Faculté de Pharmacie

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Ecole Doctorale en Sciences Pharmaceutiques

Contribution to the research on

Drug Resistant Mycobacterium tuberculosis

Karolien STOFFELS

Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques

Promoteur

Véronique FONTAINE

Unité de Microbiologie Pharmaceutique et Hygiène du Laboratoire de Chimie Biologique et Médicale et Microbiologie Pharmaceutique, ULB

Co-promoteur

Maryse FAUVILLE-DUFAUX

Tuberculose et Mycobactéries, Institut Scientifique de Santé Publique

Composition du jury

Jean-Paul DEHAYE (Président) Laboratoire de Chimie Biologique et Médicale et Microbiologie Pharmaceutique, ULB

Paule BOUSSARD (Secrétaire) Laboratoire des Biopolymères et des Nanomatériaux Supramoléculaires, ULB

Olivier VANDENBERG Service de Microbiologie, iris-LAB, CHU de Bruxelles (ULB)

Bouke DE JONG Dienst Mycobacteriën, Instituut voor Tropische Geneeskunde, Antwerpen

Alain BAULARD Centre d’Infection et d’Immunité de Lille, Institut Pasteur de Lille, France

Année académique 2014-2015

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ACKNOWLEDGEMENTS

This is more than an acknowledgement, this is a sincere

THANK YOU!

First of all, I would like to express my gratitude to my co-promotor Maryse Fauville. You supported me in my scientific development and learned me a lot about mycobacteria, a relatively unknown but fascinating species in the world of micro-organisms.

Véronique Fontaine, thank you for kindly accepting the promotor task, for your support, rapid corrections of this manuscript and your help in ULB’s administration when I wasn’t able to.

Drs. Jean-Paul Dehaye, Paule Boussard, Olivier Vandenberg, Bouke De Jong and Alain Baulard: Thank you for accepting my jury membership.

This work would never have been written without the scientific help of Caroline Allix-Béguec, Vanessa Mathys and Pablo Bifani. You might not realize how much your help stimulated me to continue analyzing data evening after evening (including “Excel-izing” pncA literature). It was a joy working with you.

To all the technicians of the Reference Laboratory of Mycobacteria and Tuberculosis: Jean-Pierre, Brigitte, Romu, Kristien, Trang, Maïté, Mehdi: Thank you! for your excellent skills, your help and kindness which made the lab a pleasant work environment.

To all other colleagues, especially the ones from the first floor: thank you for making the building a happy place to come to.

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Finally, a very very special thanks goes out to

my dearest husband Jeroen, who supported me, took care of me when needed and encouraged me to keep myself shackled to our computer even though many other tasks should have gotten my attention

and to my lovely daughter Hanne for the relaxing playtimes, your laugh energizes me every time. Big hug!!

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LIST OF FIGURES AND TABLES

A. FIGURES

p. 20 Figure I.1: Opened thorax of Egyptian mummy ~1550-1080 BC p. 21 Figure I.2: René Laennec and his cylinder like stethoscope.

p. 22 Figure I.3: Historical figures in the discovery of tuberculosis’ causative agent p. 25 Figure I.4. Sanatorium Prince Charles in Auderghem

p. 27 Figure I.5: The double-barred cross, symbol of the fight against TB

p. 29 Figure I.6: Examples of posters used in the international sensibilisation campaigns in the 20th century

p. 30 Figure I.7: Time line of key events in the history of tuberculosis p. 32 Figure I.8: Transmission of TB by airborne droplets

p. 33 Figure I.9: Tuberculosis infection by droplets and its progression to active and latent tuberculosis.

p. 35 Figure I.10: TB incidence rates of 2012 estimated by WHO

p. 41 Figure I.11: Schematic representation of a chromosome of a hypothetical strain X of Mycobacterium tuberculosis: location of the interaction of the different genotyping techniques.

p. 41 Figure I.12: IS6110-RFLP. p. 42 Figure I.13: Spoligotyping p. 42 Figure I.14: MIRU-VNTR

p. 43 Figure I.15: Scanning Electron Micrograph picture of Mycobacterium tuberculosis p. 43 Figure I.16: Taxonomy of the genus Mycobacterium

p. 46 Figure I.17: Schematic representation of a mycobacterial cell wall

p. 48 Figure I.18: Picture of Mycobacterium tuberculosis after Ziehl-Neelsen staining p. 49 Figure I.19: Picture of Mycobacterium tuberculosis after Auramine based staining p. 51 Figure I.20: Mycobacterium tuberculosis colonies on a solid Löwenstein-Jensen

medium

p. 62 Figure I.21: Picture of a positive tuberculin skin test indicating a TB infection p. 68 Figure I.22: Chemical structure of isoniazid

p. 70 Figure I.23: Pharmacokinetics of isoniazid following fasting and a high-fat meal. p. 71 Figure I.24: Chemical structure of rifampicin

p. 73 Figure I.25: Pharmacokinetics of rifampicin p. 73 Figure I.26: Chemical structure of pyrazinamide

p. 75 Figure I.27: Proposed mode of action of pyrazinamide by Zhang and Mitchison 2003. p. 76 Figure I.28: Pharmacokinetics of pyrazinamide

p. 76 Figure I.29: Chemical structure of ethambutol p. 78 Figure I.30: Pharmacokinetics of ethambutol.

p. 94 Figure III.1: Number of MDR, Pre-XDR and XDR isolates in the study cohort

p. 96 Figure III.2: Increase of resistance to additional drugs among MDR-TB primary isolates p. 96 Figure III.3: Increase of the number of drugs each primary MDR-TB isolate is resistant

to

p. 97 Figure III.4: Genetic families of the MDR-TB isolates (1994-2008) based on spoligotyping technique

p. 97 Figure III.5: Patient’s origin of the MDR-TB isolates (1994-2008)

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B. TABLES

p. 44 Table I.1: Members of Mycobacterium tuberculosis complex

p. 45 Table I.2: Most common isolated pathogenic or opportunistic NTM p. 55 Table I.3: Examples of diagnostic tests for mycobacteria

p. 59 Table I.4: Recommended concentration for DST to first line anti-tuberculous drugs p. 60 Table I.5: Classification of infective microorganisms by risk group defined by WHO p. 66 Table I.6: WHO-categorization of anti-tubercular drugs based on their efficacy p. 82 Table I.7: Overview of genomic regions associated with decreased susceptibility to

antituberculosis agents

p. 91 Table III.1: Primers and PCR conditions used for mutation analysis of M.tuberculosis isolates

p. 94 Table III.2: Characteristics of the study population (174 MDR-TB patients)

p. 95 Table III.3: Previous treatment history of 174 MDR-patients in Belgium between 1994 and 2008 (data obtained from BELTA)

p. 98 Table III.4: Characteristics of the MDR-TB clinical isolates

p. 100 Table III.5: Summary of the Resistance-determining region of the embB gene from 72 MDR-TB clinical isolates

p. 101 Table III.6: Analysis of sequencing of the Ethambutol resistance-determining region of embB gene for the prediction of resistance to ethambutol

p. 102 Table III.7: Genotypic analysis of the Resistance-determining region of the embB gene of 72 MDR-TB clinical isolates

p. 104 Table III.8: Analysis of the Resistance-determining region of the gyrA gene of 32 MDR-TB clinical isolates

p. 104 Table III.9: Analysis of sequencing of the Fluoroquinolone resistance-determining region of gyrA gene for the prediction of resistance to ofloxacin

p. 105 Table III.10: Genotypic analysis of the Resistance-determining region of the gyrA gene of 37 MDR-TB clinical isolates

p. 108 Table III.11: Evolution of the resistance profile of serial isolates

p. 111 Table III.12: Characteristics of the 58 MDR-TB patients in strain-clusters and results of epidemiological investigation

p. 114 Table III.13: Treatment outcome of MDR (first-line drug resistance only), pre-XDR and XDR-TB patients in Belgium (1994-2008)

p. 115 Table III.14: Patients with XDR-TB in their first isolate examined in Belgium, 1994-2008.

