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70-RUBINAAJB-11-2248

70-RUBINAAJB-11-2248

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Published by Muhammad Akram
rubina ghani
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Published by: Muhammad Akram on Mar 22, 2014
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African Journal of Biotechnology Vol. x(xx), pp. xxx-xxx, xx xxxxx, 2011 Available online at http://www.academicjournals.org/AJB DOIXXXXXX ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Rapid detection of typical and atypical
Mycobacterium tuberculosis 
 by polymerase chain reaction (PCR) and its comparison with Ziehl Neelsen staining technique
Rubina Ghani
1
, Hafeez ul Hassan
2
, Hasan Ali
3
, Mohammad Usman
4
, Mulazim Hussain Bukhari
5
, M. Akram
6
* and Asif Iqbal
6
 
1
Department of Biochemistry, Baqai Medical University, Karachi, Pakistan.
2
Department of Physiology, Baqai Institute of Hematology, Baqai Medical University, Karachi, Pakistan.
3
Department of Biochemistry, Bahria University, Medical and Dental College, Karachi Campus
 
Pakistan.
4
Department of Hematology, Baqai Medical University, Karachi, Pakistan.
5
Department of Pathology, King Edward Medical University Lahore, Pakistan.
6
Faculty of Eastern Medicine, Hamdard University, Karachi, Pakistan.
Accepted 28 October, 2011
Tuberculosis is a challenging problem and it represents the major health problem in developing countries like Pakistan. In view of the importance of early diagnosis of tuberculosis, the efficiency of polymerase chain reaction (PCR), one of the most reliable and sensitive DNA based assays, was compared with conventional method for the detection of
mycobacterium tuberculosis 
. We hypothesize that PCR is a more sensitive method for the detection of acid fast bacilli (AFB) as compared to Ziehl Neelsen (ZN) staining. To affirm this, a total of 200 sputum samples were analyzed. A simultaneous analysis of the sputum samples was done using ZN staining and PCR to compare the two methods. PCR was also performed on AFB negative cases using another specific primer for atypical mycobacteria. Results indicate that on both methods, by ZN staining and PCR, 46/200 (23%) sputum were positive for mycobacterium tuberculosis in both sexes. From 154 negative samples for AFB, on ZN staining, 4/154 (2.6%) samples had the positive atypical mycobacteria by PCR using specific primers. We conclude that PCR is a more sensitive method for the detection of AFB as compared to ZN staining Key words:
 Sputum, PCR, acid fast bacilli (AFB), atypical mycobacterium.
INTRODUCTION
Tuberculosis (TB) is one of the leading infectious diseases of the world and is responsible for 2.9 million worldwide deaths and 8.8 million new cases of active TB occur per year (Dye et al., 1999; Kwara et al., 2008). Whereas, WHO has also declared tuberculosis a global emergency as there has been a 20% increase in its
incidence over past decades (Corbett et al., 2003
; Mehnaz et al., 2005). It is one of the commonest infectious disea-ses of the developing countries, resulting in high
*Corresponding author. Email: makram_0451@yahoo.com. Tel: 9221-4552661 or 9221-5862603.
morbidity and mortality in these areas. It is estimated that 95% cases occur in under-developed world where diag-nostic and treatment facilities are inadequate (Khan et al., 2006). In Pakistan, it is estimated that 2,680,000 new cases and 64,000 deaths occur due to tuberculosis each year (Saeed et al., 2002). The diagnosis of mycobacterial infection is accom-plished by
 
culture-based identification. Primary culture of slowly growing
 
mycobacteria without using the BACTEC culture system, usually
 
takes four to six weeks or longer (Yeager et al., 1967). Conventional bacteriology such as direct microscopy and culture are not sufficient for the early diagnosis of tuberculosis because there are few bacilli in the sputum to be demonstrated by direct
 
 microscopy and on the other hand, successful cultural identification of tubercle bacilli takes about seven to eight weeks (Rafi et al., 2003). The initial diagnosis of tuber-culosis is usually based on clinical grounds, but definite diagnosis would require the isolation and identification of the infecting organism. The usual laboratory procedures are Ziehl-Neelsen (ZN) staining and microscopic exami-nation for acid-fast bacillus (AFB) by oil immersion lens. The presence of acid-fast bacteria in sputum is a rapid presumptive test for tuberculosis. Subsequently, when cultured,
Mycobacterium tuberculosis 
 would grow very slowly producing distinct non-pigmented colonies after several weeks.
M. tuberculosis 
 can be differentiated from most other mycobacteria by the production of niacin (Soini et al., 2001). However, recent methodological
 
