Introduction

Gestational diabetes mellitus (GDM) is defined as glucose intolerance of varying severity that occurs first time, or is first detected during pregnancy [1]. Prevalence of GDM is 7% of all pregnancies in the world [2]. GDM is one of the leading causes of morbidity and mortality in both mother and new-borns worldwide [3]. In India, its prevalence is found to vary in urban and rural areas. However, a recent study by Choudhary et al reported a prevalence of 9% in India [4].GDM is commonly associated with infections of the urinary tract.

Asymptomatic bacteriuria (ASB) refers to the presence of bacteria in the urine without any symptoms of a UTI, while pyuria is the presence of an elevated number of white blood cells in the urine, indicating potential inflammation or infection in the urinary tract. Both conditions are important to consider when evaluating urinary tract health and may require further investigation and management, depending on the individual's clinical characteristics and risk factors.

Urine culture is considered the gold standard for diagnosing ASB. It involves incubating urine samples on culture media to detect bacterial growth. The presence of a significant colony count of a single uropathogenic organism typically indicates ASB. Urine culture is generally highly sensitive, capable of detecting low bacterial loads, and is considered the benchmark for assessing sensitivity of other techniques. The specificity of urine culture is also high as it can differentiate between pathogenic and commensal bacteria.

However, urine culture has some limitations. It is time-consuming, usually requiring 24 to 48 hours for results. Additionally, some fastidious bacteria may not grow on standard culture media, leading to false negatives. Moreover, it may not be practical in certain settings, such as point-of-care testing.

Heparin-binding protein (HBP) is a biomarker associated with inflammation and tissue damage. ELISA-based assays can measure the concentration of HBP in urine samples, which may indicate the presence of inflammation in the urinary tract. Specificity for UHBP has been reported to be higher than sensitivity in some studies, suggesting that false positives might be less frequent. One limitation of UHBP measurement is that elevated levels can be seen in other inflammatory conditions, not specific to ASB. Therefore, the diagnostic accuracy of UHBP needs to be carefully assessed.

Multiplex PCR is a molecular technique that can amplify and detect DNA sequences of multiple bacterial species simultaneously. It is highly sensitive and can detect even low bacterial loads.

Urinary tract infections (UTI) are one of the most common infections resulting in a significant healthcare expenditure. Pregnant women because of the physiological changes are highly susceptible to UTI [5]. During pregnancy, the treatment of UTI is recommended even if there no accompanying symptoms, i.e., Asymptomatic bacteriuria (ASB).

It has been claimed ASB is three to four times more common in women with diabetes than in healthy women [6]. Prevalence of ASB is reported to be as high as 30% in diabetic women [7]. ASB is considered clinically significant and worth treating during pregnancy because treatment will effectively reduce the risk of pyelonephritis and preterm delivery [8]. UTI are dependent on the accurate identification of pathogens. Even though urine culture is the gold standard test, it takes 24-48 hours to give results. Some organisms can be fastidious and therefore difficult to grow in culture. So it is need of the hour to look for an alternative urinary biomarker which correlates with the urine culture results. It is also important to look for a technique which gives accurate results in a short time interval. Further, PCR results can be obtained in a day or less, while culture can require 2 or more days [9]. In the present study, we compared three different methods such as urine culture, Urinary Heparin Binding Protein (UHBP) assay by ELISA and identification of pathogens by multiplex PCR for the diagnosis of ASB/UTI in GDM. Gestational diabetes, which is a combination of pregnancy and diabetes are at the high risk of ASB as well as UTI. Therefore, it is justifiable to detect a biomarker or technique which can detect ASB/UTI in a short span of time.

