INVITED REVIEW SERIES: TUBERCULOSIS

SERIES EDITORS: WING WAI YEW, GIOVANNI B. MIGLIORI AND CHRISTOPH LANGE

Advances in the diagnosis of tuberculosis

CHRISTOPH LANGE1 AND TORU MORI2
1

Clinical Infectious Diseases, Research Center Borstel, Borstel, Germany, and 2Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo, Japan

ABSTRACT
Tuberculosis ranges among the leading causes of morbidity and mortality worldwide. A diagnostic approach to a patient with possible tuberculosis includes a detailed medical history and clinical examination as well as radiological, microbiological, immunological, molecular-biological and histological investigations, where available. Recently, important advances have been achieved in these elds that have led to substantial improvements in the accuracy and the timing of the diagnosis of tuberculosis. Novel methods allow for a better identication of latently infected individuals who are at risk of developing active tuberculosis, they also offer the possibility for a rapid diagnosis of active tuberculosis in patients with negative sputum smears for acid-fast bacilli and enable prompt identication of drug-resistant strains of Mycobacterium tuberculosis directly from respiratory specimen with a high accuracy. In addition, promising methods that will further optimize the diagnosis of tuberculosis are under development. In the future, therapeutic interventions based on the results of novel diagnostic procedures can be made earlier leading to improvements in patient care. Key words: diagnosis, imaging microbiology, immunology, tuberculosis.

INTRODUCTION

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Tuberculosis ranges among the leading causes of morbidity and mortality worldwide. Based on surveillance and survey data, the World Health Organisation (WHO) estimates in the latest report from the year

C.L. is a Pulmonologist and Infectious Diseases specialist. He is leading the Division of Clinical Infectious Dieseases and the Center for Clinical Studies at the Medical Clinic of the Research Center Borstel, Germany, and he is chairing the Tuberculosis section of the European Respiratory Society (ERS) and the Tuberculosis Network European Trials group (TBNET). T.M. is a tuberculosis expert with a focus on epidemiological research and public health, and has been involved with tuberculosis control in Japan as well as in the developing world for the past 40 years. Correspondence: Toru Mori, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24, Matsuyama, Kiyose, Tokyo 204-8533, Japan. Email: tmori-rit@jata.or.jp Received 27 October 2009; invited to revise 30 October 2009; revised 5 November 2009; accepted 5 November 2009. 2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology

2009 that 13.7 million individuals were living with active tuberculosis in the year 2007 (206 per 100 000 population) and 9.27 million people (139 per 100 000 population) developed tuberculosis in that year. Among the 1.76 million persons who died from tuberculosis in the year 2007, 1.3 million were seronegative and 455 000 seropositive for HIV infection.1 In clinical practice the rapid detection of individuals with tuberculosis can be difcult,2 as only 44% of all new cases (and only 1520% of children3) are identied by presence of acid-fast bacilli (AFB) on sputum smears.1 The gold standard for the diagnosis of tuberculosis is the detection of Mycobacterium tuberculosis, the causative microorganism of tuberculosis. In fact, whenever M. tuberculosis is recovered from human specimens by microbiological culture the diagnosis of active tuberculosis is regarded as denite. However, culture growth of M. tuberculosis may take 2 or more weeks on average. The ad hoc decision to initiate anti-tuberculosis treatment can be difcult in cases where AFB are not found on sputum smear microscopy despite the clinical suspicion of tuberculosis. The clinical diagnosis of active tuberculosis then classically relies on the results of different methods, including the tuberculin skin test (TST), chest radiography, amplication of M. tuberculosis nucleic acids and/or pathological examinations from biological specimens (Fig. 1). In this article we review the epidemiology and clinical manifestation of tuberculosis and we discuss recent advances that allow a better and earlier diagnosis of active pulmonary tuberculosis in clinical practice. A continuous update on evidenced-based tuberculosis diagnosis can also be found at http:// www.tbevidence.org

CURRENT TRENDS IN THE EPIDEMIOLOGY OF TUBERCULOSIS

The world can be divided into two parts based on the extent of tuberculosis epidemics. One part is the lowprevalence areas. They are composed of countries that experienced serious tuberculosis epidemics after the 18th century but have gradually overcome them and have nally reduced the incidence rate to 100 per 100 000 or less. The other part is the high-prevalence
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Figure 1 Flow diagram for the diagnosis of tuberculosis in clinical practice. *NTM NAAT may be helpful, when available. In accordance with WHO recommendations (WHO. Treatment of tuberculosis. Guidelines for national programmes. Geneva; 2003), clinical response to antibiotic therapy may be considered before further investigations; however, in countries of low TB incidence immediate further diagnosis with bronchoscopy can be indicated at this stage to better rule out other diseases. BAL, bronchoalveolar lavage; IGRA, interferon-g release assay; MTB, Mycobacterium tuberculosis; NAAT, nucleic acid amplication test; NTM, non-tuberculous Mycobacteria; TB, tuberculosis; TBB, tubercle bacilli; TST, tuberculin skin test; WHO, World Health Organisation.

areas comprising countries with an incidence rate exceeding 100 per 100 000 that have suffered tuberculosis epidemics after the turn of the 20th century. The low-prevalence countries are industrialized countries, while the high-prevalence countries are mostly developing counties or areas. The latter accounts for two-thirds of the world population, but as much as 95% of the estimated number of newly occurring tuberculosis patients (of all forms) globally. Furthermore, 98% of tuberculosis deaths occur in these highprevalence areas.1 Tuberculosis accounts for 2.7% of the total disability-adjusted life-years in low- and middle-income countries.4 In addition to the difference in its level, there are clear differences in characteristics of tuberculosis disease. In high-prevalence countries, most tuberculosis patients are in their 20s to 40s, resulting in tremendous socioeconomic loss as this is the most productive generation. In contrast, among low-prevalence countries, tuberculosis is drifting to involve the elderly, socioeconomically marginalized people, medical high-risk groups (e.g. diabetics5 and those treated with immunosuppressive agents, such as TNF-alpha blockers6), which presents a challenge to both medical and welfare services.
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As a consequence of the global efforts in tuberculosis control under the Directly Observed Treatment Short-course strategy since the 1990s, the incidence of tuberculosis is estimated to have started to decline for the rst time around 2003, although very slowly.1 At the same time, issues that had been given only lower priority in the developing world have emerged as unavoidable challenges. One of these issues is multi-drug resistant (MDR) tuberculosis that strikes a half million people annually and is a malignant burden to the patients and community, as well as to national tuberculosis programmes with its poor treatment outcome.7 In line with this problem, extensively drug resistant (XDR) strains of M. tuberculosis are emerging recently.8 The use of effective secondary drugs based on the result of high-performance drug sensitivity tests is necessary in order to address these issues, which requires technical innovation.7 A second newly emerging issue is co-infection of the HIV and M. tuberculosis. Currently, 15% of the new tuberculosis patients are infected with HIV, and in some areas or countries this proportion exceeds 50%. One quarter of the global tuberculosis deaths are due to HIV, and this is equal to one-third of new
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HIV-positive tuberculosis cases and to 23% of the estimated two million HIV-related deaths in 2007.1 Diagnosing tuberculosis in these subjects with sputum smear examination alone cannot prevent their infectiousness and save their lives; more aggressive case-nding and treatment of smear-negative cases are required. Another issue is tuberculosis in children for whom Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccination has been virtually the only control measure in developing countries. This also requires accurate diagnosis in the early stage of tuberculosis.9 As seen above, tuberculosis appears as a typical south-north problem of health, but currently in many developed countries over half of the new tuberculosis cases are foreign-born, that is, immigrants from highprevalence areas, or spill-over of tuberculosis.10,11 It is actually argued that to further reduce tuberculosis in the low-prevalence countries it is necessary to strengthen control efforts in the high-prevalence, developing countries.12

