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European Review for Medical and Pharmacological Sciences

Abstract. – Background: Tuberculousspondylodiscitis is one the many manifestationsof active tuberculosis (TB) and can result follow-ing primary infection or, more frequently, fromreactivation of active TB in subjects with latentTB. Definitive diagnosis of tuberculous spondy-lodiscitis requires the identification of Mycobac-terium tuberculosis in the biological sample fol-lowing microbiological analysis.

Aim: To summarize the recent advancementin the diagnosis of TB, focusing on classical andmolecular microbiological procedures, providingan overview on the recent advancements in theunderstanding of TB pathogenesis and their im-plications for the immunological diagnosis

Materials and Methods: Isolation in cultureof the bacilli and detection using moleculartools are the gold standards, though sensitivityof these assays is significantly lower comparedto what observed for pulmonary TB, making di-agnosis of spinal TB challenging.

Results: The use of the interferon-γ releaseassays (IGRAs) for the immunological diagnosisof TB infection could be of help and shall pre-cede the invasive techniques, such as biopsy orsurgery, required to obtain the biological sample.IGRAs measure the presence of effector T cellsin the blood that can readily respond to an anti-genic stimuli by secreting cytokines, and that arean indication of the presence of the bacilli invivo. IGRAs are more sensitive and specific thanthe intradermic reaction of Mantoux, though boththese immunological tests cannot distinguish be-tween latent TB infection and active TB.

Conclusions: A modern diagnosis of TBspondylodiscitis should rely on the use of mi-crobiological and immunological assays and thelatter could potentially be of great help in moni-toring therapy effectiveness.

Key words:

Tuberculosis, Spinal TB, Pott’s Disease.

Introduction

TB is one of the ancient human scourge thathas afflicted mankind since millennia and is still

Microbiological and immunological diagnosisof tuberculous spondylodiscitisG. DELOGU, A. ZUMBO, G. FADDA

Institute of Microbiology, School of Medicine, Catholic University of the Sacred Heart, Rome (Italy)

Corresponding Author: Giovanni Delogu, PhD; email gdelogu@rm.unicatt.it

one of the deadliest infectious disease agents,causing 1,7 million deaths and with 8,4 millionnew cases annually1. Most of the TB cases occurin developing countries where TB poses a majorhealth problem, with relevant social and econom-ic implications.

Mycobacterium (M.) tuberculosis is a humanpathogen that belongs to the species Mycobacteri-um tuberculosis (MTB) complex, which compris-es M. africanum, which causes TB in humans butis restricted to Africa, M. bovis, which is the etio-logic agent of TB in animals and M. microtiwhich infects voles and does not infect humans2.The complete sequencing of the M. tuberculosisgenome has provided a new understanding on thebiology of the tubercle bacillus, leading to a bet-ter understanding of the mechanism of pathogen-esis and the definition of the genetic determinantsof drug resistance, opening new avenues to com-bat the disease through more effective prophylac-tic and therapeutic measures3-5. The 80 years oldlive and attenuated vaccine strain Bacillus of Cal-mette and Guerin (BCG), while ineffective in pre-venting TB in adults, remains the only tool avail-able to reduce the risk of fatal and systemic TBdisease in children. There is an urgent need for anew and improved vaccine against TB and manyefforts are underway using traditional and highlyinnovative strategies6,7. The same is true for thetherapeutic regimens that still rely on more thanforty years old drugs such as isoniazid, rifampin,streptomycin, pyrazinamide, ethambutol and fewother second line drugs8.

The new interest in TB research, that coincid-ed with the spread of the HIV pandemic and there-emergence of TB in western countries, had animpact primarily on the diagnosis of TB, with theintroduction of more sensitive, specific and rapidmethods that have contributed significantly to thecontrol of TB in the last ten years.

Microbiological diagnosis of TB is always achallenging task, particularly when non-pul-monary TB is suspected, as in the case ofspondylodiscitis9,10, since the biological samples

2012; 1(Suppl 2): 73-78

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available to detect M. tuberculosis are difficult toobtain. At the same time, several causative agentsof infectious spondylodiscitis have been identi-fied, which require different antimicrobial thera-pies. For these reasons, it is recommended thatthe start of the antimicrobial therapy in case ofinfectious spondylodiscitis should be delayed un-til a microbial diagnosis is established10.

