The role of cell‐free DNA in non‐invasive diagnosis of urinary tract infection

Abstract

Urinary tract infection (UTI) constitutes a pervasive health concern. UTIs are dichotomously classified into upper and lower strata, and further categorized as either complicated or uncomplicated. Upper UTIs predominantly afflict the renal and ureteral domains, typified by conditions exemplified in pyelonephritis, whereas lower UTIs manifest within the confines of the urethra and bladder. The conventional diagnosis of UTIs relies upon clinical manifestations and urine culture. Common symptoms encompass dysuria, frequent micturition, hematuria, and suprapubic discomfort. Urine culture serves to identify the specific pathogens instigating the infection and assess their antibiotic susceptibility. However, this procedural protocol generally necessitates an approximate duration of 24 h, coupled with an additional 24‐h interval for antibiotic susceptibility testing. In contradistinction, the detection of cell‐free DNA (cfDNA) in the circulatory system presents a potential avenue for non‐invasive diagnostic modalities. Notwithstanding, cfDNA emanates from deteriorating cells, affording insights into the extant cellular demise within the organism. Extensive scholarly inquiry has established a positive correlation between cfDNA concentration in the bloodstream and the incidence of cell death in conditions, such as severe traumas, sepsis, aseptic inflammation, myocardial infarction, and stroke. However, limited investigative effort has been devoted to elucidating the diagnostic potential of cfDNA in the context of UTIs. Consequently, the concentration and constitution of plasma cfDNA harbor substantial promise for non‐invasively diagnosing UTIs. In summation, the utilization of cfDNA in UTI diagnosis remains an incipient area of scholarly pursuit, underscoring the imperative for further research to elucidate the prospective role of plasma cfDNA in non‐intrusively discerning UTIs.

Graphical abstract

Keywords

cell‐free DNA / diagnosis / DNA methylation / urinary tract infection

1 INTRODUCTION

Urinary tract infection (UTI) is a highly prevalent medical condition in the general population[1]. In a prospective study involving 1497 patients catheterized for a minimum of 24 h and subjected to daily monitoring over a 14‐day span, the determined incidence of UTI was 15.6%[2]. The prevailing benchmark for diagnosing bacterial UTI remains in vitro urine culture[3]. Small fragments of DNA freely circulate in various bodily fluids of both healthy and diseased individuals, including blood and urine. These fragments, known as cell‐free DNA (cfDNA), are postulated to emanate from expiring cells, thereby reflecting the ongoing cellular demise within the organism[4]. After the presence of cfDNAs was initially observed in 1948[5], heightened concentrations were subsequently noted in the serum of systemic lupus erythematosus patients[6]. Subsequent research demonstrated elevated concentrations of cfDNAs in cancer patients compared with healthy individuals[7]. Later on, further investigations revealed that cfDNAs in cancer patients encapsulate tumor‐derived DNA[8], with subsequent identification of tumor‐specific DNA alterations in cfDNA[9–15]. These cfDNA molecules represent genetic material debris from deceased cells, offering opportunities for precise diagnosis based on omics principles, which have transformative implications for various medical fields[16–19]. For instance, next‐generation sequencing of fetal DNA circulating in maternal blood facilitates non‐invasive prenatal testing (NIPT) for fetal chromosomal abnormalities[16,17]. Additionally, the identification of donor‐derived DNA in the circulation of organ transplant recipients aids in the early detection of graft rejection[18,20]. Concurrently, the assessment of mutated DNA in circulation supports the detection, genotyping, and monitoring of cancer[4,21]. The concentration of plasma cfDNAs correlates positively with the rate of cell death, offering insights into outcomes related to severe injury, septic shock, sepsis, myocardial infarction, aseptic inflammation, and stroke[22]. However, studies exploring the diagnostic role of cfDNA in UTIs are scarce. Consequently, both the concentration and constitution of plasma cfDNAs hold significant potential for non‐invasive UTI diagnosis. This review aims to discuss the role of cfDNA in the non‐invasive diagnosis of UTIs.

