Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Tuberculosis research in South Africa over the past 30 years: From bench to bedside.

The South African Medical Research Council Centre for Tuberculosis Research has a rich history of high-impact research that has influenced our understating of this hyper-epidemic which is further exacerbated by the emergence and spread of drug-resistant forms of the disease. This review aims to summarise the past 30 years of research conducted in the Centre which has influenced the way that tuberculosis (TB) is diagnosed and treated. The review includes the development of new technologies for rapid screening of people with probable TB and the repurposing of human diagnostics for wildlife conservation.

Generation and characterization of thiol-deficient Mycobacterium tuberculosis mutants.

Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization of Mycobacterium tuberculosis (M.tb) mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficient M.tb mutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.

Proteomic analysis reveals that sulfamethoxazole induces oxidative stress in M. tuberculosis.

The emerging resistance of tuberculosis (TB) to current first line drugs (isoniazid, rifampicin, pyrazinamide, ethambutol) warrants alternative treatment approaches with broad-spectrum efficacy. Previously, we have shown that sulfamethoxazole (SMX) has synergestic activity with rifampicin against Mycobacterium tuberculosis. The primary target of SMX is folP1 in mycobacteria; however, SMX may affect other secondary targets in M. tuberculosis. This study investigated the potential additional targets of SMX in a clinical isolate of M. tuberculosis using Orbitrap mass spectrometry to identify differentially expressed proteins following treatment with a sub-lethal concentration of SMX. Raw data have been deposited as ProteomeXchange accession PXD009315. Our proteomic analysis identified approximately 1500 proteins in total of which 45 proteins were differentially regulated as a result of SMX treatment. These included 25 upregulated and 20 downregulated proteins. The oxidative stress proteins (Rv2428, AhpC and Rv2394, GgtB) and an enzyme from the electron transport chain (Ndh-II, Rv1854c) were found to be upregulated. Gene expression analysis correlated with the observed proteomic changes. In conclusion our results show that SMX treatment of a drug sensitive M. tuberculosis clinical isolate resulted in the regulation of proteins involved in the oxidative stress response, indicating the induction of oxidative stress by SMX in mycobacteria.

Antigen-specific interferon-gamma release is decreased following the single intradermal comparative cervical skin test in African buffaloes (Syncerus caffer).

Effective disease management of wildlife relies on the strategic application of ante-mortem diagnostic tests for early identification and removal of M. bovis-infected animals. To improve diagnostic performance, interferon-gamma release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST). Since buffaloes are major maintenance hosts of M. bovis, optimal application of bovine TB diagnostic tests are especially important. We aimed to determine whether the timing of blood collection relative to the TST has an influence on IFN-γ production and diagnostic outcome in African buffaloes. Release of IFN-γ in response to bovine purified protein derivative (PPD), avian PPD and PC-HP® and PC-EC® peptides was measured by Bovigam® and an in-house IGRA in a group of Bovigam®-positive and - negative buffaloes at the time the TST was performed and three days later. There was significantly lower IFN-γ release in response to these antigens post-TST in Bovigam®-positive buffaloes, but no significant changes in Bovigam®-negative buffaloes. Also, a significantly greater proportion of buffaloes were Bovigam®-positive prior to the TST than three days later. We therefore recommend that blood samples for use in IGRAs be collected prior to or at the time the TST is performed to facilitate the correct identification of greater numbers of IGRA-positive buffaloes.

Detection of tuberculosis by automatic cough sound analysis.

OBJECTIVE: Globally, tuberculosis (TB) remains one of the most deadly diseases. Although several effective diagnosis methods exist, in lower income countries clinics may not be in a position to afford expensive equipment and employ the trained experts needed to interpret results. In these situations, symptoms including cough are commonly used to identify patients for testing. However, self-reported cough has suboptimal sensitivity and specificity, which may be improved by digital detection. APPROACH: This study investigates a simple and easily applied method for TB screening based on the automatic analysis of coughing sounds. A database of cough audio recordings was collected and used to develop statistical classifiers. MAIN RESULTS: These classifiers use short-term spectral information to automatically distinguish between the coughs of TB positive patients and healthy controls with an accuracy of 78% and an AUC of 0.95. When a set of five clinical measurements is available in addition to the audio, this accuracy improves to 82%. By choosing an appropriate decision threshold, the system can achieve a sensitivity of 95% at a specificity of approximately 72%. The experiments suggest that the classifiers are using some spectral information that is not perceivable by the human auditory system, and that certain frequencies are more useful for classification than others. SIGNIFICANCE: We conclude that automatic classification of coughing sounds may represent a viable low-cost and low-complexity screening method for TB.

