Advances in the diagnosis of tuberculosis

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 fields that have led to substantial improvements in the accuracy and the timing of the diagnosis of tuberculosis. Novel methods allow for a better identification 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 identification 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.

INTRODUCTION

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 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 difficult,² as only 44% of all new cases (and only 15–20% of children³) are identified 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 definite.

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 difficult 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, amplification of M. tuberculosis nucleic acids and/or pathological examinations from biological specimens (Fig. 1).

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-γ release assay; MTB, Mycobacterium tuberculosis; NAAT, nucleic acid amplification test; NTM, non-tuberculous Mycobacteria; TB, tuberculosis; TBB, tubercle bacilli; TST, tuberculin skin test; WHO, World Health Organisation.

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 low-prevalence areas. They are composed of countries that experienced serious tuberculosis epidemics after the 18th century but have gradually overcome them and have finally reduced the incidence rate to 100 per 100 000 or less. The other part is the high-prevalence 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 high-prevalence 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.

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 first 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 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-finding 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 high-prevalence 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

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

In general, clinical manifestations of illness are the first clue to the diagnosis of tuberculosis. However, they are often non-specific 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.

Table 1.  Clinical presentations and laboratory examinations for diagnosis and differential diagnosis for major types of extra-pulmonary tuberculosis^134,302

Site	Symptoms & signs	Laboratory tests	Differential diagnosis
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.
Pleurisy	Primary infection cases have generally an acute course of symptoms than reactivation-type TB. Cough (non-productive) and chest pain (pleuritic—sharp, stabbing, associated with respiration). Feverishness, dyspnea, chills, sweats, weight loss in more advanced cases. TB pleural effusions are generally exudates.	Radiography, sputum bacteriology (for undiagnosed pulmonary TB), thoracocentesis with pleural fluid for cell profile, protein, pH, glucose, LDH, smear, culture, NAAT,^13–16 ADA,^17–20 IFN-γ,^20,21 IGRA,^22–26 pleural biopsy for histology, culture and NAAT.^27,28	Effusions due to congestive heart failure, carcinoma, other types of infections and rheumatological disorders.
Lymphadenopathy	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 superficial LN, lesion starts as a painless enlargement, with no inflammation over the skin, then may undergo pustulation and fistulation over several weeks or months. In a case with limited lesion, general symptoms are rare.	Culture isolation of MTB, chest radiography, biopsy (total excisional) followed by bacteriological culture/PCR^28 and histology. Fine needle aspiration.^29	NTM infections, lymphoma, sarcoidosis, Kikuchi's disease, Castleman's disease, Kimura's disease, corynebacterium pseudotuberculosis lymphadenitis.^30
Bone & Joint	Commonest in vertebral column, followed by hip and knee. Fever and wasting may appear in large inflammatory 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).	MTB from aspirate (abscess, synovial fluid) and biopsy specimen (e.g. synovia), ^31–34 CT & MRI.^35–38	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.
Disseminated or miliary TB	Diverse depending on the organs involved. Feverishness, weakness or debility, anorexia, weight loss, headache (meningeal complication), abdominal pain/swelling (peritoneal involvement), cough.^39,40	Chest radiography (often no abnormality in the beginning), CT (HRCT),^41,42 fiberoptic bronchoscopy, TBLB,^43 haematology (anaemia, leukopenia or leukocytosis, rarely leukemoid reaction). Liver function, bone marrow biopsy, liver biopsy (including NAAT^44), fundoscopy.^45	Alveolar microlithiasis, disseminated carcinoma, sarcoidosis, NTM infections, hypersensitivity pneumonitis.
Central nervous system	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 deficit.	Cerebrospinal fluid for pressure, cellularity, protein, glucose,^46 MTB (microscopy, culture^47,48 and PCR^49,50), immunology (ELISA, IgG immune complex, antibody assays and IGRA^51–56), and ADA. Radiography, CT, MRI.^57–61 Meningeal biopsy (histology, MTB).	Other infections (fungal, viral, trypanosomal, bacterial), vascular (multiple emboli, SBE, thrombosis of sagittal vein), collagen vascular (SLE, polyarteritis, and others).
Abdominal	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	Peritonitis: ultrasound,^64–66 laparoscopy (with guided biopsy),^67–70 paracentesis of ascites for culture and IGRA^71,72 and ADA.^73	Malignant ascites, cirrhosis with spontaneous bacterial peritonitis, starch peritonitis, sarcoidosis, NTM peritonitis.
Pericarditis	Dyspnoea, tachycardia, neck vein distension, oedema, hepatomegaly, paradoxical pulse, pericardial rub, fever.^74,75	Pericardial tissue/fluid for bacteriology, histology,^76,77 IGRA,^78,79 and ADA.^80–82 Echocardiography,^83–85 CT and MRI (pericardial effusion and thickening),^86 ECG (low voltage, inversion of T).^87	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.
Genitourinary	Dysuria, frequency, nocturia, urgency, pain in the back, flank or abdomen, tenderness/swelling of the testis or epididymis, haematuria. Superimposed urinary tract infection with other bacteria in urinary stasis cases.^88–93	Urine or secretion (early morning specimen) for MTB (smear, culture, PCR),^94 ultrasound,^95 plain abdominal radiograph, i.v. urography (high-dose), image-intensified endoscopy, percutaneous antegrade pyelography,^96–102 biopsy for suspect of genital lesion.^103–105	Benign and malignant tumor, cystic kidney, pyelonephritis, xanthogranulomatous pyelonephritis, urinary malakoplakia.

Tuberculosis in children

Because of its paucibacillary nature, tuberculosis of children is difficult to diagnose. Bacteriological confirmation seldom exceeds 30–40% 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 HIV-infected, 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 infiltration 100%.¹⁰⁸ Based on these observations, several point-scoring systems, diagnostic classifications 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%; specificity 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 resource-poor settings with high HIV epidemics.¹⁰⁹ However, given this set of sensitivity and specificity, 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 confirmed 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 findings, 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 findings 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.

Delays in diagnosis

One of the basic indicators of quality in diagnosing tuberculosis is the delay in diagnosis (‘doctor's delay’, or ‘health system's delay’), that is, the time from the first visit of a patient until the establishment of tuberculosis diagnosis. Figure 2 depicts the delay separately for high-, intermediate- and low-prevalence settings, together with the patient's delay, that is, the time from the onset of clinical symptoms until the first 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.

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) Patient's delay (time a patient needs from the occurence of the first symptoms to seeking healthcare) and (b) health system's delay (time to establish the diagnosis of tuberculosis after the first presentation of a patient to a healthcare facility).

Sasaki et al. reviewed the diagnostic process of private practitioners with Japanese patients and concluded that insufficient 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 Rozovsky-Weinberger et al. compared the management of suspect tuberculosis cases at three public hospitals and seven not-for-profit 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,^117–119 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.

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 air-space consolidation and/or pleural effusion,^121–123 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 defined nodules, linear opacities and cavitations.^123–125

While the classification in ‘primary’ and ‘reactivation’ tuberculosis is still widely en vogue, evidence from genotype fingerprinting studies confirms 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.^128–131

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-fluoro-deoxy-D-glucose positron emission tomography has been investigated as a promising non-invasive method to monitor disease activity and responses to anti-tuberculosis 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.

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 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 benefit of fluorescent microscopy for enhancing sensitivity over that of ordinary light microscopy without any loss in specificity.¹³⁵ The fluorescence microscopy widely used in resource-rich countries has been accepted as more sensitive than ordinary microscopy, although with a concern of loss of specificity, especially under conditions in the developing world. Nonetheless, a recent literature review has confirmed that it may be also beneficial in the latter as well. This could be further improved by attaching a stronger light source called an ultra-bright (Lumin^TM, LifeEnergy^®, Germany) light-emitting 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 chemical processing is more sensitive with similar specificity.¹³⁷ Operational studies are needed to determine the balance between the benefit 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 first two examinations, at least in laboratories with good-quality control.^138–141 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 beneficial to ensure high sensitivity of sputum smear tests in resource-poor settings where such drastic methods as gastric washing or fibro-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 flute’ has been developed and may be worth trying¹⁴⁵ (refer to Table 2 for collecting and processing specimens for the diagnosis of tuberculosis).

