Multi Drug Resistant Mycobacterium Tuber Culosis "Molecular – Perspective"

Tuberculosis

Is an infectious disease caused by bacteria belonging to the mycobacterium tuberculosis complex

It affect the lung

In 1/3^rd cases :- Other organs involved

Etid - pathogenesis :-

1. M. tuberculosis is a rod shaped, AFB
2. Transmission : Droplet nuclei
3. Crowding and poor ventilation
4. Endogenous factor

Transmission through respiratory route

Bacilli enter into the lung

Bacilli ingested by macrophage and transported to regional lymph node

Bacilli reaches to the blood steam

Dissemination ocuure

(primary infection)

Tuberculosis develops within wks after primary infection.

Problem of tuberculosis

TB has reemerged as one of leading cause of death

Global incidence of TB

• Estimated 8.8 million new cases every yrs. In world
• Nearly 3 million deaths annualy
• 52,000 death / wk
• > 7000 death each day

Factors :-

1. Productive age 15 – 49 yr.
2. HIV infection

Incidence and mortality is high in developing country (India)

Clinical presentation :- Patient presents with

• Fever
• Bight sweats, wt. Loss
• Anorexia, weakness

Majority of Cases

• Cough with expectoration
• hemoptysis

Clinical manifestation :-

Classifies as :-

• Pulmonary TB
• Extra – pulmonary TB

Pulmonary TB :-

• Primary disease
• Secondary (Post primary)

Primary pulmonary TB :- it result from initial infection with tubercle baulci.

• localized to middle and lower lung zones.
• Seen in children

Secondary TB :- Result from endogenous reactivation of latent infection

• Localized to upper lobe
• Occur in adult

Extra pulmonary TB :- Common sites are lymph node, pleura, U.T. bones, ioint

Pharmacotherapy

I^st Line Drugs :-

1. INH
2. Streptomycin
3. Rifamicin
4. Pyrazinamide
5. Ethambutol
6. Thiacetazone

II^nd Line Drugs :-

1. Ethionamide
2. Cycloserine
3. Kanamycin
4. Amikacin
5. Caprecmycin

Short course chemotherapy :- For 6 – 9 month duration

Goals :- Who regimens for tuberculosis

It consists of following category

Category I :-

1. New SP. + VE pulmonary TB
2. SP. – VE + extensive parenchymal involvement
3. New cases of extra pul TB

Category II :- SP. + VE failure, relapse

Category III :- New SP. – VE + parenchymal + less extra pul.

Category IV :- Chronic cases MDR

Category – wise treatment regimens

R + H – ZE + S + Cipro

INH :- Primary drug for chemotherapy

Bacteriostatic and bacteriocidal

Mech :- Inhibit the biosynthesis of mycolic acid

Kinetic :- orally absorb

• Peak plasma conc^N 3 – 5 ug / ml – 1 – 2 hrs.
• INH acetylation shows genetic variation fast and slow acetylators

AD effect :- Skin Rashes

• Iaundice peripheral neuritis

Interaction :- Phenytion – Hepatic biotransfor mation by INH

Dose :- 300 mgs daily

Rifampioin :- Semisynthitic derivative of rifamycin B

• It is bacteriostatic.

Mech :- Inhibit DNA dependent RNA polymerase of mycobacteria forming stable drug enzyme complex leading to suppression of chain formation in RNA synthesis.

Kinetics :- Absorb orally

• Peak plasm concⁿ :- 2 + hrs about 7 g / ml
• T½ - 1.5 – 5 Hrs.
• 80 % bound to plasma protein
• Desacetyl rifampicin – active metabolite

AD effect :- Nausea, Hepatitis, Flu – like syndrom

Interaction :- Drug is inducer of hepatic microsonal enzymes t½ fo many compound

1. Yarapamil, OC
2. Propranolol, sleroids

Streptomycin :- Bctericidal durg

Toxic in nature least used

Pyrazinamide :- Synthetic pyrazine analog of nicotinamide bactericidal in nature.

Kinetic :-

• Absorb from G.I.T.
• PPC – 45 mg / ml at 2 hrs. – after dose of 2 gms.

