Marker for assisting identification of klebsiella pneumoniae related urinary tract infection in patients with spinal cord injury and application thereof

Abstract

The application provides a marker for assisting identification of Klebsiella pneumoniae related urinary tract infection of a spinal cord injury patient and application thereof, and relates to the technical field of medical examination and molecular diagnosis. The risk of symptomatic urinary tract infection of a patient is evaluated by detecting the iroN gene carrying state in Klebsiella pneumoniae separated from urine culture. Research finds that the carrying rate of the iroN gene in the strain from a symptomatic urinary tract infection patient is significantly higher than that from an asymptomatic bacteriuria patient, which is helpful for distinguishing Klebsiella pneumoniae colonization from symptomatic urinary tract infection. The application further provides primers, a kit and a detection method for detecting the iroN gene. The method is rapid in detection, simple in operation, and can provide a basis for the auxiliary differential diagnosis of urinary tract infection of a spinal cord injury patient with positive Klebsiella pneumoniae in urine culture, the rational use of antibacterial drugs and the infection risk assessment, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the fields of medical testing and molecular diagnostics, specifically to biomarkers for assisting in the identification of Klebsiella pneumoniae-associated urinary tract infections in patients with spinal cord injuries and their applications. Background Technology

[0002] Spinal cord injury (SCI) patients often develop neurogenic bladder, and urinary tract infection (UTI) is one of the most common and important complications. Due to factors such as long-term indwelling catheters, urinary dysfunction, and repeated hospitalizations, these patients are highly susceptible to recurrent and persistent UTIs, severely impacting their quality of life and long-term prognosis. In clinical practice, patients with UTIs can present with two distinct clinical states: symptomatic UTI (urinary tract infection) with symptoms such as urinary frequency, urgency, dysuria, or fever; and asymptomatic bacteriuria (ASB) with a positive urine culture but no symptoms of UTI.

[0003] The differentiation between UTI and ASB mainly relies on clinical symptoms and routine urinalysis. However, this differentiation process faces several challenges: asymptomatic bacteriuria is extremely common in patients with spinal cord injury, and due to sensory impairment caused by spinal cord injury, patients often lack typical urinary tract irritation symptoms, making it difficult to accurately distinguish between bacterial colonization and true infection in clinical practice; and routine urinalysis indicators such as white blood cells and nitrite may also be positive in ASB patients, so relying solely on routine urinalysis indicators often leads to a large number of ASB patients being misdiagnosed as having UTI.

[0004] Because current diagnostic methods lack specific identification techniques, numerous misdiagnoses directly lead to the overuse of antibiotics, increasing the risk of adverse drug events, exacerbating the financial burden on patients, and, more seriously, further promoting the spread of multidrug-resistant bacteria. Particularly in SCI patients with positive urine cultures for Klebsiella pneumoniae, current detection methods struggle to determine whether the isolated strain is more likely to infect related strains or colonize existing strains. Therefore, it is necessary to develop auxiliary molecular biomarkers for this specific scenario. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide biomarkers and their applications for assisting in the differentiation between symptomatic urinary tract infections and asymptomatic bacteriuria in spinal cord injury patients with positive urine cultures for Klebsiella pneumoniae, thereby solving the problem that existing detection methods are insufficient to help differentiate between Klebsiella pneumoniae-related infections and colonization in such patients. To achieve the above-mentioned objective, the present invention provides the following technical solutions: A first aspect of the present invention provides the use of a reagent for detecting a molecular target in a test sample in the preparation of a diagnostic product for differentiating between symptomatic urinary tract infections associated with Klebsiella pneumoniae and asymptomatic bacteriuria in patients with spinal cord injury, wherein the molecular target is the Klebsiella pneumoniae virulence gene iroN.

[0006] In this invention, the Klebsiella pneumoniae-associated symptomatic urinary tract infection, or simply symptomatic infection, refers to the presence of a certain number of pathogenic bacteria in the urine (Klebsiella pneumoniae isolated by urine culture), and the patient having at least one of the following clinical manifestations: fever, urinary frequency, urinary urgency, dysuria, worsening spasms, new onset or significantly worsening of urinary leakage, autonomic hyperreflexia, foul-smelling flocculent urine, pain in the kidney or bladder area, and general malaise.

[0007] In this invention, asymptomatic bacteriuria refers to the presence of a certain number of bacteria in urine without any symptoms of urinary tract infection. The diagnostic criteria are the isolation of a single microorganism from the specimen, but the patient has no urinary tract symptoms indicative of a urinary tract infection.

