22% radial keratectomy (RK) (n = 10). In 37% of cases, ORange predicted IOL power to within ±0.50 diopters (D) of emmetropia, compared to 30% for IOLMaster keratometry, 26% for Avg K, and 17% for ASCRS web site. In eyes after myopic treatment, ORange, IOLMaster, Avg K, and ASCRS web site predicted within ±0.50 D of emmetropia in 39%, 27%, 24%, and 18%, respectively, and within ±1.0 D in 60%, 39%, 39%, and 51%, respectively. In eyes after RK, ORange, Avg K, and ASCRS web site predicted to within ±0.50 D of emmetropia in 14% and the IOLMaster in 43% cases. CONCLUSIONS: Although the ORange most often predicted to within ±0.5 D of emmetropia, no method was able to achieve this accuracy more than 50% of the time. Predictions for eyes after RK were worse than for other types of refractive procedures.

3D printing of microencapsulated Lactobacillus rhamnosus for oral delivery.

3D Printing is an innovative technology within the pharma and food industries that allows the design and manufacturing of novel delivery systems. Orally safe delivery of probiotics to the gastrointestinal tract faces several challenges regarding bacterial viability, in addition to comply with commercial and regulatory standpoints. Lactobacillus rhamnosus CNCM I-4036 (Lr) was microencapsulated in generally recognised as safe (GRAS) proteins, and then assessed for robocasting 3D printing. Microparticles (MP-Lr) were developed and characterised, prior to being 3D printed with pharmaceutical excipients. MP-Lr showed a size of 12.3 ± 4.1 µm and a non-uniform wrinkled surface determined by Scanning Electron Microscopy (SEM). Bacterial quantification by plate counting accounted for 8.68 ± 0.6 CFU/g of live bacteria encapsulated within. Formulations were able to keep the bacterial dose constant upon contact with gastric and intestinal pH. Printlets consisted in oval-shape formulations (15 mm × 8 mm × 3.2 mm) of ca. 370 mg of total weight, with a uniform surface. After the 3D printing process, bacterial viability remained even as MP-Lr protected bacteria alongside the process (log reduction of 0.52, p > 0.05) in comparison with non-encapsulated probiotic (log reduction of 3.05). Moreover, microparticle size was not altered during the 3D printing process. We confirmed the success of this technology for developing an orally safe formulation, GRAS category, of microencapsulated Lr for gastrointestinal vehiculation.

Encapsulated Phytomedicines against Cancer: Overcoming the "Valley of Death".

P2Et is the standardized extract of Caesalpinia spinosa (C. spinosa), which has shown the ability to reduce primary tumors and metastasis in animal models of cancer, by mechanisms involving the increase in intracellular Ca++, reticulum stress, induction of autophagy, and subsequent activation of the immune system. Although P2Et has been shown to be safe in healthy individuals, the biological activity and bioavailability can be increased by improving the dosage form. This study investigates the potential of a casein nanoparticle for oral administration of P2Et and its impact on treatment efficacy in a mouse model of breast cancer with orthotopically transplanted 4T1 cells. Animals were treated with either free or encapsulated oral P2Et orally or i.p. Tumor growth and macrometastases were evaluated. All P2Et treatments significantly delayed tumor growth. The frequency of macrometastasis was reduced by 1.1 times with P2Et i.p., while oral P2Et reduced it by 3.2 times and nanoencapsulation reduced it by 3.57 times. This suggests that nanoencapsulation led to higher doses of effective P2Et being delivered, slightly improving bioavailability and biological activity. Therefore, the results of this study provide evidence to consider P2Et as a potential adjuvant in the treatment of cancer, while the nanoencapsulation of P2Et provides a novel perspective on the delivery of these functional ingredients.

Epithelial ingrowth after LASIK: clinical characteristics, risk factors, and visual outcomes in patients requiring flap lift.

