U.S. patent application number 11/774487 was filed with the patent office on 2008-02-07 for iconic colorimetric test device with reduced susceptibility to false positive and false negative readings.
This patent application is currently assigned to Quidel Corporation. Invention is credited to Aulena Chaudhuri, Paul J. Lawrence.
Application Number | 20080032411 11/774487 |
Document ID | / |
Family ID | 34375444 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080032411 |
Kind Code |
A1 |
Lawrence; Paul J. ; et
al. |
February 7, 2008 |
Iconic Colorimetric Test Device with Reduced Susceptibility To
False Positive and False Negative Readings
Abstract
Laminated test devices in which positive results are detected by
the appearance of a symbol on the surface of the device due to a
color change in an indicator in the device are improved by placing
the indicator lamina on one side of a light-transmissive substrate
sheet and a pigment lamina on the other side, the indicator lamina
forming the icon and the pigment lamina forming the background, the
pigment being the color of the indicator prior to the color change.
This arrangement reduces false positive readings by obscuring the
outlines of the symbol prior to the color change. False negative
readings are reduced by further changes in the arrangement of the
laminae.
Inventors: |
Lawrence; Paul J.; (Pacific
Grove, CA) ; Chaudhuri; Aulena; (San Jose,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Quidel Corporation
San Diego
CA
|
Family ID: |
34375444 |
Appl. No.: |
11/774487 |
Filed: |
July 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10944283 |
Sep 17, 2004 |
7255832 |
|
|
11774487 |
Jul 6, 2007 |
|
|
|
60504066 |
Sep 19, 2003 |
|
|
|
Current U.S.
Class: |
436/111 |
Current CPC
Class: |
G01N 33/521 20130101;
Y10T 436/17 20150115; Y10T 436/173845 20150115 |
Class at
Publication: |
436/111 |
International
Class: |
G01N 33/52 20060101
G01N033/52 |
Claims
1. A method for the identification of a condition indicative of
bacterial vaginosis in a patient by analyzing a sample of vaginal
fluid from said patient, said method comprising applying said
sample to a solid alkali and a gas-permeable indicator lamina of
material permeable to gas but impermeable to aqueous liquids, said
gas-permeable indicator lamina impregnated with an indicator that
undergoes a detectable transition upon contact with amines, the
penetration of said amines into said gas-permeable indicator lamina
correlating, without also analyzing the pH of said sample or
analyzing said sample for the presence of clue cells, to bacterial
vaginosis.
2. The method of claim 1 wherein said indicator is a visual
indicator that changes color upon contact with gaseous amines.
3. The method of claim 1 wherein said indicator is bromocresol
green.
4. The method of claim 1 wherein said solid alkali is a member
selected from the group consisting of alkali and alkaline earth
metal aluminates, carbonates, and hydroxides.
5. The method of claim 1 wherein said solid alkali is a member
selected from the group consisting of sodium aluminate, sodium
carbonate, and magnesium hydroxide.
6. The method of claim 1 wherein said material permeable to gas but
impermeable to aqueous liquids is ethyl cellulose.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of co-pending
U.S. patent application Ser. No. 10/944,283 filed Sep. 17, 2004
which claims benefit from U.S. Provisional Patent Application No.
60/504,066, filed Sep. 19, 2003, the contents of which are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention resides in the field of test devices for
clinical use that provide visual readouts, and is particularly
concerned with test devices that utilize a colorimetric indicator
to display an icon or symbol indicating either the presence or
absence of a particular substance in a sample, a high or low
concentration of a substance, a high or low pH, or any such
indication of either a desired or undesired condition of the
sample.
[0004] 2. Description of the Prior Art
[0005] Test devices that produce visually detectable plus signs to
indicate the presence of bacterial vaginosis in a sample of vaginal
fluid are disclosed in the following United States patents
(inventors: Paul J. Lawrence et al.): U.S. Pat. No. 5,660,790,
issued Aug. 26, 1997; U.S. Pat. No. 5,897,834, issued Apr. 27,
1999; U.S. Pat. No. 5,910,447, issued Jun. 8, 1999; U.S. Pat. No.
6,099,801, issued Aug. 8, 2000; U.S. Pat. No. 6,113,856, issued
Sep. 5, 2000; and U.S. Pat. No. 6,200,817, issued Mar. 13, 2001.
Two distinct tests are disclosed in these patents: a pH test of
vaginal fluid in which a pH of 4.7 or greater serves as one
indication of an infection, and an amine test in which the presence
of volatilizable amines serves as another indication of an
infection. The devices in these patents are laminated test cards
and the tests are conducted by applying a specimen of vaginal fluid
to the surface of a card with a swab and checking the card for the
appearance of symbols on the card surface. In each of these two
tests, a negative test result appears as a "minus" sign formed by a
single horizontal bar and a positive result appears as a "plus"
sign formed by two bars crossing at right angles. The plus sign is
formed by two separately operable but intersecting bars: one
horizontal (the positive control); and, the second vertical (the
test response). The horizontal bar thus appears regardless of the
outcome of the test, and thereby serves as a positive control
indicating that the indicator that causes the color change in both
bars is functioning properly. Thus, if no bar at all appears after
application of a sample, the test device is deemed not usable and
should be discarded; the appearance of a single bar (a "minus"
sign) means that the device is functional and the test result is
negative; and the appearance of both bars (to form the "plus" sign)
indicates that the device is functional and the test result is
positive.
[0006] Plus and minus signs are also produced by the test devices
disclosed in U.S. Pat. Nos. 4,916,056, issued Apr. 10, 1990, and
5,008,080, issued Apr. 16, 1991, both to inventors William E.
Brown, III, et al. The tests in these patents involve immunological
binding rather than pH changes, but like the Lawrence et al.
patents, the tests results are displayed as a plus sign formed when
two separately operable but intersecting bars become visible and a
minus sign formed from when only one of the two bars becomes
visible. All patents in this and the preceding paragraph are
incorporated herein by reference in their entirety.
[0007] Licensed physicians and laboratory technicians find products
like these useful and efficient because of their compact nature and
their simple visual readout. Since they are easy to use, the
devices are also attractive for consumers and other individuals who
are not clinically trained. This is particularly true of the
Lawrence et al. devices where the test procedures consist of simply
applying a swab of the sample to the surface of the device, and
watching the device to see what symbols appear. In some cases,
however, users, particularly untrained individuals, may read the
devices incorrectly, reporting results that are either false
negatives or false positives.
[0008] One source of an incorrect reading arises from the
independent responses of the two independent, separable but
intersecting bars and the fact that the response of each bar can
vary to some degree with the amount of sample applied to the device
or the amount or concentration of analyte in the sample. Slight
differences in color intensity between the two intersecting bars
can prompt the user to treat one bar as a color comparator for the
other, and thereby wait for the color of the vertical bar to
precisely match that of the horizontal bar before declaring a
positive result. A positive result can thus be interpreted as
negative (i.e., a false negative).