In this review, we will summarize the recentadvancement in the diagnosis of TB, focusing onclassical and molecular microbiological proce-dures, providing an overview on the recent ad-vancements in the understanding of TB patho-genesis and their implications for the immuno-logical diagnosis and usefulness that these newtests may have in guiding the diagnostic query.

Pathogenesis of Tuberculosis

TB is an airborne transmitted disease, with thebacilli released by a patient with active pul-monary TB inhaled by a healthy person11. Thebacilli once in the alveoli are ingested bymacrophages which can kill the bacteria, thoughin 20-50% of the exposed subjects, M. tuberculo-sis resists to the innate immune response and ac-tively multiply within the macrophages. Nearbycells are infected by the replicating bacilli, whichcause a local inflammation that activates the sec-ondary host immune response12,13. It is at theseearly times of the infectious process, when thehost cannot yet contain bacterial multiplication,that M. tuberculosis spreads through the lym-phatics, but most importantly can translocate tothe blood stream and spread haematogenously topotentially reach any organ14,15. The mounting ac-quired cell mediated immune response then be-gins to contain the infection, starting at the site ofprimary infection when the cellular infiltrate or-ganize in the typical granuloma. Only in ≅ 5% ofcases, the host immune response fails to controlprimary infection which then progresses to overtpulmonary TB and it is the immune response it-self that is responsible for the extensive tissuedamage and necrosis which is the hallmark of ac-tive TB in immune-competent patients11.

Conversely, it is estimated that in 90-95% ofinfected subjects, the host immune response cancontain bacterial multiplication, leading to latentinfection (latent TB). Latent TB is clinicallysilent, with no outward signs or symptoms of dis-ease, and is characterized by the presence of a

specific cell mediated immune response specificfor M. tuberculosis, classically highlighted by theMantoux test16. Latent TB can last for decadesand people with latent TB have a ≅ 5% chance ofdeveloping active TB during lifetime, indicatingthat the host immune response cannot completelyeradicate the bacteria in vivo. These observationshave puzzled scientists since the early 20th centu-ry and only recently we are gaining a new under-standing of the immunological processes associat-ed with latent TB and on the metabolic status ofthe bacteria during this stage17,18. The old viewthat latent M. tuberculosis exists in classic TB le-sions such as the primary complex has been con-tradicted by many findings, including the demon-stration that bacilli can be found in non-profes-sional phagocytic cells such as endothelial cells,pneumocytes and fibroblast that are scattered inthe lung tissue19. M. tuberculosis also persists inadipose tissue where it can remain in a non-repli-cating state avoiding killing by antimicrobials andrecognition by the effector immune cells20. Thestatus of the bacilli during latent TB may varygreatly from a persistence non-replicating state, todormancy, to active replication, and M. tuberculo-sis can either use aerobic and anaerobic metabo-lism16-18. What is remarkable is that M. tuberculo-sis can resist the attack from many immune cells,including CD4 and CD8 T cells, which providean harsh and oxidizing environment for the bacte-ria21. In line with these findings, Fortune et al22

have recently demonstrated that the mutation rateof M. tuberculosis during latent TB is similar tothat observed during active TB, as a results of theactive bacilli replication and of the oxidativedamage to the M. tuberculosis DNA exerted bythe antimicrobials produced by activatedmacrophages.

As a result of these findings, the traditional di-chotomy of TB status (latent TB and active TB)is being revisited, with the introduction of theconcept of TB spectrum, which identify a dy-namic equilibrium between the host immune re-sponse and the bacilli16,18. The TB spectrum isseen as a gradient that may potentially vary fromlatent TB in an almost dormant state to overt ac-tive TB, and any event that can affect this equi-librium may lead to the progression of latent TBto overt disease.

Hence, tuberculous spondylodiscitis, alsocalled Pott’s disease, occurs when the bacteriathat has reached the spine, starts replicating in thetissue forming a secondary lesion that progressesto cause the typical immunopathogenesis and tis-

G. Delogu, A. Zumbo, G. Fadda

sue damage that may evolve in complicationssuch as vertebral collapse, paraplegia andparaspinal abscesses. Since haematogenousspread of the bacilli occurs readily following pri-mary infection in the lung tissue, spinal TB candevelop either soon after primary infections ormany years or decades later following reactiva-tion. Moreover, since during latent TB a dynamicequilibrium between the host immune responseand actively replicating bacilli is maintained, andbacteria can be found in many tissues and cells,any event that can suppress the immune responsecan results in disease reactivation. For these rea-sons, tuberculous spondylodiscitis can occur inpatients with concurrent infections such as HIVor undergoing biological therapy with anti-TNFdrugs. Osteoarticular TB can also be associatedwith TB disease elsewhere in the body includingpulmonary TB. In any case, proper diagnosis ofspinal TB is a challenging task for the clinicianthat must identify the proper therapeutic regimen.

