8/12/2019 70-RUBINAAJB-11-2248

1/6

African Journal of Biotechnology Vol. x(xx), pp. xxx-xxx, xx xxxxx, 2011Available online at http://www.academicjournals.org/AJBDOIXXXXXXISSN 16845315 2011 Academic Journals

Full Length Research Paper

Rapid detection of typical and atypical Mycobacteriumtuberculosisby polymerase chain reaction (PCR) andits comparison with Ziehl Neelsen staining technique

Rubina Ghani1, Hafeez ul Hassan2, Hasan Ali3, Mohammad Usman4, Mulazim HussainBukhari5, M. Akram6* and Asif Iqbal6

1Department of Biochemistry, Baqai Medical University, Karachi, Pakistan.

2Department of Physiology, Baqai Institute of Hematology, Baqai Medical University, Karachi, Pakistan.

3Department of Biochemistry, Bahria University, Medical and Dental College, Karachi Campus Pakistan.

4

Department of Hematology, Baqai Medical University, Karachi, Pakistan.5Department of Pathology, King Edward Medical University Lahore, Pakistan.

6Faculty of Eastern Medicine, Hamdard University, Karachi, Pakistan.

Accepted 28 October, 2011

Tuberculosis is a challenging problem and it represents the major health problem in developingcountries like Pakistan. In view of the importance of early diagnosis of tuberculosis, the efficiency ofpolymerase chain reaction (PCR), one of the most reliable and sensitive DNA based assays, wascompared with conventional method for the detection of mycobacterium tuberculosis. We hypothesizethat PCR is a more sensitive method for the detection of acid fast bacilli (AFB) as compared to ZiehlNeelsen (ZN) staining. To affirm this, a total of 200 sputum samples were analyzed. A simultaneousanalysis 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%) sputumwere positive for mycobacterium tuberculosis in both sexes. From 154 negative samples for AFB, on ZNstaining, 4/154 (2.6%) samples had the positive atypical mycobacteria by PCR using specificprimers. We conclude that PCR is a more sensitive method for the detection of AFB as compared to ZNstaining

Key words:Sputum, PCR, acid fast bacilli (AFB), atypical mycobacterium.

INTRODUCTION

Tuberculosis (TB) is one of the leading infectiousdiseases of the world and is responsible for 2.9 million

worldwide deaths and 8.8 million new cases of active TBoccur per year (Dye et al., 1999; Kwara et al., 2008).Whereas, WHO has also declared tuberculosis a globalemergency as there has been a 20% increase in itsincidence over past decades (Corbett et al., 2003; Mehnazet 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 tha95% 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 newcases and 64,000 deaths occur due to tuberculosis eachyear (Saeed et al., 2002).

The diagnosis of mycobacterial infection is accom-plished by

culture-based identification. Primary culture o

slowly growingmycobacteria without using the BACTEC

culture system, usuallytakes four to six weeks or longe

(Yeager et al., 1967). Conventional bacteriology such asdirect microscopy and culture are not sufficient for theearly diagnosis of tuberculosis because there are fewbacilli in the sputum to be demonstrated by direct

8/12/2019 70-RUBINAAJB-11-2248

2/6

microscopy and on the other hand, successful culturalidentification of tubercle bacilli takes about seven to eightweeks (Rafi et al., 2003). The initial diagnosis of tuber-culosis is usually based on clinical grounds, but definitediagnosis would require the isolation and identification ofthe 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 rapidpresumptive test for tuberculosis. Subsequently, whencultured, Mycobacterium tuberculosis would grow veryslowly producing distinct non-pigmented colonies afterseveral weeks. M. tuberculosiscan be differentiated frommost other mycobacteria by the production of niacin(Soini et al., 2001).

