Rajiv gandhi university of health sciences, bangalore, karnataka



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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

Annexure II

Proforma for registration of subject for dissertation






1.


Name of the candidate


Dr. Jayashree.R.


2.



Name of the institution


St. Johns Medical College, Bangalore



3.



Course of study and subject


Post graduation in Microbiology MD (3yrs)



4.


Date of admission to the course


18th March 2009



5. TITLE OF THE TOPIC:

Use of liquid culture (Automated) technique for recovery and identification of Mycobacteria and Drug Susceptibility Testing of Mycobacterim tuberculosis Isolated from Clinical Specimens.


6. BRIEF RESUME OF THE STUDY
6.1 NEED FOR STUDY:

Bacteriological confirmation plays a key role in the diagnosis of mycobacterial infections. Smear microscopy is an important tool for Mycobacterial diagnosis, but has to be supplemented by culture. The conventional Lowenstein – Jensen medium used in many of the developing countries, requires 2-8 weeks for detecting growth and atleast 2- 4 weeks for the drug susceptibility test results to become available. Moreover these techniques have a limited role in the diagnosis of extrapulmonary and paucibacillary forms 1.

The use of the liquid culture methods has improved the recovery and has shortened the time needed to detect Mycobacterial growth. At present the use of BACTEC 460 TB System, which is a radiometric liquid culture method improves isolation and can detect Mycobacterial growth in 12 – 15 days of incubation. Drug susceptibility test results are available within 5 – 6 days.2,3 .Though considered ‘gold standard’ in many countries worldwide , this system requires attention to the safe use and disposal of radioisotope reagents being used.3,4. . Thus there is a need for an alternative method for rapid culture and drug susceptibility testing. Rapid and accurate detection of Mycobacterial infections is of importance for the proper management of the patients .
The Mycobacteria Growth Indicator Tube (MGIT) introduced recently by Becton Dickinson Diagnostic Instruments Systems, is a nonradiometric, liquid culture medium for the rapid identification of Mycobacteria and also for drug susceptibility testing .The reliability of this system for rapid detection and susceptibility testing has been evaluated in several studies and it is reported that this system requires 7 – 12 days for the detection of Mycobacterial growth and 5 days for the

results of drug susceptibility testing to become available 1,3,4,5,6,7,8 .However in India there is very little data regarding the use of Mycobacteria Growth Indicator Tube for drug susceptibility testing.


This study therefore aims to compare the accuracy of Mycobacteria Growth Indicator Tube with the conventionally used Lowenstein–Jensen culture method in the detection and identification of Mycobacteria and drug susceptibility testing for the Mycobactium tuberculosis isolates from the various clinical specimens .

6.2 REVIEW OF LITERATURE :

Tuberculosis is one of the oldest diseases of mankind .In 1882 , Sir Robert Koch isolated the tubercle bacilli and proved it to be the cause of the disease. In the early days , the Industrial Revolutions with its crowded living conditions favored its spread through out the world. At present ,the resurgence of tuberculosis in conjunction with HIV and the rapid spread of multidrug resistant strains is a major public health concern across the world , especially in the developing countries. Thus the need for rapid and accurate diagnosis is of paramount importance.9,4



Smear microscopy , first developed in the 1880s still remains the most widely used method in many countries .Though simple and cost effective the sensitivity is low ( 20 - 80%)10,9 and it has to be followed by culture methods.
Culture of mycobacteria is the gold standard for both diagnosis and drug sensitivity. Conventional methods using Lowenstein – Jensen or 7H11 medium are cheap and simple .However due to the time constraint and frequent negative results in paucibacillary and extrapulmonary forms with these methods ,the faster and highly sensitive radiometric liquid culture methods like Bactec 460 are now preferred 11. Due to the difficulty of working with radioactive materials used in these techniques , alternative growth detection systems like MGIT960, Bactec9000MB ,ESPII culture system, MB Redox and MB/BacT Alert 3D have been introduced .These methods are reported to have a sensitivity of 95% 10.
The Mycobacteria Growth Indicator tube contains modified MiddleBrooke 7H9 broth base with 0.25% glycerol and 10% carbon dioxide environment. A fluorescence quenching based oxygen sensor is embedded at the bottom of the tube. The level of fluorescence is detected by viewing with a 365nm UV light from a transilluminator .The fluorescence corresponds to the amount of oxygen consumed by organisms in the tube and is proportional to the number of bacteria present. Various studies done showed the sensitivity to be 95% 1,3,4,5,6,7,8,10.
The newer immunodiagnostic tests, like the antigen based detection test which give results in an hour(98% sensitive)(96% specific) are at present still at the research stage 11.
Phage-based systems require limited culture facilities and give results within 2 days. WHO studies show that they are technically complex, are labour intensive and have high rates of contamination11.
Amplification tests are the latest diagnostic methods .The conventional and real time PCR can be used directly on clinical specimens. These tests give results in a few hours. Studies done with these tests report a low sensitivity especially with extrapulmonary specimens and suggest their use in combination with microscopy and culture. At present these tests have limited use in a routine mycobacterial laboratory.9,11
Various non culture techniques like T- cell based tests, DNA Microarrays, Breath Detection methods are still at the research stage 11.
The newer identification tests introduced over the last two decades are considered to be excellent diagnostic methods for the rapid and accurate diagnosis of mycobacterial infections . However in the developing countries objective evaluation of these tests is required. Moreover the requirement of proper infrastructure , and the low specificity under field conditions make most of these techniques inappropriate for resource poor settings 9,11.


