9 February 2022
Medicine and medical technology have evolved rapidly in the past decades. Infectious diseases that were once impossible to confirm can now be diagnosed and cured efficiently. Unfortunately, in the face of advanced treatment, microorganism pathogenicity and virulence grew stronger to survive. Severe infections still take medical professionals into a race against time to accurately diagnose pathogenic microorganisms and treat patients with the right antimicrobial drug.
The fight between the pathogenic microorganisms and the host at risk of being infected
The pathogenicity of an infectious agent, either bacterial, viral, fungal, or parasitic, refers to its capability to cause disease. On the other hand, virulence is described as an ability of a pathogen to infect the host. Virulence factors are the molecules that help the microorganism enter a host, deceive the defense mechanisms of the infected organism, and cause illness. These molecules are often synthesized by the pathogens and encoded in their genome, but may also be acquired from the environment via transmissible genetic elements.
Virulence factors of pathogenic bacteria include adherence and invasion factors which help to colonize and enter host cells. Protective capsules shelter pathogenic bacteria from being tagged (opsonization) as invaders by specific proteins (opsonins), ingested, and eliminated (phagocytosis process) by the host’s immune system. Other factors include endotoxins and exotoxins responsible for severe clinical signs such as fever, inflammation, alterations of blood pressure, and shock.
The virulence factors of viruses are encoded in their genetic information. The basic structure of a virus contains one type of nucleic acid, either RNA or DNA, and a protective protein coat. The protein coating protects the virus from the destructive enzymes of the infected host (nucleases), helps it attach to the host cell, and suppresses the type I interferon (IFN) response, an inhibitor of viral replication and activator of the immune response.
Virulence factors such as toxins and toxin-producing genes, species- and strain-selective genes can be used as targets associated with a specific pathogen, helping establish a quicker, accurate diagnosis.
The susceptibility or the likelihood of infection is dependent on the integrity of the host’s immune system. Once the pathogenic microorganism enters the organism, complex mechanisms involving a series of proteins (interleukins) and mediators of inflammation (eicosanoids and complement system) and white blood cells fight against the invading pathogen. When the pathogenic bacteria breach the immune system and cause infection, early diagnosis is critical in preventing the aggravation of clinical signs 1.
The limitation of current techniques for the detection of pathogenic microorganism
Current traditional methods for pathogen detection include polymerase chain reaction (PCR), immunology-based techniques, and culture and colony counting.
PCR amplifies the genetic material (nucleic acid) or the information-carrying molecules within a microorganism or virus. Since the first development of PCR tests, the used methods have developed and improved in terms of quality, efficiency, cost savings, techniques, increase in sensitivity and specificity, required time before getting the results, and detection of more samples simultaneously. Current methods vary and include real-time PCR, multiplex PCR, and reverse transcriptase PCR (RT-PCR). PCR can also be coupled with other diagnostic techniques like acoustic wave sensor (SAW) or evanescent wave biosensors.
Unfortunately, there are a series of limitations in PCR assays. One crucial factor might be that PCR tests don’t discriminate between dead or alive pathogens, as the genetic material is still present. Other factors are that PCR testing is expensive as the protocol requires specific primers complementary to the targeted genome, special enzymes (Taq polymerase enzyme), and viral or bacterial-specific targeted primers. PCR test is highly sensitive, and sample contamination can lead to misleading results 2, 3.
Fig. 1 Schematic representation of a PCR cycle2.
Immunology-based methods include immunomagnetic separation (IMS), which is a method that only captures and extracts the suspected pathogen. The IMS needs to be combined with other optical methodologies for detection. Another essential immunology-based method is the enzyme-linked immunosorbent assay (ELISA). This procedure detects and measures the antigen or antibodies for a specific pathogen in a sample. ELISA method is an expensive procedure requiring specific antibodies developed on cell cultures. Even further, these antibodies are unstable proteins that need refrigerated transport and storage. ELISA test may fail to detect low antibodies titer in the early phases of a disease or in immunosuppressed individuals. Also, it does not differentiate active forms of illness from recovering patients who are no longer sick but still produce antibodies 4.
The culturing and plating method, although it is the oldest method, it is still the gold standard for bacterial detection. Unfortunately, it might be the method with the most limitations. The culturing and plating process is time-consuming, and in some cases, it can take four to nine days to rule out an infection and up to 16 days for confirmation of a specific bacteria (Campylobacter). Even more, the results of up to 70% of cultured samples are negative. In many patients, specifically those in critical conditions who require immediate, specific treatment, the wait is incompatible with life 5, 6, 7.
What does the future hold for early diagnosis?
Biosensors are among the newest methods developed for detecting pathogens, especially those that take a long time to culture and diagnose with the previous methods. Nanomaterials combined with the biological systems overcome most limitations of current traditional methods used to diagnose bacterial infections. Biosensors allow the measurement of chemical, biological, physiological, or biochemical analytes and biological processes.
The structure of a biosensor integrates a molecular identification or recognition component (bio-receptor), a signal generator or transducer, and a reading device or amplifier. In the field of microorganisms, bio-receptors that can be used are for antigens, nucleic acids, or antibodies. Biosensors recognize specific biological analytes and convert them into measurable electrochemical, optical, acoustic, or electronic signals. Types of biosensors include Surface Plasmon Resonance (SPR), genosensors, immunosensors, micromechanical sensors, or phage-based biosensors 6. Research on biosensors is still developing, and in the future, diagnosis methods will probably include much more complex and rapid possibilities for diagnosis and early treatment 5,8, 9.
Fig. 2 Components and measurement formats associated with electrochemical biosensors for pathogen detection 10.
Studies of virulence factors are important for better understanding microbial pathogenesis and host defense mechanisms. It also provides key information for the vaccine and drug development in preventing and treating infectious diseases. Metagenomic sequencing-based approaches offer a better way to decode the virulence factors and their contributions to overall pathogenesis by having access to the full gene repertoire of a strain.
Devin® filter and Parti-Seq®platform are amongst the newest and most advanced methods developed for the early detection of pathogenic microorganisms. This combined protocol allows the accurate identification of a pathogenic microorganism within 24 hours from the sample’s submission. Devin®Filter, as the name states, filters up to 95% of the patient’s nucleated cells from the blood sample, allowing a high passage of bacteria and viruses in just five minutes. This filtration process is essential as human genetic information (DNA) in the blood samples that need to be analyzed is a critical holdback in rapidly identifying pathogenic microorganisms. Further, pathogen identification is performed with NGS-based Pathogen Real-Time Identification by Sequencing (PaRTI-Seq®) developed by Micronbrane for rapid pathogen identification within less than 24 hours with a sensitivity of 102 genome copies /mL 11.
Fig. 3 Overview of contemporary depletion techniques 11.
While the traditional methods used for the current diagnosis of bacterial or viral infections might give accurate results, they have significant limitations that are not favoring a critically ill patient. New highly specific diagnostic methods are currently used worldwide, helping patients overcome severe infections.