REALMS study: real-world effectiveness and safety of fingolimod in patients with relapsing-remitting multiple sclerosis in Portugal.

BACKGROUND: Fingolimod, an oral sphingosine 1-phosphate receptor modulator, is approved by EMA for relapsing-remitting multiple sclerosis (RRMS). OBJECTIVES: To assess the effectiveness and safety of fingolimod in patients with RRMS in real-world clinical practice in Portugal. METHODS: Retrospective, multicentre, non-interventional study, reporting 3 years follow-up of data collected from October 2015 to July 2016. Sociodemographic data and previous treatments at baseline and data regarding disease evolution, including number of relapses, annualised relapse rates (ARR) and Expanded Disability Status Scale (EDSS), were collected. RESULTS: Two-hundred and seventy-five participants were enrolled in the REALMS study. Results showed that the main reason to switch to fingolimod was failure of previous treatment (56.7%) and only 3.6% were naïve patients. In the total population, there was a significant decrease in ARR of 64.6% in the first year of treatment, 79.7% in the second year and 82.3% in the third year, compared with baseline. More than 67.0% of patients had no relapses during the 3 years after switching to fingolimod. EDSS remained stable throughout the study. CONCLUSIONS: Therapy with fingolimod showed a sustained effectiveness and safety over the 3 years, particularly on patients switched from first-line drugs (BRACE). No new safety issues were reported.

Optical Stability of High-translucency Resin-based Composites.

This study investigated the stability of the optical properties of high-translucent shades of dental resin-based composites. Four commercial materials (Filtek Z350 XT, Opallis, Amelogen Plus, and IPS Empress Direct) and 14 non-Vita shades were tested. Disc-shaped specimens for each resin-based composite-shade combination (n=6) were evaluated at T0 (baseline), T1 (after 30 days of storage in water), and T2 (after 30 days of storage each in water and a coffee solution). Color measurements were performed according to the L'C'h' color system. Translucency Parameter (TP) and CIEDE2000 color difference (ΔE00) were calculated. Data were statistically analyzed at α = 0.05. Baseline TP values varied from 43 ± 1 to 55 ± 1. Changes in TP at T1 varied from -3.0% (Opallis T-Neutral) to 4.2% (Amelogen Plus Trans Orange), with no major differences from T0. At T2, most resin-based composites showed significantly increased opacity, with changes varying between -15.0% (Empress Direct Trans 20) and -2.7% (Z350 XT Blue). However, the TP values were ≥40 throughout the study. Storage in water caused negligible color differences, with ΔE00 values at T1 ≤ 0.9 ± 0.6. At T2, all materials tested showed significant color difference, and ΔE00 ≥ 3.2 ± 0.2. The orange shades from Opallis and Amelogen Plus showed lower color variation than did the other shades. The most significant optical changes upon storage were detected in the hue and particularly the chroma color coordinate. In conclusion, the high-translucent resin-based composites showed large variability in the stability of their optical properties among the tested brands and different shades of the same material. Regardless of the storage condition, the tested resin-based composites retained their high-translucency character over time.

Metaflumizone is a novel sodium channel blocker insecticide.

Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips-Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide.

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs.

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches.

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.

Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer.

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Right hemifield alexia without hemianopia.

We studied a 54-year-old man who suffered a stroke in the visual-association areas of the left hemisphere. He showed no right-sided visual field defect and results of a neuropsychologic examination were normal except for his performance in several tasks presented to the right visual field. Naming of drawings was normal in both hemifields but reading of letters, words, digits, and arithmetic symbols was defective in the right field. We believe that the lesion in the visual association areas of the left hemisphere that did not interrupt interhemispheric connections disconnected the primary visual areas from temporoparietal areas of the left hemisphere, affecting reading.

Maintenance of GABA receptor function of small-diameter cockroach neurons by adenine nucleotides.

Small diameter (<20 microm) neurons from the sixth abdominal ganglion of the American cockroach, Periplaneta americana, were enzymatically isolated and responses to exogenously applied gamma-aminobutyric acid (GABA) were recorded using the whole-cell patch clamp technique. With a minimal intracellular medium, responses to repeated applications of GABA decreased to zero within a few minutes. The rate of rundown of GABA responses was decreased by the intracellular inclusion of the phosphatase inhibitors microcystin and okadaic acid, suggesting that phosphorylation is necessary for the maintenance of cockroach GABA receptor function. ATP (5 mM) prevented GABA response rundown. ADP (5 mM) also slowed GABA response rundown, but responses stabilized at a level about half that seen with ATP. In the presence of protein kinase A inhibitory peptide (PKI), ATP was only as efficacious as ADP in slowing rundown. PKI had no effect on the ability of ADP to slow rundown, suggesting that the beta-phosphate of ADP is not involved in PKA-dependent phosphorylation of the GABA receptor. These results suggest that in cockroach neurons, GABA receptor function is maintained intracellularly by adenine nucleotides, not only by phosphorylation, but also possibly by an interaction with a nucleotide recognition site unrelated to PKA-dependent phosphorylation.

Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels.

The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin ShI), Condylactis gigantea (toxin CgII) and Calliactis parasitica (toxin CpI) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon. All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). ShI and CgII were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels. All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation. Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 msec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 msec) time constant. The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between -60 and -20 mV and then increase at more positive potentials. All three toxins shifted the peak sodium current-voltage relation to the left. This voltage shift was greater at 20 degrees C than at 10 degrees C. Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents. Our results indicate that the three crustacean-active sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level.

Block of neuronal voltage-dependent K+ channels by diacylhydrazine insecticides.

RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a novel insect growth regulator, also produces acute neurotoxic symptoms by selectively blocking the maintained voltage-dependent K+ current (IK) in nerve and muscle Salgado (1992). The effects of RH-5849 and an analog were examined on IK channels in internally-perfused crayfish giant axons. For bilaterally applied RH-5849, the concentration needed for 50% block (IC50) was 79 +/- 6 microM (mean +/- SEM, n = 3), with a Hill coefficient near 2. Block was independent of membrane potential, but dependent on time, with a speed proportional to concentration, suggesting an open channel block mechanism. In addition to their effects on IK, both diacylhydrazines were much weaker blockers of the voltage-dependent sodium current (INa). RH-5849 blocked IK from either side of the membrane, and was more potent when applied bilaterally. When RH-5849 was introduced inside the axon and internal perfusion was halted, IK increased within a few minutes to the control level, indicating that the compound diffused freely through the membrane and bound to a receptor within the plane of the membrane. The permeability coefficients measured in the stopped-flow experiments indicate that diacylhydrazines can diffuse readily throughout the body of a poisoned insect, consistent with the rapid onset of central nervous system symptoms following injection. The octanol:water partition coefficient of RH-5849 increased sharply from 145 to 258 at aqueous concentrations between 5 and 10 microM, suggesting that a new phase, possibly micellar, is formed in the octanol phase. This may be responsible for the anomalously high Hill coefficients for the channel blocking activity of the diacylhydrazines.

Interactions of the pyrethroid fenvalerate with nerve membrane sodium channels: temperature dependence and mechanism of depolarization.

Depolarization of nerve membranes is an important component of the mode of action of pyrethroids, and its negative temperature dependence parallels that of insecticidal activity. We studied the mechanism and temperature dependence of depolarization of crayfish giant axons by pyrethroids, using intracellular microelectrode and voltage clamp techniques. Membrane depolarization caused by tetramethrin and fenvalerate was greater at 10 degrees C than at 21 degrees C, and was reversible upon changing the temperature. Short-duration depolarizing pulses in voltage-clamped fenvalerate-treated axons induced prolonged sodium currents that are typical of other pyrethroids, but the decay of the tail current following repolarization was extremely slow, lasting several minutes at the large negative holding potential of -120 mV. At the normal resting potential, the tail current did not decay completely, and even without stimulation, a steady-state sodium current developed, which could account for the depolarization. The steady-state current induced by fenvalerate at the resting potential was much larger at 8 degrees C than at 21 degrees C, accounting for the negative temperature dependence of the depolarization. The negative temperature dependence of the steady-state current seems to be due ultimately to the great stabilizing effect of low temperature on the open-modified channel. When the steady-state current was induced at the resting potential, hyperpolarization to more negative potentials caused it to decay with exactly the same time course as tail currents induced by short-duration depolarizing pulses, indicating that both types of currents are carried by identically-modified channels. The modified channels were shown to be inactivated very slowly at potentials more positive than - 100 mV, accounting for the limited depolarization observed in micro-electrode experiments. Even when applied directly to the internal face of the membrane, the effect of fenvalerate on the sodium channel developed slowly, taking more than 90 min to reach its final level. Fenvalerate did not significantly affect potassium currents.

Effect of electrical stimulation of the baso-lateral amygdala nucleus on defensive burying shock probe test and elevated plus maze in rats.