Objectives:

• To find the prevalence of asymptomatic bacteriuria/pyuria in gestational diabetes mellitus
• To find the association between asymptomatic bacteriuria/pyuria and urinary heparin binding protein (UHBP)
• To compare sensitivities and specificities of the techniques (urine culture and UHBP by ELISA and multiplex PCR)

Methods

Study design and participants:

We included 50 GDM patients diagnosed based on 75 gm oral GTT (OGTT) as per modified DIPSI criteria who are willing to participate in this observational study. Samples were collected from OBG department (OP & IP) from Justice K. S. Hegde Charitable Hospital, Deralakatte, Mangalore and the study was conducted in Microbiology and Molecular genetics laboratory of Central Research Lab, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangaluru, Karnataka, India.

Exclusion criteria and Ethics: Women with multiple pregnancy, known pre-gestational diabetes, pregnancies complicated by major foetal malformations or known major cardiac, renal or hepatic disorders were excluded.

Institutional Ethics Committee approval was obtained prior to the study. Written Informed Consents were taken from the patients

Laboratory Investigations

Urine culture

Clean catch midstream urine samples were collected into a sterile screw capped urine container by standard method. The samples were labelled and 0.2 mg of boric acid was added to prevent the bacterial growth in urine samples. The samples were cultured on cysteine-lactose electrolyte deficient agar and blood agar using a sterile 4 mm platinum wired calibrated loop for the isolation of microorganisms. The plates were incubated for overnight at 37 °C and the samples were considered positive when an organism cultured is at a concentration of 10 ⁵ CFU/mL which was estimated through multiplying the isolated colonies by 1000. The isolates were identified up to the species level by standard biochemical tests.

Antibiotic sensitivity testing:

Antibiotic sensitivity testing was performed by the modified disc diffusion method. Inoculums adjusted to 0.5 McFarland standard was swabbed on Mueller Hinton agar plates for antibiotic sensitivity assay. Eight groups of antimicrobials such as Amoxyclav, Cephalexin, Nitrofurantoin, Erythromycin, Sulfisoxazole, Fosfomycin, Imipenem and Trimethoprim / Sulfamethoxazole were selected and sensitivity pattern was tested.

Estimation of UHBP by ELISA

Tubes were centrifuged within 1 hour of sampling at 3000 rpm for 10 minutes, and separate aliquots of the supernatant were stored at -20°C until analysis for UHBP. UHBP was estimated using ELISA.

Characterization of Pathogen by Multiplex PCR

DNA Extraction and Analysis

DNA was extracted from urine samples using the MagMAX DNA Multi-Sample Ultra Kit. One ml of urine sample was added into 2.0ml collection tube. Centrifuged for 2 minutes at 13,000 rpm. The supernatant was discarded completely. The pellet obtained was resuspended using 200μl of Resuspended Solution (1X PBS) (ML116) and mixed well. 20μl of RNase A solution was added, mixed and incubated for 2 minutes at room temperature (15-25ºC). Cell lysis was carried out by adding 20μl of Proteinase K solution followed by 200μl of Lysis solution to the re-suspended pellet. Mixed for 15 minutes using a Vertex. Incubated for 10 minutes at 70ºC. 200μl of ethanol (96-100%) was added to the lysate and mixed with help of vertex for 5 to 10 seconds. Lysate obtained was transferred to the HiElute Miniprep Spin Column. Centrifuged at 10,000 rpm for 1 minute. The flow through liquid was discarded and the column was placed in a same 2.0ml collection tube. About 500μl of diluted wash solution was added to the column and centrifuged at 10,000 rpm for 1 minute. The flow- through liquid was discarded and the same collection tube was re-used with the column. Again 500μl of diluted wash solution was added to the column and centrifuge at 13,000 for 3 minutes to dry the column. The column was centrifuged for another 1 minute at same speed to dry the residual ethanol (as required). 200μl of Elution buffer was added directly onto the column, incubated for 1 minute at room temperature (15-25ºC). Centrifuged at 10,000 rpm for 1 minute to elute the DNA. The isolated DNA was transferred to a fresh 2ml collection tube with a cap and stored -20ºC.

