Conversation between Archana Madhav and Lara Marks, 6 November 2020

Lara

Can you tell me a little bit about yourself?

Archana

My name is Archana Madhav. I am 22 years old, and a recent BSc biomedical sciences graduate from Brunel University London. As a part of my four year undergraduate degree, in my third year, I opted to do a placement year to work with Dr. Ankur Mutreja and his group at Cambridge University where I was asked to help develop a dipstick for a diagnostic kit to detect antimicrobial resistance based on one they had previously developed for cholera.

Archana Madhav (credit: Archana Madhav).

Lara

What was your role when you took up the placement in Cambridge?

Archana

My central task was to work out which antibiotic resistance genes to include as probes to attach to the dipsticks based on specific inclusion criteria and then to design primer sets for the selected genes. A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. My aim was to use the primers as different probes that could pick up on multiple antibiotic resistance genes and their closely related variants.

Lara

Can you explain how you set about to develop the primers?

Archana

Initially, the work involved a lot of research to work out the high-priority and high prevalence resistance genes. Once this was decided, I set about designing primers to act as probes on the dipstick. This involved using several computer applications and running simulations to first design primers that matched our parameters and then to check that different sets of primers did not cross-react which would reduce the efficiency of the test.

Once we had designed and ordered the primers, we first needed to check how they worked experimentally with gel electrophoresis. This was very time-consuming. The key was to work out which bands showed up on the gel when running the test. If a band consistently did not show up, despite me having done everything correctly as far as I knew, I would alter the parameter that wasn't working (e.g. temperature) for something else or design another primer set for the same gene. During this time, I continued reading through the literature and had identified some better gene targets to replace some previous ones and improve the breadth of the panel. I ordered a second set of primers that included a few new targets. We ordered three ‘generations’ of primers overall which resulted in a ‘primer bank’ containing two or three different primer sets for some of the gene targets. These could be substituted in at various stages if proven to work better. Once we found out which bands were appearing, we then had to determine the optimal conditions for running the test and write up SOPs.

Lara

What did you achieve in the project?

Archana

In the end, with the help of the team in Cambridge, I created a panel of 16 different genes, split across four dipsticks, which covers over 100 different closely related variants. Based on this, we sent off an order to a company to manufacture the tagged primers and corresponding probes on a dipstick. We only placed orders for two sets of primers, because we wanted to check they worked before going forward. We chose a group of primers for resistance genes that pop up quite often. When they came back we validated them with the same samples that we had tested out experimentally. And they came out beautifully. It was such a big moment for me to actually see the dipstick work. It was very exciting.

Complete panel of 16 genes split into 4 groups (numbers in brackets indicate amplicon lengths in base pairs). Credit: Archana Madhav.

During the month it took for the manufacturing process, we also conceptualized a data recording app to help interpret the results of the dipstick test. The app would allow a registered diagnostician to input test results by providing an interactive image of the diagnostic test where each target can be selected as being ‘positive’, ‘negative’ or ‘inconclusive’. The app then summarises the lab results in the form of a summary report for each sample with the resistance profile and corresponding antibiotics to avoid for clinicians to use as a guide to improve prescription practices. A beta version of the app is currently available.

Interface of PredicStix app showing capture of dipstick results and summary report(credit: Archana Madhav).

During this time, I was also given the incredible opportunity to contribute as first author to a book chapter exploring the evolution of microbial defence mechanisms to antimicrobial agents along with Robert Will and Ankur Mutreja. It has now been published by Springer and can be accessed online.

Lara

I understand you went to India at the end of the project. What were you doing there?

Archana

In June 2019 I went to Christian Medical College (CMC) Vellore, India to pilot-test the dipsticks. There, I spent two months carrying out sensitivity and specificity testing. The aim was to optimise the experimental conditions for the test to ensure that it worked reliably for pure DNA and on cultures and to validate the results using positive control samples. After establishing that the testing protocol was reproducible on boiled pure-culture samples in India, I then started testing it on a bulk sample set to accurately define sensitivity and specificity values. We obtained a sensitivity of 89.6% and specificity of 96%.

Towards the end of my stay in India I was presented with the chance to apply the diagnostic test on a collection of resistant pathogens from clinical samples kept by the microbiology department at CMC. I only had about a week to do the work, so did not get a chance to complete the study. In the future I aim to pick up the study where I left off.

Lara

What do you see are the challenges with AMR and how do you think an affordable diagnostic could help combat the problem?

Archana

I'm from India where the sale of antibiotics is very poorly regulated in pharmacies. I can walk into a pharmacy, right now, and ask for an antibiotic without a prescription. Also if somebody goes into any hospital outpatient department, with a cold, cough, or fever, the most common outcome is that they are prescribed antibiotics. This is also the case with COVID because not everyone knows it is caused by a virus for which antibiotics are ineffective. Another major problem in India which might also apply to other low-middle income countries is that many people can't afford a doctor's visit so they often self-medicate with antibiotics.

AMR is going to become a huge problem in the next few years, so we need to start putting into place rapid, affordable and point-of-care diagnostic tools to pick up resistance. This would speed up the process of treatment, make it more targeted and reduce the severity of future infections. Diagnostics is not the only solution. What is also important is antibiotic stewardship. Similarly vaccines are important. Vaccines for both viral and bacterial infections have been proven to reduce antibiotic prescriptions. This applies across several sectors. If we are to reduce the burden of AMR globally, it would require a huge ‘One Health’ collaboration between the World Health Organization, Food and Agriculture Organization and the Organization for Animal Health in a united effort to reduce antibiotic use, so we better get on it.

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