C Lange and T Mori

several point-scoring systems, diagnostic classications and diagnostic algorithms have been developed to support an objective diagnostic judgment. Marais et al. tested such an approach and found that combining a persistent non-remitting cough lasting over 2 weeks, documented deterioration of health (in the preceding 3 months) and fatigue provided reasonable diagnostic accuracy in HIV-uninfected children (sensitivity 62.6%; specicity 89.8%; positive predictive value 83.6%). The performance was poorer in HIV-infected children than in the low-risk group, which offers a serious challenge in resourcepoor settings with high HIV epidemics.109 However, given this set of sensitivity and specicity, the positive predictive value is calculated as only 24% in a patient population with a prevalence of tuberculosis of as high as 5%.

Tuberculosis in old ages

In low-prevalence situations, tuberculosis is a problem predominantly of the aged population and includes many more cases of clinical development in immunologically compromised subjects. This is why there are many tuberculosis cases with atypical clinical presentation(s) in older persons.110,111 Elderly patients are more likely to have extrapulmonary tuberculosis, including miliary disease.111 The proportion of bacteriologically conrmed pulmonary tuberculosis patients was higher in the elderly than in the younger patients as reported in a meta-analysis.112 Fever, sweating and haemoptysis are less frequent in older patients, but dyspnoea is more frequent.112 Laboratory ndings, such as the TST-positive rate, serum total protein level and white blood cell counts, were lower in elderly patients. Also, cavity formation was less common in elderly patients, while lesions in the upper lung were similar for both age groups.112 The most common chest X-ray ndings in the elderly or immunocompromized tuberculosis patients are lesion in the lower zone accompanied by basal effusion or thickening.110 Such atypical clinical presentation of tuberculosis in the elderly can often cause delay in diagnosis, which can be further complicated due to underlying illnesses.

CLINICAL MANIFESTATION OF TUBERCULOSIS (PULMONARY AND EXTRAPULMONARY AND CHILDHOOD TUBERCULOSIS)

In general, clinical manifestations of illness are the rst clue to the diagnosis of tuberculosis. However, they are often non-specic and misleading, and therefore the diagnosis is not always easy. This is especially the case for tuberculosis in children and in elderly subjects, as seen below. Further, extrapulmonary tuberculosis often poses a challenge to early diagnosis, especially pertaining to its variety of presentations. Clinical signs and symptoms of tuberculosis of the organs other than the lungs are summarized in Table 1.

Tuberculosis in children
Because of its paucibacillary nature, tuberculosis of children is difcult to diagnose. Bacteriological conrmation seldom exceeds 3040% among children in developed as well as developing countries.106,107 Consequently, the diagnosis of tuberculosis in children in resource-poor settings is largely dependent on a combination of a history of contact with a known tuberculosis patient, clinical signs and symptoms, and special examinations, such as chest radiography and the TST when available. Edwards and colleagues observed a total of 91 tuberculosis cases younger than 15 years, of whom about half were HIVinfected, and found the following frequency of symptoms and signs in the HIV-seronegative children: weight loss 69%, fever 100%, cough 83%, night sweat 43%, fatigue 21%, tuberculosis contact 60%, malnutrition 57%, lymphadenopathy 88%, organomegaly 31%, positive TST 89%, elevated erythrocyte sedimentation rate 79%, and chest X-ray inltration 100%.108 Based on these observations,
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Delays in diagnosis
One of the basic indicators of quality in diagnosing tuberculosis is the delay in diagnosis (doctors delay, or health systems delay), that is, the time from the rst visit of a patient until the establishment of tuberculosis diagnosis. Figure 2 depicts the delay separately for high-, intermediate- and lowprevalence settings, together with the patients delay, that is, the time from the onset of clinical symptoms until the rst visit to a health facility, based on published studies (T. Mori, pers. comm.). It is remarkable that these delays in the low-prevalence settings are always longer than those in the high-prevalence settings.
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Table 1 Clinical presentations and laboratory examinations for diagnosis and differential diagnosis for major types of extra-pulmonary tuberculosis134,302
Site
Pleurisy

Symptoms & signs

Primary infection cases have generally an acute course of symptoms than reactivation-type TB. Cough (non-productive) and chest pain (pleuriticsharp, stabbing, associated with respiration). Feverishness, dyspnea, chills, sweats, weight loss in more advanced cases. TB pleural effusions are generally exudates. Most often in the neck and head region, rare in the axillary & inguinal region. Right predominates but 1/4 have biliateral & 78% have multiple lesions. 41% have pulmonary TB. In supercial LN, lesion starts as a painless enlargement, with no inammation over the skin, then may undergo pustulation and stulation over several weeks or months. In a case with limited lesion, general symptoms are rare. Commonest in vertebral column, followed by hip and knee. Fever and wasting may appear in large inammatory collections, but the local manifestation predominates. Pain is commonest. Soft tissue collection (cold abscess) at/near the bone or joint focus. Neurological signs (weakness or numbness from compression of the spinal cord). Diverse depending on the organs involved. Feverishness, weakness or debility, anorexia, weight loss, headache (meningeal complication), abdominal pain/swelling (peritoneal involvement), cough.39,40 The presentation depends on the size and location of the tuberculoma and the pressure it produces.Early symptoms (feverishness, malaise, anorexia, irritability, headache) followed by neurological symptoms (progressive headache, lethargy, personality changes, memory disturbance, impaired cognition, confusion), and then stupor-coma with or without neurological decit. Frequency: peritonitis, followed by ileocaecal, anorectal and mesenteric lymph node infection.In peritoneal TB, abdominal swelling, fever, ascites, pain, anorexia/weight loss are common.62,63 Dyspnoea, tachycardia, neck vein distension, oedema, hepatomegaly, paradoxical pulse, pericardial rub, fever.74,75

Laboratory tests
Radiography, sputum bacteriology (for undiagnosed pulmonary TB), thoracocentesis with pleural uid for cell prole, protein, pH, glucose, LDH, smear, culture, NAAT,1316 ADA,1720 IFN-g,20,21 IGRA,2226 pleural biopsy for histology, culture and NAAT.27,28

Differential diagnosis
Effusions due to congestive heart failure, carcinoma, other types of infections and rheumatological disorders.