[0008] In this invention, the diagnostic product is used to detect whether symptomatic urinary tract infection occurs in spinal cord injury patients with positive urine cultures for Klebsiella pneumoniae.

[0009] Furthermore, the diagnostic product is used to detect the virulence gene iroN carrier status of Klebsiella pneumoniae in urine cultures to assess the risk of symptomatic urinary tract infections in patients.

[0010] In an optional embodiment, the reagent is one used based on at least one of the following detection methods: PCR, real-time quantitative PCR, nucleic acid amplification, or gene sequencing.

[0011] In an optional embodiment, the reagent is a primer pair that specifically amplifies the virulence gene iroN, and / or a probe that specifically binds to the virulence gene iroN.

[0012] In this invention, the primer pair specifically amplifying the virulence gene iroN refers to a primer pair capable of specifically recognizing and binding to the nucleic acid sequence of the virulence gene iroN under stringent conditions. Using iroN as a template, DNA synthesis is initiated via polymerase chain reaction (PCR) to generate paired oligonucleotides containing the amplification product of a specific fragment of iroN. In an optional embodiment, the primer pair includes an upstream primer and a downstream primer. The upstream and downstream primers have at least 90% homology with the target region of the virulence gene iroN, and the length of the amplification product of the primer pair is not strictly limited, as long as it can effectively amplify and be used for downstream detection. Typically, the length of the amplification product is 50 bp-1000 bp.

[0013] The probe that specifically binds to the virulence gene iroN refers to an oligonucleotide fragment capable of binding complementaryly to a specific nucleotide sequence within the amplified product of the virulence gene iroN under strict hybridization conditions. In optional embodiments, the nucleotide sequence of the probe is completely or substantially complementary to the target sequence of the virulence gene iroN (i.e., has at least 95% sequence identity), and its length is typically 15 bp-40 bp, more preferably 18 bp-30 bp.

[0014] In a specific embodiment of the present invention, the primer pair includes an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID NO:11, and the sequence of the downstream primer is shown in SEQ ID NO:12.

[0015] Those skilled in the art should understand that, due to the degeneracy of the genetic code or the single nucleotide polymorphism (SNP) between strains, without changing the core recognition function of the primers and probes, 1 to 3 protective bases may be added or deleted at their 5' or 3' ends (such as the addition of protective bases), or chemically modified nucleotides may be introduced into the primers to enhance binding affinity. These variant forms all fall within the protection scope of this invention.

[0016] In this invention, the virulence gene iroN is derived from Klebsiella pneumoniae, whose corresponding strain classification reference NCBI classification number is 272620. It should be noted that those skilled in the art, upon learning of this NCBI classification number, can accurately obtain the complete genome information of the strain corresponding to this classification number from public nucleic acid databases (such as NCBI GenBank, RefSeq, etc.) based on common knowledge in the field and conventional bioinformatics techniques, and further confirm the specific nucleic acid sequence of the iroN gene through gene annotation or sequence alignment. Given the high conservation of the functional domains of the iroN gene within Klebsiella pneumoniae, those skilled in the art, based on the NCBI classification number information and reference sequence position disclosed in this invention, can obtain iroN gene nucleic acid sequences with the same virulence function from different strains without creative effort through polymerase chain reaction (PCR) amplification, chemical synthesis, or database retrieval. All of the above methods fall within the technical scope covered by this invention.

[0017] In the context of this invention, the test sample is from a spinal cord injury patient with a positive urine culture for Klebsiella pneumoniae. The spinal cord injury patient includes a patient with traumatic spinal cord injury, the severity of which can be assessed according to the American Spinal Injury Association (ASIA) classification (AIS), including Grade A (complete injury), Grade B, Grade C, and Grade D (incomplete injury).

[0018] Furthermore, the sample to be tested is urine that has a positive urine culture for Klebsiella pneumoniae. A positive urine culture for Klebsiella pneumoniae means that the urine sample contains ≥10 Klebsiella pneumoniae colonies after culture. 5 CFU / mL.

[0019] In optional embodiments, the urine sample can be collected using methods conventional in the art. For spinal cord injury patients capable of urinating independently, a clean-catch midstream urine collection method can be used to collect morning urine or random urine samples. For spinal cord injury patients with neurogenic bladder dysfunction requiring intermittent catheterization, urine samples can be collected via intermittent catheterization. Specifically, a sterile catheter can be used to collect urine into a sterile container. The collected urine sample can be tested immediately.

[0020] Furthermore, the sample to be tested is a strain of Klebsiella pneumoniae isolated from urine culture or its genomic DNA.