Multiplex PCR

HiMedia’s Hi-PCR Sepsis Pathogen Semi-Q PCR Kit (Multiplex) was used for the amplification of specific gene using specific primers in a single tube reaction. The PCR Master mix was prepared as indicated in the Table1 . Components listed in the table 1 were added to centrifuge tube and was centrifuged at 6000rpm for about 10 seconds. The tubes were placed in MiniAmp Plus Thermal cycler and PCR program was set as listed in the Table 2 . For analysis of the PCR data, 10μL of amplicon were loaded on a 1.5% agarose gel (incorporated EtBr) along with 1μL of 6X Gel loading buffer. 3μL of 50bp DNA ladder was used. Organisms detected and their band sizes are listed in the Table 3.

Statistical Analysis

The statistical analysis was carried out with SPSS 21.0. and GraphPad Prism 9. Receiver operating characteristic (ROC) curves were constructed to compare sensitivities, specificities, positive predictive values and negative predictive values of the three techniques.

Results

Total of 50 patients with GDM were enrolled during first year with the mean age of 29.3±4.27years. Their mean FBS value was 157 ± 17.2 mg/dl. Prevalence of ASB among GDM patients on basis of urine culture is listed in Table 5. Prevalence of ASB among GDM patients on basis of Multiplex PCR is demonstrated in Table 6. A significant association was noted between detection of organisms and multiplex PCR findings compared to urine culture (p <0.0001) as indicated in Table 7 .

Median (25% - 75%) value of UHBP levels in urine was noted to be 689 (625, - 863) pg/ml. A significant association was observed between UHBP levels and multiplex PCR findings with (p= 0.017) (Table 7). No association was found between UHBP levels and urine culture reports (p<0.99) (Table 8).

Fig 1: Agarose gel image of Multiplex PCR

Fig 2: ROC for Urine culture and UHBP, taking multiplex PCR as gold standard

AUC for the Receiver operating characteristic curves (ROC) of UHBP was 0.480 and that of urine culture was 0.492. Cut off value of UHBP was 675 pg/ml.

Discussion

Studies reported the prevalence of ASB in GDM range from 4-18% which is higher compared to the prevalence of ASB in pregnancy without diabetes which range from 4.6-8.2%[10]. In our present study, the prevalence of ASB in GDM as per urine culture was found to be 6%. The prevalence of ASB in GDM as per multiplex PCR is higher compared to the urine culture. Multiplex PCR also identified polymicrobial infection (34%) accurately compared to urine culture. Polymicrobial infection can result in antibiotic resistance, therefore identification of specific organism is of great clinical significance [11]. Even though urine culture is considered as the gold standard for the diagnosis of ASB it takes 24-48 hours to get the results, whereas PCR detected pathogens even in samples which were negative for growth in urine culture in a rapid and accurate way. As indicated in Table 7,a significant association was noted between detection of organisms and multiplex PCR findings compared to urine culture (p <0.0001). Out of 47 growth negative samples from urine culture, whereas only 14 samples were showed negative growth in multiplex PCR. This further support the previously reported studies that multiplex PCR can be used effectively to detect UTI similar to urine culture and therefore multiplex PCR is noninferior to urine culture for detection of UTI [9,11].

Heparin-binding protein (HBP) is a pro-inflammatory protein stored in secretory and azurophilic granules of neutrophils. Activated neutrophils release HBP which acts as a chemoattractant, activates monocytes and induces vascular leakage [12]. UHBP was found to be more sensitive and specific for UTI than the presence of leukocyturia and can be used as a diagnostic marker for UTI in adults [13]. However the potential of UHBP as a diagnostic marker in ASB is not clear. In the present study, UHBP levels in urine was noted to be 689 (625, - 863) pg/ml. UHBP levels in urine was significantly associated with multiplex PCR results, but the association with urine culture results was found to be non-significant. These results further indicates that multiplex PCR results are more specific compared to urine culture results.

By taking multiplex PCR as gold standard AUC for the ROC of UHBP was 0.480 and that of urine culture was 0.492. Cut off value of UHBP was 675 pg/ml. These results suggest the limitation of urine culture along with delay in results. Multiplex PCR was found to be more sensitive and specific compared to Urine culture and estimation of UHBP.

