U.S. patent application number 15/681124 was filed with the patent office on 2017-12-28 for integrated method for collection and maintenance of detectability of a plurality of microbiological agents in a single clinical sample and for handling a plurality of samples for reporting a sum of diagnostic results for each sample.
The applicant listed for this patent is MEDICAL DIAGNOTIC LABORATORIES, L.L.C.. Invention is credited to MARTIN ADELSON, ELI MORDECHAI.
Application Number | 20170372041 15/681124 |
Document ID | / |
Family ID | 37767706 |
Filed Date | 2017-12-28 |
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United States Patent
Application |
20170372041 |
Kind Code |
A1 |
ADELSON; MARTIN ; et
al. |
December 28, 2017 |
Integrated Method for Collection and Maintenance of Detectability
of a Plurality of Microbiological Agents in a Single Clinical
Sample and for Handling a Plurality of Samples for Reporting a Sum
of Diagnostic Results for Each Sample
Abstract
A method and kit related thereto are described for the
collection and maintenance of detectability of a plurality of
species of microbiological agents in a single clinical sample as
well as an integral method for handling a plurality of the samples
and managing information associated therewith for reporting a sum
of diagnostic results for each sample.
Inventors: |
ADELSON; MARTIN;
(HILLSBOROUGH, NJ) ; MORDECHAI; ELI;
(ROBBINSVILLE, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDICAL DIAGNOTIC LABORATORIES, L.L.C. |
HAMILTON |
NJ |
US |
|
|
Family ID: |
37767706 |
Appl. No.: |
15/681124 |
Filed: |
August 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15252951 |
Aug 31, 2016 |
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15681124 |
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11343822 |
Jan 31, 2006 |
9464310 |
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15252951 |
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60651688 |
Feb 10, 2005 |
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60654485 |
Feb 18, 2005 |
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60654729 |
Feb 18, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/569 20130101;
C12Q 1/045 20130101; C12Q 1/6895 20130101; G16H 10/20 20180101;
C12Q 1/6893 20130101; C12Q 1/04 20130101; C12Q 1/689 20130101; G16H
10/40 20180101; C12Q 1/705 20130101 |
International
Class: |
G06F 19/00 20110101
G06F019/00; C12Q 1/68 20060101 C12Q001/68; C12Q 1/04 20060101
C12Q001/04; G01N 33/569 20060101 G01N033/569; C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method for receiving and handling at least one swab of a
gynecological specimen from a patient, said method comprising:
receiving said at least one swab disposed in a medium for
maintaining detectability of two or more microbiological agents in
said gynecological specimen; and testing DNA from said medium for
validation of the existence of said two or more microbiological
agents comprising at least one of Atopobium vaginae, Candida
krusei, erythromycin-resistant Streptococcus agalactiae, and
clindamycin-resistant Streptococcus agalactiae.
2. The method of claim 1, wherein said two or more microbiological
agents comprise at least Atopobium vaginae and a second agent
comprising at least one of Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida dubliniensis, Candida
krusei, Candida lusitaneae, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae,
Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35,
HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66,
HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
3. The method of claim 2, wherein said second agent comprises at
least one of Candida krusei, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae, HPV-16,
or HPV-18.
4. The method of claim 2, wherein said second agent also comprises
at least one of Chlamydia trachomatis, Neisseria gonorrhoeae,
Gardnerella vaginalis, Mobiluncus mulieris, Mobiluncus curtisii,
Bacteroides fragilis, Candida albicans, Candida glabrata, Candida
parapsilosis, Candida tropicalis, Ureaplasma urealyticum, at least
one of Herpes simplex virus, Treponema pallidum, or Haemophilis
ducreyi.
5. The method of claim 1, further comprising: extracting said DNA
from said medium.
6. The method of claim 1, further comprising: amplifying said
DNA.
7. The method of claim 6, wherein said amplifying comprises
real-time Polymerase Chain Reaction (PCR).
8. The method of claim 1, further comprising: providing a kit
containing said swab and said medium.
9. The method of claim 1, further comprising: culturing at least
one of said two or more microbiological agents.
10. A method for receiving and handling at least one swab of a
gynecological specimen from a patient, said method comprising:
receiving said at least one swab disposed in a medium for
maintaining detectability of two or more microbiological agents in
said gynecological specimen; and testing DNA from said medium for
validation of the existence of said two or more microbiological
agents comprising at least Atopobium vaginae.
11. The method of claim 10, wherein said two or more
microbiological agents comprises a second agent comprising at least
one of Chlamydia trachomatis, Neisseria gonorrhoeae, Gardnerella
vaginalis, Mobiluncus mulieris, Mobiluncus curtisii, Bacteroides
fragilis, Candida albicans, Candida glabrata, Candida parapsilosis,
Candida tropicalis, Candida dubliniensis, Candida krusei, Candida
lusitaneae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma
urealyticum, Treponema pallidum, Haemophilis ducreyi, Molluscum
contagiosum Virus, erythromycin-resistant Streptococcus agalactiae,
clindamycin-resistant Streptococcus agalactiae, Lymphogranuloma
venereum, and Human papillomavirus (HPV).
12. The method of claim 11, wherein said second agent comprises at
least one of Molluscum contagiosum virus, Mycoplasma genitalium,
Mycoplasma hominis, Candida dubliniensis, Candida krusei, Candida
lusitaneae, erythromycin-resistant Streptococcus agalactiae,
clindamycin-resistant Streptococcus agalactiae, Lymphogranuloma
venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45,
HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-6/11,
HPV-42, HPV-43, and HPV-44.
13. The method of claim 12, wherein said second agent comprises at
least one of Candida krusei, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae, HPV-16,
or HPV-18.
14. A method for receiving and handling at least one swab of a
gynecological specimen from a patient, said method comprising:
receiving said at least one swab disposed in a medium for
maintaining detectability of two or more microbiological agents in
said gynecological specimen; and testing DNA from said medium for
validation of the existence of said two or more microbiological
agents comprising at least one of Candida krusei,
erythromycin-resistant Streptococcus agalactiae,
clindamycin-resistant Streptococcus agalactiae.
15. The method of claim 14, wherein said two or more
microbiological agents comprises a second agent comprising at least
one of Atopobium vaginae, Chlamydia trachomatis, Neisseria
gonorrhoeae, Gardnerella vaginalis, Mobiluncus mulieris, Mobiluncus
curtisii, Bacteroides fragilis, Candida albicans, Candida glabrata,
Candida parapsilosis, Candida tropicalis, Candida dubliniensis,
Candida lusitaneae, Mycoplasma genitalium, Mycoplasma hominis,
Ureaplasma urealyticum, Treponema pallidum, Haemophilis ducreyi,
Molluscum contagiosum Virus, Lymphogranuloma venereum, and Human
papillomavirus (HPV).
16. The method of claim 15, wherein said two or more
microbiological agents comprises at least Candida krusei, and said
second agent comprises at least one of Atopobium vaginae, Molluscum
contagiosum virus, Mycoplasma genitalium, Mycoplasma hominis,
Candida dubliniensis, Candida lusitaneae, erythromycin-resistant
Streptococcus agalactiae, clindamycin-resistant Streptococcus
agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58,
HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
17. The method of claim 15, wherein said two or more
microbiological agents comprise at least erythromycin-resistant
Streptococcus agalactiae, and said second agent comprises at least
one of Atopobium vaginae, Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida krusei Candida
dubliniensis, Candida lusitaneae, clindamycin-resistant
Streptococcus agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18,
HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56,
HPV-58, HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and
HPV-44.
18. The method of claim 15, wherein said second agent comprises at
least one of clindamycin-resistant Streptococcus agalactiae, and
said second agent comprises at least one of at least one of
Atopobium vaginae, Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida dubliniensis, Candida
krusei, Candida lusitaneae, erythromycin-resistant Streptococcus
agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58,
HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
19. The method of claim 14, wherein said two or more
microbiological agents comprises a second agent comprising at least
one of Atopobium vaginae, HPV-16 or HPV-18.
20. A kit for facilitating the handling of at least one swab of a
gynecological specimen from a patient, said kit comprising: at
least one swab; a medium for receiving said swab after said
gynecological specimen has been collected and for maintaining the
detectability of two or more microbiological agents in said
gynecological specimen; and a test requisition form listing two or
more microbiological agents for validation, said two or more
microbiological agents comprising at least one of Atopobium
vaginae, Candida krusei, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae.
21. The kit of claim 20, wherein said two or more microbiological
agents comprise at least Atopobium vaginae and a second agent
comprising at least one of Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida dubliniensis, Candida
krusei, Candida lusitaneae, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae,
Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35,
HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66,
HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
22. The kit of claim 21, wherein said second agent comprises at
least one of Candida krusei, erythromycin-resistant Streptococcus
agalactiae, clindamycin-resistant Streptococcus agalactiae, HPV-16,
or HPV-18.
23. The kit of claim 21, wherein said second agent also comprises
at least one of Chlamydia trachomatis, Neisseria gonorrhoeae,
Gardnerella vaginalis, Mobiluncus mulieris, Mobiluncus curtisii,
Bacteroides fragilis, Candida albicans, Candida glabrata, Candida
parapsilosis, Candida tropicalis, Ureaplasma urealyticum, at least
one of Herpes simplex virus, Treponema pallidum, or Haemophilis
ducreyi.
