IN VITRO STUDY OF A MULTI DRUG RESISTANT MYCOBACTERIA AND EFFECT OF HERBAL DRUG ON IT
1. PRESENTED BY
SMITA D RAJANI
Lecturer
LOKHAT SARVAJANIK TRUST, RAMPURA ,SURAT.
GUIDE
DR.PRATIBHA B.DESAI
DIRECTOR & HEAD
DEPARTMENT OF MICROBIOLOGY,
SHREE RAMKRISHNA INSTI. OF COMP. EDU. & APPL. SCIENCES, SURAT.
“IN VITRO STUDY OF A MULTI DRUG
RESISTANT MYCOBACTERIA AND
EFFECT OF HERBAL DRUG ON IT”
2. INTRODUCTION
Tuberculosis is an ancient disease & it remains the leading cause of death of human
being.
It is mainly caused by Mycobacterium tuberculosis
Mycobacteria causing human disease may be classified as follows:
Mycobacteria
1.CULTIVABLE MYCOBACTERIA:
a). Typical tubercle bacilli.
Human type M.tuberculosis.
Bovine type M.bovis.
Vole type M.microti.
Human type M.africanum.
4. Group 3 Non photochromogens
M.avium-intracellular complex
M. Paratuberculosis
M.terrae-triviale
M.Shimoidae
Group 4 Rapid growers
M.fortuitum/chelonae complex
M.thermoresistible
5. C). Saprophytic mycobacteria.
M.smegmatis
M.phlei
M.stercoris
M. thermo
2) NON CULTIVABLE MYCOBACTERIA:
M.leprae (cause leprosy in human)
6. It is estimated that yearly about 9 millions people in the world are attacked with TB, with
1.7 million deaths. [1]
Airborne disease that is transmitted only after prolonged exposure to someone with
active disease.
TB usually infects the lungs but other organs can be involved
8. EPIDEMIOLOGY
The average prevalence of tuberculosis in India is estimated to be 5.05 per
thousands.
Prevalence of smear positive cases 2.7 per thousand.
Annual incidence of smear positive cases at 84 per 1, 00,000 annually. [2]
The recent survey of WHO Says 98% of cases in developing countries with an
increase of ~3% annually, 10% in African countries.
80% of cases seen in 22 countries; about half in 5 countries: India, China,
Indonesia, Nigeria and Bangladesh.
In India every day more than 5,000 develop TB disease and more than 1,000
people die. [3]
9. JUSTIFICATION OF STUDY
Find out prevalence of Tuberculosis in South Gujarat region.
Identification and characterization of MDR & XDR TB cases.
Effectivity of Herbal Drug on MDR & XDR TB Strains.
10. HIV –TB CO-INFECTION
Tuberculosis is the most common life threatening opportunistic infection in patient
with HIV.
About 25 to 65 % patient with HIV/AIDS having tuberculosis of any organ. [6, 7, 8]
About 11.5 million people were co-infected with HIV and M.tuberculosis globally at the
end of 2000. [4, 9]
HIV reactivates latent TB by weakning the natural defences of infected persons
In India 5.1 million HIV infected people, about half of them are co infected with
M.tuberculosis [10]
Thus tuberculosis is a leading cause of morbidity and mortality in patient with
HIV/AIDS [4, 5]
11. TREATMENT & CONTROL
There are various ways to prevent tuberculosis.
BCG Vaccine
The use of BCG stimulates partial immunity
The effectiveness of BCG in preventing tuberculosis in adult is limited.
Tuberculosis can be treated effectively by a combination of anti-tubercular drugs.
First line drugs Isoniazid, Rifampicin, Streptomycin, Pyrazinamide and
Ethambutol.[11]
Anti-tubercular drugs
Second line drugs Amikacin,Levofloxacin, Gatifloxacin, PAS [Para amino Salicyclic
acid], Ethionamide, Kanamycin Cycloserine, Capreomycin and Ofloxacin.
Majority of these drugs are bacteriostatic than bactericidal, therefore causes of MDR
& XDR more.
Patient with tuberculosis, who fail to complete ‘Standard’ course or irregular intake
of drug are at increased risk for treatment failure and they may play a role in both the
emergence of drug resistant strains of M.tuberculosis and further spread of
tuberculosis in the society. [11]
12. MDR-TB & XDR TB
CAUSES OF DRUG RESISTANCE
Inadequate dosage or treatment with too few drugs.
Lack of compliance
Patients fail to take medication consistently for 6-12 months necessary for
cure.
Patients feel better after 3 or 4 weeks
Drugs have unpleasant side effects
13. WHAT IS MDR-TB & XDR TB
Drug resistant TB is widespread and found in all countries surveyed. It emerges as
a result of treatment mismanagement and is passed from person to person in the
same way as drug sensitive TB.
