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.

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.

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.

Prevalence and distribution of non-tuberculous mycobacteria (NTM) in cattle, African buffaloes (Syncerus caffer) and their environments in South Africa.

It has been hypothesized that a variety of non-tuberculous mycobacteria (NTM) species to which livestock and wildlife species are naturally exposed induce broadly cross-reactive anti-mycobacterial immune responses which interfere with current standard diagnostic assays. Non-tuberculous mycobacteria have also been implicated in Mycobacterium bovis-specific immune responsiveness, hence potentially the development of tuberculosis. Cattle and African buffaloes are both maintenance hosts of bovine tuberculosis (BTB) in South Africa, yet the effective diagnosis and control in these species may be hampered by adverse effects of NTM. As part of an investigation of the role of NTM in the immune responsiveness of cattle and African buffaloes to NTM, we conducted a countrywide survey to establish the prevalent NTM species and their distribution in the natural environments of these animals. A total of 1123 samples (water, soil, nasal and pharyngeal swabs) were collected for mycobacterium isolation. In addition, NTM isolated from tissue samples between 1991 and 2011 were included in the analysis. Mycobacteria were isolated from 56% of the samples from the countrywide survey. A total of 420 NTM isolates from soil, water, animal tissues and animal-derived swab samples were genotyped with the following results: 302 belonged to 40 known NTM species, 79 were found to be closely related to 23 known NTM species, and 38 isolates were found to be potentially novel species that are not currently listed in the RIDOM and NCBI BLAST databases. The four NTM species or closely related groups most frequently isolated in this survey included Mycobacterium terrae (11.2% of isolates), a group of mycobacteria closely related to Mycobacterium moriokaense (referred to as M. moriokaense-like) (8.1% of isolates), Mycobacterium nonchromogenicum (7.4% of isolates) and Mycobacterium vaccae/M. vanbaalenii (5.2% of isolates). The phylogenetic analysis of the M. moriokaense-like isolates, based on the 16S rRNA sequences, revealed at least eight clusters, possibly associated with eight different NTM species. Our findings provide account of NTM species diversity and associated prevalences in cattle and African buffaloes and their environments in South Africa. The identification of the most prevalent NTM species in this study will allow for a targeted investigation of their effects on host immune responses.

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.

Spectrum of non-tuberculous mycobacteria identified using standard biochemical testing vs. 16S sequencing.

Non-tuberculous mycobacterial isolates from gold miners were speciated using standard biochemical testing (SBT) and 16S rDNA sequencing. Of 237 isolates tested, SBT identified 126, compared with all 237 identified using sequencing. Of 111 isolates unspeciated by SBT but identified by sequencing, 38 (34.2%) were identified as Mycobacterium gordonae and 8 (7.2%) were new species. Of 126 isolates speciated by both methods, 37 were discordant, with 14/17 M. gordonae isolates incorrectly identified as M. scrofulaceum using SBT. The majority of these were the potentially pathogenic strain D, M. gordonae. Sequencing is preferable where available to guide treatment.

The diagnostic performance of the GenoType MTBDRplus version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Detection of Mycobacterium tuberculosis infection in dogs in a high-risk setting.

Dogs infected with Mycobacterium tuberculosis can develop clinical tuberculosis (TB) but there are currently no validated immunological assays for diagnosing this infection in this species. Using a post mortem survey we investigated the prevalence of non-clinical M. tuberculosis infection and clinical TB disease in a high-risk population of dogs and developed and utilised a novel interferon-gamma release assay to determine the risk of transmission of M. tuberculosis from TB patients to contact dogs. The prevalence of clinical TB in dogs from a high-risk setting was 1% (95% CI: 0-5%) while the prevalence of immunological sensitization to M. tuberculosis antigens in dogs living in contact with sputum smear-positive TB patients was 50%. The IGRA proved a useful test of M. tuberculosis infection in dogs and the high levels of transmission of this pathogen from humans to companion dogs should be considered when assessing the zoonotic risks associated with such animals.

Mycobacterium tuberculosis Beijing genotype: a template for success.

The diverse clinico- and histopathological features, frequency of transmission and treatment outcome of Mycobacterium tuberculosis have been associated with several environmental, host and bacterial factors. Many Mycobacterium tuberculosis genotypes have been studied in an attempt to understand the genetic variations among the different genotypes and to clarify their contribution to phenotypic differences. Strains of the Beijing genotype have been extensively investigated due to their increased ability to spread and cause disease. Here we review the evidence of hypervirulence of the Beijing genotype as well as other Beijing-associated phenotypic characteristics such as alternate host immune modulation, clinical and pathological features, drug resistance, resistance to BCG vaccination and other epidemiological features to enhance our understanding of the contribution of pathogenic factors. From the data collected it is clear that the genetic background of Mycobacterium tuberculosis may influence the differential induction of the immune response, drug resistance patterns and clinical, epidemiological and pathogenic features which define disease progression following infection. This highlights the importance of ongoing research into the genetic mechanisms underlying the phenotypic and genotypic characteristics of different Mycobacterium tuberculosis genotype strains. Furthermore, these findings could help to direct future drug, vaccine and diagnostic test development towards targeting critical virulence factors and to identify persons at risk for developing active disease thereby limiting transmission and the perpetuation of the tuberculosis epidemic.

inhA promoter mutations: a gateway to extensively drug-resistant tuberculosis in South Africa?

SETTING: Western Cape and Eastern Cape Provinces, South Africa. OBJECTIVE: To assess the potential association between the evolution of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis and mutations in the inhA promoter or the katG gene. DESIGN: Analysis of the frequency distribution of isoniazid (INH) resistance conferring mutations in a population sample of drug-resistant isolates of M. tuberculosis. RESULTS: In the Western Cape and Eastern Cape Provinces, the percentage of isolates exhibiting inhA promoter mutations increased significantly from respectively 48.4% and 62.4% in multidrug-resistant tuberculosis (MDR-TB) isolates to 85.5% and 91.9% in XDR isolates. Data from the Western Cape revealed that significantly more XDR-TB isolates showed mutations in the inhA promoter than in katG (85.5% vs. 60.9%, P < 0.01), while the respective proportions were equal for INH-resistant non-MDR-TB isolates (∼30%). CONCLUSIONS: inhA promoter mutations are strongly associated with XDR-TB in South Africa. We suggest that this is due to the dual resistance to ethionamide and (low-dose) INH conferred by inhA promoter mutations. The use of molecular probe assays such as the GenoType® MTBDRplus assay, which allows the detection of inhA promoter mutations, could enable treatment regimens to be adjusted depending on the pharmacogenetic properties of the mutations detected.

Mycobacterium tuberculosis strains with the Beijing genotype demonstrate variability in virulence associated with transmission.

Phylogenetic analysis has shown that Beijing genotype strains can be grouped into at least 7 different sublineages. We aimed to test the hypothesis that the virulence of Beijing genotype strains differed among members of the different sublineages and that the level of virulence correlated with their ability to spread and cause disease. BALB/c mice were infected with Beijing strains representative of the different lineages and of different epidemiological characteristics (transmitted vs. non-transmitted). Survival times, lung pathology, bacterial load and immunology kinetics were evaluated at defined intervals post-infection. Transmissibility was determined by co-housing infected and uninfected mice in close contact for 1-2 months. The results show that mice infected with the highly transmitted Beijing strains began showing mortality 3 weeks post-infection and all had died by 5 weeks, suggesting high virulence phenotypes. In contrast, >80% of mice infected with the non-transmitted strains survived 4 months post-infection, suggesting low virulence phenotypes. Our co-housing transmission model confirmed these virulence phenotypes. Extensive tissue damage and the induction of lower levels of IFNγ and iNOS expression, as well as high but ephemeral TNFα expression were associated with the high virulence phenotype. In contrast, minimal tissue damage and progressive expression of IFNγ and TNFα were associated with the low virulence phenotype. Both virulence phenotypes induced similar levels of IL-4 expression during the early stages of infection after which the high virulence strain induced significantly higher levels of IL-4 expression. In conclusion, this study demonstrates that Beijing genotype strains display a spectrum of virulence phenotypes in mice which mimic their epidemiological characteristics. Both transmissible and non-transmissible strains may exist in the same sublineage.

Drug-resistant tuberculosis epidemic in the Western Cape driven by a virulent Beijing genotype strain.

Temporal analysis of drug-resistant tuberculosis (TB) cases in the Western Cape, South Africa, showed a 1.5-fold increase over a 2-year period, suggesting a doubling time of 8.2 years. This increase was strongly associated with multidrug resistance and the Beijing genotype. Forty-two per cent of the overall increase was due to the Beijing genotype strain R220, suggesting that this strain had evolved unique properties that allowed for both acquisition and transmission of drug resistance. To curb the drug-resistant TB epidemic in this setting, it will be essential to implement rapid diagnostics and efficient infection control measures, improve contact screening and ensure treatment adherence.

Zoonotic tuberculosis and brucellosis in Africa: neglected zoonoses or minor public-health issues? The outcomes of a multi-disciplinary workshop.

