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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
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Year : 2004  |  Volume : 22  |  Issue : 1  |  Page : 44-46
 

Comparison of rapid colorimetric method with conventional method in the isolation of mycobacterium tuberculosis


Department of Microbiology, Christian Medical College and Hospital, Ludhiana - 141 008, Punjab, India

Correspondence Address:
Department of Microbiology, Christian Medical College and Hospital, Ludhiana - 141 008, Punjab, India

 ~ Abstract 

The aim of the study was to evaluate two methods (colorimetric and conventional) for isolation of Mycobacterium tuberculosis. A total of 500 clinical specimens were processed by modified Petroff's method and then inoculated into MB/BacT-240 system bottles and on LJ medium slopes. The specimens included 242 sputum, 95 gastric aspirates, 47 pleural fluids, 45 CSF, 32 urine, 18 pus, 11 bronchoalveolar lavage, 3 tissue, 2 stool, 2 lymphnode specimens, 2 synovial fluid and 1 bronchial wash specimens. The isolation rate was 16.4% by the colorimetric method and 2.2% by the conventional method. The mean detection time was 16 days and 26 days respectively. Among 36 direct smear positive samples, 63.9%(23/36) and 30%(11/36) were positive by colorimetric and conventional methods respectively. Out of 464 direct smear negative samples 12.9%(60/464) and 0.6%(3/464) were positive by colorimetric and conventional methods respectively. Therefore, colorimetric method enables rapid detection leading to early diagnosis and drug susceptibility testing.

How to cite this article:
Oberoi A, Kaur H. Comparison of rapid colorimetric method with conventional method in the isolation of mycobacterium tuberculosis. Indian J Med Microbiol 2004;22:44-6


How to cite this URL:
Oberoi A, Kaur H. Comparison of rapid colorimetric method with conventional method in the isolation of mycobacterium tuberculosis. Indian J Med Microbiol [serial online] 2004 [cited 2014 Jul 29];22:44-6. Available from: http://www.ijmm.org/text.asp?2004/22/1/44/8061


Tuberculosis (TB) continues to be one of the leading infectious causes of death in the world today. It affects one third of the world's population, of which 95% is in the developing countries where resources are limited. In India, 13 million are infected and 3.5 million are positive for acid fast bacilli (AFB) with 2.2 million new TB cases being added every year.[2] The scenario in case of TB is quite alarming and has been further complicated by the spread of HIV as well as increasing drug resistance in improperly treated cases. Smear microscopy remains the main stay of TB diagnosis in developing countries but suffers from low specificity and variable sensitivity.[3] Laboratory cultivation of Mycobacterium tuberculosis is much more sensitive, but it is time consuming and susceptible to contamination.[4] Rapid diagnosis of tuberculosis is essential to initiate timely and appropriate treatment to curb the spread of this potentially life threatening disease.
The BACTEC (460 MTB) system was the first broth based system which could provide a rapid result[5] and has been in use for many years. But it has certain demerits. It is a radiometric method using C14 palmitate which produces[14] CO2 by mycobacterial respiration that collects within the vial and the system is semiautomated which requires constant monitoring and is labour intensive. These drawbacks have been overcome to some extent in the MB/Bact-240 system (Organon Teknika), which is a fully automated colorimetric detection system. Once the culture bottles are loaded into the system there is no handling of specimens till positive or negative results become known. The working principle of this system is based on mycobacterial growth detection by a colorimetric sensor. If the organisms are present, CO2 is produced as the organism metabolizes the substrate glycerol. The colour of the gas permeable sensor at the bottom of each culture bottle results in increase of reflectance in the unit, which is monitored every ten minutes by the system using infra red rays. As and when the results flag positive, there is beep from the corresponding cell in the unit which denotes approximately 106 to 107 organisms/mL in the bottle. The objective of this study was to evaluate mycobacterial isolation rates and mean detection time by colorimetric and conventional methods.

