Tuberculosis and the Expanding Role of the Laboratory

Harry, a 29-year-old white homeless man living in a downtown shelter is seen in the emergency room because of a five-month history of cough, 12-pound weight loss, fever, and drenching sweats. Until a few months ago, he spent 10 years in India, where he was treated twice for tuberculosis (TB) in the past three years but cannot remember the names of his medications. He notes that during the day he “hangs out” at a homeless drop-in site that provides services to the HIV-infected in the community and where an HIV- related multidrug-resistarit tuberculosis (MDR-TB) case was recently diagnosed.

Final diagnosis: Multidrug smear-positive tuberculosis by molecular beacon testing with confirmation of transmission of disease to the recently diagnosed, HIV-infected contact by DNA fingerprinting.

* Fifteen years ago, laboratory confirmation of a multidrug- resistant case of TB like Harry’s would have taken a minimum of two- and-a-half to three months between the time sputum samples were collected and the point where second-line drug-susceptibility information was obtained. With the evolution of more rapid methods and molecular-based assays, the time needed for diagnosis in patients with smear-positive disease can be shortened to a few hours. Without the widespread use of these tools, however, most physicians continue to “treat in the dark,” often with the wrong drugs, until the long wait for second-line susceptibility results return. In addition, confirming links to other cases with hard evidence was not possible until the advent of molecular epidemiology and DNA fingerprinting.

Is the TB skin test (TST) on its way out?

Keiko, an asymptomatic 19-year old Japanese exchange student has a positive TST of 15 mm when she is tested routinely before entering a nursing program. She argues with her physician that the test is positive because of two prior BCG vaccinations she received as a child and denies any exposure to active disease. Her physician gently insists that nine months of preventive treatment with isoniazid is the standard of care recommended by the Centers for Disease Control and Prevention (CDC) in Atlanta. A chest X-ray confirms that she does not have disease, and she is “cleared”; however, Keiko is worried that her refusal of treatment may jeopardize her entry into the nursing program. Her physician remembers that the local TB program is now using a new, more specific TB blood test that would remove the “BCG factor” and obtains the test for Keiko.

Final diagnosis: No TB infection (QuantiFERON-TB Gold result is negative); treatment is determined unnecessary.

* Powerful new tools are on the horizon that may eventually replace the TST because of greater specificity and equal or improved sensitivity. For instance, QuantiFERON-TB Gold (QFT-GoId) – a new blood-based T-cell assay that is being used at only a handful of U.S. locations – is quickly catching on and changing the paradigm of clinic-based diagnosis of latent TB infection (LTBI) to a laboratory diagnosis.

While TB is at an all-time low in the United States, it continues to dominate infectious diseases globally by its ability to infect, become quiescent, and then reactivate in the body later in life. An estimated 1.8 billion individuals are infected worldwide with the TB bacillus – fully one-third of the globe’s population. One death due to TB occurs every 15 seconds,1 1.7 million people the annually from this preventable and curable disease, and 8.8 million new cases occur every year, according to the World Health Organization’s (WHO’s) 2003 report. By the standards of the WHO, the United States is considered a low-incidence country, accounting for only 14,874 cases in 2003.2 These numbers are deceptive, however, because TB rates among new immigrants, undocumented individuals, and racial and ethnic minorities resemble that of the developing world. Outbreaks and transmission continue to occur all over the United States, and newer challenges of imported drug resistance and “super-bugs” are of serious concern.

In November 2004, MLO published a cover story by Dr. James W. Brown, entitled “Tuberculosis: Keeping an ancient killer at bay.” The article described TB laboratory methods currently in use. In this article, we hope to showcase the current and potential impact on tuberculosis control of the newer, rapid, and more specific laboratory testing methods. In particular, rapid methods for diagnosis and drug-susceptibility testing, surveillance tools, and the new blood-based assays for the diagnosis of TB infection will be discussed.

