New Diagnostic Tests for Tuberculosis: Bench, Bedside, and Beyond

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1 SUPPLEMENT ARTICLE New Diagnostic Tests for Tuberculosis: Bench, Bedside, and Beyond Susan E. Dorman Johns Hopkins University Center for Tuberculosis Research, Baltimore, Maryland Current tools and strategies for diagnosis of tuberculosis (TB) are inadequate, particularly in settings with a high prevalence of human immunodeficiency virus (HIV) infection. Several promising new tools are at advanced stages of development and evaluation. This review describes some of those promising new technologies and the key barriers to their effective implementation. Robert Koch s recognition in 1882 of Mycobacterium tuberculosis as the microbial cause of tuberculosis (TB) led shortly thereafter to the identification of methods to stain bacilli in clinical specimens, rendering the organisms visible with use of light microscopy. Such was the birth of TB diagnostics and of diagnostic microbiology in general. Tragically, development and implementation of TB diagnostics kept pace neither with medical technology nor with the catastrophic explosion of TB, including drug-resistant TB, in the wake of the global human immunodeficiency virus (HIV) pandemic. Inadequate tools and weak systems for laboratory-based diagnosis of active TB have contributed to (1) underdiagnosis of disease, leading to individual morbidity and mortality and to continued transmission; (2) overdiagnosis of disease, leading to unnecessary treatment with attendant consequences to the patient and inappropriate resource utilization by the health care program; and (3) delayed diagnosis of drug resistance, leading to acquisition of additional resistance and to morbidity and transmission. Besides deficiencies in diagnostic tools, access to TB diagnostics continues to be a major challenge. However, notable advances in TB diagnostic technologies have been made in the past several years, and the potential exists for translating Reprints or correspondence: Dr Susan E. Dorman, Johns Hopkins University Center for Tuberculosis Research, CRB2, 1550 Orleans St, Rm 1M-06, Baltimore, MD, (DSusan1@JHMI.edu). Clinical Infectious Diseases 2010; 50(S3):S173 S by the Infectious Diseases Society of America. All rights reserved /2010/5010S3-0014$15.00 DOI: / these developments into meaningful improvements in global TB clinical care and control. This review focuses on types of technologies currently in the TB diagnostics pipeline and identifies areas of progress and gaps in knowledge that are relevant to moving the field forward. The interferon-g release assays, which are principally for detection of M. tuberculosis infection, were reviewed recently and are not included here [1]. DIAGNOSTIC TOOLS USED ROUTINELY FOR DIAGNOSIS OF ACTIVE TB Acid fast staining of clinical material, followed by smear microscopy, remains the most frequently used microbiological test for detection of TB. The major drawback of sputum smear microscopy is its poor sensitivity, estimated to be 70% in a recent systematic review [2]. However, the sensitivity of sputum smear microscopy is clearly less in many field settings and may be as low as 35% in some settings with high rates of TB and HIV coinfection [3]. Compounding the limitation of poor test sensitivity is inadequate or absent test quality assurance in some resource-constrained settings, further driving down the yield of microscopy, driving up the laboratory workload as more sputum tests per patient are performed in an effort to reach a diagnosis, and increasing diagnostic delay and patient loss to follow-up. Drug susceptibility status cannot be ascertained from smear microscopy. The HIV pandemic has brought into focus the inadequacy, from individual and public health perspectives, of sputum smear microscopy as the cornerstone of TB diagnosis in low- and middle-income settings. New Diagnostic Tests for TB CID 2010:50 (Suppl 3) S173

2 HIV infection dramatically increases the incidence, severity, and mortality risk of active TB [4, 5, 6]. At the public health level, failure to diagnose disease in a large proportion of HIV-infected patients with smear-negative TB may contribute to transmission [7] and further stresses health care and/or personal resources, because individuals remain in the health care system without correct diagnosis and treatment or exit the system and probably die. Culture of M. tuberculosis in clinical specimens is substantially more sensitive than smear microscopy. Culture can be performed using solid media, such as Lowenstein-Jensen, or liquid media, such as that used in commercially available automated systems. Until the recent advent of molecular tests for drug resistance (described in the next section), isolation of M. tuberculosis with use of culture was a prerequisite for subsequent phenotypic drug-susceptibility. The Achilles heel of culture is the long time to results (10 14 days for