2 DIAGNOSIS METHODS OF UTI

2.1 What is UTI?

UTI stands as a prevalent global public health issue, constituting a microbial affliction affecting individuals across diverse age groups, leading to inflammation within the urinary tract. UTIs span a spectrum from mild bladder inflammation, known as cystitis, to severe instances resulting in uroseptic shock. The urethra serves a dual role as both the conduit for urine excretion and a portal for bacteria ingress into the urinary tract. Normally, bacteria inhabit the vicinity near the urinary tract outlet, flushed out during urination. However, infections occur when these bacterial colonies evade adequate elimination during urination, ascending to the bladder before micturition occurs[23]. Prevalence of UTIs is notably higher in females, with approximately 81% of reported cases occurring in females[24]. This is attributed to the unique physiological structure of the female urethra, which is shorter than that of males[25].

2.2 The diagnosis of UTI

UTIs encompass two main categories: uncomplicated UTI and recurrent UTI (rUTI). Clinical symptoms are often used to diagnose UTIs, but when rUTI is suspected, urine culture is deemed the definitive diagnosis modality. Predominant UTI symptoms encompass dysuria, frequent urination, hematuria, and suprapubic pain, with dysuria ranking as the most commonly reported symptom. Manifestations such as fever, chills, back pain, nausea, and vomiting raise concerns for pyelonephritis and warrant a more comprehensive evaluation[26,27]. Urine culture enables the identification of the causative organisms, enabling tailored therapeutic interventions and validating the presence of microbiota. A positive diagnosis of symptomatic UTI is typically defined by a bacterial count exceeding 105 colony‐forming units per milliliter in a urine sample. However, obtaining culture results usually takes approximately 24 h, and an additional 24 h is required for antibiotic susceptibility testing[28]. As some symptoms of UTI overlap with other conditions like overactive bladder, urine culture is preferred to confirm the diagnosis in suspected cases of rUTI[29]. Urinalysis, which can promptly detect the presence of pyuria, is generally processed more quickly than urine culture. It has demonstrated a sensitivity of 95% and specificity of 70% and remains a standard practice during rUTI workup. However, ensuring uncontaminated midstream urine specimens from patients can post challenging[28,30,31]. A urine dipstick test can be used as an initial screening tool to rule out acute UTI. In low‐risk patients with positive dipstick findings, UTI is considered. Nevertheless, in rUTI cases, women are more prone to yield positive dipstick results, making this test less reliable for rUTI cases[26]. Renal ultrasound imaging and computed tomography urogram are additional diagnostic modalities for UTI, particularly beneficial for assessing pyelonephritis and evaluating recurrent episodes.

Currently, urine culture remains the definitive standard for diagnosing UTIs. However, it is important to note that the culturing techniques used are primarily focused on identifying the most common organisms associated with UTIs, including Escherichia coli, Proteus, Klebsiella, Staphylococcus saprophyticus, and Enterococcus[26,32,33]. There are currently no economically viable or expeditious alternatives to agar plates and conventional culture methods[34,35]. While 16S rRNA amplicon sequencing and expanded quantitative urine culture exhibit promise in terms of improved sensitivity and specificity, they are currently limited to research settings and not widely applicable in clinical practice[32,34]. Regarding imaging studies, cystoscopy and computed tomography cystogram are not typically recommended in the initial assessment of uncomplicated rUTI. However, contemplation of these modalities arises when concern exist regarding pyelonephritis, obstruction, or other complicating factors[26,30,31]. These imaging modalities can provide valuable information in specific cases but are not routinely performed for uncomplicated rUTI diagnosis.

There are several other techniques used in the detection of UTIs. Phenotypical biochemistry and culture identification strategies are commonly employed but can be slow due to the time required for bacterial growth. Polymerase chain reaction (PCR) and immunoassay techniques offer rapid detection, but both have their limitations. PCR can be prone to background contamination from exogenous sources of DNA, while immunoassays rely on the sensitivity of detecting specific antigens and may require time for seroconversion[23]. The existing diagnostic modalities for UTIs have various disadvantages, as summarized in Table 1. These limitations highlight the need for a more preferable detection method that can overcome these challenges and provide accurate and efficient UTI diagnosis. Research efforts are ongoing to develop and improve diagnostic techniques for UTIs in order to enhance patient care and management.