However, recent methodologicaladvances in molecular

biology have provided alternative rapidapproaches, such

as the polymerase chain reaction (PCR) and PCR-linkedmethods. A rapid alternative method to culture is PCR;for the rapid detection

or identification of M. tuberculosis,

target genes specific primersto mycobacteria are used ina PCR (Eing et al., 1998; Gunisha et al.,2001; Montenegroet al., 2003). In this study, we used a simplified multiplexPCR assay

to identify mycobacterium tuberculosis

and

another strain of mycobacteria like atypical mycobacteriacomplex in our population.

MATERIALS AND METHODS

Collection of specimens

The simple descriptive study was carried out in the laboratorywhere the 200 sputum samples were collected as a routine. From

each patient, samples were collected for three consecutive dayswith brief clinical history. All 200 cases fulfilled the following criteriawith the clinical history of mycobacterial infection in the family.

Criteria of patients selection

Samples of those patients were collected whose chest x-rayshowed radiographic consolidations suspicious of pulmonary tuber-culosis; either patchy or nodular infiltrate in upper lobes or superiorsegment of lower lobes, bilateral upper lobe infiltrate with patchysoft shadows with or without cavitations. In some cases, there werebilateral upper lobe infiltrate with patchy soft shadows withoutcavitations. Besides, on the basis of chest x-rays evaluation ofrespiratory 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 (themost characteristic location, most visible in an apical lordotic view)suggest the reactivation of TB. Middle and lower lung infiltrateswere nonspecific but prompt suspicion of primary TB in patients(usually young) whose symptoms or exposure history suggestedthe recent infection, particularly if there was pleural effusion.

Direct smear preparation

For the AFB smear, morning sputum samples of three consecutivedays were collected in sterile, wide mouthed plastic bottles. All thesamples 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 wasincubated at room temperature for 10 to 15 min and shaken aregular intervals. Then, 8 ml of distilled water were added and thesamples were centrifuged at 3000 g for 15 to 20 min. The super-natant was discarded and the pellets were suspended in few dropsof the remaining fluids. Slides were prepared from the suspendedsediment, air-dried, heat fixed and stained by Ziehl Neelsenmethod.

Microscopic examination and interpretation of the result

The sputum smear were prepared and stained with ZN method foAFB. The smears were covered with tissue papers flooded slidesusing strong carbol fuchsion for 5 min while heating with steamThe paper was removed and decolorized with acid alcohol andcounter was stained with malachite green. After staining, more than100 field of each smear were examined carefully under the lightmicroscope using the oil immersion 100 X lens and interpretation ofthe 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 andatypical mycobacteria in these specimens by using PCR, and thesignificant diagnostic value of PCR were observed by comparingwith conventional methods (acid fast microscopy).

Grading of AFB

The numbers of acid-fast bacilli seen on the smears were recordedaccording to the recommendation by WHO. When there was noAFB seen per 100 oil immersion fields, it was graded as negativeWhen there were one to nine AFB seen per 100 oil immersionfields, the grade was reported scanty. When there were 10 to 99AFB seen per 100 oil immersion fields, the grade was given +1When they were 1-10 per oil immersion field, they were graded as+2 and when the number of AFB were more than 10 per oi

immersion fields, they were graded as +3-. (Aung et al., 2001).

DNA extraction

The DNA was extracted from sputum by using 4% NaOH. Equavolume of NaOH was added to the sputum in Eppendorf cup andwas incubated at room temperature for 30 min to digest the sputumsamples. Then, the digested sputum was centrifuged at 13,200 rpmfor 10 min and supernatant was discarded. The pellet was furtheprocessed following Gentra kit procedure, then the DNA pellet wasresuspended with DNA hydration solution present in Gentra Kit andPCR was performed (Drosten et al., 2003) according to thefollowing procedure; the PCR was carried out in a tube containing20 l of a reaction mixture made up of the following components: 10

pmol of each forward and reverse primers for mycobacterium andatypical mycobacteria, 500 M of four deoxynucleotides, 2 U of Taqpolymerase (Promega), 10 x PCR buffer containing and 1.5 mMMgCl2.

