Вы находитесь на странице: 1из 32







MARCH, 2016
I dedicate this research work to my parents,Mr & Mrs C.A Ogbu and my brother Mr Patrick
My profound gratitude goes to the almighty God,His son Jesus Christ(Our Saviour0 and the
Holy spirit,for giving me wisdom,knowledge,understanding and good healthnof mind and
body before and during the period of my seminar research.

I wish to appreciate all my Lecturers in the Department of Medical Laboratory

Science,Rivers State University of Science And Technology,Port Harcourt.I really appreciate
all your efforts at impacting extensively in me the knowledge and practice of Medical
Laboratory Science.

However,a special appreciation goes to my caring and active

Supervisor/HOD,DR(Mrs)Wokem, G.N whose guidelines and approval gave this research
work a reality.

Am highly indebted to all the researchers and writers to whom materials I have use in the
writing of this seminar and Google for providing an easy platform for information retrieval.

Final thanks goes to my friends and classmates for their advice and information in the course
of writing and typesetting this work

The use of antibiotics in the management of tuberculosis has been a double-edged knife. Despite the fact that
they destroy the Mycobacterium tuberculosis, they also show drug resistance against the bacteria indicating
ineffectiveness. Worldwide surveillance has depicted that drug resistant tuberculosis is widespread and is
recently a menace to tuberculosis control programs in many countries. The use of molecular techniques like the
GeneXpert and Line Probe Assays, during the last few years has greatly changed our understanding of drug
resistance in tuberculosis. The arrival of HIV/AIDS in the 1980s resulted in an increase in transmission of TB
associated with outbreaks of multidrug-resistant Tuberculosis,that is resistant to isoniazid and rifampin. In this
paper, a discuss on the challenges associated with the control of Mycobacterium tuberculosis drug resistance,
and an update on recent diagnostic and drug regimen in the management of the disease, within the context of the
new public health, as it refers to the ways in which health systems manage and monitor threats to public health.

1.0 Introduction
1.1 Scientific classification
1.2 Historical perspective of tuberculosis
1.3 Mode of transmission
1.4 Symptoms and associated risk factors
1.4.1 Tuberculosis disease risk factors
1.5 Diagnostic methods of Tuberculosis
1.5.1 WHO- recommended diagnostic technique

2.0 Evolution of drug resistance and global surveillance
2.1 Molecular mechanism of drug resistance in Mycobacterium tuberculosis
2.2 First line drugs for TB
2.3 Second line drugs for TB
2.4 Newly introduced drugs in treating TB

3.0 Challenges to public health and health administrators
3.1 Management of Tuberculosis
Conclusion and recommendation


Tuberculosis 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 a bacterial microorganism, the

tubercule bacillus or Mycobacterium tuberculosis. Mycobacterium tuberculosis and seven

very closely related mycobacterial species (M. bovis, M. africanum, M. microti, M. pinnipedi,

M. canetti, M. caprae and M. mungi) together comprise what is known as the M. tuberculosis


Fig.1.1 Mycobacterium tuberculosis

Tuberculosis(TB) remains an important infectious disease and public health concern

worldwide. According to the latest World Health Organization (WHO) report, there were an

estimated 8.6 million incident cases of TB in 2012 and 1.3 million deaths were attributed to

the disease. More than half a million cases occurred in children and 320,000 deaths were

reported among HIV-infected persons(WHO,2013). However, even more disturbing is the

emergence of drug resistance. In 2013,there were an estimated 480,000 cases of multidrug

resisitant(MDR)-TB and 170,000 deaths were due to it (WHO,2014).

1.1 Scientific classification

Kingdom :- Bacteria

Phylum :- Actinobacteria

Class :- Actinobacteria

Order :- Actinomycetales

Suborder :- Corynebacterineae
Family :- Mycobacteriaceae

Genus :- Mycobacterium

Species :- M. tuberculosis

1.2 Historical perspective of tuberculosis

Consumption, phthisis, potts disease and the white plague are all terms used to refer to

tuberculosis throughout history(Ackerknecht,1982). The term phthisis first appeared in Greek

literature around 460 B.C. Hippocrates identified the illness as the most common cause of

illness in his time(Barnes,1995).

Galen, the most eminent Greek physician after Hippocrates, defined phthisis as the

ulceration of the lungs, thorax or throat, accompanied by a cough, fever and consumption of

the body by pus(Bourdelais & Bart,2006).

The tuberculosis epidemic in Europe, which probably started in the 17th century and which

lasted two hundred years, was known as the great white plague. Death by tuberculosis was

considered inevitable and it was the principal cause of death in the mid-17th century. The high

population density, as well as the poor sanitary conditions that characterized most European

and North American cities at that time created perfect environment for its propagation.

In the nineteenth century, precisely on March 24,1882, the microbiologist Robert Koch

announced to the Berlin physiological society that he had discovered the cause of

tuberculosis. Because antibiotics were unknown at that time, the only means of controlling

the spread of infection was to isolate patients in private hospitals limited to patients with TB,

which is known as TB sanatorium a practice that continues to this day in some countries.

During the early 1940s, streptomycin, the first antibiotic effective against Mycobacterium

tuberculosis was discovered, then the infection began to come under control. Although their

was an upsurge in the disease in the late twentieth century due to the HIV/AIDS epidemic.
AIDS patients are much more likely to develop tuberculosis because of weakened immune


1.3 Mode of transmission

M. tuberculosis is carried in airborne particles, called droplet nuclei, of 1-5 microns in

diameter. Infectious droplets are generated when a TB patient exhales, coughs,sneezes or

even shouts, and are released into the air. This mist or aerosol as it is often called can be

taken into the nasal passages and lungs of a susceptible person nearby. Depending on the

environment, these tiny particles can remain suspended in the air for several hours. Unlike

many other infections, TB is not transmitted by surface contact with a patients clothing, bed

linens or dishes and cooking utensils. The most important exception is in pregnancy. The

foetus of an infected mother may contract TB by inhaling or swallowing the bacilli in the

amniotic fluid.

Fig.1.2 TB is spread from person to

person through the air. The dots in
the air represent droplet nuclei
containing tubercle bacilli.

1.4 Symptoms and associated risk factors

There are two types of tuberculosis conditions: (i)Latent tuberculosis infection(LTBI) and (ii)

Tuberculosis disease. Persons with latent tuberculosis infection have the M. tuberculosis in

their bodies, but do not have the disease or make such persons sick and cannot spread the

infection to other people.

