Abstract
Conventional and molecular techniques were applied to detect and characterize drug resistance of mycobacteria in the sputum samples of clinically confirmed tuberculosis. The sensitivities of mycobacterium detection by ZN staining, culture, multiplex PCR, and restriction fragment length polymorphism (RFLP) were 27.7%, 19.9%, 92.9%, and 95.7%, respectively, but all were 100% specific. The conventional and multiple-allele-specific PCR (MAS-PCR) methods enabled establishment of the drug resistance in 19.3% and 86.9% cases, respectively. We demonstrated that molecular techniques have potential in the accurate diagnosis of tuberculosis.
TEXT
In addition to Mycobacterium tuberculosis, nontuberculous mycobacteria (NTM) also contribute to life-threatening pulmonary tuberculosis (TB) (6, 22). Because of the differential pathogenic potentials and responses to antitubercular drugs (ATDs), it is necessary to accurately identify mycobacteria at the early stage of disease diagnosis (17, 18).
Clinically important mycobacteria rapidly develop resistance to first-line ATDs (5). Information on the comparative characteristics of these strains, their mutation patterns, and occurrence of resistance phenotypes among patients is currently lacking. Conventional methods like microscopy, biochemical tests, and drug sensitivity on culture medium are labor-intensive and time-consuming for identification of the organisms and detection of their resistance patterns. These tests may also yield ambiguous results (2). Hence, accurate methodology enabling rapid identification up to the subspecies level and assessment of drug susceptibility are necessary.
DNA-based techniques allow specific identification and characterization of drug resistance patterns with speed and high sensitivity (8, 11, 14). It is envisaged that a thorough understanding of the genetic basis of strain differentiation and their drug susceptibility will facilitate better management of the disease. The objectives of the present study were to (i) detect species and (ii) analyze resistance patterns of mycobacteria in sputum samples of clinically suspected first-line drug (rifampin, isoniazid, and ethambutol) failure in TB patients by using the molecular approach.
This study was approved by the institutional ethics committee, and written informed consents were obtained. Morning sputum samples from TB patients (n = 145) were collected on three consecutive days. Clinical diagnosis was supported by persistent symptoms, radiological abnormalities, and Mantoux test.
Digestion and decontamination of sputum samples were carried out using the N-acetyl-l-cysteine (NALC)/NaOH method (10). Briefly, NALC-NaOH solution (2% NaOH, 1.45% Na-citrate, and 0.5% NALC) and sputum samples were mixed at a 1:1 dilution, vortexed, and incubated for 15 min at room temperature. After digestion and decontamination, samples were concentrated by adjusting the volume to 50 ml with phosphate buffer (0.067 M, pH 6.8), followed by centrifugation of the mixture at 3,500 × g for 20 min. The sediments were resuspended in 2 ml of distilled water. Around 500 μl of each resuspended sediment was removed and kept at 2 to 8°C for DNA extraction. The remaining was used for Ziehl-Neelsen (ZN) staining and inoculation on Lowenstein-Jensen (LJ) solid and Middlebrook 7H9 liquid medium (10). For each sample, inoculations on both types of media were done in triplicate and incubated at 25°C, 37°C, and 42°C for 6 weeks (15). LJ slopes were examined on every alternate day for the first week and twice every week thereafter. Middlebrook 7H9 bottles were read every other day for up to 6 weeks for positive culture. Drug-resistant M. tuberculosis and other slow-growing mycobacteria grew more slowly than drug-susceptible ones, as found by earlier workers (12). In order to not miss these organisms, all culture tubes found negative at 6 weeks were further incubated up to 12 weeks for final identification. Antimicrobial drug susceptibility testing was performed by the resistance ratio method according to the manufacturer's instructions (Tulip Diagnostics Pvt. Ltd., Goa, India). Culture-positive samples were identified to the species level by biochemical tests, namely niacin, catalase, nitrate reduction, heat stable catalase, pigment production, and aryl sulfatase tests.
