Tuberculosis is an ancient disease & it remains the leading cause of death of human being.

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.

3. b). Atypical mycobacteria (Runyon’s classification)
Group 1 Photochromogen
M. kansasii
M.marinum
M. simiae
M. asiaticum
Group 2 Scotochromogen
M.Scrofulsceum
M. Szulgai
M. Xenopi
M.Gordonae
M. Celatum

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

7. TRANSMISSION
Transmission of Tuberculosis
•The spread of M. tuberculosis involves a 3-step process:
Transmission of bacteria,
Establishment of infection, and
Progression to disease.

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]

14. [19]

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

20. D. Identification Tests
PNB TEST [paranitrobenzoicacid], Niacin production test,

21. Catalase quantitative test,
OTHER BIOCHEMICAL TEST:
Nacl tolerance test,
Nitrate reduction,
Pyrazinamide [15]

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
REFERENCES
2. Epidemiology of Tuberculosis: Current status in India. A.K.Chakraborty.India J
med Res. 120, oct-2004. p.p.248-276
3. Module for Laboratory Technicians.oct-2005 Central TB Division – New Delhi –
110011
4. Harries A, Maher D, Graham S.TB/HIV: A Clinical Manual. 2nd Edition.
Geneva : WHO : 2004 WHO/HTM/TB/2004 : 329
5. Raviglione MC, Narain JP, Kochi A. HIV Associated Tuberculosis in developing
Countries : Clinical Features, Diagnosis & Treatment, Bull WHO 1992;70: 515-25
6. Narain jp, Tripathy SP, Pontali E. TB & HIV Infection, Tuberculosis : Epidemiology
& Control, New Delhi : WHO Regional office for South Ease Asia : 2002
7. Gothi D, Joshi JM, Clinical & Laboratory observations or tuberculosis at a
Mumbai [India] clinic post grad Med j 2004;80:97-100

27. 9. Corbett EL,Watt CJ, Walker N, Maher D, Williams BG, Raviglione MC, eta. The
growing burden of tuberculosis; global trends & interactions with the HIV
epidemic. Arch Intern. Med.2003;163 : 1009-21
10.Khatri GR, Friden TR, Controlling tuberculosis in India. N Engl .J med 2002; 347:
1420-5.
11.Gandhi N R, Moll A, Sturm A W , Pawinski R, Govender T, Laloo U, Redeller K,
Andrews J,Firdlnnd G,2006 Extensively drug resistant tuberculosis as a cause of
death in patient co infected with HIV and TB in a rural area in a south Africa.
Lancet 368: 1575/1580
12.WHO [2006] Global Tuberculosis Control: Surveillance, Planning, Financing, WHO,
Geneva, Switzerland.
13.Centres for Disease Control & Prevention [CDC] [2006] Emergence of
M.tuberculosis with extensive resistance to 2nd line drugs world wide, 2000-
2004. Morb,wkly Rep 55:301-305
8. Sharma SK, Kadhiravan T, Banga A, Goyal T, Bhatia I, Saha PK, Spectrum of
clinical disease in a cohort of 135 hospitalized HIV infected patients from north
India, BMC Infec.Dis.2004;4:52

28. 16.Antimicrobials in laboratory medicine edited by Ashok Rattan, BI,Churchill
Livingstone 1st Edition [156-166]
17.U Raut, P Narang, DK Mendiratta, R Narang P.Narang, V.Deotale: Evaluation of
Rapid MTT tube Method for detection of Drug Susceptibility of M.tuberculosis to
Rifampicin & Isoniazid.
18.Paramshivam CN :An overview on drug resistant tuberculosis in India, India J
tuber.1998;45:73-81
19.Anargyros, P., D. S. J. Astill, and I. S. Sim. 1990. Comparison of improved
BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical
specimens. J. Clin. Microbiol. 28:1288–1291
14.R.Vijdea, M.Stegger, A.sosnovskaja, A.B.Anderson V.Thomson, D.Bang, MDR-TB
– Rapid detection of resistance of RIF & high or low levels of INH in clinical
specimens & isolates: EURJ Clinical Micro. Infec.Dis. [2008]27: 1079-1086
15.Clinical Microbiology Procedures Handbook Editor in Chief, Henry D.Isenberg, Vol-
I, American society for microbiology/WashingtonD.C.

29. 20. RNTCP Launches Cat IV (DOTS Plus) treatment for Multi-Drug Resistant TB
www.tbcindia.com[2008]
21. In vitro Antibacterial activity of Garlic Against isolates of Acinetobacter spp.,
Journal of Biological Sciences 7 [5]: 819-822,2007 ISSN 1727-3048
22. Delaha,E.C. AND V.F. Garaqusi, 1985. Inhibition of Mycobacteria by Garlic extract
[Allium sativum]. Antimicrob agents Chemother.,27:485-486.