Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay

doi:10.1016/0962-8479(95)90009-8

Tubercle and Lung Disease

Volume 76, Issue 5, October 1995, Pages 425-430

Tubercle and Lung Di...

https://doi.org/10.1016/0962-8479(95)90009-8 Get rights and content

Abstract

Setting: Multidrug resistant Mycobacterium tuberculosis strains are threatening TB control in the world. Rapid diagnosis of resistance is essential for adequate treatment and optimal control of the disease.

Objective: Evaluation of a new technique (Line Probe Assay, LiPA) for easy and rapid detection of Rifampicin resistance (RMP^R) of M. tuberculosis.

Design: After amplification of the region of the RNA polymerase, involved in RMP^R, the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained the presence or absence of RMP^R M. tuberculosis can be assessed. 67 clinical samples positive in culture for M. tuberculosis were analyzed with LiPA and results were compared with classical susceptibility testing.

Results: In vitro drug sensitivity testing identified 46 rifampicin sensitive and 21 resistant strains. In 65 of the 67 specimens LiPA results matched classical testing. In two RMP^R cases LiPA showed a sensitive pattern.

Conclusion: In contrast to culture and sensitivity testing, where results take on average 6 weeks, LiPA testing is an easy and rapid (< 48 h) method of detecting RMP^R M. tuberculosis in clinical samples. Results correlated in 97% of the samples. In the two RMP^R samples with a sensitive LiPA pattern another mechanism of resistance is suspected.

Résumé

Cadre: Les souches de Mycobacterium tuberculosis multirésistantes aux antibiotiques constituent une menace pour la lutte antituberculeuse dans le monde. Le diagnostic rapide de la résistance est nécessaire pour un traitement adéquat et pour une meilleure maîtrise de la maladie.

Objet: Evaluation d'une nouvelle technique (Line Probe Assay, LiPA) pour la détection facile et rapide de la résistance de M. tuberculosis à la rifampicine (RMP^R).

Schéma: Après amplification de la région del ARN polymerase limpliquée dans la RMP^R, le produit amplifié est hybridé avec un groupe de 10 oligonucléotides fixés sur une bande de membrane. A partir de la réaction obtenue, la présence ou l'absence de RMP^R M. tuberculosis peut être évaluée. 67 échantillons cliniques, positifs en culture pour M. tuberculosis, ont été analysés avec LiPA et les résultats ont été comparés avec ceux d'un test de sensibilité classique.

Résultats: Des tests in vitro de sensibilité aux drogues ont identifié 46 souches sensibles et 21 résistantes à la rifampicine. Pour 65 des 67 échantillons les résultats du LiPA ont été compatibles avec ceux du test classique. Pour deux cas de RMP^R le LiPA a montré une sensibilité.

Conclusion: Par comparaison avec la culture et le test de sensibilité, où il faut en moyenne 6 semaines pour obtenir les résultats, le test LiPA est une méthode facile et rapide (< 48 h) pour la détection de RMP^R M. tuberculosis dans des échantillon cliniques. Les résultats étaient compatibles pour 97% des échantillons. Pour les deux échantillons RMP^R avec une réaction LiPA sensible, un autre mécanisme de résistance est soupçonné.

Resumen

Marco de referencia: Las cepas de Mycobacterium tuberculosis multi-resistentes a los antibióticos constituyen una amenaza para el control de la tuberculosis en el mundo. El diagnóstico rápido de la resistencia es esencial para el tratamiento adecuado y un control óptimo de la enfermedad.

Objetivo: Evaluación de una nueva técnica (Line Probe Assay, LiPA) para la detección fácil y rápida de la resistencia de M. tuberculosis a la rifampicina (RMP^R).

Método: Después de amplificación de la región de ARN polimerase involucrada en la RMP^R, el producto amplificado es hibridado con un grupo de 10 oligonucleótidos fijados sobre una banda de membrana. A partir del perfil obtenido se puede evaluar la presencia o ausencia de M. tuberculosis RMP^R. Se analizaron con LiPA, 67 muestras clínicas positivas para M. tuberculosis al cultivo y los resultados fueron comparados con los de un test clásico de sensibilidad.

Resultados: Con los tests in vitro de sensibilidad a los antibióticos se identificaron 46 cepas sensibles y 21 resistentes a la rifampicina. En 65 de las 67 muestras los resultados del método LiPA fueron compatibles con los del test clásico. En dos casos de RMP^R, LiPA mostró un perfil de sensibilidad.

Conclusión: En comparación con el cultivo y el test de sensibilidad clásico, cuyos resultados requieren un promedio de 6 semanas, el test LiPA es un método fácil y rápido (< 48 horas) para la detección de M. tuberculosis RMP^R en las muestras clínicas. Los resultados fueron compatibles para el 97% de las muestras. Para las dos muestras RMP^R con reacción LiPA sensible, se sospecha otro mecanismo de resistencia.

