rapid diagnosis of drug-resistant tuberculosis · 2018-09-04 · rapid diagnosis of drug-resistant...

Rapid diagnosis of drug-resistant tuberculosis

CHIANG Chen-Yuan MD, MPH, DrPhilos

Int J Tuberc Lung Dis 2015;19:1276–1289


Mechanism of resistance – isoniazid (INH)

• INH, a pro-drug that is activated by the catalase-peroxidase enzyme (KatG) encoded by the katG gene to generate highly reactive species, is capable of attacking multiple targets in M. tuberculosis, the primary one being the InhA enzyme (enoyl acyl carrier protein reductase).

• The active species (isonicotinic acyl radical or anion) reacts with nicotinamide adenine dinucleotide (H), forming INH-NAD adduct, which then inhibits InhA, causing inhibition of cell wall mycolic acid synthesis.


Mechanism of resistance – rifampicin (RMP)

• Rifampicin interferes with RNA synthesis by binding to the ß subunit of the RNA polymerase.

• Mutations in a defined region of the 81-bp region of rpoB (codons 507-533) in about 96% of RMP-resistant M. tuberculosis isolates.

– most frequent mutations at positions 531, 526 and 516.

– generally result in cross-resistance to all rifamycins, but some RMP-resistant strains are rifabutin (RBT)-susceptible.

– not all mutations in rpoB are associated with RMP resistance

rpoB Mutations and MICs

for Rifampin and Rifabutin

in M. tuberculosis

Jamieson FB, et al.

J Clin Microbiol 2014

Assay Procedure for the MTB/RIF Test

N Engl J Med 2010;363:1005-15

Assay Procedure for the MTB/RIF Test

• Among culture-positive patients, a single, direct MTB/RIF test identified

– 551 (98.2%) of 561 patients with smear-positive tuberculosis

– 124 (72.5%) of 171 with smear-negative tuberculosis.

• A second MTB/RIF test increased sensitivity by 12.6% points and a third by 5.1% points, to a total of 90.2%.

• As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified

– 97.6% rifampin-resistant bacteria and

– 98.1% rifampin-sensitive bacteria.

N Engl J Med 2010;363:1005-15

Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis

of tuberculosis and multidrug resistance

• One-off MTB/RIF testing detected 933 (90.3%) of 1033 culture-confirmed cases of tuberculosis, compared with 699 (67.1%) of 1041 for microscopy.

• MTB/RIF in smear-negative, culture-positive patients

– sensitivity 76.9% (296 of 385 samples),

– specificity 99.0% (846 of 2876 non-tuberculosis samples).

• MTB/RIF test for rifampicin resistance

– sensitivity 94.4% (236 of 250)

– specificity 98.3% (796 of 810).

Lancet 2011; 377: 1495–1505

Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis

of tuberculosis and multidrug resistance

• Median time to detection of tuberculosis

– MTB/RIF test 0 day (IQR 0–1),

– microscopy 1 day (0–1),

– solid culture 30 days (23–43),

– liquid culture 16 days (13–21).

• Median time to detection of resistance

– line-probe assay 20 days (10–26)

– conventional DST 106 days (30–124).

• Indeterminate rate: MTB/RIF 2.4% vs 4.6% for cultures.

Lancet 2011; 377: 1495–1505

Forest plots of Xpert sensitivity and specificity for detection of rifampicin resistance

10 Steingart KR et al. Cochrane Database of Systematic Reviews 2013, Issue 1. Art. No.: CD009593. DOI:

10.1002/14651858.CD009593.pub2

Rapid Implementation of the Xpert MTB/RIF diagnostic test

WHO March 2011

Xpert MTB/RIF SYSTEM WHO Policy Statement 2011

• The PPV is less than 70% when the prevalence of underlying rifampicin resistance falls below 5%.

• In such circumstances, positive Xpert MTB/RIF results should be confirmed by conventional DST or LPA.

13 Chiang C-Y, et al. Int J Tuberc Lung Dis 2013

GLI 2014 : Interpreting Xpert MTB/RIF results

Adapt according to NTP guidelines in your country

Silent Mutation in rpoB

• substitutions in the RIF resistance-determining region (RRDR) of rpoB that impaired hybridization with the wild-type probe but did not result in an amino acid change were not associated with resistance.

