Culture-Negative Peritonitis and the Latest Diagnostic Techniques

Peritoneal dialysis (PD), hemodialysis, and renal transplantation are common treatments for end-stage renal disease (ESRD) patients [1]. Millions of people die because of the lack of access to dialysis (including hemodialysis and PD) for the treatment of kidney failure [2, 3]. PD is an auxiliary renal function technique that uses the peritoneum of ESRD patients as a semi-permeable membrane to discharge excess waste from the body through natural ultrafiltration and penetration [4, 5]. Globally, >200,000 patients with advanced kidney diseases use PD [6]. Compared with hemodialysis, PD is more portable and simpler. It not only provides higher economic practicality and more freedom from frequent visits to hospital dialysis centers but also better preserves residual kidney function [7, 8].

Despite its advantages, PD faces various challenges, including peritonitis [3], inflammation [9], membrane failure [10], and cardiovascular disease [11]. Peritonitis is considered the primary cause of technical failure, including catheter removal and transition to hemodialysis, in PD patients [4]. Positive bacterial culture is one of the gold standards for the diagnosis of peritonitis; however, the current rates of positive bacterial cultures are always low, and patients with culture-negative peritonitis are often overlooked. This may lead to a delay in appropriate or specific antibiotic treatment. Patients would obtain a better survival status if preclinical intervention could be conducted in the early stages of peritoneal transit-related peritonitis [12]. Therefore, we aimed to summarize and collate the current state of research and advances in negative peritonitis and to explore additional clues to improve the positivity of bacterial cultures.

The classification of peritonitis is based on various etiologies, including culture-positive peritonitis caused by specific bacterial infections, culture-negative peritonitis, catheter-associated peritonitis, and peritonitis of enterogenic origin. Peritonitis of enterogenic origin is caused by enterogenic infections (pancreatitis) or noninflammatory factors (narrower enterocolitis). Culture-negative peritonitis refers to the presence of peritonitis symptoms despite negative peritoneal fluid cultures and can be further categorized as infectious or noninflammatory peritonitis. Some patients may exhibit peritonitis-like symptoms owing to peritoneal sensitivity to transudate content. This study focused specifically on bacterial culture-negative peritonitis associated with infections.

The quality of life and expectancy of ESRD patients can be greatly improved with timely diagnosis and intervention for peritoneal-associated peritonitis [5]. According to the diagnostic criteria of the International Society for Peritoneal Dialysis (ISPD) 2022, obtaining a bacterial culture for rapid and accurate identification of the causative pathogen is essential for diagnosis and selecting appropriate treatments [13, 14]. Bacterial culture remains an irreplaceable clinical gold standard for authenticating the presence of peritonitis [15]. Bacterial cultures can provide valuable information to clinical workers, such as clarifying the specific source of infection through the characteristics of bacterial colonies and providing effective clinical information, such as drug sensitivity [16], to further guide medication and achieve more precise treatment.

Culture-negative peritonitis is characterized by a turbid dialysate and/or abdominal pain, with a presence of 100 leukocytes/L of PD effluent (PDE) (at least 50% polymorphonuclear leukocytes) and no microbial growth observed within 72 h [17]. According to the 2022 ISPD guidelines, the negative rate of bacterial cultures should not exceed 20%, and in some specialized institutes, it can be as low as <10% [15]. However, the CNS accounts for 13.4%–40% of all episodes of peritonitis [18‒20]. There are multiple reasons why the actual negative rates are much higher than those required by the ISPD guidelines.

