Ana Matos

An Experimental Study of the Ant Colony System for the Period Vehicle Routing Problem

In this paper, a new Ant System approach to the Period Vehicle Routing Problem (PVRP) is presented. In PVRP, visit days have to be assigned to customers in order to find efficient routes over the period. We suggest a new technique for defining the initial solution and a novel strategy to update the pheromone trails that is especially suited for solving large scale problems. An illustrative example for a waste collection system involving 202 localities in the municipality of Viseu, Portugal, demonstrates the effectiveness of the model.

The Influence of Chemokine Receptor CCR2 Genotypes in the Route to Cervical Carcinogenesis

Invasive squamous cell carcinomas of the cervix arise from earlier, reversible precursor lesions called low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively). The aim of our study was to analyse the influence of the CCR2-64I polymorphism in the development of SIL due to its preponderant role in the cervical carcinogenesis and in the progression of these lesions to invasive cervical carcinoma. We conducted a case-control study, analyzing 565 Caucasian women from the north of Portugal. The CCR2-64I polymorphism was analysed through polymerase chain reaction followed by restriction fragment length polymorphism. The frequency of GG, GA and AA genotypes was 76.5, 23.5 and 0.0% respectively, in HSIL patients, and 87.8, 11.1 and 1.1% respectively, in the control group. The G allele frequency was 88.2% in the HSIL group and 93.4% in the control group. Regarding the A allele frequency, it was 11.8% in the HSIL group and 6.6% in the control group. Overall, the frequency of A carrier genotypes was higher in HSIL patients than in the control group (p = 0.013; OR = 2.21; 95%CI: 1.17–4.15), suggesting that CCR2-64I polymorphism might contribute to the establishment of HSIL, through the disruption of the naturally fragile immune response directed towards human papillomavirus infection.

Combined C4d and CD3 immunostaining predicts immunoglobulin (Ig)A nephropathy progression

A number of molecules have been shown recently to be involved in the pathogenesis and progression of immunoglobulin (Ig)A nephropathy (IgAN). Among these, we have selected C4d (complement lectin pathway involvement), CD3 (T cell marker, traducing interstitial inflammation), transglutaminase 2 (TGase‐2, involved in tissue fibrosis development) and p‐extracelluar‐regulated kinase (ERK)1/2 (protein kinase intracellular signaling molecule) to perform a panel of immunohistological biomarkers and assess its predictive value for disease progression. Immunohistochemical staining of these biomarkers was performed in paraffin sections from 74 renal biopsy cases with the clinical diagnosis of IgAN. Association between score analysis of these parameters and disease course was assessed through univariate and multivariate analysis, including baseline clinical and histological data. Univariate analysis showed that glomerular C4d, tubulointerstitial TGase2 and CD3 scores were associated with baseline proteinuria and disease progression. Multivariate analysis showed that only baseline estimated glomerular filtration rate (eGFR), C4d and CD3 were associated independently with progressive kidney disease (decline of at least 50% in the eGFR or progression to end‐stage renal disease (ESRD) during the follow‐up period). Establishing an accurate prediction model for IgAN progression is still a matter of research in clinical nephrology. The complement system, particularly lectin pathway activation, and T cell activation, have been shown previously to be potential modifiers of the disease course. Here we show that the combination of two histological biomarkers (C4d and CD3) can be a powerful predictor of IgAN progression and a potential useful tool for the clinical approach of this disease.

Criteria to predict carriers of a novel SCN5A mutation in a large Portuguese family affected by the Brugada syndrome

AIMS Brugada syndrome (BrS) is a life-threatening arrhythmia disorder associated with autosomal-dominant mutations in the SCN5A gene. We aimed to characterize the diagnostic challenges and clinical manifestations of a novel SCN5A mutation associated with BrS. METHODS AND RESULTS From a novel SCN5A mutation (c.664C>T; p.Arg222X) identified in a proband with the characteristic electrocardiographic pattern and the history of sudden collapse, 122 family members were studied including 40 carriers of the mutation. The electrocardiographic diagnosis of BrS requires type 1 Brugada electrocardiogram (ECG) pattern in >1 right precordial lead (V1-V3), but recently an isolated lead with coved-type ECG was proposed to be enough for the diagnosis. In this family, these proposed criteria (PC) were more sensitive in detecting mutation carriers than the conventional criteria without repercussion on the specificity. Carriers had, on average, longer P-wave duration, PR, and QRS intervals and higher transmural dispersion of repolarization. The prevalence of late potentials was higher in carriers, and individual signal average ECG (SAECG) parameters (QRSf, LAS, and RMS40) also were related to SCN5A gene mutation. Three non-carriers were found to be affected by BrS, two with a spontaneous type 1 ECG with alternative placement of the precordial electrodes, and one only after the pharmacological provocative test, suggesting that other genes may play a role in the pathophysiology of this disease. CONCLUSION The PC for BrS diagnosis should be implemented. Some parameters from the spontaneous ECG and the SAECG are more effective tools than the characteristic repolarization pattern to discriminate between carriers of SCN5A mutations.

Granuloma Coinfection with Mycobacterium bovis, Mycobacterium avium subsp. paratuberculosis, and Corynebacterium pseudotuberculosis in Five Hunted Red deer (Cervus elaphus) in Portugal

Abstract We report granulomatous lymphadenitis in red deer (Cervus elaphus) in Portugal caused by coinfection with Corynebacterium pseudotuberculosis, Mycobacterium bovis, and Mycobacterium avium subsp. paratuberculosis, as demonstrated by molecular methods.

