PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0308253 PONE-D-24-20393 Research Article Medicine and health sciencesWomen's healthMaternal healthPregnancy Medicine and health sciencesWomen's healthObstetrics and gynecologyPregnancy Biology and life sciencesAnatomyBody fluidsUrine Medicine and health sciencesAnatomyBody fluidsUrine Biology and life sciencesPhysiologyBody fluidsUrine Medicine and health sciencesDiagnostic medicine Biology and life sciencesCell biologyCellular typesAnimal cellsBlood cellsWhite blood cells Biology and life sciencesCell biologyCellular typesAnimal cellsImmune cellsWhite blood cells Biology and life sciencesImmunologyImmune cellsWhite blood cells Medicine and health sciencesImmunologyImmune cellsWhite blood cells Medicine and health sciencesUrologyGenitourinary infectionsUrinary tract infections Research and analysis methodsSpectrum analysis techniquesSpectrophotometryCytophotometryFlow cytometry Biology and life sciencesCell biologyCellular typesAnimal cellsEpithelial cells Biology and life sciencesAnatomyBiological tissueEpitheliumEpithelial cells Medicine and health sciencesAnatomyBiological tissueEpitheliumEpithelial cells Earth sciencesGeologyPetrologySediment Earth sciencesGeologySedimentary geologySediment Reduction of misdiagnosis in urinary tract infections during pregnancy: The role of adjusted urine flow cytometry parameters Reduction of misdiagnosis in urinary tract infections during pregnancy https://orcid.org/0000-0002-5140-8941 Liu Zhaojie Conceptualization Data curation Writing – original draft Liu Dan Conceptualization Data curation Formal analysis Su Guangming Conceptualization Data curation Formal analysis https://orcid.org/0009-0002-4760-3776 Yang Wei Conceptualization Data curation Formal analysis Writing – original draft Writing – review & editing * Department of Laboratory Diagnostics, First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China Domfeh Seth Agyei Editor Kwame Nkrumah University of Science and Technology, GHANA

The authors have declared that no competing interests exist

 * E-mail: yangwei6295@163.com 23 9 2024 2024 19 9 e0308253 25 5 2024 21 7 2024 2024 Liu et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction

Urinary tract infections (UTIs) pose a significant health concern, particularly among pregnant women, for whom accurate diagnosis is essential. However, the use of Urine flow cytometry (UF) for detecting UTIs in this demographic often results in misdiagnosis. The objective of this study was to explore the reasons behind these diagnostic errors and to develop a strategy to minimize the rate of UTI misdiagnosis in pregnant women.

 Material and methods

The study enrolled 1,200 women aged 18 to 40 years, categorized into pregnant and non-pregnant groups. UTIs were diagnosed using urine bacterial culture, microscopic examination, and UF, followed by statistical analysis to identify any discrepancies in diagnosis between the groups. Following the calibration of UF analyzer’s parameters, the most effective CR(WBC)-CW-FSC-P Gain setting for diagnosing UTIs in pregnant women through UF was ascertained by applying the Youden index.

 Results

The clinical diagnosis rate of UTIs was significantly higher in pregnant women (40.91%) compared to non-pregnant women (20.26%). However, urine microscopy and bacterial culture showed no significant difference in the rates of UTIs between the two groups, suggesting a potential for misdiagnosis. The false-positive rate for WBCs detected by UF was 30.43%, and adjusting the CR(WBC)-CW-FSC-P Gain value of UF reduced the false-positive rate to 9.45%.

 Conclusion

The incidence of UTIs in pregnant women may be overestimated because of the limitations inherent to UF. Adjusting the parameters of the UF analyzer, particularly the CR(WBC)-CW-FSC-P Gain value, can significantly reduce the rate of UTI misdiagnosis in pregnant women.

 The author(s) received no specific funding for this work. Data Availability All relevant data are within the manuscript and its Supporting Information files. Introduction

Urinary tract infections (UTIs) are a prevalent class of infections in clinical settings, posing a substantial health risk, particularly for pregnant women. These infections during pregnancy are correlated with preterm birth [1, 2]. Recognizing the profound impact of UTIs on both individual health and societal well-being, precision in the diagnosis of UTIs becomes indispensable [3]. However, the physiological changes that accompany pregnancy often complicate the accurate detection of UTIs, necessitating a more nuanced diagnostic approach [4, 5].

Leukocytes counts is a critical diagnostic criterion for UTIs [6, 7]. Urine flow cytometry(UF), recognized for its heightened sensitivity, has been extensively used for the detection of urinary leukocytes [8]. Obtaining a clean-catch midstream urine sample is essential for the accurate application of UF in detecting urinary leukocytes. However, urine samples from pregnant women often contain contaminants from the vaginal or anal regions, which can lead to false-positive or false-negative urinalysis results, and consequently, misdiagnoses [9]. Our previous study revealed that UF had a high false-positive rate when detecting urinary leukocytes in pregnant women [10]. The aim of this study was to elucidate the phenomenon that the high false positive rate of urinary leukocytes in pregnant women may lead to misdiagnoses of UTIs, with the ultimate goal of identifying a method to reduce the misdiagnosis rate in pregnant women with UTIs.

 Material and methods

The present study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No.2022103) and was conducted in accordance with the Declaration of Helsinki. All methods were carried out in accordance with the relevant guidelines and regulations. A written informed consent was taken from every participant of the study.