The primary reason for the low number of organisms and dose is an inappropriate sampling method, which results in a reduced positivity rate [14]. The use of substandard samples during sampling increases the rate of negative detection. Some organizations transport peritoneal dialysate in sterile bottles, aspirate it directly under a microscope, and then inoculate it into traditional enrichment media (e.g., cooked meat broth) with extended incubation times, which can significantly increase the positivity rate [21]. Second, the wide variety of microorganisms and their differing environmental requirements necessitate the selection of appropriate culture methods and techniques, particularly for bacteria that are difficult to culture and require special environments [22, 23]. Peritoneal dialysate bacterial culture techniques lack sufficient sensitivity for the management of early-stage peritonitis patients and are highly susceptible to dominant flora and mixed contaminants during the culture process. Third, the substantial quantity of dialysate present in the peritoneal cavity dilutes the concentration of pathogenic microorganisms [24]. Fourth, as reported by Szeto et al. [20], the effect of a patientʼs recent antibiotic exposure on the results of bacterial cultures is significant [25]. Owing to the easy availability of antibiotic prescriptions, many patients take oral antibiotics for noninfectious or viral diseases [16], and these procedures greatly reduce the success rate of bacterial culture. We took a short discussion on Mycoplasma peritonitis, eosinophilic peritonitis (EP), and tuberculosis-associated peritonitis.

Mycoplasma, including Mycoplasma solani, is often misclassified within the phylum Fungi, despite its distinct characteristics. These organisms typically colonize the genitourinary tract, and Mycoplasma solani is further divided into two types: Mycoplasma minima and Mycoplasma solani [26]. Detecting these pathogens in PD fluids poses significant challenges due to their specific growth media requirements for in vitro cultivation, coupled with the fact that many Mycoplasma minima infections are asymptomatic. Notably, peritonitis associated with Mycoplasma urealyticum has been infrequently reported, with only 2 cases identified through Mycoplasma culture in existing literature. Most detections have relied on molecular techniques such as polymerase chain reaction (PCR) or metagenomic next-generation sequencing (mNGS) [27, 28].

In female patients presenting with recurrent culture-negative PD-associated peritonitis, particularly in cases correlated with menstruation, sexual intercourse, or urinary tract infections, it is crucial to emphasize the detection of Mycoplasma, U. parvum, and U. urealyticum. Early identification of these organisms can facilitate timely intervention and improve patient outcomes. Given that Mycoplasma lacks a cell wall, it exhibits inherent resistance to antibiotics targeting cell wall synthesis, such as glycopeptides (e.g., vancomycin, ticlopidine) and β-lactams (e.g., penicillins, cephalosporins) [29]. Therefore, antibiotics that disrupt bacterial deoxyribonucleic acid (DNA) replication (e.g., quinolones) and inhibit protein synthesis (e.g., tetracyclines, macrolides) are recommended as first-line therapeutic options, with caution advised for use in pregnant women and neonates [30].

EP is a relatively uncommon complication of PD, characterized by eosinophilia in the dialysate. Eosinophilia is defined as the presence of more than 100 eosinophils per cubic millimeter in the dialysate or an eosinophil count exceeding 10% of the total non-red blood cell population. This condition can manifest shortly after the initiation of dialysis or may develop 1–2 years or more afterward [31]. The primary clinical presentation of EP includes cloudy peritoneal dialysate that is unresponsive to antibiotic therapy. Symptoms typically associated with peritonitis, such as abdominal pain, pressure, and rebound tenderness, are often mild or absent, and repeated cultures of peritoneal dialysate are negative, distinguishing EP from bacterial peritonitis.

In a reported case by Qingyan et al. [32], a 28-year-old male patient experienced turbid peritoneal dialysate within the first month of commencing PD, without any significant signs of infection, such as abdominal pain or fever. Cytological analysis of the dialysate revealed elevated leukocyte counts and multinucleated cells. Repeated bacterial cultures of the PDE yielded negative results, and no pathogens were identified through mNGS. An antibiotic regimen lasting 28 days did not result in significant symptom improvement. Based on the persistent eosinophilia in the peritoneal fluid, the patient was ultimately diagnosed with EP. Notably, the peritoneal dialysate cleared following a change in the dialysis solution, which retained the same buffers and electrolytes but was devoid of polyvinyl chloride.

While EP often resolves spontaneously, antihistamines or glucocorticosteroids may be required to prevent catheter obstruction in some cases. In patients without an identifiable trigger for eosinophilia, modifying the dialysis solution may provide therapeutic benefit [33].

