Risk factors for homelessness among people with psychotic disorders

  Background: Although it is widely accepted that rates of severe mental illness amongst single homeless people are high, little is known about the reasons these individuals become homeless. This study aimed to identify risk factors for homelessness among people with psychotic disorders. Methods: A matched case-control study of homeless and never homeless people with psychotic disorders was carried out, with respondents recruited from mental health services (N = 39 pairs). Data were collected by semi-structured interviews and from medical records. Results: A number of social and behavioural risk factors were identified; key factors being loss of contact with childhood carers, and substance use. Clinical and service use factors appeared less important as predictors of homelessness. Conclusions: Mental health services have a limited role in circumventing homelessness among people with psychotic disorders. An integrated approach involving other key agencies is required. Accepted: 23 May 2000     

Investigating the Use of Protein Saver Cards for Storage and Subsequent Detection of Bovine Anti-Brucella abortus Smooth Lipopolysaccharide Antibodies and Gamma Interferon

Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.     

Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.Brucellosis is a zoonosis of widespread distribution and significance caused by species of the genus Brucella. The disease is known to be especially prevalent in the Middle East and Mediterranean Basin (33), and there are disturbing signs of its reemergence across large areas of the globe, especially in central Asia. Few countries have successfully managed to eradicate the disease. In many of these countries, although the livestock sector is disease free, a significant wildlife reservoir remains and presents a risk of reintroduction (11).The principle etiologic agents of brucellosis are the classical smooth species Brucella abortus, B. melitensis, and B. suis. These species all have smooth lipopolysaccharide (sLPS), which is a major virulence factor (17, 35). Each of these species has a preferred host; however, many animals, including humans, are susceptible to each of the classical species, although the underlying mechanisms for host preference are not clearly understood (5). Brucellosis in ruminants is mainly manifested by reproductive failure due to abortion. There are few other clinical signs, and this causes difficulties with diagnosis. In humans, the disease is mainly presented as an undulating febrile condition, although there may be other, more serious complications. The symptoms of human brucellosis are particularly nonspecific, and this again presents serious diagnostic complications (2).There are many aspects to the effective control of the disease in both the human and the animal populations, including educational programs, effective animal tracing, vaccination of animals, and intersectorial cooperation. Within and, indeed, prior to the implementation of a holistic control program, establishing the prevalence of disease and identifying infected animals are crucial. Owing to a lack of specific symptoms, the most effective means of doing this is through serological testing, followed by, if possible, the isolation of Brucella from serologically positive animals. In areas where the disease has been eradicated, a surveillance system is vital in order to maintain freedom. Once again, serology also plays a vital role in this.The Organization International des Epizooties (OIE) prescribed and alternative serological tests for the diagnosis of brucellosis due to infection with smooth strains largely rely upon the detection of antibodies to the O antigen of sLPS (10, 32). The classical tests include the Rose Bengal test, the complement fixation test (CFT), and the serum agglutination test (SAT), all of which employ a whole-cell antigen as the key diagnostic reagent. More recently developed techniques, such as the indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and the fluorescence polarization assay (FPA), use purified sLPS or O antigen. The immunodominance of the sLPS O antigen is the basis for the generally good sensitivity of these assays. The use of these antigens can lead to false-positive serological test results when animals are infected with bacteria possessing O antigens with a structure similar to that of the O antigen of Brucella species (7), such as Yersinia enterocolitica O:9. Owing to the widespread use of the S19 and Rev 1 vaccines, such tests also fail to reliably differentiate between vaccinated and infected animals.In all effective brucellosis control scenarios, the number of samples tested is high, and therefore, optimizing the efficiency of the testing regimen is critical to limit costs. ELISAs are readily amenable to high-throughput testing due to the standardized nature of the technology and reagents. This allows for many efficiency savings, including the introduction of automation (20). Although ELISAs have advantages over classical tests in this regard, they still require several steps to be completed, including separation (wash) steps. Although these steps can be automated, they are a vital part of the assay yet present a frequent source of imprecision, error, mechanical breakdown, and additional cost. Assays which have the advantages of the ELISA, such as assays that use a 96-well format, and that have an objective means of assessment of the results and good sensitivities and specificities but that reduce the burden of work and opportunity for error are clearly desirable.The aim of the project described here was to improve the efficiency of serological testing by developing a homogeneous homologue of the Brucella cELISA (from the Veterinary Laboratories Agency, Weybridge, United Kingdom) by using the principles of time-resolved fluorescent resonance energy transfer (TR-FRET). FRET occurs when two fluorophores (a donor and an acceptor) with the appropriate spectral properties transfer energy between them if they are within sufficient proximity to each other (9). The degree to which complementary antigens and antibodies have bound (and are therefore within close proximity) can be detected by labeling each with an appropriate fluorophore and measuring the amount of energy transfer produced after the initial excitation of the donor.Use of a donor fluorophore with a long fluorescent lifetime enables the specific transfer of energy to persist long after the nonspecific background fluorescence, due to the initial excitation, has ceased. Initiating fluorescence intensity (FI) measurements after the background fluorescence has reduced improves the sensitivity of the FRET technique (26). The fluorescence of both the donor and the acceptor fluorophores can be measured, and this endows the method with additional resistance to variable fluorescent effects due to sample matrices, such as serum.The introduction of competing agents, such as specific antigens or serum antibodies, that alter the degree of binding between the labeled reagents will be manifested as a change in the donor and the acceptor intensities. This process can be conducted with no solid-phase reagents or wash steps and with only a single incubation and a single read step.     

Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains

Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISA_SPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.Species of the genus Brucella cause serious chronic infections, collectively known as brucellosis. Brucellosis is a mammalian disease infecting many economically important animal species as well as humans. With a global distribution, brucellosis causes considerable animal and human health problems as well as huge economic costs. Brucella species are gram-negative, nonmotile, facultative intracellular coccobaccilli belonging to the α-2 subdivision of proteobacteria. The genus consists of six classical species, namely, Brucella abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae, plus more recently discovered strains from marine mammals. Of the Brucella species, B. abortus, B. melitensis, and B. suis are of principal human health and economic importance. These species have smooth lipopolysaccharide (sLPS), which is considered a major virulence factor of disease (23), whereas B. ovis and B. canis have rough LPS (1).The World Organization for Animal Health (OIE) prescribed and alternative serological tests for diagnosis of brucellosis caused by smooth strains rely largely upon the measurement of the host antibody response to the O antigen of the sLPS (8, 22). Classical tests include the Rose Bengal test, the complement fixation test (CFT), and the serum agglutination test (SAT), all of which employ a whole-cell antigen as the key diagnostic reagent. More contemporary techniques, such as indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and fluorescent polarization assay (FPA), employ purified LPS or O antigen as the diagnostic reagent. The immunodominance of the LPS O antigen is the basis for the generally excellent sensitivity of these assays (21). However, the use of this antigen can lead to false-positive serological results when animals are infected with bacteria possessing O antigens of similar structure (6), such as Yersinia enterocolitica O:9.ELISAs are readily amenable to high-throughput testing due to the standardized nature of the technology and reagents. This allows for many efficiency savings compared to the classical assays, including the use of effective automation (16). Despite the advantages of ELISA over the classical tests in this regard, ELISAs still require several steps to complete, including separation steps. Although these steps can be automated, they are a vital part of the assay and are a frequent source of imprecision, error, and mechanical breakdown. Assays which have the advantages of ELISA, such as a 96-well format, objective assessment, and good sensitivity and specificity, but which reduce the burden of work and opportunity for error are clearly desirable.The Meso Scale Discovery (MSD) electrochemiluminescence (ECL) platform uses electrochemical stimulation of reporter molecules conjugated to biological components to generate a light signal measured by photodetectors (2, 31), such as a charge-coupled device (CCD) camera. Carbon electrodes are integrated into the bottom of 96-well microtiter plates, to which biological components (for example, LPS) from traditional assays such as ELISA can be passively adsorbed. Biological conjugates, for example, monoclonal antibodies (MAbs), can be labeled with the reporter molecule ruthenium(II) tris-bipyridal [Ru(bpy)₃²⁺], which upon electrical stimulation emits light at 620 nm if it is within sufficient proximity to the carbon electrode excitation source. The reaction is enhanced by the addition of read buffer, which contains coreactants, including tripropylamine. Nonspecific signals are minimized as the stimulation mechanism (electricity) is decoupled from the signal (light).MSD ECL assays have the potential for separation-based (“wash”) and non-separation-based (“no-wash”) immunoassays due to the proximity-based nature of signal generation, where only labels near the surfaces of the electrodes are stimulated. The non-separation-based approach allows the addition of read buffer (MSD) directly to the components of the assay; this has an advantage over separation-based assays in that it removes a source of variation and reduces the time and labor required to perform the assay. In addition, MSD ECL assays can be multiplexed by spotting up to 10 different antigens onto discrete areas of the carbon electrode within each well of a 96-well plate; thus, the potential exists for multiplexed no-wash serological assays. The signal from each spot can be detected independently by a CCD camera, providing a quantitative measurement of the amount of light detected. Furthermore, the time taken to read a 96-well plate, approximately 70 s, is comparable to that for ELISAs and compares favorably to those for some other multiplex and ECL systems.We assessed the feasibility and potential of wash and no-wash MSD ECL assays for veterinary serodiagnosis of brucellosis. Biological components (B. melitensis 16 M sLPS antigen and BM40 [10], an anti-M O-chain epitope MAb) from the brucellosis cELISA produced by the Veterinary Laboratories Agency (26) were applied to the MSD platform. As with the parent cELISA, the wash and no-wash ECL assays allow competition between serum antibodies and the MAb BM40 [labeled with Ru(bpy)₃²⁺ for the MSD ECL assay] for sLPS bound to the carbon electrode. This affects the quantity of BM40 able to bind to this target. Thus, a positive result yields a low-intensity signal and a negative result yields a high-intensity signal. The new assays were validated against existing serological methods, using sera from Brucella-infected and noninfected ruminants.     

The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes

Five candidate genes from the Brucella melitensis 16M genome were selected. Eukaryotic expression plasmids encoding these antigens were constructed and expression was verified in vitro from transfected Cos7 cells. Each vaccine was assessed for protective efficacy in a BALB/c mouse brucellosis infection model. From these experiments two protective DNA vaccines were identified: p-omp25 and p-ialB. The Omp25 antigen (BMEI1249) has previously been studied in terms of Brucella virulence, serodiagnosis and as a protective antigen. However, this study represents the first report of a significant protective effect achieved against B. melitensis 16M challenge using the Omp25 antigen in a DNA vaccine approach. The other protective vaccine identified in this study was p-ialB. The ialB candidate (BMEI1584) was selected based upon its' putative function as an invasion protein which was assigned due to shared identity with the invasion protein B (ialB) of Bartonella bacilliformis. This candidate has not previously been investigated with regard to Brucella virulence or pathogenesis. This study is the first report to identify the Brucella invasion protein B (BMEI1584) as a novel protective antigen for brucellosis.