Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

Paula Fernanda Gonçalves Dos Santos,Maria Lucia Rossetti,Leonardo Souza Esteves,Sarah Sengstake,Elis Regina Dalla Costa,Martha M Oliveira,Indra Bergval,Luciana De Souza Nunes,Miguel Viveiros,Regina Bones Barcellos,Daniela M Ramalho,Afrânio Kritski,Rodrigo Rodenbusch,Richard M Anthony

doi:10.1590/0074-02760160376

Abstract

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed.OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr).METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays.RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)].CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Highlights

MethodsSetting and routine laboratory procedures - In a retrospective study carried out at a reference TB hospital and secondary Health Unit in the Rio de Janeiro city, Brazil, between January 2004 and July 2006, 96 M. tuberculosis isolates were obtained (one sample per patient) on the basis of routine testing
To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed
Kappa value was identical between Genotype®MTBDRplus and multiplex ligaton-dependent probe amplification (MLPA) compared with the standard drug susceptibility testing (DST) and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]

Summary

Methods

Setting and routine laboratory procedures - In a retrospective study carried out at a reference TB hospital and secondary Health Unit in the Rio de Janeiro city, Brazil, between January 2004 and July 2006, 96 M. tuberculosis isolates were obtained (one sample per patient) on the basis of routine testing. The isolates were submitted to DST using the proportion method on Lowenstein-Jensen solid media (Canetti et al 1969), following the Brazilian Ministry of Health recommended breakpoints for M. tuberculosis DST on solid media [isoniazid 0,2 μg/mL; ethambutol (EMB) 2 μg/mL; streptomycin 4 μg/mL; pyrazinamide (PZA) 25 μg/mL] (MS 2010). In summary: seventeen discriminatory markers were selected, and MLPA probes were designed to provide information about drug resistance, principal genotypic group (PGG), and (mycobacterial) species (Table I) (Bergval et al 2008). (i) Drug resistance markers (targeted by probes rpoB522, rpoB-526G, rpoB- 526T, rpoB-531, rpoB-176, inhA15, katG-315, and embB-306). (iv) Discriminatory region (specific to members of the MTBC): 16S rRNA gene. (v) Probe targeting TbD1: a region that is absent in the genome of “modern” M. tuberculosis strains but present in all other members of the MTBC.

Results

Discussion

Conclusion