The key to eliminating TB will be through social mobilization and maintaining the public interest and commitment necessary to provide sufficient resources for the effort
In conjunction with contact tracing, early diagnosis1 and effective treatment of infection and disease are the major strategies for limiting transmission of tuberculosis (TB).2 Beyond the clinician’s primary concern for administering successful therapy to the patient lies a collective responsibility to prevent further infection and transmission of the disease. An important first step toward controlling TB transmission is an organized effort to trace patient contacts accurately and rapidly. TB may involve a long incubation period between infection and disease onset, thus adding to the burdens of epidemiologic investigations aimed at identifying the sources of infection and sites of transmission.
The merger of modern molecular protocols for strain identification at the DNA level3 and conventional epidemiological methodologies has given birth to an enhanced collaborative strategy, known as molecular epidemiology, for studying the dynamics of disease movement. Components of this new strategy, including genetic fingerprinting, have already been used to identify numerous suspected and unsuspected transmission links that would have been overlooked only 10 years ago.
Genetic Markers as Strain Identifiers
Strain-specific differentiation of members of the Mycobacterium tuberculosis complex was difficult in the past. Only unique strain characteristics (biochemical reactions or characteristic resistance patterns) could support epidemiological data in assessing whether an infection was a reactivation or an exogenous reinfection,4 or whether an unexpectedly positive culture result was actually a false positive, perhaps due to cross-contamination in the laboratory.5,6 Strain discrimination based on differential susceptibility to mycobacteriophage invasiveness has proven to be technically challenging and has resulted, at best, in offering a limited number of strain types.
Molecular biological methods and the publishing of the complete DNA sequence of M. tuberculosis,7 in conjunction with an increased understanding of the molecular genetics of mycobacteria, have provided the means necessary to differentiate reliably among strains of M. tuberculosis at the DNA level.
For any DNA-based system to work, strains of M. tuberculosis must exhibit a certain degree of genetic polymorphism. The different rates of change among different fingerprinting markers can be exploited by linking together the results of multiple fingerprinting methods. Another feature of the M. tuberculosis genome is the existence of a large family of repeated DNA sequences. Several types of stable, repetitive DNA sequences are now known to be present on the chromosome in variable copy numbers and at different locations.
The majority of TB fingerprinting studies have focused on the insertion sequence 6110 (IS6110), one of a number of mycobacterial mobile genetic elements. IS6110 is a naturally occurring, transposable genetic element that appears to be detectable only in species belonging to the M. tuberculosis complex. IS6110 is capable of self-replication and transposition to different positions on the M. tuberculosis chromosome in a relatively unbiased fashion. Consequently, the number of copies of IS6110 elements and their chromosomal locations vary to such a degree that this forms the basis of a unique genetic fingerprint. Little is known about the factors affecting the frequency of IS6110 transposition. It appears that this frequency produces considerable background diversity in epidemiologically unlinked strains, yet IS6110 is a stable molecular marker of strain identity over the time period spanned by most epidemiologic investigations.
Technology based on M. tuberculosis restriction fragment length polymorphism (RFLP) has incorporated the properties of IS6110 into an internationally standardized molecular protocol dedicated specifically to the DNA typing of M. tuberculosis complex strains.8 M. tuberculosis strain differentiation by RFLP requires availability of a cell mass large enough to yield sufficient DNA for molecular manipulation, preferably luxuriant growth on a solid culture medium. Chromosomal DNA is isolated from harvested cells and enzymatically digested. A major role in the RFLP protocol is played by a restriction enzyme (PvuII) produced by and refined from Proteus vulgaris and its corresponding restriction site on the DNA molecule. PvuII recognizes a specific six-nucleotide palindromic sequence in the DNA and cleaves it at each occurrence of the sequence. (The enzyme does not cleave the DNA at these sites in the cell producing the enzyme due to specific DNA modifications that inhibit cleavage.) The DNA fragments are then separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed with a portion of IS6110, resulting in different patterns for unrelated strains. RFLP patterns are routinely entered into a computer database for both intergel and intragel pattern comparison9 and permit collaborative projects between laboratories (Figures 1 and 2).