[0009] Another source of an incorrect reading occurs when the
outlines of the intersecting horizontal and vertical bars are
visible, even when only faintly so, before any color change has
occurred. This problem can occur with any iconic readout, the terms
"iconic" and "icon" referring herein to any graphical symbol whose
form suggests its meaning. In the case of the Lawrence et al. and
Brown et al. devices, the icon is either a minus sign indicating a
negative result when only one bar appears or a plus sign indicating
a positive result when both bars appear. The icon can also be a
unitary plus sign that appears only as a whole and lacks the
capability of allowing only the minus sign portion to appear. Icons
of other forms or shapes will be readily apparent. With any of
these icons, incorrect readings can arise from the construction of
the device, which typically involves several laminae one or more of
which forms the outlines of the icon. In some cases, for example,
the icon is formed by a channeling lamina with an icon-shaped
opening, overlying an indicator lamina that extends over the full
lateral dimensions of the device, or at least beyond the boundaries
of the opening. The channeling lamina permits the sample (and any
analyte present) to contact only the portion of the indicator that
is directly below the opening. Even if the channeling lamina is
completely transparent, the edges of the opening can be visible to
one who looks closely at the device, since the light reflectivity
of the channeling lamina may differ from that of the indicator
lamina. In other cases, the icon is formed by the indicator lamina
itself which is in the shape of the icon and rests above any
underlying layers. Although the filed behind the icon is the same
color as the icon (prior to the test), the edges of the icon are
faintly visible due to the difference in height, even though that
difference may be very small. To the unskilled or untrained user,
this visibility can suggest a positive test result when the result
is actually negative (i.e., a false positive result). The thickness
difference can be minimized or eliminated by depositing a material
in the areas adjacent to the icon, using a material that does not
change color upon contact with either the sample or the analyte.
The icon and the surrounding material will then be of different
chemical compositions, however. This by itself can produce enough
of a color difference, however slight, to make the borders between
the two visually distinguishable. Furthermore, if there is a risk
of the indicator diffusing into the surrounding material and
thereby obscuring the readout, precautions or structural features
to prevent this from happening can also make the outline
visible.
SUMMARY OF THE INVENTION
[0010] It has now been discovered that false readings of icons on
test devices such as those described above can be reduced or
eliminated by constructing the device as a laminate that includes
among its laminae a light-transmissive, liquid-impermeable lamina
coated on one side with an indicator lamina and on the other side
with a pigment lamina that is the same color as the indicator
before the color change that indicates a positive test result. The
light-transmissive, liquid-impermeable lamina is also referred to
herein as a "substrate sheet" since it can serve as a convenient
base for applying the other laminae on both sides and since it is
typically flat and thin. The icon is formed either by a channeling
lamina over the indicator lamina or by limiting the size and shape
of the indicator lamina so that it forms the icon by itself. In
either case, the pigment lamina extends beyond the icon area, and
preferably over the icon area as well. As described herein, the
test device contains a delineated test region that contains the
colored areas, including the icon area that changes color upon the
occurrence of a positive test result and the non-changing area (or
field) surrounding the icon area that makes the icon substantially
invisible until the icon area changes color. The icon area is thus
a restricted area within, and smaller than, the test area. The test
area itself can extend across the entire length and width of the
device, although in preferred embodiments, the test area will be a
smaller area covering only a portion of the total area of the
device.
[0011] In further preferred embodiments of the invention, the test
device includes a positive control area within the test area but
spatially separated from, and significantly smaller than, the icon
area. In these embodiments, the positive control serves the same
purpose as the positive control on the devices disclosed in the
Lawrence et al. and Brown et al. patents referenced above but does
not form part of the positive test result icon. In addition to
their spatial separation, the positive control and the test icon
preferably differ in size and shape, with the separation, the size,
and the shape differences tending to eliminate any confusion in the
mind of the user between a control signal and a positive test
signal.
[0012] It has also been discovered that a test for volatile amines
utilizing the color-change methodology of the Lawrence et al.
patents cited above, utilizing either the laminate arrangement
disclosed in those patents or those disclosed herein, when
performed on a sample of vaginal fluid, is sufficiently indicative
of bacterial vaginosis that the volatile amines test alone can
replace the more traditional diagnosis based on the Amsel criteria.
The Amsel criteria for bacterial vaginosis (BV) are based on a
report by Amsel, R., et al., Am. J. Med. 74:14-22 (1983), which
sets forth four criteria that collectively result in a diagnosis of
BV: elevated pH (originally 4.5 or above, now 4.7 or above) of a
vaginal fluid specimen, a "whiff" test (treatment of the specimen
with alkali followed by an olfactory test to detect an amine odor),
vaginal fluid homogeneity, and the presence of clue cells. To
detect a positive result in the Amsel criteria, the elevated pH
must be met, together with at least two of the remaining three
criteria. The present invention resides in the discovery that the
use of the amines test alone will serve as an effective
diagnosis.
[0013] Further features, embodiments, and benefits of the invention
will be apparent from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a plan view of a test device in accordance with
the present invention and containing two test regions, one to
determine whether a particular pH threshold has been passed in a
liquid sample and the other for the presence of volatilizable
amines in the sample.
[0015] FIG. 2 is a plan view of the test device of FIG. 1 after a
sample that is positive for both tests has been applied to both
regions.
[0016] FIG. 3 is an expanded view in perspective of one arrangement
of laminae forming the pH test region of the test device of FIGS. 1
and 2.
[0017] FIG. 4 is an expanded view in perspective of an alternative
arrangement of laminae for the pH test region of the test device of
FIGS. 1 and 2.
[0018] FIG. 5 is an expanded view in perspective of one arrangement
of laminae forming the amines test region of the test device of
FIGS. 1 and 2.
[0019] FIG. 6 is an expanded view in perspective of an alternative
arrangement of laminae for the amines test region of the test
device of FIGS. 1 and 2.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0020] In the various embodiments of this invention, the indicator
lamina, also referred to herein as the "analyte indicator lamina"
is one that changes color to indicate a positive test result.
Samples that can be tested on the devices of this invention are
liquid samples, including biological liquid samples, and a positive
test result is a visually detectable color change in the indicator
due to a chemical reaction between the indicator and a component of
the sample. The positive test result can thus arise from the
presence of a distinct species in the sample, the absence of a
distinct species (i.e., a species that would otherwise inhibit the
reaction), a pH of the sample that is above or below a transition
point, or any such quality or characteristic of the sample that
serves as an indication of a condition whose presence is sought to
be determined. In many cases, the indicator in the analyte
indicator lamina will be a pH indicator that will undergo a visible
color change when the pH passes a transition point. Many
conventional pH indicators can be used, the choice of course
depending on the transition point and the particular analyte. The
lamina itself will preferably consist of a hydrophilic
fluid-permeable polymer that serves as a matrix for an impregnated
indicator.
[0021] The pigment lamina is a lamina that is substantially
indistinguishable colorimetrically from the analyte indicator
lamina prior to any change occurring in the analyte indicator
lamina upon application of the sample. Accordingly, the pigment
lamina can be of the same composition as the analyte indicator
lamina and prevented from changing color by the substrate sheet
which is located between the analyte lamina and the pigment lamina,
thereby keeping the two out of contact. The substrate sheet will be
impermeable to the sample liquid so that the sample will not
diffuse through the sheet. The pigment lamina can also be prevented
from contact with the sample by placing a protective sealing lamina
over the pigment lamina. Alternatively, and preferably, the pigment
lamina is a colored ink that is chemically distinct from the
indicator lamina and non-reactive with the analyte, or at least
undergoes no reaction or color change upon contact with the sample,
regardless of whether the sample contains or does not contain the
analyte. Common commercial inks or ink combinations can be
used.