24. The kit of claim 20, wherein said two or more microbiological
agents comprises at least Candida krusei, and said second agent
comprises at least one of Atopobium vaginae, Molluscum contagiosum
virus, Mycoplasma genitalium, Mycoplasma hominis, Candida
dubliniensis, Candida lusitaneae, erythromycin-resistant
Streptococcus agalactiae, clindamycin-resistant Streptococcus
agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58,
HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
25. The kit of claim 20, wherein said two or more microbiological
agents comprise at least erythromycin-resistant Streptococcus
agalactiae, and said second agent comprises at least one of
Atopobium vaginae, Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida krusei Candida
dubliniensis, Candida lusitaneae, clindamycin-resistant
Streptococcus agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18,
HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56,
HPV-58, HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and
HPV-44.
26. The kit of claim 20, wherein said second agent comprises at
least one of clindamycin-resistant Streptococcus agalactiae, and
said second agent comprises at least one of at least one of
Atopobium vaginae, Molluscum contagiosum virus, Mycoplasma
genitalium, Mycoplasma hominis, Candida dubliniensis, Candida
krusei, Candida lusitaneae, erythromycin-resistant Streptococcus
agalactiae, Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58,
HPV-59, HPV-66, HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/252,951, filed on Aug. 31, 2016, which is a continuation of
U.S. application Ser. No. 11/343,822, filed on Jan. 31, 2006 (now
U.S. Pat. No. 9,464,310), which claims the benefit under 35 U.S.C.
119(e), to U.S. Provisional Application No. 60/651,688, filed on
Feb. 10, 2005, the entire contents of which are hereby incorporated
by reference, to U.S. Provisional Application No. 60/654,485, filed
on Feb. 18, 2005, the entire contents of which are hereby
incorporated by reference, and to U.S. Provisional Application No.
60/654,729, filed on Feb. 18, 2005, the entire contents of which
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention is in the field of clinical diagnostic
services, i.e., in the industry of identification,
characterization, `quantification of biological agents associated
with disease conditions. The present invention is particularly
directed toward a method and materials for the collection and
maintenance of detectability of a plurality of species of
microbiological agents selected from the group consisting of
bacteria, fungi, viruses, and protozoa in a single clinical sample
and managing information associated with a plurality of samples for
reporting a sum of diagnostic results for each sample.
2. Description of the Related Art
[0003] Clinical diagnostics provide an essential aid to the
physician for the diagnosis and monitoring of numerous pathologies
and infectious diseases. Rapid and accurate identification of
causative agents of a myriad of different human pathophysiological
conditions is a paramount requisite to effective treatment.
[0004] A biological sample generally is taken from the patient,
most often at the request of a physician, and sent to a medical
laboratory for analysis to establish or confirm a diagnosis of
clinical symptoms. A physician may suspect a particular causative
agent upon physical examination. However, certain symptoms may be
characteristic of a plethora of different causative agents.
Therefore, due to misdiagnoses of causative agents, patients may be
treated non-efficaciously. Moreover, in other instances, a
physician may request a certain diagnostic test to be performed on
a clinical specimen wherein the test subsequently produces a
negative result. Then, of course, further clinical samples and
diagnostic testing are required. In many instances, due to the lack
of timely and accurate diagnoses, patients' original conditions
progress to the further detriment of treatability and to the
well-being of the patient. Accurate clinical diagnosis is critical
to specifically identify causative agents in a timely manner which
mediates pathophysiological conditions. Accordingly, a need indeed
exists for materials and a method, for example, to collect an
accurate clinical sample, e.g. a "snapshot," representative of
vaginal flora, i.e., a certain gynecological microbiological
environment, and maintain the detectability of a plurality of
species in a single gynecological sample.
[0005] Diagnostic kits are available, for example, capable of
detecting several specific species. However, in many instances
current diagnostic products and services are inadequate to identify
the causative agent or are inoperable under clinical
circumstances.
[0006] Advances in the detection of C. trachomatis and N.
gonorrhoeae, for example, have included the development of nucleic
acid amplification tests from cervical as well as urine samples.
The Roche COBAS AMPLICOR.TM. CT/NG Test, for example, is an in
vitro multiplex diagnostic test that can detect either or both
Chlamydia trachomatis or Neisseria gonorrhoeae in endocervical or
urethral swabs and/or urine samples. The COBAS AMPLICOR.TM.
Analyzer is an instrument which automates amplification and
detection of the PCR process. The test utilizes polymerase chain
reaction (PCR) nucleic acid amplification and nucleic acid
hybridization (Roche Diagnostic Systems, Branchburg, N.J.). APTIMA
COMBO 2 Assay is a Gen-Probe nucleic acid amplification test that
uses target capture for in vitro qualitative detection and
differentiation of rRNA from C. trachomatis and N. gonorrhoeae in
endocervical and male urethral swab specimens and in urine
specimens. The assay uses target capture (TC),
Transcription-Mediated Amplification (TMA) and Dual Kinetic Assay
(DKA) (Gen-Probe, Inc., San Diego, Calif.).
[0007] However, in view of the myriad of different pathological
agents that mediate disease conditions, a need indeed exists for
materials and methods to collect an accurate clinical sample, e.g.
a "snapshot," representative of a certain gynecological
microbiological environment, for example, and maintain the
detectability of the diversity of pathological agents in a single
gynecological sample. Methods are particularly needed for receiving
and handling a plurality of single gynecological swab clinical
samples, each having identity and test requisition information
associated therewith, wherein the test requisition information
indicates a test for at least one causative agent from a plurality
of listed agents, and managing information associated therewith for
reporting a sum of diagnostic results for each sample.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention is directed to methods and materials
for the collection and maintenance of detectability of a plurality
of species of microbiological agents indicative of a gynecological
disorder selected from the group consisting of bacteria, fungi,
viruses and protozoa, in a single gynecological sample comprising
providing transport media in a resealable container, a sterile
swab, and instructions for preparation and handling of a
gynecological sample, and an indication of the detectability of the
plurality of species. The current invention is particularly
directed to receiving a plurality of these gynecological swab
samples and managing material and information associated with the
samples for reporting a sum of diagnostic results for each
sample.
[0009] Embodiments of the present invention are preferred wherein
at least one species within the plurality of species is indicative
of at least one gynecological disorder, e.g., wherein at least one
species within the plurality of species is selected from the group
consisting of Bacteroides fragilis, Candida albicans, Candida
glabrata, Candida parapsilosis, Candida tropicalis, Chlamydia
trachomatis, Gardnerella vaginalis, Haemophilis ducreyi, Herpes
simplex virus subtype 1 (HSV-1), Herpes simplex virus subtype 2
(HSV-2), Human papillomavirus (HPV), Mobiluncus mulieris,
Mobiluncus curtisii, Molluscum contagiosum Virus, Mycoplasma
genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Treponema
pallidum, Trichomonas vaginalis, Ureaplasma urealyticum, and
Streptococcus agalactiae (Group B Streptococcus). Methods are
preferred wherein at least two (2), three (3), four (4), five (5),
or six (6) species within the plurality of species are selected
from the group.
[0010] The present invention is directed to a method of receiving
and handling a plurality of clinical samples and managing
information associated therewith for generating and reporting any
of a plurality of different diagnostic results from each sample in
a timely manner, particularly within about thirty (30) hours.
[0011] The present invention is directed to a method for the
collection and maintenance of detectability of a plurality of
species of microbiological agents selected from the group
consisting of bacteria, fungi, and viruses, in a single clinical
sample and for handling a plurality of samples and managing
information associated therewith for reporting a sum of diagnostic
results for each sample comprising the steps of: [0012] (a)
providing transport media in a resealable container with
instructions for preparation and handling of a sample and an
indication of the detectability of the plurality of species; [0013]
(b) receiving a plurality of samples, each having identity and test
requisition information associated therewith wherein the test
requisition information indicates a test for at least one species
from a plurality of species; [0014] (c) entering the information
into a system to create a requisition file for each sample; [0015]
(d) processing the information to create a list of samples to be
tested for each species; [0016] (e) dispensing an aliquot
corresponding to each sample into an individual vessel, to create a
secondary sample, for each designated test for different species on
each sample; [0017] (f) assembling a general supply of master
reagent mix for each different test; [0018] (g) combining an
aliquot of master reagent mix for each test with each corresponding
secondary sample to produce a diagnostic test reaction for each
secondary sample; [0019] (h) determining the presence or absence of
a certain product of each reaction to produce a result, recording
the result of each reaction; [0020] (i) combining the result of
each reaction derived from each primary sample into the requisition
file for each sample on the system, thereby producing a sum of
results for each sample; and [0021] (j) reporting the results;
[0022] wherein the plurality of species comprises at least one
species selected from the group consisting of Molluscum contagiosum
virus, Mycoplasma genitalium, Mycoplasma hominis, Candida
dubliniensis, Candida krusei, Candida lusitaneae, Atopobium
vaginae, erythromycin-resistant Streptococcus agalactiae,
clindamycin-resistant Streptococcus agalactiae, Lymphogranuloma
venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45,
HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-6/11,
HPV-42, HPV-43, and HPV-44.
