U.S. patent application number 09/940860 was filed with the patent office on 2004-01-08 for molecular diagnosis of bactermia.
Invention is credited to Majmudar, Maulik D., Rothman, Richard E..
Application Number | 20040005555 09/940860 |
Document ID | / |
Family ID | 30002740 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005555 |
Kind Code |
A1 |
Rothman, Richard E. ; et
al. |
January 8, 2004 |
Molecular diagnosis of bactermia
Abstract
A highly specific assay can be used for the detection of
bacteremia in the clinical setting. The ubiquitous background
endogenous DNA present in all PCR reagents is eliminated using a
restriction endonuclease digestion. Universal primers for
eubacteria are used for detection, and specific primers or probes
for bacterial species can be used for identification of
species.
Inventors: |
Rothman, Richard E.;
(Cockeysville, MD) ; Majmudar, Maulik D.;
(Germaintown, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
30002740 |
Appl. No.: |
09/940860 |
Filed: |
August 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60229376 |
Aug 31, 2000 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/91.2; 536/24.3 |
Current CPC
Class: |
C07H 21/04 20130101;
C12Q 1/6806 20130101; C12Q 1/689 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Claims
We claim:
1. A pair of polymerase chain reaction primers for amplifying a 16S
rRNA gene of eubacteria selected from the group consisting of: PEU
7 and PEU 8 (SEQ ID NO 1 and 2); and PEU 4 and PEU 5 (SEQ ID NO: 3
and 4).
2. A method of performing polymerase chain reaction comprising:
digesting reagents for polymerase chain reaction with a restriction
endonuclease, wherein the reagents comprise Taq DNA polymerase,
deoxynucleotide triphosphates, reaction buffer, and primers,
wherein the restriction endonuclease does not cleave said primers
and said primers have no recognition sites for the restriction
endonuclease; inactivating said restriction endonuclease but not
said Taq DNA polymerase; mixing test sample and the reagents for
polymerase chain reaction to form a mixture; subjecting the mixture
to conditions such that any templates present in the test sample
which hybridize to both primers are amplified; detecting
amplification product, wherein a detected amplification product
indicates the presence of template which hybridizes to both primers
in the test sample.
3. The method of claim 2 wherein the restriction endonuclease is
AluI.
4. The method of claim 2 wherein the step of inactivating comprises
heating to a temperature which inactivates the restriction
endonuclease but not the Taq DNA polymerase.
5. The method of claim 2 wherein the test sample is a treated blood
sample.
6. The method of claim 5 wherein the blood sample is from a patient
suspected of systemic bacteremia.
7. The method of claim 2 wherein the primers are PEU7 and PEU8.
8. The method of claim 3 wherein the step of inactivating is
performed at about 65.degree. C. for about 20 minutes.
9. The method of claim 2 wherein the step of detection employs an
agarose gel.
10. The method of claim 9 wherein amplification product is labeled
with ethidium bromide and visualized under ultraviolet light.
11. The method of claim 5 wherein the blood sample was treated to
extract DNA therefrom.
12. The method of claim 2 wherein the sample is urine.
13. The method of claim 2 wherein the sample is cerebrospinal
fluid.
14. The method of claim 2 wherein the primers hybridize to at least
10 eubacterial species' DNA in regions which are highly
conserved.
15. The method of claim 2 wherein the primers hybridize to 16S RNA
genes.
16. The method of claim 2 further comprising the step of:
identifying a bacterial species as a source of the templates by
sequencing the amplification product.
17. The method of claim 2 further comprising the step of:
identifying a bacterial species as a source of the templates by
restriction endonuclease digestion of the amplification product and
determining sizes of products of said digestion.
18. The method of claim 2 further comprising the step of:
identifying a bacterial species as a source of the templates by
amplification of the amplification product using primers which
hybridize to a single eubacterial species 16S RNA.
19. The method of claim 2 further comprising the step of:
identifying a bacterial species as a source of the templates by
amplification of the templates in the test sample using primers
which hybridize to a single eubacterial species 16S RNA.
20. The method of claim 2 wherein the Taq DNA polymerase is not
active under the conditions used for the step of digesting.
21. The method of claim 2 wherein the amplified product comprises
at least one recognition site for the restriction endonuclease.
22. The method of claim 2 wherein the amplified product comprises
at least two recognition sites for the restriction
endonuclease.
23. A method of performing polymerase chain reaction comprising:
digesting reagents for polymerase chain reaction with AluI
restriction endonuclease, wherein the reagents comprise Taq DNA
polymerase, deoxynucleotide triphosphates, reaction buffer, and a
pair of primers selected from the group consisting of PEU7 and PEU
8 (SEQ ID NO: 1 and 2); and PEU 4 and 5 (SEQ ID NO: 3 and 4);
inactivating said AluI restriction endonuclease by heating said
reagents to a temperature which inactivates AluI but does not
inactivate Taq DNA polymerase; mixing a test sample of DNA isolated
from a patient's blood sample and the reagents for polymerase chain
reaction to form a mixture; subjecting the mixture to conditions
such that any templates present in the test sample which hybridize
to both primers are amplified; detecting an amplification product
of 416 or 811 basepairs, wherein a detected amplification product
indicates the presence of template which hybridizes to both primers
in the patient's blood, which indicates bacteremia in the
patient.
24. A kit for detecting bacteremia in a patient sample, comprising:
a pair of primers which hybridize to opposite strands of 16S RNA of
at least 10 eubacterial species; a restriction endonuclease which
has a four base pair recognition site; wherein the recognition site
does not occur in either of the primers.
25. The kit of claim 19 further comprising: deoxyribonucleotides,
Taq DNA polymerase, and buffer.
26. The kit of claim 19 wherein the primer sequence has been
designed to eliminate a recognition site for the restriction enzyme
present in said 16S RNA gene.
27. A pair of primers which hybridize to opposite strands of 16S
RNA genes of at least 10 eubacterial species at conserved regions,
wherein said primers prime synthesis of an amplification product
comprising a non-conserved region of said 16S RNA which is
distinctive for each of said at least 10 eubacterial species.
