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