Canine and Phocine Distemper Viruses: Global Spread and Genetic Basis of Jumping Species Barriers.

Canine distemper virus (CDV) and phocine distemper (PDV) are closely-related members of the Paramyxoviridae family, genus morbillivirus, in the order Mononegavirales. CDV has a broad host range among carnivores. PDV is thought to be derived from CDV through contact between terrestrial carnivores and seals. PDV has caused extensive mortality in Atlantic seals and other marine mammals, and more recently has spread to the North Pacific Ocean. CDV also infects marine carnivores, and there is evidence of morbillivirus infection of seals and other species in Antarctica. Recently, CDV has spread to felines and other wildlife species in the Serengeti and South Africa. Some CDV vaccines may also have caused wildlife disease. Changes in the virus haemagglutinin (H) protein, particularly the signaling lymphocyte activation molecule (SLAM) receptor binding site, correlate with adaptation to non-canine hosts. Differences in the phosphoprotein (P) gene sequences between disease and non-disease causing CDV strains may relate to pathogenicity in domestic dogs and wildlife. Of most concern are reports of CDV infection and disease in non-human primates raising the possibility of zoonosis. In this article we review the global occurrence of CDV and PDV, and present both historical and genetic information relating to these viruses crossing species barriers.

Disruption of redox homeostasis leads to oxidative DNA damage in spermatocytes of Wolbachia-infected Drosophila simulans.

Molecular interactions between symbiotic bacteria and their animal hosts are, as yet, poorly understood. The most widespread bacterial endosymbiont, Wolbachia pipientis, occurs in high density in testes of infected Drosophila simulans and causes cytoplasmic incompatibility (CI), a form of male-derived zygotic lethality. Wolbachia grow and divide within host vacuoles that generate reactive oxygen species (ROS), which in turn stimulate the up-regulation of antioxidant enzymes. These enzymes appear to protect the host from ROS-mediated damage, as there is no obvious fitness cost to Drosophila carrying Wolbachia infections. We have now determined that DNA from Wolbachia-infected mosquito Aedes albopictus (Aa23) cells shows a higher amount of the base 8-oxo-deoxyguanosine, a marker of oxidative DNA damage, than DNA from uninfected cells, and that Wolbachia infection in D. simulans is associated with an increase in DNA strand breaks in meiotic spermatocytes. Feeding exogenous antioxidants to male and female D. simulans dramatically increased Wolbachia numbers with no obvious effects on host fitness. These results suggest that ROS-induced DNA damage in sperm nuclei may contribute to the modification characteristic of CI expression in Wolbachia-infected males and that Wolbachia density is sensitive to redox balance in these flies.

Integrating the implementation of the European Union Water Framework Directive and Floods Directive in Ireland.

Water Framework Directive (WFD) statutory authorities and stakeholders in Ireland are now challenged with the issue of how the proposed programmes of measures in the newly required River Basin Management Plans - designed to protect and restore good ecology by reverting as closely as possible back to natural conditions - are to be implemented in a way that concurrently complies with other existing and emerging intersecting European Union legislation, such as the Floods Directive (FD). The WFD is driven largely by ecological considerations, whereas the FD and other legislation are more geared towards protecting physical property and mitigating public safety risks. Thus many of the same waterbodies, especially heavily modified waterbodies, arguably have somewhat competing policy objectives put upon them. This paper explores the means by which Ireland might best achieve the highest degrees of cost effectiveness, economic efficiency and institutional durability in pursuing the common and overarching objective of the WFD and FD - to ensure Irish waterways are put to their highest valued uses.

Phocine distemper virus in seals, east coast, United States, 2006.

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

Adrenomedullin gene delivery is cardio-protective in a model of chronic nitric oxide deficiency combining pressure overload, oxidative stress and cardiomyocyte hypertrophy.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling. RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension. CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Persistent measles virus infection of mouse neural cells lacking known human entry receptors.

AIMS: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry. METHODS: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction. RESULTS: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections. CONCLUSIONS: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.

Antibacterial activity of honey against community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA).

