Diagnostics from a printer

“As much as necessary, as little as possible” should be the watchword when using antibiotics. But that is often not the case. The consequence: the drugs, once hailed as a miracle weapon, are failing in the fight against more and more disease-causing bacteria. Multi-resistant germs have become a dreaded problem, especially in hospitals. They lead to wound infections, blood poisoning or other diseases that are difficult or impossible to treat.

“According to projections, by 2050 more people will be dying from infections caused by resistant germs than from cancer,” says Dieter Spiehl, researcher at the Merck Lab at the TU Darmstadt. In addition to the excessive use of antibiotics in mass animal husbandry, he also criticises the prescribing practice in human medicine. In a patient with cystitis, for instance, the type of bacteria causing the infection is not usually investigated. Most of the time coliforms are behind it, but sometimes staphylococcus or other bacterial pathogens are involved. This could be tested – classically with a culture in a Petri dish – and a specific remedy then prescribed. But only hospitals perform this as standard.

“For practising doctors, the examination is too laborious and expensive. Instead, they prefer to prescribe broad-spectrum antibiotics,” laments Spiehl. These preparations may fight the most diverse pathogens, but they also at-tack the body’s own harmless bacteria. This not only leads to unwanted side effects, but also promotes the increase in resistant germs. Because whenever antibiotics are used, bacteria develop survival strategies. The most resilient survive and continue to spread.

The Merck Lab researchers now want to contain the problem with simplified diagnostic tools. “We want to replace the classic Petri dish with test cards,” explains Gerhard Schwall of the Darmstadt-based science and technology company Merck, who heads the Merck Lab at the TU. Petri dishes are relatively large and, when filled with culture media, have only a limited shelf life. The cultivation of a culture also requires specialists trained in microbiology. Alternative analytical tools are already established in food production, where bacterial detection is a routine matter: test cards with printed culture media. They can be stored dry, require no refrigeration, and save space. Spiehl and his colleagues have applied this concept to medical diagnostics.

The test cards developed at the Merck Lab not only identify bacteria, but also detect antibiotic resistance. Their use is very simple. A member of staff at the laboratory or in the practice drips the patient’s sample, for example a little urine, onto the card, covers it with a protective film and places it in a incubator overnight. The bacterial pathogens multiply and form bacterial colonies – as in the standard test in a Petri dish – that can be seen by the naked eye. The test field contains various detection reagents so that, for example, coliform bacteria appear as red dots, while staphylococcal colonies turn green. For the purpose of testing resistance, the test cards also contain various antibiotics. If the bacteria multiply in an area prepared like this, also identifiable by discolouration of the field, it means: attention – this antibiotic will not fight this pathogen!

Various printing methods are used in the production of the test cards. A screen printing machine using a viscous ink containing the nutrient medium and dyes for bacterial detection applies the test field onto a film. The liquid sample is later dripped onto this area. To prevent it from seeping beyond the field, a 3D printer prints a plastic outline around the test field. For resistance testing, droplets of various antibiotics are printed by ink jet onto certain spots inside the test field. Special printing strategies ensure that the antibiotics do not seep or even mix arbitrarily on the test field when the liquid sample is applied. Short legends and comparison fields for evaluation can also be printed directly onto the card. Finally, the system is given a transparent protective film that is folded up when the sample is applied. “We have already printed and used about 1500 test cards,” estimates Spiehl.

With a doctorate in mechanical engineering Spiehl was already very familiar with printing techniques. His doctoral thesis focused on printing electronics, the focus of the Merck Lab at the time. Parallel to the current project, he is also leading a research group at the Institute of Printing Science and Technology (IDD) at the TU. He quickly became familiar with the topic of infectious diseases and their diagnosis in the multidisciplinary team of biologists and physicians. For three years, he and his colleagues tinkered with the design and production of the test cards. The result is impressive: “We have produced prototypes for various applications and demonstrated the feasibility of the concept,” says Spiehl.

“The tests are so simple and can be carried out without high-tech laboratory equipment, which makes them ideal for small, less automated laboratories, even in developing countries,“ emphasises the head of the laboratory, Gerhard Schwall. The problem is serious in the world’s poorest regions. Bacterial infections affect many more people there, while at the same time antibiotics are often used indiscriminately, thereby promoting resistance. “We discussed their needs regarding simplified diagnostics with doctors and laboratory technicians from European and African countries,” says Spiehl. There is much interest from countries such as Nigeria and Zimbabwe: “We had already planned trials with two laboratory chains in Africa, but then the Corona pandemic came and we had to postpone the project indefinitely.”

The new diagnostic tools have already been tested at Frankfurt University Hospital. Hundreds of samples, including from patients with urinary tract infections, were examined there with the test cards from the Merck Lab, always in parallel to the standard procedure in a Petri dish. “Step by step, we optimised our system and ultimately showed that it works,” reports Spiehl with pleasure. Together with Merck, he and his colleagues are currently investigating commercial use. For the TU researchers, the project ends this year. It is to be hoped that the test cards will be developed quickly to market maturity and thus make a valuable contribution in the fight against antibiotic-resistant germs.