SAFIKAMAL MANDAL
Bengal School of Technology(a college of pharmacy)
INTRODUCTION
Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis.
Tuberculosis (TB) remains as 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. However, even more disturbing is the emergence of drug resistance. In 2012, there were an estimated 450,000 cases of multidrug resistant (MDR)-TB and 170,000 deaths were due to it.
MDR-TB is caused by strains of Mycobacterium tuberculosis that are resistant to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. Since 2006, it has been recognized the presence of even more resistant strains of M. tuberculosis labelled as extensively drug resistant (XDR)-TB. These strains in addition to being MDR are also resistant to any fluoroquinolone and to at least one of the injectable second-line drugs: kanamycin, capreomycin or amikacin. More recently, a more worrying situation has emerged with the description of M. tuberculosis strains that have been found resistant to all antibiotics that were available for testing, a situation labelled as totally drug resistant (TDR)-TB.
Early detection of all forms of drug resistance in TB is a key factor to reduce and contain the spread of these resistant strains. A better knowledge of the mechanisms of action of anti-TB drugs and the development of drug resistance will allow identifying new drug targets and better ways to detect drug resistance. The following sections will review the mode of action and resistance mechanisms of the main anti-TB drugs as well as new drugs recently described with anti-TB activity.
TB SPEARD METHOD
Drug-susceptible TB and drug-resistant TB are spread the same way. TB bacteria are put into the air when a person with TB disease of the lungs or throat coughs, sneezes, speaks, or sings. These bacteria can float in the air for several hours, depending on the environment. Persons who breathe in the air containing these TB bacteria can become infected.
TB is not spread by
Shaking someone’s hand
Sharing food or drink
Touching bed linens or toilet seats
Sharing toothbrushes
Kissing
Symptoms of TB disease::
The general symptoms of TB disease include feelings of sickness or weakness, weight loss, fever, and night sweats. The symptoms of TB disease of the lungs may also include coughing, chest pain, and coughing up blood. Symptoms of TB disease in other parts of the body depend on the area affected. If you have these symptoms, you should contact your doctor or local health department.
1. First-Line Anti-TB Drugs
1.1. 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 constitutes the basis of the multidrug treatment regimen for TB. Rifampicin is active against growing and non-growing (slow metabolizing) bacilli. The mode of action of rifampicin in M. tuberculosis is by binding to the β-subunit of the RNA polymerase, inhibiting the elongation of messenger RNA. The majority of rifampicin-resistant clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the β-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 regionl of 81-bp spanning codons 507–533 of the rpoB gene. This region is also known as the rifampicin resistance-determining region. Mutations in codons 516, 526 and 531 are the most commonly associated mutations with rifampicin resistance in the majority of studies. Although less frequent, some reports have also noted the occurrence of mutations outside of the hot-spot region of rpoB. Cross-resistance with other rifamycins can occur. Mutations in some codons (e.g., 518 or 529) have been associated with low-level resistance to rifampicin but still susceptible to other rifamycins, such as rifabutin or rifalazil. This is important for TB patients that need to receive antiretroviral therapy since rifabutin is a less effective inducer of the cytochrome P450 CYP3A oxidative enzyme. On the other hand, monoresistance to rifampicin is quite rare and almost all rifampicin-resistant strains are also resistant to other drugs, especially to isoniazid. This is the reason why rifampicin resistance is considered as a surrogate marker for MDR-TB.
Recent genome sequencing studies have uncovered the acquisition of compensatory mutations in rpoA and rpoC, encoding the α and β' subunits of RNA polymerase, in rifampicin-resistant strains with mutations in rpoB. These compensatory mutations would be responsible for restoring the fitness of these strains in vivo and have also been associated with a higher transmissibility in some settings.
1.2. 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. Isoniazid acts by inhibiting the synthesis of mycolic acids through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase, encoded by inhA. 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. Among these, the most prevalent gene mutation has been identified as S315T in katG resulting in an isoniazid product deficient in forming the isoniazid-NAD adduct needed to exert its antimicrobial activity. This mutation has been consistently associated with high-level resistance (MIC > 1 μg/mL) to isoniazid and occurs more frequently in MDR strains. The second most common mutation occurs in the promoter region of inhA causing an overexpression of InhA or less frequently,
a mutation in its active site, which decreases its affinity for the isoniazid-NAD adduct. The most prevalent mutation found is at position −15C/T and is more commonly associated with low level resistance to isoniazid (MIC < 1 μg/mL). Mutations in inhA not only cause resistance to isoniazid but also to the structurally related drug ethionamide, which shares the same target. A recent study found that a mutation in the inhA regulatory region together with a mutation in the inhA coding region produced high-level isoniazid resistance and also cross-resistance to ethionamide.
One recent interesting finding showed that the 4R isomer of the isoniazid-NADP adduct causes inhibition of the dihydrofolate reductase (DfrA) in M. tuberculosis, suggesting that mutations in dfrA could possibly play a role in resistance to isoniazid. Moreover, an analysis of the proteome of isoniazid targets in M. tuberculosis identified 16 other proteins, in addition to InhA and DfrA, that were bound by these adducts with high affinity, which could signal other not yet clearly defined actions of isoniazid on the bacteria. Two recent studies, however, have failed to identify any mutation in dfrA associated with resistance to isoniazid.
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. 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. Moreover, overexpression of AhpC does not confer resistance to isoniazid.
Several studies have found single nucleotide polymorphisms in other genes in isoniazid resistant clinical isolates of M. tuberculosis, including kasA and the oxyR-ahpC and furA-katG intergenic regions. However, their direct role as a cause of isoniazid resistance has not been fully demonstrated. On the other hand, co-resistance to isoniazid and ethionamide has been clearly demonstrated to be caused by mutations in ndh in M. smegmatis and M. bovis BCG, by altering the NADH/NAD ratios inside the cell, leading to a competitive inhibition of the INH-NAD adduct. A recent study has also found that a silent mutation in mabA conferred isoniazid resistance through upregulation of inhA in M. tuberculosis
1.3. 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. 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.
The recognized mechanism of resistance to ethambutol has been linked to mutations in the gene embB with mutations at position embB306 as the most prevalent in most of the studies performed. Some studies, however, have also found mutations in embB306 in ethambutol susceptible isolates. 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. In fact, allelic exchange studies have shown that individual mutations causing certain amino acid substitutions produced ethambutol resistance, while other amino acid substitutions had little or no effect on ethambutol resistance. The same authors have more recently reported that mutations in the decaprenylphosphoryl-B-D-arabinose (DPA) biosynthetic and utilization pathway genes, Rv3806c and Rv3792, together with mutations in embB and embC accumulate, giving rise to a range of MICs of ethambutol depending on mutation type and number. These findings could have influence on the correct detection of ethambutol resistance by current molecular methods. Mutations in embB306 then, cause variable degrees of ethambutol resistance and are required but are not enough to cause high-level resistance to ethambutol. 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. Second-Line Anti-TB Drugs
2.1. 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. 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.
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. In M. tuberculosis, only type II topoisomerase (DNA gyrase) is present and, thus, is the only target of fluoroquinolone activity. Type II topoisomerase is a tetramer formed by two α and β subunits, coded by gyrA and gyrB, respectively, which catalyzes the supercoiling of DNA. 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. The most frequent mutations found are at position 90 and 94 of gyrA but mutations at position 74, 88 and 91 have also been reported. A recent systematic review of fluoroquinolone-resistance-associated gyrase mutations in M. tuberculosis has been published.
One interesting finding in M. tuberculosis is the presence of a natural polymorphism at position 95 in gyrA that is not related to fluoroquinolone resistance since it is also found in fluoroquinolone-susceptible strains. Another interesting finding has been the report that the simultaneous occurrence of mutations T80A and A90G in gyrA led to hypersusceptibility to several quinolones. This finding could point out that the problem of fluoroquinolone resistance in M. tuberculosis might be more complex than was thought initially.
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. Also, the involvement of efflux mechanisms has been suggested as a possible cause for fluoroquinolone resistance in M. tuberculosis.
2.2. Kanamycin, Capreomycin, Amikacin, Viomycin
These four antibiotics have the same mechanism of action by inhibiting the protein synthesis but, while kanamycin and amikacin are aminoglycosides, capreomycin and viomycin are cyclic peptide antibiotics. All four are second-line drugs used in the management of MDR-TB.
