U.S. patent application number 14/812128 was filed with the patent office on 2015-11-12 for primers for detecting plasmodium.
This patent application is currently assigned to OTSUKA PHARMACEUTICAL CO., LTD.. The applicant listed for this patent is Ehime University, OTSUKA PHARMACEUTICAL CO., LTD.. Invention is credited to Eun-Taek HAN, Takafumi TSUBOI.
Application Number | 20150322534 14/812128 |
Document ID | / |
Family ID | 39683432 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322534 |
Kind Code |
A1 |
TSUBOI; Takafumi ; et
al. |
November 12, 2015 |
PRIMERS FOR DETECTING PLASMODIUM
Abstract
The present invention provides an easy and rapid method for
detecting/identifying the presence or absence of specific
Plasmodium parasites and four species of malaria parasites in a
human specimen, an anti-malaria measure support system, and a
malaria infection-prevention/treatment system, which can contribute
to practical diagnosis in a malaria endemic area. According to the
present invention, using a genus-specific primer set that can
detect four Plasmodium parasites that infect humans at a time, and
the primer sets each specific to each of four species of Plasmodium
parasites (P. falciparum, P. vivax, P. malariae, and P. ovale), the
presence or absence of infection with these parasites can be
detected/identified easily and rapidly.
Inventors: |
TSUBOI; Takafumi;
(Matsuyama-shi, JP) ; HAN; Eun-Taek;
(Chuncheon-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ehime University
OTSUKA PHARMACEUTICAL CO., LTD. |
Ehime
Tokyo |
|
JP
JP |
|
|
Assignee: |
OTSUKA PHARMACEUTICAL CO.,
LTD.
Tokyo
JP
EHIME UNIVERSITY
Ehime
JP
|
Family ID: |
39683432 |
Appl. No.: |
14/812128 |
Filed: |
July 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14571888 |
Dec 16, 2014 |
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14812128 |
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13495736 |
Jun 13, 2012 |
8936920 |
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14571888 |
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12602113 |
Feb 26, 2010 |
8309702 |
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PCT/JP08/60115 |
May 27, 2008 |
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13495736 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 33/02 20180101; C12Q 1/6893 20130101; A61P 33/06 20180101;
C12Q 2600/158 20130101; Y02A 50/58 20180101; A61P 37/04 20180101;
C12Q 2600/16 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2007 |
JP |
2007-140525 |
Claims
1-27. (canceled)
28. A method of detecting Plasmodium ovale in a biological sample
with a malarial infection, the method comprising: (a) extracting
DNA from the biological sample; (b) amplifying a region of a
Plasmodium 18S rRNA gene sequence by reacting the DNA extracted in
step (a) in a reaction mixture containing a strand displacement DNA
polymerase and a sequence-specific primer set which is an
oligonucleotide set containing nucleic acid sequences represented
by SEQ ID NOs: 25 to 30; and (c) detecting the presence or absence
of an amplified product of step (b); wherein the presence of said
amplification product is indicative of the presence of Plasmodium
ovale in the biological sample.
29. The method according to claim 28, wherein the DNA extraction
from the specimen in step (b) is carried out by boiling the
specimen containing the DNA, and performing centrifugation.
30. The method according to claim 29, wherein the boiling time is
up to ten minutes.
31. The method according to claim 28, wherein the amplification in
step (b) of the region of Plasmodium 18S rRNA gene sequence is
performed at about 60.degree. C. for about 1 hour using a
constant-temperature water bath or an amplifier specially designed
for loop-mediated isothermal amplification (LAMP).
32. The method according to claim 28, wherein the presence or
absence of the amplification product is identified by visual
observation of turbidity or a real-time turbidimeter.
33. The method according to claim 28, which is performed in a
malaria endemic area.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a Divisional of application Ser. No. 12/602,113
filed Feb. 26, 2010 (now U.S. Pat. No. 8,309,702), which is a
National Stage of International Application No. PCT/JP2008/060115
filed May 27, 2008, claiming priority based on Japanese Patent
Application No. 2007-140525, filed May 28, 2007, the contents of
all of which are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to a primer set capable of the
rapid and accurate detection/identification of malaria parasites of
the genus Plasmodium in malaria-endemic areas, a method for
detecting and identifying thereof, a detection kit thereof, an
anti-malaria measure support system, and a malaria
infection-prevention/treatment measure system.
BACKGROUND ART
[0003] In many countries where malaria parasites are endemic, the
rapid and accurate diagnosis of malaria parasites presents
challenges. Of four Plasmodium species, P. falciparum, which can be
fatal, must be identified promptly and distinguished from the other
Plasmodium species that produce the disease in humans (Moody, A.,
Clin. Microbiol. Rev. 15 (2002): 66-78).
[0004] In addition, most malaria-endemic areas feature infections
involving two or more of these species; these mixed infections
often go unrecognized or underestimated (Zimmerman, P. A., et al.,
Trends Parasitol. 20 (2004): 440-447). Failure to detect mixed
infection could result in inadequate treatment, and may result in
severe disease (Mayxay, M., et al., Trends Parasitol. 20 (2004):
233-240). There is, therefore, an urgent need to develop malaria
diagnostic methods that are operable in endemic areas, easy, rapid,
highly sensitive and species-specific.
[0005] Currently the easy diagnostic method for malaria is
microscopic examination of blood smears. Given a high density of
parasites, such microscopy has relatively high sensitivity and
specificity and provides developmental stage and species
determination. However, in endemic areas where parasite density is
generally low, this method is labor-intensive, requires
well-trained experts, and may result in therapy being delayed.
[0006] To improve the speed and precision of malaria diagnosis in
regions where standard laboratory diagnosis is not available,
researchers have developed rapid diagnostic tests (RDTs) for
malaria based on immunoreaction (Moody, A. Clin. Microbiol. Rev. 15
(2002): 66-78; Ndao, M., et al., J. Clin. Microbiol. 42 (2004):
2694-2700). However, the sensitivity varies between products
(Murray, C. K., et al., Trop. Med. Int. Health. 8 (2003): 876-883),
and a species-specific product is available only for P. falciparum.
Very long observation times and considerable expertise are required
for correct diagnosis by microscopy under several circumstances:
when parasitemia is low, during mixed infection, after drug
treatment, and during the chronic phase of the infection.
Therefore, this situation can lead to false negative results or
unreliable species diagnosis (Coleman, R., et al., Thailand. Malar.
J. 14 (2006): 121).
[0007] Subsequently, molecular-biological methods based on DNA
amplification, such as nested PCR and real-time quantitative PCR,
were developed for malaria diagnosis. Compared to microscopy, these
methods have demonstrated higher sensitivity and greater
specificity for mixed infections (Kimura, K., et al., Parasitol.
Int. 46 (1997): 91-95; Perandin, F., et al., J. Clin. Microbiol. 42
(2004): 1214-1219; Rougemont, M., et al., J. Clin. Microbiol. 42
(2004): 5636-5643; Singh, B., et al., Am. J. Trop. Med. Hyg. 60
(1999): 687-692; Singh, B., et al., Lancet. 363 (2004): 1017-1024;
Snounou, G., et al., Mol. Biochem. Parasitol. 58 (1993): 283-292;
Snounou, G., et al., Mol. Biochem. Parasitol. 61 (1993): 315-320).
However, the long turnaround time, high cost, and availability only
in well-equipped laboratories render this PCR technology inadequate
for routine diagnosis in the hospital laboratories and on-site
clinics of endemic areas (Hanscheid, T., and Grobusch, M. P.,
Trends Parasitol. 18 (2002): 395-398).
[0008] Regarding malaria detection, Examples 8 and 10 in patent
document 1 disclose a method of extracting nucleic acids from blood
samples and conducting nested PCR to detect four species of
Plasmodium. Example 8 discloses each forward primer and the reverse
primer sequences, which are different from the primer sequences of
the present invention (patent document 1).
[0009] Patent documents 2 and 4 Patent Publication disclose methods
for detecting one or multiple species of malaria infection based on
a solid phase method or nested PCR, in which one or a plurality of
multiple types of primers are used to clinically detect P.
falciparum, P. vivax, P. malariae or P. ovale. However, those
primers have different primer sequences to those of the present
invention.
[0010] Patent documents 3 Patent Publication discloses a method for
detecting P. falciparum and/or P. vivax, in which P. falciparum
and/or P. vivax specific primers are bound to labelor solid
supports. However, these specific primer sequences are different
from the oligonucleotide sequences of the primer sets of the
present invention.
[0011] Recently, a novel, easy and highly sensitive technique
called loop-mediated isothermal amplification (LAMP) was developed
(Notomi, T., et al., Nucleic Acids Res. 28 (2000): e63; WO
2000/28082).
[0012] LAMP is a nucleic acid amplification method that relies on
auto-cycle strand-displacement DNA synthesis performed by Bst DNA
polymerase. The amplified products are stem-loop structures with
several repeated sequences of the target, and have multiple
loops.
[0013] The principal merit of this method is that denaturation of
the DNA template is not required, (Nagamine, K., et al., Clin.
Chem. 47 (2001): 1742-1743), and thus the LAMP reaction can be
conducted under isothermal conditions (ranging from 60 to
65.degree. C.) LAMP requires only one enzyme and four types of
primers that recognize six distinct target regions. The method
produces a large amount of amplified product, resulting in easier
detection, such as detection by visual judgment of the turbidity or
fluorescence of the reaction mixture (Mori, Y., et al., Biochem.
Biophys. Res. Commun. 289 (2001): 150-154). LAMP in which a
fluorescent substance such as fluorescein, fluorescein
isothiocyanate (FITC), X-rhodamine (ROX) or the like is used to
measure the fluorescence polarization values of the reaction
mixture, and LAMP in which SYBR Green 2, a green dye, is used as an
intercalator are known (Japanese Unexamined Patent Publication No.
