Detection Of EmbB Gene Mutation For Ethambutol-resistance In Mycobaterium Tuberculosis In Sputum Specimens And Appliacation

Background: In recent years, the mycobacterium tuberculosis(MTB) with high drug- resistance has become one of three difficulties of controlling tuberculosis. Ethambutol (EMB) is a first-line drug used for antituberculosis therapy. It is often used in combination with isoniazid, rifampin, pyrazinamide, and streptomycin. The action of EMB in antituberculosis mechanism is the destruction of cell wall of tubercle bacilli via binding the target molecule -- arabinosyl transferases. These enzymes are involved in the polymerization of the cell wall arabinan. Inhibition of arabinan synthesis by EMB results in the accumulation of mycolic acids, leading to cell death.The EMB-resistance of MTB is involved with the mutation of emb operon or the over expression of emb operon. The Mycobacterium tuberculosis emb operon is a gene cluster of three contiguous genes, namely, embC, embA, and embB, which encode mycobacterial arabinosyl transferases among which the length of embB is about 3246bp. The mutation of embB causes the alterations in the structure of arabinosyl transferase which could not be recognized by EMB, producing the EMB-resistance of MTB. Alterations at codon 306 of embB have been identified as being the most common mutation in EMB-resistant M. tuberculosis clinical isolates.The traditional cultivation and drug-susceptibility test of MTB is a time-consuming work ( 6~8weeks). The drug-susceptibility test sometimes could not provide any valuable information for the selection of drug with efficacy of antituberculosis. The recently established methods are to detect MTB with EMB-resistance from clinical isolates. It usually takes 4 to 6 weeks to isolate clinical strains of MTB from the sputum. So far, EMB-resistance has not found in the active tuberculosis with negative result in smear stain and cultivation. It is very important to establish a rapid and efficient test to detect MTB with EMB-resistance with the aim to carry out the chemo-therapy against tuberculosis early.This study explored the nested PCR to amplify the embB gene directly. The amplified PCR products were be sequenced for the analysis of mutation sites in embB gene. The aim is to identify the infection of MTB with EMB-resistance and providing the useful information to clinical doctors for the selecting the efficient drug for the therapy of tuberculosis and active tuberculosis with negative stain and cultivation of sputum.Objective: This study will explore the nested PCR and sequence to test the mutation associated with embB gene in MTB from sputum in order to establish a direct, rapid and accurate methods to detect the infection of MTB with EMB-resistance.Methods: The sputum specimen of 130 cases with active tuberculosis and 20 cases with pulmonary illness but tuberculosis were used for the acid-fast stain, cultivation in Roch medium, EMB susceptibility test, nested PCR and sequence analysis.Results: In the sputum specimen from 130 cases with active TB, the positive rate in acid-fast stain is about 29.2%(38/130) and the positive cultivation in Roch medium was 60.8%(79/130) among which there were 29 isolates with EMB-resistance in drug-susceptibility test. EMB-resistance in 18 isolates was caused by the mutation at coden 306 in embB gene with positive rate of 62.1%. The PCR result was negative in 20 specimen from pulmonary diseases without active TB and 5 negative control strains of no MTB. The data in this study showed high specificity of 100%. Only one specimen in 7 specimen with negative result in acid-fast stain and cultivation was shown alteration in coden 306 in embB gene.Conclusion: The nested PCR combine DNA sequence would become alternative method with rapid, high specificity and accuracy to detect the infection of MTB with EMB-resistance from the sputum specimen.