Introduction
Tuberculosis (TB) has been one of the oldest infectious diseases
affecting human. It was identified 4000 years ago, from
the Middle East and Europe, as the cause of death, suggesting
that this disease has been already a widespread health problem
back then. In a detailed history, Hippocrates wrote about
patients with wasting away associated with coughing and chest
pain, often with blood in sputum. These symptoms had been
allowed Hippocrates to diagnose TB, which at that fourth
dimension has been called “consumption”. The occurrence of
descriptions of patients with these signs indicated that the disease
was already well entrenched in early times.1,2
TB is a communicable and deadly infectious disease
caused by mycobacteria, essentially Mycobacterium tuberculosis.
3 TB affects 8.8 million people each year, most of whom
dwell in low economic society.4 Additionally, an estimated
2 billion people are believed to be latently infected, providing a
great reservoir.5 Tuberculosis typically attacking the lungs
(as pulmonary TB) but can affect the central nervous system,
the lymphatic system, the circulatory system, the genitourinary
system, the gastrointestinal system, bones, joints, and even the
skin (extra pulmonary TB).6 Although in that respect other
mycobacteria such as Mycobacterium bovis, Mycobacterium
africanum, Mycobacterium canetti, and Mycobacterium microti
also can cause tuberculosis, these species are less common. The
usual symptoms of pulmonary tuberculosis are a chronic cough
with blood-tinged sputum, fever, night sweats and weight loss.
Tuberculosis considers an immunological disease and the
pathology of disease mediates by the host immune response.7
Diagnostic tests devoted to the rapid, sensitive, and specific
identification of the causative agent are key components
of successful wellness plans directed at disease control. Moreover,
the precise determination of Mycobacterial burden might
be beneficial for fast assessment of patient response to standard
therapy, particularly in those patients suspected of harboring
antibiotic resistant M. tuberculosis strains.8
Chest radiographs may be applied to exclude pulmonary
TB disease in a person with a normal immune system who has
a positive TST (tuberculin skin test) reaction or IGRA (IFN-?
release assay) and has no symptoms or signs of TB disease.9
Gain techniques for the diagnosis of tuberculosis have
attracted considerable interest in diagnosis, particularly with
the hope of reducing the time taken to find and identify Mycobacterium
tuberculosis in respiratory and non-respiratory
specimens.10 Real-time PCR has further advantages, including
precision, reproducibility, accuracy, quality control procedures
and reduced pollution. In addition, real-time PCR eliminates
the need for electrophoresis after the cycling reaction.
Furthermore, this technique cuts the analysis time for 3 or 4
days, which is important in lengthy microbiological diagnoses,
such as those needed for TB.11 The Gene X-pert system, a realtime
PCR that simultaneously detects both Mycobacterium
tuberculosis complex (MTBC) and rifampin resistance, was
developed.12 In contrast to some real-time PCR instruments,
the X-pert MTB/RIF is an on-demand assay described as a
simple method that can be performed by personnel with minimal
grooming and can provide answers within 2 h.13
The detection of rifampin resistance, as a surrogate for multidrug-
resistant TB (MDR-TB), directly from smear-positive respiratory
specimens from patients bearing a high risk of MDR-TB
has recently been advocated by the World Health Organization.14
Chemotherapy of drug susceptible TB consists of three or four
drug regimen, administered for 6 months. The long duration of
therapy solutions in poor compliance leading to the emergence
of multi-drug resistant forms of M. tuberculosis.15

Materials and Methods
Study Population
A total of 60 patients suspected with tuberculosis infection, in
the Specialized Chest and Respiratory Center, Hilla City
during the period from February to June 2015. The clinical
signs were recorded for each patient, including the night
sweating, fever, loss of weight, and history of dry cough.
Checklist sheets were drawn out for each patient including
age, gender, radiological finding, and incidence area. Sputum
samples were collected from TB according to the stated
procedure.