2002-272475, and WO 2002/103053).
[0014] Several investigators have reported LAMP methods for the
rapid identification of Plasmodium, Trypanosoma, Babesia, Fusarium,
Listeria and Legionella, and have recommended the usefulness of
LAMP assay (Ikadai, H., et al., J. Clin. Microbiol. 42 (2004):
2465-2469; Kuboki, N., et al., J. Clin. Microbiol. 41 (2003):
5517-5524; Thekisoe, O., et al., Mol. Biochem. Parasitol. 122
(2002): 223-236; Japanese Unexamined Patent Publication No.
2005-245257, Japanese Unexamined Patent Publication No. 2007-61061,
Japanese Unexamined Patent Publication No. 2003-219878 and Poon,
L., et al., Clin. Chem. 52 (2006): 303-306).
[0015] Poon et al., estimated that the cost of running a LAMP assay
is about one tenth that of normal PCR for P. falciparum detection
(Poon, L., et al., Clin. Chem. 52 (2006): 303-306). The biggest
reduction in cost and time came from simple sample preparation
without previous DNA extraction (Iwasaki, M., et al., Genome Lett.
2 (2003): 119-126).
[0016] For the preparation of samples, simply heating the infected
blood at 99.degree. C. for 10 minutes was enough to prepare a DNA
template for LAMP (Poon, L., et al., Clin. Chem. 52 (2006):
303-306). However, to date, LAMP for the detection of malaria
parasites in clinical diagnosis has been validated only in acute P.
falciparum patients (Poon, L., et al., Clin. Chem. 52 (2006):
303-306). Although P. falciparum is the most important cause of
severe disease, its geographic distribution overlaps with those of
P. vivax, P. malariae and P. ovale infection, and therefore a
method allowing the rapid detection and identification of all four
species infecting humans would be desirable.
[0017] In recent years, in malaria-endemic areas, the development
of drug-resistant strains has been a major problem for appropriate
malaria treatment. Practicing medical personnel or hospital doctors
desire rapid and highly sensitive differentiation methods to obtain
information on whether a patient with a fever is infected with a
particular malaria parasite or multiple species of malaria
parasites, to thereby appropriately treat said malaria patient with
a fever. [0018] [Patent document 1] WO2006/88895 [0019] [Patent
document 2] Japanese Unexamined Patent Publication No. 1994-261758
[0020] [Patent document 3] Japanese Unexamined Patent Publication
No. 1993-227998 [0021] [Patent document 4] Japanese Unexamined
Patent Publication No. 2003-250564 [0022] [Non-patent document 1]
Poon, L., et al., Clin. Chem. 52 (2006): 303-306
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0023] To solve the above-mentioned problems, the development of
easy and rapid methods are desired for the detection and
identification of the four species of malaria parasites of the
genus Plasmodium (particularly, the presence of mixed infections),
which are different from known methods such as microscopy or
PCR-reaction-mediated methods.
[0024] An object of the present invention is to provide a rapid and
highly sensitive detection and identification method for clinically
detecting and specifying P. falciparum, P. vivax, P. malariae or P.
ovale using LAMP. Further, according to the malaria parasite
detection and identification method of the present invention, using
blood samples obtained at clinics in malaria-endemic areas, a
primer set for detecting the four species of malaria parasites of
the genus Plasmodium, where the primer set has been evaluated
through comparison of microscopy and LAMP; a detection method for
the four species of malaria parasites of the genus Plasmodium using
the primer set; an identification method; and a detection kit are
provided.
[0025] Therefore, an object of the present invention is to solve
the above-mentioned problems, and to provide an easy and rapid
detection/identification method for the presence or absence of
Plasmodium parasites or any one of the 4 specific species of
malaria parasites in human specimens, which is capable of
contributing to medical care in malaria-endemic areas, and further
to provide an anti-malaria measure support system and a malaria
infection-prevention/treatment measure system.
Means for Solving the Problems
[0026] The present inventors, in aiming to solve such problems,
conceived of using a Loop-mediated isothermal amplification (LAMP)
method, which is an isothermal gene amplification reaction: (WO
00/28082). However, in general, nucleic acid sequences of malaria
parasite genes differ greatly from those of other organisms, being
rich in AT content. Therefore, existing primer design software was
unable to find optimal primer sets, demanding a repetitive process
of trial and error with difficulties in designing each type of
primer. Finally, among the many combinations of synthetic primer
sets, particularly useful primer sets having both high sensitivity
and high specificity were found. Thus, it was found that the use of
genus-specific primer sets capable of detecting the four
human-infecting species of Plasmodium parasites at the same time
and primer sets each specific to the four species of parasites (P.
falciparum, P. vivax, P. malariae and P. ovale) allows the easy and
rapid detection/identification of the presence or absence of such
infections.
[0027] It was also found that these results can be effectively used
for anti-malaria measure support system and the malaria
infection-prevention/treatment measure system can be effectively
used.
[0028] That is, the present invention can provide the following as
described in Items 1 to 27 below.
[0029] Item 1. A method for detecting or identifying infection with
the genus Plasmodium and/or one or more of Plasmodium falciparum,
Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale in a
specimen; the method comprising the following steps (a) to (c):
a) extracting DNA from the specimen; b) amplifying a particular
region of a Plasmodium 18S rRNA gene sequence by reacting the DNA
extracted in the step (a) in a reaction mixture containing a strand
displacement DNA polymerase and a sequence-specific primer set; and
c) detecting or identifying the presence or absence of an amplified
product of the genus Plasmodium and/or one or more of Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium
ovale, amplified in the step (b);
[0030] the sequence-specific primer set being an oligonucleotide
set containing nucleic acid sequences represented by SEQ ID NOs: 1
to 6 for amplifying a particular region of a Plasmodium 18S rRNA
gene sequence; and/or
one or more of a primer set comprising an oligonucleotide set
containing nucleic acid sequences represented by SEQ ID NOs: 7 to
12 for amplifying a particular region of a Plasmodium falciparum
18S rRNA gene sequence, a primer set comprising an oligonucleotide
set containing nucleic acid sequences represented by SEQ ID NOs: 13
to 18, SEQ ID NOs: 31 to 36, or SEQ ID NOs: 37 to 42 for amplifying
a particular region of a Plasmodium vivax 18S rRNA gene sequence, a
primer set comprising an oligonucleotide set containing nucleic
acid sequences represented by SEQ ID NOs: 19 to 24 for amplifying a
particular region of a Plasmodium malariae 18S rRNA gene sequence,
and a primer set comprising an oligonucleotide set containing
nucleic acid sequences represented by SEQ ID NOs: 25 to 30 for
amplifying a particular region of a Plasmodium ovale 18S rRNA gene
sequence.
[0031] Item 2. A detection or identification method according to
Item 1, wherein the DNA extraction from the specimen is carried out
by boiling the specimen containing the DNA, and performing
centrifugation.
[0032] Item 3. A detection or identification method according to
Item 2, wherein the boiling time is from several minutes to ten and
several minutes.
[0033] Item 4. A detection or identification method according to
any one of Items 1 to 3, wherein, in the step (b) of amplifying a
particular region of a Plasmodium 18S rRNA gene sequence, the DNA
amplification reaction is performed at about 60.degree. C. for
about 1 hour using a constant-temperature water bath or an
amplifier specially designed for LAMP.
[0034] Item 5. A detection or identification method according to
any one of Items 1 to 4, wherein, in the step (C), the presence or
absence of an amplification product of the genus Plasmodium, and/or
one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium
malariae, and Plasmodium ovale is detected or identified using
visual observation or a real-time turbidimeter.
[0035] Item 6. A detection or identification method according to
any one of Items 1 to 5, which is performed in a malaria endemic
area.
[0036] Item 7. A detection or identification method according to
any one of Items 1 to 6, wherein infections with the genus
Plasmodium and/or one or more of Plasmodium falciparum, Plasmodium
vivax, Plasmodium malariae, and Plasmodium ovale are detected or
identified simultaneously or separately.
[0037] Item 8. A detection or identification method according to
any one of Items 1 to 6, wherein infection with the genus
Plasmodium is detected or identified using, as the
sequence-specific primer set, a primer set comprising an
oligonucleotide set containing nucleic acid sequences represented
by SEQ ID NOs: 1 to 6 for amplifying a particular region of a
Plasmodium 18S rRNA gene sequence.
[0038] Item 9. A detection or identification method according to
any one of Items 1 to 6, wherein infection with Plasmodium vivax is
detected or identified using, as the sequence-specific primer set,
a primer set for detecting Plasmodium vivax that comprises an
oligonucleotide set containing nucleic acid sequences represented
by SEQ ID NOs: 13 to 18, SEQ ID Nos: 31 to 36, or SEQ ID Nos: 37 to
42 and that is capable of amplifying a particular region of a
Plasmodium vivax 18S rRNA gene sequence.
[0039] Item 10. A detection or identification method according to
any one of Items 1 to 6, wherein infection with Plasmodium malariae
is detected or identified using, as the sequence-specific primer
set, a primer set for detecting Plasmodium malariae that comprises
an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 19 to 24 and that is capable of
amplifying a particular region of a Plasmodium malariae 18S rRNA
gene sequence.
[0040] Item 11. A detection or identification method according to
any one of Items 1 to 6, wherein infection with Plasmodium ovale is
detected or identified using, as the sequence-specific primer set,
a primer set for Plasmodium ovale that comprises an oligonucleotide
set containing nucleic acid sequences represented by SEQ ID NOs: 25
to 30 and that is capable of amplifying a particular region of a
Plasmodium ovale 18S rRNA gene sequence.
