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BRIEF RESEARCH ARTICLE
Year : 2022  |  Volume : 66  |  Issue : 4  |  Page : 487-489  

Spectrofluorimetric-based approach to screen urine contamination in drinking water: A step toward the development of screening method for leptospirosis


1 Senior Demonstrator, Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
2 PhD Student, Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
3 Research Associate, Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
4 Tutor (Bioinformatics), Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
5 Professor, Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India

Date of Submission28-Apr-2022
Date of Decision11-Jun-2022
Date of Acceptance22-Oct-2022
Date of Web Publication31-Dec-2022

Correspondence Address:
Dibyajyoti Banerjee
Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh -160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijph.ijph_587_22

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   Abstract 


Hygiene hypothesis and sanitization are two important pivots of modern civilization. The drinking water should be free from urine and stool contamination. Coliform test is popular for understanding feces contamination. However, understanding urine contamination in drinking water is a difficult task. On the other hand, urine contamination can cause disease like leptospirosis. It occurs mainly in animals and infects humans through contaminated water, food, and soil and causes serious consequences. Rat urine is the most common source of such disease outbreaks. Further, sophisticated laboratories with high-end technologies may not be present at the site of disease outbreaks. In this context, we have proposed a spectrofluorimetric approach to screen urine contamination in water. The screening method can sense up to 156 nl/ml of rat urine.

Keywords: 2-naphthol, fluorescence, leptospirosis, public health, rat urine, water contamination


How to cite this article:
Kumar D, Yadav D, Kaur S, Chowdhary S, Bhattacharyya R, Banerjee D. Spectrofluorimetric-based approach to screen urine contamination in drinking water: A step toward the development of screening method for leptospirosis. Indian J Public Health 2022;66:487-9

How to cite this URL:
Kumar D, Yadav D, Kaur S, Chowdhary S, Bhattacharyya R, Banerjee D. Spectrofluorimetric-based approach to screen urine contamination in drinking water: A step toward the development of screening method for leptospirosis. Indian J Public Health [serial online] 2022 [cited 2023 Feb 4];66:487-9. Available from: https://www.ijph.in/text.asp?2022/66/4/487/366587



Hygiene and sanitization are important for the good health and development of humankind. Hygiene is to keep ourselves clean while sanitation deals with the management of waste generated. Many developing countries face adequate challenges in maintaining the same. The drinking water should be free from urine, stool, and other contaminants. Water monitoring technologies are widely used to detect such contaminants.[1] Coliform test is popular for understanding feces contamination. However, understanding urine contamination in drinking water is a difficult task. On the other hand, urine contamination can cause disease like leptospirosis.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genera leptospira.[2] It occurs more predominantly in hot and humid regions of tropical and subtropical world.[3] Homo sapiens is an incidental host. It occurs more in other animals, including livestock. Nevertheless, it can have severe consequences when humanity is affected and causes multisystem failure if left untreated.[4] The disease spread through contaminated rat urine. When the contaminated rat urine comes in contact with drinking water, it may result in outbreak of the disease.[5] Therefore, there should be some way to screen whether the drinking water is contaminated with rat urine or not.

Further, sophisticated laboratories with high-end technologies may not be present at the site of disease outbreaks.[6],[7] Therefore, the screening method should be user-friendly and straightforward to use in the practical realities of the developing world, where leptospirosis is common.[8] It is in this context; our observed results are important.

2-Naphthol, disodium hydrogen phosphate, and potassium dihydrogen phosphate were from HiMedia, Mumbai, and ethanol was from Changshu Hongsheng Fine Chemical Co. Ltd., Multimode reader (TECAN) was used for taking fluorescence emission readings setting the instrument at default. 96-well black fluorescence plates (TECAN), reverse osmosis (RO) water (Kent RO water system), and double distilled water were used throughout the studies. One millimeter stock solutions of 2N were prepared in ethanol. The stock solutions were kept at 4°C and used the same day. 0.1M phosphate buffer (pH 7.38) and RO water were used for making the reaction mixtures.


   Human Urine Top


Urine sample of healthy individuals (n = 6) was collected in a sterile urine container after obtaining informed consent. Pooled urine samples obtained from six normal individuals were used throughout the study. Similarly, the urine sample of Wistar rats was collected in a sterile container. Pooled urine samples obtained from six normal rats (three males and three females) were used throughout the study.

Institute Ethics Committee approves the study protocol vide reference No. NK/5874/Res/535 and Institute Animal Ethics Committee (IAEC) vide ref. No. 729/IAEC/107/106.

The fluorescence emission spectra of 2N (5μM) were studied in PB (pH 7.38) by exciting it at a different wavelength ranging from 240 to 350 nm. A similar scan was performed for the appropriate blank (i.e., PB and ethanol). The total reaction mixture volume was 250 μl.

After understanding the working excitation and emission wavelength of 2N, the fluorescence emission of increasing concentrations of 2N (0–10 μM at pH 7.38 in PB Ex240/Em 414 nm) was recorded. The same experiment was also repeated in RO water.

For understanding the fluorescence of 2N in urine, the fluorescence emission scan of 2N in both human and rat urine was performed at the selected excitation wavelength. Similarly, the effect of gradient of the urine sample (both human and Wistar rat) on 2N fluorescence was also studied at selected excitation and emission wavelength.

To understand the detection limit, rat urine dilution with PB (pH 7.38) and RO water was done. An unpaired Student's t-test was used to analyze the data. All the values were represented as mean ± standard deviation (n = 6). A P < 0.05 was considered to be statistically significant.

