Mixed tuberculosis infections in rural South Vietnam

Abstract

Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as "mixed infections." These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥ 2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.

Fig 1

Mixed infections detected by IS6110 RFLP and spoligotyping. Spoligotypes and IS6110 RFLP patterns of the M. tuberculosis isolates that were identified as mixed infections on the basis of discordant genotyping results. The dendrogram (shown on the left) shows the similarity of the spoligotype patterns, as determined by using the Dice coefficient and unweighted-pair group method using average linkages for clustering. To the right of the IS6110 RFLP patterns, the interpreted genotype families are indicated, as determined by spoligotyping and IS6110 RFLP typing, respectively.

Fig 2

Correlation between the heterogeneity in VNTR patterns and the detection of mixed infections by combined IS6110 RFLP and spoligotyping among 1,248 M. tuberculosis isolates. The percentage of mixed infections by (IS6110 RFLP/spoligotyping) is indicated for isolates showing VNTR patterns with no double alleles (n = 1,126), a double allele at 1 VNTR locus (n = 62), or double alleles at 2 to 9 different VNTR loci (n = 60).