[0041] Item 12. A primer set for detecting the genus Plasmodium,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 1 to 6, the primer set being capable of
amplifying a particular region of a Plasmodium 18S rRNA gene
sequence.
[0042] Item 13. A primer set for detecting Plasmodium vivax,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 13 to 18, SEQ ID NOs: 31 to 36, or SEQ
ID NOs: 37 to 42, the primer set being capable of amplifying a
particular region of a Plasmodium vivax 18S rRNA gene sequence.
[0043] Item 14. A primer set for detecting Plasmodium malariae,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 19 to 24, the primer set being capable
of amplifying a particular region of a Plasmodium malariae 18S rRNA
gene sequence.
[0044] Item 15. A primer set for detecting Plasmodium ovale,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 25 to 30, the primer set being capable
of detecting a particular region of a Plasmodium ovale 18S rRNA
gene sequence.
[0045] Item 16. A primer set for detecting the genus Plasmodium
and/or any one of P. falciparum, P. vivax, P. malariae, and P.
ovale, comprising an oligonucleotide primer set containing nucleic
acid sequences selected from those defined in Items 12 to 15 and
nucleic acid sequences represented by SEQ ID NOs: 7 to 12, the
primer set being capable of amplifying particular regions of 18S
rRNA gene sequences of the genus Plasmodium and various species of
Plasmodium including a particular region of a Plasmodium falciparum
18S rRNA gene sequence.
[0046] Item 17. A detection kit for the genus Plasmodium and/or any
one of P. falciparum, P. vivax, P. malariae, and P. ovale;
comprising at least a primer set selected from the primer sets
defined in Items 12 to 16, a strand displacement DNA polymerase,
dNTPs and a reaction buffer.
[0047] Item 18. A detection kit according to Item 17, wherein the
detection kit detects the genus Plasmodium and/or P. falciparum, P.
vivax, P. malariae, and P. ovale, simultaneously or separately.
[0048] Item 19. An anti-malaria measure support system
comprising:
[0049] means for inputting and storing malaria-infected patient
information including the number positive for the genus Plasmodium
parasites that cause malaria in a malaria endemic area, and the
carrier rate in the area;
[0050] a malaria infection-prevention public health measure guide
database that specifies public health measures for the malaria
endemic area based on the malaria-infected patient information,
into which database public health measure selection information for
a malaria parasite-infected area to be inputted together with the
measure priority indicating which of the public health measures
should be given priority in selection has been inputted;
[0051] a public health measure extraction section that extracts
public health measures for the malaria-infected endemic area and
the priority thereof from the malaria infection-prevention public
health measure guide database, according to malaria-infected
patient information about malaria parasites in a subject; and
[0052] a public health measure display section that displays the
public health measures extracted in the public health measure
extraction section, together with the priority thereof.
[0053] Item 20. An anti-malaria measure support system according to
Item 19, wherein the malaria-infected patient information about
malaria parasites in the malaria endemic area is obtained by
identifying the presence or absence of infection with the genus
Plasmodium using a detection or identification method according to
Item 1 and/or a primer set according to Item 12, or the Plasmodium
detection kit according to Item 17.
[0054] Item 21. An anti-malaria measure support system
comprising:
[0055] means for inputting and storing malaria parasite therapeutic
agent information for specifying a malaria therapeutic agent that
acts on infection with one or a plurality of four species of
malaria parasites;
[0056] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria endemic
area;
[0057] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria
non-endemic area;
[0058] a treatment guide database into which, according to indices
of efficacy against malaria parasites detected in a subject,
malaria parasite therapeutic agent selection information to be
inputted together with a priority that indicates which malaria
therapeutic agent should be given priority in selection for use
against the malaria parasites;
[0059] a malaria therapeutic agent extraction section that
extracts, according to the information about the pathogen of
malaria infection in the subject, malaria therapeutic agents to be
administered and the priority thereof, from the treatment guide
database; and
[0060] a malaria therapeutic agent display section that displays
the malaria therapeutic agents extracted in the above malaria
therapeutic agent extraction section, together with the priority
thereof.
[0061] Item 22. An anti-malaria measure support system according to
Item 21, wherein the information about the pathogen of malaria
infection in a patient with a fever is obtained by identifying
infection with one or a plurality of four species of malaria
parasites using a detection or identification method according to
any one of Items 1 to 11 and/or a primer set according to any one
of Items 12 to 16, or a detection kit according to Item 17 or
18.
[0062] Item 23. An anti-malaria measure support system in which a
public health measure relating to an anti-malaria measure and a
malaria parasite treatment measure relating to an anti-malaria
measure are carried out in combination;
[0063] the public health measure comprising:
[0064] means for inputting and storing malaria-infected patient
information including the number positive for the genus Plasmodium
that causes malaria in a malaria endemic area, and the carrier rate
in the area;
[0065] a malaria infection-prevention public health measure guide
database that specifies public health measures for the malaria
endemic area based on the malaria-infected patient information,
into which database public health measure selection information for
a malaria parasite-infected area to be inputted together with a
priority of measures that indicates which of the public health
measures should be given priority in selection has been
inputted;
[0066] a public health measure extraction section that extracts
public health measures for the malaria-infected endemic area and
the priority thereof, from the malaria infection-prevention public
health measure guide database according to malaria-infected patient
information about malaria parasites in the subject; and
[0067] a public health measure display section that displays the
public health measures extracted in the public health measure
extraction section, together with the priority thereof; and
[0068] the malaria parasite treatment measure comprising:
[0069] means for inputting and storing malaria parasite therapeutic
agent information for specifying a malaria therapeutic agent that
acts on infection with one or a plurality of four species of
malaria parasites;
[0070] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in the malaria endemic
area;
[0071] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria
non-endemic area;
[0072] a treatment guide database into which, according to indices
of efficacy against malaria parasites detected in a specimen,
malaria parasite therapeutic agent selection information to be
inputted together with a priority that indicates which malaria
therapeutic agent should be given priority in selection for use
against the malaria parasites;
[0073] a malaria therapeutic agent extraction section that
extracts, according to the information about the pathogen of
malaria infection in the subject, malaria therapeutic agents to be
administered and the priority thereof, from the treatment guide
database; and
[0074] a malaria therapeutic agent display section that displays
the malaria therapeutic agents extracted in the above malaria
therapeutic agent extraction section, together with the priority
thereof.
[0075] Item 24. An anti-malaria measure support system according to
Item 23, wherein the malaria-infected patient information about
malaria parasites in the malaria endemic area is obtained by
identifying the presence or absence of infection with the genus
Plasmodium using a detection or identification method according to
Item 1 and/or a primer set according to Item 12, or the genus
Plasmodium detection kit according to Item 17 or 18; and/or
information about the pathogen of malaria infection in a patient
with a fever is obtained by identifying infection with one or a
plurality of four species of malaria parasites using a detection or
identification method according to any one of Items 1 to 11 and/or
a primer set according to Item 16, or a Plasmodium species
detection kit according to Item 17 or 18.
[0076] Item 25. A malaria infection-prevention measure system for
persons who plan to travel to a malaria endemic area, the system
comprising:
[0077] means for obtaining the state of the implementation of
public health measures in the malaria endemic area, information
about the pathogens of malaria infection in the endemic area, and
the state of the treatment of infected patients, from the
anti-malaria measure support system according to Item 23;
[0078] means for selecting a malaria prophylactic/therapeutic agent
from a malaria parasite treatment guide database; and
[0079] means for administering the selected agent before
travel.
[0080] Item 26. A malaria infection-prevention/treatment measure
system for returnees from a malaria endemic area, the system
comprising:
[0081] means for obtaining the state of the implementation of
public health measures in the malaria endemic area, information
about the pathogens of malaria infection in the endemic area, and
the state of the treatment of infected patients;
[0082] means for selecting a malaria prophylactic/therapeutic agent
from a malaria parasite treatment guide database; and
[0083] means for administering the selected agent after returning
from the malaria endemic area, according to Item 23.
[0084] Item 27. A malaria infection-prevention/treatment measure
system according to Item 26, wherein, when a returnee from the
malaria endemic area has a fever, identification of Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium
ovale is performed to select and administer a malaria therapeutic
agent that should be given priority in selection.
[0085] Use of one of the primers or primer set of the above Items
(1) to (5) for LAMP allows the annealing of a particular region of
the genus Plasmodium 18S rRNA gene, a P. falciparum 18S rRNA gene,
a P. vivax 18S rRNA gene, a P. malariae 18S rRNA gene or a P. ovale
18S rRNA gene. Amplifying this under the amplification conditions
of the LAMP method allows amplification of a specific gene region.
The presence or absence of such an amplification product is
analyzed by electrophoresis or an easy detection method. In such a
method, infections by the specific genus Plasmodium and each of the
four species of malaria parasites can be simultaneously or
separately detected and differentiated.
[0086] When the detection method of the present invention is used
for specific specimens (for example, human blood), DNA samples can
be isolated from the specimens, with these DNA samples, any one of
the primer sets of Item (1) to Item (5) were reacted for
amplification, thereby confirming the presence or absence of any
amplified DNA products. In such a way, any of the genus Plasmodium
or the four species of malaria parasites: P. falciparum, P. vivax,
P. malariae or P. ovale can be detected simultaneously or
separately.
Effects of the Invention
[0087] The present invention allows the use of a primer set for
LAMP, wherein the primer set comprises an oligonucleotide set
containing the nucleic acid sequences of SEQ ID NOs: 1 to 6, 7 to
12, 13 to 18 (31 to 36 or 37 to 42), 19 to 24 or 25 to 30; and
allows the simultaneous or separate amplification of a common
region of the genus Plasmodium 18S rRNA genes, or a particular
region of the 18S rRNA genes of each of the four species of human
malaria parasites: P. falciparum, P. vivax, P. malariae or P.
ovale; thereby allowing simultaneous or separate detection or
differentiation of the presence or absence of any human malaria
parasite infection or one of the four species of human malaria
parasites.
