INT J TUBERC LUNG DIS 4(12):S171–S175 © 2000 IUATLD
Access to newer laboratory procedures: a call for action Y. M. Hale,* E. P. Desmond,† K. C. Jost, Jr, ‡ M. Salfinger§¶ * Bureau of Laboratories, Florida Department of Health, Jacksonville, Florida, † Microbial Diseases Laboratory, California Department of Health Services, Berkeley, California; ‡ Bureau of Laboratories, Texas Department of Health, Austin, Texas; § Wadsworth Center, New York State Department of Health, Albany, New York; ¶ Department of Medicine, Albany Medical College, Albany, New York SUMMARY
Healthy People 2010, an initiative from the federal government, calls for action from tuberculosis controllers and tuberculosis laboratories in the fight to eliminate tuberculosis. Many patients, such as immunocompromised patients and those infected with multidrugresistant tuberculosis strains, pose a challenge for care and diagnosis. Fortunately, many changes have occurred in the last decade to facilitate more rapid and accurate testing to assist with the care of these patients. California, Florida, New York and Texas have almost 50% of the tuberculosis cases in the United States, and their
public health laboratories utilize different approaches to meet the same goal of rapid and accurate testing of specimens. With the targets of Healthy People 2010 (e.g., to reduce the average time for a laboratory to confirm and report tuberculosis cases to 2 days for 75% of cases) already looming on the horizon, innovative methods for achieving these goals should be evaluated. Using these public health laboratories as models, rapid, gold-standard testing methods should be provided to all patients in the United States. Soon it will be the year 2010 . . . , are you ready to swiftly move forward?
THE MYCOBACTERIOLOGY LABORATORY plays an important role in public health and must respond to changes in patient population as well as the expectations of the patients and physicians for shorter turnaround time. This urgency has been brought on especially by an increase in the number of immunocompromised patients and those in congregate settings. The laboratory diagnosis of tuberculosis must be expedited for better patient management and to save limited resources within the health care system. The most urgent questions that need to be addressed rapidly by the microbiology laboratory are: 1) Is the acid-fast stained smear positive for acidfast bacilli (AFB)? 2) Are tubercle bacilli involved? and 3) If tuberculosis organisms are identified are they resistant to rifampin? The tuberculosis laboratory has passed through several phases in its effort to answer these questions more rapidly, from using radiometric susceptibility testing in the mid-1980s, to using DNA probes in the late 1980s and nucleic acid amplification (NAA) in the mid-1990s, and to DNA sequencing now being available in a ‘kit’ format.
and susceptibility testing of the tubercle bacilli.1,2 Rapid growth detection, including the use of radiometric detection or similar broth-based automated detection systems, is recommended as the primary method for the isolation of tubercle bacilli. Once growth is obtained, a swift identification can be obtained, using methods such as DNA hybridization for Mycobacterium tuberculosis complex, fluorescence detection high performance liquid chromatography (FL-HPLC),3 and polymerase chain reactionrestriction enzyme pattern analysis (PRA).4,5 Rapid detection of M. tuberculosis complex can also be achieved directly from the submitted patient specimen using FL-HPLC3 as well as NAA.6,7 These methods do not require an actively growing culture, and can be completed in as little as half a day from receipt of the specimen. Rapid susceptibility methods currently include radiometric as well as non-radiometric methods newly approved by the US Food and Drug Administration.8,9 These methods can produce accurate and timely results in the testing of the first-line anti-tuberculosis drugs (isoniazid [INH], rifampin [RMP], pyrazinamide [PZA], ethambutol [EMB], and streptomycin [SM]) as recommended by the American Thoracic Society (ATS) and the CDC.10 Other methods, such as the direct conventional agar proportion method, can be utilized successfully, with the main objective being the return of timely and accurate results to the physician.
