The following procedures were used to identify patients with positive Mycobacterium tuberculosis cultures and obtain isolates for RFLP analysis. In 1996, a survey of hospitals and private physicians was conducted to ascertain where specimens were being sent for mycobacterial culture. This survey allowed the TB Division to determine which laboratories inside and outside of the state were processing clinical specimens for Massachusetts residents. In addition, a letter was sent to directors of all laboratories in Massachusetts that are licensed under the Clinical Laboratory Improvement Act (CLIA) to perform mycobacteriology services and to other laboratories that were identified through the survey, asking for their cooperation with the TB genotyping network project. Most (71%) hospitals and physicians sent specimens to the Massachusetts State Laboratory Institute (MSLI) for culture identification, susceptibility testing, or both. The TB Division and the Mycobacteriology Laboratory,MSLI, share a joint database where all bacteriology reports, including drug susceptibility information, are automatically linked to suspected and confirmed cases of TB. For M. tuberculosis specimens that were processed elsewhere, the epidemiologists on the TB genotyping network project identified laboratories by attending routine TB case and cohort reviews conducted monthly by the state TB nurses and the Boston Public Health Commission TB Program. Laboratories were then contacted and arrangements were made for shipment of specimens to the MSLI and the Wadsworth Center.

RFLP analysis by using IS6110 is a powerful tool for discerning one strain of M. tuberculosis from another when there are many copies of IS6110. However, for strains of M. tuberculosis with low copy numbers of IS6110, RFLP analysis has less discriminating power, and therefore a secondary typing method is used to help differentiate strains (7,8). For the TB genotyping network project, isolates were considered to be clonally related (i.e., were the same strain of TB) if they had identical IS6110 patterns containing seven or more bands or they had identical IS6110 patterns containing six or fewer bands with identical spoligotyping. A cluster was defined as containing two or more patients with clonally related TB strains.

In 1998, CDC funded the Cluster Investigation Study to evaluate epidemiologic links between clustered cases in a more formal manner. Cluster investigations consisted of standardized medical record reviews wherever a patient was seen for TB, and standardized interviews with the patient (or a proxy) if the patient was unable to participate. All patients were eligible for interview, unless strong epidemiologic links were found between all members of the cluster. In that situation, interviews were considered unnecessary. Written informed consent was obtained from all subjects, and interpreters were used as needed. Information collected through medical record reviews and patient interviews included the estimated period of infectivity, demographics, employment history, and social connections and activities during the 2 years before diagnosis. Each patient in a genetic cluster was examined to determine the following: 1) the period of infectivity (by reviewing date of diagnosis, disease type, smear status, chest radiology results, and date treatment started), 2) name of contacts identified, and 3) how and where the patient spent his or her time during the period of infectivity. If a patient identified another patient in the same cluster, or if patients were found to be in the same place at the same time when one was infectious, the likelihood of transmission was classified as “definite.” Transmission was “possible” if patients were thought to be at the same place, at the same time up to 2 years before diagnosis, or if patients identified the same contact as being the source of TB. A final category, “unlikely,” was designated when no common place or other epidemiologic connection was identified or when patients had arrived so recently in the country that transmission was unlikely to have occurred. Further details about the formal cluster investigation study are provided elsewhere (9). Data were analyzed by using Epi Info version 6.03 (10). The study was reviewed and approved by the, Human Research Review Committee, Massachusetts Department of Public Health.

From July 1, 1996, to December 31, 2000, a total of 1,281 cases were reported and verified as TB by the TB Division, of which 1,032 (81%) were confirmed with positive culture for M. tuberculosis. Genotype results were obtained for 984 (95%) of the culture confirmed cases. For the remaining 48 cases, genotype results were not obtained for a variety of reasons, including inability to obtain M. tuberculosis isolates from private laboratories and too little growth on culture. Of the 984 TB patients for whom DNA fingerprinting results were obtained, epidemiologic analyses were conducted for 983 patients whose country of origin was known. The greatest risk for developing TB in Massachusetts was being born outside the United States.

Six hundred eighty four (70%) of the TB patients were foreign-born (from 78 different countries). Most (295; 43%) foreign-born patients were from Asia, followed by the Caribbean region (118;17%) and Africa (116;17%). Countries with the highest number of cases included: Vietnam: 87 cases (13%); Haiti, 83 (12%); China, 59 (9%), India, 54 (8%); Cambodia, 31 (5%), and the Dominican Republic, 30 (4%). Analyses of intervals between arrival into the United States and diagnosis of TB indicated that 176 (26%) patients were diagnosed with TB within 1 year of arrival and 353 (52%) were diagnosed with TB within 5 years of arrival (Table 1).

Foreign-born patients were likely to be younger than U.S.-born TB patients (Table 2). Three hundred twenty-seven (48%) of the foreign-born patients were ages 25–44, as compared to 75 (25%) of U.S.-born patients; 103 (15%) of foreign-born patients were >65 years, as compared with 108 (36%) of U.S.-born patients. Foreign-born patients were also more likely to have extrapulmonary disease: 232 (34%) of foreign-born patients had extrapulmonary TB compared with 61 (20%) of U.S.-born patients. TB patients born in the United States were more likely to have been homeless within the year before diagnosis, and drug use and excessive alcohol use were higher among U.S.-born patients than among foreign-born TB patients. Definition of drug use (injecting drug use and noninjecting drug use), homelessness, and excessive alcohol use are based on CDC criteria as contained in the instruction for the completion of the CDC TB cases reporting forms (11).