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honey's ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n=6 isolates), was examined for its susceptibility to natural honey (n=3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 10(6) colony-forming units (cfus) (mean = 6.46 log10 cfu/g) to none detectable within 24h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

Measles virus and classical Hodgkin lymphoma: no evidence for a direct association.

A proportion of Hodgkin lymphoma (HL) cases are causally associated with the Epstein-Barr virus (EBV) but the aetiology of the remaining cases remains obscure. Over the last 3 decades several studies have found an association between HL and measles virus (MV) including a recent cohort study describing the detection of MV antigens in Hodgkin and Reed-Sternberg cells, the tumour cells in HL. In the present study we looked at the relationship between history of MV infection and risk of developing HL in a population-based, case/control study of HL. In addition we used immunohistochemistry and RT-PCR to look for direct evidence of MV in HL biopsies. There was no significant difference in the proportion of cases reporting previous measles compared to controls in the entire data set or when young adults were considered separately. Using a robust immunohistochemical assay for MV infection, we failed to find evidence of MV in biopsies from 97 cases of HL and RT-PCR studies similarly gave negative results. This study therefore provides no evidence that MV is directly involved in the development of HL. However, when age at first reported MV infection was investigated, significant differences emerged with children infected before school-age having higher risk, especially of EBV-ve HL, when compared with children infected at older ages; the interpretation of these latter results is unclear.

Speciation of Bacillus spp. in honey produced in Northern Ireland by employment of 16S rDNA PCR and automated DNA sequencing techniques.

Phenotypic speciation of foodborne Bacillus spp. remains problematic in terms of obtaining a reliable identification. In this study, we wished to identify several bacterial isolates from honey produced in Northern Ireland, and which belonged to the genus Bacillus, through employment of a molecular identification scheme based on PCR amplification of universal regions of the 16S rRNA operon in combination with direct automated sequencing of the resulting amplicons. Seven samples of honey and related materials (propolis) were examined microbiologically and were demonstrated to have total viable counts (TVC) ranging from <100 to 1700 colony-forming units/g. No yeasts or filamentous fungi were isolated from the honey materials. Several bacterial isolates were identified using this method, yielding two different genera (Paenibacillus and Bacillus), as well as four Bacillus species, namely Bacillus pumilus, B. licheniformis, B. subtilis and B. fusiformis, with B. pumilus the most frequently identified species present. When the use of molecular identification methods is justified, employment of partial 16S rDNA PCR and sequencing provides a valuable and reliable method of identification of Bacillus spp. from foodstuffs and negates associated problems of conventional laboratory and phenotypic identification.

A phase I trial of stereotactic external beam radiation for subfoveal choroidal neovascular membranes in age-related macular degeneration.

Effective treatment for neovascular age-related macular degeneration (AMD) is currently limited. Radiation therapy, a therapeutic approach with known antiangiogenic properties, has been investigated as a modality to prevent severe visual loss in AMD. Most of the studies using external beam radiation employed <25 Gy to the whole eye, which is below the dose of radiation that is toxic to the retina and optic nerve ( approximately 50 Gy and approximately 59 Gy, respectively). Stereotactic fractionated external beam radiation (St-EBR) is a method that allows radiation to be delivered to a small, defined area. We investigated the effects of St-EBR in incremental doses up to 40 Gy on neovascular AMD. Patients with clinical signs and fluorescein angiography demonstrating neovascular AMD, visual acuity (VA) better than 20/400 and ineligible for laser treatment (MPS criteria) or who refused to have laser photocoagulation were enrolled in the study. Each patient was treated with radiation at incremental dosages from 20 Gy to 40 Gy. After completion of the radiation course, all patients were followed-up at 3 and 7 weeks and 3, 6, and 12 months. Best-corrected VA (ETDRS), slit-lamp and fluorescein angiographic evaluations were performed at each visit. 94 eyes of 89 patients were treated from October 1997 to April 2000. The VA was 0.82+/-0.35 before treatment, 0.83+/-0.36 at 6 months, and 0.89+/-0.33 at 12 months. No patients suffered any significant acute side effects. No significant benefits in either VA or in membrane size were derived from increasing the doses of radiation. Our results are consistent with trends of a palliative benefit of radiotherapy in neovascular AMD and support further investigation of radiotherapy. Since there is no evidence that therapeutic effectiveness is dose dependent, our data provide no justification for potentially dangerous escalations in radiation dosage for treating neovascular AMD.

