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A Study of Screening Tests for the Presumptive Diagnosis of Significant Bacteriuria in Urinary Tract Infections

Urinary tract infections (UTIs) are the inflammatory disorders of the urinary tract caused by the abnormal growth of pathogens. 1 Urinary tract infection (UTI) is an acute bacterial infection of the urinary tract, which is accounting for around 8 million patient visits annually. 2 Urinary tract infections can be community acquired or nosocomial. 3,4 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 09 (2019) Journal homepage: http://www.ijcmas.com


Introduction
Urinary tract infections (UTIs) are the inflammatory disorders of the urinary tract caused by the abnormal growth of pathogens. 1 Complications of UTI include pyelonephritis requiring hospitalization and can result in permanent renal damage. UTI is most commonly caused by uropathogens from faecal flora that ascend the urethra to infect the bladder. 5 Young women who are sexually active are particularly vulnerable. In pregnancy, bacteriuria can lead to complications such as pyelonephritis, sepsis, and increased risk of preterm birth. 6 All individuals are susceptible to UTI, but the prevalence depends on age, gender and certain predisposing factors. The infections may range from asymptomatic infection to simple acute symptomatic infection with a susceptible organism to a more serious recurring infection such as pyelonephritis which may be caused by resistant and often difficult to treat organisms. 7 From a microbiological perspective, UTI exists when pathogenic microorganisms are detected in the urine. Thus Microscopy of urine is of diagnostic value and culture is still the gold standard for isolation of bacteria. It is now universally accepted that cultural isolation of the particular causative agent is important for a definite diagnosis of urinary tract infection. However presumptive approach with non-culture rapid screening tests is more practical and cost-effective to manage UTI. A positive result for bacteriuria provides enough evidence of infection to omit urine culture and susceptibility testing and treat the patients empirically with presumptive approach. The various screening methods would reliably and economically separate specimens, which contain evidence of clinical infection from those which do not. Thus, this can help to avoid culture of all irrelevant specimens.
Rapid detection of significant bacteriuria can have profound effect on patient care as well as laboratory economics. This is because it avoids unnecessary antibiotic therapy to patients with normal urine. Secondly, it prevents the cost of labor and material by helping to avoid culturing of negative urine specimens. Hence, the need for rapid and more accurate methods for detection of significant bacteriuria is very important. Several biochemical urine screening methods have been described such as assays for leucocyte esterase activity, nitrate reduction and glucose detection. Physical methods, such as impedance, filtration, particle counting and microscopic examination have also been reported, in addition to semiautomatic systems. 8 The present study aims at establishing the usefulness of several laboratory based rapid screening tests, such as gram staining, determination of pyuria and bacteriuria by wet mount examination and biochemical tests like leucocyte esterase. Finally relevant information drawn from this study was compared with those of other workers, both within and outside our country.

Materials and Methods
The present study was undertaken in the Department of Microbiology, Goa Medical College, Bambolim Goa, over a period of 5 months, from January to May, 2019. The material for the study included two hundred samples that were collected randomly from patients, attending the Out Patient Department of this Institution.

Selection of subjects
Urine samples were collected from patients with a provisional diagnosis of urinary tract infection, based on clinical history alone. Only those patients who had not received any antimicrobial treatment were included in the study.

Collection of sample
Subjects were instructed to collect an early mid stream clean catch urine in a sterile container. The males were instructed to first clean genitals with soap and water and then collect urine after retracting prepuce and the glans penis. The women were asked to collect urine after careful anogenital toilet with soap and water. The female patients were instructed to separate the labia with fingers, while collecting the urine. The samples were refrigerated when a delay of more than 1-2 hours was anticipated in transportation of the samples. Semi quantitative culture was performed by using a calibrated platinum loop with a diameter of 1.45 mm calibrated to deliver 0.001 ml of urine by a streak plate method. A loopful of uncentrifuged urine was inoculated on blood agar and MacConkey agar. It was streaked on MacConkey agar plate employing an inverted cone technique. 10 The streaking on blood agar plate employed was using primary, secondary and tertiary streak. After overnight incubation at 37°C, colony count was done and significant bacteriuria was reported at a count of ≥10 5 CFU/ml.

