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Original Article
Detection of ascitic uid infections in patients with liver cirrhosis and ascites
Marwa S. Mostafa a, Eman A. El-Seidi a, Abdel Meguid Kassem b,, Mohamed A. Shemis c, Mohamed Saber c,
Michael N. Michael a
a
Medical Microbiology and Immunology Department, Faculty of Medicine, Cairo University, Cairo, Egypt
Tropical Medicine Department, Faculty of Medicine, Cairo University, Cairo, Egypt
c
Biochemistry Department, Theodor Bilharz Research Institute, Giza, Egypt
b
a r t i c l e
i n f o
Article history:
Received 29 June 2010
Accepted 24 December 2010
Keywords:
Ascites
Spontaneous bacterial peritonitis
PCR
Bacterial translocation
a b s t r a c t
Background and study aims: Ascitic uid infections (AFIs) are the frequent complications of advanced liver
disease. Bacterial translocation is considered a key step in the pathogenesis of gut-derived bacterial infections; mainly spontaneous bacterial peritonitis (SBP) in cirrhotic patients. Bacterial DNA (bactDNA) in
ascitic uid and serum has been suggested as a surrogate marker for bacterial translocation. We
attempted at the isolation and identication of bacteria in ascitic uid in cirrhotic patients and the
assessment of polymerase chain reaction (PCR) in ascitic uid and serum.
Patients and methods: Fifty cirrhotic patients having ascites with no signs of infection were included.
Ascitic uid cultures were obtained from patients. Ascitic uid and serum were subjected to DNA extraction and PCR for the universal amplication of a region of the 16S ribosomal RNA (16S rRNA) gene to
detect bactDNA.
Results: Bacteria were isolated from 9 (18%) of the ascitic uid samples, and were mainly Gram-positive
bacteria. BactDNA was detected simultaneously in the ascitic uid and serum of 17 (34%) patients and in
the ascitic uid of only 2 patients. In a single patient with positive ascitic uid culture no bactDNA was
detected in ascitic uid or serum. By considering AFIs as a positive ascitic uid culture and/or the presence of bactDNA in the ascitic uid and/or serum, ascitic uid culture could detect 9 out of 20 patients
with AFIs (45%), PCR of ascitic uid could detect 19 out of 20 (95%) while PCR of serum could detect
17 out of 20 (85%). In 10 patients with culture negative non-neutrocytic ascites (CNNNA) bactDNA could
be detected in serum and ascitic uid.
Conclusion: AFI can be caused by Gram positive as well as Gram negative organisms. A substantial percentage of cases with CNNNA show bactDNA in serum and ascitic uid. PCR of ascitic uid should, therefore, be used in the diagnostic workup of suspected cases of ascitic uid infections.
2011 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
Introduction
Ascitic uid infections (AFIs) are considered serious complications in cirrhotic patients and are associated with high morbidity
and mortality. AFIs include spontaneous bacterial peritonitis (SBP)
with polymorphnuclear (PMN) count P250 mm3 and positive
ascitic uid culture without any evidence of external or intraabdominal source of infection [1] as well as culture negative neutrocytic ascites (CNNA) with PMN >250 mm3 and a negative
ascitic uid culture [2]. The reported incidence of AFIs was found
to be 830% [1]. SBP has a very high recurrence rate of up to 70%
at 1 year [3] and in-hospital mortality ranges from 20% to 40%
[4,5] and may reach up to 78% [1]. SBP is considered to be the
Corresponding author.
E-mail address: kassem@git.eg.net (A.M. Kassem).
1687-1979/$ - see front matter 2011 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ajg.2011.01.004
21
1000
900
800
700
600
500
400
300
200
100
Total DNA was extracted from the ascitic uid samples and sera
using Genomic DNA purication kit (Promega, Madison, USA),
according to manufacturers instructions. Positive and negative
controls were included in each PCR run. Positive control DNA
was obtained by extraction of bacterial DNA from overnight broth
culture of Staphylococcus aureus (ATCC 25213) and Escherichia coli
(ATCC 35320) according to Fang and Hedin [14], whereas sterile
distilled water was used as a negative control.
Data were analyzed using SPSS, version 15. Mean and standard
deviation were used for quantitative variables, whereas number
and percent were used for qualitative variables. Comparisons between groups were done using Chi-square test for qualitative variables and independent sample t-test for normally distributed
quantitative variables, while non-parametrical MannWhitney test
was used for quantitative variables with no normal distribution. p
Values of 5% or less were considered as statistically signicant.