Table 2.  Biological specimen for the diagnosis of tuberculosis

Specimen	Amount	Application	Preservation/transport	Comment
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.
Sputum	2–5 mL	A, B, C	Unprocessed	3× in the morning on an empty stomach
Induced sputum	2–5 mL	A, B, C	Unprocessed	Expectoration following inhalation of 3% NaCl solution
Bronchial secretion or bronchoalveolar-lavage	2–5 mL	A, B, C, D	Unprocessed	BAL-ELISPOT should be performed on the day of sample collection
Gastric aspirate	>2 mL	A, B, C	In 1–2 mL phosphate buffer (trinatrium phosphate)	Only when sputum cannot be obtained and bronchoscopy (BAL) is not indicated
Biopsy, survival specimen (e.g. lmph nodes)	2 separate portions (1) and (2)	A, B, C, E	(1) In 0,9% NaCl for microbiological examination; (2) in formalin for histopathological examination	(1) Not in formalin
Pleural effusion, ascites	20 mL	A, B, C, D,	Unprocessed	ELISPOT should be performed on the day of sample collection
Cerebrospinal fluid	2–3 mL	A, B, C, D	Unprocessed	ELISPOT should be performed on the day of sample collection
Urine	30 mL	A, B, C	Unprocessed	– 3×– First specimen of urine in the morning – Fluid restriction the evening/night before
Stool	5–10 mL	A, B, C	Unprocessed	– 3×
Blood	5–10 mL	A, B, C, D	Heparin- or lithium-citrate tubes	– Indicated only in immunosuppressed patients – Do not use EDTA blood
Bone marrow	2 separate portions (1) and (2)	A, B, C, E	(1) In heparin- or lithium-citrate tubes; (2) air-dried smears and/or formalin preserved biopsies	– Indicated only in immunosuppressed patients – Biopsy or aspirate for (1) not in EDTA or formalin

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

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 specificity (range 83% to 100%), but lack sufficient 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 insufficient 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 confirmed 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 specificity, and the mean time to results was 21–23 days.¹⁵⁸

Given the technical difficulty 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 proficiency 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

MICROBIOLOGICAL DIAGNOSIS: MOLECULAR METHODS

Nucleic acid amplification techniques

The M. tuberculosis-specific nucleic acid amplification 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) fluid and can have important implication for the management of a patient. Unfortunately, NAAT amplification 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.^161–164

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 confirmed. 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 amplification target are related to a higher diagnostic accuracy.

In individuals with a negative sputum smear, the specificity of NAAT for the diagnosis of active tuberculosis has been 97% and 98% in an earlier meta-analysis and an independant recent study.^161,165 A positive result in a M. tuberculosis-specific NAAT performed on a respiratory specimen is therfore highly indicative of pulmonary tuberculosis. However, in our experience far less than 50% of patients with smear-negative 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.

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.^168–170 The Genotype MTBDRplus assay detects several mutations in the rpoB gene, in the katG gene and the inhA gene promoter regions.^{168,171–174} 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% (72–92%).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 fluoroquinolones 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

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 specific 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 specificity. Sometimes the systems are specifically 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 profile 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 specificity 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).

Table 3.  Types and nature of serodiagnostic methods (based on^{134,177–182})

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
Compound	Protein, lipid, polysaccharide (and their complex)
Composition	Single antigen, multiple antigen
Source	Native, recombinant
Ig class	IgG, IgA, IgG (single or combined)
Laboratory technique	Enzyme-linked immunosorbent assay, immunochromatography, immunodot rapid test, kaolin agglutination test

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 multiple-antigen 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

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 specificity, from 53% to 99%; and in general, tests with high specificity 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 specificity, 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 specificity, 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-γ release assays (IGRA) evaluate in vivo (TST) and ex vivo (IGRA) the presence of persistent mycobacteria-specific 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

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 identification of persons with a positive immune response against M. tuberculosis.¹⁸⁸

A standard preparation of purified protein derivate (PPD), an extract of the sterile supernatant of M. tuberculosis-cultered filtrate, 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 48–72 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 immunodeficiencies (e.g. those with HIV infection^194–196), 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 specificity 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 specificity of the TST increases with the increase of the cut-off used to define 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 filtrate 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.