[0022] The substrate sheet separating the analyte indicator lamina
from the pigment lamina, in addition to being impermeable to the
liquid sample, is light-transmissive to render the pigment lamina
visible through the sheet, i.e., from the indicator side of the
sheet. Preferably, the substrate sheet is transparent and
colorless. A presently preferred material for the substrate sheet
is a polyethylene terephthalate fill such as MYLAR.RTM. (Du Pont de
Nemours & Co., Wilmington, Del., USA). In many cases, a coating
over the substrate sheet can improve the adherence of the laminae
that are applied over it, particularly when the pigment and
indicator laminae are susceptible to damage during the handling of
the device as it is being manufactured or during use. This type of
damage can occur, for example, when the laminae are applied as
solutions in solvents that do not penetrate the substrate sheet.
Laminae that are deposited using aqueous and alcohol solvents, for
example, do not adhere well to MYLAR. A preferred coating material
that can improve this adherence is polyamide.
[0023] The icon is formed by restricting the color change in the
analyte indicator lamina to a restricted area within the test
region. This is accomplished either by the use of a channeling
lamina overlying the analyte indicator lamina, the channeling
lamina containing an icon-shaped opening to expose an icon-shaped
area of the analyte indicator lamina, or by forming the analyte
indicator lamina itself in the shape of the icon, leaving the
adjacent areas free of indicator. When a channeling lamina is used,
the channeling lamina is preferably transparent. As noted above,
the icon can assume any shape or form, preferably one that conveys
the message of a positive test result to the user. A plus sign is
preferred, particularly one that, once visible due to the color
change, is fully surrounded by areas that are the same color as the
icon before the color change.
[0024] In embodiments that contain a positive control in addition
to the test icon, the positive control as noted above is preferably
spatially separated from the test icon and of a different size and
shape. The positive control is itself an icon, and in embodiments
having a plus sign-shaped test icon, a convenient positive control
icon is a circular dot of a diameter substantially smaller than the
plus sign, in keeping with the secondary role of the positive
control. The positive control icon is itself an indicator, but one
that changes color upon contact with any sample regardless of
whether the sample contains the analyte (or the absence of the
analyte, or other chemical condition) that will produce the color
change in the test icon. Prior to application of the sample, the
positive control icon will display a color that is identical both
to that of the test icon and to that of the background in the test
region. The color change in the positive control icon is preferably
the same as that in the test icon, although produced in a different
manner to respond to the sample rather than the analyte. The
difference may be a different transition point or any other
characteristic that will differentiate between serving as a
positive control and indicating a positive test result.
[0025] The test icon and the positive control icon are preferably
contained in a common test region on the test device, and the user
is instructed to apply the sample over the entire test region so
that both the test icon and the positive control are contacted with
the sample. The preferred test region is therefore small enough
that it can easily be covered with the amount of sample that can be
contained in a single swab, such as a Q-tip or any other common
implement that the consumer may keep in a medicine cabinet. To
accommodate both icons and allow the sample to be applied by a
circular motion, a convenient shape for the test area is an
ellipse, an oval, or a wider bar with circular arc-shaped ends.
[0026] Further assurance of a consistent and unchanging background
color beneath the test device to afford a more consistent detection
of the color change in the icons, i.e., both the test icon and the
positive control icon, can be achieved by the optional placement of
an opaque white lamina over the entire outer side of the pigment
lamina (i.e., on the side of the pigment lamina opposite the side
occupied by the substrate sheet). This is particularly useful when
the pigment lamina, the analyte indicator lamina, and the positive
control allow small amounts of light to pass.
[0027] It has also been found that when the pigment lamina is
formed from the same material as the analyte indicator lamina, the
background color will appear more consistent with that of the test
icon (prior to a color change in the icon), and the icon outlines
less visible, when an outer transparent coating layer is placed
over the pigment lamina, i.e., once pigment material is applied to
the bottom surface of the substrate sheet, the outer transparent
coating is applied over the pigment material. Although the
inventors do not intend to be bound by this explanation, it is
believed that the solvent system for the outer transparent coating,
which in the preferred embodiments herein is n-propanol, penetrates
the pigment layer to a small degree and produces a slight color
change that further obscures the outlines of the test icon.
[0028] As a still further option when the pigment lamina is formed
from the same material as the analyte indicator lamina, the pigment
lamina can be protected from damage or color change due to exposure
to or contact with objects or liquids during handling by a
protective coating. This coating can be the outermost lamina on the
bottom surface of the device, and can be of the same material as
the channeling lamina or any other inert, non-porous lamina of the
device. The protective lamina can be light-transmitting or opaque.
Polyamide is a preferred material for this lamina.
[0029] Two examples of tests that can be performed on devices that
meet the above description are the pH test and the amine test that
are described in the Lawrence et al. patents referenced above.
These are two distinct tests that are performed on samples of
vaginal fluid and are used in the diagnosis of bacterial
vaginosis.
pH Test
[0030] The pH test indicates whether the sample has a pH of 4.7 or
higher, as one of several indications of bacterial vaginosis. The
analyte indicator lamina in the pH test is a deposited material
that includes a combination of (i) a pH indicator with an ionizable
phenol group and a negatively charged group and (ii) a polymer
matrix the polymer of which contains quaternary ammonium groups
which immobilize the indicator in the matrix. A wide variety of pH
indicators with ionizable phenol groups and negatively charged
groups are known. Preferred groups of negative charge are sulfate
and sulfonate groups. Examples of indicators with ionizable phenol
groups and negatively charged groups are: [0031] acid blue 92
(anazolene sodium, CAS No. 3861 73 2) [0032] acid blue 29 (CAS No.
5850 35 1) [0033] acid alizarin violet N (CAS No. 2092 55 9) [0034]
bromophenol blue (3',3'',5',5''-tetrabromophenolsulfonephthalein,
CAS No. 15539 9) [0035] bromochlorophenol blue
(3',3''-dibromo-5',5''-dichlorophenolsulfonephthalein, CAS No.
102185 52 4) [0036] bromocresol green
(3',3'',5',5''-tetrabromo-m-cresolsulfonephthalein, CAS No. 76 60
8) [0037] chlorophenol red (3',3''-dichlorophenol sulfonephthalein,
CAS No. 4430 20 0) [0038] bromocresol purple
(5',5''-dibromo-o-cresolsulfonephthalein, CAS No. 115 40 2) [0039]
alizarin complexone dihydrate
((3,4-dihydroxy-2-anthraquinolyl)-methyliminodiacetic acid, CAS No.
3952 78 1) [0040] alizarin red S monohydrate
(3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt,
CAS No. 130 22 3) [0041] bromothymol blue
(3',3''-dibromothymolsulfonephthalein, CAS No. 76 59 5) [0042]
brilliant yellow (CAS No. 3051 11 4) [0043] phenol red
(phenolsulfonephthalein, CAS No. 34487 61 1) cresol red
(3',3''-dimethylphenolsulfonephthalein, CAS No. 1733 12 6) [0044] m
cresol purple (2',2''-dimethylphenolsulfonephthalein, CAS No. 2303
01 7) [0045] thymol blue
(2',2''-dimethyl-3',3''-diisopropylphenolsulfonephthalein, CAS No.
76 61 9) [0046] nitrazine yellow
(2-(2,4-dinitrophenylazo)naphthol-3,6-disulfonic acid, disodium
salt, CAS No. 5423 07 4)
[0047] All of these indicators are readily available from
commercial suppliers. Preferred indicators are bromophenol blue,
bromochlorophenol blue, bromocresol green, bromocresol purple,
bromothymol blue, brilliant yellow, and nitrazine yellow.
