National Centerfor Infectious Diseases' and National Centerfor Prevention Services,2. Centers for ... ing timely species identification and susceptibility testing.
Vol. 31, No. 4
OF CLINICAL MICROBIOLOGY, Apr. 1993, p. 767-770 0095-1137/93/040767-04$02.00/0 Copyright © 1993, American Society for Microbiology
JOURNAL
GUEST COMMENTARY The Resurgence of Tuberculosis: Is Your Laboratory Ready? FRED C. TENOVER,1* JACK T. CRAWFORD,' ROBIN E. HUEBNER,2 LARRY J. GEITER,2 C. ROBERT HORSBURGH, JR.,1 AND ROBERT C. GOOD' National Center for Infectious Diseases' and National Center for Prevention Services,2 Centers for Disease Control and Prevention, Atlanta, Georgia 30333
tuberculosis include medically underserved, low-income populations, immigrants from countries with a high prevalence of tuberculosis, and residents of long-term-care and correctional facilities. Those at increased risk of developing disease following infection include individuals with HIV infection; close contacts of infectious cases; children less than 5 years old; patients with renal failure, silicosis, and diabetes mellitus; and individuals receiving treatment with immunosuppressive medications. In the United States, the number of tuberculosis cases reported annually declined steadily between 1953 and 1985; however, in 1986, the rate for newly diagnosed cases increased 1.1% over the preceding year (5). This upward trend has continued; in 1991, a total of 26,283 cases were reported to the Centers for Disease Control (CDC), representing an increase of 18.4% over the number reported in 1985 (8). Since 1990, nosocomial outbreaks of multidrug-resistant tuberculosis (MDR-TB) involving over 200 patients have been reported to CDC. These outbreaks have included transmission of M. tuberculosis to patients, health care workers, and inmates and employees of correctional facilities. Investigation of four such outbreaks that occurred in hospitals in Florida and New York City demonstrated that
INTRODUCTION After years of declining case rates, tuberculosis is again a major public health problem in the United States. To compound the problem, serious outbreaks involving both patients infected with the human immunodeficiency virus (HIV) and HIV-infected and non-HIV-infected health care workers have been noted in several major metropolitan areas. Cases have also increased in other population groups, including the homeless, prisoners, migrant farm workers, and immigrants. The definitive diagnosis of tuberculosis depends on the isolation and identification of the etiologic agent, Mycobacterium tuberculosis, while design of an appropriate therapeutic regimen depends on the results of antituberculosis susceptibility testing. With this information in hand, the necessary infection control procedures and contact tracings can be initiated and informed decisions can be made regarding therapy. If laboratories cannot screen specimens rapidly for acid-fast bacilli, identify isolates in a timely manner, and provide drug susceptibility data in a short period of time, patient care may suffer and infectious patients may continue the chain of transmission. The laboratory has a major role to play in breaking this chain; the importance of completing thorough evaluations of clinical specimens for mycobacteria and, if mycobacteria are present, the importance of obtaining timely species identification and susceptibility testing results cannot be overemphasized. To halt the continuing spread of tuberculosis across the United States and to control transmission within hospitals, laboratories must recognize the urgency and optimize their procedures in reporting results of acid-fast smears, cultures, and drug susceptibility tests to clinicians.
most cases of MDR-TB occurred in individuals known to be
infected with HIV (3, 6, 7, 9, 11, 13, 17). The
fatality
inmates and one prison guard have died of MDR-TB. All of these outbreaks have been characterized by the transmission of strains of M. tuberculosis resistant to at least isoniazid and rifampin (MDR), with some strains showing additional resistance to other drugs including ethambutol, streptomycin, ethionamide, kanamycin, and rifabutin. Delays in the laboratory diagnosis and reporting of drugresistant tuberculosis contributed to the magnitude of these outbreaks since cases were not rapidly identified, the organism was not isolated, or the patients were not put on adequate therapy. seven
THE TUBERCULOSIS PROBLEM Tuberculosis is a bacterial disease caused by organisms of the M. tuberculosis complex (i.e., M. tuberculosis, M. bovis, and M. africanum). It is transmitted primarily by airborne droplet nuclei produced when individuals with pulmonary or laryngeal tuberculosis sneeze, cough, or speak. Individuals are particularly infectious if they are excreting sufficient bacilli to produce a positive acid-fast-stained preparation of their sputum (1). Infection occurs when susceptible individuals inhale these droplet nuclei. Tuberculosis can occur in any organ of the body (1), although only 5 to 15% of infected individuals will develop active disease within 2 years of primary infection (1, 20). The population groups in the United States that are at increased risk for infection with M. *
case
rate among patients with active MDR-TB was exceptionally high, 72 to 89%, and the median interval from the time of diagnosis to death was very short, 4 to 16 weeks. Three subsequent outbreaks, involving two additional hospitals and a state correctional facility, are under investigation in New York state (12, 17). At the correctional facility, at least
SAFETY IN THE MYCOBACTERIOLOGY LABORATORY
Specimens received in the mycobacteriology laboratory for staining and culture should routinely be considered to contain mycobacteria and, therefore, must be handled in a safe manner. Such specimens may be from patients who are infected with MDR strains of M. tuberculosis, which would
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GUEST COMMENTARY
make treatment of laboratory personnel difficult should an accident occur and a staff member become infected. The incidence of tuberculosis in those who work with M. tuberculosis in the laboratory is three times higher than the incidence among laboratory personnel who do not work with the bacterium (10). Safety procedures as proposed in the safety guide for laboratories (10) must be enforced. The guidelines recommend Biosafety Level 2 practices, containment equipment, and facilities for preparing acid-fast smears and culturing specimens. All aerosol-generating procedures must be conducted in a class I or II biological safety cabinet. Biosafety Level 3 practices, containment equipment, and facilities are recommended for propagation and manipulation of cultures of M. tuberculosis. In practice, this means that workers wear special laboratory clothing and protective gloves, decontaminate all infectious wastes, and work in controlled-access areas clearly labeled with biohazard warn-
ing signs. In addition to wearing protective clothing, using equipment designed to prevent aerosol formation, and working in facilities constructed as recommended to meet the safety requirements, personnel should be monitored for tuberculin hypersensitivity at yearly intervals; they should be monitored more often if a conversion has been documented (10, 16). It is the responsibility of all laboratory personnel to work in such a manner as to protect themselves and others from infection. Proper training in laboratory procedures and safety must be provided for all employees who work in such laboratories.
