Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13.

Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PAPERCLIP.

A multi-phenotype genome-wide association study of clades causing tuberculosis in a Ghanaian- and South African cohort.

Despite decades of research and advancements in diagnostics and treatment, tuberculosis remains a major public health concern. New computational methods are needed to interrogate the intersection of host- and bacterial genomes. Paired host genotype datum and infecting bacterial isolate information were analysed for associations using a multinomial logistic regression framework implemented in SNPTest. A cohort of 853 admixed South African participants and a Ghanaian cohort of 1359 participants were included. Two directly genotyped variants, namely rs529920 and rs41472447, were identified in the Ghanaian cohort as being statistically significantly associated with risk for infection with strains of different members of the MTBC. Thus, a multinomial logistic regression using paired host-pathogen data may prove valuable for investigating the complex relationships driving infectious disease.

Development of a cytokine gene expression assay for the relative quantification of the African elephant (Loxodonta africana) cell-mediated immune responses.

Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.

CYTOKINE-RELEASE ASSAY FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN CHEETAH (ACINONYX JUBATUS).

The lack of species-specific assays for the diagnosis of infectious diseases, such as bovine tuberculosis, poses a threat to the management of wildlife populations, especially for vulnerable species such as cheetah (Acinonyx jubatus). The aim of this study was to identify and develop a cell-mediated immunological cytokine-release assay that could distinguish between Mycobacterium bovis-infected and uninfected cheetahs using commercially available feline cytokine ELISA and domestic cat (Felis catus) recombinant proteins. Antibodies against domestic cat cytokines, tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and interferon gamma (IFN-γ), were screened for cross-reactivity with plasma cytokines from cheetah whole blood stimulated using QuantiFERON®-TB Gold Plus (QFT) tubes. Evidence of cytokine production in response to QFT mitogen stimulation was observed in all four ELISA assays. However only the Mabtech Cat IFN-γ ELISABasic kit could distinguish between M. bovis-infected (n = 1) and uninfected (n = 1) cheetahs and was therefore selected for further evaluation. A preliminary cheetah specific cutoff value (11 pg/ml) for detecting M. bovis infection using the Mabtech Cat IFN-γ release assay was calculated using a M. bovis uninfected cheetah cohort. Although this study only included one confirmed M. bovis culture-positive and one M. bovis culture-negative cheetah, the Mabtech Cat IFN-γ release assay demonstrated its potential for diagnostic application in this species.

A regulatory variant in the C1Q gene cluster is associated with tuberculosis susceptibility and C1qA plasma levels in a South African population.

Several genetic studies have implicated genes that encode for components of the innate immune response in tuberculosis (TB) susceptibility. The complement system is an early player in the innate immune response and provides the host with initial protection by promoting phagocytosis of apoptotic or necrotic cells. The C1q molecule is the first component of the classical pathway that leads to the activation of complement by binding to immune complexes and is encoded by the C1Q gene cluster. We investigated variants in this region to determine its association with TB susceptibility. Five single nucleotide polymorphisms (SNPs) (rs12033074, rs631090, rs172378, rs587585, and rs665691) were genotyped using TaqMan® SNP assays in 456 TB cases and 448 healthy controls and analysed by logistic regression models. The rs587585 variant showed a significant additive allelic association where the minor G allele was found more frequently in TB cases than in controls in both the discovery (p = 0.023; OR = 1.30; 95% CI, 1.04-1.64) and validation cohort (p = 0.038; OR = 1.31; 95% CI, 1.22-1.40). In addition, we detected increased C1qA expression when comparing cases and controls (p = 0.037) and linked this to a dosage effect of the G allele, which increased C1qA expression in TB cases. This is the first study to report the association of C1Q gene polymorphisms with progression to tuberculosis.

Identification of a novel WAS mutation in a South African patient presenting with atypical Wiskott-Aldrich syndrome: a case report.

BACKGROUND: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in the WAS gene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes. CASE PRESENTATION: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identified c. 397 G > A as a novel hemizygous missense mutation located in exon 4 of WAS. CONCLUSION: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome.

The VetMAX™ M. tuberculosis complex PCR kit detects MTBC DNA in antemortem and postmortem samples from white rhinoceros (Ceratotherium simum), African elephants (Loxodonta africana) and African buffaloes (Syncerus caffer).