liquid culture and 3 4 weeks for solid culture), which is a consequence of the long doubling time of M. tuberculosis. Currently available culture methods are technically demanding, require implementation of biosafety practices and equipment to prevent inadvertent infection of laboratory personnel, and have relatively high per-test prices. The Global Tuberculosis Report 2008 documents the stunning lack of culture facilities in the government health sector in most developing countries, some of which have a single laboratory that may or may not be well resourced or quality assured [8]. Tuberculin skin using purified protein derivative and chest radiographs are used as adjuncts to smear microscopy (and culture, if available) in some settings; however, the former have poor sensitivity and specificity for active TB, and the latter are often not available at the point of primary patient care. Trials of antibiotics directed against common bacterial pneumonia pathogens are often recommended in TB program diagnostic algorithms but are also fraught with problems and may lead to lengthy diagnostic delay. NEW DIAGNOSTIC TECHNOLOGIES AND TOOLS There is a clear need for development, introduction, and effective implementation of cost-effective new tools that contribute to improvement in patient-centered outcomes and public health and that perform well for HIV-infected and HIV-uninfected individuals. The Stop TB Partnership Working Group on New TB Diagnostics has placed priority on accurate, simple new tools for TB case detection, rapid identification of drug-resistant TB, and reliable detection of latent TB infection [9]. Against a backdrop of increased need fueled by HIV infection and drug-resistant TB, advances in biology (including the solution of the M. tuberculosis genome in 1998 [10]), and emergence of public-private partnerships involved in global health, engagement in TB diagnostics development and evaluation has increased among public health and academic groups, government funding agencies, and importantly, the industry sector. The result has been an expansion in the number of promising diagnostic tests under development, including 2 new tests (in addition to liquid culture) that have been endorsed for use by the World Health Organization. Table 1 lists some of the more promising new technologies and tests currently in demonstration or late-stage evaluation phase [11, 12]. BEYOND NEW TECHNOLOGY Successful implementation of new tools will depend on more than technological innovation (Figure 1). At the research level, rigorous implementation of well-designed, bias-minimized studies and complete and accurate reporting are essential for appropriate decision making by the health care community charged with implementing tests for individual patient evaluation or recommending tests for TB program use. The Standards for Reporting of Diagnostic Accuracy Initiative has developed standards and tools for improving the quality of reporting of diagnostic accuracy studies, to best allow the reader to detect the potential for bias in a study and to assess the generalizability and applicability of study results [13]. Assessment of test impact on relevant clinical outcomes should become a routine component of late-stage evaluation research. Diagnostic accuracy is arguably just a surrogate for patientand public health oriented outcomes. Economic assessments are important during the continuum of diagnostics research. During device development, cost estimates can guide the need for device modifications to facilitate use in settings with the greatest need. Later, during the evaluation and demonstration phase, cost-effectiveness and cost-benefit analyses can provide information critical to policy development and implementation. Operations and health systems research is also needed to understand how to effectively implement new tools in relevant settings where existing access to and delivery of health care are weak. New programmatic approaches, including revised clinical algorithms for TB diagnosis, may be needed to maximize the impact of new tools. For example, should rapid molecular tests for drug resistance be performed for all persons with suspected TB during initial evaluation, be reserved for use in the initial evaluation only of persons with suspected TB with risk factors for drug resistance, or be used in some other place in a diagnostic algorithm? In populations with a high prevalence of HIV infection, should urine-based antigen detection tests be used solely for evaluation of symptomatic persons with suspected TB, or should they also play a role in routine screening of HIV-infected persons [14]? To date, most TB diagnostic test development has focused on maximizing sensitivity and spec- S174 CID 2010:50 (Suppl 3) Dorman