Peritoneal tuberculosis is caused by infection with M. tuberculosis. Due to the special culture conditions required for M. tuberculosis and the nonspecific nature of clinical and laboratory findings associated with peritoneal tuberculosis, utilizing a 150 mL sample of dialysate in clinical practice is recommended for patients presenting with culture-negative peritonitis or culture-positive peritonitis that is resistant to appropriate antibiotic therapy, especially in the absence of other suggestive findings [34]. This approach enhances the sensitivity of smear and culture tests. Furthermore, the use of high-throughput DNA sequencing can significantly improve the diagnostic sensitivity for tuberculosis peritonitis [35, 36]. In cases of suspected peritoneal tuberculosis, laparoscopy and biopsy should be considered at an early stage. Clinical judgment should guide the decision to perform a laparoscopic biopsy early in the evaluation of patients suspected of having peritoneal tuberculosis [37].

In the context of PD, the possibility of peritonitis should prompt the immediate acquisition of a bacterial culture from the peritoneal fluid, regardless of the presence or absence of typical physical symptoms. Abdominal fluid is stored in the abdomen for at least 4 h. It is of paramount importance that specimen collection be completed before the patient is exposed to antibiotics. During the collection process, the principle of asepsis must be rigorously adhered to, and the first tube of peritoneal fluid should be discarded to avoid any potential errors that might affect the culture results [38]. The specimens should be sealed after collection and delivered to the laboratory within 6 h [39]. If immediate transport to the laboratory is not possible, the inoculated culture bottles should not be refrigerated or frozen as this may kill or retard the growth of some microorganisms. Whenever possible, inoculated culture bottles are preferably incubated at 37°C, with the exception of some patients in whom infection by specific pathogens is considered [26]. Samples can also be pretreated before the inoculation of cultures to compensate for the low biomass of PDE, thereby increasing culture success [40].

Early intervention for peritonitis and timely initiation of antibiotic treatment are keys to successful treatment of peritonitis. Therefore, the clinical need for culture tests are urgently required. Traditional culture techniques can result in a negative rate of 58.1% [20], which is much higher than the <20% required by the ISPD guidelines. Microbial culture technology is critical to produce negative bacterial cultures. Some improvements have been made to the traditional methods of directly culturing bacteria.

Peritoneal dialysate (3–5 mL) was inoculated into the bacterial culture on a BACTEC (Johnston Laboratories, Inc., Towson, MD, USA). Blood culture bottles (aerobic, hypertonic, and anaerobic media) were incubated for up to 7 days according to the manufacturerʼs instructions. Specimens were extracted directly from the blood culture bottles, and Gram-stained membranes were prepared to produce a positive growth index (radioactivity detection).

Tan et al. [41] collected 72 samples from 46 patients during the study period and compared bacterial growth results from peritoneal dialysate in the BACTEC blood culture system versus conventional culture. However, they did not find a statistically significant difference in culture positivity using these different culture methods, which contrasts with previous studies that showed a higher positivity of culture yield in blood culture bottle systems. They found that the BACTEC method had a significantly shorter time to detection (TTD) (rounded down to the nearest hour) than that of the conventional culture method (18.9 ± 24.4 vs. 37 ± 16.5 h, p = 0.014). Chow et al. [42] also found that the BACTEC method has a shorter TTD (31.2 h compared than that of the water lysis method). This finding aligns with the results of Tan et al.’s [41] study. Both BACTEC and conventional culture methods have comparable sensitivities and yields in identifying pathogens in PD cultures, partly because of the enrichment medium and prolonged incubation period. The BACTEC method confers the additional benefit of a shorter TTD, allowing targeted antimicrobial treatment and improved clinical outcomes.

A 500 mL specimen of dialysate was collected and passed through an Addi-Chek filtration system (Millipore Corporation, Bedford, MA, USA). The system consists of an enclosed sterile chamber containing a 0.45 µm (pore size) membrane filter used to capture microorganisms present in the dialysate. The pore diameter of a membrane filter determines the maximum amount of effluent that can be processed by each filtration unit. Each Addi-Chek unit was supplied with sterile, prepackaged 100 mL of bacterial growth medium (tryptic soy meat solution), and incubated at 35°C for 10 days. The presence of organisms was determined by turbidity development, and graphite-stained membranes were prepared from the turbid broth. The final recovery and identification of microorganisms were conducted in accordance with the standard methods for each system.

