RAJIV GANDHI UNIVERSITY FOR HEALTH SCIENCES, KARNATAKA, BANGALORE-41

ANNEXURE-2

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / Name of the Candidate / SUSHIL KUMAR CHAUDHARY
2 / Address / SUSHIL KUMAR CHAUDHARY,
M.Sc, M.L.T (Microbiology)
St. John’s Medical College,
Bangalore-34
3 / Course of Study and Subject / M.Sc, MLT (Microbiology)
4 / Date of Admission to the Course / September 2008
5 / Subject / Clinical Microbiology and Immunology

TITLE:

“Comparison of hypertonic saline-sodium hydroxide (HS-SH)decontamination method with modified Petroff’s method for concentrating sputum sample”

NEED FOR STUDY:

The social economic burden of tuberculosis has been the subject of much study and major efforts are under way to try to achieve its control.

It is estimated that 2 billion people world wide are infected with Mycobacterium tuberculosis and at least 10% of these people (200 Million) will develop active tuberculosis in their lifetime1.For establishing a definitive diagnosis of pulmonary tuberculosis and identification of tubercle bacilli in the sputum either direct microscopy or culture is essential2.

Concentrated sputum techniques are much better, than direct sputum microscopy. Modified Petroff’s method is one of the popular method. Unfortunately, this technique is too cumbersome in field conditions2.

The spinning time low (15 minute) and the Relative Centrifugal Force (RCF ) high (4000rpm) have sedimentation effect of 95%. But the RCF is low (i.e. 3000rpm) in modified Petroff’s method. And 4% of NaOH method of Petroff’s kills 60%- 70% of the mycobacteria3.

While in hypertonic saline-sodium hydroxide (HS-SH) method only 1.33% NaOH (final concentration w/v in 3 ml) is used for decontamination1. RCF for HS-SH method is high (4000rpm) and the sensitivity for acid fast bacilli smear is about 71.4% which is more than the N-acetyl-L-cysteine-Sodium hydroxide (66.7%) decontamination and concentration method.

Hence there is a need to use hypertonic sodium hydroxidedecontamination method for sputum concentrating technique in pulmonary tuberculosis.

REVIEW OF LITERATURE:

Tuberculosis is one of the major health problems, particularly in developing countries. Presently about 1/3rd of the world’s population is infected with Mycobacterium tuberculosis.

Currently about 10 million new cases of tuberculosis every year with 3 million deaths world wide4.

In India each year about 2 million people develop active disease and upto half a million die3.

India is estimate to have 3.5 million HIV patients and about 1.8 million of these are co-infected with tuberculosis5.

Diagnostic methods available to detect tuberculosis are6:-

  1. By direct approach:

-Microscopy

-Culture

-Biochemical tests

-Nuclear techniques

  1. By indirect approach:

Detection of Antibodies for diagnosis of tuberculosis

-TB STAT PAK

-Enzyme immunoassay (EIA) for detection of antimycobacterial super oxide dismutase antibody.

-Insta Test TB

In developing countries diagnosis of acid fast bacilli is performed by microscopic examination of Ziehl-Neelsen (Z-N) stained sputum smear, because it is simple, inexpensive & provides rapid results. But this technique has a low sensitivity 22-43% for single smear7.

And upto 60% under optimal conditions8, when compare with cultures so that culture remains as gold standard to diagnose tuberculosis.

The threshold for detection of acid fast bacilli in sputum samples under optimal condition is between 104-105 bacilli/ml 9, which can be obtained by decontamination & concentration techniques.

Various decontamination methods are listed below3:-

N-Acetyl-L-Cysteine- sodium Hydroxide (NALC- NaOH) Method

Zephiran- Trisodim Phosphate (Z-TSP) method

Modified Petroff’’s Sodium Hydroxide (NaOH) Method

Oxalic Acid Method

Sulphuric Acid Method

Cetylpyridinium chloride- sodium chloride (CPC) Method

Modified Petroff’s method is a simple, inexpensive method for decontamination & concentration of sputum and also control the growth of contaminants. Here specimen exposure time must be strictly followed to prevent over kill of tubercle bacilli. The initial kill (due to 4% NaOH) is independent of additional contributory factors, such as heat build up in the centrifuge and centrifugal efficiency.

In hypertonic saline-NaOH method, hypertonic saline is used for mucolysis and sodium hydroxide for decontamination10. Literature survey has shown HS-SH technique substantially increased the sensitivity of AFB smears compared with the routine direct smear for AFB and correlated well with the NALC-NaOH decontamination method for sputum culture in suspected Tuberculosis patients.1,11

Hypertonic saline has been used successfully for the symptomatic treatment of cystic fibrosis11.

AIM ANDOBJECTIVES:

1)Sputum sample will be decontaminated and concentrated by using 2

procedures.

a) Hypertonic Saline – Sodium hydroxide decontamination method.

b) Modified Petroff’s method.

2) The result of the microcopy and culture will be analyzed and compared by using student’s t test.

MATERIAL AND METHODS:

1)Source of Data:

A comparative study of Hypertonic Saline-NaOH decontamination method and modified petroff’s method for concentrating sputum will be done. A total of 50 sputum samples collected in the Mycobacteriology laboratory from suspected tuberculosis cases will be processed.

This study will be done during the period between 2009-2010 at St. John’s Medical College and Hospital, Bangalore.

2)Sample Collection :

Sputum:

The successful isolation of the organism depends on the quality of the specimen obtained and the appropriate processing techniques employed by the mycobacteriology laboratory.

To obtain a desirable sputum sample, the patients should be instructed to –

  1. Rinse the mouth with water to minimize residual food particles, mouth wash or oral drugs.
  2. Saliva and nasopharyngeal discharges should be not be collected.
  3. Only the exudative material brought up from the lungs after a deep productive cough will be collected.