Completion of the sequence of a cetacean morbillivirus and comparative analysis of the complete genome sequences of four morbilliviruses.

The gene encoding the large (L) protein and the genome termini of the dolphin strain of cetacean morbillivirus (CeMV) were sequenced. The CeMV genome is 15702 nucleotides long and has been compared with other available morbillivirus genome sequences in regards to the "rule of six" and the "phase" of any particular nucleotide, defined as its position within a given hexamer, which here is defined as a group of six nucleotides starting from the 3' end of the genomic RNA. With exception of the position of the start of the F gene, the phase of the transcription start sites of each gene is strictly conserved between the morbilliviruses, but each gene is in a different phase. The lengths of gene transcripts differ between viruses by multiples of six nucleotides with exception of the M and F transcripts. The differences between the various morbilliviruses result from deletions or insertions of multiples of six nucleotides in the 3' and 5' UTRs of the different viral genes. The four bases were distributed non-randomly over the six positions in the hexamer boxes. However, the distribution patterns of each of the four bases indicated that multiples of three were more prevalent than those of six nucleotides. This reflected the positions of nucleotides in codons and codon usage in the reading frames. The L protein of CeMV was found to be 2183 amino acids in length and similar to that of MV and RPV. The CeMV L protein sequence was found to be equidistant between those of the CDV/PDV and MV/RPV subgroups of the morbilliviruses. This concurs with the analyses carried out on the other structural proteins.

Long-term follow-up of radiotherapy for prostate cancer.

PURPOSE: To determine the long-term outcome of radiotherapy for prostate cancer. METHODS AND MATERIALS: A total of 136 consecutive patients with prostate cancer underwent primary radiotherapy. All but 4 patients received 6000 cGy to the prostate. The minimal follow-up was 22.9 years. RESULTS: Of the 136 patients, 93 had Stage B (T2), 9 Stage A (T1), and 34 Stage C (T3). Sixty-nine percent of the patients developed recurrence, and 51% of all patients died of prostate cancer. The recurrences developed at a steady state throughout the length of follow-up. One half the recurrences occurred after 10 years, and recurrence was still observed >20 years after treatment. The survival rate at 5, 10, 15, 20, and 25 years was 81%, 59%, 37%, 16%, and 10%, respectively. The recurrence-free survival rate at 25 years was 17%. The median survival for Grade 3-4 patients was 6.3 years and for Grade 1-2 patients was 13.0 years. The median survival for those with T1 tumors was 12.9 years; T2 tumors, 12.4 years; and T3 tumors, 9.5 years. CONCLUSION: Despite favorable early results, with long-term follow-up, patients continued to experience prostate cancer recurrence. Unless they died an intercurrent death, they were highly likely to develop recurrence and die of prostate cancer. The conclusions from treatment studies with <15 years of follow-up should be viewed as preliminary.

The Three Rivers Project--water quality monitoring and management systems in the Boyne, Liffey and Suir catchments in Ireland.

The "Three Rivers Project" is a government initiative and one of a series of catchment based water quality monitoring and management systems being developed throughout Ireland since 1997. The establishment of these multi-sectoral, basin-wide and community based systems is a response to historically perceived disjointed, legalistic and non-participative approaches to water resource management and purports to transcend the restrictions of traditional local authority administrative boundaries. The new management model embodies the concepts and objectives contained in the European Union (EU) Water Framework Directive (WFD) enacted in December 2000. Ireland, in common with many EU countries, has failed to halt decades of increasing levels of eutrophication of surface waters due principally to phosphorus loading. The "Three Rivers Project" is promoting the benefits of an integrated and cooperative approach to the management of three important river systems in Ireland, namely, the Boyne, Liffey and Suir. The project objective is to protect and improve water quality to conform with "good ecological status". The implications of the Project findings for agricultural, municipal and industrial policy are grave and one of the greatest challenges now is to organise and fund Irish River Basin Management Systems as envisaged by the WFD to continue and build on the work which the "Three Rivers Project" has undertaken.