Sensitivity, specicity, positive predictive value (PPV), negative
predictive value (NPV), and total accuracy were calculated to assess
the validity of different tests, while phi symmetric measures were
used to assess the agreement between the tests.
DNA amplication
Results
DNA extraction
22
SBP (No. = 2)
CNNA (No. = 1)
>250 mm3
>250 mm3
E. coli
Negative
2
1
4
2
BA (No. = 7)
<250 mm3
CoNS
A. lwofi
Diphtheroids
Anthracoids
4
1
1
1
<250 mm3
Negative
10
Isolated bacteria
No.
BactDNA in serum
Positive
Positive
Positive
Positive
14
Positive
Negative
Positive
Positive
Positive
Negative
Negative
Negative
20
Positive
Positive
PMN: Polymorphnuclear leucocytes; SBP: Spontaneous bacterial peritonitis; CNNA: Culture negative neutrocytic ascites; BA: Bacteriascites; CNNNA: Culture negative non-neutrocytic ascites; CoNS: Coagulase negative staphylococci.
for bactDNA (DNA extraction and PCR were repeated three times).
The remaining 11 bactDNA positive ascitic uid samples were
culture negative. The phi coefcient of correlation between ascitic
uid culture and PCR was 0.491, i.e. moderate correlation
(p-value = 0.001).
Out of 50 serum samples, bactDNA was detected in 17 samples
(34%). Six out of nine patients who had culture positive ascitic uid
samples were found to have bactDNA in their sera; these also had
bactDNA in their ascitic uid samples. The single patient who had
culture positive ascitic uid (A. lwofi) and bactDNA negative ascitic uid sample had also repeatedly negative bactDNA in the serum
sample. The other two patients, who had culture positive ascitic
uid (having diphtheroids and anthracoids) and bactDNA positive
ascitic uid, had also repeatedly negative bactDNA in their sera.
The remaining 11 patients were the same patients who had culture
negative ascitic uid and bactDNA positive ascitic uid samples.
Table 1 summarizes the microbiological results in the study
patients.
Results of serum and ascitic uid bactDNA show a signicant
correlation. The phi coefcient of correlation between PCR of
bactDNA in ascitic uid and PCR of bactDNA in serum was 0.917
(p-value <0.001) (Table 2). Taking ascitic uid bactDNA as a gold
standard, the accuracy of diagnosing AFI was 76% for ascitic uid
culture and 96% for serum bactDNA (Table 3).
According to the aforementioned results, all patients were classied into two groups: group I patients who had AFIs (including
20 patients, 40%; who had positive ascitic uid culture and/or
Table 2
PCR of bactDNA in serum vs. PCR of bactDNA in ascitic uid.
Ascitic uid PCR
Total
Negative
Positive
Serum PCR
Negative No. (%)
Positive No. (%)
31 (100%)
0 (0%)
2 (10.5%)
17 (89.5%)
33 (66%)
17 (34%)
31 (100%)
19 (100%)
50 (100%)
The phi coefcient of correlation between PCR of bactDNA in ascitic uid and PCR of
bactDNA in serum was 0.917 (p value <0.001).
Table 3
Sensitivity, specicity, PPV, NPV and total accuracy of ascitic uid culture and PCR of
bactDNA in serum, considering PCR of bactDNA in the ascitic uid as the gold
standard.
Test
Sensitivity
(%)
Ascitic uid
culture
PCR of bactDNA in
serum
42.1
89.5
Specicity
(%)
96.8
100
PPV
(%)
88.9
100
NPV
(%)
Total
accuracy (%)
73.2
76
93.9
96
Table 4
Demographic and laboratory data of group I and group II patients.
Variable (mean SD)
Group I
(No. = 20)
Group II
(No. = 30)
51 5
54 9
0.149
Sex
Male
Female
15 (75%)
5 (25%)
23 (76.7%)
7 (23.3)
1.000
7077.5 5565.6
6157.9 3450.9
0.760
2.5 0.3
78.4 121.2
1.2 0.4
3.4 4
1.8 3
96.2 9.1
2.5 0.6
50 31.6
1.2 0.6
2.4 1.6
1.2 1.2
59.6 37.8
0.925
0.807
0.760
0.992
0.984
0.04*
41.2 28.5
36 28.5
0.272
131 38.4
135 57
0.777
1.3 0.5
0.5 0.2
211 427.6
1.4 0.6
0.5 0.3
49 151
0.611
0.625
0.300
There was no statistically signicant difference of any of the values except for
AST; which was signicantly higher in group I than in group II.