Diagnosis of TB

The laboratory diagnosis of tuberculosis underwent a radical change following the resurgence of TB in the United States in the late 1980s and early 1990s.3 During this period, outbreaks of TB included deadly MDR-TB strains. These patients, many of whom were HIV-infected, often died of TB before culture identification and drug-susceptibility testing were completed by the mycobacteriology laboratory. In response, the CDC made recommendations for laboratory methods with the specific purpose of improving turnaround time.4 These recommendations included the use of fluorescent, acid-fast microscopy; selective broth medium with a sensitive growth- detection system; a rapid-identification system, such as DNA probes; and drug-susceptibility testing using a rapid broth system. Rapid culture and susceptibility testing reduced the time from sputum collection to growth from eight to 12 weeks to two to six weeks.

While these were definite improvements, the slow growth of Mycobacterium tuberculosis continues to require physicians who do not have the luxury of culture or susceptibility results to empirically treat TB. Furthermore, inexperienced physicians commonly continue to make the error of waiting for results before deciding to treat. This has resulted in delayed diagnosis and treatment, increased morbidity and transmission, and sometimes death.

In recent years, exciting new rapid nucleic acid amplification (NAA) testing has been adopted by a growing number of labs to detect and identify the presence of M tuberculosis directly in clinical specimens within six to 12 hours. Because of this, they are often called “direct amplification tests.” The U.S. Food and Drug Administration (FDA) approved two commercial assays for respiratory specimens: the Mycobacterium Tuberculosis Direct (MTD) from Gen- Probe and the AMPLICOR from Roche Molecular Systems. Limitations of these tests have been their relatively high costs, lack of national guidelines for their specific use, lower sensitivity than bacteriologic culture, and occasional false-positive results. They are useful in confirming tuberculosis when the pretest probability of TB is high, but poor at ruling out disease because of their lower sensitivity and negative predictive value.5 The potential use of NAA tests in nonrespiratory specimens has also been demonstrated in the difficult diagnosis of tuberculous meningitis by early confirmation, although the low sensitivity precludes ruling out the disease.6 Therefore, public health experts strongly encourage dialogue with providers who use their laboratories to maximize the benefit of these tests.

Development of criteria for use of NAA testing is currently being discussed at the national level. At issue is the utility of NAA testing for shortening the time-to-confirmation of active TB and for determining bacillary burden in smear-negative disease as it relates to releasing patients from high-level isolation rooms during hospitalization. The most cost-effective approach to using these tests is yet to be determined.

Treatment in the dark and drug susceptibility

Currently, drug-susceptibility testing is routinely performed after subculture of a positive specimen and causes a delay of 20 to 30 days from the date of sputum collection to reported results. This translates to “treatment in the dark” until results reveal whether patients have been placed on the appropriate drugs.

A recent innovation for mycobacteriology laboratories has been the development of molecular methods that detect drugresistant mutations within hours of specimen receipt. One of these methods, molecular beacons, was described in the November 2004 issue of MLO.7 This method uses fluorescent-labeled, hairpin-shaped DNA probes in a real-time PCR assay, with unfolding of the probes and development of fluorescence if the PCR products have the normal, nonmutated sequence. Other methods have been described, including a dual- probe, real-time PCR assay; DNA sequencing; DNA arrays to determine sequence changes in PCR products; and a commercially available line- probe assay, InnoLiPA Rif.TB, from Belgian biotechnology company Innogenetics.

Transmission of TB

The Inno-LiPA method is in routine use in some European countries, but has not been cleared by the FDA for use in the United States. Despite the number and variety of methods described in published studies, routine detection of drug resistance by molecular methods is not widely available in the United States. The Microbial Diseases Laboratoiy of the California Department of Health Services is offering molecular beacons testing for California patients when there is a specific need based on suspicion of drug resistance, exposure of a susceptible population that may need preventive therapy, or critical illness of a TB patient who may have difficulty tolerating seco\nd-line drug regimens.

