Rapid and specific detection of Mycobacterium tuberculosis directly from sputum specimens using IS 6110 and pncA through multiplex-PCR

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis complex, is the second leading cause of death. One third of the world’s population is infected with TB. Every day more than 23000 people develop active TB and nearly 5000 die. Approximately 20-50% of patients with TB are smear negative, accounting for as much as 17% of TB transmission, posing an important public health hazard. In the objective of improved TB diagnosis and rapid detection of M. tuberculosis complex species particularly from paucibacillary sputum specimens, the sensitivity of Polymerase Chain Reaction (PCR) using IS6110 and pncA as genetic targets was compared with the conventional fluorescent microscopy. The study also aimed to check the sensitivity of two different genetic markers IS6110 and pncA for the detection of M. tuberculosis. Sputum specimens were collected from TB suspected subjects (N= 200; males=108; females=92; mean age=47±18.4) attending outpatient department at Fatima Jinnah General and Chest Hospital, Quetta. All the samples were decontaminated using N-acetyl-Lcysteine (NALC)-NaOH method and were subjected to auramine-O fluorescent microscopy and multiplex PCR using TB1 and TB2 primers specific for M. tuberculosis complex and pncATB1.2 and pncAMT-2 primers specific to M. tuberculosis. Out of 200 specimens, M. tuberculosis around 15 (7.5%) was detected by fluorescent microscopy and 33 (16.5%) were detected through


Introduction
Tuberculosis is an ancient, extensively prevalent, and more often deadly, contagious disease caused by M. tuberculosis complex species, usually M. tuberculosis [1].TB has proceeded to be a "slate wiper" in the human history throughout centuries and was titled "consumption" and "white plague" in the 20 th century.It was world's major cause of death from all causes, responsible for one death in every seven cases [2].Today's statistics are still alarming.Globally, TB ranks 2 nd major cause of death after HIV by an infectious disease and WHO estimates that some 2 billion individuals, 1/3 rd population of the world, are infected with M. tuberculosis [3].Each day some 23000 new TB cases develop and nearly 5000 people die.Annually, approximately 8.7 million active TB cases and 1.7 million deaths are reported.If TB was not massively controlled by TB control programs, another estimated 1 billion people globally will become infected and 40 million will die of TB by 2020.Mortality is highest in developing countries, where over three-quarters of TB-cases occur [2,4].TB is prevalent and a serious national health problem in Pakistan.Globally, Pakistan ranks 5 th among the 22 High Burden Countries (HBCs) and 4 th among the 27 countries with highest burden of Multi Drug Resistant (MDR) TB [3].About 420,000 new cases of TB develop every year, with an incidence of 231/100,000 and 60,000 people die of the disease.Of these (420,000), around 175,000 cases are sputum smear positive and 9000 are the cases of MDR TB while remaining of the cases are "sputum smear negative

Collection of the sputum specimens
A total of 200 sputum samples (male=102 and female=92) were collected in sterile prelabeled containers.Specimens were collected from all TB suspects showing the classical symptoms of disease such as the history of prolonged more than two weeks' chronic cough, fever and chest pain.Gender and age of the TB-suspects were also recorded.

Sample processing
All the sputum specimens were decontaminated and liquefied using the standard N-Acetyl-L-cysteine (NALC)-NaOH method [16] and concentrated by centrifugation in Biosafety Level III Provincial Reference Laboratory for TB.
The resuspended sediments were used for preparation of AFB smears and rest (500 µl) of the sediments were rendered noninfectious by heat killing.The heat killed samples were then transported to CASVAB and stored at -20°C until used for PCR.