3 APPLICATION OF cfDNA IN NON‐INVASIVE DIAGNOSIS

3.1 Cell‐free DNA

Cell‐free DNA, also known as circulating free DNA (cfDNA), refers to degraded DNA molecules that are released by cells into the blood. Results about the size of cfDNA are quite diverse, but electrophoretic studies indicate that it varies between 180 base pairs (bp) and 10 000 bp[36]. The half‐life of cfDNA is around 16 min, and it can form complexes with cellular or non‐cellular components, such as glycoproteins, which increase its stability. cfDNA can also act as a signaling molecule between different cells and tissues[37,38]. Under normal physiological conditions, cfDNA is mainly derived from the degradation of genomic DNA in aging apoptotic cells. However, in various disease states, such as malignant tumors, trauma, organ transplant rejection, tissue and organ failure, and infections, abnormal necrotic cells can release a significant amount of DNA[4,18,19]. The presence of circulating free DNA was first described by Mandel and Metais in 1948, and subsequent research has shown its increased concentration in cancer patients compared with healthy individuals, with further increases observed after radiation therapy[5,7,39,40]. Various types of cfDNA isolated from human blood for diagnostic and screening purposes include circulating tumor DNA, mitochondrial DNA, and fetal DNA. With the continuous accumulation and support of clinical research, it has found wide applications in liquid biopsy, non‐invasive prenatal screening (NIPT), medication guidance, and diagnosis of infectious diseases[41–48].

The composition of cfDNA includes the tissues of origin (TOOs) and their corresponding proportions, which can vary depending on an individual's physiological state. In healthy individuals, plasma cfDNA is primarily derived from deceased hematopoietic cells, with tiny contributions from other tissues. Nonetheless, in pathological states, the contributions from specific disease tissues can differ from those in healthy conditions. Accurately identifying the TOOs of cfDNA and predicting their proportions is crucial for non‐invasive diagnosis. Recently, several methods have been proposed to aid the cfDNA‐based non‐invasive diagnoses, and these methods can be classified into three main groups based on the signals use: cfDNA mutations, methylation patterns, and cfDNA fragmentation patterns[49]. Methylation patterns are one of the most commonly used approaches in this regard.

3.2 DNA methylation

DNA methylation is indeed a critical epigenetic modification involving the addition of covalent methyl groups to cytosine residues, particularly in CpG dinucleotides. This epigenetic process serves as a regulatory system that marks genetic information, providing instructions on when and how to interpret DNA and control transcription. Unlike the inherited DNA sequence, methylation patterns are established in a programmed manner that continues throughout development, creating stable gene expression profiles[50]. The process of DNA methylation undergoes dynamic changes during development. Methylation patterns derived from gametes are erased before embryo implantation, and a new profile is established in each individual[51,52]. This resetting process occurs in two stages. During implantation, the majority of the genome undergoes de novo methylation, except for specific promoter sequences known as CpG islands, remaining unmethylated[52]. Despite occurring only once, the resulting bimodal methylation pattern is maintained in all subsequent embryonic cell divisions through a simple semiconservative mechanism[53] (Figure 1). DNA methylation is primarily a repressive mechanism that leads to gene silencing and allele inactivation, including X‐chromosome inactivation. It contributes to the diversity of gene expression in different cells and is associated with tissue‐ and cell‐specific patterns[54,55]. Additionally, diseased tissues often exhibit distinct DNA methylation profiles[56]. An interesting aspect is that DNA methylation modifications on cytosine residues are not erased when cfDNAs are released from dead cells. This property makes DNA methylation a valuable indicator of the TOOs of cfDNAs[54]. On the basis of tissue‐specific methylation patterns, methylation pattern‐based methods can be categorized into two groups: methods based on de novo regions and methods based on the methylation level of genomic sites[49]. These methods play a crucial role in non‐invasive diagnostic approaches, particularly in identifying the origin of cfDNAs and understanding their potential implications in health and disease.

