0270-9139/89/0904-0662$02.00/0 HEPATOLOGY Copyright 0 1989 by the American Association for the Study of Liver Diseases

Vol. 9, No. 4, pp. 662-665, 1989 Printed in U. S.A.

Correspondence

More on Ascitic Fluid Culture Technique

To the Editor:

In connection with the letters published by Drs. Edel- stein (Edelstein PH. Hepatology 1988; 8:983, Corre- spondence) and Runyon (Runyon BA. Hepatology 1988; 8:983-985, Correspondence) about the ascitic fluid (AF) culture techniques, we would like to report the results of our own prospective study.

From January, 1982, to December, 1986, two AF cul- ture methods were simultaneously assessed (i) the con- ventional method, consisting of direct inoculation of 10 ml of centrifuged AF in plates of aerobic and anaerobic blood-agar and chocolate-agar incubated at 37°C for 48 hr and inoculation of 0.5 to 1 ml of AF in thioglycollate broth, also incubated at 37°C for 48 hr; and (ii) the blood culture bottle method, consisting of the inoculation of 10 ml of AF in two commercial blood culture bottles (tryptic soy broth and thioglycollate broth, Cromatest'"', Knick- erbocker Laboratories). Both bottles were incubated at 37°C and, if macroscopic signs of growth were not ob- served in 48 hr, a blind subculture in plates was per- formed according to the technique described above.

We obtained 87 positive AF cultures. Thirty-six of them were from patients with secondary peritonitis and therefore excluded. Spontaneous bacterial peritonitis

TABLE 1. Results of AF culture by conventional and blood culture bottle methods

CC (n = 49) P" BCB (n = 49)

Positive culture 33 <0.001 49 SBP 26 <0.001 31 Bacterascites 7 NSb 12

CC = conventional culture; BCB = blood culture bottles. x 2 test. NS = not significant.

(SBP) was defined by a positive AF culture and more than 250 neutrophils per ml in AF (1). When the AF culture was positive with less than 250 neutrophils per ml, the patient was considered to have bacterascites (I).

Among the 49 positive AF cultures, 47 were monomi- crobial and two were polymicrobial. Enterobacteriaceae were the most commonly isolated organisms (83.7%). None of the isolated bacteria was considered to be a contaminant.

Table 1 shows the results. AF cultures were positive with both techniques in 67.3% (33/49) of cases, but in 32.7% (16/49), the bacteria were only isolated in blood culture bottles (p < 0.001). Of the 33 positive AF cultures with the conventional method, seven were bacterascites, whereas of the 49 positive cultures with blood culture bottle method, 12 were bacterascites (NS).

We conclude that in our study AF culture in blood culture bottles detected about 30% more bacteria in SBP, without a significant increase in the diagnosis of bacter- ascites. Therefore, we are in agreement with Dr. Run- yon's recommendation that the blood culture bottle method be used for AF culture.

SERGIO SAINZ GERMAN SORIANO CARLOS GUARNER PERE COLL FRANCISCO VILARDELL Escuela de Patologia Digestiva and Servicio de Microbiologia Hospital de la Santa Creu i Sant Pau Barcelona, Spain 08025

REFERENCE 1. Hoefs JC, Runyon BA. Spontaneous bacterial peritonitis. Disease-

a-Month 1985; 31:l-48.

Reply: method-which involves transport of the sample to the laboratory in a syringe or tube and subsequent inocula- tion of small volumes of unprocessed fluid (or centrifuged

I would like to thank Dr. Sainz et al. for providing us sediment) onto selective agar plates and into broth! The with the results of their ascitic fluid culture study. Al- keys to the success of the blood culture bottle method though their study design was slightly different than that are (i) the capacity of these bottles to accept large vol- of our two previous studies, the conclusions are the umes of fluid and (ii) the formulation of the medium, same-Inoculation of blood culture bottles with ascitic which provides a nutritious environment and protects fluid is far superior to the conventional method of culture the bacteria from further complement- and phagocyte- (1,2).All five studies of this subject (including Dr. Sainz's mediated killing once the bacteria enter the bottle. Al- study) have demonstrated the superiority of the blood though no studies have been published specifically re- culture bottle method (1-3; Dalmau B, et al. Arch Intern garding the superiority of bedside vs. laboratory inocu- Med 1987; 147:1849, Correspondence). The details of lation of the blood culture bottles, we favor bedside optimal volume of ascites to be inoculated, types of inoculation because (i) theoretically, bacteria may die in bottles, etc., are available in Reference (2). No study has transit to the laboratory in a syringe or tube and (ii) the demonstrated the superiority of the conventional preliminary analysis of our ongoing trial indicates that

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VOI. 9, No. 4, 1989 CORHESPON1)ENCE 668

bedside inoculat.ion is superior ( Runyon HA, Antillon MR, McHutchison J, et al., unpublished observations).

