Team:IISER Pune/Practices


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Visit to Genepath Dx Labs:

To know more about mycobacteria and the real world diagnostic techniques, we visited GenePath Dx lab, Pune. Here we were shown the state-of-the-art technology in TB diagnosis- The Cepheid GeneXpert system. Recently, India purchased 300 units of this machine to improve TB diagnosis in our country.

In Dec 2010, WHO endorsed the Cepheid GeneXpert and declared it a major milestone in TB diagnosis. It is a PCR based technique that targets the rpoB gene of mycobacterium tuberculosis. rpoB gene encodes for the β subunit of bacterial RNA polymerase. Mutations in this gene confer resistance to rifampicin. The test involves just loading the sample in the cartridge and loading this into the machine. The system consists of 5 molecular probes each having different fluorescence. Each probe binds to a specific sequence of the rpoB gene. If any of the sequences is mutated, the specific probe does not bind and the specific fluorescence is not seen. The end result is a graph of fluorescence vs Cycles of PCR. The graph has 5 lines for the 5 probes and 2 as control. Depending on when we see rise in fluorescence, the intensity of infection can also be predicted.

Now, Cephied also provides extra primers to target drug resistances other than rifampicin. The limitations of this method are the high cost of this setup. Also, some cases of have been reported where the PCR showed a positive result but clinical tests showed negative results. This was because even though mutations was present, expression levels of the gene were not enough to actually give the bacteria resistance to rifampicin. Thus, even with all these advancements in diagnosis, we still need a quicker yet more efficient and simple test for TB.

We also visited Jehangir Hospital Pune where Dr. Vikram Padbidri, the head of Clinical Microbiology Dept. introduced us to a new technique called the MGIT system. We also got the opportunity to see traditional methods of diagnosis like staining, culturing, etc. Traditionally, TB is diagnosed by a combination of chest X-rays, culturing and staining. The following methods are most commonly used in India:

  • Ziehl-Neelsen staining - This method is based on stains taken up by the mycolic acid layer of mycobacteria. The limitations of this method are its specificity (since the stains are taken up by all mycobacteria and not Mycobacterium tuberculosis specifically) and sensitivity (requires minimum 5000 Acid Fast Bacilli per ml to give clear results). Image: www.scilogs.com
  • Culture based diagnosis - The Lowenstein-Jensen medium is a egg-based solid medium that contains various chemical compounds that prevent growth of other bacteria and enhance the growth of mycobacteria tuberculosis. The specimen from the patient undergoes decontamination and is then inoculated onto the medium. This is then kept at 37°C for 4-8 weeks, till growth is observed. This test is limited by the time taken for growth to be observed.

Apart from these 2 common techniques, a new technique that has recently come up in India is the Mycobacterium Growth Indicator Tube : a technique based on fluorescence. As the bacteria grow, the oxygen is used up and carbon dioxide is produced. Due to depletion of free oxygen, the fluorochrome is no longer inhibited and we see fluorescence under UV light. The tubes are incubated at 37°C for 1-6 weeks. The machine is fully automated, which checks for fluorescence every 60 minutes. It indicated when a tube has a positive result and this tube is then taken out for further tests. This method also enables drug susceptibility testing. The limitations of this method are that it considers a tube having positive result only if the concentration is more than 100 cfu/ml. If it is less, then it is considered as negative result. Errors in decontamination can cause false fluorescence since it is basically an oxygen sensor system. Also, a loss of about 60-70% mycobacteria can occur during decontamination. The cost of this machine also is a limiting factor. Image: www.euco-net.eu www.tbonline.info

The MGIT system based on fluorescence

Though both MGIT and Cephid techniques are faster and more accurate than the staining and culturing techniques the cost of the setup required for these tests is very high. These visits to Jehangir Hospital and GenePath Dx Labs not only helped us about the diagnostic techniques used, but also that not many clinical labs have funds to purchase these new high end instruments. Also, the lab technicians just follow the protocols given on a sheet. Thus, there is a need for a simple technique that does not require expensive instruments and has an easy-to-follow protocol. The number of TB tests carried out in countries like India require a cost-effective, quick yet accurate and simple test for TB diagnosis.

TB Awareness and Foldscope Demonstration

Aware & Inspire

After the visits to Genepath Lab & Jehangir Hospital, we decided to propagate the TB literacy that we had acquired, to the societal section, which needs it the most. We spent the Sunday afternoon of 13 September with students at Disha.

Disha is a voluntary organisation, initiated by IISER students that functions to educate children who lack resources, through innovation. Disha has now grown beyond IISER and has officially achieved the status of an NGO. It runs various programs for students studying in classes 4 to 12, who study in corporation schools. One of the pivotal programs of Disha is the ‘weekend program’ which enrolls students of classes 7 to 10. These students meetup in IISER on every Sunday to discuss their academics with each other and to get help from Disha volunteers. A necessary epilogue to academics, on which Disha volunteers emphasise, is exposing these students to the approach of rational inquiry, to the fun and technique of arts and craft and to the developments in science and technology; to give them that, what they usually don’t get.

We conducted an afternoon long session with them, which was sectioned in two parts.

  • The first part was a discussion on TB involving the following issues -

    • Biology & bacteria
    • Infection & disease
    • Introduction to TB as a bacterial disease
    • Signs & symptoms of TB
    • Distinguishing TB from regular cough
    • Treatment : Availability, accessibility & affordability of TB cure
    • Societal perception : A change needed

    Our motive behind this was to make them aware about the gravity of the issue and equip them with one of the greatest mitigation tool in public health - awareness itself. This session was particularly relevant to these students, as almost all of them reside in localities that are potential sites of such epidemics due to negligence towards health & illiteracy towards hygiene.

  • The second part of the session was a more fun one. We demonstrated to them construction of Foldscope, which was then used to perform videography of few bacterial slides by connecting the Foldscope to an iPad. The students then were provided materials (cardboard paper & PDMS lens) to make simple paper-based microscopes of & on their own. Our team members along with the volunteers, explained to them -

    • Need for microscopy
    • Optics behind it
    • Revolutionary impact on science
    • Different microscopes : as function of resolution & cost
    • Advantages of Foldscope

    The basic idea was to instill in them an idea of simple & elegant solution to “big” problems. The program was conducted in Marathi which is the native language of these students. The support from volunteers & enthusiasm of students intertwined to generate a response that helped us realise the social & human relevance of our project.

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