The thermal cycler (Master Gradient PCR System, EppendorAG, Germany) was programmed to first incubate the sample for 5min for 95C followed by 30 cycles consisting of 94C for 45 s, 56Cfor 45 s and 72C for 1 min with final extension for 7 min at 72CThe PCR amplified products were identified by electrophoreses ona 2% agarose gel, stained with ethidium bromide, and evaluatedunder transilluminator. The sizes of PCR amplified product wereestimated according to the migration pattern of a 100-bp DNAladder (Gibco BRL Life Technologies) (Kox et al., 1994; Noordhoeket al., 1994; Kocagoz et al., 1993).

8/12/2019 70-RUBINAAJB-11-2248

3/6

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 samplesstained 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 onthree 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'-CGTACG GTC GGC GAG CTG ATC CAA-3') and 5'-C CAC CAG TCG

GCG CTT GTG GGT CAA-3' were designed to amplify a 541bpfragment while the primers used for atypical mycobacteria were 5'-G GAG CGG ATG ACCACC CAG GAC GTC-3' and 5'-CAG CGGGTT GTT CTG GTC CAT GAA

C-3' to amplify 200 bp.

RESULTS

A total of 200 sputum samples were collected fromsuspected cases of pulmonary tuberculosis selected onthe bases of history, clinical examination and chestroentogram. All the cases were between the ages of 15to 70 years with mean age 46.5 2 for both sexes, out ofwhich 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, onlyone to nine AFB per 100 oil immersionfields was detected;only on third day specimen was AFB positive and gradedas +1, after a long search. It was also noted that in 20cases all the three samples were AFB positive by stainingwith ZN stain; in each field >10 AFB per oil immersion,field was seen and were graded as +3. In 14 cases onday 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, whereasin 154 patients, the AFB was negative in all the three

sputum samples and all the results were interpretatedaccording to National TB Control Program as summarizedin Table 3.

AFB positive and negative cases were further confirmedby multiplex PCR for the detection of mycobacteriumtuberculosis and atypical mycobacteria. Among the 200cases, PCR assay correctly identified 46 cases whichwere as well smear positive. However, this assay failed topick 154 smear negative cases giving a sensitivity o92%. There was no false positive case in these smeapositive specimens, giving a specificity of 100% for thetest by PCR.

Similarly, in 154 samples, AFB was negative although

the symptoms were identical to M. tuberculosis. Furthemolecular assay was used for the identification of anothestrain of mycobacteria causing the same symptoms(atypical mycobacteria) by PCR using specific primersThere were 4/154 (2.6%) atypical mycobacteria detectedby PCR from AFB negative smears using specificprimers. In comparison to AFB among samples fromclinical TB patients, sensitivity, specificity, and predictivevalue of positive test by PCR was 100.0%. In Figure 1gel shows the 541 bp positive cases for mycobacteriumtuberculosis. Figure 2 indicates that the AFB negative in

8/12/2019 70-RUBINAAJB-11-2248

4/6

N P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

541 bp

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

DNA ladder of 50 bp.

1 2 3 4 5 6 7 8 9 10 11 12 N P P M

541 bp

200 bp

Figure 2.The analysis of Mycobacterium tuberculosisDNA 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 arethe positive control with both atypical and Mycobacterium tuberculosisdetected in the sputum sample; lane 16 is the DNAladder of 50 bp.

the sputum samples of patients with same symptoms, asM. tuberculosis were atypical mycobacteria amplified at200 bp. These could be efficiently differentiated usingPCR by specific primers.

