Self-administered vaginal swabs are used to sample vaginal contents for a range of clinical applications including tests for sexually transmitted diseases,1–3 cervical cancer,4 condom failure,5–7 and vaginal microbicide use and safety.8,9 Validation of proper collection of self-obtained vaginal samples is of critical importance for assessing subject adherence in clinical and research settings. For example, confirmation of vaginal exposure of swabs that are negative for a semen biomarker is critical in the evaluation of new female condoms.10
Detection of vaginal cells is important in forensic investigation, and several attempts have been made to validate tests for this purpose.11–14 Detection of glycogen-positive cells has been used to prove vaginal penetration in sexual assault cases.12,15–18 Glycogen serves as an energy source and is stored in adipose tissue. It is made primarily by the liver and muscle tissue, but during the reproductive period of women, high estrogen levels stimulate the vaginal epithelium to produce large amounts of glycogen.19 Mucosal epithelial cells in the mouth, penile urethra, and rectum also synthesize glycogen, but to a much lesser extent.15–17 Thus, glycogen provides an attractive marker for the detection of vaginal cells. Most methods for the detection of vaginal glycogen have been based on the detection of glycogen-rich vaginal cells in stained cell smears. However, this process is time-consuming and subjective, requires a high level of expertise, and has limited specificity and sensitivity. An alternative approach is to take advantage of the fact that glycogen is released from vaginal epithelial cells into vaginal secretions where it serves as an important energy source for lactobacilli.20 Commercial quantitative colorimetric glycogen assays have been developed to detect alterations in blood glycogen concentrations caused by metabolic abnormalities associated with diabetes and several genetic glycogen storage diseases. These assays are inexpensive (<$2/test), straightforward, and objective. The purpose of the current study was to validate the use of a quantitative soluble glycogen assay to confirm that a swab has been inserted into the vagina.
Four different sets of specimens were collected and tested: (1) the Centers for Disease Control and Prevention (CDC) provided (n = 27) de-identified swab eluates (from vaginal, buccal, hand/finger, and empty swabs); (2) California Family Health Council (CFHC) provided (n = 26) archived de-identified remnant vaginal swabs from a condom clinical trial for assay development; (3) CONRAD provided de-identified vaginal swabs (n = 8) from 2 reproductive-aged women, 3 perimenopausal women, and 3 postmenopausal women; and (4) in addition, Boston University Medical Campus (BUMC) recruited healthy nonpregnant reproductive-aged (18–40 years old) female volunteers (n = 13) to provide vaginal samples. Fresh semen samples were obtained as discarded de-identified specimens with normal semen parameters (n = 10) from BUMC Urology Department. This study was approved by the institutional review board of BUMC, and all volunteers provided written informed consent for the study.
Specimen Collection and Processing
Vaginal and control specimens were obtained using Falcon Swubes (Becton, Dickinson & Co, Franklin Lakes, NJ). Each Swube consists of a 6-in. (15.2-cm) cotton-tipped swab attached to the screw cap of a 1.6 × 15-cm clear plastic tube containing a Minipax (Multisorb Technologies Inc) desiccant. For vaginal sampling, the participants were instructed to introduce the swab into the vagina until resistance was felt, rotate it 5 times, and then remove it. Control samples were obtained by placing the swab against the inner cheek buccal mucosa or hand/finger skin and rotating 5 times. The participants were instructed to reinsert the swab into the Swube tube immediately after the sample was taken and to tighten the cap. The swabs were kept in the tubes for a minimum of 24 hours for drying. For elution of swab contents, dried swabs were transferred to Eppendorf tubes containing 1.5 mL of phosphate-buffered saline (PBS) with 0.05% Tween 20 (polysorbate 20; Sigma), vortexed for 10 seconds, and then incubated for 30 minutes at 4°C. This elution protocol was selected because it is widely used to elute vaginal swabs for prostate-specific antigen (PSA; semen) testing,21 allowing us to evaluate the feasibility of using 1 eluate to test for both glycogen and PSA in the future. Swabs were squeezed against the side of the tube as they were removed, and 250 μL of aliquots of the eluates was pipetted into labeled cryotubes and stored at −80°C until used for testing.
