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Keywords:

  • tuberculosis;
  • central nervous system;
  • diagnosis;
  • novel diagnostics;
  • cerebrospinal fluid

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Tuberculous meningitis (TBM) comprises a significant proportion of TB cases globally and causes substantial morbidity and mortality, especially in children and HIV-infected patients. It is a challenging condition to diagnose due to its non-specific clinical presentation and the limited sensitivity of existing laboratory techniques. Smear microscopy and culture are the most widely available diagnostic tools yet are negative in a significant proportion of TBM cases. Simplified and more affordable nucleic acid amplification tests (NAATs) are increasing in use in resource-limited settings but have not been optimised for cerebrospinal fluid (CSF) samples. Novel diagnostic methods such as CSF interferon-gamma release assays and various biomarkers have been developed but require further evaluation to establish their utility as diagnostic tools. There is an urgent need for further research into optimal diagnostic strategies to decrease the morbidity and mortality as a result of delayed or missed diagnosis of TBM. In this review, we discuss current and novel diagnostic tests in TBM and areas where future research should be prioritised.

La méningite tuberculeuse (MTB) représente une proportion importante des cas de TB dans le monde et entraîne une morbidité et une mortalité importantes, surtout chez les enfants et les patients infectés par le VIH. C'est une condition difficile à diagnostiquer en raison de sa présentation clinique non-spécifique et la sensibilité limitée des techniques de laboratoire existantes. L'examen microscopique des frottis et la culture sont les outils de diagnostic les plus largement disponibles et donnent pourtant des résultats négatifs dans une proportion importante des cas de MTB. Des tests d'amplification d'acides nucléiques (TAAN) simplifiés et plus abordables sont de plus en plus utilisés dans les régions à ressources limitées, mais n'ont pas été optimisés pour les échantillons de liquide céphalo-rachidien (LCR). De nouvelles méthodes de diagnostic comme les tests de libération d'interféron-gamma sur LCR et des biomarqueurs divers ont été développés, mais nécessitent une évaluation plus approfondie pour établir leur utilité comme outils de diagnostic. Des recherches supplémentaires sont urgemment nécessaires sur les stratégies optimales de diagnostic pour diminuer la morbidité et la mortalité suite à un diagnostic retardé ou manqué de MTB. Dans cette revue, nous discutons des tests diagnostiques actuels et nouveaux pour la MTB et les domaines où des recherches futures devraient être prioritaires.