Study population

A total of 1,200 women, aged between 18 and 40 years, who visited the obstetrics and gynecology outpatient department from June 1, 2023, to January 1, 2024, participated in this study. All participants included in this study underwent urine human chorionic gonadotropin (HCG) testing and abdominal ultrasound examination. Participants were categorized as pregnant if the urine HCG test was positive, or if the abdominal ultrasound revealed the presence of a gestational sac and fetal heartbeat within the uterine cavity. Conversely, if the urine HCG test was negative and the abdominal ultrasound did not detect a gestational sac or fetal heartbeat, participants were classified as non-pregnant. According to this criterion, we anticipate that there will be 600 participants in the pregnant group and an equal number in the non-pregnant group. Detailed selection and screening process is shown in Fig 1 Exclusion criteria included the use of indwelling catheters, immunosuppressive agents, and antimicrobial drugs, as well as an inability to express symptoms. For a diagnosis of UTI, patients should present with recent-onset urinary tract irritative symptoms, which include increased frequency of urination, a sudden compelling need to urinate, or painful urination. In addition to these clinical manifestations, the diagnostic criteria were satisfied by the fulfillment of at least one of the following objective parameters:

The detection of five or more leukocytes per high-power field (≥5/HPF) within the urine sediment.

A positive urine culture that confirms the presence of a single uropathogen with a concentration of 104 or more colony-forming units per milliliter (≥104 CFU/mL).

 10.1371/journal.pone.0308253.g001 Overview of screening and selection process. Urinalysis

A 20mL midstream urine sample was randomly collected, mixed evenly, and then divided equally into two tubes, with each tube containing 10 mL. One tube was used for the urine bacterial culture and urinary sediment examination, and the other tube was used for UF.

 Microbiological examination

10 microliters of non-centrifuged urine were placed on blood agar plates, and the results were observed after 24 hours. A culture result was deemed positive in case of growth of 104 CFU/mL or more and defined as a monoculture if 90% or more of the cultured colonies were of 1 microorganism. The primary pathogens isolated from the urinary tract are predominantly Escherichia coli, Enterococcus species, Klebsiella spp., and Proteus mirabilis. The growth of more than three bacterial species on the agar plate was indicative of contamination.

 Microscopic examination

The remaining urine specimens were centrifuged in a horizontal centrifuge at 400g for 5 minutes. The supernatant was discarded, and the remaining sediment was mixed. Next, 0.2 mL of the urine sediment was collected using a pipette and placed on a glass slide, which was then covered with an 18 × 18 mm coverslip. Under a light microscope, the number of white blood cells (WBCs) and epithelial cells (ECs) were counted at high magnification for 10 consecutive visual fields, and the average number per visual field was calculated. Each sample was counted by two veteran morphologists under double-blind conditions. For the microscopic method, the following diagnostic cut-off values were used for WBCs and ECs: negative, ≤5/HPF; positive, >5/HPF.

 Urine flow cytometry

UF was conducted utilizing the Sysmex UF-5000 analyzer (Sysmex Corporation, Kobe, Japan). The Sysmex UF-5000’s initial settings for the compensated forward scatter (CW-FSC-P), side scatter height (CW-SSH-P), fluorescence height (CW-FLH-P), and differential side scatter (CW-DSS-P) parameters within the coefficient ratio (CR) for the WBCs were established at 0.88, 1551, 2428, and 2600, respectively. Subsequently, an assessment of the UF-5000’s diagnostic performance was executed with a range of CW-FSC-P Gain values, specifically at 0.78, 0.98, 1.08, and 1.18, under the condition that all other parameters remained unchanged. The Gain value was adjusted mainly according to the change of Youden index. In the UF5000 analyzer, the initial setting for the gain value was established at 0.88. Upon adjusting the gain value to 0.78, we observed an increase in the false positive rate and a concurrent decrease in the Youden index. This finding indicated that reducing the gain value did not effectively lower the false positive rate of the UF5000. Subsequently, we incrementally increased the gain value to 0.98, 1.08, and 1.18. The selection of urine samples for analysis was predicated on predefined thresholds, where samples with a WBC count ≥25/μL and an epithelial cell (EC) count of 31 or higher per microliter (≥31/μL) were categorized as positive. Samples that did not meet these thresholds were designated as negative.

 Statistical analysis

Statistical analysis was conducted using SPSS version 26 (SPSS, Inc., Chicago, USA). The data were presented as the number of cases (n), percentages, and means with standard deviations. Microscopic examination was used as the gold standard for diagnosis in this study. When a sample was positive by other tests but negative by microscopy, it was called a false positive. Conversely, when a sample was negative by other tests but positive by microscopy, this was called a false negative. The positive rates of UTIs between pregnant and non-pregnant women were compared using chi-square tests. WBC results from false positive samples and ECs results from false negative samples, as determined by the UF-5000 and the microscopic method, were compared utilizing Pearson’s correlation coefficient. The receiver operating characteristic (ROC) curves were plotted using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, California, USA) to assess the diagnostic performance of the index tests with different Gain values for CW-FSC-P. The area under the curve (AUC) was calculated to determine the discriminatory power of these tests. The optimal cutoff values, balancing sensitivity and specificity, were identified using the Youden index. For all analyses, P < 0.05 was defined as statistically significant.

 Results

From an initial screening of 1,200 participants, 1,121 individuals met the inclusion criteria and were categorized into two groups: the pregnancy group with 545 participants and the non-pregnancy group with 576 participants (Fig 1). A comprehensive summary of the baseline characteristics for the study groups is presented in Table 1. The groups were found to be demographically similar with respect to age, representation of certain ethnic groups (Han, Korean, Mongolian), and prevalence of hypertension. However, significant disparities were observed between the two groups in terms of other ethnic backgrounds (Manchu, Hui, Xibe, and Daur), smoking habits, alcohol consumption, and incidence of diabetes. Notably, the pregnancy group reported no cases of alcohol consumption, in contrast to 45 instances in the non-pregnancy group.

