Prevalence and resistance gene mutations of multi-drug resistant and extensively drug resistant mycobacterium tuberculosis in the Eastern Cape
- Authors: Hayes, Cindy
- Date: 2014
- Subjects: Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Microbial mutation
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10125 , http://hdl.handle.net/10948/d1020374
- Description: The emergence and spread of multi-drug resistant (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) are a major medical and public problem threatening the global health. The objectives of this study were to (i) determine the prevalence of MDR-TB and XDR-TB in the Eastern Cape; (ii) analyze patterns of gene mutations in MDR-TB and (iii) identify gene mutations associated with resistance to second line injectable drugs in XDR-TB isolates. A total of 1520 routine sputum specimens sequentially received within a period of 12 months i.e. February 2012 to February 2013 from all MDR-TB and XDR-TB patients treated by Hospitals and clinics in the Eastern Cape were included in this study, of which 1004 had interpretable results. Samples were analyzed with the Genotype MTBDRplus VER 2.0 assay kit (Hain Lifescience) for detection of resistance to Rifampicin and Isoniazid while solid and liquid culture drug susceptibility tests were used for ethambutol, streptomycin, ethionamide, ofloxacin, capreomycin and amikacin. PCR and sequence analysis of short regions of target genes gyrA, (encode subunit of DNA topoisomerase gyrase), rrs (16S rRNA) and tlyA (encodes a 2’-O-methyltransferase) were performed on 20 XDR-TB isolates. MTBDRplus kit results and drug susceptibility tests identified 462 MDR-TB, 284 pre-XDR and 258 XDR-TB isolates from 267 clinics and 25 hospitals in the Eastern Cape. There was a high frequency of resistance to streptomycin, ethionamide, amikacin, ofloxacin and capreomycin. Mutation patterns indicated differences between the health districts as well as differences between the facilities within the health districts. The most common mutation patterns observed were: (i) ΔWT3, ΔWT4, MUT1 [D516V+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG), ΔWT1 [C15T] (inhA) [39 MDR, 204 XDR-TB and 214 pre XDR-TB isolates], (ii) ΔWT8, MUT3 [L533P+S531L] (rpoB), ΔWT, MUT1 [S315T1] [145 MDR, 18 pre-XDR and 3 XDR-TB solates] and (iii) ΔWT3, WT4 [D516Y+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG) [75 MDR, 1 pre-XDR and 7 XDR-TB isolates]. Mutations in inhA promoter regions were strongly associated with XDR-TB isolates. Two thirds (66.6 percent (669/1004) of the isolates had inhA mutations present with 25.4 percent (170/669) found among the MDR isolates, 39.2 percent (262/669) among the pre-XDR isolates and 35.4 percent (237/669) among the XDR-TB isolates, which implies that these resistant isolates are being spread by transmission within the community and circulating in the province. There was good correlation between XDR-TB drug susceptibility test results and sequence analyses of the gyrA and rrs genes. The majority of XDR-TB isolates contained mutations at positions C269T (6/20) and 1401G (18/20) in gyrA and rrs genes respectively. Sequence analysis of short regions of gyrA and rrs genes may be useful for detection of fluoroquinolone and amikacin/ kanamycin resistance in XDR-TB isolates but the tlyA gene is not a sensitive genetic marker for capreomycin resistance. This study highlighted the urgent need for the development of rapid diagnostics for XDR-TB and raised serious concerns regarding ineffective patientmanagement resulting in ongoing transmission of extremely resistant strains of XDRTB in the Eastern Cape suggesting that the Eastern Cape could be fast becoming the epicenter for the development of Totally Drug-resistant Tuberculosis (TDR-TB) in South Africa.
