M A J O R A R T I C L E
Clinical Evidence for the Role of Trichomonas
vaginalis in Regulation of Secretory Leukocyte
Protease Inhibitor in the Female Genital Tract
Jill S. Huppert,1Bin Huang,2Chen Chen,2Hassan Y. Dawood,3and Raina N. Fichorova3
1Division of Gynecology and2Division of Epidemiology and Biostatistics, Cincinnati Children’s Hospital Medical Center, Ohio; and3Laboratory of
Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School and Brigham and Women’s Hospital,
to and innate and adaptive immune responses in the vaginal mucosa. Depressed cervicovaginal SLPI levels have
been correlated with both Trichomonas vaginalis infection and poor reproductive health outcomes.
Methods. We measured levels of SLPI in 215 vaginal specimens collected from adolescent and young adult
females aged 14–22 years. Log-transformed SLPI values were compared by analysis of variance or by an unpaired
t test before and after adjustment for confounding effects through the propensity score method.
Results. Females receiving hormonal contraceptives and those with an abnormal vaginal pH had lower SLPI
levels as compared to their peers. After propensity score adjustment for race, behavioral factors, hormonal use,
and other sexually transmitted infections (STIs), SLPI levels were lower in females with a positive T. vaginalis
antigen test result, a vaginal pH >4.5, vaginal leukocytosis, and recurrent (vs initial) T. vaginalis infection, with
the lowest levels observed in those with the highest T. vaginalis loads.
Conclusions. The SLPI level was reduced by >50% in a T. vaginalis load–dependent manner. Future research
should consider whether identifying and treating females with low levels of T. vaginalis infection (before they
become wet mount positive) would prevent the loss of SLPI and impaired vaginal immunity. The SLPI level could
be used as a vaginal-health marker to evaluate interventions and vaginal products.
Secretory leukocyte protease inhibitor (SLPI) is responsible for regulating inflammatory damage
SLPI protein; human; Trichomonas vaginalis; Vaginosis; Bacterial; Sexually Transmitted Diseases;
Secretory leukocyte protease inhibitor (SLPI) is abun-
dantly produced in human mucosal epithelia, includ-
ing the female reproductive tract, and plays multiple
roles in controlling inflammation and in antibacterial
and antiviral innate and adaptive immune responses .
An elevated SLPI level appears to protect against
human immunodeficiency virus (HIV) acquisition ,
whereas a depressed SLPI level has been associated
with failure of vaginal microbicides to protect against
HIV acquisition [3, 4].
Depressed cervicovaginal levels of SLPI have been
associated with the most common curable vaginal in-
fections: trichomoniasis, caused by sexually transmit-
ted pathogen Trichomonas vaginalis, and bacterial
vaginosis [5–7]. T. vaginalis and bacterial vaginosis
often coexist  and are established risk factors for
adverse reproductive outcomes, including pregnancy
loss, premature rupture of membranes, and preterm
labor . Interestingly, decreased levels of SLPI in am-
niotic fluid have been associated with premature
rupture of membranes . Thus, decreased SLPI
levels may be one of the reasons for the poor repro-
ductive outcomes associated with vaginal infections.
have an increased risk of poor pregnancy outcomes,
Received 25 September 2012; accepted 2 December 2012; electronically pub-
lished 25 January 2013.
Correspondence: Jill S. Huppert, MD, MPH, Division of Pediatric and Adoles-
cent Gynecology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave,
MLC 2026, Cincinnati, OH 45229-3039 (firstname.lastname@example.org).
The Journal of InfectiousDiseases2013;207:1462–70
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
1462 • JID 2013:207 (1 May) • Huppert et al
by guest on November 24, 2015
antibiotic treatment of T. vaginalis does not alter the risk [11, 12],
suggesting that immunologic changes triggered by T. vaginalis
infection may persist after parasitological cure. T. vaginalis is
known to cause vaginal disruption, as evidenced by an elevated
vaginal pH, vaginal leukocytosis, and altered vaginal mucosal
immune response [4, 9, 13, 14]. Elevated vaginal pH is associated
with sexually transmitted infections (STIs), bacterial vaginosis
, and poor pregnancy outcomes .Vaginal leukocytosis is
associated with endometritis . The SLPI level is lower in
women infected with T. vaginalis as compared to women with
Chlamydia trachomatis infection, Neisseria gonorrhea infection,
bacterial vaginosis, or no infection . In vitro, SLPI is digested
by T. vaginalis cysteine proteases .