10.1371/journal.pone.0308253.t001 Baseline characteristics.
Pregnancy group (n = 545) Non-pregnancy group (n = 576)
Age (Years) 31.38 (4.29) 33.09(3.98)
Gestational age (Weeks) 29.02 (8.20) --
Race(n)
    Han 514 532
    Korean 11 14
    Manchu 8 13
    Mongolian 5 6
    Hui 5 11
    Xibo 1 0
    Daur 1 0
Smoking habit (n) 1 11
Alcohol consumption (n) 0 45
Diabetes mellitus(n) 92 51
Hypertension(n) 54 59
The prevalence of UTIs among pregnant women is observed to be higher than that among non-pregnant women

Clinical diagnosis within the pregnant cohort revealed a significantly higher rate of UTIs (40.91%), when juxtaposed with a 20.26% rate observed in the non-pregnant cohort (P < 0.001), as detailed in Table 2. UF demonstrated a positive rate for WBCs of 41.83% in the pregnant group, which was markedly higher than the 20.16% rate in the non-pregnant group, with this discrepancy being statistically significant (P < 0.001). These findings are congruent with clinical diagnoses and underscore the diagnostic value of WBC presence in urine for UTIs. However, urine microscopy, which is traditionally considered the gold standard for urine sediment analysis, showed a positive rate for WBCs of 22.75% in the pregnant group and 19.51% in the non-pregnant group, indicating no significant difference between the two cohorts (P > 0.05) (Table 2). These findings suggest that the increased rate of UTI diagnosis in pregnant women could be attributable to diagnostic misattribution, potentially associated with the outcomes of the UF examination. This hypothesis is supported by the data presented in Table 3, which shows that UF produced 126 false positives for WBC detection in the pregnant group, corresponding to a false positive rate of 30.43%. This indicates that the elevated incidence rate of UTIs in pregnant women may be a result of false positives during WBC detection by UF, consequently leading to clinical misdiagnosis. The diagnostic accuracy of UF in the context of UTIs within the pregnant population, as illustrated in Fig 2A, was inferior to that in the non-pregnant population. Furthermore, Fig 2B demonstrates that UF is less effective than urine sediment microscopy examination, highlighting the suboptimal diagnostic efficacy of UF in this specific demographic.

10.1371/journal.pone.0308253.g002 Receiver operating characteristic (ROC) curves.

ROC curves for urine flow cytometry in diagnosing Urinary Tract Infections (UTIs) in both pregnancy and non-pregnancy groups (a). ROC curves for urine flow cytometry and the microscope method in diagnosing UTIs in pregnancy (b).

 10.1371/journal.pone.0308253.t002 Diagnosis of urinary tract infections in pregnant and non-pregnant women using different methods.
Positive rate (%) Negative rate (%)
Pregnancy group Non-pregnancy group P-value Pregnancy group Non-pregnancy group P-value
Clinical Diagnosis 40.91 20.26 <0.001 59.09 79.74 <0.001
urine flowcytometry 41.83 20.16 <0.001 58.17 79.84 <0.001
microscope 22.75 19.51 >0.05 77.25 80.49 >0.05
bacteriological culture 17.92 16.09 >0.05 82.08 83.91 >0.05
10.1371/journal.pone.0308253.t003 The performance of urine flowcytometry (UF) and microscopic method in screening urinary WBC and EC of pregnant women.
Positive for both UF and microscopic (n) Negative for both UF and microscopic (n) UF positive/microscopic negative (n) UF negative/microscopic positive (n) Positive predictive value (%) False positive* rate (%) Negative predictive value (%) False negative# rate (%)
WBC 102 288 126 29 44.73 30.43 90.85 22.13
EC 257 139 19 130 93.11 14.84 51.67 33.59

*False positive: Positive by urine flowcytometry but negative by microscopy.

#False negative: Negative by urine flowcytometry but positive by microscopy.

 Misdiagnosis potentially stems from urine sediment analyzer misidentifying epithelial cells as white blood cells

UF has been found to identify 130 instances of false negatives in the detection of ECs within urine samples of pregnant women, resulting in a false negative rate of 33.59%. This rate is notably similar to the false positive rate for WBCs, which is 30.43%, as shown in Table 3. This congruence suggests that there may be a correlation between the false positive rates for WBCs and false negative rates for ECs in UF detection. To empirically examine this hypothesis, a linear correlation analysis was conducted. This study assessed the relationship between the number of WBCs detected by UF and the corresponding count of ECs observed through microscopy in a sample of 126 cases that resulted in false positive WBC readings (Fig 3A). The analysis produced a correlation coefficient of r = 0.63 (P < 0.0001), indicating a significant positive correlation. In parallel, another linear correlation analysis was executed to evaluate the relationship between the number of ECs observed microscopically and the number of WBCs identified by UF in 130 cases that were false negatives for ECs (Fig 3B). This analysis yielded a correlation coefficient of r = 0.53 (P < 0.0001), also denoting a significant positive correlation. The results of these analyses collectively suggest that the misattribution of ECs as WBCs by UF may be a contributing factor to the elevated false positive rate for WBCs in urinalysis of pregnant women.

10.1371/journal.pone.0308253.g003 Correlation analysis.

Correlations between the number of WBCs detected by UF and the corresponding number of epithelial cells observed microscopically in 126 cases of false positive WBCs samples (a). Correlations between the number of epithelial cells observed microscopically and the number of WBCs detected by UF in 130 cases of false negative epithelial cells samples (b).

 Adjusting the CR(WBC)-CW-FSC-P Gain value of UF reduces the misdiagnosis rate of UTIs in pregnant women

The elevated rate of false positives may correlate with the utilization of suboptimal settings for the instrument’s parameters. A potential resolution to the inflated false positive rate, which arises from the misclassification of epithelial cells as WBCs by UF, involves the recalibration of the detection parameters. Post-adjustment of the CR(WBC)-CW-FSC-P Gain value on the UF instrument, the consequent enhancement in the analyzer’s efficacy for the detection of urinary WBCs in pregnant women is depicted in Fig 4.

10.1371/journal.pone.0308253.g004 The performance of urine flow cytometry in screening for urinary WBCs in pregnant women among different CR(WBC)-CW-FSC-P Gain values.

Our analysis revealed that the AUC, a metric for diagnostic efficacy, escalates with an increase in the Gain value, attaining an apex of 0.91 at a Gain value of 1.08, beyond which there is a subsequent decline. Table 4 provides a detailed breakdown of the false positive rate for WBCs in relation to various CR(WBC)-CW-FSC-P Gain values. Notably, upon setting the Gain value to 1.08, the false positive rate for WBCs was substantially reduced to 9.45%, concomitant with an optimal Youden’s index of 65.33%.

