Asymptomatic Bacteriuria or Urinary Tract Infection? New and Old Biomarkers

: Urinary tract infections (UTIs) are among the most common infective disease in the adult population. UTI diagnosis is based essentially on the presence of lower urinary tract symptoms (e.g., dysuria, urgency, and frequency) and the evidence of bacteriuria (by dipstick testing and/or urine culture). UTI diagnosis is not always easy because symptoms can be vague, or patient basal conditions can interfere negatively with the diagnostic process, whereas urine culture is still ongoing. In those cases, the differential diagnosis among UTIs and asymptomatic bacteriuria (ABU) may be challenging, while the clinician has to decide whether to start an antibiotic treatment shortly. The purpose of the present review is to analyze the biomarkers that could help in UTI diagnosis. Some biomarkers, such as procalcitonin, interleukin-6, neutrophil gelatinase-associated lipocalin, chemokines, lactoferrin, and bone morphogenetic protein-2, seem promising in UTI diagnosis, while other biomarkers failed to show any utility. Whereas a single biomarker was not enough, a combination of biomarkers could have more chances to help in the diagnosis.


Introduction
Urinary tract infections (UTIs) are among the most common infections in the adult population [1]. In non-pregnant healthy women, the diagnosis of UTI is straightforward, based on urinary symptoms, such as dysuria, frequency, and urgency in the absence of vaginal discharge [2]. In such patients, urine culture is not recommended to diagnosticate UTI, not improving the diagnosis accuracy of the clinical assessment [3]. In other cases, including recurrent UTIs (more than two episodes in the last six months or more than three UTIs in one year), male gender, pregnant women, patients with relevant anatomical/functional abnormalities of the urinary tract, immunocompromised patients, indwelling urinary catheters, renal diseases, and diabetes mellitus, the urine culture has a pivotal role in the diagnosis of UTIs [2]. Therefore, in cases such as complicated UTIs and recurrent UTIs, the presence of urinary symptoms and the detection of a pathogen by urine culture impact antibiotic treatment. In co-morbid patients and the elderly, UTI symptoms could be atypical or related to other conditions. For example, in patients with a cognitive impairment, it is not easy to determine the presence of lower urinary tract symptoms (LUTS), whereas in those patients with prostatic enlargement, non-infectious urethritis, bladder dysfunction, bladder tumors, and distal ureteric stone, LUTS could be present without concomitant UTIs. Finally, the results of urine cultures are not immediately available and could take 1-3 days. In this scenario, the diagnosis and the related antibiotic treatment can be a challenge. An inappropriate antibiotic therapy increases the risk of antibiotic resistance [4], and it is a financial burden for the community [5]. Conversely, a delay of an appropriate treatment could impact patient conditions, considering that mortality rates due to urosepsis can be as high as 16% [6]. Consequently, there is a strong need for biomarkers able to ease the clinical diagnosis of UTIs. The purpose of the present review is to assess the role of biomarkers in the diagnosis of UTIs. Specifically, we focus on the uncertain diagnosis when the symptoms are vague or difficult to interpret, evaluating the biomarkers that can help to discriminate between asymptomatic bacteriuria and UTIs.

Search Strategy
We performed a non-systematic literature search using the following strategy on Pubmed and Scopus in September 2021. Specifically, we searched for the terms "Urinary tract infection" and "biomarker" in the Pubmed Mesh Resources. Moreover, we performed a free text search in Pubmed and Scopus adopting the following search terms: "Procalcitonin", "Interleukin 6", "Neutrophil Gelatinase associated Lipocalin", "Proadenomedullin", "adenosine-5-triphosphate", "Tamm-Horsfall protein", "Bone morphogenetic protein-2", "beta2-Microglobulin", and "lactoferrin". Finally, we restricted our results by adopting the following limits: "English" language and "adults". We screened all the abstracts to identify all the relevant studies. Appendix A shows the details for every biomarker term.

