Tuberculosis is one of the most investigated diseases on earth but the bacteria-driven malady continues to be one of the major killers. Kashmir microbiologist, Dr Beenish Rufai, has studied the TB ecosystem for a very long time and is now busy improving the vaccine. In this long interview with Humaira Nabi, the young Kashmir scientist details the challenges that the evolved bacteria have posed to mankind.
KASHMIR LIFE (KL): Over the past decades, Tuberculosis (TB) has been immensely studied by scientists globally. Has the disease’s prior research been a failure? What motivates researchers like you to continue studying the disease?
DR BEENISH RUFAI (DBR): TB has always been concomitant with a high mortality rate over the centuries and since then tremendous research has been done in various areas like epidemiology and control, operational research, case findings, operation research, and basic science as well. Until now, the disease remains among the thirteenth leading causes of death and the second leading infectious killer.
Concerned with higher mortality rates of disease, the United Nations General Assembly’s (UNGA) first-ever high-level meeting, United to end tuberculosis: an urgent global response to a global epidemic was set to accelerate efforts in ending TB and reach all affected people with prevention and care. Of the five-year target (2018-22) to treat 40 million people with TB treatment only 50 per cent of people were reached. Also, preventive TB treatment was also provided to 29 per cent of the target of 30 million for 2018-22. This indicates the need for more efforts in all high-priority research areas to help find, detect and treat TB cases. There is a dire need to work on TB priority areas like the development of affordable and diagnostic tests for rapid detection and drug resistance, new drugs and effective vaccines. This challenge motivates me to continue working on Mycobacterium tuberculosis.
I cannot say that we have failed completely but we have not been able to achieve some of our set targets in the last couple of years. In 2015, WHO set certain milestones including treating 40 million people up to 2020 and lowering the mortality rates. But we have not met any of our set goals so far. Covid19 may be one of the hindrances that only 37 per cent of set targets for treating people were met. TB report 2022 released by India’s Health Minister reported a sharp 11per cent rise in TB cases in 2021 over the previous year. This is coupled with the rise in Multidrug-resistant TB (MDR TB) rates.
So, over the past few years, we do not know exactly what has happened as each one of us got with Covid19. So, there are a lot of things that we need to put effort into in terms of estimating the targets, targeting the incident rate, starting with the people who are having multidrug resistance and to reach to the populations who have TB and are not getting treated specially in the impoverish zones of India.
KL: What is the state of TB in Jammu and Kashmir?
DBR: The Health Ministry report for 2021, says that three districts from the Kashmir division – Anantnag, Kupwara and Pulwama – have shown a 60-80 per cent reduction in TB cases in the last five years and have secured gold medals under the programme.
KL: Guide us through your academic journey.
DBR: I did my early schooling at Woodlands House School, Shivpora Srinagar. Then, I joined Kothibagh Higher Secondary School for my eleventh and twelfth grades. I graduated in Medical Microbiology from Dehradun because I was always fascinated to see tiny creatures under the microscope, which are a cause of much of the disturbance in the world. I pursued a master’s in microbiology from the same institute.
I then enrolled in AIIMS Delhi to begin my doctoral studies. I joined the clinical microbiology division under Dr Sarman Singh, who is a very well-known clinical microbiologist. After my doctorate, I spent a year as a senior resident at AIIMS Bhopal because I wanted to study all aspects of microbiology. I was assigned to work in a number of microbiological departments there, including parasitology, mycology, and bacteriology. Handling the clinical strains and observing how Nosocomial infections affect people in hospitals was a very positive experience. Besides, I learned how to diagnose and report them.
It was followed by my postdoctoral training under Dr Marcel Behr, at McGill Canada. My research focus was estimating India’s zoonotic TB burden, constructing of first complete reference genome of Mycobacterium orygis and understanding the pathogenicity of the organism in animal models. After that, I applied for the Ramanujan fellowship as I wanted to come back to Kashmir to utilize the learning and experience here. So, I joined the CSIR IIIM after getting the Ramanujan fellowship and here I work on vaccine development for TB.