Lymphadenopathy

Culture isolation of MTB, chest radiography, biopsy (total excisional) followed by bacteriological culture/ PCR28 and histology. Fine needle aspiration.29

NTM infections, lymphoma, sarcoidosis, Kikuchis disease, Castlemans disease, Kimuras disease, corynebacterium pseudotuberculosis lymphadenitis.30

Bone & Joint

MTB from aspirate (abscess, synovial uid) and biopsy specimen (e.g. synovia), 3134 CT & MRI.3538

Disseminated or miliary TB

Central nervous system

Chest radiography (often no abnormality in the beginning), CT (HRCT),41,42 beroptic bronchoscopy, TBLB,43 haematology (anaemia, leukopenia or leukocytosis, rarely leukemoid reaction). Liver function, bone marrow biopsy, liver biopsy (including NAAT44), fundoscopy.45 Cerebrospinal uid for pressure, cellularity, protein, glucose,46 MTB (microscopy, culture47,48 and PCR49,50), immunology (ELISA, IgG immune complex, antibody assays and IGRA5156), and ADA. Radiography, CT, MRI.5761 Meningeal biopsy (histology, MTB).

For arthritis: pyogenic, rheumatoid, gout, regional osteoporpsis, idiopathic chondrolysis. For cystic bone lesions: eosinophilic granuloma, sarcoidosis, cystic angiomatosis, plasma cell myeloma, fungal infection, metastatic malignancy. Alveolar microlithiasis, disseminated carcinoma, sarcoidosis, NTM infections, hypersensitivity pneumonitis.

Other infections (fungal, viral, trypanosomal, bacterial), vascular (multiple emboli, SBE, thrombosis of sagittal vein), collagen vascular (SLE, polyarteritis, and others).

Abdominal

Peritonitis: ultrasound,6466 laparoscopy (with guided biopsy),6770 paracentesis of ascites for culture and IGRA71,72 and ADA.73 Pericardial tissue/uid for bacteriology, histology,76,77 IGRA,78,79 and ADA.8082 Echocardiography,8385 CT and MRI (pericardial effusion and thickening),86 ECG (low voltage, inversion of T).87

Malignant ascites, cirrhosis with spontaneous bacterial peritonitis, starch peritonitis, sarcoidosis, NTM peritonitis. Bacterial (e.g. Pneumococcus), viral (e.g. CMV, HSV, Coxsackievirus) or fungal (e.g. Aspergillus) infections; collagen vascular diseases; uremia; post-myocardial infarction or post-pericardiotomie; malignancy; trauma. Benign and malignant tumor, cystic kidney, pyelonephritis, xanthogranulomatous pyelonephritis, urinary malakoplakia.

Pericarditis

Genitourinary

Dysuria, frequency, nocturia, urgency, pain in the back, ank or abdomen, tenderness/swelling of the testis or epididymis, haematuria. Superimposed urinary tract infection with other bacteria in urinary stasis cases.8893

Urine or secretion (early morning specimen) for MTB (smear, culture, PCR),94 ultrasound,95 plain abdominal radiograph, i.v. urography (high-dose), image-intensied endoscopy, percutaneous antegrade pyelography,96102 biopsy for suspect of genital lesion.103105

ADA, adenosine deaminase; CT, computed tomography; ECG, electrocardiogram; HRCT, high resolution computed tomography; IGRA, interferon-gamma release assay; LDH, lactate dehydrogenase; LN, lymph node; MRI, magnetic resonance imaging; MTB, Mycobacterium tuberculosis; NTM, non-tuberculous mycobacterium; PCR, polymerase chain reaction; SBE, subacute bacterial endocarditis; SLE, systemic lupus erythematosus; TB, tuberculosis; TBLB, transbronchial lung biopsy. 2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology Respirology (2010) 15, 220240

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ADVANCES IN THE IMAGING DIAGNOSIS OF TUBERCULOSIS

None of the radiological abnormalities seen in pulmonary tuberculosis are pathognomonic for this disease. Nevertheless, several features are typical of tuberculosis. Classical radiographic abnormalities have recently been reviewed.120 They include features of primary tuberculosis, for example, unilateral hilar lymphnode enlargement, parenchymal airspace consolidation and/or pleural effusion,121123 or features of reactivation tuberculosis, for example, focal or patchy heterogeneous consolidation involving the apical and posterior segments of the upper lobes and the superior segments of the lower lobes, poorly dened nodules, linear opacities and cavitations.123125 While the classication in primary and reactivation tuberculosis is still widely en vogue, evidence from genotype ngerprinting studies conrms that the radiographic feature in tuberculosis following recent and remote infection are very similar and that integrity of the immune system predicts the appearance of the patterns of active tuberculosis on chest imaging: immunocompromized individuals (e.g. those with advanced HIV infection) having the appearance of primary tuberculosis and immunocompetent individuals having the appearance of reactivation tuberculosis.126,127 The conventional chest X-ray is still the most commonly used method for screening, diagnosis and follow up of treatment responses in patients with pulmonary tuberculosis. However, chest computed tomography (CT), in particular high-resolution CT, is more sensitive than conventional chest X-ray to identify early parenchymal lesions or mediastinal lymph node enlargements and to determine disease activity in tuberculosis.128131 Radiographic features on CT that are suggestive of active tuberculosis include cavitations and parenchymal abnormalities and/or centrilobular nodules and the tree-in-bud pattern.120 Recently, serial pulmonary [(18)F]-2-uoro-deoxyD-glucose positron emission tomography has been investigated as a promising non-invasive method to monitor disease activity and responses to antituberculosis chemotherapy.132,133 Although highly expensive, this technique could be useful and even be cost-effective for the managment of patients with MDR and XDR tuberculosis in selected cases.

Figure 2 Box plots of delay in case detection for countries with high, intermediate and low prevalence of tuberculosis. Figures in parentheses indicate the number of studies analysed. (a) Patients delay (time a patient needs from the occurence of the rst symptoms to seeking healthcare) and (b) health systems delay (time to establish the diagnosis of tuberculosis after the rst presentation of a patient to a healthcare facility).