In some people, the tubercle bacilli overcome the immune system and multiply, resulting in

progression from latent tuberculosis infection(LTBI) to tuberculosis(TB) disease. Persons

who have TB disease are usually infectious and may spread the bacteria to other people.
The symptoms of the disease include:-

* A persistent cough that last 3 weeks or longer

* Pain in the chest

* Coughing up blood or sputum(mucus from deep inside the lungs)

* Weakness and weight loss

* No appetite

* Chills and fever

* Sweating at night

1.4.1 Tuberculosis disease risk factors :-

Anyone can get TB, but persons at high risk generally fall into several categories:-

* People recently infected with M. tuberculosis

* People with medical conditions that weaken the immune system (immunocompromised),for

instance: persons with HIV infection has a very high risk of developing TB disease, HIV and

tuberculosis are so closely connected that there relationship is often described as a co-


* People who have spent time with a TB patient.

* Health-care workers who works with patients suffering the disease.

* Children younger than 5 years of age.

1.5 Diagnostic methods of tuberculosis

An unprecedented effort to improve and expand TB laboratory capacity is currently

underway, spearheaded by World Health Organization (WHO) and its network of

international collaborators (WHO,2013).

Genotypic (molecular) methods have considerable advantages for scaling up programmatic

management and surveillance of drug-resistant TB, offering speed of diagnosis, standardized

testing, potential for high throughput and fewer requirements for laboratory biosafety. The
development of the Xpert MTB/RIF assay for the Gene-Xpert platform was completed in

2009 and is considered an important breakthrough in the fight against TB.(WHO,2013)

1.5.1 WHO- recommended diagnostic techniques

*Conventional light microscopy:

Ziehl-Neelsen(ZN) light microscopy performed directly on sputum has been the primary

diagnostic technique for over 100years, and is suitable for all laboratory service levels,

including peripheral laboratories at primary health care centres or zonal hospitals.

*Conventional fluorescent microscopy:

Conventional fluorescence microscopy typically uses quartz-halogen or high-pressure

mercury vapour lamps as light sources. A lower magnification objective is used to scan

smears allowing a much larger area of the smear to be seen and therefore taking less time

than the light microscopy.

*Drug susceptibility testing(DST):

Drug susceptibility testing provides a definitive diagnosis of drug-resistant TB. A number of

different DST methods are available which are:

---Phenotypic method involves culturing of M. tuberculosis in the presence of anti-TB drugs

to detect growth(indicating drug resistance) or inhibition of growth(indicating drug


---Genotypic method involves targeting specific molecular mutation associated with

resistance against individual drugs. DST is essential for identifying patients at risk of MDR-

TB, as the first priority(WHO,2012).

*Molecular testing:

Molecular line-probe assays(LPAs) focused on rapid detection of rifampicin and isoniazid

resistance have been endorsed by WHO in 2008 with detail policy guidance on its

introduction at country level. The amplification of nucleic acids (DNA or RNA) for the
diagnosis of TB or to detect drug resistance is a sensitive method that can produce faster

results than conventional culture methods. Polymerase chain reaction (PCR) is the most

common method of amplification. The Xpert MTB/RIF cartridge is a disposable plastic

cartridge that contains all reagents required for the detection of M. tuberculosis and

rifampicin resistance with PCR. Rifampicin resistance is an indicator of MDR-TB. The Xpert

MTB/RIF cartridge uses Cepheids fully automatic GeneXpert device. The assay can be

performed directly on a sputum sample. The test has similar sensitivity to culture, is specific

for M. tuberculosis and results can be obtained within two hours.(WHO,2013)

Fig 1.3
A Med Lab. Scientist using GeneXpert machine.

2.0 Evolution of drug resistance and global surveillance

Drug resistance has being a major issue in the treatment of tuberculosis cases, which has

resulted in the issue of multidrug resistance. Multidrug resistance-tuberculosis (MDR-TB),

is defined as resistance in invitro to at least isoniazid and rifampicin, while Extensively drug-

resistant (XDR-TB) is resistant to at least one fluoroquinolone and one injectable second line

anti-TB drug like kanamycin or capreomycin in addition to rifampicin and isoniazid(Gandhi


Shortly after the first anti-tuberculosis (TB) drugs were introduced, streptomycin(STR), para-

aminosalicylic acid(PAS), isoniazid(INH) resistance to these drugs was observed in clinical

isolates of Mycobacterium tuberculosis(Crofton&Mitchison,1948). This led to the need to

measure resistance accurately and easily. By the end of the 1960s rifampicin(RIF) was

introduced and with the use of combination therapy, there was a decline in funding and

interest in TB control programs. As a result, no concrete monitoring of drug resistance was

carried out for the following 20 years. The arrival of HIV/AIDS in the 1980s resulted in an

increase in transmission of TB associated with outbreaks of multidrug-resistant TB (Fischl et

al.,1992) i.e resistant to INH and RIF. In the early 1990s drug resistance surveillance was

resumed in developed countries, but the true incidence remained unclear in the developing


Since 2005, the worldwide incidence rate has dropped slowly. In 2011, an estimated 8.7

million cases occurred globally including 1.1 million cases among the HIV-infected

population. The global incidence rate is 125 cases per 100 000 population. The 22 high-

burden countries account for 82% of the global TB burden(WHO,2012). These countries are

all low-and-middle-income countries (LMIC) and the incidence rates in 14 of these countries
are declining, six countries have stable rates and two countries have an increasing TB

incidence rate. The number of TB deaths was approximately 1.4 million in 2011. Globally the

treatment success rate has reached 85% in 2009(WHO,2013).

Since the 2004 Priority Medicines report the world has seen an increase in the incidence of

M. tuberculosis resistant to first-line drugs (Multi-Drug Resistant TB; MDR-TB) and the

emergence of Extensively-Drug Resistant TB (XDR-TB) in 2006. Since 2006, several

countries (e.g. Italy, Iran and India), reported TB strains with severe patterns of drug

resistance. The term Totally-Drug Resistant TB is used for these TB strains, but has not

been accepted by the WHO for several reasons.(WHO,2010).

Global trends in MDR-TB rates are unclear due to the lack of data in several countries.