La meningitis tuberculosa (MT) comprende una proporción significativa de los casos de TB a nivel global y causa una morbilidad y mortalidad sustanciales, especialmente en niños y pacientes infectados con VIH. Se trata de una enfermedad difícil de diagnosticar debido a su presentación clínica inespecífica y a la sensibilidad limitada de las técnicas de laboratorio actualmente disponibles. La microscopía y el cultivo son las principales herramientas diagnósticas disponibles, y sin embargo son negativas en una proporción significativa de casos de MT. Las pruebas de amplificación de ácidos nucleicos (PAAN), simplificadas y más baratas, son cada vez más utilizadas en emplazamientos con recursos limitados, pero no se han optimizado para ser utilizadas con muestras de líquido cefalorraquídeo (LCR). Se han desarrollado nuevas pruebas diagnósticas tales como los ensayos de liberación de interferón gamma y varios marcadores biológicos en LCR, pero aún han de evaluarse con el fin de establecer su utilidad como herramientas diagnósticas. Existe una necesidad urgente de investigar sobre estrategias óptimas de diagnóstico que disminuyan la morbilidad y mortalidad que resulta de un retraso o error en el diagnóstico de la MT. En esta revisión se discuten las pruebas actualmente disponibles así como las novedades en el diagnóstico de la MT en áreas en las que debería priorizarse la investigación.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Tuberculous meningitis (TBM) results in the highest rates of morbidity and mortality out of all forms of tuberculosis (WHO 2012). It is of particular concern in young children, in whom it comprises up to 33% of all cases of TB (Soeters et al. 2005). Outcomes of infection include death in up to 50% of cases (Ruslami et al. 2013). Survivors can suffer substantial neurological sequelae including developmental delay in children, seizures, hydrocephalus, and cranial nerve palsies (Thilothammal et al. 1994; Yarmis et al. 1998). Improved clinical outcomes are dependent on timely diagnosis and initiation of appropriate treatment at optimal doses (Kennedy & Fallon 1979; Garg 1999; Ruslami et al. 2013). Despite this, current diagnostic methods are limited in sensitivity due to the paucibacillary nature of this form of TB, and hence, the need for large volumes of cerebrospinal fluid (CSF) that are impractical in most cases especially in children. Smear microscopy and culture are often negative in TBM, and a combination of consistent CSF findings (moderate pleocytosis with a lymphocyte predominance, increased protein content and low glucose concentration), clinical and radiological criteria are required to optimise the diagnosis (Thwaites et al. 2002; Marais et al. 2010). Atypical CSF findings have been described in children (Yarmis et al. 1998; Stark 1999), and the sensitivity and specificity of neurological symptoms and signs are particularly low in this vulnerable population (Kumar et al. 1999). A large proportion of TBM cases remain undetected and probably die without access to appropriate treatment (WHO 2011a). Molecular techniques are routinely employed in developed countries and increasingly in less wealthy settings. The sensitivity of these assays is highly variable, however, and, in many cases, offers little advantage over smear microscopy performed by an experienced technician (Pai et al. 2003; Thwaites et al. 2004a). The inadequacy of current laboratory diagnosis of TBM has encouraged a plethora of studies investigating novel diagnostic assays for TBM in the past decade. While many are promising, studies evaluating these assays are often small, and methods may be less suitable in resource-constrained, TB endemic settings. Comparisons of TBM diagnostic studies are hindered by the lack of a clear reference standard, low study subject numbers and heterogeneity in both study design and specific diagnostic platforms. There is an urgent need for simple, affordable and effective techniques to enhance detection and treatment of this debilitating disease. Our objective in this review is to describe the utility and limitations of current laboratory methods for TBM, discuss recent advances, and propose best practice approaches and future research priorities.

Search strategy and selection criteria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

References were obtained using PubMed from the last 15 years using the search strategy ‘tuberculous meningitis’ and ‘diagnosis’. References that included laboratory diagnostic techniques were included. Relevant references cited from these initial references were also reviewed. Studies with low subject numbers (<10 cases) or unsuitable study design, such as no controls, were excluded. Additional references concerning specific aspects of laboratory diagnosis (microscopy, interferon-gamma release assay, etc.) were further obtained by searching PubMed using these subject headings specifically combined with ‘tuberculous meningitis’.

Microscopy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Detection of acid fast bacilli (AFB) in patient samples using Ziehl–Neelsen (ZN) staining is the most widely employed technique for diagnosing TB. It remains the only laboratory test for TB diagnosis in many resource-limited TB endemic regions and forms an integral part of the WHO diagnosis and treatment algorithm (WHO 2010a). Fluorescent microscopy using fluorochrome dye (auramine-O or auramine-rhodamine) has improved the sensitivity of microscopy over conventional ZN staining (by approximately 10%) and significantly decreased the time required to examine each slide (Steingart et al. 2006). Cheaper light-emitting diode fluorescent microscopes have enabled wider adoption of this technique (WHO 2011b; Das & Selvakumar 2012). AFB microscopy is, however, insensitive in extrapulmonary TB especially TBM, with sensitivity rates of about 10–20% (Thwaites et al. 2000), although this figure varies considerably (0–87%) (Kennedy & Fallon 1979; Hosoglu et al. 2002; Bhigjee et al. 2007), and is highly influenced by the clinical case definition used for diagnosis, the volume of CSF sent and the skill of the technician examining the slide. The sensitivity of smear microscopy in TBM can be maximised by examination of the spun deposit of large volume CSF samples (>6 ml), several CSF specimens collected over a few days and prolonged slide examination (30 min) (Thwaites et al. 2004b). However, these criteria are rarely achieved in practice. Despite being the most practical and universally adopted test for TB diagnosis, there have been few advances towards improving smear microscopy. Recently described fluorescent microscopy using small membrane filters (Fennelly et al. 2012) and modified ZN stain using cytospin and Triton (a detergent) processing of CSF samples (Chen et al. 2012) have provided promising avenues for further developments in this field.

