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The Nitrite Reaction as an Indicator of Urinary Infection1
THE NITRITE REACTION AS AN INDICATOR OF URINARY INFECTION 1 ABRAHAM HYMAN
AND
LEWIS T. MANN
From the service of Dr. Edwin Beer, Mount Sinai Hospital, New York
The purpose of this contribution is to present a simple method for the rapid determination of infection of the urinary tract, anq one especially applicable to use in the office. This test, which depends upon the finding of nitrites in the urine, has proved of value in a quite extensive use abroad since its advancement in 1925 by Weltmann and Haslinger. They used the Ilosvay modification of the well-known Griess test for nitrites as applied to the determination of the potability of a water supply. The presence of nitrites in the water is an indication of pollution with products of the action of bacteria, generally the colon bacillus, on human or animal excreta. If not acted upon by nitrite forming bacteria, the urine is normally free from nitrites per se. A freshly excreted specimen, if uninfected, contains only nitrates, but ferments if allowed to stand in a contaminated container with the production of nitrites. If such a urine is preserved under sterile precautions, or if the acidity of the urine is so great as to inhibit bacterial growth below pH 5, no nitrite will be formed, as proven by a negative test. A positive nitrite reaction in an infected urine depends upon the presence of nitrites formed from nitrates normally present, by the action of certain nitrite forming bacteria (B. coli, staphylococcus, pyocyaneous, etc.-the cause of the majority of infections of the urinary tract). Those that will not form nitrites are gonococci, streptococci, and tubercle bacilli, the latter two being rare as infective agents of the urinary tract. 1 Read before the Genito-Urinary Section of the New York Academy of Medicine, December 21, 1927. 521
522
ABRAHAM HYMAN AND LEWIS T. MANN
The following is a description of the preparation of the reagent: I. Dissolve 0.5 gram of sulphanilic acid in 150 cc. of 10 per cent acetic acid. 2. Dissolve 0.2 gram of alpha-naphthylamine in 20 cc. of boiling water, and filter into 150 cc. of 10 per cent acetic acid. 3. Mix solutions 1 and 2. Store in a brown bottle.
This solution should be colorless, and in case it changes to pink on standing, it should be discarded. It is advisable to test it against a dilute sodium nitrite solution, adding equal quantities of each, which will give a deep pink color if the reagent is satisfactory. The technique of the test is very simple. The urine must be freshly voided into a sterile chemically clean container. As a normal urine contains only nitrates, and as a container that is not sterile may contain nitrite forming bacteria, this precaution must be strictly adhered to, as nitrite will be formed from the nitrates and we may fall into error. The specimen of urine having been properly obtained, we add to 2 cc. of the urine an equal quantity of the reagent. If there are nitrite forming bacteria, a pink color develops immediately in a majority of instances. The presence of such bacteria can be proven by culturing a urine giving a positive reaction. There may be very little pus in such a urine, which will be slightly hazy and show but a few pus cells microscopically; nevertheless a positive reaction means infection with nitrite forming bacteria. Occasionally on adding the reagent one gets a red color, which fades out immediately. If more reagent is added in excess, the red color will be permanent. This condition, f,o und in a urine which has gone along to an advanced stage of fermentation, indicates that nitrites are present in very small amounts and will disappear shortly. The reaction of the urine is of no importance, as the acetic acid in the reagent assures one of an acid reaction. In phosphaturia the cloudiness of the urine disappears as the precipitated salts dissolve, and no interference is found with the test. There are a few exceptions to obtaining an immediate positive test, when freshly passed infected urine will give no color reaction. First, the urine may be infected with non-nitrite forming bacteria.