PURPOSE: To describe clinical characteristics, risk factors, and visual outcomes in patients requiring flap lift for epithelial ingrowth following LASIK. METHODS: Consecutive, noncomparative, retrospective case series of eyes requiring flap lift for epithelial ingrowth following LASIK from June 2003 through July 2011 at a tertiary care, university-based eye hospital. Main outcome measures were uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA) at 1 and 3 months and recurrence of epithelial ingrowth. RESULTS: Forty-five eyes were included. Laser in situ keratomileusis retreatment was the most common etiologic factor for epithelial ingrowth (28/45 eyes [62%]). All patients were treated with flap lift and scraping. Mean logMAR UDVA at presentation was 0.28 (Snellen equivalent 20/38). Mean logMAR UDVA at 3 months was 0.17 (Snellen equivalent 20/30) with 53% of eyes 20/25 or better. Mean logMAR CDVA at 3 months was 0.06 (Snellen equivalent 20/23) with 78% of eyes 20/25 or better. Epithelial ingrowth into the central cornea portended a trend towards UDVA worse than 20/25 or worse at 3-month follow-up (hazard ratio [HR] 5.54, 95% confidence interval [CI]: 0.98-31.3, P=.05) and CDVA worse than 20/25 at 3-month follow-up (HR 4.32, 95% CI: 0.85-21.9, P=.08). Recurrence after treatment was 31% at 3 months and 36% at 1 year. Risk factors for recurrence included: infectious etiology of ingrowth (HR 5.7, 95% CI: 1.11-29.1, P=.04), use of microkeratome for primary LASIK (HR 4.64, 95% CI: 1.07-20.1, P=.04), and hyperopic primary LASIK (HR 2.49, 95% CI: 0.98-6.31, P=.06). CONCLUSIONS: Patients undergoing flap lift for the treatment of epithelial ingrowth have a relatively high rate of recurrence but good visual acuity outcomes.

Evidence for classification of c.1852_1853AA>GC in MLH1 as a neutral variant for Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS. METHODS: The following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers. RESULTS: Twenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients. CONCLUSIONS: The p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives.

Zein Nanoparticles Improve the Oral Bioavailability of Curcumin in Wistar Rats.

Curcumin is a natural compound obtained from turmeric root with high antioxidant and anti-inflammatory activities. However, clinical application of curcumin has been limited due to its low solubility and bioavailability and rapid metabolism and degradation. This study was conducted to evaluate the effect of curcumin incorporation in zein nanoparticles on the pharmacokinetic parameters of systemic curcumin in plasma. Wistar rats were administered a single oral dose of 250 mg/kg of standard curcumin (control) or nanocurcumin (zein-based nanoparticles, Nucaps). The proposed new formulation was also compared with two commercially available curcumin complexes. Blood samples were collected at different times, and plasma levels were determined using HPLC-MS/MS. Overall, nanocurcumin (Nucaps) formulation was well tolerated and showed a 9-fold increase in oral bioavailability when compared to the standard curcumin natural extract. In addition, the nanoparticles prepared in this study demonstrated a bioavailability profile superior to that of other bioavailability-enhanced curcumin complexes currently available in the marketplace. Thus, our nanoparticle-based formulation has shown great potential as a nutraceutical for the oral administration of curcumin.

Corneal epithelial thickness profile in dry-eye disease.

BACKGROUND/OBJECTIVES: To characterize and evaluate the use of corneal epithelial profile maps generated by an ultrahigh-resolution optical coherence tomography (UHR-OCT) in the diagnosis and management of dry-eye disease (DED). SUBJECTS/METHODS: This prospective, interventional case-control study included 115 eyes of 71 subjects (52 DED and 19 controls) imaged using an UHR-OCT. Average, maximum, and minimum, range of corneal epithelial thicknesses were extracted from epithelial profile maps. Surface regularity was quantified using the range and variance of the epithelial thickness measured along a horizontal UHR-OCT scan. The variance of thickness measurements along a scan was named epithelial irregularity factor (EIF). Symptoms of 31 DED patients (55 eyes) were quantified by questionnaire and correlated to epithelial profile findings, fluorescein staining, tear breakup time, and Schirmer's test. Twenty-one DED eyes were administered autologous serum drops and follow-up UHR-OCT images were captured. RESULTS: DED patients had a highly irregular corneal epithelial surface compared with controls. Epithelial thickness profile variance (EIF) and range were significantly higher in DED as compared with controls (5.79 vs. 0.77, p < 0.001 and 7.6 vs. 4.6 µm, p < 0.001). Both parameters were highly significantly correlated with questionnaire scores (EIF: r = 0.778; p < 0.001, range: r = 0.737; p < 0.001). Follow-up showed a statistically significant reduction in epithelial thickness profile variance and range of treated patients ( p < 0.001). CONCLUSIONS: DED patients have irregular epithelial surface that can be quantified using UHR-OCT generated CEP maps. Epithelial thickness profile range and EIF correlate accurately with patients' symptoms and could be used to follow-up patients and response to treatment.

