Major Mycobacterium tuberculosis lineages associate with patient country of origin

Abstract

Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that "clustered" tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with >or=6 IS6110 copies) or RFLP in combination with spoligotyping (for isolates with <6 IS6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS6110-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.

FIG. 1.

Global distribution of countries and lineages represented by Montreal TB patients. Each color represents a distinct LSP-defined TB lineage. Large circles indicate where ≥10 cases were detected in patients originating from a single country. Small circles indicate <10 cases. Symbols incorporating multiple colors indicate that more than one lineage accounts for ≥25% of all cases originating from that particular country. Only those cases identified as being “unique” through IS6110 RFLP or spoligotype analysis (i.e., isolates not involved in forming a cluster of recent transmission) have been included. (The world outline map used in the preparation of the figure was obtained from WorldAtlas.com and is used with permission.)

FIG. 2.

Major countries and lineages represented among Montreal TB cases. The “top 10” countries of origin with the highest numbers of TB cases are shown and are stratified according to LSP-defined TB lineage. The upper panel consists of only the isolates identified as being IS6110 RFLP or spoligotype “unique.” The lower panel consists of isolates involved in chains of recent transmission (“clustered”). Note that the relative scales of the two panels differ.

FIG. 3.

Lineage-related trends over time for patients from “mixed-lineage” countries. (A) For foreign-born patients with either “early” (≤5 years following arrival in Canada) or “late” (>5 years after arrival in Canada) TB, the sum of the lineage-specific trends for each of the countries listed in panel B is presented. The former are most likely to represent patients infected outside Canada; the latter could also include patients infected within Montreal, where general exposure is most likely to be due to the Euro-American/African lineage. (B) The Euro-American/African lineage data presented in panel A, arranged by country. Only those countries of origin where multiple lineages are present (each of which represents a significant proportion of cases within the countries involved) have been included. For both panels, all TB cases are presented (unique plus clustered).

FIG. 4.

Major M. tuberculosis lineages and primary site of disease among foreign-born TB patients residing in Montreal. The proportions of foreign-born patients infected with each of the major LSP-defined lineages and displaying either pulmonary or extrapulmonary disease are presented. Both unique and clustered TB cases have been included in the analysis.