Kanamycin and amikacin inhibit protein synthesis by alteration at the level of 16S rRNA. The most common mutations found in kanamycin-resistant strains are at position 1400 and 1401 of the rrs gene, conferring high-level resistance to kanamycin and amikacin. However, mutations at position 1483 have also been reported. Full cross-resistance between kanamycin and amikacin is not complete, as previously thought. Some studies have shown variable levels and patterns of resistance suggesting that other mechanisms of resistance might be possible. In concordance with this, a low-level resistance to kanamycin has been associated with mutations in the promoter region of the eis gene, encoding an aminoglycoside acetyltransferase. Mutations at position −10 and −35 of the eis promoter led to an overexpression of the protein and low-level resistance to kanamycin but not to amikacin. These mutations were found in up to 80% of clinical isolates showing low-level resistance to kanamycin.
Capreomycin and viomycin, on the other hand, have a similar structure and bind at the same site in the ribosome, at the interface of the small and large subunits. They show full cross-resistance as reported in previous studies. Mutations in the tlyA gene have also been associated with resistance to capreomycin and viomycin. TlyA is an rRNA methyltransferase specific for 2'-O-methylation of ribose in rRNA. Mutations in tlyA determine the absence of methylation activity. Although some studies did not find this association, a recent meta-analysis, evaluating the association of genetic mutations and resistance to second-line drugs, has confirmed the presence of tlyA mutations in addition to mutations in rrs and eis.
2.3. 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. EthA is regulated by the transcriptional repressor EthR. Resistance to ethionamide occurs by mutations in etaA/ethA, ethR and also mutations in inhA, which cause resistance to both isoniazid and ethionamide. 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.
2.4. Para-Amino Salicylic Acid
Although it was one of the first anti-tuberculosis drugs used in the treatment of the disease, together with isoniazid and streptomycin, para-amino salicylic acid or PAS is now considered as a second-line drug part of the treatment regimen for MDR-TB. Until recently, its mechanism of action was not completely defined. It has been proposed that being an analog of para-amino benzoic acid, it must compete with it for dihydropteroate synthase, interfering in the process of folate synthesis. A study using transposon mutagenesis identified mutations in the thyA gene associated with resistance to PAS that were also present in clinical isolates resistant to PAS. A recent study has also identified various missense mutations in folC encoding dihydrofolate synthase that conferred resistance to PAS in laboratory isolates of M. tuberculosis. In a panel of 85 clinical MDR-TB isolates, mutations in folC were identified in five isolates resistant to PAS. Nevertheless, just less than 40% of PAS-resistant strains had mutations in thyA indicating that still other mechanisms of resistance to the drug might exist.
2.5. 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. 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. 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.
2.6. 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.
3. New Anti-TB Drugs
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.
3.1. 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. Bedaquiline was discovered after a high-throughput evaluation of thousands of compounds using Mycobacterium smegmatis in a whole-cell assay. 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. A ―black box‖ warning is, however, accompanying this authorization due to the reported unexplained deaths and QT interval prolongation. Recent reviews and evaluation of this new drug have been published. 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.
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. By sequencing the genome of these mutants and comparing to that of the susceptible strains, the only mutation found was in the atpE gene, which encodes the c part of the F0 subunit of the ATP synthase. This is a complex structure that generates the ATP needed by the mycobacterial cell for which bedaquiline has a favored specificity compared to mitochondrial ATP synthase.
Structure of Bedaquiline
The most prevalent mutation in the atpE gene found in bedaquiline resistant mutants is A63P but also I66M has been found. The latter introduces a modification that interferes the proper binding of bedaquiline to its target. Nevertheless, in a study to further assess the mechanisms of resistance to bedaquiline in M. tuberculosis, it was found that only 15 out of 53 resistant mutants had mutations in atpE. The other 38 strains lacked mutations in atpE or even in the F0 or F1 operons, which suggests that other mechanisms of resistance are still possible
3.2. 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. Delamanid was previously shown to have a very good in vitro and in vivo activity against drug-susceptible and drug-resistant M. tuberculosis, as well as good early bactericidal activity comparable to that of rifampicin. Delamanid has more recently shown its safety and efficacy in a clinical evaluation 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 α-mycolic acid.
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.
3.3. PA-824
PA-824 is a bicyclic derivative of nitroimidazole that showed specific activity against M. tuberculosis. This small-molecule compound showed a very good in vitro and in vivo activity in animal models and it also showed to be safe and well tolerated. PA-824 is currently undergoing further clinical evaluations
Structure of PA-824
PA-824 needs to be activated by a nitroreductase to exert its activity and it inhibits the synthesis of protein and cell wall lipids. The mechanism of resistance to PA-824 has been shown to be most commonly associated with loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or the dezaflavin cofactor F420. More recently, a nitroimidazo-oxazine-specific protein causing minor structural changes in the drug has also been identified.
3.4. SQ-109
Compound SQ-109 is a synthetic analogue of ethambutol that has shown in vitro and in vivo activity against drug-susceptible and drug-resistant M. tuberculosis. 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. SQ-109 is currently being evaluated in a phase II clinical trial.
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. Moreover, by producing spontaneously generated resistant mutants to SQ-109 analogs and performing whole-genome sequencing, mutations in the mmpL3 gene were
identified, suggesting MmpL3 as the target of SQ-109 and signaling MmpL3 as transporter of trehalose monomycolate.
3.5. Benzothiazinones
A new class of drug with antimycobacterial activity, 1,3-benzothiazin-4-one or benzothiazinone (BTZ), was recently described. The lead compound, 2-[2-S-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (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.
Structure of BTZ043
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. DprE1 and DprE2 were proposed as names for these two key enzymes. More recent studies have characterized more precisely the mechanism of action of BTZ043 by showing that the drug is activated in the bacteria through reduction of an essential nitro group to a nitroso derivative, which can react with a cysteine residue in DprE1. In studies with M. smegmatis, an alternative mechanism of resistance has been suggested. The overexpression of a nitroreductase NfnB led to the inactivation of the drug by reducing an essential nitro group to an amino group. 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.
Just recently a series of piperazine-containing BTZs has been reported. The lead compound PBTZ169 has improved activity, safety and efficacy in animal models and has shown in vitro synergy with bedaquiline signaling it as an attractive new candidate for further clinical development.
Fig: High burden TB countries
Drug resistance in tuberculosis
Definition of drug resistance
Drug resistance in mycobacteria is defined as a decrease in sensitivity to a sufficient degree to be reasonably certain that the strain concerned is different from a sample of wild strains of human type that have never come in contact with the drugs.
Guidelines for Management of Multidrug-Resistant TB
A single new drug should never be added to a failing regimen.
When initiating or revising therapy, always attempt to employ at least 3 previously unused drugs to which there is demonstrated in vitro susceptibility. One of these should be an injectable agent.
Sufficient numbers of oral drugs should be started at the onset of therapy to make sure there is an adequate regimen once the injectable agent is discontinued.
Do not limit the regimen to 4 agents if other previously unused drugs that are likely to be active are available.
Patients should receive either hospital-based or domiciliary directly observed therapy (DOT).
Intermittent therapy should not be used in treating TB caused by multidrug-resistant organisms, except perhaps for injectable agents after an initial period (usually 2 to 3 months) of daily therapy.
The use of drugs to which there is demonstrated in vitro resistance is not encouraged because there is little or no efficacy of these drugs (assuming the test results are accurate). In the case of low-level resistance to INH, high doses are sometimes given intermittently to complement the regimen.
Resistance to RIF is associated in most cases with cross-resistance to rifabutin and in all cases to rifapentine.
Cross-resistance between amikacin and kanamycin is nearly universal. There is emerging data that certain mutations may confer cross-resistance between amikacin, kanamycin and capreomycin .
Determination of resistance to PZA is technically problematic and thus, is not determined in all laboratories. However, resistance to PZA is uncommon in the absence of resistance to other first-line drugs. PZA monoresistance in vitro is essentially universal for Mycobacterium bovis isolates.