Wet mount examination
A wet mount examination was done for detection of both pyuria and bacteriuria after centrifugation. 11 Approximately 12 ml of urine was centrifuged in a tube at 2500 to 3000 rpm for 5 minutes.
The supernatant was discarded and a drop of the deposit was put on a slide. The drop was covered by a coverslip and observed under a high power objective. 11 The number of organisms per high power field was examined (least 10 organisms/HPF) for bacteriuria, and the number of pus cells per 20 HPF were counted (more than10 pus cells/ 20 HPF) for pyuria.

Gram stain 11
A smear was prepared on a slide, heat fixed and stained by Gram staining technique. The sediment obtained by centrifuging urine. The slide was then observed under oil immersion objective for presence of bacteria. Presence of ≥ 1 organism/OIF was considered positive. At least 50 fields were examined before declaring the smear negative.

Griess nitrite test 12
Preparation of Reagents: Solution A -Sulphanilic acid solution and Solution Balpha -Naphthylamine solution. Sulphanilic acid solution was prepared by dissolving 0.5 gm of sulphanilic acid in 150 ml of 10% acetic acid. α -Naphthylamine solution was prepared by dissolving 0.2 gm of α -Naphthylamine in 20 ml of distilled water which was heated to boiling.
After thorough stirring, the solution was filtered through filter paper. Equal amounts of solution A and solution B were mixed to obtain a working reagent. The combined reagent if pink in colour was unfit for use.

Method of urine testing for nitrites
A few ml of the urine was placed in a testtube. To the urine sample, one-fourth to onehalf volume of the combined reagent solution was added. In the presence of nitrite, a red colour developed instantaneously and remained stable for several hours. This was taken as a positive test result.

Triphenyl tetrazolium chloride test (TTC) 13
Preparation of the reagent Stock solution was prepared by dissolving 750 mg of TIC in 100 ml of a saturated solution of disodium hydrogen phosphate. The stock solution was stored in the dark and was stable for at least two months. Working Solution was prepared by taking 4 ml of the stock solution and diluting it to 100 ml with saturated solution of disodium hydrogen phosphate. The working solution was also stored in the dark and was stable for at least two weeks.

Test procedure
Two ml of well mixed urine was taken in a clean test-tube and 0.5 ml of the working solution of TIC was added to it with a sterile pipette. The tube was then incubated for four hours at 37°C. At the end of four hours, the deposit was examined with the naked eye. A positive result was shown by a red precipitate of triphenyl formazan while a negative result by the absence of this precipitate. A concave mirror was used to read the result.

Catalase test 14
The catalase test was performed by mixing equal volumes of urine and freshly prepared 3% hydrogen peroxide (weight in volume) in a sterile test tube. The tube was left standing in a rack at room temperature and the presence of a definite layer of foam on the surface of the mixture at the end of 15 minutes was regarded as a positive reaction to the catalase test.

The leucocyte esterase dipstick test 15
The Leucocyte Esterase dipstick test was performed using the Multistix ® 10 SG Reagent strips, manufactured by Bayer Diagnostics Manufacturing Ltd, South Wales. These strips contained solid phase reagent areas affixed to a plastic support and provided in a dry reagent format.

Procedure
The test was performed on well mixed uncentrifuged urine, according to the manufacturer's instructions. The strip was completely immersed in the urine and removed immediately to avoid dissolving out reagents. While removing, the edge of the entire length of the strip was run against the rim of the urine container to remove excess urine. The strip was held in a horizontal position to prevent possible mixing of chemicals from adjacent reagent areas. The test results were carefully compared with the colour chart on the bottle label in good day light. Time reading was 60 seconds for nitrites and 2 minutes for leucocytes. The positivity of the LE dipstick test was decided when either leucocyte esterase was positive, or nitrite was positive or both leucocyte esterase and nitrites were positive.

Statistical analysis
The sensitivity, specificity and positive and negative predictive values were calculated by using the following formulae.