A sufficient volume of peritoneal dialysate was collected according to strict aseptic standards using physical methods such as centrifugation or adsorption to dislodge antibiotics and then cultured for bacteria.

Peritoneal dialysate (50 mL) was washed in 50 mL of saline to remove antibiotics and centrifuged, and the suspended sediment was injected directly into the medium for direct culture [43].

Peritoneal dialysate (50 mL) was injected directly into BACTEC 16B and 17D antibiotic-removal resin bottles for direct bacterial culture without washing and centrifugation [43].

Using a large volume of peritoneal fluid (at least 50 mL), disturbances were quelled by passing it through an adsorption filter (pore size 0.22 µm; Bedford) or by direct centrifugation. The precipitate was then rinsed with 100 mL of sterile saline. After removal of antimicrobials from the peritoneal fluid, the resuspended precipitate was inoculated by re-centrifugation into sulphoethanolic acid medium (135/C medium [BBL Microbiology Systems, Cockeysville, MD, USA], supplemented with 0.05% sodium polycyanidazole sulfonate) to support aerobic and anaerobic growth and culture. Standard microbiological methods were used to identify these microorganisms [24, 43].

When changing the peritoneal dialysate, the concentrated culture solution used for bacterial culture was added directly to the draining dialysate exchange bag, and the exchange bag with added culture medium was incubated at 35°C and observed for turbidity. Bacterial cultures were performed on 78 dialysate exchange bags from nonclinical peritonitis patients (negative controls) and 48 dialysate exchange bags from clinical peritonitis patients. All cultures from clinical peritonitis patients were positive (100% sensitivity), and bacteria were recovered from 5 patients in the negative control group (94% specificity). The majority of the cultured bacteria were Gram positive (86%), with a minority being Gram negative (14%) [44].

In addition to improving traditional culture methods, the yield of peritoneal dialysis fluid (PDF) culture can also be improved by direct inoculation of liquid into rapid culture bottle kits, such as BACTEC. However, when Males et al. [45] compared Addi-Chek filtration and BACTEC with the traditional 10 mL culture technique, there was no significant difference between the results [8]. Vas Law suggested that incorporating an additional washing step in the PDF bacterial culture method to eliminate the influence of antibiotics on sample liquid detection could enhance the PDF culture yield. Furthermore, Vas Law proposed in the study that the highest recovery of PDF was achieved when all improvements were implemented simultaneously.

When cultures remain negative after 3–5 days of incubation, the PDE should be sent for repeat cell counts, differential counts, and fungal and mycobacterial cultures. In addition, subculturing on media with aerobic, anaerobic, and microaerophilic incubation conditions for an additional 3–4 days may help identify slow-growing fastidious bacteria and yeasts that are undetectable in some automated culture systems. Furthermore, the culture of PD catheters can improve diagnostic yield, especially for the detection of fungi and enterococci [46].

To overcome the limitations of traditional culture techniques in detecting microorganisms, including uncultivable or difficult-to-culture ones in the PDF of patients receiving antibiotics, clinical laboratories are increasingly using molecular techniques. In the last decade, bacterial DNA amplification and detection methods for PCR have had a strong diagnostic significance for the diagnosis of infectious diseases [47, 48]. PCR technology can rapidly and simultaneously detect a variety of microbial species, has a high sensitivity, and is not easily affected by patients receiving pre-antibiotics; these aspects undoubtedly establish PCR as the preferred method in clinical diagnosis of peritoneal-related peritonitis [49].

DNA PCR. Kim et al. [50] compared a variety of different DNA extraction methods and 16S DNA to evaluate the accuracy of wide-range PCR in CAPD dialysates. Wide-range DNA sequencing and conventional culture methods are highly consistent [51]. Although response inhibitors in clinical specimens can affect final results, different primer species have an equal impact on the amplification results. However, considering many factors, wide-range DNA PCR can be used to supplement traditional bacterial culture methods [52].