KL: What was the major takeaway from your PhD programme?
DBR: During my PhD programme, I worked on the clinical strains of Mycobacterium Tuberculosis. We know TB just as a disease but there is a lot in bacteria that is going around it. We know about human evolution that we evolved from apes and in the future, we might evolve into some other lineages. Mycobacterium Tuberculosis has undergone an evolution as well from its ancestor into various lineages and is adapted to diverse geographical regions. Some lineages have adapted to be more virulent, prone to acquire mutations ultimately causing drug resistance, higher transmission rates, rapid conversion of later to active cases, and higher pathogenicity in animal models.
My research focus was to see the transmission pattern of these lineages across India and understand the genomic factors that make them prone to drug resistance. Our team worked in different centres in India, cultured sample isolates from suspected TB patients and screened for their drug resistance. We found 22 per cent of strains as resistant to isoniazid and rifampicin drug (MDR), 30.6 per cent of the strains as resistant to Fluoroquinolones and 2.7 per cent of strains resistant to both fluoroquinolones as well as amino-glycosides (XDR) (Rufai et al; Scientific reports 2018).
Our study found that 52 per cent of strains were significantly associated with drug resistance belonging to lineage 2 (Beijing modern lineage). While every strain seemed to have occupied a specific place, the Beijing lineage was supposed to be found in the North Eastern region because of its Chinese origin but during the epidemiological transmission survey, we found that these strains are circulating all over India. In a separate genomic transmission study, we also found that in North East same strains are present in drug-resistant TB cases and endemic in the state leading to aggressive TB cases (Mudliar et al; Genes 2021).
We also tried to work on comparative genomics is just like aligning the genome of all the strains of TB and studying their commonalities. We found that a particular gene known as CRISPR, which is known to provide some adaptive immunity to the bacterial cell is deleted in the Beijing strain. We are unsure if that is the cause of the vulnerable resistance. It needs further research.
KL: You talked about the evolution of bacteria. Do viruses also undergo evolution or mutation?
DBR: The viruses mutate very fast but the evolution of TB bacteria has taken millions of years. There are a lot of factors which come into play for genomic architecture to evolve. Their adaptation is linked to environmental pressure, epigenetic changes and many other factors.
KL: Which strain of Mycobacterium Tuberculosis is found in Kashmir?
DBR: We have not done any epidemiological transmission studies yet. But in future, we can plan and work on them.
KL: Your PhD was followed by spending a year as Senior Resident at AIIMS Bhopal. Tell us about the experience.
DBR: I worked in a Microbiology lab where we received the samples from a patient, followed by the culturing of the samples by the technical staff. We then had to identify the organisms and look for the type of drug resistance they had. The senior residency was more focused on the identification and detection of pathogenic microbes in parasitology, virology, bacteriology as well as mycology. I was trained to take responsibilities like creation, optimization and implementation of assays for use in clinical diagnostics, supervising clinical microbiology lab, maintaining standard operating procedures and reporting test results and consulting with physicians on tests requested.
I was also working on Omics data of M. tuberculosis isolates (proteomics and genomics) from Arunachal Pradesh, colistin resistance in Carbapenem-resistant Klebsiella pneumonia and serology of zika virus in Central India.
KL: What was your postdoc all about?
DBR: Our research group was working on the hypothesis that due to two independent cattle domestication events another variant of Mycobacterium tuberculosis complex known as Mycobacterium orygis is endemic in cattle stock of South Asia. On further follow-up, we reported the presence of M. orygis from human isolates suspected of having TB and no presence of M. bovis isolates from India. This raises a possibility of M.orygis as a zoonotic cause of TB transmission in humans and further needs to be investigated.