[0021] A second aspect of the present invention provides a kit for distinguishing between symptomatic urinary tract infections and asymptomatic bacteriuria associated with Klebsiella pneumoniae in patients with spinal cord injury, the kit comprising reagents for detecting a molecular target in a test sample, the molecular target being the Klebsiella pneumoniae virulence gene iroN.

[0022] Furthermore, the kit comprises: (a) Primer pairs or probes that specifically amplify the virulence gene iroN; (b) PCR amplification reagents, wherein the PCR amplification reagents include one or more of the following: DNA polymerase, PCR reaction buffer, dNTPs, Mg 2+ Ion source; (c) Positive control; (d) Negative control.

[0023] In an optional embodiment, the positive control is a template containing the iroN gene target sequence or known iroN-positive Klebsiella pneumoniae DNA; the negative control is a template-free control or known iroN-negative DNA.

[0024] In an optional embodiment, the kit may further comprise one or more of the following: nucleic acid extraction reagent, sterile nuclease-free water, fluorescent dye, sample pretreatment reagent, and instructions for use.

[0025] In optional embodiments, the kit may further include primers or probes for detecting one or more virulence genes among rmpA, rmpA2, irp2, iutA, peg-344, or magA for expanded detection or research purposes.

[0026] A third aspect of the present invention provides a detection system for differentiating between symptomatic urinary tract infections associated with Klebsiella pneumoniae and asymptomatic bacteriuria in patients with spinal cord injury, the detection system comprising: The sample processing module is used to extract nucleic acids from the sample to be tested; The detection module includes the primer pairs and / or probes described in the first aspect for amplifying and detecting the nucleic acid; The analysis module is used to determine whether the amplification product of the Klebsiella pneumoniae virulence gene iroN is present in the sample to be tested based on the output of the detection module; if the amplification product is present, the virulence gene iroN in the sample to be tested is determined to be positive.

[0027] In this invention, those skilled in the art should understand that if the virulence gene iroN amplification product is detected in the sample to be tested (e.g., a typical S-shaped amplification curve appears in real-time fluorescence PCR, or a clear band appears at the expected molecular weight position in gel electrophoresis after conventional PCR), then the virulence gene detection result in the sample is determined to be "positive". This positive result suggests that the patient from whom the sample was obtained has a higher risk of developing Klebsiella pneumoniae-related symptomatic urinary tract infection and can be used as an auxiliary basis for judgment based on clinical symptoms, urine culture, and routine urine results. If the virulence gene iroN amplification product is not detected in the sample to be tested (no amplification curve or no expected band), then the virulence gene iroN detection result in the sample is determined to be "negative". This negative result indicates that the Klebsiella pneumoniae strain isolated from the patient's urine culture does not carry this virulence gene, which tends to suggest asymptomatic bacteriuria (ASB) or non-invasive colonization.

[0028] Advantages and beneficial effects of this invention: This invention is the first to discover a significant correlation between the carrier status of the virulence gene iroN and symptomatic urinary tract infection in a specific population, namely SCI patients with positive urine cultures of Klebsiella pneumoniae. The iroN gene carrier status can serve as a molecular marker to aid in differentiating the risk of Klebsiella pneumoniae colonization and symptomatic urinary tract infection. This invention helps in the auxiliary diagnosis of Klebsiella pneumoniae-related urinary tract infections in spinal cord injury patients, reduces unnecessary antibiotic use, and lowers the risk of drug-resistant bacteria development.

[0029] This invention can be used to develop rapid molecular diagnostic kits, which have the advantages of rapid detection, simple operation, and high specificity. They can provide a basis for the auxiliary diagnosis of urinary tract infections in patients with spinal cord injury, the rational use of antimicrobial drugs, and the assessment of infection risk, and have good clinical application prospects. Attached Figure Description

[0030] Figure 1 The results of PCR detection of virulence gene carrying status of Klebsiella pneumoniae strains in the UTI and ASB groups are shown in the figure.

[0031] Figure 2 Electrophoresis images of representative iroN gene PCR amplification products from the training set. Lane M is the DL2000 Marker, lanes 1, 2, 3, 5, 6, 7, 12, and 13 are iroN (665 bp) and positive controls, and lanes 4, 8, 9, 10, and 11 are negative controls.

[0032] Figure 3 This is a comparison of the distribution of the iroN gene in the training set across the UTI and ASB groups.