28. The pair of primers of claim 24 wherein said primers prime
synthesis of an amplification product in each of said at least 10
eubacterial species which contains at least one recognition site
for a restriction endonuclease which is not present in said
primers.
29. The pair of primers of claim 24 wherein the non-conserved
region in the eubacterial species other than Chlamydia trachomatis
comprises at least 10 base pair differences with respect to the
sequence amplified with said primers using Chlamydia trachomatis
16S RNA gene (SEQ ID NO: 5) as a template.
30. The pair of primers of claim 24 wherein the non-conserved
region in the eubacterial species other than Chlamydia trachomatis
comprises at least 20 base pair differences with respect to the
sequence amplified with said primers using Chlamydia trachomatis
16S RNA gene (SEQ ID NO: 5) as a template.
31. The pair of primers of claim 24 wherein the conserved regions
comprise at least 18 contiguous base pairs which are at least 80%
identical among said 10 eubacterial species.
32. The pair of primers of claim 24 wherein the conserved regions
comprises at least 18 contiguous base pairs which are at least 80%
identical to a pair of primers selected from the group consisting
of: PEU7 and PEU 8 (SEQ ID NO: 1 and 2), and PEU 4 and 5 (SEQ ID
NO: 3 and 4).
Description
[0001] This application claims priority to provisional U.S.
Application Ser. No. 60/229,376, filed Aug. 31, 2000, the
disclosure of which is expressly incorporated herein.
FIELD OF THE INVENTION
[0002] The invention relates to the field of clinical diagnostics.
In particular, it relates to the field of detection of bacteremia
in patients.
BACKGROUND OF THE INVENTION
[0003] There are an estimated 500,000 patients in the United States
who develop bacteremia, with mortality rates ranging from
25-50%..sup.1 Early recognition and aggressive therapeutic
intervention is known to significantly improve outcomes for those
with systemic bacterial infections..sup.1 Unfortunately, no
definitive clinical parameters or diagnostic assays currently exist
that allow clinicians to rapidly and accurately identify patients
with bacterial infections among those in whom systemic infections
are suspected.
[0004] Patients with fulminant bacteremia are usually easily
recognized by the presence of fever and significant vital sign
abnormalities, described as sepsis syndrome. Early systemic
bacterial infections, or those which occur in vulnerable or
immunosuppressed hosts may be more subtle however, leading to
potential delays in diagnosis and treatment with associated
increased risk for morbidity and mortality. Further, inherent
limitations of the `gold standard` diagnostic test for bacteremia,
blood culture, renders it ineffective for guiding acute management
decisions. These limitations include significant time delays
associated with reporting of positive findings (typically at least
24-48 hours), relatively low sensitivity ranging from 30-50% among
patients who meet criteria for sepsis syndrome, and diminished
sensitivity in patients already on antibiotics.
[0005] The failure of either clinical judgment or diagnostic
technology to provide quick and accurate data for identifying
patients with bacteremia, leads most clinicians to follow a
conservative management approach for those in whom systemic
infection is suspected. Empiric intravenous antibiotic therapy
offers the advantage of maximizing patient safety and improving
outcomes for those later found to be bacteremic. Well known
clinical examples in which patients are routinely hospitalized and
given antibiotics while awaiting blood culture results include
febrile episodes in infants, due to the high mortality associated
with unrecognized septicemia, and any febrile illness in
intravenous drug users due to the high risk of life-threatening
infective endocarditis which is principally characterized by the
presence of bacteremia. The benefits of conservative management may
be offset however, by added costs and potential iatrogenic
complications associated with treatment and hospital days for those
later found not to be bacteremic, as well as increased rates of
antimicrobial resistance. A rapid accurate assay for blood-borne
bacterial infections which could be used to screen patients
considered at risk would thus be invaluable for clinicians.
[0006] PCR, or polymerase chain reaction, is a technique which
allows for rapid nucleic acid amplification and detection of small
amounts of a target pathogen (e.g. bacterial RNA or DNA).
Development of PCR diagnostics for clinical use have shown promise
when primers for a specific pathogen are used in selected clinical
settings. Examples include detection of Mycobacterium tuberculosis
in patients with suspicious pulmonary infections, and
identification of Streptococcus pneumoniae in children and infants
with suspected septicemia from this organism. Numerous
investigations have also been carried out employing a universal
probe for more broad based bacterial identification. Findings
published to date have principally been restricted to detection of
bacteria in highly infected tissue specimens, e.g. resected heart
valves in patients with suspected infective endocarditis, or
clinical samples from an infected site such as an abscess.
Unfortunately, less success has occurred with universal screening
of blood samples, principally due to technical problems of the
assay, most commonly related to contaminant bacterial DNA.
[0007] There is a need in the art for a rapid and sensitive test
for detecting specific pathogens in clinical samples.
BRIEF SUMMARY OF THE INVENTION
[0008] According to one embodiment of the invention a pair of
polymerase chain reaction primers for amplifying a 16S rRNA gene of
eubacteria is provided. The primer pairs are selected from the
group consisting of: PEU 7 and PEU 8 (SEQ ID NO 1 and 2), and PEU 4
and PEU 5 (SEQ ID NO: 3 and 4).
[0009] Another embodiment of the invention provides a method of
performing polymerase chain reaction. Reagents for polymerase chain
reaction are digested with a restriction endonuclease. The reagents
comprise Taq DNA polymerase, deoxynucleotide triphosphates,
reaction buffer, and primers. The primers have no recognition sites
for the restriction endonuclease. The restriction endonuclease is
inactivated under conditions that do not inactivate the Taq DNA
polymerase. A test sample and the reagents for polymerase chain
reaction are mixed to form a mixture. The mixture is subjected to
conditions such that any templates present in the test sample which
hybridize to both primers are amplified. Amplification product is
detected. A detected amplification product indicates the presence
of template which hybridizes to both primers in the test
sample.
[0010] According to yet another embodiment of the invention a
method of performing polymerase chain reaction is provided.
