Diagnosis of Drug Resistant TB in South

Africa

Dr Keeren Lutchminarain
Pathologist: Department of Medical Microbiology
UKZN & NHLS
Burden of Disease in South Africa
• WHO 2014: SA
 Highest Incidence globally: 834 (736 – 936) per 100,000
 High co-infection with HIV > 60%
 Increasing cases of drug resistant TB: 2nd highest

• KwaZulu-Natal:
 Third of the country’s burden of drug resistant TB
 TB concentrated in urban areas: highest TB in eThekwini,
Umgungundlovu and Uthungulu
 Districts with the highest MDR TB: Umkhanyakude and
Zululand
 District with highest XDR TB: Umzinyathi and eThekwini
 Introduction
• Early identification of drug resistance profiles has become
increasingly important need in the management of patients
with Tuberculosis.

• With the availability of new drugs e.g. Bedaquiline, early

diagnosis assists in optimizing regimens

• Also important - programmatically in preventing

transmission of the disease.
Currently available TB diagnostic tests
Genotype MTBDRplus Xpert MTB/RIF
(LPA) (GeneXpert)

= endorsed by WHO = still under research

Slide adapted from FIND (Foundation for Innovative New Diagnostics)

Sensitive test = Early case finding
Early case finding = Decrease in mortality and transmission

+
Sensitivity of sputum sample testing
-
MGIT LJ PCR FM ZN
Xpert MTB/RIF

1+ 2+ 3+ 4+

101 102 103 104 105 106 107

-
Severity of TB pulmonary disease/bacterial load

Jose A. Caminero (The Union)

+
 Available Platforms
• The diagnosis of Drug Resistant TB in South Africa is
currently performed 2 main methodologies: Genotypic and
Phenotypic methods

• Genotypic methods: Xpert MTB/RIF

Line probe assay

• Phenotypic methods: Direct Susceptibility testing

(Liquid/Solid Agar)
 Safe Sputum Collection
• TB is spread by infected droplet nuclei (<5 microns)
• Droplet nuclei remain suspended in the air
• Especially bad in confined spaces with minimal air
exchanges
• All health care workers must protect themselves and
other patients when supervising sputum collection

Image courtesy of www.scienceblogs.com

 SPUTUM COLLECTION
1. Let person rinse mouth with water
2. Ask patient to direct sputum into container without contaminating
outside of container, in well ventilated space
3. Give patient container without the lid
4. Hold lid yourself. If with patient stand behind them
5. Demonstrate cough from bottom of chest after a few deep
breaths.
6. Encourage person to produce specimen after deep coughing, as
much as possible
7. Replace lid immediately
8. Secure lid, screw and press on centre till click is heard
9. Wash hands after collection
10. Label correctly with your contact information
8
Label the container first, very clearly with:

• Name of clinic/hospital
• Name of patient and clinic/hospital number
• Indicate whether the specimen is pre-treatment,
follow-up or end of treatment specimen
• Clear instructions regarding what investigations are
required
• Date and time of collection of the specimen
 Transport and Storage
• TRANSPORT
• Transport to laboratory as soon as possible
• Prevent spillage
• If not same day store in fridge

• STORAGE
• Store in fridge/cooler box (do NOT freeze)
• Protect against heat and sunlight
• Place in plastic bag to prevent contamination
Molecular TB Diagnosis
• PCR-based
• Detect presence of MTB complex DNA
• Detect changes (mutations) in the DNA that may be
associated with drug resistance.
• Offering speed of diagnosis
 DR-TB. Frequency of Mutations

100
90 ahpC

80
70
60
inhA
rrs gyrA

50
40
rpoB
pcnA
embB
30 katG

20 rpsL
10
0
INH RMP PZA SM EMB FQ

Courtesy: F. Alcaide
 Isoniazid (INH)
• Rapidly bactericidal: Kills rapidly multiplying TB
• Prodrug: Must be activated by enzyme catalase peroxidase
to be affective against TB
• The enzyme is regulated by the katG gene
• Mutation in the katG gene=high level resistance
• Once activated INH acts on inhA promoter region
• inhA is also a target for Ethionamide
• Mutations in inhA = low level resistance to INH and
resistance to Ethionamide
 Isoniazid: katG and inhA mutations
• katG mutations: High level resistance to INH, therefore
INH cannot be used to treat TB
• inhA mutations: low level resistance to INH and resistance
to Ethionamide, therefore high dose INH (10-15mg/kg)
can be used but Ethionamide cannot be used
• Line probe assay (Genotype MTBDRplus) can be used
to differentiate between the two INH resistance conferring
mutations
 Rifampicin
• Inhibits DNA-dependent RNA polymerase.
• Sterilizing: Prevention of relapses
• Resistance: mutations in a defined rpoB region for
the RNA polymerase. Responsible for > 95% of
resistance
• Both LPA and Xpert can detect these mutations
Pyrazinamide
• Only active at acid pH: active against semi-dormant forms
(slowly multiplying)
• Prodrug: activated by Pyrazinamidase : encoded by pncA
gene
• Resistance: mutations in the pncA gene
• The susceptibility testing method for Pyrazinamide has not
been established
Ethambutol
• Bacteriostatic
• Acts on the MTB cell wall
• Mainly used to protect other first line drugs against
acquired resistance
• Poor reliability of DST
 Xpert MTB/RIF
• The instrument is called GeneXpert, the test is the Xpert
MTB/RIF test
• Automated molecular platform for:
 Diagnosis of MTB
 Detection of rifampicin resistance.
 Directly on clinical samples including extrapulmonary
samples