advances in molecular biology have provided alternative rapid
 
approaches, such as the polymerase chain reaction (PCR) and PCR-linked methods. A rapid alternative method to culture is PCR; for the rapid detection
 
or identification of
M. tuberculosi
, target genes specific primers
 
to mycobacteria are used in
a PCR (Eing et al., 1998; Gunisha et al.,
 2001; Montenegro et al., 2003). In this study, we used a simplified multiplex PCR assay
 
to identify mycobacterium tuberculosis
 
and another strain of mycobacteria like atypical mycobacteria complex in our population.
MATERIALS AND METHODS Collection of specimens
The simple descriptive study was carried out in the laboratory where the 200 sputum samples were collected as a routine. From each patient, samples were collected for three consecutive days with brief clinical history. All 200 cases fulfilled the following criteria with the clinical history of mycobacterial infection in the family.
Criteria of patient’s selection
Samples of those patients were collected whose chest x-ray showed radiographic consolidations suspicious of pulmonary tuber-culosis; either patchy or nodular infiltrate in upper lobes or superior segment of lower lobes, bilateral upper lobe infiltrate with patchy soft shadows with or without cavitations. In some cases, there were bilateral upper lobe infiltrate with patchy soft shadows without cavitations. Besides, on the basis of chest x-rays evaluation of respiratory symptoms (cough > 3 week, hemoptysis, chest pain, and dyspnea), an unexplained illness and flu were also considered. In adults, a multinodular infiltrate above or behind the clavicle (the most characteristic location, most visible in an apical lordotic view) suggest the reactivation of TB. Middle and lower lung infiltrates were nonspecific but prompt suspicion of primary TB in patients (usually young) whose symptoms or exposure history suggested the recent infection, particularly if there was pleural effusion.
Direct smear preparation
For the AFB smear, morning sputum samples of three consecutive days were collected in sterile, wide mouthed plastic bottles. All the samples were digested using sodium hydroxide (NaOH). The remaining sputum (1 to 2 ml) was transferred to 10 ml screw cap-ped tube and mixed with equal volume of NaOH. The mixture was incubated at room temperature for 10 to 15 min and shaken at regular intervals. Then, 8 ml of distilled water were added and the samples were centrifuged at 3000
g
for 15 to 20 min. The super-natant was discarded and the pellets were suspended in few drops of the remaining fluids. Slides were prepared from the suspended sediment, air-dried, heat fixed and stained by Ziehl Neelsen method.
Microscopic examination and interpretation of the result
The sputum smear were prepared and stained with ZN method for AFB. The smears were covered with tissue papers flooded slides using strong carbol fuchsion for 5 min while heating with steam. The paper was removed and decolorized with acid alcohol and counter was stained with malachite green. After staining, more than 100 field of each smear were examined carefully under the light microscope using the oil immersion 100 X lens and interpretation of the result was done according to the National TB control program (Aung et al., 2001). After initial investigation for the AFB, the DNA were extracted from all specimens for detecting mycobacterium tuberculosis and atypical mycobacteria in these specimens by using PCR, and the significant diagnostic value of PCR were observed by comparing with conventional methods (acid fast microscopy).
Grading of AFB
The numbers of acid-fast bacilli seen on the smears were recorded according to the recommendation by WHO. When there was no AFB seen per 100 oil immersion fields, it was graded as negative. When there were one to nine AFB seen per 100 oil immersion fields, the grade was reported scanty. When there were 10 to 99 AFB seen per 100 oil immersion fields, the grade was given +1. When they were 1-10 per oil immersion field, they were graded as +2 and when the number of AFB were more than 10 per oil immersion fields, they were graded as +3-. (Aung et al., 2001).
DNA extraction
The DNA was extracted from sputum by using 4% NaOH. Equal volume of NaOH was added to the sputum in Eppendorf cup and was incubated at room temperature for 30 min to digest the sputum samples. Then, the digested sputum was centrifuged at 13,200 rpm for 10 min and supernatant was discarded. The pellet was further processed following Gentra kit procedure, then the DNA pellet was resuspended with DNA hydration solution present in Gentra Kit and PCR was performed (Drosten et al., 2003) according to the following procedure; the PCR was carried out in a tube containing 20 µl of a reaction mixture made up of the following components: 10 pmol of each forward and reverse primers for mycobacterium and atypical mycobacteria, 500 µM of four deoxynucleotides, 2 U of Taq polymerase (Promega), 10 x PCR buffer containing and 1.5 mM MgCl
2
. The thermal cycler (Master Gradient PCR System, Eppendorf AG, Germany) was programmed to first incubate the sample for 5 min for 95°C followed by 30 cycles consisting of 94°C for 45 s, 56°C for 45 s and 72°C for 1 min with final extension for 7 min at 72°C. The PCR amplified products were identified by electrophoreses on a 2% agarose gel, stained with ethidium bromide, and evaluated under transilluminator. The sizes of PCR amplified product were estimated according to the migration pattern of a 100-bp DNA ladder (Gibco BRL Life Technologies) (Kox et al., 1994; Noordhoek et al., 1994; Kocagoz et al., 1993).
 