Interferon-γ release assays

Introduction of IGRA into clinical practice is regarded by many as the most important development in the 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 specific to M. tuberculosis,²¹⁵ and the development of simplified technologies of measuring interferon-γ. There are two commercialized systems for the latter technology. QuantiFERON-Gold (QFT-G) (Cellestis Ltd, Carnegie, Australia²¹⁶) measures interferon-γ in IU/mL using an enzyme-linked immunosorbent assay (ELISA) and T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK²¹⁷) counts the cells releasing interferon-γ 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 findings 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. Specificity 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 specificity 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 specificity. 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.

Table 4.  Summary sensitivity and specificity of IGRA (meta-analysis)

Series	Diagnostics	Subject	No. studies	Summary	Range
Figures in parentheses in column ‘Summary’ indicate 95% confidence limits. For specificity, 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.
Sensitivity
1	QFT-G	TB patients, adult	21	0.80 (0.78–0.82)	0.62–0.95
2	QFT-G-IT	TB patients, adult	6	0.74 (0.69–0.78)	0.64–0.93
3	QFT-G/G-IT	TB patients, child	9	0.82 (0.75–0.87)	0.53–1.00
4	QFT-G/G-IT, T-SPOT.TB	HIV-infected TB patients	5	0.70 (0.60–0.79)	0.63–0.85
7	T-SPOT.TB	TB patients	13	0.90 (0.86–0.93)	0.83–1.00
8	TST	Healthy subjects	20	0.77 (0.71–0.82)	0.57–1.00
Specificity
1	QFT-G/G-IT	Healthy young adults	12	0.98 (0.97–0.99)	0.92–1.00
2	QFT-G/G-IT	Healthy young adults, BCG(−)	8	0.99 (0.98–1.00)	0.95–1.00
3	QFT-G/G-IT	Healthy young adults, BCG(+)	8	0.96 (0.94–0.98)	0.89–0.99
4	T-SPOT.TB	Predominantly BCG vaccinated	8	0.93 (0.86–1.00)	0.85–1.00
5	TST	BCG not vaccinated	6	0.97 (0.95–0.99)	0.93–1.00
6	TST	BCG vaccinated	6	0.59 (0.46–0.73)	0.35–0.79

The performance of the IGRA for the immunodiagnosis of M. tuberculosis infection has been investigated in immunocompromised hosts, such as in HIV-infected,^{195,220–233} elderly,^234–236 chronic-renal-failure^237–239 patients and those taking corticosteroids^240–242 or TNF-alpha blockers.^243–248 In general, the responses to IGRA (T-SPOT.TB > QFT-G-IT) 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 reflect the dynamics of infection immunity more sensitively, so that the interferon-γ level may fluctuate 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 confirmed and the discussion should be expanded to the level of response that persists after many years of infection.

For the diagnosis of tuberculosis in non-immunocompromised 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

Other biomarkers for tuberculosis disease status and diagnosis

Evaluation of other biomarkers for active tuberculosis or LTBI is a research priority in the field of tuberculosis. The details on the advances that have been achieved in the search for new biomarkers have recently been reviewed^255–262 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).