Results and Discussion
The prospective study included a bacteriological analysis of 200 clean catch early morning midstream, randomly selected urine samples from patients with clinical suspicion of urinary tract infection. The study was carried out over a period of 5 months from January to May 2015.      The wide variation in the UTI prevalence among various studies is probably due to selection of different categories of patients, which extended from complicated and uncomplicated UTI, patients with obstruction, antenatal mothers and general population. A high UTI rate in the present study could be due to selection of patients, with careful history undertaken and exclusion of patients who had received antimicrobial therapy.

Evaluation of screening tests
Although cultural isolation remains the gold standard for arriving at a diagnosis of UTI, result of urine culture requires a minimum time of 24 to 48 hours to be ready. Rapid biochemical urine screening method, which would indicate a high suspicion of UTI could help in arriving at a presumptive diagnosis earlier. These tests could eliminate culturing of those samples which would yield no growth on culture. In the present study, various screening tests were evaluated in comparison with semi-quantitative urine culture, to determine the value of any screening method that would be sensitive without compromising on the specificity.

Pyuria
Wet mount examination for detection of pus cells on a centrifuged deposit was used. The sensitivity and specificity of this test 87.9% and 82.9% respectively. This test did not detect UTI in 12.1% culture confirmed cases, while false positivity was to the tune of 17.1%. In a study by thakre and Supriya urine pus cells count could diagnose 72.41% culture positive UTI cases. 19 Their false positivity was 5.53%.
Pyuria may not be a specific finding. It may be present in cases without infection, as in cases with stones, following operations of urinary tract or in cases after treatment with antibiotics as stated by Brumfitt and Percival (1964). 20 Further, scientifically, sterile pyuria may be attributed to infections caused by fastidious organisms, such as chlamydia, which fail to grow on routinely employed urine culture media. A false negative result may be seen in UTI caused by organisms like Proteus, Klebsiella and Pseudomonas, which make the urine alkaline by virtue of the urease enzyme they possess. The alkaline urine could cause disintegration of pus cells.

Bacteriuria
Methodology employed and interpretative standards vary for detection of bacteriuria in unstained smears of whole or sediment urine.
In the present study, unstained sediment from centrifuged urine was analysed for bacteriuria and the presence of at least 10 organisms/HPF was considered suggestive of infection.
The sensitivity of bacteriuria in the present study was 82.3% and specificity was 86.8%, false positive result was seen in 13.2% cases, while false negative result was obtained in 17.7% subjects. Littlewood and Jacobs used the similar criteria as in the present study and observed a sensitivity of 87% and a specificity of 96%. 21 Although the sensitivity increased to 95%, the specificity reduced to 50%. Jenkins and Fenn (1986) opined that viewing more than 10 organisms/HPF should be the criteria to be employed as it correlates with 95% specificity. This gives a high confidence of bacteriuria at a level of 10 5 or more CFU/ml. 11

Gram staining
The sensitivity of gram staining to predict UTI in the present study was 85.5% i.e. True positivity. This finding is in accordance with the result of studies conducted by Shaw and Karin (1988) who obtained a sensitivity 89% respectively. 22 In the present study, the specificity was 89.5% ; similar to that obtained by Shaw and Karin (1998) i.e. 81%. 22 False positive results with gram staining can be due to deposits in the gram stains. However, gram staining is a quick procedure and offers information of the type of organism, be it gram positive or gram negative, bacilli or cocci. It may also aide in separating low count bacteriuria from mixed morphology of normal urethral flora.

Griess nitrite test
The sensitivity and specificity of the  19 A false negative result may be obtained if the patient has frequent bladder emptying and diuresis, infection with nitrate negative bacteria, lack of sufficient substrate and inhibition of metabolic activity of bacteria by antibiotics.
The practical significance of the Nitrite test is increased by the fact that Escherichia coli, a strong nitrate reducer is the most commonly encountered UTI associated pathogen. The striking simplicity of this test is an indication for this test to be employed routinely as tests for albumin and sugar as opined by Smith and Thayer (1961). 23

TTC test
The TTC test is a salt reduction test, used for presumptive detection of evidence of UTI. In the present study, the sensitivity and specificity of TTC test was 79.8% and 78.9% respectively. This is in accordance with the finding of Agarwal and Das (1986) 24 False positive result was obtained in 21.1% cases, in the present study. Simmons and William (1962) opine that contamination of urine with lactobacilli from the vagina may account for a positive test in bacteriologically significant cases. 25 Escherichia coli and Klebsiella which account for maximum number of urinary tract infection are TTC test positive and therefore help to improve the usefulness of the test. Since bacteria reduce colourless TTC to an insoluble red compound at an alkaline pH a false result may be obtained at a low pH equal to or less than 6.5.

