Ascitic fluid analysis for diagnosis and monitoring of spontaneous bacterial peritonitis

Abstract

Polymorphonuclear (PMN) cell count in the ascitic fluid is essential for the diagnosis and management of spontaneous bacterial peritonitis (SBP). To date, PMN cell count is routinely performed by traditional manual counting. However, this method is time-consuming, costly, and not always timely available. Therefore, considerable efforts have been made in recent years to develop an alternative test for a more rapid diagnosis and monitoring of SBP. The use of urinary reagent strips was proposed to achieve an “instant” bedside diagnosis of SBP. A series of reports evaluated the urine strip test for SBP diagnosis and reported promising results. However, a recent large multicenter study revealed a surprising lack of diagnostic efficacy of the urine screening test for SBP diagnosis. Another method, more recently proposed as an alternative to the manual PMN count, is the measurement of lactoferrin in ascitic fluid, but the data available on the diagnostic value of this test are limited to a single study. However, both urinary reagent strips and ascitic lactoferrin tests are qualitative methods and need, therefore, to be further confirmed by standard cytology of the ascitic fluid. To date, the only quantitative method proposed as a valid alternative to manual PMN counting is automated blood cell counters, commonly used in all laboratories for blood cell counting. Data available in the literature on the diagnostic performance of this method are limited but very promising, and this tool seems to have the potential to replace the manual counting method.

Key Words: Ascites; Ascitic fluid lactoferrin; Automated blood cell counter; Paracentesis; Polymorphonuclear cell count; Urinary reagent strips

INTRODUCTION

Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of decompensated cirrhosis. Since its first description in the 1970s, when the mortality rate exceeded 80%, a significant and substantial improvement in the prognosis of SBP has been noted. In more recent prospective studies the mortality rate was reported to be around 20%[1–3]. The widespread use of diagnostic paracentesis, as well as the prompt initiation of empiric antibiotic therapy, based on the results of ascitic polymorphonuclear (PMN) cell count, has contributed to an improvement in the survival of these patients.

DIAGNOSIS AND TREATMENT OF SBP

The percentage of SBP in hospitalized cirrhotic patients with ascites ranges between 10% and 30%[4–9], whereas the prevalence of SBP among outpatients with cirrhosis is markedly lower (approximately 3.5%)[10]. Four practical guidelines and experts’ consensus reports on the diagnosis and management of SBP are currently available[11–14], all of which suggest that the diagnosis of SBP should be based on PMN cell count in the ascitic fluid. The detection of a PMN cell count greater than 250 cells/mm³ is highly suspicious of SBP and provides an indication to initiate empirical antibiotic treatment. Only a few SBP patients, in fact, have typical symptoms suggestive of peritoneal infection such as fever, abdominal pain and a high blood leukocyte count. More often, SBP is suspected when the patient develops signs of hepatic encephalopathy or a rapid impairment of renal function without any apparent precipitating factor. In addition, in a significant percentage of cases, SBP may be completely asymptomatic and the diagnosis is made only by diagnostic paracentesis. The presentation of SBP recently observed in a consecutive series of hospitalized patients[15] is reported in Table 1. Following hospitalization of any cirrhotic patient with newly diagnosed ascites, even in the absence of symptoms or if admitted for other reasons, a diagnostic paracentesis is therefore advised, aimed at investigating the presence of SBP. To rule out the possible presence of SBP, a paracentesis tap should also be repeated in all ascitic cirrhotic patients with gastrointestinal bleeding or hepatic encephalopathy, and whenever patients develop clinical symptoms and signs of infection, renal impairment or an unexplained worsening of their clinical condition[11].

Table 1 Signs and symptoms of SBP at the time of hospital admission in a recently published paper[15].

Patients/No. of episodes of SBP (32/38)	n
Clinical manifestations at hospital admission
Fever	12
Abdominal pain	6
Leukocytosis	11
Hepatic encephalopathy	8
Renal failure	14