As reported earlier, it was observed that the excitation maximum of 2N (5 μM) is at 240 nm.[9] The fluorescence emission scans of 2N (5μM) at pH 7.38 exciting at various wavelengths are provided in [Supplementary Figure 1].



From the fluorescence emission spectra of 2N (5 μM), it was observed that when excited at λEx240nm, the maximum fluorescence emission of 2N occurs at 414.00 ± 1.41 nm (n = 6). The fluorescence emission scan of the appropriate blank, i.e., PB and ethanol, is given in [Supplementary Figure 2].



Based on these observations, the excitation and emission wavelength of 2N were selected as λEx240 nm/λEm414 nm. The gradient experiment with 2N shows that the fluorescence intensity of 2N increases with an increase in its concentration in both PB (pH 7.38) and RO water [Figure 1]a and [Figure 1]b.
Figure 1: The gradient of 2N in (a) PB and (b) RO water at Ex240nm/Em414nm. (c-h) the fluorescence emission spectra of 2N (5μM) in PB, rat urine, and human urine at E × 240nm.

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When a fluorescence emission study of 2N was carried out in urine, it was observed that at λEx240nm, both the rat and human urine quenches the fluorescence of 2N (5 μM) [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f, [Figure 1]g, [Figure 1]h.

The gradient experiment clearly shows that the quenching of fluorescence intensity of 2N increases with an increase in rat and human urine samples [Figure 2]a and [Figure 2]b.
Figure 2: The effect of (a) Wister rat and (b) human urine on fluorescence intensity of 2N (5 μM) at E × 240nm/Em414nm in PB. Effect of rat urine on 2N (5 μM) in (c) PB and (d) RO water.

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A similar observation was observed in RO water also. The developed method can sense rat urine up to 156 nanolitre/millilitre in PB (pH 7.38) and RO water [Figure 2]c and [Figure 2]d.

It is known that human urine quenches 2N fluorescence.[9] In the instant study, we have reproduced such finding. Further, we have found that such property is not limited to human urine and even rat urine quenches 2N fluorescence. 2N shows fluorescence in drinking water, i.e., generated by RO. RO is a standard method of making potable drinking water.[10] Therefore, it is expected that 2N will exhibit fluorescence emission at 414 nm when excited at 240 nm in drinking water. Such emission is expected to be less when urine or rat urine is mixed with drinking water, somehow more than 156 nl/ml. If periodically the spectra of 2N in drinking water are recorded, then quenching of fluorescence emission can be easily recorded using a fluorimeter. In this way, whether the drinking water is contaminated with urine or not can be screened easily. There may be several factors in drinking water that will quench 2N fluorescence. However, if some quenching phenomenon is observed, water samples can be sent to a sophisticated laboratory to understand urine contamination.[6] In this work, we report that rat urine quenches 2N fluorescence and propose further exploitation of this observation to understand rat urine contamination in drinking water in regions where leptospirosis outbreaks occur.

Acknowledgements

DB acknowledges PGIMER, Chandigarh (File No: 71/2-Edu-16/1915 dated July 31, 2020).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Zulkifli SN, Rahim HA, Lau WJ. Detection of contaminants in water supply: A review on state-of-the-art monitoring technologies and their applications. Sens Actuators B Chem 2018;255:2657-89.  Back to cited text no. 1
    
2.
Karpagam KB, Ganesh B. Leptospirosis: A neglected tropical zoonotic infection of public health importance-an updated review. Eur J Clin Microbiol Infect Dis 2020;39:835-46.  Back to cited text no. 2
    
3.
Ehelepola NDB, Ariyaratne K, Dissanayake WP. The correlation between local weather and leptospirosis incidence in Kandy district, Sri Lanka from 2006 to 2015. Glob Health Action 2019;12:1553283. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327921/ [Last accessed on 2022 Nov 09].  Back to cited text no. 3
    
4.
Narkkul U, Thaipadungpanit J, Srisawat N, Rudge JW, Thongdee M, Pawarana R, et al. Human, animal, water source interactions and leptospirosis in Thailand. Sci Rep 2021;11:3215.  Back to cited text no. 4
    
5.
Jittimanee J, Wongbutdee J. Prevention and control of leptospirosis in people and surveillance of the pathogenic Leptospira in rats and in surface water found at villages. J Infect Public Health 2019;12:705-11.  Back to cited text no. 5
    
6.
Budihal SV, Perwez K. Leptospirosis diagnosis: Competancy of various laboratory tests. J Clin Diagn Res 2014;8:199-202.  Back to cited text no. 6
    
7.
Bierque E, Thibeaux R, Girault D, Soupé-Gilbert ME, Goarant C. A systematic review of Leptospira in water and soil environments. PLoS One 2020;15:e0227055.  Back to cited text no. 7
    
8.
Soo ZMP, Khan NA, Siddiqui R. Leptospirosis: Increasing importance in developing countries. Acta Trop 2020;201:105183.  Back to cited text no. 8
    
9.
Kumar D, Bhattacharyya R, Banerjee D. Fluorimetric method for specific detection of human serum albumin in urine using its pseudoesterase property. Anal Biochem 2021;633:114402.  Back to cited text no. 9
    
10.
Abd El-Salam MH. MEMBRANE TECHNIQUES | Applications of Reverse Osmosis. In: Caballero B, editor. Encyclopedia of Food Sciences and Nutrition. 2nd ed. Oxford: Academic Press; 2003. p. 3833-7. Available from: https://www.sciencedirect.com/science/article/pii/B012227055X007628. [Last accessed on 2022 Jun 11].  Back to cited text no. 10
    


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