Reagents for polymerase chain reaction are digested with AluI
restriction endonuclease. The reagents comprise Taq DNA polymerase,
deoxynucleotide triphosphates, reaction buffer, and a pair of
primers selected from the group consisting of PEU7 and PEU 8 (SEQ
ID NO: 1 and 2), and PEU 4 and 5 (SEQ ID NO: 3 and 4). The AluI
restriction endonuclease is inactivated by heating the reagents to
a temperature which inactivates AluI but does not inactivate Taq
DNA polymerase. A test sample of DNA isolated from a patient's
blood sample is mixed with the reagents for polymerase chain
reaction to form a mixture. The mixture is subjected to conditions
such that any templates present in the test sample which hybridize
to both primers are amplified. Amplification product of 416 or 811
basepairs is detected. A detected amplification product indicates
the presence of template which hybridizes to both primers in the
patient's blood. This in turn indicates bacteremia in the
patient.
[0011] The present invention also provides a kit for detecting
bacteremia in a patient sample. The kit contains (1) a pair of
primers which hybridize to opposite strands of 16S RNA of at least
10 eubacterial species, and (2) a restriction endonuclease which
has a four base pair recognition site. The recognition site does
not occur in either of the primers.
[0012] Another embodiment of the invention is a pair of primers
which hybridize to opposite strands of 16S RNA genes of at least 10
eubacterial species at conserved regions. The primers prime
synthesis of an amplification product comprising a non-conserved
region of the 16S RNA which is distinctive for each of the at least
10 eubacterial species.
[0013] These and other embodiments of the invention provide the art
with methods and tools for sensitively and accurately detecting
bacterial microbes in a variety of sample types.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A and 1B provide a description and homology of PCR
primers. FIG. 1A. Oligonucleotides designed to target the highly
conserved 16S rRNA region for universal amplification of bacterial
DNA. FIG. 1B. Alignment of target sequences of primer set PEU7/PEU8
designed from the 16rRNA gene of S. aureus with the 16S rRNA genes
of other pathogens. A dot indicates the same base, and a letter
indicates a base different from that in the primer set.
[0015] FIG. 2 shows the effect of restriction enzyme digestion on
contamination in the PCR system. PCR amplification using 16S rRNA
primers. Molecular weight marker is indicated MW. Lane 1 is a PCR
amplification using the PEU 7/8 primer pair, with no added
bacteria. Lane 2 is the identical amplification with Alu I
predigestion of the PCR cocktail. Lane 3 is a PCR amplification
using DNA purified from whole blood taken from a healthy human
donor, again with an Alu I predigestion step. Lane 4 is the same as
Lane 3 with purified E. coli bacteria spiked into human blood.
[0016] FIG. 3 shows PCR amplification of common bacterial isolates.
Healthy donor blood spiked with known bacterial isolates: DNA was
extracted and PCR amplification was carried out using conserved
16SrRNA primers. Molecular weight marker is indicated by MW. Lane 1
is whole blood with no bacteria added; Lane 2-5 contain whole blood
spiked with E. coli, E. faecalis, S. aureus and S. pneumonia
respectively. Lane 6 is whole blood with no bacteria added; Lane 7
is a positive control in which bacterial DNA was purified from E.
Coli colonies and subjected to PCR amplification. All PCR reactions
were pretreated with AluI prior to amplification.
[0017] FIG. 4 shows detection limits of PCR system. E. coli
bacteria were diluted in dd H.sub.2O; OD was measured. Serial
dilutions were then performed, followed by DNA isolation and PCR
amplification using PEU 7/8 primer pair. All PCR reactions were
pretreated with AluI prior to amplification. Concentrations are
500, 50, 10 and 5 colonies/PCR, for lanes 1-4. Molecular weight
marker is indicated as MW. Lane 5 is a negative control. Lane 6 is
a positive control.
[0018] FIG. 5 shows a representative gel taken from clinical trial.
Whole blood was taken from febrile patients with suspected
bacteremia. DNA was isolated and PCR amplification carried out
using PEU 7/8 primer pair. All PCR reactions were pretreated with
AluI prior to amplification. Lanes 1-7 were taken from patient
samples; `+` refers to positive control, which is a PCR
amplification of DNA isolated from purified E. coli bacteria; `-`
refers to negative control, which is a PCR amplification of a mock
DNA isolate. Blood culture findings for the seven clinical samples
are indicated as + (positive) or - (negative), below the
figure.
DETAILED DESCRIPTION OF THE INVENTION
[0019] It is a discovery of the present invention that ubiquitous
contamination of reagents for nucleic acid amplification can be
eliminated or reduced by treatment of the reagents with a
restriction endonuclease which does not cleave or recognize a site
within the implication primers, but desirably does cleave and
recognize a site within the desired amplicon. Such a technique
improves the sensitivity and specificity of PCR for detecting
bacteremia in clinical samples, for example. The methods and tools
disclosed can be used in other contexts, for example in the
detection of environmental pathogens and food borne pathogens.
Samples which are expected to harbor bacteria at low concentrations
can be advantageously analyzed using the methods disclosed
herein.
[0020] One of the major obstacles in transferring PCR technology
from the laboratory to the clinical setting is the presence of
ubiquitous `contaminant` DNA, leading to frequent false-positive
results. We have addressed this problem by incorporating a
`decontamination` step in our assay which decontaminates all
reagents. Optionally one can use hot start Taq polymerase
amplification, which also reduces contaminating amplification
products. Any of a variety of techniques can be used to achieve a
hot start amplification. In all methods Taq polymerase is prevented
from synthesizing DNA until a suitable temperature is achieved that
insures sufficient stringency of hybridization of primers to
template. In one method enzyme is modified so that the increase in
temperature during the first denaturation step of PCR causes a
conformation change which activates the modified Taq. A second type
of hot start technology employs antibody inhibition. An antibody
cocktail specific for the polymerase active site can be added, and
the steric hindrance of the antibody prevents polymerase activity.
The antibody irreversibly denatures during the initial denaturation
step. Still another method employs a physical barrier of wax to
separate the primers from the polymerase; the wax is melted during
the initial denaturation step.
