Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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This invention relates to a method oE assaying Gramrnegative
bacteria in biological fluids and to a kit for conducting such an assay.
It has previously been proposed to employ gelation of the lysate
of amebocytes of Limulus polyphemus (L~L) as a test for the presence of bacterial
endotoxins in biological fluids, but studies have shown that sevexal pyrogenic
compounds other than such endotoxins give positive test results, as reported
by Elin et al., J. of Infectious Diseases, Vol. 128, 349-352 (1973). Cther
studies have shown that the L~L test for endotoxin in blood samples in many
cases fails to correlate with blood cultures showing the presence of a variety
of Gram-negative bacteria. M~rtinez-G et al., J. of Infectious Diseases Vol. 127,
102-105 (1973). For these reasons, the I~L test has not been effective for
assay of biological fluids such as blood even though it has been quantified
for determining biomass of Gram-negative bacteria in sea water by Watson et al.,
Applied and Environm~ntal Microbiol., Vol. 33, 940-946 (1977).
The present invention is based on the discovery that if Gram-
negative bacteria in the biological fluid are separated from extraneous
sources of pyrogen as well as fram free endotoxin, the amount of lipopoly-
saccharide produced by incubation of the Gram-negative bacteria with LimMlus
polyphemus amebocyte lysate can be taken as a quantitative measure of the
amount of bacteria present in the specimen fluid. me present invention is
particularly useful for assaying Gram~negative bacteria in blood but is also
applicable to other biological fluids such as cerebro-spinal fluid, urine,
ascites, etc.
The present inven~ion accordingly provides a method of assaying
Gram-negative bacteria content in biological fluids containing blood cells
which comprises lysing blood cells present in the fluid at pH 6 to 8, separating
cellular debris from the bacteria-containing liquid phase, separating the bacteria
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from the liquid phase, preferably by subjecting the liquid phase to a force
of at least 5,000 G to separate microorganisms from said liquid by sedimentation,
washing the separated bacteria to remove pyrogens therefrom, incubating said
bacteria with Limulus polyphemus amebocyte lysate, and measuring the amount of
turbidity formed during said incubation.
The invention also provides a kit for assay of Gram-negative bac-
teria in a biological fluid comprising a container of blood-cell-lysing surface-
active agent, a container of mulus polyphemus amebocyte lysate, and one or
more standard dilutions containing known amounts of Gram-negative bacteria.
The first step of the process, the lysing of the cells present in
the biological fluid at pH 6-8, is preferably brought about by mixing the
fluid specimen with an aqueous solution of a surface-active agent; a clear,
sparkling lysate is produced in a few minutes on standing at room temperature.
The surface-active agents employed are preferably non-ionic, such as condensates
of ethylene oxide with hydrophobic bases formed by condensing propylene oxide;
or alkylphenoxy polyethoxylates such as octylphenoxy polyethoxy ethanol,
iso-octylphenoxy polyethoxy ethanol, decylphenoxy polyethoxy ethanol, and the
like. Particularly preferred is octylphenoxy polyethoxy (9-10) ethanol sold
under the trade mark Triton X-100. The concentration of surface-active agent
may vary over a wide range, e.g. from 0.1 to 10% by weight of the biological
fluid, preferably from 0.5 to 1.5% by weight. The surface-active agent is
preferably first dissolved in water (pyrogen-free) to provide a solution
containing 0.01 to 1% by weight of surface-active agent, and the biological
fluid is mixed with the aqueous solution in appropriate proportions to provide
the desired ratio of surface-active agent to biological fluid. The solution
of surface-active agent preferably also contains a buffer such as sodium
carbonate-sodium bicarbonate at O.OIM to maintain the pH not only of the
solution, but also of the mixture of the solution with the biological fluid,
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at 6 to 8.
After lyslng of the cells as described above, the relatively coarse
cellular debris is separated from the liquid phase, preferably by filtration.
The filter used must be pyrogen-free and have a pore size sufficiently large
~5 to 60 micrometers, preferably 40-60 micrometers) to pass microorganisms
with the liquid phase while retaining the largerparticle size solid cellular
debris.
The microorganisms are then separated rom the liquid phase, pre-
ferably by subjecting the liquid phase to a force of at least 5000 G, prefer-
ably 5000 to 12,000 G, for example by centrifugation, to cause sedimentation
and separation o the microorganisms from the liquid phase on to a collection
surface such as the surface of a glass or plastic centrifuge tube. Following
sedimentation, the supernatant liquid phase is removed, preferably by aspir-
ation, and the sedimented microorganisms are washed at least once with
pyrogen-free water from which they are again separated by sedimentation. The
deposit or pellet of microorganisms is then ready for the next stage of the
method.
After aspiration o the supernatant, the washed sedimented micro-
organisms are preerably further treated by resuspending in a small quantity
o pyrogen-free water and ~iltering through a pyrogen-free filter having a
pore size of 0.4 to 0.6 micrometers which retains the microorganisms while
permitting residual small partlcles o blood cell stroma to pass through as
waste. The microorganisms are then suspended in a suitable aqueous medium
such as a pyrogen-free 3% aqueous saline ~sodium chloride) solution to form
a suspension of the microorganisms. This suspension Cor an aliquot of the
suspension~ is then incubated with Limulus polyphemus amebocyte lysate, pre-
ferably a~ a constant temperature between 35 and 45C. for about one hour.
