|Year : 2019 | Volume
| Issue : 1 | Page : 154-159
Causative agents of urinary tract infections and their antimicrobial susceptibility patterns at a referral center in Western India: An audit to help clinicians prevent antibiotic misuse
Harshkumar B Patel1, Sumeeta T Soni2, Aroor Bhagyalaxmi3, Neev M Patel1
1 B. J. Medical College and Civil Hospital, Ahmedabad, Gujarat, India
2 Department of Microbiology, B. J. Medical College and Civil Hospital, Ahmedabad, Gujarat, India
3 Department of Community Medicine, B. J. Medical College and Civil Hospital, Ahmedabad, Gujarat, India
|Date of Web Publication||31-Jan-2019|
Harshkumar B Patel
A/1, Hardwar Society, Thalota Road, Visnagar - 384 315, Gujarat
Source of Support: None, Conflict of Interest: None
Background: Urinary tract infections (UTIs) remain one of the most common infections in community and susceptibility of uropathogens to commonly used antimicrobials has declined over years. It is important to periodically study susceptibility patterns of uropathogens, so that empiric treatment can be determined using recent data, helping improve patient outcomes. Methods: Urine samples received by the laboratory for culture and susceptibility testing over a period of 3 months were analyzed and included in this study. Antimicrobial susceptibility testing was done on cultured isolates. Results: Of total 3,151 urine samples received, 3,066 were processed, and organisms were isolated from 1,401 (45.69%) samples. Isolation rate from male and female urine samples was 45.29% and 46.32%, respectively. The most commonly isolated organism was Escherichia coli (36.11%), followed by Candida spp. (18.56%), and Klebsiella spp. (18.06%). E. coli was most susceptible to meropenem (91.89%) and imipenem (91.69%). Klebsiella spp. was most susceptible to imipenem(75.89%) and meropenem(75.49%). Susceptibility of E. coli and Klebsiella spp. to nitrofurantoin, cotrimoxazole, and ciprofloxacin was 72.33%, 32.02%, and 18.97%, and 51.77%, 27.27%, and 22.13%, respectively. Candida spp. was most susceptible to amphotericin B (97.30%). Conclusion: Treatment for UTIs should be determined based on current local antimicrobial susceptibility patterns of uropathogens to minimise therapeutic failures and prevent antibiotic misuse.
Keywords: Antimicrobial susceptibility, Escherichia coli, urinary tract infections, uropathogens
|How to cite this article:|
Patel HB, Soni ST, Bhagyalaxmi A, Patel NM. Causative agents of urinary tract infections and their antimicrobial susceptibility patterns at a referral center in Western India: An audit to help clinicians prevent antibiotic misuse. J Family Med Prim Care 2019;8:154-9
|How to cite this URL:|
Patel HB, Soni ST, Bhagyalaxmi A, Patel NM. Causative agents of urinary tract infections and their antimicrobial susceptibility patterns at a referral center in Western India: An audit to help clinicians prevent antibiotic misuse. J Family Med Prim Care [serial online] 2019 [cited 2020 Nov 26];8:154-9. Available from: https://www.jfmpc.com/text.asp?2019/8/1/154/251128
| Introduction|| |
Urinary tract infection (UTI) describes microbial colonization and infection of structures of the urinary tract. UTI is categorized by infection site as pyelonephritis (kidney), cystitis (urinary bladder), and urethritis (urethra), and can also be classified as uncomplicated or complicated. UTIs are among the most prevailing infectious diseases in the community with substantial clinical and financial burden. Almost 95% of all UTIs are caused by bacteria, most of them by Escherichia More Details coli (30%–90%, depending on clinical setting). Klebsiella, Enterobacter, Proteus, Pseudomonas, Enterococcus, Staphylococcus, and others can also cause UTI.,
Antibiotic resistance among bacteria causing common infections is increasing in all regions of the world. It is interesting that pattern of resistance observed varies from hospital to community, large hospital to small hospital, state to state, and even vary from country to country. Emergence of resistance to antibiotics illustrates importance of using evidence-based strategies for treatment. In UTI cases, antibiotic treatment is often started empirically before the results of urine culture and susceptibility testing are available. Appropriate antibiotic use in patients with UTI seems to reduce length of hospital stay and therefore favors patient outcomes and healthcare costs. Hence, it becomes important to regularly monitor the resistance or susceptibility patterns of uropathogens, so that the guidelines for empirical antibiotic therapy can be improved to include antibiotics with low resistance, aiding clinicians in proper management of UTIs with minimal therapeutic failures.,
Taking all these into consideration a need was felt for a study to know causative agents of UTI and their antimicrobial susceptibility patterns, in a referral hospital in Gujarat, Western India. This study can help us take a step towards evidence-based medicine and help us keep track of antimicrobial susceptibility trends.