6.3 OBJECTIVES OF THE STUDY:

This study aims to compare Mycobacteria Growth Indicator Tube with conventional Lowenstein – Jensen medium for

1.) The detection and identification of mycobacteria from clinical specimens.

2.) Biochemical identification of mycobacterial isolates.

3.) Drug susceptibility testing for the Mycobacterial tuberculosis isolates.
7. MATERIALS AND METHODS:

7.1 SOURCE OF DATA:

The study will be conducted at the Mycobacterial Laboratory ,Department of Microbiology, St. John’s Medical College Hospital, Bangalore.

In order to estimate 95% sensitivity with a 3% precision and 95% confidence interval the sample size is 200.
7.2 METHODS OF COLLECTION OF DATA:

Method of Sample collection

Samples will be collected in sterile, disposal, leak proof containers without any preservatives


Inclusion criteria

Irrespective of the age and gender , consecutive pulmonary and extrapulmonary specimens(expect blood) of patients suspected of tuberculosis, from the departments of Chest Medicine, Medicine, Surgery, Pediatrics and various other departments will be included in the study.

Following are the pulmonary and extrapulmonary specimens to be included in the study.



Pulmonary samples.

Extrapulmonary samples.

Sputum

Biopsy specimens

.

Bronchoalveolar lavage



Aspirated fliuds

-

Urine and Fecal samples

-

Pus

The period of study –September 2009- January 2011.
Exclusion criteria

1.samples less than 2ml.

2.pulmonary samples consisting mainly of saliva

3.swabs


4.samples preserved in formalin
PROCEDURE

a. The samples received at the mycobacteriology laboratory will be processed

immediately. However if there is delay of more than 24hours the samples will be

stored at 40C in the refrigerator.


b. Processing of the samples will be as follows :-
S

ample



sss

AFB smear N-Acetyl L- Cysteine

Zeihl-Neelson staining Sodium Hydroxide Decontamination( for samples

Contaminated with bacterial flora)





  1. Centrifuge

  2. AFB smear

  3. Inoculate culture media


MGIT LJ Medium

1.inoculate with enrichment supplement 1.inoculate 0.2ml of sediment in 2 LJ

.and antibiotic mixture(PANTA) slants

then


2.incubate at 370Cfor 8 weeks

2. inoculate 0.5ml of the sediment 3.examine twice weekly x 2weeks

3.incubate at 370C for 8 weeks then weekly x 6 weeks

4.examine daily for growth

5.if fluorescence detected considered 4.appearance of colonies on the

growth positive surface considered positive

6.AFB smear and inoculation 5.AFB smear

of blood agar plate 6.further identification by

7.further identfication by standard biochemical tests

PNBA assay 8

a.growth rate

b.pigment production

c.niacin test

d.nitrate reduction test

e.PNBA medium

The sterile samples will be directly inoculated into the culture medium without prior decontamination

Drug Susceptibility Testing


a. The strains isolated and identified as Mycobacterium tuberculosis will be subjected to drug susceptibility testing by the following two methods – MGIT and Proportion methods
. The following drugs will be used

First line drugs Second line drugs
1.Streptomycin 1.Kanamycin

2.Isoniazid 2. Cycloserine

3.Rifampicin 3. Ofloxacin

4.Ethambutol 4. Ethionamide



Procedure

1. MGIT Antimycobacterial susceptibility testing (3,4,5,7,12)

Principle


Based on the principle used for primary isolation. Susceptibility is indicated

by absence of fluorescence in the drug containing tubes .Resistance is

indicated by the increase in fluorescence when compared to the growth

control tubes.



Preparation of antimicrobial solution

MGIT drug susceptibility kit contains lyophilised antibiotics in the powder form .The drugs

are rehydrated with distilled water as per manufacturer’s instructions. The first-line drugs

are aliquoted and stored at – 20 0 C till the day of use . However the second- line drugs

have to be prepared on the day of use.14

Inoculum preparation for MGIT drug susceptibility testing

1. each positive culture from MGIT is used within 2 days of showing fluorescence.

