Assessment of InCheck™ TB platform for detection of drug-resistant TB in high TB incidence area

Vladyslav Nikolayevskyy1, Irina Kontsevaya2, Paolo Miotto3, Daniela Cirillo3, Olga Ignatyeva2, Svetlana Mironova2, Yulia Chinkova2, Alexander Kovalyov2, Yanina Balabanova1, Francis Drobniewski1

1 – Queen Mary and Westfield College, University of London, London, UK 2 – Samara Oblast TB Service, Samara, Russian Federation

3 – Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy

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J ABSTRACT

Background. The emergence of multidrug resistant tuberculosis (MDR-TB) is a major threat to global TB control. Rapid diagnosis of drug resistance is of vital importance in high TB and drug resistant TB incidence settings. Aim. To test and validate a new lab-on-chip-based platform (In-Check™) for rapid identification of TB and resistance to rifampicin (RIF) and isoniazid (INH) on mycobacterial cultures and primary specimens collected in high TB incidence area. Methods. A total of 459 patients with bacteriologically confirmed pulmonary TB have been recruited into the study in Samara, Russian Federation. Baseline epidemiological data was collected using the structured questionnaires. Molecular drug susceptibility tests (DST) for RIF and INH were performed on decontaminated sputum using the Hain GT MTBDRPlus. Phenotypic DST was performed on cultures derived from these specimens using standard methods on solid and liquid media. Crude DNA specimens extracted from sputum and cultures were tested using the In-Check™ TB platform according to manufacturers protocols. Results. A total of 160 and 116 culture DNA extracts were analysed using two versions of chips, TB1.0 and TB4.0, respectively. In addition, 18 and 10 DNA specimens extracted from sputum were analysed using the same versions. Strains tested included in the most common mutations (S531L, H526Y, H526D, D516V in the rpoB gene, S315T in the katG gene, and C15T, A16G in the regulatory region of the inhA gene). Each chip was read at 250, 500, and 1000 ms acquisition times to adjust signal intensity and provide data for setting cut-off values and the development of interpretation rules. Evaluation of the TB1.0 version demonstrated that many probes needed to be re-designed and PCR and hybridization conditions required adjustment to equalize signal intensities. Using the TB4.0 version, the best probes have been identified, including S315Tm2,m4,m5; C15Tm2; inhAw2; D516Vm1,m2,m4; S531Lm2,m3 and all ID probes. Although performance of the katG and rpoB wild type probes was still suboptimal, it was possible to identify sensitive strains with no mutations using negative signal for mutation probes. Conclusion. In-Check™ TB platform has the potential to be used as a new, non-invasive and reliable test for the rapid diagnosis of MDR-TB in high TB incidence settings. Further validation and development is needed to assess the suitability of the platform for routine testing on clinical specimens.

J Patients recruitment and drug susceptibility testing on isolates

Samara, Russian Federation

Patients 459 M.tuberculosis cultures 459

MTB DNA (crude extracts from cultures) 459

MTB DNA (crude extracts from sputum) 459

Proportion of MDR strains in the collection 30.3%

Genotyping results available 459

(24 loci VNTR + spoligotyping)

Phenotypic DST results available 459 Molecular DST results available 459

J - Development and implementation of infrastructural changes in Samara, Russian Federation (middle TB incidence and high TB drug resistance site) - Ethical permissions obtained in London and Samara, study protocol developed and implemented - Personnel recruited and trained in Samara and London - Databases and analysis algorithms developed and validated - Procedures for specimens transportation (Samara-London) established - InCheck instruments (x2) were installed in London - Three staff members from Samara have been trained and performed testing in London on Samara specimens

J RESULTS

Two versions of chips tested in London: TB1.0 (RIF+INH) - initial validation, optimization of protocols and probes selection TB4.0 (RIF+INH+ID) – further validation Four PCR and/or hybridization (developed by the HSR) tested and validated

J MUTATIONS

associated with RIF and INH resistance

35.5%

8.1% 3.2% 4.8%

47.6%

0.8% rpoB

wt D516V H526D H526Y S531L L511P

40.5%

58.2%

1.3%

katG

wt S315T1 S315T2 92.4%

5.1% 2.5%

inhA

wt C15T T8C

J INITIAL selection of probes for detection of mutations

associated with RIF and INH resistance

D516V_w3

D516V_w1b

D516V_w2b

D516V_w3b

D516V_w4b

0.66

0.68

0.7

0.72

0.74

0.76

0.78

0.8

0.82

0.84

250 ms 500 ms 1000 ms

H526_w1

H526_w2

H526_w5

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

250 ms 500 ms 1000 ms

S531L_w1

S531L_w4

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

250 ms 500 ms 1000 ms

D516V_m1

D516V_m2

D516V_m4

D516V_m1b

0

0.2

0.4

0.6

0.8

1

250 ms 500 ms 1000 ms

H526Y_m1

H526D_m1

0.75

0.8

0.85

0.9

0.95

1

250 ms 500 ms 1000 ms S531L_m1

S531L_m2 S531L_m3

S531L_m4 L533P_m1

L533P_m4

0

0.2

0.4

0.6

0.8

1

250 ms 500 ms 1000 ms

J FINAL selection of probes for detection of mutations associated

with RIF and INH resistance and identification

Class Target Wt  or  Mut Good Medium   Badm1,m2,m4,m5 m2,m4,m5 m1

w2,w4 w2,w4

Wid Type w2,w3,w5 w2 w3,w5m1,m2,m4 m1,m2,m4

w1,w2

w1,w2 * Number of exp shoul be

increasedm1 m1

w1,w2,w5 w2,w5 w1m1,m2,m3 m2,m3 m1

w1,w4 w1,w4ropW,ropW2 ropW,ropW2

IDMYC3a,MYC10a;MYC15a,

MYC16aMYC3a,MYC10a;MYC15a,M

YC16a

m2m2,m3,m4 m3,m4

m2,m3 m2 m3

ropB

D516V

H526Y

S531L

KatG S315T1

C15T

T8CInhA