Ribonucleic Acid (RNA) PCR. Rampini et al. [53] compared the PCR results of 16S RNA of 394 samples with conventional culture and found a concordance rate as high as 90%. They prospectively studied 237 samples with negative conventional culture and found that the sensitivity, specificity, positive test value, and negative test value of 16S ribosomal RNA (rRNA) PCR were 42.9%, 100%, 100%, and 80.2%, respectively. In clinical studies, patients who had received previous antibiotic treatment showed superior results compared to conventional culture techniques [54]. Because of the high specificity of 23S rRNA in the genes of many species, Yoo et al. [55] compared the results of 23S rRNA with the overall coincidence rate of 23S rRNA PCR and standard culture techniques. When 23S rRNA PCR was combined with conventional bacterial culture technology and used simultaneously, the microbial detection rate reached 93.3%.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is a DNA sequencing technology derived from PCR and GeneChip [56]. NGS pioneered the introduction of reversible termination ends and realized sequencing by synthesis during DNA replication. In NGS, individual DNA molecules are amplified into gene clusters composed of the same DNA, which are then replicated synchronously to determine the DNA sequence by capturing special markers (usually fluorescent molecular markers) carried by the newly added bases [57]. NGS is characterized by high sensitivity, resolution, and throughput. With advancements in technology and cost reduction, NGS is gradually transitioning from professional laboratories to clinical applications, providing clinicians with more diagnosis and treatment insights.

NGS can detect polymicrobial infections, including nondominant bacteria (i.e., those comprising less than 1% of the total bacterial population), and has been used in several human microbiome studies. Furthermore, NGS highlights complex interactions between causative organisms during infection process [58]. Peritonitis was diagnosed in 25 cases using 16S rRNA NGS technology. Weinstock et al. [59] observed that the results of traditional bacterial cultures were identical to those of NGS. NGS was found to be more sensitive, identifying 33 different bacterial species (including one non-culturable bacterium) from the same batch of specimens, compared to the bacterial culture technique, which identified only 13 dominant bacterial groups. However, because of the small size of the sequenced 16S rRNA gene fragments, NGS results lacked specificity [58].

mNGS is a high-throughput sequencing technology that utilizes NGS of both DNA and RNA extracted directly from clinical samples [60]. This innovative approach bypasses the need for pathogen cultivation, enabling the simultaneous detection of various pathogens, including bacteria, fungi, viruses, parasites, and others. Following sequencing, bioinformatics analyses and database comparisons are performed to identify the pathogens present in the samples.

In Ye et al. [61] study, 30 patients with peritonitis were included, and their peritoneal dialysate was analyzed using both mNGS and conventional culture methods to identify pathogens. The results demonstrated a significantly higher detection rate of pathogens using NGS (86.67%) compared to conventional bacterial culture methods (60.00%). Notably, mNGS also identified specific pathogens, such as Kochia pallidum, in a small subset of patients. Similarly, Guo et al. [62] conducted a study involving 37 patients with PF-associated peritonitis, randomly divided into two groups. One group’s samples were cultured using conventional blood culture bottles, while the other group’s samples were tested for pathogenicity using the mNGS method. The mNGS approach yielded a positivity rate of 96.77%, in contrast to 70.97% for the blood culture bottle method. The sensitivity of mNGS was 96.77%, with a specificity of 83.33%, compared to 70.97% sensitivity and 100% specificity for the blood culture bottle method.

While traditional single-target molecular methods can obtain a large amount of validated data, greatly improving their ability to detect bacterial pathogens, multiplexed methods offer even greater possibilities. Kaleta et al. [63] employed a novel multiplex approach – PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS). A team extracted DNA directly from 234 BACT-ALERT blood culture bottles from septicemia patients and used PCR/ESI-MS to search for the genera and species of microorganisms that cause bloodstream infections in humans. When compared with the clinical reference standard method, the results showed the concordance was 98.7% and 96.6% at the genus and species levels, respectively. This approach allowed for the acquisition of more information about pathogenic organisms without the need for prior knowledge and facilitated the implementation of targeted antimicrobial therapy by detecting almost all bloodstream infection pathogens in a single rapid assay. In Chang et al. [64] study cohort, 15 double-positive samples used both PCR/ESI-MS and cultures; the concordance at the genus and species levels was 100% and 87.5%, respectively. Chang et al. [64] also applied PCR/ESI-MS technology to detect PDE microbial yield, which can also detect the presence of antibiotic genes and can be used to guide clinicians to convert empirical broad-spectrum antibiotics into target antibiotics.

