Biomarkers innovation in urinary tract infections: Insights into pathophysiology, antibiotic resistance, and clinical applications

Abstract

Urinary tract infections (UTIs) are the most common bacterial infections. Escherichia coli is the most common cause of UTIs, accounting for 50% of hospital-reported and 90% of community-reported cases. Also, this includes species of Klebsiella, Proteus, Acinetobacter, Pseudomonas, Staphylococcus, Streptococcus, and Enterococcus. Patients experience cystitis, polyuria, and dysuria. If untreated, this affects the kidneys, further leading to septicemia. UTIs majorly affect adult females (40%-60%). Microbiological culture has been proven to be the standard method. However, it takes 48-72 hours for the tests to be reported. In cases of recurrent UTI, it is mandatory to have a quick, sensitive, and specific diagnostic procedure. Dipstick tests are considered early methods for diagnosing UTIs; however, they have limitations. Recently, biomarkers are being used to assess the severity of the disease. To achieve the United Nations Sustainable Development Goals 3 and 8, the expertise from General Medicine, Biotechnology, and Microbiology come together in achieving the set targets by 2030. In addition to diagnosis of UTI, resistance to antibiotics should not be neglected. This review aimed to examine the clinical relevance of biomarkers such as neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, interleukin (IL) 6, IL-8, heparin-binding protein, procalcitonin, lipopolysaccharide-binding protein, xanthine oxidase, cell-free DNA, and transrenal DNA.

Key Words: Urinary tract infection; Pathogens; Diagnosis; Antibiotic sensitivity; Host-derived biomarkers; Pathogen-derived biomarkers; Point-of-care testing; Sustainable development goals 3; Sustainable development goals 8

Core Tip: Urinary tract infections (UTIs) are prevalent and often require timely diagnosis to prevent complications. The emerging urinary biomarkers, including neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, interleukin (IL)-6, IL-8, heparin-binding protein, procalcitonin, lipopolysaccharide-binding protein, xanthine oxidase, cell-free DNA, and transrenal DNA, have shown potential in identifying infection severity, organ dysfunction, and antibiotic resistance. The review further examines how integrating these biomarkers with advanced biosensor-based diagnostic tools can enhance diagnostic sensitivity, facilitate point-of-care testing, and improve clinical outcomes. The review also underscores the importance of validating and standardizing biomarker-based diagnostics to bridge laboratory innovations with clinical application, ultimately supporting more accurate, rapid, and personalized UTI management.

INTRODUCTION

The urinary system is an excretory system made up of the kidneys, ureters, urinary bladder, and urethra. Its main functions are to filter blood to remove waste and to regulate water and electrolyte balance. Infections of the urinary tract can affect any part of the urinary system. Most urinary tract infections (UTIs) involve the lower urinary tract, which includes the bladder and urethra. However, the infection can ascend to affect the kidneys also[1]. Each year, approximately 150 million people are affected by these infections, resulting in significant health issues and economic costs[2]. In 2019, India had the highest number of UTI-related deaths with 55558 fatalities[3]. UTIs can impact individuals of any age, but are common in pediatric and elderly populations. Among adults, females are the most affected due to differences in the anatomical structure and the proximity of the urethra to the anus. In clinical practice, nearly 25% of infections in women are UTIs. Between 50% and 60% of women will experience at least one episode of UTI during their lifetime. About 80% of recurrent UTIs are reinfections, with recurrences often occurring within three months of the initial infection[4]. Predisposing factors of recurrent UTIs may be structural abnormalities, poor personal hygiene, or the presence of disorders such as diabetes mellitus or an impaired immune system.

COMMONLY DETECTED PATHOGENS IN UTIS

Generally, the microbiota present in the urinary tract system tend to cause impacts on other systems, such as the gastrointestinal and genitourinary systems. These microbiotas tend to control these intricate interactions through the immune system, both locally and systemically. Whether alterations in the microbiota and related metabolites are a cause or an effect of UTI is still unknown[5].

Escherichia coli is a common facultative anaerobe responsible for 85% of UTIs diagnosed at primary health facilities and approximately 50% of hospital-acquired cases[6]. Other uropathogens include Proteus mirabilis, Klebsiella pneumoniae, and Enterococcus faecalis. Various virulence factors in these uropathogens enable them to adhere to the mucosal surfaces of the urinary tract, leading to infection[6]. Candida species are the most common cause of fungal UTIs, particularly among hospitalized patients, accounting for approximately 10% to 15% of all UTIs. This illness is common in diabetic patients with uncontrolled hyperglycemia and those having indwelling urinary catheters[7]. Some of the common microorganisms associated with UTIs are displayed in Figure 1 and Table 1[8,9].