GOLD STANDARD TESTING The current recommendations from the Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories call for rapid testing of specimens for the isolation, identification,
Correspondence to: Max Salfinger, MD, Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA. Tel: (11) 518-474-2196. Fax: (11) 518-474-6964. e-mail:
[email protected]
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PRIORITIZATION OF SPECIMENS AFB smear positivity usually dictates what happens to the patient in the first few days of care. Patients are rapidly placed on therapy using four first-line drugs, including PZA, until susceptibility test results are available.10 Rapid detection of RMP resistance is essential for proper patient care, as RMP resistance will dictate longer and more complicated treatment to prevent negative outcomes.11,12 The emergence of strains of tubercle bacilli that are resistant to anti-tuberculosis agents has received increased attention, largely owing to dramatic outbreaks of multidrug-resistant (MDR) tuberculosis in human immunodeficiency virus (HIV) infected patients in New York and Florida in the late 1980s to early 1990s.13 Delayed diagnoses, inadequate treatment regimens, high mortality and significant rates of nosocomial transmission characterized these outbreaks.14 However, drug-resistant tuberculosis is a worldwide problem in both immunocompetent and HIV-infected populations.15,16 This again brings into the forefront the need for rapid testing in clinical mycobacteriology laboratories. A description is given of the organization of laboratory services in the four states whose tuberculosis cases constituted 48% of the 17 528 cases in the US in 1999.17 Each state laboratory has developed a different method for achieving rapid, quality testing for their populations. Organization of public health laboratory services, California A survey of mycobacteriology laboratories in California in 1994/1995 indicated that approximately 500 000 specimens per year were cultured for mycobacteria. In California, there are 39 public health laboratories (PHL) at a county or city level, and one State Microbial Diseases Laboratory in Berkeley. Approximately 20% of mycobacteriology cultures are performed in all the PHL. The remaining cultures are performed in hospital, independent commercial and health maintenance organization-associated laboratories. Nine PHL in rural areas, however, collaborate with the State Laboratory in Berkeley in the ‘BACTECs by mail’ program. These local laboratories receive uninoculated BACTEC 12B vials from the Berkeley laboratory, process specimens for mycobacterial smear and culture, inoculate decontaminated specimens into the BACTEC vials, and mail the inoculated vials, unincubated, to Berkeley. Solid media are incubated and examined locally. The Berkeley laboratory incubates the BACTEC vials received in the mail, identifies positive cultures by rapid methods (DNA probe and/or HPLC), and performs drug susceptibility testing by the radiometric BACTEC method. California regulations require: 1) that drug susceptibility testing be done on new, culture-positive TB
patients; 2) that one culture from each culture-positive patient be sent to the local PHL; and 3) that initial MDR patient isolates be sent to the State PHL in Berkeley for possible confirmation and DNA fingerprinting. Organization of public health laboratory services, Florida In 1997, approximately 155 000 specimens were cultured for mycobacteria in 103 laboratories according to a laboratory survey instituted by the Florida Department of Health. The PHL tested about 20% of all specimens submitted for AFB, and performed about 90% of all drug susceptibility tests. In Florida, there are two PHL processing specimens for tuberculosis. The main facility is located in Jacksonville, and the other is on the campus of A G Holley State Tuberculosis Hospital in Lantana. The Jacksonville laboratory performs testing 6 days/week including AFB smear, sputum processing, and NAA assay. Currently, all smear positive, newly diagnosed patients are automatically tested using NAA, placing these specimens in a Fast Track system.18,19 The decontaminated specimens are set up using BACTEC and solid media daily. The Jacksonville laboratory identifies positive cultures by rapid methods on a daily basis, predominantly using PRA and HPLC, or DNA probes. All