Following the diagnosis of SBP, ascitic fluid collection and blood cultures should be carried out before initiating antibiotic administration. The commonest organisms isolated in patients with SBP include Escherichia coli, Gram-positive cocci (mainly Streptococcus species) and Enterococci. These organisms account for approximately 70% of all cases of SBP[1617]. Antibiotic therapy, however, should not be delayed to the moment when microbiological results are available; with conventional culture techniques, the ascitic fluid culture may be negative in up to 60% of patients with SBP[11]. Antibiotic therapy should instead be initiated empirically immediately after evidence of SBP at diagnostic paracentesis, without the need to wait for identification of the causative organism and in vitro susceptibility. Cefotaxime is the antibiotic most extensively investigated in patients with SBP[18–20], since it covers 95% of the flora isolated from the ascitic fluid and achieves high ascitic fluid concentrations during therapy[1221]. The dose of cefotaxime is 4-8 g/d given intravenously for a minimum of five days. Other studies[22–26] have evaluated the efficacy of cephalosporins (such as ceftriaxone and ceftazidime) administered once daily; although data are still limited, they appear to be as effective as cefotaxime. The association between amoxicillin and clavulanic acid also proved to be as effective as cefotaxime, with a similar security profile[2728]. In patients with uncomplicated SBP (i.e. no gastrointestinal bleeding, hepatic encephalopathy or renal failure), oral treatment with ofloxacin or other quinolones for eight days can be administered[29]. An intravenous → oral step-down schedule with ciprofloxacin (switch therapy) allows completion of the antibiotic treatment at home, once the patient is discharged. This regimen was recently shown to be as efficacious but more cost-effective than intravenous cefotaxime[30]. With the regimen described above, the resolution of SBP is achieved in approximately 90% of cases and 30-d survival is at least 80%[11].

The empirical antibiotic treatment chosen should be continued until symptom resolution or should be modified according to the results of the microbiological test. However, since SBP is often asymptomatic and the ascitic fluid culture outcome is often negative, once again the PMN cell count should be considered as a diagnostic tool to objectively demonstrate the efficacy of the ongoing antibiotic treatment and, finally, resolution of the infection. According to the International Ascites Club guidelines published in 2000[11], a diagnostic paracentesis should be repeated after two days during antibiotic therapy to assess treatment response. A PMN cell count reduction greater than 25% of the initial value was in fact suggested as the main criterion for establishing the efficacy of the antibiotic and the need for switching the therapy. This recommendation is very important, since there have been suggestions that the type and etiology of bacterial infections in cirrhosis may have changed during recent years[3132]. An increasing incidence of SBP caused by Gram-positive bacteria in cirrhotic patients with ascites has been observed by different authors[3334]. In addition, an increased frequency of bacteria resistant to multiple antibiotics was shown[35]. Our recent study[15] reported that, in clinical practice, a switch from cefotaxime to another antibiotic was necessary in more than 40% of SBP cases, thus supporting the possibility that the microbial etiology of SBP, as well as sensitivity to antibiotic therapy, is changing. A method aimed at objectively establishing resolution of the infection would be extremely useful; once again, the PMN cell count in the ascitic fluid obtained at diagnostic paracentesis was revealed to be the most reliable tool.

THE DETERMINATION OF PMN CELLS IN THE ASCITIC FLUID

Unfortunately, in clinical practice, ascitic PMN cell count is not always possible within a few hours, thus causing an unacceptable delay in the diagnosis and treatment of this potentially fatal infection. At present, PMN cell count is routinely performed by a traditional hematological method with a light microscope in a manual counting chamber. For this method, 10 mL of ascitic fluid are collected in tubes containing ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1500 r/min for 10 min. Nine milliliters of the supernatant are discharged and 40 μL of the remaining ascitic fluid are diluted with 800 μL of Turk’s fluid, gently shaken and used to fill the counting chamber. The cells are counted (40 × objective) in one of the nine large squares and the number of white blood cells per cubic millimeter calculated. Another 10 mL sample of ascitic fluid is used for the PMN percentage determination (100 × objective), after centrifugation and May Grünwald-Giemsa staining. This method is presently considered the “gold standard” for the evaluation of ascitic PMN count, but this procedure is laborious and time-consuming. This results in the laboratory often providing the PMN count too late in the day, or sometimes even on the next day, when the patient may have progressed from an early stage to a fatal stage of infection. Moreover, the manual method is not always timely available in all hospitals, especially in those small patient care units with limited laboratory facilities, and the method cannot frequently be performed on an emergency basis (at night or on week-ends). This manual system, therefore, often delays the initiation of antibiotic therapy and may expose patients to a higher risk of death.