p. 121 Table III.15: Frequency of spontaneous pyrazinamide resistant M.tuberculosis (CDC1551)

p. 125 Table III.16: Clustered clinical isolates sharing same DNA fingerprint and pncA mutations.

p. 126 Table III.17: Strains with distinct genotype but with common pncA mutations p. 127 Table III.18: PZA susceptible clinical isolates encoding mutations in the pncA gene p. 130 Table III.19: Overview of the observed mutations in the pncA gene in this study p. 131 Table III.20: Minimum Inhibitory Concentration determination using the

BACTECTMMGIT960 system of 24 spontaneous mutants

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ABBREVIATIONS

ATS American Thoracic Society

AUC Area Under Curve

BCG Bacillus Calmette-Guérin

BELTA Belgian Lung and Tuberculosis Association

bp base pair

CAMHB Cation Adjusted Muller Hinton Broth CDC Center for Disease Control and Prevention CFU Colony Forming Unit

CLSI Clinical and Laboratory Standards Institute DNA Deoxyribonucleic acid

DPI Dry Powder Inhaler

DST Drug Susceptibility Testing ELF Epithelial Lining Fluid

EURO-TB European tuberculosis surveillance program

ECDC European Centre for Disease Prevention and Control FARES Fonds des Affections Respiratoires

FIC Fractional Inhibitory Concentration HAART Highly Active Anti-Retroviral Therapy IGRA Interferon Gamme Release Assay

INSERM Institut national de la santé et de la recherche médicale IPCS International Program on Chemical Safety

IS6110 Insertion Sequence 6110

IUATLD International Union Against Tuberculosis and Lung Disease L-J Löwenstein-Jensen (solid egg-based medium)

LTBI Latent TB Infection M. (tuberculosis) Mycobacterium MDR Multi-drug resistant

MIC Minimum Inhibitory Concentration

MIRU-VNTR Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeats

NALC N-Acetyl-L-Cysteine

NTM Non Tuberculous Mycobacteria

OADC Oleic Albumin Dextrose Catalase growth supplement PAS para-aminosalicylic acid

PCR Polymerase Chain Reaction

POA pyrazinoic acid

PPD Purified Protein Derivative preXDR pre eXtensively Drug Resistant PZAR pyrazinamide resistant

PZAS pyrazinamide susceptible PZase pyrazinamidase

QRDR Fluoroquinolone resistance-determining region RFLP Restriction Fragment Length Polymorphism RIVM RijksInstituut voor Volksgezondheid en Milieu RTI Recent Transmission Index

SNP Single Nucleotide Polymorphism Spoligotyping spacer oligonucleotide typing

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TST Tuberculin Skin Test

VRGT Vlaamse vereniging voor Respiratoire Gezondheidszorg en Tuberculosebestrijding

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Tuberculosis (TB) is a potentially fatal contagious disease that can affect almost any part of the body but is mainly an infection of the lungs. It is caused by micro-organisms of the Mycobacterium tuberculosis complex. It is the second greatest killer worldwide due to a single infectious agent, after the Human Immunodeficiency Virus (HIV). Without treatment, fatality is 50% in immune competent persons. TB remains the leading cause of death among HIV positive persons, causing one fifth of the deaths. The World Health Organization estimates that one third of the world population is infected by this micro-organism but only 5 to 10% develop TB disease. Nevertheless, this enormous reservoir leads to around 1.4 millions deaths annually. Standard curative treatment lasts at least 6 months and includes 4 different drugs. Toxicity of the drugs leading to (severe) adverse events and the long duration of the daily administration challenges patient’s compliance. Subinhibitory concentration of the drugs (due to poor adherence) can induce resistance of the mycobacteria to the provided drugs. Unlike most bacteria where resistance is acquired by plasmids, drug resistance of mycobacteria is obtained by genomic mutations. “Multi drug-resistant tuberculosis (MDR-TB)” is strictly defined as TB resistant to specifically isoniazid and rifampicin, the two main first line drugs. “Extensively drug resistance (XDR)” is defined as MDR-TB with additional resistance to any of the fluoroquinolones (such as ofloxacin or moxifloxacin) and to at least one of three injectable second-line drugs (amikacin, capreomycin or kanamycin). The increase of MDR-TB represents an enormous challenge to Public Health globally. This research examined different aspects of tuberculosis resistance performed in the Belgian National Reference Center, a clinical laboratory setting.

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obtained for 67.8% of the MDR-TB cases. Drug susceptibility testing (DST) of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) in liquid culture medium has a turn around time of at least two weeks, after identification of the positive culture (obtained after 2 to 4 weeks) from the patient’s clinical isolate. In order to provide the clinician with valuable information about the isolated mycobacteria leading to patient adapted therapy before bacteriological DST results are available, resistance is predicted by detection of mutations in certain genes of the mycobacteria. It is common practice for rifampicin (rpoB gene) and isoniazid (katG gene and/or inhA promoter region). In this MDR-TB collection, rifampicin resistant related mutations were found in 97.1% (168/173) of the clinical isolates and isoniazid resistant related mutations in 94.1% (160/170). The pncA, embB and gyrA genes have been sequenced to identify possible mutations because of their possible involvement with resistance to pyrazinamide, ethambutol and the fluoroquinolones respectively. However, little is known about the resistance prediction value of the mutations in these genes.

The study is also the first study on the molecular epidemiology of MDR-TB in the country. DNA fingerprinting showed a large diversity of strains (67% of the patients were infected by a strain with a unique pattern) and further epidemiological examination revealed limited local transmission of MDR-TB in Belgium.

The second part investigated the pncA gene and its association with pyrazinamide resistance in MDR-TB isolates from Belgium and in vitro cultured spontaneous mutants. The genetic analysis showed that 98.3% (59/60) of the Belgian clinical MDR pyrazinamide resistant (PZAR) isolates present a mutation in the pncA gene. We found 1.7% (1/60) of the PZAR MDR-isolates encoding wild type pncA and flank. A total (PZAR and PZAS) of 41 different amino acid changes, 3 protein truncations and 5 frameshifts were observed including eight novel mutations: 8Asp>Ala, 13Phe>Leu, 64Tyr>Ser, 107Glu>stop, 143Ala>Pro, 172

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frequency of in vitro mutagenesis to pyrazinamide at pH 6.0 was determined and found to be relatively high at 10-5 CFU/ml.

