Sepsis is one of the most ambiguous disorders in medicine due to its early onset. Previously, sepsis was thought to be the process through which flesh decomposes, swamps acquire a putrid odour, and wounds deteriorate [1]. Later, it was renamed as systemic infection, which is commonly referred to as “blood poisoning” and was acknowledged as a result of pathogenic organisms growing within the circulation and evading the host’s immune system. As a result, it was established that the pathogen, not the host, is the perpetrator in the pathophysiology of sepsis [2]. Pathogens interact in the host immune system during infection, triggering a downstream inflammatory cascade including cytokines and other mediators, eventually producing immunosuppression, which leads to various types of organ failure and subsequent clinical degeneration [3].

Originally, sepsis was thought to be the result of internal organs rotting or decaying, with sepsis being defined as a result of the host’s systemic inflammatory response syndrome (SIRS) to infection, severe sepsis (sepsis associated with organ dysfunction, hyperfusion, or hypotension), and septic shock (sepsis induced hypotension that persists despite adequate fluid resuscitation) [4]. However, due to a lack of effective antimicrobials and supportive treatment, the definition of sepsis has evolved with time, preventing patients with sepsis from living long enough to be analyzed or they acquire a sequel of organ failure. As a result, the American College of Chest Physicians (ACCP) and the Society of Critical Care Medicine (SCCM) announced SIRS and published new agreements as a definition of sepsis and associated medical criteria (Sepsis-3) with deadly organ dysfunction caused by a dysregulated host response to an infection, which can be accepted as a shift in complete Sequential Organ Failure Assessment (SOFA) score points ≥2 subsequent toward the disease ^[3][4][5][3,4,5]. The guidelines of the SOFA score were presumed to be zero in the patients having no infection or organ failure whereas a SOFA score ≥2 mirrors a general fatality danger of approx. 10% in a common emergency population with suspected infection [6]. This novel and advanced definition focus on the power of the non-homeostatic response of the host to infection, the potential fatality of the infection that is impressively more than a direct disease, and the requirement for dire acknowledgement. As depicted later, even an unpredictable level of organ dysfunction when it was suspected earlier is related to an in-emergency mortality rate of nearly 10%.

According to the World Health Organization (WHO), in hospitals, sepsis is not just the costliest condition to treat as an additional main source of death, but certain reports have assessed a great many cases at a greater expense and death rates influencing more than 30 million individuals worldwide, thus steadily prompting 6 million deaths with rates of mortality between 20% and 50% [7]. The weight of sepsis is probably most noteworthy in low-income countries [8]. A reported 31.5 million people have are diagnosed with sepsis annually around the world, of which, 19.4 million individuals suffer from severe sepsis while 5.3 million individuals experience death, 3 million cases are estimated in neonates worldwide annually and there are 1.2 million cases in children, with a death rate of 11–19% [9], as well as 75,000 annual deaths in females due to puerperal sepsis globally [5]. Additionally, as per the ongoing safety information of Centers for Disease Control and Prevention (CDC) reports, sepsis inpatient admissions stay high for septic shock, roughly 60%; for severe sepsis, around 36%; for sepsis credited to a particular creature, roughly 31%; and for unknown sepsis, roughly 27% [10]. The latest in-hospital mortality estimates for sepsis patients has decreased from 28% to 18% [11]. Patients with severe sepsis admitted to the ICU increased from 7.2% to 11.1% and hospital mortality in severe sepsis decreased from 35% to 18%, according to a recent study of prospectively collected data from more than 90% of all intensive care unit (ICU) hospitalizations, confirming these trends in both incidence and mortality. Finally, the best epidemiologic evidence shows that severe sepsis is becoming more common while simultaneously becoming less fatal [12].

Because of its narrow window, early and systemic diagnosis of sepsis is a crucial task which may deviate towards shock, organ failure or even death. Delay in every hour in providing the correct treatment results in a decline in the survival rate of sepsis patients by 7.6% [13]. To combat this, non-specific broad-spectrum antibiotics are administered immediately in suspected cases which results not only in poor patient outcomes but also in the development of multi-drug resistance (MDR) ^[14][15][14,15]. Hence, the diagnostic method should be rapid and applicable to detect pathogens along with drug resistance (preferably within 3–5 h of patient admission) ^[16][17][16,17]. It should be capable enough to diagnose polymicrobial infections along with unknown and emerging pathogens. Furthermore, the results of the diagnosis should be able to provide appropriate decisions and antibiotic stewardship within a key time window of hours in order to limit morbidity and death [14]. The procedures that are sensitive, specific and quick for identifying the pathogen are therefore the major operational instruments for critical care units ^[18][19][18,19].

