Evaluating the Accuracy of Imputation Methods in a Five-Way Admixed Population.

Genotype imputation is a powerful tool for increasing statistical power in an association analysis. Meta-analysis of multiple study datasets also requires a substantial overlap of SNPs for a successful association analysis, which can be achieved by imputation. Quality of imputed datasets is largely dependent on the software used, as well as the reference populations chosen. The accuracy of imputation of available reference populations has not been tested for the five-way admixed South African Colored (SAC) population. In this study, imputation results obtained using three freely-accessible methods were evaluated for accuracy and quality. We show that the African Genome Resource is the best reference panel for imputation of missing genotypes in samples from the SAC population, implemented via the freely accessible Sanger Imputation Server.

A pilot study evaluating the utility of commercially available antibodies for flow cytometric analysis of Panthera species lymphocytes.

BACKGROUND: The immune response against tuberculosis in lions is still poorly defined and our understanding is hampered by the lack of lion specific reagents. The process for producing antibodies against a specific antigen is laborious and not available to many research laboratories. As the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine, we have investigated the use of commercially available antibodies to characterize T cell subsets in African lions (Panthera leo). RESULTS: Commercially available antibodies were screened and investigated the influence of two different sample processing methods, as well as the effect of time delay on cell surface marker expression on lion lymphocytes. Using commercially available antibodies, we were able to identify CD4+, CD5+, CD8+, CD14+, CD25+, CD44+ and CD45+ T lymphocytes in samples obtained by density gradient centrifugation as well as red cell lysis of lion whole blood. Two distinct lymphocyte populations, which differed in size and phenotype, were observed in the samples processed by density gradient centrifugation. CONCLUSION: Commercially available antibodies are able to differentiate between T lymphocyte subsets including immune effector cells in African lion whole blood, and possibly give insight into unique specie phenotypes.

Papanicolaou induced fluorescence, Ziehl-Neelsen and Auramine O stains on lymph node fine needle aspiration biopsy specimens from children: A comparative study.

BACKGROUND: The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria. METHODS: Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture. RESULTS: PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests. CONCLUSION: This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries.

Exome Sequencing Identifies a Novel MAP3K14 Mutation in Recessive Atypical Combined Immunodeficiency.

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met- versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches.

Progenitor strain introduction of Mycobacterium bovis at the wildlife-livestock interface can lead to clonal expansion of the disease in a single ecosystem.

Mycobacterium bovis infects multiple wildlife species and domesticated cattle across South Africa, and negatively impacts on livestock trade and movement of wildlife for conservation purposes. M. bovis infection was first reported in the Kruger National Park (KNP) in South Africa during the 1990s, and has since spread to infect numerous animal host species throughout the park and across South Africa. Whole genome sequencing data of 17 M. bovis isolates were analyzed to investigate the genomic diversity among M. bovis isolates causing disease in different animal host species from various locations in South Africa. M. bovis strains analyzed in this study are geographic rather than host species-specific. The clonal expansion of M. bovis in the KNP highlights the effect of an introduction of a transmissible infectious disease leading to a rising epidemic in wildlife, and emphasizes the importance of disease control and movement restriction of species that serve as disease reservoirs. In conclusion, the point source introduction of a single M. bovis strain type in the KNP ecosystem lead to an M. bovis outbreak in this area that affects various host species and poses an infection risk in neighboring rural communities where HIV prevalence is high.

Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta).

BACKGROUND: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. RESULTS: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. CONCLUSION: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.

Optimizing the use of cadavers by integrating pathology during anatomy dissection.

An accurate knowledge of anatomy, especially natural variation within individuals, is of vital clinical importance. Cadaver dissection during anatomical training may be a valuable introduction to pathology for undergraduate students, which can contribute greatly to a successful medical career. The purpose of this study was to determine the extent and type of pathology lesions in a cadaver population (n = 127) used for medical dissection. This was done to gauge whether sufficient pathology lesions representative of all the organ systems were present in the cadaver population to warrant the use of cadavers as an additional pathology learning resource. This study demonstrated a wide variety of pathology lesions in different organ systems. The respiratory system was most affected with pulmonary tuberculosis (TB) lesions being the most common finding (seen in 76% of cadavers) followed by bronchopneumonia and emphysema. Other common pathology findings included atherosclerosis, colonic diverticula, and chronic pyelonephritis. Skeletal fractures and degenerative joint disease were also noted. This study shows that cadaveric dissection offers a chance to alert and expose students to a wide variety of gross pathology and histopathology. It has been suggested that most medical students will practice in primary health care and as such more attention should be given to the pathology of commonly encountered diseases. This is particularly true for developing countries, where diseases such as TB are commonly encountered. The integration of pathology into the dissection hall may therefore be beneficial to student learning while simultaneously optimizing the use of cadaver material. Anat Sci Educ 9: 575-582. © 2016 American Association of Anatomists.