- Full Text:
- Date Issued: 2014
- Authors: Hayes, Cindy
- Date: 2014
- Subjects: Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Microbial mutation
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10125 , http://hdl.handle.net/10948/d1020374
- Description: The emergence and spread of multi-drug resistant (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) are a major medical and public problem threatening the global health. The objectives of this study were to (i) determine the prevalence of MDR-TB and XDR-TB in the Eastern Cape; (ii) analyze patterns of gene mutations in MDR-TB and (iii) identify gene mutations associated with resistance to second line injectable drugs in XDR-TB isolates. A total of 1520 routine sputum specimens sequentially received within a period of 12 months i.e. February 2012 to February 2013 from all MDR-TB and XDR-TB patients treated by Hospitals and clinics in the Eastern Cape were included in this study, of which 1004 had interpretable results. Samples were analyzed with the Genotype MTBDRplus VER 2.0 assay kit (Hain Lifescience) for detection of resistance to Rifampicin and Isoniazid while solid and liquid culture drug susceptibility tests were used for ethambutol, streptomycin, ethionamide, ofloxacin, capreomycin and amikacin. PCR and sequence analysis of short regions of target genes gyrA, (encode subunit of DNA topoisomerase gyrase), rrs (16S rRNA) and tlyA (encodes a 2’-O-methyltransferase) were performed on 20 XDR-TB isolates. MTBDRplus kit results and drug susceptibility tests identified 462 MDR-TB, 284 pre-XDR and 258 XDR-TB isolates from 267 clinics and 25 hospitals in the Eastern Cape. There was a high frequency of resistance to streptomycin, ethionamide, amikacin, ofloxacin and capreomycin. Mutation patterns indicated differences between the health districts as well as differences between the facilities within the health districts. The most common mutation patterns observed were: (i) ΔWT3, ΔWT4, MUT1 [D516V+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG), ΔWT1 [C15T] (inhA) [39 MDR, 204 XDR-TB and 214 pre XDR-TB isolates], (ii) ΔWT8, MUT3 [L533P+S531L] (rpoB), ΔWT, MUT1 [S315T1] [145 MDR, 18 pre-XDR and 3 XDR-TB solates] and (iii) ΔWT3, WT4 [D516Y+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG) [75 MDR, 1 pre-XDR and 7 XDR-TB isolates]. Mutations in inhA promoter regions were strongly associated with XDR-TB isolates. Two thirds (66.6 percent (669/1004) of the isolates had inhA mutations present with 25.4 percent (170/669) found among the MDR isolates, 39.2 percent (262/669) among the pre-XDR isolates and 35.4 percent (237/669) among the XDR-TB isolates, which implies that these resistant isolates are being spread by transmission within the community and circulating in the province. There was good correlation between XDR-TB drug susceptibility test results and sequence analyses of the gyrA and rrs genes. The majority of XDR-TB isolates contained mutations at positions C269T (6/20) and 1401G (18/20) in gyrA and rrs genes respectively. Sequence analysis of short regions of gyrA and rrs genes may be useful for detection of fluoroquinolone and amikacin/ kanamycin resistance in XDR-TB isolates but the tlyA gene is not a sensitive genetic marker for capreomycin resistance. This study highlighted the urgent need for the development of rapid diagnostics for XDR-TB and raised serious concerns regarding ineffective patientmanagement resulting in ongoing transmission of extremely resistant strains of XDRTB in the Eastern Cape suggesting that the Eastern Cape could be fast becoming the epicenter for the development of Totally Drug-resistant Tuberculosis (TDR-TB) in South Africa.
- Full Text:
- Date Issued: 2014
Molecular characterization of the Mycobacterium tuberculosis complex (MTC) of raw milk from selected dairy farms in the Eastern Cape
- Authors: Komani, Nosiphiwo
- Date: 2013
- Subjects: Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Milk -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Dairy farming -- South Africa -- Eastern Cape , Tuberculosis in cattle -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11276 , http://hdl.handle.net/10353/d1013157 , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Milk -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Dairy farming -- South Africa -- Eastern Cape , Tuberculosis in cattle -- South Africa -- Eastern Cape
- Description: Tuberculosis (TB) is an ancient infectious disease that has been infecting different populations around the globe and it has also been considered as one of the most successful human and animal disease. TB found in animals such as cattle and other known bovids is known as bovine tuberculosis. Bovine tuberculosis (BTB) is an infectious disease found in cattle mainly caused by Mycobacterium bovis. M. bovis is a member of the Mycobacterium tuberculosis complex (MTC) together with M. tuberculosis, M. africanum, and M. canetti where the natural host is humans; whereas M. caprae, M. microti and M. pinnipedii usually have animals as their natural host. In this study the molecular characterization of the MTC from cow milk in the Eastern Cape was investigated. One hundred and twenty samples (40 ml each) were collected from three dairy farms in the Eastern Cape, South Africa. These samples were processed using a modified Petroff decontamination method. Sample processing was followed by DNA isolation using a Zymo Bacterial/Fungal DNA Kit and the amplification and detection of the MTC was done using the Seeplex MTB Nested ACE assay. The drug susceptibility tests were done using GenoTypeMTBDRplus assay which detects mutations and resistance to INH (isoniazid) and RMP (rifampicin). The milk isolates were further analyzed using a spoligotyping method which is based on the PCR amplification of a highly polymorphic direct repeat locus in the M. tuberculosis genome which detects and types the MTC. A percentage of 20.8 % samples were found to be positive for MTC using the Seeplex MTB Nested ACE assay. There were 42.1 % samples that were resistant to both INH and RMP with the rest sensitive to either INH or RMP. The spoligotyping method showed that 78.3 % samples resembled Family 33 strains and the rest (21.7 %) resembled a spoligotyping signature known to be that of M.africanum. Both these strains belong to the Ancestral lineage with Indo-Oceanic and West Africa 2 lineage. The outcomes of our study showed that molecular methods for detection of MTC can be applied directly on milk samples without the need for culturing.
- Full Text:
- Date Issued: 2013
- Authors: Komani, Nosiphiwo
- Date: 2013
- Subjects: Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Milk -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Dairy farming -- South Africa -- Eastern Cape , Tuberculosis in cattle -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11276 , http://hdl.handle.net/10353/d1013157 , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Milk -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Dairy farming -- South Africa -- Eastern Cape , Tuberculosis in cattle -- South Africa -- Eastern Cape
- Description: Tuberculosis (TB) is an ancient infectious disease that has been infecting different populations around the globe and it has also been considered as one of the most successful human and animal disease. TB found in animals such as cattle and other known bovids is known as bovine tuberculosis. Bovine tuberculosis (BTB) is an infectious disease found in cattle mainly caused by Mycobacterium bovis. M. bovis is a member of the Mycobacterium tuberculosis complex (MTC) together with M. tuberculosis, M. africanum, and M. canetti where the natural host is humans; whereas M. caprae, M. microti and M. pinnipedii usually have animals as their natural host. In this study the molecular characterization of the MTC from cow milk in the Eastern Cape was investigated. One hundred and twenty samples (40 ml each) were collected from three dairy farms in the Eastern Cape, South Africa. These samples were processed using a modified Petroff decontamination method. Sample processing was followed by DNA isolation using a Zymo Bacterial/Fungal DNA Kit and the amplification and detection of the MTC was done using the Seeplex MTB Nested ACE assay. The drug susceptibility tests were done using GenoTypeMTBDRplus assay which detects mutations and resistance to INH (isoniazid) and RMP (rifampicin). The milk isolates were further analyzed using a spoligotyping method which is based on the PCR amplification of a highly polymorphic direct repeat locus in the M. tuberculosis genome which detects and types the MTC. A percentage of 20.8 % samples were found to be positive for MTC using the Seeplex MTB Nested ACE assay. There were 42.1 % samples that were resistant to both INH and RMP with the rest sensitive to either INH or RMP. The spoligotyping method showed that 78.3 % samples resembled Family 33 strains and the rest (21.7 %) resembled a spoligotyping signature known to be that of M.africanum. Both these strains belong to the Ancestral lineage with Indo-Oceanic and West Africa 2 lineage. The outcomes of our study showed that molecular methods for detection of MTC can be applied directly on milk samples without the need for culturing.
- Full Text:
- Date Issued: 2013
Major spoligotype families of Mycobacterium tuberculosis strains isolated from tuberculosis patients in Port Elizabeth, Eastern Cape, South Africa
- Authors: Nqini, Babalwa J
- Date: 2012
- Subjects: Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11271 , http://hdl.handle.net/10353/d1006877 , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
- Description: South Africa is burdened with tuberculosis (TB) which is aggravated by the concurrent epidemic of HIV as well as the emergence of drug resistance. In most developed countries molecular techniques have been used to look at the dynamics of the TB epidemic however, despite the prevalence that is high in sub-Saharan Africa, there is little data on strain types that are available in Port Elizabeth. This study aims to find the major clades of M. tuberculosis that are circulating in Port Elizabeth. Two hundred MDR-TB DNA samples were obtained from the National Health Laboratory Services TB laboratory in Port Elizabeth. Spoligotyping and MIRU-VNTR were used to genotype the strains. Two hundred strains were sent to the University of Stellenbosch for spoligotyping and 179 of those were typed. Spoligotype defined families were further typed by MIRU-VNTR typing, so as to further differentiate and assess clonal diversity within the spoligotype families. The Beijing family was the dominant family and the MANU family being the least dominant, with percentages of 71 percent and 0.5 percent respectively. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 15 shared international types. Forty four percent (44 percent) of the isolates that were typed by MIRU-VNTR showed similarities, suggesting epidemiological relatedness. Thirty eight percent of isolates from spoligotyping were from the same family, the Beijing family, with the same shared international type STI1, but when typed by 12 MIRU-VNTR they showed no epidemiological relatedness and 18 percent of the isolates showed no relatedness when typed by 12 MIRU-VNTR but spoligotyping showed that they were from the LAM family. Results from our study illustrate the effectiveness of MIRU-VNTR typing together with spoligotyping in epidemiological studies in the region of Port Elizabeth.
- Full Text:
- Date Issued: 2012
- Authors: Nqini, Babalwa J
- Date: 2012
- Subjects: Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11271 , http://hdl.handle.net/10353/d1006877 , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
- Description: South Africa is burdened with tuberculosis (TB) which is aggravated by the concurrent epidemic of HIV as well as the emergence of drug resistance. In most developed countries molecular techniques have been used to look at the dynamics of the TB epidemic however, despite the prevalence that is high in sub-Saharan Africa, there is little data on strain types that are available in Port Elizabeth. This study aims to find the major clades of M. tuberculosis that are circulating in Port Elizabeth. Two hundred MDR-TB DNA samples were obtained from the National Health Laboratory Services TB laboratory in Port Elizabeth. Spoligotyping and MIRU-VNTR were used to genotype the strains. Two hundred strains were sent to the University of Stellenbosch for spoligotyping and 179 of those were typed. Spoligotype defined families were further typed by MIRU-VNTR typing, so as to further differentiate and assess clonal diversity within the spoligotype families. The Beijing family was the dominant family and the MANU family being the least dominant, with percentages of 71 percent and 0.5 percent respectively. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 15 shared international types. Forty four percent (44 percent) of the isolates that were typed by MIRU-VNTR showed similarities, suggesting epidemiological relatedness. Thirty eight percent of isolates from spoligotyping were from the same family, the Beijing family, with the same shared international type STI1, but when typed by 12 MIRU-VNTR they showed no epidemiological relatedness and 18 percent of the isolates showed no relatedness when typed by 12 MIRU-VNTR but spoligotyping showed that they were from the LAM family. Results from our study illustrate the effectiveness of MIRU-VNTR typing together with spoligotyping in epidemiological studies in the region of Port Elizabeth.
- Full Text:
- Date Issued: 2012
Prevalence, seasonal trends and incidences of cattle tuberculosis and brucellosis in the Cacadu municipality, Eastern Cape, South Africa
- Authors: Nombebe, Thobeka
- Date: 2012
- Subjects: Tuberculosis in cattle -- South Africa -- Eastern Cape , Brucellosis in animals -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Biostatistics and Epidemiology)
- Identifier: vital:11783 , http://hdl.handle.net/10353/d1016200 , Tuberculosis in cattle -- South Africa -- Eastern Cape , Brucellosis in animals -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape
- Description: A cross-sectional study was conducted from February 2011 to August 2012 to investigate the status and identify risk factors for Bovine Tuberculosis (BTB) and cattle Brucellosis. A total of 1277 cattle from 6 selected dairy farms were tested. The farms were purposively selected on suspected risk. Samples were analysed using Rose Bengal Test (RBPT) and Complement Fixation Test (CFT) for Brucellosis and Comparative Intradermal Tuberculin Test (CITT) for BTB. The results revealed the overall prevalence of 1.6% for Brucellosis and no BTB was found. Logistic regression analysis revealed that age of cattle, herd size and number of calves a cow has had significant association with Brucellosis with p-values 0.0071, 0.0490 and 0.000 respectively. The role of location (p>0.05), gender (p>0.05) and breed (p>0.05) in the occurrence of the disease was not statistically significant at α=0.05, although higher rates were obtained in females and Jersey breed. Questionnaires were also administered to 47 individuals a total of 12 females and 35 males. Most respondents were not aware about cattle brucellosis and cattle tuberculosis. Consumption of raw or unpasteurized milk still occurs with 74.75% of respondents revealed.
- Full Text:
- Date Issued: 2012
- Authors: Nombebe, Thobeka
- Date: 2012
- Subjects: Tuberculosis in cattle -- South Africa -- Eastern Cape , Brucellosis in animals -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc (Biostatistics and Epidemiology)
- Identifier: vital:11783 , http://hdl.handle.net/10353/d1016200 , Tuberculosis in cattle -- South Africa -- Eastern Cape , Brucellosis in animals -- South Africa -- Eastern Cape , Dairy farms -- South Africa -- Eastern Cape , Mycobacterium tuberculosis -- South Africa -- Eastern Cape
- Description: A cross-sectional study was conducted from February 2011 to August 2012 to investigate the status and identify risk factors for Bovine Tuberculosis (BTB) and cattle Brucellosis. A total of 1277 cattle from 6 selected dairy farms were tested. The farms were purposively selected on suspected risk. Samples were analysed using Rose Bengal Test (RBPT) and Complement Fixation Test (CFT) for Brucellosis and Comparative Intradermal Tuberculin Test (CITT) for BTB. The results revealed the overall prevalence of 1.6% for Brucellosis and no BTB was found. Logistic regression analysis revealed that age of cattle, herd size and number of calves a cow has had significant association with Brucellosis with p-values 0.0071, 0.0490 and 0.000 respectively. The role of location (p>0.05), gender (p>0.05) and breed (p>0.05) in the occurrence of the disease was not statistically significant at α=0.05, although higher rates were obtained in females and Jersey breed. Questionnaires were also administered to 47 individuals a total of 12 females and 35 males. Most respondents were not aware about cattle brucellosis and cattle tuberculosis. Consumption of raw or unpasteurized milk still occurs with 74.75% of respondents revealed.
- Full Text:
- Date Issued: 2012
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