Since 1997, a new TB DNA fingerprinting method known as spacer oligonucleotide type analysis (spoligotyping) has been applied, allowing the fingerprinting of priority cases within 1 to 2 working days. Spoligotyping is a polymerase chain reaction (PCR)-based assay that does not require large cell masses. The spoligotyping PCR reaction can be performed on cells directly from a growing culture at its early stage, from an old nonviable culture,10 or from a culture of M. tuberculosis mixed with nontuberculous mycobacteria. The rapidity and lower cost of this assay, versus RFLP analysis, have greatly popularized this fingerprinting method.
The direct repeat region is present in all members of the M. tuberculosis complex. The region consists of multiple copies of a conserved 36–base-pair DNA sequence separated by “spacer” sequences that are 34 to 41 base pairs long.11 The presence or absence of these spacer sequences can be used as another epidemiological marker. As with RFLP, the spoligotyping assay and nomenclature are performed according to internationally standardized protocols.12,13 Spoligotyping is first accomplished using PCR to amplify the direct repeat region from the strain of interest. The labeled PCR-products are then hybridized to 43 synthetic DNA oligonucleotides that have been bound to a nylon membrane. Spoligotyping results are digital in that they can readily be converted to a series of numbers that describe the pattern of hybridization.
To expedite DNA fingerprinting results and prioritize samples for RFLP, a spoligotyping assay will, most often, be performed first.6 In many cases, spoligotyping provides sufficient information to satisfy the request without performance of the more labor-intensive confirmatory RFLP.
Currently, no single DNA fingerprinting method is sufficiently discriminating for every M. tuberculosis strain. The combination of spoligotype and RFLP analyses provides excellent results in studying recent transmissions and in laboratory cross-contamination investigations.14
DNA Fingerprinting Services
In response to several nosocomial outbreaks, the US Centers for Disease Control and Prevention, Atlanta, established the National Tuberculosis Genotyping and Surveillance Network in 1992. Part of the responsibility of this network is to provide M. tuberculosis DNA fingerprinting services at no charge to state TB control offices. Through this program, DNA fingerprinting services are available, when required, to every TB control office in the United States. Requests for DNA typing may include investigation of cases of suspected TB transmission, of suspected false-positive laboratory results, or of suspected reinfection (in contrast to relapse or treatment failure). Furthermore, DNA fingerprinting services may be available in most local areas of the United States and Canada that have access to large public health or research laboratories. Caregivers and clinical laboratories that may require M. tuberculosis DNA fingerprinting services are encouraged to contact their state TB control offices; their requests will be forwarded to the appropriate laboratories for approval and testing.
Application of DNA Fingerprinting
TB Outbreak in a Low-Incidence Area
Toward the end of 1998, the Florida Bureau of TB Control and Refugee Health recognized a high number of TB cases in residents of a rural county in northern Florida. This resulted in a TB case rate of 14.8 per 100,000 population for that year, a rate 70.8% higher than the state average of 8.7 per 100,000. Because of this localized higher rate, fingerprinting by the Florida Department of Health was requested on 15 patients’ isolates identified from November 1995 through September 1998 (Figure 3, Panel A). Eight of these 15 isolates demonstrated identical fingerprints, with a ninth isolate having an extra band (Lane 10), and six having unique patterns. RFLP analysis warranted an expanded contact investigation, since, until this time, the contact investigation had been unproductive. By October 1999, three additional patients were linked to the original cluster of six (Figure 3, Panel B). In November 1999, a similar event unexpectedly occurred in a neighboring county, where four cases developed. The county medical director requested RFLP analyses. Three of the four cases displayed the same pattern as those in the first county (Figure 3, Panel C). The fourth demonstrated a unique pattern. In early December 1999, four additional patients were connected: one from the first county, one from the second county, and two from a southern Florida county (Figure 3, Panel D). In January 2000, five more patients were identified in the second county, and isolates were analyzed using RFLP (Figure 3, Panel E). Three patients had a pattern identical to that of the cluster, one had an extra band (Lane 30), and the fifth had a unique pattern. In summary, during this 14-month investigation, 30 patients were identified, resulting in 21 identical IS6110 fingerprints and two closely related strains.