[0033] Figure 4 To validate the receiver operating characteristic (ROC) curves for predicting UTIs based on the iroN gene in the dataset. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0035] Example 1: Screening and efficacy verification of diagnostic biomarkers 1. Screening of key virulence genes Based on previous transcriptomics screening and literature review, this invention identified seven Klebsiella pneumoniae virulence genes as candidate biomarkers: rmpA, rmpA2, and magA (capsule synthesis regulation genes), and iutA, iroN, irp2, and peg-344 (siderophore uptake related genes). PCR amplification was used to detect Klebsiella pneumoniae in the urine of SCI patients in a previous independent cohort as a preliminary experimental validation. The PCR reaction system consisted of a total volume of 20 μL, 10 μL of 2×taq mix enzyme, 1 μL of 10 μmol / L upstream primer, 0.5 μL of 10 μmol / L downstream primer, and 2 μL of template DNA. Sterile distilled water was then added to bring the total PCR volume to 20 μL. PCR amplification and detection: Pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s (annealing temperatures for different primers are shown in Table 1), extension at 72℃ for 30 s, for a total of 34 cycles; final extension at 72℃ for 5 min to amplify the target sequence, followed by incubation at 12℃. PCR primers are shown in Table 1.

[0036] Table 1. Primer sequences for PCR amplification of 7 virulence genes

[0037] Test results as follows Figure 1 As shown, only the iroN gene showed a significant difference between the UTI and ASB groups (P < 0.001), suggesting that iroN may be a key candidate molecular marker for distinguishing between UTI and ASB.

[0038] 2. Validation of the efficacy of iroN as a diagnostic biomarker To determine whether iroN can differentiate SCI patients into UTI and ASB, 77 patients with Klebsiella pneumoniae (KP) strains who were hospitalized at Beijing Bo'ai Hospital of the China Rehabilitation Research Center from October 2024 to December 2025 and had positive urine cultures for Klebsiella pneumoniae were collected. The inclusion criteria were: (1) no gender restriction; (2) age ≥18 years; (3) meeting the diagnosis of spinal cord injury and AIS grade A, B, C, or D; (4) presence of neurogenic bladder; (5) positive urine culture for Klebsiella pneumoniae; and (6) full consent to participate in the study. The exclusion criteria were: (1) history of sexually transmitted diseases or other urogenital infectious diseases; (2) bladder dysfunction unrelated to neurogenic bladder after spinal cord injury; (3) severe liver and kidney dysfunction; and (4) refusal to sign the informed consent form.

[0039] Patients were divided into two groups based on their clinical manifestations: symptomatic urinary tract infection (UTI) group (n=45) and asymptomatic bacteriuria (ASB) group (n=32).

[0040] The diagnostic criteria for symptomatic urinary tract infection include: (1) at least one symptom or sign related to urinary tract infection: fever, urinary frequency, dysuria, urinary urgency, increased spasms, newly occurring or significantly worsened urinary leakage, autonomic hyperreflexia, foul-smelling cloudy urine, pain in the kidney or bladder area, or general malaise; (2) white blood cells > 10 / HPF in routine urine tests; (3) Klebsiella pneumoniae count ≥ 10 in urine culture. 5 CFU / mL.

[0041] The diagnostic criteria for asymptomatic bacteriuria include: the isolation of a single microorganism from the specimen, with a count ≥10. 5 CFU / ml; The patient had no urinary tract symptoms indicative of urinary tract infection, such as fever, urinary frequency, dysuria, urinary urgency, difficulty urinating, pain in the kidney or bladder area, chills, etc.

[0042] The 77 samples collected were randomly divided into training and validation sets in a 6:4 stratified ratio. The stratified random partitioning was performed with a random seed of 1, resulting in: a training set (n=47, 20 ASB cases and 27 UTI cases) for model building, and a validation set (n=30, 12 ASB cases and 18 UTI cases) for diagnostic efficacy validation.

[0043] Based on the PCR results, Fisher's exact test revealed that, in the training set, the iroN gene carrier rate in the UTI group (15 / 27) was significantly higher than that in the ASB group (1 / 20) (P=0.0004). Representative test results are shown below. Figure 2 As shown. Figure 3 The chart shows a comparison of the distribution of the iroN gene in the UTI and ASB groups in the training set, indicating a significant difference in gene distribution between the two groups. A logistic regression model was established with clinical status as the dependent variable and the presence or absence of the iroN gene as the independent variable. The results showed that iroN positivity was significantly associated with UTI (OR=23.75, 95%CI 4.02–458.06, P=0.00387).