Figure 1 Pathogens causing urinary tract infections. UTI: Urinary tract infections.

Table 1 Commonly detected pathogens causing urinary tract infections.

Bacteria	Factors of UTI
Escherichia coli	The most common cause of UTI; the pathogen ascends from the urethra to the bladder or descends from the kidneys to lower urinary tract
Staphylococcus saprophyticus	Present as skin flora; cause UTIs, especially in young women
Enterococcus faecalis	It is an opportunistic pathogen; associated with complicated UTIs, especially in hospitalized patients
Pseudomonas aeruginosa	Cause antibiotic resistance; form biofilms, infect patients with catheters or those who are immunocompromised
Klebsiella pneumoniae	Affect individuals with underlying health conditions that are resistant to multiple antibiotics
Proteus mirabilis	Associated with catheter use and urinary retention; produces urease, which contributes to struvite stones
Enterobacter spp.	Cause UTIs in hospitalized patients; resistant to multiple antibiotics
Coagulase-negative Staphylococci spp.	Present in skin flora; cause UTIs in immunocompromised individuals or those with catheters

UTI: Urinary tract infections.

DIAGNOSIS OF UTI

To date, the diagnosis of UTI involves both clinical features and identifying the causative organism through a urine culture[10]. Clinical features of UTI include dysuria, frequent micturition, nocturia, urgency, cloudy urine, hematuria, pyuria, low back pain, high temperature, etc. The symptom-based approach is appropriate in most situations, except in specific patient groups, such as small children, the elderly, long-term catheter users, and individuals with urologic diseases. This group of patients tends to present with nonspecific clinical features[11].

White blood cells, nitrites, leukocyte esterase, or other substances present in urine also indicate the presence of infection. Timely antibiotic administration may prevent complications and facilitate a quicker recovery. Analyzing various urine constituents aid in diagnosing UTIs, identifying their source, and assessing the severity of kidney impairment[1]. Dipstick urine analysis and direct microscopy are the most popular tests used in patients with suspected UTI, which are fast, inexpensive, and require little technical experience[12]. Figure 2 illustrates a few diagnostic tools used to diagnose UTIs.

Figure 2 Laboratory methods used in the diagnosis of urinary tract infections. MALDI-TOF MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; WBC: White blood cell; RBC: Red blood cell.

Urine dipstick

The dipstick test is a widely used point-of-care test for detecting substances in urine such as sugar, hemoglobin, proteins, nitrites, and leukocyte esterase[13]. It is a valuable tool for screening bacteriuria, especially when both nitrites and leukocyte esterase are positive. A negative result for either marker can effectively rule out infection in the general population. However, this is less reliable in the elderly, pregnant women, or patients who have undergone surgery. While identification of proteinuria is common, data on its diagnostic accuracy are lacking. Even though the dipstick test is reliable, cost-effective, and practical in emergency settings, it has decreased accuracy, especially for nitrites in symptomatic UTIs. Dipstick testing has little value in screening healthy children to assess the severity of kidney diseases[14].

Urine microscopy

Microscopic evaluation primarily focuses on identifying white and red blood cells, epithelial cells, protein casts, and microorganisms[15]. In clinical practice, the presence of white blood cells and nitrites indicates bacteriuria, and the presence of red blood cells indicates severe inflammation[15].

Microbiological urine culture

The culture is considered to be the standard method for the identification of pathogens causing UTIs. The culture takes at least 48 hours to 72 hours to produce definite results[16]. Urine culture using midstream urine is used to identify the pathogen. This also quantifies the bacterial load in the patient, necessitating the type and dosage of the appropriate antibiotic. Nevertheless, neither scientific literature nor microbiological labs have established the minimal amount of bacteriuria required to indicate a UTI[17]. The threshold for culture-contaminated urine is usually set at more than 10⁵ colony-forming units per milliliter (CFU/mL) for diagnosing a UTI. According to the American Urological Association Core Curriculum, patients should be diagnosed with a UTI if they exhibit symptoms and their urine culture reveals more than 10³ CFU/mL. A count of less than or equal to 10⁴ CFU/mL is found in 20%-40% of women with UTI. A single organism with more than or equal to 10³ CFU/mL in a symptomatic patient is regarded as a UTI[18]. The threshold of 10⁵ CFU/mL, however, ignores a large number of pertinent infections. Therefore, depending on the types of bacteria found, some recommendations suggest diagnosing a UTI based on a level of 10³ CFU/mL[17]. Culture is slow, and therefore, the diagnosis happens with a considerable delay, which is not desirable. In women, if two consecutive adequately collected midstream urine samples exceed the upper limit of ≥ 10⁵ CFU/mL, in the absence of clinical features of a UTI, the patient is considered to have asymptomatic bacteriuria (ABU). In men, one detection is sufficient[10].

