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 Ghani1, Hafeez ul Hassan2, Hasan Ali3, Mohammad Usman4, Mulazim Hussain
Bukhari5, M. Akram6* and Asif Iqbal6
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. 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 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
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 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
 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 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.

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 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 false-
positives 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 manage-
ment 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 diag-
nosis is needed to establish a patient's prognosis or in
organ transplant cases (Morán Moguel et al., 2000).
Finally, one-tube regular-PCR, a variant which used
specific 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 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. There-
fore 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, tuber-
culosis 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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