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Chapter 1

Microbiology of

Mycobacterium

tuberculosisand a new

diagnostic test for TBVera Katalinic-Jankovic*, Lucinda Furci# and Daniela Maria Cirillo#

SUMMARY: Tuberculosis (TB) has been one of the mostimportant human diseases for centuries now. It is mainly causedby Mycobacterium tuberculosis, a highly elusive bacillus. Thisintracellular pathogen does not possess the classic bacterialvirulence factors. However,M. tuberculosis efficiently evades theimmune response by complex and manipulative mechanisms,which enable survival for as long as decades. The fight with such

a smart rival gives rise to the necessity for early diagnosisand appropriate treatment. The ability to rapidly detect M.tuberculosis in clinical specimens, as well as drug resistance, isessential for the appropriate treatment of TB patients and theprevention of spread of drug-resistant strains. New moleculartools are now used in many countries as part of a standardlaboratory diagnosis. It is clear that important advances in TBdiagnosis have recently been made and potentially useful newtools are emerging. Nevertheless, there is still a lot to be done,

especially in high-burden countries where fast identification andearly treatment are needed.

KEYWORDS: Diagnostic tools, drug resistance, immunity,latency, Mycobacterium tuberculosis, pathogenesis

*Croatian National Institute of PublicHealth, TB NRL and SNRL, Zagreb,Croatia.#Emerging Bacterial Pathogens Unit,San Raffaele Scientific Institute,Milan, Italy.

Correspondence: D.M. Cirillo,Emerging Bacterial Pathogens Unit,Division of Immunology,Transplantation and InfectiousDiseases, San Raffaele Scientific

Institute, Via Olgettina 58, Milan,Italy.Email: [email protected]

Eur Respir Monogr 2012; 58: 113.Copyright ERS 2012.DOI: 10.1183/1025448x.10022311Print ISBN: 978-1-84984-027-9Online ISBN: 978-1-84984-028-6Print ISSN: 1025-448xOnline ISSN: 2075-6674

In 1882, Robert Koch discovered Mycobacterium tuberculosis, the bacillus responsible fortuberculosis (TB), thus identifying TB as an infectious disease [1]. This discovery led shortlythereafter to the identification of methods to stain bacilli in clinical specimens, making the organisms

identifiable with the use of light microscopy. Such was the birth of TB diagnostics and of microbialdiagnostics in general. The name Mycobacterium, which means fungus bacterium, was introduced in1896 [2]. It describes the way that the tubercle bacillus grows on the surface of liquid media as mould-like pellicles [2]. These aerobic, asporogenous rods have been referred to as the ducks of the microbialworld due to their thick, waxy outer coating. The genus Mycobacterium comprises a number ofaerobic bacteria and is the only member of the family Mycobacteriaceae, sharing an unusually highgenomic DNA G+C content (6270%) and the production of mycolic acids with the closely related

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genera Nocardiaand Corynebacterium within the order Actinomycetales. The most important memberof the genus, M. tuberculosis, is an intracellular pathogen that does not possess the classic bacterialvirulence factors such as toxins, capsules or fimbriae. Several structural and physiological properties ofthe bacterium are recognised for their contribution to virulence and pathology of the disease. M.tuberculosisis an aerobic, non-spore forming, non-motile bacillus with a high cell wall content of highmolecular weight lipids, which comprise approximately 60% of the cell wall structure. Due to this cellwall composition, mycobacteria stain poorly with Gram stain but are described as acid-fast, as once

stained with hot carbol-fuchsin it resists decolourisation with acidified organic solvents (ZiehlNeelsenstain) [3]. The high lipid concentration in the cell wall accounts for its impermeability and resistanceto antimicrobial agents, and resistance to killing by acidic and alkaline compounds in both the intra-and extracellular environment. M. tuberculosis has the ability to form serpentine structures (cords).The cord factor is primarily associated with virulent M. tuberculosisstrains. Although its exact role inM. tuberculosisvirulence is unclear, it is known to be toxic to mammalian cells and to be an inhibitor ofpolymorphonuclear leucocyte migration. M. tuberculosisgrows successfully under aerobic conditionsbut it is also able to survive in oxygen-deprived environments. In vivo, M. tuberculosisgrows better intissues with a high oxygen content, such as the lungs. The bacillus divides every 2022 hours, and thisslow replication rate and the ability to persist in a latent state means that individuals infected with M.

tuberculosisrequire long periods of drug and preventive therapies.

TB is caused by M. tuberculosis and TB complex members (Mycobacterium bovis, M. bovis bacilleCalmetteGuerin (BCG), Mycobacterium africanum, Mycobacterium canettii, Mycobacterium

pinnipedii and Mycobacterium microti) and is one of the most intensively studied human diseases.It can target practically any organ of the body and clinical microbiological studies have beenperformed for decades. Humans are the only reservoir for the M. tuberculosis species, althoughmany animals are also susceptible to infection [4]. M. bovis was responsible for about 6% of allhuman deaths in Europe before the introduction of milk pasteurisation and attenuation of alaboratory strain of M. bovis led to the development of the BCG vaccine in 1921.

