Line probe assay in the rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from clinical specimens.

The sensitivity of a PCR-based line probe assay (Inno-LiPA Rif. TB Assay; Innogenetics NV Zwijndrecht, Belgium) was studied by using nested-PCR technique. A total of 75 specimens, representing various body locations from 70 suspected tuberculosis patients were obtained. LiPA yielded 30 Mycobacterium tuberculosis complex positive results (sensitivity 58.8%, compared with final diagnoses) whereas culture for M. tuberculosis was positive in 18 specimens (sensitivity 35.3%). Genotypic rifampin resistance testing by LiPA showed that 7 specimens contained rpoB mutations associated with RMP resistance, and sequencing data of the rpoB gene and LiPA patterns agreed in 29 of 30 M. tuberculosis positive specimens (96.7%). This indicates reliable performance, which makes the test suitable for the rapid determination of resistance to rifampin directly in clinical samples. However, the best results are obtained if LiPA is combined with conventional staining and culture methods.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from northwestern Russia.

Thirty-six pyrazinamide-resistant and eight pyrazinamide-susceptible Mycobacterium tuberculosis isolates from Russia were analyzed for their pncA mutations. Thirty-one (86.1%) of the resistant isolates had a mutation either in pncA or upstream of the gene. Twenty of the 23 different mutations found in this study had not been described earlier. pncA genotype correlated well with pyrazinamidase activity and BACTEC 460 susceptibility test results.

A Ser315Thr substitution in KatG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St. Petersburg area in Russia.

Parts of katG and rpoB from 27 Russian Mycobacterium tuberculosis isolates were sequenced to detect mutations causing resistance to isoniazid (INH) and rifampin (RMP), respectively. All 24 INH-resistant isolates had a mutated katG, and 22 of them (91.7%) carried a mutation coding for a Ser315Thr shift. An rpoB mutation was noted for each of the 21 RMP-resistant isolates, with Ser531Leu being the most prevalent change encoded. Only two isolates had identical IS6110 fingerprints.

Rapid detection of rifampin-resistant Mycobacterium tuberculosis by sequencing and line probe assay.

Resistance to rifampin (RMP) is associated with mutations in the rpoB gene. Disk elution method, direct DNA sequencing and line probe assay were compared in rapid detection of rpoB mutations from 30 Mycobacterium tuberculosis isolates. Concordance between LiPA and culture was 93.3%, whereas sequencing yielded 100% concordance with culture. These results indicate that line probe assay is nearly as sensitive a method as sequencing in rapid detection of RMP-resistance of M. tuberculosis isolates, and can be applied in laboratories working with standard PCR equipment.

Survey of drug-resistant tuberculosis in northwestern Russia from 1984 through 1994.

The morbidity, mortality, and relapse rates of tuberculosis have increased in the Russian Federation since 1991. Increased drug resistance may be one reason for the weakened efficacy of local tuberculosis treatments. Laboratory data on tuberculosis resistance were collected from a survey area that included two republics and seven other administrative regions (oblasts) with a total population of more than 14 million. Susceptibility data from 1991 through 1994 were available from all nine regions; data on resistance to individual drugs and data from 1984 through 1994 were available from the Leningrad region and the city of St. Petersburg. From 1991 through 1994. the annual notification rate of tuberculosis increased in the survey area by 53.7% (from 25.1 to 38.6 cases per 100000 inhabitants), tuberculosis mortality doubled (from 4.4 to 9.2 deaths per 100000), and primary resistance to at least one drug increased from 17% (95% CI, 14.9-19.9) to 24% (95% CI, 22.2.-25.8). The prevalence of primary resistance to at least isoniazid and rifampin (multidrug resistance) was 5.1% in the Leningrad region in 1992 through 1994. The proportion of pulmonary isolates with secondary multidrug resistance increased from 21.6% (95% CI 7.9-25.3%) in the period 1984-1994 to 33% (95% CI 29.7-36.3%) in 1989-1994. Even if these figures are biased upwards because of selection, it can be concluded that secondary resistance to tuberculosis drugs was already prevalent in northwestern Russia ten years ago. Since then, a distinct shift towards multidrug resistance has occurred. The lower prevalence of primary multidrug resistance raises hopes that the resistance problem can be controlled with properly designed and monitored therapeutic measures.