Tuberculosis (TB) is an ancient infectious disease, which is now spread to nearly every part of the globe1. Tuberculosis is a major public health problems2. About one third of the world's populations is infected with M.tuberculosis3. The emergence of Human Immunodeficiency Virus (HIV) infection and the Acquired Immunodeficiency Syndrome (AIDS) ended this optimism and fuelled the resurgence of TB worldwide4. Recognizing the importance of the impact of TB globally, in April, 19935, the World Health Organization (WHO) declared tuberculosis a global public health emergency.
There were an estimated 9.6 million new TB cases in 2014. Also, 1.5 million TB deaths. The number of TB deaths is unacceptably high. In India, one fourth of the global incidence TB cases occur annually. Out of estimated global annual incidence of TB cases, 2.2 million (167 person per lakh of population) were estimated to have occurred in India. Whereas prevalence was 2.5% (195 person per lakh of population). Also, India had the largest number of TB cases i.e. 23% of the global6. Globally, 3.3% of new TB cases and 20% of previously treated cases have MDR-TB. Whereas In India, 2.2 % of new TB cases and 15% of previously treated cases have MDR-TB in 20146.
M.tuberculosis is aerobic, gram positive rod shape bacterium and non-motile. It has no capsule and does not form spore, slightly curved or straight rods, 0.2 to 0.6''m by 1.0 to 10''m, which may branch7. It is characterized by a thick and complex lipid rich cell wall which is commonly associated with pathogenesis8 and considerably slow growth9.
In most of the cases, M.tuberculosis is the causal agent of TB in human. This illness can be caused by other mycobacteria in some cases with special conditions10. These mycobacteria belong to member of MTB complex. The member of MTB complex include M.tuberculosis, M.bovis, M.africanum, M.pinnipedi, M.caprae, M.microti and M.canetti11.
It is worth mentioning that there are also other mycobacteria called Non Tuberculous Mycobacterium (NTM). These are omnipresent in environment and in extreme circumstances like immune-compromised, some of them become pathogenic for humans and can induce opportunistic infections12.
Currently there are 200 known mycobacterial species7. Most of them being saprophytic soil bacteria and a minority of these species is pathogenic to human causing mycobacterial disease13.
TB is spread from person to person through the air by droplet nuclei. Which are produced when pulmonary or laryngeal TB cough, sneeze, sing or speak. Also, by aerosol treatments, sputum induction and aerosolization during bronchoscopy, through manipulation of lesion or processing of tissue or secretion in the hospital or laboratory14.
Droplet nuclei are so small that air currents normally present in any indoor space can keep them airborne for long periods of time15. After inhalation, droplet nuclei are carried down the bronchial tree and implant in respiratory bronchiole or alveolus. Whether or not an inhaled tubercle bacillus establishes an infection in the lung depends on both the bacterial virulence and the inherent microbicidal ability of the alveolar macrophage that ingests it16-17. Though TB is predominantly a disease of respiratory system but it can also affect other parts of the body.
Individuals with inactive TB infection are not infectious and this can't transmit M.tuberculosis. Approximately 10 % of persons who are infected with TB infection and are not given appropriate therapy will develop active TB disease, with the highest risk occurring in the first two years after infection18.
The increasing number of mycobacterial infections has made it important to quickly identify mycobacteria. The diagnosis of pathogenic species not only has epidemiological implications but is also applicable for patient management.
Although, diagnosis of tuberculosis can be made by different methods like clinical, immunological, and radiological methods. But, the identification of causative organism M.tuberculosis in the clinical samples is the most accurate and reliable method. 19.
Various approaches are being used in the diagnosis of MTB complex20. Lower sensitivity and longer incubation period of tests especially with the routine diagnostic procedures still poses a major problem in an effort to detect this organism.
Smear microscopy is still one of the most rapid and most inexpensive ways to diagnose tuberculosis. However, the reliability of smear microscopy is highly dependent not only on the experience of the laboratory technician but also on the AFB present in the specimen. Usually 106 AFB/ml of specimen required for positive result. Only 60% of the smear are positive if 104 AFB/ml are present21. Therefore, the numbers of AFB below the minimum limit of detection may not detected causing potential source of transmission to the communities. The overall sensitivity of the smear has been reported to range from 22% to 80%22. Also, this techniques can't differentiate between mycobacterium tuberculosis complex and other species of the genus mycobacterium that may be acid fast.
On the other hand, isolation of M.tuberculosis on solid media is most sensitive than sputum smear examination for diagnosis of active TB infection. It can differentiate between species of mycobacteria. But it can take up to 8 weeks to recover mycobacteria from clinical specimens due to slow growth. The sensitivity of culture is generally between 80% to 85% and specificity of about 98%23-24. Sometime bacilli fail to grow or often become contaminated is another problems. Several liquid culture techniques have been developed for the rapid detection of MTB but it can reduce bacterial growth time 1 to 3 weeks23 that is on average it takes 14 days to become positive. It is expensive and required sophisticated equipment and also required skilled personnel.
To overcome this problems, laboratories have assess the usefulness of the detection of MTB-DNA in clinical samples by PCR method. PCR is a very sensitive method for detecting mycobacterial DNA or RNA directly in clinical sample25.
For detection of specific sequences of MTB26, so many PCR methods have been developed. These PCR could be based on conventional DNA based, nested PCR and RT-PCR. Targets include insertion and repetitive elements, various protein encoding genes, ribosomal rRNA27. Development have been very rapid in this area. For targeting different MTB genes, there are a lots of PCR assay have been described28-40. Separate gene targets like MPB 6431, repetitive sequences34, GC repeats35, devR36, 38kD37, TRC 438, IS 108139 have used in different Indian studies. But none of the method are universal due to region specific variation in the genome of mycobacteria41-42.
Detection of MTBC by PCR method has varying results, especially in the sensitivity of the test43-45. Lots of molecular tests reported in the literature are based on the amplification of gene IS6110, an insertion element is believed to be limited to members of the M.tuberculosis complex46.
By targeting only IS6110 gene may not be sensitive enough to diagnose 100% of the cases. The sequence is found to be absence or presence in few copies has been reported in some strains of Southeast Asia region47. A large number of clinical isolates of M.Tuberculosis from South India had either a single copy (40%) or no copy (4%) of IS6110 gene48 and existence of homology between an IS6110 derive probe and DNA isolated from potentially pathogenic mycobacterium strains have been reported49. Also, monoplex PCRs have the limitation of detecting only one target gene. In this situation the use of other targets for PCR in addition to IS6110 for the detection of TB can be helpful50-53. Variations harbored in IS6110 insertion sequences were found by Sankar et.al54 when they analyzed the sequence diversity of IS6110. They also reported that there are divergences within the copies of one strain54. Also, a list of primers were collected by him of those which were successfully used in the conventional PCR54. Sensitivity and specificity for different region of IS6110 were found to be variant in reported data. The recommended develop multiplex PCR assay targeting more than one region of the genome of MTB54-55.
We standardized a multi targeted PCR assay using three different well establish DNA target genes i.e. IS6110, MPB64 and 38kDa (Pab b) for establishing a diagnosis of TB also assess the role of this multi targeted PCR in the detection of MTBC in sputum sample.