AD effect :- Hepatotoncity

Hyperuricemia

Dose :- 2 gms daily

Ethambutol :- Bacteriostatic drug

• Inhibit protein synthesis in cell
• It prevent from development of resistance

Dose – 250 mgs OD

S.E. :- Postural hypotension

Olfactory disturbance, blurred vision

Thiaceazone :- Static drug low efficacy

• hepatotoxic

II^nd Line drugs

1. Ethionamide – Structurally similar to INH

• Has short duration of action

S.E. – hepatitis intolerence

Dose – 1 gm / day.

1. Cycloserin :- Antibiotic, analouge of D. alanine

• It inhibit bacterial cell wall synthesis
• CNS toxicity is high

Dose – 100 mg / day

1. Other aminoglycoside :- More toxic

• Kanamycin
• Amikacin
• Capreomycin

Increased death rate is due to emergence of new strains of M. Tuberculosis resistant to some or all current anti – TB drugs.

50 – 80 death raies associated with MDR – TB.

"MDR State" in mycobactesiology refers to simultaneous resistance to at least rif and INH with or without resistance to other drugs.

Etiopathogenesis of MDR

1. Improper prescriptiions
2. Improper Dosage
3. Non compliance of patient
4. Associated HIV infection
5. The drug fail to reach the target site
6. Inactivation of drug.

The discovery of drug resistant was made by middle book and yegian 1947

1. Streptomycine resistance – Prolonged exposure
2. Pas – Bacteriostatic agent.

Less resistant then streptomycine

3. INH resistant demonstrated in trials
4. Rostogi reported a case of MDR at institute pasteur serial isolates of myco. TB were coltured from patient who fail to responds chemo therapy.

1^st Isolates – INH, RIF

2^nd Isolates – ENB

3^rd Isolates – PZA

Last Isolates – Cipro, sparfloxacin.

5. – Genetic and molecular analysis – Resistance is acquired by the bacilli

• Alteration of drug target through mutation

Resistance is high for less effective drugs

• Thibacetazone
• Etbiconamide
• Capreomycine, cycloserin

Intermedate :- INH, SM, EMB

• PAS, Kanamycin

Lowest :- Rifampicin

Tupes of drug resistants :-

Primary drug Resistant :- Is that encountered in patient who have never HID any chemotherapy arising either from mixed population of both sensitive and resistant organisms.

Secondary :- Result from inadequate drug therapy

Acquired :- patient who were previously infected with sensitive organsisms but later developed resistance due to faulty treatment.

Initial :- Patient reporting for the first time with drug resistance strain.

Epidemiology :-

1. Prevelence rate of MDR :- 48 % in developing countries.
2. In Nagpur – Study was done 1997 shows that – 22.11 % MDR TB

Chart showing resistant and sensitive strain

Mechanism of Resistance :-

In the past, few years, genetic and molecular insight have unravelled the mech. of drug reststance.

But now with the development of various molecular strategies to rapidly detect MDR – TB.

Resistance to INH

Resistance of – INH ethionamide :-

Inha locus is responsible for co-resistance to INH and ethionamide

• Inha locus composed of two open reading frames (ORFs) disignated as orf1 and inha seperated by 21 – bp noncoding region Inha an – ACP reductase
• Mutation in the putative prometer region hyper-expression of inha, result in INH resistance.
• Inha mutation occure along with katg mutation in relation to lipid metabglism of INH resistant isqlates which play the tole of the respective loci in the mechanisms of action of INH and acquisition of drug resistance.

Resistance to rif

Mechanisms of action

RNA Polymerase Composed of and different subint

• Rpr gene in E-coli demonstrated that rif interacted with the B subunit of RNA polymerase so mutation occure in rpob locus, leading to defective binding of the drug and resistance occure.

Mutation – rpob lacus acts as surrogate market for KDR – TB

• Shows single A.A. substitution in bp core region of rpob gens.
• Change in codon se – 531 and his 52 account for more than 70 % mutation with rif. Resistance
• RIF pemeability and mutation in alternate subunits of RNA polymerase – resistance.

Mutation can be detected by following techniges

• PCR
• Finger printing
• Line probe hubidization

Resistance to EMB :-

1. Indentificaition of arabinosm transferasc as the primary traget for EMB resistance.
2. Locus consists of 3 complete ORF's EMB EmbA, EmB

• EMB interects with Emb CAB proteins coded by Embs, Emba, Embb leads to inactivation of arabino – galaction synteesis.
• EmbB lacks A potenential ribosomes binding site so it coupled to Emb A – Leads to resistance.
• Lyperexpression of the EMB – CAB protein.