[0044] Based on the results from the training set, the above model is applied to the validation set for evaluation. Figure 4 The validation set ROC curve showed an area under the curve (AUC) of 0.764 (95% CI 0.622–0.906). The sensitivity was 0.6111, and the specificity was 0.9167.

[0045] The above results indicate that the iroN gene carrier status helps in assessing the risk of Klebsiella pneumoniae colonization and symptomatic urinary tract infection (UTI), and can serve as an auxiliary diagnostic biomarker for UTIs in SCI patients with positive Klebsiella pneumoniae urine cultures. This invention addresses the clinical challenge of poor specificity in existing technologies. In the validation set, this invention achieved a high specificity of 91.67%, significantly reducing the risk of clinical misdiagnosis; simultaneously, its sensitivity meets the basic requirements for clinical auxiliary screening and can be used to differentiate between symptomatic UTIs and asymptomatic bacteriuria in patients with spinal cord injury. The combined AUC results demonstrate that this method has good auxiliary diagnostic efficacy and promising prospects for widespread application in actual clinical samples.

[0046] Example 2: A kit based on iroN for differentiating between symptomatic urinary tract infections and asymptomatic bacteriuria associated with Klebsiella pneumoniae in patients with spinal cord injury. The following is a specific implementation of the kit.

[0047] The kit includes at least one primer pair for the specific detection of the iroN gene (specific sequence is SEQ ID NO: 11-12).

[0048] The kit also includes: PCR amplification reagents (including DNA polymerase, PCR reaction buffer, dNTPs, Mg...) 2+ (One or more of the ion sources); positive control and negative control.

[0049] Optionally, the kit may further include one or more of the following: nucleic acid extraction reagent, sterile nuclease-free water, fluorescent dye, sample pretreatment reagent, and instructions for use.

[0050] Optionally, the kit may also include primers or probes for detecting one or more virulence genes among rmpA, rmpA2, irp2, iutA, peg-344, or magA for expanded detection or research purposes.

[0051] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of a reagent for detecting molecular targets in a test sample in the preparation of a diagnostic product for differentiating between symptomatic urinary tract infections associated with Klebsiella pneumoniae and asymptomatic bacteriuria in patients with spinal cord injury, characterized in that, The molecular target is the Klebsiella pneumoniae virulence gene iroN.

2. The application according to claim 1, characterized in that, The reagents are those used in at least one of the following detection methods: PCR, real-time quantitative PCR, nucleic acid amplification, or gene sequencing.

3. The application according to claim 1, characterized in that, The reagents are primer pairs that specifically amplify the virulence gene iroN, and / or probes that specifically bind to the virulence gene iroN.

4. The application according to claim 3, characterized in that, The primer pair includes an upstream primer and a downstream primer, the sequence of which is shown in SEQ ID NO:11 and the sequence of which is shown in SEQ ID NO:

12.

5. The application according to claim 1, characterized in that, The sample to be tested was urine that tested positive for Klebsiella pneumoniae.

6. The application according to claim 5, characterized in that, The sample to be tested is a strain of Klebsiella pneumoniae isolated from urine culture or its genomic DNA.

7. A kit for differentiating between symptomatic urinary tract infections associated with Klebsiella pneumoniae and asymptomatic bacteriuria in patients with spinal cord injury, characterized in that, The kit contains reagents for detecting a molecular target in the sample to be tested, the molecular target being the Klebsiella pneumoniae virulence gene iroN.

8. The reagent kit according to claim 7, characterized in that, The kit contains: (a) Primer pairs or probes that specifically amplify the virulence gene iroN; (b) PCR amplification reagents, wherein the PCR amplification reagents include one or more of the following: DNA polymerase, PCR reaction buffer, dNTPs, Mg 2+ Ion source; (c) Positive control; (d) Negative control.

9. The reagent kit according to claim 8, characterized in that, The kit also includes one or more of the following: nucleic acid extraction reagent, sterile nuclease-free water, fluorescent dye, sample pretreatment reagent, and instructions for use.

10. A detection system for differentiating between symptomatic urinary tract infections associated with Klebsiella pneumoniae and asymptomatic bacteriuria in patients with spinal cord injury, characterized in that, The detection system includes: The sample processing module is used to extract nucleic acids from the sample to be tested. A detection module comprising the primer pair and / or probe as described in claim 3 or 4, for amplifying and detecting the nucleic acid; The analysis module is used to determine whether the amplification product of the Klebsiella pneumoniae virulence gene iroN is present in the sample to be tested based on the output of the detection module; if the amplification product is present, the virulence gene iroN in the sample to be tested is determined to be positive.

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