Indian Pediatrics 1999;36: 887-890

An Indigenously developed nitrite kit to aid in the diagnosis of urinary tract infection

From the Departments of Microbiology and Pediatrics⁺. All India Institute of Medical Sciences, New Delhi 110 029, India and Center for Science Education and Communication*. Delhi University. Cavalary Lane, Delhi, India.
Reprint requests: Dr. Seema Sood, Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India.
Manuscript received: June 26, 1998; Initial review completed: August 21, 1998; Revision accepted: March 11, 1999.

Objective: To evaluate the utility of an indigenously developed nitrite kit for the rapid diagnosis of urinary tract infection (UTI) Methods: 1018 urine specimens were collected from all cases where there was clinical suspicion of UTI. Samples were cultured as per standard microbiological protocol. Presence of nitrites was indicated by the development of purple color on addition of color developing solution and compared with the set of graded positive and negative controls also provided in the Kit. Results: The results of the nitrite kit were compared with the semi-quantitative urine culture as the gold standard. The sensitivity, specificity, positive predictive and negative predictive values were 47%, 87%, 31% and 93%, respectively. Conclusion: Nitrite kit as a screening test can decrease the work load in the clinical bacteriology laboratory. More importantly in a field set up that is devoid of culture facilities, it can be used to correctly predict the absence of UTI.

Key words: Bacteriuria, Nitrite test, Urinary tract infection.

The diagnosis of urinary tract infection (UTI) is often difficult. A quantitative culture of the urine specimen is necessary for confirming the diagnosis of UTI. Facilities for urine culture are often not available. Also, about 70-80% of the urine samples received in the laboratory are found to be free from evidence of bacterial infection(1). Microscopic examination of fresh urine helps to make a diagnosis of UTI. It also reduces the work load of the clinical bacteriology laboratory(2). Apart from micro-scopy, urinalysis also provides information more rapidly than urine culture to decide whether the patient has UTI(3). Urinary nitrites produced by coliform bacteria which reducenitrates present in urine can be detected by quantitative and qualitative chemical methods. Dipstick analysis of the urine for the presence of nitrites and leukocyte esterase have been used to identify patients of UTI(3-7). These imported tests are neither available nor inexpensive to allow their routine use as a diagnostic test in India. In the present study results of urine nitrites using an indigenously developed diagnostic kit were compared to assess its reliability in detecting bacteriuria.

Material and Methods

We examined 1018 urine specimens submitted in sterile containers to the Microbiology services of All India Institute of Medical Sciences, New Delhi during July 1997 to October 1997. Approximately 20% of samples were from children. The samples were collected from all cases where there was clinical suspicion of urinary tract infection. All specimens were collected by clean catch midstream urine method and processed for culture and presence of nitrites within 60 minutes of collection.

Urine nitrites were measured with an indi-genously developed kit, developed at the Center for Science Education and Communication. One drop of urine was added into a well of a microtiter plate and mixed with one drop of color developing solution. The color developing solution was prepared by reconstituting 0.11 g sulphanilamide in phosphoric acid and 10 mg N-1-naphthylenediamine in equal volumes. The solution has a limited shelf life of 2 weeks at 4�C. The presence of nitrites was indicated by the development of a purple color. This could be semiquantitatively measured by comparison with a set of standards with increasing concentration of nitrites from 1 mg/ml to 1000 mg/ml as positive controls. For the purpose of analysis, a positive test was taken as a nitrite concentration of 1 mg/ml. No test was interpreted without comparing with the graded positive control and negative control that was put up with each run to minimize the observer error.

Urine was cultured using a 0.001 ml calibrated wire loop to inoculate CLED (Cystine Lactose Electrolyte Deficient) agar plates and incubated at 37�C for 24 hours. Isolates were identified and colony counts determined by standard microbiologic techniques. Significant bacteriuria was defined as greater than 10⁵ colony forming units/ml of a single pathogen. Colony counts between 10³-10⁴ cfu/ml of a single organism were considered as doubtful bacteriuria. In order to remove any bias in reporting, the results of nitrite test were not available to the observer who examined the culture plates.

The sensitivity, specificity, positive and negative predictive values of urinary nitrite for positive urine culture were calculated.

Results

Significant bacteriuria was found in 113 specimens (11.3%). The organisms isolated included Escherichia coli (39.8%), Klebsiella sp. (19.4%). Pseudomonas sp. (13.2%). Proteus sp. (8.8%), Enterococcus faecalis (7.07%), Citrobacter sp. (6.1%). Acinetobacter sp. (1.7%), Staphylococcus aureus (1.7%) and Coagulase negative Staphylococcus (1.7%). An additional 88 specimens (7.9%) showed doubtful bacteriuria. Of all the 113 specimens with >10⁵ cfu/ml, 53 (47%) showed positive nitrite test. Of the 88 specimens with 10³-10⁴ cfu/ml, 23 (26%) were positive for nitrite.

The sensitivity, specificity and predictive values of urinary nitrite in relation to bacteriuria are given in Table I. The sensitivity and specificity of the test were 47% and 87%, respectively. The negative predictive value of the test was 93%, therefore a negative nitrite test may correctly predict the absence of UTI in situations where the possibility of UTI is low.