OPTIMIZING LABORATORY METHODS Specimens must be delivered to the laboratory promptly, ideally within 30 min of collection, but at least within 24 h of collection. They should not be held until an "adequate" number is available for shipment, since results from even one specimen, if positive, will have an impact on infection control procedures and may be extremely important for controlling the spread of the disease and for treatment of the patient. Although procedures for mycobacterial staining, culture, and drug susceptibility testing have undergone radical changes in the last few years, some laboratories have been unable to incorporate these new methodologies. The initial step in the laboratory diagnosis of tuberculosis is microscopic examination of smears stained by an acid-fast procedure. Although the sensitivity of microscopy is relatively low, requiring about 5 x 103 bacilli per ml of specimen for detection (19), the number of tubercle bacilli in pulmonary secretions is directly related to the risk of transmission. Thus, microscopy is an important tool for screening patients who may require isolation on admission to a hospital. Two types of acid-fast stains are commonly used. The first type is a basic fuchsin stain (Ziehl-Neelsen or Kinyoun methods) combined with light microscopy. Examination of smears requires an average of 15 min; 300 fields must be viewed before the smear is judged to be negative (19). The second and preferred type uses fluorescent microscopy and an auramine-rhodamine fluorochrome stain. The yellowish fluorescence of this acid-fast stain when it is taken up by mycobacteria makes the organisms easier to detect, allowing lower magnifications to be used. This, in essence, increases the sensitivity of the method and allows much faster screening of smears. The definitive diagnosis of tuberculosis by isolation of M. tuberculosis from clinical specimens is hampered by the slow growth rate of tubercle bacilli. Standard culture meth-
ods with solid media require incubation for 3 to 6 weeks before sufficient growth is obtained to initiate identification methods (16, 18). Primary culture in a selective broth medium is preferred since mycobacteria grow more rapidly in a liquid medium. One of the new procedures that has undergone extensive testing is the BACTEC radiometric system (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.), which uses a liquid growth medium containing radiolabeled palmitic acid as the substrate. Growth of mycobacteria is detected within 7 to 14 days by measuring the released 14C02. Once growth is detected, the organisms can be identified as M. tuberculosis by using specific inhibition
byp-nitro-a-acetylamino-o-hydroxypropiophenone (NAP; a precursor in the synthesis of chloramphenicol), DNA probes
(18), high-performance liquid chromatography (HPLC) (4), or the standard biochemical tests for production of niacin,
catalase, and nitrate reductase (16, 18). Use of biochemical identification tests may require an additional 3 to 6 weeks for completion after the organism is isolated on a solid medium, while NAP tests can be completed in 3 to 5 days. DNA probes and HPLC analysis can each be completed in 2 to 4 h; however, up to 107 bacilli are required for reliable, reproducible results. Nucleic acid probe assays are simple to perform, and the accuracy of such assays is nearly 100%; i.e., all species in the M. tuberculosis complex, including M. bovis, are positive while other species of bacteria, including other mycobacteria, fail to react with the probe. The CDC laboratory has developed an HPLC method for identification of all clinically relevant Mycobactenum species by detecting differences in the spectrum of mycolic acids present in the cell wall. The method is more versatile than probe assays and is equally rapid. An advantage of the procedure is that the initial one-time cost of $35,000 to $50,000 for the equipment may be offset by minimal longterm reagent costs.
Under the best of circumstances, using the radiometric system for primary culture and a rapid identification method, about 2 weeks are still required to detect and identify M. tuberculosis in clinical specimens. This is an improvement over the time required for identification by classical biochemical methods (16, 18); however, the number of laboratories using these rapid methods is relatively low (15a). Although DNA probe assays and HPLC are considered rapid methods, they still require the isolates to be cultivated in a solid or liquid medium. Newer nucleic acid amplification methods are aimed at direct detection of M. tuberculosis in clinical material. The most widely studied technique for this purpose is the polymerase chain reaction. Assays specific for M. tuberculosis have been developed and, in small trials, have been shown to be relatively sensitive and highly specific. More efficient methods of extracting DNA from the organisms in the clinical specimen will be required before the assay will be acceptable for widespread clinical use. The procedure also requires special equipment and facilities and additional training for personnel. It is clear, however, that there is an urgent need for an assay that allows direct detection of tubercle bacilli in clinical material without culture. Such procedures should be available soon.
SUSCEPTIBILITY TESTING OF M. TUBERCULOSIS Strains of M. tuberculosis isolated from patients prior to 1989 were generally susceptible to antituberculosis drugs;