At present, there are few studies of SLPI that include large
numbers of young nonpregnant females, many of whom use
hormonal contraception, which, on its own, has controversial
effects on the mucosal immune function in adolescents [20,
21]. In addition, T. vaginalis infections vary from asymptom-
atic infections detected by nucleic amplification testing only to
infections associated with severe vaginal symptoms and a pos-
itive result of wet mount testing. Although no lasting acquired
immunity has been associated with T. vaginalis , initial
T. vaginalis infections may behave differently than recurrent
or persistent infections in term of innate immune environ-
ment. We sought to determine whether vaginal levels of SLPI
correlated with T. vaginalis loads, initial versus recurrent T.
vaginalis infections, hormonal contraceptive use, and tradi-
tional measures of vaginal disruption.
Clinical Study Design
This study was part of a larger cross-sectional study assessing
the accuracy and acceptability of self-testing for T. vaginalis,
the results of which are detailed in our previous work .
This analysis and the original study were approved by the hos-
pital’s institutional review board. Briefly, adolescent and
young adult females ages 14–22 years were recruited from an
adolescent clinic and the emergency department. A clinician
performed a pelvic examination and collected multiple vaginal
swab specimens for immediate testing. One polyester swab
was frozen dry at −80°C and stored for future use and testing.
Participants completed questionnaires encompassing demo-
graphic characteristics, sexual history (including prior STI
history, number of sex partners, and use of contraceptives),
and vaginal or gynecological symptoms.
Clinical bacterial vaginosis was defined using modified
version of the Amsel criteria (ie, an abnormal vaginal pH
[>4.5], a high clue cell percentage [>20% of epithelial cells per
field], and a positive result of an amine test). Compared with
Nugent Gram staining, these objective criteria provide sensi-
tivity and specificity similar to that of the full Amsel criteria
[23, 24]. Hormonal contraception in the prior 3 months was
characterized as none; combined estrogen and progestin (pills,
ring, or patch); or progestin only (depot medroxyprogesterone
acetate or progestin-containing intrauterine device).
As described previously , vaginal swab specimens were
used for wet mount diagnostic testing and for assessment of
pH, amines, sialidase, and STIs. Leukocytosis on wet mount
testing (magnified at 400×) was categorized as low (≤5 leuko-
cytes per field) or high (≥6 leukocytes per field) . Clue
cells (vaginal epithelial cells with adherent bacteria) were cate-
gorized as present (>20% of epithelial cells per field) or absent
. Vaginal pH (pHydrion, Mikro Essentials Laboratories,
Brooklyn, NY) was classified as >4.5 (elevated) or ≤4.5
(normal) .Amines were recorded if a fishy odor was detect-
ed after applying potassium hydroxide to the specimen .
The wet mount test was deemed positive for T. vaginalis if
motile trichomonads were observed on direct microscopy. Sia-
lidase testing (BVBlue, Sekisui Diagnostics, Cambridge, MA),
the rapid antigen T. vaginalis test (OSOM T. vaginalis Tricho-
monas Rapid Test, Sekisui Diagnostics), and T. vaginalis
culture (InPouch T. vaginalis culture, BioMed, White City,
OR) were performed following manufacturers’ directions. On
the basis of prior work, we considered any positive T. vaginalis
test to be a true positive T. vaginalis infection .
