1 Joint Departments of Epidemiology and Biostatistics and Occupational Health, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
2 Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
3 Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada.
4 Direction de la Santé Publique, Montréal-Centre, Montréal, Québec, Canada.
5 Laboratoire de Santé Publique du Québec, Montréal, Québec, Canada.
Received for publication July 1, 2003; accepted for publication October 3, 2003.
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ABSTRACT |
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disease transmission; DNA fingerprinting; emigration and immigration; tuberculosis; variation (genetics)
Abbreviations: Abbreviations: CI, confidence interval; IS, insertion sequence; RFLP, restriction fragment length polymorphism.
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INTRODUCTION |
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In low-incidence contexts, tuberculosis is consistently concentrated among persons born in countries where the disease remains endemic. In the United States, foreign-born patients most often have unique M. tuberculosis isolates and thus appear to be less likely than US-born residents to be implicated in outbreaks (5, 6). However, researchers in Amsterdam, the Netherlands, documented a high frequency of clustering among tuberculosis patients born in Surinam and the Netherlands Antilles, which they related to ongoing transmission (7). In another study in Norway, identical fingerprints among East African-born residents were, according to clear epidemiologic links, the result of local transmission (8).
The presence of M. tuberculosis isolates with matching DNA fingerprints can only imply a high probability of recent transmission when these isolates are observed against a background of isolates with sufficient genetic diversity. This reduces the likelihood of detecting false clusters, since isolates not truly linked by new transmission are likely to be distinct. For example, investigators in Copenhagen, Denmark, attributed the high frequency of clustering among Somalia-born tuberculosis patients partly to limited mycobacterial diversity within Somalia and time spent in refugee camps prior to departure, rather than exclusively to ongoing transmission after the patients arrival in Denmark. The finding of additional matching isolates in the same subgroup in the Netherlands provided additional support for this hypothesis (9).
In Montreal, Quebec, Canada, the overall incidence of tuberculosis has been stable at approximately 10 per 100,000 population annually (10), and 80 percent of tuberculosis patients are foreign-born. Their most frequent country of birth is Haiti. Haitian-born persons accounted for 18 percent of all tuberculosis cases observed in Montreal during 19961998, though they represent less than 3 percent of the population. The estimated incidence of tuberculosis is 54 per 100,000 population among the 50,800 Montrealers born in Haiti (based on the 1991 census (11)). Moreover, Haitian-born immigrants are more likely than other Montreal immigrants to share isolates by IS6110 typing (12).
The seeming genetic similarity of M. tuberculosis isolates among Haitian-born Montrealers could reflect a lack of diversity among strains endemic to Haiti or, alternatively, a restricted diversity in strains circulating in the communities from which emigrants to Canada originate. With either scenario, a limited diversity of strains would be imported by Haitian-born persons into Canada, potentially resulting in a bacterial founder effect in Montreal. We wished to determine whether frequent clustering among the Haitian-born was due to limited genetic diversity of M. tuberculosis organisms or recent transmission.
We supplemented the categorical approach to classifying DNA fingerprints (identical/near-identical vs. unique) with a quantitative descriptor of genetic similarity between isolates, called the "genetic distance" (13). Using these two techniques, we investigated the genetic diversity of IS6110 isolates in the Montreal Haitian population.
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MATERIALS AND METHODS |
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We obtained clinical and epidemiologic information from a review of public health records, supplemented by the public health reporting database. As part of mandatory case reporting in Canada, country of birth is recorded for virtually all patients; however, no information on ancestral origin is recorded for Canadian-born patients. We selected Haitian-born and other foreign-born tuberculosis patients and compared their demographic, clinical, and fingerprint patterns produced in RFLP analysis.
A unique identifier, the public health reporting number, was used to link individual demographic and clinical data with laboratory results for each patient. All personal identifiers were removed prior to use of reporting data for this study. This study was approved by the research ethics committees of the Montreal General Hospital, the Quebec Public Health Laboratory, and the regional public health board.
DNA fingerprinting
We typed all culture-positive M. tuberculosis isolates using standard insertion sequence (IS) 6110-based RFLP analysis (15). Only isolates with five or more copies of IS6110 bands (termed adequate copy isolates) were included in the analysis. We set the minimum number of copies at five because of the previously reported poor specificity of low-copy-number fingerprints (13).
Matching versus unique isolates
We used a categorical classification scheme to identify matched and unique isolates: Two or more isolates for which gel electrophoresis demonstrated identical numbers of IS6110 bands of matching molecular weights, as well as those for which there was no more than a single band difference (band addition, band deletion, or band shift), were considered matched. Other isolates were classified as unique (unmatched).