AFB Smear
The slides were flooded with carbol fuchsin dye. The slides are
heated slowly until it is steaming with direct flame for at least
5 minutes. The smear was decolorized with a decolorizing
agent and left until the color of the solution is gone. Then it
was washed with tap water. Methylene blue was applied to
slides by which the dye covered the entire slides surface and
left for maximum 60 seconds than washing with water. Microscopically
examination: The positive acid fast bacilli smear
appears as pink or red rods in a blue background.16 The
number of AFB was calculated by country number of cell in
smear according to standard method.17
Real Time PCR Assay
Extraction of DNA
The required number of 1.5 ml disposable polypropylene
micro centrifuge tubes was developed, including single tube
for Negative Control of Extraction (Negative Control, c-). Ten
microliter of the MTB IC (Internal Control) and 300 ?l of Lysis
Solution was added to each pipe. One hundred microliter of
samples was added to the appropriate tubes using pipette tips
with aerosol barriers. The control was prepared as follows:
Add 100 ?l of Negative Control C- to the labeled Cneg. Vortex
the tubes and incubated for 5 min at 65?C. Centrifuged for 10
s. Four hundred microliter of Prec solution was added and
mixed by vortex. Centrifuged all tubes at 13,000 rpm for 5 min
and using a micropipette with a plugged aerosol barrier tip,
carefully removed and discarded supernatant from each tube
without disturbing the pellet. Changed tips between the tubes.
Five hundred microliter of Wash Solution was added into each
tube. Vortex vigorously rinsed to ensure pellet washing. Centrifuged
all tubes at 13,000 rpm for 60 s using a micropipette
with a plugged aerosol barrier tip. Carefully removed and discarded
supernatant from each tube without disturbing the
pellet. Changed tips between the tube. Two hundred microliter
of Wash Solution 4 was added to each tube. Vortex vigorously
rinsed to ensure the pellet washing. Centrifuge all tubes
at 13,000 rpm/min for 60 s and using a micropipette with a
plugged aerosol barrier tip. Carefully removed and discarded
supernatant from each tube without disturbing the pellet. The
tubes were incubated with open caps at 65? C for 5 min. Resuspended
the pellet in 50 ?l of RE- buffer (the elution volume
can be increased upwards to 90 ?l). Incubate for 5 min at 65?C
and vortex periodically. The tubes were centrifuged at 13000 g
for 60 s. The supernatant contains RNA/DNA ready for amplification.
If amplification is not performed on the same day of
extraction, the processed samples can be stored at 4?C for a
maximum period of 5 days or frozen at –20?C for a long time.

Amplification Mix Preparation
In the new sterile tube each sample was prepared 10* (n+1) ?l
of PCR-mix-1, 5* (n+1) ?l of PCR Buffer Flu, 0,5* (N+1) ?l
Taq DNA Polymerase and 0,5* (N+1) ?l of UDG Enzyme.
Vortex rinsed gently and centrifuged briefly. Fifteen microliter
of Reaction Mix was added to each tube. Ten microliter of
extracting DNA was added to appropriate tube. Two controls
were prepared for each panel: 10 ?l of DNA-buffer was added
to the tube labeled Amplification Negative Control. 10 ?l of
C-MTB&IC was added to the tube labeled Amplification
Positive
Control. The tubes were ready to insert in to the thermalcycler.
Data analysis: The fluorescent single intensity was
detected in two channels: Mycobacterium tuberculosis was
detected on the FAM (Green) channel, IC DNA on the JOE
(Yellow) / HEX/Cy3 channel.
Gene X-Pert MTB/RIF Assay
Preparing the Sputum Samples
Each X-pert MTB/RIF cartridge was labelled with the sample ID.
In a leak-proof sputum collection container, a. The lid of the
sputum collection container was carefully opened. b. Approximately
2 times the volume of the SR was poured to the sputum
(2:1 dilution, SR:sputum). c. The lid was replaced and secured. d.
Shaked vigorously 10 to 20 times by vortex for at least 10 s.
The sample was incubated for 10 minutes at room temperature,
and then shaken the specimen vigorously 10 to 20
times or vortex for at least 10 seconds. The sample was
incubated
at room temperature for an additional 5 minutes.
The Test Procedure
The computer was turned on, farther Turn on the Gene
X-pert instrument. Open the Software shortcut icon of the
Gene X-pert in the Windows® desktop by double-click on it.