The New York State Department of Health’s Wadsworth Center also is using molecular methods for detection of rifampin resistance in cultures from selected TB patients as a routine service. It is regrettable that this service is not more widely available. Early detection of rifampin resistance in a time frame of hours to days rather than the approximate four weeks turnaround time required by culture-based methods could prevent inappropriate treatment and reduce transmission and development of additional drug resistance. Rifampin resistance is a key indicator of multidrug resistance and requires a prolonged and fortified treatment of additional anti-TB drugs.

DNA fingerprinting and connecting the dots of transmission

Traditional molecular assays are being refined, while rapid strain genotyping is being studied and is becoming more available. Some of these methods have been described recently in MLO.7 Genotyping by IS6110 restriction fragment length polymorphism (RFLP) has been used for more than 10 years8 and has proven to be valuable for detecting outbreaks, particularly when traditional “shoe leather” contact investigations have failed to reveal contacts or locations linked to transmission.1′ Suspected laboratory cross- contamination incidents can be confirmed or refuted by RFLP, and the determination of the extent of cases caused by contamination, as indicated by the extent of clustered or matching fingerprints, has been useful in developing TB-control-program strategies.

RFLP, however, has its limitations in that a large cell mass is required, a week is necessary to perform the assay and evaluate the results, and results are band patterns that are difficult to convert into a digital format and reliably compare in order to establish differences or identity. For this reason, PCR-based strain-typing methods have been developed that require only a very small cell mass and yield numerical results that can be compared easily either within a laboratory or between laboratories to determine whether two strains are the same. These methods are:

* Spacer oligonucleotide typing (spoligotyping), which is based on differences between strains in the spacer sequences in the direct repeat locus of the M tuberculosis genome. An important technical advance was the adaptation of spoligotyping to the Luminex (Austin, TX) multianalyte profiling system.10 The Luminex-based method yields digital results that can be downloaded automatically to a database.

* Mycobacterial interspersed repetitive units (MIRU), which is based on variable numbers of tandem repeats at 12 loci in the genome of M tuberculosis.” Each locus is amplified using a set of specific primers, and the number of repeated sequences is evaluated by determining the size of the amplified product using a DNA sequencer. Again, the results are digital and downloadable into a database.

The CDC has funded two regional laboratories, the California Microbial Diseases Laboratory in Richmond, CA, and the state public health laboratory in Lansing, MI, to perform rapid strain typing. Use of the PCR-based spoligotyping and MIRU methods enables these laboratories to work with light growth (e.g., early growth in broth cultures) and to generate results quickly and efficiently. The goal for these laboratories is to produce initial strain-typing results within two weeks of receiving a pure, viable culture. It is hoped that with rapid detection of outbreaks and rapid identification of false-positive results due to laboratory cross-contamination, TB- control programs will be able to focus their efforts on critical opportunities for intervention in the spread of this disease. Genotyping is available at no charge by submitting cultures through the Michigan state public health laboratory.

Replacing the test we love to hate: the TST

The TST is nearly 100 years old and is still the standard of practice in the United States in diagnosing LTBI. This method suffers from problems with reproducibility and false-positive results in many patients who have been vaccinated with BCG vaccine or are colonized with nontuberculous mycobacteria. The need for two clinic visits – one to implant the antigen and a second to observe, measure, and record the result – often leads to many unread TSTs and reader errors. For many years, a more reliable and specific alternative has been desired to overcome the many operational limitations of the TST Those tests are finally here.

There are two commercial blood tests that have been developed recently for the diagnosis of TB infection, but only one of these is available for use in the United States. In December 2004, the FDA approved a new diagnostic test for M tuberculosis-complex infection: the QuantiFERON-TB Gold test (Cellestis Ltd., Melbourne, Australia). The other diagnostic, the T SPOT-TB test (Oxford Immunotec, Oxford, UK) is available in Europe and uses a different method of detecting interferon gamma (IFN-γ) from peripheral mononuclear cells, a procedure not yet established in clinical laboratories in the United States.