Finally, the in vitro activity of tobramycin and clarithromycin (with unclear efficacy against M. tuberculosis) was evaluated on 25 M. tuberculosis clinical isolates with various resistance profiles. The effect of the drugs administered together was examined for possible synergistic effect. The median minimum inhibitory concentration (MIC) of 8 µg/ml obtained for both drugs in this study is rather high but are beyond the concentrations obtained in lung tissues. This suggests that both drugs should be investigated further as potential adjuncts to the treatment of resistant TB when other alternatives have failed; in particularly through new drug delivery systems such as the Dry Power Inhaler which allows local drug deposition with high drug concentrations in the lungs but low toxicity due to limited systemic absorption. In addition, for 36% of the tested isolates a decrease of the MIC of clarithromycin by a single or twofold dilution was observed in the presence of a subinhibitory concentration of tobramycin and no antagonistic effect was seen for the remaining isolates.

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1. TUBERCULOSIS: A BRIEF HISTORY

1.1. Origin of the tuberculosis bacteria

Tuberculosis (TB) is a pulmonary disease which can cause death if not treated. Archeological research proved that this disease already infected humans for at least 5000 years. Skeletal abnormalities typical for tuberculosis (Pott’s disease) have been found in Egyptian mummies and artworks [Daniel 2006]. DNA of Mycobacterium tuberculosis complex -the bacteria that cause TB infection- was found in tissues of Egyptian mummies for the first time in 1997 (Figure I.1) [Nerlich et al. 1997][Crubezy et al. 1998][Zink et al. 2003] and in Peruvian mummies from the pre-Columbian era in 1994 [Salo et al. 1994].

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Figure I.1: Opened thorax of Egyptian mummy ~1550-1080 BC, from the tombs of the nobles at Thebes-West, Upper Egypt

The big arrow indicates pleural adhesions of the right lung and the small arrow points at destruction of boney elements of lumbar vertebral bodies L4 and L5, possibly due to TB spread.

DNA from M.tuberculosis was identified in extracts of the right lung, while the visibly unaffected left lung remained negative in TB DNA analysis [Nerlich et al. 1997].

1.2. Discovery of the causative agent

In the classic Greek timeframe tuberculosis was known as ‘phtisis’, consumption. It is believed that Hippocrates was the first to use this word to describe the disease. Tuberculosis flourished worldwide in the 17-19th century and was one of the leading causes of death with death rates of 800-1000 per 100 000 capita per year in London, Stockholm and Hamburg. Worldwide around one third of adult mortality was caused by TB [Palomino and Ritacco 2007] [Kaufmann 2003]. In the current history books this phenomenon is known as the Great White Plague.

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In 1819 René Théophile Laennec, known for his invention of the stethoscope, published “D’Auscultation Mediate” where he clearly described the pathology of tuberculosis and physical signs of the pulmonary form. Some of his pathological description is still used today [Daniel 2006].

Figure I.2: René Laennec and his cylinder like stethoscope.

[Encyclopedia Brittanica]

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Louis Pasteur 1822-1895 Jean-Antoine Villemin 1827-1892 Robert Koch 1843-1910 [Portraits from the Dibner

Library of the History of Science and Technology]

[Science photo library: www.sciencephoto.com]

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1.3. Diagnostic tools for tuberculosis

1.3.1. Microscopic examination

Paul Ehrlich (1854-1915) assisted Koch’s historical seminar and he received a pure culture of the tubercle bacilli to optimize the coloration technique. He experimented with several chemicals including 30% nitric acid and alcohol to decolorize the surrounding tissues while the tubercle bacilli remained colored (hence the name acid-alcohol-fast bacilli). He also showed that the red tubercle bacilli would appear more clearly by counterstaining with a blue or yellow dye. As many scientists have experienced in the past, Ehrlich discovered a significant finding by accident. In his laboratory there was a small stove in which the fire had gone out for some time. Before going home he left colored slides on the cold stove to dry. The next day he frustratingly found the stove lit, but when he examined the slides he was amazed to find the bacilli even more clearly. Later Franz Ziehl (1859-1926) and Friedrich Neelsen (1854-1898) adapted Ehrlich’s technique by using the chemicals carbolic for colorization and sulphuric acid mixed with alcohol for decolorization of the surrounding tissues to produce the Ziehl-Neelsen staining method [Sakula 1982]. Even today, staining of suspected tissues or positive cultures obtained from clinical tissues is still performed by Ziehl-Neelsen based colorization methods. It remains an important tool for the diagnosis of tuberculosis in many countries.

1.3.2. Tuberculin skin test

Robert Koch dedicated all his research to infectious diseases. In 1890 he announced to have found a compound not only inhibiting the growth of the tubercle bacilli in infected laboratory animals (treatment) but also making the animals insensible to inoculation of tubercle bacilli (prevention). He named this compound tuberculin. It was prepared from extracts of glycerol based liquid cultures of these germs. Clinical trials did not however yield the results everybody hoped for, it even made patients worse. Improved derivatives did not deliver the magical cure the world was waiting for either.

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diagnostic technique using tuberculin: if a drop of tuberculin is applied on some scratches, a red inflammatory reaction will occur after a few days if the person has been previously infected with tubercle bacilli (Pirquet’s test). Around the same time, Charles Mantoux described the intradermal injection of tuberculin leading to a local induration, swelling and red skin color at the site of injection in TB infected persons, after 24 to 72 hours (Mantoux test). Non-infected people do not respond to the intradermal injection, as they have not been in contact with the TB specific antigens. Optimization of tuberculin production was established by Seibert and colleagues in 1941. This purified form called Purified Protein Derivative (PPD) has become the international standard compound used in the tuberculin skin test (TST) [Palomino and Ritacco 2007] [Shashidhara and Chaudhuri1990].

1.3.3. Chest X-ray

In 1895, a German physics professor called Wilhelm Conrad Röntgen (1845-1923), discovered the X-Ray. He published his findings in December 1895 in a paper called “W. C. Roentgen: On A New Kind of Ray (preliminary communication)” – translated by Arthur Stanton in Nature 53, 274-276 (1896). This radiation allows the recognition of internal body parts as they absorb X-Ray beams. The absorption by each part is specific and depends on its density. An X-Ray sensitive film can capture the image: when X rays are able to reach the film, the exposed spots turn black. In the chest X-ray for example, the calcium density of the spine and ribs blocks most X rays leaving white areas on a film. The water densities of the stomach and liver give a grayish image. Finally, the air spaces in the lungs allow penetration of most of the X-ray beam, and look almost black on the ﬁlms. Tubercles and scar tissue of the tubercle lesions can be visualized as grey zones in the lung area [Linton 1995].