Who is at risk for getting MDR TB-Drug resistance is more common in people who:
Do not take their TB medicine regularly
Do not take all of their TB medicine as told by their doctor or nurse
Develop TB disease again, after having taken TB medicine in the past
Come from areas of the world where drug-resistant TB is common
Have spent time with someone known to have drug-resistant TB disease
Fig:Cross-resistance for anti-TB drug
Types of drug resistance
Drug resistance in TB may be broadly classified as primary or acquired. When drug resistance is demonstrated in a patient who has never received anti- TB treatment previously, it is termed primary resistance. Acquired resistance is that which occurs as a result of specific previous treatment. The level of primary resistance in the community is considered to reflect the efficacy of control measures in the past, while the level of acquired resistance is a measure of on-going TB control measures. However, the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Diseases (IUATLD), in the light of discussions in several international fora, have replaced the term primary resistance by the term “drug resistance among new cases” and acquired resistance by the term “drug resistance among previously treated cases .
Causes of drug resistance
The emergence of drug resistance in M.tuberculosis has been associated with a variety of management, health provider and patient-related factors. These include-
(i)deficient or deteriorating TB control programmes resulting in inadequate administration of effective treatment;
(ii) poor case holding, administration of sub-standard drugs, inadequate or irregular drug supply and lack of supervision;
(iii) ignorance of health care workers in epidemiology, treatment and control; (iv) improper prescription of regimens;
(v) interruption of chemotherapy due to side effects;
(vi) non-adherence of patients to the prescribed drug therapy;
(vii) availability of anti-TB drugs across the counter, without prescription;
(viii) massive bacillary load;
(ix) illiteracy and low socio-economic status of the patients;
(x) the epidemic of HIV infection;
(xi) laboratory delays in identification and susceptibility testing of M. tuberculosis isolates;
(xii) use of nonstandardized laboratory techniques, poor quality drug powders and lack of quality control measures; and
(xiii) use of anti-TB drugs for indications other than tuberculosis.
Mechanism and transmission of drug resistance
Drug resistance in M. tuberculosis occurs by random, single step, spontaneous mutation at a low but predictable frequency, in large bacterial populations. The probability of incidence of drug resistant mutants is 10-8 for rifampicin, while for isoniazid and some of the other commonly used drugs it is 10-6. Therefore, the probability for resistance to both isoniazid and rifampicin to develop is 10-14, which is much larger than the number of organisms present in a medium sized cavity in a patient with open pulmonary TB. Although for several years, drug resistant strains of M. tuberculosis were considered to be less infectious than the drug susceptible ones, recent studies have demonstrated that the drug resistant mutants are equally infectious and can cause severe disease in an individual exposed to
Fig: Drug resistance Mechanism of TB
Modern methods
BACTEC Radiometric methods:
Radiometric method have been developed for rapid drug-susceptibility testing of M. tuberculosis. In the BACTEC-460 (Becton- Dickinson) radiometric method, 7H12 medium containing palmitic acid labelled with radioactive carbon (14C-palmitic acid) is inoculated. As the mycobacteria metabolise these fatty acids, radioactive carbon dioxide (14CO2) is released which is measured as a marker of bacterial growth. The proportions method has been modified by incorporating the BACTEC technique in place of the conventional Lowenstein-Jensen culture. With this modification, sensitivity results will be available within 10 days. The mycobacteria growth indicator tube (MGIT) system (Becton-Dickinson) is a rapid, nonradioactive method for detection and susceptibility testing of M. tuberculosis. The MGIT system relies on an oxygen-sensitive fluorescent compound contained in a silicone plug at the bottom of the tube which contains the medium to detect mycobacterial growth. The medium is inoculated with a sample containing mycobacteria and with subsequent growth mycobacteria utilise the oxygen and the compound fluoresces. The fluorescence thus produced is detected by using a ultraviolet transilluminator. Studies carried out both with cultures and direct clinical samples showed comparable results with the BACTEC and the proportions method60. Restriction fragment length polymorphism (RFLP) patterns used to categorise isolates of M. tuberculosis and to compare them, has facilitated the elucidation of molecular epidemiology of TB61.
X-RAY METHOD:
In this technique, DNA is extracted from the cultured bacilli. A restriction endonuclease such as PvuII cleaves the element at base pair 461. Subsequent steps involve separation of DNA fragments by electrophoresis on an agarose gel, transfer of the DNA to a membrane (Southern blotting), followed by hybridisation and detection with a labelled DNA probe. The DNA from each mycobacterial isolate is depicted as a series of bands on an X-ray film to create the fingerprint. A banding pattern reflecting the number and position of copies of IS6110 (a 1361 base pair insertion sequence) within the chromosomes is obtained and this depends on the number of insertion sequences and the distance between them. As the DNA fingerprints of M. tuberculosis have been observed not to change during the development of drug resistance, RFLP analysis has also been used to track the spread of drugresistant strains61.
FLUORESCENCE TEST: Recently, Goulding et al62 determined the value of fluorescent amplifiedfragment length polymorphism (FAFLP) analysis for genetic analysis of M. tuberculosis and suggested that FAFLP can be used in conjunction with IS6110 RFLP typing to further understand the molecular epidemiology of M. tuberculosis. Ligase chain reaction (LCR) involves the use of an enzyme DNA ligase which functions to link two strands of DNA together to continue as a double strand. This can occur only when the ends are complementary and match exactly, and this method facilitates the detection of a mismatch of even one nucleotide.
PCR DETECTION TECHNIQUE:
Luciferase reporter assay is a novel reporter gene assay system for the rapid determination of drug resistance. It is based on the gene coding for luciferase, an enzyme identified as the light producing system of fireflies. In the presence of adenosine triphosphate (ATP), it interacts with luciferin and emits light. The luciferase gene is placed into a mycobacteriophage.
Once this mycobacteriophage attaches to M.tuberculosis, the phage DNA is injected into it and the viral genes are expressed. If M. tuberculosis is infected with luciferase reporter phage and these organisms are placed in contact with antituberculosis drugs, susceptibility can be tested by correlating the generation of light with conventional methods of testing. This technique has the potential to identify most strains within 48 hr. FASTPlaqueTB-RIF, a rapid bacteriophage-based test, to identify rifampicin susceptibility in clinical strains of M.tuberculosis after growth in the BACTEC-460 semi-automated liquid culture system has also shown potential to rapidly aid in the diagnosis of MDR-TB Polymerase chain reaction (PCR) based sequencing has often been employed to understand the genetic mechanisms of drug resistance in mycobacteria. This technique allows for detection of both previously recognised and unrecognised mutations. The PCR-based methods are not readily applicable for routine identification of drug resistance mutations because several sequencing reactions need to be performed for each isolate. However, for targets such as rpoB, where mutations associated with rifampicin resistance are concentrated in a very short segment of the gene, PCR-based sequencing is a useful technique
Monitoring response to treatment
Patients receiving treatment for MDR-TB should be closely followed up. Clinical (e.g., fever, cough, sputum production, weight gain), radiological (e.g., chest radiograph) , laboratory (erythrocyte sedimentation rate) and microbiological (e.g., sputum smear and culture) parameters should be frequently reviewed to assess the response to treatment. In addition, considerable attention must be focussed on monitoring the adverse drug reactions which often develop with the second-line antituberculosis drugs. A detailed description of these adverse drug reactions is beyond the scope of this review. Majority of the patients who respond to treatment begin to show favourable signs of improvement by about four to six weeks following initiation of treatment. Failure to show positive trend may alert the clinician to resort to other measures outlined below.