2. Conventional Methods for Management of Sepsis

2.1. Microbiological Methods

The detection of pathogens using these techniques is based on the apparent growth of microorganisms on a suitable culture media (solid agar and broth). There are two different methods and they have been discussed in detail in the section below.

2.1.1. Identification through Blood Culture/Gram Staining

For detection as well as identification of causative pathogens, sampling of blood/urine/lavage from patients, their routine culture followed by the Gram staining test remains the gold standard method ^[20][21][22][20,21,22]. To enhance the diagnosis, a minimum requirement of blood samples collected aseptically is 0.5–1 mL. Preferably, blood is drawn for blood cultures from two distinct venipunctures sites. One is the central venous catheters, which allows blood to be obtained concurrently from a peripheral and a vascular catheter, allows for faster detection of peripheral bacteremia vs. catheter-related bloodstream infections and appropriate dosing for clinical treatment [23]. Since some microbes can only be identified at the collection site and not in the blood, continuous monitoring of the collection sites is necessary when a positive culture is detected, which facilitates the further processing for pathogen identification in proper sepsis assessment [24]. These are the confirmatory tests to detect the potentiality of microbes in the given sample [25]. Comparatively, gram staining analysis is rapid (<15 min), economical, and provides information about the categorization of the infectious microbe as either Gram-positive, Gram-negative, Gram variable or Gram indeterminate ^[26][27][26,27].

2.1.2. Identification through Bactec Fx/VITEK 2

This automated system is built on sensors that detect any change in pressure within the blood culture bottle or track the CO₂ emitted by actively metabolizing species. Gram-negative microbes take 14 to 24 h in blood culture bottles to detect microbial growth, while Gram-positive bacteria take 24 to 48 h. It is now feasible to eliminate enough pathogenic load for direct identification in Bactec FX without first growing on an agar plate until a blood culture bottle has been marked positive and is suitable for traditional diagnosis ^[28][29][28,29]. Blood culture bottles are prepared, checked and incubated for 5 days at 35 °C with shaking agitation every 10 min in the instrument. By spreading 0.1 mL of consecutive 10-fold dilutions on a blood agar plate, the quantitative plate count technique is used to determine microbial fixation. They are then tested after overnight incubation at 350 °C on plates containing the colonies [30]. The following are the three main issues with using this culture-based method: Firstly, the conventional microbiological tests require 5 days to detect and identify the pathogens involved in sepsis. As a result, the procedure does not provide results promptly, which may be a significant source of anxiety for patients not having the infection. Secondly, the blood culture/Gram stains analysis has a lower sensitivity. Only 30% to 60% of the overall positive result of sensitivity has been estimated despite using it in the proper analytical manner, standardized procedures and accurate collection of amounts of blood sample ^{[28][29][30][31]}[28,29,30,31]. These recommended false-negative results which range from 40% to 70% might be owing to a scarcity of particular microbial species that flourish in laboratory culture medium, or distantly similar microbial strains ^[32][33][32,33]. Another reason for the false-negative result is self-medication and administration of antibiotics to the patients before the sampling for diagnosis [34]. Thirdly, there is the possibility of false positives due to non-adherence with the sterile condition during sample processing. As a consequence, patients receive antibiotics for those bacteria of which they are not infected. This misuse has led to the prolonged exposure of antibiotics, resulting in allergic reactions, toxicity, development of MDR, a long stay of patients in hospitals and hence increased medical costs ^[34][35][36][34,35,36]. As a result, certain points should be proposed for laboratory experts, concerned bodies and other stakeholders to improve the laboratory service by applying and using refined and emerging technology to classify and assess sensitivity to drugs for various microorganisms. However, there is a need for more intensive interventional trials to assess the effect of these technologies on sepsis treatment in clinical practice.

