Pattern Recognition in Pulmonary Tuberculosis Defined by High Content Peptide Microarray Chip Analysis Representing 61 Proteins from M. tuberculosis

Background: Serum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M.tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations. Method: We constructed a high-content peptide microarray with 61 M.tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/2. Findings: Three distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB2, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB2 does no

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

The emerging human disease Buruli ulcer, caused by Mycobacterium ulcerans, is of increasing challenge for public health systems in many countries, mainly in West and Central sub-Saharan Africa. Genetic differentiation of patient isolates, a prerequisite for scientific studies on and intervention of disease transmission and dispersal, is hampered by an exceptional lack of genetic diversity within this species. Comparative genomics on M. ulcerans of worldwide geographical origin has already allowed for distinguishing several haplotypes separated into two distinct lineages. Differences in prevalence and incidence of Buruli ulcer were already suspected, but biological relevance for this was unclear. Here, we show newly identified hot spot regions of genomic instability, a biased silencing of coding sequences belonging to distinct functional groups, and a differential gene repertoire across M. ulcerans strains. Gene inactivation mediated by different mechanisms in M. ulcerans adds to the concept of anti-virulence genes observed in an increasing number of bacterial species. According to this concept, loss of such genes—in addition to gain of function—may confer a selective advantage for a pathogen radiating into a new niche. In the case of M. ulcerans, a distinct set of disrupted genes may enhance virulence, particularly in the classical lineage

Unconventional Low-Cost Fabrication and Patterning Techniques for Point of Care Diagnostics

The potential of rapid, quantitative, and sensitive diagnosis has led to many innovative ‘lab on chip’ technologies for point of care diagnostic applications. Because these chips must be designed within strict cost constraints to be widely deployable, recent research in this area has produced extremely novel non-conventional micro- and nano-fabrication innovations. These advances can be leveraged for other biological assays as well, including for custom assay development and academic prototyping. The technologies reviewed here leverage extremely low-cost substrates and easily adoptable ways to pattern both structural and biological materials at high resolution in unprecedented ways. These new approaches offer the promise of more rapid prototyping with less investment in capital equipment as well as greater flexibility in design. Though still in their infancy, these technologies hold potential to improve upon the resolution, sensitivity, flexibility, and cost-savings over more traditional approaches

Protection against Tuberculosis in Eurasian Wild Boar Vaccinated with Heat-Inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines

A Preliminary Analysis of the Immunoglobulin Genes in the African Elephant (Loxodonta africana)

The genomic organization of the IgH (Immunoglobulin heavy chain), Igκ (Immunoglobulin kappa chain), and Igλ (Immunoglobulin lambda chain) loci in the African elephant (Loxodonta africana) was annotated using available genome data. The elephant IgH locus on scaffold 57 spans over 2,974 kb, and consists of at least 112 VH gene segments, 87 DH gene segments (the largest number in mammals examined so far), six JH gene segments, a single μ, a δ remnant, and eight γ genes (α and ε genes are missing, most likely due to sequence gaps). The Igκ locus, found on three scaffolds (202, 50 and 86), contains a total of 153 Vκ gene segments, three Jκ segments, and a single Cκ gene. Two different transcriptional orientations were determined for these Vκ gene segments. In contrast, the Igλ locus on scaffold 68 includes 15 Vλ gene segments, all with the same transcriptional polarity as the downstream Jλ-Cλ cluster. These data suggest that the elephant immunoglobulin gene repertoire is highly diverse and complex. Our results provide insights into the immunoglobulin genes in a placental mammal that is evolutionarily distant from humans, mice, and domestic animals

Evaluation of the CervidTB STAT-PAK for the Detection of \u3ci\u3eMycobacterium bovis\u3c/i\u3e Infection in Wild Deer in Great Britain

Deer are acknowledged as hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), and determining the prevalence of infection in deer species is one of the key steps in understanding the epidemiological role played by cervids in the transmission and maintenance of bTB in the United Kingdom. This study evaluated a rapid lateral-flow test for the detection of bTB in samples from wild deer species in the United Kingdom. Fallow deer (Dama dama), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) from areas in Wales, the Cotswolds, and southwestern England were necropsied for a bTB survey. Serum samples from individual deer were tested with the CervidTB STAT-PAK, and the results were evaluated against the culture of M. bovis from tissues (n = 432). Sensitivity and specificity were 85.7% (95% confidence interval [CI], 42.1 to 99.6%) and 94.8% (95% CI, 92.3 to 96.7%), respectively, with an odds ratio of 109.9 (95% CI, 12.7 to 953.6%) for a positive STAT-PAK result among culture-positive deer. The low prevalence of infection (3.8%, n = 860) affected the confidence of the sensitivity estimate of the test, but all culture-positive fallow deer (n= 6) were detected by the test. In addition, antibodies to M. bovis could be detected in poor-quality serum samples. The results suggest that the CervidTB STAT-PAK could be deployed as a field test for further evaluation

Antibody Responses in Reindeer (\u3ci\u3eRangifer tarandus\u3c/i\u3e) Infected with \u3ci\u3eMycobacterium bovis\u3c/i\u3e

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer

Evaluation of the CervidTB STAT-PAK for the Detection of Mycobacterium bovis Infection in Wild Deer in Great Britain▿

Deer are acknowledged as hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), and determining the prevalence of infection in deer species is one of the key steps in understanding the epidemiological role played by cervids in the transmission and maintenance of bTB in the United Kingdom. This study evaluated a rapid lateral-flow test for the detection of bTB in samples from wild deer species in the United Kingdom. Fallow deer (Dama dama), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) from areas in Wales, the Cotswolds, and southwestern England were necropsied for a bTB survey. Serum samples from individual deer were tested with the CervidTB STAT-PAK, and the results were evaluated against the culture of M. bovis from tissues (n = 432). Sensitivity and specificity were 85.7% (95% confidence interval [CI], 42.1 to 99.6%) and 94.8% (95% CI, 92.3 to 96.7%), respectively, with an odds ratio of 109.9 (95% CI, 12.7 to 953.6%) for a positive STAT-PAK result among culture-positive deer. The low prevalence of infection (3.8%, n = 860) affected the confidence of the sensitivity estimate of the test, but all culture-positive fallow deer (n = 6) were detected by the test. In addition, antibodies to M. bovis could be detected in poor-quality serum samples. The results suggest that the CervidTB STAT-PAK could be deployed as a field test for further evaluation

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas