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February 2005, Volume 55, Issue 2

Original Article

Diagnostic Yield of Fast Plaque TBTM Test for Rapid Detection of Mycobacterium

Abstract

Objective: To compare the diagnostic yield of FAST Plaque TB™ test with the conventional methods for detection of mycobacterium tuberculosis in sputum of Tuberculosis suspects at Jinnah Postgraduate Medical Center Karachi Pakistan.
Methods: A comparative study of diagnostic yield of FAST Plaque TB™ test with the culture and ZN staining, conducted from January to June 2004.
Results: The study was completed on 48 samples, 31 (64.58%) male and 17 females (35.42%). Half of the cases were sputum positive. Culture positive was in 17 (35.41%) and negative in 28 (58.3%) wereas 3 (6.25%) were contaminated. FAST Plaque TB™ test was positive in 16 (33.33%) and negative in 32 (66.6%) specimens. Out of 17 culture positive, 2 (11.7%) were negative and in 28 culture negative, 1 (3.57%) specimen was positive for FAST Plaque TB™ test. Out of 24 smear positive, 11 (45.83%) were negative and in 24 smear negative, 3 (12.5%) were positive, for FAST Plaque TB™ test. Compared to culture it has sensitivity of 86.23% and specificity of 96.42%, positive predictive value of 93.75% and negative predictive value of 93.1%.
Conclusion: FAST Plaque TB™ test is a simple test that can detect viable mycobacterium in 2 days. It has a good sensitivity and specificity. The cost is three times less than the other available tests like PCR. Thus it can be useful in the diagnosis of tuberculosis as an adjunct to sputum microscopy in endemic countries (JPMA 55: 57;2005).

Introduction

Tuberculosis is a serious health problem. It is estimated by WHO (World Health Organization) that approximately one third of the world population is infected with Mycobacterium tuberculosis resulting in more than 23000 people developing active tuberculosis and about 5000 deaths daily. The disease is especially prevalent in developing countries, where it accounts for more than a quarter of all preventable adult deaths.1
Prevalence of tuberculosis in Pakistan is 178/1,00,000.2 New cases that are sputum smear positive are only 51%.3 Microscopic examination to detect Mycobacterium tuberculosis is specific but is not very sensitive as more than 103 to 104 organisms per ml are required for the direct smear to be positive4,5 Starting treatment on this ground takes care of the patients who are more infectious. Nevertheless a smear negative pulmonary tuberculosis patient is also capable of transmitting disease.6
Drawback of relying on smear examination is that most of them are left untreated. Currently, the only sure criterion for definite diagnosis of tuberculosis is the demonstration of the presence of Tubercle bacillus in clinical specimen by gold standard culture on LJ (Lowenstein-Jensen) method which takes 6-8 weeks to reach the diagnosis and having sensitivity and specificity of more than 90%.7
Although new more rapid diagnostic methods have developed that are based either on liquid culture technique such as BACTEC or on molecular technique, their expense plus the requirement for special personnel and equipment limited their use.8,9 There is a need of a test, which can rapidly detect Tubercle bacilli. Any test that will be broadly accepted by the global diagnostic community needs to be simple, accurate, rapid, sensitive, low cost and easy to implement within the current infrastructure, especially for the developing world. The Fast- plaque TBTM test utilizing Mycobacteriophage claims the same. Mycobacteriophage are viruses that infect mycobacteria. First discovered 50 years ago, there are now 250 known Mycobacteriophages.10 Mycobacteriophage D29 was isolated from the soil.11 It is a lytic phage, which is able to infect and replicate in the slow growing pathogenic strains such as mycobacterium tuberculosis and mycobacterium ulcerans and fast growing environmental strain such as mycobacterium smegmata. David and colleagues demonstrated the utility of mycobacteriophage D29 for testing susceptibility of mycobacterium to anti-tuberculosis drugs in1980 by David and colleagues.12
Phage Amplification Technology is simple, rapid, requires 2 days for results. No expensive delicate equipment or specialised facilities are required and results are read by naked eye.
Therefore it is well suited for use in those countries which have high prevalence of tuberculosis. Use of this technology as a diagnostic tool for the detection of bacterial pathogens has been well documented.12

Materials and Methods

This study was conducted in the department of Chest Medicine at Jinnah Postgraduate Medical Center Karachi. Jinnah Postgraduate Medical Center Karachi is a tertiary care hospital in the public sector. This institute is imparting training to under- graduate and postgraduate medical students in various specialties. The aim of the study was to compare the diagnostic yield of FAST Plaque TB™ test with the conventional methods for detection of Mycobacterium tuberculosis in sputum of tuberculosis suspects.
In the department of Thoracic medicine Jinnah Postgraduate medical Centre an approximate of 175 patients attend each OPD. The estimated prevalence of TB patients is about 4-5%. For the purpose of estimating sample size the desire precision is taken as 5% with a-risk of 5%. A sample size came out as 56 subjects. For this study a sample of 50 patients had been taken using convenience sampling technique.
This was also in accordance with research method for promotion of lung health, a guide to protocol development for low-income countries by Enarson AD.13
Sputum samples were randomly collected from 50 patients from the out patient department of chest medicine unit Jinnah Postgraduate Medical Center Karachi. These patients had cough for at least 3 weeks. According to standard procedure each sample was subjected to Ziehl Neelsen acid fast staining method, two independent microbiologists examined the slides. Specimen was inoculated on two Lowenstein Jensen (LJ) media slopes. When no colonies were seen after 8 weeks incubation the results were recorded as negative. FAST Plaque TB™ test was performed according to manufacturer\'s instruction, the number of plaques were recorded by naked eye. With each batch negative and positive controls were also run.14
The FAST Plaque TB test (Biotec Laboratories Ltd., Ipswich, Suffolk, U.K.), utilizes specific Mycobacteriophage or Phage (Actiphage™) to reflect the presence of viable Mycobacterium tuberculosis in sputum samples. After Phage infection, a veridical (Virusol) solution destroys all phage that have not infected the tubercle bacilli. The phage replicate in the infected bacilli until new progeny phage is released as the cells lyses. The new phages are amplified by the addition of a nonpathogenic rapid growing mycobacterium host (Sensor™ cells) that is also able to support phage replication. The resulting phage can be visualized as clear areas (plaques) in a lawn of Sensor cells. The number of plaques visualized from a given sample is related to the number of viable tubercle bacilli in the original sample.

Results

Samples from 50 patients were included in the study. Two samples could not be processed due to power failure. The study was completed in 48 samples, 31 (64.58%) male and 17 females (35.41%). Over all smear positive cases were 24 and same number were smear negative (50% each). Number of culture positive and culture negative were 17 (35.41%) and 28 (58.3%) respectively, 3 (6.6%) were contaminated. FAST Plaque TB™ test was positive in 16 (33.33%) and negative in 32 (66.6%) specimens. FAST Plaque TB™ test was compared with culture, it was observed that out of 17 specimens which were culture positive, 2 (11.76%) were negative for FAST Plaque- TB™ test, where as among 28 cultures negative specimen 1 (3.57%) specimen was positive for FAST Plaque TB™ test (Table 1).


When FAST Plaque TB™ test was compared with smear, 11 (45.8%) out of 24 smear positive yielded a negative result. Out of 24 smear negative, 4 (16.6%) reacted positive to FAST Plaque TB™ test (Table 2). This was similar to the results when smear was compared with gold standard sputum culture (Table 2).
simple and require little or no specimen processing. Results should be available on the same day. The cost of test should be comparable to costs currently incurred for diagnosis of smear negative tuberculosis, including a chest radiograph and culture.18



Data presented in this study demonstrates that FAST Plaque TB™ test meets the criteria for sensitivity and specificity. The test is simple, safe and can be easily performed in routine microbiology laboratory. Regular laboratory staff can be trained easily to perform this test. The cost is three times less than the other available tests like PCR.5 This test can be useful in the diagnosis of tuberculosis suspects, as an adjunct to sputum microscopy in highly endemic countries.

References

1. Marei AM, El-Behedy AM,Mahtadi HA, et al. Evaluation of a rapid bacteriophage-based method for the detection of mycobacterium tuberculosis in clinical samples, J Med. Microbiol 2003;52:331-5.
2. World Health Organization. Tuberculosis country profile for Pakistan. WHO report 2004.
3. World Health Organization. Global tuberculosis control. WHO report 1999.
4. Hall GS, Howard BJ. Mycobacterium. In: Howard BJ, Keiser JF, Smith TF, Weisfeld AS, Tilton RC. Clinical and pathogenic microbiology. St: Louis: Mosby Year Book Inc. 1994, pp.503-28.
5. Muzaffar R, Batool S, Aziz F, Naqvi A, Rizvi A. Evaluation of the FAST Plaque TB assay for direct detection of mycobacterium tuberculosis in sputum specimens. Int J Tuberc Lung Dis 2002;6:635-40.
6. Behr MA, Warren SA, Salmon H. Transmission of mycobacterium tuberculosis from patients smear negative for acid fast bacilli. Lancet 1999;353: 444-9.
7. Foulds J, O\'Brien R. New tools for the diagnosis of tuberculosis, the perspective of developing countries. Int J Tuberc Lung Dis 1998;2:778-83.
8. Roth A, Schaberg T, Mauch H. Molecular diagnosis of tuberculosis: current clinical validity and future perspectives. Eur Respir J 1997;10:1877-91.
9. Jonas V, Longiaru M. Detection of mycobacterium tuberculosis by molecular method. Clin Lab Med 1997;15:119-28.
10. McNerney, R. TB: the return of the phage: a review of fifty years of mycobacteriophage research. Int J Tuberc Lung Dis 1999;3:179-84.
11. Froman S, Will DW, Bogen E. Bacteriophage active against virulent mycobacterium tuberculosis. I. Isolation and activity. Am. J Public Health. 1954;44:1326-33.
12. David H, Clavel S, Clement F, Pereira MJ. Effects of antituberculosis and antileprosy drugs on mycobacteriophage D29 growth. Antimicrob Agents Chemother. 1980;18:357-9.
13. FAST Plaque TB™ a rapid bacteriophage assays for the detection of mycobacterium tuberculosis complex in clinical sample Product inserts. Ipswich, UK: Biotech Laboratories Ltd. 2000.
14. Enarson AD, Kenedy SM, Miller DL, Bekke P. Research method for promotion of Lung health. Int J Tuber 2004;91:5-9.
15. Albert H, Heydenrych A, Brookes R, et al. Performance of a rapid phage-based test, FAST Plaque TB ™ to diagnose pulmonary tuberculosis from sputum specimens in South Africa. J Tuber Lung Dis 2002;6:529-37.
16. Mustafa R, Barolo S, Asia F, Navy A, Rizvi A. Evaluation of FAST Plaque TB assay for direct detection of mycobacterium tuberculosis in sputum specimens. Poster presentation 32 IUATLD world conference on Lung Health November, Paris France, 2001.
17. Butt T, Ahmed RN, Karma SY, et al. Rapid diagnosis of pulmonary tuberculosis by mycobacteriophage assay. Int J Tuber Lung Dis 2004;7:899-902.
18. World Health Organization. WHO Tuberculosis Diagnostics Workshop: Product Development Guidelines. Cleveland, Ohio: 27 July 1997.

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