Fluorescent microscopy
Smears were stained with auramine-O staining technique [16].All the smears were then examined under florescent microscope using 40X objective.Polymerase chain reaction DNA was extracted using standardized cetyltrimethyl ammonium bromide (CTAB) method as described by Ausubel et al. [17].
Extracted DNA was subjected to PCR using two sets of oligonucleotide primers (Macrogen) as shown in Table 1.The multiplex PCR was carried out by amplifying 123-bp fragment of the IS6110 specific for M. tuberculosis complex and 185-bp fragment of the pncA gene for specific for M. tuberculosis (Figure 1).The multiplex PCR was performed in a total volume of 25 µl reaction mix which contained 1X Taq buffer, 2.5mM of MgCl2,50µM of each dNTP,1.25 U of Taq DNA polymerase, 10 pmol of each of both oligonucleotide primers IS6110 and pncA (Forward and Reverse), and 4µl Template DNA.Amplification was performed using automated thermal cycler (Applied Biosystems, 2720).The mixture was first denatured at 96°C for 5minutes, followed by35 cycles of PCR with denaturation at 94°C for 45 seconds, annealing at 57°C for 45 seconds, extension at 72°C for 45 seconds and the final extension at 72°C for 7 minutes.A negative control (without DNA template) and a positive control containing M. tuberculosis DNA were also included in each PCR run.The amplified PCR products were analyzed by agarose gel electrophoresis (2% W/V) at 100 Volts for 1 hour followed by staining with ethidium bromide (0.5mg/ml).The electrophoresed amplicons were visualized and documented using UV transilluminator (Wealtec Dolphin-View, USA).The 50-bp DNA marker (Vivantis, Malaysia) was used as a reference to estimate the DNA bands.) by PCR with both genetic markers (Figure 2).All the 15 smear positive specimens were also positive by PCR.Among these 33 (16.5%) positive samples 18 (16.6%)were males and 15 (16.30%) were females.The sensitivities of auramine-O fluorescent microscopy, IS6110 and pncA-PCR were 45%, 100% and 100%, respectively, whereas the specificities of all three were 100% (Table 2).Sputum sample analysis revealed that both males and females were approximately at equal risk of TB infection (OR= 1.03, 95% CI: 0.48-2.17).Therefore, the difference in the incidence of TB between males and females was statistically non-significant (χ 2 = 0.005, df= 1, p= 0.945).The proportions of study subject with age categorized as 1-20, 21-40, 41-60 and >60 were observed 15%, 33.0%, 33.55%, and 18.50%, respectively.TB patients with age 41-60 years were 1.7fold more likely to be infected with TB as compared with patients less than 20 years of age (95% Cl: 0.25-2.45).However, age was not found to be statistically associated with TB risk (χ 2 = 1.33, df=3,p= 0.722) (Table 3).cited the males as being more likely to be infected with TB infection.WHO report on TB incidence and gender demonstrated that prevalence of TB infection was higher in males at all ages except in childhood and that the gender differential incidence appears to begin at age ten and sixteen years; reasons for increased incidences and prevalence among males are poorly understood and need further investigation Age is implicated as one of the important risk factors for TB.Patients aged between 41-60 years were 1.7-fold (95% CI: 0.25 -2.45) at higher risk of acquiring the disease compared with <20 years old patients (20.89% vs. 13.0%).This may be attributed to weakened and compromised immune system in the old age.However, the age was not found significantly associated with TB infection (p>0.05).This finding is consistent with the previous studies which cited TB infection to be considerably increased with age [36-38].

Conclusion
In conclusion, the findings of our study strongly suggested the use the multiplex PCR using the "IS6110" and "pncA" in the routine analysis as a potential tool for the rapid TB diagnosis due to its increased sensitivity.

Figure 2 .
Figure 2. Bar chart showing the frequency of positive TB cases detected through fluorescent microscopy and PCR using IS6110 and pncA specific genetic markers.
Conceived and designed the experiments: MA Awan, M Rizwan & M Shafee, Performed the experiments: M Rizwan, Analyzed the data: M Naeem, SK Achakzai, S Ashraf & Z Ahmed, Contributed reagents/ materials/ analysis tools: A Samad.Wrote the paper: H Haider & M Rizwan & FS Bugti.