 ~ Materials and Methods Top

Five hundred samples were collected during the year 2001-2002 from indoor and outdoor patients, and were processed by both colorimetric and conventional methods. Among them there were 242 sputum, 95 gastric aspirate, 47 pleural fluid, 45 C.S.F, 32 urine, 18 pus, 11 bal, 3 tissue, 2 stool, 2 lymphnode specimens, 2 synovial fluid and 1 bronchial wash specimens.
Processing of specimens
All specimens were processed with in 24 hours after collection. Modified Petroff's method using double the volume of NaOH (4%) for sputum and equal volume of NaOH (2%) for extra pulmonary specimens was used for processing of all the specimens. The specimens were kept in a shaker for homogenization and then decontaminated for 20 and 10 minutes respectively. The processing was stopped by the addition of distilled water upto the brim and centrifuging in a shielded centrifuge (3000g) for 15 minutes. The supernatant fluid was then discarded and the sediment was used for inoculation for both colorimetric and conventional methods. All specimens were subjected to Ziehl-Neelsen staining and smear grading as per the guidelines under the Revised National Tuberculosis Control Programme.[6]
Inoculation
For the colorimetric method 1 mL distilled water was added to the sediment and 0.5mL of this was injected into the MB/BacT culture bottle containing Middle brook 7H9 broth, pancreatic digest of casein, bovine serum albumin, catalase and antibiotic supplement comprising amphotericin B, aziocillin, nalidixic acid, polymyxin, trimethoprim and vancomycin, bulking agent and reconstituion fluid consisting of oleic acid, glycerol and bovine albumin. For the conventional method, sediment was inoculated on two LJ slopes.
Incubation and detection of growth
For the colorimetric method, the MB/BacT culture bottles were loaded onto the identified cell in the system. The test was declared negative if the culture tube did not flag positive and give a beep upto 42 days, after inoculation. For the conventional method, the slopes (LJ) were read on a weekly basis and sent for identification as soon as sufficient growth was visible. The slopes were incubated at 37C for (sputum specimens) and 12 weeks (extra pulmonary specimens) respectively before declaring them as negative.
Positive and negative controls, for Mycobacterium tuberculosis were inoculated along with the samples in each run. The isolates grown in the colorimeteric mycobacterial isolation system were confirmed by Ziehl Neelsen's staining.

 ~ Results Top

Out of 500 specimens processed by both the methods, 36(7.2%) were smear positive and 464(92.81) were smear negative. The isolation rate was 16.4% (82/500) by colorimetric method and 2.2% (12/500) by conventional method as shown in [Table - 1].
Among 36 smear positive specimens colorimetric method detected 23(63.9%) positives and the conventional method detected 11(30.5%) positives as shown in [Table - 2]. The colorimetric method detected 60(12.9%) additional positives and conventional method detected 3(0.6%) additional positives as shown in [Table - 2] from 464 smear negative specimens.
The contamination rates by the conventional and colorimetric methods were 14.4% (72/500) and 24.4% (122/500) respectively. The mean isolation time was 16 days (14 hours - 31 days) by colorimetric method, and 26 days (11 days - 41 days) by conventional method. Out of 268 extrapulmonary specimens, colorimetric method detected 39 positives (14.55%). The mean detection time was 16 days (14 hours - 31 days) and 26 days (11 days - 41 days) by colorimetric and conventional method respectively.

 ~ Discussion Top

In the present study, of the total 500 specimens processed, 7.2% were smear positive. The yield by colorimetric method was 14% higher than by conventional culture method. The additional yield was contributed by the extra pulmonary specimens. The isolation rate by the colorimetric method was higher by 14% from extra pulmonary specimens. This was comparable to the Newcastle study which reported isolation rate by colorimetric method to be 13% higher than by the LJ method.[7] One study reported 15.3% higher yield by colorimetric method from the extra pulmonary specimens.[8] The mean detection times (MDT) of mycobacteria by colorimetric and conventional method in our study were 16 days and 26 days and contamination rates were 14.4% and 24.4% respectively.
A major constraint of MB/BacT system is the cost factor. The cost for the conventional method is a fraction of that for colorimetric method. Major use of the colorimetric method is in the smear negative and extrapulmonary specimens. In conclusion, colorimetric method will enable the clinicians to make rapid diagnosis and indirectly to control the problem of drug resistance in tuberculosis. 

 ~ References Top

1.Eltrimgham JL, Wilson SM, Drobaniewski FA. Evaluation of bacteriophage - based assay (Phage amplified biological assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, Pyrazinamide, and ciprofloxacin among clinical isolates of mycobacterium tuberculosis. J Clinical Microbiol 1999:37(11): 3528-3532.  Back to cited text no. 1    
2.Udwadia 2F. India. In clinical tuberculosis 2nd edition PDO Davis.Ed. (Chapmon & Hall, London) 1998:591-605.  Back to cited text no. 2    
3.Mole RJ, Maskell TWO. Review Phage as a diagnostic - the use of phage in TB diagnosis. J Chem Tech Biotech 2001;76:683-688.  Back to cited text no. 3    
4.Thornton CG, Maclellan KM, Brink TL, Passen S. In vitro comparison of NALC-NaoH, Tween 80 and C18 - carboxypropylbetaine for processing of specimens for recovery of mycobacteria. J Clin Microbiol 1998;36:3558-3566.  Back to cited text no. 4    
5.Hawkinds JOE. Antibacterial susceptibility tests. Manual of clinical Microbiology.Ed-5 American Society for Microbiology. Washington DC.1991:1138.  Back to cited text no. 5    
6.Rieder Hl. Public Health services National Tuberculosis Reference Laboratory and National Laboratory Network, IUATLD, 1998:72.  Back to cited text no. 6    
7.Freeman R, Magee J. Continous automated mycobacterial liquid culture systems. PHLS Microbiology Digest 1997;14(2):69.  Back to cited text no. 7    
8.B Mahadev. Comparison between Rapid colorimetric mycobacterial isolates and susceptibility testing method and conventional method using LJ medium. Indian J Tuberculosis 2001;48:129-134.  Back to cited text no. 8    
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2004 - Indian Journal of Medical Microbiology
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