Direct diagnosis

Definitive diagnosis of TB requires the detec-tion of M. tuberculosis from the biological sam-ple by at least one of current microbiologicaltechniques: microscopical analysis, isolation inculture or molecular methods. There are severalmajor problems with the direct diagnosis of TBspondylodiscitis. First, the biological sample canbe obtained only using invasive techniques, suchas biopsy or tissue sampling during surgery. Sec-ond, TB spondylodiscitis is usually paucibacil-lary, which makes microscopical analysis follow-ing acid-fast staining not very reliable do to itslow sensitivity. Moreover, microscopic analysisdoes not allows the proper identification of themycobacterial species. Isolation in culture of M.tuberculosis from the specimen is considered thegold standard, since it demonstrate the presenceof the etiologic agent and allows for the determi-nation of the susceptibility testing to first andsecond line drugs. This is becoming of para-mount importance at a time when M. tuberculo-sis strains resistant to two more drugs currentlyused in TB therapy are found responsible for TBdisease, including spondylodiscitis23. Moreover,the isolation in culture is required to extract ge-nomic DNA that could be used for the identifica-tion of the genetic determinants of drug resis-tance and the genotypic characterization and

identification of the epidemiological features ofthe M. tuberculosis strain. Unfortunately, M. tu-berculosis is a slow growing mycobacteria andisolation in culture may require up to 45 days, re-sulting in a delay of diagnosis and in the initia-tion of appropriate therapy.

A recent report by Kumar et al24, where 51 pa-tients with Pott’s disease were evaluated, indicat-ed that the sensitivity of the microscopic analysiswas only of 33%, that of culture of 43% and thetwo combined of 59%. These results underscorethe problems associated with the microbiologicaldiagnosis of TB osteomyelitis.

The use of molecular diagnosis to detect andidentify M. tuberculosis directly in the specimenis of great usefulness, given its high sensitivityand the possibility to obtain the results in one day.There are several commercially available assaysto detect M. tuberculosis in biological specimensand though these tests have not yet been licensedfor use in non-respiratory specimens, several re-ports clearly indicate that they should be consid-ered highly reliable25,26. The specimens sent to thelaboratory are directly subjected to amplificationof specific M. tuberculosis DNA sequences usingone of the many available techniques such aspolymerase chain reaction (PCR), real-time PCRor strand displacement amplification. To broadenthe search for the detection of other possible bac-terial infectious agents, it is possible to amplifyby PCR the DNA genomic fragment encoding the16S RNA fragment, using universal primers thatcan amplify DNA from any bacteria. If an ampli-fication is achieved, the DNA fragment is subject-ed to sequencing to identify the bacterial speciesdetected in the specimen27.

While it is widely accepted that moleculartools can greatly improve the diagnosis of tuber-culous spondylodiscitis, more studies are re-quired to carefully determine the sensitivity andspecificity of these assays also in comparison totraditional microscopic and culture techniques.

Immunological diagnosis

The immunological diagnosis of TB has beenhistorically performed by the Mantoux test or tu-berculin skin test (TST), which consists in the in-tradermic inoculation of the purified protein de-rivative (PPD) and in measuring 48-72 hours lat-er a delayed-type hypersensitivity which measurethe cell mediated immune response against the

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tubercle bacilli. In the last few years, more sensi-tive and specific immunological assays designedfor the immunological diagnosis of M. tuberculo-sis have been developed and are now widely usedas surrogate of TST28. These are named interfer-on-γ release assays (IGRAs) and measure the in-terferon-γ cytokine released by T cells obtainedfrom a blood sample following restimulationwith M. tuberculosis antigens. These antigens arespecific for MTB complex and few other my-cobacteria such as M. kansasii and M. szulgaiand are not found in atypical mycobacteria and inthe vaccine strain BCG, which lacks the regionof difference 1 (RD1), encoding in MTB com-plex for these antigens29. IGRAs measure thepresence of effector T cells in the blood that arespecific for M. tuberculosis. Effector T cells canreadily respond to an antigenic stimuli by secret-ing cytokines, and are different from memory Tcells that require more time (>24 hours). EffectorT cells are present only when the immune systemis currently exposed to the antigenic stimuli,which in this case is M. tuberculosis. A positiveIGRAs gives therefore an indication of an M. tu-berculosis infection, but cannot distinguish, simi-larly to TST, between an active TB from a latentinfection. The Quantiferon TB Gold In Tube(QFT-IT) is an IGRAs that measure the amountof interferon-γ secreted following restimulation,and many laboratories provide to the cliniciansthe results in UI/ml. Many researchers have at-tempted to correlate the amount of interferon-γsecreted with TB status but so far unclear corre-lation has been found.

