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,
would grow very slowly producing distinct non-pigmented colonies after several weeks.
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
, 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
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).
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
. 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).