Seroprevalence of Mycobacterium bovis infection in warthogs (Phacochoerus africanus) in bovine tuberculosis-endemic regions of South Africa.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), has been reported in many species including suids. Wild boar are important maintenance hosts of the infection with other suids, that is domestic and feral pigs, being important spillover hosts in the Eurasian ecosystem and in South Africa, warthogs (Phacochoerus africanus) may play a similar role in M. bovis-endemic areas. However, novel diagnostic tests for warthogs are required to investigate the epidemiology of bTB in this species. Recent studies have demonstrated that serological assays are capable of discriminating between M. bovis-infected and uninfected warthogs (Roos et al., ). In this study, an indirect ELISA utilizing M. bovis purified protein derivative (PPD) as a test antigen was used to measure the prevalence and investigate risk factors associated with infection in warthogs from uMhkuze Nature Reserve and the southern region of the Greater Kruger National Park (GKNP). There was a high overall seroprevalence of 38%, with adult warthogs having a higher risk of infection (46%). Seroprevalence also varied by geographic location with warthogs from Marloth Park in the GKNP having the greatest percentage of positive animals (63%). This study indicates that warthogs in M. bovis-endemic areas are at high risk of becoming infected with mycobacteria. Warthogs might present an under-recognized disease threat in multi-species systems. They might also serve as convenient sentinels for M. bovis in endemic areas. These findings highlight the importance of epidemiological studies in wildlife to understand the role each species plays in disease ecology.

Widespread use of incorrect PCR ramp rate negatively impacts multidrug-resistant tuberculosis diagnosis (MTBDRplus).

The scale-up of rapid drug resistance testing for TB is a global priority. MTBDRplus is a WHO-endorsed multidrug-resistant (MDR)-TB PCR assay with suboptimal sensitivities and high indeterminate rates on smear-negative specimens. We hypothesised that widespread use of incorrect thermocycler ramp rate (speed of temperature change between cycles) impacts performance. A global sample of 72 laboratories was surveyed. We tested 107 sputa from Xpert MTB/RIF-positive patients and, separately, dilution series of bacilli, both at the manufacturer-recommended ramp rate (2.2 °C/s) and the most frequently reported incorrect ramp rate (4.0 °C/s). Mycobacterium tuberculosis-complex DNA (TUB-band)-detection, indeterminate results, accuracy, and inter-reader variability (dilution series only) were compared. 32 respondents did a median (IQR) of 41 (20-150) assays monthly. 78% used an incorrect ramp rate. On smear-negative sputa, 2.2 °C/s vs. 4.0 °C/s improved TUB-band positivity (42/55 vs. 32/55; p = 0.042) and indeterminate rates (1/42 vs. 5/32; p = 0.039). The actionable results (not TUB-negative or indeterminate; 41/55 vs. 28/55) hence improved by 21% (95% CI: 9-35%). Widespread use of incorrect ramp rate contributes to suboptimal MTBDRplus performance on smear-negative specimens and hence limits clinical utility. The number of diagnoses (and thus the number of smear-negative patients in whom DST is possible) will improve substantially after ramp rate correction.

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress.

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.