Combined data showed that on average multidrug resistance occurs in 3.4% and 19.8% of

new and previously treated TB cases respectively(WHO 2010). Countries with a high burden

of MDR-TB cases in Europe include Estonia, Lithuania, Moldova and Belarus. Data on

XDR-TB are much scarcer, but globally approximately 9.4% of all MDR-TB cases were

extensively drug resistant with high burdens in Estonia, Latvia, South Africa and


At least one-third of the worlds HIV-positive population is infected with TB. Co-infected

TB/HIV patients are 21-34 times more likely to develop active TB than those living solely

with LTBI(WHO,2010). Tuberculosis is the leading cause of death among HIV-positive

people. In 2010, 350,000 people died due to HIV-associated TB(WHO,2010). For HIV-

positive TB patients, it is crucial to detect MDR as soon as possible due to their high risk of

mortality. Mortality rates can exceed 90% in patients co-infected with XDR-TB and HIV

(WHO, 2013).
2.1 Molecular mechanism of drug resistance in Mycobacterium tuberculosis

In order to control the drug resistance epidemic it is necessary to gain insight into how M.

tuberculosis develops drug resistance. This knowledge will help us to understand how to

prevent the occurrence of drug resistance as well as identifying genes associated with drug

resistance of new drugs. Mutations in the genome of M. tuberculosis that can confer

resistance to anti-TB drugs occur spontaneously with an estimated frequency of 3.5 10-6 for

INH and 3.1 10-8 for RIF. Because the chromosomal loci responsible for resistance to

various drugs are not linked, the risk of a double spontaneous mutation is extremely low: 9

10-14 for both INH and RIF(Brossier et al, 2011).

2.2. First line drugs for TB

2.2.1 Rifampicin

Rifampicin is a rifamycin derivative introduced in 1972 as an antituberculosis agent. It is one

of the most effective anti-TB antibiotics and together with isoniazid makes up the basis of

the multidrug treatment regimen for TB. Rifampicin is active against growing and non-

growing (slow metabolizing) bacilli(Mitchison, 1979). The mode of action of rifampicin in

M. tuberculosis is by binding to the -subunit of the RNA polymerase, inhibiting the

elongation of messenger RNA(Boshoff et al,2004). The majority of rifampicin-resistant

clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the -

subunit of the RNA polymerase. As a result of this, conformational changes occur that

decrease the affinity for the drug and results in the development of resistance. In about 96%

of M. tuberculosis isolates resistant to rifampicin, there are mutations in the so-called hot-

spot region of 81-(base polar)bp spanning codons 507533 of the rpoB gene. This region is

also known as the rifampicin resistance-determining region (Ramaswamy & Musser,1998).

2.2.2 Isoniazid
Isoniazid was introduced in 1952 as an anti-TB agent and it remains, together with

rifampicin, as the basis for the treatment of the disease. Unlike rifampicin, isoniazid is only

active against metabolically-active replicating bacilli. Also known as isonicotinic acid

hydrazide, isoniazid is a pro-drug that requires activation by the catalase/peroxidase enzyme

KatG, encoded by the katG gene, to exert its effect (Zhang et al,1992). Isoniazid acts by

inhibiting the synthesis of mycolic acids through the NADH-dependent enoyl-acyl carrier

protein (ACP)-reductase, encoded by inhA(Rawat et al,2003). Although simple in its

structure, resistance to this drug has been associated with mutations in several genes, such as

katG, inhA, ahpC, kasA and NDH.

The two main molecular mechanisms of isoniazid resistance are associated with gene

mutations in katG and inhA or its promoter region. Indeed, numerous studies have found

mutations in these two genes as the most commonly associated with isoniazid

resistance(Ramaswamy et al,2003 & Hazbon et al,2006).

In M. tuberculosis, ahpC encodes an alkyl hydroperoxidase reductase that is implicated in

resistance to reactive oxygen intermediates and it was initially proposed that mutations in the

promoter of ahpC could be used as proxy markers for isoniazid resistance(Rinder et al,1998).

It is now better understood that mutations in the promoter of ahpC are compensatory

mutations for the loss of catalase/peroxidase activity rather than the cause for isoniazid

resistance. However, the role of kasA mutations in isoniazid resistance is presently unclear,

because similar mutations were also found in isoniazid-suscetible isolates , and, in cases of

isoniazid resistance, mutations were also found in katG or inhA.

2.2.3 Ethambutol

Ethambutol was first introduced in the treatment of TB in 1966 and is part of the current first-

line regimen to treat the disease. Ethambutol is bacteriostatic against multiplying bacilli

interfering with the biosynthesis of arabinogalactan in the cell wall(Takayama &

Kilburn,1989). In M. tuberculosis, the genes embCAB, organized as an operon, code for

arabinosyl transferase, which is involved in the synthesis of arabinogalactan, producing the

accumulation of the intermediate D-arabinofuranosyl-P-decaprenol(Mikusova et al,1995) .

Recognized mechanism of resistance to ethambutol has been linked to mutations in the gene

embB. Moreover, a study with a large number of M. tuberculosis isolates found that

mutations in embB306 were not necessarily associated with resistance to ethambutol but with

a predisposition to develop resistance to increasing number of drugs and to be

transmitted(Hazbon et al,2005). These findings could have influence on the correct detection

of ethambutol resistance by current molecular methods. There remain about 30% ethambutol

resistant strains that do not present any mutation in embB stressing the need to identify other

possible mechanisms of drug resistance to this drug.

2.2.4 Pyrazinamide

Pyrazinamide was introduced into TB treatment in the early 1950s and constitutes now part

of the standard first-line regimen to treat the disease. Pyrazinamide is an analog of

nicotinamide and its introduction allowed reducing the length of treatment to six months. It

has the characteristic of inhibiting semi-dormant bacilli residing in acidic environments such

as found in the TB lesions(Mitchison,1985) . Pyrazinamide is also a pro-drug that needs to be

converted to its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase

coded by the pncA gene. The proposed mechanism of action of pyrazinamide involves

conversion of pyrazinamide to pyrazinoic acid, which disrupts the bacterial membrane

energetics inhibiting membrane transport. Pyrazinamide would enter the bacterial cell by

passive diffusion and after conversion to pyrazinoic acid it is excreted by a weak efflux

pump. Under acid conditions, the protonated pyrazinoic acid would be reabsorbed into the

cell and accumulated inside, due to an inefficient efflux pump, resulting in cellular

damage(Zhang, 2005).

A recent study, however, has challenged the previous model by proposing that pyrazinoic

acid inhibits trans-translation, a process of ribosome-sparing in M. tuberculosis(Zimhony et

al,2007) .
Mutations in the gene pncA remain as the most common finding in pyrazinamide resistant

strains. Some few studies have reported the occurrence of pyrazinamide resistant strains

without any mutation in pncA stating that the resistance could be due to mutations in another

not yet identified regulatory gene(Cheng et al, 2004). Based on the current evidence, the

contribution of mutations in rpsA to pyrazinamide resistance remains limited(Simons et


2.2.5 Streptomycin

Originally isolated from the soil microorganism Streptomyces griseus, streptomycin was the

first antibiotic to be successfully used against TB. Unfortunately, as soon as it was

prescribed, resistance to it emerged, a result of being administered as a single therapy

(Crofton and Mitchison,1948). Streptomycin is an aminocyclitol glycoside active against

actively growing bacilli and its mode of action is by inhibiting the initiation of the translation

in the protein synthesis(Moazed & Noller,1987). More specifically, streptomycin acts at the

level of the 30S subunit of the ribosome at the ribosomal protein S12 and the 16S rRNA

coded by the genes rpsL and rrs, respectively(Finken et al,1993).