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detects UTI pathogens and provides results within 18 hours to 30 hours. In the majority of clinical settings, it is essential to identify the bacteria at the species level accurately. MALDI-TOF MS can detect the strains from blood cultures, cerebrospinal fluid, and urine in addition to strains grown on solid media. Gram-positive and gram-negative bacteria, aerobes, anaerobes, mycobacteria, Nocardia, yeasts, filamentous fungus, and viruses are identified by MALDI-TOF MS[19].

Biosensors

Developments in nanoscience and sensing technologies have led to a significant increase in the development of biosensors with excellent sensitivity, specificity, and reusability, as compared to the dipstick test. Point-of-care testing devices offer quick and efficient diagnosis, ease of handling, and portability[20].

Imaging techniques

Cystoscopy and urinary tract imaging are rarely beneficial, hence not advised for simple UTIs. But in situations where infections recur, imaging could help rule out stones and structural abnormalities of the urinary system[16].

Biomarkers for UTI

Technological advancements have enhanced the detection of UTIs and related renal involvement, particularly upper UTIs. During UTI, biomarkers are produced either by the host, as part of the immune or inflammatory response, or by the pathogenic bacteria themselves. These biomarkers become elevated in biological fluids, such as urine, blood, and other fluids. They help the clinicians in early diagnosis, assess infection severity, and monitor treatment response[21,22]. As illustrated in Figure 3, urinary biomarkers can be classified based on biological origin[21,22].

Figure 3 Classification of urinary biomarkers based on biological origin. NGAL: Neutrophil gelatinase-associated lipocalin; KIM-1: Kidney Injury molecule-1; IL: Interleukin; HBP: Heparin-binding protein; LBP: Lipopolysaccharide-binding protein; PCT: Procalcitonin; XO: Xanthine oxidase; cfDNA: Cell-free DNA; trDNA: Transrenal DNA.

Nephron-derived biomarkers: In the presence of upper UTIs such as pyelonephritis, tubular epithelial cells may become injured and release proteins, including kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), into the urine. These molecules serve as indicators of tubular damage and are valuable in distinguishing between lower and upper urinary tract involvement, as well as in identifying early kidney injury[21].

NGAL: NGAL, an acute-phase protein, was initially identified in acute kidney injury cases, especially those affecting the proximal renal tubules[21]. NGAL production also occurs within the thick ascending limb of the loop of Henle and the intercalated cells of the collecting duct[21]. NGAL exists in multiple forms, including monomeric, homodimeric, and heterodimeric complexes with gelatinase, as displayed in Figure 4[23]. The monomeric form, produced by injured renal epithelial cells, is the most clinically relevant form. NGAL is also abundantly expressed by activated neutrophils during infection and inflammation[23].

Figure 4 Multiple forms of neutrophil gelatinase-associated lipocalin. NGAL: Neutrophil gelatinase-associated lipocalin.

NGAL is generally responsible for iron trafficking throughout the genitourinary tract, as well as for epithelial cell proliferation, differentiation, and the response to infection. Iron availability directly influences bacterial growth[24]. Most gram-negative bacteria, which require iron to survive, produce enterochelin to scavenge free iron and transport it into their cells[25]. NGAL binds to the enterochelin-iron complex, promoting its excretion through urine and exerting a bacteriostatic effect as part of the innate immune response. Elevated urine NGAL levels, indicate localized infection within the genitourinary system[26] (Figure 4).

Infected patients exhibit elevated plasma levels of NGAL, which enhances proteolytic activity, particularly of gelatinase B on collagen, by preventing its degradation. Under normal conditions, NGAL is present in low concentrations in blood and urine; however, its levels rise significantly during UTIs. NGAL also plays a crucial role in the pathophysiological mechanisms underlying kidney injury during UTIs[27]. After 12 hours of UTIs, NGAL levels start to rise and peak in three days[21]. NGAL is especially valuable in pediatric and critically ill patients, where conventional renal markers are unreliable. Urinary NGAL levels are increased in patients with both upper and lower UTIs, but does not differentiate between the two UTIs[28]. NGAL is also elevated in various non-renal conditions such as systemic inflammation, infections, or malignancies. Factors such as age, baseline renal function, and systemic illness significantly impact NGAL levels, underscoring the importance of clinical context in interpretation[27]. Despite many challenges, NGAL continues to hold strong potential as a rapid, non-invasive diagnostic tool for evaluating UTIs and monitoring kidney health[27].