10.1371/journal.pone.0308253.t004 The performance of urine flow cytometry among different CR(WBC)-CW-FSC-P Gain values in screening urinary WBC of pregnant women.
Gain values
0.78 0.88 0.98 1.08 1.18
False positive rate (%) 35.13 30.43 18.91 9.45 6.75
False negative rate (%) 21.25 22.13 25.58 27.9 44.18
Youden index (%) 45.88 55.92 57.45 65.33 53.71

These findings indicate that by precisely adjusting the CR(WBC)-CW-FSC-P Gain value on the UF instrument, it is possible to significantly reduce the incidence of false positives for WBCs, thereby decreasing the likelihood of misdiagnosing UTIs in pregnant women. Specifically, when the CR(WBC)-CW-FSC-P Gain value was fine-tuned to 1.08, the diagnostic accuracy of UF for UTIs in this demographic was maximized.

 Discussion

This study elucidated that the increased prevalence of UTIs among gravidae compared to non-pregnant females might be attributable to diagnostic errors. Subsequent investigations have indicated that these inaccuracies may originate from an elevated rate of false positives when utilizing UF to ascertain the presence of urinary WBCs in pregnant individuals. By calibrating the coefficient ratio (CR) of WBCs to the compensated forward scatter (CW-FSC-P) gain value within the UF protocol, the incidence of false positives during the detection of urinary WBCs in gravidae could be markedly diminished. Consequently, this adjustment could reduce the clinical misdiagnosis rate of UTIs among pregnant women. The implications of this discovery hold significant potential for clinical practice, particularly in enhancing the precision of UTI diagnoses within the obstetric population.

This study presents a pivotal finding concerning the elevated false-positive rate for UTIs in pregnant women, which was as high as 30.43% when employing UF. This heightened rate is likely due to the analyzer’s tendency to erroneously classify ECs as WBCs, a phenomenon supported by the comparable false-negative rate for ECs and a notable linear correlation between the counts of these two cell types. López et al. [11] previously highlighted that UF exhibits a significant rate of false positives in the diagnosis of UTIs among pregnant women, aligning with the current study’s observations. Notably, the research conducted by Kim et al. [12] also suggests that UF has a high false-positive rate in diagnosing UTIs within the general population. Incorrect diagnosis of UTIs carries significant implications for both individual patients and the healthcare system as a whole. The overdiagnosis of UTIs, particularly in cases of false positives, can lead to unnecessary antibiotic prescriptions. This overuse of antibiotics contributes to the growing problem of antibiotic resistance, where bacteria evolve to become less susceptible to the effects of antibiotics, making infections more difficult to treat [13, 14]. Such misdiagnosis may engender unwarranted anxiety among patients, particularly when they undergo additional, unrequired testing and therapeutic interventions under the erroneous presumption of a UTI. This scenario can engender discomfort and potential iatrogenic harm due to the imposition of superfluous medical procedures and pharmacological agents. Furthermore, misdiagnosis can precipitate augmented healthcare expenditures, attributable to the costs of superfluous diagnostics, therapeutics, and hospital stays, imposing a significant financial strain on both the patient and the healthcare infrastructure. The erosion of patient trust in healthcare providers and the medical establishment is another critical consequence of incorrect diagnoses. Broadly, the misdiagnosis of UTIs can impede public health initiatives focused on the containment of infectious diseases and the advocacy of judicious antibiotic stewardship. The precision of diagnostic practices is indispensable for the efficacious surveillance and management of UTIs within the population. It is imperative to address these challenges through rigorous clinical governance, enhanced diagnostic protocols, and the promotion of antibiotic resistance mitigation strategies. Consequently, there is an imperative need for a diagnostic approach that can diminish the false-positive rate associated with UF and enhance its diagnostic precision, thereby mitigating the misdiagnosis rate of UTIs in pregnant women. Bilsen et al. [15] identified that the current pyuria thresholds are set too low, leading to an inappropriate diagnosis of UTIs in older women. By elevating these thresholds, they demonstrated a reduction in the misdiagnosis rate of UTIs among the elderly. However, the entrenched practice of utilizing current pyuria cutoffs for UTI diagnosis by physicians presents a challenge in altering this clinical approach. Therefore, the objective of this study was to augment the accuracy of UTI diagnosis in pregnant women without necessitating departure from the traditional pyuria cutoffs.

This study provides an in-depth analysis of the diagnostic markers for UTIs, with a particular focus on the utility of UF in different patient demographics. In the field of medical diagnostics, UF, as a relatively new technique, has been extensively applied in the clinical diagnosis of UTIs. Compared to conventional methods, UF offers distinct advantages, including high-throughput processing that facilitates the simultaneous analysis of multiple samples with rapid turnaround times. This technique is adept at swiftly identifying critical urinary components such as leukocytes, erythrocytes, and epithelial cells, thereby conferring a high degree of sensitivity and specificity in the diagnosis of UTIs. The automation inherent in UF significantly diminishes human operational errors, enhancing the uniformity and dependability of the test results. However, UF is not without its drawbacks. The substantial costs associated with the equipment and requisite reagents can be prohibitive, potentially precluding its adoption in settings with economic constraints. The traditional criterion of WBC count exceeding 5/HPF in urine remains a prevalent indicator for UTIs, despite the superior accuracy offered by urine bacterial culture, which is considered the gold standard. However, the requirement for a substantial time investment and the susceptibility to false negatives make the latter less suitable for rapid clinical diagnosis [16–18].

