Esterase activity in second- and third-trimester amniotic fluid: An indicator of chorioamnionitis

Iffatb Abbasi Hoskins, MD, Joseph Katz, PhD, Steven A. Ordorica, MD, and Bruce K. Young, MD

New York, New York

Accurate and rapid diagnosis of chorioamnionitis poses a major diagnostic dilemma. We previously reported that leukocyte esterase activity in amniotic fluid, as measured by dipstick assay, could be used as an aid in the diagnosis of chorioamnionitis. This study examines the effectiveness of an in vitro spectrophotometric assay of esterase activity in amniotic fluid. We define baseline levels of esterase activity in un infected amniotic fluid and demonstrate a quantative increase when infection is present. Fifty-seven amniotic fluid samples obtained at second- and third-trimester amniocenteses were divided into three parts. one for culture and two for a comparison of esterase activitites by the dipstick and spectrophotometric methods. In this study, the spectrophotometric assay, because of its higher specificity and sensitivity in the determination of elevated esterase activity, was shown to be more reliable for predicting chorioamnionitis than either the dipstick or culture method. (AM J OesTET GVNECOL 1989;161 :1543-5.)

Key words: Leukocyte esterase, chorioamnionitis screening, in vitro assay

Chorioamnionitis plays a major role in perinatal mor­bidity and mortality. One of the basic problems in its diagnosis is the lack of uniformity and accuracy of the criteria on which this diagnosis is made. The clinical diagnosis is usually based on a syndrome of fever, ma­ternal or fetal tachycardia, uterine tenderness, foul odor of the amniotic fluid , and peripheral blood leu­kocytosis, but there is uncertainty regarding early di­agnosis and laboratory confirmation. I Since leukocytes are known to be released in response to infections, ex­amination of the amniotic fluid for their presence may prove to be useful in the evaluation of chorioamnionitis. Neutrophilic leukocytes contain several esterases that are not present in serum, urine, or vaginal secre­tions}·3 * We have previously described the reliability of leukocyte esterase activity in the rapid detection of urinary tract infections in obstetric patients4 and for the detection of chorioamnionitis in term pregnancies uncomplicated by other diseases.' In the present study, we assessed the use of an in vitro assay to establish baseline levels of esterase activity in second- and third­trimester amniotic fluid samples and to demonstrate any alterations of these levels in chorioamnionitis .

From the Dzvision of Maternal-Fetal Medu:zne, Department of Ob­stetrics and Gynecology, N ew York UmveTSIty MedIcal Center.

Presented in part at the ThIrty-fifth Annual M eeting of the Societ.~ for Gynecologtc InvestigatIOn, BaltImore, Maryland. March 17-20, 1988.

Repnnt "equests: Iffath AbbasI Hoskins, MD, Department of Obstet­ncs and Gynecology, NYU-Bellevue Hospital Center, 450 FlTSt Ave., R oom 9 North 1, New York, NY 10016

*Chemstnp 9 BIOdynamics, package Insert. 6/6/15954

Table I. Relationship between leukocyte esterase activity and other parameters used to diagnose chorioamnionitis

Unznfected Infected

Pos. I Neg. Pos. I Neg.

Amniotic fluid Culture 2 37 14 4 Gram's stain 2 37 15 3

Leukocyte esterase activity Dipstick assay 2 37 17 1 In vitro assay 0 39 18 0

Material and methods

Sterile specimens of amniotic fluid were obtained from 57 antepartum patients who had undergone am­niocenteses during the second and third trimesters for genetic studies, for rhesus isoimmunization, for fetal lung maturity studies, or to rule out chorioamnionitis. Thirty-nine samples were obtained from uninfected patients along with 18 samples from women who had chorioamnionitis as determined by the presence of at least twO of the clinical criteria used for its diagnosis, including maternal pyrexia (temperature >37.8° C), leukocytosis (white blood cell count >12,OOOlmm'), uterine tenderness, and fetal tachycardia (heart rate > 160 beats per minute). Each sample was divided into three parts: one for the in vitro leukocyte esterase assay, one for detection of leukocyte esterase activity by dip­stick, and one for Gram's stain and aerobic and anaer­obic cultures.

