Direct and early detection of Mycobacterium tuberculosis complex and rifampicin resistance from sputum smears

INT J TUBERC LUNG DIS 17(2):258–261

R. Bhutia,*† K. Narain,* K. R. Devi,* T. S. K. Singh,† J. Mahanta*

* Regional Medical Research Centre, Indian Council of Medical Research, North Eastern Region, Dibrugarh,

† Department of Microbiology, Sikkim Manipal Institute of Medical Sciences, Sikkim Manipal University, Gangtok, India



SETTING: Four district DOTS centres in Sikkim, India.

OBJECTIVE: Direct and early detection of Mycobacterium tuberculosis complex and rifampicin (RMP) susceptibility using the INNO-LiPA Rif.TB assay.

DESIGN: DNA was extracted from 44 inactivated acid fast bacilli-positive sputum slides using the Gen Elute bacterial genomic DNA kit. Eluted DNA were ethanol precipitated. The LiPA kit detected RMP susceptibility by detection of mutation in the 81 base pair (bp) rpoB gene. Culture of sputum from the same person was used for drug susceptibility testing for RMP using the proportion method.

RESULTS: Amplification of 88.6% (39 slides) was achieved from DNA extracted using kits (66.7% for 1+, 81.8% for 2+ and 100% for 3+ slides). Hybridisation using the LiPA kit was applied to 31 amplified products. There was a concordance of 96.8% (24 RMP-resistant and 6 RMP-susceptible samples) and discordance of 3.2% (one sample resistant using the proportion method and susceptible on LiPA) between LiPA and the proportion method.

CONCLUSION: LiPA proved useful on DNA extracted from smear-positive slides. Appropriate treatment regimen could be decided early. Slide smear preparation could replace sputum transport in cetylpyridinium chloride, reducing biohazard and thereby controlling the transmission of multidrug-resistant tuberculosis in the community.

PMID: 23317964



Detection of rifampicin resistance, surrogate marker for MDR-TB, lead to appropriate early TB patients treatment (1). 96%  rifampicin resistance related mutation occur in hotspot region (codons 509–534 of 81-basepair rifampicin resistance-determining region of rpoB gene) (2). Although culture and susceptibility testing using proportionate method is gold standard, facility is absent in remote peripheral health centres where diagnosis still depends upon sputum smear microscopy alone. Smear positive patients detection is crucial in fighting TB, as they spread TB in community. Resistant cases are often suspected after initial treatment failure, remain infectious for years despite treatment.

To screen rifampicin resistance, INNO-LiPA Rif.TB assay (Innogenetics, Gh ent, Belgium), reverse-based hybridization technique was used. Five wild-type oligonucleotide probes (S1–S5) sequences complementary to wild-type sequence encode amino acid codons in hotspot region. In rifampicin resistance geno-type, hybridisation doesn’t occur between amplified product and corresponding wild-type probe. Frequently occurring mutations (D516V, H526Y, H526D, and S531L) are associated with probes R2, R4a, R4b and R5.

Present study, evaluated feasibility of early detection of M. tuberculosis complex and rifampicin susceptibility directly from 3+, 2+ and 1+ grade positive sputum slides using INNO-LiPA Rif.TB assay.

Sputum samples from DOTS category-I TB patients in Sikkim were collected in sterile screw-capped containers. Muco-purulent sputum was spread onto two new, clean glass slides (area 1×2 cm), air-dried, heat-fixed and Ziehl-Neelsen stained, followed by oil immersion examination. Sputum was graded as per Revised National Tuberculosis Control Programme guideline (RNTCP). For AFB-positive samples (Figure 1), one additional sputum slide was prepared and inactivated using 5% phenol in ethanol for 5 min (3).

 Rinchenla Bhutia-1 Figure 1. show AFB (acid-fast bacilli) positive sputum seen under oil immersion in Ziehl Neelsen stained sputum slide.