SURVEY OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN ROAD-KILLED WILD CARNIVORES IN PORTUGAL

Abstract:  A survey to determine the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in wild carnivores in Portugal was conducted by testing samples from road-killed animals between 2009 and 2012. Postmortem examinations were performed and tissues were collected from wild carnivores representing four families and six different species, with a total of 74 animals analyzed. Cultures were performed by using Löwenstein-Jensen and Middlebrook 7H11 solid media and acid-fast isolates were identified by polymerase chain reaction (PCR) and mycobactin dependency characteristics. Tissues were also screened for MAP by directly extracting DNA and testing for the MAP-specific sequences. The occurrence of infected animals (an animal had at least one tissue that was positive for culture or direct PCR) was 27.0% (n = 20). MAP was isolated from culture of 25 tissue samples (3.8%) and was detected by direct PCR in 40 (6.0%) samples. Infection was recorded in 5/6 studied species: 7/49 (14.3%) red foxes (Vulpes vulpes), 3/3 (100%) beech martens (Martes foina), 2/4 (50.0%) Eurasian otters (Lutra lutra), 7/15 (46.7%) Egyptian mongooses (Herpestes ichneumon), and 1/1 (100%) European badger (Meles meles). These species represent three different taxonomic families: Canidae (14.3% were positive), Mustelidae (75.0% were positive), and Herpestidae (46.7% were positive). The results of this study confirm the presence of MAP infection in wild carnivores in Portugal.

Disseminated Mycobacterium avium subsp. paratuberculosis infection in two wild Eurasian otters (Lutra lutra L.) from Portugal.

Abstract:  Disseminated Mycobacterium avium subsp. paratuberculosis (MAP) infections were found in two Eurasian otters (Lutra lutra, L. 1758) killed by vehicular trauma in February and March 2010 in Castelo Branco, Portugal. At postmortem examination, the organs showed no significant gross alterations; however, microscopically, both animals had diffuse lymphadenitis with macrophage infiltration and deposition of hyaline material in the center of the lymphoid follicles. Acid-fast organisms were isolated from gastrointestinal tissue samples via bacteriologic culture. These organisms were identified as M. avium subsp. paratuberculosis by IS900 polymerase chain reaction (PCR). Additionally, direct IS900 PCR-positive results were obtained for multiple organs of both animals. This is the first report of MAP infection of otters in Portugal.

Mycobacterium avium Complex in Domestic and Wild Animals

Mycobacteria from the Mycobacterium avium complex (MAC) cause a variety of diseases in‐ cluding tuberculosis-like disease in humans and birds, disseminated infections in AIDS pa‐ tients and otherwise immunocompromised patients, lymphadenitis in humans and mammals and paratuberculosis in ruminants. M. avium subsp. paratuberculosis (Map) is the etiologic agent of Johne ́s disease in cattle and it has been identified in human patients with Crohn’s disease. The MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various do‐ mestic and wild mammals and birds [1].

New Insights into Mycobacterium bovis Prevalence in Wild Mammals in Portugal

A survey to determine the prevalence of Mycobacterium bovis in wild mammals in Portugal was conducted by testing samples from hunted animals and those found dead between 2009 and 2013. In this study, we investigated 2116 wild mammals. Post-mortem examinations were performed, and tissues were collected from wild mammals representing 8 families and 11 different species, with a total of 393 animals analysed. Cultures were performed, and acid-fast isolates were identified by PCR. Tissues were also screened for Mycobacterium bovis by directly extracting DNA and testing for the Mycobacterium bovis-specific sequences. Mycobacterium bovis prevalence was 26.9% (95% CI: 22.8-31.5%). Mycobacterium bovis was recorded in 106 of the 393 studied species: prevalence by species were 26.9% (95% CI: 16.8-40.2%) in red foxes, 20.0% (95% CI: 7.0-45.2%) in Egyptian mongooses, 21.4% (95% CI: 16.2-27.7%) in wild boar and 38.3% (95% CI: 29.9-47.4%) in red deer. Mycobacterium bovis infection was detected in six of eight taxonomic families. For some species, the small sample sizes obtained were a reflection of their restricted range and low abundance, making estimates of infection prevalence very difficult (1 beech marten of 4; 1 Eurasian otter of 3; 2 common genet of 3). Infection was not detected in European badgers, hedgehog, wild rabbits and hare. The results of this study confirm the presence of Mycobacterium bovis infection in wild carnivores in Portugal.

Brugada Syndrome Diagnosis: Three Approaches to Combining Diagnostic Markers

Brugada syndrome (BS) is an inherited cardiopathy that predisposes individuals without structural heart disease to sudden cardiac death. The diagnosis is performed by detecting a typical pattern in the electrocardiogram (ECG), called Type 1 Brugada pattern, but this is not always visible, so the diagnosis is not straightforward. In this study, we investigated other ECG markers, independent of the typical pattern, which exhibited a good ability to differentiate the carriers and the non-carriers of the genetic mutation responsible for this disease. The combination of these markers through linear models has led to enhancing the ability of each marker to discriminate between the two groups. We found linear combinations of these markers for which the area under the ROC curve (AUC) was greater than 0.9, which suggests an excellent ability to discriminate between the two groups. This study points towards good alternatives for diagnosing BS which may prevent searching for the Type 1 Brugada pattern in an ECG, but these alternatives should be investigated with a larger database in order to produce a good effective predictive model.