| Materials and Methods|| |
This study was approved by Institutional Ethics Committee.
Study area and population
A cross-sectional study was conducted in a teaching and public-sector referral hospital and its affiliated Bacteriology Laboratory, located in urban area of Ahmedabad, Gujarat. The hospital has around 2,000 beds and provides treatment to over 9 lac OPD (outdoor/ambulatory) patients and 1 lac IPD (indoor/hospitalized) patients annually. This hospital caters needs of patients from nearby urban and rural areas and also of patients referred from various districts of Gujarat as well as from Madhya Pradesh and Rajasthan. Patients whose urine samples were received by the Bacteriology Laboratory during 3-month period – July, August, and September of 2016 – were included in the study. Culture and susceptibility reports were obtained directly from the Bacteriology Laboratory.
Sample collection and processing
Clean catch mid-stream urine samples or those obtained by aspiration from catheter tube or suprapubic aspirate collected in sterile, wide mouth universal container, from all the suspected UTI patients (outdoor/ambulatory patients and indoor/hospitalized patients) were received and processed by the Bacteriology Laboratory. Contaminated/non-sterile samples were discarded and not processed.
Culture and identification of isolates
Urine samples were inoculated on appropriate culture media by using semiquantitative methods and inoculated media was incubated for 48 hours aerobically at 37°C. Cultures were then examined for growth and colonies counted for determination of significant or insignificant bacteriuria. A growth of ≥105 colony forming units/ml was considered as significant bacteriuria, suggestive of UTI. Identification was done based on standard biochemical and other laboratory tests.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was done by Modified Kirby Bauer Disc Diffusion method on Muller Hinton Agar as per the CLSI Standards. The antibiotics tested were amikacin (30 μg), ampicillin (10 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), cefotaxime (30 μg), cefoperazone (75 μg), co-trimoxazole (25 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), levofloxacin (5 μg), linezolid (5 μg), meropenem (10 μg), nitrofurantoin (300 μg), piperacillin + tazobactam (100 + 10 μg), polymyxin B (300 μg), tetracycline (30 μg), ticarcillin + clavulanic acid (75 + 10 μg), and vancomycin (30 μg). For Candida spp., amphotericin B, clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole and nystatin were used in antifungal susceptibility testing.
Calculations were done using Microsoft Excel. Results were presented as frequencies and percentages, and Z-test was applied where necessary and result considered significant at P ≤ 0.05.
Definition of terms
- Urinary tract infection: Infection and microbial colonization of urinary tract.,
- Cystitis: Infection limited to urinary bladder/lower urinary tract. It often presents with dysuria, urinary urgency, frequency, and/or suprapubic pain.
- Pyelonephritis: Infection of the kidney/upper urinary tract. It often presents with fever, tachycardia, chills or rigors, costovertebral tenderness, and/or flank pain with or without symptoms of cystitis.
- Uncomplicated UTI: Acute, sporadic or recurrent lower (uncomplicated cystitis) and/or upper (uncomplicated pyelonephritis) UTI, limited to nonpregnant, premenopausal women with no known relevant anatomical and functional abnormalities within the urinary tract or comorbidities.
- Complicated UTIs: UTIs in a patient with an increased chance of a complicated course, that is, all men, pregnant women, patients with relevant anatomical or functional abnormalities of the urinary tract, indwelling urinary catheters, renal diseases, and/or with other concomitant immunocompromising diseases.
| Results|| |
Of total 3,151 urine samples received, 1,901 from males and 1,250 from females, 85 contaminated/non-sterile samples were discarded. Of 3,066 samples processed, organisms were isolated from 1,401 samples yielding positive culture rate or isolation rate of 45.69%.