Culture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Culture of Mycobacterium tuberculosis (MTB) from the CSF of TBM patients is slow and insufficiently sensitive to be used as a ‘rule out’ diagnostic test, although it represents the most definitive ‘rule-in’ test – usually in retrospect (Tortoli et al. 1999; Venkataswamy et al. 2007). Culture is essential for phenotypic drug susceptibility testing (DST) and to confirm resistance detected by more rapid molecular techniques (WHO 2011c). Reduced turnaround times have been achieved using broth-based culture compared with solid media for the isolation of MTB (13 days vs. 26 days) (Tortoli et al. 1999) and DST (Tortoli et al. 2002). For CSF samples, both liquid and solid culture media should be used for optimal detection and should be incubated for longer periods (up to 8–10 weeks) than routine culture (Venkataswamy et al. 2007). The sensitivity of CSF culture for MTB varies (60–70% in adults) (Hosoglu et al. 2002; Thwaites et al. 2004b) and is considerably lower in children (Yarmis et al. 1998; Van Well et al. 2009). As with microscopy, sensitivity can be increased by culturing the deposit of relatively large volumes of CSF (Thwaites et al. 2004b). Laboratories that culture MTB and perform DST are required to meet specific biosafety level requirements beyond those of routine microbiological testing to protect staff from aerosol transmission and to prevent environmental contamination. These include negative pressure ventilation, lockable doors and restricted access, personnel protective equipment including N95 masks, specific waste management and emergency plans for spills and accidents. All specimens that are processed for MTB culture and all culture manipulation must be performed inside a certified and maintained biological safety cabinet. In addition, quality assurance systems must be in place to ensure accurate and reliable results (WHO 2004). In developing nations, these considerable infrastructure requirements are generally only met in national reference laboratories, further hindering timely identification and DST results.

In an attempt to overcome some barriers to conventional culture, WHO has endorsed the microscopic observation drug susceptibility (MODS) assay (WHO 2011d). This is a simple and inexpensive liquid culture alternative for detection of MTB and DST in which processed specimens are inoculated into broth media, with and without antibiotics and examined frequently, after 5–7 days incubation, for the presence of cording in control tubes. In respiratory samples, MTB can be detected by MODS in a median of 7–9 days (Moore et al. 2006a; Ha et al. 2010; Shah et al. 2011) with sensitivities and specificities of 85–87% and 93–97%, respectively, against standard culture (Ha et al. 2010; Shah et al. 2011). In TBM patients, MODS on CSF specimens compares favourably (sensitivity 65% and specificity 100% against clinical reference standard, with mean CSF volume 4.6 ml) with conventional culture methods in a significantly shorter detection time (median 6 days) (Caws et al. 2007). In addition to more timely identification and DST results, other advantages are cost, ease of use and the ability to perform this technique without the infrastructure required and with no greater risk of cross-contamination than with conventional culture. (Moore et al. 2006b; Ha et al. 2010; Shah et al. 2011) Further, larger studies into the diagnostic utility of MODS in TBM are required, including studies in children. Its performance for DST in CSF has not been demonstrated and is likely to be less successful than for sputum due to the paucibacillary nature of this disease and the clumping of bacilli within CSF (Thwaites et al. 2004a; Caws et al. 2007).