Sasaki et al. reviewed the diagnostic process of private practitioners with Japanese patients and concluded that insufcient medical work-ups, including AFB examinations of sputa and chest X-rays of subjects with a high suspicion for tuberculosis, was the principal cause of delayed diagnoses.113,114 In Hong Kong, general practitioners practice was reviewed, and it became clear that they depend too much on X-rays rather than sputum examinations, and that they were slow in referring tuberculosis patients to the government tuberculosis service.115 RozovskyWeinberger et al. compared the management of suspect tuberculosis cases at three public hospitals and seven not-for-prot private hospitals in the USA in terms of their rates of ordering acid-fast smears and isolations, and urged private hospitals to be more alert to tuberculosis.116 Similar reviews of hospital management were reported by several other studies,117119 the results of which illustrate the need for improved education of doctors. All of these studies urge a higher index of suspicion for tuberculosis in medical staff in low-prevalence countries.
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MICROBIOLOGICAL DIAGNOSIS: CONVENTIONAL METHODS

Remarkable efforts have been made globally to accelerate the development and expansion of new diagnostic technologies. However, tuberculosis case detection still remains dependent upon sputum smear and culture, radiography and clinical symptomatology, and currently 57% of global tuberculosis patients receive a bacteriological diagnosis. Therefore, efforts to improve the quality of existing methods
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chemical processing is more sensitive with similar specicity.137 Operational studies are needed to determine the balance between the benet from increased sensitivity and the costs in terms of complexity and potential biohazards. In order to enhance the sensitivity of sputum smear tests, the examination must be done three times, but this principle was challenged so that the third examination adds very little to the rst two examinations, at least in laboratories with good-quality control.138141 This is incorporated in the International Standards of Tuberculosis Care in routine practice.142 If a patient cannot produce sputum, any method for sputum induction is encouraged. This is especially benecial to ensure high sensitivity of sputum smear tests in resource-poor settings where such drastic methods as gastric washing or bro-optic bronchoscopy cannot be used.143 It was shown that induction performed well in developing countries with little added costs.144 Recently, a new device for sputum induction called the lung ute has been developed and may be worth trying145 (refer to Table 2 for collecting and processing specimens for the diagnosis of tuberculosis).

are necessary, and there actually have been certain achievements in this direction.

Sputum smear examination by microscopy

One recent achievement in conventional tuberculosis microscopy is the recognition of the benet of uorescent microscopy for enhancing sensitivity over that of ordinary light microscopy without any loss in specicity.135 The uorescence microscopy widely used in resource-rich countries has been accepted as more sensitive than ordinary microscopy, although with a concern of loss of specicity, especially under conditions in the developing world. Nonetheless, a recent literature review has conrmed that it may be also benecial in the latter as well. This could be further improved by attaching a stronger light source called an ultra-bright (LuminTM, LifeEnergy, Germany) lightemitting diode.136,140 Another systematic review on the sputum processing in sputum smear testing demonstrated that centrifugation combined with any of several chemical methods (including bleach) is more sensitive, and overnight sedimentation preceded by

Table 2 Biological specimen for the diagnosis of tuberculosis Specimen

Sputum Induced sputum Bronchial secretion or bronchoalveolar-lavage Gastric aspirate

Amount
25 mL 25 mL 25 mL

Application
A, B, C A, B, C A, B, C, D

Preservation/transport
Unprocessed Unprocessed Unprocessed

Comment
3 in the morning on an empty stomach Expectoration following inhalation of 3% NaCl solution BAL-ELISPOT should be performed on the day of sample collection Only when sputum cannot be obtained and bronchoscopy (BAL) is not indicated (1) Not in formalin

>2 mL

A, B, C

In 12 mL phosphate buffer (trinatrium phosphate) (1) In 0,9% NaCl for microbiological examination; (2) in formalin for histopathological examination Unprocessed Unprocessed Unprocessed

Biopsy, survival specimen (e.g. lmph nodes)

2 separate portions (1) and (2)

A, B, C, E

Pleural effusion, ascites Cerebrospinal uid Urine

20 mL 23 mL 30 mL

A, B, C, D, A, B, C, D A, B, C

Stool Blood

510 mL 510 mL

A, B, C A, B, C, D

Unprocessed Heparin- or lithium-citrate tubes (1) In heparin- or lithium-citrate tubes; (2) air-dried smears and/or formalin preserved biopsies

Bone marrow

2 separate portions (1) and (2)

A, B, C, E

ELISPOT should be performed on the day of sample collection ELISPOT should be performed on the day of sample collection 3 First specimen of urine in the morning Fluid restriction the evening/night before 3 Indicated only in immunosuppressed patients Do not use EDTA blood Indicated only in immunosuppressed patients Biopsy or aspirate for (1) not in EDTA or formalin

Application in different tests: A, microscopy; B, culture; C, NAAT; D, IGRA; E, histopathology. BAL, bronchoalveolar lavage; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot; EDTA, ethylenediaminetetraacetic acid.
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The quality of smear examination has become widely recognized as so important that the need for implementing quality assurance in every laboratory has been strongly advocated.146

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MICROBIOLOGICAL DIAGNOSIS: MOLECULAR METHODS

Nucleic acid amplication techniques
The M. tuberculosis-specic nucleic acid amplication tests (NAAT) performed on bronchopulmonary specimen are the most frequently used molecular tests for laboratory diagnosis of pulmonary tuberculosis. NAAT results can be available to the clinician within 1 day after obtaining sputum or bronchoalveolar lavage (BAL) uid and can have important implication for the management of a patient. Unfortunately, NAAT amplication targets are not standardized and the diagnostic accuracy of the tests is highly heterogenous. The clinical value of in-house and/or commercial NAAT performed on respiratory specimens for diagnosis of pulmonary tuberculosis have recently been repeatedly reviewed in meta-analyses.161164 In individuals with positive AFB sputum smears, the sensitivity of NAAT to detect M. tuberculosis nucleic acid on these specimens is greater than 95%.161,162 When AFB are found on sputum or BAL smears, the presumptive diagnosis of tuberculosis can thus be rapidly conrmed. Apart from rare exceptions, a negative NAAT result in this situation strongly indicates the presence of a non-tuberculous Mycobacteria (NTM) species in this specimen. In contrast, in individuals with negative AFB sputum smears, the estimated sensitivity of NAAT for the diagnosis of active tuberculosis is highly heterogeneous (especially when in-house assays are compared) and is not consistently accurate enough to be routinely recommended for the diagnosis of tuberculosis.163,164 In general, nested NAAT methods, and the use of IS6110 as the amplication target are related to a higher diagnostic accuracy. In individuals with a negative sputum smear, the specicity of NAAT for the diagnosis of active tuberculosis has been 97% and 98% in an earlier metaanalysis and an independant recent study.161,165 A positive result in a M. tuberculosis-specic NAAT performed on a respiratory specimen is therfore highly indicative of pulmonary tuberculosis. However, in our experience far less than 50% of patients with smearnegative tuberculosis have a positive sputum or BAL NAAT result.165 False positive results are seen in individuals with a past medical history of tuberculosis and in patients with bronchogenic carcinoma.

