Ultraviolet Spectroscopy vs Congo Red Card Test—A Step Forward toward Early Prediction of Preeclampsia: A Case–Control Study

INTRODUCTION

Over 60,000 maternal mortalities are attributed to pregnancy hypertension and related disorders in the world per year.¹ In India, the incidence of preeclampsia (PE) is reported to be 8–10%.²

Major contributors to etiopathogenesis of PE are hypoxic injury and release of inflammatory cytokines which can ultimately lead to protein misfolding.^3–5 Therefore, these conditions lead to aggregation and deposition of misfolded proteins in the placenta and body fluids such as urine. The “foldedness” and stability of the structure of proteins depend upon their three-dimensional structure. In this arrangement, the hydrophobic residues are buried in the protein core. On exposure to stress, hypoxia, and other conditions responsible for the pathogenesis of PE, the proteins lose their structural stability leading to misfolding and thereby exposure of these hydrophobic ends to the outer microenvironment. These residues mostly include aromatic amines such as tryptophan, tyrosine, and phenylalanine which have the ability to absorb light in the near-ultraviolet (UV) region (240–295 nm) when measured by ultraviolet–visible (UV–Vis) spectroscopy.⁶ These changes in the protein structure can be indirectly measured by UV–Vis spectroscopy in the form of increased absorbance in this region. This property is used to detect misfolded proteins in the urine of PE patients. Spectroscopy is a unique and novel method in the detection of preeclampsia and can be a game changer due to its cost effectiveness, convenience, and speed.

Several researches show the accumulation of amyloid precursor protein fragments and Ab amyloid plaques in the placenta, along with the presence of proteins such as α-1-antitrypsin, albumin, immunoglobulin G kappa (IgGκ)-free light chain, ceruloplasmin, and interferon-inducible protein 6-16 in different bodily fluids including urine of PE patients.⁷ The Congo red dye (CRD) is widely used to demonstrate the deposition of amyloid plaques in diseases such as Alzheimer’s disease. In the case of PE, this congophilia can be demonstrated as early as 2 weeks before the onset of symptoms of PE.⁸ Thus, facilitating early diagnosis and further management to prevent complications (Table 1).

In this study, we intend to detect misfolded proteins in PE patients by the following two methods: Spectroscopy and Congo red card test in the hope of developing a test that is cheap, non-invasive, and effective in early diagnosis and treatment of PE so that the several dreaded complications of this disease can be prevented in time and the burden of the disease could be reduced (Fig. 1).

AIM AND OBJECTIVES

• To establish the role of spectroscopy in the detection of misfolded proteins.

• Comparative evaluation of UV–Vis spectroscopy and Congo red card-based test in the detection of urinary misfolded proteins in PE patients.

MATERIALS AND METHODS

The study was conducted in the Department of Obstetrics and Gynecology., LLR Hospital, GSVM Medical College, Kanpur, Uttar Pradesh, India from January 2021 to October 2022 on patients who were admitted from the outpatient department (OPD) and emergency (Table 2).

RESULTS

The results of the UV-absorption spectra scan ranged between 250 and 400 nm is shown in Figure 5. The observations of the spectra curve showed that there was increase in UV-absorption among the cases urine samples compared to the urine samples of the control group. The absorption spectra also showed maximum absorption at 280 nm ƛ as shown in Figure 5B.

The paper-based Congo red dye test result is shown in (Fig. 4), in which the urine sample of cases represented with darker color appearance compared to the control urine samples with a lighter color. The images obtained by the Congo red paper-based test were then processed using IMAGE J software to calculate pixels of the image along with the area covered under the Congo red stain in the paper for both control and case groups.

The results of the Congo red paper-based test in both cases and control showed non-significant differences in pixels of color, in contrast to the area covered by the pixels where the p-value was below 0.0001 groups as shown in Tables 4 and 5.

The results of the present study showed that the UV-absorbance method is more sensitive and specific compared to the Congo red paper-based test in detection of the misfolded proteins present in the urine samples of the preeclamptic patients (Table 6).

Unpaired t-test was applied for both groups and p-value was found to be below 0.0001. Our findings suggest that with increase in severity associated with preeclampsia showed an increase in absorbance in the UV–Vis region and area covered under the Congo red assay that can be implicated in the source of misfolded protein.