Suggested treatment for patients with MDR-TB
Resistance pattern Initial phase Continuation phase
Drugs Minimum duration Drugs Minimum duration
(months) (months)
Resistance to Aminoglycoside 3 Ofloxacin or 18-24
isoniazid and levofloxacin
rifampicin with Ofloxacin or
or without levofloxacin Ethambutol
resistance to Ethionamide
streptomycin Pyrazinamide
Ethambutol
Ethionamide
Resistance to Aminoglycoside 3 Ofloxacin or 18-24
isoniazid, levofloxacin,
rifampicin and Ofloxacin or
ethmbutol with levofloxacin Ethionamide
or without Cycloserine
resistance to Pyrazinamide
Streptomycin Ethionamide
Cycloserine
During the ontinuation phase antituberculosis drugs are administered for a period of at least 18 months after sputum conversion
MDR Treatment
Currently available second-line drugs used to treat MDR-TB are four to ten times more likely to fail than standard therapy for drug-susceptible tuberculosis. After the introduction of rifampicin, no worthwhile antituberculosis drug with new mechanism(s) of action has been developed in over thirty years. Moreover, no new drugs that might be effective in treatment of MDR-TB are currently undergoing clinical trials. It appears that effective new drugs for tuberculosis are at least a decade away. Recently, a series of compounds containing a nitroimidazopyran nucleus that possess antituberculosis activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antituberculosis drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static bacilli. Lead compound PA-824 showed potent bactericidal activity against multidrug-resistant M. tuberculosis and promising oral activity in animal infection models. It is being hoped that these nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis
Surgery:
Various surgical procedures performed for patients with MDR-TB have ranged from segmental resection to pleuro-pneumonectomy. Based on the experience reported in the literature about surgery for MDR-TB, it can be concluded that the operation can be performed with a low mortality (<3%). However, the complication rates are high with bronchopleural fistula (BPF) and empyema being the major complications. Sputum positivity at the time of surgery, previous chest irradiation, prior pulmonary resection and extensive lung destruction with polymicrobial parenchymal contamination are the major factors affecting morbidity and mortality. Over 90 per cent of the patients achieve sputumnegative status post-operatively.
Although operation related mortality is less than three per cent, deaths due to all causes occur in about 14 per cent patients. Even this compares favourably with over 22% mortality due to TB in a similar group of patients treated medically. More liberal use of muscle flaps to reinforce the bronchial stump and fill the residual space has helped significantly in reducing the rates of BPF, air leaks and residual space problems. These must be used in patients with positive sputum, when residual post-lobectomy space is anticipated, when BPF already exists pre-operatively or when extensive polymicrobial contamination is present. Thus, resectional surgery is currently recommended for MDR-TB patients whose prognosis with medical treatment is poor.
Indications for surgery in patients with MDR-TB include:
(i) persistence of culture-positive MDR-TB despite extended drug retreatment; and/or
(ii) extensive patterns of drug resistance that are associated with treatment failure or additional resistance; and/or
(iii) local cavitary, necrotic/destructive disease in a lobe or region of the lung that was amenable to resection without producing respiratory insufficiency and/or severe pulmonary hypertension. It should be performed after minimum of three months of intensive chemotherapeutic regimen, achieving sputum-negative status, if possible. The operative risks are acceptable and the long-term survival is much improved than that with continued medical treatment alone. However, for this to be achieved, the chemotherapeutic regimen needs to continue for prolonged periods after surgery also, probably for well over a year, otherwise recrudescence of the disease with poor survival is a real possibility.
Nutritional enhancement
Tuberculosis is a wasting disease. The degree of cachexia is most profound when MDR-TB occurs in patients with HIV-infection/AIDS131. While the mechanisms involved in weight loss are not well known, current evidence points to tumour necrosis factor to be the cytokine responsible for this phenomenon. TNF, in addition to inducing immunopathological effects such as tissue necrosis and fever, is also thought to induce the catabolic response132. Further, several second-line drugs used to treat MDR-TB such as PAS, fluoroquinolones cause significant anorexia, nausea, vomiting and diarrhoea interfering with food intake, further compromising the cachectic state. Therefore, nutritional support is a key factor in the care of patients with MDR-TB, especially those undergoing major lung surgery. Though definitive evidence is not yet available, it is generally believed that malnourished patients are at a greater risk of developing post-operative complications Nutritional assessment and regular monitoring of the nutritional state by a dietician are essential for the successful management of MDR-TB patients and should be an essential part of such programmes. When the routine measures are not able to improve the nutritional status and induce weight gain, nasogastric feeding may be employed to supplement the diet. When the patients are very sick and have severe nutritional deficit, feeding gastrostomy/ jejunostomy may have to be performed.
Immunotherapy
Ever since the early attempts by Robert Koch, several workers have attempted to modify the immune system of patients with tuberculosis to facilitate cure133,134. The measure employed in the earlier days included heliotherapy, dietary supplementation including milk and cod-liver oil. It is likely that these interventions acted through 1,25 (OH)2 D3, which is now recognised to have significant effects on Tlymphocyte and macrophage function. Agents with potential for immunotherapy are detailed below. Mycobacterium vaccae vaccination: Transiently favourable results were observed when immunoenhancement using M. vaccae vaccination was used to treat drug-resistant tuberculosis patients who failed chemotherapy. It was postulated that M.vaccae redirected the host’s cellular response from a Th-2 dominant to a Th-1 dominant pathway leading to less tissue destruction and more effective inhibition of mycobacterial replication. However, subsequent reports from randomised controlled trials have not confirmed these observations. Cytokine therapy: With further understanding of the molecular pathogenetic mechanisms of tuberculosis, several attempts have been made to try cytokines in the treatment of MDR-TB. Recent data, however, suggest that interferon may improve disease evolution in subjects affected with pulmonary tuberculosis caused by multidrug-resistant (IFN-) and sensitive (IFN-) strains. The mechanisms involved are not known, even though it has been reported that IFN-gammasecreting CD4+ Th cells may possess antituberculosis effects. In addition, IFN-a can induce IFN-secretion by CD4+ Th cells, and both types of IFN may stimulate macrophage activities
Other Agents
Several agents have evoked interest as potential adjunctive treatment for patients with MDR-TB. Though very little information is available regarding their clinical usefulness, they are described here considering their therapeutic potential. Thalidomide48,140 and pentoxifylline141,142 have been shown to combat the excessive effects from TNF-. These may be useful in limiting the wasting associated with MDR-TB. Other agents which have occasionally been considered include, levamisole143,144, transfer factor145, inhibitors of transforming growth factor-(TGF-)146, interleukin- 12 (IL-12)133, interferon-(IFN-) and imiquimod an oral agent which stimulates the production of IFN-147. Though there have been anecdotal reports of their usefulness, further studies are required to clarify their role.
Providing care for patients with drug-resistant TB is a team effort and a variety of staff and community members may be involved. It is very important that roles and responsibilities are clearly delineated and understood and that effective lines of communications are maintained. Duties and responsibilities may change throughout care as the patient’s needs change.
Conclusions::
In view of the results presented above, there is no clear evidence of an increase in the prevalence of initial drug resistance in India over the years. However, relatively high prevalence of acquired resistance has been reported from Gujarat, New Delhi, Raichur and North Arcot districts. When compared to the global prevalence of drug resistance, initial drug resistance is found to be marginally less while that of acquired resistance is much higher in India in specialized settings. The magnitude of drug resistance problem to a large extent is due to acquired resistance. The prevalence of MDR-TB also is found to be at a low level in most of the regions of India. However, these studies need to be repeated in different regions and in diverse settings to reconfirm this belief. TRC, Chennai and NTI, Bangalore have been working closely with central TB division and finalized recently a protocol for carrying out drug resistance surveillance (DRS) at the state level. The central TB division has been providing assistance to investigators in carrying out DRS at their respective places. As a follow-up, DRS protocols have been finalized for two large Indian states, namely, Gujarat and Maharashtha and the results are expected to be known in 2005. Similar efforts are underway for two other states, namely, Andhra Pradesh and Orissa with funds provided by the WHO Global Fund for AIDS, tuberculosis and malaria (GFATM).
A strong tuberculosis programme that can reduce the incidence of drug resistance in the community and particularly directly observed therapy (DOTS) which is cost effective, will prove to be effective in treatment completion and in turn prove to be effective against generation of resistant strains. Newer drugs for tuberculosis are unlikely to come up in the near future and hence the key to success remains in adequate case finding, prompt and correct diagnosis and effective treatment of infective patients including careful introduction of second-line drugs to which the patient is susceptible.
Apart from a strong tuberculosis control programme, there is also need for better and more rapid diagnostic methods, a continuous or periodic survey of drug resistance, with an emphasis on internal quality control and external quality assessment, which will provide information on the type of chemotherapy to be used for the treatment of patients and also serve as a useful parameter in the evaluation of current and past chemotherapy programmes.