Alonso M, et al. J Clin Microbiol 2011

False-positive rifampicin resistance on Xpert MTB/RIF caused by a silent mutation

Mathys V, et al. Int J Tuberc Lung Dis 16:1255–1257

a silent mutation in codon533 in the rpoB gene

Mathys V, et al. Int J Tuberc Lung Dis 16:1255–1257

Xpert: Silent mutations and mutations associated

with low-level resistance

McAlister A J, et al.

Clin Microbiol 2015;53:1752–1753

Borderline RMP-resistant strains, variation of MIC by

method (averages all labs)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

RES1,

456

Leu

RES2,

436

Pro

RES3,

436

Pro

RES4,

441T

yr

RES5,

441T

yr

RES6,

441T

yr

PR1,

441T

yr

PR2,

458

Pro

PR3,

458

Pro

PR4,

451

Leu

PR5,

451

ser

PR6,

436

Pro

PR7,

440

Ile-4

41Tyr

PR8,

497

phe

PS1,

W

T

PS2,

W

T

PS3,

W

T

SEN1,

WT

SEN2,

WT

Strains, clinical classification and rpoB mutation

Rati

o M

IC t

o c

riti

cal

co

ncen

trati

on

Resistance cut-off at 1.25X MIC LJ 1%6W Agar1% BACTEC MGIT

RES = R-resistant

PR = probably R-resistant

PS = probably R-susceptible

SEN = R-susceptible

Criteria: clinical outcome & rpoB

Slide courtesy: Van Deun A

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates

with Specific rpoB Mutations

• Full agreement between LJ and MGIT-DST was observed for mutations located at codons 513 (Lys or Pro) and 531 (Leu, Trp), which were always resistant by both methods.

• For mutations 511Pro, 516Tyr, 533Pro, 572Phe, and several 526 mutations, LJ and MGIT results were highly discordant, with MGIT-DST failing to give a result or declaring the strains susceptible.

Rigouts L, J Clin Microbiol 2013;51:2641-5

MICs determined on LJ or by DST in the automated Bactec MGIT 960 system, stratified by rpoB mutation type for 129 M. tuberculosis isolates

J Clin Microbiol 2013

Suboptimal specificity of Xpert MTB/RIF among treatment-experienced patients

• Xpert false detection of active TB (“Xpert false-positive”) was defined as Xpert-positivity in absence of any of the three culture modalities being positive.

• The false-positive rate among patients with recurrent TB was 13.3% (95% CI 5.9–24.6%).

• mean cycle threshold independently predicted false detection of active TB: with values over 30 (Xpert quantitation result “very low”) having a likelihood ratio of 5.4 and a specificity of 91% for false-positivity

Metcalfe JZ, et al. Eur Respir J 2015; 45: 1504–1506

Xpert MTB/RIF Results in Patients With Previous Tuberculosis

• false positive : Xpert-positive, culture-negative

– new cases 8% [95% CI, 6%–12%]

– Retreatment cases 14% [95% CI, 10%-18%]

• Factors associated with false positivity: – fewer years since treatment completion (adjusted odds ratio [aOR],

0.85 [95% CI, .73–.99]),

– less mycobacterial DNA (aOR, 1.14 [95% CI, 1.03–1.27] per cycle threshold [CT]), and

– a chest radiograph not suggestive of active tuberculosis (aOR, 0.22 [95% CI,.06–.82])

Theron G,et al. Clin Infect Dis 2016

Discordance: Xpert MTB/RIF MTB detected, culture negative

• The Xpert MTB/RIF result should be used to guide treatment decision pending additional testing.

• Culture negative may be due to : the patient being treated for TB, transport or processing problems that inactivated the tubercle bacilli, cultures lost to contamination, or inadequate testing volume, or the discrepancy may be due to laboratory or clerical error.

• Follow-up actions may include re-evaluate the patient for TB, reassess possibility of prior or current treatment with anti-TB drugs (including fluoroquinolone use), evaluate the possibility of laboratory or clerical error, and repeat culture.

