Team:IISER Berhampur/Description
Inspiration
These kinds of posters for awareness of tuberculosis (TB) are common sightings in India under the government policy of ‘TB Mukt Bharat’ (translated as “ TB free India”). At least one out of three people in the Indian population has latent TB. When one of our team members, Nisha Kumari’s aunt, got sick with TB, she started reading about this infectious disease and realized that multidrug-resistant TB (MDR-TB) is acting as a barrier in the process of TB eradication and there is no awareness or accessible diagnostic kit for it. So, after one month of brainstorming, when the iGEM competition started this year, she along with others at Team IISER_Berhampur 2021 came up with a raw idea of improving diagnostic conditions of MDR-TB, by making a cost-effective and accessible diagnostic kit.
Background
Mycobacterium tuberculosis, a tiny rod-shaped bacteria, has been causing a staggering global impact. It is a human pathogen responsible for causing tuberculosis (TB) and it is a major health problem in India and all across the world. All over the world in 2020, 9.9 million people fell ill with TB and 1.9 million estimated deaths occurred due to this disease. India contributed 24 lakh TB cases to the global TB burden and over 79,000 deaths due to the disease.[1]
TB is highly contagious in its active stage and not so serious symptoms like bad Cough, weight Loss, fatigue, shortness of breath, fever, night sweats, and lack of appetite all together can be indicators of this disease. TB can effectively be treated by using drugs like isoniazid (INH), Rifampicin (RIF), Pyrazinamide (PZA), Ethambutol (EMB), and Streptomycin (SM). However, these drugs often fail to cure TB for several reasons like patients stopping their 6 months treatment abruptly just in 2-3 weeks. The relapse and spread of the disease contribute to the emergence of drug resistant bacteria, which leads to the development of multidrug resistant TB (MDR-TB). MDR-TB is resistant to two of the most important TB drugs, isoniazid (INH) and rifampicin (RMP), which happens due to mutation in katG gene and rpoB gene respectively.
Estimated mortality rates for MDR-TB is around 40% and India has the second highest burden of MDR-TB in the whole world after China.[1] MDR-TB is of great concern and more problematic because of its difficult diagnosis as well as treatment. The treatment options are limited, expensive, and often toxic, and drug therapy can last up to 2 years. The cure rate in MDR-TB is low (50-60%), therefore an early and accurate detection is essential for better treatment.
Existing Methods
Current detection methods of MDR-TB can be categorized into three parts: Culture methods, phenotypic methods and molecular methods.
CONVENTIONAL CULTURE-BASED METHODS:-
Culture methods work on the principle where bacteria are grown in presence of antibiotics to distinguish between susceptible and resistant strains. It includes solid culture and liquid culture methods.
Solid culture:-
Solid cultures use solid media for bacteria to grow. While it is quite affordable and requires little specialized equipment, it takes eight to twelve weeks to find drug-resistant bacteria using standardized DST techniques and traditional approaches, which can be troublesome; for example, in the case of MDR-TB, an incorrect treatment plan can result in mortality within weeks of commencement (especially in HIV-infected patients). In addition to this, delayed diagnosis of resistance leads to inadequate treatment, which may result in further drug resistance and community transmission.
Liquid culture:-
Liquid cultures use liquid media for bacteria to grow. Similar to solid cultures, Liquid culture also requires a lot of time around 2–12 weeks and it is only done in labs at urban places because of the requirement of resources and skilled technicians. Liquid culture systems are considered more sensitive than solid medium cultures and they also cut time required for the test compared to solid cultures. However, even with liquid cultures, as detection takes two to four weeks, their substantially higher cost is an issue for resource-limited countries.
Phenotypic methods:-
FAST Plaque-Response is a phage amplification-based test, and can be modified for direct use on sputum specimens; it is one of the cheap phenotypic assays but it is not that easy to perform due to the requirement of high standards of biosafety and quality control. However, there is not enough evidence available on the accuracy of this assay when performed directly on sputum samples. Safety and quality control issues related to the use of this technique are still to be addressed.
MOLECULAR METHODS:-
Nucleic acid amplification, electrophoresis, sequencing, and hybridization are all used in molecular approaches. Though these techniques were developed for the detection of drug resistance in TB complex isolates, they are being tested for direct detection of TB markers and alleles related to drug resistance in non-invasive methods like sputum samples. As there is no need for growing the organism and as results can be determined in days rather than weeks these molecular methods are considered more advantageous and a lot of research backs up that these methods are highly reliable.
(1) RT-PCR (0.5-3 hours)
Real-time PCR is a new favored tool for the development of rapid diagnostic tests. RT-PCR has two main ways for detection first is the use of non-specific fluorescent dyes to detect any double-stranded DNA generated by PCR amplification, and in a second way, sequence-specific probes tagged with a fluorescent reporter are used for the specific detection of the hybridization between probes and amplicons.
However, RT-PCR is shown to have certain disadvantages. First, it requires expensive, specific equipment and skilled technicians. Second, the number of probes that can be used in one reaction is limited, resulting in limited target numbers. This is a major drawback when used for the detection of MTB, as it can harbor many mutations.