Multidrug resistant TB [MDR-TB] is a form of TB that does not respond to the
standard six month treatment using first line drugs [i.e. resistant to Isoniazid and
Rifampicin]. It can take two years to treat with drugs that are more toxic, and 100
times more expensive. If the drugs to treat MDR-TB are mismanaged. Further
resistance can occur.
Extensively drug resistant [XDR TB] is a form of TB caused by bacteria resistant to
all the most effective drugs [i.e. MDR-TB plus resistance to any fluoroquinolone and
any of the second line anti-TB injectable drugs: Amikacin, Kanamycin or
Capreomycin]. [18]
15. MDR-TB AND HIV
MDR-TB and XDR-TB are associated with an extremely high mortality, especially in
the HIV co infected person [14]
According to the 4th WHO report [2008] on anti tuberculosis drug resistance;
MDR-TB has been shown to be almost twice as common in TB patient living with HIV
as compared to TB patient without HIV [12]
To resolve the problem of TB the National Tuberculosis Programme [NTP] in India
was implemented in 1962
To improve & strengthen tuberculosis control activities, the Government of India
launched the Revise National Tuberculosis Control Programme [RNTCP] in 1997 and
cover almost the whole country with excellent results by the end of 2005. [3]
Directly Observed Treatment, Short Course Chemotherapy [DOTS] means that the
patient swallows short course anti TB drugs in the presence of health worker or
other trained individual. [3]
There are two phases in the treatment of tuberculosis,
-The intensive phase which is of 3 months,
-The continuation phases for 4 and 5 months.
16. According to the WHO, under this programme second line anti TB drugs are used
to control the prevalence of MDR-TB & thus DOTS-PLUS should be implemented in
selected areas with moderate to high levels of MDR-TB. [13]
There have been a number of reports on drug resistance in India including state level
Drug Resistance Surveillance [DRS] surveys conducted in Gujarat & Maharashtra.
Data from these studies have found MDR-TB levels of about 3%in new cases and
12%-17% in re-treatment cases.[20]
As per the DOTS Plus strategy the diagnosis of MDR-TB will be made at the
Intermediate Reference Laboratories (IRLs) accredited to perform culture and Drug
Sensitivity Testing (DST).
RNTCP has initiated the establishment of the laboratory network in a phased manner
in all the states across the country with support from the four National reference
Laboratories.
17. PLAN OF WORK
A. Sample collection
Sputum samples
2 Consecutive days Sputum samples
Normally sputum is non-sterile clinical sample and so it contains the organisms
present as a normal flora of the respiratory tract.
B. Staining
The screening for the bacilli of M.tuberculosis will be done by acid fast staining.
18. C. Culture
The sample which will show 8-10 bacilli per field will be further studies for the
sputum culture.
Liquefication and Decontamination
For the target isolation of M.tuberculosis, the samples are to be treated with acid and
alcohol.
Method used for Digestion & Decontamination of sputum sample will be N-Acetyl-l-
cysteine sodium hydroxide. [15]
Inoculation
To perform the sputum culture the sample will be inoculated into Loweinstein Jenson
[L-J] media along with Middle Brook 7H11 [16].
19. Incubation
All the inoculated media will be incubated for 3 to 4 weeks.
Isolated colonies will be studied in detail for their morphological, cultural and
biochemical characteristics.
Growth on L.J. Medium
Rough, Buff and Tough
Colony on L.J. Medium
22. E. Drug Susceptibility Testing
The drug susceptibility testing will be performed by Two method:
Direct Susceptibility Testing [ Gold Standard Method ] [16]
MTT. [3-4,5 dimethyithiazol – 2 yl-2-5-diphenyl tetrazolium bromide] tube
method. [ Rapid Method ] [17]
List of drug to be tested
Primary Drug
Isoniazid,
Rifampicin,
Streptomycin,
Ethambutol,
Pyrazinamide
23. Those organisms showing resistance again more than two drugs will be considered
as multi drug resistant
F. Result
Those organisms showing resistance again MDR TB plus resistant to any
fluoroquinolone and any of the second line anti-TB injectable drugs will be considered
as a XDR-TB [Extensively Drug Resistant TB]
Secondary Drug
Amikacin,
Kanamycin,
Ethionamide,
Levofloxacin,
Gatifloxacin,
Ofloxacin,
PAS
24. G. Effect of herbal extracts
MDR and XDR strains are selected for the further studies.
Herbal Compound
Garlic [Allium sativum]
Study will be done by MIC [Minimum Inhibition Concentration] method. [21,22]
25. Study will determine the prevalence of MDR and XDR in south Gujarat region.
EXPECTED RESULT
Attempts will also be done to suggest the Herbal drugs and its concentration to combat
the spread of drug resistant tubercle bacilli .
26. 1. S.Singh: Scaling up Anti Mycobacterial drug susceptibility services in India. It is
high time, Indian Journal of Medical Microbio. [2008] 26 [3] : 209-11
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