KIM-1: KIM-1 is a multifunctional protein that is rarely found in normal kidney tissues, but is expressed in proximal renal tubular epithelial cells that recover following injury. It is a transmembrane glycoprotein with an extracellular domain, a single transmembrane domain, and a short intracellular domain, as shown in Figure 5. Following injury, metalloproteinases cleave and release the extracellular domain into the urine[29,30]. The transmembrane domain not only signals renal injury but also contributes to tissue repair by enabling tubular epithelial cells to phagocytose apoptotic debris[31]. Thus, KIM-1 aids tubular repair by functioning as a phosphatidylserine receptor, allowing epithelial cells to phagocytose apoptotic bodies and debris, thereby facilitating regeneration[29] (Figure 5).

Figure 5 Structure of kidney Injury molecule-1. KIM: Kidney Injury molecule.

Clinically, KIM-1 is used to detect acute kidney injury, monitor renal function, and assess the risk of progression of kidney disease[32]. KIM-1 is highly expressed following acute ischemic, hypoxic, and toxic injuries, as well as in certain renal tubular interstitial and polycystic kidney diseases[29]. Urinary KIM-1 correlates with the severity and duration of UTIs, particularly in cases involving multidrug-resistant (MDR) uropathogens. KIM-1 levels rise dramatically within hours of UTIs, and they are associated with the severity of kidney injury[29]. But, KIM-1 lacks specificity, elevated levels are observed in non-infectious states, including renal cell carcinoma, chronic kidney disease, and other forms of toxic or ischemic injuries of kidneys[33]. Gender-related differences have also been observed in KIM-1 expression. Pediatric populations have reported higher urinary KIM-1 levels especially in females than in males, and these levels increase with age during adolescence[34].

Immune/inflammatory biomarkers: These biomarkers are produced by activated immune cells during infection or inflammation, thus reflecting the inflammatory response of the host to bacterial invasion[35]. Upon detecting pathogen invasion, urinary tract epithelial cells quickly mobilize resident tissue cells or circulating immune cells to the infection site to clear the bacteria. The primary innate immune cells that combat UTIs, in the early stages, are neutrophils. In infected tissue, toll-like receptor 4 (TLR4) on bladder epithelial cells initiates intracellular signaling, leading to the release of inflammatory mediators and cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8, which are correlated with the removal of infectious agents. Following infection, the adhesion molecules, P-selectin and E-selectin, are rapidly upregulated to facilitate neutrophil recruitment[36]: (1) IL-6: As a multifunctional cytokine, it controls several processes, including inflammation, organ development, and the acute phase response. T-helper cells, neutrophils, macrophages, hepatocytes, and podocytes express the IL-6 receptor, to which IL-6 directly binds. Additionally, a soluble IL-6 receptor exists, which enables IL-6 to influence a wide range of target cells. Macrophages respond to TNF-α, IL-1, and IL-2 by producing the chemokine, Il-8. In the nephron, IL-6 is primarily expressed by proximal tubular epithelial cells in response to inflammatory stimuli[37]. In older persons, urine IL-6 levels differentiate individuals with ABU from symptomatic UTI[38]; (2) IL-8: IL-8 functions primarily as a chemoattractant for neutrophils, facilitating their migration to sites of infection within the urinary tract. Its expression is typically localized and correlates with inflammation of the lower urinary tract[39]. Urinary levels of IL-6 and IL-8 are significantly higher in children with febrile UTIs compared to those with other febrile illnesses[40]. The urine of healthy individuals contains negligible levels of both IL-6 and IL-8. Urinary IL-6 and IL-8 levels may be helpful in the acute phase of UTI, but are not able to differentiate upper from lower UTI[39]. Systemic inflammation can cause serum IL-8 levels to increase, even when the urinary tract is not involved. Hence, using IL-8 alone may not be sufficient. When used in conjunction with clinical symptoms or in combination with NGAL or KIM-1, the likelihood of obtaining an accurate diagnosis is high[39]; and (3) Heparin-binding protein: The 37 kDa heparin-binding protein (HBP) is found in the azurophilic and secretory granules of human neutrophils. HBP works as a chemoattractant and monocyte activator while also causing vascular leakage. HBP possesses a wide range of antibacterial activity and might aid in the direct opsonization of bacteria. In children with UTIs, HBP shows elevated levels in urine. Urine HBP may distinguish pyelonephritis from cystitis. HBP is a potential UTI biomarker in ABU, as well as in those who have urogenital pathology[41,42]. Urine HBP have proven to have a higher diagnostic value than WBC, IL-6, and nitrite in children to differentiate bacterial from non-bacterial UTI. In contrast to IL-6, it exhibits a modest discriminating value between elderly patients with UTI and those who have ABU[43].