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1.4. Treatment

Before the antibiotic era, a complete cure from tuberculosis was rarely obtained. The key to success was bed rest, good nutrition and fresh air. Rehabilitation centers were built worldwide for tuberculosis patients and carried the name sanatorium, from the Latin word sano (to heal). The first was established in Germany, now Poland, in 1854 by Hermann Bremer [FARES 2006][Daniel 2006]. Some architectural features are typically found in these buildings: high ceilings with high windows and porches. The patients spent most of their time resting outside on the porch (Figure I.4).

Figure I.4. Sanatorium Prince Charles in Auderghem [FARES 2006]

In 1944 the fight against tuberculosis made a revolutionary step forward with the discovery of the first drugs active against tuberculosis: streptomycin and para-aminosalicylic acid (PAS). A few years later, the combined use of streptomycin and PAS showed a clear advantage over single drug use with an increased treatment success rate [Daniels and Hill 1952].

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Since then, only few other drugs made their entry in the TB treatment regimen. Due to their poor efficacy they are only introduced as second (e.g. amikacin, moxifloxacin) or third line (e.g. linezolid, thioacetazone) drugs when the first line regimen fails. The need to develop new anti-TB drugs remains in order to shorten and simplify treatment of drug-susceptible TB and to provide shorter, safer, more effective and cheaper treatment alternatives for resistant TB. Several new anti-tuberculosis molecules are in the pipeline. The most promising ones for the near future are [Working group on new TB drugs. http://www.newtbdrugs.org][TB Alliance, Global Alliance for TB Drug Development. http://www.tballiance.org][Lobue and Menzies 2010][Zumla et al. 2013]:

- TMC207 (R207910): bedaquiline (a diarylquinoline), inhibiting the mycobacterial ATP synthase. FDA approval has been obtained and the European Commission has provided a conditional licence because comprehensive benefit-risk data is not yet available and further studies are required. The molecule is marketed by Janssen Pharmaceutica (Belgium) under the name Sirturo. [http://www.sirturo.com/dosage-and-administration][European Medicines Agency, www.ema.europa.eu]

- OPC-67683 and PA-824: nitroimidazo-oxazole drugs, both inhibiting among others cell wall lipid synthesis and evaluated in clinical phase III.

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1.5. Prevention

At the beginning of the 20th century sensibilisation campaigns were organised to educate people how to stop the spread of tuberculosis disease and improve hygienic living conditions. The most memorable actions were books, posters in the streets and special printed letter stamps (figure I.6). TB-conferences were held by official and/or non-governmental agencies and in 1902, Dr Gilbert Sersiron suggested adopting the symbol of a crusader as he compared the fight against tuberculosis to a crusade. The Duke of Lorraine, Godfrey of Bouillon, was the first Christian ruler of Jerusalem and his banners displayed a double-barred cross as a symbol of courage and success to the crusaders. This cross became the worldwide symbol of the fight against TB (figure I.5).

Figure I.5: The red double-barred cross, symbol of the fight against TB.

On scientific level, a breakthrough in the prophylactic field was documented with the development of the (semi-efficient) vaccine “BCG” (Bacille Calmette-Guérin). The laboratory of Albert Calmette and Camille Guérin managed to grow a weakened, non-virulent form of the bovine tuberculosis germ Mycobacterium bovis: the strain lost its virulence after 230 subcultures on medium of cooked potatoes and glycerinated bile. In 1921 the first clinical test was performed by giving a child 6 mg of BCG orally and over the next 7 years, more than 100 000 children were vaccinated with 30 mg of orally administrated BCG [Rieder 2002].

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USA USA France

France Italy Poland

China _USA

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2. TUBERCULOSIS: INFECTION AND TRANSMISSION

2.1. Primo-infection and transmission of pulmonary tuberculosis

TB is caused by microorganisms of the Mycobacterium tuberculosis complex. They usually affect the lungs where the bacteria cause tubercles (tuberculous nodules) that can develop into cavities - due to necrosis of the center of the tubercles - when remained untreated. This means that lung tissue is locally disintegrated causing holes which lead to respiratory failure. The affected lung tissue is replaced by scarring and the cavities are filled with white necrosis material that can be coughed up (~White Plague). Without treatment, fatality is 50% in immune competent persons.

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uncommon (~paucibacillary TB, i.e. smear negative but culture positive) [Inserm 2004].

In addition to these social factors, bacterial characteristics may play a role in infectivity of the microorganism: some strains are thought to be more viable, fit or transmittable than others.

Figure I.8: Transmission of TB by airborne droplets [http://www.FARES.be]

If the bacteria reach the lungs, alveolar macrophages play an essential role in the elimination of the bacteria by phagocytosis. Sometimes, the bacteria can however defeat the elimination process and will survive and multiply in the macrophage. This might be the case if the infected person is immunocompromised (HIV, cancer, diabetes, malnutrition,…), if the bacteria are very virulent or if a large bacterial load is inhaled. After proliferation of the bacteria in the macrophage, it bursts open and the released bacteria invade other macrophages. This way, lesions are formed. The cellular immune response of the infected host can react on this “primo-infection” by forming a granuloma that consists mainly of infected macrophages and T cells. The granuloma prevents further dissemination of the bacteria. The bacteria inside the granuloma are believed to be non-active and (almost) non-replicating and can become dormant (but not eliminated). This is called Latent TB Infection (LTBI) which is clinically asymptomatic and not contagious. This dormant stage of the infection is not yet well-understood. Especially the bacterial survival during transition of an oxygen rich environment (alveolars) to the oxygen poor environment of the granuloma is puzzling.

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lifetime but for HIV patients, the probability increases to 5-10% each year [FARES 2006][Koul et al. 2011]. The World Health Organization (WHO) estimated that one third of the global population has latent TB, an enormous reservoir of this killing disease.

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2.2. Extra-pulmonary tuberculosis

If the inhaled bacteria access the bloodstream or lymph system, they can be disseminated throughout the entire body. This is more likely to happen in immuno-suppressed persons and children. A special form is miliary tuberculosis where little lesions are formed in the entire body.

If extra-pulmonary tuberculosis occurs, it often remains locally and is not communicable:

- infection of the bones, typically the vertebrates close to the lungs. The bacteria can further travel to the intervertebral discs causing malformations of the spine (Pott’s disease)

- Infection of the brain causing tubercular meningitis - Renal infection

- Intestinal infection - …

2.3. Risk factors for developing TB disease after infection by M.tuberculosis

Certain people have a higher risk of developing TB disease after infection than other. Medical conditions as well as social circumstances can influence the progression to disease.

- Medical conditions: infection with HIV, AIDS stadium, organ transplantation with immunosuppressive treatment, treatment with corticosteroids or anti-TNF α, silicosis, chronic renal insufficiency, cancer, Type I diabetes, …

- Other circumstances: alcoholism, smoking, underweight, malnutrition, younger than 5 years of age, …

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3. EPIDEMIOLOGY OF TUBERCULOSIS

3.1. Global burden

The World Health Organization (WHO) estimated the prevalence (i.e. number of cases) of TB disease at 12.0 million cases in 2012. This is equivalent to 169 per 100 000 humans globally. TB mortality was estimated around 1.3 million (equivalent to 18 per 100 000 humans globally) of which 940 000 deaths were among HIV negative persons. Among HIV positive cases, TB is still the leading cause of death. Approximately 75% of total TB deaths (HIV-negative and –positive) occurred in the African and South-East Asia Regions in 2012. India and South Africa accounted for about one-third of global TB deaths.