At study closure, the stored vaginal swab sample was
brought to room temperature and eluted with 1.0 mL of phos-
phate-buffered saline for 10 minutes. One half of the eluent
was transferred to a vaginal swab collection kit (Aptima, Gen-
Probe, San Diego, CA) and transported on ice to the Interna-
tional Sexually Transmitted Diseases Research Laboratory
(Baltimore, MD). C. trachomatis and N. gonorrhea were de-
tected using nucleic acid amplification testing (NAAT; Aptima
Combo2, Gen-Probe). T. vaginalis and Mycoplasma genitalium
were detected on the same NAAT platform, using research-
The remaining eluent (0.5 mL) was transferred to a sterile
vial, stored frozen at −80°C, and shipped frozen to the Labo-
ratory of Genital Tract Biology at Brigham and Women’s Hos-
pital, where samples were assayed for total protein content
and SLPI. The use of these samples for biomarker analysis was
approved by the Brigham and Women’s Institutional Review
Board for Human Subject Research. The Laboratory of Genital
Tract Biology is accredited by the College of American Pathol-
ogists for immunoassay analysis and operates under strict
quality control procedures. SLPI concentrations were mea-
sured by the Quantikine enzyme-linked immunosorbent assay
(R&D Systems, Minneapolis, MN), using a Victor2 reader
(Perkin Elmer Life Sciences, Boston, MA), as previously de-
scribed [30, 31]. For initial screening, all samples were diluted
40-fold in the sample diluent provided by R&D Systems, and
Trichomoniasis and Innate Immunity • JID 2013:207 (1 May) • 1463
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all samples showing levels below or above the assay detection
range were repeatedly tested at dilutions of 4-fold or 400-fold,
respectively, to obtain accurate measurements. All measure-
ments were performed in duplicate. A split quality control
pool prepared from cervicovaginal samples was tested on each
plate showing an interplate variation of <25%.The total
protein concentration in each eluted sample was determined
by the BCA assay (Thermo Scientific, Rockford, IL), using the
Victor2 counter, and the SLPI concentrations were normalized
to milligrams of total protein. For the BCA assay, all samples
were screened at a 2-fold dilution in phosphate-buffered
saline, and testing was repeated without dilution or at a 5-fold
dilution if values were below or above the standard curve, re-
spectively. The coefficient of variation between duplicate
values for each sample was <10%.
As the closest surrogate measure of T. vaginalis load avail-
able, we used a 4-level grading system based on previously es-
tablished sensitivities of each T. vaginalis diagnostic test, with
the highest load assumed to be corresponding to a positive
result wet mount testing (least sensitive test), a moderate load
corresponding to a negative result of wet mount testing but
positive results of culture and/or antigen testing, and the lowest
load corresponding to a positive result of transcription-mediated
amplification (TMA) analysis but negative results of all other
tests [29, 32, 33]. Theoretically, each of the diagnostic tests can
give false-negative results because of testing methods, strain dif-
ferences, or sample handling. In the literature, the reported
false-negative rates are as follows: wet mount testing, 31%–58%
[34–36]; culture, 7%–28% [32, 36]; rapid antigen testing, 2%–
18% [29, 38]; and TMA analysis, 1%–6% [29, 39, 40]. In addi-
tion, the organism load required for a positive test result is
reported as 10000 organism/mL for wet mount testing, 100–
500 organisms/mL for culture, 800–2500 organism/mL for rapid
antigen testing, and 1–40 organisms/mL for NAAT [41,42].
We evaluated the distribution of important covariates by
T. vaginalis infection status, using χ2tests for dichotomous
variablesand analysisofvariance (ANOVA)forcategorical vari-
ables. SLPI results were not normally distributed, so they were
log-transformed (natural log) before comparison by means of
the Student t test. Differences in mean log SLPI values are pre-
sented as the risk ratio (covariate present vs covariate absent),
where a value of >1 represents the degree to which the SLPI
level is increased, and a value of <1 indicates the degree of
decrease when the covariate is present.
Modeling to Adjust for Confounding
In this observational study, there are many potential con-
founders, such as age, race, sexual behavior, and types of con-
traception. Failure to adequately consider these confounding
effects may bias the results. For example, African American
race is overrepresented in the T. vaginalis–infected group and
thus may bias the comparison of SLPI levels between the in-
fected and noninfected females. Traditionally, confounding
effects are adjusted for by including confounders in the regres-
sion model. However, this approach is limited by modeling
assumptions, such as distribution and linear additive assump-
tions, and often requires increases in sample size as the
number of confounders increase. The propensity score
method is a relatively new technique that does not require the
imposition of any distribution or modeling assumptions and
helps boost study power. It was proposed to statistically evaluate
causal effects free from confounding effects, by mathematically
refashioning an observational study into a randomized study;
therefore, studies using the propensity score approach are
sometimes considered as pseudo-randomized studies . The
most commonly used propensity score techniques are matching
and inverse weighting, which is considered more appropriate
for small sample sizes. Confounders were predetermined on the
basis of the current literature and clinical knowledge. We
created propensity scores by using both the matching and the
inverse weighting technique. Since they gave the same results,
we chose to show the weighted results. The distributions of the
confounders were compared before and after application of the
propensity score methods. For each hypothesis, propensity
scores are first created to balance the distribution of confound-
ers between the exposure of interest (present vs absent groups).