I joined as a post-doc with Dr Marcel Behr at McGill University Health Centre Canada in February 2020, where my research focus was the construction of the first circular genome of M. orygis (Rufai et al MRA; 2021) and working on the Type VII secretion system and secretome of Mycobacterium orygis in comparison with other members of Mycobacterium tuberculosis complex. I constructed the reference genome of M. orygis using long reads PacBio sequencer and polishing using short illumine reads. After genomic and proteomic profiling of this newly evolved pathogen, we further stepped to look for the pathogenicity of this pathogen in animal models. We identified certain virulent genes in M. orygis.
So, overall it was a very good learning experience. I got very good training there and then I planned to utilise all this here in Kashmir.
KL: What are you currently working on?
DBR: During the course of my training, I realized that we need a vaccine against TB. The BCG strain, which is used as a vaccine against the disease, was developed in 1921. Different countries took that strain and now we have BCG-Denmark, BCG-Russia, and BCG-Japan. The repeated sub-culturing of the strain results in losing some of the characteristics of the strain. The BCG vaccine underwent the same reverberation.
The genetic architecture of the strains was not fully understood in 1921 due to the state of science at that time. Therefore, we were unsure about the precise contents of the organism. Slow efficiency of the BCG strain to provoke our immune system and give protection against TB rained down. Right now we do not have an effective TB vaccine, which is a need of the hour, especially in countries with high burden TB countries.
So, I planned to work on the same concept. I work on membrane vesicles of TB that are released from the bacteria inside the host body. These vesicles released by TB are already known to have a role in immune invoke evasion. On this wise, I thought to engineer the BCG strain to incorporate the strain. If there are some vesicles that are actually helping our immune system in a positive manner against infection, we can engineer this BCG strain so that they release these vesicles. My aim is to work on the BCG strain to engineer it with such genes that aid in the secretion of these vesicles thus enhancing the efficacy of the strain.
KL: You must also be having researchers working under you. Are they also working on the same thing?
As of now, I am in the capacity to have some students with me to work on various parts of the project. But before that, I had to do efforts to create an infrastructure and lab that will be sufficient enough, to begin with. I would really like to teach students here; that would be helpful for their future perspective. Our ultimate goal is to enhance the efficacy of the BCG strain.
KL: Do you have the adequate infrastructure here for your research?
DBR: We do not have any infrastructure here. When you engineer the gene knocking of even a non-infectious bacterium, you have no idea how it can turn out; as you are actually messing up with the genome of a specie. These types of experiments and research are done in a Level 3 facility. A slight gaffe has the potential of galvanizing another pandemic. So, a Level 3 facility is a prerequisite.
I work on TB and for my final engineering strain, I need to infect mice and then vaccinate them with the sample. I have to do all this in the BSL-3 laboratory but since we don’t have any BSL-3 laboratory facility here, we are making a lot of effort for this. As of now, I have collaborated with the TB state facility, to begin with mycobacterial culture and also we have a BSL3 facility in CSIR IIIM Jammu which I will be utilizing for my animal experiments.
KL: Is it possible that you and many others may be working on the same issue at the same time? If so, is there a connection that would enable you to better understand one another and prevent everyone from spending their time recreating the wheel?
DBR: Yes, I agree with you. Many scientists are currently working on a TB vaccine globally; however, the approaches are different. It is always wise in research to collaborate with the labs for facilities, discussing ideas, utilizing each other’s expertise and ultimately reaching the goal that will be beneficial for the community,
If you see the previous vaccines that failed in phases two and three, they were having a different approach. This is one of the ethics of science and the concept that you are working on should not be a stolen concept. It should not be a concept that you have seen some scientists working on. Your idea should be novel. When we apply for any fellowships, the funding agencies make sure that the concept is novel.
KL: The Ramanujan fellowship is a five-year programme. What’s next?
DBR: The department of science and technology has this fellowship to attract people from overseas to come here. Then, the institute you are working in has to absorb you within this time frame. If there are few positions they need to give preference to people who are having this fellowship.
KL:: How soon should we expect a path-breaking research paper from you?
DBR: I will be working hard on this but it takes a minimum of four or five years to get a very good research paper or good findings that are actually useful for the community.