Neural transplantation in Huntington's disease: the NEST-UK donor tissue microbiological screening program and review of the literature.

Neural transplantation of human fetal tissue for Huntington's disease (HD) is now entering the clinical arena. The safety of the procedure has now been demonstrated in a number of studies, although the efficacy of such an approach is still being investigated. Stringent but practicable screening of the donor tissue for potential pathogens is an essential prerequisite for successful implementation of any novel transplant program that uses human fetal tissue. In this article we summarize the UK-NEST protocol for the screening of human fetal tissue being grafted to patients with mild to moderate HD. We describe the results of microbiological screening of 87 potential tissue donors in a pilot study, and of the first four donor-recipient patients included in the UK-NEST series. The rationale for the adoption and interpretation of the various tests is described and our methodology is compared with those previously used by other centers. This article therefore presents a comprehensive, logical yet pragmatic screening program that could be employed in any clinical studies that use human fetal tissue for neurotransplantation.

Candidaemia in a large teaching hospital: a clinical audit.

BACKGROUND: Candidaemias are associated with significant morbidity and mortality. The British Society of Medical Mycology and Infectious Diseases Society of America recently published audit standards, to address the changing epidemiology of candidaemia and to improve outcomes. AIM: To investigate the local epidemiology of candidaemia and the standard of care in a large teaching hospital. DESIGN: Retrospective audit. METHODS: Data were obtained for all candidaemia episodes over the 4-year period ending July 2004, from the medical and nursing notes, laboratory computer and patient administration system. RESULTS: We identified 92 episodes in 90 patients. The main predisposing factors were being on an intensive care unit, having a central venous catheter, and (for neonates) prematurity. Central venous catheters were removed at a mean 1.8 days following candidaemia; 79% (37/47) were removed within 48 h (the audit standard). Identification and susceptibility tests were performed for 94.7% of isolates. All were susceptible to amphotericin B; 87% were susceptible to fluconazole. Antifungal treatment was started within 24 h of a positive blood culture in 84% of episodes. Initial antifungal therapy was appropriate in 95% (61/64) of treated cases. Most patients (81%) who survived or completed their intended course of treatment before death received at least 2 weeks treatment. However, only 45% of those transferred to other hospitals had accompanying guidance on the intended further duration of therapy. Thirty-day mortality was 41%. After adjustment for age, the presence of Candida-related complications was associated with an odds ratio for mortality of 6.5 (95% CI 1.2-36.5, p = 0.03). DISCUSSION: Overall the audit standards set by the BSMM and IDSA were met, and discrepancies did not lead to a change in outcome. Improved intravenous catheter care, a more pro-active approach to searching for complications, and improvement in the inter-hospital transfer process, will assist in reducing morbidity and mortality.

Regional specificity of HTLV-I proviral integration in the human genome.

The location of HTLV-I (human T-cell leukemia virus type 1) proviral sequences in the genome of infected human cells was explored by hybridization of a viral probe with compositional fractions of host-cell DNAs. In the twelve cases examined, HTLV-I sequences were absent from the GC-poorest 40% of the host genome (namely, from isochores that are below 39% GC). Transcriptionally inactive proviral sequences were localized in GC-poor isochores (comprised between 39% and 42-44% GC) of the human genome, which are characterized by a constant and low gene concentration. In contrast, transcriptionally active proviral sequences were found in the GC-rich and very GC-rich isochores, which are gene rich, transcriptionally and recombinationally active, and endowed with an open chromatin structure. Since GC-rich isochores are present in R'-bands and very GC-rich isochores form T-bands, these results also provide information on the location of HTLV-I proviral sequences in human chromosomes. The results obtained with HTLV-I are in agreement with the non-random, compartmentalized integration of animal retroviral sequences that had been previously observed in other viral-host systems. They provide, however, much more detailed information on the regional location of proviral sequences in the host genome and on the correlation between their transcription and their location.

Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1.