The isolation rate from samples received from OPD (30.23%) was lower than from samples received from IPD (51.62%) and the difference was statistically very significant (Z-value = 10.64, P < 0.0001) [Table 1]. Isolation rates in samples from males and females were 45.29% and 46.32% respectively, without statistically significant difference (Z–value = 0.55, P = 0.58). Isolation rate from IPD + OPD (overall) and IPD samples was lower in males than in females. Isolation rate from OPD samples was higher in males than in females [Table 2].
|Table 1: Frequency of positive culture or isolation, out of all urine samples received by the laboratory from OPD and IPD|
Click here to view
|Table 2: Isolation frequency of uropathogens based on gender and source of sample - OPD or IPD|
Click here to view
The most common isolated organism was E. coli with 506 isolations (36.11%), followed by Candida spp.(18.56%), Klebsiella spp. (18.06%; K. pneumoniae = 17.15% and other Klebsiella spp. = 0.91%), Pseudomonas spp. (14.65%; P. aeruginosa = 13.19% and other Pseudomonas spp. = 1.46%), Acinetobacter spp. (5.06%; A. baumannii = 4.25% and other Acinetobacter spp.=0.81%), Enterococcus spp. (4.14% ; E. feacalis = 2.76% and E. faecium = 1.38% ), Proteus spp. (1.78%; P. mirabilis = 1.07% and other Proteus spp. = 0.71%), Staphylococcus spp. (0.78%; S. aureus = 0.67% and coagulase-negative staphylococci = 0.11%), Providencia spp. (0.43%), and Morganella morganii (0.43%). Due to low number of Providencia and Morganella isolates, their antibiotic susceptibility results were excluded from the study. In males, followed by E. coli (33.29%), Klebsiella spp. (19.12%) was the second and Candida spp. (17.59%) was the third most isolated organism. In females, however, after E. coli (40.43%), Candida spp. (20.03%) was the second and Klebsiella spp. (16.42%) was the third most isolated organism [Table 3].
|Table 3: Sex based distribution of various uropathogens in culture positive samples|
Click here to view
E. coli was most susceptible to meropenem (91.89%) closely followed by imipenem (91.69%). E. coli had least susceptibility to cefuroxime (18.18%) and ciprofloxacin (18.97%). Klebsiella was most susceptible to imipenem (75.89%) closely followed by meropenem (75.49%). Klebsiella spp. was least susceptible to ampicillin (2.37%). Pseudomonas spp. was most susceptible to polymyxin B (92.19%) followed by aztreonam (68.29%). Pseudomonas spp. was least susceptible to ciprofloxacin (25.85%). Gram-positive bacteria Enterococcus spp. and Staphylococcus spp. were most susceptible to linezolid at 96.55% and 100%, respectively followed by vancomycin at 87.93% and 100%, respectively [Table 4].
|Table 4: Susceptibility rates of isolated bacteria to various tested antibiotics|
Click here to view
Susceptibility of Candida spp. was maximum to amphotericin B (97.30%), followed by nystatin (89.61%). Candida had minimal susceptibility to clotrimazole (50.38%) [Table 5].
|Table 5: Susceptibility rates of isolated Candida spp. to various tested antifungals|
Click here to view
| Discussion|| |
Positive culture rate or isolation rate of 45.69% obtained in this study was close to that obtained by similar studies conducted across India.,,, The data obtained from this study shows that spectrum of organisms causing UTI is also similar to that reported by other studies across India. However, isolation rates of various organisms varied from study to study.,,, Isolation rates in male and female samples were not statistically different which was in stark contrast to other studies conducted across India, in which the isolation rates were found to be higher in females.,, This discrepancy could in part be because of higher number of complicated UTIs in males resulting in higher number of requests for culture and susceptibility reports and because treatment of most uncomplicated UTIs is usually done empirically without requesting culture and susceptibility reports.