1. Quantitative colorimetric assay
Samples were thawed and centrifuged in a microfuge at 400g for 1 minute. The supernatants were tested for glycogen content using the EnzyChrom Glycogen Assay kit (BioAssay Systems, Hayward, CA) per manufacturer’s instructions. Briefly, 10 μL of swab eluate was added to individual wells in a 96-well flat bottomed plate, followed by the addition of a single working reagent that enzymatically converts glycogen into glucose and detects glucose with a colorimetric assay read at 570-nm absorbance. For the preliminary studies, the samples were read on a spectrophotometer and were scored as greater than 200 μg/mL or less than 200 μg/mL (semiquantitative assay). For quantitative glycogen assessment, the samples were read on an automated BioTek Synergy HT plate reader, and glycogen values were calculated from a standard curve run in each assay, with a linear range from 50 to 200 μg/mL. Selected samples with glycogen values above the linear range of the standard curve (>200 μg/mL) were serially diluted in PBS/Tween and retested to determine the glycogen concentration.
2. Detection of glycogen-positive cells
Cell pellets from swab eluate samples were processed for detection of glycogen-positive epithelial cells using the classical periodic acid–Schiff (PAS) stain.17 Cells were resuspended in 20 μL of PBS/Tween, and drops of suspended cells were applied to individual spots of Teflon-coated 8-well microscope slides (Roboz Surgical Instruments, Washington, DC), dried, and fixed in absolute acetone. Slides were processed through the PAS stain for detection of cells containing accumulations of polysaccharides (i.e., glycogen), which appear bright red.17
Data were analyzed by 1-way analysis of variance. Significant analysis of variance was followed by pairwise post hoc testing by Bonferroni multiple comparison test. Differences were considered statistically significant when P < 0.05. StatView (version 5.0.1; SAS Institute, Cary, NC) and Prism (version 3.0; GraphPad Software, Inc, La Jolla, CA) statistical software were used to perform the statistical computations. Sensitivity and specificity calculations were performed using a statistics Web site available through Vassar College (http://faculty.vassar.edu/lowry/clin1.html).
Detection of Glycogen in Vaginal and Control Swab Eluates: Preliminary Studies
Initially, buccal, hand/finger, and vaginal swab eluates from 3 healthy female volunteers (recruited at BUMC) were evaluated. All vaginal samples contained greater than 200 μg/mL of glycogen (above assay range), whereas all controls contained less than 200 μg/mL of glycogen by semiquantitative analysis. All 3 vaginal samples contained numerous PAS-positive epithelial cells, whereas the control samples had few, if any, PAS-positive cells (Fig. 1). After these encouraging results, a series of 27 coded vaginal and control swab eluates (provided by the CDC) were tested using both the quantitative glycogen assay and PAS test. The 10 vaginal swab samples were successfully differentiated from the 17 control swabs (7 never used, 5 used in mouth [buccal], and 5 used on hand/finger skin). All 10 vaginal swab eluates contained greater than 200 μg/mL of glycogen and numerous PAS (glycogen)-positive cells. In contrast, all negative control samples had less than 200 μg/mL of glycogen, and only 1 sample (buccal) had numerous PAS (glycogen)-positive cells.
Detection of Glycogen in Archived Vaginal Swabs From a Condom Efficacy Trial
A total of 26 archived de-identified remnant vaginal swabs (provided by the CFHC) from samples collected before and after condom uses as part of a condom efficacy trial were tested for glycogen to determine if the assay works for aged (>1 year) archived specimens. Eluates from all of these samples contained greater than 200 μg/mL of glycogen (data not shown).