The plasmid-mediated evolution of the mycobacterial ESX (Type VII) secretion systems.

BACKGROUND: The genome of Mycobacterium tuberculosis contains five copies of the ESX gene cluster, each encoding a dedicated protein secretion system. These ESX secretion systems have been defined as a novel Type VII secretion machinery, responsible for the secretion of proteins across the characteristic outer mycomembrane of the mycobacteria. Some of these secretion systems are involved in virulence and survival in M. tuberculosis; however they are also present in other non-pathogenic mycobacteria, and have been identified in some non-mycobacterial actinomycetes. Three components of the ESX gene cluster have also been found clustered in some gram positive monoderm organisms and are predicted to have preceded the ESX gene cluster. RESULTS: This study used in silico and phylogenetic analyses to describe the evolution of the ESX gene cluster from the WXG-FtsK cluster of monoderm bacteria to the five ESX clusters present in M. tuberculosis and other slow-growing mycobacteria. The ancestral gene cluster, ESX-4, was identified in several nonmycomembrane producing actinobacteria as well as the mycomembrane-containing Corynebacteriales in which the ESX cluster began to evolve and diversify. A novel ESX gene cluster, ESX-4EVOL, was identified in some non-mycobacterial actinomycetes and M. abscessus subsp. bolletii. ESX-4EVOL contains all of the conserved components of the ESX gene cluster and appears to be a precursor of the mycobacterial ESX duplications. Between two and seven ESX gene clusters were identified in each mycobacterial species, with ESX-2 and ESX-5 specifically associated with the slow growers. The order of ESX duplication in the mycobacteria is redefined as ESX-4, ESX-3, ESX-1 and then ESX-2 and ESX-5. Plasmid-encoded precursor ESX gene clusters were identified for each of the genomic ESX-3, -1, -2 and -5 gene clusters, suggesting a novel plasmid-mediated mechanism of ESX duplication and evolution. CONCLUSIONS: The influence of the various ESX gene clusters on vital biological and virulence-related functions has clearly influenced the diversification and success of the various mycobacterial species, and their evolution from the non-pathogenic fast-growing saprophytic to the slow-growing pathogenic organisms.

Whole genome sequence analysis of Mycobacterium suricattae.

Tuberculosis occurs in various mammalian hosts and is caused by a range of different lineages of the Mycobacterium tuberculosis complex (MTBC). A recently described member, Mycobacterium suricattae, causes tuberculosis in meerkats (Suricata suricatta) in Southern Africa and preliminary genetic analysis showed this organism to be closely related to an MTBC pathogen of rock hyraxes (Procavia capensis), the dassie bacillus. Here we make use of whole genome sequencing to describe the evolution of the genome of M. suricattae, including known and novel regions of difference, SNPs and IS6110 insertion sites. We used genome-wide phylogenetic analysis to show that M. suricattae clusters with the chimpanzee bacillus, previously isolated from a chimpanzee (Pan troglodytes) in West Africa. We propose an evolutionary scenario for the Mycobacterium africanum lineage 6 complex, showing the evolutionary relationship of M. africanum and chimpanzee bacillus, and the closely related members M. suricattae, dassie bacillus and Mycobacterium mungi.

Mycobacterium bovis at the animal-human interface: a problem, or not?

Mycobacterium bovis is a pathogen of significant importance in livestock and a wide range of wild animal species worldwide. It is also known to cause tuberculosis disease in humans, a fact which has raised renewed concerns regarding the zoonotic risk for humans, especially those living at the animal-human interface. This review consolidates recent reports in the literature mainly on animal and zoonotic tuberculosis with an emphasis on evolution, epidemiology, treatment and diagnosis. The information presented reveals the fundamental differences in the complexity and level at which the disease affects the economy, ecosystem and human population of regions where animal tuberculosis control is achieved and regions where little or no control is implemented. In conclusion the review suggests that bovine tuberculosis has essentially been reduced to a disease of economic importance in the developed world, while low-income countries are facing a multifaceted impact which potentially affects the health of livestock, humans and ecosystems and which is likely to increase in the presence of debilitating diseases such as HIV/AIDS and other factors which negatively affect human livelihoods.