In the 1950s, it became clear that other Mycobacterium spp. in addition to those causing TB and leprosywere also human pathogens. In 1959, RUNYON [5] proposed a classification of these non-tuberculousmycobacteria (NTM) into four major groups, based on growth rates and colony pigmentation. NTMsare generally free-living organisms that are ubiquitous in the environment around the world, and canbe found in deserts, under rocks and among dried roots of vegetation [6]. Their optimal habitat in theenvironment is close to fresh water, both flowing and static. Currently, more than 150 NTM specieshave been identified. Phylogenetic trees are available that depict genetic relatedness based on homologyof the 16S ribosomal RNA (rRNA) gene sequence. Mycobacteria that have highly homologous rRNAsequences are closely related and are on neighbouring branches of the tree [7]. The mycobacterialphylogenetic tree can be further subdivided into fast- and slow-growing bacteria. The fast growers form

colonies on selective media in less than 7 days, whereas the slow growers take more than 7 days. Inaddition, within the genus Mycobacterium a number of species are grouped into complexes (e.g.Mycobacterium avium and M. tuberculosis complexes) that include bacterial species that have a highdegree of genetic similarity and cause similar disease syndromes. The M. tuberculosiscomplex speciesshare 99.9% sequence identity and probably evolved from a single clonal ancestor.

Advances in mycobacterial genomics are providing evidence that the amount of sequence variation inthe M. tuberculosisgenome might have been underestimated and that some genetic diversity does haveimportant phenotypic consequences. Studies of the phylogeny and biogeography of M. tuberculosishave revealed six main strain lineages that are associated with particular geographical regions [8].

Pathogenesis and immunity

Early steps of infection

M. tuberculosis is a highly successful bacterial pathogen that mainly targets host macrophages, keymediators of both innate and adaptive immune response. In lung infections, M. tuberculosis is

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typically inhaled into the body, passes through the airways and reaches the alveolar space. Here, itinteracts with dendritic cells [9, 10], alveolar macrophages and pulmonary epithelial cells, but itsoptimal hosts are alveolar macrophages and other mononuclear phagocytes [11]. M. tuberculosisgains entry into alveolar macrophages through receptor-mediated phagocytosis, a normal feature ofthe innate immune system. Two main routes are exploited: bacterial cell surface molecules activatecomplement proteins present in the alveolar space, which are then recognised by complementreceptors on macrophages; or alveolar macrophages recognise bacterial mannose residues

(particularly mannose-capped lipoarabinomannan), directly through binding with macrophagemannose receptors (fig. 1) [12]. Alveolar macrophages are attractive targets for M. tuberculosisbecause they are adapted to the task of removing small airborne particles through phagocytosis, and

Inhaled M. tuberculosis

M. tuberculosisuptake

receptors

M. tuberculosis

recognition receptors

1

TLR2

TLR9

TLR4

NLRP3

Alveolar space

DC-SIGN

Phagocyte

NOD2

Mannose

receptors

Scavengerreceptors

Alveolarmacrophages

Dendritic

cells

Complementreceptors

(CR1, CR2, CR4)

4

3

2

Figure 1. Early steps of phagocyte infection. 1) Mycobacterium tuberculosis is inhaled through the airways andtravels all the way to the alveoli of the lower portion of the lungs where it establishes stage I infection. 2) In

the alveolar space, mycobacteria are actively phagocytosed by resident macrophages and dendritic cells.3) Complement receptors (CR) are primarily responsible for uptake of opsonised M. tuberculosis and mannosereceptors, and scavenger receptors for the uptake of nonopsonised M. tuberculosis. 4) Recognition receptors

and Toll-like receptors (TLRs) are expressed not only at the cell surface but also in phagosomes; therefore,

immune activation may occur with or without phagocytosis. NOD: nucleotide-binding oligomerisation domainprotein; NLRP3: NOD, LRR and pyrin domain containing 3; DC-SIGN: dendritic cell-specific intercellular

adhesion molecule-3 grabbing nonintegrin.

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do not induce strong inflammatory responses. Their ability to produce anti-microbial chemicalssuch as nitric oxide (NO) and reactive oxygen intermediates (ROI) is blunted [13].

Innate immunity

The recognition ofM. tuberculosiscomponents by multiple pattern-recognition receptors on alveolarmacrophages initiates innate immunity. Of the Toll-like receptors (TLRs), TLR2 has the largest

number of identified mycobacterial agonists, including lipoproteins (as many as 99 of them),phosphatidylinositol mannans and lipomannan [14]. In addition, TLR9 senses mycobacterial DNAand contributes to the production of cytokines by macrophages and dendritic cells [15]. Additionalrecognition ofM. tuberculosisis mediated by specific members of the C-type lectin receptor family,including the lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin(DC-SIGN) [16] and the mannose receptor [17]. Of the cytosolic pattern-recognition receptors,nucleotide-binding oligomerisation domain protein 2 (NOD2) [18] and NOD, NOD-like receptors(NLRs) and pyrin domain containing 3 (NLRP3) [19] recognise the peptidoglycan subunit N-glycolyl muramyl dipeptide and one or more extraembryonic, spermatogenesis, homeobox 1homologue (ESX1)-secreted substrates (such as the 6-kDa early secretory antigenic target (ESAT6)),

respectively. Stimulation of these pattern recognition receptors, individually or collectively, inducesthe expression of pro-inflammatory cytokines.

Circulating blood monocytes are recruited through chemokine signals produced by infectedalveolar macrophages, and migrate rapidly across the blood vessels to the site of infection. Withinthe tissue, they differentiate into macrophages with the ability to ingest and kill the bacteria.Interaction between macrophages and T-cells (and in particular, the activation of macrophages byinterferon (IFN)-c secreted by T-cells) is considered central in the elimination of M. tuberculosis[20]. However, as these cells are recruited, they become infected by the expanding populationof mycobacteria and establish early granulomas. In other infectious diseases, the recruitment of

phagocytic cells restricts and even eliminates invading pathogens, whereas the recruitment ofphagocytes to sites of mycobacterial infection actually benefits the pathogen during the early stagesof infection, by providing additional cellular niches for bacterial population expansion [21].