Specimen will be collected in a sterile, leak-proof screw-capped container, clearly labeled with patient’s name and /or identification number.

Inclusion Criteria:

Consecutive sputum sample collected from suspected pulmonary tuberculosis patients.

Exclusion Criteria:

  1. Patient already underanti-tubercle drug treatment.
  1. Inadequate sample less than 2ml.
  1. Saliva only.

DECONTAMINATION METHOD:

# Hypertonic Saline - Sodium Hydroxide Method :

Procedure:

Take 1 ml of sputum + 1 ml 7% NaCl + 1 ml 4% NaOH in a sterile 15

ml centrifuge tube.

Homogenize for 15-20 second using a vortex mixer.

(Final concentration (w/v) in 3 ml : 2.33% NaCl, 1.33% NaOH).

Incubate at 370C for 30 minutes.

After incubataion neutralize the mixture with sterile PBS (PH 6.8)

bringing the total volume to 15 ml.

Vortexed the mixture for 5 second and centrifuge at 4000 rpm for

15 minutes.

Discard the supernatants into a splash-proof discard container with

5% hypochlorite solution.

Suspend the pellet in 200 micro liter of sterile PBS(Phosphate Buffer

Saline) .Homogenize for 5 second with a vortex mixer and inoculate

into L-J(Lowenstein-Jensen) medium (200µl) and prepare a smear for

Z-N-staining.

# Modified Petroff’s Method:

Procedure:

Add an equal volume of 4% NaOH to 2 ml sputum sample in a

tightly fitted screw capped container and mix thoroughly by shaking.

Incubate the container at 370C for 20 minutes and further mixing by

shaking at intervals.

Centrifuge at 3000 rpm for 30 minutes then pipette off the

supernatant and discard into strong disinfectant.

Add a drop of Bromothymol blue to the deposit, then neutralize it to

about pH7 by adding 8% hydrochloric acid drop by drop. (If too much

acid is added, then adjust the pHby adding 0.4% NaOH.)

Transfer (200µl) of the deposit on 2 L-J media and also use for

microscopic examination of sputum smear.

Culture:

Immediately after the decontamination and concentration process, 200µl each re-suspended pellet will be used to inoculate a tube of L-J medium.

AFB Smears:

One drop of each suspended pellet is used to prepare slide for AFB microscopy using the Ziehl-Neelsen stain. Each slide will be coded, examined and graded according to the RNTCP guidelines.

Smears are reported as follows-

Negative : No AFB

Less than 1 + : 1-9 AFB in 100 Microscopic fields.

1+ :10-99 AFB in 100 fields.

2+ : 1-10 AFB per field in at least 50 fields.

3+ : more than 10 AFB per field in at least 20

fields.

RESULT:

The results of microscopy finding and culture will be analysed and compared.

REFERENCE:

  1. Ganoza C A, Ricaldi J. N, Chauca J, Rojas G, Munayco C, Agapito J et

al. Novel hypertonic saline- sodium hydroxide (HS-SH) method for

decontamination and concentration of sputum samples for

Mycobacterium tuberculosis microscopy and culture. Journal of Medical

Microbiology( 2008); 57: 1094-98.

  1. Indian Journal of Tuberculosis (1988);35; 80.
  1. Kent, P. T . & Kubica , G. P.(1985); 21-44.
  1. Dye,C, Scheele, S, Pathania, V & Raviglione, M.C. Global Burden of

Tuberculosis estimated incidence and mortality by country. JAMA

282:677 ,1999.

  1. Global TB/HIV Working Group. Tuberculosis and HIV.
  1. ICMR Bulletin August (2002), Vol. 32, No.8, Part I : Techniques for

diagnosis of T.B.

  1. Toman,K. (2004) Tuberculosis Case-Finding & Chemotherapy. Case

Detection, treatment and monitoring – Questions & Answers, 2nd

Edition Geneva WHO.

  1. Asper,L.,Mutsvangwa,J.,Magwenzi,J.,Chigara,N.,Butterworth,A.,Maso

N,P.and Van der Stuyft,P.(2003).A comparison of direct microsopy,the

concentration method and the Mycobacteria Growth Indicator Tube for

the examination of sputum for acid fast bacilli. Ent J Tuberc Lung Dis

7, 376-381.

  1. IUATUD(2005). Tuberculosis bacteriology-priorities and indications in

high prevalence countries: position of the technical staff of the

Tuberculosis Division of the International Union Against Tuberculosis

and Lung Disease. International Journal of Tuberculosis and Lung

disease9, 355-361.

10. Ricaldi, J.N and Guerra, H.(2008).A simple and improved

method for diagnosis of Tuberculosis using hypertonic

saline and sodium hydroxide (HS-SH) to concentrate and

decontaminate sputum.Trop doct38, 97-99.

11. King, M., Dasgupta, B., Tomkiewiz, R. P. & Brown, N. E.

(1997).Rheology of cystic fibrosis sputum after in vitro

treatment with hypertonic saline alone and in combin

ation with recombinant human deoxyribonuclease I.

Am J Respir Crit Care Med 156, 173 –177.

signature of the Candidate:

remarks of the Guide:

Name and designation of:
Guide: / Dr. BAIJAYANTI MISHRA. MD.
ASSOCIATE PROFESSOR,
DEPT. OF MICROBIOLOGY,
ST. JOHN’S MEDICAL COLLEGE,
BANGALORE.
Signature:
Head of the department: / Dr. H. SRINIVASA. MD.
PROFESSOR AND HEAD,
DEPT. OF MICROBIOLOGY,
ST. JOHN’S MEDICAL COLLEGE,
BANGALORE.
Signature :
Remarks of the Chairman and Principal:
Signature

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