Systemic biomarkers: Acute pyelonephritis and febrile UTIs trigger a systemic host response, whereas ABU usually elicits no response or only a localized one. Afebrile, symptomatic lower UTIs produce local mucosal reactions. Because C-reactive protein (CRP), urine nitrite, leukocyte esterase, pyuria, and proteinuria have limited sensitivity and specificity, UTI diagnosis may be inaccurate, leading to overtreatment or undertreatment, which in turn risks kidney damage, antibiotic resistance, recurrent infections, or septicemia. Studies on HBP, Lactoferrin, heat-shock protein-70, human defensin-5, procalcitonin (PCT), and lipopolysaccharide binding protein (LBP) show promising results[21]: (1) PCT: PCT is a calcitonin precursor consisting of 116 amino acids. PCT is primarily secreted by the thyroid gland, and trace amounts are found in the blood. Also, PCT is secreted by spleen, liver, and kidneys. The expression of the CALC1 gene, which produces PCT, is stimulated by inflammatory cytokines in infectious conditions, such as pneumonia, acute pyelonephritis, and UTI. PCT level has been shown to predict the severity and prognosis of the disease. PCT is a useful biomarker to distinguish bacterial from viral infections because the PCT level is not elevated in viral infections[21]. Thus, in individuals with acute pyelonephritis, PCT is a helpful marker for the early detection of bacteremia. PCT levels help in guiding the type and dosage of antibiotic in UTI[44]. PCT exhibits a plasma concentration of 6 mg/L; with higher levels in inflammatory conditions. A PCT of less than 0.25 ng/mL may rule out UTI. The upper and lower UTIs in children can be distinguished by serum PCT and CRP. In senior nursing home residents, simultaneous measurement of PCT and CRP helps to distinguish between bacterial and fungal UTIs. While a CRP of less than 20 mg/L does not indicate pyelonephritis, a serum PCT level of ≥ 0.5 ng/mL indicates the presence of pyelonephritis[22]. PCT in UTI serves as an indicator of severity of the infection, and identifying patients at risk for complications[45].

(2) LBP: LBP is an acute-phase protein that recognizes lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria. By binding to LPS and presenting it to receptors such as a cluster of differentiation (CD)14 and TLR4, LBP initiates signaling pathways that activate the innate immune response against bacterial infection. In the kidney, pericytes are found in the glomeruli (where they function as mesangial cells) and around peritubular capillaries and vasa recta in both cortex and medulla[46,47]. In disease states like sepsis, elevated LBP can interact with pericytes, making them essential targets of injury and pathology[48]. LBP is primarily secreted from the liver with substantial concentrations also released from pulmonary and gut epithelial cells. LBP facilitates the transfer of multimers of LPS to its sensing receptor consisting of CD14, TLR4, and myeloid differentiation factor (MD)2, initiating the inflammatory response. It plays a role in detoxification of LPS by transferring LPS to lipoproteins. LBP is also able to bind lipopeptides originating from both Gram-negative and Gram-positive bacteria and to mediate their pro-inflammatory effects. It is associated with bacterial translocation in the gut, potentially adding function to this molecule by sensing/scavenging bacterial material entering the body through a deranged gut wall[49]. LBP serves as an indicator of systemic inflammation caused by bacterial infection, including gut dysbiosis. When LPS is released into the bloodstream, it leads to endotoxemia[48]. Serum LBP concentration constitutes a reliable biologic marker for the diagnosis of afebrile UTI in children[49]. However, in cases of upper UTIs, such as pyelonephritis, LBP accumulates in the urine due to release and filtration in the kidneys. Elevated levels of urinary LBP can indicate the severity of the infection in the kidney tissue, but the infection does not produce LBP[50]. Since LBP levels are influenced by systemic infections, inflammatory conditions, and liver function, LBP may not be used in isolation to diagnose UTI[50].