Our research findings, corroborated by the analysis of ROC curves, revealed that the diagnostic efficacy of UF is attenuated in pregnant women when compared to non-pregnant women. Nonetheless, the WBC count criterion of greater than 5/HPF in urine maintains substantial diagnostic relevance for UTIs in non-pregnant women. The heightened sensitivity of UF could be a contributing factor to the elevated false-positive rate [19, 20]. Modulating the sensitivity of UF presents a potential strategy to mitigate the false-positive rate of WBC detection, thereby reducing the misdiagnosis rate of UTIs in pregnant women, without the need to modify existing cutoffs. Considering the superior diagnostic performance of UF in non-pregnant women with UTIs and its diminished effectiveness in pregnant women, it is plausible that the analyzer’s parameters necessitate fine-tuning for distinct patient populations. By adjusting the Gain value of CR(WBC)-CW-FSC-P, we have successfully achieved a marked reduction in the false-positive rate for WBC detection in pregnant women. This modification has led to an enhanced Youden’s index, signifying an improved equilibrium between sensitivity and specificity, and consequently, a more precise diagnosis of UTIs within this demographic.

Our results indicate that a Gain value of 1.08 is optimal for minimizing the false-positive rate and maximizing the diagnostic efficacy of UF in the detection of UTIs in pregnant women. This targeted optimization offers a straightforward and efficacious solution to enhance the accuracy of UTI diagnosis during pregnancy, thereby potentially improving patient outcomes and reducing unnecessary antibiotic use.

Limitations and future research

The sample size, though substantial, may not be fully representative of all pregnant women, and the study was conducted at a single medical center. Future research should aim to validate our findings in a larger population and across multiple centers. Additionally, the long-term clinical outcomes associated with the misdiagnosis of UTIs in pregnancy warrant further investigation.

 Conclusions

In conclusion, our study underscores the possibility that the incidence of UTIs among pregnant women is potentially overstated due to the inherent limitations of UF. Through the identification of the misclassification of ECs as WBCs as a contributing factor, and by proposing a refinement in the parameters of the UF analyzer, we have delineated a methodological approach to mitigate the occurrence of misdiagnosis. This adjustment not only enhances the diagnostic accuracy of UF but also has significant implications for optimizing clinical management and reducing unnecessary treatment in this patient population.

 Supporting information Correlations between age, gestational age, red blood cells, white blood cells, yeast, bacteria, epithelium.

(DOCX)

 References Liu L, Oza S, Hogan D, Perin J, Rudan I, Lawn JE, et al. Global, regional, and national causes of child mortality in 2000–13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet. 2015;385(9966):430–40. doi: 10.1016/S0140-6736(14)61698-6 25280870 Werter DE, Kazemier BM, Schneeberger C, Mol BWJ, de Groot CJM, Geerlings SE, et al. Risk Indicators for Urinary Tract Infections in Low Risk Pregnancy and the Subsequent Risk of Preterm Birth. Antibiotics (Basel). 2021;10(9). doi: 10.3390/antibiotics10091055 34572637 Kwok M, McGeorge S, Mayer-Coverdale J, Graves B, Paterson DL, Harris PNA, et al. Guideline of guidelines: management of recurrent urinary tract infections in women. BJU Int. 2022;130 Suppl 3(Suppl 3):11–22. doi: 10.1111/bju.15756 35579121 Wallin HP, Gissler M, Korhonen PE, Ekblad MO. New insights into smoking and urinary tract infections during pregnancy using pregnancy-pair design: A population-based register study. Acta Obstet Gynecol Scand. 2023;102(1):25–32. doi: 10.1111/aogs.14473 36263583 Johnson CY, Rocheleau CM, Howley MM, Chiu SK, Arnold KE, Ailes EC. Characteristics of Women with Urinary Tract Infection in Pregnancy. J Womens Health (Larchmt). 2021;30(11):1556–64. doi: 10.1089/jwh.2020.8946 34491115 Ansaldi Y, Martinez de Tejada Weber B. Urinary tract infections in pregnancy. Clin Microbiol Infect. 2023;29(10):1249–53. doi: 10.1016/j.cmi.2022.08.015 36031053 Ebell MH, Gagyor I. Diagnosis of Urinary Tract Infection in Women. Am Fam Physician. 2022;106(3):335–6. 36126022 Oyaert M, Maghari S, Speeckaert M, Delanghe J. Improving clinical performance of urine sediment analysis by implementation of intelligent verification criteria. Clin Chem Lab Med. 2022;60(11):1772–9. doi: 10.1515/cclm-2022-0617 36069776 Wang T, Wu G, Wang J, Cui Y, Ma J, Zhu Z, et al. Comparison of single-dose fosfomycin tromethamine and other antibiotics for lower uncomplicated urinary tract infection in women and asymptomatic bacteriuria in pregnant women: A systematic review and meta-analysis. Int J Antimicrob Agents. 2020;56(1):106018. doi: 10.1016/j.ijantimicag.2020.106018 32417205 Yang W, Yu X, Liu D, Guan X. High false positive rate of white blood cells in urine samples of pregnant women may be caused by epithelial cells being misclassified by the sysmex UF-1000i urine flow cytometer. Cytometry B Clin Cytom. 2019;96(6):464–8. doi: 10.1002/cyto.b.21727 30302910 Angulo Lopez I, Urrutikoetxea-Gutierrez M, Aragon-Diez J, Fraca Padilla M, Diaz de Tuesta Del Arco JL. [Evaluation of Sysmex UF-1000i(R) flow cytometer as a screening method for asymptomatic bacteriuria and detection of Group B Streptococcus in pregnancy]. Rev Esp Quimioter. 2020;33(3):193–9. Kim SY, Park Y, Kim H, Kim J, Koo SH, Kwon GC. Rapid Screening of Urinary Tract Infection and Discrimination of Gram-Positive and Gram-Negative Bacteria by Automated Flow Cytometric Analysis Using Sysmex UF-5000. J Clin Microbiol. 2018;56(8). doi: 10.1128/JCM.02004-17 29769277 Byron JK. Urinary Tract Infection. Vet Clin North Am Small Anim Pract. 2019;49(2):211–21. doi: 10.1016/j.cvsm.2018.11.005 30591189 Smaill FM, Vazquez JC. Antibiotics for asymptomatic bacteriuria in pregnancy. Cochrane Database Syst Rev. 2019;2019(11). doi: 10.1002/14651858.CD000490.pub4 31765489 Bilsen MP, Aantjes MJ, van Andel E, Stalenhoef JE, van Nieuwkoop C, Leyten EMS, et al. Current Pyuria Cutoffs Promote Inappropriate Urinary Tract Infection Diagnosis in Older Women. Clin Infect Dis. 2023;76(12):2070–6. doi: 10.1093/cid/ciad099 36806580 Iezzi P, Cappellini F, Intra J, Carnicelli S, Fossati L, Basta F, et al. The diagnostic performances of the Atellica(R) 1500 automated urinalysis system for ruling out bacterial urinary tract infection. Clin Chim Acta. 2023;548:117494. Karakukcu C, Kayman T, Ozturk A, Torun YA. Analytic performance of bacteriuria and leukocyturia obtained by UriSed in culture positive urinary tract infections. Clin Lab. 2012;58(1–2):107–11. 22372352 Allen KJ, Leslie SW. Autonomic Dysreflexia. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Stephen Leslie declares no relevant financial relationships with ineligible companies.2024. Klarkowski D, O’Brien DP, Shanks L, Singh KP. Causes of false-positive HIV rapid diagnostic test results. Expert Rev Anti Infect Ther. 2014;12(1):49–62. doi: 10.1586/14787210.2014.866516 24404993 Kobayashi R, Murai R, Moriai M, Nirasawa S, Yonezawa H, Kondoh T, et al. Evaluation of false positives in the SARS-CoV-2 quantitative antigen test. J Infect Chemother. 2021;27(10):1477–81. doi: 10.1016/j.jiac.2021.06.019 34238663 10.1371/journal.pone.0308253.r001 Decision Letter 0 Domfeh Seth Agyei Academic Editor 2024 Seth Agyei Domfeh This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