Resistance to FQ's

Mechanisms of action :- AQ's binds with greater affinity to single stranded DNA. Leads to inhibit DNA regulation leads to effective transcription block and cellular death.

• Mutation in 9 yr A and 9yrB genes leads to resistance
• Amind acids substitution at position 88, 94
• Mutation in 9yrA – Change in permeibicity (cell wall)

Resistance to SM :-

Mechanisms of Action :_ SM disarupts the decoding of amino – acyl RNA and thus inhibit mRNA translation.

1. Resistance :- Due to acetylation of the drug by dmino glycoside modifyni enzymes.
2. Mutation –

1. S12 ribosonal protein – rpsl gene
2. 12S rRNA encoded by rrs.

1. There occure two distinct classes of mutation

1. Point mutation in S12 ribasomal protein encoded by rpsl gene
2. Mutation in the rrs encoding the 16S rRNA.

Point mutation in the rpsl gene result in single amino – acid substitution that affect the higher under structure of 16S rRNA and resistance occure

• Mution affect lysing residues of position 43, 88 leads to substitution with either arginine at 88 or arginine and theonine at 43.

2. Mutation in the rrs lacus have been mapped to two region

1. 530 loop and 915 region of amino acid.

3. Role of cell wall permeability.

Resistance to PZA :-

1. Point mutation in the pncA gene of PZA – resistant MTB strains – leads to resistance
2. Substitution of Cys 138 – Ser.

Asp 63 – His.

Deletion of G. nucleotide at position 162, 288 resulted in defective pzoase.

Gene loci involved in conferring drug resistance in mycobacterium tuberculosis.

Treatment of Nor

Basic principles to treat MDR – TB as follows

1. Drugs to be given in adequate doses and for adequate duration.
2. Drugs used must not have been given in past.
3. First line drugs are preferred. As they are more effective and less toxic.
4. INH must be used in all regimens.

Potential regimens for patients with MDR – TB

b :- If resistance to amikacin, kanamycin, SM, then capreomycin is good alternative. Given daily new anti – TB drugs.

c :- Drugs like clofazimine, amoxicillin – clavulanate, clarithro xycin, azithromycin, rifabutin are useful but unproved utility.

2. Dot programene (Directly observed treatment)

• The NTCP helps patient getting best medicine available.

Under DOT, A patient is required to ingest the medicine in presence of physician or trained person.

• SSC is used under DOT programme.

MDR infection inHIV + VE patient

Initial reposts shows that MDR TB and Aids has high and rapid mortality.

Mortality – 85 % after diagnosis.

• DOT
• ANTI – HIV therapy
• Life time fallow up

Prevention of MDR – TB

Two main approahes

1. Indentification and treatment of MDR
2. Indentification of person with tubercular infection and their prophylatic treatment – the aim is to prevent 5- 10 % risk of diseases development.

Chemoprophyaxi of MDR

INH is the acsepted drug

If resistance to INH, RIF then – PZA + EMB PZA with or without ciprofloxin

Summary :-

• MDR strains of mycobacterium tuberculosis seriously threaten tuberculosis control and prevention efforts.
• Molecular studies of the mechnism of action of anti – tubercular drus have elucidated the genetic basis of drug resistance in myco – TB.
• Drug resistance of M. Tuberculosis is attributed primarly to the accumulation of mutation in the drug target genes, these mutation leads to an altered target or to change in titration of drug.
• Development of specific mechanism – Based inhibitors and techniques to rapidly detect multi – drug resistance will require further studies addressing the drug target interaction.

References :-

1. Satoskar
2. Thripathi
3. Good – man – gilman's
4. The Indian iournal of tuberculosis

Jan 99, 46, 1

5. Livette S. Johnson, Kent A. Sepkawitz
6. Published paper of Dr. V. A. Rajadnya

N. J. medical College, Pune.

7. Drugs 94, 48 (5)
8. Harrissons
9. Unpublished paper from dept. of TB and chest diseases, GMC, Nagpur.