Then the groups are compared on the basis of their log SLPI
values, adjusted by the inverse weight of propensity scores,
using a 2-group t test. To adjust for multiple comparisons, we
adopted the false-discovery rate (FDR) procedure .
For ease of interpretation and to establish normative ranges
in adolescent females, we also present the summary statistics
in picograms of SLPI per milligram of total protein, derived
by calculating the antilog (exponential) of the log-transformed
Of the 249 females recruited for the original trichomoniasis
study, 215 had samples available for SLPI testing and com-
posed the current study sample. The subjects’ characteristics
are summarized in Table 1. The mean age was 18.3 years
(range, 14–22 years), and the majority self-identified as
African American. T. vaginalis was detected (by any T. vagina-
lis test) in 53 females (24%), C. trachomatis in 49 (22.6%),
N. gonorrhea in 21 (9.7%), and M. genitalium in 30 (14%).
Many females had sexual risk factors such as multiple sex
partners, a history of prior STI, and a current laboratory-
confirmed STI. Females with a positive result of any T. vagina-
lis test were more likely to have a positive result of an amine
test, an abnormal pH, or gonorrhea, compared with those
1464 • JID 2013:207 (1 May) • Huppert et al
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who were negative for T. vaginalis. Of the 53 T. vaginalis
infections, 20 (38%) had a high load, 27 (50%) had a mod-
erate load, and 6 (11%) had low load. Additionally, 99 (46%)
had no past history of T. vaginalis infection and no current
infection and were considered to be T. vaginalis naive; 62
(28.8%) reported prior T. vaginalis infection and were cur-
rently uninfected, 21 (9.8%) had current infection only, and
32 (14.9%) had both a past history of and current T. vagina-
lis infection (these females were considered to have recurrent
Figure 1 shows the direction and magnitude of each covari-
ate effect on SLPI concentration, unadjusted by propensity
score. The bars represent the ratios of mean SLPI concentra-
tions in females with the factor present versus those with the
factor absent, illustrating the risk magnitude and direction of
change. A risk ratio of <1.0 represents a decrease in SLPI level
with the factor present, and a risk ratio of >1.0 represents an
increase in SLPI level with the factor present. To interpret the
risk ratio, a risk ratio of 0.47 means that SLPI levels are de-
creased by 53%. This value can also be derived by calculating
the antilog (exponential) of the difference between the log-
transformed levels. SLPI levels were significantly (P<.05, by
t tests) lower for those with any hormonal contraception use,
a positive result of a T. vaginalis test (TMA, culture, antigen,
or wet mount), an elevated white blood cell (WBC) count on
wet mount testing, and an abnormal vaginal pH of >4.5.
There was also a trend for SLPI levels to be higher in females
>18 years of (ratio 1.36) and lower in females with C. tracho-
matis infection (ratio 0.64), but the differences did not reach
statistical significance (P> .05 and P<.1, respectively).
We compared mean log SLPI concentration across categorical
variables (hormonal contraception use, T. vaginalis test results,
and T. vaginalis infection history), using ANOVA (Table 2).
When hormonal contraception was modeled as a dichotomous
variable, any hormonal contraception use was associated with a
reduced SLPI concentration; however, as a categorical variable,
progestin-only contraception appeared to depress the SLPI level
more than a combined estrogen-progestin method (ratio 0.61).