[0048] The quaternary ammonium groups in the polymer can be any
such groups capable of asserting a positive charge sufficient to
form an ionic attraction with the negatively charged group(s) in
the indicator. Preferred quaternary ammonium groups are lower alkyl
ammonium groups in which the alkyl groups are C.sub.1-C.sub.4 alkyl
groups. Trimethylammonium groups are particularly preferred. The
quantity of quaternary ammonium groups in the polymer formulation
can vary widely, depending on where (i.e., at what pH value) the
color transition point is intended to be. In many applications of
this invention, the amount of quaternary ammonium groups will be
selected to lower the mid-range transition point of the indicator
by about 1.0 to about 3.0 pH units, and preferably by about 1.5 to
about 2.5 pH units, with about 2.0 pH units preferred. The amount
of quaternary ammonium groups can also be expressed in terms of the
alkali value of the resulting polymer, i.e., the milligrams of KOH
equivalent to the basicity of the quaternary ammonium groups
contained in 1 g of the dry polymer. In most implementations of
this invention, the alkali value will range from about 5 to about
50, with values ranging from about 15 to about 40 preferred.
[0049] The polymer forming the matrix is preferably porous (or
otherwise water permeable) and hydrophilic. The hydrophilic
character of the polymer arises either from the quaternary ammonium
groups or from other groups on the polymer structure. Any polymer
which is inert to the sample components, solid and capable of being
formed into a thin layer, coating or lamina can be used. The
polymer should be largely insoluble in aqueous media, particularly
in vaginal fluid for those embodiments of the invention that are
designed for testing vaginal fluid, and preferably transparent.
Examples are acrylic polymers, particularly copolymers of acrylic
and methacrylic acid esters, and diethylaminoethyl cellulose. Two
presently preferred polymers that are readily available from
commercial suppliers are EUDRAGIT.RTM. R: PO and EUDRAGIT.RTM. RS
PO Ammonio Methacrylate Copolymers, which are copolymers of methyl,
ethyl and trimethylammonioethyl esters of acrylic and methacrylic
acids, as chloride salts. The quaternary ammonium groups are
present on these polymers as trimethylammonioethyl esters of
methacrylate units that constitute 10.4% (RL PO) or 5.6% (RS PO) by
weight of the polymer. The alkali values are 28.1 and 15.2,
respectively. These polymers are available from Rohm Tech Inc.,
Malden, Mass., USA.
[0050] The combination of nitrazine yellow and the
trimethylammonioethyl esters of EUDRAGIT RL PO illustrates how
different pH transition points can be achieved. The transition
point of nitrazine yellow in the absence of quaternary ammonium
groups is about pH 6.6. Using a liquid solution of which the
polymer constitutes 25.0% and nitrazine yellow constitutes 0.2% to
0.34%, the transition point will be pH 4.7. With 25.0% polymer and
0.36% nitrazine yellow, the transition point will be pH 4.4. With
25.0% polymer and 0.4% nitrazine yellow, the transition point will
be pH 4.4. With 25.0% polymer and 0.6% nitrazine yellow, the
transition point will be pH 4.3. All percents are by weight.
[0051] For nitrazine yellow and EUDRAGIT RL PO, the ratio,
expressed as the weight ratio of KOH equivalent in the polymer to
nitrazine yellow, has a preferred range of about 1.0:1 to about
6.0:1, and a more preferred range of about 1.5:1 to about 3.5:1.
For EUDRAGIT RL PO, a preferred weight ratio range of polymer to
nitrazine yellow is about 50 to about 250, and more preferably
about 75 to about 125. If the polymer and indicator are applied as
a liquid solution, a preferred range of the polymer in the solution
is about 12% to about 35%, and a preferred range of the nitrazine
yellow is about 0.05% to about 1.0%, all by weight.
[0052] The indicator in the positive control icon is a pH indicator
whose transition point is at least about 0.7 pH unit lower than the
transition point of the test indicator. When the positive control
is contacted with a liquid whose pH lies between its transition
point and the transition point of the test indicator, a color
change in the positive control informs the user that the indicator
in the positive control is in functional condition, which is
logically extended to mean that both indicators are in functional
condition. A color change in the positive control also indicates
that the amount of sample applied to the device was adequate.
Preferably, the same color change occurs in both indicators. If the
test indicator has a transition point of pH 4.7, for example, the
positive control preferably has a transition point of pH 4.0 or
below, and most preferably pH 3.5 or below.
[0053] An example of an indicator that is useful for the positive
control when the pH indicator lamina has a transition point of 4.7
is a mixture of nitrazine yellow and bromocresol green. The weight
ratio of nitrazine yellow and bromocresol green in such a mixture
can vary and different ratios will produce different transition
points. In general, however, best results will be obtained with a
weight ratio (bromocresol green to nitrazine yellow) of about 0.05
to about 20.0, and preferably from about 0.5 to about 5.0.
[0054] The polymer and indicator combinations for both the test and
positive control icons preferably form solid, thin laminae that are
homogeneous, transparent, and water permeable, and that neither
dissolve nor disintegrate when placed in contact with aqueous
liquids. Additional components can be included in each lamina or in
the composition from which the lamina is formed, for a variety of
purposes. For example, a vehicle for spreading or applying the
lamina components to a surface can be included, as well as drying
agents, penetrants, agents to facilitate wetting of the polymer by
the sample, and agents to facilitate depositing the mixture on, or
adhering it to, the surface of the substrate sheet.
[0055] The substrate sheet that serves as a support for both the
analyte indicator lamina and positive control can be formulated to
stabilize the indicators and render them easier to apply. An
example of a substrate sheet that serves these purposes is a
polyethylene terephthalate film such as MYLAR.RTM. (Du Pont de
Nemours & Co., Wilmington, Del., USA) coated with ethyl
cellulose or a similar coating to improve the adherence of the
indicators. The coating may also contain acid to further control
the pH of the indicator to be deposited over the coating. Different
amounts of acid may be present in different regions of the coating
to assist in the independent pH control of the test indicator and
the control indicator.
[0056] Although the tests and test devices of this invention can be
used for testing aqueous liquids from any source, the invention
will be of primary interest in biological fluids, such as urine,
saliva, blood, cerebrospinal fluid, and vaginal fluid. In vaginal
fluid, as indicated above, a prime example of the use of this
invention is in the detection of bacterial vaginosis. Vaginal fluid
can also be tested for other purposes by this invention, such as
for example for the pH change (a rise from 4.5 to 7.0 and higher)
accompanying the rupture of the amniotic membrane. The invention
will be of interest not only in testing human fluids, but also
fluids from animals, such as livestock and pets. One example is the
detection of mastitis in cattle by determining whether the pH of
the milk has reached or surpassed a threshold value of
approximately 6.8.
Amine Test
[0057] In addition to its analyte indicator and positive control
laminae, the amine test includes a solid gas-releasing lamina
immediately accessible to the fluid specimen, and the analyte
indicator lamina is a gas-permeable lamina that is impermeable to
the aqueous liquid sample itself or to any of its components that
are not in gaseous form. The gas-releasing lamina is a solid lamina
of alkali that reacts with amine salts in the specimen and converts
them to volatile amines. The volatilized amines then penetrate the
gas-permeable analyte indicator lamina where they cause a color
change in the indicator.
[0058] The terms "volatile amines" and "volatilized amines" include
amines that are only slightly volatile as well as those that are
sufficiently volatile to escape into the atmosphere at substantial
rates. Slightly volatile amines are those that form only a thin
film of gas at the liquid surface without significant amounts
diffusing into the atmosphere. This thin film of gas however is
sufficient to penetrate the gas-permeable indicator lamina. The
choice of solid alkali for the gas-releasing lamina is not critical
and can vary. In general, alkali and alkaline earth metal
aluminates, carbonates and hydroxides can be used. Best results
will most often be achieved with the use of either sodium
aluminate, sodium carbonate, or magnesium hydroxide. Sodium
aluminate is particularly preferred.