E. coli was the most common isolated organism responsible for causing UTIs in our setup, in trend with other studies across India.,, High E. coli isolation rate of 69.8% was seen in study conducted by George et al. in Karnataka compared to 36.11% seen in our study. High susceptibility of E. coli to meropenem (91.89%) and imipenem (91.69%) was similar to that seen in other studies across India.,,,, However, a study done in Lahore, Pakistan by Sabir et al. reported low E. coli susceptibility rate of 39.5% to imipenem. Susceptibility of E. coli to nitrofurantoin was found high at 72.33%, in trend with other studies across India.,,, Susceptibility of E. coli to cotrimoxazole was 32.02% in this study, while in other studies across India it varied from 15.15% to 52.3%., Susceptibility of E. coli to ciprofloxacin was 18.97% which lower compared to susceptibility rates seen in other studies across India and Pakistan.,,, High susceptibility rates of E. coli to ciprofloxacin were reported in studies done in Iran (68.1%), Poland (65.8%) and Ethiopia (54.8%) showing geographical variations in antibiotic susceptibility trends ,, [Table 6].
|Table 6: Comparison of susceptibility rates of Escherichia coli to various antibiotics, found in various studies across India and abroad|
Click here to view
Klebsiella spp. was the third most commonly isolated uropathogen at rate of 18.06%, similar to isolation rate of 18.71% reported in a study from Meerut, India. Klebsiella spp. however, was the second most commonly isolated organism in various studies across India.,, Susceptibility to nitrofurantoin, cotrimoxazole and ciprofloxacin was found to be 51.77%, 27.27% and 22.13% respectively. Susceptibility of Klebsiella isolates to nitrofurantoin varied from 38% to 67% in studies across India., Susceptibility of Pseudomonas isolates to anti-pseudomonas cephalosporin ceftazidime was found to be 40.48%.
Candida spp. was the second most common isolated organism in this study at rate of 18. 56% which was higher compared to other studies across India, with isolation rate as low as 8% reported in a study in Kerala, India. The isolation rate of Candida spp. in the current study could be influenced by various patient factors such as urinary catheterisation and stenting, diabetes, immunocompromised status, hospitalization and use of broad spectrum antibiotics. Candida isolates were most susceptible to amphotericin B (97.30%) which was close to susceptibility rate of 91% observed in a study done in Mangalore, India.
Resistance to antibiotics is higher in India compared to nations like UK, USA, Australia and South Africa. Decreased susceptibility of uropathogens to empiric antibiotics for UTI like cotrimoxazole and ciprofloxacin and even to broad spectrum antibiotics in India, as evident by this and other studies, could be because of rampant use of antibiotics predisposed by many factors. Tendency to self-medicate, noncompliance to treatment, financial constraints and lack of education on part of patient, sale of antibiotic drugs without proper prescription and failure to educate patient on part of pharmacists, negligible surveillance of susceptibility patterns, poor regulatory controls over antibiotics and lack of will to make change on part of health care system, and administering antibiotics before obtaining sample for culture, failure to educate patient and poor prescribing practices on part of physicians are among many factors that lead to injudicious and inappropriate use of antibiotics in India, hence causing rapid development of resistance.,,,
Based on this study, it can be recommended that nitrofurantoin be preferred instead of cotrimoxazole and ciprofloxacin for use as empiric antibiotic for uncomplicated cystitis., For uncomplicated and complicated pyelonephritis, aminoglycosides and carbapenems should be preferred over fluoroquinolones and cephalosporins., In all cases, urine sample for culture and susceptibility testing should be collected before administration of antibiotics and then therapy should be modified to narrow spectrum agent as per urine culture and susceptibility report. This study emphasizes the need for hospital or regional antibiograms in order to combat the problem of antibiotic resistance. Antibiograms help monitor antimicrobial resistance trends and help clinicians select appropriate antibiotic therapy.