Isolation and Potential for Transmission of Mycobacterium bovis at Human-livestock-wildlife Interface of the Serengeti Ecosystem, Northern Tanzania.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a multihost pathogen of public health and veterinary importance. We characterized the M. bovis isolated at the human-livestock-wildlife interface of the Serengeti ecosystem to determine the epidemiology and risk of cross-species transmission between interacting hosts species. DNA was extracted from mycobacterial cultures obtained from sputum samples of 472 tuberculosis (TB) suspected patients and tissue samples from 606 livestock and wild animal species. M. bovis isolates were characterized using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) on 24 loci. Only 5 M. bovis were isolated from the cultured samples. Spoligotyping results revealed that three M. bovis isolates from two buffaloes (Syncerus caffer) and 1 African civet (Civettictis civetta) belonged to SB0133 spoligotype. The two novel strains (AR1 and AR2) assigned as spoligotype SB2290 and SB2289, respectively, were identified from indigenous cattle (Bos indicus). No M. bovis was detected from patients with clinical signs consistent with TB. Of the 606 animal tissue specimens and sputa of 472 TB-suspected patients 43 (7.09%) and 12 (2.9%), respectively, yielded non-tuberculous mycobacteria (NTM), of which 20 isolates were M. intracellulare. No M. avium was identified. M. bovis isolates from wildlife had 45.2% and 96.8% spoligotype pattern agreement with AR1 and AR2 strains, respectively. This finding indicates that bTB infections in wild animals and cattle were epidemiologically related. Of the 24 MIRU-VNTR loci, QUB 11b showed the highest discrimination among the M. bovis strains. The novel strains obtained in this study have not been previously reported in the area, but no clear evidence for recent cross-species transmission of M. bovis was found between human, livestock and wild animals.

Tuberculosis in Rhinoceros: An Underrecognized Threat?

Historical evidence of tuberculosis (TB) affecting primarily captive rhinoceroses dates back almost two centuries. Although the causative Mycobacterium tuberculosis complex (MTC) species has not been determined in many cases, especially for those that occurred before bacterial culture techniques were available, the spectrum of documented reports illustrates the importance of TB as cause of morbidity and mortality in different rhinoceros species across continents. In more recent years, sporadic suspected or confirmed cases of TB caused by Mycobacterium bovis (M. bovis) have been reported in semi-free or free-ranging rhinoceroses in South Africa. However, the true risk TB may pose to the health and conservation of rhinoceros populations in the country's large conservation areas where M. bovis is endemic, which is unknown. Underlying the current knowledge gap is the lack of diagnostic tools available to detect infection in living animals. As documented in other wildlife species, TB could establish itself in a rhinoceros population but remain unrecognized for decades with detrimental implications for wildlife conservation at large and should such animals be moved to uninfected areas or facilities. This paper reviews the current state of knowledge regarding TB in rhinoceros including critical gaps that need to be addressed to effectively assess the threat that this disease may present to rhinoceros.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

Disease Control in Wildlife: Evaluating a Test and Cull Programme for Bovine Tuberculosis in African Buffalo.

Providing an evidence base for wildlife population management is difficult, due to limited opportunities for experimentation and study replication at the population level. We utilized an opportunity to assess the outcome of a test and cull programme aimed at limiting the spread of Mycobacterium bovis in African buffalo. Buffalo act as reservoirs of M. bovis, the causative agent of bovine tuberculosis (BTB), which can have major economic, ecological and public health impacts through the risk of infection to other wildlife species, livestock and surrounding communities. BTB prevalence data were collected in conjunction with disease control operations in Hluhluwe-iMfolozi Park, South Africa, from 1999 to 2006. A total of 4733 buffalo (250-950 per year) were tested for BTB using the single comparative intradermal tuberculin (SCIT) test, with BTB-positive animals culled, and negative animals released. BTB prevalence was spatially and temporally variable, ranging from 2.3% to 54.7%. Geographic area was a strong predictor of BTB transmission in HiP, owing to relatively stable herds and home ranges. Herds experiencing more intensive and frequent captures showed reduced per capita disease transmission risk and less increase in herd prevalence over time. Disease hot spots did not expand spatially over time, and BTB prevalence in all but the hot spot areas was maintained between 10% and 15% throughout the study period. Our data suggest that HiP's test and cull programme was effective at reducing BTB transmission in buffalo, with capture effort and interval found to be the crucial components of the programme. The programme was thus successful with respect to the original goals; however, there are additional factors that should be considered in future cost/benefit analyses and decision-making. These findings may be utilized and expanded in future collaborative work between wildlife managers, veterinarians and scientists, to optimize wildlife disease control programmes and mitigate conflict at the interface of conservation, agricultural and urban areas.