WHO estimated TB incidence (i.e. number of new cases) at 8.6 million in 2012. This is equivalent to 122 cases per 100 000 humans globally. Most of these incidence cases resided in Asia (58%) and Africa (27%). The European regions accounted for only 4% of this incidence and the Region of the Americas for 3% (Figure I.10)[WHO Global tuberculosis Report 2013]. Even though the percentage of our European region is low, the increase in world mobility and migration makes TB a global burden with significant local consequences.

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3.2. Multi-drug resistance

Resistant tuberculosis is classified as ‘primary drug resistant’ when bacterial resistance was observed with no history of previous treatment or as ‘acquired (formerly known as ‘secondary’) resistance’ where resistance is acquired during treatment. Primary resistance is assumed to be due to transmission of drug-resistant strains as natural resistance is rare: M.tuberculosis has the ability to undergo spontaneous mutations leading to drug resistance at a frequency of 10-6 to 10-8 mycobacterial replications [Zhang and Yew 2009][Alangaden et al.1995]. Thus the probability to obtain mutations causing resistance to for instance three drugs simultaneously can be approximated at 10-18 to 10-24. Theoretically, this probability is considered virtually non-existent, hence the recommendations to include at least three effective drugs in the initiation phase of TB treatment.

A major concern in the control of tuberculosis (TB) is the spread of multi-drug resistant tuberculosis (MDR-TB) caused by Mycobacterium tuberculosis resistant to at least isoniazid and rifampicin, the two most important first line drugs in TB treatment. A particularly dangerous form is the so-called XDR-TB (extensively drug resistance) [Van Rie 2006], with resistance to at least isoniazid, rifampicin, to any fluoroquinolone and one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin) [CDC 2006]. WHO estimated 450 000 new MDR-TB cases in 2012 and 170 000 deaths from MDR-TB [WHO 2013]. It expects to treat 1.6 million MDR and XDR-TB cases in 2015 [WHO and Stop TB Partnership 2007].

Globally, an estimated 3.6% of new cases and 20.2% of previously treated cases had MDR-TB in 2012. In Europe, the percentage of MDR-TB remains alarming with 16 % among the new TB patients and 21 % among the previously treated ones. Eastern European citizens contributed particularly to these high percentages and to the proportion of XDR-TB [WHO and ECDC 2009].

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3.3. Situation in Belgium

According to the latest report of the FARES/VRGT (Fonds des Affections Respiratoires/Vlaamse vereniging voor Respiratoire Gezondheidszorg en Tuberculosebestrijding), Belgium is a low TB incidence country with a TB incidence rate of 8.9 cases per 100 000 habitants in 2012 (n=987), the lowest incidence rate ever documented (e.g. incidence of 9.6 per 100 000 habitants in 2009 [FARES 2009]). Incidence rates were higher in the cities because of the higher concentration of the population at risk. The 3 cities with highest incidence rate were Brussels (27.4/100 000; n=312), Liège (22.5/100 000; n=44) and Antwerp (20.7/100 000; n=104). Among the 987 declared TB patients in 2012, 31.6% resided in the Brussels agglomeration [FARES 2012].

Among the 687 declared patients with pulmonary TB in Belgium, 49.2% provided a smear positive sputum sample. This means that TB bacilli were detected at microscopic investigation of the sputum (indicating a high bacillary load) and those patients are considered highly contagious. In 83.7% of the cases, a positive culture was obtained.

In Belgium, a surveillance network of MDR-TB was implemented in 1994 by the Belgian Lung and Tuberculosis Association (BELTA) in collaboration with the laboratories performing first line drug susceptibility testing of M. tuberculosis and the National Reference Laboratory performing drug susceptibility to second line drugs and genotyping. Since 2005 the project BELTA-TBnet ensures the free of charge treatment of every MDR-TB patient and particularly the purchase of second line drugs. This program enters within the framework of the European project monitoring MDR-TB.

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2012]. A difference in MDR-TB numbers between the official national register and the BELTA-TB net project is due to the fact that the national register only counts the MDR-TB cases at time of diagnosis while the BELTA-TB net project counts all MDR-TB patients, including secondary resistance cases.

3.4. Molecular epidemiology: laboratory tools to detect ongoing transmission

Molecular techniques have been developed to investigate on-going transmissions. In industrialized countries, a special interest goes to the molecular surveillance of MDR-TB. In Europe, successive networks on molecular surveillance of MDR-TB were implemented by RIVM (Rijksinstituut voor Volksgezondheid en Milieu), Institut Pasteur, EURO-TB (European tuberculosis surveillance program) and ECDC (European Centre for Disease Prevention and Control). Genetic fingerprints are collected through various countries in order to identify international strain-clusters. The objective is to allow coordinated measurements aiming to eliminate these particularly dangerous strains [EuroTB 2006]. Several databases of fingerprints were created based on one or more of following genotyping techniques:

- IS6110-RFLP (Insertion Sequence 6110 - Restriction Fragment Length Polymorphism)

- Spoligotyping (spacer oligonucleotide typing)

- MIRU-VNTR on 24 loci (Mycobacterial Interspersed Repeat Unit – Variable Number of Tandem Repeats).

The first M. tuberculosis genotyping method IS6110-RFLP is now replaced by the latter two methods. If two Mycobacterium tuberculosis strains have identical genotyping results, an epidemiological link can be assumed between the strains and thus the patients.

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strains were mainly found in Central America and Caribbean (about 25%), suggesting a link of Haarlem to the post-Columbus European colonization [Brudey et al. 2006].

3.4.1. IS6110-RFLP (Insertion Sequence 6110-Restriction Fragment Length Polymorphism)

IS6110 is a transposon only present in the genome of the M.tuberculosis complex [Thierry et al. 1990]. The number of IS6110 elements and their position in the genome are variable from strain to strain. This transposon contains a restriction sequence for the restriction enzyme PvuII. This means that PvuII can cut the DNA at this specific site. When the M.tuberculosis chromosome (containing many cleavage sites for PvuII) is digested with this PvuII enzyme, a series of DNA strands of different length are generated, including pieces with a partial (cut) IS6110 (represented by the black arrows in Figure I.11). These strands are separated by gel electrophoresis. Probes specific to the partial IS6110 are hybridized and made visible by e.g. chemi-luminescence method. The obtained pattern (presence of a number of bands at specific lengths depending on the number of base pairs (bp)) is strain specific [van Embden et al. 1993] (Figure I.12).

3.4.2. Spoligotyping (spacer oligonucleotide typing)

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presence/absence of specific spacers in this Direct Repeat region, e.g. based on microbeads [Abadia et al. 2011] or on a DNA chip [Song et al. 2007].

3.4.3. MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats)

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Figure I.11: Schematic representation of a chromosome of a hypothetical strain X of Mycobacterium tuberculosis: location of the interaction of the different genotyping

techniques (adapted from Barnes and Cave 2003).