1543

1544 Hoskins et al. December 1989 Am J Obslel Gynecol

Table II. Reliability of parameters used to detect chorioamnionitis

Sensitivity (%)

Amniotic fluid Culture 78 Gram's stain 83

Leukocyte esterase activity Dipstick assay 94 In vitro assay 100

For in vitro leukocyte esterase assay, known amounts (10 >.) of amniotic fluid were added to the solutions containing 20 >. of as ILmol/ L solution of p-nitrophenyl hexanoate in dimethyl sulfoxide and 1 ml of buffer (0.1 mollL phosphate, pH 7.4). Activity was calculated from the initial slopes of the change in absorbance at 405 nm as a function of time. One unit of activity was defined as a change of 1 absorbance unit at 405 nm/min. Each sample was then centrifuged at 2000 rpm for 10 min­utes and the assay was repeated.

Aliquots of the same amniotic fluid samples were tested for leukocyte esterase activity by immersion of the dipstick (Chemstrip 9, Boehringer-Mannheim Di­agnostics, Bio-Dynamics Division) in the fluid and com­parison of the color reaction with the color chart after I minute.

Gram's stain test was performed with commercial re­agents (crystal violet, safranine, Gram's iodine; Difco Laboratories, Detroit) under standard conditions.6 The presence of white blood cells and microorganisms was noted, and a semiquantitative assessment was made af­ter examination of 20 high-power fields. A positive Gram's stain was defined as one in which there were any microorganisms per high-power field. The amni­otic fluid aliquots destined for cultures were collected in plastic-capped syringes and transported to the lab­oratory immediately after collection for inoculation into the culture media.

Positive cultures were defined as those containing one or more types of organisms in excess of 104

cfu/ml. The leukocyte esterase activity test results were then compared with the results obtained from Gram's stains and aerobic and anaerobic cultures.

Results

Table I represents the relationship between leukocyte esterase activity, as measured by dipstick and in vitro assay, and results of Gram's stain culture in both the un infected and infected samples of amniotic fluid. There were 39 uninfected samples for analysis. Twenty-one of these were obtained during the second trimester after amniocenteses for genetic indications. Eighteen were obtained during the third trimester-4 for Rh sensitization and 14 for studies of lung maturity.

Predzctive values

SpecificIty (%) PosItive 1 Negative

95 90 80 95 93 88

95 97 90 100 100 100

No differences were found between the Rh-sensitized and normal samples. Two samples from the third trimester showed positive cultures with Escherichia coli organisms> 104 cfu/m!. Both of these samples were also positive on Gram's stain analysis and showed leu­kocyte esterase activity, as measured by dipstick. How­ever, when the in vitro enzyme assay was performed, the leukocyte esterase activity was within the ranges we had defined as baseline normal (5.8 and 6.3 enzyme activity units) for un infected samples (Fig. 1).

There were 18 infected samples of amniotic fluid obtained from women with chorioamnionitis. All of these samples were obtained during the third trimesters from patients who had undergone amniocenteses to

rule out chorioamnionitis (spontaneous rupture of membranes, 8; preteI'm labor, 10). Fourteen of these women had positive amniotic fluid cultures, with or­ganism counts> 10' cfu/ml (E. coli, 10; group B strep­tococci 2; Enterobacteriaceae 2). However, if a positive culture was defined as one containing organisms > 102