Non-nephron kidney-origin biomarkers: These biomarkers are released from kidney cells or tissues outside the nephron structure. These markers include xanthine oxidase (XO) and myeloperoxidase (MPO): (1) XO: XO is predominantly found in the liver, kidney, and endothelium within the human body. In the kidney, it originates from the renal vasculature, including the renal arteries and veins. The breakdown of purines by XO generates reactive oxygen species (ROS) and hydrogen peroxide, contributing to oxidative stress and inflammation. This oxidative activity makes XO a promising biomarker for assessing oxidative stress associated with UTIs[51]. Measurement of either urinary or blood XO levels provides critical diagnosis, monitoring, and prediction of severity for a UTI. An increase in urinary XO activity is associated with UTIs, with a significant elevation observed when bacterial counts exceed 10⁵ CFU/mL[52]. UTIs appear to reduce the activity of NADPH oxidase, ultimately impairing its function. This reduction indirectly inhibits the activity of XO, affecting oxidative stress pathways. The primary cause of superoxide anion production during UTI is XO. ATP loss during UTI causes hypoxanthine and xanthine accumulation, thereby increasing the substrates for XO, which eventually generate ROS. Increased ROS production, damages the urothelial lining of the urinary tract, making it more susceptible to bacterial invasion. This damage exacerbates inflammation, further promoting the development of UTIs[53]. Various factors, including dietary intake, smoking, alcoholism, altered renal function, and the presence of other comorbidities, influence the activity of XO and decrease its specificity[53]; and (2) MPO: MPO is an enzyme produced by neutrophils and monocytes that uses hydrogen peroxide and chloride ions to generate powerful oxidants like hypochlorous acid. Its primary function is to kill invading microorganisms as part of the innate immune system. However, MPO also contributes to tissue damage and inflammation by modifying lipids and proteins, making it a key biomarker for inflammatory diseases such as atherosclerosis[54]. The antibacterial activity is supported by the upregulation of MPO gene expression in activated phagocytes[53]. Urinary MPO levels may serve as a promising marker for monitoring the treatment effects of UTIs[55]. The activities of MPO are elevated in a series of kidney diseases. The urinary MPO/creatinine ratio may be a prospective marker for observing the treatment response in UTI. MPO is a marker of urinary neutrophil extracellular traps and are markedly higher in concentrations in children with UTI[22].

Molecular markers: Molecular markers such as cell-free DNA (cfDNA), transrenal DNA (tr-DNA), and 16S ribosomal RNA (rRNA) have been applied to the diagnosis of UTI[22]. In UTI, cfDNA originates from host cells in the urinary tract or blood, while trDNA is a subset of cfDNA that crosses the kidney barrier from the blood into the urine. 16S rRNA comes from bacterial DNA and is used to identify the specific types of bacteria present in the urine, aiding in the diagnosis and treatment of UTIs. The detection of certain pathogen substances may facilitate the early diagnosis of UTIs. Their advantages include assessing the effectiveness of treatment, identifying any remaining bacteria, and helping to tailor medical care. Standardizing cfDNA/trDNA and ensuring its compatibility with 'omics' technologies could significantly transform the molecular diagnosis of infections, including UTIs[22]. cfDNA, a promising non-invasive biomarker widely used in oncology and pregnancy, has shown great potential in clinical applications for diagnosing infectious diseases. cfDNA from both the pathogen and host perspectives is being used as a sensitive diagnostic tool[56]. cfDNA in urine comes from two primary sources: Shedding of DNA from apoptotic or necrotic cells of the urinary tract lining, and trDNA that enters the bloodstream and subsequently filters from the blood into the urine[57]. The origin of the cfDNA influences its size, with locally shed DNA being longer and blood-derived DNA being smaller after it passes through the kidney[58]. cfDNA is typically longer in size with a minimum of 1000 base pairs. The blood-derived DNA that passes into the kidney is between 40 and 250 base pairs, which are small enough to be filtered by the glomerular basement membrane to appear in the urine[59,60]. The choice of DNA extraction affects the integrity of cfDNA during UTI detection. Standardized protocols for urine collection, cfDNA stabilization, and analysis are needed to ensure reproducibility and accuracy across different studies and clinical settings[60].

The 16S rRNA gene, present only in the bacterial chromosomal genome, contains nine hypervariable regions that allow differentiation of bacterial species through evolutionary polymorphisms[61]. This approach offers higher sensitivity and specificity compared with conventional diagnostic methods. A study demonstrated that 16S rRNA amplicon sequencing can characterize the diversity of the female urinary flora and improve species-level identification of pathogenic bacteria, even in healthy women[62]. Furthermore, it has potential utility in children when urine culture results are inconclusive, as well as in identifying urinary microbiota genera associated with UTIs in vesicoureteral reflux[63,64]. However, 16S rRNA amplicon sequencing is limited by its reliance on primer-specified amplicons, which restricts detection across all taxa (Table 2).

