In vitro inhibition of esterase activity in amniotic fluid: Comparison with bacterial cultures

Iffath Abbasi Hoskins, MD, Joseph Katz, PhD, Faith J. Frieden, MD, Steven A. Ordorica, MD, and Bruce K. Young, MD

New York, New York

Assessment of leukocyte esterase activity in amniotic fluid for the rapid and reliable diagnosis of chorioamnionitis has been demonstrated previously. We compared in vitro inhibition of esterase activity in amniotic fluid with bacterial cultures to identify the origins of the specific esterases released by the infecting organisms. One hundred forty-one samples were tested (90 uninfected. 51 infected). Each sample was evaluated for Gram stain. cultures. and an in vitro esterase assay followed by ebelactone inhibition. Forty-two patients had positive amniotic fluid cultures. Ebelactone produced varying degrees of inhibition of esterase activity (range. 20% to 60%) in the uninfected samples and in those infected with gram-negative organisms. There was no inhibition in the samples infected with gram-positive organisms. Thus different groups of bacteria may elicit the production of different and specific esterases in infected amniotic fluid. as shown by the differences in in vitro inhibition. (AM J OBSTET GVNECOL 1990;163:

1944-7.)

Key words: Chorioamnionitis, leukocyte esterase, inhibition. bacterial cultures

Chorioamnionitis is a common diagnostic dilemma for the obstetrician. Rapid and accurate diagnosis poses the most significant problem. The clinical diagnosis is usually based on a constellation of symptoms that in­clude maternal fever, tachycardia, uterine tenderness, foul odor of amniotic fluid. leukocytosis, and fetal tachycardia. However, there is uncertainty with regard to accurate and early diagnosis and laboratory confir­mation.'

Because leukocytes are known to be released in re­sponse to infections, examination of the amniotic fluid for their presence may be useful in the evaluation for chorioamnionitis. The neutrophilic leukocytes released contain several esterases that are not present in serum, urine, or body tissues!"

We have previously described the reliability of an in vitro spectrophotometric assay of leukocyte esterase activity in amniotic fluid for the prediction of chorioam­nionitis. In this study we compared in vitro inhibition of this activity with the results of bacterial cultures in an attempt to identify the origins of the specific ester­ases that are released by the infecting organisms.

Material and methods

Sterile specimens of amniotic fluid were obtained from 141 antepartum patients who had undergone

From the Department of Obstetrics and Gynecology, New York Uni­versity Medical Center, Presented at the Thirty-seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: Iffath Abbasi Hoskins, MD, Department of Obstet­rics and Gynecology, New York University Medical Center, 550 First Ave" Room 9N1, New York, NY 10016. 6/6/24775

1944

transabdominal amniocenteses during the second and third trimesters for genetic studies, rhesus isoimmu­nization, and fetal lung maturity studies, or to rule out chorioamnionitis. All patients included in the study had intact membranes. Ninety samples were obtained from uninfected patients and 51 from women with cho­rioamnionitis as determined by the presence of at least two of the following criteria: maternal pyrexia (tem­perature >37.80 C), leukocytosis (white blood cell count > 12,000/mm'), uterine tenderness, and fetal tachycar­dia (heart rate >160 beats/min). Each sample was di­vided into three parts: one each for Gram stain, aerobic and anaerobic cultures, and the in vitro esterase activity. The in vitro assay was performed by adding known amounts of amniotic fluid (lOA) to solutions containing 20A of a 5 j.Lmol/ L solution of p-nitrophenyl hexanoate in dimethyl sulfoxide and 1 ml of buffer (0.1 mol/L phosphate. pH 7.4). The change in optical density at wavelength 405 nm (IlOD4o) 10-') 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! min. Then, lOA of a nonspecific esterase inhibitor ebelactone (1 mg/ml) was added to each aliquot, and the OD4o) 10-' was again calculated to measure the degree of inhibition of es­terase activity in the sample. The results were compared with those obtained from amniotic fluid aerobic and anaerobic cultures.

Gram staining was performed with commercial re­agents (crystal violet, safranin, and Gram's iodine; Difco Laboratories, Detroit) under standard conditions. The presence of white blood cells and microorganisms was noted, and a semiquantitative assessment was made

Volume 163 Number 6, Part I

Inhibition of esterase activity in amniotic fluid 1945

Table I. Results of parameters used to detect chorioamnionitis

Uninfected (n = 90) Second trimester (1S-20 wk)

(n = 58) Third trimester (28-40 wk)

(n = 32) Infected (n = 51)

Second trimester (1S-20 wk) (n = 19)

Third trimester (28-38 wk) (n = 32)

* Absorbance units, tp < O,OS.

