Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2016. / The reference phenotypic methods for Mycobacterium tuberculosis drug susceptibility
testing are qualitative and based on drug critical concentrations. Limitations include
lack of standardization and variations in laboratory preparation of drug stock solutions.
The recommended critical concentrations are determined by consensus and experience
rather than scientific data. Consequently incorrect and inadequate susceptibility
breakpoints are used and patients receive ineffective antimicrobial therapy. The
determination of wild-type minimal inhibitory concentration distribution is an important
tool used by European Committee for antimicrobial susceptibility Testing (EUCAST) to
establish clinical breakpoints in Europe. This could be applicable in South Africa.
Aim
To determine wild-type minimal inhibitory concentration distributions of first and secondline
drugs against Mycobacterium tuberculosis complex clinical isolates and compare
these with the recommended critical concentration in Limpopo province.
Methods
A sample of 101 Mycobacterium tuberculosis complex positive cultures were collected
from National Health Laboratory Services in Polokwane (Limpopo province) and subcultured
on BACTEC MGIT 960 system. The isolates were inoculated on MYCOTB MIC
plates to determine the wild-type MIC distributions of first and second-line drugs. The
data were compared with currently recommended critical concentrations. DNA was
extracted and amplified by PCR. Genotypic drug susceptibility testing was performed
using GenoType MTBDRplus version 2.0 and GenoType MTBDRsl version 2.0 for the
first- and second-line drugs, respectively. Genotyping of clinical isolates was performed
to determine M. tuberculosis strain families using spoligotyping.
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Results
Wild-type MIC distributions range reported in this study are as follows rifampin (≤ 0.12 -
0.5 μg/μg/ml), isoniazid (≤ 0.3 - 2.00 μg/ml), rifabutin (≤ 0.12 - 0.25 μg/ml), ethionamide
(≤ 0.12 - 5 μg/ml), ethambutol (≤ 0.5 - 2 μg/ml), streptomycin (≤ 0.25 - 0.5 μg/ml), paraaminosalicylic
(≤ 0.5 - 4.0 μg/ml), cycloserine (≤ 2 -16 μg/ml), amikacin (≤ 0.12 - 0.5
μg/ml), kanamycin (≤ 0.6 -2.5 μg/ml), moxifloxacin (≤ 0.6 - 0.5 μg/ml), ofloxacin (≤ 0.25 -
1 μg/ml).
GenoType MTBDRplus detected (n= 68, 67%) rifampin resistance (MUT 3=26, MUT
2=18, MUT 2B=8) on the rpoB gene. Isoniazid resistant (n=20, 19.8%) was detected
katG MUT (n=20, 19.8%) on katG gene (S315T1).
Genotypic resistance to second-line drugs determined by GenoType MTBRsl detected
no mutations in (n= 98, 97%) isolates on gyrA, gyrB rrs and eis gene and (n=3, 2.9%)
isolates non mycobacterium tuberculosis complex were detected.
The frequency and percentage of Mycobacterium tuberculosis family strain were
identified in (n= 81, 80%) of the clinical isolates which matched 18 pre-existing shared
types. The results showed high genotype diversity with the Beijing strain (n= 30, 29.7%)
and T family (n= 19, 18.8%) dominating. Twenty isolates (19.8%) had no shared types
thus reported as orphan.
Conclusion
The findings obtained in this study suggest wild-type Minimal Inhibitory Concentration
distributions may be considered when setting clinical breakpoints. Discordant results
were observed between phenotypic and genotypic DST for rifampin, isoniazid,
streptomycin, rifabutin and ethambutol, suggesting that breakpoint concentrations for
some drugs are set too high while others are too low. The Mycobacterium tuberculosis
clinical isolates displayed diverse family strain with Beijing and T strain predominate
breakpoints for first-line and second-line drugs used in Mycobacterium tuberculosis
treatments.
Poster Presentations
Poster presented at faculty of Health science first annual research day on Second-line
drug susceptibility breakpoints for Mycobacterium tuberculosis using MYCOTB MIC
plate. University of Limpopo Tiro hall 16th to 17th September 2014.
Poster presented at National Health Laboratory Service Pathology Research and
Development Congress (PathReD) on Determination of families strains of
Mycobacterium tuberculosis circulating in Limpopo Province, South Africa. Emperors
Palace 14th April-16th April 2015.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/1712 |
| Date | January 2016 |
| Creators | Seloma, Ngwanamohuba Mologadi |
| Contributors | Maguga-Phasha, N. T. C., Makgatho, M. E., Mbajiorgu, E. F., Da Silva, M. P. M. F. |
| Publisher | University of Limpopo |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xix, 95 leaves |
| Relation |
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