Nucleic acid amplification tests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

The first NAATs for use on CSF specimens were described over two decades ago (Kaneko et al. 1990; Shankar et al. 1991), and there have been numerous in-house polymerase chain reaction (PCR) assays developed since. The design and performance of these are heterogeneous, however, which makes comparing them difficult. This issue was highlighted by a meta-analysis of the diagnostic accuracy of commercial and in-house NAATs for TBM diagnosis (Pai et al. 2003). Commercial assays were found to be insensitive at detecting MTB in CSF samples (sensitivity 56% and specificity 98%), whereas no useful comparative information could be obtained for in-house PCRs (Pai et al. 2003). Subsequently, a number of in-house PCRs using a variety of targets (IS6110, INS, protein b, rpoB, MPB64) and testing platforms have described, with improved overall performance, especially with the use of multiplex PCRs, compared with commercial assays (sensitivity 71–94% and specificity 88–100%) (Kulkarni et al. 2005; Bhigjee et al. 2007; Rafi et al. 2007; Huang et al. 2009; Sharma et al. 2010; Kusum et al. 2011; Chaidir et al. 2012). NAATs have a distinct advantage over microscopy and culture, once antituberculous therapy has commenced with DNA remaining detectable for up to a month after starting treatment (Donald et al. 1993; Thwaites et al. 2004a).

Limitations of PCR tests for MTB detection include cost, the need for laboratory infrastructure, trained technical staff and careful quality control to prevent cross-contamination, detect inhibition and monitor assay performance. In 2010, the WHO endorsed the use of Xpert® MTB/RIF (Cepheid, CA, USA), a rapid fully automated NAAT for use on sputum specimens in resource-constrained TB endemic countries, at concessional pricing (WHO 2011e). The main advantages of Xpert® MTB/RIF over pre-existing molecular assays are its simplicity of use – it requires minimal technical expertise and biosafety requirements and the low rates of cross-contamination (Banada et al. 2010; Boehme et al. 2010; Van Rie et al. 2010; Lawn & Nicol 2011). Another key advantage is its ability to rapidly detect rifampicin resistance and hence predict multidrug resistant disease (MDR). Of note however, increasing rates of rifampicin monoresistance have been reported in recent studies in more than one-third of total rifampicin resistant cases (Anisimova et al. 2012; Mukinda et al. 2012). Moreover, although initial validation studies reported 100% specificity for the detection of rifampicin resistance (Boehme et al. 2010), more recent studies have shown specificity to be much lower than this (Carriquiry et al. 2012), prompting modifications in product software (FIND 2011). So, while Xpert® MTB/RIF technology has allowed increased decentralisation of drug-resistance detection, all detected rifampicin resistant isolates should ideally be confirmed with conventional DST to detect false-positive results and in addition, concomitant isoniazid testing is required to avoid potential misassignment of MDR disease (WHO 2011c).

Xpert® MTB/RIF has been extensively evaluated for MTB detection in sputum specimens and performs well on smear-positive samples (sensitivity 98% compared with 68% in smear-negative samples; specificity 98%) (Steingart et al. 2013). Preliminary studies on a range of extrapulmonary TB samples have also been promising in smear-positive samples (sensitivity 96–100% vs. 37–90% in smear-negative specimens; specificity 98–100%) (Armand et al. 2011; Causse et al. 2011; Hillemann et al. 2011; Vadwai et al. 2011; Tortoli et al. 2012). For CSF samples specifically, further studies are required as the few that have been performed have small subject numbers, variable results (sensitivity 27–86%, specificity 99–100%) (Vadwai et al. 2011; Tortoli et al. 2012) and have not been performed in high TB-burden settings. The use of a composite reference standard, combining microbiological, clinical, radiological and/or histological findings may enable a more accurate interpretation of the utility of Xpert® MTB/RIF in diagnosing extrapulmonary TB, where culture confirmation, the historical ‘gold standard’, may be absent in true TB disease (Vadwai et al. 2011). An important emerging challenge, given the wide global rollout of Xpert® MTB/RIF for use on pulmonary specimens, is to demonstrate how access to this assay can impact on patient outcomes, as the existing paradigm in high TB-burden settings is to institute TB therapy for cases of clinically suspected TBM even in the absence of microbiological confirmation. Whether Xpert® MTB/RIF can, cost-effectively, improve time to initiation of appropriate treatment, patient outcomes, recognition of alternative diagnoses, resource allocation and epidemiological understanding through improved case notification, requires further investigation.