Adaptive immunity

A peculiar characteristic of the adaptive immune responses to M. tuberculosis infection is the longdelay in onset. Measurable adaptive immune responses emerge in humans approximately 42 daysafter M. tuberculosis exposure [22] and a similar delay is observed with hepatitis C virus infection[23]. In contrast, immune responses can be activated within ,20 hours postinfection with theinfluenza virus [24]. It is currently unclear why this step is so prolonged, although there isevidence that M. tuberculosis infection of myeloid dendritic cells inhibits their migration inresponse to ligands for CC chemokine receptor 7 (CCR7) [25]. After the onset of adaptiveimmunity with the accumulation of effector CD4+ and CD8+ T-cells in the lungs, the growth ofthe bacterial population is arrested and most patients become asymptomatic, do not shed bacteriaand are considered to have latent TB infection (LTBI). The delayed adaptive immune responsecould be significant in establishing latency by giving the bacteria time to establish sufficientnumbers to evade complete elimination. Multiple mechanisms probably contribute to the limitedability of adaptive immune responses to kill M. tuberculosis, some of which are well characterisedand include: 1) impaired major histocompatibility complex (MHC) class II-mediated antigenpresentation [26]; 2) induction of the anti-inflammatory mediator lipoxin A4 [27]; 3) restrictionby regulatory T-cells [28]; 4) down-regulation of bacterial antigen gene expression and, therefore,failure to induce antigen-specific CD4+ T-cells [29]; and 5) resistance to the macrophage-activating effects of IFN-c [30]. Moreover, a recent study in non-human primates revealed that M.tuberculosis also accumulates mutations during latency [31]. Taken together, these data provideconvincing evidence that LTBI is not simply a state of bacterial stasis, but a state of dynamicbacterial and immunological equilibrium. Thus, inflammation is a double-edged sword in the host

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response to M. tuberculosis. On the one hand, it is required for the initial control of infection tobring the bacteria into homeostasis with the human host. Failure to mount an adequateinflammatory response leads to progressive disease upon infection in both animal models andgenetically susceptible humans. On the other hand, the bacteria exploit the host inflammatoryresponse to spread to susceptible individuals and to fill as completely as possible their ecologicalniche.

Mechanisms of immune evasion

Considerable evidence indicates that M. tuberculosis and other pathogenic mycobacteria of the M.tuberculosis complex have evolved multiple mechanisms to manipulate their cellular niches fortheir own advantage (fig. 2). First, pathogenic mycobacteria modulate the trafficking and

Macrophage

4

2

NO

Agpresentatio

n

T-cell

IFN-

IFN-

ROI

DC-SIGN

CCR7

Dendritic cell

M. tuberculosisgene expression Dormancy regulon

Toxinantitoxin pairs Resuscitating factors

ManLAM

RNI

MHC II +

M. tuberculosis

Ag

pH 3

51

8

6

9

10

8

7

Figure 2. Mechanisms of immune evasion by Mycobacterium tuberculosis. 1) M. tuberculosis makes themacrophages a sanctuary and enables the bacteria to resist killing by nitric oxide (NO), reactive nitric oxide

intermediates (RNI) and reactive oxygen intermediates (ROI); 2) by blocking the phagosomelysosome fusion;

and 3) by inhibiting lysosome acidification. 4) Virulent strains of M. tuberculosis can translocate from thephagosome to the cytosol, bacille CalmetteGuerin cannot. This capacity is linked to the ESX-1 coding region

(within region of deletion (RD)1). 5) M. tuberculosishas evolved specific mechanisms to adopt and recover from a

state of latency and that latency is not merely the suppressive effect of the host immune response on bacterial

replication. 6) M. tuberculosis disrupts antigen (Ag) presentation through the downregulation of majorhistocompatibility complex (MHC) class II and 7) consequently causes a decrease in the antigen-specific CD4

T-cell population. 8) Insufficient interferon (IFN)-c is essential for both macrophage and dendritic cell (DC)maturation and development of an efficient cellular immune response against M. tuberculosis infection.9) Ligation of dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) by

M. tuberculosis virulence factor mannose-capped cell wall component lipoarabinomannan (ManLAM) reduces

Toll-like receptor 4-triggered DC maturation and enhances the production of interleukin-10, thus resulting ininhibition of DC maturation. 10) Moreover, the migration of infected DCs to lymph nodes in response to CC

chemokine receptor (CCR)7 ligands is impaired by M. tuberculosis induced downmodulation of CCR7.

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maturation of the phagosomes in which they reside [32], allowing them to evade lysosomalmechanisms of restriction, killing and degradation. Secondly, mycobacteria use several virulencemechanisms to optimise their spread from cell to cell. For example, the ESX1 type VII secretionsystem, the absence of which attenuates the strain of M. bovis used in the BCG vaccine [33],promotes the necrotic death of infected cells and the recruitment of macrophages. This allows theintracellular bacteria to be released from the cell for uptake by the freshly recruited adjacentphagocytes, resulting in subsequent intracellular growth and bacterial population expansion [21].

Thirdly, M. tuberculosis possesses multiple mechanisms for inhibiting host cell apoptosis [34];among other benefits to the bacteria, such inhibition allows for the prolonged survival of infectedcells and for a larger number of bacteria to accumulate in a given cell before they are released bycell death [25].

Strong evidence exists that the mycobacteria are also active contributors to the immunologicalequilibrium state in LTBI. First, a well-characterised bacterial regulon that is controlled by DosRDosS, a two-component signal transduction system in mycobacteria, is induced by several stimulithought to prevail during LTBI, including local hypoxia [35], NO [36] and carbon monoxide [37].This dormancy regulon controls the expression of genes that allow the bacteria to use alternative

energy sources, especially lipids, and genes encoding factors that are selectively recognised by T-cellsfrom humans with LTBI (but not active TB infection) [38]. The expression of this gene networkimplies that M. tuberculosis has evolved specific mechanisms to adopt a state of latency, and thatlatency is not merely the suppressive effect of the host immune response on bacterial replication. Inaddition, M. tuberculosis encodes five proteins that resemble the well-characterised Micrococcusluteus resuscitation-promoting factor (Rpf), which is a secreted protein that has the ability toresuscitate bacteria from a nutrient-starved dormant state [39]. Deletion of one or more of the M.tuberculosis rpfgenes generates bacteria that have an impaired recovery from dormancy, indicatingthat these genes may participate in the progression from latency to reactivation [40]. Finally, M.tuberculosis encodes 88 toxinantitoxin gene pairs, the expression balance of which regulates

multiple phenomena, including whether the bacteria replicate or remain static [41]. Thus, M.tuberculosis possesses at least three systems (the dormancy regulon, resuscitation promoting factorsand toxinantitoxin gene pairs) that regulate its metabolic and growth state.

Mechanisms of TB reactivation

Most cases of TB in adults are attributable to reactivation that can occur decades after the initialinfection [42]. Reactivation of TB is widely attributed to weakened immunity, although only aminority of cases can be linked to well-characterised defects in immunity. In humans, only twomechanisms have been identified that explain reactivation of TB. 1) The quantitative andqualitative CD4+ T-cell defects that occur in people infected with HIV [43]; although the precisemechanisms that these cells use to establish and maintain immune control in the latent state of thedisease remain to be identified. 2) The therapeutic neutralisation of tumour necrosis factor (TNF)[44], that results in decreased macrophage-mediated anti-mycobacterial activity and thesubsequent death of macrophages [45]; the induction of a higher frequency of regulatoryT-cells [46]; and the depletion of a subset of CD45RA+ effector memory CD8+ T-cells that containgranulysin [47].

As noted previously, M. tuberculosis has specific programmes for initiating a state of dormancy inresponse to certain environmental signals (some of which are imposed by adaptive immuneresponses) and this state manifests as clinical latency. In turn, M. tuberculosis also has specific

programmes for recovering from dormancy, suggesting that the bacteria may assume a primaryrole in some cases of reactivation TB that are not explained by immune defects or deficiencies.

The development of efficacious vaccines against TB presents unique challenges that demand abetter understanding of protective and pathological immune responses in TB. Clear correlates ofprotective immunity have not yet been identified, especially in humans, making surrogate end-points inadequate for evaluating TB vaccine efficacy.

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New diagnostic tests for TB

TB still remains a serious public health threat. More than 9 million new cases are reportedannually, and the incidence rate is falling at less than 1% per year [48]. The current TB diagnosticpipeline is vastly better than the portfolio available 10 years ago when smear microscopy, a 100-

year old test, was the only option for most resource-limited settings (fig. 3) [49]. Despite being thecornerstone of TB diagnosis in low-resource settings, smear microscopy has only modest

sensitivity for TB disease and particularly low sensitivity in patients with advanced HIV disease(fig. 4). In settings with a high prevalence of HIV infection, current tools and strategies fordiagnosis of TB are inadequate. Additionally, poor access to TB diagnostics continues to be amajor challenge contributing to under-diagnosis of disease, which leads to individual morbidityand mortality and to continued transmission and delayed diagnosis of drug resistance, leading toacquisition of additional resistance and to morbidity and transmission. The lack of accurate andrapid diagnostics remains a major obstacle in the progression of the detection rate for new sputumsmear-positive and smear-negative pulmonary cases of TB, childhood TB and extrapulmonary(EP)TB. This is of importance for the control of both sensitive and drug-resistant (DR)-TB atnational and global level [50]. Development of resistance to antituberculotic drugs, and especially

multidrug-resistant (MDR)-TB (defined as TB resistant to isoniazid and rifampicin) and exten-sively drug-resistant (XDR)-TB (defined as MDR-TB resistant to second-line injectables andfluoroquinolones) are threats to the elimination of TB worldwide. The ability to rapidly andaccurately detect M. tuberculosisin clinical specimens, as well as drug resistance, is essential for theappropriate treatment of TB patients and the prevention of spread of drug-resistant strains.

Point-of-care tests

There is a great need for rapid point-of-care tests that can be readily used at all levels of the healthsystem and in the community [51]. These techniques are often unsatisfactory and unavailable at

patients first point of contact with the health system. Notable advances in TB diagnostictechnologies have been made in the past several years, and the potential exists for translating thesedevelopments into meaningful improvements in global TB clinical care and control. Unfortunatelyno real point-of-care tests will be commercially available in the next few years.

Technologies or methods endorsed by WHO Technologies at late stages of development Technologies at early stages of development

Liquid culture and DSTRapid speciationLPA for MDR-TBNon-commercial culture and DST (MODS, NRA, CRI)

Manual NAAT 1st generationRapid colorimetric DST

POC test (detection of TB)Prediction (LTBI)

Manual NAAT 2nd generation

2-specimen approaches

Distancefrompatients

LED microscopySame-day diagnosis

201520142013201220112010200920082007 2016

1040

Accessafter5years%

70

95Peripheral level

Intermediate level

New SS+case definition

LPA for XDR-TBReference level

XpertMTB/RIF

Figure 3. The pipeline for new diagnostics. Xpert1 MTB/RIF is manufactured by Cepheid (Sunnyvale, CA, USA).DST: drug-susceptibility test; LPA: line probe assay; MDR: multidrug-resistant; TB: tuberculosis; MODS:microscopy observed drug susceptibility; NRA: nitrate reductase assay; CRI: colorimetric redox indicator assay;

XDR: extensively drug-resistant; SS+: sputum smear positive; LED: light-emitting diode; NAAT: nucleic acid

amplification techniques; POC: point-of-care; LTBI: latent TB infection; WHO: World Health Organization.Reproduced and modified from [48] with permission from the publisher.