In recent years, researchers have been exploring the potential role of urinary heparin binding protein (HBP) as a biomarker for UTIs and inflammatory conditions. HBP is a protein involved in the immune response and is released by activated neutrophils, a type of white blood cell, during inflammation. Its presence in the urine may indicate inflammation and tissue damage in the urinary tract. The association between ASB/pyuria and urinary HBP in the context of gestational diabetes is an area of interest, as it could provide insights into the pathophysiology of UTIs during pregnancy and potentially lead to the development of better diagnostic and management strategies.

A study published by Mardh et al reported the association between urinary HBP and ASB in pregnant women. The researchers collected urine samples from pregnant women with and without ASB and measured the levels of urinary HBP using an ELISA-based assay. The study found that pregnant women with ASB had significantly higher levels of urinary HBP compared to those without ASB, suggesting a potential link between urinary HBP and the presence of bacterial colonization in the urinary tract during pregnancy[14].

Another study by Cobo et al, examined the association between urinary HBP and pyuria in pregnant women [15]. The study found a positive correlation between the levels of urinary HBP and the presence of pyuria, indicating that HBP may be a potential biomarker for inflammation and infection in the urinary tract during pregnancy. (Reference:

Mardh et al have shown variable sensitivities for UHBP in detecting ASB, reporting a sensitivity of 84% for UHBP in pregnant women with ASB [14].

Studies have shown high sensitivities for multiplex PCR in detecting ASB. For instance, A study Park et al reported a sensitivity of 91% for multiplex PCR in detecting ASB in a diverse population [16]. One drawback of PCR-based methods is that they do not distinguish between live and dead bacteria, potentially leading to false positives in cases of past infections or contamination.

Comparative studies have been conducted to assess the diagnostic performance of these three techniques. A study by Jo et al compared the diagnostic accuracy of UHBP measurement by ELISA and multiplex PCR for detecting ASB in elderly patients [17]. The researchers found that UHBP had a sensitivity of 81.5%, while multiplex PCR had a sensitivity of 93.4%. The specificity of UHBP and multiplex PCR was 72.1% and 79.2%, respectively. The study concluded that multiplex PCR had higher sensitivity but lower specificity compared to UHBP measurement.

Another study by Mardh et al compared the diagnostic performance of urine culture, UHBP measurement by ELISA, and multiplex PCR in detecting ASB in pregnant women[18]. The researchers reported a sensitivity of 84% for UHBP measurement, 100% for multiplex PCR, and 81% for urine culture. The specificities for UHBP measurement, multiplex PCR, and urine culture were 83%, 82%, and 100%, respectively. The study suggested that UHBP measurement and multiplex PCR had comparable sensitivities, but urine culture had lower sensitivity (Reference:

Overall, multiplex PCR appears to have higher sensitivity compared to UHBP measurement and urine culture, making it a promising technique for detecting ASB. However, further research is needed to validate and compare these methods in diverse populations and clinical settings. Moreover, considering the cost, availability, and turnaround time of each technique is essential when choosing the most suitable method for ASB detection.

Limitations

Limitations of the existing research include relatively small sample sizes, potential confounding factors, and the cross-sectional nature of some studies, which limits the ability to establish causality. Additionally, the diagnostic accuracy and threshold values for urinary HBP in detecting ASB and pyuria need to be validated in larger and more diverse populations.

Conclusion

In conclusion, the study suggest a potential association between urinary HBP and ASB/pyuria in gestational diabetes, more comprehensive research is required to confirm and elucidate the nature of this relationship. Future studies should focus on larger cohorts, prospective designs, and standardized measurement techniques to further explore the role of urinary HBP as a potential biomarker for UTIs during pregnancy and its implications for gestational diabetes management.

Funding: Research society for the study of diabetes in India and Indian council of medical research.

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