Another simple, rapid and cost-effective NAAT that has been used for TB diagnosis is loop-mediated isothermal amplification (LAMP) (Eiken Chemical Co., Ltd, Tokyo, Japan). This technology operates at isothermal conditions without the need for sophisticated equipment or skilled personnel, making it an attractive and feasible option in resource-limited settings (Boehme et al. 2007). It has been used for the diagnosis of a variety of infectious diseases (Parida et al. 2008; Mori & Notomi 2009). In smear-positive sputum, samples LAMP has good sensitivity and specificity (98% and 94–100%, respectively), but it is less sensitive in smear-negative samples (49–56%) (Boehme et al. 2007; Mitari et al. 2011). A small study evaluating the use of LAMP on CSF for TBM diagnosis demonstrated good performance (sensitivity 88% and specificity 90%) with better sensitivity than nested-PCR (Nagdev et al. 2011). Unlike PCR however, LAMP cannot be multiplexed, hence simultaneous drug-resistance detection is only possible with selected isothermal amplification methods. It is unclear how LAMP technology would be incorporated into TB testing algorithms in laboratories that have existing Xpert® MTB/RIF instruments, but, given preliminary data suggesting superior sensitivity to PCR in extrapulmonary samples (Nagdev et al. 2011; Yang et al. 2011), further larger studies will be valuable to determine its role in this setting.

Adenosine deaminase and interferon-gamma activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Adenosine deaminase (ADA) is an enzyme that is widely distributed in tissues and body fluids and has been used in the diagnosis of pleural, meningeal and pericardial TB (Segura et al. 1989). Measurement of this enzyme is simple and affordable, but the evidence to support its utility in TB diagnosis is inconclusive. Measurement of ADA has not been standardised, and the cut-off level that defines a positive result has not been determined (Tuon et al. 2010; Xu et al. 2010). For TBM diagnosis, ADA measurements on CSF may contribute to other supportive diagnostic findings once other pathogens have been ruled out and, by optimising the defined cut-off value used (Tuon et al. 2010; Xu et al. 2010). However, in HIV patients, this test is not useful for distinguishing TBM from other clinically similar neurological illnesses (Corral et al. 2004).

Interferon-gamma (IFN-γ) plays a major role in the immune response to MTB, stimulating macrophage activity and lymphocyte Th1 differentiation (Farrar & Schreiber 1993). It has been used in a similar fashion to, and often in conjunction with, ADA to aid in the diagnosis of pleural (Jiang et al. 2007; Khan et al. 2013), pericardial (Burgess et al. 2002; Reuter et al. 2006) and peritoneal (Sharma et al. 2006) TB with good sensitivity and specificity. The quantification of IFN-γ in CSF as a diagnostic tool in TBM holds much promise, yet has only received preliminary attention in recent years (Juan et al. 2006; Patel et al. 2011). The measurement of unstimulated IFN-γ, on unprocessed CSF, when combined with cryptococcal antigen testing and gram stain, in a high HIV-prevalence setting, has been shown to be 92% sensitive and 100% specific in diagnosing TBM (Patel et al. 2011). Further, larger studies in different settings are required to validate these findings. IFN-γ measurements share similar accuracy and may have a complementary role to ADA in diagnosing TB, however, may be limited in low-resource settings due to technical and financial constraints (Reuter et al. 2006; Sharma et al. 2006).

Interferon-gamma release assays (IGRAs)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