susceptibility testing is centralized at the Jacksonville facility and performed 7 days/week (using the radiometric BACTEC system for the four first-line drugs and PZA) on all newly diagnosed patients. All positive reports are faxed, mailed, and/or downloaded to the provider and county health departments, as well as to the State TB Control. The hospital, independent commercial, and other laboratories can refer specimens, including raw sputum, to the PHL. AFB smear positive sputum can be sent for Fast Track testing with same-day turnaround time for NAA results. In addition, the Jacksonville laboratory performs DNA fingerprinting in its facility on request. Florida State regulations require that laboratories report findings suggestive of tuberculosis to the local health department. It has no specific regulations regarding specimen handling. Organization of public health laboratory services, New York In 1998, about 230 000 specimens were examined for tuberculosis in New York. Twelve per cent were tested in five PHL. However, 85% of all susceptibility tests were performed in three PHL (New York City, New York State, and Westchester County). The model Fast Track program for tuberculosis18,19 was initiated by the New York State Department of Health in 1993 to expedite testing for highly infectious tuberculosis patients. As of January 1998, more than 160 institutions were enrolled in this program in New York State. The program consists of the follow-
Optimizing TB laboratory services
ing five main characteristics: 1) broader accessibility to state-of-the-art tuberculosis laboratory procedures enhancing the health care of highly infectious smearpositive patients by identifying them early. This includes the use of gold standard technologies (radiometric detection, NAA, and radiometric susceptibility testing) with rapid specimen transport by overnight courier to a central laboratory. 2) For patients infected with nontuberculous mycobacteria, the period of exposure to potentially toxic and unnecessary antituberculosis drugs is dramatically shortened; these patients can also be released from respiratory isolation earlier, conserving health care resources. 3) Results are transmitted by mail and fax to the sender, and reported immediately to the Bureau of Tuberculosis Control for New York City, or New York State, as appropriate. Additionally, reports are downloaded daily to the New York State Bureau of Tuberculosis Control. 4) The mail report is accompanied by current information on case management to enhance the likelihood of appropriate care. 5) The implementation of new procedures is usually time consuming and costly, especially with the increasing financial constraints being experienced by laboratories. Upon availability, new technologies are quickly incorporated into the Fast Track program, thus providing immediate benefits to all enrolled hospitals, clinics, and medical centers. The results of the New York State Fast Track program show that susceptibility test results are available to the clinician in less than 2 weeks after sputum collection for about 50% of the smear-positive cases. A recent addition to the New York State Fast Track system includes the use of a PCR-DNA sequencing assay for the identification of RMP resistance. It has been shown that resistance to RMP is associated with mutations within an 81-bp region of the gene for the b subunit of RNA polymerase (rpoB) in RMP-resistant isolates of M. tuberculosis.20 This molecular assay is used to expedite detection of resistance to RMP in patient specimens when drug resistance is suspected. New York State additionally plays a pivotal role in the assurance of the quality of the laboratories. Laboratories testing for tuberculosis are required to adhere to a set of 22 minimal tuberculosis standards. Organization of public health laboratory services, Texas Six PHL in Texas (Austin, Harlingen, San Antonio, Houston, Dallas, and El Paso City) processed a total of 50 714 tuberculosis specimens in 1999. The number of specimens processed in Texas by hospital and private laboratories has not been surveyed recently. However, the Austin laboratory receives a high volume of mycobacterial isolates from these institutions for identification and drug susceptibility testing. Texas PHLs test at least one isolate from approximately 80% of the new tuberculosis cases in the state.
