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.

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.

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.

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.

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.

Phage-based detection of bacterial pathogens.

Bacterial pathogens cause significant morbidity and mortality annually to both humans and animals. With the rampant spread of drug resistance and the diminishing effectiveness of current antibiotics, there is a pressing need for effective diagnostics for detection of bacterial pathogens and their drug resistances. Bacteriophages offer several unique opportunities for bacterial detection. This review highlights the means by which bacteriophages have been utilized to achieve and facilitate specific bacterial detection.

Evaluation of the AID TB resistance line probe assay for rapid detection of genetic alterations associated with drug resistance in Mycobacterium tuberculosis strains.

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n=104; South Africa, n=52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n=5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.

Mutation rate and the emergence of drug resistance in Mycobacterium tuberculosis.

The emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis remains a major concern of tuberculosis control programmes worldwide, as treatment depends on low-efficacy, toxic compounds that often lead to poor outcomes. M. tuberculosis develops drug resistance exclusively through chromosomal mutations, in particular single-nucleotide polymorphisms. Moreover, in laboratory assays the organism exhibits a spontaneous mutation rate that is at the lower end of the bacterial spectrum. Despite this, whole-genome sequencing technology has identified unexpected genetic diversity among clinical M. tuberculosis populations. This suggests that the mycobacterial mutation rate may be modulated within the host and, in turn, implies a potential role for constitutive and/or transient mutator strains in adaptive evolution. It also raises the possibility that environmental factors might act as key mutagens during M. tuberculosis infection. Here we consider the elements that might influence the mycobacterial mutation rate in vivo and evaluate the potential roles of constitutive and transient mutator states in the generation of drug resistance mutations. In addition, we identify key research questions that will influence future efforts to develop novel therapeutic strategies for a disease that continues to impose a significant global health burden.

Mycobacteria and disease in southern Africa.

The genus Mycobacterium consists of over 120 known species, some of which (e.g. M. bovis and M. tuberculosis) contribute extensively to the burden of infectious disease in humans and animals, whilst others are commonly found in the environment but may rarely if ever be disease-causing. This paper reviews the mycobacteria found in southern Africa, focussing on those in the M. tuberculosis complex as well as the non-tuberculous mycobacteria (NTM), identifying those found in the area and including those causing disease in humans and animals, and outlines some recent reports describing the distribution and prevalence of the disease in Africa. Difficulties in diagnosis, host preference and reaction, immunology and transmission are discussed.

Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission.

Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P < 0.01). Moreover, these putative compensatory mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P < 0.001). Our study supports a role for rpoC mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.