The measurement of interferon-gamma release in whole blood (QuantiFERON-TB© Gold In Tube [(QFT-IT); Cellestis Limited Chadstone, Vic., Australia] or peripheral blood mononuclear cells (T-SPOT.TB; Oxford Immunotec, Abingdon, UK) in response to stimulation with specific MTB antigens, to diagnose latent TB, has been incorporated into screening guidelines in many low-incidence, high-income countries instead of, or together with, tuberculin skin testing (TST) because of its high specificity and need for only one healthcare visit (Denkinger et al. 2011; National Institute for Health & Clinical Excellence 2011). However, evidence to support the use of IGRAs to diagnose active TB has been less compelling. Meta-analyses have concluded that IGRAs cannot be used to rule out active disease or to reliably distinguish latent from active TB (Diel et al. 2010; Sester et al. 2011; Rangaka et al. 2012). The use of IGRAs directly on CSF specimens has been evaluated for the diagnosis of TBM, based on the premise that mononuclear cells localised to infected sites produce more interferon than peripheral blood mononuclear cells (PBMC) (Kim et al. 2008), which has also been demonstrated in pleural (Losi et al. 2007) and alveolar fluid (Jafari et al. 2006; Dheda et al. 2009) in thoracic TB. There is some evidence that comparison of CSF vs. PBMC IGRA levels (Kim et al. 2008), or combinations of CSF IGRA with other CSF tests such as ADA (Kim et al. 2010), gram stain and cryptococcal antigen (Patel et al. 2010a) or with MTB PCR (Juan et al. 2006) may improve the diagnosis of TBM. Further studies are warranted to confirm these findings. It will be necessary to overcome barriers such as expense, availability and fastidious sample handling requirements before widespread use of IGRAs becomes feasible in most TB endemic countries. The value of other cytokine readouts (such as TNF alpha, IL-2 and IL-17, etc.) is currently under investigation and may add value compared with an isolated interferon-gamma read-out.

Novel biomarkers

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

The search for novel biomarkers for TB screening, diagnosis and treatment monitoring has come to the forefront of attention in recent years (Parida & Kaufmann 2010; McNerney et al. 2012). The deficiencies in our existing strategies for diagnosing TBM make research into diagnostic biomarkers for this condition appealing. Biomarkers are defined as measurable characteristics that indicate normal biological or pathogenic processes, or pharmacological responses to a therapeutic intervention (Biomarkers working group 2001), and encompass ADA, IFN-γ and IGRAs, as described above. Other biomarkers such as MTB-specific antigen, antibody (Katti 2001, 2002; Kashyap et al. 2004, 2005; Mudaliar et al. 2006) cytokine measurements (Kashyap et al. 2010; Misra et al. 2010), gene-expression profiles and metabolomics (Kumar et al. 2012; Maertzdorf et al. 2012), in TBM patient have been described, but their feasibility and clinical utility as diagnostic tools are unknown.

The detection of lipoarabinomannan (LAM), a MTB cell wall lipopolysaccharide antigen, in urine is useful in the diagnosis of TB in HIV patients with advanced immunodeficiency and has recently been developed as a low-cost lateral flow assay for use as a point-of-care test [(Determine TB-LAM) Alere, Waltham, MA, USA] (Minion et al. 2011; Lawn 2012; Peter et al. 2012). Preliminary investigations measuring CSF LAM for TBM diagnosis in immunosuppressed HIV-infected patients have shown improved diagnostic value (sensitivity 64% and specificity 69%) compared with smear microscopy (Patel et al. 2009, 2010b).

A pragmatic approach

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

Table 1 summarises the accuracy and recommendations for use of current and novel diagnostic techniques in TBM, and a summary of the optimal use of existing techniques is provided in Table 2. While optimising current techniques is desirable and logical, in the majority of regions most affected by TBM, clinicians are limited to smear microscopy as the sole microbiological diagnostic test, and clinical acumen governs empiric antimicrobial treatment. The use of clinical prediction rules for differentiating TBM from other forms of meningitis is sensitive and specific (Kumar et al. 1999; Thwaites et al. 2002; Hristea et al. 2012) and can significantly enhance the utility of point-of-care diagnostic tests such as CSF LAM (Patel et al. 2010a,b) and Xpert MTB/RIF (Vadwai et al. 2011). A standardised clinical case definition, which is applicable irrespective of patient's age, HIV status or resource limitations, has been developed by a group of experts in this field, and enables more accurate diagnosis of TBM and consistency for study comparisons (Marais et al. 2010). Components of this case definition are summarised in Table 3. Sampling non-central nervous system (CNS) sites such as sputum, lymph nodes, urine, bone marrow and ascitic fluid can be particularly helpful in diagnosing TBM (Hosoglu et al. 2002; Van Well et al. 2009), especially in children, where up to 68% of cases in one study were MTB culture positive from gastric aspirates (Doerr et al. 1995). Similarly, chest radiography provides valuable additional information in TBM with up to 60% of cases demonstrating changes consistent with active TB (Schutte 2001; Hosoglu et al. 2002; Van Well et al. 2009). These adjuvant tests may also be diagnostic in TBM cases where CSF cannot be obtained, for example, when lumbar puncture is contraindicated, consent for lumbar puncture is not provided, or there is technical failure resulting in inability to obtain CSF.