Remaining sputum was transported in 1% cetylpyridinium-chloride in 2% sodium chloride for culture on two Lowenstein-Jensen medium slants at 37°C for up to 8 weeks. M. tuberculosis complex was identified based on granular buff-colour colony appearing after 2 weeks incubation, colony ZN staining, nitrate reduction test, 68°C heat-stable catalase and para-nito-benzoic acid test. Rifampicin sensitivity was performed using proportion method, followed in RNTCP. H37RV was rifampicin susceptible control and MDR-TB sample was rifampicin resistant control. Slides for DNA were randomly selected and extracted using Gen Elute Bacterial Genomic DNA kit (Sigma-Aldrich, St-Louis, MO, USA; Catalogue no NA2120). Eluted DNA was concentrated using ethanol precipitation. LiPA assay was done as per manufacturer’s instruction. Amplified 260-bp product was visualized on 2% agarose gel (Figure 2).

Rinchenla Bhutia-2Figure 2. INNO LiPA Rif.TB amplification of 260bp bands of the rpoB gene characteristic for MTBC in 7 isolates.  From Left to right, Lane 1= NC, lane 2= PC, lane 3 to 9 = patients samples. Lane 10=100bp DNA ladder (Takara) (code 341 0A Lot A401-1), NC= negative control for MTBC   (added milli-Q water in place of MTBC   DNA), PC= positive control for MTBC   (H37RV)


To increase amplification efficiency by removing PCR inhibitors, DNA with negative amplification products was re-purified using ethanol precipitation and subjected to amplification. With second-round ethanol purification of  DNA, amplification success rate increased from 79.5% (n = 35) to 88.6% (n = 39; 66.7% for 1+, 81.8% for 2+ and 100% for 3+ slide smears). Rifampicin sensitive control (H37RV strain), rifampicin-resistant control (MDR-TB sample) and negative control (non-amplified product using sterile water instead of target DNA) included in assay gave appropriate result. On hybridization, 31 amplified products were reactive for M. tuberculosis complex. We achieved results, from DNA extraction to hybridization (Figure 3), within 1–2 days.

Rinchenla Bhutia-3Fig 3. LiPA hybridization strip from left to right showing result for samples 1=Positive control (MDR sample), 2=wild type, 3=R5 mutation, 4= R5 mutation, 5= R5 mutation, 6= wild type, 7= R5 mutation, 8= R5 mutation, 9= R5 mutation, 10=Negative control


96.8% concordance (24 RMP-resistant and 6 RMP-susceptible) of LiPA with proportionate test obtained. 3.2% discordance (one sample resistant using proportion method and susceptible using LiPA) might be mutation occurring outside hotspot region (4).

The importance of this study is two-fold: Firstly, this technique would be useful for transporting samples from peripheral health centres, by preparing inactivated sputum slide, without transporting sputum in cetylpyridinium-chloride, thus reducing laboratory infection risks among technicians as well as biohazard risk during samples transport to reference laboratory. Secondly, this would help in molecular confirmation of M. tuberculosis complex and early multidrug-resistant tuberculosis prediction.



  1. Watterson SA, Wilson SM, Yates MD, Drobniewski FA. Comparison of three molecular assays for rapid detection of rifampin resistance in Mycobacterium tuberculosis. Journal of clinical microbiology. 1998;36(7):1969-73.
  2. Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993;341(8846):647-50.
  3. Suresh N, Singh UB, Gupta C, Arora J, Rana T, Samantaray JC. Rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from stained sputum smears using single-tube nested polymerase chain reaction deoxyribonucleic acid sequencing. Diagnostic microbiology and infectious disease. 2007;58(2):217-22.
  4. Rossau R, Traore H, De Beenhouwer H, Mijs W, Jannes G, De Rijk P, et al. Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrobial agents and chemotherapy. 1997;41(10):2093-8.


Acknowledgements: Regional Medical Research Centre (ICMR), Dibrugarh, Assam, India for use of TB laboratory, Sikkim Manipal University and State TB Cell of Sikkim, India.



Dr J.Mahanta, M.D

Director and scientist G

NE Region, Regional Medical Research Centre (ICMR)

Dibrugarh, Assam, India


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