Figure I.12: IS6110-RFLP. The genome is cut by the restriction enzyme PvuII at the specific sites represented by the arrows in Figure I.11, including one site at each

IS6110 element. The obtained fragments are separated by gel electrophoresis. Probes are hybridized to the right half of the IS6110 elements and visualized. The obtained pattern is strain specific (adapted from Barnes and Cave 2003) [Barnes and

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Figure I.13: Spoligotyping of 10 isolates. The spacers of the Direct Repeat locus (DR locus in Figure I.11) are amplified by PCR with biotine labeled primers. The amplified

fragments are hybridized to synthetic spacer oligonucleotides immobilized on a membrane, corresponding to 43 spacers of the Direct Repeat locus. The obtained pattern (absence or presence of a spacer) is strain specific [Kamerbeek et al. 1997]

[Dale et al. 2001] M IR U 0 4 M IR U 1 0 M IR U 1 6 M IR U 2 6 M IR U 3 1 M IR U 4 0 V N T R 1 95 5 V N T R 2 16 3b V N T R 2 16 5 V N T R 4 05 2 V N T R 0 42 4 V N T R 0 57 7 V N T R 2 40 1 V N T R 3 69 0 V N T R 4 15 6 V N T R 2 34 7 V N T R 2 46 1 V N T R 3 17 1 M IR U 0 2 M IR U 2 0 M IR U 2 3 M IR U 2 4 M IR U 2 7 M IR U 3 9 3 2 3 2 2 1 3 4 2 6 4 4 2 1 2 4 2 1 2 2 5 1 3 2 2 4 2 6 3 3 3 2 3 7 2 3 4 3 2 2 2 3 2 2 3 1 3 2 2 4 3 5 3 6 2 2 2 4 3 4 1 2 1 4 2 3 1 2 6 1 3 2

Figure I.14: MIRU-VNTR. The number of repeats in certain loci can be derived by the length of the amplified product. The length of these fragments can be determined by gel electrophoresis or automated capillary electrophoresis (adapted from Barnes and

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4. THE GENUS MYCOBACTERIUM

Mycobacteria are slim rod organisms of 1-10 µm long and 0.2-0.6 µm wide (Figure I.15). They are aerobic, nonmotile, nonspore forming and do not have a capsule. They have a replication time of 2-24 hours (and 36h for M.ulcerans), which is very long compared to other bacteria where it might take 15 to 20 minutes, or even less than a minute, to replicate [Manual of Clinical Microbiology, 10th Edition, 2011].

The genus Mycobacterium belongs to the family of Mycobacteriaceae of the order Actinomycetales of the class Actinobacteria (Figure I.16).

Figure I.15: Scanning Electron Micrograph picture of Mycobacterium tuberculosis magnification of 15549x (http://phil.cdc.gov/phil/details.asp)

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4.1. Group 1: Mycobacterium tuberculosis complex

The causative agent of tuberculosis is a member of the Mycobacterium tuberculosis complex (Table I.1). They are all slowly growing bacteria. With a replication time of 12-24h, a colony is only visible on solid media after 7 days.

Table I.1: Members of Mycobacterium tuberculosis complex

Mycobacterium tuberculosis most common infectious agent in humans

Mycobacterium africanum usually found in humans of the Western African region (subtype I) and Eastern Africa (subtype II) [Mostowy et al. 2004] Mycobacterium canetti thought to be closely related to the

common ancestor strain of Mycobacterium tuberculosis complex strains [Brosch et al. 2002]

Mycobacterium bovis usually found in cattle, but also pathogenic for humans

Mycobacterium microti, M. caprae and M.pinnipideii

cause tuberculosis in mice, goats and seals respectively. M.microti has been shown to be pathogenic for humans [van Soolingen et al. 1998]

4.2. Group 2: Mycobacterium leprae

Another specific infectious pathogen of this genus is Mycobacterium leprae. It provokes leprosy and laboratories have not been able to culture it in vitro yet. It is usually cultured in vivo in a mouse foot-pad without provoking leprosy-like lesions in these animals [Levy and Ji 2006].

4.3. Group 3: Non-Tuberculous Mycobacteria

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Table I.2: Most common isolated pathogenic or opportunistic NTM (adapted from Griffith et al. 2007)

Mycobacterium ulcerans responsible for Buruli ulcers (necrosis of the skin and soft tissues); it is the 3rd most common mycobacterial disease in immuno-competent persons, after TB and leprosy. It is encountered in subtropical and tropical climates. Mycobacterium avium Mycobacterium avium complex (MAC)-disease:

lymphadenitis, pulmonary and intestinal infections; spreads quickly through the bloodstream causing disseminated disease in HIV positive patients in AIDS stadium, especially common before the Highly Active Anti-Retroviral Therapy (HAART)-era

Mycobacterium intracellulare belongs to the Mycobacterium avium complex group Mycobacterium kansasii respiratory infections

Mycobacterium marinum can cause skin tissue infections, usually occurs in people who work with fish and aquaria

Mycobacterium abscessus respiratory infections especially in patients with cystic fibrosis; skin, soft tissue and bone disease

Mycobacterium chelonae skin, soft tissue and bone disease Mycobacterium xenopi pulmonary disease

NTM can be divided in

- slowly growing mycobacteria: replication time of 12 to 24 hours; it takes more than 7 days to obtain visible colonies in in vitro cultures on solid media - rapidly growing mycobacteria: replication time of 2 to 6 hours; visual culture

in less than 7 days

The Runyon classification is based on phenotypic characteristics, namely reproduction time and pigmentation [Runyon E.H. 1959]:

- Group I: slowly growing, photochromogenic species where the colonies show a yellow-orange pigmentation in the presence of light

(e.g. M. kansasi, M. marinum)

- Group II: slowly growing, scotochromogenic species where the colonies are pigmented in the absence or presence of light

(e.g. M. gordonae, M. xenopi…)

- Group III: slowly growing, nonchromogenic species where the colonies are not pigmented, i.e. white-cream colored

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- Group IV: rapidly growing, with or without pigmentation (e.g. M. abscessus, M. chelonae)

4.4. Characteristics of mycobacteria

4.4.1. Cell wall

Mycobacteria have a unique cell wall. They cannot be called Gram-positive or Gram-negative (but are classified under Gram-positive even though Gram staining can not be used to visualize these bacilli because of their specific cell wall):

- in Gram staining they do not retain the purple coloration well like Gram-positive bacteria do

- they do not have an outer lipopolysaccharide cell wall like Gram-negative bacteria.

The mycobacteria a more complex structure with a high lipid content. The inner region of the cell wall contains peptidoglycan linked to a second polysaccharide polymer, arabinogalactan. The mycolic acids are long chain length branched fatty acids, typically containing 70-90 carbon atoms, which are characteristic of mycobacteria and account for up to 60% of the whole cell dry weight. They are linked to arabinogalactan polymer. In the outer region different kind of other lipids are present, like glycopeptidolipids and lipooligosaccharides (Figure I.17) [PA Lambert 2002][Alsteens et al. 2008].

