Introduction of Drug Resistant Reflex
Testing in KZN & Descriptive Analysis of
Second line mutations
Dr Keeren Lutchminarain
Department of Medical Microbiology
IALCH, Durban
Background
WHO Recommendations
2016
Background
All patients placed on the
shorter MDR regimen must
be tested for resistance to
second line drugs
4
Kwazulu Natal – DR-TB Reflex testing
• 11 MDR initiation sites
• 88 GXP machines
• GXP performed at patients initial visit → 2nd sample sent from the same site
→ result available within 28 days at the MDR initiation site on arrival of
patients.
• Laboratory workflow → All GXP Rif R samples arrive → decontamination →
microscopy →culture →PCR 1st line → PCR 2nd line → Phenotypic
susceptibility testing on selected isolates
• Provincial training completed in May 2017.
Descriptive analysis of MTBDRsl Ver 2
mutations Month Total Reflex
Samples
Total second line resistance
detected (%pre-XDR /XDR from
samples directly)
August 457 52 (11.3%)
September 710 73 (10.28%)
October 651 35 (5.4%)
7
gyr A mutations – Total number 127
WT absent Total No.
(freq-%)
Associated Mutation
(frequency-%)
WT 1 1 (0.7%) Mut 3C (100%)
WT 2 75 (59%) Mut 1 (41) (54%)
Mut 2 (18) (24%)
Mut 3C (5) (6.6%)
No mutation (11) (14.6%)
WT 2 & 3 3 (25%) No mutation (1) (33.3%)
Mut 3C (2) (66.6%)
gyr A mutations – Total number 127
WT Absent Total No
(freq-%)
Associated Mutation
(frequency-%)
WT 3 32 (25%) Mut 1 (2) (6.25%)
Mut 3A (1) (3.1%)
Mut 3B (8) (25%)
Mut 3C (18) (56%)
Mut 3D (1) (3.1%)
No mutations (1) (3.1%)
Mut 3A & 3B (1) (3.1%)
gyr A mutations – Total number 127
WT Absent Total No.
(frequency-%)
Associated Mutations
(frequency-%)
All WT + 15 (11.8%) Mut 3C (8) (53%)
Mut 3B (4) (26.6%)
Mut 2 (1) (6.6%)
Mut 1 (2) (6.6%)
Summary: gyrA
• WT in order of decreasing frequency :
WT 2
MUT 1
WT3
MUT3C
WT ++
MUT3C
19 isolates displayed heteroresistance/mixed
infection
13 isolates had WT missing with no mutation
bands
A90V / S91P
gyr B Mutations - Total number 4
WT Absent Total Number Associated Mutation
WT absent 4 Locus control only (4)
No associated mutations
rrs Mutations – Total 103 WT Absent Total Number
(frequency-%)
Associated mutations
(frequency-%)
All WT absent 37 (35.9%) Locus control only (37) (100%)
No associated mutations
WT1 61 (59.2%) No mutation (10) (16%)
Mut 1 (49) (80%)
Mut 2 (2) (3.3%)
WT 2 2 (1.9%) Mut 2 (1) (50%)
No mutation (1) (50%)
All WT present 3 (2.9%) Mut 2 (3)
Summary: rrs
• Mutations in order of decreasing frequency
WT1 MUT 1Locus control
only All WT
present/MUT2
Significant amount of strains are not covered by the WT
and mutation probes in the assay.
Commonest rrs mutation in KZN - A1401G ffd by
C1402T
Heteroresistance /mixed strain infection noted in 3
isolates.
eis Mutations – Total 11
WT Absent Total Number
(frequency-%)
Associated Mutation
WT 1 7 (63.6%) No mutation (7) band
WT 2 3 (27.2%) Mut 1 (3)
All WT present 1 (9.09%) Mut 1 (1)
Conclusion
• DR-TB reflex testing is fully operational in KZN –
challenges still exist
• Mutations frequently noted belong to gyrA and rrs
genes
• Heteroresistance is noted in all 4 gene loci
• Circulating mutations may not be covered in the
assay.
• Phenotypic susceptibility testing is still required.
Acknowledgements
• Professor K.P. Mlisana
• Afsana Kajee
• Priyanka Ramlal
• Dr. Y. Mahabeer
• TB laboratory staff at IALCH
• Hain Lifescience Representatives
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