 
Table 1.
 The percentage of female/male cases of pulmonary tuberculosis.
Sex
 
Total (n = 200)
 
Percentage (%)
 Female 72 36 Male 128 64 Total 200 100
Male to female ratio was 1:1.7.
Table 2.
The summary of AFB positive cases of sputum samples stained with Ziehl-Nelsen (Z N.) stain in both sexes.
S/N
 
Sex
 
Positive
 
Percentage (%)
 1 Female 14 7 2 Male 32 16 3 Total 46 23 The ratio of positivity was 1: 2.3.
Table 3.
 The grading of AFB examined in 100 x fields present in sputum samples collected on three consecutive days according to National TB Control program.
Number of sample (n = 200) Day 1 Day 2 Day 3
Sputum (20) ++ (2+) +++ (3+) +++ (3+) Sputum (14) - ++ (2+) +++ (3+) Sputum (12) - - + (1+) Sputum (154) - - -
The primers used for the detection of
M. tuberculosis 
 5'-CGT ACG GTC GGC GAG CTG ATC CAA-3') and 5'-C CAC
 
CAG TCG GCG CTT GTG GGT CAA-3' were designed to amplify a 541bp fragment while the primers used for atypical mycobacteria were 5'-G GAG CGG ATG ACC
 
ACC CAG GAC GTC-3' and 5'-CAG CGG GTT GTT CTG GTC CAT GAA
 
C-3' to amplify 200 bp.
RESULTS
A total of 200 sputum samples were collected from suspected cases of pulmonary tuberculosis selected on the bases of history, clinical examination and chest roentogram. All the cases were between the ages of 15 to 70 years with mean age 46.5 ± 2 for both sexes, out of which 72 were females and 128 were males with female to male ratio 1.7:1 (Table 1). There were 46 cases, out of which only 14 female and 32 male were AFB positive (Table 2). In 12 patients, only
one to nine AFB per 100 oil immersion
 fields was detected; only on third day specimen was AFB positive and graded as +1, after a long search. It was also noted that in 20 cases all the three samples were AFB positive by staining with ZN stain; in each field >10 AFB per oil immersion, field was seen and were graded as +3. In 14 cases on day one, AFB was negative after examining 100 fields. When second and third day samples were collected from these patients, they were graded as +2 and +3, whereas in 154 patients, the AFB was negative in all the three sputum samples and all the results were interpretated
according to National TB Control Program
 as summarized in Table 3. AFB
positive and negative cases were further
 confirmed by multiplex PCR for the detection of
mycobacterium tuberculosis 
 and atypical mycobacteria. Among the 200 cases, PCR assay correctly identified 46 cases which were as well smear positive. However, this assay failed to pick 154 smear negative cases giving a sensitivity of 92%. There was no false positive case in these smear positive specimens, giving a specificity of 100% for the test by PCR. Similarly, in 154 samples, AFB was negative although the symptoms were identical to
M. tuberculosis 
. Further molecular assay was used for the identification of another strain of mycobacteria causing the same symptoms (atypical mycobacteria) by PCR using specific primers. There were 4/154 (2.6%) atypical mycobacteria detected by PCR from AFB negative smears using specific primers. In comparison to AFB among samples from clinical TB patients, sensitivity, specificity, and predictive value of positive test by PCR was 100.0%. In Figure 1, gel shows the 541 bp positive cases for
mycobacterium tuberculosis 
. Figure 2 indicates that the AFB negative in

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