In the present report, the putative effect of a single electrical stimulation (75, 150 or 300 microA) to the baso-lateral amygdala (BLA) nucleus was assessed in shock probe defensive burying behavior test (DB) and elevated plus maze (EPM). These models have been used for measuring anxiety levels and screening putative anxiolytic compounds. A group of 28 rats were randomly divided for the following experimental conditions: Control-control, sham-operated, BLA stimulated groups: 75, 150 and 300 microA tested for DB. The cumulative defensive burying in a 15 min-test, the latency of burying, the number of shock received and the height of the bedding material in the probe were recorded. Another group of 28 individuals was also randomly distributed for the following experimental conditions: Control-control, sham-operated, BLA stimulated animals: 75, 150, 300 microA and tested in the EPM. The time the subjects spent in the open arms, the crosses and the faeces number excreted during the test were recorded. Decreased levels of defensive burying were observed in 75, 150 and 300 microA stimulated groups. The 150 and 300 microA groups reached statistical significance. The fact that 300 microA stimulated group showed statistically significant increase in the latency of defensive burying, in the number of shock received and decreased amount in bedding material suggests a sedative action of electrical stimulation. Increased time in the open arms and augmented number of crossings in 150 microA group was observed. No changes in the number of faeces were observed in any group. The evidence supported the notion of an inhibitory amygdaline mechanism triggered by sub-threshold electrical stimulation.

[Neuroleptic malignant syndrome. Analysis of 9 cases]. / Síndrome neuroléptico maligno. Análisis de 9 casos.

The malignant neuroleptic syndrome (MNS) is an idiosyncratic reaction to neuroleptic drugs. Although it was described in the sixties, it is still a poorly known condition. Controversy still exists about its conceptual frame, and its true incidence is still unknown. We have evaluated 9 cases of MNS seen in a 37 month period. There were 5 females, with a mean age of 50 years. Haloperidol, either single or in association, was the most commonly implicated drug. The estimated frequency of MNS in our population at risk was 1.5%. Eight patients had complications and 5 died from them (55%). We discuss the pathogenetic mechanisms, the conceptual characterization and the current diagnostic criteria. We evaluate the causes of the high mortality in our series. We think that a higher suspicion index of this condition would be desirable, and we recommend early treatment with vigorous supportive measures and drug therapy. We think that prospective studies are required to assess the true incidence of MNS.

[Acute cerebrovascular disorder and arterial hypertension. Prospective study with 248 patients]. / O acidente vascular cerebral agudo e a hipertensão arterial. Estudo prospectivo com 248 doentes.

OBJECTIVE: To evaluate the hypertension associated to different types and sub-types of cerebrovascular disease (stroke), with particular reference to the frequency of hypertension, the values of blood pressure, the risk factors and the involvement of other target organs. DEFINITION: Prospective study in 248 patients with acute stroke admitted to a Clinical Medicine Unit in three independent time periods. SETTING: Internal Medicine Clinic of University Hospital in Lisbon. METHODS: Medical, neurological and cardiologic examination were performed and all patients were also submitted to different complementary tests, including a computer tomography scan of the brain, and an echocardiogram. The values of blood pressure were measured in the admission at the urgent service and 24 h after in the the ward. We identified three sub-types of stroke: intracerebral hemorrhage (IH), ischaemic stroke (IS) and lacunes (L). For each sub-type and for those with hypertension or not, we evaluated: age, sex, duration of stay in hospital and mortality. We also compared for each sub-type the values of blood pressure, the risk factor and the repercussion on other target organs. PATIENTS: Two hundred and forty eight patients (52% were men) with mean age 68.0 +/- 10.2 years, and ages among 40 and 92 years. Thirty-seven patients (15%) died. MAIN RESULTS: In the entire population (n = 248) hypertension were more prevalent in IH 83% and L 82% than in IS 59% (p < 0.0005). Hypertension was present in 172 patients (69%) and 81 (47%) were IS, 58 (34%) L and 33 (19%) IH. Sixty six percent of the 172 patients with hypertension had at least another risk factor and the most aged ones (> 65 years old) were more frequent in IS 75% than in HI 45% or L 58% (p < 0.001). For all subtypes blood pressure measurements were higher in admission than in ward and they were also higher in IH than in IS (p < 0.05). Hypertensive cardiopathy was more prevalent in IH 76% and L 61% than in IS 49% (p < 0.05). Renal failure was more frequent in IS 37% than in IH 28% and L 17% (p < 0.05). CONCLUSIONS: Hypertension is very frequent, and like advanced age is a major risk factor of stroke. Hypertension is frequently associated with one or more risk factors. Hypertensive cardiopathy is more related with IH and L and the renal involvement with IS.

[Acute cerebrovascular accident and heart disease. Prospective study of 248 patients]. / O acidente vascular cerebral agudo e a doença cardíaca. Estudo prospectivo com 248 doentes.