GLI model TB diagnostic algorithms 2017

Discordance: Xpert MTB/RIF MTB not detected, culture positive

• Treatment decision should be based on the culture result

• Using a sputum specimen, Xpert MTB/RIF has a pooled sensitivity of 89% for detecting MTB compared to culture

• False-positive cultures can result from a variety of causes such as cross contamination in the laboratory or from sample labelling problems.

• Follow-up actions may include re-evaluation of the patient for TB and response to anti-TB therapy; conduct additional testing using Xpert MTB/RIF; process and culture additional samples; and evaluate the possibility of laboratory or clerical error.


Discordance: Xpert MTB/RIF MTB detected, rifampicin resistance detected; rifampicin susceptible by phenotypic DST.

• The Xpert MTB/RIF result should be used to guide treatment decisions pending additional testing.

• particularly in the BACTECTM MGITTM system (i.e., a false-susceptible phenotypic result).

• In some low MDR-TB prevalence settings, silent mutations have been observed that generate a false-resistant Xpert MTB/RIF result but these tend to be very rare.

• Follow-up actions may include DNA sequencing, phenotypic DST using solid media, and evaluating the possibility of laboratory or clerical error.


Discordance: Xpert MTB/RIF MTB detected, rifampicin resistance not detected; rifampicin resistant by phenotypic DST.

• Treatment decisions should be based on the phenotypic DST result.

• False rifampicin-susceptible Xpert MTB/RIF results are rare but have been observed in 1–5% of TB cases tested in various epidemiologic settings.

– mutations outside the sampled region of the rpoB gene , which produce an Xpert MTB/RIF result of rifampicin resistance not detected.

• Follow-up actions may include DNA sequencing, repeating the phenotypic DST, and evaluating the possibility of laboratory or clerical error.


An automated, cartridge-based molecular assay for the detection of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid

Xie YL, et al N Engl J Med 2017;377:1043-54.

GenoType® MTBDRplus test procedure

1) DNA

Extraction From NALC/NaOH

Processed sputum

2) Amplification

by PCR

3) Hybridization Reverse hybridization of

amplified nucleic acids

to specific DNA probes

bound on strips

4) Evaluation

Rapid Molecular Screening for MDR-TB in a High-Volume Public Health Laboratory in South Africa

• the Genotype MTBDRplus assay: detects mutations directly from smear positive sputum in

– the rpoB gene (RIF resistance),

– the katG gene (high-level INH resistance),

– the inhA gene (low-level INH resistance).

• Sensitivity, specificity, and positive and negative predictive values

– 98.9, 99.4, 97.9, and 99.7%, for rifampicin resistance;

– 94.2, 99.7, 99.1, and 97.9%, for isoniazid resistance;

– 98.8, 100, 100, and 99.7%,for MDR-TB

Barnard M, et al. Am J Respir Crit Care Med 2008;177:787-92

INH-resistant strains

• 33.8% (37.7% of MDR strains and 10% of INH-mono-resistant strains) had a mutation in the katG

• 67.6% (63.9% of MDR strains and 90% of INH-mono-resistant strains) had a mutation in the inhA.

Buyankhishig B. Int J Mycobacteriol 2012;1:40-4

Evaluation of GenoType MTBDR Line Probe Assay, India

INH resistance and gene mutations :

• katG gene, 72/87 (83%)

• inhA gene, 10/87 (11%)

• combined katG and inhA 5/87 (6%)

Yadav RN, et al. PLoS ONE 8(9): e72036. doi:10.1371/journal.pone.0072036

Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant

tuberculosis

three LPAs: GenoType MTBDRplus, GenoType MTBDRplus V2,

Nipro NTM+MDRTB Detection Kit 2

Nathavitharana RR, et al. Eur Respir J 2017

Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis

Nathavitharana RR, et al. Eur Respir J 2017

Characteristics of Genotype MTBDRsl versions 1.0 and 2.0 as per manufacturer

The use of molecular line probe assays for the detection of resistance to second-line

anti-tuberculosis drugs: Policy Guidance. WHO 2016

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs

• The inclusion of probes for the detection of mutations in the eis promoter region resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively)

• the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively).