Consequently, mutations in rpsL and rrs are the major mechanisms of resistance to

streptomycin but account for 60%70% of the resistance found(Gillespie,2002). There are an

important percentage of strains resistant to streptomycin that lack mutations in either of these

two genes, suggesting additional mechanisms of resistance.

2.3 Second line drugs for TB

According to the WHO the following drugs can be classified as second line drugs:

fluoroquinolones(ofloxacin,ciprofloxacin and gatifloxacin),aminoglycosides(kanamy

-cin,capreomycin and amikacin),ethionamides,D-cycloserine and thiocetazone(WHO,2010).

Unfortunately,second-line drugs are inherently more toxic and less effective than first line-

line drugs(WHO,2010). Second line drugs are mostly used in the treatment of MDR-TB and

as a result prolong the total treatment time from 6 to 9 months(Cheng et al,2004).

2.3.1 Fluoroquinolones

Fluoroquinolones are currently in use as second-line drugs in the treatment of MDR-TB. Both

ciprofloxacin and ofloxacin are synthetic derivatives of the parent compound nalidixic acid,

discovered as a by-product of the antimalarial chloroquine(Goss et al, 1965). Newer-

generation quinolones such as moxifloxacin and gatifloxacin are being evaluated in clinical

trials and proposed as first-line antibiotics with the purpose of shortening the length of

treatment in TB (Palomino & Martin,2013).

The mode of action of fluoroquinolones is by inhibiting the topoisomerase II (DNA gyrase)

and topoisomerase IV, two critical enzymes for bacterial viability. These proteins are

encoded by the genes gyrA, gyrB, parC and parE, respectively (Fabrega et al,2009). In M.

tuberculosis, only type II topoisomerase (DNA gyrase) is present and, thus, is the only target

of fluoroquinolone activity(Aubry et al,2004). Type II topoisomerase is a tetramer formed by

two and subunits, coded by gyrA and gyrB, respectively, which catalyzes the supercoiling

of DNA(Takiff et al,1994) . The main mechanism of development of fluoroquinolone

resistance in M. tuberculosis is by chromosomal mutations in the quinolone resistance-

determining region of gyrA or gyrB. A recent systematic review of fluoroquinolone-

resistance-associated gyrase mutations in M. tuberculosis has been published(Maruri et


Cross-resistance is assumed to occur between fluoroquinolones although isolated reports have

acknowledged the presence of strains resistant to gatifloxacin and moxifloxacin that were still

susceptible to ofloxacin(VonGroll et al,2009). Also, the involvement of efflux mechanisms

has been suggested as a possible cause for fluoroquinolone resistance in M.

tuberculosis(Escribano et al,2007).

2.3.2 Aminoglycosides

Kanamycin(KAN) and Aminokacin(AMI) are aminoglycosides which inhibit protein

synthesis and thus cannot be used against dormant M. tuberculosis. Aminoglycosides bind to
bacterial ribosomes and disturb the elongation of the peptide chain in the bacteria. Mutations

in the rrs gene encoding for 16s rRNA are associated with resistance to KAN and AMI.

Nucleotide changes at positions 1400,1401 and 1483 of the rrs gene have been found to be

specifically associated with KAN resistance (Suzuki et al,1998).

2.3.3 Ethionamide

Ethionamide is a derivative of isonicotinic acid structurally similar to isoniazid. It is also a

pro-drug requiring activation by a monooxygenase encoded by the ethA gene. It interferes

with the mycolic acid synthesis by forming an adduct with NAD that inhibits the enoyl-ACP

reductase enzyme. Resistance to ethionamide occurs by mutations in etaA/ethA, ethR and also

mutations in inhA, which cause resistance to both isoniazid and ethionamide(DeBarber et

al,2000; Brossier et al,2011). Moreover, studies with spontaneous isoniazid- and

ethionamide-resistant mutants of M. tuberculosis found that they map to mshA, encoding an

enzyme essential for mycothiol biosynthesis (Vilcheze et al, 2008).

2.3.4 Cycloserine

Cycloserine is an oral bacteriostatic second-line anti-tuberculosis drug used in MDR-TB

treatment regimens. It is an analog of D-alanine that by blocking the activity of D-alanine: D-

alanine ligase inhibits the synthesis of peptidoglycan. It can also inhibit D-alanine racemase

(AlrA) needed for the conversion of L-alanine to D-alanine(Zhang,2005). Although the actual

target of cycloserine in M. tuberculosis is not completely elucidated, in previous studies in M.

smegmatis it was shown that overexpression of alrA led to resistance to cylcoserine in

recombinant mutants(Caceres et al,1997) . More recently, it has also been shown that a point

mutation in cycA, which encodes a D-alanine transporter, was partially responsible for

resistance to cycloserine in M. bovis BCG(Chen et al,2012).

2.3.5 Thioacetazone

Thioacetazone is an old drug that was used in the treatment of TB due to its favourable in

vitro activity against M. tuberculosis and its very low cost. It has toxicity problems, however,
especially in patients co-infected with HIV. It belongs to the group 5 drugs of the WHO and

acts by inhibiting mycolic acid synthesis(Grzegorzewicz et al,2012).

2.4 Newly introduced drugs in treating TB

Notwithstanding the alleged lack of interest of the pharmaceutical industry for the

development of new antibiotics, there are several anti-tuberculosis drugs in the pipeline and

some of them are already being evaluated in clinical trials and in new combinations with the

purpose of reducing the length of TB treatment.

2.4.1 Bedaquiline

Formerly known as TMC207 or R207910, bedaquiline is a new antibiotic belonging to the

class of diarylquinolines with specific activity against M. tuberculosis, which has also shown

in vitro activity against other non-tuberculous mycobacteria(Huitric et al, 2007). Bedaquiline

was discovered after a high-throughput evaluation of thousands of compounds using

Mycobacterium smegmatis in a whole-cell assay(Andries et al,2005). The drug showed in

vitro and in vivo activity against M. tuberculosis and then entered into clinical evaluation for

drug susceptible and MDR-TB. Based on the results of two phase II clinical trials,

bedaquiline has recently received conditional approval for the treatment of MDR-TB under

the trade name Sirturo. Recent reviews and evaluation of this new drug have been published

(Chahine et al, 2014). A phase III clinical trial was scheduled to begin in 2013 but has not yet

started. Bedaquiline is also being evaluated in new combination regimens with the purpose of

reducing the length of treatment(Diacon et al,2012).