[0021] There are four notable features which may be used to achieve
a `universal` PCR assay as disclosed herein. (1) Selection of a
primer pair from the highly conserved 16S rRNA sequence, allowing
for broad range bacterial amplification. (2) The presence of at
least one recognition and/or cleavage site for a restriction
endonuclease is desirable in the amplicon but not in the PCR primer
set. This allows digestion or decontamination of all components of
the PCR cocktail prior to PCR amplification. (3) Multiple copies of
this restriction site within the amplified product is desirable
because it increases the probability of cleaving contaminating DNA
into `nonamplifiable` product, thus diminishing the likelihood of
amplifying background DNA which might contain a single point
mutation in the restriction site. (4) Hot Start Taq DNA polymerase
reaction is only activated at high temperatures (about 95.degree.
C.), preventing DNA polymerization from occurring during the
decontamination step, which is performed at about 65.degree. C.
[0022] A pair of primers can be used which hybridizes to opposite
strands of 16S RNA genes of at least 10 eubacterial species at
conserved regions. Such primers can hybridize to 5, 8, 12, 15, 20,
25, or 30 such species. Preferred species for hybridization are
those which are prevalent in the context being analyzed. If
clinical specimens are analyzed, then desirably the primers
hybridize to the most important pathogens. Desirably the primers
prime synthesis of an amplification product comprising a
non-conserved region of said 16S RNA which is distinctive for each
of the eubacterial species. The non-conserved regions provide the
ability to determine which species is actually present and serving
as the template for the amplification product. Preferably the
amplification products contain at least one, two, three, or four
recognition sites for a restriction endonuclease which is not
present in said primers. The primers can be naturally devoid of
such sites or can be designed to eliminate such a site(s).
Optionally the non-conserved region in the amplicon comprises at
least 10 base pair differences with respect to the sequence
amplified with the same primers using Chlamydia trachomatis 16S RNA
gene (SEQ ID NO: 5) as a template. More preferably the
non-conserved region comprises at least 20 base pair differences
with respect to the sequence amplified with the same primers using
Chlamydia trachomatis 16S RNA gene (SEQ ID NO: 5) as a template.
The conserved regions to which the primers hybridize comprise at
least 18 contiguous base pairs which are at least 80% identical
among the at least 10 eubacterial species. More preferably the
conserved regions comprises at least 18 contiguous base pairs which
are at least 80% identical to PEU7 and PEU 8 (SEQ ID NO: 1 and 2),
or PEU 4 and 5 (SEQ ID NO: 3 and 4). Primers which are particularly
suitable in the practice of the present invention are PEU 7 and PEU
8 (SEQ ID NO 1 and 2), and PEU 4 and PEU 5 (SEQ ID NO: 3 and 4).
These produce amplification products of 416 or 811 basepairs,
respectively.
[0023] One can amplify without contamination or with much reduced
contamination by digesting all of the reagents for amplification
with a restriction endonuclease. Alu I is a preferred enzyme
although others can also be used. The considerations for selecting
an endonuclease are that it should not recognize and cleave within
the primer sequences, but it should cleave at least once, twice, or
thrice within the amplicon. Any Taq polymerase can be used as is
convenient in the particular context. Preferably the polymerase
will not be active until heated to about 95 degrees centigrade.
Thus non-specific amplification will not occur, or will not occur
to appreciable extent during the restriction digestion phase of the
process. The restriction endonuclease can be inactivated using any
means known in the art, however, it needs to be a selective means
which does not adversely affect the activity of Taq DNA polymerase.
Heating the restriction endonuclease for a time and at a
temperature sufficient to inactivate it but not the polymerase is
preferred. For Alu I, an about 20 minute incubation at about 65
degrees C. may be sufficient. Suitable temperatures range from 55
to 75.degree. C., preferably from 60 to 70.degree. C., more
preferably from 62 to 67.degree. C. Suitable times range from 5 to
35 minutes, preferably 10 to 30 minutes, more preferably 15 to 25
minutes. All features need not be used to achieve excellent
results. Determination of appropriate inactivation times and
conditions for any particular enzyme and reagent mixture is well
within the skill of the art.
[0024] A test sample is mixed with the reagents for polymerase
chain reaction and the mixture is subjected to conditions such that
any templates present in the test sample which hybridize to both
primers are amplified. Suitable temperatures and times for cycling
PCR are known in the art and can be selected by those of skill in
the art, depending upon the length and base composition of the
template and primers, for example. Amplification product is
detected using any techniques known in the art, including
electrophoresis, fluorescence of degraded probes (real-time PCR),
and detection of incorporated radioactive mononucleotides. A
detected amplification product indicates the presence of template
which hybridizes to both primers in the test sample.
[0025] Suitable test samples include blood, urine, cerebral spinal
fluid, stool, tears, saliva, food samples, water samples, samples
of ventilation systems, etc. The samples are treated to liberate
DNA from any bacteria which may be present. Means for isolating DNA
from bacterial samples are well known in the art and any such
technique can be used as is desired by the routineer.
[0026] If an amplification product is detected, bacteria are
indicated as present in the test sample. The identity of the
bacteria can be determined by a variety of means. The amplification
product can be sequenced and the determined sequence matched with
the known sequence of 16S RNA genes from bacteria. Alternatively, a
bacterial species can be identified as a source of the templates by
restriction endonuclease digestion of the amplification product and
determining sizes of products of said digestion. Patterns of
digests can be uniquely identified with particular bacterial
species. Another alternative technique is to amplify the
amplification product or original template in the test sample using
primers which are specific, rather than universal, i.e., primers
which hybridize to a single eubacterial species' 16S RNA.
[0027] Kits for detecting bacteremia in a patient sample can
comprise any number of the reagents necessary for carrying out the
assay. Some kits contain only primers and a restriction
endonuclease. As discussed above, these are selected or designed in
tandem so that the primers are not cleaved by the endonuclease but
the amplicon is preferably cleaved at least once. Suitable enzymes
are those that recognize sites of four, five, or six base pairs.
The primers preferably hybridize to and amplify 16S RNA genes from
at least 10 eubacterial species. Other reagents which can be
included in the kit include deoxyribonucleotides, Taq DNA
polymerase, and buffer. Additionally control template can be
included, such as from Chlamydia trichomatis. The reagents in the
kit can be separately packaged or packaged in groups.
EXAMPLES
[0028] In these examples we describe a method for removing
background bacterial DNA from a PCR assay which employs a highly
conserved region of the 16s rRNA (allowing for universal
amplification of bacterial species)..sup.1 Results from a
prospective clinical study using human whole blood specimens taken
from patients with suspected bacteremia in an urban emergency
department are reported.