Gram stain

Pas, I Neg, Pas,

57 o

3 29

7 12 13

18 14 28

Cultures

I Neg,

58

31

6

4

Leukocyte esterase activity* (range)

2.8-7.0t

3.1-6.8t

17.0-18.3t

17.3-19.6t

E belactone* (range)

0.9-3.7t

1.3-3.9t

9.0-17.8t

7.6-18.2t

Table II. Reliability of amniotic fluid diagnostic parameters

Sensitivity Specificity Positive predictive Negative predictive (%) (%) value (%) value (%)

Second Third Second Third Second Third Second Third trimester I trimester trimester I trimester trimester I trimester trimester I trimester

Gram stain Cultures

36.8 68.4

S6.3 87.5

98.3 100

after examination of 20 high-power fields. A positive Gram stain was defined as one containing any micro­organisms per high-power field.

The amniotic fluid aliquots 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 101 cfu/m!. For purposes of this study, if more than one organism was isolated on culture, the predominant organism was labeled the causative one. A positive leukocyte esterase enzyme assay was defined as a value > 12 enzyme activity units.'

The sensitivity, specificity, and predictive values for the diagnostic parameters were calculated. A X2 analysis was performed to compare the results of the esterase assay and the inhibition assay between the uninfected and infected groups. Statistical significance was defined as a p value <0.05.

Results

The relationship between the Gram stain, cultures, leukocyte esterase activity, and inhibition in the unin­fected and infected samples is shown in Table I. There were 90 uninfected amniotic fluid samples; 58 were obtained during the second trimester. One sample showed gram-positive cocci on Gram stain, there were no positive amniotic fluid cultures, and the leukocyte esterase activity of all samples was within the normal

90.6 96.9

87.S 100

8S.7 96.6

82.6 90.6

Table III. Pathogens identified on amniotic fluid cultures

Uninfected Organism (n = 90)

Aerobic gram-positive Group B streptococci I Enterococci 0

Anaerobic gram-positive Streptococci 0

Aerobic gram-negative E. coli 0 Enterobacteriaceae 0

Anaerobic gram-negative B. fragilis 0 F. nucleatum 0

67.4 88.6

Infected (n = 51)

6 10

0

21 0

3 I

ranges as previously defined (> 12 enzyme activity units). Three of the 32 uninfected samples obtained in the third trimester had positive Gram stains (two cases with gram-positive cocci and one with gram-negative rods). One of these samples also showed a positive am­niotic fluid culture with organisms> 104 cfu/ml (group B streptococcus).

Nineteen of the 51 infected samples were obtained from second-trimester amniotcenteses. Of these, only seven had positive Gram stains (gram-positive cocci, three cases; gram-negative rods, three; gram-negative cocci, one). All seven samples also had positive amniotic fluid cultures (group B streptococcus, two; Escherichia coli, four; Bacteroides fragilis, one). An additional six samples had positive amniotic fluid cultures but neg-

1946 Hoskins et al. December 1990 Am J Obstet Gynecol

Table IV. Comparison of bacterial cultures and inhibition of leukocyte esterase activity

n

Uninfected 90 Infected 51 Positive cultures 41 Aerobic gram-positive

Group B streptococcus 6 Enterococcus 10

Aerobic gram-negative E. coli 21

Anaerobic gram-negative B. tragilis 3 F. nucleatum 1

* Absorbance units.

ative Gram stains (group B streptococcus, two; E. coli, two; B. fragilis, two). Thirty-two samples were obtained from third-trimester amniotcenteses. Of these, 18 had positive results on both Gram stains and amniotic fluid cultures, and 10 had negative Gram stains but positive amniotic fluid cultures.

Forty-five of the 51 infected samples had positive leukocyte esterase activity (> 12 enzyme activity units). Two of the six fluids with negative leukocyte esterase activity were second-trimester samples. One of these also had a positive amniotic fluid culture for group B streptococcus. The remaining four were from third­trimester samples. All four had negative amniotic fluid cultures.

The reliability of the diagnostic indices is shown in Table II. Gram stains ofthe amniotic fluid samples were not found to be highly sensitive (36.8%, second trimes­ter; 56.3% third trimester). This finding is similar to a previous study in which we found Gram stains to be only 62% sensitive in third-trimester amniotic fluid samples.5 The specificity of the Gram stains in second­and third-trimester fluids was 98.3% and 90.6%, re­spectively. In this study the sensitivity of amniotic fluid cultures was only 68.4% in the second-trimester sam­ples and 87.5% in the third-trimester samples.