2.2. Biochemical Test

Biochemical tests including the mannitol test, citrate tests, triple sugar iron (TSI) test, indole test, methyl red test and enzymatic tests such as oxidase tests, urease tests and coagulase tests are used to distinguish pathogenic organisms that depend on the various diverging biochemical processes of different bacteria. For the identification of extracellular and intracellular bacterial enzymes, biochemical tests are used. Hence, they are distinguished based on biochemical activities [37].

3. Modern Methods for Management of Sepsis

There is a paradigm shift from conventional culture and biochemical-based detection of pathogens to modern techniques including molecular as well as emerging methods which rely on detection at the strain level in much less time (20 min to 3 h). System biology has been thrust into the spotlight in molecular research due to technological advances and the knowledge provided by the human genome project, which has accelerated multiple methods that may not only produce an expanded understanding of complicated sepsis pathophysiology but can also articulate undetermined methodologies ^[38][39][38,39]. Reviewed here are the modern advancements in early detection, including inventive high-throughput approaches.

3.1. Implementing Molecular Detection for the Identification of Pathogens

Molecular detection using PCR depends upon the amplification of the pathogen’s target nucleic acid region using gene-specific primers or probes. For the identification of different pathogens, a variety of molecular targets are used ^{[40][41][42][43]}[40,41,42,43].

3.1.1. PCR

In 1983, Kary Mullis [44] devised the standard PCR, which allows the detection of a single bacterial pathogen by identifying a specific target DNA sequence [45]. A sensitive examination is portrayed here to classify the microorganisms in the entire bloodstream by the PCR. A particular primer–probe set is intended to reproducibly detect bacteria of purified DNA from whole blood. This assay framework was demonstrated to be comprehensive for all strains, equally from all bacteria. This unique PCR-based test was created to assist amplification from a range of human, bacterial and yeast genomic DNAs due to its inefficiency. A broad sample preparation methodology was designed that was applicable for the DNA purification from various bacteria in whole blood. With the help of this method, it was feasible to distinguish every particular bacterial DNA from whole blood samples inoculated with a minimum of 4 CFU/mL. Co-purified human blood DNA did not influence the sensitivity of detection by PCR [46]. PCR can identify even a single copy of a target DNA sequence under ideal conditions in a given sample. Therefore, prior multiplication enrichment of the microbe is not needed, all things considered with basic DNA probe tests. Thus, PCR-based diagnostic tests have made a significant advanced improvement for infectious agents subsequently [47]. The technology has been updated to expand the usage of traditional PCR. The use of the primers pair in parallel reactions with simultaneous amplification for different target DNA sequences, known as multiplex-PCR, is the first absolute shift. As a result, several DNA sequences replicated in the same processor might be amplified [48]. Another change is nested PCR, which uses two sets of primers with a preferred target for amplification of an internal DNA sequence. The first reaction is carried out using the first set of primers, and the results are subsequently subjected to a second amplification with various primer sets [49].

