Novel diagnostic and therapeutic approaches in tuberculosis

(Inventors: S. E. Hasnain et al.) 

Brief description:  

The direct observed treatment short course (DOTS) has been the most effective intervention against tuberculosis taking into account the number of lives saved and relapses avoided. However, the proportion of relapsed cases still is alarming with several cases going undetected. Of the total cost, 5-10% is spent on diagnosis that often lack sensitivity and can be time consuming, leading to delay in the treatment procedure and consequently result in increase in relapsed cases.  

Treatment of infectious and asymptomatic cases by early and accurate detection is the best way today to intervene against tuberculosis. The bottleneck in TB management is the proper and early detection of the disease. Understanding its priority, one of the central goals of WHO is to ensure detection of 70% of TB case worldwide. 

Mycobacterium tuberculosis can practically remain undetected in lungs for decades, efficiently escaping detection by the host immune system and the drug therapy. Only in 10% of the infected people, the number being higher in immuno-compromised patients, TB erupts as a full-blown disease. Delay in diagnosis of the asymptomatic patients and hence their treatment impedes the downstream management and control of the disease. These patients act as potential reservoir of M. tb and spread infection in the community, unless effectively treated.  With the increasing emergence of multi drug resistant strains and co-infection with HIV the problem is getting further compounded. Early diagnosis, therefore, is a matter of utmost concern for TB disease management and epidemiological investigations. Current diagnostic tools for tuberculosis often lack sensitivity and can be time consuming. TB diagnosis largely banks upon tuberculin skin test and staining and culture methods. The epidemiological relevance of tuberculin test with purified protein derivative (PPD) is questionable in areas where BCG vaccination is compulsory because PPD is not sensitive enough to distinguish between vaccinated and infected individual. Often the BCG vaccinated population is scored positive leading to a wrong line of treatment.  Microscopic determination of the bacilli in the sputum samples requires high titers of bacilli (5000 – 10000 / ml) in sputum – a condition seen only in full blown tuberculosis patients. Culture techniques can detect very low titers but are time consuming, taking approximately 3 – 6 weeks. This necessitates development of more promising and rapid diagnostic methods for successful TB management. 

We have developed several rapid ELISA based strategies that target these problems of TB diagnosis, namely, a) effective avoidance of false positives where BCG vaccinated healthy population can be differentiated from infected ones and b) differentiation between the fresh and relapsed cases of tuberculosis.  Both the points are very important in launching a proper drug regime to avoid recurrence of the disease and develop MDR-TB.  In particular, avoiding relapses can have important public health implications, by reducing number of cases of TB with subsequent reduction of cost associated with relapses. Also, since the method is ELISA based it can be performed in any diagnostic lab. Further, it can be extended into a time-saving high-throughput screening set-up for both diagnostic and epidemiological investigations. In the present scenario of available detection techniques we believe these will be of high commercial potential. 

We have established in an ELISA based study:

1.   M. tb ICDs could elicit strong B cell response and significantly distinguish BCG-vaccinated healthy individuals from TB-patients. This observation is highly significant as PPD, predominantly used for tuberculin skin test for TB detection, lacks the sensitivity to distinguish between BCG vaccinated and TB-infected populations. Also, M. tb ICDs do not cross react with the sera of NTMs (there are more than 82 recognized species of mycobacteria that occasionally infect mammalian hosts. These are referred to as nontuberculous mycobacteria) and non-TB patients. These results, for the first time,  highlight the potential of M. tb ICDs as a diagnostic marker for TB and at the same time discriminate TB from BCG as well as NTM background.  

2.   Rv2608 a PPE_MPTR protein and the peptides corresponding to regions of high antigenic index can differentiate between fresh tuberculosis and the relapsed cases, with the latter category patients demonstrating the highest B cell responses to the peptides. Therefore these peptides and the full length protein, for the first time, can be used as diagnostic markers for rapid detection of a relapsed case and instant initiation of an appropriate drug management for the patient.  

3.   We have recently shown that a member of the hypothetical PE/PPE ORF 2430c mounts a strong B-cell response in sera of TB patients. What is more interesting about this is the fact that the response is significantly pronounced for category one patient (fresh infection or so called activation cases) as opposed to the relapse case. Furthermore, this response was higher than that generated by M.tb. HSP10 or PPD. This, therefore, provides a strong diagnostic for active infection.  


In summary, the diagnostic antigens identified by us will be able to detect active infection (Rv2430c), relapse cases (Rv2608) and finally M.tb from NTM and also BCG background. We believe this is the first time that we have a powerful system for diagnostics which will have huge commercial potential. Accordingly we have filed applications to protect our IPR.

Research publications:

Banerjee, S., Nandyala, A., Podili, R., Katoch, V.M., Murthy, K.J.R., and Hasnain, S.E. 2004. Mycobacterium
           tuberculosis isocitrate dehydrogenases display strong B cell response and differentiate BCG-vaccinated healthy
           controls from TB patients. Proc. Natl. Acad. Sci. USA. 101: 12652-12657.[PDF]

Chakhaiyar, P., Nagalakshmi, Y., Aruna, B., Murthy, K.J.R., Katoch, V.M., and Hasnain, S.E. 2004. Regions of
           high antigenicity within the hypothetical PPE_MPTR Rv2608 ORF show a differential humoral response and
           a low T cell response in various categories of patients with Tuberculosis.  J. Inf. Dis. 190: 1237-1244.[PDF]

Choudhary, R.K., Mukhopadhyay, S., Chakhaiyar, P., Sharma, N., Murthy, K.J.R., Katoch, V.M., and
           Hasnain, S.E. 2003. PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response.
           Inf. Imm. 71: 6338-6343.[PDF] 

Patents filed: 

Patent applications on this invention have been filed under two different titles, namely: 

(i)   Antigenic Peptides (from Rv 2608) 

       Patents applications :    [PCT]

       Search Reports         :   [International]      

(ii)  A method of diagnosing tuberculosis (based on using anti-ICD antibodies) 

      Patents applications   :   [PCT]    

       Search Reports         :   [International]