The TGFBR1*6A allele is not associated with susceptibility to colorectal cancer in a Spanish population: a case-control study.

BACKGROUND: TGF-beta receptor type I is a mediator of growth inhibitory signals. TGFBR1*6A (rs11466445) is a common polymorphic variant of the TGF-beta receptor I gene and has been associated with tumour susceptibility. Nevertheless, the role of this polymorphism as a risk factor for colorectal cancer is controversial. The aim of this study was to assess the association between TGFBR1*6A and colorectal cancer, age, sex, tumour location and tumour stage in a Spanish population. METHODS: The case-control study involved 800 Spanish subjects: 400 sporadic colorectal cancer patients and 400 age-, sex-, and ethnic-matched controls. The odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A polymorphism were calculated using unconditional logistic regression adjusted for age and sex. Analysis of somatic mutations at the GCG repeat of TGFBR1 exon 1 and germline allele-specific expression were also conducted to obtain further information on the contribution of the TGFBR1*6A allele to CRC susceptibility. RESULTS: There was no statistically significant association between the TGFBR1*6A allele and CRC (p > 0.05). The OR was 1.147 (95% CI: 0.799-1.647) for carriers of the TGFBR1*6A allele and 0.878 (95% CI: 0.306-2.520) for homozygous TGFBR1*6A individuals compared with the reference. The frequency of the polymorphism was not affected by age, sex or tumour stage. The TGFBR1*6A allele was more prevalent among colon tumour patients than among rectal tumour patients. Tumour somatic mutations were found in only two of 69 cases (2.9%). Both cases involved a GCG deletion that changed genotype 9A/9A in normal DNA to genotype 9A/8A. Interestingly, these two tumours were positive for microsatellite instability, suggesting that these mutations originated because of a deficient DNA mismatch repair system.Allele-specific expression of the 9A allele was detected in seven of the 14 heterozygous 9A/6A tumour cases. This could have been caused by linkage disequilibrium of the TGFBR1*6A allele with mutations that cause allele-specific expression, as was recently suggested. CONCLUSION: Our results suggest that the TGFBR1*6A allele does not confer an increased risk of colorectal cancer in the Spanish population.

The Int7G24A variant of transforming growth factor-beta receptor type I is a risk factor for colorectal cancer in the male Spanish population: a case-control study.

BACKGROUND: The Int7G24A variant of transforming growth factor-beta receptor type I (TGFBR1) has been shown to increase the risk for kidney, ovarian, bladder, lung and breast cancers. Its role in colorectal cancer (CRC) has not been established. The aims of this study were to assess the association of TGFBR1*Int7G24A variant with CRC occurrence, patient age, gender, tumour location and stage. METHODS: We performed a case-control study with 504 cases of sporadic CRC; and 504 non-cancerous age, gender and ethnically matched controls. Genotyping analysis was performed using allelic discrimination assay by real time PCR. RESULTS: The Int7G24A variant was associated with increased CRC incidence in an additive model of inheritance (P for trend = 0.005). No significant differences were found between Int7G24A genotypes and tumour location or stage. Interestingly, the association of the Int7G24A variant with CRC risk was significant in men (odds ratio 4.10 with 95% confidence intervals 1.41-11.85 for homozygous individuals; P for trend = 0.00023), but not in women. We also observed an increase in susceptibility to CRC for individuals aged less than 70 years. CONCLUSION: Our data suggest that the Int7G24A variant represents a risk factor for CRC in the male Spanish population.

Mannosylated Nanoparticles for Oral Immunotherapy in a Murine Model of Peanut Allergy.