Progress in culture examinations

Since the 1990s, a series of culture examination systems has been developed using liquid media for rapidly detecting M. tuberculosis. A systematic review demonstrates that these liquid cultures are more rapid and sensitive than solid medium cultures.147,148 The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460, compared with 27.0 days with Lowenstein Jensen solid medium.148 Thus, WHO recently endorsed the use of liquid tuberculosis culture and drug susceptibility testing for M. tuberculosis in low-resource settings.149 The newly developed rapid liquid culture systems have unique sensing systems to detect a small amount of bacterial growth, such as by radioactivity or oxygen concentration changes, as quickly as possible. These systems can also be used for drug susceptibility testing as well as detecting M. tuberculosis.150,151 Novel diagnostic test using mycobacteriophages to identify M. tuberculosis from biological specimen require only 2 days of turnaround time in the laboratory. They have a high specicity (range 83% to 100%), but lack sufcient sensitivity (range 21% to 88%) to substitute conventional culture techniques.152 Bacteriophage-based assays have also been developed for the rapid detection of rifampicin resistance in M. tuberculosis. However, the diagnostic accuracy of these assays is insufcient when applied directly to clinical isolates.153 Still other systems based on phenotypes have been devised mainly for drug susceptibility testing. One such system is the microscopic observation drug susceptibility,154 where the characteristic growth of M. tuberculosis in the liquid medium in a well is checked under an inverted light microscope. In another system, bacterial growth is conrmed from the bacterial activity to reduce nitrate to nitrite in the liquid media, which is indicated by the change in colour of the media (nitrate reductase assay).155,156 Other colorimetric redox indicator methods are also being evaluated for the rapid detection of MDR in M. tuberculosis.157 These DST methods can be applied to clinical sputum samples and have been shown useful with performance comparable to genetic methods, in their sensitivity and specicity, and the mean time to results was 2123 days.158 Given the technical difculty of the procedure and the wide variation of the results, the need for quality assurance is indeed very pressing for drug susceptibility testing, especially with solid media. WHO/ IUATLD established a system of prociency testing to quantify the technical level of local laboratories159 and introduced the system of a supra-national reference laboratory network in which a laboratory so designated should technically support the nearby local laboratories.160
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LINE PROBE ASSAYS

Line probe assays are NAAT to detect common genomic mutations responsible for antibiotic resistance from a biological probe or culture by DNA hybridization (GenoType MTBDR assay, Hain Lifescience, Nehren, Germany166 or INNO-LiPA Rif. TB kit, Innogenetics, Zwijndrecht, Belgium167). In brief, the tests involve DNA extraction, multiplex NAAT, solid phase reverse hybridization on the test strip and detection of the resistance mutations.168170 The Genotype MTBDRplus assay detects several mutations in
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Table 3 Types and nature of serodiagnostic methods (based on134,177182)
Antigens 38 kDa, 16 kDa, 88 kDa, MPT51, malate synthase, CFP-10, TbF6 polyprotein, antigen 85B, antigen A60, antigen 5, alpha-crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6-tetraacyltrehalose 2-sulfate (SL-1), cord factor, tuberculophosphatide, lipoarabinomannan, Rv3425 Protein, lipid, polysaccharide (and their complex) Single antigen, multiple antigen Native, recombinant IgG, IgA, IgG (single or combined) Enzyme-linked immunosorbent assay, immunochromatography, immunodot rapid test, kaolin agglutination test

the rpoB gene, in the katG gene and the inhA gene promoter regions.168,171174 In a meta-analysis, the pooled sensitivity in resistance detection on clinical specimen for rifampicin was similar to conventional DST following culture. However, the pooled sensitivity for isoniazid-resistance testing was less optimal at 85% (7292%).175 The latest version of the line probe assays, the Genotype MTBDRsl assay in addition can detect genetic mutations that are related to drug resistance of strains of M. tuberculosis, including those for uoroquinolones and injectable drugs (amikacin or capreomycin) enabling the rapid diagnosis of XDR tuberculosis in >85% of all cases, including direct testing on clinical specimen.176

Compound Composition Source Ig class Laboratory technique

IMMUNOLOGICAL DIAGNOSIS
Avances in serology for antibody/antigen detection
There has been a long history of developing systems to diagnose tuberculosis based on the serological reaction, that is, detection of a specic antibody. Currently, the development of such systems is very urgently needed due to the pressure for strengthening earlier diagnosis of diseases in the paucibacillary stage, including pulmonary tuberculosis with negative sputum smears of adults, extrapulmonary tuberculosis, childhood tuberculosis and tuberculosis patients with HIV coinfection. The system must be operationally simple for use at the point of care in the developing world and must have rapidity, in addition to diagnostic accuracy in terms of sensitivity and specicity. Sometimes the systems are specically expected to detect latent tuberculosis infection (LTBI) and monitor the progress of tuberculosis treatment. However, in contrast to many cases of other acute bacterial and viral infections, there are several barriers to the successful application of the serological reactions for diagnosing tuberculosis, including the gap between active disease and latent infection, the wide prole of the disease from one with extensive cavitary lesions to an almost inactive, minimal disease, and distinction from NTM infection.134 These characteristics of tuberculosis comprise formidable factors against sensitivity and specicity of the expected diagnostics. There is a long list of systems so far proposed and developed as serological diagnostics, each having different characteristics in terms of antigens used and other characteristics (Table 3). Recently, Steingart and colleagues conducted a systematic review and meta-analysis of the published studies of these techniques. They found 254 studies evaluating 51 distinct single antigens and 30 multipleantigen combinations in terms of their performance in diagnosing pulmonary tuberculosis.177 The authors noted that the data in sputum smear-negative or pediatric patients were not enough but concluded that none of the antigens sensitivity was high enough to replace sputum smear microscopy. In a separate review, the same authors group made a similar conclusion for extrapulmonary tuberculosis.178
2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology

Furthermore, WHO/TDR evaluated commercially available tuberculosis tests with regard to their performance, reproducibility and operational characteristics.180 They used 355 well-characterized archived serum samples to evaluate 19 rapid tuberculosis tests at one laboratory. The sensitivity of these rapid tests ranged from 1% to 60%; the specicity, from 53% to 99%; and in general, tests with high specicity had very low sensitivity. Test performance was poorer in patients with sputum smear-negative tuberculosis and in HIV-positive patients. Again they concluded that none of the assays performed well enough to replace microscopy. As suggested by the above reviews, combinations of existing potential candidate antigens may enhance sensitivity without losing much specicity, and therefore related research should be pursued, as well as efforts to discover novel antigens. At the same time, the quality of assessing new techniques should be improved, including a better study design and development of effective performance indicators beyond sensitivity and specicity, and establishment of a tuberculosis specimen bank, such as the one used in the WHO/TDR project above. Currently available serological tests cannot be recommended for the diagnosis of tuberculosis.