The clinical application of DNA methylation in diagnostics, particularly through liquid biopsy, is an emerging and exciting area in medical research. When cells die, they release fragments of DNA into the bloodstream, forming circulating cfDNA. This cfDNA can serve as a valuable diagnostic tool, commonly referred to as a liquid biopsy[57]. One notable application of liquid biopsy is the analysis of cfDNA to identify fetal chromosome aberrations in maternal plasma[16,58]. This non‐invasive approach has proven to be effective in prenatal diagnostics. Additionally, liquid biopsy has been instrumental in the detection of specific mutations in circulating tumor DNA, enabling the monitoring of cancer progression and response to therapy. Importantly, this can be achieved even in cases where the tumor is inaccessible or its location is unknown[21,48,59,60]. Another innovative application of the liquid biopsy concept involves using DNA methylation patterns to identify the cell‐type origin of cfDNA. This allows for the monitoring of cell death in specific tissues[61–63]. Each cell type in the human body is characterized by a unique DNA methylation profile that remains consistent in every individual and is stably maintained throughout life in both healthy and diseased cells. This stability makes the DNA methylation profile an ideal barcode for identifying dying‐cell‐derived DNA from specific tissues. The ability to distinguish the tissue origin of cfDNA through DNA methylation patterns opens up new possibilities for diagnosing and monitoring various diseases. It provides insights into the health status of specific tissues and organs, allowing for the early detection of diseases and personalized treatment strategies. This non‐invasive and dynamic approach to diagnostics holds great promise for advancing precision medicine and improving patient outcomes.

3.3 The application status of cfDNA in non‐invasive diagnosis

The use of cfDNA in plasma has become a powerful tool for non‐invasive diagnostics, as it collects fragments of DNA released from dead cells in various tissues throughout the body. This approach has found applications in numerous areas, showcasing the versatility and potential of cfDNA in medical research and diagnostics (Figure 2).

In the plasma of expectant mothers, around 10% of cfDNAs are of fetal origin, enabling NIPT. Subsequent to Lo et al.'s discovery[16,56] of fetal‐derived cfDNA in maternal plasma and serum, a consecutive array of methodologies[64–72] for NIPT emerged, encompassing screening for fetal chromosomal aneuploidy[17,66,67,73–77] and monogenic diseases[64,65,68,70,71]. Within the context of screening for fetal chromosomal aneuploidy, the premise lies in the deviation of cfDNA proportions from a specific chromosome, indicating a potential aneuploid chromosomal state.

In the plasma of individuals with malignancies, the presence of tumor‐derived cfDNAs has been harnessed for non‐invasive cancer screening. Investigational findings indicate that a substantial proportion of somatic mutations identified in solid tumor samples are likewise discernible in cfDNAs[48,69]. Consequently, the prospect of screening cancers based on cfDNAs, particularly in asymptomatic or early‐stage scenarios, progressively garners appeal. Additionally, the dynamics of cancer development can be scrutinized through the alterations in the composition of cfDNAs.

In the plasma of recipients of transplantations, the cfDNAs originating from deceased cells within the transplanted tissue afford clinicians the means to surveil instances of transplantation rejection. In the scrutiny of plasma cfDNAs among individuals undergoing heart transplantation, Snyder et al. precisely quantified the proportions of donor‐derived DNAs utilizing donor‐specific single nucleotide polymorphisms and sequencing technology. Their observations delineate a notable escalation in the levels of donor‐derived cfDNAs during episodes of acute cellular rejections, as diagnosed through endomyocardial biopsies[78]. Employing digital PCR to quantify donor‐derived cfDNAs in renal transplantation subjects, Lee et al. ascertained a heightened abundance of these elements in urine compared with plasma. However, no discernible variance in the quantity of donor‐derived cfDNAs was evident across patients with distinct clinical conditions, attributed to the pronounced variability in urinary cfDNAs[79].