[0059] Laminae that are permeable to gas but not to aqueous liquids
can be formed, for example, from hydrophobic polymers solidified to
porous solids. Suitable polymers are those that are solid,
insoluble in aqueous fluids, particularly vaginal fluid, and
readily formed into a layer, coating or lamina that does not
dissolve, disperse into particulate form, or otherwise disintegrate
upon contact with the sample. Examples of such polymers are ethyl
cellulose, cellulose acetate and cellulose nitrate. Ethyl cellulose
is particularly preferred. Alternatively, the indicator can reside
in a hydrophilic water-permeable polymer that is covered by a
hydrophobic, gas-permeable lamina.
[0060] Any indicator that changes color upon exposure to
unprotonated amines, and preferably amines in a fluid specimen that
would otherwise be acidic, may be used. Bromocresol green is one
example of such an indicator, and may be used both here and in the
pH test. Other examples are bromophenol blue, bromocresol purple,
bromochlorophenol blue, nitrazine yellow, and various other
indicators among those listed above.
[0061] The gas-releasing lamina and the gas-permeable indicator
lamina are arranged in the test device such that the alkali in the
gas-releasing lamina and the indicator in the gas-permeable lamina
can be brought into contact only by the application of a fluid
specimen. The laminae are arranged to permit the user to first
contact the gas-releasing lamina with the specimen (preferably
using a swab), and then to contact the gas-permeable indicator
lamina with the same specimen, so that gas released in the specimen
upon contact with the gas-releasing lamina will penetrate the
gas-permeable lamina and thereby reach the indicator.
[0062] The substrate sheet for the amine test can be the same as
that for the pH test, particularly when the two are included on a
common test device. A coating can be applied to the substrate sheet
for the same reasons and effects as disclosed above in the
description of the pH test.
[0063] As further protection of the indicator in the gas-permeable
lamina from liquid amines, an additional lamina that is permeable
to gas but impermeable to liquid can be placed over the test
indicator lamina. This protective or barrier lamina can be
constructed of the same polymer used in the gas-permeable indicator
lamina. Here again, ethyl cellulose is preferred.
[0064] The positive control indicator for the amine test is any
indicator that will change color upon application of the sample
regardless of the presence or absence of volatilizable amines in
the sample. The color change may be due to the nonvolatile amines
in the sample, or to the solid alkali in the gas-releasing lamina
as the alkali is drawn into the sample by the applicator swab. The
immobilizing matrix can be a hydrophilic polymer, and the same
polymers cited above as preferred for use in the pH test can be
used here as well. The indicator can be the same indicator used in
the gas-permeable indicator lamina. One method of applying a
positive control indicator that will change color regardless of
whether the sample contains volatilizable amines is by
incorporating the indicator in a matrix that is permeable to liquid
rather than being permeable only to gas, and by placing the control
in a position where it is not protected by the gas-permeable
liquid-impermeable lamina.
[0065] While the present invention is susceptible to a wide range
of configurations and embodiments, an understanding of the
underlying concepts and principles of the invention and its novel
aspects is best gained by a detailed review of specific
embodiments. These are depicted in the drawings and described
below.
DETAILED DESCRIPTIONS OF THE DRAWINGS
[0066] FIGS. 1 and 2 illustrate a single test device 11 containing
separate test regions for a pH test and an amine test. The device
is approximately the size of a common credit card (enlarged in the
figure for ease of illustration) and is made of laminated plastic.
FIG. 1 shows the device before any samples have been applied, while
FIG. 2 shows the device after a sample has been applied to both
test regions in succession and the test result in both cases is
positive. The card includes a delineated area 12 of oval shape as
the test region for application of a vaginal sample for the pH test
and a second delineated area 13, also of oval shape, as the test
region for application of a vaginal sample for the amines test.
Within each test region is a test icon 14, 15, and a positive
control icon 16, 17, the test icons appearing as plus signs and the
positive control icons appearing as circular dots, all visible only
in FIG. 2. Various indicia (not numbered) are printed on the
surface of the device to guide the user, indicating that the sample
should be collected on a cotton swab and that the swab should then
be applied to the pH test region first and the amine test region
second. The indicia also indicate that the swab should be applied
in a circular motion in each case beginning at the periphery of the
test region and moving toward the center in a spiral motion.
[0067] FIG. 3 depicts, in an expanded view, one example of an
arrangement of laminae for the pH test portion of the device. The
base over which the laminae are applied is the substrate sheet 31,
which is a transparent solid sheet of nonporous material that is
chemically inert to all substances coming in contact with it either
during application of the laminae or in use in as the test is being
performed. The test icon is created by the combination of an
indicator lamina 32 and a transparent channeling lamina 33, the
channeling lamina allowing liquid to pass only through a plus
sign-shaped opening 34 in the channeling lamina. The pigment lamina
35 is applied to the underside of the substrate sheet 31, and when
the pigment lamina is the same material as the indicator lamina 32,
a layer of transparent polyamide 36 is applied to the underside of
the pigment lamina. A positive control icon is applied as a
separate lamina 37 above the channeling lamina 34. Additional,
optional laminae include side-by-side coatings 38, 39, both
transparent, over the substrate sheet (on the same side as the
indicator lamina 32, channeling lamina 33, and control icon lamina
37), and an opaque white lamina 40 on the underside of the
substrate sheet. The coatings 38, 39 are applied as solutions of
different pH to help control the transition pH for the test
indicator (the indicator lamina 32) at 4.7 and for the control
reagents (the positive control 37) at 4.0. Optionally, as noted
above, a single coating, preferably one that matches the pH of the
indicator lamina 32, can be used in place of the two 38, 39 that
are shown. The channeling lamina 33, indicator lamina 32, and
pigment lamina 35 are all shaped as the oval test area 12 of FIGS.
1 and 2.
[0068] An alternative arrangement of laminae for the same pH test
is depicted in FIG. 4. This arrangement differs from that shown in
FIG. 3 as follows: the indicator lamina 32 of FIG. 3, which fills
the oval of the test region, is replaced by a smaller, icon-shaped
indicator lamina 41, and the channeling lamina 34 is eliminated
entirely.
[0069] Examples of materials used in forming the various laminae
shown in FIGS. 3 and 4 are as follows (all percents are by weight):
[0070] the substrate sheet 31 is a sheet of MYLAR, 10 mils (0.001
inch, 0.00254 cm) in thickness [0071] the 38, 39 for enhanced
control of the pH transition points are each ethyl cellulose
applied as a 15% solution in n-propanol, the solution containing
maleic acid in sufficient concentration to give the solution a pH
of 2.2 for the test icon portion 38 and 4.6 for the positive
control portion 39 [0072] the adherence-promoting layer 36 is
polyamide applied as a 20% solution of polyamide in a mixture of
n-propanol acetate and n-propanol (50:30 weight ratio) [0073] the
indicator laminae 32, 41 are yellow and transparent and each is
applied as a solution of the following composition: 25.0% EUDRAGIT
RL PO, 0.34% nitrazine yellow, 8.6% sorbitol (70% aqueous
solution), 10.0% 2-ethoxy ethanol, 12.4% deionized water, and
43.66% 1-propanol [0074] the positive control lamina 37 is also
yellow and transparent and is applied as a solution of the
following composition: 25.0% EUDRAGIT RL PO, 0.17% nitrazine
yellow, 0.30% bromocresol green, 8.6% sorbitol (70% aqueous
solution), 30.0% 2-ethoxy ethanol, 12.4% deionized water, 0.65%
2-sulfobenzoic acid anhydride (for moisture resistance), and 22.8%
1-propanol [0075] the channeling lamina 33 is transparent ethyl
cellulose, applied as a 15% solution in 30% n-propanol [0076] the
pigment lamina 35 is an inert yellow ink, consisting of a mixture
of 99.3% Pantone Yellow, 0.145% Process Blue, 0.29% Black, and
0.28% Warm Red the opaque white lamina 40 is CUSTAGLOSS.RTM. pure,
opaque, white ink #1010
[0077] In a currently preferred embodiment, each oval test area is
0.391 inch (0.99 cm) in width and 0.2975 inch (0.756 cm) in height,
the test indicators ("plus" signs) are bars 0.17 inch (0.43 cm) in
length and 0.0553 inch (0.140 cm) in width, the positive control
dots are 0.075 inch (0.190 cm) in diameter.