The hard data from the RFLP testing enabled the contact investigators to obtain more detailed information from the patients and ultimately led to the discovery of an illegal gambling, drug-trafficking, and prostitution industry that appeared to be the focal point for the spread of TB.
Laboratory Cross-Contamination: Case 1
A 38-year-old male presented with a 3-month history of progressive shortness of breath and nonproductive cough. He was a nonsmoker who worked as a correctional officer. The patient denied fever, weight loss, and night sweats (symptoms normally encountered in TB patients), and had no other significant medical history. Chest radiography and chest CT revealed a posterior mediastinal mass. A bronchoscopy and a mediastinoscopy were performed; both were nondiagnostic. They were followed by a thoracotomy with biopsies of the hilar and mediastinal lymph nodes. The hilar lymph nodes showed noncaseating granulomas with calcification and multinucleated giant cells. The mediastinal lymph node demonstrated noncaseating granulomas, in addition to silver-methenamine-stain-positive organisms consistent with Histoplasma capsulatum. Both specimens were cultured for mycobacteria, but revealed no acid-fast bacilli on the smears. Six weeks later, cultures from both specimens grew out M. tuberculosis in both liquid and solid media. Based on the culture results, a four-drug regimen was prescribed for the patient. After 3 weeks of treatment, the patient claimed that taking the medications resulted in nausea and diarrhea, and he refused further treatment. A court order was requested to confine the patient to the state TB hospital and ensure that he continued treatment for TB. The state TB controller reviewed the case and suspected that the cultures were falsely positive, triggering a request for DNA fingerprinting of the two isolates at the Florida Department of Health. In the local hospital, three other patients’ specimens had been processed the same day as the suspected case’s specimens. The RFLP performed on all five specimens revealed three identical patterns (both of the case’s specimens and one of the three other patients’ specimens) and two nonmatching patterns (Figure 4). A second typing assay was performed on the three identical specimens, resulting in matching patterns. Since the state TB controller’s suspicion of false-positive results was confirmed, the legal court-order proceedings were halted. The final diagnosis was established as H. capsulatum infection.
Laboratory Cross-Contamination: Case 2
The pleural fluid of a 53-year-old man was culture positive for isoniazid-resistant M. tuberculosis. The TB control staff of a New England state found this diagnosis inconsistent with the patient’s clinical presentation and initiated an investigation. It was later learned that the original testing laboratory had worked with an isoniazid-resistant proficiency-testing sample from the College of American Pathologists (CAP) on the same day. The patient’s and CAP’s TB isolates were sent to the New York State Health Department for DNA fingerprinting. Spoligotype analysis on both isolates produced a matching pattern for both that had also been previously observed for the common laboratory-control strain H37Ra (Figure 5).15
Laboratory cross-contamination is a menacing, expensive problem for medical and laboratory staff, as well as suspected patients.5,6 The probability of recognizing false-positive results is highest when the culture report is questioned, either by the caregiver or by the diagnostic laboratory. The sources of cross-contamination include another patient’s specimen (Case 1), a laboratory-control strain, or a proficiency-testing sample (Case 2). While the estimated incidence of laboratory cross-contamination is low, the cost can be great in unnecessary medical care, TB control investigations, and patient suffering. Therefore, surveillance for, and verification of, false positives should be a priority for TB control programs.
Outlook
In the 1990s, DNA typing became an indispensable tool for TB control programs. The molecular approach, however, is only an adjunct to conventional investigation and clinical diagnosis; therefore, a close relationship among caregivers, public-health officials, and the laboratory performing the DNA typing is warranted. With the development of automated genetic analyses and microarray technology, DNA fingerprinting may become a standard tool in the clinical mycobacteriology laboratory in the near future, enabling TB control programs to perform real-time epidemiology.