cfu/ml, then all 18 samples were found to be positive (E. coli, 13; group B streptococci, 2; Enterobacteriaceae, 3). Only 15 ofthese samples were positive by the Gram's stain test, and the organisms revealed by this test were similar to those revealed by cultures. Seventeen of the 18 infected specimens were positive by leukocyte es­terase dipstick assay, but only 15 of these showed evi­dence of bacteria on Gram's stain analysis. The two samples with positive leukocyte esterase activity re­vealed by dipstick but negative Gram stains showed varying numbers of white blood cells on the slides. The one sample that tested negative by dipstick assay grew organisms> 104 cfu/ml (Enterobacteriaceae). When the in vitro enzyme assay was performed, the observed es­terase activity was severalfold higher (range, 17.8 to 18.2 enzyme activity units) than that of the uninfected normal samples (Fig. I). When the uninfected samples were centrifuged and assayed by the in vitro leukocyte esterase method, there was a severalfold increase in observed esterase activity (range, 14.6 to 17.2 enzyme activity units) to levels similar to those seen in the in­fected samples. This increase was found to be due to cell disruption and lysis that allowed the release of azu­rophilic, esterase-containing granules in the leukocytes.

Volume 161 Number 6, Part 1

I"') 0 .... )(

10 0 ..

c::i 0 <I

Esterase as indicator of chorioamnionitis 1545

20 T .L

15

10

5 T 1

o NORMAL INFECTED

Fig. 1. Esterase activity in normal and infected samples of centrifuged amniotic fluid.

Table II shows the sensitivity, specificity, and predic­tive values of the various parameters used in the com­parison ofleukocyte esterase activity by the dipstick and the in vitro methods. In our study, the in vitro assay had a 100% sensitivity and a 100% specificity for cho­rioamnionitis, whereas the leukocyte esterase dipstick had a sensitivity of 94% and a specificity of 95%. The cultures of these samples had shown a sensitivity of only 78% and a specificity M 95%.

Comment

Acute chorioamnionitis remains a serious infection for the mother and her baby. Despite the seriousness of this condition, there are major problems associated with its diagnosis. At present it is impossible to deter­mine what constitutes an optimal diagnostic finding, and attempts to improve our ability to diagnose intra­partum infections have met with little success. To iden­tify an optimal screening test for chorioamnionitis, we reported previously" on the dipstick method of mea­suring leukocyte esterase activity as an inexpensive and reliable (sensitivity 91 %, specificity 95%) test for diag­nosing chorioamnionitis in term patients in the ab­sence of significant numbers « 1 04 cful ml) of organ­isms on culture. However, the results of the dipstick assay, like those of the aerobic and anerobic cultures, can sometimes be misleading or equivocal. When that is the case, in vitro spectrophotometric assay of esterase activity may be more useful in identifying infected am-

moUe fluid samples and eliminating any confusion, since this test has both a sensitivity and a specificity much higher (100% each) than those of the other tests.

Thus a simple, rapid, and inexpensive regimen for the evaluation and diagnosis of chorioamnionitis could involve the use of the dipstick assay of esterase activity as an initial screening test (before culture results have been obtained), with in vitro spectrophotometric assay as a backup for samples that yield equivocal or negative results by the dipstick method. The use of this test may serve to eliminate the need for any additional testing or aid management decisions in situations in which waiting for culture results is not feasible.

REFERENCES 1. Gibbs RS, Castillo MS, Rodgers PJ. Management of acute

chorioamnionitis. AM J OBSTET GYNECOL 1980; 136:709. 2. Rindler-Ludwig R, Schmazl F, Braunsteiner H. Esterases

in human neutrophilic granulocytes: Evidence for their protease nature. Br J Haematol 1974;27:57.

3. Li CY, Lam KW, Yam LT. Esterases in human leukocytes. J Histochem Biochem 1973 ;21: 1.

4. Abbasi lA, Hess LW, Johnson TRB, et al. Leukocyte es­terase activity in the rapid detection of urinary tract and lower genital tract infections in obstetric patients. Am J Perinatol 1985;2:311.

5. Hoskins I, Johnson TRB, Winkel CA. Leukocyte esterase activity in human amniotic fluid for the rapid detection of chorioamnionitis. AM J OBSTET GYNECOL 1987;157:730.

6. Hendrickson DA. Reagents and stains. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ, eds. Manual of clinical microbiology. 4th ed. Washington DC: American Society for Microbiology, 1985: 1093.