The advantages of the T-cell blood-based assays include results from a single patient visit and, because it is a blood test performed in a qualified laboratory, the elimination of the variability associated with the TST. Other advantages of this newer- generation blood test are that it is not affected by past BCG vaccination and it can eliminate unnecessary treatment resulting from TST false-positive results in would-be treatment-eligible patients. The elimination of the second visit for reading the TST is also likely to make the QFT-GoId competitive in cost-benefit considerations.

The basis of these new blood tests is the detection of IFN- γ released on stimulation of sensitized T-cells by a specific cocktail of M tuberculosis antigens that are absent from BCG strains. In die QFT-GoId test, whole-blood samples are drawn in heparinized tubes and mixed with these antigens in addition to a nil and mitogen control. T-cells in the blood of patients who have been infected with M tuberculosis will respond to the presence of these antigens by making IFN-γ, which is then detected by an enzyme immunoassay. In the T SPOT-TB assay, T-cells releasing IFN-γ to ESAT-6 and CFP-10 are detected and quantified as spot-forming cells.

Numerous studies that have been conducted throughout the world using these methods show higher specificity and better correlation with exposure to M tuberculosis.12-14 In Japan, QFT-GoId showed greater sensitivity in 118 confirmed active cases of TB (89% vs. 66% with the TST), rare positive reactions in 216 subjects who had received BCG vaccine, and minimal risk of exposure to TB when compared to the TST (specificity 98% and 35%, respectively, for QFT- GoId and TST).13 Remaining questions about QFT-GoId are in regard to the true sensitivity in detecting LTBI in contacts of active cases and its performance in immunocompromised hosts and children. Without a gold standard for LTBI, perhaps the question of sensitivity will be answered by long-term cohort studies of patients that determine risk of progression to active disease in untreated TB contacts with negative QFT-GoId results. Many studies are underway to address these issues.

Current limitations of QFT-GoId are that the cost of LTBI testing is shifted to the laboratory and that a fresh blood sample must be stimulated with antigen within 12 hours to assure viability of the T- cells. The time limit can create a logistical problem for reference laboratories that might wish to provide QFT-GoId for a broad region. A future generation of QFT-GoId, QuantiFERON-TB Gold InTube, is now available in Europe and is undergoing FDA evaluation. This version of the test provides direct antigen stimulation in the blood- drawing tubes and eliminates the maximum 12-hour processing requirement.

The adoption of this test requires a dramatic shift as the diagnosis of TB infection will be made in the laboratory instead of the clinic. This is a welcome shift in that laboratories are more likely to give reliable results because of routine quality assurance and proficiency testing, something that is not possible in all clinical settings in a given locale. The CDC is currently developing interim guidelines for the use of QFT-GoId that will be finalized in the coming months.

The future of TB laboratory services

A task force of the Association of Public Health Laboratories has published a report entitled “The Future of TB Laboratory Services,” which is available online at www.aphl.org. This report calls for integration of services provided by public and private laboratories with tuberculosis-control programs to provide an effective system for diagnosis, treatment, and control of TB. In instances where some of the newer molecular methods are not available or are not costeffective in most mycobacteriology laboratories, a system must be developed so that testing can be referred in a timely manner to a reference laboratory. A critical component of such a system will be to assure that results are sent to the appropriate TB-control program, as well as to the healthcare provider. Development and promotion of this effective public/private system is a challenge to and the responsibility of all mycobacteriology laboratory staff.

Moving to a new era of TB diagnostics

Because TB is an airborne infectious disease, rapid diagnosis and treatment of individuals with TB is the cornerstone of cutting the line of transmission and controlling disease in communities. This requires targeted screening of high-risk groups for the purpose of case finding and identification of individuals with latent TB infection at most risk of breakdown with disease. Reliance on symptom and risk-factor screening, TB skin testing, and sputum examination have been the current standard. While progress has moved us out of the “stone age” of TB diagnostics, there is still a long way t\o go before available rapid genetic probes, genotyping, and cell-mediated blood tests are fully implemented. Realizing this higher standard of practice will greatly impact patient care and public safety by allowing earlier, more accurate, and appropriate diagnosis and treatment of this age-old disease.