[0088] The method for detecting or differentiating the presence or
absence of malaria parasite infection or one of the four species of
human malaria parasites of the present invention comprises:
amplifying DNA samples obtained from specimens by LAMP (isothermal
gene amplification) using a primer set comprising the
oligonucleotide set containing the nucleic acid sequences of SEQ ID
NOs: 1 to 6, 7 to 12, 13 to 18 (31 to 36 or 37 to 42), 19 to 24 or
25 to 30; and analyzing the presence or absence of any
amplification products. Such a detection or differentiation method
allows easy, rapid and reliable detection or differentiation of the
presence or absence of any malaria parasite infections, or one of
the four species of malaria parasites: P. falciparum, P. vivax, P.
malariae or P. ovale simultaneously or separately. The present
invention also provides a kit for the simultaneous or separate
detection or identification of such human malaria parasites or the
four species of malaria parasites.
[0089] The development of drug-resistant strains has become a major
issue for appropriately treating malaria. The method for
simultaneously differentiating the four species of malaria
parasites: P. falciparum, P. vivax, P. malariae and P. ovale of the
present invention provides, in malaria-endemic regions, practicing
medical personnel or hospital doctors with rapid and highly
sensitive information on whether a patient with a fever is infected
with a particular malaria parasite or multiple malaria parasites,
allowing rapid and appropriate treatment for a malaria patient with
a fever.
[0090] The use of the genus Plasmodium or the four species of
malaria parasite detection/identification method of the present
invention makes it possible to monitor the therapeutic effects of
malaria therapeutic agent administration to malaria-infected
patients.
[0091] According to the anti-malaria measure support system of the
present invention, clinical practitioners and hospital doctors can
check information about the pathogen of malaria infection in a
patient with a fever against the malaria therapeutic agent guide
database in a PC or via a cellular phone to have a conventional
software to operate, thereby obtaining a display of which malaria
therapeutic agent should be given priority in selection, or which
malaria therapeutic agents should be used in combination.
[0092] Using the malaria infection-prevention measure system of the
present invention, it is possible for a person who plan to travel
to a malaria endemic area to know beforehand the malaria infections
that are endemic in the area and the state of appearance of
drug-resistant strains. Thus, to prevent malaria infection, the
person can take, before travel, a preferable malaria therapeutic
agent to be given priority in selection for use against the malaria
infections that are endemic in the area, so that even if the person
should be infected with malaria, symptoms due to malaria infection,
such as a fever, can be reduced at an early stage and serious
conditions can be prevented, making it possible to exterminate
malaria parasites from the person's blood at an early stage.
BRIEF DESCRIPTION OF THE DRAWING
[0093] FIG. 1 shows the locations of LAMP targets and priming sites
for Plasmodium genus (A) and four Plasmodium species (B), and the
18S rRNA gene nucleotide sequences. (A) The locations of the
priming sites by the Plasmodium genus-specific primer set in the
reference sequence (GenBank Accession No. M19173.1) are indicated
by arrows. (B) Partial sequence alignment of the 18S rRNA genes of
four human malaria parasites, P. falciparum (Pf; GenBank Accession
No. M19173.1), P. vivax (Pv; GenBank Accession no. U03079), P.
malariae (Pm; GenBank Accession No. M54897), and P. ovale (Po;
GenBank Accession No. L48986), along with the species-specific
primer annealing sites.
[0094] FIG. 2 is a schematic diagram of the anti-malaria measure
support system using LAMP.
[0095] FIG. 3 is a flow chart of the process of the anti-malaria
measure support system based on the principle of the present
invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0096] Preferred embodiments of the present invention are explained
in detail below.
[0097] The present invention is an easy and highly reliable
detection system for routine malaria parasite screening, both at
hospitals and laboratories, or at malaria clinics in endemic areas.
For example, the genus Plasmodium or any one of the four species of
malaria parasites: P. falciparum, P. vivax, P. malariae or P. ovale
when present in human blood samples are determined by an isothermal
gene amplification method that employs LAMP using oligonucleotide
primers specific to the genus Plasmodium and each one of the four
species of malaria parasites. Specifically, the invention is based
on a method comprising the steps of targeting a particular region
of the genus specific Plasmodium 18S rRNA gene sequence, the P.
falciparum 18S rRNA gene sequence, P. vivax 18S rRNA gene sequence,
P. malariae 18S rRNA gene sequence, or P. ovale 18S rRNA gene
sequence; amplifying the particular region of the Plasmodium 18S
rRNA gene sequence, the P. falciparum 18S rRNA gene sequence, P.
vivax 18S rRNA gene sequence, P. malariae 18S rRNA gene sequence or
P. ovale 18S rRNA gene sequence using the primer set of Item 16;
and analyzing the presence or absence of any amplification
products.
[0098] The detection/identification method of the present invention
can be applied to malaria parasites present in human blood that is
infected with malaria parasites.
[0099] As used herein, the term "specimen" may include the genus
Plasmodium or one of the four specific species of malaria
parasites: P. falciparum, P. vivax, P. malariae or P. ovale,
implying human blood samples to be targeted by the
detection/identification method of the present invention. As used
herein, the term "detection" implies, for example, determining
whether malaria parasites present in a blood sample are malaria
parasites of a specific Plasmodium species, and is sometimes used
as a synonym for determination.
[0100] As used herein, the term "identification" sometimes implies
distinguishing between specific Plasmodium species such as P.
falciparum, P. vivax, P. malariae or P. ovale and the other
Plasmodium species; and detecting simultaneously or separately; in
specimens where at least one species of Plasmodium species is
present. However, the term "identification" generally implies
identifying a particular malaria parasite to be detected amongst
multiple malaria parasites. Hence, the term "identification"
includes the detection of single malaria parasite infections and
multiple malaria parasite infections.
[0101] The LAMP method used in the present invention is a gene
amplification method in which, unlike PCR, thermoregulation
(thermal cycle) is not required in the amplification steps, one
kind of DNA polymerase is used, and genes are amplified at a
constant temperature (isothermal temperature) (WO 2000/28082 and
Notomi, T., et al., Nucleic Acids Res. 28 (2000): e63).
[0102] The above DNA polymerase may be any template-dependent
nucleic acid synthetic enzyme that processes strand displacement
activity. Such enzymes include Bst DNA polymerase (Large Fragment),
Bca (exo-) DNA polymerase, the Klenow fragment of Escherichia coli
DNA polymerase I, Vent (Exo-) DNA polymerase (Vent DNA polymerase
without exonuclease activity), DeepVent (Exo-) DNA polymerase
(DeepVent DNA polymerase without exonuclease), KOD DNA polymerase
and the like.
[0103] Bst DNA polymerase (Large Fragment) is preferably used. When
this enzyme is used, the reaction is preferably performed around at
65.degree. C., which is an optimum temperature for the enzyme
reaction.
[0104] An amplified product of the LAMP method can be detected by
known techniques. For example, it can be detected using a labeled
oligonucleotide that specifically recognizes the amplified gene
sequence; or the reaction mixture can be directly subjected to
agarose electrophoresis after completion of the reaction for easy
detection. The LAMP method produces a ladder of multiple bands
having different base lengths.
[0105] Furthermore, the white suspension caused by magnesium
pyrophosphate accumulation as a by-product of amplification can be
detected by the naked eye or with a turbidimeter (Mori, Y., et al.
Biochem. Biophys. Res. Commun. 289 (2001): 150-154).
[0106] Alternatively, amplification can be simply inspected with
the naked eye using SYBR Green I (product of Applied Biosystems),
which turns green in the presence of amplified DNA. However, SYBR
Green I results were consistent with those deduced from a real-time
turbidimeter (Parida, M., et al., J. Clin. Microbiol. 43 (2005):
2895-2903 and Japanese Unexamined Patent Publication No.
2001-242169). Since the turbidity assay can be carried out in a
closed system, the risk of contamination is lower than that for
agarose gel electrophoresis. This is an additional merit of LAMP in
clinical use (Enosawa, M., et al. J. Clin. Microbiol. 41 (2003):
4359-4365; Seki, M., et al., J. Clin. Microbiol. 43 (2005):
1581-1586; Poon, L., et al., Clin. Chem. 52 (2006): 303-306).
[0107] Thus, LAMP diagnosis, in principle, does not require
expensive reagents for DNA extraction, a turbidimeter, a thermal
cycler, or a skilled technician). The template can be prepared by
direct heat-treatment of blood samples, without time-consuming and
expensive DNA extractions using a commercial kit.
[0108] For example, in the field in malaria-endemic areas, only
boiling and separating a very small amount of blood from a
subject's fingertip allows the preparation of a desired DNA sample
enough for the LAMP reaction.
[0109] Moreover, LAMP requires only a simple incubator, such as a
heat block or a water-bath that provides a constant 60.degree. C.,
which makes it more economical and practical than nested PCR or
real-time PCR.
[0110] A genus specific primer set common to the four species of
human malaria parasite 18S rRNA genes, and each specific primer set
of the present invention for the P. vivax 18S rRNA gene, P.
malariae 18S rRNA gene, and P. ovale 18S rRNA gene, target
particular regions of each 18S rRNA gene. The primer sets are
designed to have a total of 4 kinds of primer as a set, in which
two are loop forming 2 kinds of inner primers: (FIP(F1c-F2) and
BIP(B1-B2c)), and the other two are 2 kinds of outer primers (F3,
B3c). Particular regions of each amplified gene range between about
70 to 500 base pair length.