Procalcitonin
Procalcitonin (PCT) is a precursor of the hormone calcitonin and belongs to acute phase reactants. In inflammatory conditions, PCT levels increase significantly. Specifically, PCT increases during bacterial infections and seems to provide supplemental information in the diagnosis of some bacterial infections, such as respiratory tract infection and urosepsis [7]. Furthermore, PCT levels correlate to bacterial load, and consequentially its levels decrease with the improvement in clinical conditions. Such peculiarities provide prognostic information in patients with infections, suggesting the management of bacterial infection, especially in septic disease [7]. On the other hand, two recent meta-analyses in children disputed this promising ability in UTI diagnosis, showing little accuracy for cystitis (AUC around 0.71 in ROC curve) [8,9], and limited evidence supports its utility in pyelonephritis diagnosis [10]. Consequently, PCT could not be recommended in clinical practice as a predictive marker of cystitis or pyelonephritis in children. Table 1 summarizes the characteristics and the main results of the five studies evaluating the role of PCT in the diagnosis of UTIs. Specifically, the evaluated trials showed the good negative predictive value of PCT in UTIs diagnosis [11,13]. This aspect is relevant in clinical practice because a PCT value < 0.25 ng/mL suggests the low likelihood of having UTIs. Thus, negative PCT supported general practitioners in the decision to wait for urine culture results or follow-up patient conditions before administering empirical antibiotic treatments. Conversely, high PCT levels at the moment of the clinical onset of a suspicious UTI are not a sensible marker for the diagnosis of UTIs. However, an elevated PCT should be evaluated carefully by the clinicians, especially in fragile people, because its value accurately predicts the presence of bacteraemia and bacterial load [16] and seems correlated to disease severity [14,15], regardless of the origin of the primary infection. Finally, three studies showed low accuracy and neglectable PCT changes in lower UTIs [12,14,15]. Those results suggest a marginal role of PCT during lower UTIs and seem congruent with the idea that PCT increases significantly during bacteremia.

Interleukin-6
Interleukin-6 (IL-6) is a cytokine and participates in different biological activities, including immunoregulation, inflammation, and oncogenesis. In an acute-phase reaction, IL-6 levels increase quickly in response to inflammation and infective stimuli [17]. For example, during a septic episode, IL-6 concentration can arise by 100 times over the basal concentration, which in healthy people is under 5 pg/mL [18]. Table 2 summarizes the characteristics and the main results of the 11 studies evaluating the role of IL-6 in the diagnosis of UTIs.   UTIs induce a significant increase in serum IL-6, and its level could differentiate lower and upper UTIs. Unfortunately, only two small studies evaluated this aspect and in two different clinical settings [15,21] Urine IL-6 was explored by eleven small studies [11,12,15,[19][20][21][22][23][26][27][28], usually performed in the elderly. Not all studies suggested a significant increase in urine IL-6 during UTIs, and the studies were too heterogenous to obtain a univocal recommendation. Currently, some studies have shown a significant increase in urine IL-6 in the case of pyelonephritis and bacteriemia [15,21]; others suggested a significant increase also in lower UTIs [11,[22][23][24][25][26]29]; others concluded there are no significant differences in urine IL-6 be-tween UTIs and asymptomatic bacteriuria [20,21,27,28]. Probably, urine IL-6 increases proportionally to inflammatory status related to urinary infection. This hypothesis could explain the heterogeneity in the results among the studies, which evaluated urine IL-6 levels in a different clinical setting of UTI.
Finally, only two studies evaluated its accuracy [11,21], and only one [21] proposed a possible cut-off for urine IL-6 level in the diagnosis of UTIs. Therefore, there is insufficient evidence to support its use as UTI predictor in clinical practice. Although considering those results promising, new studies should be performed to estimate its actual accuracy in UTI diagnosis.

Neutrophil Gelatinase-Associated Lipocalin
Neutrophil gelatinase-associated lipocalin (NGAL) is a 21-kD protein of the lipocalin superfamily. During bacterial infections, NGAL is secreted by neutrophils to limit bacterial growth. Specifically, NGAL binds bacterial siderophores limiting the iron supply and consequently the bacterial cellular process. Furthermore, NGAL seems to promote differentiation of renal epithelial cells and its predictive value in acute kidney injury has been proven in recent years [30]. Table 3 shows the characteristics and the main results of the four studies evaluating the role of urine NGAL value in the diagnosis of UTIs.  The first reports about urine NGAL accuracy in UTI diagnosis seem promising, especially in women. Specifically, Price et al. showed how u-NGAL has excellent accuracy in UTI diagnosis in a cohort of 100 women, 50 adult women with UTIs and 50 healthy adult women [31]. However, in our opinion, such remarkable results are related to the nature of the control group (constituted by healthy women), which can magnify the difference with UTI women and the accuracy of uNGAL as a biomarker of UTIs. Moreover, Gadalla et al. showed how u-NGAL performance could be implemented by combining its use with other immunological biomarkers (IL-8, IL-1alfa, and matrix metalloproteinase-8), even comparing symptomatic women with UTIs and without UTIs [20].
Nevertheless, currently, the number of studies and the number of patients enrolled do not allow to suggest its clinical use in UTI diagnosis.

Urinary Adenosine-5 -Triphosphate
Urinary adenosine-5 -triphosphate (ATP) is an organic compound that has the primary role to provide energy in many biological intracellular processes. Specifically, during UTIs, ATP release in a urine sample is increased by inflammatory cells and bacteria [34]. Consequently, its detection in the urine could be a useful biomarker of the infective process. Table 4 summarizes the characteristics and the main results of the three studies evaluating the role of urine ATP levels in the diagnosis of UTIs. These studies have ambiguous results. Specifically, whereas older studies suggested a good performance of ATP as a surrogate marker of bacteriuria [36,37], the latest reports failed to prove its utility as a surrogate marker of UTIs [35]. Likely, these inconclusive results could be related to differences in the methods of ATP analysis and the clinical setting.

