We analyzed culture-positive cases of MDR TB reported to the California TB registry from January 1, 2004, through December 31, 2007. California state law (Health and Safety Code Title 17 §2505) requires reporting of all verified cases of TB, submission of all M. tuberculosis isolates to local public health laboratories, and submission of all MDR M. tuberculosis isolates to the California Department of Public Health Microbial Diseases Laboratory. Testing for first- and second-line drug susceptibilities was performed at local laboratories or at the Microbial Diseases Laboratory by using BACTEC 460 (Becton Dickinson Diagnostic Instruments, Sparks, MD, USA), MGIT 960 (Becton Dickinson), or the agar proportion method. Some isolates were forwarded to the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA) for additional second-line drug testing or for confirmation of drug resistance. Patients were included in the study if MDR M. tuberculosis was identified on >1 isolate. Demographic and clinical information for all patients with MDR TB was abstracted from state TB surveillance forms (Report of a Verified Case of Tuberculosis), which have high validity (18). All protocols were approved by the California Committee for the Protection of Human Subjects and University of California, San Francisco, Committee for the Protection of Human Subjects.

During the study period, 11,395 cases of TB were reported in California, of which 9,037 (79%) had positive cultures. Of these, 8,899 (98%) had isoniazid and rifampin susceptibility results available. Of the 141 (2%) incident MDR TB cases, 123 (87%) had isolates available for MIRU, spoligotyping, and IS6110 RFLP analysis. Isolates unavailable for genotyping (n = 18) were more often from Los Angeles County; other demographic and clinical characteristics were similar to those of analyzed cases.

Twenty-five MDR M. tuberculosis isolates were aggregated in 8 clusters (1 cluster of 10 cases, 1 cluster of 3 cases, and 6 clusters of 2 cases) for an overall cluster proportion of 20% (25/123). Excluding the 8 index cases, 14% (17/123) of all cases were considered to have resulted from recent transmission and rapid progression to disease (secondary cases). Within clusters, a median of 3 months elapsed between successive secondary cases (range 0–20 months). Of the 123 total cases, 113 (92%) occurred among foreign-born patients, with more than half (56%) occurring among immigrants from Mexico, Philippines, the People’s Republic of China, or Vietnam (Table 1). Seven of 8 index cases occurred either in Mexican immigrants (3/8) or recent refugees from Thailand, Lao People’s Democratic Republic, or India (4/8). Median time from patient arrival in the United States to TB diagnosis was approximately twice as long for clustered as for nonclustered cases (4.3 years vs. 2.4 years) and 3 times as long for cases in Mexican-born persons as for cases in persons from other countries (7.3 years vs. 2.3 years).

Younger persons were more likely than older persons to harbor strains involved in MDR TB clusters (Table 1). HIV infection was unusual in this patient population; only 3 (4%) of 75 patients with known HIV status were HIV-infected. Twenty-eight percent (35/123) of patients reported a history of active TB; this proportion did not vary between clustered and nonclustered cases (p = 0.75). Eight patients had documented previous treatment in California. Time to culture negativity (2.4 vs. 2.8 months, p = 0.95), treatment failure, or death did not differ between clustered and nonclustered cases (p = 0.57) among 105 (85%) of 123 cases for which data were available.

Seventy-five percent of MDR M. tuberculosis strains harbored the isoniazid resistance–conferring mutation katG S315T, including all (100%) clustered strains (Table 2). When we controlled for strain lineage and refugee status, katG S315T was inversely associated (OR 0.28, 95% CI 0.09–0.89, p = 0.03) with sputum smear positivity (Table 3). Most rifampin resistance–conferring mutations were found between positions 529 and 534 of the rpoB gene, likely indicative of the serine to leucine substitution at position 531 (S531L) of the rpoB gene (27). The association between this mutation and clustering did not reach statistical significance (OR 2.2, 95% CI 0.8–7.4; p = 0.16).

MDR M. tuberculosis isolates were distributed among East-Asian (47%), Euro-American (30%), Indo-Oceanic (20%), and East African–Indian (3%) phylogeographic lineages. Lineage could not be established for 11 cases. On univariate analysis, East-Asian strain lineage was associated with clustering (Table 4), but not with adverse outcome (death or treatment failure). Indo-Oceanic strains produced no secondary cases.

