Building pharmacogenetics into a pharmacovigilance program in Singapore: using serious skin rash as a pilot study.

To study the possible genetic associations with adverse drug reactions (ADR), the Singapore Health Sciences Authority (HSA) has piloted a program to collect DNA and phenotype data of ADR cases as part of its pharmacovigilance program. Between 2009 and 2012, HSA screened 158 cases of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). To assess the association between HLA-B*1502 and carbamazepine (CBZ)-induced SJS/TEN, 13 cases and 26 drug-tolerant controls were analyzed. All 13 CBZ-SJS/TEN cases and 3/26 controls were HLA-B*1502 positive (odds ratio 181, 95% confidence interval: 8.7-3785, P=6.9 × 10(-8)). Discussions of the finding with the Ministry of Health and an expert panel led to the decision to make HLA-B*1502 testing the standard of care prior to first use of CBZ in Asians and to subsidize the genotyping test at public hospitals. This program illustrates the role of a regulatory authority in advancing the use of pharmacogenetics for drug safety.

Advance care planning and the older patient.

Making treatment decisions for older people is difficult, because of the complex interplay of their multiple co-morbidities, but also because of the fine balance of risks vs. benefit in any chosen management plan. This becomes even more difficult when they lose the capacity to tell us what they want, and often in such situations we have to rely on information from others in order to make decisions based on their best interests. Advance care planning should help with making these decisions clearer, based on the documented preferences of what the patient would have wanted while capacity was still present. However, such documents are still very rarely used, and even if they are, health-care professionals are often wary of them for the multitude of ethical and legal problems that can arise.

Intracranial vasospasm with delayed ischaemic deficit following epidermoid cyst resection.

Intracranial vasospasm following surgical removal of intracranial tumours is rare. To our knowledge there have been no previously reported cases of delayed vasospasm occurring post debulking of epidermoid cysts. We report a case in which vasospasm led to established cerebral infarction 2 weeks postoperatively. MRI and serial magnetic resonance angiography (MRA) reliably show initial multiple stenoses in the vertebral and internal carotid arteries followed by their spontaneous normalisation. MRA imaging is now of sufficient quality to enable it to be a safe and effective means of both looking for vasospasm and monitoring its resolution.

The effect of age on the phenotype and function of developing thymocytes.

The immune system declines with age leading to a progressive deterioration in the ability to respond to infection and vaccination. Age-associated thymic involution is one of the most recognized changes in the ageing immune system and is believed to be a major contributor towards immunosenescence; however, the precise mechanisms involved in age-associated thymic involution remain unclear. In order to gain further insight into the effect of ageing on T-cell development, steady-state thymopoiesis was studied in mice ranging from 1 to 18 months of age. There was a decrease in thymic cellularity with age, but the most dramatic loss occurred early in life. Although there were no alterations in the proportion of the major thymocyte subsets, there was a significant decline in the expression of other key molecules including CD3 and CD24. There was a decline in the ability of thymocytes from older mice to respond to mitogens, which was demonstrated by a failure to up-regulate expression of the activation marker CD69 and to enter the G(2)--M phase of the cell cycle. This was concurrent with an increased resistance to apoptosis in thymocytes from aged animals. Together, these results suggest that T cells may be flawed even before exiting to the periphery and that this could contribute to the age-associated decline in immune function.

Oral pemphigus vulgaris: a case report and literature update.

INTRODUCTION: Pemphigus vulgaris is a rare cause of oral mucosal ulceration. CLINICAL PICTURE: A 47-year-old Chinese man presented with a 3-month history of oral ulceration. There were no lesions on the skin or other mucosal sites. Histology and immunostaining were consistent with pemphigus vulgaris. TREATMENT: Systemic and topical corticosteroids were instituted, together with topical antifungals. Conventional periodontal therapy was carried out to improve gingival/oral health. OUTCOME: Control of oral ulceration was achieved with re-establishment of normal oral function. No other sites to date have been involved. CONCLUSIONS: Chronic oral ulceration can be the sole manifestation of pemphigus vulgaris, at least initially. Early recognition of this lesion may prevent delayed diagnosis and inappropriate treatment of a potentially chronic dermatological condition.

Delusional parasitosis: case series of 8 patients and review of the literature.

INTRODUCTION: Delusional parasitosis (DP) is a condition in which a person has the unshakeable and mistaken belief of being infested with parasites. PATIENTS AND METHODS: All patients with DP seen by the Division of Dermatology, Department of Medicine, National University Hospital were reviewed. The case histories of 8 Chinese patients are discussed. RESULTS: The patients with DP were predominantly women above 50 years of age, with medical co-morbidities and presented with a variety of symptoms and signs. The patients were prescribed various antipsychotics including the atypical antipsychotics. Response was variable, and the patients often defaulted follow-up. An approach to the management of DP is proposed together with a review of the literature.

Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.

Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR.

Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. There is a need for improved tests to identify Rhodococcus species. PCR-based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species-specific sequences in the 16S rRNA gene, were successfully developed for R. globerulus, R. erythropolis, R. opacus and R. ruber. These tests gave positive results with all or most strains of target species but did not generally cross-react with other species. Cases of apparent cross-reaction were shown to be due to prior misclassification of strains of R. opacus as R. erythropolis and of strains of R. ruber as R. rhodochrous. A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R. erythropolis in an environmental sample. Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze-thaw cycles. Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL-6B in a spun-column format. Extracts prepared using a tris-EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds. A detection limit of 104 cfu g-1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g-1.

Identification of Rhodococcus equi using the polymerase chain reaction.

Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.

Detection of unintegrated HIV type 1 DNA in cell culture and clinical peripheral blood mononuclear cell samples: correlation to disease stage.

This article reports on the development of PCR as a sensitive method of detecting both linear and circular forms of HIV-1 unintegrated viral DNA (UVD). The method was developed in a cell line study designed to follow the sequential synthesis of these forms over time. In all T lymphoid lineage cell lines, the full-length linear UVD (LUVD) was synthesized prior to both 1 and 2 LTR forms of circular UVD (CUVD), although all forms were detected by 12 hr postinoculation. Analysis of unstimulated PBMC samples from HIV-positive patients showed a significant difference in the presence of detectable CUVD forms and CDC groups II and IV (p < 0.001) and CDC groups III and IV (p < 0.001). No significance was demonstrated between CDC groups II and III (p > 0.5), linking the presence of CUVD forms to clinical disease and immunodeficiency. We propose that circular unintegrated forms of HIV-1 DNA may play a role in the development of acquired immunodeficiency syndrome.

Susceptibility of human monoclonal antibody-producing B cell lines to infection by human immunodeficiency virus.

Seven immortalized B cell clones, five of which secreted specific human monoclonal antibodies (MAbs) against hepatitis B, tetanus toxoid, and Rhesus D antigens, were evaluated for their susceptibility to infection by human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Infection was confirmed in three human MAb-producing lines by detection of infectious virus and p24 antigen in culture supernates, by immunofluorescence, and by detection of viral DNA in cells by polymerase chain reaction. The infectable lines were as susceptible to HIV-1 infection as several T cell lines and remained persistently infected for several months, but in contrast to T cell controls, viral cytopathic effects were not observed. Levels of unintegrated viral DNA in the HB1 B cell line were significantly lower than in the HUT78 T cell line. Cell lines that were susceptible to HIV expressed HLA DR, CD20, and CD21, whereas the uninfectable cell lines did not express any of the markers tested. CD4 was undetectable or present on a small percentage of cells in two of the infectable cell lines. However, infection with HIV-1 was blocked more efficiently in B cells than in T cells by soluble CD4, anti-CD4 MAb, and dextran sulphate. The effect of HIV infection on human MAb secretion was variable, being reduced on a per-cell basis in one line, increased in another, and unchanged in a third.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction.

Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.

Human immunodeficiency virus detection: correlation with clinical progression in the Edinburgh haemophiliac cohort.

HIV p24 antigenaemia and virus detection in cultures of peripheral blood lymphocytes were examined in 16 of 18 haemophiliacs infected with HIV by a single batch of Scottish National Blood Transfusion Service factor VIII concentrate. Six (38%) had p24 antigenaemia and 11 (69%) had positive lymphocyte cultures. All seven patients with serious HIV disease (CDC group IV) had positive lymphocyte cultures whereas four (57%) had p24 antigenaemia. Four of nine (44%) patients with asymptomatic HIV disease (CDC groups II and III) had positive cultures and two (22%) had p24 antigenaemia. Twenty-eight of 36 samples from the symptomatic group were HIV culture positive compared with nine of 30 samples from the asymptomatic group (P less than 0.001). None of 14 antibody negative haemophiliacs who also received the implicated batch of factor VIII had p24 antigenaemia or positive HIV cultures. The ability to detect HIV in cultured lymphocytes correlates with the clinical severity of HIV disease in this cohort.

Immunohistology of the skin rash associated with acute HIV infection.

An immunohistological study of the rash associated with acute HIV infection in a homosexual man was undertaken. The salient feature was an infiltrate of mononuclear cells predominantly of the CD4+ phenotype around the superficial dermal vessels. HIV p24 antigen was detected in occasional cells, possibly Langerhans' cells, of the infiltrate. Their presentation of HIV antigen to local CD4+ cells may lead to a delayed type hypersensitivity reaction.