Long-term follow-up of radical retropubic prostatectomy for prostate cancer.

OBJECTIVES: The ultimate outcome of patients after radical prostatectomy is often predicted from statistical projections of short-term follow-up. Only actual long-term follow-up can demonstrate true outcome. METHODS: One hundred thirty-one patients underwent retropubic prostatectomy for clinically organ confined prostate cancer and have been followed for a minimum of 22.5 years. Preoperatively, all but 12 had clinically palpable cancer. RESULTS: Overall survival in these patients was similar to an age-matched population, with 65% alive at 15 years, and 23% alive at 25 years. Thirty-seven percent of the patients recurred and 24% of all the patients died of prostate cancer. For patients with pathologically organ confined disease, 27% recurred, while those with extension outside the gland or positive nodes had an 83% recurrence rate. Although, the median time to recurrence was 7 years, recurrences occurred at a steady-state throughout the length of follow-up. Patients with higher grade tumors, even if organ confined, were significantly more likely to recur. CONCLUSIONS: In a cohort of patients treated with radical prostatectomy for predominantly palpable disease, long-term follow-up (79% deceased) reveals that 37% will recur and 24% will die of prostate cancer. Almost half the recurrences occurred after 10 years, indicating that reports with shorter follow-up will underestimate the recurrence rate.

Human influenza viral infection in utero alters glial fibrillary acidic protein immunoreactivity in the developing brains of neonatal mice.

Epidemiological reports describe a strong association between prenatal human influenza viral infection and later development of schizophrenia. Postmodern human brain studies, however, indicate a lack of gliosis in schizophrenic brains presumably secondary to absence of glial cells during the second trimester viral infection in utero. We hypothesized that human influenza infection in day 9 pregnant mice would alter the expression of glial fibrillary acidic protein (GFAP, an important marker of gliosis, neuron migration, and reactive injury) in developing brains of postnatal days 0, 14 and 35 mice. Determination of cellular GFAP immunoreactivity (IR) expressed as cell density in cortex and hippocampus of control and experimental brains showed increases in GFAP-positive density in exposed cortical (P = 0.03 day 14 vs control) and hippocampal cells (P = 0.035 day 14, P = 0.034 day 35). Similarly, ependymal cell layer GFAP-IR cell counts showed increases with increasing brain age from day 0, to days 14 and 35 in infected groups (P = 0.037, day 14) vs controls. The GFAP-positive cells in prenatally exposed brains showed 'hypertrophy' and more stellate morphology. These results implicate a significant role of prenatal human influenza viral infection on subsequent gliosis, which persists throughout brain development in mice from birth to adolescence.

Prospective randomized trial of low- versus high-dose radiation therapy in adults with supratentorial low-grade glioma: initial report of a North Central Cancer Treatment Group/Radiation Therapy Oncology Group/Eastern Cooperative Oncology Group study.

PURPOSE: To compare survival and toxicity in adult patients treated with low-dose (50.4 Gy/28 fractions) versus high-dose (64.8 Gy/36 fractions) localized radiation therapy (RT) for supratentorial low-grade astrocytoma, oligodendroglioma, and mixed oligoastrocytoma. PATIENTS AND METHODS: From 1986 to 1994, 203 eligible/analyzable patients were randomized: 101 to low-dose RT, 102 to high-dose RT. Almost half were younger than 40 years, and 95% had grade 2 tumors. Histologic subtype was astrocytoma (or mixed oligo-astrocytoma with astrocytoma dominant) in 32% of patients and oligodendroglioma (or oligoastrocytoma with oligodendroglioma dominant) in 68%. Tumor diameter was less than 5 cm in 35% of patients, and 41% of tumors showed some degree of contrast enhancement. Extent of resection was gross total in 14% of patients, subtotal in 35%, and biopsy only in 51%. RESULTS: At the time of the present analysis, 83 patients (41%) are dead, and median follow-up is 6.43 years in the 120 who are still alive. Survival at 2 and 5 years is nonsignificantly better with low-dose RT; survival at 2 and 5 years was 94% and 72%, respectively, with low-dose RT and 85% and 64%, respectively, with high-dose RT (log rank P =.48). Multivariate analysis identified histologic subtype, tumor size, and age as the most significant prognostic factors. Survival is significantly better in patients who are younger than 40 years and in patients who have oligodendroglioma or oligo-dominant histology. Grade 3 to 5 radiation neurotoxicity (necrosis) was observed in seven patients, with one fatality in each treatment arm. The 2-year actuarial incidence of grade 3 to 5 radiation necrosis was 2.5% with low-dose RT and 5% with high-dose RT. CONCLUSION: This phase III prospective randomized trial of low- versus high-dose radiation therapy for adults with supratentorial low-grade astrocytoma, oligodendroglioma, and oligoastrocytoma found somewhat lower survival and slightly higher incidence of radiation necrosis in the high-dose RT arm. The most important prognostic factors for survival are histologic subtype, tumor size, and age. The study design of the ongoing intergroup trial in this population will be discussed.

