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D Howells

Publications and source records attributed to D Howells.

8 recordsLinked to original sources
PubMed2000

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Bacterial Proteins↗
PubMed2000

Localisation of HHV-8 in AIDS related lymphadenopathy.

BACKGROUND: Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposi's sarcoma (KS), malignant lymphoma (Hodgkin's and non-Hodgkin's), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. AIM: To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposi's sarcoma development. METHODS: A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). RESULTS: Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. CONCLUSIONS: The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

AIDS-Related Complex↗
PubMed2000

Cellular localisation of HHV-8 in Castleman's disease: is there a link with lymph node vascularity?

AIMS: Human herpesvirus 8 (HHV-8) has been identified in multicentric Castleman's disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis. METHODS: Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as "low" or "high". RESULTS: Five multicentric Castleman's disease and two solitary Castleman's disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10-50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. CONCLUSION: The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.

Adult↗
PubMed1998

A novel, rapid in cell RNA amplification technique for the detection of low copy mRNA transcripts.

Growing interest now focuses on improvements of in situ polymerase chain reaction (PCR) technology for the detection of DNA and RNA cellular sequences. In this study, reverse transcription PCR in situ hybridisation (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripheral blood lymphocytes (PBLs). The success of in cell RNA amplification depends on the type of cell/tissue fixation, cell permeabilisation, and the efficiency of reverse transcription and cDNA amplification. This paper presents new approaches to overcome the critical aspects of fixation, permeabilisation, and reverse transcription when performing in cell RNA amplification. A novel fixative, "Permeafix", possessing fixative and permeabilisation properties, was used for cell fixation procedures. "Permeafix" obviated the need for pre-amplification proteolysis, facilitating entry of PCR reagents to target sequences within the cell. In addition, a simple on step RNA in cell amplification protocol using recombinant Thermus thermophilus (rTth) DNA polymerase, which reverse transcribes mRNA efficiently to cDNA and then catalyses cDNA amplification, was used. The value of a semi-junctional primer system for in cell gene expression studies, without the need to perform DNase digestion, is demonstrated.

DNA Primers↗
PubMed1995

Laryngograph: speech pattern element tools for therapy, training and assessment.

The clinically based, real-time analysis of speech into physically definable elements, which are of direct perceptual and productive importance, has become more readily possible in recent years as the result of microprocessor developments. The combination of the acoustic signal of speech derived from a microphone together with the accompanying Laryngograph signal provides the basis for a highly reliable set of facilities. The paper describes methods and results for the analysis of voice, frication and timbre for both quantitative analysis and teaching and therapy using interactive visual displays. A brief discussion is given of links to work in stroboscopy, electropalatography and the associated use of additional sensors. Finally reference is made to a complete clinical work station combining these different facilities together with the quantitative analytical procedures of the speech pattern audiometer.

Humans↗
PubMed1995

Speech pattern audiometry for clinical use.

A new PC-based speech perception testing system ('speech pattern audiometer') is described which aims to provide a simple and efficient clinical tool to assess patients' ability to make use of the acoustic speech pattern information which is fundamental to speech perception. The speech pattern audiometer is likely to find its main application in audiology and speech and language therapy clinics with those who are deaf or who have developmental or acquired speech-perceptual disorders. This system constitutes a module of a complete speech and hearing assessment and teaching/therapy workstation.

Audiometry, Speech↗