On the basis of these considerations, considerable efforts have been made in recent years to develop an alternative test for a more rapid diagnosis of SBP. This represents an interesting and promising area of investigation, which could determine the further optimization of SBP management and further improvement in its prognosis. The use of urinary reagent strips-which identify leukocytes by detecting their esterase activity via a colorimetric reaction-was proposed to achieve an “instant” bedside diagnosis of SBP. Their employment was tested for the rapid diagnosis of bacterial meningitis, pleural or synovial infections, thus suggesting that they could also be used for the rapid diagnosis of SBP. The first study[36], published in 2002, involved 72 cirrhotic patients, nine of whom had SBP, reported a sensitivity and a specificity of 100% for the urinary strips in the diagnosis of SBP. After this successful result, in the following years a number of reports[37–51] evaluated the urine strip test for the diagnosis of SBP (Table 2). These studies reported promising and encouraging results, with the sensitivity of urinary strips for the diagnosis of SBP ranging between 85% and 100% and the specificity between 90 and 100%. These published studies were, however, limited by the small number of SBP episodes and were restricted to one or two centers. Therefore, the need to validate this method in a larger population of cirrhotic patients in a multicenter study arose. A prospective multicenter (70 centers) study with a total of 2123 paracenteses performed in 1041 patients was published in 2007 by Nousbaum et al[52]. Surprisingly, this paper did not confirm the good and promising results of the previous studies, but confirmed a very high specificity for reagent strips in the diagnosis of SBP, and revealed a poor sensitivity of this test (45%). This study, therefore, reported the lack of diagnostic efficacy of the urine screening test and the authors concluded that a routine cytological examination remains mandatory for the diagnosis of SBP. After initial great enthusiasm, the use of reagent strips for SBP diagnosis has been disappointing.

Table 2 Results of the urine reagent strips for the diagnosis of SBP.

Authors	Samples/SBP (n)	Strips/cut-off grade	Sensitivity	Specificity	PPV	NPV
Vanbiervliet et al[36] 2002	72/9	Multistix ≥ 1	100	100	100	100
Castellote et al[37] 2003	228/52	Aution ≥ 3	89	99	98	97
		Aution ≥ 2	96	89	74	99
Thevenot et al[38] 2004	100/9	Multistix ≥ 3	89	100	100	99
		Combur ≥ 2	89	100	100	99
Butani et al[39] 2004	136/12	Multistix ≥ 2	83	99	91	98
Sapey et al[40] 2005	184/13	Center 1/Nephur ≥ 1	86	100	100	99
		Center 1/Multistix ≥ 1	100	100	100	100
		Center 2/Nephur ≥ 1	100	92.5	75	100
		Center 2/Multistix ≥ 1	83	96	83	96
Sapey et al[41] 2005	245/17	Nephur ≥ 3	88.2	99.6	93.8	99.1
		Multistix ≥ 1	64.7	99.6	91.7	97.4
Kim et al[42] 2005	75/18	Uriscan ≥ 2	100	100	100	100
		Multistix ≥ 3	50	100	100	87
Sarwar et al[43] 2005	214/38	Combur ≥ 3	97.7	89.4	90	97.7
Wisniewski et al[44] 2005	47/6	Multistix ≥ 1	83	83	97	42
Campillo et al[45] 2006	443/46	Multistix ≥ 1	69.6	94.9	64	96
		Combur ≥ 1	80.4	90.4	52	97.3
		Multistix ≥ 2	45.7	98	75	93.3
Rerknimitr et al[46] 2006	200/42	Combur ≥ 1	88	81	55	96
		Combur ≥ 2	63	96	82	81
Braga et al[47] 2006	100/9	Combur ≥ 2	100	98.9	92.3	100
Torun et al[48] 2007	63/15	Aution ≥ 2	93	100	100	98
Gaya et al[49] 2007	173/14	Multistix ≥ 1	100	91	50	100
Nobre et al[50] 2008	109/9	Combina ≥ 2	78	88	37	98
		Combina ≥ 3	67	98	75	97
Ribeiro et al[51] 2007	200/14	Multistix ≥ 1	86	96	60	99
		Multistix ≥ 2	71	99	91	98
Nousbaum et al[52] 2007	1041/117	Multistix ≥ 3	45.3	99.2	77.9	96.9

Another method, more recently proposed as an alternative to the manual PMN count, is the measurement of lactoferrin in ascitic fluid. Lactoferrin is released by PMN leukocytes on the activation of these cells and its presence in body fluids is proportional to the flux of neutrophils. It was therefore hypothesized that its use could also be useful in the detection of SBP. The data available on the diagnostic value of this test for SBP diagnosis are limited to a single study[53]. This study, in which ascitic fluid lactoferrin was measured in a total of 218 ascites samples, reported that the sensitivity and specificity of the assay for SBP diagnosis were 95% and 97%, respectively. However, the quantitative lactoferrin assay used in this study is not commercially available. Qualitative and rapid tests are already commercially available for bedside measurement of lactoferrin concentration in the stool (to distinguish inflammatory from non-inflammatory bowel conditions). The authors hypothesized that qualitative tests able to detect lactoferrin levels in excess of a predetermined level for bedside diagnosis can easily be developed and with limited costs. They concluded that, although further confirmatory studies are needed, ascitic fluid lactoferrin could serve as an initial screening test for the detection of SBP in cirrhotic patients.