3 Table 1. Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health Organization (WHO), and in Later Stages of Development Method Products Intended and/or typical use Level of health system Main strengths Main weaknesses In use Smear microscopy for acid-fast bacilli (light microscopy) Culture on solid media Many commercialized prepared media and reagents Noncommercial Rapid, point-of-care test for TB case detection TB case detection and as prerequisite to drug-susceptibility Community Requires moderate training; minimal infrastructure; minimal equipment Low sensitivity Referral laboratory Good sensitivity Slow time to growth Chest radiograph NA TB case detection (pulmonary TB) Referral Indications and use not restricted to TB Tuberculin skin test Many commercialized reagents Detection of M. tuberculosis infection Interferon-g release assays QuantiFERON-TB Gold (Cellestis); T-SPOT.TB (Oxford Immunotec) Trial of antibiotics directed against routine bacterial pneumonia pathogens Automated, nonintegrated NAAT Endorsed by the WHO Detection of M. tuberculosis infection NA TB case detection for persons with suspected pulmonary TB whose sputum smear results are negative Amplified Mycobacterium tuberculosis direct test (Gen-Probe); Amplicor M. tuberculosis test (Roche) Culture in liquid media MGIT (Becton Dickinson); BacT/ ALERT (BioMérieux); others Strip-based species identification (detects TB-specific antigen in positive cultures) Line probe manual amplification and hybridization Community Extensive practical and published experience Low specificity; low sensitivity; requires equipment, trained interpreter Sensitivity decreases with increasing immunocompromise; cross-reaction with BCG vaccine Referral to rference laboratory Highly specific for M. tuberculosis Requires moderate training and equipment; imperfect sensitivity, especially for immunocompromised persons Community May be clinically beneficial to patients with bacterial pneumonia TB case detection (pulmonary TB) Reference laboratory Sensitivity between that of smear and culture; highly specific for TB TB case detection and as prerequisite to drug-susceptibility Capilia TB (Tauns) Species identification (TB versus not TB) in cultures positive for mycobacterial growth Genotype MTBDRplus (Hain Lifescience); INNO-LiPA Mycobacteria (Innogenetics) TB case detection and drug-susceptibility Referral laboratory High sensitivity (higher than culture on solid media) Referral laboratory (with culture) Accurate; requires minimal training; minimal equipment; minimal consumables Reference laboratory Poor sensitivity in smear-negative specimens; relatively short time to result Poor discriminatory power; engenders time delay in further evaluation and care for patients with TB Requires moderate training and equipment; labor intensive; potential for cross-contamination among specimens Slow time to detection (although faster than culture on solid media); high contamination rates in some settings Labor intensive; potential for cross-contamination; requires extensive training NOTE. Adapted from [11, 12]. BCG, bacille Calmette-Guérin; ELISA, enzyme-linked immunosorbence assay; LAMP, loop-mediated isothermal amplification; LED, light emitting diode; MODS, microscopic observation drug susceptibility; NA, not applicable; NAAT, nucleic acid amplification test.

4 Figure 1. assurance. Components of the post research-and-development process for promising new tuberculosis (TB) diagnostic technologies. QA, quality ificity to rule in or confirm a TB diagnosis. On the other hand, a test with an exceedingly high negative predictive value might have use in ruling out TB and, thereby, allowing efficient triage of patients and resources; such a test would require careful assessment to determine its optimal use in clinical algorithms. Laboratory capacity needs to be strengthened, especially in resource-limited settings. Although some aspects of laboratory strengthening will vary according to the characteristics of the new tests, there are, nevertheless, general unmet needs, including those for training at technologist and management levels, retention of trained personnel, enhancement of quality-assurance systems, enhancement of results-reporting mechanisms, and reliable mechanisms for instrument maintenance and supply procurement. Strengthening of HIV (and in some instances TB) laboratory capacity under the US President s Emergency Plan for AIDS Relief and related programs serves as a useful model, as does the successful collaborative program undertaken by the Foundation for Innovative New Diagnostics, Partners in Health, the World Health Organization, and the Lesotho Ministry of Health and Social Welfare to strengthen the National Reference Laboratory of Lesotho [12]. A number of important barriers exist with respect to the full engagement of industry and investors in diagnostics development. Barriers include uncertainty about the size and/or accessibility of the TB diagnostics market, especially in developing countries; complex regulatory processes; unfavorable intellectual property rights protections; and in some instances, lack of knowledge