Sepsis – microscopic invader in the blood

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The words "sepsis" or "septicaemia" continue to appear in newspaper headlines and are repeated by journalists causing shivers of terror for masses of readers, listeners and viewers. Both terms mean the same, i.e., an infection of the whole body, spreading in the bloodstream.

However, this does not mean that we are all in danger of sepsis. The human body is exposed to the influence of millions of microorganisms every day, but this does not cause a systemic infection, as our immunological (immune) system is still active. Sepsis is caused by pathogens such as bacteria, or (more rarely) fungi. It occurs when the human body is weakened (e.g., devastated by cancer, HIV infection or chronic diseases such as untreated diabetes, etc.) and is unable to effectively defend itself from the microorganisms that penetrate into the bloodstream, e.g., during surgery or after an infection. Unfortunately, sepsis is fatal for many patients, in spite of treatment in intensive care units and the application of a whole array of antibiotics. Every year, approximately 750,000 people in the U.S. suffer from sepsis and it is the cause of more than 215,000 deaths. Surprisingly, 146,000 patients die from sepsis annually in the European Union.

Uncertain tests

In order to give the patient a chance to fight the infection, it is necessary to find out if sepsis was caused by a microorganism and then to determine the type of this microorganism. Such information enables the physician to introduce an appropriate therapy using antimicrobial drugs and thus increase the chance of survival. For many years, the only available method of diagnosis was taking blood cultures to grow the microorganisms (bacteria or fungi) causing the infections from the blood sample. Unfortunately, this procedure, although common throughout the world, does not allow for the detection of the microbiological pathogen in as many as 60% of the cases, in spite of the presence of sepsis. This type of analysis is limited by the sensitivity of the culture method. Another problem is the time needed to obtain laboratory test results – in extreme cases the results are only available after a week from the date of sample collection, which is definitely too long for a dying patient.

The application of methods based on the detection of the DNA or RNA of microorganisms directly in the blood of patients with symptoms of sepsis provides a chance to eliminate these hindrances. At present, there are only three commercial tests available on the market that use genetic methods, allowing for the confirmation of the presence of several or more than ten most likely species of bacteria or fungi. This does not satisfy the needs connected with the diagnosis of the infection, because these tests are designed to detect a panel of the microorganisms that are most commonly found in the blood. Thus, a negative result does not guarantee that there really are no pathogens. Thus the reason why further research on this issue is needed.

Image from a fluorescence microscope obtained with use of the FISH method
on whole, venous blood. Visible Escherichia coli bacteria – EC probe was used, magnification 1000x

Only six hours

Research aimed at the development of innovative diagnostic methods used in microbial infections has been conducted at the Department of Microbiology (Jagiellonian University in Kraków Medical College) since 2010. The project was financed from a grant from the National Science Centre (NSC). "At the moment we already know how to detect bacteria and fungi directly in blood samples taken from patients with symptoms of sepsis without the need to conduct a time-consuming culture process. The method developed allows for the detection of infection in the blood within six hours from taking the blood sample. We have two types of methods: one enables us to visualize microbial cells in the blood sample with use of a fluorescence microscope, or FISH (Fluorescent In Situ Hybridization), and the other, known as PCR (Polymerase Chain Reaction) is based on the amplification (multiplication) of DNA marker fragments of microorganisms in the genetic material isolated from a sample of the patient's blood," said Tomasz Gosiewski, PhD, who coordinates research on fast methods of diagnosing sepsis.

The sensitivity of the FISH method enables the confirmation of the presence of microbial cells in blood if there are at least 1000 such cells in 1 ml of blood, while PCR enables detection on the level of 100 cells/ml. The cost of conducting a test using the FISH method is approximately three times lower than that of the PCR method. This results from the specific features of the analysis as it does not require the use of enzymes to multiply the DNA of microorganisms in vitro, which is necessary to confirm their presence. Both methods enable the differentiation of the detected microorganisms into Gram-negative and Gram-positive bacteria, yeast and mold fungi, i.e., into groups covering all pathogenic species of bacteria and fungi. This information enables the doctor to use more targeted antibiotic treatment.

The methods developed have been presented in specialized English-language journals (e.g., "Current Microbiology", 2014, or "BMC Microbiology", 2014) and during local and international scientific conferences (e.g., 23rd European Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, 2013). Moreover, applications for patent protection of the PCR analysis in Poland and abroad have been filed. Further development of the methods that will allow for detection of specific species of bacteria and fungi and to detect resistance to antibiotics is also possible. This will make it possible to eliminate ineffective treatment right from the start.

Research team (Department of Microbiology, Jagiellonian University Medical College): Tomasz Gosiewski, PhD; Monika Brzychczy-Włoch, PhD; Małgorzata Bulanda, PhD; Agata Pietrzyk, PhD