In addition to the advancement of new technologies aimed at enhancing bacterial culture techniques, various research teams are currently exploring methods to expedite the provision of information regarding the identification of causative pathogens in peritoneal infections. This effort is crucial to obtain swift and dependable diagnostic data that can inform therapeutic decision-making. Furthermore, these researchers proposed that patients at a heightened risk of complications associated with PD should be expeditiously transferred to alternative renal therapies such as hemodialysis [65].

Owing to the unique nature of abdominal peritoneal operation, the abdominal cavity serves as a special space, offering a distinctive perspective on the tissue-resident immune system. When the concentration of interferon (IFN-γ) in the dialysis solution of acute peritonitis patients reaches a certain level, the neutral granulocyte count in the peritoneal fluid is correlated with the level of IL-17. The specific β1 glycoprotein (SP1) serves as the necessary transcription factor for the production of C-X-C motif chemokine ligand 1 (CXCL1). When PD patients are in a state of peritoneal infection, IL-17 levels in the peritoneal fluid increase and quickly initiate the transcription and translation of SP1. After the HPMCS is stimulated by both TNF-α and IFN-γ, CXCL1 is released from the cells and combines with extracellular neutrophils to regulate the transmissions of the neutral granulocytes [66] (Fig. 1a). Hautem et al. [67] reported that lipopolysaccharide (LPS) and E. coli activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome and IL-1b in macrophages to induce PD-associated peritonitis (Fig. 1b). In the peritoneal cavity of these patients, macrophages stimulated by pathogens resulting in higher levels of IL-6 and TGF-β appear in the abdominal transit fluid. This stimulation constantly induces the production of vascular endothelial growth factor by c-Fos proto-oncogene and SP4 transcription factors. Moreover, IL-17 continuously induces inflammation. The induction of inflammation promotes small-molecule solute transport, leading to repeated damage of mesothelial cells and gradual fibrosis [68] (Fig. 1c).

In this localized region, the cellular composition of the peritoneal outflow includes all relevant cellular components and humoral mediators that are involved in local inflammation. Under peritoneal and luminal active complex immune interactions, outflow fluid cell and cytokine profiles can counter-react to the conditions of PDF exposure and concurrent infection. Theoretically, cytokines or cells involved in peritoneal inflammation can serve as biomarkers for predicting PD-related complications. There is growing evidence of differences in patient responses to conspecific invasive microorganisms and/or differences in inflammatory responses triggered by different species [69]. This discrepancy may be attributed to the involvement of various inflammatory mediators.

IL-6 is present in the PDE and is a key regulator of acute peritoneal inflammation in response to infections [70]. Acute dialysate IL-6 levels were higher in peritonitis patients than in non-peritonitis patients, and there was also a significant correlation between the number of peritonitis episodes and IL-6 levels after 1 year (upper-20). Recently, a novel IL-6-based point-of-care diagnostic device was developed for rapid diagnosis of peritonitis. This test mainly detects the elevation of two pro-inflammatory markers, IL-6 and MMP-8, in the PDF using a lateral flow experiment [71]. As the peritoneal fluid was absorbed and passed through the nitrocellulose membrane in the device, the antibody and binding reagents located on the detection line combined with the target marker to produce visually interpretable results. When this device is used, the diagnosis or exclusion of peritonitis requires approximately 5 min [72]. Goodlard et al. [73] evaluated 107 PDE samples collected in a real-world clinical setting. Of the 107 samples, 49 and 58 tested positive and negative, respectively, using this device [73]. The device had a high negative predictive value of 98.3% (95% CI: 89.1–99.8) and 83.7% (95% CI: 72.8–90.8) and sensitivity and specificity of 97.6% (95% CI: 87.4–99.9) and 87.7% (95% CI: 77.2–94.5), respectively. In addition to IL-6 [74], IL-1b, IL-4, IL-16 [75], and IL-17a [76] are potential predictors of peritonitis. IL-1β regulates mitochondrial function in mesothelial cells, sensitizes mesothelial cells, and induces inflammation, thus significantly amplifying the inflammatory response of the body [77].