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All Texas PHL utilize rapid test methods. Results are reported by electronic dial-in connection, fax, or mail. Additionally, PHL customers receive telephone reports for smear-positive specimens, direct specimen identifications, culture identifications of M. tuberculosis complex and detection of drug resistance. Reports are sent to providers, the local Public Health Region, and the State Tuberculosis Elimination Division. The Austin laboratory performs most of the Texas PHL AFB culture work and offers several unique services. On a daily basis (Monday–Friday), all smearpositive specimens, and smear-negative specimens upon request, from patients without recent tuberculosis history are tested directly by FL-HPLC and identified as either M. tuberculosis complex, M. avium complex, M. kansasii, or nontuberculous mycobacteria. In instances where the direct FL-HPLC is inconclusive, NAA is performed. Cultures are tested daily by FLHPLC at the first evidence of positivity. The Austin laboratory testing algorithm for both direct specimen and culture identification emphasizes a rapid distinction between M. tuberculosis complex and nontuberculous mycobacteria. Conventionally, many laboratories report AFB-positive smear results from cultures because specific species identification may not be available until days later. Such identifications, at least to the level of M. tuberculosis or nontuberculous mycobacteria species, are available on the same day as culture AFB smear results. Consequently, AFB-positive smear results from culture are not reported routinely. This has substantially reduced the number of reports and calls issued by the laboratory and improved patient care by substituting a rapid M. tuberculosis complex identification for a non-specific AFB culture smear result. Texas regulations require that confirmed and suspected cases of tuberculosis and susceptibility results be reported to the local health authority or to the office of the regional director.
THOUGHTS FOR THE FUTURE: A CALL FOR ACTION The tuberculosis laboratory is no longer an entity by itself with its own pace; it is part of a virtual organization aimed at the elimination of tuberculosis. As seen by the illustration of the four states’ PHL networks, these services can be achieved in a variety of manners. No single approach by itself may be best. But the ultimate goal of timely and adequate services must be provided, and continually evaluated. A highly infectious tuberculosis patient must have access to stateof-the-art laboratory services even though the patient may reside in an area where the laboratory may not have the training or the funding to implement newer technologies. In order to achieve this, innovative ideas may be required, such as centralized testing by a
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Table 1 Turnaround times required for at least 80% of specimens/isolates tested
Smear Diagnostic specimens ,24 hours Diagnostic isolates NA
Growth detection and M. tuberculosis complex vs. NTM
Susceptibility testing
,2–3 weeks
,2–5 weeks
,1 week
,1.5–2 weeks
NTM 5 nontuberculous mycobacteria; NA 5 not applicable.
large public health laboratory where many specimens are already being tested. The impact of adding additional specimens would be minimal, but the benefits of providing this service to a wider area would be significant. Recently, the CDC incorporated more stringent turnaround times into the FY2000 Cooperative Agreement for state and PHLs in major cities. The guidelines require these turnaround times for at least 80% of specimens/isolates tested (Table 1). In January 2000, the US Department of Health and Human Services published ‘Healthy People 2010— Understanding and Improving Health’,21 a wide range of public health opportunities that exist in the first decade of the 21st century. Healthy People 2010 covers 467 objectives in 28 focus areas, including the following four tuberculosis targets: 1) to reduce tuberculosis, 2) to increase the proportion of all tuberculosis patients who complete curative therapy within 12 months, 3) to increase the proportion of contacts and other high-risk persons with latent tuberculosis infection who complete a course of treatment, and 4) to reduce the average time it takes for a laboratory to confirm and report tuberculosis cases (Table 2). State and local TB controllers working with public health laboratorians will have to decide how to incorporate changes in order to achieve these goals. The