[0023] The present invention is directed to a method for the
collection and maintenance of detectability of a plurality of
species of microbiological agents selected from the group
consisting of bacteria, fungi, and viruses, in a single
gynecological sample and for handling a plurality of samples and
managing information associated therewith for reporting a sum of
diagnostic results for each sample comprising the steps of: [0024]
(a) providing a kit comprised of transport media in a resealable
container, a sterile swab, and instructions for preparation and
handling of a gynecological sample and a writing which indicates
the detectability of the plurality of species; [0025] (b) receiving
a plurality of samples, each derived from the kit, having identity
and test requisition information associated therewith wherein the
test requisition information indicates a test for at least one
species from the plurality of species; [0026] (c) entering the
information into a system to create a requisition file for each
sample; [0027] (d) processing the information to create a list of
samples to be tested for each species; [0028] (e) dispensing an
aliquot corresponding to each sample into an individual vessel, to
create a secondary sample, for each designated test for different
species on each sample; [0029] (f) assembling a general supply of
master reagent mix for each different test; [0030] (g) combining an
aliquot of master reagent mix for each test with each corresponding
secondary sample to produce a diagnostic test reaction for each
secondary sample; [0031] (h) determining the presence or absence of
a certain product of each reaction to produce a result, recording
the result of each reaction; [0032] (i) combining the result of
each reaction derived from each primary sample into the requisition
file for each sample on the system, thereby producing a sum of
results for each sample; and [0033] (j) reporting the results,
wherein the plurality of species comprises at least one species
selected from the group consisting of Molluscum contagiosum virus,
Mycoplasma genitalium, Mycoplasma hominis, Candida dubliniensis,
Candida krusei, Candida lusitaneae, Atopobium vaginae,
erythromycin-resistant Streptococcus agalactiae,
clindamycin-resistant Streptococcus agalactiae, Lymphogranuloma
venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45,
HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-6/11,
HPV-42, HPV-43, and HPV-44.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is an example of a Test Requisition Form.
[0035] FIG. 2 displays validation data for Bacteroides fragilis
wherein the PCR amplicon is 842 bp in which each sample was
independently inoculated and extracted in triplicate. Lanes 2-4
represent detectability after storage at room temperature for zero
days. Lanes 5-7 represent detectability after storage at room
temperature for one day. Lanes 8-10 represent detectability after
storage at room temperature for two days. Lanes 11-13 represent
detectability after storage at room temperature for three days.
Lanes 14-16 represent detectability after storage at room
temperature for four days. Lanes 17-19 represent detectability
after storage at room temperature for five days. Lanes 20 and 21
are the positive and negative controls, respectively.
[0036] FIG. 3 displays validation data for Mobiluncus mulieris
wherein the PCR amplicon is 1015 bp in which each sample was
independently inoculated and extracted in triplicate. Lanes 2-4
represent detectability after storage at room temperature for zero
days. Lanes 5-7 represent detectability after storage at room
temperature for one day. Lanes 8-10 represent detectability after
storage at room temperature for two days. Lanes 11-13 represent
detectability after storage at room temperature for three days.
Lanes 14-16 represent detectability after storage at room
temperature for four days. Lanes 17-19 represent detectability
after storage at room temperature for five days. Lanes 20 and 21
are the positive and negative controls, respectively.
[0037] FIG. 4 displays validation data for Candida albicans.
[0038] FIG. 5 displays validation data for Candida glabrata.
[0039] FIG. 6 displays validation data for Candida
parapsilosis.
[0040] FIG. 7 displays validation data for Candida tropicalis.
[0041] FIG. 8 displays validation data for Chlamydia
trachomatis.
[0042] FIG. 9 displays validation data for Gardnerella
vaginalis:
[0043] FIG. 10 displays validation data for Haemophilis
ducreyi.
[0044] FIG. 11 displays validation data for HSV-1.
[0045] FIG. 12 displays validation data for HSV-2.
[0046] FIG. 13 displays validation data for Trichomonas
vagina/is.
[0047] FIG. 14 displays validation data for Ureaplasma
urealyticum.
DETAILED DESCRIPTION
[0048] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All
publications and patents referred to herein are incorporated by
reference.
[0049] Physicians are generally faced with observing patients'
symptoms, obtaining biological samples, and ordering clinical
diagnostic tests to determine the identity of causative agents
which mediate pathological conditions. Since methods of treatment
of pathophysiological conditions are intimately related to the
identity of the causative agent(s) of the condition, rapid and
accurate identification and reporting of the causative agent(s) is
of paramount importance to the practice of medicine today. The
present invention enables the accurate and rapid reporting of the
detection of any of a plurality of biological agents from each
clinical sample.
[0050] It is an object of the present invention to provide methods
and materials for the collection and maintenance of detectability
of a plurality of species of microbiological agents indicative of a
gynecological disorder in a single gynecological sample comprising
providing transport media in a resealable container, a sterile
swab, instructions for preparation and handling of a gynecological
sample, and an indication of the detectability of a plurality of
species. It is also an object of the present invention to integrate
a method of handling a plurality of these samples and managing
information associated therewith for reporting a sum of diagnostic
results for each sample.
[0051] The present invention is fundamentally a method for
providing certain materials for the collection and maintenance of
detectability of a plurality of species of microbiological agents
indicative of a gynecological disorder in a single gynecological
sample and receiving and handling a plurality of the clinical
samples resulting therefrom and managing material and information
associated therewith for generating and reporting a sum of
diagnostic results for each sample.
[0052] The flow of information and reporting of results from a
clinical laboratory is a fundamental aspect of the present
invention. The present invention enables the accurate and rapid
reporting of the detection of any of a plurality of biological
agents from each clinical sample. The present invention is also a
method for receiving and handling a plurality of clinical samples
and managing information associated therewith to effect rapid
diagnostic testing for any of a plurality of agents in each sample.
Although the invention defined by the claims appended hereto are
not necessarily so limited, preferred methods of the invention are
for handling a plurality of single gynecological swabs (e.g., each
from a different patient) and managing information associated
therewith.
[0053] A basic embodiment of the method of the present invention
involves a rapid method of handling a plurality of single
gynecological swab samples and managing information associated
therewith for reporting any of a plurality of different diagnostic
results for each sample within about fifty (50) hours of receiving
the sample (preferably within forty eight (48) hours, more
preferably within about thirty (30) hours, most preferably within
about twenty four (24) hours). Single gynecological swab samples
each have identity and test requisition information associated
therewith, wherein the test requisition information indicates a
test for at least one causative agent, from a choice of a plurality
of agents (for example, between about 5 and about 25 different
microbiological agents). The term "causative agent" as used herein
refers to biological entities that mediate disease conditions,
including, but not limited to, microorganisms, e.g., bacteria,
fungi, and viruses. Preferred agents, referred to herein as
causative agents, include but are not limited to microbiological
species associated with pathological gynecological conditions, for
example, collected in a single swab specimen (clinical sample).
Causative agents referred to herein include, but are not limited to
Bacteroides fragilis, Candida albicans, Candida glabrata, Candida
parapsilosis, Candida tropicalis, Chlamydia trachomatis,
Gardnerella vaginalis, Haemophilis ducreyi, Herpes simplex virus
subtype 1 (HSV-1), Herpes simplex virus subtype 2 (HSV-2), Human
papillomavirus (HPV), Mobiluncus mulieris, Mobiluncus curtisii,
Molluscum contagiosum Virus, Mycoplasma genitalium, Mycoplasma
hominis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas
vaginalis, Ureaplasma urealyticum, and Streptococcus agalactiae
(Group B Streptococcus), for example.
[0054] Proper and timely identification of causative agent(s) of
vaginosis, a massive microecologic alteration of the vaginal flora,
is a continuous problem for obstetrician-gynecologists. Bacterial
vaginosis (BV), for example, is related to considerable and
preventable infectious morbidity in non-pregnant women. Bacterial
vaginosis mediates endometritis, pelvic inflammatory disease,
post-surgical abortion infections, post-hysterectomy infections, an
increased risk of HIV acquisition and cervical intraepithelial
neoplasia. The diagnosis of BV is complicated by the polymicrobial
nature of the condition. Bacterial Vaginosis (BV), for example, is
characterized by a logarithmically increased concentration of
Gardnerella vaginalis and logarithmically increased concentrations
of pathogenic bacteria, including Bacteroides spp., Mobiluncus
spp., along with Ulreaplasma urealyticum and Mycoplasma spp.
Bacteroides fragilis is an anaerobic bacterium that is commonly
associated with BV. Mobiluncus species, e.g., Mobiluncus mulieris
and Mobiluncus curtisii, are anaerobic bacteria that are commonly
associated with BV. Fifty percent of patients diagnosed with BV,
however, display no symptoms.
[0055] Mycoplasmas are small (0.2-0.3 nm) membrane bound organisms
capable of independent self-replication. The most prevalent strains
recoverable from the genital tract are Ureaplasma urealyticum,
Mycoplasma hominis and Mycoplasma genitalium. In some pregnant
women, Ureaplasma infections are considered to be the cause of
chorioamnionitis and premature delivery. They are frequently
transmitted from mothers to their infants, which may cause a
variety of disorders including pneumonia, persistent pulmonary
hypertension, and chronic infection of the central nervous system.
M hominis is associated with pyelonephritis, pelvic inflammatory
disease (PID), spontaneous abortion, and postpartum septicemia and
fever. M genitalium has been associated with non-gonoccocal
urethritis, acute endometritis, cervicitis, and pelvic inflammatory
disease (PID). Infants become colonized with genital mycoplasmas
during birth. Genital mycoplasma infections are usually diagnosed
by culture. However, due to its fastidious slow-growing nature, M
genitalium, for example, may take up to eight (8) weeks to
culture.
[0056] Candida Vaginitis (CV) is currently the second most common
cause of vaginal infections, with bacterial vaginosis the most
common diagnostic entity. However, CV is misdiagnosed in as much as
50% of all cases. Most studies indicate that CV is a frequent
diagnosis among young women, affecting as many as 15% to 30% of
symptomatic women visiting a clinician. Candida albicans is one of
the major causes of Candida Vaginitis (CV). The widespread use of
topical antifungals appears to contribute to selection for
non-albicans yeasts, e.g., C. glabrata accounts for 7% of all
vaginal fungal infections and about 10% of vaginal yeast isolates.
Candida tropicalis is isolated from 1% to 5% of vaginal yeast
isolates and may be associated with a higher rate of recurrence
after standard treatment. C. parapsilosis accounts for 1% of
vaginal yeast isolates.