It is important to underline that both urinary reagent strips and ascitic lactoferrin tests are qualitative methods and need, therefore, to be further confirmed by standard cytology of the ascitic fluid. These tests could merely be a screening modality providing only a presumptive diagnosis, but they do not have the potential to replace the manual counting method. Moreover, a qualitative test does not have applicability in the clinical management of SBP, since a reduction of more than 25% of the initial PMN cell count value is, according to the current guidelines, the criterion to establish the efficacy of the ongoing antibiotic treatment. To date, the only quantitative methods proposed as a valid alternative to manual PMN counting are automated blood cell counters, commonly and largely used in all laboratories for blood cell counting. They offer accurate and rapid differential counts of leukocytes, including PMN cells. The published data available on the diagnostic performance of this method are limited but very promising (Table 3). The first study[54], which tested an automated cell counter for the diagnosis of SBP, was published in 2003 by our group. This study, in which 130 ascitic fluid samples obtained from 74 cirrhotic patients were analyzed, demonstrated that the use of an automated cell counter provides an accurate determination of ascitic fluid PMN and has a reliable diagnostic accuracy when compared with manual counting. In fact, the automated blood cell counter had a sensitivity of 94% and a specificity of 100%, while positive and negative predictive values were 100% and 99.1%, respectively (Table 3). These findings were also confirmed by Cereto et al[55] (Table 3). More recently, a further study[56], in which we analyzed 112 samples of ascitic fluid, showed that the manual and automated methods had good agreement not only in the first diagnostic paracentesis, but also in those performed during the follow-up. These results suggested that the automated cell counter is a useful and reliable tool not only for the diagnosis of SBP, but also in its management for monitoring the therapeutic efficacy of antibiotic treatment, as well as for establishing resolution of the infection. The use of automated PMN counting may provide several advantages for the diagnosis and management of SBP: it is commonly available in all patient care units, it is routinely accessible in emergencies (i.e. at night or on week-ends) and provides the results in a few minutes, thus allowing the clinician to quickly decide whether to start empiric antibiotics or to change the ongoing antibiotic therapy. Finally, it is important to remember that all this involves a very low cost (only €1.38 for a single ascitic cell determination). Another, although less important, advantage of the automated cell counting method over urinary reagent strips, the manual method and the ascitic lactoferrin test, is that it is possible to precisely assess the amount of PMN in bloody (for a traumatic tap or a condition inducing bleeding) ascitic fluid. With the other methods, the amount of PMN derived directly from the blood spilled over into the ascitic fluid cannot be differentiated from the amount of PMN due to the infection. A correction factor of 1 PMN per 250 red blood cells has been proposed[11], because this is the maximum expected ratio of PMN to red blood cells normally present in the peripheral blood. With automated cell counters, a measure of the amount of red blood cells and PMN in both the peripheral blood and the ascitic fluid can be obtained simultaneously. The amount of ascitic PMN due to the infection can therefore be calculated by the real PMN-to-red blood cells ratio in the blood and by the real erythrocytes and PMN number in the ascitic fluid.

Table 3 Results of the automated cell counter for the diagnosis of SBP.

Authors	Samples/SBP (n)	Automated cell counters	Diagnosis of SBP
			Sensitivity	Specificity	PPV	NPV
Angeloni et al[54] 2003	130/11	Technicon System H*1	94	100	100	99.1
		Coulter	100	92	87	100
Cereto et al[55] 2004	133/48	UF-100	98	98	96	99
		ADVIA	100	95	95	100
Riggio et al[56] 2008	112/16	Technicon System H*1	100	97.7	94.1	100

CONCLUSION

Although further confirmatory data are needed, automated cell counters offer a reliable, easier and quicker PMN count, they may also considerably simplify the diagnostic approach and clinical management of SBP and should be routinely adopted. The automated method has the potential to be an optimal substitute for manual PMN counting and could definitely replace it.