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Bengal School of Technology(a college of pharmacy)
TUBERCULOSIS
INTRODUCTION
Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis.
Tuberculosis (TB) remains as 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. However, even more disturbing is the emergence of drug resistance. In 2012, there were an estimated 450,000 cases of multidrug resistant (MDR)-TB and 170,000 deaths were due to it.
MDR-TB is caused by strains of Mycobacterium tuberculosis that are resistant to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. Since 2006, it has been recognized the presence of even more resistant strains of M. tuberculosis labelled as extensively drug resistant (XDR)-TB. These strains in addition to being MDR are also resistant to any fluoroquinolone and to at least one of the injectable second-line drugs: kanamycin, capreomycin or amikacin. More recently, a more worrying situation has emerged with the description of M. tuberculosis strains that have been found resistant to all antibiotics that were available for testing, a situation labelled as totally drug resistant (TDR)-TB.
Early detection of all forms of drug resistance in TB is a key factor to reduce and contain the spread of these resistant strains. A better knowledge of the mechanisms of action of anti-TB drugs and the development of drug resistance will allow identifying new drug targets and better ways to detect drug resistance. The following sections will review the mode of action and resistance mechanisms of the main anti-TB drugs as well as new drugs recently described with anti-TB activity.
TB SPEARD METHOD
Drug-susceptible TB and drug-resistant TB are spread the same way. TB bacteria are put into the air when a person with TB disease of the lungs or throat coughs, sneezes, speaks, or sings. These bacteria can float in the air for several hours, depending on the environment. Persons who breathe in the air containing these TB bacteria can become infected.
TB is not spread by
Shaking someone’s hand
Sharing food or drink
Touching bed linens or toilet seats
Sharing toothbrushes
Kissing
Symptoms of TB disease::
The general symptoms of TB disease include feelings of sickness or weakness, weight loss, fever, and night sweats. The symptoms of TB disease of the lungs may also include coughing, chest pain, and coughing up blood. Symptoms of TB disease in other parts of the body depend on the area affected. If you have these symptoms, you should contact your doctor or local health department.
 |
| Add Fig: Sign and symptoms of TB |
1. First-Line Anti-TB Drugs
1.1. 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 constitutes the basis of the multidrug treatment regimen for TB. Rifampicin is active against growing and non-growing (slow metabolizing) bacilli. The mode of action of rifampicin in M. tuberculosis is by binding to the β-subunit of the RNA polymerase, inhibiting the elongation of messenger RNA. The majority of rifampicin-resistant clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the β-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 regionl of 81-bp spanning codons 507–533 of the rpoB gene. This region is also known as the rifampicin resistance-determining region. Mutations in codons 516, 526 and 531 are the most commonly associated mutations with rifampicin resistance in the majority of studies. Although less frequent, some reports have also noted the occurrence of mutations outside of the hot-spot region of rpoB. Cross-resistance with other rifamycins can occur. Mutations in some codons (e.g., 518 or 529) have been associated with low-level resistance to rifampicin but still susceptible to other rifamycins, such as rifabutin or rifalazil. This is important for TB patients that need to receive antiretroviral therapy since rifabutin is a less effective inducer of the cytochrome P450 CYP3A oxidative enzyme. On the other hand, monoresistance to rifampicin is quite rare and almost all rifampicin-resistant strains are also resistant to other drugs, especially to isoniazid. This is the reason why rifampicin resistance is considered as a surrogate marker for MDR-TB.
Recent genome sequencing studies have uncovered the acquisition of compensatory mutations in rpoA and rpoC, encoding the α and β' subunits of RNA polymerase, in rifampicin-resistant strains with mutations in rpoB. These compensatory mutations would be responsible for restoring the fitness of these strains in vivo and have also been associated with a higher transmissibility in some settings.
1.2. 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. Isoniazid acts by inhibiting the synthesis of mycolic acids through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase, encoded by inhA. 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. Among these, the most prevalent gene mutation has been identified as S315T in katG resulting in an isoniazid product deficient in forming the isoniazid-NAD adduct needed to exert its antimicrobial activity. This mutation has been consistently associated with high-level resistance (MIC > 1 μg/mL) to isoniazid and occurs more frequently in MDR strains. The second most common mutation occurs in the promoter region of inhA causing an overexpression of InhA or less frequently,
a mutation in its active site, which decreases its affinity for the isoniazid-NAD adduct. The most prevalent mutation found is at position −15C/T and is more commonly associated with low level resistance to isoniazid (MIC < 1 μg/mL). Mutations in inhA not only cause resistance to isoniazid but also to the structurally related drug ethionamide, which shares the same target. A recent study found that a mutation in the inhA regulatory region together with a mutation in the inhA coding region produced high-level isoniazid resistance and also cross-resistance to ethionamide.
One recent interesting finding showed that the 4R isomer of the isoniazid-NADP adduct causes inhibition of the dihydrofolate reductase (DfrA) in M. tuberculosis, suggesting that mutations in dfrA could possibly play a role in resistance to isoniazid. Moreover, an analysis of the proteome of isoniazid targets in M. tuberculosis identified 16 other proteins, in addition to InhA and DfrA, that were bound by these adducts with high affinity, which could signal other not yet clearly defined actions of isoniazid on the bacteria. Two recent studies, however, have failed to identify any mutation in dfrA associated with resistance to isoniazid.
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. 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. Moreover, overexpression of AhpC does not confer resistance to isoniazid.
Several studies have found single nucleotide polymorphisms in other genes in isoniazid resistant clinical isolates of M. tuberculosis, including kasA and the oxyR-ahpC and furA-katG intergenic regions. However, their direct role as a cause of isoniazid resistance has not been fully demonstrated. On the other hand, co-resistance to isoniazid and ethionamide has been clearly demonstrated to be caused by mutations in ndh in M. smegmatis and M. bovis BCG, by altering the NADH/NAD ratios inside the cell, leading to a competitive inhibition of the INH-NAD adduct. A recent study has also found that a silent mutation in mabA conferred isoniazid resistance through upregulation of inhA in M. tuberculosis
1.3. 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. 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.
The recognized mechanism of resistance to ethambutol has been linked to mutations in the gene embB with mutations at position embB306 as the most prevalent in most of the studies performed. Some studies, however, have also found mutations in embB306 in ethambutol susceptible isolates. 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. In fact, allelic exchange studies have shown that individual mutations causing certain amino acid substitutions produced ethambutol resistance, while other amino acid substitutions had little or no effect on ethambutol resistance. The same authors have more recently reported that mutations in the decaprenylphosphoryl-B-D-arabinose (DPA) biosynthetic and utilization pathway genes, Rv3806c and Rv3792, together with mutations in embB and embC accumulate, giving rise to a range of MICs of ethambutol depending on mutation type and number. These findings could have influence on the correct detection of ethambutol resistance by current molecular methods. Mutations in embB306 then, cause variable degrees of ethambutol resistance and are required but are not enough to cause high-level resistance to ethambutol. 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. Second-Line Anti-TB Drugs
2.1. 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. 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.
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. In M. tuberculosis, only type II topoisomerase (DNA gyrase) is present and, thus, is the only target of fluoroquinolone activity. Type II topoisomerase is a tetramer formed by two α and β subunits, coded by gyrA and gyrB, respectively, which catalyzes the supercoiling of DNA. 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. The most frequent mutations found are at position 90 and 94 of gyrA but mutations at position 74, 88 and 91 have also been reported. A recent systematic review of fluoroquinolone-resistance-associated gyrase mutations in M. tuberculosis has been published.
One interesting finding in M. tuberculosis is the presence of a natural polymorphism at position 95 in gyrA that is not related to fluoroquinolone resistance since it is also found in fluoroquinolone-susceptible strains. Another interesting finding has been the report that the simultaneous occurrence of mutations T80A and A90G in gyrA led to hypersusceptibility to several quinolones. This finding could point out that the problem of fluoroquinolone resistance in M. tuberculosis might be more complex than was thought initially.
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. Also, the involvement of efflux mechanisms has been suggested as a possible cause for fluoroquinolone resistance in M. tuberculosis.
