Diagnosis of spontaneous bacterial peritonitis in cirrhotic patients in northeastern Brazil by use of rapid urine-screening test

ABSTRACT CONTEXT AND OBJECTIVE: Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of cirrhotic patients with ascites. It has been proposed that the reagent strip for leukocyte esterase designed for the testing of urine (Combur test® UX) could be a useful tool for diagnosing SPB. The aim of this study was to assess the sensitivity and specificity of urine test strips for diagnosing SBP in cirrhotic patients with ascites. DESIGN AND SETTING: Prospective study, at a university hospital in northeastern Brazil. METHODS: Forty-two unselected consecutive cirrhotic patients (32 males; mean age: 51.7 ± years) were included, and a total of 100 paracenteses were performed. All ascitic fluid samples were analyzed using the reagent strip and cytology, neutrophils, lymphocyte count, appropriate biochemical tests and culturing. The strips were considered positive if the color became purple on a colorimetric scale. RESULTS: Nine patients were diagnosed with SBP using cytology (> 250 neutrophils/mm3), and the strips were positive for all these nine patients with SBP. In one sample, the strip was positive but the neutrophil count was less than 250 cells/mm3. For 86 samples, both the strips and cytology were negative. At the threshold of 250 neutrophils/mm3 in ascitic fluid, the sensitivity, specificity, positive predictive value and negative predictive value for the strips were respectively 100%, 98.9%, 92.3% and 100%. CONCLUSION: The Combur test® UX urine screening test is a very sensitive and specific method for diagnosing SBP in cirrhotic patients with ascites.


INTRODUCTION
Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of cirrhotic patients with ascites.7][8][9] Improved survival in SBP episodes might be obtained through rapid diagnosis and treatment.
1][12][13][14][15] SBP is highly likely when the PMN cell count in the ascitic fl uid reaches a cutoff of 250/mm 3 . 10Once this cut-off has been reached, antibiotic therapy must be started immediately, without waiting for a culture from the ascitic fl uid.However, total and PMN leukocyte counts from the ascitic fl uid are not always available everywhere, or it may be impossible to obtain them in emergency situations.
Reagent strip testing for leukocyte esterase has been found to be a sensitive and accurate predictor for the presence of PMN in body fl uids such as urine, [16][17][18] cerebrospinal fl uid, seminal and peritoneal fl uid. 19This test is based on the esterase activity of granulocytes present in the biological fl uid, which reacts with a chemical compound on the reagent strip to cause a color change in the azo dye (purple).
It has been proposed that reagent strip testing for leukocyte esterase could be utilized to reduce the time between performing paracentesis and obtaining a presumptive diagnosis of SBP from a few hours to a few seconds.Moreover, such strips would be available everywhere, and could be a useful tool for diagnosing SBP, especially in developing countries.Therefore, it seems very important to validate the use of such reagent strips in a country like Brazil, and especially in the northeast of this country.

OBJECTIVE
The aim of this prospective study was to evaluate the accuracy of reagent strips for rapidly diagnosing SBP in cirrhotic patients with ascites in the university hospital in Fortaleza, State of Ceará, Brazil.

Patients Patients
This study was carried out in Hospital Universitário Walter Cantídio between March and August 2004 and it was approved by the Ethics Committee of Hospital Universitário Walter Cantídio, Universidade Federal do Ceará.Forty-two consecutive unselected cirrhotic patients (32 males, 10 females, mean age 51.7 years) were included and a total of 100 paracenteses were performed.The diagnosis of cirrhosis was established by histological criteria or by clinical criteria (splenomegaly, ascites and/or esophageal varices) and ultrasonography fi ndings.All these patients had evidence of advanced liver disease: 23 patients (54.7%) were in Child-Pugh class B and 19 patients (45.3%) in Child-Pugh class C. The cirrhosis was related to alcoholism in 27 patients (64.2%), and to chronic hepatitis B or C in six patients (14.2%).The characteristics of the study population are summarized in Table 1.
The diagnosis of SBP was defi ned as a PMN count in the ascitic fl uid greater than 250/mm 3 , in the absence of a contiguous source of intra-abdominal infection, with or without a positive culture, 10 and after excluding other causes of elevated PMN in ascitic fl uid such as tuberculosis, peritoneal carcinomatosis or pancreatitis.Antibiotic therapy was started empirically in all cirrhotic patients CONTEXT AND OBJECTIVE: Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of cirrhotic patients with ascites.It has been proposed that the reagent strip for leukocyte esterase designed for the testing of urine (Combur test ® UX) could be a useful tool for diagnosing SPB.The aim of this study was to assess the sensitivity and specifi city of urine test strips for diagnosing SBP in cirrhotic patients with ascites.