3.1.2. Real-Time PCR

Despite several advances, high-throughput science research laboratories can redirect standard PCR methods, including nested PCR techniques, due to the immense exposure of excess contamination with amplified products [50]. Traditional PCR procedures rely on automated detection of fluorescence from PCR amplicons, while real-time PCR systems are faster, less sensitive to contamination and require less labour ^[51][52][51,52]. Real-time PCR techniques also allow for infinite and comparable calibration of the desired sequence, which may help to increase the gap in critical microbial potential ^[53][54][53,54]. In addition, compared to traditional PCR, real-time PCR has several novel advantages, including simplicity, quantitative capacity and speed [55]. Fluorescence-based real-time PCR is based on the detection of the fluorescent signal generated during DNA amplification. After a specific number of cycles, the real-time assays synthesize a quantity of target DNA. When the amplification of a PCR product is controlled, it is initially observed during cycling by determining the cycle number at which the reporter dye’s discharge energy dominates the background noise. As a result, the threshold cycle is named after this cycle number (Ct). This Ct value is determined during the PCR exponential phase and is inversely proportional to the target’s copy number. As a result, the difference in fluorescence signal is observed before the incident when the beginning of the copy number of the target DNA is higher, and the Ct value is lower [56]. SYBR Green, TaqMan, molecular beacons and scorpions are the four types of fluorescent DNA probes currently available for real-time PCR product detection. All of these probes emit a fluorescent signal that can be used to detect the PCR products [57]. The ability to quantify the process is a key function of RT-PCR [58]. An internal calibrant is put to each well of the assay plates as a reference in each experiment, and PCR is used to examine each well for quantification. The peak heights in the mass spectrum for amplicons determine the proportionate ratios of calibrant to microorganisms ^[59][60][59,60]. The concentration of the pathogen can be thoroughly evaluated using the known initial concentration of calibrant. Having a significant effect on pathogen detection in diagnostic microbiology, real-time PCR is primarily focused on the pathogen genotype [61]. The role of microbes in various human diseases is examined in these essays, which is the most critical use of real-time technology in microbiology. Due to the strength of complexity the technology has positively evaluated the various microbes and their nucleic acid targets present in a single sample, also it has allowed the differentiation of various forms of microbial genotypes in a single reaction tube [62]. The significance of RT-PCR in microbial load detection is that these techniques are extremely useful since they actively reveal the spectrum of increasing infection, host–pathogen interaction and antimicrobial medication effectiveness. It also enables the administration of antibiotics promptly. Real-time assays are useful for distinguishing serotypes within a particular microbial population ^[63][73], diagnosing pathogens in clinical samples ^[64][65][74,75] and bacteria (viruses, bacteria, fungi, protozoa or toxins produced by them that cause diseases) used as biological warfare agents ^[66][76]. Despite these, molecular techniques including PCR do not provide any information about AMR among pathogens ^[67][77].

3.1.3. Surface-Enhanced Raman Spectroscopy (SERS)

Surface-enhanced Raman spectroscopy (SERS) is emerging as an important technology for the identification which amplifies the Raman dispersing of the objective particles on a superficial layer of metal made of graphene or other different materials ^{[68][69][70][71][72]}[78,79,80,81,82]. This technique has the ability to facilitate the label-free nucleic acid identification ^[73][83]. Surface plasmons are generated by applying an excitation frequency that is in phase with the particle’s plasmon assimilation profile, resulting in a solid electromagnetic field on the metal surface. The emission of the Raman dispersed light is radiated in every direction of the particle is gathered through a microscope and consequently identified. In addition, the Raman dispersed light is then coordinated against a reference profile of microorganisms to be recognized ^{[74][75][76][77]}[84,85,86,87]. Consequently, SERS can efficiently recognize the occupancy of microbial cells on the outer surface, thus yielding a data-rich spectrum that can be used for microorganism identification ^{[78][79][80][81][82][83][84]}[88,89,90,91,92,93,94]. In addition to the pathogen identification, SERS has also been utilized for antibiotic susceptibilities in urosepsis ^[81][91]. The primary reason the SERS procedure has not been set up as a routine scientific strategy is that it does not withstand its high sensitivity and specificity, which are the significant disadvantage of SERS, and restricted ability in investigating polymicrobial tests which are because of the lower reproducibility of the SERS signal ^[85][95]. Thus, SERS combined with different methods should be incorporated which outlines the wide uses of this incredible method.

3.1.4. MALDI-TOF

Matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-MS) is another newly discovered procedure that is now being used in clinical research to detect bacterial species. This technique guarantees fast detection of causative bacterial microorganisms demonstrated to be viable in the positive blood cultures that can rapidly recognize bacterial development, hence accelerating the general process of the antimicrobial resistance report ^[86][96]. MALDI is a protein identification ionization technique in which the analyte crystallizes in a strong lattice matrix crystal that absorbs laser light, allowing it to ionize and desorb from the matrix. The ionized atoms are isolated and dependent on their mass to charge (m/z) ratio from the entire microscopic organism’s test when it flies through a vacuum tube produces an m/z profiles of the apparent multitude of proteins in the sample ^[87][97]. Mass spectroscopy has been utilized for the inoculation of different species, along with the microbial suspension. Because of the low microbial concentration, MS cannot conduct direct examinations on human blood samples, which is a major disadvantage. Because of the low reproducibility and changeability in preliminary methodology and the matrix composition, blood culture is typically needed to improve the microscopic organisms to a detachable level ^[88][98]. Another drawback of MS is its restricted capacity in arranging various microbes from the polymicrobial samples ^[89][90][91][99,100,101] as the spectral profiles formed by this technique are more complicated, making it difficult to deconvolute the composite spectra gathered at the same time from different microbial species in the poly-microbial samples.

