© Borgis - Postępy Nauk Medycznych 4/2015, s. 249-254
*Anna Zabost, Ewa Augustynowicz-Kopeć
Zastosowanie testu GenoType MTBDR plus do wykrywania molekularnych mechanizmów oporności na ryfampicynę i izoniazyd wśród prątków gruźlicy
Use of GenoType MTBDR plus assay for the detection of mycobacteria molecular rifampicin and isoniazid resistance
Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw
Head of Department: prof. Ewa Augustynowicz-Kopeć, MD, PhD
Streszczenie
Wstęp. Jedną z przyczyn występowania gruźlicy lekoopornej jest długotrwała diagnostyka chorych i trudności w akceptacji schematów leczenia przez chorych. Szybka identyfikacja lekoopornych szczepów M. tuberculosis complex pozwala na włączenie odpowiedniego schematu leczenia, co w konsekwencji zapobiega transmisji szczepów. Izoniazyd (INH) i ryfampicyna (RMP) są głównymi lekami przeciwprątkowymi, stosowanymi w schemacie leczenia rekomendowanym przez WHO. Wystąpienie oporności na te dwa podstawowe leki uznaje się za jedną z kilku przyczyn niepowodzenia leczenia chorych na gruźlicę.
Cel pracy. Celem pracy było porównanie wyników testu lekooporności szczepów M. tuberculosis complex na izoniazyd i ryfampicynę uzyskanych metodą fenotypową i molekularną.
Materiał i metody. Analizie poddano 60 szczepów Mycobacterium tuberculosis complex, u których wykonano test lekooporności na RMP i INH metodą molekularną GenoType MTBDR plus oraz metodą fenotypową z zastosowaniem systemu Bactec MGIT 960.
Wyniki. W analizowanej puli 60 szczepów prątków gruźlicy mutacje w genie rpoB warunkujące oporność na RMP wykryto u 40 z 42 szczepów M. tuberculosis fenotypowo opornych na RMP (92,9%). W puli 51 szczepów fenotypowo opornych na INH mutacje w genach katG i inhA warunkujące oporność stwierdzono u 46 szczepów (90,2%). Czułość, specyficzność oraz dodatnia i ujemna wartość predykcyjna testu GenoType MTBDR plus dla szczepów o oporności MDR (MDR-TB) wynosiły odpowiednio: 82,9, 94,7, 97,1 i 72%.
Wnioski. Test GenoType MTBDR plus jest testem molekularnym wykrywającym najczęściej występujące mutacje w genach odpowiedzialnych za oporność na RMP i INH. Ponieważ jednak nie wykrywa on wszystkich mutacji związanych z opornością na oba leki, wyniki badań molekularnych muszą zostać potwierdzone metodą fenotypową.
Summary
Introduction. The main causes of drug-resistant TB is the use of improper treatment regimens, and incorrect diagnosis of patients. The rapid identification of drug resistance strain is an important challenge to ensure a rapid and adequate therapy of tuberculosis and to limit the dissemination of multidrug resistant strains. Isoniazid (INH) and rifampicin (RMP) are the main antituberculosis drugs, used in the treatment regimen recommended by the WHO. The occurrence of resistance to these two basic medicines are considered to be one of several causes of treatment failure in patients with tuberculosis.
Aim. The aim of this study was to compare the results of resistance to isoniazid and rifampicin obtained with the molecular and the phenotypic method.
Material and methods. The MTBDR plus assay was performed on 60 strains Mycobacterium tuberculosis complex, and the results were compared with the results of conventional drug susceptibility testing (Bactec MGIT 960).
Results. In the analyzed group of 60 strains of mycobacterium tuberculosis, mutations in the rpoB gene were detected in 40 of 42 strains of M. tuberculosis phenotypically resistant to RMP (92.9%). In the group of 51 M. tuberculosis strains phenotypically resistant to INH, mutations in the katG gene and inhA gene were found in 46 (90.2%). The sensitivity, specificity and positive and negative predictive values of the MTBDR assay were respectively 82.9, 94.7, 97.1 and 72% for multidrug-resistant TB (MDR-TB).
Conclusions. The GenoType MTBDR plus assay is a molecular test which detecting the most common mutations in strains resistant to RMP and INH. However the test does not detect all mutations associated with resistance to isoniazid and rifampicin, therefore, the results of molecular must be confirmed by phenotype.
Introduction
The most important reasons for the worsening situation of the epidemiology of tuberculosis in the world include: poor disease control programs and their inadequate implementation, ignoring the problem of tuberculosis in developed countries, the lack of resources for treatment of patients in developing countries, the spread of HIV. The phenomenon of drug resistance of mycobacteria has been recognized by WHO experts as one of the major reasons for the severity of tuberculosis in the modern world.
The World Health Organization reported, in 2013, 480 000 new cases of MDR TB (MDR-TB) resistant to at least isoniazid (INH) and rifampicin (RMP). It is estimated that, worldwide, TB patients excreting MDR (multidrug resistant) tubercle bacilli represent 3.5% of the new patients and 20.5% of the previously treated group. The incidence of drug resistance is a primary indicator of the effectiveness of epidemiological surveillance and treatment of tuberculosis in societies and acquired drug resistance shows the correctness of the treatment of various groups of patients. The largest number of cases of MDR-TB is recorded in Eastern Europe and Central Asia. There, patients with MDR-TB represent over 20% of the new patients and more than 50% of the previously treated patients (1). In Poland, according to the Central Register of Tuberculosis, in 2013, 40 patients with MDR tuberculosis were registered, representing 0.9% of all of the registered cases of tuberculosis (2). Tuberculosis resistant to drugs, and particularly MDR-Tb and XDR-Tb, is a serious problem affecting the health of humans and implementation of programs to combat tuberculosis. MDR-TB and XDR-TB are highly fatal diseases, with a mortality rate of about 60%.