The major limitation of this study is that since direct laboratory data was used, it does not take into account risk factors that can cause drug resistant and complicated UTIs like diabetes, compromised immunity, cancer chemotherapy, HIV, prolonged urinary catheterisation, recent antibiotic use, incomplete treatment of prior UTIs, urinary tract malformations and old age., This study is also limited by the fact that those patients who were treated on outpatient basis might have had uncomplicated UTIs and physicians treating them might not have requested urine culture and susceptibility reports.
| Conclusion|| |
This study provides important data to monitor and compare with other studies, the trend of antimicrobial susceptibility of uropathogens and helps us towards deciding empirical treatment of UTIs at this referral healthcare center. Similar studies should be done on a larger scale periodically in different regions, so that empiric antibiotic therapy guidelines can be framed according to local antimicrobial susceptibility trends improving patient outcomes and minimizing anitbiotic misuse.
Authors are thankful to Dr. Sandhya Pillai Nair, PhD, Associate Professor, Department of Biochemistry, Dr. M. K. Shah Medical College and Research Centre, Ahmedabad for her guidance on critical concepts, designing, and methodology of this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ejrnæs K. Bacterial characteristics of importance for recurrent urinary tract infections caused by Escherichia coli
. Dan Med Bull 2011;58:B4187.
Cardwell SM, Crandon JL, Nicolau DP, McClure MH, Nailor MD. Epidemiology and economics of adult patients hospitalized with urinary tract infections. Hosp Pract (1995) 2016;44:33-40.
Gupta S, Kapur S, Padmavathi D. Comparative prevalence of antimicrobial resistance in community-acquired urinary tract infection cases from representative states of Northern and Southern India. J Clin Diagn Res 2014;8:DC09-12.
Fair RJ, Tor Y. Antibiotics and bacterial resistance in the 21st
century. Perspect Medicin Chem 2014;6:25-64.
Farajnia S, Alikhani MY, Ghotaslou R, Naghili B, Nakhlband A. Causative agents and antimicrobial susceptibilities of urinary tract infections in the Northwest of Iran. Int J Infect Dis 2009;13:140-4.
Nickel JC. Management of urinary tract infections: Historical perspective and current strategies: Part 2 – modern management. J Urol 2005;173:27-32.
Spoorenberg V, Hulscher ME, Akkermans RP, Prins JM, Geerlings SE. Appropriate antibiotic use for patients with urinary tract infections reduces length of hospital stay. Clin Infect Dis 2014;58:164-9.
Sharma N, Gupta A, Walia G, Bakhshi R. Pattern of antimicrobial resistance of Escherichia coli
isolates from urinary tract infection patients: A three year retrospective study. J Appl Pharm Sci 2016;6:62-5.
Naber KG, Schito G, Botto H, Palou J, Mazzei T. Surveillance study in Europe and Brazil on clinical aspects and antimicrobial resistance epidemiology in females with cystitis (ARESC): Implications for empiric therapy. Eur Urol 2008;54:1164-75.
Kass EH. Bacteriuria and the diagnosis of infections of the urinary tract; with observations on the use of methionine as a urinary antiseptic. AMA Arch Intern Med 1957;100:709-14.
Collee JG, Miles RS, Watt B. Test for identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th
ed. London: Churchill Livingstone Inc.; 1996. p. 433.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI Document M100- S24. Wayne, Pennsylvania, USA: CLSI; 2014. Available from: https://www.clsi.org/standards/products/microbiology/
. [Last accessed on 2018 Aug 01].
George CE, Norman G, Ramana GV, Mukherjee D, Rao T. Treatment of uncomplicated symptomatic urinary tract infections: Resistance patterns and misuse of antibiotics. J Family Med Prim Care 2015;4:416-21.
] [Full text]
Prakash D, Saxena RS. Distribution and antimicrobial susceptibility pattern of bacterial pathogens causing urinary tract infection in urban community of Meerut city, India. ISRN Microbiol 2013;2013:749629.
Somashekara SC, Deepalaxmi S, Jagannath N, Ramesh B, Laveesh MR, Govindadas D, et al.
Retrospective analysis of antibiotic resistance pattern to urinary pathogens in a tertiary care hospital in South India. J Basic Clin Pharm 2014;5:105-8.
Singhal A, Sharma R, Jain M, Vyas L. Hospital and community isolates of uropathogens and their antibiotic sensitivity pattern from a tertiary care hospital in North West India. Ann Med Health Sci Res 2014;4:51-6.