The mode of action of bedaquiline is by inhibiting the ATP synthase of M. tuberculosis,

which was a completely new target of action for an antimycobacterial drug. This mode of

action was discovered by analyzing M. tuberculosis and M. smegmatis mutants resistant to

bedaquiline. The minimal inhibitory concentration (MIC) of bedaquiline against M.

tuberculosis is very low, and its bactericidal activity in the murine model is superior to that of

that of isoniazid and rifampicin(Andries et al,2005).

The results of two trials (phase II) suggested that a standard 2-month treatment regimen with

bedaquiline yielded high culture conversion rates, rapid sputum culture conversion and low

acquired resistance to companion drugs in newly diagnosed MDR-TB cases(Diacon et

al,2012) .

2.4.2 Delamanid

Delamanid, previously known as OPC-67683, is a derivative of nitro-dihydro-

imidazooxazole with activity against M. tuberculosis that acts by inhibiting the synthesis of

mycolic acid and is undergoing clinical evaluation in a phase III trial(Palomino and

Martin,2013). Delamanid was previously shown to have a very good in vitro and in vivo

activity against drug-susceptible and drug-resistant M. tuberculosis(Matsumoto et al, 2006),

as well as good early bactericidal activity comparable to that of rifampicin(Diacon et

al,2011). Delamanid at present has shown its safety and efficacy in a clinical management for

MDR-TB. The specific mode of action of delamanid is by inhibition of the mycolic acid

synthesis but it differs from isoniazid in that, it only inhibits methoxy- and keto-mycolic acid

while isoniazid also inhibits -mycolic acid (Matsumoto et al, 2006).

Delamanid also requires reductive activation by M. tuberculosis to exert its activity. In

experimentally generated delamanid-resistant mycobacteria, a mutation was found in the

Rv3547 gene, suggesting its role in the activation of the drug(Matsumoto et al,2006).

2.4.3 SQ-109

Compound SQ-109 is a synthetic equivalent but independent type of ethambutol that has

shown in vitro and in vivo activity against drug-susceptible and drug-resistant M.

tuberculosis(Protopopova et al,2005). It has also been shown to possess synergistic in vitro

activity when combined with first-line drugs, and more interestingly, when combined with
bedaquiline and the oxazolidinone PNU-10048(Reddy et al,2010). The mode of action of SQ-

109 is by interfering with the assembly of mycolic acids into the bacterial cell wall core,

resulting in accumulation of trehalose monomycolate, a precursor of the trehalose

dimycolate. Transcriptional studies have shown that, similar to other cell wall inhibitors such

as isoniazid and ethambutol, SQ-109 induces the transcription of the iniBAC operon required

for efflux pump functioning (Boshoff et al,2004).

2.4.4 Benzothiazinones

A new class of drug with antimycobacterial activity, 1,3-benzothiazin-4-one or

benzothiazinone (BTZ), was recently described(Makarova et al,2009). The lead compound of

(BTZ043) was found to have in vitro, ex vivo and in vivo activity against M. tuberculosis. It

was also found to be active against drug-susceptible and MDR clinical isolates of M.

tuberculosis (Pasca et al,2010).

By transcriptome analysis, the mode of action of BTZ043 was initially spotted at the cell wall

biogenesis level. By further genetic analysis, using in vitro generated mutants, the target of

the drug was identified at the level of the gene rv3790, which together with rv3791 encode

proteins that catalyze the epimerization of decaprenylphosphoryl ribose (DPR) to

decaprenylphosphoryl arabinose (DPA), a precursor for arabinan synthesis needed for the

bacterial cell wall(Mikusova, 2005). DprE1 and DprE2 were proposed as names for these two

key enzymes (Makarova,2009). Although M. tuberculosis apparently lacks nitroreductases

able to reduce the drug, this finding could be important for development of new BTZ

analogues with improved activity.


3.0 Challenges to public health and health administrators

Mortality due to infectious disease has fallen dramatically in the past centuries as a result of

sanitary and food safety development along with vaccines, antibiotics and other advances in

societal conditions and medical sciences. However, the challenges of population growth,

urbanization, deforestation, pollution, global climate change and global movement of

populations have been associated with a shift in geographical distribution and accelerated

diffusion of old and new pathogens resulting in an increased number of outbreaks of

Mycobacterium tuberculosis cases.

The health, social, economic, and political consequences associated with these emerging

diseases are still significant to public health. In the context of the New Public Health, they are

still central issues because of the enormous unfulfilled potential to reduce morbidity and

mortality globally. The public health community has learned much about facing new

challenges of newly emerging or as yet widespread infectious diseases, as well as about

infections as causes of chronic disease.

Global anticipation of the most recent TB epidemic has required international, national and

local health authorities to undertake extensive preparation. Despite the preparation, public

awareness and acceptance of immunization were insufficient and the pandemic eventually

faded from public view by itself. Hence, much effort is still needed to better understand the
biological mechanisms of HIV transmission and diffusion as well as peoples

behaviours/attitudes in the face of risk before we can hope to better combat tomorrows

outbreaks of Tuberculosis resistance to antibiotics.

New knowledge concerning micronutrients, oral disease and infectious diseases will become

part of public health methodology. New methods of producing vaccines against infectious

agents like Tuberculosis will bring huge public health gains in the coming decades, but the

urgency is great for new vaccines, such as that for Tuberculosis. Infections are unequally

spread and unpredictably transferred around the world. Rapid mass transportation and human

mobility helps introduce and spread organisms to previously unexposed populations, which

can then become resistant to treatments available, thus, rendering the disease Tuberculosis

even more dangerous.

Optimism but not complacency is justified. Political and financial support is needed to

maintain and develop the gains achieved in the past century and to transmit the latest

knowledge and technology to many parts of the world where preventable deaths measure in

the hundreds of thousands. The New Public Health calls for fair distribution of resources and

the timely application of existing knowledge and tools; funding ,initiative and training, all

with the aim of eradicating the resistance of Mycobacterium tuberculosis to antibiotics.

3.1 Management of tuberculosis

General measures such as avoidance of overcrowded and unsanitary conditions are also

necessary aspects of prevention. Hospital emergency rooms and similar locations can be

treated with ultraviolet light, which as an antibacterial effect.

