Manormoney Pillay

Evolution of the Extensively Drug-Resistant F15/LAM4/KZN Strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa

BACKGROUND Although several hot spots of multidrug-resistant tuberculosis have been identified on the African continent, extensive drug resistance (XDR) has not been reported until recently, when a large number of XDR cases were identified in KwaZulu-Natal. The majority of the patients involved were infected with the same strain of Mycobacterium tuberculosis (F15/LAM4/KZN). We report this strains development from multidrug resistance to XDR. METHODS We searched databases for studies performed during the period 1994-2005 that involved the resistance patterns of isolates of M. tuberculosis with the F15/LAM4/KZN strain fingerprint. RESULTS As early as 1994, the F15/LAM4/KZN strain was responsible for a number of cases of multidrug-resistant tuberculosis, indicating the ability of the strain to cause cases of primary resistant tuberculosis. Some of the isolates were also resistant to streptomycin. From 1994 onwards, multidrug-resistant isolates with resistance to additional drugs were found, and the first XDR isolate was discovered in 2001. CONCLUSIONS Drug resistance to as many as 7 drugs developed in a local strain of M. tuberculosis in slightly more than a decade. This coincided with the introduction of the directly observed therapy-based and directly observed therapy-plus-based tuberculosis-control programs. It is postulated that the introduction of these programs in the absence of susceptibility testing or drug resistance surveillance has been instrumental in the development of XDR in this highly transmissible F15/LAM4/KZN strain. The expanding pool of human immunodeficiency virus-infected, tuberculosis-susceptible individuals has likely contributed to this development.

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion.

Population Structure of Multi- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in South Africa

ABSTRACT Genotyping of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhA promoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.

Emergence and treatment of multidrug resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in South Africa

Drug resistant tuberculosis (TB) has reached alarming proportions in South Africa, draining valuable resources that are needed to fight drug susceptible TB. It is currently estimated that 9.6% of all TB cases have multi-drug resistant (MDR)-TB, thereby ranking South Africa as one of the highest MDR-TB burden countries in the world. Molecular epidemiological studies have demonstrated the complexity of the epidemic and have clearly shown that the epidemic is driven by transmission as a consequence of low cases detection and diagnostic delay. The latter has in turn fueled the amplification of drug resistance, ultimately leading to the emergence of extensively drug resistant (XDR)-TB. Despite the introduction of new drugs to combat this scourge, culture conversion rates for XDR-TB remain below 20%. Failure to achieve cure may be explained from DNA sequencing results which have demonstrated mutations in 7 genes encoding resistance to at least 8 anti-TB drugs. This review shows how molecular epidemiology has provided novel insights into the MDR-TB epidemic in South Africa and thereby has highlighted the challenges that need to be addressed regarding the diagnosis and treatment of MDR-TB. An important step towards for curbing this epidemic will be collaboration between clinicians, laboratories and researchers to establish scientific knowledge and medical expertise to more efficiently guide public health policy.

Programmatically Selected Multidrug-Resistant Strains Drive the Emergence of Extensively Drug-Resistant Tuberculosis in South Africa

Background South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known. Methods We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations. Results Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin. Conclusions XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.

Adhesion to and invasion of pulmonary epithelial cells by the F15/LAM4/KZN and Beijing strains of Mycobacterium tuberculosis

Globally, specific genotypes of Mycobacterium tuberculosis have been shown to dominate in patients, suggesting that these are more successful pathogens. One such genotype, the F15/LAM4/KZN (KZN) family of M. tuberculosis, has predominated in KwaZulu-Natal, South Africa, since the early 1990s. This strain recently evolved from multidrug-resistant to extensively drug-resistant (XDR). The ability of M. tuberculosis strains belonging to the Beijing family, the KZN family, strains with unique DNA fingerprint patterns and laboratory strains (H37Rv and H37Ra) to adhere to and invade a human alveolar (A549) and a human bronchial (BBM) epithelial cell line was investigated. All strains displayed greater adhesion to and invasion of A549 cells as compared to BBM cells. The Beijing and KZN strains combined showed greater adhesion (28 %) than the unique strains (5 %) (P <0.05). The XDR variant of KZN invaded A549 cells more effectively than the other isolates. These results suggest that the successful spread of the Beijing and KZN strains might be related to their interaction with alveolar epithelium.

Pentacyclo-undecane derived cyclic tetra-amines: Synthesis and evaluation as potent anti-tuberculosis agents

As part of an ongoing effort to develop highly potent anti-tuberculosis agents, fourteen pentacyclo-undecane (PCU) tetra-amine compounds were synthesized and screened for their in vitro anti-mycobacterial activity against two TB strains, H37Rv and XDR 194 [an extensively drug-resistant strain of tuberculosis]. Using the broth macrodilution method, nitrofuranylamide based compounds (6a and 6b) showed almost similar activities against the H37Rv strain of Mycobacterium tuberculosis when compared with the control drug, ethambutol. N-Geranyl piperazine PCU (8a) and trans-trans farnesyl piperazine PCU (8b) were 3.2 and 3.7 times more potent than commercially available ethambutol. Both isoprenyl PCU tetra-amine derivatives and N-decyl piperazine PCU (9a) were highly active against the XDR 194 strain of tuberculosis with MICs in the range of 0.63-3.02 microM. Cytotoxicities (IC(50)) of isoprenyl based compounds (8a, 8b) and compound 9a were tested on a mammalian cell line [MDBK (Madin Darby bovine kidney epithelium)] with values of 30, 24 and 25 microM respectively.

Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets

Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the hosts defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.

Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p=0.047) and 56.20% (p=0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p=0.033). Furthermore, at 72h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p=0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is a rapid, cheap, screening test for the in vitro anti-tuberculous activity of chalcones

Rapid and reliable drug susceptibility testing facilitates replenishment of the TB drug pipeline in the fight against drug resistant Mycobacterium tuberculosis. This study compared the performance of the MTT and MABA assays on the anti-tuberculous activity of a set of chalcones. Twenty seven chalcones and chromenochalcones were screened against the laboratory strain M. tuberculosis H37Rv, using a microtitre plate MTT assay at 7 days. The MIC for 20 active compounds was subsequently determined using the MABA, MTT and the Macroscopic broth assays at 7, 14 and 21 days. No significant difference in the MICs, or increase in the MICs was observed over time between the MABA (p=0.209) and the MTT (p=0.207) assays, in contrast to the gold standard, the Macroscopic broth assay (p=0.000). The MICs (16 to >128μg/ml) were much higher than the currently used TB drugs. In conclusion, the MTT assay is a cost effective method (R0.06/well) for the rapid in vitro screening of chalcones against M. tuberculosis, producing reliable results in 8 days. The chalcone with a MIC of 16μg/mL shows promise as a potential lead compound and should be investigated further.