2.2. Kanamycin, Capreomycin, Amikacin, Viomycin
These four antibiotics have the same mechanism of action by inhibiting the protein synthesis but, while kanamycin and amikacin are aminoglycosides, capreomycin and viomycin are cyclic peptide antibiotics. All four are second-line drugs used in the management of MDR-TB.
Kanamycin and amikacin inhibit protein synthesis by alteration at the level of 16S rRNA. The most common mutations found in kanamycin-resistant strains are at position 1400 and 1401 of the rrs gene, conferring high-level resistance to kanamycin and amikacin. However, mutations at position 1483 have also been reported. Full cross-resistance between kanamycin and amikacin is not complete, as previously thought. Some studies have shown variable levels and patterns of resistance suggesting that other mechanisms of resistance might be possible. In concordance with this, a low-level resistance to kanamycin has been associated with mutations in the promoter region of the eis gene, encoding an aminoglycoside acetyltransferase. Mutations at position −10 and −35 of the eis promoter led to an overexpression of the protein and low-level resistance to kanamycin but not to amikacin. These mutations were found in up to 80% of clinical isolates showing low-level resistance to kanamycin.
Capreomycin and viomycin, on the other hand, have a similar structure and bind at the same site in the ribosome, at the interface of the small and large subunits. They show full cross-resistance as reported in previous studies. Mutations in the tlyA gene have also been associated with resistance to capreomycin and viomycin. TlyA is an rRNA methyltransferase specific for 2'-O-methylation of ribose in rRNA. Mutations in tlyA determine the absence of methylation activity. Although some studies did not find this association, a recent meta-analysis, evaluating the association of genetic mutations and resistance to second-line drugs, has confirmed the presence of tlyA mutations in addition to mutations in rrs and eis.
2.3. 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. EthA is regulated by the transcriptional repressor EthR. Resistance to ethionamide occurs by mutations in etaA/ethA, ethR and also mutations in inhA, which cause resistance to both isoniazid and ethionamide. 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.
2.4. Para-Amino Salicylic Acid
Although it was one of the first anti-tuberculosis drugs used in the treatment of the disease, together with isoniazid and streptomycin, para-amino salicylic acid or PAS is now considered as a second-line drug part of the treatment regimen for MDR-TB. Until recently, its mechanism of action was not completely defined. It has been proposed that being an analog of para-amino benzoic acid, it must compete with it for dihydropteroate synthase, interfering in the process of folate synthesis. A study using transposon mutagenesis identified mutations in the thyA gene associated with resistance to PAS that were also present in clinical isolates resistant to PAS. A recent study has also identified various missense mutations in folC encoding dihydrofolate synthase that conferred resistance to PAS in laboratory isolates of M. tuberculosis. In a panel of 85 clinical MDR-TB isolates, mutations in folC were identified in five isolates resistant to PAS. Nevertheless, just less than 40% of PAS-resistant strains had mutations in thyA indicating that still other mechanisms of resistance to the drug might exist.
2.5. 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. 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. 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.
2.6. 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.
3. New Anti-TB Drugs
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.
3.1. 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. Bedaquiline was discovered after a high-throughput evaluation of thousands of compounds using Mycobacterium smegmatis in a whole-cell assay. 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. A ―black box‖ warning is, however, accompanying this authorization due to the reported unexplained deaths and QT interval prolongation. Recent reviews and evaluation of this new drug have been published. 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.
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. By sequencing the genome of these mutants and comparing to that of the susceptible strains, the only mutation found was in the atpE gene, which encodes the c part of the F0 subunit of the ATP synthase. This is a complex structure that generates the ATP needed by the mycobacterial cell for which bedaquiline has a favored specificity compared to mitochondrial ATP synthase.
Structure of Bedaquiline
The most prevalent mutation in the atpE gene found in bedaquiline resistant mutants is A63P but also I66M has been found. The latter introduces a modification that interferes the proper binding of bedaquiline to its target. Nevertheless, in a study to further assess the mechanisms of resistance to bedaquiline in M. tuberculosis, it was found that only 15 out of 53 resistant mutants had mutations in atpE. The other 38 strains lacked mutations in atpE or even in the F0 or F1 operons, which suggests that other mechanisms of resistance are still possible
3.2. 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. Delamanid was previously shown to have a very good in vitro and in vivo activity against drug-susceptible and drug-resistant M. tuberculosis, as well as good early bactericidal activity comparable to that of rifampicin. Delamanid has more recently shown its safety and efficacy in a clinical evaluation 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 α-mycolic acid.
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.
3.3. PA-824
PA-824 is a bicyclic derivative of nitroimidazole that showed specific activity against M. tuberculosis. This small-molecule compound showed a very good in vitro and in vivo activity in animal models and it also showed to be safe and well tolerated. PA-824 is currently undergoing further clinical evaluations
Structure of PA-824
PA-824 needs to be activated by a nitroreductase to exert its activity and it inhibits the synthesis of protein and cell wall lipids. The mechanism of resistance to PA-824 has been shown to be most commonly associated with loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or the dezaflavin cofactor F420. More recently, a nitroimidazo-oxazine-specific protein causing minor structural changes in the drug has also been identified.
3.4. SQ-109
Compound SQ-109 is a synthetic analogue of ethambutol that has shown in vitro and in vivo activity against drug-susceptible and drug-resistant M. tuberculosis. 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. SQ-109 is currently being evaluated in a phase II clinical trial.
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. Moreover, by producing spontaneously generated resistant mutants to SQ-109 analogs and performing whole-genome sequencing, mutations in the mmpL3 gene were
identified, suggesting MmpL3 as the target of SQ-109 and signaling MmpL3 as transporter of trehalose monomycolate.
3.5. Benzothiazinones
A new class of drug with antimycobacterial activity, 1,3-benzothiazin-4-one or benzothiazinone (BTZ), was recently described. The lead compound, 2-[2-S-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (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.
Structure of BTZ043
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. DprE1 and DprE2 were proposed as names for these two key enzymes. More recent studies have characterized more precisely the mechanism of action of BTZ043 by showing that the drug is activated in the bacteria through reduction of an essential nitro group to a nitroso derivative, which can react with a cysteine residue in DprE1. In studies with M. smegmatis, an alternative mechanism of resistance has been suggested. The overexpression of a nitroreductase NfnB led to the inactivation of the drug by reducing an essential nitro group to an amino group. 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.
Just recently a series of piperazine-containing BTZs has been reported. The lead compound PBTZ169 has improved activity, safety and efficacy in animal models and has shown in vitro synergy with bedaquiline signaling it as an attractive new candidate for further clinical development.
Fig: High burden TB countries
Drug resistance in tuberculosis
Definition of drug resistance
Drug resistance in mycobacteria is defined as a decrease in sensitivity to a sufficient degree to be reasonably certain that the strain concerned is different from a sample of wild strains of human type that have never come in contact with the drugs.
Guidelines for Management of Multidrug-Resistant TB
A single new drug should never be added to a failing regimen.
When initiating or revising therapy, always attempt to employ at least 3 previously unused drugs to which there is demonstrated in vitro susceptibility. One of these should be an injectable agent.
Sufficient numbers of oral drugs should be started at the onset of therapy to make sure there is an adequate regimen once the injectable agent is discontinued.
Do not limit the regimen to 4 agents if other previously unused drugs that are likely to be active are available.
Patients should receive either hospital-based or domiciliary directly observed therapy (DOT).
Intermittent therapy should not be used in treating TB caused by multidrug-resistant organisms, except perhaps for injectable agents after an initial period (usually 2 to 3 months) of daily therapy.
The use of drugs to which there is demonstrated in vitro resistance is not encouraged because there is little or no efficacy of these drugs (assuming the test results are accurate). In the case of low-level resistance to INH, high doses are sometimes given intermittently to complement the regimen.
Resistance to RIF is associated in most cases with cross-resistance to rifabutin and in all cases to rifapentine.
Cross-resistance between amikacin and kanamycin is nearly universal. There is emerging data that certain mutations may confer cross-resistance between amikacin, kanamycin and capreomycin .
Determination of resistance to PZA is technically problematic and thus, is not determined in all laboratories. However, resistance to PZA is uncommon in the absence of resistance to other first-line drugs. PZA monoresistance in vitro is essentially universal for Mycobacterium bovis isolates.
Who is at risk for getting MDR TB-Drug resistance is more common in people who:
Do not take their TB medicine regularly
Do not take all of their TB medicine as told by their doctor or nurse
Develop TB disease again, after having taken TB medicine in the past
Come from areas of the world where drug-resistant TB is common
Have spent time with someone known to have drug-resistant TB disease
Fig:Cross-resistance for anti-TB drug
Types of drug resistance
Drug resistance in TB may be broadly classified as primary or acquired. When drug resistance is demonstrated in a patient who has never received anti- TB treatment previously, it is termed primary resistance. Acquired resistance is that which occurs as a result of specific previous treatment. The level of primary resistance in the community is considered to reflect the efficacy of control measures in the past, while the level of acquired resistance is a measure of on-going TB control measures. However, the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Diseases (IUATLD), in the light of discussions in several international fora, have replaced the term primary resistance by the term “drug resistance among new cases” and acquired resistance by the term “drug resistance among previously treated cases .
Causes of drug resistance
The emergence of drug resistance in M.tuberculosis has been associated with a variety of management, health provider and patient-related factors. These include-
(i)deficient or deteriorating TB control programmes resulting in inadequate administration of effective treatment;
(ii) poor case holding, administration of sub-standard drugs, inadequate or irregular drug supply and lack of supervision;
(iii) ignorance of health care workers in epidemiology, treatment and control; (iv) improper prescription of regimens;
(v) interruption of chemotherapy due to side effects;
(vi) non-adherence of patients to the prescribed drug therapy;
(vii) availability of anti-TB drugs across the counter, without prescription;
(viii) massive bacillary load;
(ix) illiteracy and low socio-economic status of the patients;
(x) the epidemic of HIV infection;
(xi) laboratory delays in identification and susceptibility testing of M. tuberculosis isolates;
(xii) use of nonstandardized laboratory techniques, poor quality drug powders and lack of quality control measures; and
(xiii) use of anti-TB drugs for indications other than tuberculosis.
Mechanism and transmission of drug resistance
Drug resistance in M. tuberculosis occurs by random, single step, spontaneous mutation at a low but predictable frequency, in large bacterial populations. The probability of incidence of drug resistant mutants is 10-8 for rifampicin, while for isoniazid and some of the other commonly used drugs it is 10-6. Therefore, the probability for resistance to both isoniazid and rifampicin to develop is 10-14, which is much larger than the number of organisms present in a medium sized cavity in a patient with open pulmonary TB. Although for several years, drug resistant strains of M. tuberculosis were considered to be less infectious than the drug susceptible ones, recent studies have demonstrated that the drug resistant mutants are equally infectious and can cause severe disease in an individual exposed to
Fig: Drug resistance Mechanism of TB
Modern methods
BACTEC Radiometric methods:
Radiometric method have been developed for rapid drug-susceptibility testing of M. tuberculosis. In the BACTEC-460 (Becton- Dickinson) radiometric method, 7H12 medium containing palmitic acid labelled with radioactive carbon (14C-palmitic acid) is inoculated. As the mycobacteria metabolise these fatty acids, radioactive carbon dioxide (14CO2) is released which is measured as a marker of bacterial growth. The proportions method has been modified by incorporating the BACTEC technique in place of the conventional Lowenstein-Jensen culture. With this modification, sensitivity results will be available within 10 days. The mycobacteria growth indicator tube (MGIT) system (Becton-Dickinson) is a rapid, nonradioactive method for detection and susceptibility testing of M. tuberculosis. The MGIT system relies on an oxygen-sensitive fluorescent compound contained in a silicone plug at the bottom of the tube which contains the medium to detect mycobacterial growth. The medium is inoculated with a sample containing mycobacteria and with subsequent growth mycobacteria utilise the oxygen and the compound fluoresces. The fluorescence thus produced is detected by using a ultraviolet transilluminator. Studies carried out both with cultures and direct clinical samples showed comparable results with the BACTEC and the proportions method60. Restriction fragment length polymorphism (RFLP) patterns used to categorise isolates of M. tuberculosis and to compare them, has facilitated the elucidation of molecular epidemiology of TB61.
X-RAY METHOD:
In this technique, DNA is extracted from the cultured bacilli. A restriction endonuclease such as PvuII cleaves the element at base pair 461. Subsequent steps involve separation of DNA fragments by electrophoresis on an agarose gel, transfer of the DNA to a membrane (Southern blotting), followed by hybridisation and detection with a labelled DNA probe. The DNA from each mycobacterial isolate is depicted as a series of bands on an X-ray film to create the fingerprint. A banding pattern reflecting the number and position of copies of IS6110 (a 1361 base pair insertion sequence) within the chromosomes is obtained and this depends on the number of insertion sequences and the distance between them. As the DNA fingerprints of M. tuberculosis have been observed not to change during the development of drug resistance, RFLP analysis has also been used to track the spread of drugresistant strains61.
FLUORESCENCE TEST: Recently, Goulding et al62 determined the value of fluorescent amplifiedfragment length polymorphism (FAFLP) analysis for genetic analysis of M. tuberculosis and suggested that FAFLP can be used in conjunction with IS6110 RFLP typing to further understand the molecular epidemiology of M. tuberculosis. Ligase chain reaction (LCR) involves the use of an enzyme DNA ligase which functions to link two strands of DNA together to continue as a double strand. This can occur only when the ends are complementary and match exactly, and this method facilitates the detection of a mismatch of even one nucleotide.
PCR DETECTION TECHNIQUE:
Luciferase reporter assay is a novel reporter gene assay system for the rapid determination of drug resistance. It is based on the gene coding for luciferase, an enzyme identified as the light producing system of fireflies. In the presence of adenosine triphosphate (ATP), it interacts with luciferin and emits light. The luciferase gene is placed into a mycobacteriophage.
Once this mycobacteriophage attaches to M.tuberculosis, the phage DNA is injected into it and the viral genes are expressed. If M. tuberculosis is infected with luciferase reporter phage and these organisms are placed in contact with antituberculosis drugs, susceptibility can be tested by correlating the generation of light with conventional methods of testing. This technique has the potential to identify most strains within 48 hr. FASTPlaqueTB-RIF, a rapid bacteriophage-based test, to identify rifampicin susceptibility in clinical strains of M.tuberculosis after growth in the BACTEC-460 semi-automated liquid culture system has also shown potential to rapidly aid in the diagnosis of MDR-TB Polymerase chain reaction (PCR) based sequencing has often been employed to understand the genetic mechanisms of drug resistance in mycobacteria. This technique allows for detection of both previously recognised and unrecognised mutations. The PCR-based methods are not readily applicable for routine identification of drug resistance mutations because several sequencing reactions need to be performed for each isolate. However, for targets such as rpoB, where mutations associated with rifampicin resistance are concentrated in a very short segment of the gene, PCR-based sequencing is a useful technique
Monitoring response to treatment
Patients receiving treatment for MDR-TB should be closely followed up. Clinical (e.g., fever, cough, sputum production, weight gain), radiological (e.g., chest radiograph) , laboratory (erythrocyte sedimentation rate) and microbiological (e.g., sputum smear and culture) parameters should be frequently reviewed to assess the response to treatment. In addition, considerable attention must be focussed on monitoring the adverse drug reactions which often develop with the second-line antituberculosis drugs. A detailed description of these adverse drug reactions is beyond the scope of this review. Majority of the patients who respond to treatment begin to show favourable signs of improvement by about four to six weeks following initiation of treatment. Failure to show positive trend may alert the clinician to resort to other measures outlined below.