3.1.1 Vaccination

Vaccination is one major preventive measure against TB. A vaccine called BCG(Bacillus

Calmette-Gurin,named after its French developers) is made from a weakened

Mycobacterium that infects cattle. Vaccination with BCG does not prevent infection by M.

tuberculosis but it does strengthen the immune system of first-time TB patients. As a result,

serious complications are less likely to develop. BCG is used more widely in developing
countries. The effectiveness of vaccination is still being studied, it is not clear whether the

vaccines effectiveness depends on the population in which it is used or on variations of


3.1.2 Designing a regimen to treat MDR-TB

Treatment of MDR-TB in developing countries is a particular dilemma because the

susceptibility testing and second-line agents are usually insufficient. Most national TB-

control programs endorse algorithms for treating patients whose infections fail to respond to

treatment that rely on the addition of new line drugs to the standard first/second-line

regimen.In settings with limited amounts of MDR-TB, these algorithms may be appropriate.

However, in settings where MDR-TB is prevalent,such an approach may actually increase

levels of resistance by selecting for additional mutants during therapy. Rational MDR-TB

treatment should include a minimum of four active drugs: a later-generation fluoroquinolone

(moxifloxacin, gatifloxacin or levofloxacin) plus an injectable aminoglycoside

(amikacin,capreomycin or kanamycin) plus any first-line drug to which the isolate is

susceptible (e.g., pyrazinamide) plus the addition of one drug from group 4(cycloserine or


Drug susceptibility testing (DST) (rapid and/or conventional) is strongly recommended by

WHO in all cases and particularly for those previously treated(Falzon et al,2013). While

awaiting DST results, in settings with a medium or low probability of MDR-TB, retreatment

cases could initially be treated with an empiric regimen including isoniazid, rifampicin,

pyrazinamide, ethambutol and streptomycin for 2 months, followed by isoniazid, rifampicin,

pyrazinamide and ethambutol for 1 month, and isoniazid, rifampicin, and ethambutol for 5


3.1.3 Prophylactic use of isoniazid

Isoniazid can be given for the prevention as well as the treatment of TB. Isoniazid is

effective when given daily over a period of 6 to 12 months to people in high-risk categories.

Isoniazid appears to be most beneficial to persons under the age of 25. Because isoniazid
carries the risk of side-effects(liver inflammation, nerve damage, changes in mood and

behaviour), it is important to give it only to persons at special risk.


Drug resistance is a worldwide problem that threatens to undermine effective control of TB.

As shown by the recent report of WHO/IUALTD, hotspots of MDR-TB have appeared in

regions with weak TB-control programs and misuse of anti-TB drugs. Prevention of drug

resistance depends on appropriate treatment of all patients with TB with combination drug

regimens and early detection of resistance followed by tailored treatment with second-line

agents and most recent, the newly introduced drugs. In countries with low levels of MDR-

TB, efforts should be concentrated on preventing acquired MDR-TB by endorsing and widely

implementing the WHO DOT strategy. In regions with high levels of MDR-TB, although

concentration on detecting and treating new susceptible TB cases remains critically

important, MDR-TB management efforts should tailor treatment by performing drug

susceptibility testing. In countries with limited resources, more operational research is needed

to define the best cost-effective strategies for individual as against standardized patient

management of MDR-TB under national program conditions. The ultimate goals are

provision of timely, appropriate, and adequate services. These must be provided, and

continually evaluated and updated. A highly infectious tuberculosis patient must have access

to state-of-the-art laboratory services, even if the patient resides in an area where a local

laboratory is not capable of providing those services. Also coordinated efforts to monitor and

prevent misuse of antibiotics are crucial to reducing the spread of microbial resistance, which

can arise due to factors such as self medication, prescription of antibiotics for viral infections

and general overuse of antibiotics.


The components of the STOP TB strategy:

Pursuing high quality directly observed therapy

Strengthening the primary health care system

Engage all healthcare providers

Enable and promote research

(WHO, 2013)


AIDS :-Acquired Immune Deficiency Syndrome

AFB :-Acid Fast Bacilli

AMI :-Aminokacin

DCS :-D-cycloserine

DOT :-Directly Observe Therapy

ETH :-Ethionamide

EMB :-Ethambutol

HIV :-Human Immuno-deficiency Virus

INH :-Isoniazid

KAN :-Kanamycin

MDR-TB :-Multi Drug Resistance Tuberculosis

STR :- Streptomycin

TDR-TB :-Total Drug Resistance Tuberculosis

WHO :World Health Organisation

XDR-TB :-Extensive Drug Resistance Tuberculosis


Ackerknecht, E. H. (1982).A Short History of Medicine. Retrieved from

http://www.jhupbooks.press.jhu.edu/content/ (Retrieved 19 January,2016).

Andries K, Verhasselt P, Guillemont J,(2005). A diarylquinoline drug active on the ATP synthase of
Mycobacterium tuberculosis. Science. 307(9), 223227.

Andries, K.; Verhasselt, P.; Guillemont, J.; Ghlmann, H.W.; Neefs, J.M.; Winkler, H.; van Gestel, J.;
Timmerman, P.; Zhu, M.; Lee, E.(2005). A diarylquinoline drug active on the ATP synthase of
Mycobacterium tuberculosis. Science 30(7), 223227.

Aubry, A.; Pan, X.S.; Fisher, L.M.; Jarlier, V.,and Cambau, E.(2004). Mycobacterium tuberculosis
DNA gyrase: Interaction with quinolones and correlation with antimycobacterial drug activity.
Journal of Antimicrobial Agents and Chemotherapy, 48(9), 12811288.

Barnes, D.S. (1995). The Making of a Social Disease: Tuberculosis in Nineteenth-century France.
Retrieved from http://www.amazon.com/books/content/ (Retrieved 7 February,2016).

Blanchard, J.S.(1996).Molecular mechanisms of drug resistance in Mycobacterium tuberculosis.

Annual Review in Biochemistry. 65(5), 215239.

Boshoff, H.I.; Myers, T.G.; Copp, B.R.; McNeil, M.R.; Wilson, M.A.; Barry, C.E.(2004). The
transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: Novel
insights into drug mechanisms of action. Journal of Biological Chemistry. 279(2), 40174

Bourdelais, Patrice; Bart K. Holland (2006). Epidemics Laid Low: A History of what Happened in
rich Countries. JHU Press. ISBN 080188294X.