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4.4.2. Microscopic examination

Due to this special cell wall, the common Gram colorization is not suitable for mycobacteria as they may remain invisible and appear as ghosts (clear zones) on the microscope slide [Trifiro et al. 1990][Atsukawa et al. 2011]. Rapidly growing mycobacteria however might emerge like Gram-positive rods [Manual of Clinical Microbiology, 10th edition][Natsag et al. 2012].

Specific staining techniques are used for microscopic visualization of mycobacteria, called acid-fast or acid-alcohol-fast staining. Acid fast staining refers to the bacteria’s ability to retain the primary stain even after decolorizing with an acid alcohol solution. The mycolic acid residues of the mycobacterial cell wall retain the primary color fuchsin.

Microscopic examination is still used as a primary tool to diagnose TB in developing countries. It is a rapid and inexpensive method to examine the patient’s expectorated sputum but has a low sensibility compared to detection with a culture method (sensitivity between 55% and 70%). The number of acid fast bacilli needs to be high in order to be detected with microscopic examination. Usually 106 bacilli/ml specimen result in a positive examination while 104 bacilli/ml specimen only result positive in 60% of the cases [Wright et al. 1998][Hooja et al. 2011][Manual of Clinical Microbiology, 10th edition]. These patients are considered as highly contagious.

Mycobacterium tuberculosis cultured in liquid medium has the specific feature to form cords which are easily recognized in microscopic examination (Figure I.18). The orientation of the long axis of each cell is parallel to the long axis of the cord. It is considered to be related with virulence. In cultures of other mycobacteria, these cord formations are rarely observed [McCarter et al. 1998][Kadam et al. 2010][Staropoli and Branda 2008][Julian et al. 2010].

The Ziehl-Neelsen staining was developed by Franz Ziehl (1859-1926) and Friedrich Neelsen (1854-1898). It allows visualizing the acid-alcohol-fast bacilli like mycobacteria as red rods under an optical microscope (Figure I.18). Principal steps:

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- Heat the slide gently and allow to steam for 5 min

- Wash the slide gently with running water and flood with acid alcohol (decolorizer)

- Counterstain with methylene blue - Wash gently with running water - Dry over gentle heat

This technique has been adapted by Kinyoun in 1915 in order to remove the inconvenient heating step. This still forms the basis for the cold staining method used in laboratories these days.

Figure I.18: Picture of Mycobacterium tuberculosis after Ziehl-Neelsen staining (regular microscopy, 1000x magnified), showing the cord formation

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Auramine O based staining is more sensitive than the Ziehl-Neelsen staining and is used to visualize acid-fast bacilli under a fluorescence microscope as yellow rods (Figure I.19). However, not every laboratory can afford this expensive equipment, especially not in developing countries where microscopic examination of sputum samples is a primary diagnostic tool. The lower-cost new Light Emitting Diode (LED) based fluorescent microscopes offer perspectives in this field.

The two main techniques currently used are the Auramine O staining (Morse method) and Auramine O – Rhodamine B staining (Truant staining) [Wright et al. 1998][Manual of Clinical Microbiology, 10th edition].

Figure I.19: Picture of Mycobacterium tuberculosis after Auramine based staining of a sputum sample using fluorescence microscopy

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4.5. Cultivation of mycobacteria

Mycobacteria need specific media (neutral pH) to grow. A combination of solid and liquid media should be used for optimal mycobacterial recovery from clinical samples. As few as (10 to) 100 viable bacilli per ml sample can engender a positive culture [Manual of Clinical Microbiology, 10th edition].

Due to the slow replication time of mycobacteria, the clinical samples need to be carefully treated before inoculation: the few mycobacteria need to be recovered and all other contaminating bacterial species need to be eliminated or they would rapidly overgrow the mycobacteria, leaving them undetected. The most common digestion-decontamination procedure employs NALC-NaOH (N-Acetyl-L-Cysteine – NatriumHydroxide) solution, as it is compatible with most of the commercially available culturing systems. In addition to the selective decontamination of the sample, some selective drugs might be incorporated to eliminate remaining contaminants.

4.5.1. Solid medium

The three most common solid media used in laboratories are Löwenstein-Jensen (L-J), Middlebrook 7H10 and Middlebrook 7H11. L-J is a green solid egg-based medium where malachite green is added to suppress growth of other (contaminating) bacteria. 7H10 and 7H11 are transparent agar-based media where OADC (Oleic Albumin Dextrose Catalase) is added as a growth supplement. Medium 7H11 differs from 7H10 by the addition of casein hydrolysate, which improves the growth of some difficult M. tuberculosis strains.

Incubation of the cultures requires an aerobic environment. Growth can be stimulated by 5-10% CO2 in the air. Temperature for optimal growth is species specific.

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enhanced by replacing the glycerol of the media by pyruvate or by using the egg and pyruvate based Stonebrink medium [WHO Grange JM et al. 1996]. Non tuberculous Mycobacteria (NTM) have species specific optimal growing temperatures; e.g. M.abscessus at 30°C and M.xenopi at 42°C. The colonies of NTM can be rough or smooth and the color is species (and sometimes light) dependent, going from cream colored over yellow to orange (e.g. M.avium, M.xenopi, M.gordonae respectively) [Runyon E.H. 1959].

Figure I.20: Mycobacterium tuberculosis colonies on a solid Löwenstein-Jensen medium (http://www.bacteriainphotos.com)

4.5.2. Liquid medium

Mycobacteria often grow faster in broth medium than on solid medium. This is especially true for M.tuberculosis. In the commercially available systems, a positive culture can be obtained in 7-10 days (versus 4 weeks on solid media). The most common broth is Middlebrook 7H9. Commercially available systems have been developed and implemented worldwide in clinical laboratories. They (semi-) automatically follow growth of micro-organisms in the broth. Most systems have also a CE and FDA approved method for drug susceptibility testing (DST) where growth is monitored in the presence of certain drugs at specific concentrations.

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specimen. They would rapidly overgrow the present mycobacteria causing a false negative mycobacterial result [Dinnes et al. 2007].

4.5.2.1. BACTEC MGIT960® (Becton Dickinson)

Mycobacteria Growth Indicator Tubes (MGIT) contain 7ml of modified 7H9. At the bottom of each tube there is an orange silicon compound, the fluorescence-quenching-based oxygen sensor. The oxygen dissolved in the medium inhibits the emission of fluorescence under UV light of 365nm. When organisms are growing and consuming oxygen, the inhibition disappears and the silicon emits an orange fluorescent glow. This detection can be performed manually (using a long wave UV light source) or automated with the BACTEC MGIT960® system where growth is followed continuously at 37°C [package insert BD BACTEC™ MGIT™ Barcoded 7mL Tube; Becton Dickinson].