] [Full text]
Kulkarni SR, Peerapur BV, Sailesh KS. Isolation and antibiotic susceptibility pattern of Escherichia coli
from urinary tract infections in a tertiary care hospital of North Eastern Karnataka. J Nat Sci Biol Med 2017;8:176-80.
Sabir S, Ahmad Anjum A, Ijaz T, Asad Ali M, Ur Rehman Khan M, Nawaz M, et al.
Isolation and antibiotic susceptibility of E. coli
from urinary tract infections in a tertiary care hospital. Pak J Med Sci 2014;30:389-92.
Kashef N, Djavid GE, Shahbazi S. Antimicrobial susceptibility patterns of community-acquired uropathogens in Tehran, Iran. J Infect Dev Ctries 2010;4:202-6.
Stefaniuk E, Suchocka U, Bosacka K, Hryniewicz W. Etiology and antibiotic susceptibility of bacterial pathogens responsible for community-acquired urinary tract infections in Poland. Eur J Clin Microbiol Infect Dis 2016;35:1363-9.
Mamuye Y. Antibiotic resistance patterns of common gram-negative uropathogens in St. Paul's hospital millennium medical college. Ethiop J Health Sci 2016;26:93-100.
Yashavanth R, Shiju MP, Bhaskar UA, Ronald R, Anita KB. Candiduria: Prevalence and trends in antifungal susceptibility in a tertiary care hospital of Mangalore. J Clin Diagn Res 2013;7:2459-61.
Laxminarayan R, Chaudhury RR. Antibiotic resistance in India: Drivers and opportunities for action. PLoS Med 2016;13:e1001974.
Porter G, Grills N. Medication misuse in India: A major public health issue in India. J Public Health (Oxf) 2016;38:e150-7.
Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, et al.
International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the infectious diseases society of America and the European society for microbiology and infectious diseases. Clin Infect Dis 2011;52:e103-20.
National Centre for Disease Control, DGHS, MOHFW, Government of India. National Treatment Guidelines for Antimicrobial Use in Infectious Diseases. New Delhi, India: Ministry of Health and Family Welfare, Government of India; 2016. Available from: http://www.apps.who.int/medicinedocs/documents/s23118en/s23118en.pdf
. [Last accessed on 2018 Aug 01].
Joshi S. Hospital antibiogram: A necessity. Indian J Med Microbiol 2010;28:277-80.
] [Full text]
Sotto A, De Boever CM, Fabbro-Peray P, Gouby A, Sirot D, Jourdan J, et al.
Risk factors for antibiotic-resistant Escherichia coli
isolated from hospitalized patients with urinary tract infections: A prospective study. J Clin Microbiol 2001;39:438-44.
Tenney J, Hudson N, Alnifaidy H, Li JT, Fung KH. Risk factors for aquiring multidrug-resistant organisms in urinary tract infections: A systematic literature review. Saudi Pharm J 2018;26:678-84.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
|This article has been cited by|
||Assessment of multidrug resistance in bacterial isolates from urinary tract-infected patients
| ||Muhammad Imran Khan,Surui Xu,Malik Mubashar Ali,Rizwan Ali,Ahsan Kazmi,Naeem Akhtar,Muhammad Bilal,Yi Hu,Fenfen Li |
| ||Journal of Radiation Research and Applied Sciences. 2020; 13(1): 267 |
|[Pubmed] | [DOI]|
||A prospective, follow up study to assess guidelines compliance in uncomplicated urinary tract infection
| ||PreetinderSingh Manshahia,Manisha Bisht,Ankur Mittal,Mohit Bhatia,ShailendraS Handu |
| ||Journal of Family Medicine and Primary Care. 2020; 9(8): 4292 |
|[Pubmed] | [DOI]|
||Microbiologic profile and clinical practices in urinary tract infections in a tertiary care center in Southern India
| ||Keithellakpam Kiranmala,Reuben Johnson,Jayanthi Savio,Jyothi Idiculla |
| ||Journal of Family Medicine and Primary Care. 2019; 8(9): 2888 |
|[Pubmed] | [DOI]|