The T-helper (Th) 17-mediated inflammatory response, particularly that mediated by the cytokine IL-17, has recently been shown to play a central role in peritoneal damage. Experimental modulation of the Th17 response and/or enhancement of the regulatory T-cell response may preserve membrane function.

Increased total white blood cell and absolute neutrophil counts in the peritoneal fluid of patients undergoing PD during an acute episode of peritonitis can serve as strong indicators of peritonitis. The use of a leukocyte esterase reagent enables prompt bedside detection of leukocyte levels in the peritoneal effluent of patients undergoing PD. Rathore et al. immersed a LERS (Multistix 10SG) directly into the PDE, and the paper was detected for 2 min; A “+” reading was considered highly suggestive of peritonitis [78]. Peritonitis occurred in 21 of 166 (12.6%) acute PD patients, of whom 20 peritonitis patients had highly suspicious LERS results [79].

The NLR proposed by Zhang et al. [80] could also predict the incidence of peritonitis, although an elevated NLR often serves as a valid predictor of cardiovascular events in PD patients [81]. NLR is associated with renal failure and endothelial dysfunction in chronic kidney disease patients [82‒84] and can be used as a sensitive marker of systemic inflammation [85]. Low serum parathyroid hormone, high calcium, and low phosphorus levels can also predict the clinical status of anterior peritonitis in patients undergoing PD [86]. In Fung et al. study, among the 364 patients with culture-negative peritonitis, those in the tuberculous peritonitis group exhibited the lowest levels of peritoneal fluid neutrophil-to-lymphocyte ratio (NLR) at the onset of the disease [87]. This was followed by patients in the nontuberculous mycobacteria, culture-negative, and methicillin-sensitive Staphylococcus aureus groups. These findings suggest that the peritoneal fluid NLR can serve as a valuable and readily available biomarker for differentiating peritonitis caused by M. tuberculosis from nontuberculous mycobacterial infections and bacterial PD-associated peritonitis.

Vibrational spectroscopy techniques such as Fourier transform infrared (FTIR) spectroscopy are rapid, noninvasive, and cost-effective analytical tools with high-throughput capabilities. These techniques have been successfully employed to differentiate between normal and pathological populations in various tissues and/or cell types, as well as in biological fluids, including plasma, serum, urine, saliva, and synovial fluid. PDE is an easily accessible biofluid that provides extensive insights into peritoneal biology and membrane status through its dissolved molecules. The most important spectral regions of biomolecules in the dialysate, including polysaccharides, proteins, and fatty acids, can be determined through FTIR spectroscopy. Grunert et al. [88] linked FTIR spectroscopy with targeted metabolomics to provide a simpler and more cost-effective way to obtain overall microbial marker information. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also used to identify the pathogenic microorganisms, including Candida tropicalis, in a 22-year-old woman presenting with sepsis and high fever in para [89].

A comprehensive range of cellular and soluble parameters, including various local immune cells, inflammatory and regulatory cytokines, chemokines, and tissue damage-associated factors, has been detected in the peritoneal fluid of patients diagnosed with acute peritonitis. Utilizing advanced mathematical modeling techniques, Lin et al. [90] found that certain crucial pathways are responsible for integrating pathogen-specific inflammatory responses at the infection site. Analysis of the final findings revealed that different types of bacteria elicit distinct patterns of local immune responses, with specific biomarker profiles associated with Gram-negative and Gram-positive bacteria as well as culture-negative cases of unknown origin. They initially proposed the use of “immune fingerprinting” as a diagnostic tool for peritonitis.

Furthermore, Zhang et al. [75] utilized mathematical machine learning algorithms to identify “integrated immune fingerprints” capable of accurately predicting the three primary categories of organisms responsible for acute peritonitis: Gram-negative, streptococcal, and coagulase-negative staphylococcal infections. The combinations of multiple biomarkers, obtained from peritoneal effluent samples, were analyzed using a systematic approach that integrates multicolor flow cytometry and multiplexed enzyme-linked immunosorbent assays. The results demonstrated that unique combinations of immune biomarkers could differentiate between streptococcal and non-streptococcal species within the Gram-positive group, including coagulase-negative Staphylococcus spp., Streptococcus acidophilus spp., and Streptococcus basophilus spp. This finding adds significance to pathogen-specific diagnosis of acute peritonitis in patients undergoing PD. Future AI/ML devices will likely have the capability to predict dialysis complications using simple clinical variables and monitor the entire dialysis process.