Table 1. Comparison of conventional and novel diagnostic tests for tuberculous meningitis performed on CSF specimens
Diagnostic test in CSF specimensLevel of evidenceaSensitivity%Specificity%Reference standardbCommentsReferences
  1. NAATs, Nucleic acid amplification tests; ADA, Adenosine deaminase; MODS, Microscopic observation drug susceptibility assay; LAMP, Loop-mediated isothermal amplification; IGRAs: Interferon-gamma release assays; PBMC, Peripheral blood mononuclear cells; LAM, Lipoarabinomannan; CSF, cerebrospinal fluid.

  2. a

    Level of evidence: strength of recommendation and the quality of evidence (Merlin et al. 2009).image

  3. b

    Reference standard: 1 = Culture, microscopy, clinical and radiological diagnosis; 2 = Culture, microscopy and clinical diagnosis; 3 = Culture and microscopy; 4 = Clinical diagnosis; 5 = Culture and clinical diagnosis; 6 = Microscopy and clinical diagnosis.

  4. c

    Dependent on cut-off value used for positive result and method of ADA measurement.

  5. d

    Heterogeneous studies: IFN-γ-based assays used alone or in combination with PBMC IFNγ values or other CSF tests.

  6. e

    Various Mycobacterium tuberculosis – specific antigens and antibodies including 65kD heat-shock protein and antigen 85 complex.

Conventional diagnostic tests
Ziehl–Neelsen (ZN) smear microscopyA III-210–201005

Highly dependent on volume of sample plus time to examine slides and may be lower in clinical practice than research settings

Use of fluorescent microscopy increases sensitivity

Steingart et al. (2006); Thwaites et al. (2000)
Culture: liquid and solid mediaA III-260–701006

Shorter time to detection with liquid media

Increased sensitivity with larger volumes

Thwaites et al. (2004b); Hosoglu et al. (2002)
NAATs
In-houseA III-271–9488–1001,2 or 4Assays highly heterogeneousRafi et al. (2007); Sharma et al. (2010); Kusum et al. (2011); Huang et al. (2009);Kulkarni et al. (2005); Bhigjee et al. (2007)
CommercialB I56981,2,3 or 4Majority of commercial assays not licensed for extrapulmonary samplesPai et al. (2003)
Xpert®MTB/RIFC III-227–8699–1001Requires further evaluation; very few studies, small subject numbersTortoli et al. (2012); Vadwai et al. (2011)
ADAC I79–84c84–91c2Variable results; possible additional benefit to other diagnostic findingsXu et al. (2010); Tuon et al. (2010)
Newer and novel diagnostic tests
MODSB III-26598–1004Requires further evaluation; shortens time to achieve positive culture resultCaws et al. (2007)
LAMPD III-288902Requires further evaluationNagdev et al. (2011)
IGRAsD III-250–82d89–100d1 or 2Requires further evaluation; may have a role when combined with other rapid diagnostic tests, for example, gram stain and cryptococcal antigenKim et al. (2008, 2010); Patel et al. (2010a,b); Juan et al. (2006)
Antigens and antibodieseD III-284–9492–992Requires further evaluationKashyap et al. (2004); Katti (2002); Mudaliar et al. (2006); Kashyap et al. (2005); Katti (2001)
LAMD III-264692Requires further evaluationPatel et al. (2009, 2010a,b)
Table 2. Best practice recommendations for laboratory diagnosis of tuberculous meningitis
Diagnostic testBest practice recommendation
  1. CSF, cerebrospinal fluid; TBM, tuberculous meningitis.

