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* From the Department of Internal Medicine (Dr. Dobbs), Division of Pulmonary, Allergy and Critical Care Medicine (Drs. Lok and Dunlap), and Department of Microbiology (Dr. Benjamin), University of Alabama at Birmingham, Birmingham; and Division of Tuberculosis Control (Mr. Bruce and Ms. Mulcahy), Department of Public Health, Montgomery, AL.
Correspondence to: Nancy E. Dunlap, MD, PhD, Professor of Medicine, 398 DREB, University of Alabama at Birmingham, Birmingham, AL 35294; e-mail: ndunlap{at}uabmc.edu
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Abstract |
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Methods: IS6110 DNA fingerprinting was performed on isolates of M tuberculosis from verified cases of tuberculosis in Alabama from 1994 to 1998. A statewide database groups isolates into "clusters" and tracks them cumulatively over time. A large cluster was identified and was secondarily investigated using traditional epidemiologic methods.
Results: Twenty-five isolates were found to be identical by fingerprinting analysis. Patients were living within 10 counties across the state, and 12 cases were localized to a single county. This represented an ongoing, statewide tuberculosis outbreak previously unrecognized by local and state health officials. Secondary investigation of the cases revealed the primary sites of transmission to be a correctional facility and two homeless shelters.
Conclusions: Population surveillance using M tuberculosis fingerprinting was successfully utilized to detect a significant and smoldering tuberculosis outbreak. Measures are currently in place to identify and prevent further transmission in the involved locations.
Key Words: DNA fingerprinting • epidemiology • surveillance • tuberculosis
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Introduction |
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Tuberculosis is endemic in Alabama. Although Alabama has a relatively low incidence of tuberculosis among known high-risk groups, such as foreign-born or HIV-positive individuals, in 1998 the annual case rate per 100,000 population was the sixth highest in the nation.12 Tuberculosis is scattered across the state, affecting all segments of the population (Fig 1 ). In populations such as ours where tuberculosis is endemic, there are many patients in whom reactivation tuberculosis develops several years after infection with the Mycobacterium tuberculosis organism. In this setting, recent transmission of tuberculosis may become obscured by the high background incidence of reactivation tuberculosis, thus escaping detection by traditional epidemiologic investigations. Surveillance using DNA fingerprinting serves as a useful tool to denote ongoing transmission of a single strain. This study describes the investigation of a statewide tuberculosis outbreak initially detected by routine surveillance using DNA fingerprint clustering.
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Materials and Methods |
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Alabama is a predominantly rural state, with a population of
approximately 4 million. From 1994 to 1998, the period investigated in
this study, tuberculosis case rates in Alabama ranged from 8.8 to 10.6
per 100,000 population.15
Unlike most states with high
case rates of tuberculosis, the age group with the highest percentage
of cases in 1998 was the group aged 
65 years. Furthermore, in 1998,
only 5.8% of tuberculosis patients in Alabama were foreign born (US
average, 41.3%); 12.9% of tuberculosis patients aged 25 to 45 years
were HIV positive, and no tuberculosis cases were found to be multidrug
resistant. Five percent of tuberculosis patients in 1998 were defined
as homeless in the past year (US average, 6.3%).12
The
lack of significant association of tuberculosis in Alabama with known
risk factors, as evidenced by the preceding sociodemographic
characteristics, creates a setting in which detection of ongoing
transmission may be quite difficult.
Molecular Analysis
In Alabama, by law, all initial M tuberculosis
cultures must be sent to the state laboratory for verification and
susceptibility testing. Since January 1, 1994, isolates of all positive
culture findings of verified cases of tuberculosis available to the
state laboratory have been sent to the University of Alabama at
Birmingham (UAB) for DNA fingerprinting. These isolates represent 80%
of all tuberculosis cases within the state. Restriction fragment length
polymorphism (RFLP) analysis using the mycobacterial insertion sequence
IS6110 as a probe is performed on all viable samples by an
internationally standardized method.16
Films are
subsequently scanned into a computerized database. The resulting DNA
fingerprints are analyzed using Whole Band Analyzer, version 4.01
(Genomic Solution; Ann Arbor, MI), and clusters with identical patterns
or patterns differing by one band are confirmed by visual inspection.