WHO Strong Recommendation: “The new automated DNA test for TB

should be used as the initial diagnostic test in individuals suspected of MDR-
TB or HIV/TB”

Therefore in South Africa Xpert MTB/RIF:

•Replaces Smear Microscopy for the initial diagnosis of TB
•One Xpert test replaces 2 smear microscopy for diagnosis of TB
Current limitations in TB diagnosis
• The burden of TB bacilli increases with time
• Infection progresses from latency through sub-clinical, then
disease
2-24 hours, 1-2 visits
XPERT
MTB/Rif*
50-150 CFU/ml

Xpert

Graphic courtesy of FIND

 GeneXpert Technology

GX1 – GX2 – GX4 – GX16 GeneXpert Infinity 80

Xpert® MTB/RIF Assay

4 8 16 64 320 throughput/8 hr day

Slide courtesy of Prof Wendy Stevens and graphics taken from www.cepheid.com
Xpert: Initial diagnosis of TB in SA
Advantages
• Automated
• Closed system: low
contamination risk
• Rapid (2 hours)
• Cartridge based
• More sensitive than smear
(>70% of smear-TB)
• Specific for MTB complex
• Easy interpretation
• Result
• Positive / Negative TB
• Resistance Yes / No to
Rifampicin
Disadvantages
• Not for follow up (detects
both live and dead bacilli)
• Sensitivity in smear
negative decreased(not
100%)
• Does not detect INH
resistance
• Detect only known
mutations
• May detect false Rif
resistance
• Expensive
 TB SUSPECTS
TB and DR-TB contacts, non-contact symptomatic individuals, re-treatment after relapse, failure and default
Collect one sputum specimen at the health facility under supervision

GXP positive GXP positive GXP positive GXP negative GXP unsuccessful
Rifampicin sensitive Rifampicin resistant Rifampicin
unsuccessful

Treat as TB Treat as MDR-TB Treat as TB HIV positive HIV negative Collect one sputum
Start on Regimen 1 Refer to MDR-TB specimen for a
Send one specimen for Unit Start on Regimen 1 repeat GXP
microscopy Collect one specimen
for microscopy and
LPA

Collect one specimen for culture and Treat with antibiotics

LPA or culture and DST (for R and H)
Treat with antibiotics and review after 5
days
Do chest x-ray
Follow up with Collect one specimen for
microscopy microscopy, culture and
DST for Rifampicin, Good response Poor response
Isoniazid, fluoroquinolone
and Aminoglycoside Poor response to LPA/ DST results
No further follow Consider other
antibiotics
up diagnosis
Clinically TB Resistant to R
TB on chest x-ray and H/ R only Advise to return
when symptoms Refer for further
recur investigation

Follow up with Treat as TB Treat as MDR-TB

microscopy and culture Start on Regimen 1 Refer to MDR-TB Unit
Review culture results
 Line Probe Assay: Genotype MTBDR plus
• Diagnoses TB and simultaneously detects resistance to RIF
and INH
• Smear positive sputum, culture positive
• Short turnaround time
• Specific for MTB complex
• Includes Probes for:
 rpoB: Rifampicin resistance
 katG: High level INH resistance = INH resistance
 inhA: low level INH resistance = Ethionamide Resistance
but can use high dose INH
 2 Amplification (PCR)
1
DNA Extraction

3
Hybridization (Detection) 4
Result interpretation

27
 LPA Limitations
• Detects only known mutations
• Detects up to 85% of INH mutations (depending on
geographical location)
• Smear positive clinical samples or culture positive MTB
• Level of skill and infrastructure requirements
• Mixed infections may be difficult to interpret
• Contamination risk
Line Probe Assay: Genotype MTBDRs l
version 2

• Same principle as MTBDR plus

• Detection of MTB and resistance to
 Fluoroquinolones: gyrA and gyrB mutations
 Second line injectables: rrs and eis mutations
• Not all resistant mechanism detected
• Validation completed in SA
 Genotype® MTBDRsl Version 2