Glycogen Concentrations in Vaginal Swab Eluates From Women Representing Different Hormonal Conditions and Negative Controls
Eighteen women (from BUMC and CONRAD) provided self-administered vaginal swabs for further validation of the glycogen quantitation assay. Twelve were nonpregnant female volunteers between the ages of 18 and 40 years, and 6 were perimenopausal/postmenopausal women. Vaginal swab eluates from all 18 women contained detectable glycogen concentrations (>50 μg/mL). Glycogen concentrations were significantly higher in vaginal swab eluates from the younger women (mean ± SE, 876.6 ± 146.4 μg/mL) than from the perimenopausal/postmenopausal women (235.3 ± 112.6 μg/mL; P < 0.01). Two of the women reported using hormonal contraception; their glycogen values were 1110 and 987 μg/mL. Five of the reproductive-aged women also provided buccal (n = 5) and hand/finger (n = 5) swabs to serve as negative controls. Glycogen levels were below assay linear detection range (<50 μg/mL) in eluates from all but 1 of the negative control swabs (buccal: mean ± SE, 40.3 ± 4.7 μg/mL; median, 45 μg/mL; range, 24–50 μg/mL; hand/finger: mean ± SE, 18.6 ± 5.4 μg/mL; median, 22 μg/mL; range, 3–31 μg/mL). Thirteen eluates from empty swabs were also tested for glycogen in the quantitative assay and were all below detection range (mean ± SE, 14.8 ± 2.5 μg/mL; median, 14 μg/mL; range, 1.3–31) (Fig. 2). All vaginal swab eluates had numerous glycogen-positive cells by PAS stain; 2 of the buccal swabs had faintly positive cells, whereas all the other negative controls were negative in the PAS assay.
Effects of Human Semen and Seminal Plasma on the Glycogen Assay
Glycogen assessments were performed with semen and soluble semen components, which can be present in vaginal secretions and swab eluates. To determine whether semen contains amounts of glycogen that could contribute to a false-positive vaginal swab result, eluates from swabs that were dipped into fresh whole human semen, dried, and extracted according to the vaginal swab elution protocol were tested in the glycogen assay. These samples all had low glycogen levels (n = 5; mean ± SE, 32.4 ± 10.7 μg/mL; median/range, 31/6–68 μg/mL) (Fig. 2), suggesting that a semen specimen would not be scored as positive in the glycogen assay. To determine whether soluble seminal factors that could be present in eluates from vaginal swabs collected after intercourse interfere with glycogen detection contributing to false-negative results, 10 μL of seminal plasma was added to vaginal swab eluates or directly to glycogen standards. Figure 3 shows that soluble semen factors do not interfere with glycogen detection in the quantitative assay. In other experiments, whole semen did not affect glycogen detection when added to vaginal swabs before elution (data not shown).
Sensitivity/Specificity of the Quantitative Glycogen and PAS Assays for Detection of Vaginal Swabs
Combined vaginal swab eluate data from 30 reproductive-aged women and data from 28 control swabs were used to determine the sensitivity and specificity for detection of vaginal samples using different cutoffs in the quantitative glycogen assay. Vaginal swab data used for this determination were from the following: (a) 3 healthy female volunteers recruited from BUMC used for assay development, (b) 12 of the 18 women from the prospectively recruited group (BUMC/CONRAD), and (c) 15 others (de-identified eluates from CDC and remnant specimens from CFHC as described earlier). Control samples represented various types of nonvaginal sources: 5 buccal, 5 hand/finger, 5 semen, and 13 empty swabs. At a cutoff of 50 μg/mL, 30 of 30 vaginal samples and 2 of 28 control samples were positive for a sensitivity of 1 (95% confidence interval [CI], 0.86–1) and specificity of 0.93 (95% CI, 0.75–0.99). At an assay cutoff of 100 μg/mL, 30 of 30 vaginal versus 0 of 28 control samples were positive for a sensitivity of 1 (95% CI, 0.86–1) and specificity of 1 (95% CI, 0.85–1). At an assay cutoff of 200 μg/mL, 29 of 30 vaginal versus 0 of 28 control samples were positive for a sensitivity of 0.97 (95% CI, 0.81–1) and a specificity of 1 (95% CI, 0.85–1).