DISCUSSION

Before the introduction of molecular typing methods, therewas little to aid the distinction between individual strains

of M. tuberculosis (Malik et al., 1998). Traditionalmethodsof diagnosing tuberculosis have been the isolation obacilli in culture or recognition of AFB in clinicaspecimens. The diagnostic value of various methodswidely used in microbiological diagnosis of tuberculosisdirect smear examination for acid-fast bacilli, culturaidentification in Lowenstein-Jensen (L-J) medium, theradiometric BACTEC 460 system and PCR, to evaluatethe time factor and the sensitivity of the clinical methodhas been reported. AFB staining lacks sensitivity. So far,

8/12/2019 70-RUBINAAJB-11-2248

5/6

the gold standard has been culture with a dividing timeof 48 h, up to 10 weeks. However, the highnumber of false-positives that we found suggests that results obtainedshould be confirmed with BACTEC, which considerablyreduces the time required for identification, and makes itpossible to carry out an antibiotic assay rapidly (Ginesu

et al. 1998).This study was conducted in molecular laboratories forthe early and cost effective diagnosis for the manage-ment of TB. We demonstrated the genetic technology;polymerase chain reaction. M. tuberculosis is one of themost successful bacterial

pathogens in the history of

mankind. In this study, out of 200 sputum samples, 46AFB positive sputum samples clinically showed TB byboth methods; PCR and AFB Stain. Our findings areconsistent with that of Moran Moguel et al. (2000) whoaccording to WHO, reported that PCR is a sensitive andspecific technique for detecting the M. tuberculosiscomplex in both positive and negative bacilloscopysamples. A controlled PCR procedure makes it possibleto establish or to exclude the diagnosis of tuberculosis ina time that is reduced from more than six weeks to just24 to 48 h. This is particularly useful when an early diag-nosis is needed to establish a patient's prognosis or inorgan transplant cases (Morn Moguel et al., 2000).

Finally, one-tube regular-PCR, a variant which usedspecific primers for M. tuberculosis and atypical myco-bacteria, 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 earlymicrobiological diagnosis of tuberculosis, given its highsensitivity and specificity and unparalleled rapidity. In thisstudy, PCR for M. tuberculosisand atypical positive in the

sputum were to ascertain an etiological diagnosis, withspecificity and sensitivity of the AFB positive resultsshown in Figure 1. In 154 negative AFB cases, only fourcases were detected as atypical mycobacteria afterperforming multiplex PCR as shown in Figure 2. There-fore rapid and sensitive tests for diagnosis of tuberculosisby using molecular methods are recommended foraccurate treatment, and as well, direct detection of otherstrains mycobacterium by nucleic acid amplificationtechniques represents the most dramatic improvement inthe field of diagnosis.

Conclusion

The development of molecular tools has added a newdimension

to the classical epidemiology of tuberculosis

and greatly enhanced

the understanding of complextransmission dynamics within populations

and between

hosts. In the process, molecular epidemiology has

demonstrated inadequacies in tuberculosis controlprogrammes

and helped accumulate motivation and

resources for their improvement. Other technologies basedon knowledge of the complete genome

sequence of

mycobacterium tuberculosis, which will provide newer

toolsfor probing the epidemiology of tuberculosis are now

emerging.In spite of recent research advances, tuber-

culosis continues

to remain a devastating infectiousdisease, disproportionately

impacting on the world's

poorest countries. The future challengefor molecula

epidemiology is to provide better and early understanding

of the transmission dynamics of tuberculosis in thosecountrieswith the greatest burden of disease, and to

stimulate urgencyof improving control measures on a

more global scale.

REFERENCES

Aung WW, Nyein MM, Ti T, Maung W (2001). Improved method odirect microscopy for detection of acid-fast bacilli in sputumSoutheast Asian J. Trop. Med. Public Health, 32(2): 390-393.

Corbett EL, Watt CJ, Walker N, Maher D, Williams BG, Raviglione MC(2003). The Growing Burden of Tuberculosis Global Trends andInteractions With the HIV Epidemic. Arch. Int. Med. 163(9): 10091021.

Drosten C, Panning M, Kramme S (2003). Detection of Mycobacteriumtuberculosis by Real-Time PCR Using Pan-Mycobacterial Primersand a Pair of Fluorescence Resonance Energy Transfer ProbesSpecific for the M. tuberculosis Complex. America Association ClinicChemical, pp. 1659-1661.

Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC (1999)Consensus statement. Global burden of tuberculosis: estimatedincidence, prevalence, and mortality by country. WHO GlobaSurveillance and Monitoring Project. JAMA. 282(7): 677-686.

Eing BR, Becker A, Sohns A, Ringelmann R (1998). Comparison oRoche Cobas Amplicor Mycobacterium tuberculosis Assay with InHouse PCR and Culture for Detection of M. tuberculosis. J. ClinMicrobiol. 36(7): 2023-2029.

Ginesu F, Pirina P, Sechi LA, Molicotti P, Santoru L, Porcu L(1998)Microbiological diagnosis of tuberculosis: A comparison of old andnew methods. J. Chemother.10(4): 295-300.

Khan MA, Mirza SH, Abbasi SA, Butt T, Anwar M (2006). Periphera

blood-based Polymerase Chain Reaction in diagnosis of pulmonaryTuberculosis. J. Ayub. Med. Coll. Abbottabad, 18(2): 25-28.

Kocagoz T, Yilmaz E, Ozkara S, Kocagoz S, Hayran M, Sachedeva M(1993). Detection of Mycobacterium tuberculosis in sputum samplesby polymerase chain reaction using a simplified procedure. J. ClinMicrobiol. 31(6): 1435-1438.

Kox LF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, VanLeeuwen J (1994). A more reliable PCR for detection oMycobacterium tuberculosis in clinical samples. J. Clin. Microbiol32(3): 672-678.

Kwara A, Herold JS, Machan JT, Carter EJ (2008). Factors AssociatedWith Failure To Complete Isoniazid Treatment for LatenTuberculosis Infection in Rhode Island. Chest, 133(4): 862.

Malik N, Karamat KA, Butt T, Abbasi S, Usman J (1998). Prevalenceand drug susceptibility pattern of typical and atypical mycobacteria inRawalpindi/Islamabad. Pak. J. Pathol. 9: 4-8.

Mehnaz A, Arif F (2005). Applicability of scoring chart in the early

detection of tuberculosis in children. JCPSP, J. College of PhysSurg. Pak. 15(9): 543-546.

Montenegro SH, Gilman RH, Sheen P, Cama R, Caviedes L, Hopper T(2003). Improved Detection of Mycobacterium tuberculosis inPeruvian Children by Use of a Heminested IS6110 Polymerase ChainReaction Assay. Clin. Infect. Dis. 36(1): 16-23.

Morn Moguel MC, Aceves Hernndez D, Pea Montes De Oca PMGallegos Arreola MP, Flores Martnez SE, Montoya Fuentes H(2000). Detection of Mycobacterium tuberculosis with polymerasechain reaction in a selected population in northwestern MexicoRevista Panamericana de Salud Pblica, 7: 389-394.

Noordhoek GT, Kolk AH, Bjune G, Catty D, Dale JW, Fine PE (1994)Sensitivity and specificity of PCR for detection of Mycobacteriumtuberculosis: a blind comparison study among seven laboratories. J.

8/12/2019 70-RUBINAAJB-11-2248

6/6

Clin. Microbiol. 32(2): 277-284.Rafi A, Naghily B (2003). Efficiency of polymerase chain reaction for the

diagnosis of tuberculous meningitis. Southeast Asian J. Trop. Med.Public Health, 34(2): 357-360.

Saeed W, Ahmad J, Naseem A (2002). Endobronchial tuberculosis:clinical and diagnostic aspects. Pak. Armed Forces Med. J. 52(2):154-158.

Soini H, Musser JM (2001). Molecular Diagnosis of Mycobacteria. Clin.

Chem. 47(5): 809-814.Yeager Jr H, Lacy J, Smith LR, LeMaistre CA (1967). Quantitative

studies of mycobacterial populations in sputum and saliva. Am. Rev.Respir. Dis. 95(6): 998-1004.