3.2. Broad-Spectrum Genomic Detection of AMR

3.2.1. High Resolution Melting Analysis Technology

High resolution melt (HRM) analysis depends on the detection of differences in melting temperature (Tm) due to the presence of a mutation in a previously amplified target that produces melt curve profiles specific to pathogens. The size and the sequence of the PCR amplicon is the major reason on which the melting curve profile depends ^[92][102]. It is so sensitive that even a single point mutation resulting in a Tm shift can be detected ^[93][103]. Therefore, it allows molecular detection of resistant genes and hereditary mutations rapidly with a higher output of post-PCR examination which allows the researchers to identify and classify the new hereditary mutations and variations along with single nucleotide polymorphisms without sequencing (gene scanning) or before sequencing in a population ^[94][104]. Several antibiotic resistance marker genes conferring to a bacterium have been usually detected. A study reported real-time PCR-based rapid identification of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Proteus mirabilis using 16S rRNA gene-specific primers. Furthermore, HRM and machine learning algorithm approaches were used to determine the antimicrobial susceptibility test within 6.5 h ^[95][105].

3.2.2. Sequencing

In the diagnostic field, the aim of the utilization of genomic rather than gene-based techniques for both bacterial species detection and AMR detection is growing. There is a need for more effective and rapid AMR preventive measures driving the shift to whole-genome sequencing (WGS). Bacterial and AMR gene identification using automated bioinformatics examination methods are easy to perform after an organism has been isolated by culture ^[96][114]. WGS allows all genes involved in resistance to be tracked, allowing all genomic data of resistant factors to be present in a bacterial cell to be analyzed. Next-generation sequencing (NGS), which has revolutionized the biological sciences, is another emerging tool. NGS makes large-scale whole-genome sequencing (WGS) affordable and realistic for the average researcher with its super high throughput, versatility and speed. It also allows scientists to sequence the entire human genome in a single experiment, allowing them to research biological processes at a level never before possible. Furthermore, in the age of complex genomic science, which necessitates a deeper understanding of details outside the boundaries of conventional DNA technology, it has filled the gap and become a routine research method to resolve those issues ^[97][115]. NGS combined with meta-genomic approaches that essentially include genome sequencing of infectious biological samples such as blood, urine and lavage without culturing them and provide the diverse profile of all species including those that are targeted and untargeted present in the sample. This approach has revolutionized the identification of all including new resistance genes in a single specimen ^[98][99][116,117]. The sequencing-based metagenomic approach has analyzed the kinetics of gut microbiota before, during and after antibiotic treatment ^[100][118].

3.2.3. DNA Microarray

Microarrays have been updated as useful methods for bacterial detection and identification due to their strong parallelism in screening for the expression of a wide variety of genes after specific gene amplification by either a broad-range or a multiplex-PCR before microarray analysis ^[101][121] Microarrays employ surface-immobilized DNA and RNA probes to collect and categorize DNA/RNA of microorganisms via sequence-specific complementary hybridization, decreasing sample and reagent consumption and costs while permitting precise segregation down to the species or strain level. A study conducted by Ballarini et al. utilized an oligonucleotide-based microarray (BactoChip) for culture-independent detection, quantification as well as differentiation from 21 different bacterial genera among clinical isolates ^[102][122]. Additionally, the Verigene stage from Luminex Corporation can distinguish the Gram-positive board of nine species of bacteria and three genes of AMR against methicillin and vancomycin and five species and six AMR genes for carbapenemase and expanded range beta-lactamases ^{[103][104][105]}[123,124,125]. Being independent of culture, microarray-based detection is rapid, hence gaining importance in clinics in combination with antimicrobial stewardship ^[106][107][126,127]. Despite these, not a single microarray platform has been commercialized so far to effectively recognize all microorganisms in polymicrobial diseases ^[108][128].