[0078] FIG. 5 depicts, in an expanded view, one example of an
arrangement of laminae for the amine test portion of the device.
Here, as in the pH test portion, the base over which the laminae
are applied is a transparent solid substrate sheet 51 of nonporous
material that is chemically inert to all substances coming in
contact with it either during application of the laminae or in use
in performing the test. The test icon is created by the combination
of an indicator lamina 52 and a transparent channeling lamina 53,
the latter having a plus sign-shaped opening 54 for the passage of
liquid. On the underside of the substrate sheet 51 is a pigment
lamina 55 applied over a layer of transparent polyamide 56, and
when the pigment lamina is the same material as the indicator
lamina 52, a layer of transparent polyamide 56 is applied to the
underside of the pigment lamina. Above the channeling lamina 34 are
a gas-permeable, liquid-impermeable lamina 57, a positive control
icon 58, and a gas releasing lamina 59. An opaque white lamina 60
resides on the underside of the substrate sheet 51. The
gas-permeable, liquid-impermeable lamina 57, channeling lamina 53,
indicator lamina 52, and pigment lamina 55, are all shaped as the
oval test area 13 of FIGS. 1 and 2.
[0079] The alternative construction for the amine test is shown in
FIG. 6. As in the alternative pH test, the test icon in this
construction is formed from the indicator lamina alone rather than
the combination of an indicator lamina and a channeling lamina. The
differences between this arrangement and that of FIG. 5 are
therefore the replacement of the indicator lamina 52 of FIG. 5,
which fills the oval of the test region, by a smaller, icon-shaped
indicator lamina 61, and the elimination of the channeling lamina
54. All other laminae are the same as their like-numbered
counterparts in FIG. 5.
[0080] Examples of materials used in forming the laminae shown in
FIGS. 5 and 6 are as follows (all percents are by weight): [0081]
the substrate sheet 51 is a sheet of MYLAR, 10 mils (0.001 inch,
0.00254 cm) in thickness [0082] the indicator laminae 52, 61 are
yellow and transparent and each is applied as a solution of the
following composition: 1.8% bromocresol green, 12% ethyl cellulose,
and 86.2% ethanol [0083] the channeling lamina 53 is clear
polyamide, applied as a 20% solution in a mixture of n-propanol
acetate and n-propanol (50:30 weight ratio) [0084] the
gas-permeable lamina 57 is colorless and transparent, and is
applied as a 10% solution of ethyl cellulose in n-propanol [0085]
the positive control lamina 58 is yellow and transparent and is
applied as a solution of the following composition: 25.0% EUDRAGIT
RL PO, 0.17% nitrazine yellow, 0.1% bromocresol green, 30.0%
2-ethoxy ethanol, 6.0% deionized water, 36.9% 1-propanol, and 2.0%
2-sulfobenzoic acid anhydride (for moisture resistance) [0086] the
pigment lamina 55 is an inert yellow ink, consisting of 99.3%
Pantone Yellow, 0.145% Process Blue, 0.29% Black, and 0.28% Warm
Red [0087] the gas-releasing lamina 59 is sodium aluminate applied
as a solution containing 28% sodium aluminate, 18% polyethylene, 3%
maltodextrin, and 51% deionized water [0088] the opaque white
lamina 60 is CUSTAGLOSS.RTM. pure, opaque, white ink #1010
[0089] The dimensions of the plus sign and the positive control dot
in this preferred embodiment are the same as those cited above for
the pH test, plus the gas-releasing lamina which is formed of
curved lines of solid alkali containing sodium aluminate in an
amount equivalent by titration to 120-150 microliters of 0.01 N
hydrochloric acid.
[0090] The invention is further illustrated by the following
examples.
Example 1
[0091] This example illustrates the benefit of the presence of a
pigment lamina on the bottom surface of the substrate sheet in
reducing the visibility of the incipient test icon formed on the
top surface of the sheet. By "incipient" is meant that the
observations were made on test device constructions that had not
undergone any color change that would have resulted from reaction
with an analyte. The device used was an amines test device as
described above, and the test icon was formed by a channeling
lamina placed over an indicator lamina covering the entire test
region. The channeling lamina had an opening exposing a portion of
the indicator lamina in the shape of the test icon. A construction
having the configuration of FIG. 5 without the pigment lamina 55
and the opaque white lamina 59 was compared with a construction
that lacked only the opaque white lamina 59. The difference between
the two constructions was therefore the pigment lamina 55 which was
absent in one and present in the other. The pigment used in the
pigment lamina was the same material used as the indicator in the
indicator lamina.
[0092] Visual examinations of the constructions were made without
the application of any test samples or analytes. Thus the
comparisons were made on the basis of the indicator prior to any
color change. When the two constructions were compared by visual
examination, the plus-sign lamina was faintly visible in the
construction that did not contain the pigment lamina, the
visibility due to the edges of the opening in the channeling
lamina. This visibility was significantly less in the construction
that contained the pigment lamina.
[0093] Two additional constructions were prepared, both containing
the pigment lamina both lacking the opaque white lamina, but one
containing a transparent protective polyamide lamina as an outer
coating over the pigment lamina. Comparison of these two
constructions, again by visual examination without the influence of
any samples or analytes, revealed that the visibility of the icon
outlines was reduced even more in the construction containing the
transparent protective lamina.
Example 2
[0094] This example illustrates the benefit of the presence of a
transparent protective lamina underneath a pigment lamina on the
bottom surface of the substrate sheet in reducing the visibility of
the incipient test icon formed on the top surface of the sheet. The
amines test device as described above was used here as well, and
the test icon was formed by a plus-sign-shaped indicator lamina.
Several constructions having configurations similar to that of FIG.
6 were prepared, including some with the opaque white lamina 59 and
some lacking the opaque white lamina. Among each of these groups
were constructions that also contained the polyamide layer and
those that did not. In constructions that contained both the opaque
white lamina and the polyamide layer, the polyamide was positioned
between the pigment lamina and the opaque white lamina. As in
Example 1, the pigment used in the pigment lamina in all cases was
the same material used as the indicator in the indicator
lamina.
[0095] Comparison of the various constructions by visual
examination, without exposure to any samples or analytes, revealed
that the polyamide layer reduced the visibility of the outlines of
the plus-sign icon in both cases, i.e., both with and without the
additional presence of the opaque white lamina.
