The molecular epidemiology of ethambutol-resistant Mycobacterium tuberculosis in Henan Province, China.

The incidence of tuberculosis (TB) in China is high, according to the Nationwide Random Survey for the Epidemiology of Tuberculosis, 1990, conducted by the Beijing Tuberculosis and Thoracic Tumor Research Institute (Tongzhou, Beijing, China). It is important to obtain fundamental data about drugresistant TB in China to enable successful treatment of this disease. In 1994, WHO launched the global drug resistance surveillance (DRS) project. Henan province was chosen as the first site in China for collection of data for the DRS in accordance with WHO/IUATLD guidelines. Thirty counties in Henan province were selected as survey sites. The samples chosen comprised 1,372 cases of TB, including 916 new cases and 456 relapsed cases. The enrollment period of the TB patients was from April 1 to December 31, 1996. Only genotypic detection of ethambutol (EMB) resistance was performed due to tight restrictions on the research budget; there have been few reports on EMB resistance involving large numbers of Mycobacterium tuberculosis isolates (1-4). As the embB operon, a gene cluster of M. tuberculosis, is involved in resistance to EMB (5,6), the study focused on the detection of a point mutation in embB codon 306 by DNA sequencing. The samples of 171 M. tuberculosis isolates were recovered from 30 counties in Henan province. The sex ratio (M:F) of the TB patients was 2.2:1, and the mean age was 43.7 years. The mycobacteria were recovered from diseased patients with a variety of distinct clinical manifestations, including pulmonary and extrapulmonary infections. Seventeen reference strains (15 resistant and 2 sensitive) were provided by the Korean Institute of Tuberculosis, Seoul, Korea. The sample included 133 EMB-resistant and 38 EMB-susceptible isolates. The isolates were initially tested for EMB susceptibility in routine diagnostic laboratories by the proportion method with Middlebrook 7H10 medium. The critical concentration of EMB was 2 μg/ml. Every series of EMB susceptibility tests included the two drug-susceptible reference strains of M. tuberculosis. The results of EMB susceptibility tests on the clinical isolates were cross-checked at the Korean Institute of Tuberculosis, with 98% of the results confirmed (laboratory accuracy: 90.8%). Seven hundred and five of the 1,372 isolates were resistant to one of the anti-TB drugs, isoniazid, rifampicin, EMB and streptomycin, by drug susceptibility testing. Ninety-four of the 916 new cases (10.3%) and 93 of the 456 retreated cases were EMB-resistant (20.4%). The 133 EMB-resistant M. tuberculosis isolates were investigated by EMB susceptibility testing for embB point mutations. Next, DNA from the M. tuberculosis isolates was purified as described previously (7). Primer sets were designed to amplify regions of the embB gene; the amplification size was 260 bp (8). Five microliters (300 ng) of genomic DNA was used as the template for amplification in 100 μl of reaction mixture. Reaction mixtures were subjected to PCR in a thermal cycler (Perkin Elmer, Fremont, Calif., USA) as follows: 93°C for 3 min followed by 35 cycles at 93°C for 1 min, annealing at 65°C for 1 min, and an extension step at 72°C for 2 min. Final extension was done at 72°C for 10 min. Efficient amplification was confirmed by gel electrophoresis on 12% polyacrylamide gels. Care was taken to prevent false results due to amplicon contamination. Direct sequencing of amplified PCR products was performed with an ABI PRISM (Model 377; Perkin Elmer). Because the previous reports suggested that mutations at codon 306 of the embB gene (the gene encoding arabinosyl transferase) were important in EMB resistance, a 260-bp embB region was sequenced in 133 EMBresistant and 38 EMB-susceptible organisms. A major point mutation was detected in codon 306 with five different kinds of base changes. Five distinct missense mutations were identified in codon 306 (ATG, Met): GTG (Val), ATA (Ile), ATT (Ile), CTG (Leu) and ATC (Ile) (Table 1). Three point mutations were detected in codons 319 and 333 (TAT,Tyr): TGT, GAT and, CAT (Cys). When only epidemiologically distinct organisms were considered, 45.2% of EMBresistant mycobacteria had an amino acid change in the region of embB studied, and most replacements (72%) occurred at position 306. We investigated the relationship between multi-