SUNScholar - Browsing by Author "Klopper, Marisa"

Browsing by Author "Klopper, Marisa"

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Emergence and spread of extensively and totally drug-resistant tuberculosis, South Africa
(Centers for Disease Control and Prevention, 2013-03) Klopper, Marisa; Warren, Robin Mark; Hayes, Cindy; Gey van Pittius, Nicolaas Claudius; Streicher, Elizabeth M.; Muller, Borna; Sirgel, Frederick Adriaan; Chabula-Nxiweni, Mamisa; Hoosain, Ebrahim; Coetzee, Gerrit; Van Helden, Paul David; Victor, Thomas Calldo; Trollip, Andre Phillip
ENGLISH ABSTRACT: Factors driving the increase in drug-resistant tuberculosis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-susceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008–July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in contrast to restricted genetic backgrounds among pre–extensively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progenitor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-aminosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.
Geospatial distribution of Mycobacterium tuberculosis genotypes in Africa
(Public Library of Science, 2018-08-01) Chihota, Violet N.; Niehaus, Antoinette; Streicher, Elizabeth M.; Wang, Xia; Sampson, Samantha L.; Mason, Peter; Kallenius, Gunilla; Mfinanga, Sayoki G.; Pillay, Marnomorney; Klopper, Marisa; Kasongo, Webster; Behr, Marcel A.; Van Pittius, Nicolaas C. Gey; Van Helden, Paul D.; Couvin, David; Rastogi, Nalin; Warren, Robin M.
Objective: To investigate the distribution of Mycobacterium tuberculosis genotypes across Africa. Methods: The SITVIT2 global repository and PUBMED were searched for spoligotype and published genotype data respectively, of M. tuberculosis from Africa. M. tuberculosis lineages in Africa were described and compared across regions and with those from 7 European and 6 South-Asian countries. Further analysis of the major lineages and sub-lineages using Principal Component analysis (PCA) and hierarchical cluster analysis were done to describe clustering by geographical regions. Evolutionary relationships were assessed using phylogenetic tree analysis. Results: The SITVIT2 global repository and PUBMED were searched for spoligotype and published genotype data respectively, of M. tuberculosis from Africa. M. tuberculosis lineages in Africa were described and compared across regions and with those from 7 European and 6 South-Asian countries. Further analysis of the major lineages and sub-lineages using Principal Component analysis (PCA) and hierarchical cluster analysis were done to describe clustering by geographical regions. Evolutionary relationships were assessed using phylogenetic tree analysis. Results: A total of 14727 isolates from 35 African countries were included in the analysis and of these 13607 were assigned to one of 10 major lineages, whilst 1120 were unknown. There were differences in geographical distribution of major lineages and their sub-lineages with regional clustering. Southern African countries were grouped based on high prevalence of LAM11-ZWE strains; strains which have an origin in Portugal. The grouping of North African countries was due to the high percentage of LAM9 strains, which have an origin in the Eastern Mediterranean region. East African countries were grouped based on Central Asian (CAS) and East-African Indian (EAI) strain lineage possibly reflecting historic sea trade with Asia, while West African Countries were grouped based on Cameroon lineage of unknown origin. A high percentage of the Haarlem lineage isolates were observed in the Central African Republic, Guinea, Gambia and Tunisia, however, a mixed distribution prevented close clustering. Conclusions: This study highlighted that the TB epidemic in Africa is driven by regional epidemics characterized by genetically distinct lineages of M. tuberculosis. M. tuberculosis in these regions may have been introduced from either Europe or Asia and has spread through pastoralism, mining and war. The vast array of genotypes and their associated phenotypes should be considered when designing future vaccines, diagnostics and anti-TB drugs.
A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic
(BMC (part of Springer Nature), 2020-02-04) Klopper, Marisa; Heupink, Tim Hermanus; Hill-Cawthorne, Grant; Streicher, Elizabeth M.; Dippenaar, Anzaan; De Vos, Margaretha; Abdallah, Abdallah Musa; Limberis, Jason; Merker, Matthias; Burns, Scott; Niemann, Stefan; Dheda, Keertan; Posey, James; Pain, Arnab; Warren, Robin Mark
Background: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era
(BMC, 2020-11-25) Beckert, Patrick; Sanchez-Padilla, Elisabeth; Merker, Matthias; Dreyer, Viola; Kohl, Thomas A.; Utpatel, Christian; Koser, Claudio U.; Barilar, Ivan; Ismail, Nazir; Omar, Shaheed V.; Klopper, Marisa; Warren, Robin M.; Hoffmann, Harald; Maphalala, Gugu; Ardizzoni, Elisa; De Jong, Bouke C.; Kerschberger, Bernhard; Schramm, Birgit; Andres, Sonke; Kranzer, Katharina; Maurer, Florian P.; Bonnet, Maryline; Niemann, Stefan
Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods: We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results: MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion: The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.
Molecular characterization of the drug resistant tuberculosis epidemic in the Eastern Cape, South Africa
(Stellenbosch : Stellenbosch University, 2015-03) Klopper, Marisa; Victor, Thomas C.; Warren, Robin M.; Streicher, Elizma M.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : South Africa has a high burden of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), with the Eastern Cape (EC) being one of the worst affected provinces in the country. This study provides the first in-depth analysis of the molecular epidemiology of drug-resistant TB in the EC. A convenience sample of drug-sensitive and drug-resistant isolates was collected over one year by the National Health Laboratory Services in Port Elizabeth. These isolates were characterized by various molecular techniques. Our results were compared to data from three additional provinces, to contextualise the population structure of MDR-TB strains. Each province had a distinct population structure. The population structure of XDR-TB cases in the Western Cape was significantly influenced by strains originating from the EC. A high degree of clustering of drug resistance mutation patterns was detected in each setting, suggestive of transmission. Clustering was particularly pronounced in the EC, with 93% of pre-XDR and XDR-TB isolates belonging to the Atypical Beijing genotype. We showed that this genotype was programmatically selected through a weakened MDR-TB regimen that failed to recognise inhA defined ethionamide resistance. This weakened regimen has facilitated transmission and is the underlying cause of mortality. We propose that existing molecular assays which detect inhA mutations should be used to identify patients at risk of XDR-TB and to adjust treatment. Through spoligotyping, restriction fragment length polymorphism typing and mutation analysis we demonstrated that the EC Atypical Beijing isolates evolved from a common progenitor, giving rise to two sub-groups, each with unique features, including mutations that confer resistance to up to 11 anti-TB drugs. This finding was supported by whole genome sequencing (WGS) and RNA sequencing demonstrating close relatedness and suggests the emergence and spread of totally drug-resistant TB in the EC. We showed that isolates harbouring the rrs A1401G mutation displayed a decreased susceptibility to capreomycin, thereby questioning the utility of this drug in the treatment of XDR-TB when amikacin resistance was already noted. Importantly, strains harbouring the rpoB516 mutation were shown to be susceptible to rifabutin, despite low-level resistance to rifampicin (RIF). Therefore the use of rifabutin in the EC may improve therapeutic success and limit transmission of XDR-TB. WGS was used to investigate molecular features that may confer a selective advantage to the EC Atypical Beijing genotype strains. These analyses revealed that all represented Atypical Beijing genotype strains – including those diagnosed as pan-susceptible – harboured a mutation in ethA, conferring phenotypic ethionamide resistance. This surprising finding may explain the apparent increased ability of the Atypical Beijing genotype strains to develop higher drug-resistance in the context of an ethionamide-containing MDR-TB treatment regimen. It is unclear why some strains additionally acquire inhA promoter mutations. This requires further investigation. A large number of genes were shown by RNAseq to be differentially regulated, however, their influence on the physiological properties of the bacillus remain to be determined. Together these findings have challenged the use of standardised MDR-TB treatment without comprehensive DST. This view is now widely recognised but has not influenced the South African TB guidelines (2014) which promote treatment of RIF resistance without relevant knowledge of drug resistance. We propose that the effective treatment of highly resistant TB can only be achieved with the development of new drugs, new drug combinations and comprehensive rapid DST.
Programmatically selected multidrug-resistant strains drive the emergence of extensively drug-resistant tuberculosis in South Africa
(Public Library of Science, 2013-08-23) Muller, Borna; Chihota, Violet N.; Pillay, Manormoney; Klopper, Marisa; Streicher, Elizabeth M.; Coetzee, Gerrit; Trollip, Andre; Hayes, Cindy; Bosman, Marlein E.; Gey van Pittius, Nicolaas C.; Victor, Thomas C.; Gagneux, Sebastien; Van Helden, Paul D.; Warren, Robin M.
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.
The rationale for using rifabutin in the treatment of MDR and XDR tuberculosis outbreaks
(Public Library of Science, 2015) Sirgel, Frederick A.; Warren, Robin M.; Bottger, Erik C.; Klopper, Marisa; Victor, Thomas C.; Van Helden, Paul D.
Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.

Browsing by Author "Klopper, Marisa"

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