Brossier, F.; Veziris, N.; Truffot-Pernot, C.; Jarlier, V.; Sougakoff, W.(2011) Molecular investigation
of resistance to the antituberculous drug ethionamide in multidrug-resistant clinical isolates of
Mycobacterium tuberculosis. Antimicrobial Agents Chemotherapy 55, 355560.

Cceres, N.E., Harris, N.B., Wellehan, J.F., Feng, Z., Kapur, V., Barletta, R.G.(1997). Overexpression
of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium
smegmatis. Journal of Bacteriology 179, 50465055.
Crofton, J.; Mitchison, D.A.(1948) Streptomycin resistance in pulmonary tuberculosis. British
Medical Journal 2(5), 10091015.

Cheng, A.F.; Yew, W.W.; Chan, E.W.; Chin, M.L.; Hui, M.M.; Chan, R.C(2004). Multiplex PCR
amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-
resistant Mycobacterium tuberculosis clinical isolates. Journal of Antimicrobial Agents
Chemotherapy 48(4), 596601.

Chen, J.M.; Uplekar, S.; Gordon, S.V.; Cole, S.T.(2012). A point mutation in cycA partially
contributes to the D-cycloserine resistance trait of Mycobacterium bovis BCG vaccine strains.
PLoS One 7, e43467.

Chahine, E.B.; Karaoui, L.R.; Mansour, H.(2014). Bedaquiline: A novel diarylquinoline for
multidrug-resistant tuberculosis. Annals of Pharmacotherapy, 48(8), 107115.

Caws, M.; Duy, P.M.; Tho, D.Q.; Lan, N.T.; Hoa, D.V.; Farrar, J(2006). Mutations prevalent among
rifampin and isoniazid-resistant Mycobacterium tuberculosis isolates from a hospital in
Vietnam. Journal of Clinical Microbiology, 44, 23332337.
DeBarber, A.E.; Mdluli, K.; Bosman, M.; Bekker, L.G.; Barry, C.E.(2000) 3rd. Ethionamide
activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc. National
Academic of Science. USA 97, 96779782.

Diacon, A.H.; Dawson, R.; Hanekom, M.; Narunsky, K.; Venter, A.; Hittel, N.L.; Geiter, J.; Wells,
C.D.; Paccaly, A.J.; Donald, P.R.(2011). Early bactericidal activity of delamanid (OPC-67683)
in smear-positive pulmonary tuberculosis patients. International Journal on Tuberculosis and
Lung Disease, 15(6), 949954.

Diacon, A.H.; Dawson, R.; du Bois, J.; Narunsky, K.; Venter, A.; Donald, P.R.; van Niekerk, C.;
Erondu, N.; Ginsberg, A.M.; Becker, P.(2012). Phase II dose-ranging trial of the early
bactericidal activity of PA-824. Journal of Antimicrobial Agents and Chemotherapy. 56, 3027

Diacon AH, Donald PR, Pym A,(2012). Randomized pilot trial of eight weeks of bedaquiline
(TMC207) treatment for multidrug-resistant tuberculosis: longterm outcome, tolerability, and
effect on emergence of drug resistance. Journal of Antimicrobial Agents and Chemother. 56(8),

Escribano, I.; Rodrguez, J.C.; Llorca, B.; Garca-Pachon, E.; Ruiz, M.; Royo, G.(2007) Importance of
the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones
and linezolid. Chemotherapy 53, 397401.

Fbrega, A., Madurga, S., Giralt, E., Vila, J.(2009). Mechanism of action of and resistance to
quinolones. Microbial Biotechnology. 2, 4061.

mpson,L.M.,Clearly,T.J., and Lai,S.(1992). An outbreak of tuberculosis caused by
multiple-drug-resistant tubercle bacilli among patients with HIV infection.Annals of internal
Finken, M., Kirschner, P., Meier, A., Wrede, A., Bttger, E.C.(1993). Molecular basis of streptomycin
resistance in Mycobacterium tuberculosis: Alterations of the ribosomal protein S12 gene and
point mutations within a functional 16S ribosomal RNA pseudoknot. Molecular Microbiology.
9(7), 12391246.

Falzon D, Gandhi N, Migliori GB,(2013). Resistance to fluoroquinolones and second-line injectable

drugs: impact on multidrug-resistant TB outcomes. European Respiratory Journal. 42: 156

Gandhi, N.R.; Moll, A.; Sturm, A.W.; Pawinski, R.; Govender, T.; Lalloo, U.; Zeller, K.; Andrews, J.;
Friedland, G.(2006). Extensively drug-resistant tuberculosis as a cause of death in patients co-
infected with tuberculosis and HIV in a rural area of South Africa. Lancet.368, 15751580

Gillespie, S.H.(2002). Evolution of drug resistance in Mycobacterium tuberculosis: Clinical and

molecular perspective. Journal of Antimicrobial Agents and Chemotherapy, 46, 267274.

Goss, W.A.; Deitz, W.H.; Cook, T.M.(1965). Mechanism of action of nalidixic acid on Escherichia
coli. II. Inhibition of deoxyribonucleic acid synthesis. Journal of Bacteriology, 89(2), 1068

Grzegorzewicz, A.E.; Kordulkov, J.; Jones, V.; Born, S.E.; Belardinelli, J.M.; Vaqui, A.; Gundi,
V.A.; Madacki, J.; Slama, N.; Laval, F.(2012) A common mechanism of inhibition of the
Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone. J.
Biol. Chem. 287, 3843438441.

Huitric, E.; Verhasselt, P.; Andries, K.; Hoffner, S.E.(2007) In vitro antimycobacterial spectrum of a
diarylquinoline ATP synthase inhibitor. Journal of Antimicrobial Agents and Chemotherapy,
51(4), 42024204.

Makarov, V.; Manina, G.; Mikusova, K.; Mllmann, U.; Ryabova, O.; Saint-Joanis, B.; Dhar, N.;
Pasca, M.R.; Buroni, S.; Lucarelli, A.P.(2009) Benzothiazinones kill Mycobacterium
tuberculosis by blocking arabinan synthesis. Science 324, 801804.

Maruri, F.; Sterling, T.R.; Kaiga, A.W.; Blackman, A.; van der Heijden, Y.F.; Mayer, C.; Cambau, E.;
Aubry, A.(2012) A systematic review of gyrase mutations associated with fluoroquinolone-
resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. Journal on
Antimicrobial Chemotherapy, 67(6), 819831.

Matsumoto, M.; Hashizume, H.; Tomishige, T.; Kawasaki, M.; Tsubouchi, H.; Sasaki, H.;
Shimokawa, Y.; Komatsu, M.(2006) OPC-67683, a nitro-dihydro-imidazooxazole derivative
with promising action against tuberculosis in vitro and in mice. PLoS Med. 3, e466.

