The dynamic course of psychological outcomes following the Victorian Black Saturday bushfires.

OBJECTIVES: To profile the long-term mental health outcomes of those affected by the 2009 Black Saturday bushfires and to document the course of mental health since the disaster. METHOD: The longitudinal Beyond Bushfires study included 1017 respondents (Wave 1; 3-4 years after the fires), 736 (76.1%) at Wave 2 (5 years after the fires) and 525 (51.6%) at Wave 3 (10 years after the fires). The survey indexed fire-related and subsequent stressful events, probable posttraumatic stress disorder, major depressive disorder, alcohol use, severe distress and receipt of health services for mental health problems. RESULTS: Relative to their status 3-4 years after the fires, there were reduced rates of fire-related posttraumatic stress disorder (6.2% vs 12.2%), general posttraumatic stress disorder (14.9% vs 18.7%) and severe distress (4.4% vs 7.5%) at 10 years. There were comparable rates between Wave 1 and Wave 3 for depression (10.9% vs 8.3%) and alcohol abuse (21.8% vs 18.5%). Of people in high-affected regions, 22.1% had posttraumatic stress disorder, depression or severe distress at Wave 3. One-third to one-half of participants who reported probable posttraumatic stress disorder or depression at any assessment did not display the disorder at the next assessment. Worsening of mental health at Wave 3 was associated with the extent of property loss, exposure to recent traumatic events or recent stressful life events. Only 24.6% of those with a probable disorder had sought professional help for this in the previous 6 months. CONCLUSION: Approximately one-fifth of people from high-affected areas have a probable psychological disorder a decade after the fires. Mental health appears to fluctuate for those who are not consistently resilient, apparently as a result of ongoing stressors. The observation that most people with probable disorder are not receiving care highlights the need for further planning about managing long-term mental health needs of disaster-affected communities.

Delayed Disaster Impacts on Academic Performance of Primary School Children.

Social disruption caused by natural disasters often interrupts educational opportunities for children. However, little is known about children's learning in the following years. This study examined change in academic scores for children variably exposed to a major bushfire in Australia. Comparisons were made between children attending high, medium, and low disaster-affected primary schools 2-4 years after the disaster (n = 24,642; 9-12 years). The results showed that in reading and numeracy expected gains from Year 3 to Year 5 scores were reduced in schools with higher levels of bushfire impact. The findings highlight the extended period of academic impact and identify important opportunities for intervention in the education system to enable children to achieve their academic potential.

Longitudinal study of changing psychological outcomes following the Victorian Black Saturday bushfires.

OBJECTIVES: To map the changing prevalence and predictors of psychological outcomes in affected communities 5 years following the Black Saturday bushfires in Victoria. METHOD: Follow-up assessment of longitudinal cohort study in high, medium and non-affected communities in Victoria, Australia. Participants included 1017 respondents (Wave 1) interviewed via telephone and web-based survey between December 2011 and January 2013, and 735 (76.1%) eligible participants were retested between July and November 2014 (Wave 2). The survey included measures of fire-related and subsequent stressful events, probable posttraumatic stress disorder, major depressive episode, alcohol use and severe distress. RESULTS: There were reduced rates of fire-related posttraumatic stress disorder (8.7% vs 12.1%), general posttraumatic stress disorder (14.7% vs 18.2%), major depressive episode (9.0% vs 10.9%) and serious mental illness (5.4% vs 7.8%). Rates of resilience increased over time (81.8% vs 77.1%), and problem alcohol use remained high across Wave 1 (22.1%) and Wave 2 (21.4%). The most robust predictor of later development of fire-related posttraumatic stress disorder (odds ratio: 2.11; 95% confidence interval: [1.22, 3.65]), general posttraumatic stress disorder (odds ratio: 3.15; 95% confidence interval: [1.98, 5.02]), major depressive episode (odds ratio: 2.86; 95% confidence interval: [1.74, 4.70]), serious mental illness (odds ratio: 2.67; 95% confidence interval: [0.57, 1.72]) or diminished resilience (odds ratio: 2.01; 95% confidence interval: [1.32, 3.05]) was extent of recent life stressors. CONCLUSION: Although rates of mental health problems diminished over time, they remained higher than national levels. Findings suggest that policy-makers need to recognize that the mental health consequences of disasters can persist for many years after the event and need to allocate resources towards those who are most at risk as a result of substantive losses and ongoing life stressors.