Example 1
[0029] Study Design
[0030] This was a prospective identity-unlinked investigation. This
technique, is a sampling method which allows single point in time
patient related data collection with anonymous testing of patient
blood samples taken for various blood borne pathogens. In brief,
excess serum was retained from patients' 18 years of age and older
who presented to The Johns Hopkins Hospital Emergency Department
and had blood drawn for blood culture. Enrolled patients were
assigned a unique study number, which was used to code the excess
sera, as well as the laboratory and descriptive data. Descriptive
data included demographics, clinical data, discharge diagnosis, and
blood culture findings. After coding, all patient identifiers were
stripped. In this way, results of the PCR analysis could not be
directly linked to a patient by name or history number. The study
was approved by the Johns Hopkins University Institutional Review
Board.
[0031] Patients and Sample Collection
[0032] Practice at our hospital involves collection of at least 2
sets of blood cultures for all patients in whom the treating
physician suspects systemic bacterial infection. During a 4-month
period 2000 febrile intravenous drug users (all of whom are
considered at risk for bacteremia) whom had blood cultures drawn
simultaneously had a 3 mL aliquot of whole blood collected for PCR
analysis. All patients who had blood drawn for the study had their
skin prepped with 2 betadine swabs. Samples were collected in a
sterile fashion. Blood obtained for culture was inoculated (5 cc
each) into aerobic and anaerobic bottles (BACTEC; Beckton
Dickinson, Inc.), and were then sent immediately to the clinical
microbiology laboratory for processing.
[0033] Samples for PCR analysis were collected by inoculating 3 cc
of whole blood into sterile Na.sub.2-EDTA tubes. These samples were
immediately placed in storage at 4.degree. C. for batch processing
at 3-4 weeks intervals. PCR analysis was carried out in the
laboratory without knowledge of blood culture results or clinical
course of the patient. For purposes of assay refinement and
control, 3 ml of blood was also collected from healthy volunteers
using standard sterile techniques described above.
[0034] DNA Extraction
[0035] Bacterial colonies from Staphylococcus aureus, Streptococcus
pneumoniae, Escherichia coli and Enterococcus faecalis were
obtained from clinical isolates and resuspended in DEPC H.sub.20
(courtesy of Jim Dick, Director, Clinical Microbiology Laboratory,
The Johns Hopkins Hospital). These pathogens were chosen because
they are among the more common organisms observed in bacteremic
patients who present to the Emergency Department. Each of the 4
bacterial samples were then spiked into whole blood specimens taken
from healthy volunteers. These samples were used to optimize the
DNA extraction technique and the PCR assay.
[0036] DNA was extracted from whole blood samples (taken either
from healthy volunteers spiked with bacterial isolates, or from
patient specimens) using a phenol-chloroform-isoamyl alcohol
extraction procedure. An aliquot of 200 .mu.l of whole blood was
diluted in 450 .mu.l DEPC water. The solution was incubated at room
temperature for 10 min with occasional mixing. 40 .mu.l proteinase
K and 15 .mu.l 5.0 mM NaCl was added to the solution and incubated
at 55.degree. C. for 30 min. Subsequently, one ml
phenol-chloroform-isoamyl alcohol (25:20:1) was added and gently
mixed, followed by centrifugation at 13000 rpm for 15 min.
Approximately 300 ul of the aqueous layer was aliquoted into 1 ml
of 100 proof ethanol. Samples were centrifuged at 13,000 rpm for 15
min, washed with 1 mL 70% ethanol, and air-dried. The DNA pellet
was then re-suspended in 100 .mu.l DEPC water.
[0037] Primer Selection
[0038] PCR primers were designed from conserved regions of the 16S
rRNA gene. Oligonucleotides were synthesized and purified on a DNA
synthesizer 380 (Applied BioSystems, CT) at The Johns Hopkins
University Genetic Core Laboratory. FIG. 1a shows the primer pairs,
sequences, and size of the amplified product after PCR.
[0039] Restriction Endonuclease Digestion
[0040] Prior to amplification of the desired target DNA, all PCR
reagents (HotStarTaq DNA polymerase, primers, dNTPs, MgCl.sub.2,
and PCR buffer) were treated with the restriction endonuclease,
AluI. The targeted product contains four sites for AluI, which is
extremely specific for the nucleotide sequence AG.Arrow-up bold.CT,
cleaving the sequence in the center as indicated by an arrow. No
sites for AluI exist in the PCR primers themselves. Predigestion of
PCR reagents thus digests all potential contaminant or background
DNAin the PCR master mixture while leaving the primer set
intact.
[0041] The 100 ul PCR reaction cocktail contained 2.5 units of
HotStarTaq DNA polymerase (QIAGEN, Inc.) 25 pmol of each primer,
200 .mu.M of each nucleotide, 4 mM MgCl.sub.2, and 1.times. PCR
buffer (QIAGEN, Inc.). Restriction digestion with AluI was carried
out by adding 10.0 units of enzyme to each 100 .mu.l PCR reaction
and incubating at 37.degree. C. for 1.5 hours, to allow for
complete digestion of endogenous background DNA. Inactivation of
restriction enzyme was achieved by heating to at 65.degree. C. for
30 min. prior to the addition of target DNA for PCR
amplification.
[0042] PCR Amplification and Detection of Products
[0043] PCR with broad-range 16S rRNA gene primers was performed
using 10 .mu.l target DNA in each 100 .mu.l PCR reaction. The
reaction consisted of a heat-mediated enzyme activation step at
95.degree. C. for 10 min, and 35 cycles of amplification in a
Perkin-Elmer Gene-Amp 2400 Thermal Cycler with the following
conditions for each cycle: denaturation at 95.degree. C. for 30
sec, annealing at 58.degree. C. for 45 sec, and extension at
72.degree. C. for 90 sec. Finally, the elongation step was
performed at 72.degree. C. for 7 min and the final amplified
product stored at 4.degree. C., until further processing. Amplified
products were detected by gel electrophoresis on a 3% agarose gel
stained with ethidium bromide. The gel was visualized under
UV-light for the presence of an 416-bp band and photographed for
documentation.