[0057] Chlamydia trachomatis is the causative agent of a variety of
diseases including trachoma and urogenital infections. It is the
most common sexually transmitted bacterial agent and in women it
causes cervicitis, urethritis, endometritis and salpingitis. In
more complicated cases it may result in tubal scarring,
infertility, and ectopic pregnancy. In men it causes urethritis and
proctatitis. Other forms of infection also seen are trachoma, the
most preventable form of blindness, and conjunctivitis in
neonates.
[0058] Neisseria gonorrhoeae is the causative agent of the sexually
transmitted disease gonorrhea. It is the most frequently reported
communicable disease in the United States. In women, the most
common presentation is endocervical infection. If left untreated it
may develop into vulvovaginitis, salpingitis, and pelvic
inflammatory disease (PD). Infections in men range from
uncomplicated lower genital tract involvement such as urethritis,
to the more serious epididymitis, prostatitis, and urethral
stricture. Untreated asymptomatic infections may, in certain
instances, develop into disseminated gonococcal infection
(DGI).
[0059] Genitourinary tract infections due to C. trachomatis and N.
gonorrhoeae are a major cause of morbidity in sexually active
individuals. In males they may cause epididymitis and urethritis.
In females, they can cause pelvic inflammatory disease (PID),
ectopic pregnancy, and infertility. If left untreated, N.
gonorrhoeae may develop into a disseminated gonococcal infection
(DGI). Coinfection with C. trachomatis and N. gonorrhoeae is not
uncommon. In fact, up to half of patients diagnosed with infection
of one of these pathogens may be infected with the other and,
therefore, it is important to test all sexually active individuals
for both. Both sexually transmissible pathogens are detectable by
swabbing the urethra and/or the cervix (for women) and performing
either a culture and/or a nucleic acid amplification assay (see Van
Doornum et al., 2001, Journal of Clinical Microbiology
39(3):829-835).
[0060] Chlamydia trachomatis, Neisseria gonorrhoeae, and
Trichomonas vaginalis are the major causes of leukorrhea. Neisseria
gonorrhoeae is the causative agent of the sexually transmitted
disease gonorrhea. In women, the most common symptom of N.
gonorrhoeae infection is endocervical infection, and if left
untreated, may develop into vulvovaginitis and pelvic inflammatory
disease. As a protozoan parasite, Trichomonas vaginalis is the
causative agent of the sexually transmitted disease trichomoniasis.
T vaginalis infection is the primary cause of vaginitis, cervicitis
and urethritis in women. Routine clinical diagnosis usually depends
on microscopic identification of the parasite in wet mount
preparations, which are only 600/% sensitive as compared to
culture-positive women.
[0061] The three major causes of Genital Ulcer Disease (GUD) are
Herpes simplex virus, Treponema pallidum (syphilis), and
Haemophilis ducreyi (chancroid). As treatment options vary, it is
medically necessary to identify the causative agent of GUD.
Currently, the diagnosis of GUD is based primarily on the clinical
presentation of the ulcer itself. However, agent-specific diagnosis
based solely on the clinical evaluations are often obscured by
multiple and mixed infections. T pallidum is the causative agent of
the sexually transmitted disease syphilis. T pallidum is one of the
few major bacterial pathogens of humans that cannot be cultivated
on artificial medium.
[0062] Human Papillomavirus (HPV) subtyping is of clinical
significance in view of the growing evidence for the association of
Human Papillomavirus (HPV) subtypes (i.e., HPV-16 and HPV-18) with
cervical and ovarian cancer. Particularly, 90% of individuals with
major grade cervical intraepithelial neoplasia (CIN 2 and C1N 3)
and invasive carcinoma of the cervix are also infected with HPV-16
or HPV-18. Moreover, as much as 10% to 20% of women in the United
States have tested positive to HPV by Papanicolaou (Pap) smear.
Since Pap smears cannot differentiate among HPV subtypes,
asymptomatic individuals that are HPV positive are being
disregarded by the clinicians.
[0063] Molluscum contagiosum virus (MCV) is a member of the human
pox viruses which produces small raised papules or lesions with
central umbilications and a white, firm, curd-like core. Infection
occurs during sexual intercourse. MCV is a common infection in the
United States and accounts for approximately 1% of all undiagnosed
skin disorders. Many physicians find it necessary to differentiate
MCV from Human papillomavirus (HPV) or Herpes simplex virus (HSV)
infections which have greater mortality and morbidity.
[0064] Vaginal Group B Streptococcus (Streptococcus agalactiae)
(GBS), the most common cause of life-threatening infection in
newborns, is a common cause of neonatal septicemia, pneumonia and
meningitis. One out of every twenty babies with GBS dies from the
infection. In pregnant women, GBS can cause bladder infections,
womb infections, and stillbirth. Many adults are asymptomatic
carriers of GBS in the bowel, vagina, bladder or throat. Diagnosis
by traditional cultures may take several days to complete. However,
once properly diagnosed, GBS can be treated with antibiotics to
prevent the spread from mother to baby.
[0065] The term "clinical sample," as used herein, refers to
biological samples known in the art. "Clinical sample" includes,
for example, but is not limited to a gynecological swab sample.
Particularly, the method of the present invention provides
materials for the collection and maintenance of a "snapshot" of a
gynecological environment for the detectability of a plurality of
species of microbiological agents in a single, gynecological
sample. Clinical samples are generally labeled or otherwise clearly
associated, e.g., packaged, with I information that distinctly
identifies the origin, source and/or destination for the results
for each sample. Each sample is associated with an identifier,
e.g., a patient's name, date of birth, and/or social security
number, for example, or information otherwise provided by the
source to indicate the distinct origin of each sample.
[0066] Methods described herein also comprise (1) providing a
transport media in a resealable container, a sterile swab, and
instructions for preparation and handling of a gynecological sample
and a written indication of the detectability of the plurality of
species, e.g., a test requisition form (see FIG. 1), (2) receiving
the completed gynecological sample in a package with a completed
test requisition form, and (3) handling a plurality of these
clinical samples and managing information associated therewith for
reporting any of a plurality of different diagnostic results for
each sample in a timely manner. Reporting may be accomplished by
means of facsimile to an attending physician who ordered the
test(s), for example, or other means, electronic or otherwise,
e.g., posting on a private-access internet web site, including all
means that are usual and customary in the health-care industry.
[0067] The present invention is not drawn to methods for the
detection, identification or diagnosis, per se, of any particular
microbiological species, or series of species, or disease
condition. The tests, per se, however, whatever tests are used, are
not relevant to the subject matter of the present invention. In
contrast, methods described herein are specifically directed toward
collection and maintenance of detectability of a plurality of
species of microbiological agents and handling a plurality of
clinical samples and managing material and information associated
therewith. Particularly, methods of the present invention are for
providing a compilation of certain materials, managing samples and
information related to the samples proximal in time, i.e., before,
during, and after, a determination of any of a plurality of
different possible diagnostic results for each sample and reporting
the results.
EXEMPLARY EMBODIMENT
[0068] The Applicants describe herein methods and materials to
collect and maintain the detectability of multiple pathogens from a
single gynecological swab. Specimen viability has been validated
for up to five days, for example, as illustrated herewith. Longer
viability, however, e.g., about 5-10 days, is reasonably expected
in view of the data presented herein. No refrigeration is required.
The present invention is not drawn to methods for the detection,
identification or diagnosis, per se, of any particular
microbiological species, or series of species, or disease
condition. In contrast, methods and articles of manufacture, i.e.,
kits, of the present invention are particularly provided for the
collection and maintenance of detectability of a plurality of
different microbiological species associated with pathological
gynecological conditions so that a clinical diagnostic service
provider, for example, may enable a physician to collect a single
swab specimen (clinical sample) and order any of a plurality of
tests on that sample from the clinical diagnostic service provider,
for example. The tests, per se, however, whatever tests are used,
are not relevant to the subject matter of the present invention.
The Applicants indeed present validation data herewith merely to
demonstrate the viability of the species exemplified herein using
the materials and methods of the present invention. Since the
species exemplified herein are well characterized in the art and
are, as discovered and illustrated by the Applicants, viable under
the conditions described herein, they are indeed detectable by an
array of different methods, e.g., nucleic acid amplification, known
to those of skill in the art.
[0069] A method is particularly preferred for the collection and
maintenance of detectability of a plurality of species of
microbiological agents selected from the group consisting of
bacteria, fungi, and viruses, in a single gynecological sample
comprising providing transport media in a resealable container, a
sterile swab, and instructions for preparation and handling of a
gynecological sample and an indication of the detectability of the
plurality of species.
[0070] Microbiological agents that are causative or are otherwise
associated with gynecological disorders are preferred. Since many
different species of microbiological agents mediate, or are
associated with, or are indicative of gynecological disorders, the
present invention provides a means for handling a plurality of
clinical gynecological swab samples and managing information
associated therewith in the process leading up to the
identification of at least one causative agent in each sample and
reporting the results representative of the ambient population of
microbiological agents in each sample at the time each sample was
taken. Particularly, the method of the present invention enables a
"snapshot" of details corresponding to each a single gynecological
sample, within a plurality of samples, to be provided in a valuable
period of time by means of information management. A single
gynecological swab sample is generally received in transport media,
between about 1 ml and about 5 ml, for example, in a resealable
container along with a test requisition form.
[0071] Transport media for use in methods of the present invention
is a universal transport media in which viability of a plurality of
organisms, e.g., bacteria, fungi, and viruses, can be sustained
under normal conditions without refrigeration for at least 48
hours. Transport media for use in methods of the present invention
is preferred wherein the detectability of species selected from the
group consisting of Bacteroides fragilis, Candida albicans, Candida
glabrata, Candida parapsilosis, Candida tropicalis, Chlamydia
trachomatis, Gardnerella vaginalis, Haemophilis ducreyi, Herpes
simplex virus subtype 1 (HSV-1), Herpes simplex virus subtype 2
(HSV-2), Human papillomavirus (HPV), Mobiluncus mulieris,
Mobiluncus curtisii, Molluscum contagiosum Virus, Mycoplasma
genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Treponema
pallidum, Trichomonas vaginalis, Ureaplasma urealyticum, and
Streptococcus agalactiae (Group B Streptococcus) is maintained at
room temperature for five (5) days.
[0072] An example of a general support media for a variety of
organisms in a clinical sample is Stuart's medium (see, e.g.,
Stuart et al., 1954, The problem of transport of specimens for
culture of Gonococci. Canadian Journal of Public Health.
45(2):73-83). Stuart's medium is a well-known buffered transport
medium which includes the component sodium glycerophosphate to
permit minimal multiplication and sodium thioglycollate as a
reducing agent to prevent oxidation within the sample. Stuart's
medium, however, contains no nutrients. The absence of nutrients
retards the growth of commensal organisms within the sample which
can multiply and overgrow the less hardy pathogens. However, the
absence of nutrients in Stuart's media can be detrimental to the
viability of less hardy pathogens. Preferred transport medium
comprises an aqueous balanced salt solution buffered at
approximately physiological pH, at least one protein stabilizer,
and combinations of carbohydrate and amino acid nutrient sources.
The medium is buffered to maintain physiological pH and may include
a pH indicator in order to indicate variation of pH outside the
physiological pH range. The medium further comprises antimicrobial
and antifungal agents and can include gelatin. Transport media, for
example, is liquid media wherein detectability of a plurality of
species of microbiological agents including bacteria, fungi, and
viruses is maintainable under normal conditions, without
refrigeration, for at least 72 hours. Example transport media
herein is demonstrated to be suitable for maintaining the
detectability, for example, of a plurality of species of
microbiological agents including bacteria, fungi, and viruses under
normal conditions, without refrigeration, for about five (5) days
(e.g., UTM-RT Transport Medium, Copan Diagnostics Inc., Corona,
Calif.).
[0073] Transport medium, for example, consists of modified Hank's
balanced salt solution supplemented with bovine serum albumin,
cysteine, gelatin, sucrose, and glutamic acid. The pH is buffered
with HEPES buffer, for example. Phenol red is used to indicate pH.
Vancomycin, amphotericin B, and colistin are incorporated in the
medium to inhibit growth of competing bacteria and yeast. The
medium is isotonic and non-toxic to human cells. Example components
comprise, for example, Hank's Balanced Salts, Bovine Serum Albumin,
L-Cysteine, Gelatin, Sucrose, L-Glutamic Acid, HEPES Buffer,
Vancomycin, Amphotericin B, Colistin, and Phenol Red; pH 7.3+/-0.2
@ 25.degree. C. Antimicrobial compositions can include vancomycin,
gentamicin, colistin, or amphotericin B.
[0074] A general transport medium of about 1.0 liter (total volume)
may be prepared in the following aqueous composition: gelatin,
0-20.0 g; sugar, 65-75 g; HEPES, 5-7 g; KCl, 0.3-0.6 g; L-glutamic
acid, 0.5-1.0 g; phenol red, 5-15 mg; CaCl.sub.2, 0.1-0.5 g;
MgSO.sub.4.7H.sub.20, 0.1-0.3 g; bovine serum albumin V, 1.0-20.0
g; vancomycin, 0.01-0.05 g; colistin, 100,000-250,000 units; and
amphotericin B, 0.5-3.0 mg.
[0075] An example may include the following ingredients in about
995 ml of water q.s. 1 liter: sucrose, 68.46 g; HEPES, 5.96 g; KCl,
0.4 g; L-glutamic acid, 0.72 g; phenol red, 11.0 mg; CaCl.sub.2,
0.27 g; MgSO.sub.4.7H.sub.2O, 0.20 g; BSA, 5.0 g; gelatin, 5.0 g;
vancomycin, 0.025 g; colistin, 200,000 units; and amphotericin B,
1.0 mg. The pH of the medium can be adjusted with acidic or basic
solutions to arrive at a final pH within physiological limits (see,
e.g., U.S. Pat. No. 5,702,944, entitled Microbial Transport Media,
which is herein incorporated by reference in its entirety).
[0076] Preferred commercially available and validated examples of
transport medium for use in methods and articles of the present
invention include, for example, the following: UTM-RT Transport
Medium, BD Cellmatics Viral Transport Medium.RTM. (Becton,
Dickinson & Company, Sparks, Md.), Multitrans Culture
Collection and Transport System (Starplex Scientific, Etobicoke,
Ontario, Canada), The ThinPrep.RTM. Pap Test Preservcyt.RTM.
Solution (CYTYC Corporation. Boxborough, Mass.), SurePath.RTM.
(Tripath Imaging Inc., Burlington, N.C.).
[0077] Preferred methods of the present invention provide a
transport media in a resealable container, a sterile swab,
instructions for preparation and handling of a gynecological sample
and a written indication of the detectability of the plurality of
species.
[0078] A labeled screw-cap tube is preferred, for example, as a
resealable container, which contains a volume of transport medium,
between about 1 ml and about 5 ml, for example, suitable for
accurate collection and maintenance of a population of
microbiological agents representative of a gynecological
environment. A resealable container optionally contains glass beads
(three 3 mm beads, for example). Although not required to be
provided, per se, in methods and the specified compilation of
materials described herein, at least one sterile swab, well-known
in the art of gynecology, is preferred in the materials of a
packaged kit described herein for obtaining the gynecological
sample. At least one sterile swab for obtaining the gynecological
sample, for example, is preferably supplied with the transport
media in a package along with written instructions for preparation
and handling of a gynecological sample in the media, and a written
indication of the detectability of a plurality of species, e.g., a
test requisition form. The transport medium may be supplied,
however, alone in a package which comprises a writing, i.e., a
written indication of the detectability of a plurality of species,
for example, a test requisition form which lists a plurality of
species of microbiological agents described herein for selection,
for example, by the attending physician. The compilation of
materials described herein is preferably provided in methods
described herein in a container, i.e., a package. The package
preferably contains the materials in a kit-format. Written
instructions for preparation and handling of a gynecological sample
in the media are preferred to be included in the compilation of
materials otherwise described herein as a packaged kit intended,
designated, and prepared for the specific purpose of collection and
maintenance of detectability of a plurality of species of
microbiological agents described herein. Once a swab sample is
collected, it should be placed immediately into the transport tube
where it comes into contact with transport medium.
[0079] A first set of example instructions are as follows:
1. Collect the single specimen with a swab (Polyester (Dacron)
tipped swabs are suitable). 2. Aseptically remove the cap from the
transport media. 3. Insert swab into the tube with the transport
medium. 4. Break swab shaft by bending it against the tube wall. 5.
Replace cap to tube and close tightly. 6. Label the tube with
appropriate patient information. 7. Complete the test requisition
form included herewith. 8. Send these items in the pre-addressed
postage materials included herewith to the laboratory for immediate
analysis.
[0080] A second set of example instructions are as follows:
1. Visualize cervix with speculum without lubricant. 2. Remove
mucus and/or secretions from the cervix with a swab, discard swab.
3. Firmly, yet gently, sample the endocervical canal with sterile
swab for 10 seconds. 4. Place the swab into the transport vial. 5.
Be sure the cap is sealed tightly.
[0081] Specimen collection swab options include, for example: one
regular size plastic shaft swab with polyester fiber tip; two
regular size plastic shaft swabs with polyester fiber tips; one
regular size plastic shaft swab and one Minitip plastic shaft swab
pre-scored for easy breakage, both with polyester fiber tips; one
Minitip plastic shaft swab with polyester fiber tip pre-scored for
easy breakage; one Combo stainless steel wire-plastic shaft Minitip
swab with polyester fiber tip: one regular size plastic shaft swab
and one Combo stainless steel wire-plastic shaft Minitip swab, both
with polyester fiber tips. These different swab applicator shafts
facilitate the collection of specimens from various sites on a
patient.
Receiving a Plurality of Single Gynecological Swab Samples
[0082] A "plurality of samples" is an inclusive term which refers
to a plurality of single samples from different patients. A
"plurality" of samples generally refers to a substantial number of
biological samples received by a clinical lab within a twenty four
(24) hour period, for example. A plurality of samples, however, as
used herein may refer to as few as several samples, e.g., about ten
(10), or about five thousand (5,000) samples, for example, to be
processed. Each sample has identity and test requisition
information associated therewith, wherein the test requisition
information indicates a test for at least one causative agent, from
a choice of a plurality of agents, from a list of between about 5
and about 25 different microbiological agents, for example. For the
purpose of illustration of the complexity of information associated
with a plurality of single gynecological swabs and test requisition
information associated therewith, each sample with a test
requisition form which indicates the detectability of twenty (20)
different agents, for example, has the possibility of about 400
different diagnostic results, for that single sample. This,
combined with the fact that a plurality of samples (e.g., 500) are
received to be processed together, that day, illustrates the
complexity of information associated therewith to be managed in
order to handle the plurality of clinical samples for reporting any
of a plurality of different diagnostic results for each sample in a
timely manner. The current invention is particularly directed to
methods wherein the test requisition information indicates a test
for at least one (1), preferably at least two or three (2 or 3),
e.g., between 4 and about 6, causative agents from a choice (list)
of a plurality of agents. Embodiments of the present invention
include, for example, wherein the plurality of species comprise at
least one species selected from the group consisting of Molluscum
contagiosum Virus, Mycoplasma genitalium, and Mycoplasma
hominis.
[0083] The information in the system, i.e., the identity of the
sample (e.g., sample identifier or identification tag) and test
requisition information, i.e., tests specifically requested to be
performed on that sample, is processed to designate a test on each
sample for at least one causative agent. Accordingly, methods
described herein comprise entering identity and test requisition
information associated with each sample into a system to create a
requisition file for each sample. The term "system" as used herein
refers generally to a system of recording and managing information,
a computer implemented information management system to manage the
flow of information and, in certain embodiments, to control
instrumentation, throughout the process of the present invention.
This system is preferred, but, however, is not required. A computer
is generally employed to receive the identity and test requisition
information associated with each sample. The information may be
entered manually into a server, for example, to create a test
requisition file for each sample which comprises the sample
information and the test requisition information. A listing, file
for example, of the identity of all samples for each test is
created. If twenty different tests are to be performed (for twenty
different pathological agents), for example, twenty different lists
of sample identifiers are created. If a certain sample will be
subject to three different tests, for example, that sample
identifier will be on at least three separate lists corresponding
to those three different tests. In some embodiments of the present
invention a computer implemented system performs calculations
and/or controls instrumentation.
[0084] A method for the collection and maintenance of detectability
of a plurality of species of microbiological agents selected from
the group consisting of bacteria, fungi, and viruses, in a single
clinical sample and for handling a plurality of samples and
managing information associated therewith for reporting a sum of
diagnostic results for each sample is preferred which comprises
providing transport media in a resealable container with
instructions for preparation and handling of a sample and an
indication of the detectability of the plurality of species,
receiving a plurality of samples, each having identity and test
requisition information associated therewith wherein the test
requisition information indicates a test for at least one species
from a plurality of species, entering the information into a system
to create a requisition file for each sample, processing the
information to create a list of samples to be tested for each
species, dispensing an aliquot corresponding to each sample into an
individual vessel, to create a secondary sample, for each
designated test for different species on each sample, assembling a
general supply of master reagent mix for each different test,
combining an aliquot of master reagent mix for each test with each
corresponding secondary sample to produce a diagnostic test
reaction for each secondary sample, determining the presence or
absence of a certain product of each reaction to produce a result,
recording the result of each reaction, combining the result of each
reaction derived from each primary sample into the requisition file
for each sample on the system, thereby producing a sum of results
for each sample, and reporting the results. Methods are
particularly preferred wherein at least one species within the
plurality of species is indicative of at least one gynecological
disorder. Methods of the present invention are preferred wherein
the plurality of species comprises Chlamydia trachomatis and
Neisseria gonorrhoeae. Methods of the present invention are
preferred wherein the plurality of species comprises Gardnerella
vaginalis, Mobiluncus mulieris, Mobiluncus curtisii, and
Bacteroides fragilis. Methods of the present invention are
preferred wherein the plurality of species comprises Candida
albicans, Candida glabrata, Candida parapsilosis, and Candida
tropicalis. Methods of the present invention are preferred wherein
the plurality of species comprises Mycoplasma genitalium,
Mycoplasma hominis, and Ureaplasma urealyticum. Methods of the
present invention are preferred wherein the plurality of species
comprises Herpes simplex virus, Treponema pallidum, and Haemophilis
ducreyi. Methods of the present invention are preferred wherein at
least one (1) species of the plurality of species is selected from
the group consisting of Molluscum contagiosum Virus, Mycoplasma
genitalium, and Mycoplasma hominis.
Nucleic Acid is Extracted from Each Sample
[0085] Established procedures for DNA extraction are used (see
Example II). In brief, swabs are thoroughly mixed in the transport
media contained within the transport vials. 470 .mu.I of transport
media is mixed with 25 .mu.I of 10% sodium dodecyl sulfate, and 12
.mu.I of freshly prepared DNase-free proteinase-K (10 mg/ml), then
incubated for 2 hours at 55.degree. C. DNA is
phenol:chloroform:isoamyl alcohol extracted and recovered by
ethanol precipitation. DNA is pelleted, dried in a speed vacuum,
and resuspended in 20 .mu.l TE buffer. DNA concentration is
calculated by absorbance 260/280 readings and was adjusted to 0.2
.mu.g/.mu.l prior to PCR analysis. Quantitation, however, is
preferred using a fluorometer. Quantitation, however, is preferred
using a fluorometer such as one available from Turner BioSystems,
Inc., Sunnyvale, Calif.
[0086] The nucleic acid from each sample is diluted to about 200
ng/.mu.l, for example, with water, for example, to provide a
standardized primary nucleic acid solution corresponding to each
sample. An aliquot of nucleic acid from each sample is dispensed
into a separate individual vessel to create a secondary sample
corresponding to each designated test on each sample. A general
supply of master reagent mix, e.g., real-time PCR mix, for each
test for each different causative agent is prepared. An aliquot of
each master reagent mix is combined with each, corresponding
secondary nucleic acid sample for each test to produce a diagnostic
test reaction for each secondary sample. Each reaction is incubated
and preferably monitored in real-time. The presence or absence of a
certain product of each reaction to produce a result is determined.
The result of each reaction is recorded in the system. The result
of each reaction derived from each primary solution is combined
into the requisition file for each sample on the system, thereby
identifying at least one causative agent in each sample, and the
results of the identification are reported.
[0087] A rapid method of handling a plurality of clinical samples
and managing information associated therewith for identifying at
least one causative agent in each sample and reporting results
comprises receiving a plurality of samples, each having identity
and test requisition information associated therewith wherein the
test requisition information indicates a test for at least one
causative agent, entering the information into a system to create a
requisition file for each sample, processing the information to
designate a test on each sample for at least one causative agent,
dispensing an aliquot corresponding to each sample into an
individual vessel to create a secondary sample for each designated
test, assembling a general supply of master reagent mix for each
test for a different causative agent, combining an aliquot of each
master reagent mix with each corresponding secondary sample for
each test to produce a diagnostic test reaction for each secondary
sample, incubating each reaction, determining the presence or
absence of a certain product of each reaction to produce a result,
recording the result of each reaction, combining the result of each
reaction derived from each primary sample into the requisition file
for each sample on the system, thereby identifying at least one
causative agent in each sample, and reporting the results of the
identification.
[0088] A preferred method of handling a plurality of clinical
samples and managing information associated therewith for
identifying at least one causative agent in each sample and
reporting results comprises receiving a plurality of samples, each
having identity and test requisition information associated
therewith wherein the test requisition information indicates a test
for at least one causative agent, entering the information into a
system to create a requisition file for each sample, extracting
nucleic acid from each sample, quantitating the nucleic acid,
diluting the nucleic acid from each sample to provide a
standardized primary nucleic acid solution corresponding to each
sample, processing the information in the system to designate a
real-time PCR test on each nucleic acid for at least one causative
agent, dispensing an aliquot of the primary solution from each
sample into a separate individual vessel to create a standardized
secondary nucleic acid sample for each designated test on each
sample, assembling a general supply of master reagent mix for each
test for a different causative agent, combining an aliquot of each
master reagent mix with each corresponding secondary nucleic acid
sample for each test to produce a diagnostic test reaction for each
secondary sample, incubating each reaction, determining the
presence or absence of a certain product of each reaction to
produce a result, electronically recording the result of each
reaction, in the system, combining the result of each reaction
derived from each primary solution into the requisition file for
each sample on the system, thereby identifying at least one
causative agent in each sample, and reporting the results of the
identification. Preferred methods of the invention described herein
employ quantitating nucleic acid from each sample by means of a
fluorometer. Methods of the present invention preferably comprise
generation of diagnostic results by means of real-time PCR.
[0089] To Maximize the Success of Clinical Diagnostic Methods
Described Herein
[0090] A clinical diagnostic laboratory should be physically set up
so that specimen separation and extractions occur in a separate
room, using a "Class II Biohazard Safety Hood." PCR preparation
should occur in a separate room, within one of many PCR cabinets
which are dedicated solely to PCR preparation. The PCR
amplification should occur in thermocyclers located in an enclosed
room. For post-amplification process of conventional PCR reactions,
gel electrophoresis should be performed in yet another physically
separate room. UV lights should be used in the PCR hoods and
commercial solvents, such as DNAway (Molecular Bio Products, San
Diego, Calif.), to decontaminate all work surfaces prior to and at
the completion of any procedures occurring in that area. Sterile,
disposable plasticware should be used wherever possible. All
glassware should be autoclaved. All PCR reactions should be
performed in individual closed tube systems as opposed to 96-well
microtiter plates to eliminate cross contamination. Real-time PCR
assays, for example, do not require gel electrophoresis and
therefore eliminate post-amplification specimen handling. All
technicians should only manipulate one specimen at a time. This
means when a reagent is added to a batch of specimens, it occurs
one tube at a time. The next patient's reaction tube is not opened
until the previous patient's tube has been closed. Pipette tips
used when dispensing reagents should be filtered to prevent aerosol
contamination and are also replaced between all specimens. Reagents
used during PCR preparation may be aliquoted into 1.5 ml
microcentrifuge tubes, for example, as opposed to dispensation into
stock bottles of greater.cndot.volumes. This enables the laboratory
to monitor potential contamination closely and discard any
reagents, if ever necessary. The use of separate rooms is
recommended to decontaminate an entire room if contamination is
suspected. Positive and negative controls should be employed to
assess false positives as well as false negatives.
Uracil-N-glycosylase is recommended in every reaction to minimize,
if not eliminate, any possible carry-over contamination.
Primers
[0091] Any pair of PCR primers may be employed in methods of the
present invention that function to amplify target nucleic acids.