MLO and Northern Illinois University (NIU), DeKalb, IL, are co- sponsors in offering continuing education units (CEUs) for this issue’s article on TUBERCULOSIS AND THE EXPANDING ROLE OF THE LABORATORY. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. program (Provider No. 0001 ) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 12, answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1.0 contact hour of P.A.C.E. credit. Participants should allow four to six weeks for receipt of certificates.

The fee for each continuing education test will be $20.

All feature articles published in MLO are peer-reviewed.

Learning objectives and CE questions were prepared by Jeanne M. Isabel, MSEd, CLSpH(NCA), MT(ASCP), associate professor, School of Allied Health Professions, Northern Illinois University, DeKalb, IL.

1. Most physicians treat suspected cases of TB without waiting for drug-susceptibility information.

a. True

b. False

2. Rapid methods and molecular-based assays have shortened the time needed for diagnosis of smear-positive patients to

a. one month.

b. one week.

c. one day.

d. a few hours.

3. Standard lab confirmation of a multidrug-resistant case of TB would take six months for susceptibility information.

a. True

b. False

4. The traditional TB skin test may soon be replaced by a blood- based T-cell assay.

a. True

b. False

5. One reason TB continues to dominate infectious diseases globally is its ability to

a. infect.

b. become quiescent.

c. reactivate in the body later in life.

d. All of the above.

6. The estimated incidence of TB infected individuals worldwide is

a. 1.5million.

b. 1.7 million.

c. 1.2 billion.

d. 1.8 billion.

7. Resurgence of TB in the United States caused the CDC to recommend

a. changing drug susceptibilities.

b. reducing turnaround time.

c. changing the BCG vaccination.

d. performing chest X-rays only.

8. Rapid identification systems include all of the following, except

a. DNA probes.

b. rapid broth media.

c. T-cell assays.

d. fluorescent acid-fast microscopy.

9. Rapid nucleic acid amplification testing is useful in ruling out disease.

a. True

b. False

10. A method for detection of drug-resistant mutations within hours of specimen receipt is known as

a. molecular beacons.

b. Belgian beacons.

c. NAA assays.

d. RNA probes.

11. Rifampin resistance is a key indicator of multidrug- resistant TB.

a. True

b. False

Match the definitions to the correct term:

12. differences between strains in spacer sequences

13. latent TB infection

14. genotyping by IS6110 restriction fragment

15. variable numbers of tandem repeats at 12 loci

a. RFLP

b. MIRU

c. spoligotyping

d. LTBI

e. Inno-LiPA Rif.TB

16. Advantages of performing a blood test instead of the TST include all of the following, except

a. elimination of variability in skin test reading.

b. single patient visit.

c. elimination of unnecessary treatment.

d. affected by past BCG vaccination.

17. The new blood test for diagnosing TB infection is based on the detection of IFN-γ production, which is measured by enzyme immunoassay.

a. True

b. False

18. Adaptation of the QuantiFERON-TB Gold or T SPOT-TB assays would shift the diagnosis of TB infection from the clinic to the lab.

a. True

b. False

19. An effective tuberculosis-control program relies on services provided by both public and private labs.

a. True

b. False

20. Rapid culture and susceptibility testing reduce the time from sputum collection to growth to

a. six to 12 hours.

b. two to six days.

c. two to six weeks.

d. six to 12 weeks.

CE CONTINUING EDUCATION

To earn CEUs, see test on page 20

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Describe standard methods of lab confirmation of TB.