Table 2 Clinical applications of biomarkers in urinary tract infections.

Biomarkers	Clinical application	Ref.
NGAL	Guides UTI diagnosis and therapy, reduces unnecessary antibiotic use, and serves as a marker for disease severity	[23]
KIM-1	Used for diagnosis and prognosis of renal diseases	[34]
IL-8	Aids in diagnosing neonatal sepsis and serves as a marker for inflammation	[41]
IL-6	Evaluate impact of therapeutic agents on urinary bacteria and systemic inflammation	[41]
XO	Measures nitrite levels during UTI, allowing real-time monitoring of bacterial activity and treatment efficacy	[53]
cfDNA and Tr-DNA	Enables early identification of bacterial infections, particularly in UTIs and post-transplant monitoring	[59]
HBP	Functions as an auxiliary marker for detecting bacteremia and assessing severity of infection	[44]
PCT	Facilitates early diagnosis and differentiation of bacterial UTIs	[63]
LBP	Serves as a biomarker for diagnosing febrile UTIs, particularly in pediatric patients	[51]

NGAL: Neutrophil gelatinase-associated lipocalin; KIM-1: Kidney Injury molecule-1; IL: Interleukin; HBP: Heparin-binding protein; LBP: Lipopolysaccharide-binding protein; PCT: Procalcitonin; XO: Xanthine oxidase; cfDNA: Cell-free DNA; trDNA: Transrenal DNA; UTI: Urinary tract infections.

ROLE OF BIOMARKERS IN THE MANAGEMENT OF UTI

UTI treatment varies depending on the location of the infection and the causative agent, as illustrated in Figure 6. Acute uncomplicated cystitis is treated with oral antibiotics. Complicated UTIs have the risk of multi-organ dysfunction and carry high morbidity and mortality when complicated by septic shock. They are treated with injectable antibiotics, such as Ceftriaxone, Cefepime, Piperacillin-Tazobactam, and Carbapenems. A delay in appropriate antibiotic initiation can result in acute kidney injury and renal replacement therapy in severe cases[65] (Figure 6).

Figure 6 Treatment of urinary tract infections. UTI: Urinary tract infections.

Antibiotic resistance and clinical utility

Globally, resistance has been developing against antibiotics used to treat bacterial infections linked to UTIs, particularly for commonly used antimicrobial medicines. Thus, it is crucial to start the appropriate empirical antibiotic treatment based on the typical etiological agents that are present in particular geographic areas. Imipenem, meropenem, amikacin, and gentamicin are the best antibiotics for treating isolates of Escherichia coli and Klebsiella spp., the most often isolated bacterial species in pediatric UTIs. Pseudomonas species exhibit resistance to imipenem, meropenem, amikacin, gentamicin, ceftazidime, cefepime, and ciprofloxacin. The best antibiotics for Enterococcus faecalis are ampicillin, vancomycin, and linezolid; for Enterococcus faecium, the best antibiotics are vancomycin, teicoplanin, and linezolid. In terms of empirical treatment, it is crucial that each center ascertains its unique resistance rates. It should be remembered that switching to a more appropriate antibiotic and reevaluating each empirically administered drug based on sensitivity and culture results would both significantly lower resistance rates[66]. To achieve the United Nations Sustainable Development Goals 3 and 8, the expertise from General Medicine, Biotechnology, and Microbiology all come together in achieving the set targets by 2030. Resistance to antibiotics should not be neglected to promote positive healthy and financial impact in dealing with UTI[67]. Table 3 lists the uropathogens and their susceptibility to antibiotics.

Table 3 Commonly encountered uropathogens with their susceptible antibiotics.