Table 2 The four tuberculosis targets covered by Healthy People 2010 1 Reduce tuberculosis Target: 1.0 new case per 100 000 population Baseline: 6.8 new cases of TB per 100 000 population in 1998 2 Increase the proportion of all TB patients who complete curative therapy within 12 months Target: 90% of patients Baseline: 74% in 1996 3 Increase the proportion of contacts and other high-risk persons with latent TB infection who complete a course of treatment Target: 85% Baseline: 62.2% in 1997 4 Reduce the average time for a laboratory to confirm and report TB cases. Target: 2 days for 75% of cases. Baseline: 21 days were needed for a laboratory to confirm and report 75% of TB cases in 1996
technology is already at hand: it is only a matter of organizing the most efficient laboratory service to meet the fourth target by 2010. However, it is important to differentiate between specimens where the diagnosis has not yet been made, and which therefore warrant priority handling, and less-urgent follow-up specimens after a diagnosis has been established. With these targets in mind, the following patients should be fast-tracked: 1) those with newly diagnosed AFB smear-positive sputum; 2) those with clinical and radiological signs of tuberculosis in spite of AFB smear-negative sputum; and 3) those with suspicion of drug-resistant tuberculosis. In March 1982, to commemorate Koch’s discovery of the tubercle bacillus, John Sbarbaro wrote about the fact that tuberculosis provides us with a basis for gaining meaningful insight into the behavior of our human institutions, our human organizations, and our human society.22 He described the discovery of streptomycin and INH and continued “. . . it took until 1961, nine years after the presentation of evidence that we had achieved a significant scientific breakthrough, for the Committee on Therapy of the American Thoracic Society to issue a statement that essentially eliminated bed rest as a therapeutic principle . . . Now, please understand that I am not ridiculing or criticizing the professionals of the past . . . Clearly, we professionals change slowly.” Soon it will be the year 2010 . . ., are you ready to swiftly move forward? References 1 The Association of State and Territorial Public Health Laboratory Directors/Centers for Disease Control and Prevention. Mycobacterium tuberculosis: assessing your laboratory. Atlanta, GA: CDC, 1995. 2 Tenover F C, Crawford J T, Huebner R E, Geiter L J, Horsburgh C R Jr, Good RC. The resurgence of tuberculosis: Is your laboratory ready? J Clin Microbiol 1993; 31: 767–770. 3 Kost K C, Dunbar D F, Barth S S, Headley V L, Elliott L B. Identification of Mycobacterium tuberculosis and Mycobacterium avium complex directly from smear-positive sputum specimens and BACTEC 12B cultures by high-performance liquid chromatography with fluorescence detection and computer-driven pattern-recognition models. J Clin Microbiol 1995; 33: 1270–1277. 4 Telenti A, Marchesi F, Balz M, Bally F, Bottger E C, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 1993; 31: 175–178. 5 Taylor T B, Patterson C, Hale Y, Safranek W W. Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media. J Clin Microbiol 1997; 35: 79–85. 6 Pfyffer G E, Kissling P, Jahn E M, Welscher H-M, Salfinger M, Weber R. Diagnostic performance of amplified Mycobacterium tuberculosis direct test with cerebrospinal fluid, other non-respiratory, and respiratory specimens. J Clin Microbiol 1996; 34: 834–841. 7 Catanzaro A, Perry S, Clarridge J E, et al. The role of clinical suspicion in evaluating a new diagnostic test for active tuberculosis: Results of a multicenter prospective trial. JAMA 2000; 283: 639–645.
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14 Dooley S W, Jarvis W R, Martone W J, Snider D E. Multidrugresistant tuberculosis. Ann Intern Med 1992; 117: 257–259. 15 Cohn D L, Bustreo F, Raviglione M C. Drug-resistant tuberculosis: review of the worldwide situation and the WHO/ IUATLD Global Surveillance Project. Clin Infect Dis 1997; 24 (Suppl 1): S121–S130. 16 Harvard Medical School/Open Society Institute. The global impact of drug-resistant tuberculosis. Boston, MA: Harvard Medical School, 1999. 17 March 24, 2000—World TB Day. Centers for Disease Control and Prevention. Atlanta, GA. 18 Salfinger M, Pfyffer G. The new diagnostic mycobacteriology laboratory. Eur J Clin Microbiol Infect Dis 1994; 11: 961–979. 19 Salfinger M, Hale Y M, Driscoll J R. The diagnostic tools in tuberculosis: Present and future. Respiration 1998; 65: 163–170. 20 Telenti A, Imboden P, Marchesi F, et al. Detection of rifampin resistance mutations in Mycobacterium tuberculosis. Lancet 1993; 341: 647–650. 21 US Department of Health and Human Services. Healthy People 2010 (Conference Edition, in 2 Volumes). Washington, DC: January 2000. 22 Sbarbaro J A. Tuberculosis: a portal through which to view the future. Am Rev Respir Dis 1982; 125: 127–132.