Table 5.  Selected methods under investigation for the diagnosis of tuberculosis

Method	Investigational target	Specimen	Sensitivity/specificity^†	Test results available within	Citation
BCG, Bacille Calmette Guerin; CD, cluster of differentiation; FACS, fluorescence activated cell sorting; IFN-γ, interferon-γ; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; RD, region of difference. ^†  The reported sensitivities and specificities from these pilot studies may not reflect the diagnostic accuracy of the tests in clinical practice.
FACS analysis of short-term stimulated blood or cell cultures	Frequency of CD27+ lymphocytes	Blood	100%/86%	24 h	^263
	Identification of intracellular IFN-γ	Sputum	89%/80%	24 h	^264
	Identification of IFN-γ- and IL-2-secreting cells	Blood	Not yet determined	24 h	^265
Chemokine production following stimulation with ESAT-6/CFP10/ TB7.7	IFN-γ-inducible protein IP-10	Blood	82%/97%	24 h	^266
IFN-γ-production following stimulation with ESAT-6/CFP10	Identification of IFN-γ-secreting cells	BAL	91%/80%	24 h	^165
	Identification of IFN-γ-secreting cells	Pleural fluid	95%/76%	24 h	^22
	Identification of IFN-γγ-secreting cells	Cerebrospinal fluid	90%/100%	24 h	^51
	Identification of IFN-γ-secreting cells	Peritoneal fluid	89%/78%	24 h	^71
IFN-γ-production following stimulation with antigens different from ESAT-6/CFP10	IFN-γ production following stimulation with heparin-binding hemagglutinin	Blood	92%/94%	4 days	^267
	IFN-γ production following stimulation with Rv3879c peptides in addition to ESAT-6 and CFP10	Blood	89%/69%	24 h	^253
	IFN-γ production following stimulation with RD-1 selective peptides	Blood	73%/71%	24 h	^254
Proteomics	Mass spectrometry analysis of serum proteins to identify a TB-specific fingerprint	Blood	94%/95%	Several days	^268
Detection of MTB-specific cell wall components	Lipoarabinomannan	Urine	18%/88%	4 h	^269
Gold nanoparticle probe assay	Hybridization of MTB-specific DNA with gold nanoparticle probes	Sputum	95%/100%	24 h	^270
Loop mediated isothermal amplification assay	Hybridization of MTB-specific DNA	Sputum	100%/94%	24 h	^271
Breath test	Identification of volatile biomarkers	Breath	83%/100%	24 h	^272

Based on the IGRA technology, other combinations of M. tuberculosis-specific antigens^{253,254,273–275} and cytokine readouts^265,276 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.^{266,276–279} When quantified 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-specific T cells clonally expand and are concentrated at the site of infection.^{22,51,71,78,282–286} 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.^{165,287–289} 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-specific T cells by BAL-ELISPOT had a sensitivity and specificity 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 sputum AFB smear-negative tuberculosis than M. tuberculosis-specific 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 anti-tuberculosis 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.

Fluorescence-activated cell sorting

Immunophenotyping of antigen-stimulated cells by fluorescence-activated cell sorting of BAL cells^286,292,293 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 flow cytometry analysis allows for a better identification 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 PPD-specific lymphocytes, and flow cytometry assays stimulated with PPD do not distinguish individuals with active tuberculosis and LTBI. Unfortunately, the frequency of region of difference-1 M. tuberculosis-specific T cells is too low in the sputum to be used as stimulants for flow cytometry cultures and other immune based assays.^264,295 However, where available, flow cytometry is a very promising tool to further improve the diagnostic accuracy of local immunodiagnostic assays for the diagnosis of AFB smear-negative tuberculosis.²⁸⁶

Other possibilities of tuberculosis diagnosis

Being the science of the structure and function of proteins, proteomics has identified proteins significant as diagnostic or prognostic biomarkers, or as therapeutic targets in a range of illnesses, including tuberculosis.^296–298 Agranoff et al. applied this technique to analysis of serum proteomic fingerprinting, or individual recognition of proteomic profile, for the purpose of distinguishing subjects with and without tuberculosis.²⁶⁸ Its diagnostic efficacy was fairly good with sensitivity of 93.5% and specificity of 94.9%. Also, to translate from proteomic signatures to conventional test formats, they identified amyloid A and transthyretin from highly informative peaks, and measured their levels by immunoassay, together 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 efficacy to use it in combination with sputum smear examination in HIV-infected patients.

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 riser's alertness to the problem. The balance between developments in the diagnosis and in the treatment of tuberculosis has changed. Recent diagnostic advances overweigh the inefficient 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 fitted to various situations. Only in that way can we combine our case-finding activities well with treatment services that are still progressing in order to make our tuberculosis control maximally effective.