The PAS stain was performed on cell smears from 22 of the vaginal swab eluates and 27 controls (10 buccal, 10 hand/finger, and 7 never-used swabs). All 22 of the vaginal samples, 3 of 10 buccal samples, and none of the other 17 control samples had glycogen-positive cells in the PAS assay, for a sensitivity of 1 (95% CI, 0.81–1) and a specificity of 0.86 (95% CI, 0.64–0.96) (Table 1).
The vaginal environment contains a number of factors that could potentially be used as markers for validating swab sample collection. Properly used vaginal swabs should contain exfoliated cells, bacterial flora and a broad array of proteins, polysaccharides, and low-molecular-weight molecules. Recent proteomic studies have identified more than 1000 proteins in cervicovaginal secretions, 136 of which have been classified as core proteins, found in high abundance in most subjects regardless of physiological status.22 Among the most abundant core proteins are those involved in immune defense such as antimicrobial peptides, cytokines, and chemokines. Several of these have been quantified in vaginal swab samples,9 but their concentrations can vary widely, even in swabs carefully collected by trained medical staff, and specificity is low because these factors are also abundant in other types of mucosal samples such as saliva23 and semen.24 Concentrations of other potential markers in cervicovaginal secretions, such as mucins, are influenced by female reproductive hormones (reviewed in Gipson25). The presence of characteristic vaginal flora species such as Lactobacilli spp. offer another possible marker for swab use validation, but vaginal flora profiles vary considerable between women26; for example, women with bacterial vaginosis have little or no lactobacilli in their vaginal secretions.27
Protein stains have been successfully applied to identify used applicators in a vaginal microbicide study28,29 but will not work well with swabs because cotton absorbs protein stain. We selected glycogen detection because high glycogen levels are fairly specific for vaginal secretions. The glycogen detection assay worked very well for our study. Soluble glycogen at levels higher than 100 μg/mL was detected in vaginal swab eluates from all reproductive-aged women, whereas eluates from control swabs (empty, buccal, hand/finger, semen) all had glycogen concentrations higher than 100 μg/mL. Likewise, the PAS cell glycogen stain positively identified all of the vaginal swab samples and was negative or weakly positive for the control samples.
A potential caveat not addressed in this study is the possibility that bacterial vaginosis, sexually transmitted infections, or use of hormonal contraceptives or certain vaginal products can affect glycogen levels or detection of glycogen in vaginal swabs. A recent study suggests that glycogen levels in vaginal secretions are significantly lower in women with BV,30 and another study that used a protein stain to detect vaginal applicator use in a vaginal microbicide gel trial showed that the presence of gel in the vagina interfered with the detection of protein on the applicators.31 More studies are needed to determine whether the glycogen test can be broadly applied, especially in clinical studies in different geographical locations. Further research should also be conducted to validate the use of the glycogen test with other types of swabs used for vaginal sampling such as Dacron, rayon, and nylon-flocked swabs.
This glycogen test is promising and is currently being used to verify exposure to the vagina of swabs that tested negative for PSA in a study of a new female condom (C. Mauck, personal communication). Without the glycogen test, a PSA-negative swab that had not been exposed to the vagina for whatever reason would be categorized as providing evidence that the female condom had worked as a successful barrier, biasing results. By using the glycogen test, such swabs would be removed from the analysis of condom performance. A similar approach could be used to validate vaginal swab use in studies in which PSA is used as a biomarker for unprotected intercourse. The low cost, ease of use, and stability of samples support the feasibility of using this glycogen test in low-resource settings.
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