21 Jun 2024

PONE-D-24-20393

Reduction of misdiagnosis in urinary tract infections during pregnancy: the role of adjusted urine flow cytometry parameters

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Reviewer #1: The idea of the study is a very promising one. Indeed, there are a lot of false positive and false negative results. For the diagnosis of urinary tract infection both in pregnant woman and in the female population without this physiological stage.

The 2 study groups were correctly chosen. I appreciate the fact that the urine samples were examined double blind by people with special experience. However, despite the design of the study, the idea is at a very beginning because it does not elaborate a clear algorithm for a single person in the conditions of consultation for urinary infection, regardless of the person's pregnancy status or not.

For this I belive that a much larger, possibly multicenter study is necessary that includes a major cohort from both groups that allows for some relevant conclusions and the individualization for each individual of what is of interest to clinicians.

The bias in this study may also come from the fact that twice as many patients with diabetes are included in the cohort of pregnant women than in the other cohort.

Therefore, the study could be of interest in the conditions of a large cohort that can this externalize the minuses or the interpretation deficiency.

Even in these conditions, the clinician will decide on antibiotic therapy or not for each individual case, including in the clinical judgement several element, not only age and pregnancy status.

The problem being a particularly serious one and on the edge and on one side and the other of the problem in the conditions in which you make a mistake by excess antibiotic therapy when it is not needed or in the minus with the risk of sepsis with urinary entrance in the conditions of physiological immunosuppression during pregnancy.

Reviewer #2: This is a study conducted in one center from China and reporting on how to improve urine flow cytometry parameters for detecting leukocyturia in pregnancy. The data are important as they discuss potential erroneous findings that might lead to inappropriate treatment. However the current manuscript is limited by the insufficient description of participant selection and by the unclarities with regards to UTI versus leukocyturia. Specifically, the authors often describe the presence of leukocyturia as indicative for UTI which may not always be the case. The authors should also consider presenting the diagnostic performance of flow cytometry separately for those who have bacteriologically confirmed UTI and those who do not. Further, the discussion would benefit from a more in-depth reflection on the benefits of using this diagnostic test and the implications of misdiagnosis in this patient population. More specific comments below.

Introduction

- Paragraph 1: suggest leaving out the mortality due to preterm births as the contribution of UTIs to this is not that substantial. The authors could just leave the association with pregnancy complications

- Paragraph 2: I would not expect that pregnant women would have much more difficulties in collecting a urine sample than any young adult (I expect it would be much more difficult in the elderly) – consider rephrasing

Methods:

- Provide more details on how the study participants were selected. Given that they were evenly split into the two groups, I assume that they were not included consecutively

- If the study aims to evaluate diagnostic accuracy of UF (as suggested by calculating sensitivities and specificities), then UF should not be included in the definition of the condition (criterion #3)

- Microbiological assessment: please be more specific with regards to which bacteria had to be isolated – the bacteria would have to be a uropathogen and there would be one/few bacterial species growing on the plate (otherwise it would be considered contaminated)

- The subheading “microbiological examination” has a typographical error

- Statistics: would the authors be able to clarify in the methods how false positives and false negatives were defined

- Please clarify in the manuscript text how the different gain values were selected

Results

- The authors should explain why leukocyturia in the absence of bacterial growth in culture was considered as UTI. This should be rephrased, as leukocyturia in the absence of infection can occur in pregnancy. This comment also applies to the methods

- The variables should have complete names (e.g. instead of microscope WBC>… ). Also the top information should be filled (replace “+” and “-“ by description)

- “Parallel, urine bacterial culture, another gold standard diagnostic method for UTIs, exhibited no significant difference in positive rates between pregnant (17.92%) and non-pregnant women (16.09%) (P > 0.05)” consider removing as this is not clinically meaningful

- Define in the footnote of table 3 false positives and negatives

Discussion

- How do the authors explain the low proportion of positive urine cultures among the study participants given that in other settings bacterial cultures are often positive among patients with UTI symptoms. Please discuss