The dead cells of parasites in human bodies also release DNAs into human plasma. Therefore identifying the cfDNAs derived from parasites has been applied to detect human parasitic infections. Baraquin et al. employed quantitative PCR and droplet digital PCR to detect Echinococcus multilocularis‐derived cfDNAs in patients with alveolar echinococcosis[80]. The study conducted by Wan et al. represents an application of targeted sequencing for the detection of Echinococcus‐derived cfDNAs in the plasma of individuals with echinococcosis. This methodology allows for a sensitive and specific detection of parasite‐derived cfDNAs, contributing to the development of diagnostic tools for parasitic infections and potentially improving the monitoring and management of such diseases[81]. Similarly, Wichmann et al. employed real‐time PCR to detect Schistosoma‐derived cfDNAs in human plasma. Across these investigations, the crux of cfDNA‐based parasite detection methodologies resides in the identification of parasite‐specific sequences, such as repeats, RNA genes, or genes specific to the parasites themselves[82].

Absolutely, the common thread in all these applications is the identification of specific sequences or mutations associated with the target condition or tissue of origin. This is a fundamental principle in the field of liquid biopsy and the use of cfDNA for non‐invasive disease detection and monitoring. Whether it is detecting somatic mutations in cancer, identifying donor‐derived cfDNAs for transplantation monitoring, or pinpointing specific sequences related to parasitic infections, the key lies in characterizing unique genetic signatures associated with the condition of interest. This can involve looking for specific mutations, variations, or genomic regions that are indicative of the presence of a disease or the origin of the cfDNA. The methodologies mentioned earlier, such as quantitative PCR, droplet digital PCR, targeted sequencing, and real‐time PCR, are all tools that enable the identification and quantification of these specific genetic elements. By understanding the molecular characteristics associated with a particular disease or condition, researchers and clinicians can leverage cfDNA analysis for early detection, monitoring treatment response, and assessing disease progression, all in a minimally invasive and accessible manner.

4 THE OUTLOOK OF cfDAN IN NON‐INVASIVE DIAGNOSIS OF UTI

A research conducted at Cornell University represents a significant advancement in understanding the origin and composition of cfDNA in various infection statuses, particularly focusing on BK polyomavirus nephropathy (BKVN) and bacterial UTIs. Patients diagnosed with BKVN‐exhibited heightened levels of kidney‐specific cfDNA in their urine compared with a normal control group. Additionally, individuals experiencing BK polyomavirus (BKV) reactivation without nephropathy also displayed increased levels of kidney‐specific DNA, though not to the same extent as those with BKVN[62,63,83–86]. The researchers utilized whole‐genome bisulfite sequencing (WGBS) to analyze methylation marks within urinary cfDNA. They aligned WGBS reads to a human reference genome using bwa‐meth and projected the genome‐wide cfDNA CpG methylation profiles onto two‐dimensional (2D) feature spaces generated by principal component analysis (PCA) and Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) using 112 public references. The observed organization of cfDNA profiles between the cluster representing kidney tissue and the white blood cell cluster in PCA and UMAP 2D projections suggested that urinary cfDNA primarily originates from blood cell types and kidney tissue, with less significant contributions from other cell types in the urinary tract. The study found associations between the composition of cfDNA derived from different cell and tissue types and infection status. Specifically, kidney‐derived cfDNA was elevated in samples from patients with BKV infection compared with those diagnosed with bacterial UTI. Additionally, samples from patients with bacterial UTIs showed an enrichment of leukocytes, particularly neutrophils, consistent with their role as first responders to infection. The researchers tested the utility of this assay for monitoring viral and bacterial infections of the urinary tract in a cohort of kidney transplant recipients. They found that the concentration of cfDNA derived from various cell and tissue types correlated with infection status in these patients, particularly in those diagnosed with BKVN[83–87]. This research highlights the potential utility of analyzing cfDNA composition, particularly the relative contributions of different cell and tissue types, as a means of monitoring infections, specifically in BKVN and bacterial UTIs, demonstrating its relevance in clinical diagnostics and disease monitoring among kidney transplant recipients. In addition to this, cfDNA cellular and tissue components were associated with infection status. For example, the relative contribution of renal‐derived cfDNA was elevated in BKV‐infected patients compared with patients diagnosed with bacterial UTI (P = 2.0 × 10⁻⁴; mean 48.6%, average 12.5%). Leukocytes were enriched in samples from patients diagnosed with bacterial UTIs by routine culture, and neutrophils were the major contributor to the difference in leukocyte content, as would be expected from their role as first responders to infection.

