Laboratory Methods

Cowan and Crawford (15) and Crawford et al. (16) describe the laboratory protocol and design and methods of the genotypying network. The Wadsworth Center performed IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping according to standardized procedures (16–18).

Genotype Cluster Identification

The Centers for Disease Control and Prevention (CDC) developed a standard protocol for cluster investigations. For IS6110 strains with more than six copies in their fingerprint patterns (high-copy strains), a single cluster designation was allocated if two or more patients had isolates with identical RFLP patterns. Strains with six or fewer copies in their patterns (low-copy strains) were assigned a single cluster designation if two or more patients had both identical RFLP and spoligotype patterns. The cluster investigation by the genotyping network took place from January 1, 1998, to December 31, 2000.

Epidemiologic Investigation of Clusters

To identify sources of transmission, we defined an epidemiologic link as two persons who shared space or time. For example, if a cluster included three persons (the source [A] and two subsequent patients [B and C]), the epidemiologic relationships identified for inclusion in the database would be A to B and A to C. However, when a common source was determined from outside the study, each relationship was counted only once without reference to direction (B and C, representing one link with no reference to A). An “expected link” was one found through conventional contact investigation, and an “unexpected link” was identified as a result of an RFLP cluster investigation.

To determine epidemiologic links, we reviewed medical records from health departments, hospitals, and clinics for each genotypically clustered case. Patients or their proxies were interviewed. Collected data included medical information (i.e., tuberculin skin-test results and BCG vaccine status, potential exposures, and previous diagnoses), demographics, and a 2-year social and work history.

CDC collaborated with the genotyping network sentinel sites in developing forms for data collection, patient consent, and interviews. The Massachusetts Department of Public Health institutional review board approved the protocol and forms. The patient consent form was translated into Spanish, Haitian Creole, Portuguese, Khmer, Vietnamese, and Chinese. Interpreters were present during interviews, when needed.

Laboratory Cross-Contamination

For the period 1994–1997, laboratory cross-contamination or other error was suspected if more than one specimen processed at the same time was positive for M. tuberculosis. At the time, we considered that contamination was less likely to occur between specimens processed in different batches on the same day; therefore, this type of contamination was not addressed in the study. Contamination in different batches was, however, addressed in a broader 1998 genotyping network study on laboratory cross-contamination. Data and methods from that study are described elsewhere (19).

Transmission Dynamics and Public Health Implications

From July 1, 1996, to December 31, 2000, a total of 1,281 TB cases were counted in Massachusetts. Of these, 1,043 (81%) were culture confirmed as TB. Positive cultures were obtained from 1,032 patients in Massachusetts, and 95% (984) of the isolates obtained from these patients were genotyped. The remaining isolates were not genotyped for various reasons, including inability to obtain growth when subcultured, contamination with other bacteria or fungi, and inability to acquire isolates from outside laboratories. Of the 984 isolates, 776 (79%) had high-copy strains (seven or more IS6110 copies in their patterns); 712 (72%) had unique DNA fingerprints; 272 (28%) aggregated into 82 clusters. Of 208 cases with low-copy strains, 100 (48%) met the study definition of genotype cluster.

Links Established by Conventional Contact Investigation

Overall, 129 expected epidemiologic relationships in Massachusetts were identified. Of these, 37 relationships were between a person in the study and a person without a genotyped isolate (e.g., children, clinical patients). Of 92 relationships analyzed (88 persons), 67 (72%) demonstrated exact or similar (±1 hybridizing band) RFLP matches. Twenty-five relationships between 38 persons were not supported by genotyping, i.e., the isolates had different genotypes.

The 25 expected relationships not supported by genotyping were as follows: 11 in persons staying at the same homeless shelter at the same time; 5 in household contacts; 2 in nonhousehold family members, 3 in friends and social contacts, and 4 in co-workers. The five relationships involving household contacts were in persons from countries with a high prevalence of TB. These relationships included roommates from Mali, siblings from Kenya and Ethiopia, and parents and adult children from China and India.

Additional Epidemiologic Links in RFLP Cluster Investigation

In addition to the 129 epidemiologic relationships identified before RFLP results, 11 unexpected epidemiologic relationships involving 21 persons were identified. We also identified additional and some previously unrecognized places of transmission (Table). Several of these are believed to have been caused by either casual contact or unsuspected settings. For example, cluster 5 in the table consisted of three patients who ostensibly had nothing in common. However, RFLP cluster investigation established that one patient had been the hairdresser of the second patient, who was a college student. The third patient was a security guard who had worked in a college dormitory frequented by the second case. At the onset of the guard’s illness, he was working at another facility.