Clustering was independently associated with katG S315T (OR 11.2, 95% CI 2.2-∞; p = 0.004) and refugee status (OR 6.0, 95% CI 1.2–36.2; p = 0.03) in exact multivariate analyses in which strain lineage and sputum smear positivity were controlled for (Table 5). The estimated association between katG S315T and case clustering did not appreciably change in sensitivity analyses that excluded either all recently arrived refugees or the single largest patient cluster.

In this 4-year population-based molecular epidemiologic study, transmission followed by secondary case generation contributed to ≈14%, or 1 of every 7, MDR TB cases in California. Clustered cases occurred more often among younger persons and persons who had emigrated from Mexico and refugee settings in Southeast Asia. In addition, pathogen-specific factors were associated with clustering of MDR TB cases, independent of traditional clinical and demographic risk factors.

The proportion of MDR TB cases attributed to transmission in California was higher than that reported by other investigators in most low incidence settings (28–32). The largest clusters in our study resulted from MDR TB outbreaks in California after resettlement of Hmong refugees in 2005–2006 (33) and resettlement of Tibetan refugees in 2001–2006. The associations between pathogen-specific factors and case clustering could be due to regional differences in strain prevalence and preferential migration of persons with specific strains to California. We attempted to control for these factors by using highly stringent criteria to define clustered cases and by adjusting for refugee status in our multivariate model. However, without detailed contact information and contact tracing, we cannot be certain of the extent to which transmission or progression to active disease occurred within or outside California. Given that most clustered cases occurred among persons residing in the United States for >3 years and that US-born persons were involved in 3 of 8 clusters, at least some proportion of MDR TB transmission is likely to have occurred in California. This observation suggests that although most MDR TB cases in the United States are related to the migration of persons already infected with drug-resistant M. tuberculosis, a small but notable proportion may be due to ongoing transmission.

Heterogeneity in the reproductive success of drug-resistant M. tuberculosis is now well established (34,35). In this study, the katG S315T mutation was the only isoniazid resistance–conferring mutation found among clustered MDR TB cases. The high prevalence of katG S315T among MDR strains (12,14,36–38) and the association of this mutation with increased secondary case generation among isoniazid-monoresistant strains (6,7) have been documented. We report that the katG S315T isoniazid resistance–conferring mutation retains an independent effect on clustering of MDR TB cases despite the presence of mutations that confer resistance to additional drugs. In particular, secondary case generation did not significantly vary according to site of rpoB mutations that confer rifampin resistance, which supports the hypothesis that these are no-cost mutations or that compensatory mutations commonly exist (39).

The katG S315T mutation is thought to preserve fitness through the relative preservation of catalase-peroxidase production (9), although whether this mutation is associated with different clinical phenotypes is unknown. In this study, we noted an inverse association between presence of the katG S315T mutation and sputum smear positivity. In addition, the katG S315T mutation was associated with case clustering, independent of sputum smear status. These findings suggest that the katG S315T mutation may preserve the ability of M. tuberculosis to transmit and cause secondary cases through mechanisms unrelated to conventional indices of disease severity, such as the presence of abundant acid-fast bacilli in sputum.

Our findings are clinically useful for at least 2 reasons. First, MDR TB in California is occurring predominantly among patients who were not born in the United States, with some cases from recent transmission and rapid progression to disease. Our study suggests that in California, younger persons and persons who have emigrated from Mexico and from refugee settings may be at higher risk for transmitting MDR M. tuberculosis. Likewise, our findings reinforce the need for giving priority to screening and prevention activities in immigrant communities and US investment in international TB control (40). Second, if our results are verified in other settings, TB-control programs should consider pathogen-specific factors such as isoniazid resistance–conferring mutations when planning the intensity of contact investigation and secondary case-finding activities.

This study has several limitations. First, our estimates of case clustering are imprecise because of the limited number of MDR TB cases observed during the study period. However, these estimates are the best currently available, given that the data make up the largest population-based TB registry in the United States. Second, although our definition of genotypic clustering was highly rigorous, the lack of detailed epidemiologic information precluded confirmation of transmission within California. Third, because our study did not include pan-susceptible or isoniazid-monoresistant strains, we cannot comment directly on MDR M. tuberculosis pathogenicity relative to these groups. Lastly, our analyses implicitly assume independence of outcome events, and household or community-level factors potentially associated with clustering were not available. Future studies should be designed so that statistical methods can be used that are able to accommodate the possible effects of within-household clustering.

We found a substantial proportion of MDR TB cases and case clustering in California among non–US-born persons, and the katG S315T mutation was independently associated with clustering. Validation of these findings in larger cohorts and in different population settings may have crucial public health consequences.