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Microscopy and culture

Smear microscopy and solid culture have been the cornerstone of TB laboratory diagnosis fordecades. Some new technologies have been endorsed by the World Health Organization (WHO),such as implementation of automated liquid cultures, fluorescence microscopy and light-emittingdiode (LED) fluorescence microscopy [52, 53]. The WHO is also recommending new policies forsputum collection and the number of samples to be examined by smear microscopy for thedefinition of a TB case. Culture of M. tuberculosis in clinical specimens is substantially moresensitive than smear microscopy. Culture can be performed using solid media, such asLowensteinJensen, or liquid media, such as the ones used in commercially available automatedsystems. Until the recent advent of molecular tests for drug resistance, isolation of M. tuberculosisby culture was a prerequisite for subsequent phenotypic drug-susceptibility testing (DST). The

Achilles heel of culture is the time it takes to acquire results (1014 days for liquid culture and 34 weeks for solid culture), which is a consequence of the long doubling time of M. tuberculosis.Currently available culture methods are technically demanding, require implementation ofbiosafety practices and equipment to prevent inadvertent infection of laboratory personnel, andhave relatively high per test prices. Automated liquid culture systems are now the gold standard forthe diagnosis of TB; they are substantially faster and have a 10% greater yield than solid media. In2007, these systems were recommended by WHO to be used in combination with antigen-basedspecies confirmation for diagnosis and DST in low-income and middle-income countries.However, such systems are expensive and prone to contamination. Alternative inexpensivenoncommercial culture and DST methods were endorsed by WHO in 2009 for use as an interim

solution in resource-constrained settings [53]. These alternatives include microscopy observeddrug susceptibility (MODS) and the nitrate reductase assay [54].

Molecular tests for diagnosis of DR-TB

To enhance the capacity for rapid diagnosis of MDR-TB, in 2008, WHO approved the use of lineprobe assays (LPAs) for the rapid molecular detection of drug resistance in smear-positive

Log cfu.mL-1543210 6

Liquid culture10100 cfu.mL-1

Solid culture1001000 cfu.mL-1

Immunochromatographyfor speciation

1000000 cfu.mL-1

AutomatedNAAT

100150 cfu.mL-1

LAMP-TB1001000 cfu.mL-1

Line probe

assay100010000 cfu.mL-1

Fluorescent/LED microscopy

500010000 cfu.mL-1

Figure 4. Sensitivity of the current diagnostics: liquid cultures performed by automated systems are the mostsensitive tool available today, automated nucleic acid amplification techniques (NAAT) (Xpert1 MTB/RIF;

Cepheid, Sunnyvale, CA, USA) shows a sensitivity comparable to solid cultures. LAMP: loop-mediatedamplification; TB: tuberculosis; LED: light-emitting diode.

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specimens or culture isolates. These molecular assays reduce the time to diagnosis of MDR- andXDR-TB from weeks or months to a matter of days. However, it has yet to be shown whether theuse of such assays improves patient outcomes. Nucleic acid amplification techniques (NAATs) arethe most promising development in TB diagnostics. These tests have been shown to have highspecificity, but limited and variable sensitivity, especially for sputum smear-negative disease.Simplified versions of these assays with higher sensitivity are being developed. A simplified manualNAAT that uses loop-mediated isothermal amplification with a simple visual colorimetric readout

is being assessed in peripheral laboratory facilities in resource-constrained settings [55]. A newrapid test that overcomes many of the current operational difficulties was endorsed by WHO inDecember 2010, the Xpert1 MTB/RIF assay is a sensitive and specific fully automated real-timenucleic acid amplification technology run on the multi-disease platform GeneXpert1 (Cepheid,Sunnyvale, CA, USA). The Xpert1 MTB/RIF assay that simultaneously detects M. tuberculosisandrifampicin resistance-conferring mutations, in a closed system, in less than 2 hours, directly fromsputum samples has been developed for use outside reference laboratory centres. This system usesa series of molecular probes and real-time PCR technology to detect M. tuberculosis and the rpoBrifampicin resistance mutation [5660]. The Xpert1 MTB/RIF assay has proven a valid tool alsofor the diagnosis of EPTB [61].

LPA technology remains a valid tool for fast detection of the MDR phenotype in smear-positivesamples [6264]. The newest generation of LPAs for detection of resistance to rifampicin andisoniazid shows an increased sensitivity also in paucibacillary specimens. A second-line drug LPAtest is commercially available; the test targets the main mutations causing resistance to injectables,fluoroquinolones and ethambutol. The test shows a high positive predictive value (PPV) forinjectables and fluoroquinolones; however, the negative predictive value is low and the test cannotbe used for excluding XDR-TB [63, 64].

For ethambutol, the correlation of the molecular test targeting the embB306 mutation withphenotypic tests performed in vitrois low, this could also be due to the suboptimal performance ofin

vitrotesting for ethambutol resistance performed by BactecTM

MGITTM

(BD Bioscience, Erebodegem,Belgium) [64].