- Discuss in more detail the implications of incorrect UTI diagnosis

- Discuss the advantages and disadvantages of using flow cytometry over more conventional methods for diagnosing UTIs (high throughput vs cost)

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Reviewer #1: No

Reviewer #2: No

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 10.1371/journal.pone.0308253.r002 Author response to Decision Letter 0 Submission Version 1

30 Jun 2024

Response letter

Dear editors and reviewers:

Thank you for your letter and for the reviewers' comments concerning our manuscript entitled “Reduction of misdiagnosis in urinary tract infections during pregnancy: the role of adjusted urine flow cytometry parameters” (ID: PONE-D-24-20393). Those comments are all valuable and very helpful for revising and improving our paper, as well as important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in manuscript (Tracked Changes). The main corrections in the paper and the responds to the reviewer's comments are as following:

Response to Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdfand https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: Special thanks to you for your comments. We made modifications and ensure that our manuscript meets PLOS ONE's style requirements, including those for file naming.

2. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods…….

Response: Thanks for your comments. We confirm that our submission contains all raw data required to replicate the results of our study. We had uploaded all raw data as Supporting Information files.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

Response: Thanks for your comments. I have an ORCID iD and that it is validated in Editorial Manager.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Response: Special thanks to you for your suggestion, and we have made correction according to the reviewer's suggestion.

5. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Response: Thanks for your suggestion, and we have made correction according to your suggestion.

Response to Review Comments to the Author:

Response to Reviewer #1

Thank you for your insightful and constructive comments on our manuscript. We have taken your feedback seriously and have made the following revisions and clarifications to address your concerns:

1. Promising Study Idea: We are grateful for your recognition of the potential of our study. We acknowledge the prevalence of false positives and negatives in the diagnosis of urinary tract infections (UTIs) and believe our research contributes to addressing this issue.

2. Study Design and Algorithm Development: You are correct in noting that our study does not yet provide a clear diagnostic algorithm. In response, we are planning a more extensive study that will focus on developing a robust algorithm for UTI diagnosis, tailored to individual patient needs, regardless of pregnancy status.

3. Need for a Larger, Multicenter Study: We concur with your suggestion for a larger, possibly multicenter study. We are currently exploring collaborations to expand our research and include a significant cohort from both groups to provide more generalizable conclusions.

4. Bias Due to the Representation of Diabetic Patients: We have noted the potential bias you mentioned regarding the overrepresentation of diabetic patients in the pregnant cohort. In our subsequent studies, we will ensure a more balanced representation to mitigate this bias.

5. Clinical Decision-Making: We appreciate your emphasis on the complexity of clinical decision-making. Our study aims to provide clinicians with additional data points to inform their judgment on antibiotic therapy, considering a range of factors beyond just age and pregnancy status.

6. Risk of Sepsis and Antibiotic Overuse: We are acutely aware of the serious implications of both sepsis risk and antibiotic overuse. Our research is designed to contribute to a more nuanced understanding of UTI diagnosis and treatment, helping to avoid unnecessary antibiotic use while promptly identifying and treating genuine infections.

Response to Reviewer #2

We greatly appreciate the detailed and thoughtful feedback provided on our manuscript. Your comments have been instrumental in guiding our revisions, and we have addressed each point as follows:

Introduction:

1. Paragraph 1: suggest leaving out the mortality due to preterm births as the contribution of UTIs to this is not that substantial. The authors could just leave the association with pregnancy complications

Response: We have removed the reference to mortality due to preterm births, focusing instead on the association with pregnancy complications, as you suggested.

2. Paragraph 2: I would not expect that pregnant women would have much more difficulties in collecting a urine sample than any young adult (I expect it would be much more difficult in the elderly) – consider rephrasing

Response: Thanks for your suggestion. We have rephrased this section.

Methods:

3. Provide more details on how the study participants were selected. Given that they were evenly split into the two groups, I assume that they were not included consecutively

Response: Thanks for your comments. We have expanded the section on participant selection to provide a clearer description of the process and criteria used to distribute participants in Study population section.

4. If the study aims to evaluate diagnostic accuracy of UF (as suggested by calculating sensitivities and specificities), then UF should not be included in the definition of the condition (criterion #3)

Response: Special thanks to you for your suggestion. UF has been removed from the definition of the condition.

5. Microbiological assessment: please be more specific with regards to which bacteria had to be isolated – the bacteria would have to be a uropathogen and there would be one/few bacterial species growing on the plate (otherwise it would be considered contaminated)

Response: Thanks for your comments. For microbiological assessment, we have specified the criteria for uropathogens and clarified the conditions under which a culture would be considered contaminated in Microbiological examination section.

6. The subheading “microbiological examination” has a typographical error

Response: The typographical error under the subheading "microbiological examination" has been corrected

7. Statistics: would the authors be able to clarify in the methods how false positives and false negatives were defined

Response: Thanks for your comments. We have added the definition to the statistics section. In our study, we used microscopic examination as the gold standard for diagnosis. When a sample is positive by other tests but negative by microscopy, it is called a false positive. Conversely, when a sample is negative by other tests but positive by microscopy, this is called a false negative. We have added the above definition to the statistics section.

8. Please clarify in the manuscript text how the different gain values were selected

Response: We have added an explanation of how different gain values were selected in the manuscript text. In the UF5000 analyzer, the initial setting for the gain value was established at 0.88. Upon adjusting the gain value to 0.78, we observed an increase in the false positive rate and a concurrent decrease in the Youden index. This finding indicated that reducing the gain value did not effectively lower the false positive rate of the UF5000. Subsequently, we incrementally increased the gain value to 0.98, 1.08, and 1.18. It was upon setting the gain value to 1.08 that a significant reduction in the false positive rate was achieved, with the Youden index reaching its optimal value. Consequently, we concluded that a gain value of 1.08 represents the most effective setting for the UF5000 in detecting leukocytes in urine samples from pregnant women, thereby optimizing the balance between sensitivity and specificity.