A blank or control procedure is carried out in parallel using sterile 3%
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aqueous saline in place of the specimen obtained from the biological fluid,
to provide a zero standard for the spectro-photometer~ During the incubation
period, ~urbidity develops in the dispersion because of the formation of
a precipitate or coagulate) and the amount of this turbidity is determined
by measuring optical density by conventional procedures, as described for
example by Watson et al., Appliod and Environmental MiGrobiol., Vol. 33,
940-946 tl977). A series of standard solutions containing known amounts of
Gram-negative bacteria such as scherichia ~E. coli), Klebsiella CK. pneumonia),
Bacteroides ~B. fragilis) or the like, or containing ~nown amounts of
endotoxins such as E. coli endotoxin, are also incubated separately with
L. polyphemus amebocyte lysate. After subtraction o the optical density
values for the blank from those o the unknown specimen samples and from those
for the standard solutions, the quantity of Gram-negative bacteria in the
unknown can readily be determined by comparing the optical density for the
unknown sample with a standard curve constructed from the results obtained
with the known standards. When the standard used is an endotoxin, a correct-
ion factor is employed to convert the results into biomass of Gram-negative
bacteria. The procedure of the present invention provides a sensitivity as
low as 20 Gram-negative bacteria per milliliter of biological fluid.
The assay of the present invention is of particular importance not
only because of its sensitivity but because it can be completed in a short
time, less than 4 hours. The invasion of the bloodstream with Gram-negative
bacteria is a serious and often life-threatening complication to many clinical
disorders as well as precursor to post-operative surgical sepsis. The ser-
iousness of bacteremia is further emphasized by the increasing numbers of
patients with immunod~ficient conditions brought on by anti-neoplastic treat-
ment, since bacteremia is associated with a high patient mortality. In the
light of these facts, the rapid and accurate diagnosis of bacteremia made
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possible by the assay of the present invention is an important advance in
the art.
The ollowing specific example wi;Ll serve to illustrate more fully
the nature of the present invention without acting as a limitation upon its
scope.
Example
A 4 ml aliquot of a venous blood sample collected in a heparinized
syringe by venipuncture after skin decontamination is added to a pyrogen free
glass flask containing 36 ml of a lyslng solution consisting of pyrogen-free
water containing 0.1% by weight of octylphenoxy polyphenoxy ~9-10~ ethanol
~sold under the trade mark - Triton X-100) and O.OlM sodium carbonate-sodium
bicarbonate having a pH of 6-8. After gentle mixing of the contents, the
flask is allowed to stand at room temperature until complete sparkling lysis
is observed, which occurs in about 3-5 minutes. A known sterile blood sample
is treated in the ldentical manner to serve as a control. Each lysed sample
is then subjected to vacuum filtration through a 60 ml coarse Allihn ilter-
ing tube having a sintered glass filter surface ~40-60 micrometer pore dia-
meter).
The filtra~e from each specimen is then divided into two 20 ml.
aliquots which are plaeed in 30 ml pyrogen-free centriuge tubes, covered with
anodized aliminum caps and subjected to centrifugation in a T-30 fixed angle
rotor at 10,000 G for 15 minutes ln an ultracentrifuge at ambient temperature.
At the completion of centrifugation, the supernatant liquid was removed by
careful aspiration leaving an almost invisible pellet composed of micro-
organisms and small blood cell fragments on the inner surface of the tube.
The pellet was resuspended in 10 ml of sterile distilled pyrogen-free water
and after gentle mixing was again subjected to centrifugation and separation
of the supernatant under the same conditions. This washing procedure was
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repeated a second time~
After the second washing and removal of the supernatan~ liquid,
the pellet was suspended in 1 ml of a saline solution containing 3% by weight
sodium chloride in pyrogen-free water.
Each 1 ml aliquot sample was then transferred to a separate test
tube. Into other test tubes, there were introduced as standards 1 ml each o
various dilutions in 3% aqueous saline of stock E. coli to provide known
concentrations ranging from 0 to 100 organisms/ml. A blank was prepared by
adding 1 ml of 3% saline to anothcr test tube.
To each tube there was added 0.2 ml of reconstituted Limulus
polyphemus amebocyte lysate obtained from a commercial source. After gentle
shaking, all the tubes were incubated in a water bath at 40C for 60 minutes.
After incubation, the tubes were again gently shaken and the optical density
of the liquid was read lndividually ln a small volume cuvette a~ 360 nm. The
optical density of the blank was subtracted rom the optical density of each
unknown and of each standard. A standard curve was drawn from the optical
densities of the known standards so that by interpola~ion of the optical
density for the unknown sample on the standard curve, the number of organisms
present in the unknown could be dete-rmined.
Similar results can be achieved by applying the assay procedure to
other biological fluids.
A kit suitable for carrylng out the assay of the present invention
can be provided in the form of a container, e.g., a vial containing the cell-
lysing agent or solution described above, a container of Limulus amebocyte
lysate and one or more standard dilutions of Gram-negative bacteria or Gram-
negative endotoxin such as E. coll endotoxln. All apparatus including filters
and test tubes and all water employed in the assay must be pyrogen-free.
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