IFN-c release assays

For the past century, the tuberculin skin test (TST) using purified protein derivative has been theonly screen available for the diagnosis of LTBI [51]. Together with chest radiographs, TST is used asan adjunct to smear microscopy (and culture, if available) in some settings; however, the former havepoor sensitivity and specificity for active TB, and the latter are often not available at the point ofprimary patient care. In detecting LTBI, the TSTs major failing is its inability to reliably distinguish

individuals infected with M. tuberculosisfrom individuals sensitised to other mycobacteria, includingBCG [51]. A decade ago IFN-c release assays (IGRAs) were developed whereby IFN-c titres weremeasured after in vitro stimulation of peripheral blood mononuclear cells with antigens such asESAT-6 and the 10-kDa culture filtrate antigen (CFP-10) (immunodominant antigens expressed bymembers of the M. tuberculosiscomplex) [4, 65]. The IGRAs are used principally for detection ofM.tuberculosis infection. Use of these tests for the diagnosis of active disease is based on thepresumption that one must have TB infection in order to have TB disease. The greater problem indiagnosing active TB is their poor specificity for disease, because these tests cannot distinguish animmune response to reactivated TB from a response to TB infection that remains latent.Nevertheless, IGRAs have now become the gold standard in industrialised countries for identifying

individuals whose immune system has previously encountered M. tuberculosis and have beenextensively tested in many clinical situations and in individuals infected with HIV. The assessment ofthese test results for detection of LTBI has been difficult because of the absence of a gold standard forTB latency [51]. A meta-analysis of these studies showed that IGRAs are at least as sensitive as andmore specific than TST [4]. Longitudinal studies have shown that the predictive value of IGRAs forreactivation of TB in immunosuppressed individuals is better than that provided by the TST inindividuals vaccinated with BCG. High levels of IFN-c release are detected by these assays in about

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7090% of individuals with active disease and these levels decrease after treatment is completed,although such reductions are not consistently recorded [4, 66].

Beyond new technology

Successful implementation of new tools will depend on more than technological innovation. At theresearch level, rigorous implementation of well-designed, bias-minimised studies and complete andaccurate reporting are essential for appropriate decision making by the healthcare communitycharged with implementing tests for individual patient evaluation or recommending tests for TBprogramme use.

Molecular approaches for diagnosis of drug resistance will certainly be implemented in the futureand tests designed using information made available by the large scale use of new-generationsequencing will allow the design of more specific and sensitive assays.

New programmatic approaches, including revised clinical algorithms for TB diagnosis, may be neededto maximise the impact of new tools. For example, should rapid molecular tests for drug resistance beperformed for all persons with suspected TB during the initial evaluation, be reserved for use in theinitial evaluation only of persons with suspected TB and risk factors for drug resistance, or be used insome other place in a diagnostic algorithm? In populations with a high prevalence of HIV infection,should urine-based antigen detection tests be used solely for evaluation of symptomatic persons withsuspected TB, or should they also play a role in routine screening of HIV-infected persons? To date,most TB diagnostic test development has focused on maximising sensitivity and specificity to rule inor confirm a TB diagnosis. However, a test with an exceedingly high negative predictive value mighthave use in ruling out TB and, thereby, allowing efficient triage of patients and resources; such a testwould require careful assessment to determine its optimal use in clinical algorithms.

Laboratory capacity needs to be strengthened, especially in resource-limited settings [67]. Although

some aspects of laboratory strengthening will vary according to the characteristics of the new tests,there are, nevertheless, general unmet needs, including those for training at technologist andmanagement levels, retention of trained personnel, enhancement of quality-assurance systems,enhancement of result-reporting mechanisms, and reliable mechanisms for instrument maintenanceand supply procurement.

Funding estimates aside, it is clear that important advances in TB diagnosis have recently been made,and potentially useful new tools are emerging; continued and augmented investment will be requiredto successfully implement the most promising of these tools in the settings where they are most neededand to maintain a robust pipeline that will ultimately yield the tools that revolutionise TB diagnosis.

Conclusions

Whats new in TB diagnostics? There are a lot of new developments, but not enough. The future isbrighter as several promising new tools enter the demonstration and late evaluation stages, but thereis a great need for improvement and important barriers still remain in translating technical advancesinto meaningful and sustainable improvements in individual and public health in settings hardest hitby TB.

Statement of Interest

None declared.

References1. Koch R. Die Aetiologie der Tuberculose. [The aetiology of Tuberculosis.] In: Berliner Klinische Wochenschrift. Bd.

19, Nr. 15, 1882; S. 221230.

2. Gandadharam PRJ, Jenkins PA. Mycobacteria: Basic Aspects. Chapman and Hall, New York, 1997.

10

MICROBIOLOGYOF

M.TUBERCU

LOSIS

7/28/2019 SEC5.body

11/13

3. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem 1995; 64: 2963.

4. Pai M, Zwerling A, Menzies D. Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis

infection an update. Ann Intern Med 2008; 149: 177184.

5. Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am 1959; 43: 273290.

6. Stanford J, Stanford C. Mycobacteria and their world. Int J Mycobacteriol 2012; 1: 312.

7. Tortoli E. Phylogeny of the genus Mycobacterium: many doubts, few certainties. Infect Genet Evol 2012; 12:

827831.

8. Gagneux S, Small PM. Global phylogeography ofMycobacterium tuberculosis and implications for tuberculosis

product development. Lancet Infect Dis 2007; 7: 328337.

9. Wolf AJ, Linas B, Trevejo-Nunez GJ, et al. Mycobacterium tuberculosis infects dendritic cells with high frequency

and impairs their function in vivo. J Immunol 2007; 179: 25092519.

10. Kang DD, Lin Y, Moreno JR, et al. Profiling early lung immune responses in the mouse model of tuberculosis.

PLoS ONE 2011; 6: e16161.