[0111] The inner primers amplify the nucleic acid sequence of the
target region, and are characterized by including: (a) as a first
segment, a nucleic acid sequence that anneals the target gene and
functions as a primer; and (b) as a second segment, a nucleic acid
sequence that is complementarily to the 3' nucleic acid sequence of
the first segment, and positions at the 5' side of the first
segment.
[0112] Further, use of loop primers (LPB, LPF) having a
complementary sequence to the single strand portion of the dumbbell
structure's 5' terminus loop, where the dumbbell structure
functions as the origin of the amplification reaction, can increase
the number of origins in DNA synthesis. Therefore, use of loop
primers can increase amplification efficacy, and shorten the time
required for amplification to about one third to a half. Outer
primers recognize the 3' terminus nucleic acid sequence of the
target region, and have a nucleic acid sequence functioning as the
origin of the synthesis.
[0113] The inventors attempted to design each inner primer
((FIP(FIc-F2), BIP(B1-B2c)), outer primer (F3, B3c) and loop primer
(LPB, LPF) using the LAMP designing software, PRIMEREXPLORER V3
(product of Fujitsu Limited), based on a genus-specific nucleic
acid sequence of the 18S rRNA genes common to the four species of
human malaria parasites, and each species-specific nucleic acid
sequence of the 18S rRNA genes of P. falciparum, P. vivax, P.
malariae and P. ovale. However, in general, the nucleic acid
sequences of malaria parasite genes differ greatly from those of
other organisms, being rich in AT content. Therefore, existing
primer design software was unable to find optimal primer sets,
demanding a repetitive trial-and-error process, and causing
difficulties in designing each type of primer. Finally, among many
combinations of such synthesized primer sets, usable primer sets
both high in sensitivity and specificity were found. Thus, the
inventors have successfully designed a genus-specific primer
capable of detecting the four species of human-infectious malaria
parasites at the same time, and specific primers for each of the
four species of malaria parasites (P. falciparum, P. vivax, P.
malariae and P. ovale) from each 18S rRNA gene.
[0114] Once the nucleic acid sequence of a primer oligonucleotide
is determined, the oligonucleotide can be synthesized by known
means, for example, by an automatic DNA synthesizer produced by
PerkinElmer, Inc.
[0115] According to the present invention, examples of a plasmodium
genus-specific primer set specific to a nucleic acid sequence of
the 18S rRNA genes common among the four species of human malaria
parasites, and specific primer sets for each of the malaria
parasite 18S rRNA gene of P. falciparum, P. vivax, P. malariae and
P. ovale include, for example, seven primer sets as follow primer
sets for the genus plasmodium and the four species of malaria
parasites:
A primer set for the genus Plasmodium [F3(SEQ ID NO: 1), B3c(SEQ ID
NO: 2), FIP(F1c-F2)(SEQ ID NO: 3), BIP(B1-B2c)(SEQ ID NO: 4),
LPF(SEQ ID NO: 5), LPB(SEQ ID NO: 6)]; A primer set for P.
falciparum [F3(SEQ ID NO: 7), B3c(SEQ ID NO: 8), FIP(F1c-F2)(SEQ ID
NO: 9), BIP(B1-B2c)(SEQ ID NO: 10), LPF(SEQ ID NO: 11), LPB(SEQ ID
NO: 12)]; A primer set for P. vivax [F3(SEQ ID NO: 13), B3c(SEQ ID
NO: 14), FIP(F1c-F2)(SEQ ID NO: 15), BIP(B1-B2c)(SEQ ID NO: 16),
LPF(SEQ ID NO: 17), LPB(SEQ ID NO: 18)]; [PvFIP-9 (F1c+F2)(SEQ ID
NO: 31), PvBIP-9(B1+B2c) (SEQ ID NO: 32), PvF3-9 (SEQ ID NO: 33),
PvB3c-9 (SEQ ID NO: 34), PvLPF-9 (SEQ ID NO: 35), PvLPB-9 (SEQ ID
NO: 36)]; or [PvFIP-7(F1c+F2) (SEQ ID NO: 37), PvBIP-7(B1+B2c) (SEQ
ID NO: 38), PvF3-7(SEQ ID NO: 39), PvB3c-7 (SEQ ID NO: 40), PvLPF-7
(SEQ ID NO: 41), PvLPB-7(SEQ ID NO: 42)]; A primer set for P.
malariae [F3(SEQ ID NO: 19), B3c(SEQ ID NO: 20), FIP(F1c-F2)(SEQ ID
NO: 21), BIP(B1-B2c)(SEQ ID NO: 22), LPF(SEQ ID NO: 23), LPB(SEQ ID
NO: 24)]; and A primer set for P. ovale [F3(SEQ ID NO: 25), B3c(SEQ
ID NO: 26), FIP(F1c-F2)(SEQ ID NO: 27), BIP(B1-B2c)(SEQ ID NO: 28),
LPF(SEQ ID NO: 29), LPB(SEQ ID NO: 30)].
[0116] Here, primers each specific to the genus Plasmodium, P.
falciparum, P. vivax, P. malariae and P. ovale are primers capable
of specifically amplifying a particular region of the 18S rRNA
genes common to said Plasmodium species and each particular region
of each 18S rRNA gene of P. falciparum, P. vivax, P. malariae and
P. ovale.
[0117] Examples of sequences characterized by the genus Plasmodium
and the four species of malaria parasites subjected to
detection/identification using LAMP of the present invention
include the 18S rRNA gene sequences of P. falciparum (P.
falciparum: GenBank Accession No. M19173.1, M19173.2, M19172), P.
vivax (P. vivax: GenBank Accession No. U03079, U03080, X13926), P.
malariae (P. malariae: GenBank Accession No. M54897), and P. ovale
(P. ovale: GenBank Accession No. L48986, L48987) deposited at
GenBank.
[0118] The detection or identification of the genus Plasmodium or
one of the four species of malaria parasites present in the
specimen are conducted by isothermal gene amplification generally
in the range of at 60 to 65.degree. C. for 15 minutes to 1 hour,
using at least one set of the aforementioned five primer sets,
following the procedure of LAMP. That is, DNA collected from
specimens such as blood samples and the like is isolated by a known
method, and this DNA is amplified using said primer set. The
presence of the amplified DNA product can be easily detected by
LAMP, or by a general method of electrophoresis.
[0119] The aforementioned easy detection includes: 1) visual
inspection of the amplification reaction mixture for white
turbidity (WO 2001/83817); 2) a method for measuring the
fluorescence polarization values of the reaction mixture using a
fluorescent substance such as fluorescein, fluorescein
isothiocyanate (FITC), X-rhodamine (ROX) or the like (Japanese
Unexamined Patent Publication No. 2002-272475), which uses a
continuous fluorometer such as an ABI Prism 7700 (product of
Applied Biosystems) and the like, allowing for the confirmation of
amplification or kinetic analysis; and 3) visual inspection using
SYBR Green 2, which uses a fluorescent green dye as an intercalator
(WO 2002/103053). By any of these methods, the presence or absence
of any amplification products (presence or absence of the target
18S rRNA gene) can be inspected using the naked eye.
[0120] According to the present invention, a malaria parasite
detection kit capable of detecting any of the genus Plasmodium, P.
falciparum, P. vivax, P. malariae or P. ovale simultaneously or
separately can be provided.
[0121] The above malaria parasite detection kit can be prepackaged
with various types of reagents necessary for detecting nucleic acid
amplified using the primer set of the present invention.
Specifically, various types of oligonucleotides necessary for the
primers or loop primers of the present invention, four species of
dNTPs as substrates for nucleic acid synthesis, a
template-dependent nucleic acid synthase with strand displacement
activity, a buffer or a salt to provide preferable conditions for
enzymatic reaction, a protective agent for stabilizing enzymes or
templates, and, when indicated, reagent(s) necessary for detecting
a reaction product are provided as a kit.
Example of Kit Components:
[0122] (1) A reaction mixture containing a primer set for the genus
Plasmodium [F3 (SEQ ID NO: 1), B3c (SEQ ID NO: 2), FIP (F1c-F2)(SEQ
ID NO: 3), BIP(B1-B2c) (SEQ ID NO: 4), LPF (SEQ ID NO: 5), LPB (SEQ
ID NO: 6)]; (2) A reagent for the visual detection of fluorescence;
(3) An enzyme mixture solution (including Bst DNA polymerase); (4)
A positive control (for the genus Plasmodium); and (5) Distilled
water are provided.
[0123] The present invention further provides an anti-malaria
measure support system and a malaria infection-prevention/treatment
measure system.
[0124] Malaria-infected patient information includes the number
positive for the genus Plasmodium that causes malaria in a malaria
endemic area, and the carrier rate in the area; and specifically,
the information includes, as basic information, subject information
such as the name, sex, age, weight, pregnancy or non-pregnancy
status, family structure, residential address, birthplace, names of
pre-existing diseases, names of drugs being taken, history of drug
side effects, etc., of individual subjects residing in the endemic
area, together with their positivity or negativity for the genus
Plasmodium and the acquisition or non-acquisition of resistance to
malaria therapeutic agents, and the malaria parasite carrier rate
in the area. The means for inputting and storing malaria-infected
patient information are, for example, computer input devices and
storage devices.
[0125] The carrier rate for the genus Plasmodium that causes
malaria in a malaria endemic area is expressed as a percentage
obtained by dividing the number of subjects positive for the genus
Plasmodium by the number of subjects of the genus Plasmodium
detection test and multiplying the resultant quotient by 100. In
the anti-malaria measure support system of the present invention,
for example, a genus Plasmodium detection method using LAMP can be
particularly preferably used in malaria endemic areas. In a subject
who has resided in a malaria endemic area for a long period and has
acquired immunity (resistance) against malaria, the number of
parasites in the subject's blood is as small as about one hundredth
to about one thousandth of that in a patient with malarial fever.