Yan et al. [91] analyzed the LPS levels and other infectivity indicators in effluent samples from 161 PD patients with peritonitis. The findings of this study revealed that increased LPS levels in the PDE could serve as supplementary evidence for the diagnosis of culture-negative peritonitis. Additionally, heightened levels of LPS in PDE have been linked to Gram-negative bacterial infections. Yan et al. [91] postulated that when peritonitis patients fail empirical treatment, have multiple culture-negative results, and exhibit a significant increase in LPS concentration in the PDF, it is advisable to replace the initial antibiotic with a more potent one with a broader antimicrobial spectrum or to add a second antibiotic targeting Gram-negative bacteria. A high level of eryptosis was also found in PD patients with elevated concentrations of IL-6, IL-18, and IL-1β. Expanding on this, Virzì et al. [92] hypothesized that eryptosis is directly connected to peritoneal membrane injury and peritonitis.

Peritonitis has been identified as a crucial core outcome in the standard outcome initiative for PD in nephrology [93]. Peritonitis patients have an increased surface area of effective blood vessels, enhanced small solute and glucose transport, protein loss into the dialysate, and osmotic gradient dissipation, leading to ultrafiltration failure [94, 95].

During the era of rampant cholera and tuberculosis, Koch and other researchers sought to link microorganisms with the occurrence of diseases. They proposed that identifying microorganisms in body fluids should become the basis for diagnosing and treating infectious diseases. In modern times, researchers like Fredrich and Relman have suggested an updated version of Koch’s postulates, which relies on isolating bacteria-specific DNA and then sequencing [96]. Continuous efforts are being made to enhance diagnostic techniques to improve the diagnostic yield for peritonitis patients with negative cultures in peritoneal dialysate. These advancements aim to increase the accuracy and effectiveness of diagnostic methods in clinical settings.

The ease of PDF and its extremely high bioinformatics content compared to other diseases make PD monitoring a particularly promising test case for predicting biomarker efficacy in clinical settings. The proposal of disease biomarkers opens new perspectives for diagnostic strategies in the field of abdominal penetration. Biomarkers, which represent disease mechanisms in clinical settings, provide relevant data for decision-making regarding patient diagnosis and treatment. Another classical definition of biomarkers is “characteristics objectively measured and assessed as indicators of normal biological processes, pathogenic processes, or pharmacological response to a therapeutic intervention.” Some biomarkers can predict the risk of PD-related complications. For high-risk patients lacking obvious clinical response, timely switching to other renal replacement methods, such as hemodialysis, may be necessary. After commencing the initial empirical antimicrobial treatment regimen, stratifying patients based on early biomarkers and appropriately adjusting the regimen according to microbiological isolation results can effectively improve clinical outcomes of PD.

The authors have no conflicts of interest to declare.

This work was supported by grants from the National Natural Science Foundation of China (No. 81570657, No. 30900682, No. 82200808).

Conceptualization: Yumei Wang, Huajun Jiang, and Jiajia Ye; data curation: Jiajia Ye, Yi Lv, Jing Sun, and Huanhuan Cao; formal analysis: Jiajia Ye, Yi Lv, and Huanhuan Cao; funding acquisition and supervision: Yumei Wang and Huajun Jiang; investigation: Jiajia Ye, Dan Chen, and Chen Ye; project administration: Dan Chen and Chen Ye; software: Jiajia Ye; writing – original draft: Jiajia Ye, Jing Sun, Chen Ye, Yumei Wang, and Yi Lv; and writing – review and editing: Jiajia Ye, Huajun Jiang, Yumei Wang, and Huanhuan Cao.

Jiajia Ye, Jing Sun, and Dan Chen contributed equally to this work.