'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis.

SUMMARY: Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.

A consensus process on management of major burns accidents: lessons learned from the café fire in Volendam, The Netherlands.

PURPOSE: The optimum response to the different stages of a major burns incident is still not established. The fire in a café in Volendam on New Year's Eve 2000 was the worst incident in recent Dutch history and resulted in mass burn casualties. The fire has been the subject of several investigations concerned with organisational and medical aspects. Based on the findings in these investigations, a multidisciplinary research group started a consensus study. The aim of this study was to further identify areas of improvement in the care after mass burns incidents. DESIGN/METHODOLOGY/APPROACH: The consensus process comprised three postal rounds (Delphi Method) and a consensus conference (modified nominal group technique). The multidisciplinary panel consisted of 26 Dutch-speaking experts, working in influential positions within the sphere of disaster management and healthcare. FINDINGS: In response to the postal questionnaires, consensus was reached for 66 per cent of the statements. Six topics were subsequently discussed during the consensus conference; three topics were discussed within the plenary session and three during subgroup meetings. During the conference, consensus was reached for seven statements (one subject generated two statements). In total, the panel agreed on 21 statements. These covered the following topics: registration and evaluation of disaster care, capacity planning for disasters, pre hospital care of victims of burns disasters, treatment and transportation priorities, distribution of casualties (including interhospital transports), diagnosis and treatment and education and training. ORIGINALITY/VALUE: In disaster medicine, the paper shows how a consensus process is a suitable tool to identify areas of improvement of care after mass burns incidents.

Medical management after indoor fires: a review.

UNLABELLED: Fires involving mass burn casualties require extreme efforts and flexibility from the regular health care system. The café fire in Volendam, which occurred shortly after midnight on the first of January 2001, resulted in the worst indoor mass burns incident in Dutch history. During the extensive medical evaluation of this disaster, it became obvious that information on similar incidents is relatively scarce in the literature. This article systematically reviews the existing information in the medical literature on indoor fires and provides findings and knowledge used in the evaluation of the medical management after indoor fires and for future mass burn casualty preparedness, mitigation and response. METHODS: A literature review was undertaken for burn disasters with characteristics similar to the indoor Volendam fire disaster. In all fires, the following aspects were investigated: characteristics of the fire; the initial emergency response; triage and on-site treatment; primary and secondary distribution; hospital admission; severity of the sustained injuries and mortality. RESULTS: A total of nine similar indoor fires were selected. The number of people involved was reported in seven fires (range 137-6000). All reports provided the mortality rate (range 1.4% to over 50%). Data regarding the emergency response could be collected in half of the studies. On-scene triage was performed in five fires. The number of hospitals participating in the primary distribution ranged from 1 to 19. Except for the Volendam fire, all patients were primarily distributed to general hospitals. CONCLUSION: Characteristics of indoor fires, which are relevant for disaster preparedness, mitigation and response are not frequently reported in medical literature. The current articles on indoor fires, mainly report on numbers of casualties and the mortality. Limited data are available to provide insight in the characteristics of management and medical treatment and to come up with suggestions for improvement of future burn incidents management. The evaluation of disasters should be based on uniform methods and structured reports and effective record keeping is essential to achieve this.

The café fire on New Year's Eve in Volendam, the Netherlands: description of events.

AIM OF STUDY: The café fire at Volendam occurred shortly after midnight on the first of January 2001 and resulted in one of the worst mass burn incidents in recent Dutch history. The aim of this study was to provide insight into medical and organisational requirements of a major burns incident. METHODS: Shortly after the fire, two university hospitals and a burn center in the region of the accident developed a plan for evaluation of medical care given during and after this major burn incident. A multidisciplinary research group investigated the management of victims at the scene, in the emergency departments (ED) and during admission in the hospitals. All 245 casualties were included in this study. RESULTS: A brief severe fire occurred in a crowded cafe with around 350 young visitors on a small embankment of a relatively isolated town, resulting in a unusually high number of severely injured burn victims. Four died immediately. The ensuing rescue effort was hampered by poor access and chaotic circumstances. At the scene of the incident, mobile medical teams ensured orderly transport and treatment priority for the injured. There were 245 victims with a median total body surface area burned of 12%. Inhalation injury was present in 96 patients. A total of 182 victims were admitted, with 112 to intensive care. Ten patients died in the hospital. Seventy-eight patients were secondarily transported, many to specialised centers in the Netherlands and abroad. In total, 36 hospitals in three countries participated. CONCLUSION: An incident with high numbers of burn victims poses a challenge to any health care system. The difficult circumstances at the site demonstrated the need for robust organisational structures. The primary and secondary distribution of patients required coordination, general hospitals were able to provide initial medical care to these major burn casualties.

Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.

Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2.