Effect of milk fermentation by kefir grains and selected single strains of lactic acid bacteria on the survival of Mycobacterium bovis BCG.

Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to determine the effect of the fermentation process on the survival of M. bovis BCG in milk. M. bovis BCG at concentrations of 6 log CFU/ml was added to products of kefir fermentation. The survival of M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar plates enriched with 2% BD BACTEC PANTA™. M. bovis BCG was increasingly reduced in sterile kefir that was fermented for a period of 24h and longer. In the milk fermented with kefir grains, Lactobacillus paracasei subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24h and by 2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation. Results from this study show that long term fermentation under certain conditions may have the potential to inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation should be combined with other hurdle technologies such as boiling and milk pasteurisation.

Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug-Resistant Tuberculosis in South Africa.

We show that the interpretation of molecular epidemiological data for extensively drug-resistant tuberculosis (XDR-TB) is dependent on the number of different markers used to define transmission. Using spoligotyping, IS6110 DNA fingerprinting, and DNA sequence data, we show that XDR-TB in South Africa (2006 to 2008) was predominantly driven by the acquisition of second-line drug resistance.

Selective breeding: the future of TB management in African buffalo?

The high prevalence of bovine tuberculosis (BTB) in African buffalo (Syncerus caffer) in regions of southern African has a negative economic impact on the trade of animals and animal products, represents an ecological threat to biodiversity, and poses a health risk to local communities through the wildlife-cattle-human interface. Test and cull methods may not be logistically feasible in many free-range wildlife systems, and with the presence of co-existing BTB hosts and the limited effectiveness of the BCG vaccine in buffalo, there is a need for alternative methods of BTB management. Selective breeding for increased resistance to BTB in buffalo may be a viable method of BTB management in the future, particularly if genetic information can be incorporated into these schemes. To explore this possibility, we discuss the different strategies that can be employed in selective breeding programmes, and consider the implementation of genetic improvement schemes. We reflect on the suitability of applying this strategy for enhanced BTB resistance in African buffalo, and address the challenges of this approach that must be taken into account. Conclusions and the implications for management are presented.

Bovine Tuberculosis and Brucellosis in Cattle and African Buffalo in the Limpopo National Park, Mozambique.

Bovine tuberculosis (BTB) and brucellosis are prevalent in buffaloes of the Kruger National Park (KNP, South Africa). Both diseases were considered to have no or a very low prevalence in wildlife and livestock in and around the Limpopo National Park (LNP, Mozambique). The same applies for tuberculosis in Gonarezhou National Park (GNP, Zimbabwe), but just recently, BTB was detected in buffaloes in the GNP and fears arose that the disease might also spread to the LNP as a result of the partial removal of the fences between the three parks to form the Great Limpopo Transfrontier Park. To assess the status of both diseases in and around LNP, 62 buffaloes were tested for bovine tuberculosis (BTB) and bovine brucellosis. The percentage of positive BTB reactors in buffalo was 8.06% using BovidTB Stat-Pak® and 0% with BOVIGAM® IFN-γ test and IDEXX ELISA. The brucellosis seroprevalence in buffalo was found to be 17.72% and 27.42% using Rose Bengal Test (RBT) and ELISA, respectively. In addition, 2445 cattle in and around the LNP were examined for BTB using the single intradermal cervical comparative tuberculin test (SICCT), and an apparent prevalence of 0.98% was found with no significant difference inside (0.5%) and outside (1.3%) the park. This is the first published report on the presence of positive reactors to BTB and bovine brucellosis in buffalo and cattle in and outside the LNP. Monitoring the wildlife-livestock-human interface of zoonotic high-impact diseases such as BTB and brucellosis is of outmost importance for the successful implementation and management of any transfrontier park that aims to improve the livelihoods of the local communities.