Suggested treatment for patients with MDR-TB
Resistance pattern Initial phase Continuation phase
Drugs Minimum duration Drugs Minimum duration
(months) (months)
Resistance to Aminoglycoside 3 Ofloxacin or 18-24
isoniazid and levofloxacin
rifampicin with Ofloxacin or
or without levofloxacin Ethambutol
resistance to Ethionamide
streptomycin Pyrazinamide
Ethambutol
Ethionamide
Resistance to Aminoglycoside 3 Ofloxacin or 18-24
isoniazid, levofloxacin,
rifampicin and Ofloxacin or
ethmbutol with levofloxacin Ethionamide
or without Cycloserine
resistance to Pyrazinamide
Streptomycin Ethionamide
Cycloserine
During the ontinuation phase antituberculosis drugs are administered for a period of at least 18 months after sputum conversion
MDR Treatment
Currently available second-line drugs used to treat MDR-TB are four to ten times more likely to fail than standard therapy for drug-susceptible tuberculosis. After the introduction of rifampicin, no worthwhile antituberculosis drug with new mechanism(s) of action has been developed in over thirty years. Moreover, no new drugs that might be effective in treatment of MDR-TB are currently undergoing clinical trials. It appears that effective new drugs for tuberculosis are at least a decade away. Recently, a series of compounds containing a nitroimidazopyran nucleus that possess antituberculosis activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antituberculosis drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static bacilli. Lead compound PA-824 showed potent bactericidal activity against multidrug-resistant M. tuberculosis and promising oral activity in animal infection models. It is being hoped that these nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis
Surgery:
Various surgical procedures performed for patients with MDR-TB have ranged from segmental resection to pleuro-pneumonectomy. Based on the experience reported in the literature about surgery for MDR-TB, it can be concluded that the operation can be performed with a low mortality (<3%). However, the complication rates are high with bronchopleural fistula (BPF) and empyema being the major complications. Sputum positivity at the time of surgery, previous chest irradiation, prior pulmonary resection and extensive lung destruction with polymicrobial parenchymal contamination are the major factors affecting morbidity and mortality. Over 90 per cent of the patients achieve sputumnegative status post-operatively.
Although operation related mortality is less than three per cent, deaths due to all causes occur in about 14 per cent patients. Even this compares favourably with over 22% mortality due to TB in a similar group of patients treated medically. More liberal use of muscle flaps to reinforce the bronchial stump and fill the residual space has helped significantly in reducing the rates of BPF, air leaks and residual space problems. These must be used in patients with positive sputum, when residual post-lobectomy space is anticipated, when BPF already exists pre-operatively or when extensive polymicrobial contamination is present. Thus, resectional surgery is currently recommended for MDR-TB patients whose prognosis with medical treatment is poor.
Indications for surgery in patients with MDR-TB include:
(i) persistence of culture-positive MDR-TB despite extended drug retreatment; and/or
(ii) extensive patterns of drug resistance that are associated with treatment failure or additional resistance; and/or
(iii) local cavitary, necrotic/destructive disease in a lobe or region of the lung that was amenable to resection without producing respiratory insufficiency and/or severe pulmonary hypertension. It should be performed after minimum of three months of intensive chemotherapeutic regimen, achieving sputum-negative status, if possible. The operative risks are acceptable and the long-term survival is much improved than that with continued medical treatment alone. However, for this to be achieved, the chemotherapeutic regimen needs to continue for prolonged periods after surgery also, probably for well over a year, otherwise recrudescence of the disease with poor survival is a real possibility.
Nutritional enhancement
Tuberculosis is a wasting disease. The degree of cachexia is most profound when MDR-TB occurs in patients with HIV-infection/AIDS131. While the mechanisms involved in weight loss are not well known, current evidence points to tumour necrosis factor to be the cytokine responsible for this phenomenon. TNF, in addition to inducing immunopathological effects such as tissue necrosis and fever, is also thought to induce the catabolic response132. Further, several second-line drugs used to treat MDR-TB such as PAS, fluoroquinolones cause significant anorexia, nausea, vomiting and diarrhoea interfering with food intake, further compromising the cachectic state. Therefore, nutritional support is a key factor in the care of patients with MDR-TB, especially those undergoing major lung surgery. Though definitive evidence is not yet available, it is generally believed that malnourished patients are at a greater risk of developing post-operative complications Nutritional assessment and regular monitoring of the nutritional state by a dietician are essential for the successful management of MDR-TB patients and should be an essential part of such programmes. When the routine measures are not able to improve the nutritional status and induce weight gain, nasogastric feeding may be employed to supplement the diet. When the patients are very sick and have severe nutritional deficit, feeding gastrostomy/ jejunostomy may have to be performed.
Immunotherapy
Ever since the early attempts by Robert Koch, several workers have attempted to modify the immune system of patients with tuberculosis to facilitate cure133,134. The measure employed in the earlier days included heliotherapy, dietary supplementation including milk and cod-liver oil. It is likely that these interventions acted through 1,25 (OH)2 D3, which is now recognised to have significant effects on Tlymphocyte and macrophage function. Agents with potential for immunotherapy are detailed below. Mycobacterium vaccae vaccination: Transiently favourable results were observed when immunoenhancement using M. vaccae vaccination was used to treat drug-resistant tuberculosis patients who failed chemotherapy. It was postulated that M.vaccae redirected the host’s cellular response from a Th-2 dominant to a Th-1 dominant pathway leading to less tissue destruction and more effective inhibition of mycobacterial replication. However, subsequent reports from randomised controlled trials have not confirmed these observations. Cytokine therapy: With further understanding of the molecular pathogenetic mechanisms of tuberculosis, several attempts have been made to try cytokines in the treatment of MDR-TB. Recent data, however, suggest that interferon may improve disease evolution in subjects affected with pulmonary tuberculosis caused by multidrug-resistant (IFN-) and sensitive (IFN-) strains. The mechanisms involved are not known, even though it has been reported that IFN-gammasecreting CD4+ Th cells may possess antituberculosis effects. In addition, IFN-a can induce IFN-secretion by CD4+ Th cells, and both types of IFN may stimulate macrophage activities
Other Agents
Several agents have evoked interest as potential adjunctive treatment for patients with MDR-TB. Though very little information is available regarding their clinical usefulness, they are described here considering their therapeutic potential. Thalidomide48,140 and pentoxifylline141,142 have been shown to combat the excessive effects from TNF-. These may be useful in limiting the wasting associated with MDR-TB. Other agents which have occasionally been considered include, levamisole143,144, transfer factor145, inhibitors of transforming growth factor-(TGF-)146, interleukin- 12 (IL-12)133, interferon-(IFN-) and imiquimod an oral agent which stimulates the production of IFN-147. Though there have been anecdotal reports of their usefulness, further studies are required to clarify their role.
Providing care for patients with drug-resistant TB is a team effort and a variety of staff and community members may be involved. It is very important that roles and responsibilities are clearly delineated and understood and that effective lines of communications are maintained. Duties and responsibilities may change throughout care as the patient’s needs change.
Conclusions::
In view of the results presented above, there is no clear evidence of an increase in the prevalence of initial drug resistance in India over the years. However, relatively high prevalence of acquired resistance has been reported from Gujarat, New Delhi, Raichur and North Arcot districts. When compared to the global prevalence of drug resistance, initial drug resistance is found to be marginally less while that of acquired resistance is much higher in India in specialized settings. The magnitude of drug resistance problem to a large extent is due to acquired resistance. The prevalence of MDR-TB also is found to be at a low level in most of the regions of India. However, these studies need to be repeated in different regions and in diverse settings to reconfirm this belief. TRC, Chennai and NTI, Bangalore have been working closely with central TB division and finalized recently a protocol for carrying out drug resistance surveillance (DRS) at the state level. The central TB division has been providing assistance to investigators in carrying out DRS at their respective places. As a follow-up, DRS protocols have been finalized for two large Indian states, namely, Gujarat and Maharashtha and the results are expected to be known in 2005. Similar efforts are underway for two other states, namely, Andhra Pradesh and Orissa with funds provided by the WHO Global Fund for AIDS, tuberculosis and malaria (GFATM).
A strong tuberculosis programme that can reduce the incidence of drug resistance in the community and particularly directly observed therapy (DOTS) which is cost effective, will prove to be effective in treatment completion and in turn prove to be effective against generation of resistant strains. Newer drugs for tuberculosis are unlikely to come up in the near future and hence the key to success remains in adequate case finding, prompt and correct diagnosis and effective treatment of infective patients including careful introduction of second-line drugs to which the patient is susceptible.
Apart from a strong tuberculosis control programme, there is also need for better and more rapid diagnostic methods, a continuous or periodic survey of drug resistance, with an emphasis on internal quality control and external quality assessment, which will provide information on the type of chemotherapy to be used for the treatment of patients and also serve as a useful parameter in the evaluation of current and past chemotherapy programmes.
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