African Journal of Biotechnology Vol. x(xx), pp. xxx-xxx, xx xxxxx, 2011 Available online at http://www.academicjournals.

org/AJB DOIXXXXXX ISSN 16845315 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 Ghani1, Hafeez ul Hassan2, Hasan Ali3, Mohammad Usman4, Mulazim Hussain Bukhari5, M. Akram6* and Asif Iqbal6
Department of Biochemistry, Baqai Medical University, Karachi, Pakistan. 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.
2 1

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 diseases of the developing countries, resulting in high morbidity and mortality in these areas. It is estimated that 95% cases occur in under-developed world where diagnostic 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 accomplished 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

*Corresponding author. Email: makram_0451@yahoo.com. Tel: 9221-4552661 or 9221-5862603.

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 tuberculosis 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 examination 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. tuberculosis, 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 patients selection Samples of those patients were collected whose chest x-ray showed radiographic consolidations suspicious of pulmonary tuberculosis; 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 capped 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 supernatant 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 l of a reaction mixture made up of the following components: 10 pmol of each forward and reverse primers for mycobacterium and atypical mycobacteria, 500 M of four deoxynucleotides, 2 U of Taq polymerase (Promega), 10 x PCR buffer containing and 1.5 mM MgCl2. 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 94C for 45 s, 56C 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 Female Male Total

Total (n = 200) 72 128 200

Percentage (%) 36 64 100

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 1 2 3

Sex Female Male Total

Positive 14 32 46

Percentage (%) 7 16 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) Sputum (20) Sputum (14) Sputum (12) Sputum (154)

Day 1 ++ (2+) -

Day 2 +++ (3+) ++ (2+) -

Day 3 +++ (3+) +++ (3+) + (1+) -

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

10

11

12

13

14

541 bp

Figure 1. The analysis of Mycobacterium tuberculosis DNA sample. Lanes 1 and 2 are negative and positive control of Mycobacterium tuberculosis; lanes 3 to 14 are the sputum sample identified for the positive M. tuberculosis; lane 15 is the DNA ladder of 50 bp.

10

11

12

541 bp

200 bp

Figure 2. The analysis of Mycobacterium tuberculosis DNA sample. For lanes 1 to 9, cases are positive with M. tuberculosis; lanes 10 to 12 are the positive cases with atypical mycobacterium; lane 13 is the negative control, while lanes 14 and 15 are the positive control with both atypical and Mycobacterium tuberculosis detected in the sputum sample; lane 16 is the DNA ladder of 50 bp.

the sputum samples of patients with same symptoms, as M. tuberculosis were atypical mycobacteria amplified at 200 bp. These could be efficiently differentiated using PCR by specific primers. DISCUSSION Before the introduction of molecular typing methods, there was little to aid the distinction between individual strains

of M. tuberculosis (Malik et al., 1998). Traditional methods of diagnosing tuberculosis have been the isolation of bacilli in culture or recognition of AFB in clinical specimens. The diagnostic value of various methods widely used in microbiological diagnosis of tuberculosis: direct smear examination for acid-fast bacilli, cultural identification in Lowenstein-Jensen (L-J) medium, the radiometric BACTEC 460 system and PCR, to evaluate the time factor and the sensitivity of the clinical method has been reported. AFB staining lacks sensitivity. So far,

the gold standard has been culture with a dividing time of 48 h, up to 10 weeks. However, the high number of falsepositives that we found suggests that results obtained should be confirmed with BACTEC, which considerably reduces the time required for identification, and makes it possible to carry out an antibiotic assay rapidly (Ginesu et al. 1998). This study was conducted in molecular laboratories for the early and cost effective diagnosis for the management of TB. We demonstrated the genetic technology; polymerase chain reaction. M. tuberculosis is one of the most successful bacterial pathogens in the history of mankind. In this study, out of 200 sputum samples, 46 AFB positive sputum samples clinically showed TB by both methods; PCR and AFB Stain. Our findings are consistent with that of Moran Moguel et al. (2000) who according to WHO, reported that PCR is a sensitive and specific technique for detecting the M. tuberculosis complex in both positive and negative bacilloscopy samples. A controlled PCR procedure makes it possible to establish or to exclude the diagnosis of tuberculosis in a time that is reduced from more than six weeks to just 24 to 48 h. This is particularly useful when an early diagnosis is needed to establish a patient's prognosis or in organ transplant cases (Morn Moguel et al., 2000). Finally, one-tube regular-PCR, a variant which used specific primers for M. tuberculosis and atypical mycobacteria, gave us 88.80% (91.43% sensitivity and 87.78% specificity) diagnostic value. On the basis of our results, we can affirm that PCR is a good method for early microbiological diagnosis of tuberculosis, given its high sensitivity and specificity and unparalleled rapidity. In this study, PCR for M. tuberculosis and atypical positive in the sputum were to ascertain an etiological diagnosis, with specificity and sensitivity of the AFB positive results shown in Figure 1. In 154 negative AFB cases, only four cases were detected as atypical mycobacteria after performing multiplex PCR as shown in Figure 2. Therefore rapid and sensitive tests for diagnosis of tuberculosis by using molecular methods are recommended for accurate treatment, and as well, direct detection of other strains mycobacterium by nucleic acid amplification techniques represents the most dramatic improvement in the field of diagnosis. Conclusion The development of molecular tools has added a new dimension to the classical epidemiology of tuberculosis and greatly enhanced the understanding of complex transmission dynamics within populations and between hosts. In the process, molecular epidemiology has demonstrated inadequacies in tuberculosis control programmes and helped accumulate motivation and resources for their improvement. Other technologies based on knowledge of the complete genome sequence of mycobacterium tuberculosis, which will provide newer

tools for probing the epidemiology of tuberculosis are now emerging. In spite of recent research advances, tuberculosis continues to remain a devastating infectious disease, disproportionately impacting on the world's poorest countries. The future challenge for molecular epidemiology is to provide better and early understanding of the transmission dynamics of tuberculosis in those countries with the greatest burden of disease, and to stimulate urgency of improving control measures on a more global scale.
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