The SLPI level was significantly lower for females with a high
T. vaginalis load (positive result of wet mount testing, ratio
0.46) as compared to females with negative results of all T. vagi-
nalis tests. Also, recurrent T. vaginalis infection appeared to
Table 1.Subject Characteristics, by Trichomonas vaginalis (TV) Infection Status
Subjects, Proportion (%), by TV Test Result(s)
≥1 PositiveAll Negative OverallP
Age ≥18 y
History of prior STI
Vaginal symptoms present
TMA test result
C. trachomatis positive
N. gonorrhea positive
M. genitalium positive
High clue cell %b
Any history of douching
Abnormal vaginal pH (>4.5)
Hormonal contraception use
Clinical BV presentc
Multiple sex partners (≥2)
Positive result of amine test
High WBC countd
Sialidase positive (n=45)
Column percentages are calculated on the basis of the total number of subjects with a defined characteristic. Denominators differ because of missing data.
Differences were assessed with a χ2test.
Abbreviations: BV, bacterial vaginosis; C. trachomatis, Chlamydia trachomatis; M. genitalium, Mycoplasma genitalium; N. gonorrhea, Neisseria gonorrhea; STI,
sexually transmitted infection; TMA, transcription-mediated amplification; WBC, white blood cell.
aWithin the past 2 days.
bDefined as >20% of epithelial cells per field.
cDefined as an abnormal vaginal pH (>4.5), a high clue cell percentage (>20% of epithelial cells per field), and a positive result of an amine test.
dDefined as >6 cells/high-power field on wet mount.
Trichomoniasis and Innate Immunity • JID 2013:207 (1 May) • 1465
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depress SLPI levels (ratio 0.49) to a greater degree than first-
time (current only) T. vaginalis infection.
Figure 2 shows the effect of propensity weighting on the
distribution of several important covariates, compared with
T. vaginalis infection status. Variables displayed are those
known to affect either T. vaginalis infection status or SLPI
level. The y-axis shows the difference in the percentage of
females who were T. vaginalis positive, compared with those
who were T. vaginalis negative, before (light bar) and after
(dark bar) propensity weighting. For example, before weight-
ing, the proportion of females who self-reported African
American race was higher (by about 10%) among those who
were T. vaginalis positive as compared to those who were
T. vaginalis negative; after weighting, the difference was only
about 4%. Although the differences prior to weighting were
significant only for N. gonorrhea infection and a positive
result of amine testing, all covariates became more evenly dis-
tributed after weighting, with no significant differences. In ad-
dition to analyzing differences on the basis of T. vaginalis
infection status (Figure 2), we also examined the effect of
weighting on the distribution of covariates with respect to fac-
tors found to be associated with the SLPI level in our data set
(ie, abnormal vaginal pH, high WBC count on wet mount
testing, and history of T. vaginalis infection; data not shown).
In each case, propensity weighting removed the imbalance
of variables and resulted in a more even distribution of
Table 3 shows the results obtained after propensity weight-
ing. After weighting, the SLPI concentration was not asso-
ciated with the combined variable “positive result of any
T. vaginalis test.” The SLPI level was decreased in females with
a positive result of the T. vaginalis antigen test (ratio 0.64), an
elevated vaginal pH (ratio 0.6), and vaginal leukocytosis
(defined as >6 WBCs/HPF on wet mount testing; ratio 0.44).
Compared with females with an initial/current infection,
females with recurrent T. vaginalis infection had a significantly
depressed SLPI level (ratio 0.49). Among females with a posi-
tive result of any T. vaginalis test, those with a positive result
of wet mount testing had the lowest SLPI level (ratio 0.27),
compared with those with a negative result of wet mount
testing. After control by use of the FDR procedure, only the
T. vaginalis antigen test became marginally nonsignificant
(adjusted P= .058 vs unadjusted P= .048); the rest of the
results remained the same.
bars represent the risk ratios of SLPI concentrations (log10of pg of SLPI per mg of total protein) in women with the specified factor present vs those
with that factor absent. A ratio of <1.0 represents a decrease in SLPI level with the factor present, and a ratio of >1.0 represents an increase in SLPI
level with the factor present. Solid dark bars are significant at P<.05, striped bars have borderline significance (P>.05 and ≤.1), and light bars are not
significant (P>.1, by the t test).aDefined as >20% of epithelial cells per field.bDefined as an abnormal vaginal pH (>4.5), a high clue cell percentage
(>20% of epithelial cells per field), and a positive result of an amine test.cDefined as >6 cells/high-power field on wet mount. Abbreviations: Ag,
antigen; BV, bacterial vaginosis; C. trachomatis, Chlamydia trachomatis; STD, sexually transmitted disease; TMA, transcription-mediated amplification;