Advances in cellular immunodiagnosis

The TST and interferon-g release assays (IGRA) evaluate in vivo (TST) and ex vivo (IGRA) the presence of persistent mycobacteria-specic T cell responses.183,184 They are indirect marker for past or present infection. TST and IGRA performed on peripheral blood alone cannot distinguish between individuals with LTBI, active tuberculosis or past tuberculosis.185,186
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228 Tuberculin skin test

The TST was developed by the Austrian paediatrician Clemens v. Pirquet as an allergy-test for the diagnosis of tuberculosis in children.187 It has been the standard test for the immunodiagnosis of tuberculosis since the beginning of the 20th century. Despite the recent invention of IGRA, the TST is still much more widely used as a screening method for the identication of persons with a positive immune response against M. tuberculosis.188 A standard preparation of puried protein derivate (PPD), an extract of the sterile supernatant of M. tuberculosis-cultered ltrate, is administered intradermaly and results in a delayed type hypersensitivity reaction represented by a local skin induration.189 For the best test result reliability, TST reactions in humans are ascertained by the diameter of induration, measured 4872 h after antigen injection with the ballpoint technique.190 A recent meta-analysis stated that the overall sensitivity of the TST for active tuberculosis is 77%;191 however, the sensitivity of the test can be dramatically impaired, for example, in infants and toddlers192 as well as in elderly persons,193 in individuals with congenital or acquired immunodeciencies (e.g. those with HIV infection194196), patients being treated with corticosteroids197 or other immunosuppressive drugs,198 patients with chronic renal failure,199,200 malnutrition,201,202 cancer203 or overt forms of tuberculosis.204,205 The specicity of the TST is dependent on the BCG vaccination status206 and the immune status of the individual who is tested.191 Cross-reactivity of antigens may result in a positive TST reaction after exposure to NTM207 or following M. bovis BCG vaccination.206,208 TST induration reactions exceeding 15 mm are likely related to tuberculosis or LTBI,209 irrespectively of the BCG vaccination status.210 While the sensitivity of the TST decreases from a cut-off of 5 to 10 and 15 mm, the specicity of the TST increases with the increase of the cut-off used to dene a positive induration.211,212 Depending on the level of exposure to an index case in contact tracing and the immune status of the individual, different cut-offs for a positive test reaction have been recommended ranging from 5- to 15-mm induration.213 Recently, results of a phase I trial of a skin test that uses recombinant early secretory antigenic target (ESAT)-6 instead of tuberculin have demonstrated safety and tolerability of such a test.214 In combination with culture ltrate protein (CFP)-10 antigen to increase diagnostic sensitivity, such a skin test could overcome some of the obstacles currently related to the use of the TST. If clinical trials show superiority to the TST this test could be made widely available for the diagnosis of LTBI in resource-limited settings where the use of IGRA is prohibited by their costs and demands for an established laboratory infrastructure.

C Lange and T Mori

Interferon-g release assays

Introduction of IGRA into clinical practice is regarded by many as the most important development in the
Respirology (2010) 15, 220240

diagnosis of M. tuberculosis infection over the last decade. IGRA is a coupling of the discovery of antigens ESAT-6 and CFP-10, which are relatively specic to M. tuberculosis,215 and the development of simplied technologies of measuring interferon-g. There are two commercialized systems for the latter technology. QuantiFERON-Gold (QFT-G) (Cellestis Ltd, Carnegie, Australia216) measures interferon-g in IU/mL using an enzyme-linked immunosorbent assay (ELISA) and T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK217) counts the cells releasing interferon-g visualized as spots with the enzyme-linked immunospot (ELISPOT) technique. During the last several years, these systems have been approved in various countries and the ndings of their diagnostic performance have been accumulated and characterized. The QFT-G test is now available as an in tube version (QFT-G-IT), which also includes, in addition to ESAT-6 and CFP-10, the antigen TB7.7. We present a summary of the performance of these systems in various settings, based on review and meta-analysis.191,218 IGRA were originally intended to diagnose LTBI, but because there is no gold standard of tuberculosis infection, the active disease is usually used as a surrogate for the infection when quantifying sensitivity. Specicity is measured in subjects with low risk of M. tuberculosis infection, for example, healthy young subjects without known contact with tuberculosis patients. As indicated in Table 4, the specicity of IGRA is consistently high and obviously superior to TST, whereas sensitivity is rather variable between studies. This variability may be greatly ascribed to the difference in patients characteristics in terms of tuberculosis disease condition, age, extent of immunosuppression due to underlying illnesses, etc. However, IGRA generally perform better than TST in its sensitivity. Comparing QFT-G and T-SPOT.TB, T-SPOT.TB seems to be more sensitive than QFT-G, and vice versa for specicity. This comparison is clearer when they are compared head to head in the same subjects.191 The same is also true for the comparison between QFT-G and QFT-G-IT, where the latter exhibits higher sensitivity in head-to-head comparison,219 perhaps due to addition of the third antigen TB7.7, while the difference was in an opposite direction in the comparison between the different subject groups, as seen in series 1 and series 2 in Table 4. The performance of the IGRA for the immunodiagnosis of M. tuberculosis infection has been investigated in immunocompromised hosts, such as in HIV-infected,195,220233 elderly,234236 chronicrenal-failure237239 patients and those taking corticosteroids240242 or TNF-alpha blockers.243248 In general, the responses to IGRA (T-SPOT.TB > QFT-GIT) are more frequently present in individuals from these patient groups when compared with the TST. This is commonly interpreted that these assays are superior to the TST to detect LTBI.218,233 Apart from the performance using tuberculosis patients as surrogates of M. tuberculosis infection, there are arguments concerning the discordance between IGRA and TST in those suspected of recent infection.249,250 However, it is now considered that IGRA may reect the dynamics of infection immunity
2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology

Diagnosis of tuberculosis
Table 4 Summary sensitivity and specicity of IGRA (meta-analysis) Series
Sensitivity 1 2 3 4 7 8 Specicity 1 2 3 4 5 6

229

Diagnostics

Subject

No. studies

Summary

Range

QFT-G QFT-G-IT QFT-G/G-IT QFT-G/G-IT, T-SPOT.TB T-SPOT.TB TST QFT-G/G-IT QFT-G/G-IT QFT-G/G-IT T-SPOT.TB TST TST

TB patients, adult TB patients, adult TB patients, child HIV-infected TB patients TB patients Healthy subjects Healthy young adults Healthy young adults, BCG(-) Healthy young adults, BCG(+) Predominantly BCG vaccinated BCG not vaccinated BCG vaccinated

21 6 9 5 13 20 12 8 8 8 6 6

0.80 0.74 0.82 0.70 0.90 0.77 0.98 0.99 0.96 0.93 0.97 0.59

(0.780.82) (0.690.78) (0.750.87) (0.600.79) (0.860.93) (0.710.82) (0.970.99) (0.981.00) (0.940.98) (0.861.00) (0.950.99) (0.460.73)

0.620.95 0.640.93 0.531.00 0.630.85 0.831.00 0.571.00 0.921.00 0.951.00 0.890.99 0.851.00 0.931.00 0.350.79

Figures in parentheses in column Summary indicate 95% condence limits. For specicity, several studies under series 1 are included in series 2 or 3. IGRA, interferon-gamma release assay; QFT-G, QuantiFERON-TB Gold; QFT-G-IT, QuantiFERON-TB Gold In-Tube; TB, tuberculosis; TST, tuberculin skin test.

more sensitively, so that the interferon-g level may uctuate above and below the cut-off.251 Similar concern is raised about the predictability of the future clinical development according to the IGRA response level, which is the main purpose of testing contacts for possible latent infections. One report suggests the higher risk of developing tuberculosis in cases with higher response at the time of infection.252 This should be further conrmed and the discussion should be expanded to the level of response that persists after many years of infection. For the diagnosis of tuberculosis in nonimmunocompromised hosts the best use of IGRA is to rule out active tuberculosis,186 as the negative predictive value for tuberculosis is higher than 95% if combined IGRA and TST test results are negative.253,254

When quantied in the supernatants from whole blood stimulated with ESAT-6 and CFP-10 antigens in individuals with tuberculosis IP-10 > MCP-2 was found highly upregulated, when compared with presumptively uninfected controls.279 However, the diagnostic accuracy of the IP-10 or MCP-2 assay was not superior when compared with the QFT-G-IT assay and this test was also not able to identify individuals with active tuberculosis when performed with cells from the peripheral blood.