Late in 2007, veterinary, medical and anthropological professionals from Europe and Africa met in a 2-day workshop in Pretoria, South Africa, to evaluate the burden, surveillance and control of zoonotic tuberculosis and brucellosis in sub-Saharan Africa. Keynote presentations reviewed the burden of these diseases on human and livestock health, the existing diagnostic tools, and the available control methods. These presentations were followed by group discussions and the formulation of recommendations. The presence of Mycobacterium bovis and Brucella spp. in livestock was considered to be a serious threat to public health, since livestock and animal products are the only source of such infections in human beings. The impact of these pathogens on human health appears to be relatively marginal, however, when compared with Mycobacterium tuberculosis infections and drug resistance, HIV and malaria. Appropriate diagnostic tools are needed to improve the detection of M. bovis and Brucella spp. in humans. In livestock, the 'test-and-slaughter' approach and the pasteurization of milk, which have been used successfully in industrialized countries, might not be the optimal control tools in Africa. Control strategies should fit the needs and perceptions of local communities. Improved intersectoral and international collaboration in surveillance, diagnosis and control, and in the education of medical and veterinary personnel, are advocated.

The clinical relevance of Mycobacterial pharmacogenetics.

Current anti-tuberculosis (anti-TB) drug sensitivity testing methods provide a dichotomous readout: isolates are reported as either drug susceptible or drug resistant. This report demonstrates that rapid molecular methods may provide information concerning both the level of resistance and cross-resistance to other anti-TB drugs that is important for optimal clinical management. Specific mutations detected by the Hain GenoType MTBDRplus test, recently approved by the World Health Organization (WHO) for rapid TB diagnosis and drug resistance testing, could inform the decision of whether to include high dose isoniazid (INH) when treating patients with INH mono-resistant TB, MDR-TB or XDR-TB. The presence of mutations in the inhA gene or promoter region generally confers low level INH resistance that can be overcome by high dose INH. The same mutations also confer resistance to ethionamide indicating little benefit from its inclusion in second line treatment regimens in such cases. This information has high clinical relevance since inhA mutations account for a large proportion of INH resistance, and optimized therapy regimens are crucial to improve patient outcomes and reduce the spread of drug resistant TB. This hypothesis needs to be tested in well controlled clinical and pharmacokinetic studies.

'Emerging' mycobacteria in South Africa.

Disease can be caused by various species of the genus Mycobacterium. A number of reports, both published and unpublished, of rarely reported mycobacteria have surfaced in South Africa in the last few years. Some unusual hosts have also been involved, causing concern in some quarters.These include reports on Mycobacterium goodii in a spotted hyaena (Crocuta crocuta), M. xenopi in a ruffed lemur (Varecia variegata), M. intracellulare in wild-caught chacma baboons (Papio ursinus), the 'dassie bacillus' in free ranging rock hyrax (dassies; Procavia capensis) the 'oryx bacillus' from free-ranging buffalo (Syncerus caffer) and M. tuberculosis in suricates (Suricata suricatta), a domestic dog and in baboons. In this article it has been attempted to put these in context and show how improved surveillance and technologies have allowed mycobacteria to be identified to species level more easily. Most of the unusual mycobacterial species have most likely been present in the region for many years and have probably caused disease episodes before, but have been misdiagnosed. Each case must be evaluated carefully with respect to the animal species involved, the environment in which the host is found and the mycobacterial species, and operational decisions made accordingly.

Discordance between mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing and IS6110 restriction fragment length polymorphism genotyping for analysis of Mycobacterium tuberculosis Beijing strains in a setting of high incidence of tuberculosis.

IS6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis. However, due to the complexity of the IS6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR locus combinations relative to IS6110 RFLP genotyping, using a collection of Beijing genotype M. tuberculosis strains with a well-established phylogenetic history. Clustering, diversity index, clustering concordance, concordance among unique genotypes, and divergent and convergent evolution were calculated for seven combinations of 27 different MIRU-VNTR loci and compared to IS6110 RFLP results. Our results confirmed previous findings that MIRU-VNTR genotyping can be used to estimate the extent of recent or ongoing transmission. However, molecular epidemiological linking of cases varied significantly depending on the genotyping method used. We conclude that IS6110 RFLP and MIRU-VNTR loci evolve independently and at different rates, which leads to discordance between transmission chains predicted by the respective genotyping methods. Concordance between the two genotyping methods could be improved by the inclusion of genetic distance (GD) into the clustering formulae for some of the MIRU-VNTR loci combinations. In summary, our findings differ from previous reports, which may be explained by the fact that in settings of low tuberculosis incidence, the genetic distance between epidemiologically unrelated isolates was sufficient to define a strain using either marker, whereas in settings of high incidence, continuous evolution and persistence of strains revealed the weaknesses inherent to these markers.