Example 3
[0096] This example illustrates the use of commercial ink
combinations as the pigment lamina in place of the unchanged and
protected indicator lamina. The commercial ink combinations were
all chemically distinct from the indicator and not designed to
change color upon exposure to any of the test materials under the
conditions of the test. The advantage of using commercial ink
combinations is that they can be formulated in a controlled manner
to achieve a distinct color or color quality without requiring the
lamina to change color.
[0097] The experiments in this example were conducted on the amines
test device as described above, and the test icon was formed by a
plus-sign-shaped indicator lamina. The same reagents and materials
were used, except that the pigment lamina was substituted by each
of four combinations of standard, primary-color printing inks, all
commercially available and blended empirically to match as closely
as possible the color of the plus sign-shaped indicator lamina. The
four combinations were as follows (all percents are weight
percents): TABLE-US-00001 Ink Combination No.: 1 2 3 4 Transparent
white 95.6% 86.8% 48.4% 12.8% Opaque white 0.5% 0.5% 0% 0% Magenta
0.25% 0.5% 0% 0% Pantone yellow 3.65% 1.20% 51.5% 86.1% Rubin red
0% 0% 0.18% 0.7% Black 0% 0% 0% 0.3%
[0098] Each of the ink combinations was applied to a separate test
laminate in one of two formats--the first as shown in FIG. 6 with
the ink layer applied on the underside of transparent substrate
sheet and the second with the ink layer applied on the upper side
of the transparent substrate sheet immediately below the plus
sign-shaped indicator lamina. Test solutions were prepared by
dissolving solid trimethylamine hydrochloride at various
concentrations in a solvent system consisting of 7% (by weight)
propanol in distilled water. Concentrations of 1 mM and 5 mM were
used, while the solvent system without trimethylamine was used in
parallel tests as a control. The test solutions and the control
were applied to laminates constructed according to both
formats.
[0099] The visibility of the plus sign on each laminate was then
determined by visual observation, as were any color changes in the
background surrounding the plus sign. Prior to any application of
the test solutions or control liquids, the outlines of the plus
sign were almost completely invisible in both formats for all ink
combinations. Upon application of the control liquids, the outlines
of the plus sign remained almost completely invisible with all ink
combinations except Combination No. 1. Upon application of the test
solutions, the laminates of the first format (with the transparent
substrate sheet positioned between the indicator lamina and the ink
combination) produced better results than the laminates of the
second format (with the indicator lamina and the ink combination on
the same side of the substrate sheet). Laminates of the second
format showed a slight color change in the ink combination,
lessening the contrast between the plus sign and the background,
while no such change was visible in laminates of the first
format.
Example 4
[0100] This example compares the performance of a pH test of the
preferred formulation described above on test laminates in
accordance with this invention with a COLORPHAST.RTM. pH test
strip, manufactured by EM Science, Gibbstown, N.J., USA, currently
sold for use by physicians. The test card was constructed with a
plus sign-shaped indicator lamina of the preferred composition
described above, and the pigment lamina was a commercial ink
composition applied to the underside of the transparent substrate
sheet. The ink combination was 99.3% Pantone Yellow, 0.14% Process
Blue, 0.28% Black, and 0.28% Warm Red. Tests were performed on 472
vaginal fluid specimens.
[0101] Of the 472 specimens, 232 indicated a negative test result
(i.e., pH<4.7) according to the COLORPHAST pH strip, and of
these, 185 also indicated a negative result according to the test
card of the present invention, or an agreement level of 79.7%. The
positive test results (pH.gtoreq.4.7) numbered 240 for the
COLORPHAST strips, and of these, 185 tested positive on the test
elements of the invention, or an agreement level of 91.7%. The
overall accuracy was therefore 86%.
Example 5
[0102] This example utilizes the traditional Amsel criteria to
reconcile the results that were not in agreement in the tests
performed in Example 4. As noted above, the Amsel criteria for
bacterial vaginosis (BV) are based on a report by Amsel, R., et
al., Am. J. Med. 74:14-22 (1983), which sets forth four criteria
that collectively result in a diagnosis of BV: elevated pH
(originally 4.5 or above, now 4.7 or above) of a vaginal fluid
specimen, a "whiff" test (treatment of the specimen with alkali
followed by an olfactory test to detect an amine odor), vaginal
fluid homogeneity, and the presence of clue cells. To detect a
positive result in the Amsel criteria, the elevated pH must be met,
together with at least two of the remaining three criteria.
[0103] The 97 samples from Example 4 that were not in agreement
were tested under the remaining three Amsel criteria (whiff test,
vaginal fluid homogeneity, and the presence of clue cells). Of the
47 specimens that gave negative results by the COLORPHAST test
strip and positive by the test laminate of the invention, only one
was positive by the Amsel criteria. Of the 20 specimens that gave
positive test results by the COLORPHAST test strip and negative by
the laminate of the invention, 19 were negative according to the
Amsel criteria. This represents a 99.5% agreement on the positive
results, an 81.6% agreement on the negative results, and a 90%
overall accuracy.
Example 6
[0104] This example compares the performance of an amine test of
the preferred formulation described above on laminates in
accordance with this invention with the "whiff test" of the Amsel
criteria. The laminates constructed with a plus sign-shaped
indicator lamina of the preferred amine test composition described
above, and the pigment lamina was a commercial ink composition
applied to the underside of the transparent substrate sheet. The
ink combination was 99.3% Pantone Yellow, 0.14% Process Blue, 0.28%
Black, and 0.28% Warm Red. Both the test in accordance with the
invention and the whiff test were performed on 464 vaginal fluid
specimens.
[0105] Of the total number of specimens tested, 157 gave a positive
response by the whiff test, and of these, 142 gave a positive
result by the laminates of the invention, representing 90.4%
positive agreement. Of the total number tested, 307 gave a negative
result by the whiff test, and of these, 268 also gave a negative
result by the laminates of the invention, representing an 87%
agreement on the negative results. This amounts to an overall
accuracy of 88.3%.
Example 7
[0106] Amsel criteria were again used to reconcile the specimens
that were not in agreement in the amine tests of Example 6. Here as
well, if amines were detected because of vaginal infections, at
least two of the other three Amsel criteria would be expected to be
positive.
[0107] The 54 amine test samples that were not in agreement between
the two tests were subjected to the remaining three Amsel criteria.
Positive agreement rose to 94.2%, negative agreement rose to 88.7%,
and overall accuracy to 91%.
Example 8
[0108] This example compares the combined pH and amine tests of the
present invention (using the laminates of the preceding examples)
with the combined results of the COLORPHAST pH test and the
standard whiff test of the Amsel criteria. Results using the
laminates of the present invention were considered positive only
when both the pH and the amine tests gave positive results, and
negative in all other cases, i.e., when either or both of the pH
and amine test gave a negative result. The reference tests were
designated in the same manner--positive when both were positive and
negative when any one or both were negative.
[0109] Of 464 vaginal fluid specimens, 149 specimens tested
positive by the COLORPHAST/whiff tests and 140 tested positive on
the laminates of the present invention. Likewise, 315 specimens
tested negative by the COLORPHAST/whiff tests and 291 tested
negative on the laminates of the present invention. Disagreement
occurred in only 33 cases. This represents 94.0% positive
agreement, 92.4% negative agreement, and an overall accuracy of
93.0%.
Example 9
[0110] As with the individual test elements for the pH and amine
tests, discrepancies in the results between the combined tests of
the invention and the combined COLORPHAST/whiff tests were
reconciled using the Amsel criteria to presumptively diagnose BV.
Of the 24 specimens that were negative according to the
COLORPHAST/whiff tests, only 9 were clinically positive for BV
according to the Amsel criteria. Of the 9 specimens that were
positive according to the COLORPHAST/whiff tests but negative
according to the combined tests according to the present invention,
6 were clinically negative for BV according to the Amsel criteria.