The various organisms identified on cultures are shown in Table III. When more than one organism was grown from an amniotic fluid sample, the infection was referred to as being caused by "mixed aerobic or an­aerobic flora," and those samples were eliminated from analysis for this study. The paucity of anaerobic isolates obtained in this study may reflect bacterial fastidious­ness or death or the limitations of the laboratory facil­ities that provided the results. There was one positive culture in the 90 uninfected samples. This was a third­trimester amniotic fluid sample in which group B Strep­tococcus (> 10' cfu/ml) was isolated. There were 41 positive amniotic fluid cultures in the infected sam­ples. As seen in the table, E. coli and enterococci were the two most common organisms identified in this

Leukocyte esterase Ebelactone* activity* (range) (range)

2.8-7.0 0.6-1.4 17.0-19.6

17.2-18.6 17.1-18.3 17.0-19.3 17.1-19.3

17.3-19.6 3.4-7.2

17.0-18.3 3.1-6.8 17.4 10.4

group. However, if a positive culture was defined as one containing organisms> 102 cfu/ ml, then all 51 sam­ples were positive. The organisms identified as > 102

cfu/ml were as follows: group B streptococcus, one case; enterococcus, one case; streptococcus, one case; E. coli, two cases; Enterobacteriaceae, one case; B. fra­gilis, four cases.

The in vitro inhibition of the leukocyte esterase ac­tivity in the uninfected and infected samples with the nonspecific esterase inhibitor ebelactone is shown in Table I. The inhibition of the leukocyte esterase en­zyme activity units ranged from 0 to 60% in both groups of samples. Further analysis of the comparison between the types of organisms cultured and the inhibition of the enzyme activity units by ebelactone is shown in Table IV. There were varying degrees of inhibition (range, 20% to 60%) in the uninfected samples. These same results were also found in the samples infected with gram-negative organisms (E. coli, B. fragilis, Fu­sobacterium). However, there was no inhibition of leu­kocyte esterase activity (range, 17.1 to 19.3) in the sam­ples infected with gram-positive organisms (group B streptococcus, enterococci). These results were found to be statistically significant. The presence and ranges of inhibition were found to be similar in the samples with organisms> 102 cfu/ml.

Comment

Despite the continuing seriousness of acute cho­rioamnionitis, which produces major morbidity in both mother and neonate, no optimal diagnostic findings currently exist. All attempts to improve our ability to diagnose this entity have been unsuccessful to date.

We previously reported on the reliability of measur­ing leukocyte esterase activity by a simple, nonquanti­tative dipstick assay to diagnose intraamniotic infection in term and preterm gestations.5

, 6 Because the results of the dipstick assay, like those of the aerobic and an­aerobic cultures, can sometimes be misleading or equiv­ocal we suggested the use of an in vitro spectropho-

Volume 163 Number 6, Part I

tometric assay of esterase activity to eliminate some con­fusion! This assay is rapid «10 minutes) and simple to perform. It requires only a spectrophotometer and the determination of change in absorbance after the addition of substrate and inhibitor, both of which are easily available.

At present, even if clinical chorioamnionitis is reliably diagnosed, most standard protocols call for empiric choices of broad-spectrum antibiotics for immediate therapy. These choices are often based on preexisting theoretic knowledge of the pathogens that can cause such infections and not on the identification of specific organisms from the patient. This latter information is usually obtained much later (24 to 48 hours) when cul­ture results are available. The results of our preliminary study suggest that the different bacteria that cause cho­rioamnionitis may elicit the production of different and specific esterases in infected amniotic fluid as shown by the differences in the in vitro inhibition of this activity by ebelactone. Thus it may be possible to identify the

Inhibition of esterase activity in amniotic fluid 1947

causative organisms in chorioamnionitis by measuring the absence, presence, and degree of in vitro leukocyte esterase inhibition by ebelactone. This would allow the formulation of timely and directed management de­cisions even before culture results are available.

REFERENCES

l. Gibbs RS, Castillo MS, Rodgers PJ. Management of acute chorioamnionitis. AM] OBSTET GYNECOL 1980; 136:709.

2. Rindler-Ludwig R, Schmalzl F, Braunsteiner H. Esterases in human neutrophil granulocytes: evidence for their pro­tease nature. Br] Haematol 1974;27:57.

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

4. Hoskins lA, Katz], Ordorica SA, Young BK. Esterase ac­tivity in second and third trimester amniotic fluid: an in­dicator of chorioamnionitis. AM] OBSTET GYNECOL 1989; 161:1543.

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

6. Hoskins lA, Marks F, Ordorica SA, Young BK. Leukocyte esterase activity in amniotic fluid: normal values during pregnancy. Am] PerinatoI1990;7:130.

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