11. Bermudez LE, Goodman J. Mycobacterium tuberculosis invades and replicates within type II alveolar cells. Infect

Immun 1996; 64: 14001406.

12. Schlesinger LS. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is

mediated by mannose receptors in addition to complement receptors. J Immunol 1993; 150: 29202930.

13. Adams LB, Dinauer MC, Morgenstern DE, et al. Comparison of the roles of reactive oxygen and nitrogen

intermediates in the host response to Mycobacterium tuberculosis using transgenic mice. Tuber Lung Dis 1997; 78:

237246.

14. Banaiee N, Kincaid EZ, Buchwald U, et al. Potent inhibition of macrophage responses to IFN-c by live virulent

Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2.

J Immunol 2006; 176: 30193027.

15. Bafica A, Scanga CA, Feng CG, et al. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating

optimal resistance to Mycobacterium tuberculosis. J Exp Med 2005; 202: 17151724.

16. Tailleux L, Schwartz O, Herrmann JL, et al. DC-SIGN is the major Mycobacterium tuberculosisreceptor on human

dendritic cells. J Exp Med 2003; 197: 121127.

17. Court N, Vasseur V, Vacher R, et al. Partial redundancy of the pattern recognition receptors, scavenger receptors,

and C-type lectins for the long-term control of Mycobacterium tuberculosis infection. J Immunology 2010; 184:

70577070.

18. Brooks MN, Rajaram MV, Azad AK, et al. NOD2 controls the nature of the inflammatory response and

subsequent fate ofMycobacterium tuberculosisand M. bovis BCG in human macrophages. Cell Microbiol2011; 13:

402418.

19. Mishra BB, Moura-Alves P, Sonawane A, et al. Mycobacterium tuberculosis protein ESAT-6 is a potent activator ofthe NLRP3/ASC inflammasome. Cell Microbiol 2010; 12: 10461063.

20. Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009; 27: 393422.

21. Davis JM, Ramakrishnan L. The role of the granuloma in expansion and dissemination of early tuberculous

infection. Cell 2009; 136: 3749.

22. Poulsen A. Some clinical features of tuberculosis. 1. Incubation period. Acta Tuberc Scand 1950; 24: 311346.

23. Thimme R, Oldach D, Chang KM, et al. Determinants of viral clearance and persistence during acute hepatitis C

virus infection. J Exp Med 2001; 194: 13951406.

24. Ho AW, Prabhu N, Betts RJ, et al. Lung CD103+ dendritic cells efficiently transport influenza virus to the

lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells. J Immunol 2011; 187:

60116021.

25. Blomgran R, Desvignes L, Briken V, et al. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to

delayed activation of naive CD4 T cells. Cell Host Microbe 2012; 11: 8190.26. Noss EH, Pai RK, Sellati TJ, et al. Toll-like receptor 2-dependent inhibition of macrophage class II MHC

expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. J Immunol 2001; 167:

910918.

27. Divangahi M, Desjardins D, Nunes-Alves C, et al. Eicosanoid pathways regulate adaptive immunity to

Mycobacterium tuberculosis. Nat Immunol 2010; 11: 751758.

28. Shafiani S, Tucker-Heard G, Kariyone A, et al. Pathogen-specific regulatory T cells delay the arrival of effector T

cells in the lung during early tuberculosis. J Exp Med 2010; 207: 14091420.

29. Egen JG, Rothfuchs AG, Feng CG, et al. Intravital imaging reveals limited antigen presentation and T cell effector

function in mycobacterial granulomas. Immunity 2011; 34: 807819.

30. Kincaid EZ, Ernst JD. Mycobacterium tuberculosis exerts gene-selective inhibition of transcriptional responses to

IFN-c without inhibiting STAT1 function. J Immunol 2003; 171: 20422049.

31. Ford CB, Lin PL, Chase MR, et al. Use of whole genome sequencing to estimate the mutation rate ofMycobacterium tuberculosis during latent infection. Nat Genet 2011; 43: 482486.

32. Chackerian AA, Alt JM, Perera TV, et al. Dissemination ofMycobacterium tuberculosisis influenced by host factors

and precedes the initiation of T-cell immunity. Infection Immun 2002; 70: 45014509.

33. Pym AS, Brodin P, Brosch R, et al. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines

Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiol 2002; 46: 709717.

11

V.

KATALINIC-

JANKOVIC

ETAL

.

7/28/2019 SEC5.body

12/13

34. Hinchey J, Lee S, Jeon BY, et al. Enhanced priming of adaptive immunity by a proapoptotic mutant of

Mycobacterium tuberculosis. J Clin Invest 2007; 117: 22792288.

35. Park HD, Guinn KM, Harrell MI, et al. Rv3133c/dosR is a transcription factor that mediates the hypoxic response

of Mycobacterium tuberculosis. Molecular Microbiol 2003; 48: 833843.

36. Voskuil MI, Schnappinger D, Visconti KC, et al. Inhibition of respiration by nitric oxide induces a Mycobacterium

tuberculosis dormancy program. J Exp Med 2003; 198: 705713.

37. Shiloh MU, Manzanillo P, Cox JS. Mycobacterium tuberculosis senses host-derived carbon monoxide during

macrophage infection. Cell Host Microbe 2008; 3: 323330.

38. Black GF, Thiel BA, Ota MO, et al. Immunogenicity of novel DosR regulon-encoded candidate antigens of

Mycobacterium tuberculosisin three high-burden populations in Africa. Clin Vaccine Immunol2009; 16: 12031212.