Information on resistance patterns of Mycobacterium tuberculosis strains isolated from patients is an important element of supervision of tuberculosis. Research and analysis of the frequency of drug-resistant TB are helpful in detecting and monitoring the spread of MDR and XDR strains and illustrate the effectiveness of surveillance of tuberculosis in the given country. At the moment, in laboratories around the world, a major challenge is rapid identification of drug-resistant strains of Mycobacterium tuberculosis complex. Early detection and diagnosis of patients with this form of tuberculosis enable appropriate treatment regimen which reduces the amount of mycobacteria and patient infectivity and prevents the transmission of drug-resistant strains in the human environment.
The tests used for the determination of drug resistant M. tuberculosis completely differ from tests performed for other bacteria. This is mainly due to the physiological and biochemical differences of mycobacteria. Development of rapid tests for drug resistance on liquid media significantly reduced the time of waiting for the result to a few days, while obtaining results on solid media took about eight weeks. The application of molecular methods for the identification of drug resistance, based on the identification of gene mutations responsible for resistance to a drug, shortened the time to a few hours and, thus, contributed to a more rapid diagnosis of patients excreting drug-resistant strains, which is particularly important in patients with MDR and XDR tuberculosis.
XDR resistance was defined by WHO, in 2006, as MDR with resistance to fluoroquinolones and one of injectable drugs – amikacin and/or capreomycin. In 2013, XDR-TB incidence was reported to the World Health Organization by 100 countries. On average, it is estimated that 9.0% of people with MDR-TB have XDR-TB. Most of the cases were reported from Ukraine (1006), South Africa (612), India (364) and Kazakhstan (305). In the group of 1269 patients registered with XDR-TB in 2011, only 284 (22%) patients completed their treatment successfully, 438 (35%) died and there is no data on the rest.
One of the tests to quickly identify a patient with MDR-TB is a recently developed GenoType MTBDR plus test, detecting mutations associated with resistance to rifampicin and isoniazid among the strains of Mycobacterium tuberculosis complex.
The mechanism of action of rifampicin is inhibition of RNA polymerase at the β’ subunit level encoded by the rpoB gene. RMP resistance is due to point mutations which give rise to altered β’ subunit of RNA polymerase. This results in a lack of opportunity for a medication to bind to the enzyme and, as a consequence, inhibition of the life processes of bacterial cells (3, 4). Among the strains of M. tuberculosis resistant to rifampicin, the most often identified mutations are those in the rpoB Ser531Leu, His526Asp or Tyr, Asp516Val genes (5).
Isoniazid is a prodrug that, upon entering bacillus cel, is converted to an active form (isonicotinic acid) by the catalase-peroxidase enzyme encoded by the katG gene (6). The resulting isonicotinic acid forms, with the NAD+ or NADP+ coenzyme molecules, complexes that act as inhibitors of enzymes involved in the biosynthesis of nucleic acids and mycolic acids setting up the mycobacterial cell wall (6). Resistance to isoniazid is often associated with mutations in the promoter region of the inhA, gene encoding the enoyl-ACP reductase enzyme. This enzyme is essential for the mycobacterial cell to carry out a proper synthesis of mycolic acids and is sensitive to isoniazid. InhA gene mutation causes a change in the reductase structure, by which it ceases to be sensitive to isoniazid and the enzyme does not lose its enzymatic activity. The mycobacterial cells, having a inhA gene mutation synthesis of mycolic acids, occurs in the presence of isoniazid.
It should, however, be noted that commercial molecular tests only take into account those areas of the genes responsible for drug resistance in which mutations occur most frequently. Therefore, it should be borne in mind that the drug resistance associated with mutations present in other places of genes are not identified by these tests, tuberculosis drug resistance may be due to other mechanisms, for example: the efflux pump.
Aim
The aim of this study was to compare the results of drug resistant strains of Mycobacterium tuberculosis complex obtained by phenotypic and molecular INH and RMP methods.
Material and methods
Sixty strains of M. tuberculosis with different drug resistance were studied. In the case of all strains resistance test was performed for rifampicin, isoniazid, streptomycin and ethambutol in the Bactec MGIT 960 system and a molecular analysis of the GenoType MTBDR plus test.
The GenoType MTBDR plus test, using the DNA-STRIP method allows the identification of Mycobacterium tuberculosis complex strains and detection of resistance to rifampicin and isoniazid. Resistance to rifampicin is detected by identifying mutations in the rpoB gene, resistance to isoniazid – mutations in the katG and inhA genes. The procedure for identifying drug-resistant strains consists of three steps: isolation of DNA, amplification using primers labeled with biotin and a reverse hybridization. This hybridization reaction includes consecutive steps: chemical denaturation of the amplification products, hybridization of single-stranded amplicons labeled with biotin on a membrane coated with probes, washing, adding streptavidin/alkaline phosphatase conjugate, staining reaction using alkaline phosphatase. Strips stained on the test are identified in accordance with the identification model (fig. 1).
Fig. 1. Exemplified results of the GenoType MTBDR plus test.
Results
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Piśmiennictwo
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