The role of anger and ongoing stressors in mental health following a natural disaster.

OBJECTIVE: Research has established the mental health sequelae following disaster, with studies now focused on understanding factors that mediate these outcomes. This study focused on anger, alcohol, subsequent life stressors and traumatic events as mediators in the development of mental health disorders following the 2009 Black Saturday Bushfires, Australia's worst natural disaster in over 100 years. METHOD: This study examined data from 1017 (M = 404, F = 613) adult residents across 25 communities differentially affected by the fires and participating in the Beyond Bushfires research study. Data included measures of fire exposure, posttraumatic stress disorder, depression, alcohol abuse, anger and subsequent major life stressors and traumatic events. Structural equation modeling assessed the influence of factors mediating the effects of fire exposure on mental health outcomes. RESULTS: Three mediation models were tested. The final model recorded excellent fit and observed a direct relationship between disaster exposure and mental health outcomes (b = .192, p < .001) and mediating relationships via Anger (b = .102, p < .001) and Major Life Stressors (b = .128, p < .001). Each gender was compared with multiple group analyses and while the mediation relationships were still significant for both genders, the direct relationship between exposure and outcome was no longer significant for men (p = .069), but remained significant (b = .234, p < .001) for women. CONCLUSIONS: Importantly, anger and major life stressors mediate the relationship between disaster exposure and development of mental health problems. The findings have significant implications for the assessment of anger post disaster, the provision of targeted anger-focused interventions and delivery of government and community assistance and support in addressing ongoing stressors in the post-disaster context to minimize subsequent mental health consequences.

Psychological outcomes following the Victorian Black Saturday bushfires.

OBJECTIVE: We aimed to map the prevalence and predictors of psychological outcomes in affected communities 3-4 years after the Black Saturday bushfires in the state of Victoria, Australia. METHODS: Baseline assessment of a longitudinal cohort study in high-, medium-, and low-affected communities in Victoria. Participants included 1017 residents of high-, medium-, and low-affected fire communities. Participants were surveyed by means of a telephone and web-based interview between December 2011 and January 2013. The survey included measures of fire-related post-traumatic stress disorder (PTSD) and general PTSD from other traumatic events, major depressive episode, alcohol use, and general psychological distress. RESULTS: The majority of respondents in the high- (77.3%), medium- (81.3%), and low-affected (84.9%) communities reported no psychological distress on the K6 screening scale. More participants in the high-affected communities (15.6%) reported probable PTSD linked to the bushfires than medium- (7.2%) and low-affected (1.0%) communities (odds ratio (OR): 4.57, 95% confidence interval (CI): 2.61-8.00, p = 0.000). Similar patterns were observed for depression (12.9%, 8.8%, 6.3%, respectively) (OR: 1.83, 95% CI: 1.17-2.85, p = 0.008) and severe psychological distress (9.8%, 5.0%, 4.9%, respectively) (OR: 2.08, 95% CI: 1.23-3.55, p = 0.007). All communities reported elevated rates of heavy drinking (24.7%, 18.7%, 19.6%, respectively); however, these were higher in the high-affected communities (OR: 1.39, 95% CI: 1.01-1.89, p = 0.04). Severe psychological distress was predicted by fear for one's life in the bushfires, death of someone close to them in the bushfires, and subsequent stressors. One-third of those with severe psychological distress did not receive mental health assistance in the previous month. CONCLUSIONS: Several years following the Black Saturday bushfires the majority of affected people demonstrated resilience without indications of psychological distress. A significant minority of people in the high-affected communities reported persistent PTSD, depression, and psychological distress, indicating the need for promotion of the use of health and complementary services, community-based initiatives, and family and other informal supports, to target these persistent problems.

Beyond Bushfires: Community, Resilience and Recovery - a longitudinal mixed method study of the medium to long term impacts of bushfires on mental health and social connectedness.

BACKGROUND: Natural disasters represent an increasing threat both in terms of incidence and severity as a result of climate change. Although much is known about individual responses to disasters, much less is known about the social and contextual response and how this interacts with individual trajectories in terms of mental health, wellbeing and social connectedness. The 2009 bushfires in Victoria, Australia caused much loss of life, property destruction, and community disturbance. In order to progress future preparedness, response and recovery, it is crucial to measure and understand the impact of disasters at both individual and community levels. METHODS/DESIGN: This study aims to profile the range of mental health, wellbeing and social impacts of the Victorian 2009 bushfires over time using multiple methodologies and involving multiple community partners. A diversity of communities including bushfire affected and unaffected will be involved in the study and will include current and former residents (at the time of the Feb 2009 fires). Participants will be surveyed in 2012, 2014 and, funding permitting, in 2016 to map the predictors and outcomes of mental health, wellbeing and social functioning. Ongoing community visits, as well as interviews and focus group discussions in 2013 and 2014, will provide both contextual information and evidence of changing individual and community experiences in the medium to long term post disaster. The study will include adults, adolescents and children over the age of 5. DISCUSSION: Conducting the study over five years and focussing on the role of social networks will provide new insights into the interplay between individual and community factors and their influence on recovery from natural disaster over time. The study findings will thereby expand understanding of long term disaster recovery needs for individuals and communities.

Immunologically reactive M. leprae antigens with relevance to diagnosis and vaccine development.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae that can manifest a wide variety of immunological and clinical outcomes ranging from potent humoral responses among borderline lepromatous (BL) and lepromatous (LL) patients to strong cellular responses among tuberculoid (TT) and borderline tuberculoid (BT) patients. Until recently, relatively little has been known about the immune responses to individual proteins of M. leprae recognized during leprosy. METHODS: The immune reactivity to a panel of 33 M. leprae recombinant proteins was evaluated among leprosy patients and controls from a high endemic area for leprosy (Goiania/GO, Central Brazil). Serum IgG responses were measured by ELISA (45 participants/group) and T cell responses (20 participants/group) were evaluated by IFN-gamma production in 24 hours whole blood cultures with antigen (whole blood assay-WBA). Study groups were newly diagnosed, untreated TT/BT and BL/LL leprosy patients classified by Ridley Jopling criteria and household contacts of BL/LL patients (HHC). Control groups were HIV-1 negative pulmonary tuberculosis patients (TB) and healthy individuals from the same endemic area (EC). In silico predictions indicated the level of identity of M. leprae proteins with homologues in other mycobacteria and the presence of T cell and B cell epitopes. RESULTS: Despite the prediction that all proteins would be reactive, 16 of 33 (48%) of the single proteins tested were immunogenic (recognized in WBA or ELISA) and seventeen were non-immunogenic (not recognized in either assay). Among the 16 immunogenic proteins, 9 were considered leprosy specific in WBA inducing cell-mediated IFN-gamma secretion from TT/BT patients and HHC. Three of these proteins were also leprosy specific in serology being recognized by serum IgG from LL/BL patients. Seven of the immunogenic proteins were not leprosy specific. CONCLUSIONS: New M. leprae antigens recognized by antibody responses of BL/LL patients and cellular responses of TT/BT leprosy patients were identified. An improved serological diagnostic test for leprosy could be developed by incorporating these IgG-reactive antigens to the current PGL-I based tests. Moreover our data indicate that the WBA is a robust, relatively simple and user friendly format for a T cell based diagnostic test. The field use of these test formats in leprosy endemic countries could contribute to early leprosy diagnosis before the development of deformities and disabilities.

The role of the natural environment in disaster recovery: "We live here because we love the bush".

This mixed-methods study explored the role of connection to the natural environment in recovery from the 'Black Saturday' bushfires that blazed across Victoria, Australia, in February 2009. Qualitative findings demonstrated that many participants had a strong connection to the natural environment, experienced considerable grief as a result of its devastation in the fires and drew solace from seeing it regenerate over the following months and years. Quantitative analyses indicated that a strong attachment to the environment was associated with reduced psychological distress, fewer symptoms of major depression and fire-related PTSD, and higher levels of resilience, post-traumatic growth and life satisfaction. While social connections are increasingly recognized as supportive of disaster recovery, the influence of landscapes also needs to be recognized in terms of the impact of their destruction as well as their therapeutic potential.

Mental Health and Social Networks After Disaster.

OBJECTIVE: Although disasters are a major cause of mental health problems and typically affect large numbers of people and communities, little is known about how social structures affect mental health after a disaster. The authors assessed the extent to which mental health outcomes after disaster are associated with social network structures. METHOD: In a community-based cohort study of survivors of a major bushfire disaster, participants (N=558) were assessed for probable posttraumatic stress disorder (PTSD) and probable depression. Social networks were assessed by asking participants to nominate people with whom they felt personally close. These nominations were used to construct a social network map that showed each participant's ties to other participants they nominated and also to other participants who nominated them. This map was then analyzed for prevailing patterns of mental health outcomes. RESULTS: Depression risk was higher for participants who reported fewer social connections, were connected to other depressed people, or were connected to people who had left their community. PTSD risk was higher if fewer people reported being connected with the participant, if those who felt close to the participant had higher levels of property loss, or if the participant was linked to others who were themselves not interconnected. Interestingly, being connected to other people who in turn were reciprocally close to each other was associated with a lower risk of PTSD. CONCLUSIONS: These findings provide the first evidence of disorder-specific patterns in relation to one's social connections after disaster. Depression appears to co-occur in linked individuals, whereas PTSD risk is increased with social fragmentation. These patterns underscore the need to adopt a sociocentric perspective of postdisaster mental health in order to better understand the potential for societal interventions in the wake of disaster.

Dyadic effects of attachment on mental health: Couples in a postdisaster context.

Research on mental health following disasters has led to the identification of many individual protective and risk factors for postdisaster mental health. However, there is little understanding of the exact influence that disasters have on the functioning of intimate relationships. Especially relevant are attachment styles, which are likely to play an important role in the provision and perception of social support between partners, and subsequent mental health outcomes. Heterosexual couples (N = 127) affected by the 2009 Victorian "Black Saturday" Bushfires in southeastern Australia were surveyed for disaster experiences, posttraumatic stress disorder (PTSD) symptoms, depression, and attachment style between May 2012 and January 2013, approximately 3 years after the disaster. Using actor-partner interdependence models (APIM), we examined both intrapersonal and interpersonal associations of attachment anxiety and avoidance with depression and PTSD, in combination with shared disaster exposure. Male partners' attachment avoidance was associated with depression and PTSD in both partners. By contrast, a female partner's attachment avoidance was associated with greater depression and PTSD in herself, but fewer PTSD symptoms in a male partner. Amid the chronic stressors of a postdisaster setting, the attachment avoidance of the male partner may play a particularly negative role, with his tendency toward isolation and denial becoming especially maladaptive for the couple as a whole. The female partner's attachment avoidance is likewise an important factor, but its associations with negative social support and relationship breakup must be clarified to understand its impact on partnership functioning. (PsycINFO Database Record

A structure-function approach to optimizing TLR4 ligands for human vaccines.

Adjuvants are combined with vaccine antigens to enhance and modify immune responses, and have historically been primarily crude, undefined entities. Introducing toll-like receptor (TLR) ligands has led to a new generation of adjuvants, with TLR4 ligands being the most extensively used in human vaccines. The TLR4 crystal structures demonstrate extensive contact with their ligands and provide clues as to how they discriminate a broad array of molecules and activate or attenuate innate, as well as adaptive, responses resulting from these interactions. Leveraging this discerning ability, we made subtle chemical alterations to the structure of a synthetic monophosphoryl lipid-A molecule to produce SLA, a designer TLR4 ligand that had a number of desirable adjuvant effects. The SLA molecule stimulated human TLR4 and induced Th1 biasing cytokines and chemokines. On human cells, the activity of SLA plateaued at lower concentrations than the lipid A comparator, and induced cytokine profiles distinct from other known TLR4 agonists, indicating the potential for superior adjuvant performance. SLA was formulated in an oil-in-water emulsion, producing an adjuvant that elicited potent Th1-biased adaptive responses. This was verified using a recombinant Leishmania vaccine antigen, first in mice, then in a clinical study in which the antigen-specific Th1-biased responses observed in mice were recapitulated in humans. These results demonstrated that using structure-based approaches one can predictably design and produce modern adjuvant formulations for safe and effective human vaccines.

Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy.

Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.

Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Protection of mice from Mycobacterium tuberculosis by ID87/GLA-SE, a novel tuberculosis subunit vaccine candidate.

Tuberculosis is a major health concern. Non-living tuberculosis (TB) vaccine candidates may not only be safer than the current vaccine (BCG) but could also be used to boost BCG to enhance or elongate protection. No subunit vaccines, however, are currently available for TB. To address this gap and to improve the global TB situation, we have generated a defined subunit vaccine by genetically fusing the genes of 3 potent protein Mtb antigens, Rv2875, Rv3478 and Rv1886, into a single product: ID87. When delivered with a TLR4 agonist-based adjuvant, GLA-SE, ID87 immunization reduced Mtb burden in the lungs of experimentally infected mice. The reduction in bacterial burden of ID87/GLA-SE immunized mice was accompanied by an early and significant leukocyte infiltration into the lungs during the infectious process. ID87/GLA-SE appears to be a promising new vaccine candidate that warrants further development.