Analyses of RFLP distribution indicated that 208 (21%)of 983 isolates contained six or fewer copies of IS6110. Sixty-seven (22%) of the isolates from 299 U.S.-born TB patients contained few copies of IS6110, as did 141 (21%) of the 684 isolates from foreign-born TB patients. However, isolates from foreign-born patients differed substantially by geographic region and country of birth (Table 3).One hundred one (34%) of isolates from Asian patients contained few copies of IS6110 compared with 4% of isolates from persons born in South America. In addition, 42 (48%) of isolates from Vietnam contained few copies of IS6110 compared with 7 (12%) from China.

Of isolates from 983 TB patients, 711 (72%) had DNA fingerprints unique among Massachusetts isolates. The remaining 272 (27.7%) were in 82 genetic clusters. However, 171 (22%) of the 775 isolates containing more than six copies of IS6110 were in genetic clusters as compared to 100 (48%) of the 208 isolates containing few copies of IS6110. Of the 208 isolates, 158 (76%) clustered by IS6110 alone; 100 (48%) of the isolates remained clustered even with the addition of spoligotyping data to further differentiate the TB strain. The genetic clusters were relatively small in size; 52 (63%) of 82 clusters contained only 2 people, 25 clusters (30%) contained 3–5 people, and the largest cluster contained 16 people. Among the 299 U.S.-born TB patients, 119 (40%) patients had isolates in genetic clusters; 180 (60%) of those had isolates with a unique fingerprint. These figures compare with 153 (22%) of the 684 foreign-born TB patients who had isolates in genetic clusters and 531 (78%) who had unique fingerprints. U.S.-born TB patients were more likely to cluster than foreign-born TB patients (odds ratio [OR] 2.29, 95% confidence interval [CI] 1.69, 3.12). Foreign-born patients who had lived longer in the United States were more likely to have isolates that clustered than were recent arrivals (chi square for trend 6.31, p<0.05). Overall, 29 (16%) of those diagnosed with TB within 1 year of arrival had isolates that clustered with others as compared to 38 (22 %) among those diagnosed from 1 to 5 years of arrival and 26% among those diagnosed >5 years after arrival (Table 4). Stratified analyses by age group (<25, 25–44, 45–64, >65) indicated that clustering was associated with increased time spent in the United States for all age groups; however, the association was strongest among those 25–44 years of age (p<0.05).

Although the TB genotyping network was started in 1996, cluster investigation did not formally begin until 1998. Therefore, of the 272 patients found in 82 clusters overall, only 161 patients in 52 clusters were investigated for epidemiologic connections as part of the formal Cluster Investigation Study. Information regarding epidemiologic connections was obtained for 152 (94%) of 161 patients. Epidemiologic connections were established for 68 (45%) of the 152 clustered TB cases, but none were found for 84 (55%) of the clustered TB cases. Epidemiologic connections were more likely to be identified for clusters containing only U.S.-born TB patients than clusters containing some or all foreign-born TB patients (62% vs. 42% and 33%, respectively; chi square for trend, p<0.05). In addition, clustered TB patients with many copies of IS6110 were more likely to have epidemiologic connections than clusters with few copies of IS6110 (OR 8.01; 95% CI 3.45,18.93). Of the 90 clustered TB isolates with many copies of IS6110, 57 (63%) had epidemiologic connections identified, compared with the 11 (18%) epidemiologic connections that were identified among the 62 clustered TB case-patients with few copies of IS6110. Among the U.S.-born patients, 26 (79%) of the 33 patients with many copies of IS6110 had definite or possible epidemiologic connections, whereas none of the 9 patients with few copies of IS6110 had connections (Table 5).

Of the 152 clustered TB patients, 42 (28%) were in clusters containing only U.S.-born patients, 67 (44%) were in clusters with mixed U.S.-born and foreign-born patients, and 43 (28%) were in clusters containing only foreign-born patients. Analysis of the 67 TB patients in mixed clusters containing both U.S.-born and foreign-born persons indicate that 38 (57%) of the TB patients were foreign-born, and 29 (43%) were U.S.-born. Epidemiologic connections were established for 28 (42%) of the 67 TB patients in mixed clusters, and the 17 resulting relationships were analyzed to determine the direction of TB transmission between the cluster members. Results indicate that TB was transmitted from foreign-born to U.S.-born persons in 6 (35%) relationships, foreign-born to foreign-born persons in five (29%) relationships, U.S.-born to U.S.-born persons in three (18%) relationships and U.S.-born to foreign-born persons in three (18%) relationships. However, three of the six foreign-born to U.S.-born relationships involved children of foreign-born parents born in the United States. Epidemiologic relationships were established for 26 (62%) of the 42 TB patients in clusters containing only U.S.-born persons, resulting in 20 relationships. Of the 43 TB patients in clusters containing only foreign-born persons, epidemiologic connections were established for 14 patients (33%), resulting in eight relationships. Overall, of the 45 relationships established through the 68 clustered TB patients with epidemiologic connections, possible TB transmission between U.S.-born persons occurred in 23 (51%) relationships, from foreign-born to foreign-born persons in 13 (29%) relationships, from foreign-born to U.S.-born in 6 (13%) relationships and from U.S.-born to foreign-born in 3 (7%) relationships. In addition, of the 38 foreign-born TB patients in mixed U.S.-born and foreign-born clusters, 10 (26%) TB was diagnosed within 1 year of arrival, in 7 (18%), TB was diagnosed from 1–5 years of arrival, and among 21 (55%), TB was diagnosed > 5 years after the person arrived in the United States. However, TB patients in mixed clusters were no more likely than patients in clusters containing only foreign-born persons to be diagnosed with TB within 1 year, from 1–5 years, or >5 years of arrival (chi square for trend 0.038, p=0.85).