OBJECTIVE: To evaluate the heart disease associated to different type and sub-types of cerebrovascular disease with particular reference to potential cardiac sources of embolus (CPE). DESIGN: Prospective study in 248 consecutive patients with acute stroke, admitted to a Clinical Medicine Unit in three independent time periods. SETTING: Internal Medicine Clinic of University Hospital in Lisbon. METHODS: Neurologic and cardiologic examination were performed and all patients were also submitted to different complementary tests, including a Computer Tomography Scan of the brain (TAC) and a Echocardiogram (ECO). We identified two types and two sub-types: intracerebral haemorrhage (HI) or ischemic stroke (AI) and among AI, cortical (C) or subcortical (SC) ischemic stroke. For each type and sub-type we evaluated past history, heart disease, hypertension (HTA), electrocardiogram, echocardiogram (ECO) and CPE. PATIENTS: Two hundred and forty eight patients (52% were men) with mean age 68.0 +/- 10.2 years and ages between 40 and 92 years. Thirty seven (15%) died. MAIN RESULTS: Eighty four percent were AI and among them 45% were C. Previous strokes were more prevalent in AI 29% (p < 0.01). There was heart disease in 81% and hypertensive cardiopathy was more frequent in HI 63% (p < 0.002). HTA and atrial fibrillation (FA) were more frequent in HI 83% (p < 0.05) and in AI 25% (p < 0.004) respectively. ECO showed a dilatation of left atrium more frequent in AI 28% (p < 0.05) and left ventricular concentric hypertrophy index (IHCE) in HI 50% (p < 0.05). CPE, including FA, was identified in 34% of patients, was more prevalent in AI 38% (p < 0.001) and among it FA was significantly more frequent in C 32% (p < 0.02). CONCLUSIONS: Heart disease is very frequent in stroke. The diagnosis of this condition is very important for stroke prophylaxis and prognosis. HTA and hypertensive cardiopathy have a great prevalence and were more related to HI. CPE and FA were very frequent and their diagnosis are important for prevention of AI and specially for embolic stroke. ECO is useful to identify CPE in elderly patient in particular to characterize heart disease.

¿Qué significa estudiar terapia ocupacional? La perspectiva de estudiantes de la Universidad de Chile / What does studying occupational therapy means? The perspective of students of the University of Chile

El objetivo de este trabajo es analizar la percepción atribuida a estudiar Terapia Ocupacional según los estudiantes de la Universidad de Chile. La investigación es de tipo cualitativa fenomenológica, pues se pretende estudiar la dimensión subjetiva de un fenómeno en particular denominado "estudiar Terapia Ocupacional". Para recolectar los datos, se realiza una entrevista semiestructurada abierta. El análisis de los datos se inicia con la categorización de acuerdo a las dimensiones significado, sentido y propósito que se describe en el marco de referencia, y las subcategorías y códigos a partir de los relatos.En los resultados se observa que en la categoría significado, la opinión externa hace referencia a "estudiar Terapia Ocupacional" y a "estudiar en una universidad" en sí, encontrando principalmente desconocimiento y percepción positiva hacia la carrera. En la dimensión sentido se identifican factores que otorgan valor positivo y/o negativo que influyen en la autoeficacia, la importancia, la motivación, el interés y el disfrute, destacándose entre ellos, el enfoque biopsicosocial y el trabajo directo con las personas. Finalmente en el propósito se identifican metas y utilidades, tanto personales como profesionales.Dichos resultados, promueven la reflexión en torno a la visión que se tiene de la carrera y valoración de los estudios profesionales.

The main goal of this work is to analyze the attributed perception of studying occupational therapy according to the students of University of Chile. A qualitative and phenomenological methodology was used, as this investigation tries to study the subjective dimension of a particular phenomenon, such as studying Occupational Therapy. Data recollection was made with open-semistructured interviews. Data analysis initiated with a previous categorization according to meaning, sense and purpose, as described in the AOTA's framework; subcategories and codes were included from the interviews. Results show that in the Meaning category, external opinion makes reference to "studying occupational therapy" and "studying in a university", mainly finding ignorance and a positive perception of the career. At the sense dimension, factors that give positive and/or negative values that influence self-efficacy, importance, motivation, interest and enjoyment are identified; these factors included the biopsychosocial approach and working with people. Finally, in the purpose dimension, goals and utilities are identified, both in a personal and professional aspects.These results prompt reflection around the perception of the career and the value given to professional studies.

Evaluation of four DNA extraction protocols for Brucella abortus detection by PCR in tissues from experimentally infected cows with the 2308 strain.

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.