Thus, using the Amsel criteria to reconcile the disagreements
between the results obtained from the different tests, positive
agreement rose to 98.0%, negative agreement rose to 95.2% and
overall accuracy rose to 96.1%.
Example 10
[0111] This example illustrates the use of laminates of the present
invention in conjunction with the two additional Amsel criteria for
the determination of the presence or absence of BV. First, all
women in the study group were tested for the all four criteria,
using the COLORPHAST pH test strip for pH determination, the whiff
test for amines, visual examination of the fluid for adherence and
homogeneity, and microscopic examination for the presence of clue
cells. The analysis was then repeated on the same group of women,
using the same four criteria, but substituting the pH and amine
test laminates of the present invention for the COLORPHAST and
whiff tests.
[0112] The study group consisted of vaginal fluid specimens from
464 women. Based on the Amsel criteria results using the COLORPHAST
and whiff tests, 156 (33.6%) of the women were classified as BV
positive and 308 (66.4%) were classified as BV negative. Of the 156
positives, 152 were also classified positive using the laminates of
the present invention (in conjunction with determinations of the
vaginal fluid consistency and the presence of clue cells). Of the
308 negatives, 292 were also classified negative when the criteria
were retested using the laminates of the present invention (in
conjunction with the vaginal fluid consistency and presence of clue
cells). This represents 97.4% positive agreement, 94.8% negative
agreement and 95.6% overall agreement.
[0113] The 20 test results that were not in agreement were
reconciled by a Gram stain test as an independent means of
diagnosing BV, where a Gram stain score of 7 and above was
considered BV positive. Of the 16 specimens that were BV negative
by the Amsel criteria using the COLORPHAST and whiff tests and BV
positive by the Amsel criteria when the laminates of the current
invention were used, 7 were clinically positive for BV by the Gram
stain test. Conversely, of the 4 specimens that were BV positive by
the Amsel criteria when the COLORPHAST and whiff tests were used
and BV negative when the laminates of the present invention were
used, none were clinically negative by the Gram test. Thus, by
reconciling the inconsistent results with the Gram stain test
results, sensitivity (the percentage of positive test results
relative to the positives by the Gram stain test) remained at
97.4%, specificity (the percentage of negative test results
relative to the negatives by the Gram stain test) rose to 97.0% and
overall agreement rose to 97.2%.
Example 11
[0114] This example illustrates the use of the pH and amine tests
performed on laminates in accordance with the present invention by
themselves as combined test criteria for BV. Comparisons were made
against the Amsel criteria (using the COLORPHAST strip for pH and
the whiff test for amine), and the Gram stain test, in which a Gram
stain score of 7 and above was considered BV positive, was used to
reconcile results that were not in agreement between the laminates
and the COLORPHAST/whiff tests. For pH and amine tests of the
present invention, the diagnosis was considered positive only when
both tests gave positive results. When one or both gave negative
results, the diagnosis was considered negative.
[0115] Of the 464 vaginal specimens tested, 156 (33.6%) were
positive by the Amsel criteria, and of these 156, 142 were also
positive by the laminates of the present invention. Those testing
negative by the Amsel criteria were 308 in number (66.4%), and of
the 308, 286 were also negative by the laminates of the present
invention. This indicates a sensitivity of 91.0%, a specificity of
92.9%, an overall accuracy of 92.2%.
[0116] The 36 test results that were not in agreement were then
reconciled with the Gram stain test. Of the 22 that were BV
negative by the Amsel criteria and positive by the tests of the
present invention, 7 were BV positive by the Gram stain. Of the 14
that were BV positive by the Amsel criteria and negative on the
laminates of the present invention, none were clinically negative
by the Gram stain.
[0117] Thus, by reconciling non-agreeing test results with the Gram
stain, 163 specimens were BV positive by the Gram-reconciled Amsel
criteria, and of these, 149 were also positive by the laminates of
the invention. Similarly, 301 specimens were negative by the
Gram-reconciled Amsel criteria, and of these, 286 were also
negative by the laminates of the invention. This raised the
sensitivity to 91.4%, the specificity to 95.0%, and the accuracy to
93.8%.
Example 12
[0118] This example illustrates the use of the amine test performed
on a test card in accordance with the present invention as a test
for clue cells. The same amine test materials and laminate
configuration used in Example 6 above were used in this example.
Comparisons were made against microscopic detection of clue cells,
and the Gram stain was used to reconcile results that were not in
agreement between the test card conclusions and the microscopic
evaluation. The microscopic detection for clue cells was considered
positive when clue cells constituted more than 20% of the vaginal
epithelial cells present in a vaginal fluid specimen. As in the
preceding examples, an amine test on a laminate of the present
invention was considered positive when a visible plus sign appeared
and negative when no plus sign appeared.
[0119] Of the 464 vaginal fluid specimens tested in the preceding
examples, 160 were interpreted as positive for clue cells by
microscopy. Of these 160 clue cell positive specimens, 140 were
also positive by the amines test of the present invention.
Specimens testing negative for clue cells by microscopy were 304 in
number, and of these, 263 were also negative by the amine test of
the present invention. This indicates a positive agreement of
87.5%, a negative agreement of 86.5% and an overall accuracy of
87%.
[0120] The 61 test results that were not in agreement were then
reconciled by the Gram stain test. Of 41 specimens that were clue
cell negative by microscopy and positive by the amines test of the
present invention, 4 were clinically positive for BV by Gram stain
analysis. Similarly, of the 20 specimens that were positive by
microscopy criteria and negative by the amine test of the present
invention, 6 were clinically positive for BV by Gram stain testing.
Thus, by reconciliation of the above data using Gram stain scores,
the positive agreement was raised to 91.1%, the negative agreement
was raised to 87.9%, and the overall accuracy was raised to
89%.
Example 13
[0121] This example demonstrates the effectiveness of an amine test
in accordance with the present invention as a test by itself for
bacterial vaginosis, by comparing the results of the amine test
with the results of a standard bacterial vaginosis test performed
using the Amsel criteria. The test materials and configuration
representing the present invention were the same as those used in
Example 6 above. Both the tests according representing the
invention and the Amsel criteria tests were performed on the same
464 vaginal fluid specimens.
[0122] Of the total number of specimens tested, 156 specimens
tested positive by the Amsel criteria, and 142 of these tested
positive as well by the laminates of the invention, representing
91.0% sensitivity. Of the total number tested, 308 tested negative
by the whiff test, and of these, 269 also tested negative by the
laminates of the invention, representing an 87.3% negative
agreement. Overall accuracy was 88.6%. This indicates that
bacterial vaginosis can be diagnosed on the basis of the amine test
alone with a high degree of accuracy.
Example 14
[0123] The specimens in the above example that were not in
agreement were reconciled by Gram stain tests. The amine test
samples that were not in agreement between the tests were 53 in
number, and with the Gram stain analysis, sensitivity rose to
91.4%, specificity rose to 89.4% and overall accuracy to 90.0%.
Hence, the amines test laminates in accordance with this invention
provided equivalent accuracy for the detection of BV to that
determined by means of the four Amsel Criteria.
[0124] The foregoing is offered primarily for purposes of
illustration. It will be readily apparent to those skilled in the
art that the configurations, dimensions, reagents and other
materials, procedural steps and other parameters of this invention
can be further modified or substituted in various ways without
departing from the spirit and scope of the invention.
* * * * *