Microscopic detection of such a small number of parasites with high
sensitivity is very difficult. Also, after malaria infection and
the latent period, in the early stages of the period in which a
fever develops due to the appearance of malaria parasites in the
blood, the number of parasites is small and therefore microscopic
detection of the parasites is extremely difficult. Microscopic
detection with high sensitivity is also very difficult when a
malaria therapeutic agent has already been administered and the
number of parasites in the subject's blood has been remarkably
reduced by the effects of the therapeutic agent.
[0126] In many malaria endemic areas, satisfactory DNA extractors
do not exist or are not provided. In the preparation of DNA for
PCR, when highly pure DNA cannot be obtained using an extraction
kit, PCR cannot be performed because PCR does not occur due to the
inhibitor of the DNA amplification enzymes for PCR, which is
present in blood.
[0127] In the genus Plasmodium detection method used in the
anti-malaria measure support system of the present invention, the
DNA sample can be very easily obtained by, for example, collecting
a very small amount of blood from the subject's finger tip, boiling
the blood in boiling water for 10 minutes, and centrifuging the
blood at 10000 rpm for 1 minute to obtain the supernatant, which
can be used as DNA. Therefore, the method in which the genus
Plasmodium in a specimen are detected using the genus Plasmodium
detection primer set of the present invention, which can amplify a
specific region of the 18S rRNA gene sequence of Plasmodium, can be
advantageously used in malaria endemic areas. Further, when
research is conducted not only on subjects in a malaria endemic
area, but also on the rate of mosquitoes collected in an endemic
area that carry malaria parasites to obtain important data for
malaria epidemic prediction based on research on mosquitoes
carrying malaria parasites, since DNA for use in the method of the
present invention can be more easily extracted from mosquitoes than
DNA for PCR, the frequency of the detection of the genus Plasmodium
in specimens, obtained using a genus Plasmodium detection primer
set of the present invention, which can amplify a specific region
of the 18S rRNA gene sequence of Plasmodium, can be stored in a
storage device as a piece of information for malaria epidemic
prediction in the malaria infection-prevention public health
measure database.
[0128] The method for detecting or identifying the genus Plasmodium
or four species of malaria parasites according to the present
invention is simpler, less expensive, more easily operable, and has
higher sensitivity and higher specificity, than microscopy and PCR,
and therefore can be used most preferably in endemic areas.
[0129] Public health measures for malaria infection endemic areas
based on malaria-infected patient information include the following
(1) to (4): (1) eliminating, from the area, environments where
mosquitoes are likely to breed, and eliminating sources of
mosquitoes by exterminating mosquitoes and mosquito larvae bred in
stagnant water; (2) spraying an insecticide in houses, sheds, etc.,
providing doors and windows with wire screens, and preferably,
installing air-conditioners in houses if the residents of the
houses can afford them; (3) providing beds with mosquito nets
(mosquito nets with long-lasting effects: LLIN, Sumitomo Chemical
Co., Ltd.) impregnated with an insecticide (pyrethroid-based
insecticide: permethrin), applying to human skin an insect
repellent spray containing an insect repellent (diethyltoluamide:
DEET), and taking measures such as wearing long-sleeved shirts
after sunset, spraying clothes with an insecticide, etc.; and (4)
prophylactically administering a therapeutic agent, such as a
mixture of mefloquine and artesunate, chloroquine, artemisinin,
quinine, doxycycline, or the like, based on information about
malaria parasites occurring with high frequency in the endemic area
and drug-resistant strains, so that the malaria therapeutic agent
also serves as a malaria prophylactic agent.
[0130] For infection-positive patients, malaria parasite
extermination (treatment) with a malaria therapeutic agent is taken
into consideration. Information about malaria parasites occurring
with high frequency in the endemic area and drug-resistant strains
is available from the websites of the departments of health of
countries with endemic areas; the Japan Health Sciences Foundation,
policy innovative drug development general research project,
research group on chemotherapy of tropical diseases, the Guidance
for Parasitic Disease Chemotherapy, revised version 6.0 (2007); and
the guidelines of the Expert Meeting on Malaria Chemoprophylaxis
published on March, 2005. The malaria therapeutic agent can be
easily selected according to the species of malaria parasites
detected or identified by the method of the present invention,
based on known standards for selecting therapeutic agents.
[0131] The priority of measures, which indicates which of the above
public health measures (1) to (4) should be given priority in
selection, is determined considering the degree of malaria
prevalence in the area, economic conditions of the country,
environmental and economic conditions of the residents, etc. The
measure (1), which can be taken by the country, local government,
autonomous body, or the like, should be given priority, and then
the measures (2) and (3), which can be easily taken by individual
subjects and other residents, are desirable.
[0132] The measure (4), prophylactic administration of a
therapeutic agent, has a lower priority considering the general
economic conditions of residents in malaria endemic areas, and is
likely to be limited to residents with special needs, such as
infants, pregnant women, etc. The measure (4) is not sufficient at
present. The priority of measures varies depending on the endemic
area, and may be, for example, preferably (1)>(2)=(3)>(4),
and from a practical point of view, (2)=(3)>(4)>(1). Such
measure priority is extracted in a public health measure extraction
section (e.g., a program) in view of the degree of malaria
prevalence, economic conditions of the country, environmental and
economic conditions of the residents, etc., and is displayed on a
public health measure display section (e.g., display).
[0133] The information stored in the malaria infection-prevention
public health measure guide database, into which the public health
measure selection information for the inputted malaria
parasite-infected area has been inputted, may include, as described
above, the type and frequency of malaria that is actually endemic
in the area, information about drug-resistant strains, information
about the public health measures (1) to (4), instruction
information about the measures, malaria epidemic prediction,
malaria diagnosis methods, and information about drugs selected for
respective species of infecting malaria parasites.
[0134] Using DNA extracted from a subject, the malaria parasite
detection method of the present invention is carried out to obtain
infected patient information about the presence or absence of
malaria infection in subjects;
and this information is checked against the public health measures
for the malaria infection endemic area, and their priority,
obtained from the public health measure extraction section that
extracts the priority of the public health measures from the
malaria infection-prevention public health measure guide database,
and the public health measure display section that displays the
public health measures extracted in the public health measure
extraction section together with their priority. Thus, the public
health measures that should be taken by local governments or
individual subjects in the endemic area can be carried out
sequentially or simultaneously. Further, the effects of such public
health measures can be stored as information in the malaria
infection-prevention public health measure guide database. This can
update the malaria infection-prevention public health measure guide
database. The anti-malaria measure support system of the present
invention can thus be provided to a malaria endemic area.
[0135] The subject information including information about the
presence or absence of infection with malaria parasites in subjects
can be managed and obtained by a conventional technique using a PC
(personal computer) or a cellular phone, a facsimile, etc. The
information to be stored in the malaria infection-prevention public
health measure guide database can be obtained from Internet
websites using a PC or a cellular phone, or from brochures.
[0136] In the anti-malaria measure support system of the present
invention, the means for inputting and storing malaria therapeutic
agent information for specifying a malaria therapeutic agent that
acts on infection with one or a plurality of four species of
malaria parasites indicates means for storing information including
the effects of drugs, drug names, generic names, areas in which
respective drugs are preferably applied, administration periods,
dosages, types and frequencies of side effects, severity of side
effects, contraindications, information about infants and pregnant
women, information about concomitant use with other drugs, drug
prices, etc., into the "treatment guide database", considering the
actual appearance of drug-resistant strains in each malaria endemic
area, and the degree thereof, or considering the clinical symptoms
of each type of malaria, age, and pregnancy or non-pregnancy status
of the patient.
[0137] It is important that patients with a fever caused by malaria
infection be differentially diagnosed from patients with other
infections such as influenza, since malaria infection, and in
particular P. falciparum infection, results in a serious outcome if
treatment is delayed. Therefore, prompt, highly sensitive, and
highly specific differential diagnosis is desired. In particular,
as described above, satisfactory DNA extractors do not exist or are
not provided in many malaria endemic areas, and in the early stages
of a fever, the number of malaria parasites is so small that
microscopic identification of malaria parasites is not easy.
[0138] The method for identifying P. falciparum, P. vivax, P.
malariae, or P. ovale using a primer set comprising a sequence
specific to P. falciparum, P. vivax, P. malariae, or P. ovale
according to the present invention, or the identification/detection
kit therefor, makes it possible to identify infection with one or a
plurality of four species of malaria parasites more easily, more
rapidly, and with higher sensitivity and higher specificity, than
other identification methods such as PCR and microscopy.
[0139] Further, the malaria parasite detection/identification
method of the present invention enables monitoring of the
therapeutic effects of the administration of malaria therapeutic
agents to malaria-infected patients.
[0140] The system of the present invention is based on the malaria
parasite-specific detection or identification technique. The
"patient information input means for inputting and storing patient
information including information about the pathogen of malaria
infection in a patient with a fever in a malaria endemic area" is
means for inputting and memorizing/storing subject information
including information about the pathogen of malaria infection in a
patient with a fever in a malaria endemic area, and the name, age,
sex, weight, pregnancy or non-pregnancy status, family structure,
residential address, birthplace, names of pre-existing diseases,
names of the drugs being taken, drug side-effect history, etc. of
the patient with a fever, into a PC (personal computer). The input
and memorizing/storage can be performed from a PC or a cellular
phone, or as facsimile information from medical facilities and
hospitals, based on information obtained from endemic areas.
[0141] The "patient information input means for inputting and
storing patient information including information about the
pathogen of malaria infection in a patient with a fever in a
malaria non-endemic area" is the same as the "patient information
input means for inputting and storing patient information including
information about the pathogen of malaria infection in a patient
with a fever in a malaria endemic area" except that the patient
with a fever is "a patient with a fever in a malaria non-endemic
area".