Mikusov, K.; Huang, H.; Yagi, T.; Holsters, M.; Vereecke, D.; DHaeze, W.; Scherman, M.S.;
Brennan, P.J.; McNeil, M.R.; Crick, D.C.(2005). Decaprenylphosphoryl arabinofuranose, the
donor of the D-arabinofuranosyl residues of mycobacterial arabinan, is formed via a two-step
epimerization of decaprenylphosphoryl ribose. Journal of Bacteriology, 187, 80208025.

Mitchison, D.A.(1979). Basic mechanisms of chemotherapy. Chest journal, 76, 771781.

Moazed, D.; Noller, H.F.(1987) Interaction of antibiotics with functional sites in 16S ribosomal RNA.
Nature.327, 389394.

Palomino, J.C.; Martin, A.(2013). Tuberculosis clinical trial update and the current anti-tuberculosis
drug portfolio. Current Medical Chemothrapy. 20, 37853796.
Palomino, J.C.; Martin, A.(2013). TMC207 becomes bedaquiline, a new anti-TB drug. Future
Microbiology 8, 10711080.
Pasca, M.R.; Degiacomi, G.; Ribeiro, A.L.; Zara, F.; De Mori, P.; Heym, B.; Mirrione, M.; Brerra,
R.; Pagani, L.; Pucillo, L.(2010) Clinical isolates of Mycobacterium tuberculosis in four
European hospitals are uniformly susceptible to benzothiazinones. Antimicrob. Agents
Chemother. 54,16161618.

Protopopova, M.; Hanrahan, C.; Nikonenko, B.; Samala, R.; Chen, P.; Gearhart, J.; Einck, L.; Nacy,
C.A.(2005) Identification of a new antitubercular drug candidate, SQ109, from a combinatorial
library of 1,2-ethylenediamines. J. Antimicrob. Chemother. 56, 968974.

Rawat, R.; Whitty, A.; Tonge, P.J.(2003) The isoniazid-NAD adduct is a slow, tight-binding inhibitor
of InhA, the Mycobacterium tuberculosis enoyl reductase: Adduct affinity and drug resistance.
Proc. Natl. Acad. Sci. USA, 100, 1388113886
Reddy, V.M.; Einck, L.; Andries, K.; Nacy, C.A.(2010) In vitro interactions between new
antitubercular drug candidates SQ109 and TMC207. Antimicrob. Agents Chemother. 54, 2840
Rinder, H.; Thomschke, A.; Rsch-Gerdes, S.; Bretzel, G.; Feldmann, K.; Rifai, M.; Lscher,
T.(1998) Significance of ahpC promoter mutations for the prediction of isoniazid resistance in
Mycobacterium tuberculosis. European Journal of Clinical Microbiology and Infectious
Diseases, 17, 508511.

Ramaswamy, S.; Musser, J.M.(1998) Molecular genetic basis of antimicrobial agent resistance in
Mycobacterium tuberculosis: update. Tuberculosis and Lung Disease,79(2), 329.

Ramaswamy, S.V.; Reich, R.; Dou, S.J.; Jasperse, L.; Pan, X.; Wanger, A.; Quitugua, T.; Graviss,
E.A.(2003). Single nucleotide polymorphisms in genes associated with isoniazid resistance in
Mycobacterium tuberculosis. Journal of Antimicrobial Agents Chemotherapy 47(8), 1241

Simons, S.O.; Mulder, A.; van Ingen, J.; Boeree, M.J.; van Soolingen, D.(2013) Role of rpsA gene
sequencing in diagnosis of pyrazinamide resistance. J. Clin. Microbiol. 51, 382.

Somoskovi, A.; Parsons, L.M.; Salfinger, M.(2001) The molecular basis of resistance to isoniazid,
rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir. Res. 2, 164168.

Sun, Z.; Zhang, J.; Zhang, X.; Wang, S.; Zhang, Y.; Li, C.(2008) Comparison of gyrA gene mutations
between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical
isolates. Int. J. Antimicrob. Agents 31, 115121.
Suzuki, Y.; Katsukawa, C.; Tamaru, A.; Abe, C.; Makino, M.; Mizuguchi, Y.; Taniguchi, H(1998).
Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the
16S rRNA gene. Journal of Clinical Microbiology, 36(2), 12201225.
Takayama, K.; Kilburn, J.O.(1989). Inhibition of synthesis of arabinogalactan by ethambutol in
Mycobacterium smegmatis. Journal of Antimicrobial Agents and Chemotherapy 33(2),1493
Takiff, H.E.; Salazar, L.; Guerrero, C.; Philipp, W.; Huang, W.M.; Kreiswirth, B.; Cole, S.T.; Jacobs,
W.R., Jr.; Telenti, A.(1994).Cloning and nucleotide sequence of Mycobacterium tuberculosis
gyrA and gyrB genes and detection of quinolone resistance mutations. Journal of Antimicrobial
Agents and Chemotherapy. 38, 773780.

Vilchze, C.; Av-Gay, Y.; Attarian, R.; Liu, Z.; Hazbn, M.H.; Colangeli, R.; Chen, B.; Liu, W.;
Alland, D.; Sacchettini, J.C.(2008). Mycothiol biosynthesis is essential for ethionamide
susceptibility in Mycobacterium tuberculosis. Molecular Microbiology, 69(9), 13161329.

Von Groll, A.; Martin, A.; Jureen, P.; Hoffner, S.; Vandamme, P.; Portaels, F.; Palomino, J.C.; da
Silva, P.A.(2009).Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in
gyrA and gyrB. Antimicrob. Agents Chemother. 53, 44984500.
World Health Organization,(2010). Multidrug and Extensively Drug-resistant TB (M/XDR-TB);
Global Report on Surveillance and Response. Retrieved from http://www.stoptb.org/wg/gli
(Retrieved 29 January,2016).

World Health Organization,(2013). Global Tuberculosis Report. Retrieved from

http://www.finddiagnostics.com/ (Retrieved 2 February,2016).

World Health Organization,(2014). Global tuberculosis report 2. Retrieved from

http://www.who.int/th/publication/globalreport/en/ (Retrieved 9 February,2016).

Zhang, Y.; Heym, B.; Allen, B.; Young, D.; Cole, S(1992). The catalase-peroxidase gene and
isoniazid resistance of Mycobacterium tuberculosis. Nature .358, 591593.

Zhang, Y.(2005). The magic bullets and tuberculosis drug targets. Annual Review of Pharmacology
and Toxicology, 45(6), 529564.