The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described.

Immune responses in bovine tuberculosis.

Knowledge of the immune responses which develop in cattle following infection with Mycobacterium bovis is essential both to the understanding of disease pathogenesis and to the logical development of immune-dependent tools, such as diagnostic tests and vaccines, which can be used to combat the disease. Studies of field cases of bovine tuberculosis (TB) and of experimental bovine models of M. bovis infection have indicated that cell-mediated immune responses (CMI) predominate within a spectrum of immunity which exists. This paper reviews aspects of recent research and indicates how knowledge of T-cell antigenic targets in bovine TB along with increasing knowledge of T-cell subpopulations and their interactions with M. bovis -infected macrophages provides opportunities for the development of better methods for disease control.

Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains.

AIMS: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format. METHODS AND RESULTS: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E. coli O157. A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E. coli (EHEC) O157 strains and also with two enterotoxigenic E. coli O157 strains. Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected. CONCLUSION: A MAb-based sELISA to detect E. coli O157 was produced. Its application to field samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-specific interference of the cross-reacting strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay produced is not wholly specific to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157.

Characterization of the early antibody response in bovine tuberculosis: MPB83 is an early target with diagnostic potential.

A 26-kDa antigen has been shown to be a dominant antibody target in Mycobacterium bovis-infected cattle. In this study, that antigen was used as an immunogen to raise a panel of mouse monoclonal antibodies. The majority of those bound to native protein with a molecular mass of 26 kDa and to recombinant MPB83, strongly suggesting that MPB83 is an important B-cell antigenic target in bovine tuberculosis. In order to provide assessment of the potential of measuring antibody responses to the native protein, one monoclonal antibody, 1F11, was incorporated into an enzyme-linked immunosorbant assay format to trap antigen from a crude bacterial extract. Despite some disadvantages of this format, serum samples from cattle which had been infected experimentally with M. bovis, and from tuberculin skin-test-negative and -positive field cattle were tested for the presence of antibodies. Data from the skin-test-negative cattle allowed an arbitrary cut-off value to be established and, under these conditions, test sensitivity and specificity were estimated at 37.5 and 89%, respectively. These results indicate potential for MPB83 in the development of assays for serological diagnosis of bovine tuberculosis.

Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle.

Monoclonal antibodies (Mabs) were produced against Leptospira borgpetersenii serovar hardjo-type Bovis antigens. A panel of 28 Mabs were characterised. Only the nine Mabs toward a lipopolysaccharide (LPS) fraction of 18, 24 kDa bands and a 26-28 kDa smear showed agglutinating, leptospiricidal and growth-inhibition activities, and passively protected hamsters against renal infection with hardjo. They also reacted strongly in the CH-ELISA, captured killed whole hardjo leptospires, gave good fluorescence in indirect FAT against smears of hardjo culture and exhibited no cross reactivity with strains in heterologous serogroups. On the basis of optimal activity in a range of tests, one IgG class Mab (designated 25) was selected for use in an antibody-capture ELISA system for the detection of bovine anti-hardjo antibodies. The system gave a wide separation of absorbance values between positive and negative sera at a 1:10 dilution. The antibodies detected by this assay are believed to be protective anti-LPS IgG.

Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland.

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).

The prognostic factors regarding long-term functional outcome of full-thickness hand burns.

The treatment of the burned hand has always been a subject of special interest. In order to obtain a better understanding of the parameters involved in the long-term functional outcome of hand burns a retrospective study was performed on 88 consecutive patients with hand burns (143 hands), treated according to a standardised protocol. Patients were followed for at least 12 months postburn. Hand function was assessed by the seven objective test criteria (7-OTC) described by Jebsen. Logistic regression analysis produced five parameters that were found to have a significant predictive value for long-term hand function. In order of predictive value, these were finger amputations, age on admission, impaired autograft take, the full-thickness hand burn surface area and the full-thickness total body burn surface area. By fitting these five variables into an equation, a probability model was obtained, which could be applied to estimate a prognosis concerning the final hand function of an individual patient with a hand burn. No relationship was found between the postburn day of operation and the long-term hand function. This finding is inconsistent with the current consensus that functional outcome is improved by early excision and grafting. In practice, it suggests that hand function is well preserved when burns of uncertain depth are treated expectantly, followed by selective debridement and grafting. Advantages include reduced blood loss, no loss of vital tissue and a reduction of the need for donor sites.

T-cell recognition of mycobacterial proteins MPB70 and MPB64 in cattle immunized with antigen and infected with Mycobacterium bovis.