Proadrenomedullin
Proadrenomedullin (proADM) plays an important role in the inflammation and infective process and showed a good performance as a new biomarker in septic disease [38]. Whereas some studies evaluated proADM impact in the treatment management of UTIs [39][40][41], no study evaluated its possible role in the discrimination between UTIs and ABU.

Beta2-Microglobulin
Beta2-microglobulin (ß2-M) is a protein with a molecular weight of 11.8 KD, passes easily through the glomerular membrane and is almost completely reabsorbed and metabolized by the tubules. Urine ß2-M increases during tubular damage and, consequently, it could be a sensitive marker of upper UTIs. Table 5 summarizes the characteristics and the main results of the four studies evaluating the role of urine ß2-M as a biomarker of UTIs.  All the studies on ß2-M were published before 1986. All the studies focused on the ability of ß2-M to discriminate between upper and lower UTIs [42][43][44][45]. As expected, no significant difference was found between lower UTIs and ABU. Consequently, its use in the diagnosis of UTIs should not be suggested.

Chemokines
Chemokines (ChKs) are small cytokines secreted by cells that induce directional movement of specific cell populations, such as leukocytes, endothelial and epithelial cells, to inflamed tissues. There are four different subgroups of ChK: CXC, CC, C, and CX3C. Specifically, CXCs are potent neutrophil/natural killer activators, while CC induce chemotaxis of T cell and monocytes. Table 6 summarizes the characteristics and the main results of the eight studies evaluating the role of ChKs as a biomarker of UTIs.    Unfortunately, the studies are not consistent in the results, and their use in clinical practice cannot be recommended considering the little accuracy of urine Chks in UTI diagnosis. Nevertheless, the studies on CXCL-8, known as well as IL-8, showed a significant increase in the urine during UTIs [11,20,26,27,48] and good specificity [20,46]. Currently, CXCL-8 is the most promising chemokines in UTI diagnosis and should be studied in depth in future research, especially in combination with other biomarkers.

Bone Morphogenetic Protein-2
Bone morphogenetic protein-2 (BMP-2) is known for its important role in the development of bone and cartilage, but it seems to participate in the paracrine and autocrine action of organ regeneration [49]. Only one study investigated its role in UTI diagnosis and showed a significant increase in its serum levels in UTIs [50]. A cut-off point of 44 pg/mL was shown to have accuracy as high as 86% in detecting UTIs. Unfortunately, we failed to identify other studies corroborating such findings, and the number of the cases and controls included in this study was too limited to suggest its use in clinical practice without further validation.

Secretory Immunoglobulin A
Secretory immunoglobulin A (S-IgA) is the major immunoglobulin isotype in external secretions of mucosal epithelia and the first line of local immunological defence. In UTIs, S-IgA was shown to inhibit the Escherichia coli adhesion to urinary epithelial cells [51]. Table 7 summarizes the characteristics and the main results of the two studies evaluating the role of s-IgA in the diagnosis of UTIs.
Although the results showed a significant increase in the biomarker during UTIs, its clinical use could not be suggested considering the quality of the studies and the limited number of cases. (*) variable represented as mean (± standard deviation).

Lactoferrin
Lactoferrin (LF) is a multifunctional 80 KD glycoprotein of the transferrin family. Its primary biological role is antimicrobial activity, interfering with bacteria growth by free iron sequester [53]. Table 8 summarizes the characteristics and the main results of the two studies evaluating the role LF in the diagnosis of UTIs.  Arao S. et al. (1999) suggested that LF levels > 200 ng/mL were associated with accuracy as high as 94% in the diagnosis of UTIs [53]. In the other report, Gill et al. showed the potential role of LF in the patients with overactive bladder syndrome to identify UTIs not diagnosed by standard urine cultures [42], but the findings of the study seem to be inclusive, and the conclusions are not fully supported by the data presented. Likely, more studies about the role of LF in UTI diagnosis are necessary to suggest its use in clinical practice.

Conclusions
Serum biomarkers showed their weakness in UTI diagnosis considering the limited positive predictive value. Although their use in clinical practice could be helpful to exclude upper UTIs and evaluate the severity of the infectious disease, few studies assess the reliability of serum markers in UTI diagnosis. Urine biomarkers are simple to collect and could be a precious help in uncertain UTI diagnoses. Unfortunately, their clinical use in this context cannot be suggested considering the current evidence (such as contradictory results, low number of patients, the different clinical settings and the different definitions of UTIs). Future studies in this field should focus on u-NGAL, u-IL-6, u-CXCL-8 and LF, which currently seems the most promising biomarker in UTI diagnosis.  Institutional Review Board Statement: Ethical review and approval were waived for this study, due to the nature of the review.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.