[0044] Sequencing
[0045] Amplified products were sequenced using the automated
fluorescent DNA Sequencer (Perkin Elmer, Inc.) to confirm the
identity of the spiked pathogens.
[0046] Blood Culture Results and Clinical Data
[0047] Blood culture findings, final clinical data, duration of
antibiotic therapy, and final discharge diagnosis were retrieved
from the electronic patient record (EPR) system at The Johns
Hopkins Hospital. Patients with single isolates of coagulase
negative Staphylococci were excluded from analysis, as this is a
common contaminant in blood culture bottles, and it was impossible
to determine whether the source of the single positive blood
culture was identical to the blood sample used for PCR
analysis.
[0048] Statistical Analysis
[0049] Sensitivity, specificity, positive predictive value (PPV)
and negative predictive value (NPV) were calculated by the method
developed by Ransoff and Feinstein. The false-positive rate,
false-negative rate, and accuracy were also calculated. Each
calculated value was expressed as a percentage.
Example 2
[0050] This example demonstrates the contamination present in PCR
reagents and the efficacy of the subject method for destroying the
contaminants.
[0051] The PCR amplification assay was first tested in a mock
sample containing water, with no added bacteria to determine
whether the reagents themselves contained contaminating sources of
bacterial DNA that might lead to false-positive results. FIG. 2
shows results of a PCR amplification using the 16S rRNA primers
described in FIG. 1. Lane 1 shows a product of the expected size,
indicating that contaminant bacterial DNA is being amplified in our
system. The effect of a predigestion step, in which AluI at is
added to all components of the PCR cocktail is shown in Lane 2.
(Titration of AluI from 1-20 U/reaction identified 10 U/reaction to
be the optimal enzyme concentration.) Lane 3 shows results of a PCR
amplification carried out using whole blood taken from a healthy
control, with no bacteria added, again showing the absence of a
signal. Lane 4 is a positive control, with spiked bacterial
pathogen from E. coli.
Example 3
[0052] This example demonstrates the successful practice of the
method using known bacteria spiked into test samples.
[0053] Healthy human whole blood was next spiked with 1 of 4
bacterial isolates and DNA was then extracted. A series of PCR
amplifications reactions were subsequently carried out with the
decontamination step, described above included, i.e., all PCR
reagents were pretreated with the restriction enzyme, Alu I at 10
U/reaction prior to amplification. FIG. 3 shows the PCR amplified
products from the whole blood specimens, as well as the negative
control samples, in which no bacteria was added prior to PCR
amplification. The amplified product of 411 base pairs was detected
for each of the spiked reactions. DNA sequencing correctly
confirmed the identity of each spiked pathogen.
Example 4
[0054] This example demonstrates the limit of detection of bacteria
in the disclosed assay system.
[0055] A titration profile was carried out to determine the limits
of detection of the PCR assay.
[0056] Healthy human blood was spiked with E. coli at
concentrations ranging from 500 to 5 bacteria/sample. FIG. 4
demonstrates the limit of detection for this assay at 5
bacteria/PCR reaction.
Example 5
[0057] This example demonstrates the successful practice of the
assay method on actual clinical samples.
[0058] DNA was next isolated from whole blood specimens from 60
patients with suspected bacteremia. Valid data for complete
analysis was available for 51 patients; 7 were excluded due to a
single positive coagulase negative Staphylococci; 1 was excluded
due to a missing blood culture report and 1 was excluded due to
missing clinical data. The products of PCR amplification were
separated on an agarose gel for these 51 patients, and compared
with blood culture results. A representative gel with 7 patient
samples is shown in FIG. 6. Table 1 summarizes patient data and
includes PCR results, blood culture findings and clinical data.
Discrepancies between blood culture findings and PCR results
occurred in 7 cases. Table 2 shows correlation of PCR and blood
culture results in terms of sensitivity (86.7%), specificity
(86.9%), PPV (76.4%) and NPV (94.1%).
1TABLE 1 51 patients with blood culture, PCR and blood culture
results, duration of antibiotic treatment and discharge diagnosis.
No. days No. days Pt # PCR BloodCx #times+ Organism IV Ab po Ab
Discharge Diagnosis 1 N N -- -- 3 14 Cellulitis/Abscess 2 P P 4 S.