Mycobacterial DNA was extracted directly from the concentrated sputum samples followed by identification and differentiation of species using multiplex PCR and restriction fragment length polymorphism (RFLP) analysis (1, 3, 19). A single-step multiple-allele-specific PCR (MAS-PCR) assay was also performed for analysis of the most frequently occurring gene mutations of katG, rpoB, embB, and the promoter site of the mab-inhA gene (23).
One positive control (DNA of M. tuberculosis H37Rv) and one negative control (no-template DNA) were included in every set of PCR. Stringency and reproducibility of PCR were established using reference strains obtained from National Jalma Institute of Leprosy and Other Mycobacterial Diseases, Agra, and Tuberculosis Research Centre, Chennai, India. Amplicons were analyzed on an agarose gel using UVP Doc. It LS image analysis software, version 6.3.3.
We evaluated the effectiveness of multiplex PCR and RFLP in comparison to conventional diagnostic tests. Of the total 145 sputum samples from clinically suspected first-line-drug-failure TB patients, four samples were later found to be due to pneumonia (2 cases) and histoplasmosis (2 cases) by clinicians. ZN staining for acid-fast bacilli (AFB) was positive in 39 samples (26.9%), and culture was positive in 28 samples (19.3%). In contrast, multiplex PCR and RFLP confirmed a total of 131 (90.3%) and 135 (93.1%) positive samples, respectively. ZN staining, culture, multiplex PCR, and RFLP with respect to clinical assessment were 100% specific, but sensitivities were 27.7%, 19.9%, 92.9%, and 95.7%, respectively. All four diagnostic methods had a 100% positive predictive value. Negative predictive values with ZN staining and culture methods were 3.8% and 3.4%, while multiplex PCR and RFLP methods had values of 28.6% and 40%, respectively. In our case, multiplex PCR amplified three DNA targets: heat shock protein (encoded by hsp65), cold shock gene (dnaJ), and insertion sequence (IS6110) (1). However, sequences homologous to IS6110 have now also been found in mycobacteria that is not part of the M. tuberculosis complex (9, 13). Several M. tuberculosis strains that lack this insertion sequence have been isolated, and this may account for the false identification (16, 21, 24). The RFLP method involving hsp65 offers the possibility of exploring the diversity within and between members of the Mycobacterium genus. This method distinguishes individual subspecies of the M. fortuitum complex. It also separates M. avium complex (MAC) into subspecies avium, paratuberculosis, and intracellulare. Furthermore, the hsp65-RFLP also distinguishes M. kansasii and M. gastri. We applied RFLP to achieve more precise identification of NTM up to the subspecies level, which is not possible by multiplex PCR.
Resistance to first-line drugs has been linked to mutations in at least 10 genes (7). Rapid turnaround time is highly desirable in assessing mycobacterial drug sensitivity (4, 20). In this study, 28 (19.3%) culture-positive samples were tested for susceptibility to the first-line ATDs through conventional sensitivity testing. In contrast, drug resistance pattern was established in 126 cases (86.9%) using MAS-PCR (Table 1). The main advantage of MAS-PCR is simultaneous detection of resistance to three first-line drugs, which is otherwise difficult to diagnose by other molecular methods (23). It offers additional advantages of simplicity, speed, and economy.
Table 1.