Microstructural characteristics of human skin subjected to static versus cyclic pressures.

Pressure ulcers are generally associated with external pressures exceeding internal capillary pressures over bony prominences when the body cannot initiate motor movement to change positions. This investigation evaluated microstructural changes occurring in human skin, in vitro, exposed to static versus cyclic pressures, simulating those recorded for heels of human subjects on various pressure-relief mattresses. Morphological data are reported for tissues exposed to pressure in a bench-scale loading system. Dynamic (cyclic-relief) pressure induced parallel alignments of connective tissue collagen bundles, which themselves became differentially oriented to various degrees perpendicular to the surface of the tissue. Static pressure, with no relief, invariably produced alignment of the collagen bundles of the connective tissue parallel to both one another and to the compressed tissue surface. The precursor to pressure ulcer formation may be microstructural alignment in response to the pressure conditions on tissue.

Altered levels of the synaptosomal associated protein SNAP-25 in hippocampus of subjects with mood disorders and schizophrenia.

SNAP-25 levels were measured in ventral hippocampus in subjects with unipolar depression (n = 12), bipolar disorder (n = 13), schizophrenia (n = 15) and controls (n = 15) using quantitative immunocytochemistry. SNAP-25 levels were reduced significantly in stratum oriens of bipolar patients compared with controls (p < 0.05); they were also reduced significantly in st. oriens (p < 0.01 vs schizophrenia), in alveous (p < 0.01 vs schizophrenia) and in presubiculum (p < 0.05 vs depressed). SNAP-25 levels were also reduced in several layers of schizophrenics, only significantly so in st. granulosum (p < 0.05 vs controls). In contrast, depressed SNAP-25 levels increased in st. moleculare (p < 0.01 vs schizophrenics) and presubiculum (p < 0.05 vs controls and bipolars; p < 0.01 vs schizophrenics). SNAP-25 values were not affected by age, sex, race, post-mortem interval, brain pH, side of brain, age of onset of disease, family history of psychiatric disease, drug or alcohol use, antipsychotic drug treatment, or mode of death. The reported changes in SNAP-25 levels appear to be disease specific, separating synaptic pathology in unipolar depression from that observed in schizophrenia and bipolar disorders.

Molecular diagnosis of infective endocarditis--a new Duke's criterion.

The molecular approach of PCR amplification of specific gene targets and universal loci for bacteria (16S rRNA) and fungi (18S, 28S and 5.8S rRNA) and subsequent sequencing was used to identify the possible causal microbial agent(s) in blood culture (47 patients) and heart valve material (30 patients) from patients with suspected infective endocarditis (IE). Culture and molecular results were analysed with respect to the patients' clinical background and the Duke Criteria. The findings demonstrated that: (i) all patients who were definite or possible cases were positive by PCR, even patients whose blood culture and valve material were culture-negative; and (ii) all patients who were rejected as having IE were also negative by PCR, with the exception of 1 patient who had bacteraemia from another source and 5 patients whose blood culture material was believed to contain an environmental contaminant. Direct molecular identification of the aetiological agents responsible for IE from blood culture material may enable specific treatment to commence at an earlier stage of the disease and hence reduce the need for valve replacement. Such a molecular approach may aid in the diagnosis of IE and should therefore be included as an additional major criterion in the Duke's classification scheme.