DESIGN AND SETTING:
Prospective study, at a university hospital in northeastern Brazil.

METHODS:
Forty-two unselected consecutive cirrhotic patients (32 males; mean age: 51.7 ± years) were included, and a total of 100 paracenteses were performed.All ascitic fl uid samples were analyzed using the reagent strip and cytology, neutrophils, lymphocyte count, appropriate biochemical tests and culturing.The strips were considered positive if the color became purple on a colorimetric scale.

RESULTS:
Nine patients were diagnosed with SBP using cytology (> 250 neutrophils/mm 3 ), and the strips were positive for all these nine patients with SBP.In one sample, the strip was positive but the neutrophil count was less than 250 cells/mm 3 .For 86 samples, both the strips and cytology were negative.At the threshold of 250 neutrophils/mm 3 in ascitic fl uid, the sensitivity, specifi city, positive predictive value and negative predictive value for the strips were respectively 100%, 98.9%, 92.3% and 100%.

CONCLUSION:
The Combur test ® UX urine screening test is a very sensitive and specifi c method for diagnosing SBP in cirrhotic patients with ascites.kocyte count were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant.Cytological examinations and differential cell counts were performed using a conventional optical microscope.
Culturing of ascitic fl uid was performed by inoculating 5 ml of ascitic fl uid into 3 ml of BHI (brain heart infusion), and into Mac-Conkey agar and chocolate agar.

Combur Test Combur Test ®
Immediately after the paracentesis, the ascitic fl uid was tested by using a reagent strip for leukocyte esterase designed for the testing of urine (Combur Test ® UX; Roche Diagnostics GmbH, D-68298 Mannheim, Germany).The procedure utilized was the same as described by the manufacturer for urine.Briefl y, the sample of fresh ascitic fl uid was collected in a clean, dry container.All reagent areas were immersed in the ascitic fl uid and the strip was removed immediately.The strip was read by the physician who carried out the paracentesis (who was unaware of the results from the cytological and bacteriological tests).The color of the leukocyte reagent area was then compared with the color chart on the bottle, and the result was scored on a colorimetric four-grade scale (0-3).The correlation between PMN and the four-grade scale suggested by the manufacturer was as follows: grade 0: 0 PMN/mm 3 ; grade 1: 25 PMN/mm 3 ; grade 2: 75 PMN/ mm 3 ; grade 3: 500 PMN/mm 3 .The reagent strip was considered positive if the color turned to purple, i.e. if the grade was 2 or 3. We considered the strip positive when it showed grade 2 or 3 on the basis of previous studies that demonstrated that the sensitivity of the test improved with this cutoff. 20,21Since SPB is an infectious disease with high mortality, it is important to have a test with high sensitivity.

Statistical analysis Statistical analysis
Quantitative variable are expressed as the mean ± standard deviation (SD).The sensitivity, specifi city, positive predictive value and negative predictive value were determined.

RESULTS
One hundred paracenteses were performed on 42 cirrhotic patients from March 2004 to August 2004 in our primary referral hospital.
Sixty-fi ve paracenteses were performed routinely in an outpatient setting to investigate refractory ascites, and 35 during classical hospitalization.Paracenteses were systematically performed on 27 asymptomatic inpatients to investigate new onset of ascites and to confi rm symptoms suggestive of peritoneal infection in the other eight inpatients.The number of paracenteses per patient ranged from one to eight, with an average of two.
During the study period, SPB was diagnosed in nine patients by cytology (> 250 neutrophils/mm 3 ); however, only three of these nine ascitic fl uid cultures grew.The test with the strips (Combur Test ® UX) was positive (colorimetric scale 2 or 3) for all nine patients with SBP (Table 2).The paracentesis was repeated after 48 hours on antibiotics, for four patients with SBP.In three cases, the neutrophil count remained above 250/mm 3 and the strips were also positive: in these cases, the antibiotics were changed.In the other case, the neutrophil count was below 250/mm 3 and the strip was negative.In fi ve patients with SBP, it was not possible to collect ascitic fl uid after 48 hours on antibiotics, because one patient died, two patients no longer had any ascitic fl uid, and two patients abandoned the treatment.
In one sample, the test with the strip was positive but the PMN count was below 250/mm 3 .In this case, the ascitic fl uid cul ture was also negative.In the other 86 samples of ascitic fl uid, the neutrophil counts were below 250/mm 3 and the strip tests were negative in all cases.
At the threshold of 250 PMN/mm 3 in ascitic fl uid, the sensitivity, specifi city, positive predictive value and negative predictive value were respectively 100%, 98.9%, 92.3% 100%, as shown in Table 3.

