Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Molecular diagnostic cytopathology: definitions, scope and clinical utility

Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Although it is not widely used in routine molecular cytological practice, some examples are presented here of the application of molecular techniques to the routine cytopathological diagnosis of solid tumours and lymphoreticular malignancies. The term 'molecular diagnostic cytopathology' is proposed to define the application of molecular diagnosis to cytopathology, and the challenges of the introduction of molecular diagnosis into routine diagnostic histopathology and cytopathology are discussed. Finally, the importance of a combined morphological, immunophenotypic and molecular approach to maintain the diagnostic pathologist at the heart of the clinical decision-making process is emphasized.

Multiplex RT-PCR for the detection of leukemia-associated translocations - Validation and application to routine molecular diagnostic practice

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [He-mavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARalpha/t (15; 17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the

Next-generation sequencing: a change of paradigm in molecular diagnostic validation

Next-generation sequencing (NGS) is beginning to show its full potential for diagnostic and therapeutic applications. In particular, it is enunciating its capacity to contribute to a molecular taxonomy of cancer, to be used as a standard approach for diagnostic mutation detection, and to open new treatment options that are not exclusively organ-specific. If this is the case, how much validation is necessary and what should be the validation strategy, when bringing NGS into the diagnostic/clinical practice? This validation strategy should address key issues such as: what is the overall extent of the validation? Should essential indicators of test performance such as sensitivity of specificity be calculated for every target or sample type? Should bioinformatic interpretation approaches be validated with the same rigour? What is a competitive clinical turnaround time for a NGS-based test, and when does it become a cost-effective testing proposition? While we address these and other related topics in this commentary, we also suggest that a single set of international guidelines for the validation and use of NGS technology in routine diagnostics may allow us all to make a much more effective use of resources.

Molecular diagnostic methods for identifying Serrasalmid fish (Pacu, Pirapitinga, and Tambaqui) and their hybrids in the Brazilian aquaculture industry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do banho de aspersão ante-mortem na contaminação microbiana da carne bovina

To determine the effects of the pre-slaughter showering on some meat quality parameters, the bacterial changes in the Longus colli muscle and the contamination of the meat surface at three different points of the slaughter line were studied. Sixteen Nelore steers were slaughtered in a commercial slaughterhouse. Eight animals were submitted to pre-slaughter showering; a control group of eight animals were slaughtered without showering. Aseptic samples were collected for evaluations in the muscle depth, in the anterior portion of Longus colli muscle, just before chilling. The swab method was used for sampling carcass surface right after dressing, before carcasse washing, and at the beginning of chilling. Longus colli muscle samples were used to determine bacteria total count, psychrotrophic count and Enterobacteriaceae count, after 5, 24 and 48 hours from slaughtering, and in carcass surface, bacteria total count and psychrotrophic count. Multivariate methods were used to evaluate bacterial data the use of pre-slaughter showering did not affect the bacteria total counts, in the deep tissue. A significant growth of psychrotrophic bacteria was detected in both treatments. No significant differences (P>.05) were found in bacteria total count and psychrotrophic count between treatments. Also, no differences (P>.05) were detected between counts taken at different momments at the kill floor: skinning, before carcasse washing, and at the cooler, before chilling.

Influência do banho de aspersão ante-mortem na eficiência da sangria e em parâmetros bioquímicos da carne bovina

To determine the effects of the pre-slaughter showering on some meat quality parameters, the biochemical changes in the Longus colli muscle and the bleeding efficiency were studied. Thirty-six Nelore steers were slaughtered in a commercial slaughterhouse. Eighteen animals were submitted to pre-slaughter showering; a control group of eighteen animals were slaughtered without showering. Samples were collected for evaluations in the muscle depth, in the anterior portion of longus colli muscle,just before chilling. Bleeding efficiency was evaluated through the ratio of muscle haemoglobin/blood haemoglobin using blood samples taken five seconds after bleeding, and muscle sample taken before chilling. Longus colli muscle samples were also used to determine glycogen, glucose, pH and acidity, 5, 24 and 48 hours after slaughtering. Multivariate methods were used to evaluate biochemical data and the bleeding efficiency data analysis followed the randomized block design. Haemoglobin retained in the muscle and bleeding efficiency were not affected (P > .05) by pre-slaughter showering. The pre-slaughter showering did not affect (P > .05) the glycolysis. There was a significant effect of time in glycogen, glucose, pH and acidity, in the first 24 hours.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

Holsback L., Pena H.F.J., Ragozo A., Lopes E. G., Gennari S. M. & Soares R. M. 2012. Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free range chickens from Pantanal of Mato Grosso do Sul. Pesquisa Veterinaria Brasileira 32(8): 721-726. Setor de Veterinaria e Producao Animal, Universidade Estadual do Norte do Parana, Campus Luiz Meneghel, Rodovia BR 369 Km 54, Bandeirantes, PR 86360-000, Brazil. E-mail: lhsfertonani@uenp.edu.br The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolandia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1: 5, 3 (11.1%) in 1: 10, 2 (7.4%) in 1: 20, 3 (11.1%) in 1: 320, 1 (3.7%) in 1: 640, 3 (11.1%) in 1: 1280, 2 (7.4%) in 1: 2560, 4 (14.8%) in 1: 5120 and 2 (7.4%) in 1: 10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1: 5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1: 25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Differentiation of ante-mortem and post-mortem fractures with MRI: a case report

We describe a case of a fatal speed flying accident in which the victim was electrocuted, burned and fell from a great height. Post-mortem imaging revealed acute appearing fractures on CT, without bone marrow oedema on MRI. Based on the known clinical imaging findings of bone marrow oedema in acute fractures, we concluded that the speed flyer died from electrocution rather than the fall and that the fractures occurred post-mortem. Radiological imaging augmented the reconstruction of the peri-mortem events. Further research is needed to assess whether bone marrow oedema in acute fractures is a reliable vital sign.

Post-mortem radiological CT identification based on classical ante-mortem X-ray examinations

Radiological identification is important in forensic medicine. Identification using comparison of individualising structures with ante- and post-mortem conventional radiographs has been known for a long time. New radiological procedures such as computed tomography (CT) and magnetic resonance imaging (MRI) are being increasingly used for identification. In this paper, a new comparative approach using various radiological methods is described and its application demonstrated. This new approach is the comparison of ante-mortem conventional radiographs with projected images calculated from post-mortem CT data. The identification procedure will be illustrated with reference to the frontal sinus and the pelvis.

Virtopsy: fatal stab wounds to the skull--the relevance of ante-mortem and post-mortem radiological data in case reconstructions

Homicides with a survival of several days are not uncommon in forensic routine work. Reconstructions of these cases by autopsy alone are very difficult and may occasionally lead to unsatisfying results. For the medico-legal reconstruction of these cases, ante-mortem and post-mortem radiological imaging should always be included in the expertise. We report on a case of fatal penetrating stab wounds to the skull in which a case reconstruction was only possible by combining the radiological ante- and post-mortem data with the autopsy findings.

Quomodo singularis illa Jesu anxietas et tristitia ante mortem, quam Lucas [agōnian] vocat, sit explicanda, nec non cum ipsius virtute et auctoritate divina concilianda /

Text in Latin, Greek, and Hebrew.