Defined antigenic reagents and knowledge of T-cell responses are required for the design of improved diagnostic tests for bovine tuberculosis. The limited species distribution of Mycobacterium bovis antigens MPB70 and MPB64 has indicated their potential for inclusion in future tests. The strategy adopted in this study was to define bovine T-cell responses to these antigens at the epitope level, using cattle immunized with recombinant forms of the antigens, and to compare these responses with cattle which had been experimentally infected with M. bovis. Panels of synthetic peptides (20-mers with 10-residue overlaps) were used and five epitopes were identified and found to be powerful stimulators of T-cell responses in both types of animal (residues 81-100 and 174-190 for MPB70, and residues 1-20, 41-60 and 181-200 for MPB64). Further investigation in larger numbers of cattle (n = 14) of mixed breeds from tuberculosis-infected herds confirmed that each peptide produced response in several of the cattle, but no single peptide was recognized by all animals. However, the limited numbers of animals in this study suggest that peptide reagents may identify as many positive animals as the intact antigenic protein and could form components of a future diagnostic test. The use of cattle immunized with the proteins of interest has proved to be an interesting model for studying the nature of bovine T-cell responses to defined mycobacterial proteins.

Recognition of a common mycobacterial T-cell epitope in MPB59 of Mycobacterium bovis.

Bovine tuberculosis, which persists as a residual level of infection in many European countries, has implications not only for the economy of farming communities but also for human health. The aim of this study was to identify a common mycobacterial antigen which was recognized in bovine tuberculosis and to characterize the response to this antigen at the epitope level. A T-cell clone, phenotype CD4+, raised from an animal experimentally infected with Mycobacterium bovis was shown to proliferate in response to a panel of sonicates derived from different mycobacterial species indicating recognition of an antigen with broad specificity. This antigen was subsequently shown to be MPB59. Recognition of MPB59 at the epitope level was determined in experimental and field cases of bovine tuberculosis using a panel of synthetic peptides (20-mers with 10-residue overlaps) incorporating the signal sequence and mature protein. The results showed that in vitro interferon-gamma was predominantly produced in response to adjacent peptides numbers 10 and 11, suggesting that the dominant epitope was contained in the overlap, correlating to residues 101-110 (YYQSGLSIVM). This epitope was recognized by 54% of tuberculous cattle of mixed breeds, which suggests that it may be genetically permissive in terms of major histocompatibility complex presentation. Sequence analysis confirmed that there were only minor differences in the amino acid composition within this region for various mycobacterial species, which could explain the common T-cell recognition described in this study. Common recognition of this epitope indicates that it would have limited potential for use as a diagnostic reagent per se but may have potential for inclusion in a subunit vaccine.

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine.

A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10(3) to 10(2) leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.

Bovine serum transferrin concentration during acute infection with Haemophilus somnus.

A novel, competitive immunoassay based on time-resolved fluorimetry was developed, and used to measure the serum concentration of bovine transferrin during acute Haemophilus somnus pneumonia. Upper and lower limits of normality were established using serum from healthy cattle (3.72-1.37 mgmL-1). Following experimental infection with Haemophilus somnus, transferrin concentration was depressed in all calves but recovered to pre-infection levels in groups of calves which had either no lesions, or mild lesions at necropsy between 5 and 6 days after infection. In a third group, which developed extensive lesions, the transferrin concentration remained depressed. Transferrin levels remained within the normal range for all calves during the experimental period. Those calves which had low transferrin concentrations pre-infection, developed extensive lung lesions following experimental infection with Haemophilus somnus.

Production and biological activity of murine monoclonal antibodies against GnRH.

Immunization against GnRH represents a nonsurgical means of castrating domestic species. However, clear target antibody titres for bioactivity have not been established. The aims of this study were to produce characterized anti-GnRH monoclonal antibodies and to determine a threshold titre. Three murine monoclonals were developed which produced IgG2a class immunoglobulins and bound 50% I(125)-GnRH at a 10(6) to 10(7) dilution. The antibodies were specific to GnRH, showed a strong affinity (Ka values from 1.99 to 2.60 x 10(10) litres/mole), and were directed towards the amino terminus. In female mice all 3 antibody clones interrupted ovarian cyclicity, causing an extension in diestrus followed by prolonged estrus/metestrus (12 to 30 d). Throughout this period circulating titres were greater than 15% I(125)-GnRH binding at a 5 x 10(4) dilution. In male mice, immunization with 0.2 ml of ascites significantly reduced testes (P < 0.05), epididymides (P < 0.001) and seminal vesicle (P < 0.01) weights. A 0.1 ml dose (61.4 +/- 18.6% binding at a 10(6) dilution) was ineffective. A serial dilution study indicated that a titre of 50% binding at 2 x 10(6) dilution (antigen binding capacity of 268 +/- 35 ng/ml) was required to completely block GnRH activity. This is a higher tire than threshold levels determined previously. Identification of factors determining the titre required for bioactivity is needed.