2. this is vortexed for 1-2 mins and left undisturbed for 20 mins.

3. the supernatant is transferred to another sterile tube and allowed to settle down for

15mins.

4 .the supernatant from this tube is transferred to a third sterile tube and adjusted with 7H9

broth to equal the density of 0.5 Mcfarland standard and 1ml is diluted with 4ml sterile

saline(1:5).



MGIT antimycobacterial susceptibility testing (first line drugs)

5 tubes are required for each isolate – 4 tubes with the drugs , 1 drug free growth control tube .

The drugs are reconstituted as per manufacturer’s recommendations.


  1. prepare inoculum for growth control tube with 0.1ml of test inoculum in 10ml sterile saline

  2. add 0.5ml of the growth control inoculum to drug free MGIT tube

  3. incubate tubes at 370C and examine daily for fluorescence

Results

  1. results will be interpreted once growth control tube shows fluorescence

  2. resistance is indicated if drug containing tube fluoresces within 2 days of the time that growth control tube fluoresces.

  3. The final low or critical drug concentrations achieved will be as follows C1. In addition, for Streptomycin, Isoniazid and Ethambutol stock solutions at higher concentrations (C2) is prepared by dissolving the high concentration lyophilized drug in 2ml of sterile distilled water, 3,16 if found resistant at C1

  4. Drugs Low Concentration (C1 ) High Concentration (C2)

First line drugs ug/ml ug/ml

1.Streptomycin 1.0 4.0

2.Isoniazid 0.1 0.4

3.Rifampicin 1 -

4. Ethambutol 5 7.5
Second line drugs

1. Kanamycin 5

2. Cycloserine 50

3. Ofloxacin 2

4. Ethionamide 5

Drug susceptibility testing from growth on LJ slants by Proportion Method(2,13)


Principle

Compares colony counts on drug containing and drug free media. Based on the 1% proportion method to determine the susceptibilities to the final drug concentrations.



Inoculum Preparation 14,16

1. The proportion method is done from a primary M.tuberculosis isolate.

2. 20 -30 colonies is scraped from a LJ slant and transferred to sterile screw capped bottle

7ml Bijoux bottle containing 0.2ml of sterile water and sterile glass beads(3mm in

diameter )

3 this mixture is vortexed for 30 secs and if needed the turbidity is adjusted with addition of

sterile water to equal 1 Mcfarland standard.

4. the suspension is left to settle down for 30minutes


Procedure 14

A .Serial dilutions of 10-1 mg/ml to 10-4 mg/ml of the standard suspension are prepared by diluting sequentially 1.0ml of the above mentioned standard suspension (1mg/ml) in tubes containing 9ml of sterile distilled water

B .Dilutions of 10-2mg/ml and 10-4mg/ml are inoculated on drug free L -J and drug containing Lowenstein– Jensen medium.

C .For each isolate 2 bacterial dilutions of the suspension are made.

D.For each dilution of the suspension 2 control tubes are inoculated and 1 tube is inoculated for each drug concentration prepared.

Therefore for each drug used 16 tubes have to be inoculated in total.

The volume of the inoculum is 0.2ml

E. After inoculation the tubes are incubated at 370C in a slanted position with screw caps slightly loosened to allow for the evaporation of the inoculum. After 24 – 48 hours screw caps are tightened and the tubes are further incubated.


The reading of the results consists of

1. the counting of colonies grown on the different slants

2. the calculation of the proportion of resistant bacilli by comparing counts on drug free

and drug containing L-J medium.

3. greater than 1% of colonies with antibiotics are considered resistant when compared to drug free media.
h. the final drug concentrations of the drugs are as follows 14,15,17

Drugs Concentrations( ug/ml)

First line drugs 1% critical proportion 10% critical proportion

1.Streptomycin 4 10

2. Isoniazid 0.2 1

3. Rifampicin 40 -

4. Ethambutol 5 2

Second line drugs

1.Kanamycin 30 20

2.Cycloserine 40 30

3. Ofloxacin 2 -

4.Ethionamide 20 10

The reading of the results is carried out on days 28 and 40 after inoculation.

H37RV is used as the reference control.


Results


The results of the tests will be analyzed using 2 sample t-test and a p value of < 0.05 will be considered statistically significant.

7.3 Does the study requires any investigation or intervention to be conducted on patients or other humans or animals. If so please describe briefly.

No
7.4 Has ethical clearance been obtained from your institution

awaiting IEBR clearance.