Aureus 28 -- Infective Endocarditis 3 P P 2/1 S. Viridians/ 28 --
Infective Endocarditis S. Aureus 4 N P 1/1 Strep G/ 42 -- Septic
Arthritis/ Propionobacterium Pneumonia 5 N N -- 2 7 Leg ulcer 6 N N
-- 2 12 Pelvic inflammatory disease 7 N N -- 4 14 Neck abscess 8 N
N -- 1 -- Bronchitis 9 P P 2 Strep GA 42 -- Infective Endocarditis
10 P N -- 3 7 Urinary tract infection/ ?Pyelonephritis 11 P N -- 2
14 Cellulitis/Abscess 12 N N -- 4 10 Cellulitis/Abscess 13 P P 2 S.
Viridians 55 -- Infective Endocarditis 14 N N -- 7 7
Cellulitis/Abscess 15 P P 3 Strep GA 14 -- Bacteremia/Cellulitis 16
N N -- 2 12 Pneumonia 17 P P 2 S. Aureus 28 -- Infective
Endocarditis 18 N N -- 1 13 Abscess 19 P P 3 S. Aureus 42 --
Infective Endocarditis 20 P P 3 S. Aureus 42 -- Infective
Endocarditis 21 P P 2 E. coli/ 5 9 Bacteremia/Pyelonephritis
Corynebacterium 22 P N -- 2 12 Cellulitis/Abscess 23 P N -- 53 18
Abscess (+end stage renal disease) 24 N N -- 4 10 Pneumonia 25 N N
-- 2 12 Urinary tract infection 26 P P 1 S. Viridians 1 -- Fever
unknown origin; left against medical advise 27 N N -- 2 8 Pneumonia
28 N N -- 1 -- Drug fever 29 N N -- 2 8 Ulcer 30 N N -- 0 0 Drug
fever 31 N N -- 2 8 Sinusitis 32 N N -- 3 7 Pneumonia 33 N N -- 1
-- Drug fever 34 N N -- 3 11 Cellulitis/Abcess 35 N N -- 0 0 Drug
fever 36 N N -- 1 13 Cellulitis 37 N N -- 1 7 Bronchitis 38 N N --
5 6 Pneumonia 39 N N -- 2 10 Cellulitis/abscess 40 N N -- 2 2
Urinary tract infection 41 P P 1 Propionobacterium 2 -- Drug fever
42 N P 1 Coryneybacterium 1 9 Cellulitis 43 N N 2 9 --
Abscess/Catheter infection 44 P P 2 Enterobacterium Check --
Urosepsis Actinobacterium 45 N N -- 2 -- Drug fever 46 P P 3 Strep
Group A 28 -- Infective endocarditis 47 N N -- 1 -- Bronchitis 48 N
N -- 1 -- Drug fever 49 N N -- 1 -- Splenic laceration 50 N N -- 7
3 Pneumonia 51 N N -- 5 5 Pneumonia
[0059]
2TABLE 2 Correlation between PCR findings and standard blood
culture results. Number of blood samples with the following results
obtained: by Blood Culture by PCR Positive Negative Total Positive
13 (a) 4 (b) 17 Negative 2 (c) 32 (d) 34 Total 15 36 51 Sensitivity
was calculated as [a/(a + c)} = 13/15 = 86.7%; Specificity was
calculated as [d/(b + d)] = 32/36 = 88.9%; Positive predictive
value (PPV) was calculated as [a/(a + b)] = 13/17 = 76.4%; Negative
predictive value (NPV) was calculated as [d/(c + d)] = 32/34 =
94.1%; False-positive rate was calculated as [b/(a + b + c + d)] =
5/51 = 9.8%; False-negative rate was # calculated as [c/(a + b + c
+ d)] = 2/51 = 3.9%, and accuracy was calculated as [(a + d)/(a + b
+ c + d) = 45/51 = 88.2%.
[0060] Microbial contamination can be derived from multiple
sources, including laboratory equipment, tubes, Taq DNA polymerase,
or any other component of the PCR cocktail. Restriction
endonuclease digestion has previously been described as a method
for elimination of false-positives. The earliest reported use of
this technique was to eliminate nonspecific amplification, or
spurious binding of primers to nearly homologous target sites in
genomic DNA, with a PCR assay for human papilloma virus detection
in cervical specimens. Importantly, the amplification target was
not the 16S rRNA gene, where contamination is particularly
problematic due to multiple copies, and the highly conserved
character of this gene. More recently decontamination studies have
focused on the recognized presence of bacterial DNA in Taq DNA
polymerase, with Carroll et al reporting effective elimination of
false-positives, by pretreatment of the Taq DNA polymerase with a
restriction enzyme that could later be heat inactivated. While
promising, the methodology described did not permit decontamination
of all components of the PCR cocktail and, as of yet, has not been
tested in a clinical trial. Of interest a recent report from
Corless et al describing restriction endonuclease digestion for
elimination of false positives in a real-time universal 16S rRNA
PCR reaction, failed to show consistent decontamination. Possible
explanations for this finding include presence of background
contaminating DNA that were not digested by the particular
restriction enzyme chosen and relative inhibition of Taq DNA
polymerase which may have been caused during inactivation of the
restriction enzyme. Other decontamination methodologies which were
described and remain under investigation included UV irradiation
and DNase digestion, either alone or in combination with
restriction endonuclease digestion.
[0061] The restriction enzyme we chose, Alu I, was selected because
it is not encoded for in the 16S rRNA primer pair, but has several,
four, sites within the amplicon. The presence of multiple sites in
the amplicon targeted by our primer pair increases the likelihood
of complete digestion of contaminating sources of bacterial DNA.
Furthermore, selection of an enzyme which has no sites in the
primer pair itself, permits decontamination of the entire PCR
cocktail, including the primer set itself, prior to the
amplification. These 2 features may in part account for the
efficacy of the predigestion step demonstrated in FIG. 2, and the
relatively high sensitivity of our assay with detection of as
little as 5 bacteria/PCR reaction.
[0062] Previous investigators have recognized the potential impact
of PCR technology for clinical practice. A variety of assays for
species-specific infectious agents have been developed with several
reaching commercial availability, including those for Mycobacterium
tuberculosis, Chlamydia trachomatis, Mycoplasma pneumonia,
Neisseria gonorrhea, herpes simplex virus and cytomegalovirus. In
clinical practice PCR is considered the method of choice for only a
very few specific clinical circumstances (e.g. detection of herpes
simplex virus or enteroviruses in cerebrospinal fluid). More
frequent applications of PCR assays are as an adjunct to standard
culture, for example in patients with suspected tuberculosis,
allowing faster time to diagnosis and treatment. Widespread
utilization of PCR techniques in routine practice however, awaits
further improvement in assay performance and validation of
sensitivity and specificity in clinical trials.
[0063] One clinical situation where PCR could be used is for rapid
detection of bacteremia, which would be invaluable in disposition
and therapeutic decision-making. Exploiting the existence of highly
conserved regions of DNA common to all bacterial species allows one
to design primers for a PCR assay that could serve as a screening
tool for systemic bacterial infection. Published clinical trials
with such universal PCR assays, have included early detection of
bacteremia in febrile neonates and neutropenic cancer patients, as
well as critically ill patients in the intensive care unit, all
considered at high risk for systemic infection. While several of
these studies offer promising results, technical concerns have
prevented general acceptance of these assays for routine use. One
of the most frequently cited factors which have slowed the
development of universal PCR amplification assays is the problem of
ubiquitous nonspecific bacterial contamination present in the
environment or the PCR reagents resulting in significant number of
false-positive results. This nonspecific amplification of
background DNA is particularly problematic for a broad spectrum
primer, such as the 16S rRNA, due its high conservation as well and
its presence in multiple copies.