BACKGROUND: Bovine tuberculosis and tuberculosis are chronic infectious diseases caused by the Mycobacterium tuberculosis complex members, Mycobacterium bovis and Mycobacterium tuberculosis, respectively. Infection with M. bovis and M. tuberculosis have significant implications for wildlife species management, public health, veterinary disease control, and conservation endeavours. RESULTS: Here we describe the first use of the VetMAX™ Mycobacterium tuberculosis complex (MTBC) DNA quantitative real-time polymerase chain reaction (qPCR) detection kit for African wildlife samples. DNA was extracted from tissues harvested from 48 African buffaloes and MTBC DNA was detected (test-positive) in all 26 M. bovis culture-confirmed animals with an additional 12 PCR-positive results in culture-negative buffaloes (originating from an exposed population). Of six MTBC-infected African rhinoceros tested, MTBC DNA was detected in antemortem and postmortem samples from five animals. The PCR was also able to detect MTBC DNA in samples from two African elephants confirmed to have M. bovis and M. tuberculosis infections (one each). Culture-confirmed uninfected rhinoceros and elephants' samples tested negative in the PCR assay. CONCLUSIONS: These results suggest this new detection kit is a sensitive screening test for the detection of MTBC-infected African buffaloes, African elephants and white rhinoceros.

Mycobacterium bovis Infection in African Wild Dogs, Kruger National Park, South Africa.

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies.

Risk alleles for tuberculosis infection associate with reduced immune reactivity in a wild mammalian host.

Integrating biological processes across scales remains a central challenge in disease ecology. Genetic variation drives differences in host immune responses, which, along with environmental factors, generates temporal and spatial infection patterns in natural populations that epidemiologists seek to predict and control. However, genetics and immunology are typically studied in model systems, whereas population-level patterns of infection status and susceptibility are uniquely observable in nature. Despite obvious causal connections, organizational scales from genes to host outcomes to population patterns are rarely linked explicitly. Here we identify two loci near genes involved in macrophage (phagocyte) activation and pathogen degradation that additively increase risk of bovine tuberculosis infection by up to ninefold in wild African buffalo. Furthermore, we observe genotype-specific variation in IL-12 production indicative of variation in macrophage activation. Here, we provide measurable differences in infection resistance at multiple scales by characterizing the genetic and inflammatory variation driving patterns of infection in a wild mammal.

Drug-Penetration Gradients Associated with Acquired Drug Resistance in Patients with Tuberculosis.

RATIONALE: Acquired resistance is an important driver of multidrug-resistant tuberculosis (TB), even with good treatment adherence. However, exactly what initiates the resistance and how it arises remain poorly understood. OBJECTIVES: To identify the relationship between drug concentrations and drug susceptibility readouts (minimum inhibitory concentrations [MICs]) in the TB cavity. METHODS: We recruited patients with medically incurable TB who were undergoing therapeutic lung resection while on treatment with a cocktail of second-line anti-TB drugs. On the day of surgery, antibiotic concentrations were measured in the blood and at seven prespecified biopsy sites within each cavity. Mycobacterium tuberculosis was grown from each biopsy site, MICs of each drug identified, and whole-genome sequencing performed. Spearman correlation coefficients between drug concentration and MIC were calculated. MEASUREMENTS AND MAIN RESULTS: Fourteen patients treated for a median of 13 months (range, 5-31 mo) were recruited. MICs and drug resistance-associated single-nucleotide variants differed between the different geospatial locations within each cavity, and with pretreatment and serial sputum isolates, consistent with ongoing acquisition of resistance. However, pretreatment sputum MIC had an accuracy of only 49.48% in predicting cavitary MICs. There were large concentration-distance gradients for each antibiotic. The location-specific concentrations inversely correlated with MICs (P < 0.05) and therefore acquired resistance. Moreover, pharmacokinetic/pharmacodynamic exposures known to amplify drug-resistant subpopulations were encountered in all positions. CONCLUSIONS: These data inform interventional strategies relevant to drug delivery, dosing, and diagnostics to prevent the development of acquired resistance. The role of high intracavitary penetration as a biomarker of antibiotic efficacy, when assessing new regimens, requires clarification.