PRESENTED BY SMITA D RAJANI

Lecturer LOKHAT SARVAJANIK TRUST, RAMPURA ,SURAT.

GUIDEDR.PRATIBHA B.DESAI

DIRECTOR & HEADDEPARTMENT 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”

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:Mycobacteria1.CULTIVABLE MYCOBACTERIA:

a). Typical tubercle bacilli.

Human type M.tuberculosis.

Bovine type M.bovis.

Vole type M.microti.

Human type M.africanum.

b). Atypical mycobacteria (Runyon’s classification)

Group 1 Photochromogen

M. kansasiiM.marinumM. simiaeM. asiaticum

Group 2 Scotochromogen

M.ScrofulsceumM. SzulgaiM. XenopiM.GordonaeM. Celatum

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

C). Saprophytic mycobacteria.

M.smegmatis

M.phlei

M.stercoris

M. thermo

2) NON CULTIVABLE MYCOBACTERIA:

M.leprae (cause leprosy in human)

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

TRANSMISSION

Transmission of Tuberculosis

•The spread of M. tuberculosis involves a 3-step process:

Transmission of bacteria,Establishment of infection, andProgression to disease.

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]

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.

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 weaking 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]

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]

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

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]

[19]

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.

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 mannerin all the states across the country with support from the four National referenceLaboratories.

PLAN OF WORK

A. Sample collectionSputum 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.

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].

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

D. Identification Tests

PNB TEST [paranitrobenzoicacid], Niacin production test,

Catalase quantitative test,

OTHER BIOCHEMICAL TEST:

Nacl tolerance test, Nitrate reduction, Pyrazinamide [15]

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 testedPrimary Drug Isoniazid,

Rifampicin,

Streptomycin,

Ethambutol,

Pyrazinamide

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

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]

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 .

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

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

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.

20. RNTCP Launches Cat IV (DOTS Plus) treatment for Multi-Drug Resistant TBwww.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.