Local immunodiagnosis for active tuberculosis by IGRA

The inability to distinguish individuals with active tuberculosis from those with LTBI by blood tests is likely related to the fact that effector memory T cells are not highly prevalent in this compartment in active disease.254,280,281 However, in active tuberculosis antigen-specic T cells clonally expand and are concentrated at the site of infection.22,51,71,78,282286 In suspects of tuberculosis with negative AFB sputum smears, comparison of systemic (peripheral blood) and local (BAL) T cell responses against mycobacterial antigens assayed by ELISPOT are useful to rapidly distinguish cases of active pulmonary tuberculosis from those with LTBI.165,287289 As the majority of patients with active pulmonary tuberculosis have negative AFB sputum smears, local immunodiagnosis for active tuberculosis by BAL-ELISPOT can have an important impact on the early diagnosis of tuberculosis. In a recent clinical trial local immunodiagnosis for mycobacteria-specic T cells by BAL-ELISPOT had a sensitivity and specicity for the detection of sputum AFB smear-negative pulmonary tuberculosis of 91% and 80%, respectively.165 BAL-ELISPOT was markedly more sensitive for the rapid diagnosis of
Respirology (2010) 15, 220240

Other biomarkers for tuberculosis disease status and diagnosis

Evaluation of other biomarkers for active tuberculosis or LTBI is a research priority in the eld of tuberculosis. The details on the advances that have been achieved in the search for new biomarkers have recently been reviewed255262 and they are beyond the scope of this review (a selection of methods under development for the diagnosis of tuberculosis is shown in Table 5). Based on the IGRA technology, other combinations of M. tuberculosis-specic antigens253,254,273275 and cytokine readouts265,276 are being explored to improve the accuracy of these assays for the diagnosis of tuberculosis and LTBI. Among the biomarkers under investigation, interferon-induced protein IP-10, a CXC chemokine and monocyte chemoattractant protein MCP-2, a CC chemokine, have been clinically evaluated.266,276279
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230

Table 5 Selected methods under investigation for the diagnosis of tuberculosis

Method

Investigational target

Specimen

Sensitivity/specicity

Test results available within

Citation
263 264 265

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FACS analysis of short-term stimulated blood or cell cultures

Blood Sputum Blood Blood 82%/97%

100%/86% 89%/80% Not yet determined

24 h 24 h 24 h 24 h

Frequency of CD27+ lymphocytes Identication of intracellular IFN-g Identication of IFN-g- and IL-2-secreting cells IFN-g-inducible protein IP-10

266

165 22 51 71 267

Chemokine production following stimulation with ESAT-6/CFP10/ TB7.7 IFN-g-production following stimulation with ESAT-6/CFP10 BAL Pleural uid Cerebrospinal uid Peritoneal uid Blood Blood 89%/69% 91%/80% 95%/76% 90%/100% 89%/78% 92%/94%

24 h 24 h 24 h 24 h 4 days 24 h

IFN-g-production following stimulation with antigens different from ESAT-6/ CFP10

253

Blood Blood

73%/71% 94%/95%

24 h Several days

254

Proteomics

268

Identication of IFN-g-secreting cells Identication of IFN-g-secreting cells Identication of IFN-gg-secreting cells Identication of IFN-g-secreting cells IFN-g production following stimulation with heparin-binding hemagglutinin IFN-g production following stimulation with Rv3879c peptides in addition to ESAT-6 and CFP10 IFN-g production following stimulation with RD-1 selective peptides Mass spectrometry analysis of serum proteins to identify a TB-specic ngerprint Lipoarabinomannan Urine Sputum Sputum Breath 18%/88% 95%/100% 100%/94% 83%/100%

4h 24 h 24 h 24 h

269

Detection of MTB-specic cell wall components Gold nanoparticle probe assay Hybridization of MTB-specic DNA with gold nanoparticle probes Hybridization of MTB-specic DNA Identication of volatile biomarkers

270

271

Loop mediated isothermal amplication assay Breath test

272

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2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology

BCG, Bacille Calmette Guerin; CD, cluster of differentiation; FACS, uorescence activated cell sorting; IFN-g, interferon-g; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; RD, region of difference. The reported sensitivities and specicities from these pilot studies may not reect the diagnostic accuracy of the tests in clinical practice.

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with previously known parameters of C-reactive protein and neopterin. The combination of these four biomarkers provided diagnostic accuracies up to 84%.268 Apart from serological diagnosis, lipoarabinomannan is considered as an attractive urine marker in an antigen capture ELISA-based system for detecting tuberculosis. According to the evaluations of a commercial kit, both sensitivity of this lipoarabinomannan ELISA were low,269 but the sensitivity was greater in HIV-seropositive subjects in some of the studies,299,300 which suggests the possible efcacy to use it in combination with sputum smear examination in HIV-infected patients.

sputum AFB smear-negative tuberculosis than M. tuberculosis-specic NAAT. A similar diagnostic accuracy of the BAL-ELISPOT for the diagnosis of AFB smear-negative pulmonary tuberculosis was recently observed in a study performed in the Republic of South Africa, although in this study up to 1/3 of test results were inconclusive due to failure of the positive and negative controls.290 In countries of high tuberculosis incidence, pulmonary immune responses to antigens of M. tuberculosis assayed by ELISPOT may be different from those observed in individuals from areas of low incidence of tuberculosis due to the frequent exposure to M. tuberculosis.291 For clinicians, BAL-ELISPOT may thus be most applicable for a rapid decision to initiate antituberculosis treatment in countries of low tuberculosis incidence, where bronchoscopy is routinely performed for individuals suspected to be affected by sputum AFB smear-negative tuberculosis and where the technology for ELISPOT is available.