Plasma antibody profiles as diagnostic biomarkers for tuberculosis.

Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.

Antigen-specific T-cell responses of leprosy patients.

The identification of human T-cell antigens of Mycobacterium leprae could improve treatment and help to disrupt the transmission of leprosy by directing diagnosis and vaccine programs. This study screened a panel of M. leprae recombinant proteins for T-cell recall responses, measured by gamma interferon (IFN-gamma) production, among leprosy patients. After initial studies using peripheral blood mononuclear cells from leprosy patients, we transitioned our studies to simple whole-blood assays (WBA), which are more applicable in field or clinical settings. T-cell responses generated in WBA using blood from individuals in Goiânia, Brazil, demonstrated that several M. leprae antigens (ML0276, ML0840, ML1623, ML2044, and 46f) elicited >0.5 IU/ml IFN-gamma, and these proteins were classified as immunogenic and leprosy specific. Several of these individual antigens were recognized by cells from >60% of Brazilian paucibacillary (PB) leprosy patients, and ML0276, ML0840, ML1623, and 46f complemented each other such that 82% of PB patients had strong (>1.25 IU/ml IFN-gamma) responses to at least one of these proteins. These proteins were also recognized by cells from a significant proportion of the household contacts of multibacillary leprosy patients, but in contrast, few responses were observed in active tuberculosis patients or healthy control groups from areas of endemicity. Our results indicate several potential candidate antigens which may be useful for either leprosy diagnosis or vaccination and demonstrate the utility of leprosy WBA that can be applied broadly in clinical or field settings.

Selection of antigens and development of prototype tests for point-of-care leprosy diagnosis.

Leprosy can be a devastating chronic infection that causes nerve function impairment and associated disfigurement. Despite the recent reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. The diagnosis of leprosy is currently based on the appearance of clinical signs and requires expert clinical, as well as labor-intensive and time-consuming laboratory or histological, evaluation. For the purpose of developing an effective, simple, rapid, and low-cost diagnostic alternative, we have analyzed the serologic antibody response to identify Mycobacterium leprae proteins that are recognized by leprosy patients. More than 100 recombinant antigens were analyzed in a protein array format to select those with discriminatory properties for leprosy diagnosis. As expected, multibacillary leprosy patients recognized more antigens with stronger antibody responses than paucibacillary leprosy patients. Our data indicate, however, that multibacillary patients can be distinguished from paucibacillary patients, and both of these groups can be segregated from endemic control groups. We went on to confirm the diagnostic properties of antigens ML0405 and ML2331 and the LID-1 fusion construct of these two proteins by enzyme-linked immunosorbent assay. We then demonstrated the performance of these antigens in rapid test formats with a goal of developing a point-of-care diagnostic test. A serological diagnostic test capable of identifying and allowing treatment of leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Antigen-specific cellular and humoral responses are induced by intradermal Mycobacterium leprae infection of the mouse ear.

Leprosy is caused by infection with Mycobacterium leprae. The immune response of leprosy patients can be highly diverse, ranging from strong cellular responses accompanied by an apparent deficit of M. leprae-specific antibodies to strong humoral responses with a deficit of cell-mediated responses. Leprosy takes many years to manifest, and this has precluded analyses of disease and immune response development in infected humans. In an attempt to better define development of the immune response during leprosy we have developed an M. leprae ear infection model. Intradermal inoculation of M. leprae into the ear supported not only infection but also the development of a chronic inflammatory response. The inflammatory response was localized, comprising a T-cell infiltration into the ear and congestion of cells in the draining lymph nodes. The development of local chronic inflammation was prevented by rifampin treatment. Importantly, and in contrast to subcutaneous M. leprae footpad infection, systemic M. leprae-specific gamma interferon and antibody responses were detected following intradermal ear infection. These results indicate the utility of intradermal ear infection for both induction and understanding of the immune response during M. leprae infection and the identification or testing of new leprosy treatments.

Use of protein antigens for early serological diagnosis of leprosy.

Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy.

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.