Bacteria	Antibiotics
Escherichia coli	Nitrofurantoin, Trimethoprim-sulfamethoxazole, Fosfomycin
Staphylococcus saprophyticus	Nitrofurantoin, Norfloxacin Trimethoprim-sulfamethoxazole
Enterococcus faecalis	Ampicillin, Vancomycin (for resistant strains)
Pseudomonas aeruginosa	Ceftazidime, Piperacillin-tazobactam, Meropenem, Ciprofloxacin, Aminoglycosides
Klebsiella pneumoniae	Ceftriaxone, Piperacillin-tazobactam, Fluoroquinolones (if susceptible), Aminoglycosides
Proteus mirabilis	Trimethoprim-sulfamethoxazole, Ciprofloxacin
Enterobacter spp.	Cefepime (less resistant strains), Fluoroquinolones (susceptible), Aminoglycosides (serious infections)
Coagulase-negative Staphylococci spp.	Vancomycin (for resistant strains), Oxacillin (if susceptible)

CLINICAL APPLICATIONS OF BIOMARKERS IN UTI

Diagnosing, monitoring, and predicting the progression of UTIs largely depends on the presence of biomarkers. Recent studies have highlighted the crucial role of microbiology in the early detection of infections, evaluating antibiotic resistance, and facilitating more personalized treatments[22].

Diagnosis and screening at the early stage of UTI

Doctors conducting regular urine cultures must wait 48-72 hours before deciding on a course of treatment. NGAL, KIM-1, IL-6, and IL-8 have been identified as rapid diagnostic markers for diseases. Measuring NGAL in urine can distinguish between lower and upper UTIs with a 93% and a 90%accuracy, respectively. KIM-1 appears early in the kidneys when a patient develops an infectious UTI, particularly in cases of pyelonephritis[41].

Monitoring antibiotic resistance

Biomarkers help to monitor the progress when fighting MDR bacteria. High levels of IL-6 and IL-8 indicate that inflammation persists in these infections. The presence of cfDNA and trDNA in urine reflects the number of bacteria and the outcome of antibiotic administration, ensuring real-time monitoring[68].

Assessing outcomes and severity

Biomarkers such as HBP and PCT assist doctors in predicting the occurrence of urosepsis. HBP levels significantly increase in individuals with severe UTIs and bloodstream infections, making it useful for early treatment. PCT is frequently utilized in diagnosing sepsis and has proven effective in distinguishing febrile UTIs from systemic bacterial infections, thereby informing antibiotic selection[69].

Specialized strategies

Separate biosensors have been created to identify markers: NGAL, IL-6, leukocyte esterase, and nitrite. A comprehensive device that combines all four markers could enhance diagnostic efficiency and promote more precise antibiotic use in the future[70].

Treatments for pediatrics and geriatrics

Biomarkers play a crucial role in diagnosing UTIs, especially in groups where common symptoms can be ambiguous, such as infants and the elderly. Urinary LBP aids in distinguishing febrile UTIs from other fever-related conditions in children. For elderly patients experiencing recurrent UTIs, biomarkers such as NGAL and KIM-1 can facilitate the early detection of kidney damage[71]. Five urine markers: IL-6, azurocidin, NGAL, TIMP-2, and CXCL-9 show good diagnostic accuracy for UTI in older women. This biomarker panel can distinguish ABU from UTI[72].

Figure 7 depicts the clinical management timeline for these biomarkers, which depends on the intensity of the patient's disease. Screening samples using these indicators enables faster decision-making, allowing for short, targeted treatment at the early onset of UTI, while severe cases require long-term intervention.

Figure 7 Clinical impact of biomarker-based rapid screening in urinary tract infections. UTI: Urinary tract infections.

To alleviate discomfort and prevent potential UTI problems, effective time management, prompt detection, and timely treatment are considered essential. Neither existing screening procedures, urine test strip analysis, nor microscopic examination reduces the high rate of false negative results for diagnosis. Urine culture is used to provide a definitive diagnosis of a UTI, which typically takes two to three days, emphasizing the importance of developing improved biomarkers for UTI diagnosis and management[73]. The cost-effectiveness assessment of UTI biomarkers is the first critical step toward the clinical application of these biomarkers. Notably, the potential savings from avoiding long-term dialysis may outweigh the actual expenses of biomarker research, development, and clinical use. Future research should aim at well-designed clinical studies across different UTI types, possibly exploring urinary bacterial metabolites or volatile organic compounds as novel indicators. A meta-analysis might help define cut-off thresholds, though the heterogeneity of data remains a significant challenge.

CONCLUSION

This review highlighted the integration and characterization of UTI pathology, emphasizing the diagnostic applications of urinary biomarkers, including NGAL, KIM-1, IL-8, IL-6, XO, HBP, LBP, cfDNA, and trDNA. Advances in molecular profiling techniques for urine, combined with biosensors that target specific disease markers, have enhanced the precise detection and monitoring of UTIs. The development of molecular diagnostic biosensors for detecting antibiotic sensitivity at the point of care promises to transform clinical diagnostics by providing a fast, accurate, robust, and cost-effective platform for UTI diagnosis.