Clusters that have low band numbers (five or fewer bands) as well as
certain clusters that warrant further molecular characterization are
subtyped using at least one polymerase chain reaction (PCR) method. The
PCR methods that have been used for this purpose include spoligotyping
in all cases and variable number tandem repeat typing when further
characterization is desired, each performed according to previously
published methods.17
18
19
20
Laboratory cross-contamination is suspected when two specimens from different patients are processed in the same laboratory on the same day and yield identical DNA fingerprints. We monitor for statewide laboratory cross-contamination by checking culture dates and sites for all isolates with matching fingerprints. In addition, the possibility of laboratory cross-contamination is investigated when a patient produces a single isolate with low colony count (< 10) from a specimen that is negative for acid-fast bacilli on microscopy.21 Laboratory data are correlated with the patient’s clinical course, and isolates identified as contaminants are removed from our database.
Definition of Cluster
In this study, two or more M tuberculosis isolates
from different patients were considered to be the same strain if DNA
fingerprinting patterns with IS6110 revealed the following:
(1) six or more fragments of identical size; (2) six or more fragments
of identical size except one or two additional fragments, or one or two
fragments of different size and identical patterns by secondary typing;
or (3) five or fewer bands of identical size and identical patterns by
secondary typing.8
Although this method is not exact and
in some instances overestimates and in other instances underestimates
the amount of true transmission, it was a practical definition given
the large number of M tuberculosis isolates needing to be
analyzed. Initial evaluation of DNA fingerprinting patterns was
performed by individuals blinded to clinical information regarding the
cases.
Surveillance Program in Alabama
A statewide DNA fingerprint database has been established based
on IS6110 genotype; PCR-based secondary typing data are
included as a separate variable. The DNA fingerprint database is
maintained at UAB and contains the following information: patient
demographics and residence, specimen collection site and date, drug
susceptibilities, date sent to UAB, fingerprint characterization and
cluster designation, and results from secondary typing methods.
Matching fingerprints are grouped into clusters and tracked
cumulatively over time. There is no time limitation for inclusion in a
particular cluster. There are clusters that decrease over time or
disappear from the population. Other clusters persist in the population
and over time may represent ongoing transmission; from this group of
persistent clusters, cases are chosen for secondary investigation as
demonstrated by this study. Overall, since 1994, we have found a total
of 119 clusters representing 47% of all fingerprinted isolates.
Thirty-two clusters have had five or more cases since 1994. This study
represents one of six clusters with 25 cases.
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Results |
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Data Collection
Once clustering was identified, records were obtained on the 25
patients from the Alabama case registry, maintained by the Alabama
Department of Public Health. Initial data included information reported
routinely such as county, date reported, age, sex, race, sputum smear
results, homeless status, and drug sensitivities. Information obtained
from this initial review of the records revealed a suspected focus of
transmission in northeast Alabama, possibly involving the homeless
population. As a result, we conducted a retrospective survey of the
registries of the two local homeless shelters and the jail, focusing on
the cluster cases. This aspect of the investigation was performed in
conjunction with the local health department officials. Information on
homeless status and time in jail from 1994 to 1998 was obtained on all
but five individuals.
Data Analysis
The dates that the tuberculosis cases were reported ranged from
April 1994 to July 1998. Ten counties across the state were involved,
and 12 of the 25 cases (48%) were reported in Madison County (Fig 2 ). This represented a possible statewide outbreak previously
unrecognized by local and state officials. Characteristics of the 25
patients in this cluster are presented in Table 1
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, 14 of the 25 patients (56%) had been homeless at some point during the past 6 years. Studies in urban centers with large homeless populations have shown that conventional contact investigations among the homeless often fail to detect clustered patients, perhaps because contacts in this setting are frequently multiple and transient.8 9 Given the inherent difficulties of establishing epidemiologic associations in this setting, information provided by molecular techniques yielded an important clue to the presence of this ongoing outbreak.
Several interesting points are suggested by this study. First of all, initial review of patient records indicated that only seven patients had been homeless within the past 1 year. This information was obtained from official patient records (report of verified case of tuberculosis), completed according to Centers for Disease Control and Prevention guidelines that limit the definition of "homeless" to 1 year. However, when further investigation of the cluster cases was pursued, 14 patients were found to have been homeless during the past 6 years. Because tuberculosis infection can persist in an individual without detection for many years, it is imperative to identify known risk factors for transmission such as homelessness, even if this occurred longer than 1 year prior to clinical detection of disease.