The art of selection and synthesis of PCR primers in order to
amplify a particular target sequence is indeed well-known to those
of ordinary skill in the art. Typically, oligonucleotide primers
are about 8 to about 50 nucleotides in length. Primers 12 to 24
nucleotides in length are preferred. Primer pairs that amplify
particular nucleic acid molecules can be designed using, for
example, a computer program such as OLIGO (Molecular Biology
Insights, Inc., Cascade, Colo.). A biotin moiety, for example, is
preferably attached to the 5' end of one of the primers to
facilitate sample preparation for "pyrosequencing," a term which
denotes the nucleotide sequencing method described in U.S. Pat.
Nos. 6,210,891 and 6,258,568; Ronaghi et al., 1998. A sequencing
method based on real-time pyrophosphate. Science 281:363-365; and
Ronaghi, 2001, Pyrosequencing sheds light on DNA sequencing. Genome
Research 11:3-11. Other entities, however, well known to those of
skill in the art, may similarly be incorporated, integrated, or
attached to one of the primers to facilitate the isolation of the
resulting amplicon for pyrosequencing.
Real-Time PCR
[0092] Quantitative real-time PCR is a preferred method of
amplification of a target nucleic acid. Products used to accomplish
the methods are commercially available from several manufacturers
including, but not limited, to Corbett Research (Mortlake,
Australia), Cepheid (Sunnyvale, Calif.), BioRad (Hercules, Calif.),
and Applied Biosystems (Foster City, Calif.). The Corbett Research
(Melbourne, Australia) Rotor-Gene''' 3000, for example, is a
centrifugal, real-time DNA amplification system.
[0093] Validation studies exemplified herein are merely a general
demonstration of the utility and value of the present invention,
namely a method for the collection and maintenance of detectability
of a plurality of species of microbiological agents in a single
gynecological sample, in the grand scheme of clinical diagnostics.
The legitimacy of the PCR method is not a relevant factor, as its
utility as an invaluable molecular biological tool has already been
well established in the international scientific literature through
the publication of thousands of peer-reviewed articles.
Particularly, molecular amplification of nucleic acids by means of
PCR is well-known to those of ordinary skill in the art, i.e., the
ability of the PCR method to detect genetic sequences specific to a
target pathogen within a given clinical specimen. The Applicants
particularly highlight, however, that the methods described herein,
which comprise providing transport media in a resealable container
to a physician, clinical lab, or medical institution, with
instructions for preparing and handling a gynecological sample,
along with a test requisition form which indicates the
detectability of a plurality of species described herein, affect
the ability of a physician, for example, to collect a single swab
sample of a gynecological environment for the maintenance of
detectability of a plurality of species of microbiological agents.
Example assays designed to test sensitivity, specificity,
interference and optimization were performed to validate the
operability of the methods and materials described herein, as
claimed. In other words, PCR methods or reagents employed to detect
microbiological agents are not relevant to the scope of the subject
matter of the claims appended hereto. In contrast, the present
invention is solely drawn toward methods and certain materials for
collection and maintenance of detectability of a plurality of
species of microbiological agents in a single gynecological swab
sample.
[0094] Sensitivity refers to a method's ability to detect very
minute amounts of a substance or organism. The frequency of a
positive test result in patients who have the disease the test is
designed to detect, is expressed mathematically as follows:
Sensitivity = True Positives .times. 100 True Positives + False
Negatives ##EQU00001##
[0095] Sensitivity studies were initially performed by purchasing
well-characterized, validated organisms from the American Tissue
Culture Collection (ATCC, Manassas, Va.). The DNA of the virus,
bacteria, or fungi is then extracted and quantitated. Standards of
known concentrations are used to determine the assay's ability to
detect varying concentrations of genetic material. The extracted
DNA is serially diluted to concentrations of 1:10, 1:100, 1:1,000
and 1:10,000. By evaluating the presence of bands in these
dilutions of known concentrations, the sensitivity of a particular
test can be established. For real-time PCR assays, the fluorescence
acquisition profile generated from the amplification of the serial
dilutions is analyzed. A region encompassing the genetic target of
the assay is generally subcloned into a vector system. Through
quantitation of the vector and the optimization of the assay as
described infra, as few as 10 genomic equivalents of the pathogen
can be reproducibly detected.
[0096] Specificity studies were used to assess the quality of the
primer selection for the assay by determining if their organisms'
DNA will cross-react in any way leading to false positives.
Initially as a theoretical test, primers are cross-referenced
against the billions of other genetic sequences which have been
deposited in the public databases by international researchers and
any potential conflicts are avoided. Next as an experimental
confirmation, the primers and probes are assayed for their
inability to amplify dozens of other known bacterial, viral, and
fungal organisms which have been identified as human pathogens. An
aliquot of the characterized positive control is also spiked in a
suspension consisting of the DNA of numerous other organisms to
ensure that the particular pathogen target genome is in no way
masked or inhibited by other genomic sequences.
[0097] Interference studies are used to determine if other
substances inherent to the specimen type will interfere with
detection by PCR. Certain effects, such as masking the organism's
target to produce a false negative, or cross-reactivity to produce
a false positive are analyzed. Characteristics, such as the
microcosm of normal flora of the genital tract, the abundance of
various proteins found in blood, and natural inhibitors commonly
found in other body fluids, such as urine, can all have detrimental
effects on the PCR process, unless accounted for during the initial
processing and extraction procedures.
[0098] Optimization studies are the final step of the validation
process. In these assays, the concentrations of various reagents
are varied such as template DNA, MgCl.sub.2, and primers, and
probes as well as the temperature and duration of each step of the
thermocycling parameters to improve the clarity of bands or the
intensity of signals, as well as eliminate streaks, multiple
banding, or haziness, which can impede the visualization of the PCR
products or interpretation of real-time PCR results. A method is
preferred wherein a progress of at least one reaction is optically
monitored by means of the system and/or wherein the presence or
absence of a product of at least one reaction is optically
determined and electronically recorded by the system.
ADDITIONAL REFERENCES
[0099] 1. Adelson et al., 2005, Simultaneous detection of herpes
simplex virus types 1 and 2 by real-time PCR and pyrosequencing.
Journal of Clinical Virology 33:25-34. (manuscript published online
on Nov. 14, 2004). [0100] 2. Trama et al., 2005, Detection of
Candida species in vaginal samples in a clinical.cndot.laboratory
setting. Infectious Diseases in Obstetrics and Gynecology
13(2):63-67. [0101] 3. Trama et al., 2005, Detection and
identification of Candida species associated with Candida vaginitis
by real-time PCR and pyrosequencing. Molecular and Cellular Probes
19(2):145-152. [0102] 4. Trama et al. Analyzing Candida albicans
gene mutations that contribute to azole resistance by
pyrosequencing. American College of Obstetricians and Gynecologists
52nd Annual Clinical Meeting, May 1-5, 2004, Philadelphia, Pa.
[0103] 5. Trama et al. Novel technique for identification of
vulvovaginal candidiasis by real-time PCR and pyrosequencing.
American College of Obstetricians and Gynecologists 52'1 Annual
Clinical Meeting, May 1-5, 2004, Philadelphia, Pa. [0104] 6.
Adelson et al., Diagnosis of Neisseria gonorrhea, Chlamydia
trachomatis, and Trichomonas vaginalis by real-time PCR. American
College of Obstetricians and Gynecologists 52nd Annual Clinical
Meeting, May 1-5, 2004, Philadelphia, Pa. [0105] 7. Mordechai et
al., Prevalency of Candida species associated with Candida
vaginitis in the United States. American Society of Microbiology
104th General Meeting, May 23-27, 2004, New Orleans, La., Poster
C-108. [0106] 8. Adelson et al., Development of a real-time PCR
assay for the simultaneous detection of herpes simplex virus types
1 and 2 with confirmation by pyrosequencing technology. American
Society of Microbiology 104th General Meeting, May 23-27, 2004, New
Orleans, La., Poster C-273. [0107] 9. Naurath et al., Detection and
quantification of Gardnerella vaginalis by real-time PCR. American
College of Obstetricians and Gynecologists 53rd Annual Clinical
Meeting. May 7-11, 2005, San Francisco, Calif. [0108] 10. Trama et
al, Detection of molluscum contagiosum virus by real-time PCR and
pyrosequencing. American Society of Microbiology 105th General
Meeting, Jun. 5-9, 2005, Atlanta, Ga. [0109] 11. Feola et al.,
Detection of Ureaplasma urealyticum, Mycoplasma hominis, an
Mycoplasma genitalium by real-time PCR and pyrosequencing. American
Society of Microbiology 105th General Meeting, Jun. 5-9, 2005,
Atlanta, Ga. [0110] 12. Gygax et al., Erythromycin and clindamycin
resistance in Group B Streptococcal clinical isolates. Presented by
Dr. Martin E. Adelson at the 45th ICAAC (Interscience Conference on
Antimicrobial Agents and Chemotherapy) Meeting in Washington D.C.
on Dec. 16, 2005. [0111] 13. Adelson et al., Evaluation of UTM-RT
for the molecular detection of a plurality of OB/GYN related
pathogens. Presented by Dr. Martin E. Adelson at the 45th ICAAC
(Interscience Conference on Antimicrobial Agents and Chemotherapy)
Meeting in Washington D.C. on Dec. 17, 2005.