Microscopy on CSF

Collect an adequate volume of CSF (>6 ml in adults)

Concentrate sample through cytospin technique (or small membrane filter)

Use fluorescent microscopy

Ensure adequate time to examine each slide by an experienced technician

Culture on CSF

Collect an adequate volume of CSF (>6 ml in adults)

Use liquid in addition to solid media culture

NAAT on CSFUse an in-house or commercial PCR which has been validated for use in CSF
Non–CSF specimensTest non-CSF specimens (e.g. sputum, gastric aspirate, urine) to aid diagnosis of disseminated TB, or TBM with other sites of infection
Table 3. Components of a standardised clinical case definition for tuberculous meningitis (Marais et al. 2010)
ComponentFeatures suggestive of tuberculous meningitis
  1. AFB, acid fast bacilli; TB, tuberculosis; MTB, Mycobacterium tuberculosis; CT, computer tomography; MRI, magnetic resonance imaging; CNS, central nervous system; NAAT, nucleic acid amplification test; CSF, cerebrospinal fluid.

Clinical criteria

Duration of neurological symptoms >5 days

Systemic symptoms suggestive of TB

History of recent (within 1 year) close contact with an individual with pulmonary TB

Focal neurological deficits or altered consciousness

CSF findings

Clear in appearance

Pleocytosis (10–500 cells/μl) with lymphocyte predominance (>50%)

Raised protein concentration (>1 g/l)

Low glucose concentration (<2.2 mm or CSF/plasma ratio <50%)

Cerebral imaging (CT or MRI)Hydrocephalus, basal meningeal enhancement, tuberculoma, infarct or pre-contrast basal hyperdensity)
Evidence of TB elsewhere

Chest radiograph suggestive of active tuberculosis

Other radiological findings suggestive of TB outside the CNS

AFB identified or MTB cultured from another source, e.g. sputum, lymph node, gastric aspirate, urine, bone marrow or blood

Positive MTB NAAT from non-CNS site

Exclusion of alternative diagnosisAlternative diagnosis confirmed using gram stain, culture, NAAT, antigen test (e.g. cryptococcus), serology (e.g. syphilis) or histopathology (e.g. lymphoma)

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

While there have been encouraging developments in the diagnosis of TB in general, none has translated directly into significant improvements in TBM diagnosis. There remains an important need for additional research to identify optimal diagnostic strategies. In contrast to sputum in pulmonary TB, CSF will invariably contain low organism numbers and limit our current diagnostic modalities. Concentrating bacilli in CSF specimens and/or designing methods to enhance the sensitivity of culture and NAATs may overcome this.It seems logical that research priorities be focused on improving the sensitivity of Xpert®MTB/RIF for use in TBM, given the expanding distribution and utilisation of this simple to use technology in TB endemic regions, many of which previously relied solely on smear microscopy to guide patient management. Although the discovery of specific biomarkers or gene-expressions profiles associated with TBM will herald an innovative and exciting era, existing methods for pathogen identification and DST will still be required for optimal case management.

Tuberculosis is a challenging disease. It is entangled in and highlights many of the complex factors contributing to global socio-economic inequalities and barriers to healthcare access. While the development of robust diagnostic tools for TBM is imperative, they will only be as good as the healthcare systems in which they are implemented. Concerted effort from all involved stakeholders together with the necessary political commitment will be fundamental to achieving global control over this age old debilitating disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References

We would like to thank Peter Jelfs, head of the TB laboratory at Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, for his contribution to several aspects of this paper.

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  2. Abstract
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  4. Search strategy and selection criteria
  5. Microscopy
  6. Culture
  7. Nucleic acid amplification tests
  8. Adenosine deaminase and interferon-gamma activity
  9. Interferon-gamma release assays (IGRAs)
  10. Novel biomarkers
  11. A pragmatic approach
  12. Conclusion
  13. Acknowledgements
  14. References
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