User name and password have been used to log on the
Gene-
X-pert Dx System software. Click Create, Test, in the
Gene X-pert Dx System window. The Scan Sample ID dialog
box appeared. In the Sample ID box, scan or type the sample
ID. Make sure you type the correct sample ID (sample ID is
associated with the test results and is shown in the View
Results window and all the reports). The Scan Cartridge Barcode
dialog box appears. The barcode on the X-pert MTB/
RIF cartridge has been scanned. The Create Test window
appears. Using the barcode information, the software
automatically
fills the boxes for the following fields: Select
Assay, Reagent Lot ID, Cartridge SN, and Expiration Date.
Click Start Test. Enter your password if requested. The instrument
module door with the blinking green light has been
opened, and loaded the cartridge. The door was closed. The
test starts and the green light stop-blinking.
When the test is finished, the light turns off. Wait until the
system releases the door lock at the end of the run, then open
the module door and remove the cartridge. Reading the Results:
The Gene X-pert Instrument system generates the results from
measured fluorescent signals and embedded calculation algorithms.
The results can be seen in the View Results window.

Results
Among 60 samples, only 10 samples which give a positive AFB,
50 samples give a negative AFB result, 39 samples positive,
21 samples negative results for Real-Time PCR, 43 samples positive,
17 negative results for Gene X-pert respectively as shows
in Table-1.
Discussion
Gene X-pert MTB/RIF technique, Real-Time PCR recording the
high sensitivity for AFB positive (100%) for both, for AFB negative
(66%, and 58%) respectively, AFB sensitivity was (16.6%),
and the specificity was (100%) as shown in Table 1. The sensitivity
of real-time PCR and X-pert MTB/RIF are given similar results
with smears positive, but it is low in real-time technique for
smears negative. These are matching with Armand et al.,18 who
suggested that both methods were highly specific and exhibited
excellent sensitivity (100%) with smear-positive specimens, but
does not match in the sensitivity of the X-pert MTB/RIF test with
the smear-negative specimens were more reduced than that of
the real-time PCR assay (48 versus 69%, P < 0.005).
American Thoracic Society Workshop,19 who estimated
that the Gene X-pert assay was introduced lately. Its advantage
lies in higher sensitivity and specificity for the diagnosis of TB,
together with the detection of drug resistance, and in
smear-positive specimens. The sensitivity and specificity of
PCR are in the range 90–100%, with a positive prognostic
value of > 95%, whereas in smear-negative specimens, the
sensitivity
of PCR was reduced to < 50%.
Our study found that the sensitivity of Gene X-pert for
AFB smear- negative was (66%). These results agreed with the
work reported by Zeka et al.,20 who suggested that the X-pert
MTB/RIF assay have reported test sensitivities of 57 to 76.9%
in cases of smear-negative.
In our study, real-time PCR has a second value of sensitivity
(69%) for smear negative after the Gene X-pert.
These results were in accordance with those results reported
by Raviglion,21 who proposed that the real-time PCR assay
with internal control achieved a sensitivity of 96.2 % and
specificity of 99.2%. Templeton et al.,22 explains that
internal control reaction has been included to determine
the robustness of the PCR resulting by monitoring the
nucleic acid extraction as well as the presence of
inhibitors.
Rajpal et al.,23 stated that direct microscopy may give
negative results if the number of AFB less than 5000
bacilli/ml, consistently positive specimens would have to
contain 105 bacilli per ml varying with the extent of the
wound or the presence citations. The scanty or numerous
AFB smear test, which differs according to the number of
bacilli in microscopy filed.
In our study, the sensitivity for the diagnosis of M. tuberculosis
of Gene X-pert and real-time PCR higher than AFB
because the Gene X-pert and real-time PCR depends on diagnosis
on DNA amplification and have ability to detect less than
10 of acid fast bacilli per ml in a sputum sample while AFB
smear depending on the ability of M. tuberculosis to take stain
and, may give a negative result if there few number of acid fast
bacilli less than 5000 bacilli per ml in the field.
Conclusions
The comparison between advance technique (Gene X-pert
and Real time PCR) and classical technique (AFB) for the
diagnosis of MTB, show that genetic technique is the best with
high sensitivity, and specificity.
Consent
All authors declare that written informed consent was obtained
from the patients for publication of this research article.
Competing Interests
Authors have declared that no competing interests exist.