2. Describe new tests developed to replace the TB skin test.

3. Identify the incidence of TB worldwide.

4. Describe evolving methods for diagnosis of TB.

5. Define types of molecular assays being used byTB-control programs.

Traditional molecular assays are being refined, while rapid strain genotyping is being studied and is becoming more available.

The TST is nearly WO years old and is still the standard of practice in the United States in diagnosing LTBI.

References

1. Dolin PJ, Raviglione MC, Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ. 1994;72:213-220.

2. CDC. Reported Tuberculosis in the United States, 2003. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention, September 2004.

3. Cantwell MF, Snider DE Jr, Cauthen GM, Onorato IM. Epidemiology of tuberculosis in the United States, 1985 through 1992. JAMA. 1994;272:535-539.

4. Tenover FC, Crawford JT, Huebner RE, Geiter LJ, Horsburgh CR Jr, Good RC. The resurgence of tuberculosis: Is your laboratory ready? J Clin Microbiol. 1993:31:767-770.

5. Forbes BA, Pfyffer G, Eisenach KD. Molecular diagnosis of mycobacterial infections. In: Cole ST, Eisenach KD, McMurray DN, Jacobs WR, eds. Tuberculosis and the Tubercle Bacillus. Washington, DC: ASM Press. 2005:85-98.

6. Pai M, Flores LL, Pai N, Hubbard A, Riley LW, Colford JM Jr, et al. Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. Lancet Infect Dis. 2003;3:633-643.

7. Brown JW. TB: Keeping an ancient killer at bay. MiO Med Lab Obs. 2004; 36(111:8-19.

8. van Embden JDA, 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. Barnes PF, Cave MD. Molecular epidemiology of tuberculosis. N Engl J Med. 2003;349:1149-1156.

10. Cowan LS, Diem L, Brake MC, Crawford JT. Transfer of a Mycobacteriiim tuberculosis genotyping method, spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system. J Clin Microbiol. 2004;42(11:474- 477.

11. Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. MoI Microbiol. 2000:36:762-771.

12. Pai M, Riley LW, Colford JM Jr. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis. 2004;4:761-776.

13. MoriT, Sakatani M, Yamagishi F, et al. Specific detection of tuberculosis infection with an interferon-gamma-based assay using new antigens. XIm J RespirCritCare Med. 2004;170(1 ):59-64.

14. Kang YA, Lee HW, Yoon HI, et al. Discrepancy between the tuberculin skin test and the whole-blood interferon gamma assay for the diagnosis of latent tuberculosis infection in an intermediate tuberculosis-burden country. JAMA. 2005:293:2756-2761.

15. Ferrara G, Losi M, Meacci M, et al. Routine hospital use of a commercial whole blood interferon-gamma assay for tuberculosis infection. Am J Respir Crit Care Med. June 16 2005; (Epub ahead of print).

By L. Masae Kawamura, MD, and Edward Desmond, PhD

L. Masae Kawamura, MD, has served as director of the Tuberculosis Control Section of the San Francisco Department of Public Health for seven years. She is an assistant professor of Medicine at the University of California-San Francisco, and is co-primary investigator of the Francis J. Curry National Tuberculosis Center, one of four regional training and medical consultation centers for tuberculosis in the United States funded by the CDC’s Division of Tubercuosis Elimination. Dr. Kawamura is currently chair of the Advisory Council for the Elimination of TB, which reports to the U.S. Department of Health and Human Services.

Edward Desmond, PhD, is chief of the Mycobacteriology and Mycology section, Microbial Diseases Laboratory (MDL), California Department of Health Services. MDL performs strain typing of Mycobacterium tuberculosis complex cultures for the western half of the United States, with funding by the CDC. Dr. Desmond’s laboratory also performs molecular beacons testing on acid-fast, smear- positive sputum samples and cultures to detect M tuberculosis and drug resistance. A graduate of Santa Clara University, Dr. Desmond has worked in laboratories in a community hospital, a Veterans Administration hospital, and a large urban medical center.

Copyright Nelson Publishing Aug 2005