The possibility to identify an immunologicalcorrelate of TB disease would be of paramountimportance in the case of non-pulmonary TBsuch as spondylodiscites, where the possibility tolink a clinical suspicion with an immunologicaldata strongly indicative of active TB would cer-tainly help the implementation of an effective an-ti-TB therapy. To address this issue modified ver-sion of IGRAs have been developed, includingthe use of different mycobacterial antigens to res-timulate T cells in vitro, the measurements ofmore and/or different cytokines such as TNF andIL-2 or chemokines such as IP-1030. So far, theseattempts have been only partly successful andmore studies are required to identify immunolog-ical correlates of TB disease or latent TB. One ofthe most promising evolution of these IGRAs in-volves the use of another M. tuberculosis antigen,named the heparin binding haemagglutinin (HB-HA)14. It has been shown that T cells obtained

from blood drawn from subjects with latent TBrespond to the HBHA stimuli by secreting inter-feron-γ while patients with active TB are unableto deploy a similar response31,32. Interestingly, pa-tients with active TB develop antibodies againstHBHA while subjects with latent TB do not31-33.Recent advances in the purification protocol ofHBHA34 have made possible to use this proteinin parallel with the QFT-IT assay and recent re-sults have indeed demonstrated the usefulness ofthe HBHA-based assay to discriminate, amongsubjects infected with M. tuberculosis, those withactive TB from those with latent TB35. It wouldbe of great interest to investigate whether in pa-tient with TB spondylodiscitis the immune re-sponse against HBHA is similar to what seen inpatients with pulmonary TB, since this may openthe possibility of using IGRA to guide in the di-agnosis of spondylodiscitis.

In any case, the use of the IGRAs currently li-censed provides more specific and sensitive re-sults compared to TST for the diagnosis of TBinfection and shall be used to inquiry whether aTB spondylodiscitis is suspected.

In a recent report, the sensitivity of theseIGRAs in patient with Pott’s disease was foundat 84%, with a specificity of 95%, which washigher than the 30% sensitivity measured for thecombined smear plus culture classical microbio-logical tests24. Interestingly, when the immuno-logical assay such as IGRA where combinedwith the smear and culture, sensitivity reached88%. These results, which certainly deserve to beconfirmed in larger studies including more pa-tients, underscore the usefulness of these im-munological assay in the diagnosis of spinal TB.

Moreover, it has been demonstrated the disap-pearance of the interferon-γ responses in patientswho received effective therapy and persistent re-sponse in patients who experienced failure oftherapy36. Hence, IGRAs may also be useful inmonitoring TB therapy in patients with spinalTB.

Conclusions

TB is still one of the most important infectiousdisease in the world, with a very high incidencein developing countries. The new migratorytrends, with the arrival of workers from countrieswhere TB is endemic to nations where TB wasthought to be conquered, is determining the

G. Delogu, A. Zumbo, G. Fadda

reemergence of TB disease, including less com-mon clinical manifestations such as Pott’s dis-ease. Diagnosis of tuberculous spondylodiscitisis a challenging task, and the clinician has to de-fine a correct diagnostic protocol to maximizethe chances to identify the etiologic agent. Theintroduction of the IGRAs for the immunologicaldiagnosis of TB could be of great usefulness andshall be used before biopsy or surgery is per-formed. A definitive diagnosis requires the iden-tification of the bacilli in the biological sampleby at least one of the currently used methods(microscopy, culture or molecular). When TB issuspected, molecular diagnosis shall always beconsidered and can provide rapid and reliable re-sults that could guide the adoption of proper ther-apeutic regimes.

––––––––––––––––––––Acknowledgements

This work has been funded by MIUR Prin 2008Y8RZTF.

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G. Delogu, A. Zumbo, G. Fadda