We have previously mapped the sequences required for dimerisation of the 5' leader of the human T-cell leukaemia virus type-1 (HTLV-1) genome. The smallest sequence necessary and sufficient for dimer formation, in vitro, was ascertained to be a 37 nucleotide (nt) region downstream of the splice donor and just upstream of the primer binding site. Deletion of a 32 base-pair sequence encompassing this region within the provirus was associated with a minor decrease in infectivity of the virus in an in vitro system. To further map and help elucidate the nature of the dimer linkage, we used RNA and DNA oligonucleotide competition assays to define the nucleotides involved. These experiments revealed that a 14 nt sequence containing a potential stem loop structure, formed from a palindromic sequence, is important for dimer formation. This was confirmed by the ability of this RNA sequence to form heterodimers with larger RNA transcripts from the same region, while sequences lacking this motif could not. RNA transcripts containing the reverse sequence, the same nucleotides in a random arrangement, and complementary DNA oligos, all failed to form heterodimers with the 14 nt sequence. The primary dimer initiation site of HTLV-1 has thus been located to a 14 nt palindrome containing sequence, and dimerisation is shown to be dependent on specific sense-sense RNA interactions.

Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production.

Reverse transcription is a key aspect of the retroviral life cycle. The enzyme reverse transcriptase requires divalent cations, manganese or magnesium, for function. In some cation-dependent systems substitution of a physiological metal by a nonphysiological metal has been shown to work. We investigated the effect of different cations on HIV reverse transcriptase activity. The studies established reaction conditions for assaying different cations. A variety of transition metals were used in in vitro assays with HIV recombinant RT homodimer and some were delivered to HIV-infected cells in vitro to study effects on virus production. Most metals substituted adequately for magnesium. However, palladium showed a marked nonreversible inhibition of RT activity in vitro that correlated with reduced HIV virus production in tissue culture. A more extensive range of transition metals and divalent cations was tested for their effects on detection of HIV RT from infected cell supernatants. In these complex phenotypes were seen. In some cases the RT activity appeared to be more easily detectable. This may relate to calcium-dependent nucleases in cell supernatants being inhibited, leading to an apparent enhancement of RT activity, or may be due to direct effects on RT processivity.

Human lymphoblastoid interferon does not increase survival when added to mitozantrone in the treatment of hepatocellular carcinomas.

Human lymphoblastoid interferon, in an initial dose of 2.5 MU m-2 weekly i.m., was given with mitozantrone 12 mg m-2 i.v. every 3 weeks to 15 patients with hepatocellular carcinoma. The survival curve for these patients was worse than that of 15 patients previously treated with mitozantrone alone; there were more long-term survivors in those not given interferon; more side-effects were seen in the group given interferon. The addition of interferon to mitozantrone in the management of hepatocellular carcinoma is not recommended.

Lentiviral vectors: progress and potential.

Lentiviral vectors are continuing to generate great interest as gene vectors for possible gene therapy in vivo. Over the past year, considerable progress has been made in demonstrating gene delivery to a wide variety of tissues, taking advantage of their unique capability of integrating the desired gene into the chromosomes of the target cell. The basic science of lentivirus vector production has expanded and their relative ease of utility is reflected by the broadening range of laboratories reporting successful gene transfer using them. Important new findings on RNA processing within the cell have come from studies of vectors which have direct relevance to the molecular biology of HIV itself. Although none have been used in clinical trials in vivo, it can only be a matter of time before this occurs. This article reviews publications over the last 12 months, highlighting the major achievements in this field.

The impact of new diagnostic methodologies in the management of meningitis in adults at a teaching hospital.

