Molecular diagnostics, genetic tools offer new hope for sepsis management by pinning down causes behind the irrepressible host response
Sepsis is among the most common causes of death in hospitals the world over. Diagnosis of this condition, which arises from the host’s response to an infection, so far relied purely on nonspecific physiological criteria and culture-based pathogen detection. Obviously, this has often led to diagnostic uncertainty and therapeutic delays, which have been a key challenge for physicians across specialities.
However, recent breakthroughs in finding new biomarkers for pathogen detection and their genetic analysis have resulted in not only better therapeutic decisions, but also hope for developing new drugs for targeted therapy.
In sepsis, the host response involves hundreds of mediators and single molecules. These mediators and single molecules have now been proposed as biomarkers, though it is unlikely that one single biomarker will be able to satisfy all the needs of sepsis screening and management.
According to earlier studies, procalcitonin (PCT) had shown some usefulness as an infection marker among the biomarkers that are measurable by assays approved for clinical use. But several recent studies have found possible new approaches, including molecular strategies, to improve pathogen detection and molecular diagnostics, and prognostics based on transcriptomic, proteomic, or metabolic profiling.
These novel approaches to diagnosing sepsis promise to transform sepsis from a physiologic syndrome into a group of distinct biochemical disorders and help in the development of better diagnostic tools and effective adjunctive sepsis therapies.
Since sepsis arises from the host response to infection, which is directed to kill the invading pathogens, patient outcomes are determined not only by the viability of the invading pathogen but also by the exuberance of host response. While the growth of the pathogen can be directly toxic and destructive to tissues, the intensity of host response results in collateral organ and tissue damage due as the highly potent effectors do not discriminate between microbial and host targets.
So, the development of sepsis-specific biomarkers and molecular diagnostics has become essential not only for the assessment of the host response and detection of pathogen, but also for drug development and improved management of sepsis.
The complex pathophysiology of sepsis involves almost all cell types, tissues, and organ systems. Researchers have currently identified nearly 180 distinct molecules as potential biological markers of sepsis, though just one fifth of them have been assessed specifically in the diagnosis of sepsis.
Nevertheless, this improved understanding of the condition has increased the possibility of much better management and of overcoming the prevailing diagnostic uncertainty.
These new findings are more relevant as they could also help overcome key challenges like similarity of clinical phenotype of a patient with sepsis and a patient with a systematic inflammatory response caused by sterile inflammation, such as pancreatitis, trauma, burns, or intoxication.
Over the last few decades, it has become evident that the immune system is concerned more with entities that do damage than with those that are foreign, referred to as endogenous alarmins and danger signals or danger-associated molecular patterns (DAMPs). Recent researches confirmed that cellular necrosis from major physical injury and trauma releases mitochondrial DNA into the circulation, where it is capable of eliciting inflammatory signals.
Sepsis biomarkers typically reflect the biology of sepsis, as evidenced by the biochemical changes that are characterized as the host response to infection at the cellular and the subcellular levels. Inflammatory mediators are classified generally into seven groups according to their biochemical properties: vasoactive amines, vasoactive peptides, fragments of complement components, lipid mediators, cytokines, chemokines, and proteolytic enzymes, all of which comprise hundreds of distinct, single molecules.
The search for sepsis biomarkers were focused primarily on the biochemical changes at the plasma level (complement system, coagulation system, and kallikrein-kinin system) and the indicators of the activation or downregulation of cellular elements such as neutrophils, monocytes/macrophages, and endothelial cells, which may lead to the release of a number of mediators and molecules including cytokines, chemokines, and acute-phase proteins.
Key requirement for sepsis biomarkers is the time benefit that they should offer for the detection of a systemic inflammatory response to an infection before clinical signs and organ damage become apparent. Such biomarkers could facilitate earlier supportive treatment and should lower sepsis mortality rates. It would also be helpful to have biomarkers that allow the monitoring of the immune status, thereby identifying patients who might benefit from a certain immunomodulatory intervention and ruling out those who would not.
Thus, a biomarker that can rapidly detect elevated levels of a specific target of an adjunctive treatment or reduced levels of a critical factor for replacement therapy is a prerequisite for drug development and the evaluation of novel and specific sepsis therapies.
Blood culture (BC) reflects the current gold standar
d for detection of bloodstream infection as viable microorganisms isolated from the blood can be analyzed to identify species and susceptibility to antimicrobial therapy. The practical value of BC in the diagnosis of sepsis is however impaired by the delay in the time to results and the fact that positive blood cultures can be found for only about 30 per cent of these patients. Furthermore, it is known that the sensitivity for many slow-growing and fastidious organisms is low.