[0142] In the above, "comprising a treatment guide database into
which, according to indices of efficacy against malaria parasites
detected in a subject, malaria therapeutic agent selection
information to be inputted together with a priority that indicates
which malaria therapeutic agent should be given priority in
selection for use against the malaria parasites; a malaria
therapeutic agent extraction section that extracts, according to
the information about the pathogen of malaria infection in the
subject, malaria therapeutic agents to be administered and the
priority thereof, from the treatment guide database; and a malaria
therapeutic agent display section that displays the malaria
therapeutic agents extracted in the above malaria therapeutic agent
extraction section, together with the priority thereof" indicates a
process in which a clinical practitioner or a hospital doctor who
has obtained information about the pathogens of malaria infection
in a patient with a fever in a malaria endemic or non-endemic area
displays which malaria therapeutic agent should be given priority
in selection for use against the infecting malaria parasites,
considering the species of malaria parasites infecting the patient
with a fever, the symptoms of the patient, and other conditions,
using an index of the efficacy of each therapeutic agent, such as
the specificity of effects on infections with P. falciparum, P.
vivax, P. malariae, P. ovale, or on complex infections with two or
more species of these malaria parasites, and further checking the
pathogen information including the source of malaria infection and
other conditions of the patient with a fever, against the malaria
therapeutic agent information stored in the "treatment guide
database" including, for example, the effects of drugs, drug names,
generic names, areas in which respective drugs are preferably
applied, specificity of action, administration periods, dosages,
types and frequencies of side effects, severity of side effects,
contraindications, information about infants and pregnant women,
information about concomitant use with other drugs, drug prices,
etc.
[0143] According to the anti-malaria measure support system of the
present invention, clinical practitioners and hospital doctors can
check information about the pathogen of malaria infection in a
patient with a fever against the malaria therapeutic agent guide
database in a PC or via a cellular phone to have a conventional
software to operate, thereby obtaining a display of which malaria
therapeutic agent should be given priority in selection, or which
malaria therapeutic agents should be used in combination.
[0144] The above-mentioned malaria therapeutic agent information
can be stored in the "treatment guide database", referring to the
Japan Health Sciences Foundation, policy innovative drug
development general research project, research group on
chemotherapy of tropical diseases; the Guidance for Parasitic
Disease Chemotherapy, Revised Version 6.0 (2007); the guideline of
Expert Meeting on Malaria Chemoprophylaxis published on March,
2005; and the malaria treatment guideline of the CDC (Centers for
Disease Control and Prevention) in the U.S.
[0145] For example, since increasing numbers of P. falciparum
strains have become chloroquine-resistant, concomitant
administration of mefloquine and artesunate is considered the
malaria treatment to be given priority in selection in Thailand and
developed countries. On the other hand, in areas where drug prices
are reflected, second-generation drugs of fansidar take priority in
selection in some cases. Against P. vivax and P. ovale, chloroquine
takes priority in selection in the erythrocytic stage that causes
clinical symptoms such as a fever, and then a two-week course of
primaquine administration is selected for complete cure and
recurrence prevention. Further, priority is given to the selection
of chloroquine for use against P. malariae.
[0146] Such information is also stored in the "malaria
infection-prevention public health measure guide database".
[0147] The information about the results of the malaria treatment
of a patient with a fever with the drug selected as a malaria
therapeutic agent to be given priority in selection, using the
anti-malaria measure support system of the present invention, is
fed back to the "malaria parasite treatment guide database" and the
"malaria infection-prevention public health measure guide
database", and stored as updated information.
[0148] Thus, an anti-malaria measure support system can be provided
in which information about the pathogen of malaria infection in a
patient with a fever can be obtained easily, rapidly, and with high
sensitivity and high specificity, by subjecting a specimen
extracted from a patient with a fever in a malaria endemic area, or
a patient with a fever in a non-endemic area, to the method for
identifying infection with a single or a plurality (complex) of
four species of malaria parasites according to the present
invention; and in which a clinical practitioner or a hospital
doctor in a malaria endemic area or non-endemic area can check such
information against the malaria parasite treatment guide database
to select a preferable therapeutic agent and administration method
that should be given priority in selection, for a patient who has
developed a fever due to malaria infection, and can apply such a
drug and method to the treatment of the malaria-infected patient
with a fever.
[0149] From the overall viewpoint of malaria infection-prevention
public health measures in a malaria endemic area and the treatment
of a malaria-infected patient with a fever in a malaria endemic
area or non-endemic area, the present invention can also provide an
anti-malaria measure support system for malaria eradication in the
endemic area, by combining: the obtainment of malaria-infected
patient information in a malaria endemic area using the method for
detecting malaria parasites according to the present invention; the
obtainment of information about the pathogen of malaria infection
in a patient with a fever in a malaria endemic area or non-endemic
area using the anti-malaria measure support system for local
governments, autonomous bodies, or residents in a malaria endemic
area, in which a malaria infection-prevention public health measure
database is used, and the method or kit for identifying infection
with a single or plurality of four species of malaria parasites
according to the present invention; and an anti-malaria measure
support system in which a clinical practitioner or a hospital
doctor in a malaria endemic area or non-endemic area using the
malaria parasite treatment guide database can select a preferable
therapeutic agent and administration method that should given
priority in selection for a malaria-infected patient with a fever
and apply the drug and method to the treatment of the
malaria-infected patient with a fever.
[0150] The above pieces of information can be inputted and stored
using a PC, and can be displayed through a PC or a cellular phone,
or through a paper brochure, in the area.
[0151] The present invention can further provide a malaria
infection-prevention measure system for travelers from a malaria
non-endemic area to a malaria endemic area. The present invention
can provide a malaria infection-prevention measure system for
persons who plan to travel to a malaria endemic area, the system
comprising means for obtaining, from the anti-malaria measure
support system described above, the state of the implementation of
public health measures in the malaria endemic area, information
about the pathogens of malaria infection in the endemic area, and
the state of the treatment of infected patients; means for
selecting a malaria prophylactic/therapeutic agent from a malaria
parasite treatment guide database; and means for administering the
selected agent before travel.
[0152] The above pieces of information are easily available from
Internet websites using a PC or a cellular phone, or as information
provided in brochures from the malaria endemic area or national
governmental organizations. Using the malaria infection-prevention
measure system for persons who plan to travel to a malaria endemic
area, it is possible for persons who plan to travel to a malaria
endemic area to know beforehand the malaria infections that are
endemic in the area and the state of the appearance of
drug-resistant strains. Thus, to prevent malaria infection, the
persons can take, before travel, a preferable malaria therapeutic
agent to be given priority in selection for use against the malaria
infections that are endemic in the area, so that even if the
persons should be infected with malaria, symptoms due to malaria
infection, such as fever, can be reduced at an early stage and
serious conditions can be prevented, making it possible to
exterminate malaria parasites in the blood of such persons at an
early stage.
[0153] The present invention can further provide a malaria
infection-prevention/treatment measure system for returnees from a
malaria endemic area, the system comprising: means for obtaining,
from the anti-malaria measure support system, the state of the
implementation of public health measures in the malaria endemic
area, information about the pathogens of malaria infection in the
endemic area, and the state of the treatment of infected patients;
means for selecting a malaria prophylactic/therapeutic agent from a
malaria parasite treatment guide database; and means for
administering the selected agent after returning from the malaria
endemic area. According to the present invention, since the latent
period before the appearance of clinical symptoms, such as a fever,
varies from 1 week to 40 days depending on the species of the
infecting malaria parasites, when a returnee has been infected with
malaria parasites immediately before returning from a malaria
endemic area and in the case where the returnee is aware of having
been bitten by a mosquito immediately before returning, a
preferable malaria therapeutic agent against malaria parasites that
may cause infection can be selected and administered referring to
information from the anti-malaria measure support system, so that
symptoms due to malaria infection, such as a fever, can be reduced
at an early stage and serious conditions can be prevented even if
the returnee has been infected with malaria, thereby making it
possible to exterminate malaria parasites in the blood of the
returnee from a malaria endemic area at an early stage.
[0154] The present invention can also provide a malaria
infection-prevention/treatment measure system for returnees from a
malaria endemic area, in which, in the above-mentioned malaria
infection-prevention/treatment measure system for returnees from a
malaria endemic area, when a returnee from a malaria endemic area
has a fever, the identification of P. falciparum, P. vivax, P.
malariae, or P. ovale is performed to select and administer a
malaria therapeutic agent that should be given priority in
selection.
[0155] Further, the use of the malaria parasite
detection/identification method of the present invention in the
malaria infection treatment measure system of the present invention
makes it possible to monitor the therapeutic effects of malaria
therapeutic agent administration to malaria-infected patients.
[0156] In the above, when the returnee develops a fever before
administration of a malaria therapeutic agent, a specimen from the
returnee with a fever is subjected to the method for identifying
four species of malaria parasites according to the present
invention, to identify P. falciparum, P. vivax, P. malariae, P.
ovale, or a plurality (complex) thereof, and select and administer
a malaria therapeutic agent that should be given priority in
selection for use against the malaria parasites. The malaria
infection treatment measure system of the present invention can
thus provide a suitable malaria infection treatment method, and a
method for monitoring the therapeutic effects thereof.
[0157] As shown in FIG. 3, information about the results of public
health measures, information about the results of malaria treatment
for patients with a fever, and information about the results of
prophylactic/therapeutic agent administration are fed back into
respective databases.
EXAMPLES
[0158] The following Examples illustrate the present invention in
further detail, but are not intended to limit the scope of the
present invention.