DISCUSSION
This prospective study confi rmed the high accuracy of reagent strips for the diagnosing of SBP in cirrhotic patients with ascites.Our  with an ascitic fl uid PMN cell count greater than 250/mm 3 and consisted of 5-10 days of ceftriaxone (2 g/day). 10One follow-up paracentesis was performed after two days of antibiotic therapy to determine the PMN count in the ascitic fl uid.

Paracentesis Paracentesis
Paracentesis was performed under aseptic conditions in the cirrhotic patients at the time of admission to hospital, or routinely in an outpatient setting, to investigate ascites.Three samples of ascitic fl uid were obtained from each patient.Ascitic fl uid was classically processed including investigation of cytology, PMN leukocytes, lymphocyte count and appropriate biochemical tests.The ascitic samples for PMN leukocytes and total leu- and specifi city (89%) for detecting SBP in cirrhotic patients with ascites. 20In another study, Théovenot et al. tested the reagent Combur-2 test ® LN, and showed a sensitivity of 89% and a specifi city of 100%. 21n the present study, one false positive result was found.Despite the fact that a false positive result could indicate antibiotic therapy for a patient without SBP, this therapy would be discontinued if the PMN count were below 250 cell/mm 3 .Considering the high mortality from SBP episodes, a method with high sensitivity is essential for the management of these patients.
The reagent strips are easy to use, do not require expertise, are rapid, can be performed everywhere, and have high sensitivity and specifi city for diagnosing of SBP, thereby allowing antibiotic therapy to immediately start.Another noteworthy point is the low cost of hospitals in our country have limited laboratory facilities, or are unable to perform PMN counts in ascitic fl uid at night or over the weekend.Considering the mortality from SBP, this test will help to improve the management of SBP.

CONCLUSIONS
In summary, this is the first study using this low-cost method for diagnosing SBP in northeastern Brazil.Our results are comparable with those reported previously, thus demonstrating that this accurate method could be used everywhere, thereby reducing the time from paracentesis to a presumptive diagnosis of SBP from a few hours to a few seconds.these strips (R$ 0.15 per strip).This new diagnostic method is also useful for determining the effectiveness of the antibiotic therapy.In fact, there were three SBP episodes in which we observed that the PMN count remained above 250/mm 3 after 48 hours of ceftriaxone therapy, and these positive results were always confi rmed by the PMN cell count.
The incidence of SBP in our study was 10.4%, which was lower than previously reported. 10This difference can be explained by the greater number of outpatients in our study (82%).
In the present study, only three positive cultures were observed among the nine cases of SBP (33%).It is possible that this low rate was due to suboptimal culturing methods and techniques.However, like what was found in this study, low rates of positive SBP cultures have been reported in other studies, with proportion ranging from 39% to 59%, 12,[23][24][25] and these fi ndings probably refl ect earlier diagnosis of the infection.All three patients for whom the cultures were positive were inpatients and had advanced liver disease, and in one of them Pseudomonas aeruginosa was isolated, a germ that is resistant to ceftriaxone.The source of the SBP seems to have to the therapy planning, since nosocomial organisms are usually more resistant to antibiotics than those acquired in the community.Therefore, it is worthwhile culturing ascitic fl uid in inpatients with advanced diseases.

Table 1 .
Characteristics of 42 patients with cirrhosis who underwent 100 paracenteses SD = standard deviation.

Table 2 .
Details regarding of spontaneous bacterial peritonitis detected by cytology and strip test in nine cirrhotic patients

Table 3 .
Sensitivity, specifi city, positive and negative predictive value for the diagnosis of spontaneous bacterial peritonitis in 42 cirrhotic patients (100 paracenteses) by using the Combur Test ® UX reagent strip