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

	Method	Advantage	Disadvantage	Duration	Clinical significance
AFB, acid-fast bacilli; BAL, bronchoalveolar lavage; BCG, Bacille Calmette Guerin; CFP-10, culture filtrate protein-10; CSF, cerebrospinal fluid; CXR, chest X-ray; ESAT-6, early secretory antigenic target-6; IFN-γ, interferon-γ; IGRA, interferon-γ release assay; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; NAAT, nucleic acid amplification techniques; NTM, non-tuberculous mycobacteria; TST, tuberculin skin test.
Medical history	Conversation, review of medical records	Information on the individual risk for TB	Risk for tuberculosis may not be obvious, clinical symptoms may be non-specific	<1 h	Very important
Clinical examination	Physical examination	Identification of the severity of the illness	Clinical signs may not be obvious or specific	<1 h	Very important
Imaging	CXR	Inexpensive, rapid, low exposure to radiation	Wide spectrum of differential diagnoses	Minutes	Standard diagnostic
	Chest computed tomography	Superior quality compared with CXR (higher resolution). May identify minimal active lesions	Images are not pathognomonic for TB. Higher exposure to radiation compared with CXR	<1 h	Improves differential diagnostic ability, improves the evaluation of treatment success
TST	Intracutaneous injection of tuberculin	Inexpensive, widely available	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	48–72 h	Standard procedure for the diagnosis of LTBI in non-BCG-vaccinated individuals from countries of low incidence of tuberculosis
IGRA	Quantiferon-Gold-IT assay: IFN-γ-release in whole blood following ex vivo stimulation with ESAT-6, CFP-10 and TB 7.7	Very high specificity (T-SPOT.TB < QTF-G-IT) High sensitivity (T.Spot.TB > QTF-G-IT) for LTBI	IGRA responses from peripheral blood do not allow a discrimination of active TB and LTBI. More expensive than the TST	1 day	Substitutes TST in routine clinical practice, especially where BCG vaccination is prevalent
	T-SPOT.TB assay: IFN-γ-release by peripheral blood mononuclear cells following ex vivo stimulation with ESAT-6 and CFP-10	Can be adapted to comparatively assess immune responses in the periphery (blood) and at the site of disease (e.g. BAL)	Requires appropriate lab-facility	1 day	Local immunodiagnosis of MTB-specific cells in the BAL, pleural effusion, peritoneal fluid or CSF can distinguish active TB from LTBI
Microscopy	Staining for acid-fast bacilli (Ziehl–Neelsen or Kinyoun method)—Fluorescence	Inexpensive, rapid, low technical demands	No differentiation between MTB and NTM species	2 h	Standard diagnostic procedure
NAAT	Identification of MTB-specific genomic sequences	Differentiates MTB from NTM. Very high specificity, very high sensitivity when acid-fast bacilli are seen on sputum smears. Rapid amplification of genes that are related to drug resistance forms the basis of line probe assays	Diagnostic accuracy of ‘in-house’ methods may be highly variable. Limited sensitivity in smear-negative cases	1–2 days	Requires special laboratory facilities. Not sensitive enough in smear-negative cases
Culture	Growth of MTB on solid or in liquid media	Definitive proof of active disease	Results are not readily available	2–6 weeks	Gold standard for active tuberculosis
Histology	Caseating granuloma in biopsy specimen	Very supportive of active TB	Histology does not distinguish TB (or NTM infection) from other granulomatous diseases (except with presence of stainable AFB)	∼1–2 days	Important when sputum acid-fast bacilli smear are negative
Serology	Identification of MTB-specific antibodies	High specificity	Low sensitivity	2 h	Currently not advocated
Other	Adenosine deaminase on pleural or cerebrospinal fluid	Inexpensive, high diagnostic accuracy	Not indicated for active TB	2 h	Inexpensive and useful for the rapid diagnosis of pleural or meningeal tuberculosis

ACKNOWLEDGEMENTS

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