J Clin Microbiol 53:2961–2969

Accuracy of MTBDRsl (version 1.0) for fluoroquinolone and second-line injectable drug resistance and XDR-TB, indirect and direct testing (smear-

positive specimens), phenotypic culture based DST reference standard



Accuracy for the detection of fluoroquinolone resistance Direct testing MTBDRsl version 1.0

The indeterminate rates for direct testing for each smear-grade

0%

5%

10%

15%

20%

25%

30%

35%

Negative Scanty 1+ 2+ 3+

Smear

Accuracy for the detection of second-line injectable drug (SLID) resistance

Direct testing MTBDRsl version 1.0

The indeterminate rates for direct testing for each smear-grade

0%

10%

20%

30%

40%

50%

Negative Scanty 1+ 2+ 3+

Smear

WHO’s policy recommendations testing of patients with confirmed rifampicin-resistant TB

or MDRTB using second-line lineprobe assays (SL-LPA)

• 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 (Conditional recommendation; moderate certainty in the evidence for test accuracy for direct testing of sputum specimens; low certainty in the evidence for test accuracy for indirect testing of Mycobacterium tuberculosis cultures).



WHO’s policy recommendations testing of patients with confirmed rifampicin-resistant TB

or MDRTB using second-line lineprobe assays (SL-LPA)

• 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 the second-line injectable drugs (Conditional recommendation; moderate certainty in the evidence for test accuracy for direct testing of sputum specimens; low certainty in the evidence for test accuracy for indirect testing of Mycobacterium tuberculosis cultures).



Testing of patients with confirmed rifampicin-resistant TB or MDRTB using second-line lineprobe assays (SL-LPA)



• 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 extra-pulmonary sites. – SL-LPA are designed to detect TB and resistance to fluroquinolones and second-line

injectable drugs from sputum samples. Other respiratory samples (e.g. bronchoalveolar lavage and gastric aspirates) or extrapulmonary samples (tissue samples, CSF or other body fluids) have not been adequately evaluated

• These recommendations apply to the direct testing of sputum specimens from rifampicin-resistant TB or MDR-TB, irrespective of the smear status, – acknowledging that the indeterminate rate is higher when testing

smear-negative sputum specimens compared with smear-positive sputum specimens




• These recommendations do not eliminate the need for conventional phenotypic DST capacity which will be necessary to confirm resistance to other drugs and to monitor the emergence of additional drug resistance;

• Conventional phenotypic DST can still be used in the evaluation of patients with a negative SL-LPA result, particularly in populations with a high pre-test probability for resistance to fluoroquinolones and/or SLID;




• Resistance conferring mutations detected by SL-LPA are highly correlated with phenotypic resistance to ofloxacin and levofloxacin. However, the correlation of these mutations with phenotypic resistance to moxifloxacin and gatifloxacin is unclear and the inclusion of moxifloxacin or gatifloxacin in a MDR-TB regimen is best guided by phenotypic DST results

• Resistance conferring mutations detected by SL-LPA are highly correlated with phenotypic resistance to SLID and are an indication to use a MDR-TB regimen which is appropriately strengthened

Shortened multidrug-resistant tuberculosis regimens overcome low-level fluoroquinolone resistance

Eur Respir J 2017; 49: 1700223 [https://doi.org/10.1183/

13993003.00223-2017]

Ajileye A, et al. Antimicrob Agents Chemother 61:e02169-16.

https://doi.org/10.1128/AAC.02169-16.

Shortened multidrug-resistant tuberculosis regimens overcome low-level fluoroquinolone resistance

LPA banding pattern - gyrA,

• low- to medium-level resistance : MUT 1 or MUT3A

• high-level alerts

– the absence of WT1 without MUT1 showing,

– MUT2, MUT3B, C or D,

– absence of WT3 without a MUT band.

LPA banding pattern - gyrB

• Both MUT1 and MUT2 bands indicate high resistance, as do multiple mutations at one or both loci.

Eur Respir J 2017; 49: 1700223 [https://doi.org/10.1183/

13993003.00223-2017]

rapid diagnosis of drug-resistant tuberculosis · 2018-09-04 · rapid diagnosis of drug-resistant...

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