In the period January 2003 to June 2007, a total of 2,664 M. tuberculosis strains were isolated from eligible patients, of which 1,795 were successfully typed in RFLP and spoligo typing. Of these, 819 (45.6%) were of the Beijing genotype based on the spoligo patterns; the remaining 976 (54.4%) were of other genotypes. Among the 819 Beijing genotype strains, 41 (5.0%) most likely belonged to Atypical Beijing lineage, as they missed one or more spacers of the characteristic 9 spacer signature.
Of the 819 Beijing strains, 353 (43.1%) were isolated from patients in the Cailay district; 221 strains (27%) isolated from patients in the Caibe district, and the remaining 245 strains (29.9%) from patients in the Chauthanh district. Regarding the gender of patients, 592/819 (72.3%) of the strains were isolated from male patients, the remaining 227 (27.7%) from females.
From these 819 Beijing genotype strains, 100 strains were randomly selected as described above, of which 5% were most likely Atypical Beijing strains because they missed one or two spacers of the characteristic nine spacer panel 35–43.
Among the 100 selected Beijing genotype strains, 38 (38%) were isolated in the Cailay district, 32 (32%) in the Caibe district and 30 (30%) in the Chauthanh district. The gender distribution was 71% males and 29% female patients. Therefore, the distribution of sex, districts, age (data not shown) in the representative Beijing strains was similar to that in the total collection of 819 Beijing strains (P>0.05).
Among the 100 Beijing isolates that were subjected to 24 loci VNTR typing, 95 yielded results suitable for analysis (including 88 that yielded results for all 24 loci and 7 with double alleles or that missed one locus only), the remaining 5 strains were excluded because ambiguous PCR results were obtained or double alleles were observed in at least two loci. Among 3/5 isolates at least two loci could not be amplified and two isolates had double alleles in two or more loci (Figure ).
Of the 24 loci analyzed, VNTR 154, VNTR 2461 and VNTR 3171 had no discrimination power and hence, the HGDI was zero. Loci VNTR 2347, VNTR 580, VNTR1644, VNTR 0577, VNTR 2531, VNTR 2401 and VNTR 802 had a HGDI of less than 0.2. The loci having a HGDI of more than 0.4 were VNTR 424, VNTR 960, VNTR 2996, VNTR 4052, VNTR1955, VNTR 2165 and VNTR 2163b. Locus VNTR 2163b had the highest allelic diversity, with a HGDI of 0.64 (Figure and Table ).
Hunter Gaston discrimination index of 24 VNTR loci among 95 Beijing strains. Y axis: Hunter Gaston discrimination index.
Hunter Gaston discrimination index (HGDI) values obtained for each locus in the present study compared to two other studies
In the 24 loci VNTR typing of the remaining 95 isolates, 14 clusters of isolates were found; one cluster of four isolates, four clusters of three isolates and nine clusters of two strains. Sixty-one isolates revealed a unique VNTR pattern. The HGDI amounted to 0.994. Twelve of the VNTR clusters were subdivided in RFLP typing.
In RFLP typing, 13 clusters of isolates were found, comprising in total of 31 strains. There were ten clusters of two, two clusters of three and one cluster of five isolates. Sixty-four isolates had a unique RFLP pattern (HGDI = 0.994). Nine RFLP clusters were subdivided in VNTR typing.