BACKGROUND: Suspected meningitis is a frequent reason for admission to hospital in the UK. While bacterial meningitis requires prompt antibiotic therapy to reduce mortality and morbidity, enteroviral meningitis, the most frequent viral cause, is almost invariably a benign disease. AIM: To determine the clinical presentation, laboratory findings and outcome of meningitis by microbiological aetiology and patient age, and to assess the clinical management of adults presenting with meningitis, with reference to national guidelines. DESIGN: Retrospective case-note review. METHODS: Adult (>14 years) admissions to Addenbrooke's Hospital with meningitis or meningococcal septicaemia March 1996-September 2001 were audited retrospectively. The case definition was: symptoms compatible with meningitis, and either abnormal CSF (leukocytes >5x10(9)/ml) or meningococcal disease. The only exclusion criterion was the presence of a ventricular shunt. RESULTS: Only 30% of patients seen by a General Practitioner were given pre-admission antibiotics. In a substantial number of cases, including those with bacterial meningitis, antibiotic administration was delayed either because patients were sent for CT head scans (delaying a lumbar puncture) or because the diagnosis was not considered, especially in elderly patients with reduced conscious levels. There were no confirmed cases of H. influenzae meningitis. Overall outcomes in terms of mortality and disability were similar to UK national data. A surprising number of patients (40%) were afebrile on admission. DISCUSSION: The proportion of patients with meningitis given pre-hospital antibiotics by GPs is still worryingly low, although early hospital management has improved. Improved diagnostic facilities, particularly viral PCR assays, reduce antibiotic usage and hospital stay, with considerable financial savings.

Serum factors for opsonisation of non-typable Haemophilus influenzae.

Neutrophil chemiluminescence was used to assess the opsonins required for phagocytosis of non-typable Haemophilus influenzae isolated from sputum samples of patients with hypogammaglobulinaemia. Immunoglobulin was the major opsonin, whereas complement was relatively unimportant. Evidence was found for a heat-labile opsonin other than complement that enhanced phagocytosis of these organisms. Tuftsin was shown to aid phagocytosis of H. influenzae without triggering chemiluminesence.

Chromosomal positioning of human T-lymphotropic type 1 proviruses by fluorescence in situ hybridisation.

Fluorescence in situ hybridisation (FISH) was employed to identify the chromosomal integration site of the human T-cell lymphotropic virus, type 1 (HTLV-1) present in T-cell clones derived from HTLV-1-infected individuals and a virally transformed cell line, C8166-45. Proviral sequences were detected in C8166-45 but not uninfected Jurkat cells. Integration sites were reliably detected in T-cell clones determined previously to be infected with HTLV-1. The results indicated that the transformed cell line and some of the T-cell clones possessed more than one proviral integration site. This hybridisation system is useful for determining the number of integration events and for localising proviruses to specific chromosomal regions.

Prolonged survival in AIDS-related progressive multifocal leucoencephalopathy following anti-retroviral therapy and cidofovir.

We describe the case of a 44-year-old man who developed sub-acute cerebellar ataxia due to AIDS-related progressive multifocal leucoencephalopathy (PML) caused by JC virus. Following treatment with highly active anti-retroviral therapy (HAART) and cidofovir, he made a marked neurological improvement and is leading an independent life 18 months after the diagnosis of PML. Early recognition of AIDS-related PML and treatment with HAART improves prognosis. Cidofovir, an inhibitor of viral DNA polymerase, appears to have an additive beneficial effect and should be considered especially in patients who fail to improve despite treatment with HAART and in patients who have a high JC virus load in CSF.

Treatment of the chronic hepatitis B virus carrier state.

Chronic hepatitis B is no longer untreatable. With the advent of powerful antiviral agents such as adenine arabinoside, and, more importantly, with recombinant DNA technology and advanced culture systems able to produce large quantities of interferons, the prospects for treating patients with chronic hepatitis B virus (HBV) infection have changed completely. In the U.K., carriers not infected at birth are currently being treated with an approximately 50% chance of permanently inhibiting viral replication. In some of these, viral markers appear to be completely eliminated.

Reasons for delay in administration of antibiotics to patients with meningitis and meningococcaemia.

To determine the extent of, and reasons for, delay in treatment of patients with bacterial meningitis or meningococcal septicaemia, we reviewed the case notes of all adults admitted to Addenbrooke's Hospital, Cambridge with these diagnoses over a 3 year period. Thirty-three patients were identified. Some 70% (21/30) patients admitted via their GPs were not treated before admission. In 12 of these cases, the diagnosis was not considered. Nine patients were not treated despite the diagnosis of meningitis being considered possible or likely; in two cases this was due to suspected penicillin allergy, but no reason was given for the remaining seven. Of 24 patients untreated prior to hospital admission, only nine were given antibiotics before lumbar puncture had been performed. Failure to treat meningitis and meningococcaemia most often resulted from failure to consider the diagnosis, but treatment was delayed in a significant number of cases for no obvious reason. General practitioners and hospital doctors need to have a low threshold for administering antibiotics as soon as the diagnosis of bacterial meningitis or meningococcaemia is considered possible.

Fatal melioidosis in a tourist returning from Thailand.

Severe infections with Pseudomonas pseudomallei, the causative agent of melioidosis, is a common cause of community acquired septicaemia in South East Asia and parts of Northern Australia, but infection in travellers returning to the United Kingdom is extremely rare. We describe the case of a tourist who acquired melioidosis in Thailand. Despite intensive intravenous therapy with antibiotics to which the organism was sensitive, the patient's infection proved fatal. Ps. pseudomallei was isolated from blood cultures at a late stage in his illness by use of a commercial blood culture system that included an antibiotic removal device (Bac/TAlert, Organon-Technica).

The use of thalidomide in the treatment of intracranial tuberculomas in adults: two case reports.

We present two cases of tuberculous meningitis (TBM) in adults complicated by focal neurological deficits which showed progression whilst on steroids. In case 1 an MRI demonstrated multiple ring-enhancing lesions compressing the optic chiasm leading to a bitemporal hemianopia. After the introduction of thalidomide serial imaging and field perimetry at 6, 9, 12 and 24 months into treatment showed progressive improvement. In case 2, two months into anti-tuberculous treatment with steroids, the patient developed fluctuating right sided paralysis with the MRI demonstrating a large ring-enhancing mass encasing the left internal carotid and middle cerebral arteries. Thalidomide was introduced as an immunomodulatory adjunct and subsequently the patient made a complete neurological recovery. The immunomodulatory effects of thalidomide may have a role in the acute and chronic management of TBM complicated by intracranial tuberculomas.

Treatment of native valve Candida endocarditis with fluconazole.

Candida endocarditis of native valves is difficult to diagnose and treat. The majority of cases require valve replacement and long term amphotericin treatment. We describe a case of Candida endocarditis which was successfully managed with fluconazole without valve replacement. The patient has now been reviewed for 4.5 years from the time of diagnosis and 39 months since treatment was discontinued.

An analysis of imported infections over a 5-year period at a teaching hospital in the United Kingdom.

Background. Imported infections are an important cause of morbidity and mortality in the United Kingdom. Methods. A 5-year analysis of cases seen in a large teaching and district general hospital in the Eastern Region of the UK was performed using ward records correlated with Hospital coding data and Hospital Episode Statistics from the Department of Health. Results. A surprising number (301) and diversity of imported infections was diagnosed. Prophylactic measures were, where assessable, generally inadequate. Conclusions. These data warrant renewed efforts to educate travellers of the risks of infection acquired abroad. The continued rise in global travel along with emergence of new infectious diseases emphasises further the need for expanded infectious diseases services incorporating accessible travel advice services in the UK which are currently underprovided.

HIV RNA packaging and lentivirus-based vectors.

Since the mid-1990s, the number of publications on lentivirus-based vectors has expanded dramatically as people have realized the opportunity that they represent. High-titer helper-virus free transfer of genes to nondividing cells is a reality and it can only be a short time before clinical trials are initiated. The most efficient vector to date appears to be HIV-1 and it is no coincidence that this is the virus in which there is the greatest theoretical understanding of the encapsidation process and viral assembly. Basic studies in the other viruses are at an earlier stage and this is reflected to some extent in their relative inefficiency. Emphasis is placed in some publications on non-HIV-based vector systems having the additional safety feature of a viral vector not based on a human pathogen. As yet, this is largely a cosmetic advantage in that no system would be used which was capable of regenerating a full-length wild-type HIV and the vectors all have single round replication kinetics. More important will be elucidation of the mechanism of packaging in the different lentiviruses. Cis and trans packaging preferences may influence efficiency. Accurate delineation of packaging signals will be important. Most influential, however, will be a deeper understanding of all the viral and cellular factors involved in the packaging pathway.

Comparative antimicrobial efficacy of multi-purpose hydrogel lens care solutions.

Ten single-bottle multipurpose hydrogel lens care solutions commercially available in the European market were evaluated for antimicrobial efficacy using the International Organization for Standardization's (ISO) Stand-Alone Procedure. The results of this study indicate that ReNu Multi Plus ReNu Multi Purpose, Complete and DUA meet the ISO Stand-Alone primary acceptance criteria for stand-alone disinfectants against all challenge organisms: Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa, Candida albicans, and Fusarium solani. Solo-Care Soft, All-In-One Light, Optiplus, Opti Free Express, UniCare and Combi Comfort do not meet the ISO Stand-Alone primary acceptance criteria for one or more test organisms within their respective labelled minimum disinfection times. In addition, although not a requirement of the guidance document, only ReNu MultiPlus and ReNu Multi-Purpose exceeded the minimum ISO Stand Alone primary acceptance criteria within 25% (1 h) of their labelled minimum disinfection time for all test organisms. This evaluation provides a direct comparison of antimicrobial activity for commercially available multipurpose lens care solutions at their labelled minimum disinfection times. The results of this study should be considered when selecting appropriate lens care systems for patients.

Randomised controlled trial of lymphoblastoid interferon alfa in Europid men with chronic hepatitis B virus infection.

OBJECTIVE: To confirm the findings of pilot studies that interferon alfa is an effective treatment of Europid men with chronic hepatitis B virus infection. DESIGN: Randomised controlled trial of three months treatment with interferon alfa followed by 12 months of observation. SETTING: Outpatient clinic of a tertiary referral centre. PATIENTS: 37 Treated men (six anti-HIV positive) and 34 untreated men (nine anti-HIV positive) who met the criteria for the trial. Four controls failed to complete follow up. INTERVENTIONS: The treated group received subcutaneous injections of 5-10 MU interferon alfa/m2 daily for five days, then 10 MU/m2 thrice weekly for 11 weeks. Follow up continued at monthly intervals for 12 months. Untreated controls were monitored over the same period. MAIN OUTCOME MEASURE: Hepatitis B e antigen and hepatitis B virus DNA state after 15 months of observation. RESULTS: 12 Of the 37 treated patients cleared hepatitis B e antigen and hepatitis B virus DNA, whereas only one of 30 untreated controls seroconverted over the same period--an increased response rate of 29% (95% confidence interval 13% to 45%). The life table estimate of response at 15 months was 35% in treated patients, an increase of 32% above controls (95% confidence interval 16% to 48%). The response rates in groups by predictive pretreatment variables were 12 of 31 anti-HIV negative patients (excess response 34%; 95% confidence interval 14% to 54%), 12 of 26 with chronic active hepatitis before treatment (excess response 46%; 27% to 65%), and 12 of 21 with a pretreatment serum aspartate aminotransferase activity greater than 70 IU/l (excess response 46%; 16% to 76%). The combination of these factors predicted response with a sensitivity of 100% and a specificity of 80%. Four of the 12 responders, who had all been infected for less than two years, also lost hepatitis B surface antigen. Treatment was well tolerated. CONCLUSIONS: Interferon alfa is effective in the treatment of a proportion of Europid men with chronic hepatitis B virus infection, who might be identified before treatment. Additional strategies are required to improve the rate of response.