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Assessment of
mucinase activity in patients with and without bacterial vaginosis
showed no significant differences between clinical groups.
Application of a
novel human cervical mucin-based assay demonstrates the absence of
increased mucinase activity in bacterial vaginosis.
Wiggins R, Millar MR, Soothill PW,
Hicks SJ, Corfield AP.
Inter J STD AIDS. 2002;13:755-760
Summary:
Question
How well does a novel ELISA mucinase assay compare to assays for enzyme
activities already identified as markers for bacterial vaginosis?
Design
This study describes a blinded comparison of the results of ELISA assays
for mucinase and other enzyme activities with those of Gram stain scoring
performed on vaginal swabs for the diagnosis of bacterial vaginosis.
Participants
One hundred pregnant women <14 weeks gestational age attending for
consultation concerning surgical termination of pregnancy who had not had
antibiotic treatment in the previous 4 weeks were tested. The age range
was between 16 and 45 years with a mean of 28.8 years. Eight women were
excluded from the analyses due to poor quality of the Gram-stained slide.
Description of Tests and Diagnostic
Standard
Two high vaginal swabs were collected from each woman during a speculum
examination. One swab was placed in 2.5 mL of buffer, vortexed for 15
minutes, stored at 4oC, and tested for enzymatic activity
within 24 hr. The mucinase assay measures the activity of a group of
glycosidases and proteinases capable of degrading human cervical mucin.
The substrate for the assay was made by pooling cervical mucus from 60
patients that were negative for sexually transmitted diseases and
bacterial vaginosis. The mucus was purified, labeled with biotin in either
the protein or carbohydrate moieties, and bound to wells of ELISA plates.
After addition of the vaginal swab sample eluent to the test wells, and
incubation at 37oC for 1 hour, the mucinase activity (i.e.
cleavage of the biotin labeled protein or carbohydrate moiety from the
bound mucin) of each sample was measured by a colorimetric biotin
detection reaction. Mucinase activity was calculated as the difference
between the OD of the blank well and the OD of the sample. Previously
developed ELISA assays for sialidase, ß-D-N-acetylgalactosamine, ß-D-N-acetylglucosamine,
and proteinase were also performed.
The second vaginal swab was rolled onto a glass slide and Gram-stained for
the diagnosis of bacterial vaginosis. The vaginal flora was graded as
Grade 1 (predominantly lactobacilli), Grade 2 (mixed lactobacilli and BV
flora), or Grade 3 (BV flora) according to a modified Spiegel's criteria.
Main Outcome Measures
The mucinase ELISA assay was optimized and the mucinase activity,
sialidase, ß-D-N-acetylgalactosamine, ß-D-N-acetylglucosamine, and
proteinase activities for each clinical group determined by the Gram stain
score were compared.
Main Results
The enzymatic activities of the 92 vaginal fluid samples by grade of
bacterial vaginosis are shown in the table.
| Enzymatic activity
in vaginal swab fluid by grade of bacterial vaginosis determined by
Gram stain among 92 pregnant women |
| Assay |
Enzyme
activity* by Gram stain result |
Statistical
significance |
Group
1
BV negative (n=46) |
Group
2
Mixed flora (n=22) |
Group
3
BV positive (n=24) |
P
1 vs 3 |
P
1 vs 2 |
P
2 vs 3 |
|
Mucinase |
|
|
|
|
|
|
|
Protein
pH 4.2 |
0.219 |
0.102 |
0.175 |
NS |
NS |
NS |
Protein
pH 5.5 |
0.212 |
0.078 |
0.084 |
NS |
NS |
NS |
Carbohydrate
pH 4.2 |
0.410 |
0.261 |
0.221 |
NS |
NS |
NS |
Carbohydrate
pH 5.5 |
0.379 |
0.439 |
0.083 |
NS |
NS |
NS |
|
Sialidase** |
|
|
|
|
|
|
| AGP |
0.000 |
1.358 |
2.685 |
.0001 |
.03 |
.004 |
| BSM |
2.427 |
4.142 |
7.928 |
.0001 |
.02 |
.001 |
| ß-galactosidase |
0.000 |
0.080 |
0.300 |
.0008 |
NS |
NS |
|
ß-N-acetyl-
hexosaminidase |
0.120 |
0.200 |
0.230 |
.049 |
NS |
NS |
| Proteinse |
0.065 |
0.060 |
0.000 |
NS |
NS |
NS |
*Enzyme activity is
optical density/mL fluid/h except for sialidase, which is nmole sialic
acid released/mL fluid/h
**Sialidase assay substrates were human alpha-1-acid glycoprotein (AGP)
and bovine submaxillary mucin (BSM)
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Authors' Conclusions
No differences in mucinase activities were
detected between normal and BV groups, while significant elevation of
sialidase and other glycosidases was confirmed as reported before.
Mucinase activity is a normal event in the mucus barrier.
Source of funding:
Tommy's Campaign, Kennington Rd, London SW
For correspondence:
Anthony Corfield, University Division of Medicine, Bristol Royal
Infirmary, Bristol BS2 8HW, UK. E-mail address: tony.corfield@bristol.ac.uk.
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