NITRITE REACTION AS INDICATOR OF INFECTION
523
If streptococci, gonococci, or tubercle bacilli are the agents
causing infection, we will get a negative test. The presence of gonococci is easily determined by smear in most cases, as the infection is rare in the upper urinary tract and an urethral discharge is generally present. Infection by streptococcus is rare in the urinary tract. A negative test with a tuberculous infection is of diagnostic value, but the fact that a mixed infection with a positive test may be present must not be overlooked. Second, the urine may be sterile although it contains pus. Third, the urine, although infected with nitrite forming bacteria, due to a very great urinary frequency may not contain sufficient nitrite to give a positive test because there is not sufficient time between urinations for the conversion of nitrates into nitrites. Fourth, in a badly infected urine, the fermentation may go beyond the nitrite stage and too little nitrite be left to give a positive test. Therefore if we get a negative test with an evidently infected urine, the following modifications may be used. To get a positive test when there is great frequency of urination, add the reagent to the urine and stand the mixture in an incubator for two hours. A positive test then will prove that the urine was originally infected with nitrite forming bacteria, since a non-infected urine which has become contaminated by being collected under non-sterile precautions will give a positive reaction at the earliest only after eight hours. The time interval between the two is great enough to obviate the possibility of an error. If a urine is apparently very badly infected and gives a negative reaction, a few drops of a 10 per cent sodium nitrate solution should be added along with the reagent, and this mixture incubated for one-half to one hour. In the presence of nitrite forming bacteria, we will then get a positive reaction. If a urine gives a negative reaction after these supplementary tests have been done, it is either sterile or is infected with nonnitrite forming organisms, which can be checked up by bacteriological examinations. As the commonest infecting organism by far is the colon bacillus, the test described dispenses with bac-
524
ABRAHAM HYMAN AND LEWIS T. MANN
teriological examinations in a majority of cases, as results are obtained within two hours. From a clinical consideration, the greatest diagnostic importance of a positive test is in a case suspected of renal infection, such as an acute febrile condition with pain in the right side of the abdomen. In such an instance one must always consider the existence of a colon bacillus pyelitis, especially in a female. Ordinarily, bacteriological and microscopic examination of the urine would be done as an aid in making a diagnosis. Here the situation may be quickly cleared up without such involved and time-consuming methods, by testing for nitrites in the urine. Weltmann and Haslinger state that they have never observed an error in the diagnosis of acute B . coli pyelitis by this method. The method has especial merit as an office test of pyelitis in children. As is well known, the urine in this type of infection may contain no pus, but cultures will show the presence of colon bacilli. With this test, the presence of organisms can be determined within a few minutes. Richard Wildmann applied this test as an aid in the differential diagnosis between "cysto-pyelitis" and other conditions accompanied by fever, in infants and young children. He had 30 negative controls between the ages of twelve days and fourteen months, 99 children with febrile diseases other than "cysto-pyelitis," and 22 of the latter. Of the 99 children suffering from disease other than " cystopyelitis," 12 had infected urine which gave a positive reaction. Four gave a positive nitrite reaction, but the culture of the urine was sterile. Eighty-three gave a negative reaction with a negative culture. Of the 22 cases of "cysto-pyelitis," 17 gave a positive nitrite reaction, with a positive culture of B. coli. One case cultured streptococci, with a negative reaction which later became positive when B. coli and staphylococci were found on culture. One case cultured staphylococcus albus, and gave a negative nitrite reaction. One case had pus in the urine, but gave both a negative reaction and no growth. In 3 cases, there was an initial positive reaction, with a positive culture of B. coli, which later was negative when the culture became sterile.
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NITRITE REACTION AS INDICATOR OF INFECTION
525
We have applied the test in 2 cases of bacilluria, and like Weltmann and Haslinger have obtained prompt positive reactions for nitrite in this condition. In cases where the urine was so slightly hazy as to leave one in doubt as to whether there was an infection present, we have obtained a positive reaction. Microscopically this urine showed but little or no pus, but culture proved the presence of colon bacilli, which checked the test. The test is also of importance in an antero-posterior gonorrhea with little or no discharge, where no gonococci are demonstrable on smear but where there is a very purulent second urine. The presence of a B. coli as a secondary invader can very quickly be ascertained by applying the nitrite test. The following are the results obtained in our application of the test: Urines examined .................... ... .. . . . ..... ....... . . ........ 100 Negative controls ........... .. ... . ................ . ........ 22 (Of which 4 had no p athological lesion of the urinary tract) Positive urines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 (A) Posterior gonorrhea with cloudy urine .. .. . ...... 12 Negative ... . ...... .. . . . ... . . ... .. .. .... 11 Positive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Culture B. coli as secondary invader (B) Tuberculosis of kidney and bladder ....... . .... , 9 Negative .... .. . 5 (culture sterile) Positive .. .... . . 4 (culture B. coli) (C) Streptococcus ...... ..... ....................... 2 Both negative 1 Prostatitis with cloudy urine (Culture Streptococcus anhemolyticus) ! Pyelitis with hazy urine (Culture Streptococcus viridans) (D) 55 Cases on culture showed the presence of nitrite forming bacteria, as follows: 46 B. coli 2 B . proteus 2 B . pyocyaneus Staphylococcus aureus Immediately positive ... . .... . .... 44 Positive after incubation . .... ... 10
.±
There was a great variety of cases in this group, including bladder and renal infections with and without stone; and new growths, one carcinoma of the prostate with cystitis, and one cord bladder with infection.
