GUIDED BY: DESAI P. B
DIRECTOR & HEAD
DEPARTMENT OF MICROBIOLOGY,
SHREE RAMKRISHNA INSTI. OF COMP. EDU. & APPL. SCIENCES, SURAT.
“IN VITRO STUDY ON MULTI DRUG
RESISTANT Mycobacterium tuberculosis”
RAJANI S. D.
LOKHAT SARVAJANIK TRUST, RAMPURA ,SURAT.
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:
a). Typical tubercle bacilli.
Human type M.tuberculosis.
Bovine type M.bovis.
Vole type M.microti.
Human type M.africanum.
North Zone 95
East Zone 65
West Zone 80**
South Zone 50
The average prevalence of tuberculosis in India is estimated to be 5.05
Prevalence of smear positive cause 2.7 per thousand and average.
Annual incidence of smear positive cases at 84 per 1, 00,000 annually.
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. 
It is estimated that yearly about 9 millions people in the world are
attacked with TB, with 1.7 million deaths. 
AIM OF STUDY
To find out prevalence of Tuberculosis in South Gujarat region.
Identification and characterization of MDR in TB cases.
To find out MDR Pattern.
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].
The present study was conducted during Jan., 2009 to Nov., 2009.
We have analyzed all samples viz., sputum, pus, urine, fluid are
collected and diagnosed by ZNCF stain and result noted as RNTCP
All samples were digested and decontaminated by N-Acetyl-l-cysteine
sodium hydroxide 
and cultured in the L.J medium.
Isolated colonies were studied in detail for their morphological,
cultural and biochemical characteristics.
All positive culture samples, which were further tested for drug
susceptibility towards first line drugs (rifampicin, isoniazid,
streptomycin, ethambutol, pyrazinamide) and second line drugs
(amikacin, kanamycin, ethionamide, sparfloxacin, levofloxacin,
ciprofloxacin,) by MIC method. [15,16]
RESULT & DISCUSSION
TABLE: 1 SAMPLE WISE DISTRIBUTION:
SR.NO SAMPLE NO.OF
01 SPUTUM 43
02 PUS 30
03 URINE 09
04 FLUID 08
Table 1 shows that among total 90
samples, majority were of sputum
SPUTUM PUS URINE FLUID
TABLE 2: DISTRIBUTION OF A.F.B. GRADATION BY RNTCP METHOD
SR.NO GRADATION NO.OF SAMPLES
01 1+ 15
02 2+ 15
03 3+ 6
04 SCANTY 0
05 TOTAL 36
Table 2 shows among total
36 samples direct
samples, majority sample
were 1+ and 2+ . None of
them was scanty.
TABLE 3: DISTRIBUTION BASED ON BIOCHEMICAL TEST
GROUP NO.OF CULTURE BIOCHEMICAL TEST
PNB CATALASE NIACIN
M.tuberculosis 32 - - +
MOTT 01 + + -
Above table 3 suggested that total 33 strains, 32 strains were
M.tuberculosis and 01 strain was MOTT.
SR.NO GROUP NO.OF
01 MDR 13
02 SENSITIVE TO ALL DRUGS 02
03 MONO DRUG & OTHER THAN MDR 18
TABLE 4: DISTRIBUTION BASED ON SENSITIVITY PATTEREN:
Table 4 shows that total 33 strains, 13 strains were MDR, 02 strains were sensitive to
all drugs and 18 strains were mono drug & other than MDR.
MDR ,Means in my study resistant to Rifampicin and Isoniazid with or without any
other drug. Mono drug resistant means resistant to any one drug .
TABLE 5: MDR PATTERN [13 STRAINS]
DRUGS NO.OF STRAINS
MDR Pattern suggested equal
no of HR & HESR resistance
i.e. 4 while no of HSR
resistance was 3 and HER
resistance was 2.
HR HSR HER HESR
In present study,4 types of specimens were included. From 90 specimens, only
47.77% (n=43) were sputum samples whereas 52.23% (n=47) were non respiratory
tract category. Among them 40% were smear positive and 36.66% were culture
In our study, total 33 culture positive among them 96.96% were M.tuberculosis
and 03.04 % were atypical Mycobacteria similar results were reported by
Navarro V, Guix J, Roig P, Borras R. (10)
We found, out of the total 33 positive culture, 39.39 % (n=13) were MDR strain
found that is higher than compare with Surveillance of drug-resistant tuberculosis in
the state of Gujarat, India in 2009. (11)
Our results are similar to study conducted in Japan. At 2008 (19)
Our study shows that among isolated strains 96.96%were
M.tuberculosis & 03.04% were atypical Mycobacteria.
The study revealed that direct smear positivity was 40. % and culture
positivity was 36.36%, though tuberculosis has been clinically confirmed
by physician but detail diagnosis was required to confirm the TB cases.
Drug susceptibility testing was carried out with 33 culture positive
isolates, 02 (6.06 %) strains were sensitive to all four drugs i.e.,
Rifampicin, Isoniazid, Ethambutol, Streptomycin. 13 (
were multi drug resistance, 18 (54.55%) strains were mono resistance
and multi resist other than MDR.
So, Prevalence of MDR-TB infection was more in South Gujarat
region and is necessary to find out the cause of spread of MDR in
1. S.Singh: Scaling up Anti Mycobacterial drug susceptibility services in India. It is
high time, Indian Journal of Medical Microbio.  26  : 209-11
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 –
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 :
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:
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  Global Tuberculosis Control: Surveillance, Planning, Financing, WHO,
13.Centres for Disease Control & Prevention [CDC]  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
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. 27: 1079-1086
15.Clinical Microbiology Procedures Handbook Editor in Chief, Henry D.Isenberg,
Vol-I, American society for microbiology/WashingtonD.C.