T. vaginalis, Trichomonas vaginalis; WBC, white blood cell.
Direction and magnitude of covariate effect on the secretory leukocyte protease inhibitor (SLPI) level, unadjusted by propensity score. The
1466 • JID 2013:207 (1 May) • Huppert et al
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We have generated solid evidence from these cross-sectional
data that a depressed SLPI concentration is highly associated
with T. vaginalis infection, as determined by a positive result
of rapid antigen testing and wet mount testing. We have previ-
ously shown that the rapid antigen test is comparable to
culture and more sensitive than wet mount testing . In ad-
dition, the antigen test is clinically available, but it has not yet
been widely used to assess the risk of outcomes or response to
treatment for T. vaginalis.
Our data suggest that the mucosal immune defense is im-
paired by T. vaginalis in a T. vaginalis load–dependent
manner. Among adolescent and young adult females with any
evidence of T. vaginalis infection, the SLPI concentration was
73% lower (risk ratio 0.27) in those with positive results of wet
mount testing, compared with those with negative results of
wet mount testing but positive results of antigen testing,
culture, or TMA analysis. However, it is unknown whether
identifying and treating women with positive results of NAAT
but negative results of wet mount tests or culture (it is pre-
sumed that such individuals have low levels of infection) will
prevent the decrease in SLPI concentration. This knowledge
should be gained through a clinical trial and would be highly
useful in determining the appropriateness of asymptomatic
T. vaginalis screening, as well as for preventing SLPI-mediated
It is well known that women with 1 episode of T. vaginalis
infection are at high risk for recurrent and persistent infec-
tions [45, 46]. A recurrent infection may be facilitated by
lower SLPI levels. This hypothesis is in agreement with our
finding that the SLPI level was approximately 50% lower
among females with recurrent T. vaginalis infection than in
those with an initial infection. This is similar to our under-
standing of the immune response to C. trachomatis infection,
in which subsequent infections induce a brisker inflammatory
response that damages the host, compared with an initial in-
fection. If these cross-sectional data are confirmed in longitu-
dinal studies, it might lead us to develop a “prevention for
positives” approach to the control of trichomoniasis. For
example, it may be more cost-effective to develop strategies to
prevent future complications among women who test positive
for a first episode of trichomoniasis than to prevent the first
Another possible explanation for lower SLPI levels in
women with recurrent infection is that some of those women
could have a persistent, rather than recurrent, T. vaginalis in-
fection that may not be responding to treatment. A number of
recent studies suggest that failed treatment may be more
common than was previously reported [45, 47]. We have
Leukocyte Protease Inhibitor (SLPI) Levels
Effect of Categorical Variables on Mean Secretory
Hormonal contraception (n=207)
TV test result(s) (n= 215)
Only TMA positive
Wet mount negative,
antigen or culture
Wet mount positive
TV history (n=215)
Prior TV Only
Current TV only
Prior and current TV
20 (9.3) 10.4470.46
Abbreviations: Ref, reference; TMA, transcription-mediated amplification; TV,
aData are pg of SLPI per mg of total protein.
bValues are unadjusted and calculated by analysis of variance.
acteristics by Trichomonas vaginalis infection status. The y-axis shows the
absolute difference in the percentage of females who were T. vaginalis
positive, compared with those who were T. vaginalis negative, within each
subject group, before (light bar) and after (dark bar) propensity weighting.
For example, before weighting, the percentage of subjects who were
African American was higher (by 10%) among those who were T. vaginalis
positive, compared with those who were T. vaginalis negative; after weight-
ing, the difference was only about 4%. The weighting completely eliminat-
ed the differences that were significant at P<.05 (marked by an asterisk).
aDefined as >20% of epithelial cells per field. Abbreviations: CT, Chlamydia
trachomatis; GC, Neisseria gonorrhea; MG, Mycoplasma genitalium.
Effect of propensity weighting on distribution of subject char-
Trichomoniasis and Innate Immunity • JID 2013:207 (1 May) • 1467
by guest on November 24, 2015
recently shown that Trichomonas vaginalis can directly sup-
press SLPI expression via its surface lipophosphoglycan .