Peanut allergy is one of the most prevalent and severe of food allergies with no available cure. The aim of this work was to evaluate the potential of an oral immunotherapy based on the use of a roasted peanut extract encapsulated in nanoparticles with immunoadjuvant properties. For this, a polymer conjugate formed by the covalent binding of mannosamine to the copolymer of methyl vinyl ether and maleic anhydride was first synthetized and characterized. Then, the conjugate was used to prepare nanoparticles with an important capability to diffuse through the mucus layer and reach, in a large extent, the intestinal epithelium, including Peyer's patches. Their immunotherapeutic potential was evaluated in a model of presensitized CD1 mice to peanut. After completing therapy, mice underwent an intraperitoneal challenge with peanut extract. Nanoparticle-treatment was associated with both less serious anaphylaxis symptoms and higher survival rates than control, confirming the protective effect of this formulation against the challenge.

New pharmaceutical approaches for the treatment of food allergies.

INTRODUCTION: Allergic diseases constitute one of the most common causes of chronic illness in developed countries. The main mechanism determining allergy is an imbalance between Th1 and Th2 response towards Th2. AREAS COVERED: This review describes the mechanisms underlying the natural tolerance to food components and the development of an allergic response in sensitized individuals. Furthermore, therapeutic approaches proposed to manage these abnormal immunologic responses food are also presented and discussed. EXPERT OPINION: In the past, management of food allergies has consisted of the education of patients to avoid the ingestion of the culprit food and to initiate the therapy (e.g. self-injectable epinephrine) in case of accidental ingestion. In recent years, sublingual/oral immunotherapies based on the continuous administration of small amounts of the allergen have been developed. However, the long periods of time needed to obtain significant desensitization and the generation of adverse effects, limit their use. In order to solve these drawbacks, strategies to induce tolerance are being studied, such as the use of either adjuvant immunotherapy in order to facilitate the reversion of the Th2 response towards Th1 or the use of monoclonal antibodies to block the main immunogenic elements.

Evaluation of nanoparticles as oral vehicles for immunotherapy against experimental peanut allergy.

The aim of this work was to evaluate the potential application of an original oral immunotherapy, based on the use of nanoparticles, against an experimentally induced peanut allergy. In this context, a roasted peanut extract, containing the main allergenic proteins, were encapsulated into poly(anhydride) nanoparticles. The resulting peanut-loaded nanoparticles (PE-NP) displayed a mean size of about 150nm and a significantly lower surface hydrophobicity than empty nanoparticles (NP). This low hydrophobicity correlated well with a higher in vitro diffusion in pig intestinal mucus than NP and an important in vivo capability to reach the intestinal epithelium and Peyer's patches. The immunotherapeutic capability of PE-NP was evaluated in a model of pre-sensitized CDI mice to peanut. After completing therapy of three doses of peanut extract, either free or encapsulated into nanoparticles, mice underwent an intraperitoneal challenge. Anaphylaxis was evaluated by means of assessment of symptom scores and mouse mast cell protease-1 levels (mMCPT-1). PE-NP treatment was associated with significant lower levels of mMCPT-1, and a significant survival rate after challenge, confirming the protective effect of this formulation against the challenge. In summary, this nanoparticle-based formulation might be a valuable strategy for peanut-specific immunotherapy.

Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis of members of the Mycobacterium tuberculosis complex.

Typical CRISPR (clustered, regularly interspaced, short palindromic repeat) regions are constituted by short direct repeats (DRs), interspersed with similarly sized non-repetitive spacers, derived from transmissible genetic elements, acquired when the cell is challenged with foreign DNA. The analysis of the structure, in number and nature, of CRISPR spacers is a valuable tool for molecular typing since these loci are polymorphic among strains, originating characteristic signatures. The existence of CRISPR structures in the genome of the members of Mycobacterium tuberculosis complex (MTBC) enabled the development of a genotyping method, based on the analysis of the presence or absence of 43 oligonucleotide spacers separated by conserved DRs. This method, called spoligotyping, consists on PCR amplification of the DR chromosomal region and recognition after hybridization of the spacers that are present. The workflow beneath this methodology implies that the PCR products are brought onto a membrane containing synthetic oligonucleotides that have complementary sequences to the spacer sequences. Lack of hybridization of the PCR products to a specific oligonucleotide sequence indicates absence of the correspondent spacer sequence in the examined strain. Spoligotyping gained great notoriety as a robust identification and typing tool for members of MTBC, enabling multiple epidemiological studies on human and animal tuberculosis.

Relatedness of Mycobacterium avium subspecies hominissuis clinical isolates of human and porcine origins assessed by MLVA.

Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.