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Cited by (212)

Systematic evaluation of line probe assays for the diagnosis of tuberculosis and drug-resistant tuberculosis
2022, Clinica Chimica Acta
Line probe assays (LPAs) are PCR-based assays used for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and drug-resistant tuberculosis (DR-TB). But studies on its performance are insufficient. Thus, in this study, we conducted a systematic review and meta-analysis to evaluate the effect of LPAs in the detection of MTB and drug-resistant TB in comparison with the traditional culture and DST methods.
A systemic literature search was conducted on the Web of Science, Embase, PubMed, the Cochrane Library, Scopus, and OVID databases. All the included studies were classified according to different detecting objects. Sensitivity, specificity, Positive Likely Ratio (PLR), Negative Likely Ratio (NLR), Diagnostic Odds Ratio (DOR), corresponding 95% confidence interval, Area Under Curve (AUC), Deeks’ funnel plot, and Bivariate Boxplot was used to do the evaluation.
147 studies included 491 datasets, with 182,448 samples, were incorporated into our analysis. The sensitivity (95% CI), specificity (95% CI), PLR, NLR, DOR and AUC for MTB were 0.89 (0.86 to 0.92), 0.94 (0.90 to 0.97), 15.70, 0.11, 139 and 0.96, respectively; for rifampicin-resistant TB were 0.96 (0.95 to 0.97), 0.99 (0.98 to 0.99), 82.9, 0.04, 1994 and 1.00, respectively; for isoniazid-resistant TB were 0.91 (0.89 to 0.93), 0.99 (0.98 to 0.99), 83.4, 0.09, (0.99 to 1.00), 195.7, 0.07, 2783 and 1.00, respectively; for Multi-drug resistant TB (MDR-TB) were 0.93 (0.90 to 0.95), 1.00 (0.99 to 1.00), 195.7, 0.07, 2783 and 1.00, respectively; for extensively drug-resistant TB (XDR-TB) were 0.60 (0.33 to 0.82), 1.00 (0.95 to 1.00), 291.3, 0.4, 726 and 0.95, respectively; for (second-line drug-resistant TB) SLID-TB were 0.83 (0.78 to 0.87), 0.98 (0.97 to 0.99), 44.6, 0.17, 262 and 0.98, respectively. Sensitivity in pre-extensively drug-resistant TB (Pre-XDR-TB) was 0.67, specificity was 0.91. No publication bias existed according to Deeks’ funnel plot.
High diagnosis performance was confirmed in LPAs for the diagnosis of MTB and drug-resistant TB. LPAs might be a good alternative to culture and DST in detecting MTB, RR-TB, INH-TB, XDR-TB, SLID-TB, and MDR-TB. While more studies were still needed to explore the diagnosis performance of LPAs for Pre-XDR TB.
Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography
2020, Journal of Microbiological Methods
Citation Excerpt :
If the majority of the samples were susceptible bacteria and only small amounts of resistant bacteria were present, there would be a possibility that no resistant bacteria could be detected. Line probe assay is also a useful method to detect RR-TB (De Beenhouwer et al., 1995). As this method positively detects the mutant sequence, it is less affected by the abundance ratio of the resistant bacteria.
Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously.
LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers.
The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.
Advances in the molecular diagnosis of tuberculosis: From probes to genomes
2019, Infection, Genetics and Evolution
Tuberculosis, disease caused by Mycobacterium tuberculosis, is currently the leading cause of death by a single infectious agent worldwide. Early, rapid and accurate identification of M. tuberculosis and the determination of drug susceptibility is essential for the treatment and management of this disease. Tuberculosis diagnosis is mainly based on chest radiography, smear microscopy and bacteriological culture. Smear microscopy has variable sensitivity, mainly in patients co-infected with the human immunodeficiency virus (HIV). Conventional culture for M. tuberculosis isolation, identification and drug susceptibility testing requires several weeks owning to the slow growth of M. tuberculosis. The delay in the time to results drives the prolongation of potentially inappropriate antituberculosis therapy contributing to the emergence of drug resistance, reducing treatment options and increasing treatment duration and associated costs, resulting in increased mortality and morbidity. For these reasons, novel diagnostic methods are need for timely identification of M. tuberculosis and determination of the antibiotic susceptibility profile of the infecting strain. Molecular methods offer enhanced sensitivity and specificity, early detection and the capacity to detect mixed infections. These technologies have improved turnaround time, cost effectiveness and are amenable for point-of-care testing. However, although these methods produce results within hours from sample collection, the phenotypic susceptibility testing is still needed for the determination of drug susceptibility and quantify the susceptibility levels of a given strain towards individual antibiotics. This review presents the history, advances and forthcoming promises in the molecular diagnosis of tuberculosis. An overview on the general principles, diagnostic value and the main advantages and disadvantages of the molecular methods used for the detection and identification of M. tuberculosis and its associated disease, is provided. It will be also discussed how the current phenotypic methods should be used in combination with the genotypic methods for rapid antituberculosis susceptibility testing.
Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives
2017, Clinical Microbiology and Infection
Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.
Bio-microfluidic platform for gold nanoprobe based DNA detection-application to Mycobacterium tuberculosis
2013, Biosensors and Bioelectronics
We have projected and fabricated a microfluidic platform for DNA sensing that makes use of an optical colorimetric detection method based on gold nanoparticles. The platform was fabricated using replica moulding technology in PDMS patterned by high-aspect-ratio SU-8 moulds. Biochips of various geometries were tested and evaluated in order to find out the most efficient architecture, and the rational for design, microfabrication and detection performance is presented. The best biochip configuration has been successfully applied to the DNA detection of Mycobacterium tuberculosis using only 3 µl on DNA solution (i.e. 90 ng of target DNA), therefore a 20-fold reduction of reagents volume is obtained when compared with the actual state of the art.
Detection of isoniazid and rifampin resistance of Mycobacterium tuberculosis by a multiplex allele-specific polymerase chain reaction (PCR) assay
2012, International Journal of Mycobacteriology
Citation Excerpt :
Many conventional approaches allow for detection of these resistance strains, however, they are usually time-consuming. Therefore, tuberculosis (TB) laboratories adapted new methods, including DNA sequencing [18], line probe assay [19], single-strand conformation polymorphism [20,21] allele = specific PCR assay [3,22,23], real time PCR, molecular beacons [24,25], DGGE [26], PCR RFLP [4], single-tube heminested PCR [27], INNO-LiPA [28], and MTBDRplus [29]. The aim of this study was to develop a method for rapid identification of drug resistance and simultaneous detection of INH- and RIF-resistance associated genetic mutation.
Background: The use of molecular techniques is a major improvement for the rapid routine detection and control of multidrug-resistant tuberculosis (MDR-TB).
Materials: In this study, the multiplex allele-specific polymerase chain reaction (MAS-PCR) was developed to simultaneously detect the most frequent mutations associated with isoniazid (INH) and rifampin (RIF) resistance in a single assay.
Results: The assay was tested with 53 clinical isolates. Among them, 27 were MDR strains, 17 were mono-resistant to INH, one was mono-resistant to RIF, and eight were susceptible. The MAS-PCR assay showed a specificity of 100% in detecting drug resistance. An equivalent sensitivity of 92.6% in detecting MDR and RIF-resistance was found. The sensitivity for the detection of INH-resistance was 88.6%.
Conclusions: The MAS-PCR assay was a simple and rapid method for detecting the INH and RIF-resistance in Mycobacterium tuberculosis (MT) clinical strains. It is also easy to perform and to interpret. The assay is inexpensive and a less-demanding technique.