A number of molecular approaches to improve conventional culture-based identification, including PCR, have been suggested. These include matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, which may decrease the time to result to 4 hours once the BC has become positive. However, a broader clinical e
valuation of this approach is still missing. Another strategy is the extraction and amplification of microbial nucleic acids from a positive BC and subsequent hybridization on a microarray platfo
rm to detect the gyrB, parE, and mecA genes of 50 bacterial species, which has recently been evaluated in an observational multicenter design with conventional BC as the comparator.
Systems Biology and Omics Technologies
There is great hope that systems biology approaches using high-throughput technologies, such as transcriptomics, proteomics, and metabolomics, will contribute to a better understanding of the pathophysiology of sepsis and systemic inflammation. This will allow the development of better sepsis biomarkers too. There are recent data that suggest that transcriptomic profiling by multiplexed quantitative PCR (qPCR) and metabolite detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS) may have potential in the clinical development of diagnostic tests. These tests are capable of overcoming the limitations of single molecules to differentiate between infectious and noninfectious causes of systemic inflammation and to determine the status of the patient’s immune system.
Potential of Gene Expression Profiling
Some studies suggest that gene expression profiling may help to differentiate between sterile SIRS and early sepsis and enable the discrimination of patients with acute infections. A set of 85 leukocyte genes (circulating leukocyte RNA profiles or “riboleukograms”) detected ventilator-associated pneumonia (VAP) in 158 intubated patients after blunt trauma with a sensitivity of 57 per cent and a specificity of 69 per cent. In ventilated children, expression patterns of 48 genes appeared to discriminate children with and children without VAP. Another research group identified a molecular signature of 138 genes expressed in peripheral blood mononuclear cells that could differentiate between sepsis and SIRS among 70 critically ill patients with 80 per cent accuracy.
A recent prospective multicenter study provided a proof of concept that a set of 42 molecular markers could differentiate between postsurgical SIRS and bacteremia with an accuracy of between 86 and 92 per cent for the detection of sepsis. In that study, 145 human white blood cell (WBC) genes associated with acute infection and inflammation were identified as being abnormally expressed. From these, a panel of 42 genes linked with innate and early adaptive immune function/ activation pathways were identified a priori by using preclinical research outcomes. These pathways allow a platform transfer to a multiplex qPCR format, retaining sensitivity and specificity, with a time to result within hours. The clinical utility of both molecular tests to identify pathogens and the ensuing host response still has to be evaluated with sufficiently powered prospective clinical utility trials with a design to allow assessments of their potential to distinguish SIRS from sepsis and to guide therapy.
But, there is hope that improved molecular diagnostics and prognostics based on transcriptomic, proteomic, or metabolic profiling will not only result in a better understanding of the complexity of systemic inflammation but also lead to new diagnostic tools which help to recognize infection and sepsis earlier and differentiate between infectious and non-infectious inflammation. Such tools also hold promise for the identification of new therapeutic targets and the identification of patient populations that may benefit from specific interventions that are aimed at these targets.
Indian docs catch up with new-gen sepsis diagnosis
Arecent Indian case study suggests that latest diagnostic tools have helped quick intervention to save sepsis patient. The case —an 8-year old male child was admitted to Rainbow Hospitals at Vijaywada with on and off fever since 3 months, 2 episodes of seizures and shortness of breath since 2 weeks. On 2D ECHO, the patient was found to have heavy vegetation in both the mitral and tricuspid valves. A portion from the biopsy of the vegetation was sent for routine microbial culture and a sample of the same was sent to Mumbai-based lab iGenetic Diagnostics for the infective endocarditis panel. The microbial culture was negative while the extremely sensitive PCR method used at the lab was able to detect Staphylococcus aureus in the sample. The doctor now needed to know which antibiotic to use to treat the patient. This is traditionally done using antibiotic sensitivity assays. But since the cultures were negative, this was not possible. Then, using a very innovative in-house developed PCR-based assay, iGenetic was able to diagnose MRSA or Methicillin-resistant Staphylococcus aureus.
iGenetic’s founder and managing director Arunima Patel says that most doctors in the infection critical care are still not used to molecular methods as they have seen microbiology using only empirical methods. And the other challenge is 50 per cent of the tests do not come in time or generate any results and most in the other half there are uncertainties. “We are able to address how fast or slow do bucks grow and what are they resistant with etc. with the latest technologies, especially Multiplex PCR methods that we have developed in-house in the last three years” she added.
“We have developed multiplex PCR methods in-house in the last three years”
Founder and Director, iGenetic Diagnostics, Mumbai