Finally, we confirmed that traditional markers of vaginal
barrier disruption, such as elevated vaginal pH and leukocyto-
sis, are strongly associated with decreases in SLPI concentra-
tion and, thus, are markers of impaired vaginal immune
defense. This knowledge could have 2 uses. With others, we
have shown that women can accurately assess their vaginal pH
by using an over-the-counter device and then seek medical
care to determine the presence of bacterial vaginosis or T. vag-
inalis infection, especially if they have engaged in risky behav-
ior [21, 48]. Second, our findings support prior studies
suggesting that the SLPI level should be used to evaluate the
safety of vaginal products, ranging from tampons to microbi-
cides [49, 50].
Strengths of our study are a reasonable sample size of well-
preserved vaginal swab samples from a population of adoles-
cent and young adult females at high risk for T. vaginalis
infection and other STIs. The SLPI level was determined using
state of the art methods, and levels are standardized by milli-
grams per total protein concentration, to avoid bias by varying
levels of sample on each swab. Our statistical approach, which
used propensity weighting, was chosen to minimize the con-
founding bias inherentin
samples. We placed emphasis on the vectorial magnitude of
the differences in SLPI levels by presenting risk ratios rather
than the absolute values, since at present there are no known
cutoffs for normal and abnormal SLPI levels. Our study of 215
predominantly African American adolescents and young
adults with a prevalent STI history has limited power to estab-
lish a normative range. Therefore, the mean SLPI concentra-
tions presented here only serve as a guide for future studies
toward establishing normative ranges for this age category.
The main limitations of our study relate to its cross-sectional
design. We relied on self-reporting of past infections, recent
sexual contact, and contraception use. Because the SLPI level
has a role in alerting the host to a new infection and in modu-
lating both the innate and adaptive immune response, SLPI
Table 3. Effect of Variables on Secretory Leukocyte Protease Inhibitor (SLPI) Levels After Propensity Score Weighting
Variable, ResponseSubjects, No.
SLPI Level, Meana
Any TV test result
TV antigen test result
WBC count on wet mount
High (>6 cells/HPF)
Normal (≤6 cells/HPF)
Both past and current
Wet mount resultg
0.44 (.28–.70) <.01
Analysis is limited to significant variables. Each row represents a separate propensity analysis.
Abbreviations: CI, confidence interval; HPF, high-power field; RR, risk ratio; TV, Trichomonas vaginalis; WBC, white blood cell.
aLog values are propensity score weighted. Antilog values are pg/mg protein and are provided to facilitate interpretation.
bCalculated as [log10of SLPI level in pg]/[mg total protein] in women with the factor present vs those with the factor absent.
cTwo-group t test adjusted by inverse weighting of propensity scores.
dCompared with naive.
eCompared with past only.
fCompared with current only.
gData are for women with any positive TV test result (n =53).
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concentrations may change over time with the duration of in-
fection. With this data set, we could not determine the duration
of any T. vaginalis infections. In addition, we could only esti-
mate organism load on the basis of the type of T. vaginalis test
yielded positive results. However, our approach is reasonable,
given that others have shown by real-time, semiquantitative
PCR that culture-positive specimens have a >2 log10higher
concentration of DNA than specimens that are PCR positive
but culture negative . Some infections that we labeled as
“probable recurrent infections” might have actually represented
persistent infections due to failure of prior treatments .
In conclusion, in adolescent and young adult females, a de-
pressed SLPI level is strongly associated with T. vaginalis in-
fection, bacterial vaginosis, hormonal contraceptive use, and
younger age. Further studies should evaluate the SLPI level as
a promising marker of the vaginal immune response to T. vag-
inalis, bacterial vaginosis, and other STIs in nonpregnant and
pregnant females of all ages.
Financial support. This work was supported by the National Institute
of Allergy and Infectious Diseases, National Institutes of Health (NIH;
grant 5K23AI063182-04 to J. S. H. and grant 5R01AI079085-04 to
R. N. F.), and the National Institute of Biomedical Imaging and Bioengi-
neering, NIH (grant 1U54EB007958-01 to J. S. H.).
Potential conflicts of interest. J. S. H. has received honorarium from
Genzyme/Sekisui as a speaker. All other authors report no conflicts of
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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