CONCLUSION
The good combination of diagnosis and treament is the most critical element of tuberculosis control and it will remain so until the advent of novel vaccines or drugs powerful enough to prevent development of tuberculosis perfectly. In the middle of the 20th century the treatment of tuberculosis made a revolutionary progress with the development of a series of chemotherapeutics, while only very little change was seen in the diagnostics. This caused disruption of the above combination leading to a low case detection rate in contrast with a fairly high treatment success rate as we see today worldwide. However, tuberculosis control is not possible, if the diagnosis of active cases is delayed as M. tuberculosis continues to be transmitted from cases to contacts. In addition, false positve diagnosis of LTBI has caused unnecessary burden to individuals and healthcare systems. The urgent need for innovation in diagnostics is obvious. However, it is good to see that the changes in diagnostics have started towards the end of the last century, assisted by the progress of biotechnology and the late risers alertness to the problem. The balance between developments in the diagnosis and in the treatment of tuberculosis has changed. Recent diagnostic advances overweigh the inefcient progress of new drug development against tuberculosis by far. Today, we have the technology to rapidly identify individuals with smear-positive MDR or XDR tuberculosis, but we do not have the drugs to treat these patients adequately.8,301 This article has overviewed such changing aspects of each of the established diagnostic techniques as summarized in Table 6. Every technique listed here was discussed in respective chapters focussing on its current and possible further improvement. Also, we have reviewed the ongoing efforts of innovations for novel modalities. Many of them are quite promising, so that we will be able to make it out of the antiquated techniques and make a full use of new techniques tted to various situations. Only in that way can we combine our case-nding activities well with treatment services that are still progressing in order to make our tuberculosis control maximally effective.
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Fluorescence-activated cell sorting

Immunophenotyping of antigen-stimulated cells by uorescence-activated cell sorting of BAL cells286,292,293 or sputum cells264 has also been performed in order to obtain a rapid diagnosis of tuberculosis in suspects with negative AFB sputum smears. While multicolour ow cytometry analysis allows for a better identication of cell populations that react following antigen encounter, the method is technically more demanding than IGRA for the immunodiagnosis of tuberculosis. In individuals with LTBI, BAL cells294 and sputum T cells264 are preferentially enriched for PPDspecic lymphocytes, and ow cytometry assays stimulated with PPD do not distinguish individuals with active tuberculosis and LTBI. Unfortunately, the frequency of region of difference-1 M. tuberculosisspecic T cells is too low in the sputum to be used as stimulants for ow cytometry cultures and other immune based assays.264,295 However, where available, ow cytometry is a very promising tool to further improve the diagnostic accuracy of local immunodiagnostic assays for the diagnosis of AFB smearnegative tuberculosis.286

Other possibilities of tuberculosis diagnosis

Being the science of the structure and function of proteins, proteomics has identied proteins signicant as diagnostic or prognostic biomarkers, or as therapeutic targets in a range of illnesses, including tuberculosis.296298 Agranoff et al. applied this technique to analysis of serum proteomic ngerprinting, or individual recognition of proteomic prole, for the purpose of distinguishing subjects with and without tuberculosis.268 Its diagnostic efcacy was fairly good with sensitivity of 93.5% and specicity of 94.9%. Also, to translate from proteomic signatures to conventional test formats, they identied amyloid A and transthyretin from highly informative peaks, and measured their levels by immunoassay, together
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232

Table 6 Comparison of established methods for the diagnosis of active tuberculosis

Advantage
<1 h Very important Very important Standard diagnostic <1 h Minutes <1 h

Method

Disadvantage

Duration

Clinical signicance

Medical history Clinical examination Imaging

Conversation, review of medical records Physical examination

CXR

Risk for tuberculosis may not be obvious, clinical symptoms may be non-specic Clinical signs may not be obvious or specic Wide spectrum of differential diagnoses

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Information on the individual risk for TB Identication of the severity of the illness Inexpensive, rapid, low exposure to radiation Superior quality compared with CXR (higher resolution). May identify minimal active lesions Inexpensive, widely available 4872 h Images are not pathognomonic for TB. Higher exposure to radiation compared with CXR Positive test results in individuals with a history of BCG vaccination. Reduced sensitivity in immunocompromised individuals. Reading of the test result requires a second visit IGRA responses from peripheral blood do not allow a discrimination of active TB and LTBI. More expensive than the TST 1 day Requires appropriate lab-facility 1 day Very high specicity (T-SPOT.TB < QTF-G-IT) High sensitivity (T.Spot.TB > QTF-G-IT) for LTBI Can be adapted to comparatively assess immune responses in the periphery (blood) and at the site of disease (e.g. BAL) Inexpensive, rapid, low technical demands No differentiation between MTB and NTM species Diagnostic accuracy of in-house methods may be highly variable. Limited sensitivity in smear-negative cases Substitutes TST in routine clinical practice, especially where BCG vaccination is prevalent 2h Standard diagnostic procedure 12 days Requires special laboratory facilities. Not sensitive enough in smear-negative cases Differentiates MTB from NTM. Very high specicity, very high sensitivity when acid-fast bacilli are seen on sputum smears. Rapid amplication of genes that are related to drug resistance forms the basis of line probe assays Denitive proof of active disease Very supportive of active TB Results are not readily available 26 weeks ~12 days Gold standard for active tuberculosis Important when sputum acid-fast bacilli smear are negative High specicity Inexpensive, high diagnostic accuracy Histology does not distinguish TB (or NTM infection) from other granulomatous diseases (except with presence of stainable AFB) Low sensitivity Not indicated for active TB 2h 2h Currently not advocated Inexpensive and useful for the rapid diagnosis of pleural or meningeal tuberculosis

Chest computed tomography

TST

Intracutaneous injection of tuberculin

Improves differential diagnostic ability, improves the evaluation of treatment success Standard procedure for the diagnosis of LTBI in non-BCG-vaccinated individuals from countries of low incidence of tuberculosis

IGRA

Local immunodiagnosis of MTB-specic cells in the BAL, pleural effusion, peritoneal uid or CSF can distinguish active TB from LTBI

Microscopy

NAAT

Quantiferon-Gold-IT assay: IFN-g-release in whole blood following ex vivo stimulation with ESAT-6, CFP-10 and TB 7.7 T-SPOT.TB assay: IFN-g-release by peripheral blood mononuclear cells following ex vivo stimulation with ESAT-6 and CFP-10 Staining for acid-fast bacilli (ZiehlNeelsen or Kinyoun method)Fluorescence Identication of MTB-specic genomic sequences

Culture

Histology

Growth of MTB on solid or in liquid media Caseating granuloma in biopsy specimen

Serology

Other

Identication of MTB-specic antibodies Adenosine deaminase on pleural or cerebrospinal uid

C Lange and T Mori

2010 The Authors Journal compilation 2010 Asian Pacic Society of Respirology

AFB, acid-fast bacilli; BAL, bronchoalveolar lavage; BCG, Bacille Calmette Guerin; CFP-10, culture ltrate protein-10; CSF, cerebrospinal uid; CXR, chest X-ray; ESAT-6, early secretory antigenic target-6; IFN-g, interferon-g; IGRA, interferon-g release assay; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; NAAT, nucleic acid amplication techniques; NTM, non-tuberculous mycobacteria; TST, tuberculin skin test.

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ACKNOWLEDGEMENTS
The authors thank Dr Martina Sester (University of Saarland, Homburg, Germany) for stimulating discussions on the diagnosis of tuberculosis.

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