DNA fingerprinting provides valuable information for a statewide surveillance program in an area in which tuberculosis is endemic. However, when using clustering of tuberculosis fingerprints to identify ongoing, smoldering outbreaks, the definition of clustering must not specify a time limitation. In a recent analysis of tuberculosis trends in San Francisco, Jasmer et al22 limited the definition of clustering to 1 year. However, like our program, van Soolingen et al23 placed no time limitation on clustering in a study of tuberculosis trends in the Netherlands. Because transmission of tuberculosis may have occurred recently or more remotely, we feel that limiting the definition of clustering to 1 year would result in a substantial number of missed associations. Such associations might elucidate important sites of ongoing tuberculosis transmission as indicated by this study. For example, if the definition of cluster were limited to 1 year in this study, only one of the three cases linked to the jail outbreak by DNA fingerprinting would have been identified (Fig 3) . Furthermore, evidence linking this strain throughout the homeless population would have been missed, and the overall extent of this outbreak would have been vastly underestimated. Not only is the tracking of clusters over time useful on a statewide basis, but transmission across state borders may also be elucidated. Recently, three individuals in West Virginia were found to have the same DNA fingerprint as the cluster in this study; furthermore, these individuals are known to be homeless. Further investigation of this situation is currently in progress.
DNA fingerprinting of M tuberculosis can signal outbreaks within a population, but traditional epidemiologic techniques must be employed to identify specific connections between individuals or sites of transmission. And once smoldering outbreaks are identified, traditional and innovative public health measures must be employed to eliminate ongoing transmission. For example, in homeless shelters air sterilization with ultraviolet lights, improved ventilation, and spot sputum screening of clients have resulted in dramatic decreases in tuberculosis cases in some areas.24
In the United States, intensified public health measures, improved living conditions, and more efficacious chemotherapeutic agents have effected a substantial decline in tuberculosis case rates over the past 4 decades. Nevertheless, > 1,000 deaths per year in the United States can be attributed to this essentially treatable, preventable disease.12 As case rates of tuberculosis continue to decline, the public health resources directed to the control of tuberculosis decrease. If background rates of reactivation tuberculosis remain high, it will become increasingly difficult to recognize ongoing, smoldering outbreaks when public health resources are redirected. In endemic areas such as Alabama, new tools and strategies are required if we are to eliminate tuberculosis (defined as less than one case per 1 million population per year).25
From 1994 to 1998, the time period addressed in this investigation, patients in 94.6% of cases in Alabama completed therapy in 12 months, and 94.2% of sputum culture-positive patients converted to sputum-culture negative 3 months after initiating therapy. An average of 10 contacts were identified for each tuberculosis case patient, and 80.4% of all patients started on preventive therapy with isoniazid completed at least 6 months of treatment. Despite such outstanding indicators of program performance, the rate of decline in tuberculosis case rates has lagged behind the rest of the nation,13 thus demonstrating the need for improved strategies to control endemic tuberculosis.
If we are to eliminate tuberculosis from endemic regions of this country, as advocated by the Institute of Medicine in its 2000 report entitled, "Ending Neglect: The Elimination of Tuberculosis in the United States,"26 we must better understand the local epidemiology and transmission patterns of tuberculosis. Conventional epidemiologic methods are not adequate as demonstrated by this study. New tools must be adopted. Fingerprinting of M tuberculosis is one such tool, but it is only useful for surveillance if a large percentage of cultures are available for analysis. As more, large, regional reference laboratories are established, specimens are increasingly sent out of state for processing. Public health policies need to be established that require that positive M tuberculosis culture findings are returned to the patient’s state of residence so that fingerprinting of the isolates can be performed.
In summary, in an area in which tuberculosis is endemic, small foci of ongoing transmission may be missed by traditional epidemiologic techniques. Such microepidemics can then persist unrecognized in a community for months or years, as was the case in our study. This study demonstrates the effective use of DNA fingerprinting as part of a multifaceted control strategy to identify ongoing transmission of tuberculosis in an endemic setting.
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Footnotes |
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Received for publication April 16, 2001. Accepted for publication June 5, 2001.
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