Discussion
Infections of the ascitic uid in patients with ascites are associated with great risks and, therefore, require special attention from
the clinician. It is important to investigate the pattern of infections
to draw conclusion that may inuence treatment regimens. In the
present study, all positive ascitic uid cultures revealed growth of
a single organism, i.e. monomicrobial infection, with the predominance of Gram positive over Gram negative bacteria (66.7% vs.
33.3%, respectively). This is in concordance to other studies that revealed monomicrobial infection [8,1518]. Although only a few
species and genera of intestinal bacteria have been proposed to
translocate into the mesenteric lymph nodes, more than 70 different microbial species have been isolated in ascitic uid from
patients with bacteriologically conrmed SBP [19]. In one study,
S. aureus was the second most commonly isolated organism in patients with decompensated cirrhosis after E. coli [20].
Sixty percent of nosocomial-acquired infections are currently
caused by Gram positive bacteria [21]. Moreover, increasing number of episodes of SBP caused by Gram positive bacteria was observed, particularly in association with the increasing numbers of
invasive procedures, hospitalization particularly in intensive care
units (ICU), and treatment with quinolones [16]. There is growing
evidence by many studies that the detection of Staphylococcus spp.
in the ascitic uid is not because of sample contamination but because of bacterial translocation [8,17,22,23].
Such et al. [8] considered the simultaneous presence of bacterial
DNA in the ascitic uid and the blood a molecular evidence of bacterial translocation. Accordingly, this is applicable in our study, too.
In the present study, bactDNA was detected in ascitic uid and/
or serum in 19 out of 50 (38%) patients with liver cirrhosis and
ascites, eight of them only had culture positive ascitic uid.
BactDNA was detected simultaneously in the ascitic uid and serum in 17 (34%) patients and in the ascitic uid only in two patients.
BactDNA was not detected in one culture positive ascitic uid
sample that grew A. lwofi. Two possible explanations were considered: (1) the used primers are not specic to the 16S rRNA of this
organism and (2) contamination of the culture after collecting the
sample. Similarly, three possible explanations were suggested for
the two cases that had positive bactDNA in the ascitic uid only
and not in the serum: (1) too low concentration of the bactDNA
in the serum to be detected by PCR; (2) contamination of the ascitic
uid during collection of the sample; (3) they were not cases of
bacterial translocation, i.e. they were cases of secondary infection
of ascitic uid (secondary peritonitis), however, this latter possibility is very remote because patients with positive signs of infection
were excluded from the study. Similarly, Vieira et al. [15] detected
bactDNA in 18 out of 40 (45%) patients with liver cirrhosis and
ascites. Cultivation of ascitic uid using blood culture technique
and PCR of bactDNA in the ascitic uid was in agreement in
87.5% of cases. BactDNA was not detected in one culture positive
ascitic uid sample, a nding similar to that in our study. Bruns
et al. [17] detected bactDNA by multiplex PCR in ascitic uid in
14 out of 68 (20.6%) of patients with liver cirrhosis and ascites.
Because the presence of bactDNA in the culture negative mesenteric lymph nodes (MLN) is associated with an inammatory
reaction similar to that observed in culture positive samples, studies suggested that the concept of bacterial translocation might be
expanded to consider it as the presence of bacterial genome fragments in MLN, irrespective of the results of microbial culture [22].
The application of nucleic acid amplication techniques for
pathogen detection in the ascitic uid may provide advantages
over bacterial culture techniques, especially for microorganisms
that are hard to cultivate or in patients after antibiotic treatment
[17]. Considering that PCR of bactDNA in ascitic uid as the gold
standard, detection of bactDNA in the serum showed higher sensitivity and specicity in the detection of bacterial translocation
(89% and 100%, respectively) than the cultivation of ascitic uid
using the blood culture technique (42.1% and 96.8%, respectively).
The introduction of 16S ribosomal RNA gene-based methods for
the detection of bactDNA revolutionized the position of the microbiological studies beyond culture-based protocols [24] and provided a tool to detect bacterial DNA fragments that provided
molecular evidence of bacterial translocation (BT) in patients with
CNNNA [8,16]. Moreover, Albillos et al. [25] concluded that the def-
23
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