Table I--Sensitivity,Specificity and Predictive Value(%) of Urinary Nitrite in Relation to Bacteriuria

Figure in parentheses indicate 95% CI (Confidence Interval).

Discussion

Detection of urinary nitrites has been used as a screening test for urinary tract infections. Nitrites in urine produce dinitrogen trioxide in contact with phosphoric acid which reacts with sulfanilimide to form a nitrosamine. This nitrosamine undergoes dehydration to produce diazonium salt which reacts with N-1 napthyl-ethylenediamine to produce purple color. The major problem with this test is the high number of false negative results(8). Its specificity is known to be good. We compared urine nitrite results with urine culture, the gold standard for the detection of bacteriuria. For specimens having colony counts of >10⁵ cfu/ml, we found the sensitivity, specificity and predictive values similar to those reported by others(6,7,9) as seen in Table II.

Table II^__Comparison of Sensitivity, Specificity and Predictive Values (%).

This kit was developed indigenously. The cost of performing a single test is 5 paise, compared to Rs 50-150 for a urine culture. The time required to perform this test is less than one minute as compared to 48 hours for urine culture. The kit is very handy and can be kept on the consultation table. It does not require very scrupulous collection of urine as is necessary for culture studies. Since production of nitrites require the bacteria to have been present in urine for a few hours, skin contaminants do not affect the results of the nitrite test. However, this does not underestimate the need for performing urine cultures in any patient with suspected UTI.

The utility of the nitrite test in clinical practice is limited by its low sensitivity. However, it's high specificity allows it to be used in a few clinical situations. First, the test is likely to be useful when symptoms of UTI are not very definite. A positive result may persuade the clinician. to send urine culture and start treatment with antibiotics presumptively. Similarly, if the nitrite test is positive in a patient with fever without localization, the clinician may avoid unnecessary evaluation for other infections. Third, in clinical settings where culture facilities are not available, this test may serve as a possible substitute.

As the sensitivity of the nitrite test is low, this test has limitations as a screening test. Certain reasons can result in a lower sensiti- vity. The causes for low sensitivity are low colony counts, frequent bladder emptying in children(10), high specific gravity(6) and low nitrates in diet. Also low sensitivity will occur in bacteria that do not produce nitrite, e.g., Streptococcus sp. and Acinetobacter sp. but it is important to remember that these are not common causes of UTI. This test is primarily for the community setting where the incidence of UTI by such organisms is very low as compared to the hospital setup. A negative nitrite test may correctly predict the absence of UTI (negative predictive value) in situations where the possibility of UTI is low. For example, the negative predictive value of our test is 98% when the prevalence of UTI is 5%. However, the negative predictive value drops to 64% if the prevalence of UTI is 50%.

Leukocyte esterase is specific to host's inflammatory response in the genitourinary tract and is not affected by conditions that cause nitrite tests to be false negative. It is suggested that a combination of both nitrites and leukocyte esterase can increase the sensitivity to reach nearly 100%. Dipstick LE and nitrites are commonly used to identify pediatric patients with positive urine cultures. Many studies suggest that routine use of dipstick tests could result in substantial cost saving by decreasing the need for more expensive microscopic urinalysis and culture(4,6,7).We are currently engaged in developing and evaluating such a combined diagnostic kit for UTI.

References

1. College JG, Duguid JP, Fraser AG, Marmion BP. Laboratory strategy in the diagnosis of infective syndromes. Macke and McCartney Practical Medical Microbiology, 13th edn, London, Churchill Livingstone, 1989; pp 600-649.

2. Sood S, Singh UB, Das BK, Kapil A, Rattan A, Kumar R. Acridine orange staining for bacteriuria screening. Indian J Med Microbiol 1995; 13: 200-203.

3. Craver RD, Abermanis JK. Dipstick. Only urinalysis screen for the pediatric emergency room.Pediatr Nephrol 1997; 11: 331-333.

4. Oneson R, Grosehel DHM. Leukocyte esterase activity and nitrite test as a rapid screen for significant bacteriuria. Am J Clin Pathol 1985; 83: 84-87.

5. Hoberman A, Wald ER, Penchansky L, Reynolds EA, Young S. Enhanced urinalysis as a screenng test for urinary tract infection. Pediatrics 1993; 91: 1196-1198.

6. Shaw KN, Hexter D, McGowan KI, Schwartz JS. Clinical evaluation of a rapid screening test for unriary tract infections in children. J Pediatr 1991; 118: 733-735.

7. Goldsmith BM, Campos JM. Comparison of urine dipstick, microscopy and culture for the detection of bacteriuria in children. Clin Pediatr 1990; 29: 214-218.

8. James GP, Paul KL, Fuller JB. Urinary nitrite and urinary tract infection. Am J Clin Pathol 1978; 70: 671-678.

9. Lohr JA, Portillia MG, Gender TG, Dunn ML, Dudley SM. Making a presumptive diagnosis of urinary tract infection by using urinalysis performed in an on-site laboratory. J Pediatr 1993; 122: 22-25.

10. Shaw ST, Poon SY, Wong Et. Routine urinalysis: Is dipstick enough? JAMA 1985; 253: 1596-1600.