4.5.2.2. BACTEC 460TB® (Becton Dickinson)

These vials contain 12B medium (modified 7H9 broth) with radioactive labeled 14C palmitic acid as carbon source for the organisms. When it is metabolized by the growing bacilli, 14CO2 is formed, released in the gas part of the bottle and detected by the instrument. The rate and amount of 14

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4.5.2.3. MB/BacT ALERT® system (bioMérieux)

Each bottle of this automated system contains adapted Middlebrook 7H9 broth and a colorimetric carbon dioxide sensor. When CO2 is produced by the growing microorganisms, the sensor changes color from blue-green to yellow. This lighter color results in an increase of reflection, monitored by the instrument [package insert BacT/ALERT®MB]. Even though initial performance analyses show promising results concerning DST results, the DST kits for M.tuberculosis are not available anymore [Barreto et al. 2003][Bemer et al.2004][Garrigo et al. 2007][personal communication with bioMérieux].

4.6. Identification methods and genotypic drug resistance testing

Species-level identification of isolated (myco)bacteria is of clinical relevance. Complete cure can only be obtained with a patient adapted regimen which depends on the identified bacterial species and its drug susceptibility. Identification of mycobacteria is mainly culture-dependant due to sensitivity limits of the current methods. Because isolation by culture can take up to several days to weeks (method and species dependant), a more rapid identification can be clinically desired. The laboratory will try to identify the mycobacteria directly on the clinical sample by molecular techniques. Any positive result on the clinical specimen requires further confirmation by an identified positive culture.

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4.7. Phenotypic drug susceptibility testing

In order to optimize the patient’s treatment, in vitro drug susceptibility testing (DST) of mycobacteria is performed in specialized laboratories. If a predefined proportion of the Mycobacterium culture (=inoculum) grows in the presence of a specific drug concentration (=critical concentration), it is unlikely that the use of that drug will lead to therapeutic success. For mycobacteria, two different categories have been used to categorize the in vitro results: “critical concentration” and “minimum inhibitory concentration (MIC)”. MIC is the lowest drug concentration that prevents visible growth of the tested bacteria. Unlike DST with other bacteria, there is no “intermediate” interpretive category when the critical concentration category is applied, even when testing is performed at the critical concentration and additional higher concentrations.

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grown mycobacteria scraped from a solid medium, with a density equivalent to 1Mc Farland turbidity standard.

It is noteworthy that the critical drug concentration is dependant on the medium used (Table I.4). As a consequence the minimum inhibitory concentration (MIC) determination may result in slightly different concentrations if different media are used.

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Table I.4: Recommended concentration for DST to first line anti-tuberculous drugs Concentration (µg/ml) 7H10 agar (CLSI 2011 M24-A2) 7H11 agar (CLSI 2011 M24-A2) BACTEC MGIT960® (Becton Dickinson) BACTEC 460TB® (Becton Dickinson) Löwenstein-Jensen (Canetti 1969)b Isoniazid 0.2 1.0a 0.2 1.0 a 0.1 0.4 a 0.1 0.1 0.2 1 Rifampicin 1.0 1.0 1.0 2.0 20 40 Ethambutol 5.0 10.0 7.5 NR 5.0 7.5 1 2 3 Pyrazinamide NR NR 100 (pH 5.9) 100 (pH 6.0) 50 100 400 (pH 4.9) a

as 2nd concentration in case of resistance to the lower one in view to determine the low or high level resistance of the strains to isoniazid

b

critical concentration in bold (growth at this concentration indicates resistance) NR: Not Recommended

The guidelines from the Centers for Disease Control (CDC) [Shinnick et al. 2005] and CLSI [CLSI 2011 M24-A2] recommend that the first isolate of M. tuberculosis complex obtained from each patient should be tested for possible first line drug resistance. DST should be repeated if the cultures fail to become negative after 3 months of therapy.

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4.8. Biosafety

Laboratories require a certain degree of containment depending on the work performed. For microorganisms, Belgium proposes 3 levels of biosafety containments, based on activities involving human, animal or plant pathogens and their genetically engineered derivatives. The aim is to avoid possible adverse events on human health or environment which might arise from the manipulation of these microorganisms [www.biosafety.be]. The WHO classifies 4 biosafety levels and divides the infective microorganisms in 4 different risk groups (Table I.5). The risk group number does not necessarily equals the required Biosafety Level (e.g. certain manipulations of a risk group 2 pathogen might be restricted to a level 3 biosafety containment). Every country and/or laboratory carries out a risk assessment according to their proper activities.

Table I.5: Classification of infective microorganisms by risk group defined by WHO [WHO 2004]

Risk group 1 No or low individual and community risk

A microorganism that is unlikely to cause human or animal disease.

Risk group 2 Moderate individual risk, low community risk

A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited. Risk group 3 High individual risk, low

community risk

A pathogen that usually causes human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available.

Risk group 4 High individual and community risk

A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available.

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5. CLINICAL DIAGNOSIS

5.1. Diagnosis of latent tuberculosis

As one third of the world population is infected with TB and as 5 to 10% among them develop the disease (and up to 50% if coïnfected with HIV), screening for LTBI is important for TB containment. As LTBI is asymptomatic, specialized screening methods are necessary. The most widely used technique is still the Mantoux test with an intradermal injection of tuberculin Purified Protein Derivative (PPD). After 24 to 72 hours the site of injection is inspected by trained health care workers. In TB infected persons PPD causes a local induration, swelling and red skin color of >10mm diameter (Figure I.21) while non-infected people do not respond to the intradermal injection. However if a person’s immune system is compromised, a false negative result may be obtained. On the other hand, BCG vaccinated people usually react positive to the tuberculin skin test without being infected by the TB bacteria.

Figure I.21: Picture of a positive tuberculin skin test indicating a TB infection (picture from Minnesota Department of Health website

http://www.health.state.mn.us/divs/idepc/diseases/tb/tst.html)

Faculté de Pharmacie

Ecole Doctorale en Sciences Pharmaceutiques

Contribution to the research on

Drug Resistant Mycobacterium tuberculosis

ACKNOWLEDGEMENTS

THANK YOU!

TABLE OF CONTENTS

LIST OF FIGURES AND TABLES

ABBREVIATIONS

1.

TUBERCULOSIS: A BRIEF HISTORY

1.1.

Origin of the tuberculosis bacteria

1.2.

Discovery of the causative agent

1.3.

Diagnostic tools for tuberculosis

1.4.

Treatment

1.5.

Prevention

2.

TUBERCULOSIS: INFECTION AND TRANSMISSION

2.1.

Primo-infection and transmission of pulmonary tuberculosis

2.2.

Extra-pulmonary tuberculosis

2.3.

Risk factors for developing TB disease after infection by M.tuberculosis

3.

EPIDEMIOLOGY OF TUBERCULOSIS

3.1.

Global burden

3.2.

Multi-drug resistance

3.3.

Situation in Belgium

3.4.

Molecular epidemiology: laboratory tools to detect ongoing transmission

4.

THE GENUS MYCOBACTERIUM

4.1.

Group 1: Mycobacterium tuberculosis complex

4.2.

Group 2: Mycobacterium leprae

4.3.

Group 3: Non-Tuberculous Mycobacteria

4.4.

Characteristics of mycobacteria

4.5.

Cultivation of mycobacteria

4.6.

Identification methods and genotypic drug resistance testing

4.7.

Phenotypic drug susceptibility testing

4.8.

Biosafety

5.

CLINICAL DIAGNOSIS

5.1.

Diagnosis of latent tuberculosis