A very interesting question to be asked is: How did the conventional method of ascitic fluid culture become “etched in stone” as the accepted method worldwide’’ Weren’t any studies performed to validate this method before essentially every clinical microbiologist in the world adopted it as the routine? I have been unable to find any such studies. Apparent.ly, the recommendation to adopt this method of culture was simply the “best guess” of a committee charged with recommending tech- niques of culture of all body fluids. [Jnfortunately, the committee guessed wrong. Isn’t it time that this error be corrected? Culturing ascites as if‘ it were stool or urine simply doesn’t work. Spontaneously infected ascites is bacteriologically much like bacteremia-a monomicro- bial infection involving low concent,rations of bacteria (median colony count in spontaneous peritonit,is is 1 per ml); it makes sense that culturing the ascites as if it, were blood would be appropriate (2). It is appropriate, and the blood culture bottle method has been shown t.o be essen- tially twice as sensitive as the conventional method:

However, despite the large amount of page space de- voted to ascitic fluid culture methodology in recent months (1-4; Dalmau B, et al. Arch Intern Med 1987; 147:1849, Correspondence; Runyon BA. Arch Intern Med 1987; 147:1849-1850; Edelstein PH. Hepatology 1988; 8:983, Correspondence; Runyon BA. Hepatology 1988; 8:983-985, Correspondence 1. the message regarding the optimal technique has still not reached many hospital laboratories. Some of my colleagues tell me that their laboratories continue to discard nll ascites cultures suh- mitted i n blood culture bottles and inform the clinician that ascites should be submitted i n the “conventional”

-90% VS. 40% (1-3’)!

syringe or tube for culture in the “conventional” fashion! My only suggestion to these frustrated physicians, who are simply following data-supported published recom- mendat.ions, is to ask t,heir clinical microbiology labora- tory direct,or to compare (with an open mind) the blood culture bottle method (bottles inoculated at the bedside) “side-by-side” with the conventional method in his own 1aborat.ory for a few months. If the specimens are handled properly, the blood culture method will prove superior, as it has in the five studies which have been published to date. Subsequent, adoption of the blood culture bottle method at these hospitals will result in (i) much less confusion about, ascitic fluid infection, (ii) availability of susceptibility testing, (iii) tailoring of antibiotic therapy based on the results of susceptibility testing and (iv) hopefully, a better outcome for the patient. The patient,s and their families will be grateful.

BRUCE A. RUNYON, M.D. USC Liver CTnit Rancho Los Amigos Medical Center 7601 East Imperinl Highway Douinej), (‘alifornia 90242

REFERENCES Iiun? on HA, I Jmland ET, Merlin T. Inoculation of hlood culture Imt.tles with ascitic fluid: improved det.ection of spontaneous hac- trrial peritonitis. Arch Intern Med 1987; 147:73-75. Rrinyon BA. Canawati HN. Akriviadis EA. Optimization of ascitic fluid culture technique. Gastroenterology 1988: 95:1351-1355. Kammerer J, Dupeyron C, Vuillemin N. e t al. Apport des examens cytologiques e t bacteriologiques du liquide d’ascite cirrhotique au diagnostic de peritonite bacterienne: a propos de 610 prelevements chez 156 malades. Med Chir Dig 1982; 11:243-251. Runyon B.4. Spontaneous bacterial peritunit.is: an explosion of information. Hepatology 1988; 8:171- 175.

Selective Permeabilization of the Endoplasmic Reticulum by Monohydroxylated Bile Acids in Liver

To the Editor:

In liver, the increase in the ionized free Ca++ of the cytosol ([Ca++Ii) plays a key role in responses to hor- mones or neurotransmit,t,ers. It stimulates ionic perme- abilities and potential, mitochondria1 respiration, glucose metabolism and secretion (1, 2 ) . Two main sources are responsible for the increase in [Ca-’ I, : extracellular C a r . via augmented Cat+ influx across plasma membrane ( 2 ) and intracellular Ca++ via permeabilization of the endo- plasmic reticulum by the intracellular messenger inositol trisphosphate (Ins 1,4,5-P:i) 3 ) .

Recent studies from our laboratory (4) and from Anwer et al. (5) have shown that, other natural molecules such as bile acids were able t,o mobilize Ca++ in rat hepato- cytes. Particularly, two monohydroxylated bile acids which strongly inhibit bile secretion, t,aurolithocholate (TLC) and lithocholate (LCr, promot,e a fast and tran- sient increase in [Ca”], as detected by the Cat’ fluores-

cent indicator quin2. In the presence of 1.2 m M external + , TLC (100 p M ) increased [Ca+’Il from 196 k 20

n M to 523 k 42 n M (n = 11). We examined the role of (’a’- influx on the Ca” rise by using the chelator EGTA at a concentration (1.24 mM) sufficient to reduce exter- nal free Ca++ to 6 pM. Lowering external Ca++ from 1.2 mM to 6 pM eliminates Ca++ influx in rat liver cells (6). When EGTA was added 15 sec before the bile acid, TLC promoted a rise in [Cat+]> not significantly different (631 rt 42 nM, n = 11) from that observed in control cells. This was also observed at different concentrations of TLC The dose-response curve in 1.2 mM Ca++ (ED,,, = 22 p A 4 ) was not altered by low-Ca++ media (ED,,, = 26 p M ) (4) These results support the view of a primary action of bile acids on the internal Ca++ store of the hepatocvte.

I n contrast, in a study on Ca++ movements in liver cells. Anwer et al. (5) reported in this Journal that the [Ca++13 increase mediated by 100 to 500 p M TLC or LC wa.; ~ubstantially reduced or suppressed when cells were