Catalase test
This test showed a sensitivity of 80.6% and a specificity of 71.1% in the present study. Similar sensitivity of 83% and specificity of 70.9% was observed in a study conducted by Berger and Bianca on 500 urine samples from cases of suspected UTI. 26 A false negative result was seen in 19.4% cases in the present study. Similar, 17% cases yielded false negative result in the study of Berger and Bianca. 26 False negative result can be due to simple bacilluria which do not produce renal injury and thereby fail to produce renal catalase. Braude and Berkowitz (1961) opined that net urinary catalase represents the sum of renal, exudative and microbial catalase traces from any one of the three sources alone might not be detected. 27 False negative result can also be due to some urinary pathogens that lack catalase, such as Enterococci. Drugs like thallium, a catalase inhibitor can also contribute to a negative catalase result as stated by Braude and Berkowitz (1961). 27 A false positive result was seen in 28.9% cases, in the present study. A false positive result can be obtained because catalase is also produced by inflammatory cells and renal tissue. Presence of red blood cells can also give a false positive result as seen by Taguchi and Grad. 28

Leucocyte esterase dipstick test
The Leucocyte Esterase test is a rapid screening test for LE activity (host response specific) and a test for urinary nitrite production which is bacteria specific.  Waisman and Zerem (1999). 29 A low sensitivity of 50% was observed in the study of Teresa (1985) and Eigbefoh. 30 The specificity of the test was 90.8% in the present study and was comparable with result obtained in the studies of Teresa et al., (1985) 30 Shaw and Karin (1998) 22 It has been observed that presence of ascorbic acid, phenazopyridine or abnormal concentration of protein may interfere with the LE test and give erratic results as observed by Wagle and Vaidya. 31

Comparison of different tests used in the study
In the recent years, a number of tests have been introduced to detect bacteria and leucocytes in urine. Urine culture, which remains the gold standard for UTI diagnosis, takes 24 to 48 hours to yield a confirmatory report. Clinicians require speedy results which are accurate. Therefore, a number of rapid urine screening methods have been tried. Berger and Bianca (1990) are of the opinion that these rapid urine screening methods have often traded specificity for sensitivity and simplicity. 26 In the present study, LE test showed the highest sensitivity (92.7%) followed by nitrite test (88.7%), wet mount for pus cells (87.1%), gram staining (85.5%) and wet mount for bacteria (82.3%). The TTC test had the lowest sensitivity (79.8%). Thus the LE cell appeared to be relatively more useful in detecting UTI cases. However, taking into consideration, the cost factor of the LE test, in a setting like ours, one would resort to nitrite test, wet mount for pus cells or gram staining.
Although the nitrite test appeared to be marginally better, in terms of sensitivity, when compared with pus cells wet mount and gram staining, it is impertinent to obtain first morning sample of urine for the nitrite test. This will allow sufficient time for the enzyme production and obviate false negative result as stated by Smith and Thayer. 23 Gram staining, on the other hand, allows presumptive identification of the type of organism responsible for UTI, based on the morphology and staining properties. This information can be made available to the clinician to choose the appropriate antibiotic before obtaining the culture result.
The specificities of LE dip stick test, Griess Nitrite test, gram staining, wet mount for bacteria and wet mount for pus cells were comparable i.e. 90.8%, 86.8%,89.5%,86.8% and 85.5% respectively. This indicates that the tests picked up true positives in relatively similar frequency. The result from the present study indicates that the LE dipstick test is the most sensitive and specific test for screening cases of bacteriuria and UTI. Although, the test is rapid and simple, it is not cost effective and cannot be undertaken as a routine.
In conclusion, the present study evaluated bacteriological culture analysis of 200 urine samples from clinically suspected cases of urinary tract infections, along with rapid screening tests for diagnosis.

1.
Culture analysis by semi-quantitative loop method was as follows:a) Significant bacteriuria was seen in 62% cases (124/200).