DISCUSSION

Previously published studies were conducted to search for a reliable method for diagnosis of preeclampsia using various predictive markers, but such method may have some associated limitations such as a costlier approach, time consuming and cumbersome approach. Our study has compared two methods for the detection of urinary misfolded proteins that can be further used for the diagnosis of preeclampsia.

The present study was conducted on 196 study participants including 98 cases and 98 controls (at 95% CI, 80% power of test). This study aims to assess the sensitivity and specificity of UV-absorbance and Congo red paper-based tests using urine specimens of control and preeclamptic patients.

Xing-Min Li et al.⁸ developed a point-of-care test device that detects urinary misfolded proteins and compared it to the CRD retention test of Buhimschi et al.⁷ and demonstrated it is a useful tool for preeclampsia diagnosis. Their test had sensitivity, 71.62%; specificity, 97.11%; PPV, 72.60%; and NPV, 96.97%. Better performance was found in early-onset preeclampsia, with sensitivity, 85.71%; specificity, 96.96%; PPV, 85.71%; and NPV 96.96%, which is comparable to our results in the Indian rural population.

In another study, Rood et al. concluded CRD paper test was positive in 25% of the cases and that it outperformed measured serum and urine markers (80.2% sensitivity, 89.2% specificity, 92.1% negative predictive value, 86.7% accuracy).⁹ Nagarajappa et al. concluded that factors such as GA of onset, severity, a superimposition by eclampsia, fetal growth restriction, or stillbirth had no effect on urinary congophilia.¹⁰ However, our study shows that when the area of the image is compared a significant increase is seen when preeclampsia has added features of severity. Anjali Rani et al. in her study found that 30 out of 250 patients with positive Congo red test develop preeclampsia, so the chance of developing preeclampsia was 12% with PPV 58.8, NPV 95.4%, sensitivity 66.7%, specificity 93.6%, and accuracy 90.4%.¹¹

Proteins that have been misfolded or denatured can form associations in solution, the formation of insoluble aggregates, and also are structurally different from naturally folded proteins. The results of our study showed maximum absorption at 280 nm for the urine samples of cases compared to the control samples. This maximum absorption might be due to structural changes in the protein conformation and aggregates formation, which increases the exposure of aromatic and side chain amino acids toward the outer microenvironment so that it contributes in maximum absorption. This study has revealed an unprecedented and novel use of spectroscopy in the field of obstetrics. Our results demonstrate sensitivity, 77.53; specificity, 80.61; PPV, 80; NPV, 78.21; and accuracy 79.08% with this test which is superior to several Congo red tests done in previous studies.

On comparing the results of both techniques, it was found that UV-absorption spectroscopy is more sensitive and specific compared to Congo red paper-based test because it depends on the absorption of each aromatic and side chain amino acids that is the major limitation in CRD, which gives only staining with aggregates of protein only using calorimetric approach.

Our findings suggest that with increase in severity associated with preeclampsia showed an increase in absorbance in UV-Vis region and area covered under Congo red assay that can be implicated into the source of misfolded protein. This study confirms that urinary congophilia and UV absorbance level is elevated in preeclamptic women compared to normotensive pregnant women in the Indian population.

CONCLUSION

Preeclampsia is a major problem nowadays and is associated with adverse pregnancy outcomes. Early detection of preeclampsia needs some simple, non-invasive, express, affordable methods using body secretion for its diagnosis. Timely screening of the preeclampsia can help the patients in timely referral to the higher center for effective management. The Congo red assay is a calorimetric qualitative assay based on the detection of urinary misfolded protein and has been proven to be a good choice to detect preeclampsia within the community. However, our In-house UV–Vis absorption spectroscopy is a unique and novel test for diagnosing misfolded proteins in the urine of preeclamptic patients. It has proved to be a more specific and sensitive method for the detection of urinary misfolded proteins compared to the Congo red assay and can be a breakthrough in the diagnosis of preeclampsia.

ORCID

Garima Gupta https://orcid.org/0000-0001-5392-2015

Sonali Bisht https://orcid.org/0000-0002-1454-1268

REFERENCES

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