39. Chao MC, Rubin EJ. Letting sleeping dos lie: does dormancy play a role in tuberculosis? Annu Rev Microbiol2010;

64: 293311.

40. Tufariello JM, Mi K, Xu J, et al. Deletion of the Mycobacterium tuberculosisresuscitation-promoting factor Rv1009

gene results in delayed reactivation from chronic tuberculosis. Infect Immunity 2006; 74: 29852995.

41. Ramage HR, Connolly LE, Cox JS. Comprehensive functional analysis of Mycobacterium tuberculosis toxin

antitoxin systems: implications for pathogenesis, stress responses, and evolution. PLoS Genet 2009; 5: e1000767.

42. Lillebaek T, Dirksen A, Vynnycky E, et al. Stability of DNA patterns and evidence of Mycobacterium tuberculosis

reactivation occurring decades after the initial infection. J Infect Dis 2003; 188: 10321039.

43. Kwan CK, Ernst JD. HIV and tuberculosis: a deadly human syndemic. Clin Microbiol Rev 2011; 24: 351376.

44. Harris J, Keane J. How tumour necrosis factor blockers interfere with tuberculosis immunity. Clin Exp Immunol

2010; 161: 19.

45. Clay H, Volkman HE, Ramakrishnan L. Tumor necrosis factor signaling mediates resistance to mycobacteria by

inhibiting bacterial growth and macrophage death. Immunity 2008; 29: 283294.

46. Nadkarni S, Mauri C, Ehrenstein MR. Anti-TNF-a therapy induces a distinct regulatory T cell population in

patients with rheumatoid arthritis via TGF-b. J Exp Med 2007; 204: 3339.

47. Bruns H, Meinken C, Schauenberg P, et al. Anti-TNF immunotherapy reduces CD8+ T cell-mediated

antimicrobial activity against Mycobacterium tuberculosis in humans. J Clin Invest 2009; 119: 11671177.

48. World Health Organization. Global tuberculosis control. WHO report 2011. WHO/HTM/TB/2011.16. Geneva,

WHO, 2011.

49. Pai M. Improving TB diagnosis: difference between knowing the path and walking the path. Expert Rev Mol Diagn

2011; 11: 24144.

50. Drobniewski F, Nikolayevskyy V, Balabanova Y, et al. Diagnosis of tuberculosis and drug resistance: what can new

tools bring us? Int J Tuberc Lung Dis 2012; 16: 860870.

51. Lawn SD, Zumla AI. Tuberculosis. Lancet 2011; 378: 5772.52. Steingart KR, Henry M, Ng V, et al. Fluorescence versus conventional smear microscopy for tuberculosis: a

systematic review. Lancet Infect Dis2006; 6: 570581.

53. WHO. Report of the ninth meeting of the strategic and technical advisory group for tuberculosis. Geneva, WHO,

2009.

54. Pai M, Minion J, Sohn H, et al. Novel and improved technologies for tuberculosis diagnosis: progress and

challenges. Clin Chest Med 2009; 30: 701716.

55. Boehme CC, Nabeta P, Henostroza G, et al. Operational feasibility of using loop-mediated isothermal

amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin

Microbiol 2007; 45: 19361940.

56. World Health Organization. Roadmap for rolling out Xpert MTB/RIF for rapid diagnosis of TB and MDR-TB.

Geneva, WHO, 2010. Available at: www.who.int/tb/laboratory/roadmap_xpert_mtb-rif.pdf Date last accessed:

June, 2012.57. World Health Organization. Rapid implementation of the Xpert MTB/RIF diagnostic test. Technical and

operational How-to. Practical considerations. WHO/HTM/TB/2011.2. Geneva, WHO, 2011.

58. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and

simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system. Policy statement. WHO/

HTM/TB/2011.4. Geneva, WHO, 2011.

59. Boehme CC, Nicol MP, Nabeta P, et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of

the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation

study. Lancet 2011; 377: 14951505.

60. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance.

N Engl J Med 2010; 363: 10051015.

61. Tortoli E, Russo C, Piersimoni C, et al. Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary

tuberculosis. Eur Respir J 2012; 40: 442447.62. Mironova S, Pimkina E, Kontsevaya I, et al. Performance of the GenoType1 MTBDRPlus assay in routine settings:

a multicenter study. Eur J Clin Microbiol Infect Dis 2012; 31: 13811387.

63. Ignatyeva O, Kontsevaya I, Kovalyov A, et al. Detection of resistance to second-line antituberculosis drugs by

use of the genotype MTBDRsl assay: a multicenter evaluation and feasibility study. J Clin Microbiol 2012; 50:

15931597.

12

MICROBIOLOGYOF

M.TUBERCU

LOSIS

7/28/2019 SEC5.body

13/13

64. Miotto P, Cabibbe AM, Mantegani P, et al. GenoType MTBDRsl performance on clinical samples with diverse

genetic background. Eur Respir J 2012; 40: 690698.

65. Diel R, Goletti D, Ferrara G, et al. Interferon-c release assays for the diagnosis of latent Mycobacterium tuberculosis

infection: a systematic review and meta-analysis. Eur Respir J 2011; 37: 8899.

66. Adetifa IM, Ota MO, Walther B, et al. Decay kinetics of an interferon-c release assay with anti-tuberculosis therapy

in newly diagnosed tuberculosis cases. PLoS One 2010; 5: e12502.

67. Parsons LM, Somoskovi A, Gutierrez C, et al. Laboratory diagnosis of tuberculosis in resource-poor countries:

challenges and opportunities. Clin Microbiol Rev 2011; 24: 314350.

V.

KATALINIC-

JANKOVIC

ETAL

.