• The presence of mutations in these regions does not

necessarily imply resistance to all the drugs within a
particular class.
• Specific mutations within these regions may be associated
with different levels of resistance (i.e. different minimum
inhibitory concentrations) to each drug within these
classes, the extent of cross resistance is not completely
understood
 WHO’s policy recommendations
• For patients with confirmed Rifampicin-resistant TB or MDR-
TB, SL-LPA may be used as the initial test, instead of
phenotypic culture-based DST, to detect resistance to
Fluoroquinolones and the second-line injectable drugs.
WHO’s policy recommendations

• These recommendations apply to the use of SL-LPA for

testing sputum specimens (direct testing) and cultured
isolates of M. tuberculosis complex (indirect testing) from
both pulmonary and extrapulmonary sites.

• Direct testing on sputum specimens allows for the earlier

initiation of appropriate treatment
WHO’s policy recommendations

• Recommendations apply to the direct testing of sputum

specimens from Rifampicin-resistant TB or MDR-TB,
irrespective of the smear status

• NOTE : indeterminate rate is higher when testing smear-

negative sputum specimens compared with smear-positive
sputum specimens
 WHO’s policy recommendations

• Do not eliminate the need for conventional phenotypic DST -

necessary to confirm resistance to other drugs and to
monitor the emergence of additional drug resistance

• Phenotypic DST can still be used in the evaluation of

patients with a negative SL-LPA result.
 WHO’s policy recommendations

• Resistance conferring mutations detected by SL-LPA are

highly correlated with phenotypic resistance to ofloxacin and
levofloxacin.

• The correlation of these mutations with phenotypic

resistance to Moxifloxacin is unclear and the inclusion of
Moxifloxacin in a MDR-TB regimen is best guided by
phenotypic DST results.
 WHO’s policy recommendations

• 2012, WHO published advice about how the shorter

standardised MDR-TB regimen - may be introduced by
national programmes.

• The availability of reliable and rapid tests for

susceptibility/resistance to these two groups of second-line
drugs would be a valuable tool to decide which patients
would be eligible for shorter MDR-TB regimens
 Proposed HDOH / NHLS Algorithm

• The National Department of Health (NDOH) and the NHLS

Expert committee have agreed on the reflex testing algorithm
to standardize tuberculosis resistance testing across
laboratories within NHLS and improve overall service
efficiency (quality and turn-around times).
MTB Culture
• Detects growth of MTB, requires
viable organisms

• Current gold standard

• Highly sensitive

• Species identification

• Distinguishes between live and dead

organisms

• Well established method for direct

susceptibility testing for first and
second line drugs.
Viability Change of M. tuberculosis in
Sputums Stored at Different Temperatures
Courtesy: SJ Kim

No. of sputa n=41

tested n=41
n=36
90 90
81

Kim SJ, et al, 1986

Paramasivan CN, et al, 1983
 MGIT Culture
Positive

ZN staining ?cording

ZN pos with cording ZN positive no cording

LPA with Rif & INH

MPT65 Ag

Mtb + Rif/INH MPT pos MPT Neg

Mtb + Rif & INH resist or
Susc Inconclusive

Send out
1% proportion DST as NTM
(First & Second
Send out & Treat line
Phenotypic susceptibility testing SL drugs
• KZN Reference Tb Laboratory – perform phenotypic
susceptibility testing using the well established Agar
proportion method.

• Performed on 7H10 solid agar.

• Drugs Tested : Moxifloxacin , Ofloxacin, Kanamycin and

Capreomycin

• WHO critical concentrations used.

Culture: Limitations
• Time to reportable result 2-6weeks
• Risk of contamination
• Skill and infrastructure
• Laboratory level of biosafety
• Costly

Place for culture in TB diagnosis:

• Diagnosis of paucibacillary TB
• Distinguish live from dead organisms (monitoring of DR TB treatment)
• Confirmation of molecular/genotypic DST
• DST of additional drugs not tested by molecular assays
CASE SCENARIOS
Case 1
Xpert Negative for MTB and
Smear Microscopy shows AFBs

• Possible cause?

• Further management?
Case 2: Discordant INH Results
LPA Sensitive and Phenotypic DST Resistant

• What are the possible causes?

• How would you manage further?

Case 3
• A 25 year old patient with an initial result on Xpert
MTB/RIF showing Rifampicin sensitive TB now
presents with smear positive sputum after two
months of treatment with first line TB treatment.
 What are the possible reasons for this scenario?
 How would you manage this patient further?
Case 4
Xpert Rifampicin resistant TB
LPA Rifampicin resistant TB
Phenotypic DST Rifampicin sensitive TB

• Possible causes?
• Further Management?
Acknowledgements
• Professor Koleka Mlisana: HOD
Microbiology: UKZN & NHLS
• CAPRISA