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Tubercle and Lung Disease

Abstract

Résumé

Resumen

References (25)

Multidrug-resistant tuberculosis and its control

Res Microbiol

Drug resistance of M. tuberculosis in Rwanda

Tubercle Lung Dis

Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study

Lancet

Drug-resistant tuberculosis: laboratory issues

Tubercle Lung Dis

Tuberculosis symposium: emerging problems and promise

J Infect Dis

The emergence of drug-resistant tuberculosis in New York City

N Engl J Med

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase β subunit in rifampicin resistant Mycobacterium tuberculosis strains from New York City and Texas

J Clin Microbiol

Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory

J Clin Microbiol

The catalaseperoxidase gene and isoniazid resistance of Mycobacterium tuberculosis

Nature

InhA, a gene encoding a target for Isoniazid and Ethionamide in Mycobacterium tuberculosis

Science

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: Alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Mol Microbiol

Cloning and nucleotide sequence of the Mycobacterium tuberculosis gyrA and gyrB genes, and characterization of quinolone resistance mutations

Antimicrob Agents Chemother

Cited by (212)

Systematic evaluation of line probe assays for the diagnosis of tuberculosis and drug-resistant tuberculosis

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography

Advances in the molecular diagnosis of tuberculosis: From probes to genomes

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives

Bio-microfluidic platform for gold nanoprobe based DNA detection-application to Mycobacterium tuberculosis

Detection of isoniazid and rifampin resistance of Mycobacterium tuberculosis by a multiplex allele-specific polymerase chain reaction (PCR) assay

Original articleRapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay

Abstract

Résumé

Resumen

Res Microbiol

Tubercle Lung Dis

Lancet

Tubercle Lung Dis

Tuberculosis symposium: emerging problems and promise

J Infect Dis

The emergence of drug-resistant tuberculosis in New York City

N Engl J Med

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase β subunit in rifampicin resistant Mycobacterium tuberculosis strains from New York City and Texas

J Clin Microbiol

Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory

J Clin Microbiol

The catalaseperoxidase gene and isoniazid resistance of Mycobacterium tuberculosis

Nature

InhA, a gene encoding a target for Isoniazid and Ethionamide in Mycobacterium tuberculosis

Science

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: Alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Mol Microbiol

Cloning and nucleotide sequence of the Mycobacterium tuberculosis gyrA and gyrB genes, and characterization of quinolone resistance mutations

Antimicrob Agents Chemother

Original article
Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay