Background. Tuberculosis is increasing in the Free State and Northern Cape provinces
of South Africa, but it is not clear how much of the disease is caused by reactivated
latent infection and how much is attributed to interpersonal transmission. The discovery
of the transposable DNA insertion sequence, IS6110, provided the desired
polymorphism among different strains to track routes of transmission, study the degree
of inter-person transmission versus reactivation, to detect laboratory contamination and
disease outbreaks. Alternative methods include spoligotyping and the mycobacterial
intergenic repetitive units or variable number of tandem repeats (MIRU-VNTR).
Sustained studies performed on a small area in the Western Cape Province and some
mines in the Gauteng Province of South Africa have found person-to-person
transmission of tuberculosis to be high in these populations. In addition, resistance
determinants to key antituberculosis drugs have remained unknown among tuberculosis
causative organisms circulating in the Free State and Northern Cape. Thus, extensive
DNA fingerprinting and gene mutation studies are needed to address these problems.
Methods. An area in the Free State suitable for long-term surveillance studies was
defined using available information from the governmental database, the 1996 census
statistics, and tuberculosis (TB) case loads and transfer data obtained from the National
Tuberculosis Database. Each clinicâs catchment information was provided by clinic
managers and the population movement data from a 2002 student project. Sputum
samples were collected and Mycobacterium tuberculosis isolated from tuberculosis
positive patients from the defined area (Gamadi). Isoniazid resistant isolates received
from a representative sample from the Free State and a few strains from the Northern Cape Province were also included in the study. IS6110-directed restriction-fragment-length
polymorphism (RFLP) analysis was performed on all isolates and drug
susceptibility testing (indirect proportion method) done on the Gamadi isolates.
Subtyping of identical strains (RFLP clusters) and some of the isolates with less than six
IS6110 bands was done using spoligotyping and the MIRU-VNTR typing. DNA
sequencing analysis of the katG and rpoB genes was done in resistant isolates and a
rapid PCR-based restriction enzyme katG gene mutation detecting method evaluated.
Results. An area characterised by extreme poverty (unemployment rate 69.0%), a
relatively young population (69.0% below 35 years) of 61534 and with high incidence of
tuberculosis (840/100 000) suitable for long -term surveillance studies was identified in
the Free State. The area is served by three clinics and a hospital and is situated near
the rural town of Thaba Nchu in the Free State province. Eighty eight M. tuberculosis
isolates and a mycobacterium-other-than-tuberculosis (MOTT) were isolated from the
286 sputum specimens collected from the Gamadi area. Only two M. tuberculosis
isolates tested isoniazid (INH) resistant and no rifampicin (RIF) resistant isolates were
found. The MOTT was resistant to INH (0.2, 1 and 5 µg/ml) and to RIF.
Standard IS6110-based DNA fingerprinting of 84 of 88 (96.5%) isolates from the defined
area was performed. Four of the isolates were cultured from duplicate sputum
specimens provided by four patients. Two of these had identical fingerprint patterns to
the first isolate of the patient and two had a different profile. The latter pair could be
attributed to laboratory error. IS6110 sequences were not detected in six isolates.
Fourteen isolates had less than six IS 6110 hybridisation bands and four strains were in
clusters. The remaining 57 (88.9%) strains had distinct RFLP profiles with more than six
bands. The number of IS6110 copies varied from seven to 21. A total of five strains was
distributed in two clusters, one with two and the other with three members. Thirteen
family groups, clustered at 65.0% on the similarity dendogram, each with two to eight
strains, but no dominant groups were evident. A cluster of three isolates with five
identical IS 6110 bands each was confirmed as one strain by MIRU-VNTR typing while
two further isolates (both had three bands of different sizes) were confirmed as different
strains by MIRU typing. A total of 37 isoniazid-resistant M. tuberculosis was analysed. DNA fingerprint profiles
showed nine isolates with less than six insertions (24.3%). Six of these isolates were
from the Free State and three from the Northern Cape Province. Three of these isolates
were multidrug resistant. The remaining 28 isolates (75.7%) contained between 9 and
18 copies of the IS6110 insertion sequence. Twenty-six different IS 6110 RFLP types
were identified. Only two clusters with two isolates, respectively, were found in each
province. Eight clonally related groups (65.0% similarity) with two to four strains were
present. Three clusters of two isolates (each with more than six bands) also exhibited
identical spoligotype patterns. Spoligotyping of two of three isolates from a fourth cluster
(4 RFLP bands each) showed two different banding patterns and all were shown to be
different by MIRU-VNTR typing. The fifth cluster (2 bands) was made up of one isolate
from each province. Spoligotyping of these strains was identical, but the MIRU was
different. One isolate from Bloemfontein had identical IS 6110-RFLP and spoligotyping
patterns to a susceptible isolate from Gamadi.
Isoniazid resistance in 22/37 isolates was sequence linked to altered nucleotides of
codon 315 of the katG gene. Twenty harboured the ACC variant at the codon. One
strain carried the AAC mutation at this codon and the other GGC. The remaining 15
carried the wild type (AGC) genotype at this site. Two of the strains harbouring the
AGC315ACC mutation belonged to the same IS6110 cluster. Two mutations were found
at codon 463 (CGG ® CTG; CGG ® CCG).
Thirteen MDR strains were investigated for rpoB gene alterations. Four of these isolates
carried no mutations within the 157-bp amplified fragment while the others had various
mutations.
Analysis of an 808bp fragment of the katG gene from INH-resistant M. tuberculosis
isolates after restriction with Msp I agreed with results obtained by sequencing. Thirteen
isolates carried a pattern consisting of 228, 153, 146, 109, 79, 65 base pairs with the
153 bp fragment indicating the presence of the wild type AGC at codon 315 of the katG
gene. Seventeen isolates demonstrated the 228, 146, 132, 109, 79, 65, 21 profile with
the 132 bp fragments indicating the presence of an ACC mutation. Three isolates
contained a mixed genotype and were digested into the fragments 228 bp, 153 bp, 146 bp, 132 bp, 109 bp, 79 bp, and 65 bp. Fragments with 146 bp and 65 bp are seen in
strains with no mutation (bases CGG) at codon 463, while a 211 bp fragment shows a
mutation at this spot. Four strains had the fragments 228, 211, 153, 109, and 79 bp.
One strain was digested into six fragments of 228 bp, 211 bp, 132 bp, 109 bp 79 bp and
21 bp containing both a 315 (ACC) and 463 (CTG) codon mutation.
Discussion and conclusions. An area consisting of ten villages and characterised by a
high incidence of tuberculosis was defined for long-term surveillance studies. Resistance
in the area appears to be low and compares favourably to the situation in the Free State.
Strains received from this area were highly diverse, but the presence of a cluster of five
isolates indicated the need for continuous investigation. Recent transmission of INH
resistance in the Free State province is not a significant factor, but since the isolates
from the Northern Cape were not representative, no deduction could be made for this
province. Resistance to INH is mostly associated with mutation AGC to ACC at codon
315 of the katG gene. The absence of alterations in a proportion of isolates is in
agreement with published data implicating the involvement of more genes in causing
INH resistance. Resistance to RIF was associated with various point mutations in the
81-bp core region of the rpoB gene. The high proportion of the ACC allele found among
INH-resistant strains, cost effectiveness, ease to perform and rapid results, make PCR-RFLP
an attractive option for detection of resistance especially in resource-poor
countries.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-05162005-164642 |
| Date | 16 May 2005 |
| Creators | Mokhethi, Sehloho Zacharia |
| Contributors | Mrs Anneke van der Spoel van Dijk |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-05162005-164642/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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