Comparison of molecular techniques with conventional methods for diagnosis of pulmonary tuberculosisa
| Mycobacterium species | Conventional method |
Molecular technique |
MAS-PCR analysis |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Biochemical test |
Genotype |
Multiplex PCR |
PCR-RFLP analysis |
Organism | No.b | Resistance pattern | Mutation site | |||||
| Organism | No.b | Resistance pattern | No.b | Organism | No.b | Organism | No.b | |||||
| M. tuberculosis | M. tuberculosis | 25 | IR + RR + ER | 1 | M. tuberculosis | 99 | M. tuberculosis | 102 | M. tuberculosis | 1 | IR + RR + ER | 315, −15, 306, 516 |
| M IR | 2 | M. tuberculosis | 4 | M IR | 315 | |||||||
| ER +RR | 1 | M. tuberculosis | 4 | M IR | −15 | |||||||
| M. tuberculosis | 2 | M RR | 526 | |||||||||
| M. tuberculosis | 1 | M RR | 531 | |||||||||
| M. tuberculosis | 1 | M RR | 516, 531 | |||||||||
| M. tuberculosis | 2 | M RR | 516 | |||||||||
| M. tuberculosis | 2 | M ER | 306 | |||||||||
| M. tuberculosis | 1 | IR + RR | 315, −15, 531 | |||||||||
| M. tuberculosis | 1 | IR + RR | −15, 516 | |||||||||
| M. tuberculosis | 1 | IR + RR | −15, 526 | |||||||||
| M. tuberculosis | 1 | IR + RR | 315, 516, 526 | |||||||||
| M. tuberculosis | 1 | IR + RR | 315, 531 | |||||||||
| M. tuberculosis | 1 | IR + RR | −15, 531 | |||||||||
| M. tuberculosis | 2 | ER + IR | −15, 306 | |||||||||
| M. tuberculosis | 1 | ER +RR | 306, 526 | |||||||||
| M. tuberculosis | 1 | ER +RR | 306, 516, 531 | |||||||||
| M. bovis | M. bovis | 1 | – | – | M. bovis | 7 | M. bovis subsp. bovis | 8 | M. bovis subsp. bovis | 1 | M IR | −15 |
| M. bovis subsp. bovis | 1 | M IR | 315, −15 | |||||||||
| M. bovis subsp. bovis | 1 | RR | 516, 531 | |||||||||
| M. avium | M. avium subsp. avium | 5 | M. avium subsp. avium | 5 | M. avium subsp. avium | 1 | M IR | −15 | ||||
| M. avium subsp. intracellulare | 3 | M. avium subsp. intracellulare | 1 | M IR | −15 | |||||||
| M. kansasi | M. kansasi | 1 | IR + RR | 1 | NTM | 20 | M. kansasii | 9 | M. kansasi | 1 | M IR | −15 |
| M. kansasi | 1 | M IR | 315 | |||||||||
| M. kansasi | 1 | RR | 526 | |||||||||
| M. kansasi | 1 | IR + RR | −15, 516, 531 | |||||||||
| M. fortuitum | M. fortuitum | 1 | – | – | M. fortuitum | 2 | M. fortuitum | 1 | M IR | 315 | ||
| M. fortuitum | 1 | M IR | −15 | |||||||||
| M. chelonae | M. chelonae subsp. abscessus | 3 | M. chelonae subsp. abscessus | 1 | IR +RR +ER | 315, 306, 531 | ||||||
| M. chelonae subsp. abscessus | 1 | IR + RR | 315, −15, 526 | |||||||||
| M. chelonae subsp. abscessus | 1 | ER +IR | 315, −15, 306 | |||||||||
| M. malmoense | M. malmoense | 1 | M. malmoense | 1 | M ER | 306 | ||||||
| M. szulgai | M. szulgai | 1 | M. szulgai | 1 | M ER | 306 | ||||||
| M. terrae | M. terrae | 1 | – | – | – | |||||||
IR + RR + ER, resistance to isoniazid, rifampin, and ethambutol; M IR, monoresistance to isoniazid; IR + RR, resistance to both isoniazid and rifampin; IR + ER, resistance to both isoniazid and ethambutol; M RR, monoresistance to rifampin; RR + ER, resistance to both rifampin and ethambutol; M ER, monoresistance to ethambutol; NTM, nontuberculous mycobacteria.
No., number of isolates showing positive results.
In conclusion, our results suggest that combined application of multiplex PCR, RFLP, and MAS-PCR is a powerful strategy, enabling accurate diagnosis of mycobacterial infection from sputum sample and profiling of susceptibility to first-line drugs. This can greatly facilitate the management of pulmonary and allied mycobacterial infections.
ACKNOWLEDGMENT
This work was carried out through a grant from the Department of Science and Technology (DST) and Indian Council of Medical Research (ICMR), Government of India.
Footnotes
Published ahead of print 29 March 2012
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