The value of the continuous genotyping of multi-drug resistant tuberculosis over 20 years in Spain

Abstract

Molecular epidemiology of circulating clinical isolates is crucial to improve prevention strategies. The Spanish Working Group on multidrug resistant tuberculosis (MDR-TB) is a network that monitors the MDR-TB isolates in Spain since 1998. The aim of this study was to present the study of the MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) patterns in Spain using the different recommended genotyping methods over time by a national coordinated system. Based on the proposed genotyping methods in the European Union until 2018, the preservation of one method, MIRU-VNTR, applied to selected clustered strains permitted to maintain our study open for 20 years. The distribution of demographic, clinical and epidemiological characteristics of clustered and non-clustered cases of MDR/XDR tuberculosis with proportion differences as assessed by Pearson's chi-squared or Fisher's exact test was compared. The differences in the quantitative variables using the Student's-t test and the Mann-Whitney U test were evaluated. The results obtained showed a total of 48.4% of the cases grouped in 77 clusters. Younger age groups, having a known TB case contact (10.2% vs 4.7%) and XDR-TB (16.5% vs 1.8%) were significantly associated with clustering. The largest cluster corresponded to a Mycobacterium bovis strain mainly spread during the nineties. A total of 68.4% of the clusters detected were distributed among the different Spanish regions and six clusters involving 104 cases were grouped in 17 and 18 years. Comparison of the genotypes obtained with those European genotypes included in The European Surveillance System (TESSy) showed that 87 cases had become part of 20 European clusters. The continuity of MDR strain genotyping in time has offered a widespread picture of the situation that allows better management of this public health problem. It also shows the advantage of maintaining one genotyping method over time, which allowed the comparison between ancient, present and future samples.

Figure 1

Diverse methods used along the study. From 1998 to 2009, IS6110-RFLP was the genotyping applied method. Standard 12-MIRU-VNTR was introduced in 2003, 15-MIRU-VNTR in 2006 and 24-loci MIRU-VNTR typing in 2009. Complementary, one isolate representative of each IS6110-RFLP cluster was genotyped by the 24-MIRU-VNTR. Spoligotyping and mutations in katG gene and promoter region of inhA gene were performed on all isolates of the study. Spoligo-International-Type (SIT) labels were assigned.

Figure 2

Distribution based on the origin of the cases along the 20 years of the study. The red line represents the native-born cases, blue line shows foreign-born cases and the bars represent the total number of cases.

Figure 3

Spoligotyping based dendrogram of the 77 MDR M. tuberculosis cluster patterns from Spain. SIT, Spoligo-International Type. Mycobacterial interspersed repetitive-unit variable-number tandem-repeat (MIRU-VNTR) pattern of each cluster, mutations associated to resistance to isoniazid and rifampicine and the cluster name in TESSy are given.

Figure 4

Distribution of the cases in clusters along the period studied. Red circles represent the native clusters, blue circles show foreign clusters and green circles show mixed clusters. The size of the circles is proportional to the number of the included isolates.

Figure 5

(A) Map of the regions or Autonomous Communities with the percentage of samples studied of each region. (B) Distribution of the clusters by geo-location of the isolates, the 77 clusters were classified in Spanish Regional-Clusters, if all the isolates were detected in one region, or Inter-Regional-Clusters, if involved cases coming from two or more different regions.