[0064] In addition to testing the enzymatic decontamination step in
human blood samples spiked with bacterial isolates, we also pilot
tested the PCR assay in a clinical trial. We found that 13 of 15
culture positive blood specimens were PCR positive, with the
universal primers we used, giving a sensitivity of 86.6%. The 2
cases which were positive by blood culture, but negative by PCR
were from gram-positive organisms (one was Corynebacterium and the
other contained mixed organisms including Streptococci Group G,
Propionobacterium and coagulase negative Staphylococci. Clinically,
the patient who grew out Corynebacterium was treated for a
localized cellulitis. Organisms grew from the aerobic bottle of
only 1/3 sets of cultures sent, and was believed to be a
contaminant by the treating physicians. Thus it is likely that this
patient may not have been bacteremic. The other blood culture
positive, PCR negative patient had septic arthritis of the knee and
developed pneumonia later during the hospitalization. Streptococci
Group G organisms grew from the anaerobic bottle of one blood
culture at 2 days, and Propionobacterium grew from one anaerobic
bottle after 7 days. This was likely a case of early bacteremia
which was missed by PCR. A possible explanations for the missed
cases include inadequate lysis of the bacterial cell wall which has
been previously described. Alternative explanations which might
have led to this false-negative findings relate to specimen
handling and processing, such as loss of DNA during the DNA
extraction procedure, or DNA degradation during storage of blood.
Collection of several blood samples which could be used for both
culture and PCR testing may help to resolve such discrepancies in
future studies; additionally specimen handling errors could be
limited by restricting storage time of specimens and DNA
quantification after extraction, prior to running the PCR
assay.
[0065] For the four cases that were PCR positive, but culture
negative all had clinically identifiable infections, three with
cellulitis and/or abscess and one with urinary tract infection and
possible pyelonephritis (see Table 1). All of them received
antimicrobial therapy for at least seven days based on clinical
appearance. Possible explanations for the discordant findings in
these cases include improved sensitivity of the PCR assay over
standard blood culture, for detection of early bacteremia, or
false-positive PCR results. Since there is no true `gold-standard`
test, the approach of interpreting the clinical picture and
likelihood of systemic infection has been used as a proxy. However,
this method is inherently subjective. Alternative approaches
include sequencing the PCR product and correlating findings with
the likely pathogen, based on site of infection, or other specimen
examination such as sputum, swab or gram stain.
[0066] While the invention has been described with respect to
specific examples including presently preferred modes of carrying
out the invention, those skilled in the art will appreciate that
there are numerous variations and permutations of the above
described systems and techniques that fall within the spirit and
scope of the invention as set forth in the appended claims.
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[0092]
Sequence CWU 1
1
7 1 20 DNA Artificial Sequence synthetic amplification primer 1
gcaaacagga ttagataccc 20 2 20 DNA Artificial Sequence synthetic
amplification primer 2 ggaggaaggc gaggatgacg 20 3 22 DNA Artificial
Sequence synthetic amplification primer 3 acaaggcccg agaacgtatt ca
22 4 21 DNA Artificial Sequence synthetic amplification primer 4
gtgccagcag cagcggtaat a 21 5 1550 DNA Chlamydia trichomatis 5
ctgagaattt gatcttggtt cagattgaac gctggcggcg tggatgaggc atgcaagtcg
60 aacggagcaa ttgtttcgac gattgtttag tggcggaagg gttagtaatg
catagataat 120 ttgtccttaa cttgggaata acggttggaa acggccgcta
ataccgaatg tggcgatatt 180 tgggcatccg agtaacgtta aagaagggga
tcttaggacc tttcggttaa gggagagtct 240 atgtgatatc agctagttgg
tggggtaaag gcctaccaag gctatgacgt ctaggcggat 300 tgagagattg
gccgccaaca ctgggactga gacactgccc agactcctac gggaggctgc 360
agtcgagaat ctttcgcaat ggacggaagt ctgacgaagc gacgccgcgt gtgtgatgaa
420 ggctctaggg ttgtaaagca ctttcgcttg ggaataagag aagacggtta
atacccgctg 480 gatttgagcg taccaggtaa agaagcaccg gctaactccg
tgccagcagc tgcggtaata 540 cggagggtgc tagcgttaat cggatttatt
gggcgtaaag ggcgtgtagg cggaaaggta 600 agttagttgt caaagatcgg
ggctcaaccc cgagtcggca tctaatacta tttttctaga 660 ggatagatgg
agaaaaggga atttcacgtg tagcggtgaa atgcgtagat atgtggaaga 720
acaccagtgg cgaaggcgct tttctaattt atacctgacg ctaaggcgcg aaagcaaggg
780 gagcaaacag gattagatac cctggtagtc cttgccgtaa acgatgcata
cttgatgtgg 840 atggtctcaa ccccatccgt gtcggagcta acgcgttaag
tatgccgcct gaggagtaca 900 ctcgcaaggg tgaaactcaa aagaattgac
gggggcccgc acaagcagtg gagcatgtgg 960 tttaattcga tgcaacgcga
aggaccttac ctgggtttga catgtatatg accgcggcag 1020 aaatgtcgtt
ttccgcaagg acatatacac aggtgctgca tggctgtcgt cagctcgtgc 1080
cgtgaggtgt tgggttaagt cccgcaacga gcgcaaccct tatcgttagt tgccagcact
1140 tagggtggga actctaacga gactgcctgg gttaaccagg aggaaggcga
ggatgacgtc 1200 aagtcagcat ggcccttatg cccagggcga cacacgtgct
acaatggcca gtacagaagg 1260 tggcaagatc gcgagatgga gcaaatcctc
aaagctggcc ccagttcgga ttgtagtctg 1320 caactcgact acatgaagtc
ggaattgcta gtaatggcgt gtcagccata acgccgtgaa 1380 tacgttcccg
ggccttgtac acaccgcccg tcacatcatg ggagttggtt ttaccttaag 1440
tcgttgactc aacccgcaag ggagagaggc gcccaaggtg aggctgatga ctaggatgaa
1500 gtcgtaacaa ggtagcccta ccggaaggtg gggctggatc acctcctttt 1550 6
30 DNA Artificial Sequence synthetic amplification primer 6
agcaaacagg attagatacc ctggtagtcc 30 7 30 DNA Artificial Sequence
synthetic amplification primer 7 caggaggaag gcgaggatga cgtcaagtca
30
* * * * *