about the types of tests that are most needed and likely to be relevant. A recent analysis indicates that, worldwide, 1US $1 billion is spent annually on TB diagnostics [15]. Of interest, approximately three-quarters of all diagnostic tests are performed outside established market economies; however, this burden accounts for only one-third ( US $326 million) of the current market, because the most common tests performed are sputum smear microscopy and chest radiograph, which have relatively low per-test costs [15]. In countries with established market economies, tuberculin skin is the most frequently used TB test, in accordance with the relatively low rates of TB and relatively high prioritization of detection and treatment of latent TB in those settings. The blood-based interferon-g release assays, including the QuantiFERON-TB Gold tests (Cellestis) and T-SPOT.TB (Oxford Immunotec), can detect TB with a higher degree of specificity than can the tuberculin skin test and are now approved for use in the United States and a number of other countries. To date, in the United States, use of these tests as replacements for or adjuncts to the tuberculin skin test has not been widespread, but momentum appears to be growing. Funding for TB diagnostics research by the top 40 TB research funding institutions in 2007 was estimated at US $41.9 million, less than the amount for TB drugs (US $170 million) and TB vaccines (US $71.2 million) and more than the amount for TB operational research (US $36.8 million) [16]. This level of funding falls woefully short of the Global Plan to Stop TB s recommendations of at least US $900 million per year for research and development of new tools for TB diagnosis, treatment, and prevention [17]. Support for technical assistance to national TB programs as they implement and monitor new tools cannot be underestimated. Funding estimates aside, it is clear that important advances in TB diagnosis have recently been made, and potentially useful new tools are emerging; continued and augmented investment will be required to success- S176 CID 2010:50 (Suppl 3) Dorman

5 fully implement the most promising of these tools in the settings where they are most needed and to maintain a robust pipeline that will ultimately yield the tools that revolutionize TB diagnosis. TOOLS IN THE PIPELINE: TRANSFORMATIVE OR INCREMENTAL GAINS? Most of the tools in demonstration or late-stage evaluation are sputum based and, thus, are likely to result at best in incremental gains in TB case detection. Their yield is expected to be suboptimal for patients with TB who have only extrapulmonary TB, who have respiratory disease in which a relatively large burden of organisms is not in communication with the airways, and who cannot provide a respiratory specimen for. Nevertheless, effective implementation might, over time, have a substantial impact on TB control through detection of a very high proportion of individuals with capacity to transmit infection to others (provided diagnosis is sufficiently prompt and treatment is available). Highly accurate, simple to perform, point-of-care tests amenable to of readily available clinical specimens, such as urine or blood, and with ability to detect and predict active TB anywhere in the body, in combination with effective preventive and treatment strategies (as described in other articles in this Supplement), are needed. Truly transformative change will require more than a perfect sputum test, but a really good sputum test would be a step in the right direction. CONCLUSIONS What s new in TB diagnostics? A lot, but not enough. The future is brighter as several promising new tools enter the demonstration and late evaluation stages. But the need is great, and important barriers remain in translating technical advances into meaningful and sustainable improvements in individual and public health in settings hardest hit by TB. Acknowledgments Potential conflicts of interest. S.E.D.: no conflicts. Financial support. National Institutes of Health (HHSN C). Supplement sponsorship. This article is part of a supplement entitled Synergistic Pandemics: Confronting the Global HIV and Tuberculosis Epidemics, which was sponsored by the Center for Global Health Policy, a project of the Infectious Diseases Society of America and the HIV Medicine Association, through a grant from the Bill & Melinda Gates Foundation. References 1. Pai M, Zwerling A, Menzies D. Systematic review: T cell-based assays for the diagnosis of latent tuberculosis infection an update. Ann Intern Med 2008;149: Steingart KR, Henry M, Ng V, et al. Fluorescence versus conventional smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006;6: Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 2003;163: Page KR, Godfrey-Faussett P, Chaisson RE. Tuberculosis-HIV coinfection: epidemiology, clinical aspects, and interventions. In: Raviglione M, ed. 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