Results

9. The authors should explain why leukocyturia in the absence of bacterial growth in culture was considered as UTI. This should be rephrased, as leukocyturia in the absence of infection can occur in pregnancy. This comment also applies to the methods

Response: Thanks for your comments. Initially, it is essential to acknowledge the inherent limitations of urinary bacterial culture as a diagnostic tool for UTIs. While it serves as a pivotal method for identifying UTIs, it does not possess absolute accuracy. Occasionally, the causative bacteria may be present in insufficient quantities within the urine sample collected, or they may be inherently recalcitrant to growth under standard culture conditions, leading to a false-negative culture result.

Furthermore, it is not uncommon for UTIs to be incited by a spectrum of pathogens that extend beyond the conventional cultivable bacteria. Certain atypical microorganisms, such as mycoplasmas, chlamydiae, and viruses, may elude detection by routine bacterial culture techniques despite their potential to incite urinary inflammation and leukocytosis.

Alternatively, urine contamination during collection, transportation, and processing may also affect the results of bacterial culture.

Lastly, the presence of leukocytes in the urine, indicated by a positive leukocyte test, is a significant marker of inflammation. Even in instances where the bacterial culture yields a negative result, a significant leukocyte count suggests an ongoing inflammatory response in the urinary tract.

10. The variables should have complete names (e.g. instead of microscope WBC>… ). Also the top information should be filled (replace “+” and “-“ by description)

Response: Thanks for your comments. Variable names” WBC-UF” have been revised to “Number of WBC detected by UF”, and “EC-Microscpe” have been revised to “Number of EC detected by microscopy” in Fig 3. “+” and “-“ have been replaced by “Positive rate” and “Negative rate” in Table 2.

11. “Parallel, urine bacterial culture, another gold standard diagnostic method for UTIs, exhibited no significant difference in positive rates between pregnant (17.92%) and non-pregnant women (16.09%) (P > 0.05)” consider removing as this is not clinically meaningful。

Response: Thanks for your advice. We have removed it according to your suggestion.

12. Define in the footnote of table 3 false positives and negatives

Response: Thank you for your valuable suggestions. Definitions for false positives and negatives have been included in the footnote of Table 3.

Discussion

13. How do the authors explain the low proportion of positive urine cultures among the study participants given that in other settings bacterial cultures are often positive among patients with UTI symptoms. Please discuss

Response: Thanks for your comments. The study by Kirk J Wojno et al.[1] reported a positive urinary bacterial culture rate of 37% among patients exhibiting symptoms of lower UTIs. This rate is indeed higher than that observed in our study. However, it is essential to note the demographic differences between the two studies. Wojno's research was conducted on patients who presented with symptoms of lower UTIs, while our study population consisted of individuals visiting a gynecological outpatient clinic, half of whom were there for routine prenatal care and may not have had UTI symptoms. In the remaining half of the study population, although some individuals sought medical attention due to UTI symptoms, a significant number were present for various other pathologies, such as uterine fibroids, ovarian cysts, and menstrual irregularities. Consequently, not all participants in our study had UTI symptoms, which likely accounts for the lower positive rate in urinary bacterial cultures.

Additionally, previous research has indicated that the incidence of UTI during pregnancy ranges from 2.3% to 15% [2-4], which is consistent with the findings of our study.

14. Discuss in more detail the implications of incorrect UTI diagnosis

Response: Thanks for your suggestions. The implications of incorrect UTI diagnosis have been discussed in more detail.

15. Discuss the advantages and disadvantages of using flow cytometry over more conventional methods for diagnosing UTIs (high throughput vs cost)

Response: Thanks for your comments. We have expanded the discussion on the advantages and disadvantages of using flow cytometry for diagnosing UTIs considering factors such as high throughput and cost in comparison to the conventional methods.

We tried our best to improve the manuscript and made some changes in the manuscript. We appreciate for Editors/the reviewers' warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

Sincerely,

Wei Yang

1. Wojno KJ, Baunoch D, Luke N, Opel M, Korman H, Kelly C, et al. Multiplex PCR Based Urinary Tract Infection (UTI) Analysis Compared to Traditional Urine Culture in Identifying Significant Pathogens in Symptomatic Patients. Urology. 2020;136:119-26. Epub 2019/11/13. doi: 10.1016/j.urology.2019.10.018. PubMed PMID: 31715272.

2. Werter DE, Kazemier BM, Schneeberger C, Mol BWJ, de Groot CJM, Geerlings SE, et al. Risk Indicators for Urinary Tract Infections in Low Risk Pregnancy and the Subsequent Risk of Preterm Birth. Antibiotics (Basel). 2021;10(9). Epub 2021/09/29. doi: 10.3390/antibiotics10091055. PubMed PMID: 34572637; PubMed Central PMCID: PMCPMC8468265.

3. Friedrich W. [Measures for age adequate adjustment of work conditions]. Z Gerontol. 1985;18(5):274-80. Epub 1985/09/01. PubMed PMID: 2933889.

4. Mazor-Dray E, Levy A, Schlaeffer F, Sheiner E. Maternal urinary tract infection: is it independently associated with adverse pregnancy outcome? J Matern Fetal Neonatal Med. 2009;22(2):124-8. Epub 2008/12/17. doi: 10.1080/14767050802488246. PubMed PMID: 19085630.

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pone.0308253.r003 Decision Letter 1 Domfeh Seth Agyei Academic Editor 2024 Seth Agyei Domfeh This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

22 Jul 2024

Reduction of misdiagnosis in urinary tract infections during pregnancy: the role of adjusted urine flow cytometry parameters

PONE-D-24-20393R1

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Kind regards,

Seth Agyei Domfeh, PhD

Academic Editor

PLOS ONE

 10.1371/journal.pone.0308253.r004 Acceptance letter Domfeh Seth Agyei Academic Editor 2024 Seth Agyei Domfeh This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

25 Jul 2024

PONE-D-24-20393R1

PLOS ONE

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