Extent of an Outbreak in a Homeless Shelter

Of 18 men with TB in the homeless or associated populations in 2000, isolates from 15 persons had RFLP pattern 437 or 5309. These patterns differ by a one-copy addition. Thirteen of the men were long-term users of one shelter, although eight used the shelter sporadically over many years. Two patients had positive tuberculin skin tests in the past, and two other patients were clinical patients in past years (1997 and 1995). Eleven of the 15 patients were interviewed, and only one interview provided a definite epidemiologic link between two patients. Other interviews could not establish specific dates that the cases had overlapped. We reviewed 199 bed logs from the shelter for October 1999 through June 2000, a time period between documented negative tuberculin skin tests and diagnosis of active disease for three of the patients.

Of 14 homeless men in the cluster, 2 were not on the bed logs for the entire 9-month period. The remaining 12 patients were never all present on the same day. However, 11 of the 12 patients were present on 1 day in November, and 10 of the 12 were present on 6 nights in October and November 1999. Of 15 persons in the cluster (14 guests and 1 employee), we established that 13 were epidemiologically linked. Of the other two, we believe that one man’s diagnosis resulted from a false-positive culture, and the other man arrived at the shelter after June 2000 and was likely exposed later.

Laboratory Cross-Contamination

In April and May 1994, two related instances of cross-contamination in one laboratory were identified 3 weeks apart. Five of ten cultures processed at the same time in the first instance and three of the ten in the second were submitted for RFLP analysis; they matched a strain used as a control in the decontamination and digestion procedure. The TB Division and the laboratory sent all isolates tested 2 weeks before the first instance through 2 weeks after the second instance for RFLP typing. Clinical records were reviewed, and epidemiologic follow-up was completed on all cases. No other specimens were affected.

Limitations of Genotyping Techniques

In the cluster investigation study, 105 (66%) of 159 persons in clusters were interviewed or had epidemiologic links to everyone else in their cluster, so they were not interviewed according to the protocol. Of the remaining 54 (33%), patients refused to be interviewed (10 patients). An additional patients were defined as “administratively closed” at the end of the study. These patients did not refuse to participate in the study but could not be reached after six phone attempts or three visit attempts. Several either agreed to participate or asked to be contacted later but were unreachable. Of 45 clusters investigated as part of the official cluster investigation study with patients eligible for interview, we interviewed all persons in 13 clusters.

The RFLP technique is expensive and technically demanding; it also requires microbiologic expertise and resources to obtain cultures as well as sophisticated software for RFLP pattern comparison. Thus, turnaround time between specimen collection and availability of RFLP result may be lengthy (27). In our study, this turnaround time averaged 7 months, which may have contributed to our difficulties in obtaining patient interviews. However, identifying clusters would be a slow process even with negligible turnaround times, as latent TB infection reactivates slowly. Reopening contact investigations after receipt of RFLP results was impractical in most instances. In addition, interpreting results is more difficult because of the lack of specificity in patterns with fewer than seven IS6110 copies.

A recently reported limitation of RFLP relates to sampling (28). A certain number of cases in every population cannot be assigned RFLP types. Either the specimens do not grow M. tuberculosis, or TB is diagnosed on the basis of clinical information without bacteriological confirmation. During the study period in Massachusetts, RFLP typing provided information on 95% of our culture-confirmed cases, 77 % of our cases overall.

RFLP and any other genotyping tool can be useful for TB control if certain assumptions hold true. M. tuberculosis strains are relatively stable but not immutable; thus, if persons fall into a cluster, they probably have the same TB strain. Clustering implies recent transmission because strains change over time. A few studies (29–32) have analyzed the stability of RFLP patterns; one determined that half of the strains will demonstrate a shift in 3–4 years (29), while another study found no change (32). Neimann et al. saw almost no change in patterns among 75 isolates in chains of transmission, i.e., multiple patients (30). These studies cannot make assumptions about changes of patterns during latency, and rates of change of strains may vary. In addition, we assume that clustering in the TB control program’s population implies recent transmission and not simply endemic strains with a lack of diversity. We assume that sampling is complete and that no difference exists between the patients with or without available isolates for genotyping. For public health programs to benefit fully from a genotyping technique, it must be rapid, inexpensive, and reproducible from laboratory to laboratory. In general, the currently available DNA fingerprinting tools should be used only in conjunction with epidemiology. Universal fingerprinting, in particular, shows its greatest utility when the health department can respond to this new information by allocating additional resources to conduct investigations of unexpected clusters (33). Genotyping can then have as great an impact on public health practice nationwide as it had in Massachusetts.