Swartz, in the preface to the Institute of Medicine report Ending Neglect: The Elimination of Tuberculosis in the United States, writes that this report “reviews the lessons learned from the neglect of TB between the late 1960s and the early 1990s and reaffirms the committing to the goal of eliminating TB in the United States, defined as a case rate of less than 1 case per 1 million population per year. Clearly, to meet this goal aggressive and decisive actions beyond what is now in effect will be required.”16
The executive summary of this report goes on to say, “The key to achieving TB elimination will be through social mobilization and maintaining the public interest and commitment necessary to provide sufficient resources for the effort. The tendency to shift attention and resources away from the elimination of TB will increase as the number of cases decreases. Only an aggressive effort aimed at building political commitment can prevent the elimination of funding for TB research and before the elimination of the disease, leading to yet another period of neglect.”16
Jeffrey R. Driscoll, PhD, is associate director, Northeast Regional TB DNA Fingerprinting Center at Wadsworth Center, New York State Department of Health, Albany. Philip A. Lee is a research scientist, Bureau of Laboratories, Florida Department of Health, Jacksonville. Robert J. Jovell is a research scientist, Wadsworth Center. Yvonne M. Hale is section head of microbiology, Bureau of Laboratories, Florida Department of Health. Max Salfinger, MD, is director of the clinical mycobacteriology laboratory, Wadsworth Center.
Acknowledgments
The authors thank Mike McGarry for technical assistance and Linda Parsons, PhD, for insightful discussions. This research was supported, in part, by the Centers for Disease Control and Prevention, National Tuberculosis Genotyping and Surveillance Network cooperative agreement (JRD, RJJ, and MS).
References
1. American Thoracic Society. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med. 2000;161:1376-1395.
2. Frieden TB, Fujiwara PI, Washko RM, et al. Tuberculosis in New York City; turning the tide. N Engl J Med. 1995;333:229-233.
3. Crawford JT. Applications of molecular methods to epidemiology of tuberculosis. Res Microbiol. 1993;144:111-116.
4. Nardell E, McInnin B, Thomas B, et al. Exogenous reinfection with tuberculosis in a shelter for the homeless. N Engl J Med. 1986;315:1570-1575.
5. Small PM, McClenny NB, Singh SP, et al. Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J Clin Microbiol. 1993;31:1677-1682.
6. Nivin B, Driscoll J, Glaser T, et al. Use of spoligotype analysis to detect laboratory cross-contamination. Infect Control Hosp Epidemiol. 2000;21:525-527.
7. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998;393:537-544.
8. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol. 1993;31:406-409.
9. Heersma HF, Kremer K, van Embden JD. Computer analysis of IS6110 RFLP patterns of Mycobacterium tuberculosis. In: Parish T, Stoker NG, eds. Methods in Microbiology. Totowa, NJ: Humana Press; 1998:395-422.
10. Driscoll JR, McGarry MA, Taber HW. DNA-typing of a nonviable culture of Mycobacterium tuberculosis in a homeless shelter outbreak. J Clin Microbiol. 1999;37:274-275.
11. Hermans PWM, van Soolingen D, Bik EM, et al. The insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun. 1991;59:2695-2705.
12. Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997;35:907-914.
13. Dale J, Brittain D, Cataldi A, et al. Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardized nomenclature. Int J Tuberc Lung Dis. In press.
14. Kremer K, van Soolingen D, Frothingham R, et al. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J Clin Microbiol. 1999;37:2607-2618.
15. Bifani P, Moghazeh S, Shopsin B, et al. Molecular characterization of Mycobacterium tuberculosis H37Rv/Ra variants: distinguishing the mycobacterial laboratory strain. J Clin Microbiol. 2000;38:3200-3204.
16. Institute of Medicine (United States). Committee on the Elimination of Tuberculosis in the United States. Ending Neglect: The Elimination of Tuberculosis in the United States. Washington, DC: National Academy Press; 2000.