Genetic distance
Genetic distance analysis quantifies the relation between any pair of isolates by estimating the putative amount of time (in months) since the two isolates diverged from a hypothetical common ancestor and assigning a numerical value for the distance between each pair (13). The genetic distance measure reflects the assumption that mutations occur independently throughout the M. tuberculosis genome and that the events are exponentially distributed over time. Therefore, the time of the next mutation is assumed to be independent of the times of past mutations. Some number of such changes, beginning from a hypothetical shared ancestor, characterizes the relation between each pair of isolates. Hence, any given isolate is associated with its own distribution of genetic distancesthe set of distances to every other isolate of interest. The lowest of these distances is termed the "nearest genetic distance," and it indicates the degree of homology with the most similar isolate of interest.
Analysis of distinct patterns
To capture the full range of genotypes over the study period and to perform an analysis based on all distinct isolates, we added a single isolate from each matched group to the unique isolatesthe isolate associated with the earliest date of diagnosis. We then calculated the genetic distance from each distinct isolate to every other isolate, among the isolates from Haitian-born patients and those from other foreign-born patients. We restricted the comparison to non-Haitian foreign-born patients because the diversity in RFLP patterns from so many countries of origin should have provided a maximum standard of diversity against which to compare the patterns of the Haitian-born patients. Canadian-born tuberculosis patients were not included because of their distinctive demographic profile: Typically older, they are even more likely to reactivate remotely acquired infection than are the foreign-born. Furthermore, there is emerging evidence of a major genetic family of M. tuberculosis organisms among the Canadian-born in Quebec (16).
We calculated and compared the median nearest genetic distance among all isolates, among isolates from Haitian-born and non-Haitian foreign-born patients, and among the unique isolates from these two subgroups. We compared the nearest genetic distance for distinct isolates from Haitian-born and other foreign-born patients to evaluate the extent of background genetic diversity. A substantially lower nearest genetic distance for the set of distinct isolates from Haitian-born patients would suggest that the infecting organisms were less diverse genetically, and hence more closely related overall.
We performed DNA fingerprint analysis and genetic distance calculations according to the "align and count" method (17) using Molecular Fingerprint Analyzer software (version 2.0; available from the Stanford Center for Tuberculosis Research (http://molepi.stanford.edu)). We used SPSS for Windows (version 11.0.1; SPSS, Inc., Chicago, Illinois) for calculation of summary statistics and comparisons, including means, medians, interquartile ranges, proportions, and odds ratios and 95 percent confidence intervals, as well as for graphic illustration of the distribution of nearest genetic distances. For statistical comparison of median nearest genetic distances between isolates from Haitian-born patients and those from non-Haitian-born patients, we approximated a 95 percent confidence interval for the differences in median values using a straightforward nonparametric bootstrap (18). For high accuracy, we used 100,000 iterations programmed in S-Plus for Windows (version 6.0; Insightful Corporation, Seattle, Washington).
For purposes of visual illustration, we also displayed and printed IS6110 banding patterns using GelCompar software (Applied Maths, Austin, Texas). The GelCompar software generates accompanying dendrograms (tree diagrams). These identify possible "clusters" of matching isolates, based on the unweighted pair group method using arithmetic averages (19, 20). The graphic output from GelCompar includes a Dice coefficient for each adjacent pair of isolates in each tree, which is an index of similarity for pairs of banding patterns ranging from zero to 100 percent (19). Neither the unweighted pair group method using arithmetic averages nor the Dice coefficient has been validated for the quantification of differences between M. tuberculosis RFLP patterns. However, as a further means of comparing pattern diversity among Haitian-born patients with that observed among other foreign-born patients, we also generated a matrix of Dice coefficients to accompany the dendrogram for each group and compared their distributions using summary statistics, as previously described (16).
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RESULTS |
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Of the 361 isolates, 76 (21 percent) were from Haitian-born patients, 216 (60 percent) were from other foreign-born patients (from 70 different countries), 64 (18 percent) were from Canadian-born patients, and five were from persons whose country of birth was unknown. Figure 1 summarizes these data. Clinical and demographic data for these patient groups are presented in table 1.
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Among all 361 isolates in the study, the median nearest genetic distance was 98 months (range, 10244 months). Nearest genetic distances were lower (median, 48 months; range, 15242 months) for the 76 isolates from Haitian-born patients, reflecting the high frequency of DNA matching, while in the non-Haitian foreign-born, the median nearest genetic distance was 110 months (range, 10244 months).