526
ABRAHAM HYMAN AND LEWIS T. MANN
There were 7 cases with clear urine which on microscopical examination showed but few pus cells and which on culture were found to be infected with colon bacilli. These 7 all gave positive reactions. One urine (the 55th case) on culture showed a mixture of B. coli and B. pyocyaneus, and gave a negative reaction. It was incubated, and still gave a negative reaction; but the specimen in this case was very acid (uric acid showers) and perhaps this inhibited the action of the bacteria. No doubt dilution of the urine with an equal quantity of sterile water would have aided matters in this instance. CONCLUSION
As far as we have been able to determine from a study of 100 cases, of which 78 were infections of the urinary tract, this test seems to offer a rapid and accurate method for the determination of nitrite forming organisms in infection of the urinary tract, there being but 1 case with a positive culture of nitrite forming bacteria giving a negative test. REFERENCES (1) WELTMANN AND HASLINGER, K.: Die Nitritreaktion im Harn als Indicator der Haminfektion. Ztschr. f. urol. Chir., 1925, xviii, 73. (2) HASLINGER, K.: Der Wert der Alpha-Nephthylaminprobe zur Erkennung von Infektionen der Niere. Ztschr. f. Urologie, 1927, xxi, 647. (3) WILDMANN, RICH;ARD: Uber den Wert der Griesschen Farbenreaktion auf Nitrite in frischgelassenem Harn fiir die Diagnose der Zystopyelitis in Kindesalter. Arch. f. Kinderheil., 1925, lxxv, 185.
AND
LEWIS T. MANN
From the service of Dr. Edwin Beer, Mount Sinai Hospital, New York
The purpose of this contribution is to present a simple method for the rapid determination of infection of the urinary tract, anq one especially applicable to use in the office. This test, which depends upon the finding of nitrites in the urine, has proved of value in a quite extensive use abroad since its advancement in 1925 by Weltmann and Haslinger. They used the Ilosvay modification of the well-known Griess test for nitrites as applied to the determination of the potability of a water supply. The presence of nitrites in the water is an indication of pollution with products of the action of bacteria, generally the colon bacillus, on human or animal excreta. If not acted upon by nitrite forming bacteria, the urine is normally free from nitrites per se. A freshly excreted specimen, if uninfected, contains only nitrates, but ferments if allowed to stand in a contaminated container with the production of nitrites. If such a urine is preserved under sterile precautions, or if the acidity of the urine is so great as to inhibit bacterial growth below pH 5, no nitrite will be formed, as proven by a negative test. A positive nitrite reaction in an infected urine depends upon the presence of nitrites formed from nitrates normally present, by the action of certain nitrite forming bacteria (B. coli, staphylococcus, pyocyaneous, etc.-the cause of the majority of infections of the urinary tract). Those that will not form nitrites are gonococci, streptococci, and tubercle bacilli, the latter two being rare as infective agents of the urinary tract. 1 Read before the Genito-Urinary Section of the New York Academy of Medicine, December 21, 1927. 521
522
ABRAHAM HYMAN AND LEWIS T. MANN
The following is a description of the preparation of the reagent: I. Dissolve 0.5 gram of sulphanilic acid in 150 cc. of 10 per cent acetic acid. 2. Dissolve 0.2 gram of alpha-naphthylamine in 20 cc. of boiling water, and filter into 150 cc. of 10 per cent acetic acid. 3. Mix solutions 1 and 2. Store in a brown bottle.