8. REFERENCES;
1.Hilleman D, Richter E, and Rusch-Gerdes S. Use of the BACTEC Mycobacteria Growth Indicator Tube 960 Automated System for Recovery of Mycobacteria from 9,558 Extrapulmonary specimens, Including Urine samples. J Clin Microbiol. 2006 Nov.; 44(11): 4014 –4017.
2. Anargyros P, Astill DS J., and .Lim LSI. Comparison of Improved BACTEC and Lowenstein-Jensen Media for Culture of Mycobacteria from Clinical Specimens.J Clin Microbiol 1990 Jun.;28(6) :1288-1291.
3.Ardito F, Posteraro B, Sanguinetti M, Zanetti S and Fadda G. Evaluation of BACTEC Mycobacteria Growth Indicator Tube(MGIT 960)automated System for Drug Susceptibility Testing of Mycobacterium tuberculosis J Clin Microbiol 2001 Dec;39(12):4440-4444.
4.Palaci M,Ueki SYM, Nakamura S, D Silva Tellis MA,Curcio M,and Silva MAE. Evaluation of Mycobacteria Growth Indicator Tube for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis Isolates from Respiratory Specimens. J Clin Microbiol 1996 Mar;34(3):762-764.
5.Rusch-Gerdes S, Pfyffer GE, Casal M, Chadwic M, and Siddiqi S. Multicenter Laboratory Validation of the Bactec MGIT 960 Technique for Testing Susceptibilities of Mycobacterium tuberculosis to Classical Second – Line Drugs and Newer Antimicrobials.J Clin Microbiol 2006 Mar;44(3): 688-692.

6. Badak F Z, Kiska D L,Setterquist S, Hartley C,O’Connell MA and Hopfer RL.Comparison of Mycobacteria Growth Indicator Tube with BACTEC 460 for Detection and Recovery of Mycobacteria From Clinical Specimens.J Clin Microbiol 1996 Sept;34(9):2236-2239.


7.Kruuner A,Yates DM and Drobniewski FA. Evaluation of MGIT 960-Based Antimicrobial Testing and Determination of Critical Concentrations of First- and Second –Line Antimicrobial Drugs with Drug – Resistant Clinical Strains of Mycobacterium tuberculosis. J Clin Microbiol 2006 Mar; 44(3): 811-818.

8. Rodrigues C, Shenai S, Sadani M, Sukhadia N, Jani M, Ajban K, Sodha A, Mehta A. Evaluation of the BACTEC MGIT 960TB System for the Recovery and Identification of Mycobacterium tuberculosis Complex in a high volume tertiary Care Centre.Ind J Med Microbiol 2009 July;27(3):217-221.


9.Global Report on Tuberculosis World Health Organisation .2007. Geneva, Switerzland.
10.Betty A. Forbes,Daniel F.Sahm,Alice S.Weissfeld ,Bailey and Scott’s Diagnostic Microbiology .12ed.Mosby Inc;478-509.
11.Perkins MD. New diagnostic tools for tuberculosis. Int J Tuber and Lung dis.2000;4(12): S182 –S188.
12.Kosket KA and Katila ML. Susceptibility Testing with the Manual Growth Indicator Tube(MGIT) and the MGIT 960 System Provides Rapid and Reliable Verification of Multidrug–Resistant Tuberculosis. J Clin Microbiol 2003 Mar.; 41(3):1235-1239.
13. Walters SB and Hanna BA. Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin by Mycobacterium Growth Indicator Tube Method. J Clin Microb 1996 June;34(6):1565-1567
14. Guidelines for Drug Susceptibility Testing for Second-Line Anti-tuberculosis Drugs for DOTS – PLUS. World Health Organization 2001,Geneva,Switzerland.
15. G. Canetti, S.Froman, J Grosset. Haudroy et al. Mycobacteria;Laboratory methods for Testing Drug sensitivity and Resistence. Bull.World Health Organization: 1963;(29):565- 578.
16. Bergmann J S, MA; Fish G,Woods G L.Evaluation of the BBL MGIT(Mycobacterial Growth Indicator Tube) AST SIRE System for Antimycobacterial Susceptibility Testing of Mycobacterium tuberculosis to 4 Primary Antituberculosis Drugs. Arch Pathol Lab Med .2000 Jan: 124 ;82-86.
17. Lee C N and Heifets L B. Determination of Minimal Inhibitory Concentrations of Antituberculosis drugs by Radiometric and Conventional Methods.1987 AM Rev Respir Dis;136:349-352
9. Signature of the candidate:
10. Remarks of the guide:
11. Name and designation of:

(In block letters)



11.1 Guide : DR. BAIJAYANTI MISHRA M.D.

ASSOCIATE PROFESSOR,

DEPT OF MICROBIOLOGY,

ST. JOHNS MEDICAL COLLEGE,



BANGALORE- 560034

11.2 Signature :
11.3 Co guide (if any) :
11.4 Signature :
11.5 Head of the Department : DR. MURALIDHARAN, M.D.
11.6 Signature:
12.
12.1 Remarks of the Chairman and Principal: DR. PREM PAIS M.D.

12.2 Signature:

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