EXAMPLES
Example I
Validation Studies
[0112] To determine if Copan UTM-RT media is suitable for the
molecular amplifications diagnostic testing, the following
pathogens were purchased from ATCC and detection assays were
performed:
TABLE-US-00001 TABLE 1 Pathogen ATCC Catalogue Number 1 Bacteroides
fragilis 23745 2 Candida albicans 18804 3 Candida glabrata 2001 4
Candida parapsilosis 10233 5 Candida tropicalis 13803 6 Chlamydia
trachomatis VR-901B 7 Gardnerella vaginalis 14018 8 Haemophilis
ducreyi 27721 9 Herpes Simples Virus-1 VR-1544 10 Herpes Simples
Virus-2 VR-734 11 Mobiluncus mulieris 35243 12 Mycoplasma hominis
14027 13 Neisseria gonorrhoeae 27628 14 Trichomonas vaginalis 30246
15 Ureaplasma urealyticum 27618
Simulation of a Positive Clinical Specimen
[0113] Pathogens were purchased from ATCC in a lyophilized pellet
form. Each pellet was dissolved in five ml of TE-buffer (10 mM
Tris, pH 7.5, and 1 mM EDTA) in case of bacteria or yeast liquid
media (10 g of yeast extract, 20 g of peptone dissolved in 1 L of
distilled water, pH 7) in case of fungi. Virus cultures were
purchased from ATCC as two ml liquid cultures.
Dilutions were subsequently prepared as follows:
TABLE-US-00002 TABLE 2 Concentration (Designation) 1:1 (A) 1:10 (B)
1:100 (C) Original Resuspension 600 .mu.l 60 .mu.I 6 Ill TE Buffer
(Bacteria, Virus) or 0 .mu.I 540 .mu.I 594 .mu.I Yeast Liquid Media
(Fungi)
[0114] DNA was extracted from 500 .mu.I of A, B, and C dilutions
using standard laboratory phenollchloroform/ethanol precipitation
protocols. For positive controls, pathogen-positive clinical
specimens were identified from the initial laboratory diagnostic
tests and 500 .mu.l of the corresponding original cervical swab
media specimen was extracted. Previously validated real-time PCRs
for each set of pathogens was performed on DNA extracted from
Dilutions A, B, and C as well as the clinical samples. Rotor-Gene
software calculated CT values for the three ATCC dilutions and the
clinical specimens (Rotor-Gene 3000 instrument). The CT values of
the dilutions were compared with that obtained for the clinical
specimens and a "simulated dilution" was extrapolated for the
subsequent studies of the Copan UTM-RT transport medium. Based upon
these studies, the following was selected:
TABLE-US-00003 TABLE 3 TE Buffer ATCC (Bacteria, Resuspension
Virus) or Yeast Overall ATCC used in this Liquid Media Dilution
Resuspension experiment (Fungi) of pellet Bacteria 5 ml TE buffer 5
.mu.I 245 .mu.I 1:250 added to pellet Fungi 5 ml TE buffer 5 .mu.I
245 .mu.I 1:250 added to pellet Viruses 2 ml culture from 2 .mu.I
198 .mu.I 1:100 ATCC
[0115] The Applicants' studies suggest that spiking an ATCC culture
(pellet suspended in 5 ml of medium or buffer) diluted at 1:50
simulates the concentration of bacterial and fungal pathogens
(i.e., 250-fold dilution of ATCC culture) and 1:100 dilution
simulates the viral pathogen (100-fold dilution of ATCC culture) in
the clinical sample.
Studying the Stability of the Pathogen
[0116] For validation studies, Copan UTM-RT transport medium (Lot #
A 303CS02) as provided by the manufacturer was pooled in a sterile
bottle. Based upon the simulated dilutions described above for each
pathogen, the following cocktails were prepared:
TABLE-US-00004 TABLE 4 Per vial (A, B, & C) .mu.l Copan
Cocktail Pathogens .mu.I Pathogen* UTM-RT 1 Candida albicans 80
.mu.l 3840 .mu.l Neisseria gonorrhoeae 80 .mu.I 2 Candida
parapsilosis 80 .mu.l 3800 .mu.l Chlamydia trachomatis 80 .mu.I
Herpes Simplex Virus-1 40 .mu.I 3 Candida glabrata 80 .mu.I 3800
.mu.l Herpes Simplex Virus-2 40 .mu.l Trichomonas vaginalis 80
.mu.I 4 Candida tropicalis 80 .mu.l 3760 .mu.l Mobiluncus mulieris
80 .mu.I Ureaplasma urealyticum 80 .mu.I 5 Bacteriodes fragilis 80
.mu.I 3840 .mu.l Mycoplasma hominis 80 .mu.I 6 Gardnerella
vaginalis 80 .mu.I 38400 Haemophilis ducreyi 80 .mu.I *Dilution
prepared for each pathogen as detailed in Table 4.
[0117] Each cocktail was prepared in triplicate (15 ml tubes) and
designated A. B, or C. Pathogen culture solution was added to
obtain desired concentration which mimics the pathogen load in a
positive clinical sample (1:250-fold dilution for ATCC bacterial
and fungal culture and 1:100-fold dilution for ATCC virus culture).
500 .mu.l of the above mix was transferred to three separate
microcentrifuge tubes labeled Day 0 to 5.
[0118] Inoculated media vials of each cocktail were incubated at
room temperature. At 24 hour intervals starting with Day 0 through
Day 5, three microcentrifuge tubes were transferred to -20.degree.
C. storage. Aliquots from each vial were extracted for DNA by
standard laboratory procedures after Day 5. Conventional and
real-time PCR reactions for each pathogen on the appropriate
cocktail followed. The summary of results is as follows:
TABLE-US-00005 TABLE 5 # Positive Time Pts./ Pathogen Type of PCR #
Specimens Tested 1 Bacteriodes fragilis Conventional 18/18 (see
FIG. 2) PCR 2 Candida albicans Real-time PCR 18/18 (see FIG. 4) 3
Candida glabrata Real-time PCR 14/18 (see FIG. 5) 4 Candida
parapsilosis Real-time PCR 18/18 (see FIG. 6) 5 Candida tropicalis
Real-time PCR 18/18 (see FIG. 7) 6 Chlamydia trachomatis Real-time
PCR 18/18 (see FIG. 8) 7 Gardnerella vaginalis Real-time PCR 18/18
(see FIG. 9) 8 Haemophilis ducreyi Real-time PCR 18/18 (see FIG.
10) 9 Herpes Simples Virus-1 Real-time PCR 18/18 (see FIG. 11) 10
Herpes Simples Virus-2 Real-time PCR 18/18 (see FIG. 12) 11
Mobiluncus mulieris Conventional 18/18 (see FIG. 3) PCR 12
Mycoplasma hominis Conventional 6/6 PCR 13 Neisseria gonorrhoeae
Real-time PCR 18/18 14 Trichomonas vaginalis Real-time PCR 18/18
(see FIG. 13) 15 Ureaplasma urealyticum Real-time PCR 18/18 (see
FIG. 14)
Example II
DNA Extraction from Transport Media
[0119] For DNA extraction, see, e.g., Goessens et al., 1995,
Influence of volume of sample processed on detection of Chlamydia
trachomatis in urogenital samples by PCR. Journal of Clinical
Microbiology 33:251-253.
[0120] The following steps outline the procedure to isolate and
purify DNA from transport media. The specimen is submitted as a
self-contained unit with transport media.
Proteinase K: 100 .mu.l Tris (pH 7.5), 4.9 ml ddH.sub.2O, 5 ml
Glycerol. Dissolve well and store at -20.degree. C. as 500 .mu.I
aliquots. 10% SDS: 10 g SDS in 100 ml of ddH.sub.20.
Equipment:
[0121] Disposable pipette tips Disposable transfer pipette
Laboratory timer 1.5 ml microcentrifuge tube 55.degree. C. water
bath Pipettes to deliver a range of 1-1000 .mu.l
Procedure:
[0122] 1. Mix the swab thoroughly in the transport media. 2.
Pipette 470 .mu.l of transport media into a labeled microcentrifuge
tube. 3. Add 25 .mu.l of 10% SDS and 12 .mu.l of Proteinase K. Mix
well. 4. Incubate for 2 hours in 55.degree. C. water bath. 5. After
2 hours, place 200 .mu.l of Tris saturated phenol and 200 .mu.l of
chloroform:isoamyl alcohol (24:1) in the tube. Shake the tube to
mix the layers. 6. Centrifuge at 14,000 rpm for 5 minutes at room
temperature. This will separate the layers. 7. Remove the top
chloroform layer (containing the DNA) being careful not to pipette
any of the bottom or middle layers. Place this into another labeled
microcentrifuge tube. The first tube containing the remaining
layers may be discarded. 8. To this new tube add 0.1.times. volume
of 3 M sodium acetate. Also add 2.times. volumes of cold 100%
ethanol. Vortex and place in -20.degree. C. overnight. 9.
Centrifuge the tube at 14,000 rpm at 4.degree. C. for 10 minutes.
This will pellet the precipitated 20 DNA. 10. Remove and discard
the supernatant. Add 1000 .mu.l of 70% ethanol to wash the pellet.
Slightly resuspend the pellet. 11. Centrifuge the tube again at
14,000 rpm at 4.degree. C. for 5 minutes to form a pellet. 12.
Place the tube with the top open into the CentriVap (Labconco,
Kansas City, Mo.). Spin at 35.degree. C. for approximately 15
minutes. Spin until the pellet is dry, being very careful not to
overdry. 13. Resuspend the pellet in 20 Al of ddH.sub.2O. 14.
Quantitate the DNA using a spectrophotometer.
[0123] All publications and patents referred to herein are
incorporated by reference. Various modifications and variations of
the described subject matter will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific embodiments, it should be understood that the
invention as claimed should not be unduly limited to these
embodiments. Indeed, various modifications for carrying out the
invention are obvious to those skilled in the art and are intended
to be within the scope of the following claims.
* * * * *