(2) Xpert-MTB/RIF (1.2-3 hours)
The Xpert MTB/RIF assay utilizes molecular beacon-based technology to detect DNA sequences that are amplified using Hemi-nested RT-PCR assay. This is a multiplex reaction in which five different nucleic acid hybridization probes can be used. Each probe is complementary to a different target sequence within the rpoB gene and can also be complementary for the KatG gene of M. tuberculosis and is labeled with a differently colored fluorophore. Xpert MTB/RIF can simultaneously detect both Mycobacterium tuberculosis complex and resistance to rifampicin (RIF) and in some cases KatG in less than 2 hours. However, it has major drawbacks including costly reagents and reduced sensitivity compared to other detection methods.
(3) Truenat test (2-3 hours)
TrueNat is a portable, chip-based, and battery-operated machine and it reduces time to 2-3 hours. The World Health Organisation has approved TrueNat for detecting TB as it is cost-effective and a miniature version of the PCR test. It takes about 25 minutes to do the DNA extraction. It takes another 35 minutes to diagnose TB. It takes an additional one hour for testing for rifampicin resistance. However, the Lifetime cost of Truenat POC is higher than that of Xpert and they face similar challenges as a PCR test does.
Our Goals
Drug resistance is the next big threat to our healthcare system. MDR-TB is also rising due to inadequate facilities for patients and unaffordable detection. Currently, the culture methods to detect MDR-TB are slow and the molecular detection methods are expensive and require expertise. In low-income countries like India, detection for MDR is unavailable in peripheral regions like in rural areas and is only available in far places from the towns. Financially challenged patients usually can't afford doctors’ advice of proper diagnosis and potentially spread MDR-TB. This unavailability of proper diagnostic tests often limits the diagnosis of patients to TB while being MDR-TB patients, resulting in late diagnosis. This year we aim to develop a rapid molecular diagnostic kit for MDR-TB named CODE M. This detection kit will utilize isothermal amplification and Cas14 based mutation detection making it a cost-effective and sensitive way to detect MDR-TB.
To know more about our experiment please visit the Design part in <Proof of concept>.
We believe that our project can have a positive impact on our local community as well as in the world. Our Diagnostic kit will not only help India to achieve its freedom from TB but also help African and other developing countries grow from this similar unfortunate condition. We hope to eliminate the difficulties that MDR-TB patients face now. As our kit will be fast, easy to handle, and cost-effective, patients will have access to MDR detection in peripheral regions like rural areas. The patients of TB and MDR-TB will also be separated in the early stages, saving precious time to cure MDR patients which were earlier lost due to late detection.
The impact of COVID-19 on our Project
When our team was formed, we all were stuck at our respective homes due to the COVID-19 lockdown. We all are located in different parts of India and were not sure when we would be able to meet our team members in person or get access to our labs. Due to this, we had to change the strategy of our project and do some alterations to make it fit into this new norm of the pandemic world. With the hope that we would gain access to our labs at least once before the submission of the wiki page, we focused on some of our project modeling in which we did an epidemiological model for predicting the rate of cases in MDR-TB when detected early vs when detected late. Along with that we also did enzyme kinetics to optimize the concentrations of the reagents in this experimental reaction and fluorescence analysis to analyze the fluorescence produced by the bioreporter in our experiment. We also did a phylogenetic analysis to find out the most prevalent strains of Mycobacterium tuberculosis so that we could modify our kit accordingly in the future.
Other than this, with our masks on, we tried to do as much outreach as possible for our project in the online mode. We conducted awareness programs for school students, shared informative posts regarding our project on our Instagram page and we also released a podcast named “SynVerse” to spread information about Synthetic biology and our project to a broader audience.
When we were in the final stage of our project, some of the wet lab team members, in August, got a chance to come to the institute and gained access to the labs. The reagents for our experiments were already ordered. However, we couldn't get the delivery on time, which was a major hurdle for us. But we were not demotivated by that, instead, we did some basic optimization and characterization experiments with the reagents available in the lab. Due to the pandemic, we couldn't perform all the experiments that we expected, but we collected data and pictures for the experiments we could perform in these difficult times. We tried to complete our project as much as possible from our side before the wiki-freeze.
References
[1]Tuberculosis fact sheet.
https://www.who.int/news-room/fact-sheets/detail/tuberculosis
[2] Chatterjee, S., Jain, Y., & Poonawala, H. (2018). Drug-resistant tuberculosis: Is India ready for the challenge? In BMJ Global Health (Vol. 3, Issue 4). https://doi.org/10.1136/bmjgh-2018-000971
[3] Nguyen, T. N. A., Berre, V. A. le, Bañuls, A. L., & Nguyen, T. V. A. (2019). Molecular diagnosis of drug-resistant tuberculosis; A literature review. Frontiers in Microbiology, 10(MAR).
https://doi.org/10.3389/fmicb.2019.00794
[4] Migliori, G. B., Matteelli, A., Cirillo, D., & Pai, M. (2008). Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: Current standards and challenges. In Canadian Journal of Infectious Diseases and Medical Microbiology (Vol. 19, Issue 2).
https://doi.org/10.1155/2008/857901
Links for Images:-
These two images are taken from non copyright sources
https://www.flickr.com/photos/saad/23244652
https://iec.nhp.gov.in/category/central-tb/central-tb-central-tb/print-central-tb-central-tb/