The described diagnostic assay represents a significant advancement in the field of transplant medicine, specifically for recipients of kidney transplants. This approach involves sequencing cfDNA in plasma, allowing for the simultaneous monitoring of both acute rejection and a broad spectrum of infections in allograft organ transplant patients. The research introduces a diagnostic assay that utilizes the sequencing of cfDNA in plasma. This is a non‐invasive method that involves analyzing the free‐floating DNA in the bloodstream, which can provide valuable information about the health status of the transplanted organ. One of the major advantages of this diagnostic assay is its ability to simultaneously monitor two critical aspects of posttransplant health: Acute rejection and infections. This is crucial for clinicians because transplant recipients are at risk of both rejection by the immune system and infections due to immunosuppressive therapies. By leveraging cfDNA sequencing, this method offers comprehensive information for clinicians. The genetic material circulating in the bloodstream can provide insights into the status of the transplanted organ and potential infectious agents. This information can be instrumental in optimizing immunosuppression therapy, tailoring the treatment to the specific needs and risks of each patient. The research suggests that the assay is capable of detecting a broad spectrum of infections. This is particularly important as transplant recipients are susceptible to various pathogens, and early detection of infections is crucial for timely and effective intervention. The ability to monitor both rejection and infections in real‐time allows clinicians to fine‐tune immunosuppression therapy. Balancing the suppression of the immune response to prevent rejection while managing the risk of infections is a delicate task, and having a comprehensive diagnostic tool can greatly aid in this process. The research highlights the relevance of this method for patients undergoing allograft organ transplants, with a specific focus on kidney transplant recipients. This suggests that the assay may have broader implications for other types of organ transplants as well[88]. In summary, this diagnostic assay represents a promising and comprehensive approach for monitoring the health of transplant recipients. By simultaneously assessing the risk of acute rejection and detecting a range of infections, it provides clinicians with valuable information to make informed decisions about immunosuppression therapy, ultimately improving the overall management of posttransplant care. Their work identified urinary cfDNA as a highly versatile analyte for monitoring UTIs[84].

Moreover, cfDNA has found diverse applications in the field of medical diagnostics. It is a versatile tool used in fluid biopsy, non‐invasive prenatal screening, medication guidance, cancer diagnosis, treatment response evaluation, prognosis judgment, and much more. Furthermore, cfDNA has demonstrated its utility in diagnosing infectious diseases, particularly in cases of postorgan transplantation infections. In the general population, UTIs are among the most common medical issues encountered by patients[89]. Yet, there has been limited exploration of the use of cfDNA in the context of UTI diagnosis. Currently, studies on the use of cfDNA in UTIs are mainly focused on infections after renal transplantation, while little research has been done on UTIs due to other causes. This prompts the question of whether cfDNA could serve as a non‐invasive diagnostic method for UTIs. It is worth noting that the application of cfDNA in infection diagnosis has predominantly focused on viral infections. In contrast, UTIs are primarily caused by bacterial pathogens. This crucial difference highlights the need for further research and investigation to determine whether cfDNA can indeed play a significant role in the diagnosis of UTI. This requires a comprehensive exploration of the unique characteristics of bacterial infections, which may differ substantially from viral infections, and how cfDNA can be leveraged effectively in this specific context. This could be our future research.

In summary, while cfDNA has shown promise in various medical applications, its role in diagnosing bacterial infections like UTIs remains an area that necessitates thorough research and validation. The distinctive nature of bacterial infections in the urinary tract underscores the importance of extensive studies to ascertain the potential of cfDNA as a valuable tool in UTI diagnosis.

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