Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates

A multiplex polymerase chain reaction (PCR) assay was performed on 167 thermophilic campylobacters isolated from non-human primates. Samples were first identified by phenotypic methods resulting in 64 Campylobacter jejuni and 103 C. coli strains. Four strains identified biochemically as C. coli, were then determined to be C. jejuni by PCR. Comparison of methodologies showed that the main discrepancies were attributed to the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Analysis of data showed that the application of phenotypic methods should be supplemented by a molecular method to offer a more reliable Campylobacter identification.

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

Struggles for meaning and struggles for control: the diffusion of bandwagon technology in two institutional environments

In this thesis, I examine the diffusion process for a complex medical technology, the PET scanner, in two different health care systems, one of which is more market-oriented (Switzerland) and the other more centrally managed by a public agency (Quebec). The research draws on institutional and socio-political theories of the diffusion of innovations to examine how institutional contexts affect processes of diffusion. I find that diffusion proceeds more rapidly in Switzerland than in Quebec, but that processes in both jurisdictions are characterized by intense struggles among providers and between providers and public agencies. I show that the institutional environment influences these processes by determining the patterns of material resources and authority available to actors in their struggles to strategically control the technology, and by constituting the discursive resources or institutional logics on which actors may legitimately draw in their struggles to give meaning to the technology in line with their interests and values. This thesis illustrates how institutional structures and meanings manifest themselves in the context of specific decisions within an organizational field, and reveals the ways in which governance structures may be contested and realigned when they conflict with interests that are legitimized by dominant institutional logics. It is argued that this form of contestation and readjustment at the margins constitutes one mechanism by which institutional frameworks are tested, stretched and reproduced or redefined.

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R²) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Representing diffusion MRI in 5D for segmentation of white matter tracts with a level set method.

We present a method for segmenting white matter tracts from high angular resolution diffusion MR. images by representing the data in a 5 dimensional space of position and orientation. Whereas crossing fiber tracts cannot be separated in 3D position space, they clearly disentangle in 5D position-orientation space. The segmentation is done using a 5D level set method applied to hyper-surfaces evolving in 5D position-orientation space. In this paper we present a methodology for constructing the position-orientation space. We then show how to implement the standard level set method in such a non-Euclidean high dimensional space. The level set theory is basically defined for N-dimensions but there are several practical implementation details to consider, such as mean curvature. Finally, we will show results from a synthetic model and a few preliminary results on real data of a human brain acquired by high angular resolution diffusion MRI.

Variabilité intra/interobservateur du coefficient de diffusion apparente dans les lésions hépatiques malignes traitées

Objectifs : Le coefficient de diffusion apparente (ADC) est utilisé pour le suivi des lésions hépatiques malignes traitées. Cependant, l'ADC est généralement mesuré dans la lésion entière, alors que cela devrait être réalisé dans la zone la plus restreinte (ZLPR), cette dernière représentant potentiellement du résidu tumoral. Notre objectif était d'évaluer la variabilité inter/intraobservateur de l'ADC dans la tumeur entière et dans la ZLPR. Matériels et méthodes : Quarante patients traités par chimioembolisation ou radiofréquence ont été évalués. Après consensus, deux lecteurs ont indépendamment mesuré l'ADC de la lésion entière et de la ZLPR. Les mêmes mesures ont été répétées deux semaines plus tard. Le test de Spearman et la méthode de Bland-Altman ont été utilisées. Résultats : La corrélation interobservateur de l'ADC dans la lésion entière et dans la ZLPR était de 0,962 et de 0,884. La corrélation intraobservateur était de 0,992 et de 0,979, respectivement. Les limites de variabilité interobservateur (mm2/sec*10 - 3) étaient entre -0,25/+0,28 dans la lésion entière et entre -0,51/+0,46 dans la ZLPR. Les limites de variabilité intraobservateur étaient respectivement : -0,25/+0,24 et -0,43/+0,47. Conclusion : La corrélation inter/intraobservateur dans les mesures d'ADC est bonne. Toutefois, une variabilité limitée existe et doit être considérée lors de l'interprétation des valeurs d'ADC des tumeurs hépatiques.

Ancient medical texts, modern reading problems

The word tradition has a very specific meaning in linguistics: the passing down of a text, which may have been completed or corrected by different copyists at different times, when the concept of authorship was not the same as it is today. When reading an ancient text the word tradition must be in the reader's mind. To discuss one of the problems an ancient text poses to its modern readers, this work deals with one of the first printed medical texts in Portuguese, the Regimento proueytoso contra ha pestenença, and draws a parallel between it and two related texts, A moche profitable treatise against the pestilence, and the Recopilaçam das cousas que conuem guardar se no modo de preseruar à Cidade de Lixboa E os sãos, & curar os que esteuerem enfermos de Peste. The problems which arise out of the textual structure of those books show how difficult is to establish a tradition of another type, the medical tradition. The linguistic study of the innumerable medieval plague treatises may throw light on the continuities and on the disruptions of the so-called hippocratic-galenical medical tradition.

Reaction to novelty and spatial orientation in ALPHA1B(-/-) KO mice

Knockout mice lacking alphalb noradrenergic receptors were tested in behavioural experiments to test a possible effect of the absence of this receptor in reaction to novelty and spatial orientation. Reaction to novelty was tested in two experiments. In the first one the mice' latency to exit the first part of a two compartment set-up was measured. The knockout mice were faster to emerge then their littermate controls. Then they were tested in an open-field, in which new objects were added at the second trial. In the open-field without objects (first trial), the knockout mice showed a greater locomotor activity (path length). Then the same mice showed enhanced exploration of the newly introduced objects, relative to the control. The spatial orientation experiments were done on a homing board and in the water maze. The homing board did not yield a significant difference between the knock-out and the control mice. Both groups showed impaired results when the proximal (olfactory) and distal (visual) cues were disrupted by the rotation of the table. In the water maze however, the alphalb(-/-) mice were unable to solve the task (acquisition and retention), whereas the control mice showed a good acquisition and retention behaviour. The knockout mice' incapacity to learn to reach the submerged platform was not due to an incapacity to swim, as they were as good as their control littermates to reach the platform when it was visible.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

Sensitivity and specificity of polymerase chain reaction in Giemsa-stained slides for diagnosis of visceral leishmaniasis in children

The aim of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) in the detection of Leishmania DNA in archived Giemsa-stained bone marrow slides for diagnosis of visceral leishmaniasis (VL), and to compare PCR with conventional diagnostic techniques, like direct microscopy and parasite culture. Specimens of archived Giemsa-stained bone marrow slides from 91 patients with VL and from 79 controls with other diseases or conditions were studied. PCR showed the highest sensitivity (92.3%) and had good specificity (97.5%). Direct examination detected 79.1% and culture 59% of positive samples. In addition, PCR was able to detect VL in 16 of 19 patients (84.2%) with negative microscopy. PCR in Giemsa-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may be useful in the diagnosis of difficult cases. Slide smears are easily stored, do not require special storage conditions such as low temperatures, and can be easily mailed to centers where PCR is available, making it an excellent option for diagnosis in the field.