This solution should be colorless, and in case it changes to pink on standing, it should be discarded. It is advisable to test it against a dilute sodium nitrite solution, adding equal quantities of each, which will give a deep pink color if the reagent is satisfactory. The technique of the test is very simple. The urine must be freshly voided into a sterile chemically clean container. As a normal urine contains only nitrates, and as a container that is not sterile may contain nitrite forming bacteria, this precaution must be strictly adhered to, as nitrite will be formed from the nitrates and we may fall into error. The specimen of urine having been properly obtained, we add to 2 cc. of the urine an equal quantity of the reagent. If there are nitrite forming bacteria, a pink color develops immediately in a majority of instances. The presence of such bacteria can be proven by culturing a urine giving a positive reaction. There may be very little pus in such a urine, which will be slightly hazy and show but a few pus cells microscopically; nevertheless a positive reaction means infection with nitrite forming bacteria. Occasionally on adding the reagent one gets a red color, which fades out immediately. If more reagent is added in excess, the red color will be permanent. This condition, f,o und in a urine which has gone along to an advanced stage of fermentation, indicates that nitrites are present in very small amounts and will disappear shortly. The reaction of the urine is of no importance, as the acetic acid in the reagent assures one of an acid reaction. In phosphaturia the cloudiness of the urine disappears as the precipitated salts dissolve, and no interference is found with the test. There are a few exceptions to obtaining an immediate positive test, when freshly passed infected urine will give no color reaction. First, the urine may be infected with non-nitrite forming bacteria.
NITRITE REACTION AS INDICATOR OF INFECTION
523
If streptococci, gonococci, or tubercle bacilli are the agents
causing infection, we will get a negative test. The presence of gonococci is easily determined by smear in most cases, as the infection is rare in the upper urinary tract and an urethral discharge is generally present. Infection by streptococcus is rare in the urinary tract. A negative test with a tuberculous infection is of diagnostic value, but the fact that a mixed infection with a positive test may be present must not be overlooked. Second, the urine may be sterile although it contains pus. Third, the urine, although infected with nitrite forming bacteria, due to a very great urinary frequency may not contain sufficient nitrite to give a positive test because there is not sufficient time between urinations for the conversion of nitrates into nitrites. Fourth, in a badly infected urine, the fermentation may go beyond the nitrite stage and too little nitrite be left to give a positive test. Therefore if we get a negative test with an evidently infected urine, the following modifications may be used. To get a positive test when there is great frequency of urination, add the reagent to the urine and stand the mixture in an incubator for two hours. A positive test then will prove that the urine was originally infected with nitrite forming bacteria, since a non-infected urine which has become contaminated by being collected under non-sterile precautions will give a positive reaction at the earliest only after eight hours. The time interval between the two is great enough to obviate the possibility of an error. If a urine is apparently very badly infected and gives a negative reaction, a few drops of a 10 per cent sodium nitrate solution should be added along with the reagent, and this mixture incubated for one-half to one hour. In the presence of nitrite forming bacteria, we will then get a positive reaction. If a urine gives a negative reaction after these supplementary tests have been done, it is either sterile or is infected with nonnitrite forming organisms, which can be checked up by bacteriological examinations. As the commonest infecting organism by far is the colon bacillus, the test described dispenses with bac-
524
ABRAHAM HYMAN AND LEWIS T. MANN
teriological examinations in a majority of cases, as results are obtained within two hours. From a clinical consideration, the greatest diagnostic importance of a positive test is in a case suspected of renal infection, such as an acute febrile condition with pain in the right side of the abdomen. In such an instance one must always consider the existence of a colon bacillus pyelitis, especially in a female. Ordinarily, bacteriological and microscopic examination of the urine would be done as an aid in making a diagnosis. Here the situation may be quickly cleared up without such involved and time-consuming methods, by testing for nitrites in the urine. Weltmann and Haslinger state that they have never observed an error in the diagnosis of acute B . coli pyelitis by this method. The method has especial merit as an office test of pyelitis in children. As is well known, the urine in this type of infection may contain no pus, but cultures will show the presence of colon bacilli. With this test, the presence of organisms can be determined within a few minutes. Richard Wildmann applied this test as an aid in the differential diagnosis between "cysto-pyelitis" and other conditions accompanied by fever, in infants and young children. He had 30 negative controls between the ages of twelve days and fourteen months, 99 children with febrile diseases other than "cysto-pyelitis," and 22 of the latter. Of the 99 children suffering from disease other than " cystopyelitis," 12 had infected urine which gave a positive reaction. Four gave a positive nitrite reaction, but the culture of the urine was sterile. Eighty-three gave a negative reaction with a negative culture. Of the 22 cases of "cysto-pyelitis," 17 gave a positive nitrite reaction, with a positive culture of B. coli. One case cultured streptococci, with a negative reaction which later became positive when B. coli and staphylococci were found on culture. One case cultured staphylococcus albus, and gave a negative nitrite reaction. One case had pus in the urine, but gave both a negative reaction and no growth. In 3 cases, there was an initial positive reaction, with a positive culture of B. coli, which later was negative when the culture became sterile.