To study whether the 47 distinct genotypes among the Haitian-born reflected subtle variants of common strains or truly different isolates, we repeated the nearest genetic distance analysis with restriction to only one member of each genotype. Here the median nearest genetic distance was 130 months (interquartile range, 98201 months) among the 47 distinct isolates from Haitian-born patients; among the non-Haitian foreign-born, the median nearest genetic distance for the 191 distinct isolates was 128 months (interquartile range, 103170 months). Hence, the difference in median nearest genetic distances was 2 months among Haitians versus non-Haitians (95 percent CI: 23, 13 (by bootstrapping)). Indeed, the distributions of these nearest genetic distances were similar in the two groups, as illustrated in figure 2.
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DISCUSSION |
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We identified 47 distinct DNA fingerprint patterns among isolates from 76 Haitian-born patients who were diagnosed during 19961998. If the infecting organisms were in fact closely related, as a result of a recent common ancestor, these patterns should have been more similar than those obtained for isolates from a diverse group of foreign-born patients. The nearest genetic distance measure allowed us to further characterize the diversity of these "distinct" isolates. Their diversity was similar to that seen among isolates from patients born in 70 other countries.
These results support the interpretation that Haitian-born tuberculosis patients in Montreal whose M. tuberculosis isolates have similar patterns upon RFLP typing are involved in transmission after their arrival in Canada. It is conceivable that some of this transmission occurred in Haiti, but this is not very likely, since this group had not been living in refugee camps before immigration. In addition, half of the Haitian-born patients with matching patterns had resided in Canada for 5 or more years and therefore were not new arrivals.
While human immunodeficiency virus infection is known to promote rapid progression to active tuberculosis following initial infection with M. tuberculosis (22), rapid progression to active disease did not appear to be the reason for the greater amount of clustering among Haitian-born immigrants in Montreal (12). Moreover, the similarity or diversity of the "unclustered" patterns examined in this report is not likely to have been affected by the rate of progression to active disease.
The genetic distance measure, which simultaneously accounts for number and position of bands that form the fingerprint pattern, permits a refined assessment of the genetic relation between isolates on a continuum from identical to very different, through varying degrees of similarity. Different patterns are no longer simply classified together: Genetic distance quantifies those differences, making analysis of "unique" patterns possible.
This study is but one example of a more refined use of molecular typing data, which could be extended to other settings. We identified the number of distinct M. tuberculosis isolates within two clinical subgroups and assessed their genetic similarity. Use of the genetic distance measure allowed us to study the extent of diversity among isolates that did not match according to categorical criteria and then to explore an alternate hypothesis. Analysis based only on the traditional dichotomous approach comparing "matched" and "unmatched" isolates does not permit such characterization.
Furthermore, we were able to compare the heterogeneity of isolates among Haitian-born patients with that of isolates from patients born around the world. Because unmatched isolates from Haitian-born patients are as distinct as those from other patients, a bacterial "founder effect" is unlikely to account for the higher proportion of matching isolates. Recent transmission remains the most credible interpretation.
The results of this study have important implications for public health. The standard approach to contact identification and evaluation has clearly not been as successful in preventing tuberculosis transmission in the Montreal Haitian community as in other groups. There are several potential reasons for this limited success. In populations with a high baseline prevalence of latent tuberculosis infection, tuberculin testing of close contacts cannot reliably distinguish recent infection from remote infection. Distinctive social networks may be associated with undetected transmission in settings other than the household or workplace. Shame or stigma associated with tuberculosis may also cause patients to withhold contact information. For similar reasons, identified contacts with latent tuberculosis infection may be reluctant to accept tuberculin testing and treatment.
The Montreal Public Health Department has already taken steps to better comprehend patterns of tuberculosis in the Haitian community. However, it is not yet clear whether these steps have improved the identification of infected contacts or the delivery of treatment for latent tuberculosis. Preliminary information from more recent years suggests that transmission in Montreals Haitian community continues (Dr. Terry Tannenbaum, Montreal Public Health Department, personal communication, 2003). Therefore, there is a need to further examine current public health approaches. Potential changes might include a broader approach to contact identification, with emphasis on promoting acceptance of and adherence to treatment for latent tuberculosis.
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ACKNOWLEDGMENTS |
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The authors acknowledge the technical assistance of Michael Purdy and Robert Kozak and the administrative assistance of Esther TomKee.
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NOTES |
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REFERENCES |
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