-
- -- - - - ~
NITRITE REACTION AS INDICATOR OF INFECTION
525
We have applied the test in 2 cases of bacilluria, and like Weltmann and Haslinger have obtained prompt positive reactions for nitrite in this condition. In cases where the urine was so slightly hazy as to leave one in doubt as to whether there was an infection present, we have obtained a positive reaction. Microscopically this urine showed but little or no pus, but culture proved the presence of colon bacilli, which checked the test. The test is also of importance in an antero-posterior gonorrhea with little or no discharge, where no gonococci are demonstrable on smear but where there is a very purulent second urine. The presence of a B. coli as a secondary invader can very quickly be ascertained by applying the nitrite test. The following are the results obtained in our application of the test: Urines examined .................... ... .. . . . ..... ....... . . ........ 100 Negative controls ........... .. ... . ................ . ........ 22 (Of which 4 had no p athological lesion of the urinary tract) Positive urines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 (A) Posterior gonorrhea with cloudy urine .. .. . ...... 12 Negative ... . ...... .. . . . ... . . ... .. .. .... 11 Positive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Culture B. coli as secondary invader (B) Tuberculosis of kidney and bladder ....... . .... , 9 Negative .... .. . 5 (culture sterile) Positive .. .... . . 4 (culture B. coli) (C) Streptococcus ...... ..... ....................... 2 Both negative 1 Prostatitis with cloudy urine (Culture Streptococcus anhemolyticus) ! Pyelitis with hazy urine (Culture Streptococcus viridans) (D) 55 Cases on culture showed the presence of nitrite forming bacteria, as follows: 46 B. coli 2 B . proteus 2 B . pyocyaneus Staphylococcus aureus Immediately positive ... . .... . .... 44 Positive after incubation . .... ... 10
.±
There was a great variety of cases in this group, including bladder and renal infections with and without stone; and new growths, one carcinoma of the prostate with cystitis, and one cord bladder with infection.
526
ABRAHAM HYMAN AND LEWIS T. MANN
There were 7 cases with clear urine which on microscopical examination showed but few pus cells and which on culture were found to be infected with colon bacilli. These 7 all gave positive reactions. One urine (the 55th case) on culture showed a mixture of B. coli and B. pyocyaneus, and gave a negative reaction. It was incubated, and still gave a negative reaction; but the specimen in this case was very acid (uric acid showers) and perhaps this inhibited the action of the bacteria. No doubt dilution of the urine with an equal quantity of sterile water would have aided matters in this instance. CONCLUSION
As far as we have been able to determine from a study of 100 cases, of which 78 were infections of the urinary tract, this test seems to offer a rapid and accurate method for the determination of nitrite forming organisms in infection of the urinary tract, there being but 1 case with a positive culture of nitrite forming bacteria giving a negative test. REFERENCES (1) WELTMANN AND HASLINGER, K.: Die Nitritreaktion im Harn als Indicator der Haminfektion. Ztschr. f. urol. Chir., 1925, xviii, 73. (2) HASLINGER, K.: Der Wert der Alpha-Nephthylaminprobe zur Erkennung von Infektionen der Niere. Ztschr. f. Urologie, 1927, xxi, 647. (3) WILDMANN, RICH;ARD: Uber den Wert der Griesschen Farbenreaktion auf Nitrite in frischgelassenem Harn fiir die Diagnose der Zystopyelitis in Kindesalter. Arch. f. Kinderheil., 1925, lxxv, 185.