Example 1
Materials and Methods
[0159] Sixty-eight samples that were positive for malaria parasites
by microscopy were collected from patients who had visited malaria
clinics in Mae Sod and Mae Kasa, northwestern Thailand. In
addition, 53 samples that were negative by microscopy were
collected from residents in a malaria endemic area of Kanchanaburi,
western Thailand.
[0160] The blood samples were tested by microscopy and LAMP. Each
test was carried out by independent researchers (microscopy in the
Armed Forces Research Institute of Medical Sciences, Thailand, and
LAMP in Ehime University, Japan), blinded to the origin of the
specimens and the laboratory results, and finally the test results
were compared and analyzed.
Microscopy:
[0161] Thick peripheral blood smears were examined under
1,000.times. magnification by microscopists with extensive
experience in the identification of malaria parasites. The parasite
density was counted per 500 leukocytes and was then calculated as
the number of parasites per microliter by assuming a leukocyte
count of 7,000/.mu.l. The initial thick film was considered
negative if no parasites were seen after 500 leukocytes were
counted.
DNA Extraction:
[0162] The DNA template for LAMP was prepared as described in
Plowe, C., et al. (Am. J. Trop. Med. Hyg. (1995) Vol. 52: 565-568).
Twenty-five to fifty microliters of the human blood was blotted as
single spot and dried on filter paper. A single blood-spot from
each filter paper was excised, then incubated for 4 hours at room
temperature and/or overnight at 4.degree. C. in 1 ml of 0.5%
saponin in phosphate-buffered saline (PBS). The filter paper was
washed for 30 minutes in PBS at 4.degree. C. and transferred into
new tubes containing 200 .mu.l of 5% CHELEX-100 cation exchange
resin(Bio-Rad, Hercules, Calif.), and vortexed for 30 seconds. The
mixture was incubated at 56.degree. C. for 15 minutes, vortexed for
30 seconds, and heated at 100.degree. C. for 15 minutes to elute
the DNA, vortexed, and centrifuged (10,000.times.g for 5 minutes).
The supernatant was either used immediately after the reaction, or
stored in aliquots at -20.degree. C.
LAMP Conditions:
[0163] LAMP primer sets for P. falciparum described in Poon et al.
(Non-Patent Document 1) were used. The remaining Plasmodium genus-
and species-specific LAMP primer sets were attempted to be designed
using the LAMP Primer Designing Software PrimerExplorer V3
(manufactured by Fujitsu Ltd.). However, the nucleotide sequences
of the genes of malaria parasites are generally greatly different
from those of organisms of other species and have a high AT
content; therefore, it was impossible to find optimal primer sets
using the above primer designing software. Accordingly, many trials
and errors were necessary, encountering difficulties in designing
the primers. Finally, however, primer sets having sensitivity and
specificity sufficient for practical use were able to be found
among the synthesized primer sets with numerous combinations. Thus,
a genus-specific primer set that is capable of detecting the four
species of the genus Plasmodium that infect humans at a time, and
primer sets each specific to each of the four species of malaria
parasites (P. falciparum, P. vivax, P. malariae, and P. ovale) were
successfully designed based on the genus- and species-specific
nucleotide sequences of the 18S rRNA genes.
[0164] After the nucleotide sequences of the oligonucleotides were
designed as above, primers were synthesized by a known method, for
example, using Automated DNA Synthesizer, manufactured by
Perkin-Elmer.
[0165] The location and nucleotide sequence of each primer are
shown in FIG. 1.
[0166] More specifically, primer sets for the genus Plasmodium and
primer sets for the respective four Plasmodium species were
used:
[0167] primer sets for the genus Plasmodium [(F3 (SEQ ID NO: 1),
B3c (SEQ ID NO: 2), FIP(F1c-F2)(SEQ ID NO: 3), BIP(B1-B2c)(SEQ ID
NO: 4), LPF(SEQ ID NO: 5), LPB(SEQ ID NO: 6)];
[0168] primer sets for P. falciparum [F3 (SEQ ID NO: 7), B3c (SEQ
ID NO: 8), FIP(F1c-F2)(SEQ ID NO: 9), BIP(B1-B2c)(SEQ ID NO: 10),
LPF(SEQ ID NO: 11), LPB(SEQ ID NO: 12)];
[0169] primer sets for P. vivax [F3 (SEQ ID NO: 13), B3c (SEQ ID
NO: 14), FIP(F1c-F2)(SEQ ID NO: 15), BIP(B1-B2c)(SEQ ID NO: 16),
LPF(SEQ ID NO: 17), and LPB(SEQ ID NO: 18)];
[0170] primer sets for P. malariae [F3 (SEQ ID NO: 19), B3c (SEQ ID
NO: 20), FIP(F1c-F2)(SEQ ID NO: 21), BIP(B1-B2c)(SEQ ID NO: 22),
LPF (SEQ ID NO: 23), LPB (SEQ ID NO: 24)];
[0171] primer sets for P. ovale [F3 (SEQ ID NO: 25), B3c (SEQ ID
NO: 26), FIP(F1c-F2)(SEQ ID NO: 27), BIP(B1-B2c)(SEQ ID NO: 28),
LPF (SEQ ID NO: 29), LPB (SEQ ID NO: 30)].
[0172] The LAMP reaction was performed with a Loopamp DNA
amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan).
[0173] Reaction mixtures (25 .mu.l) contained 1.6 to 2.4 .mu.M of
each FIP and BIP, 0.2 .mu.M of each F3 and B3c, 0.8 .mu.M of each
LPF and LPB, 2.times. reaction mix (12.5 .mu.l), Bst DNA polymerase
(1 .mu.l), and 1 to 2 .mu.l of DNA sample (corresponding to
approximately 0.125 to 0.5 .mu.l of blood).
[0174] The LAMP reaction was performed at 60.degree. C. for 100
minutes, then the enzyme was inactivated at 80.degree. C. for 2
minutes.
Analysis of LAMP Products:
[0175] The LAMP reaction causes turbidity in the reaction tube,
proportional to the amount of amplified DNA. Therefore, the
turbidity was observed with the naked eye. To confirm the
sensitivity of LAMP, turbidity was also monitored by a Loopamp
real-time turbidimeter (RT-160C, Eiken Chemical Co., Tokyo,
Japan).
[0176] For further confirmation, 5 .mu.l of LAMP product was
electrophoresed at 100 V in a 3% agarose gel, followed by staining
with ethidium bromide, using MassRuler.TM. DNA ladder marker
(Fermentas Inc., Hanover, Md.). The specificity of LAMP was
evaluated by restriction enzyme digestion of the amplified
product.
[0177] Based on the restriction enzyme maps of the target sequences
of each LAMP product, restriction enzyme, DdeI was selected for
restriction enzyme analysis of the Plasmodium genus-specific LAMP
products, HpyCH4V for P. falciparum, P. vivax, and P. malariae, and
restriction enzyme, AluI for P. ovale. Following overnight
digestion at 37.degree. C., the digested products were analyzed by
agarose gel electrophoresis.
Diagnostic Threshold of LAMP Results:
[0178] The formation of LAMP reaction products was monitored using
a Loopamp real-time turbidimeter. Most of the positive samples
tested multiple times showed positivity within 1 hour. Therefore, a
sample having turbidity greater than or equal to the threshold
value by turbidimeter within 1 hour was considered positive.
Positive Control Plasmid DNA and Sequencing:
[0179] For sensitivity assessment, plasmids containing the target
region of the 18S rRNA gene for LAMP reaction were constructed for
each species. Target DNA sequence was amplified with two LAMP
primers (F3 and B3c) by PCR, then cloned into the pCR.RTM. 2.1-TOPO
TA Cloning vector (Invitrogen, Carlsbad, Calif.).
[0180] The nucleotide sequences were determined using an automated
DNA sequencer (ABI PRISM.RTM. 310 Genetic Analyzer, Applied
Biosystems).
Analytical Sensitivity and Specificity of LAMP:
[0181] To establish the minimum copy number (lower detection limit)
of target gene sequence detectable by LAMP, positive control
plasmid DNAs were used as templates. The standard curve for LAMP
was constructed using 10-fold serial dilutions of plasmid DNA
(10.sup.6 to 1 copy) into sterile water. For each standard, the
copy number was plotted against the threshold time. The resulting
plots were analyzed by linear regression, and the statistical
significance of .gamma..sup.2 values was analyzed by ANOVA (Free
Statistics and Forecasting Software v1.1.21). Probabilities less
than 0.05 were considered statistically significant. The
specificity of the genus- and species-specific LAMP was evaluated
on each control plasmid DNA and P. falciparum genomic DNA (gDNA)
purified from NF54 strain, P. vivax gDNA from Sal-I strain, P.
malariae gDNA from Uganda strain, and P. ovale CDC type gDNA from
CDC strain.
Results of Analytical Sensitivity and Specificity of Plasmodium
Genus- and Species-Specific LAMP:
[0182] The sensitivity of LAMP for the genus Plasmodium and four
species of malaria parasite, P. falciparum, P. vivax, P. malariae,
and P. ovale, was 10 copies for P. malariae and P. ovale, and 100
copies for the genus Plasmodium, P. falciparum, and P. vivax.
[0183] The specificity of each LAMP reaction was further confirmed
by restriction enzyme digestion of LAMP products. The sizes of the
resultant digestion products were in good agreement with the
predicted sizes.
Clinical Sensitivity and Specificity:
[0184] The clinical sensitivity and specificity of the Plasmodium
LAMP were calculated on 121 whole-blood samples with microscopy as
the reference standard method. Sensitivity was calculated as
(number of true positives)/(number of true positives+number of
false negatives), and specificity was calculated as (number of true
negatives)/(number of true negatives+number of false
positives).
Clinical Sensitivity and Specificity: Comparison of Microscopy and
LAMP:
[0185] The results of microscopy and LAMP are given in Table 2.
[0186] Among 68 patients who were positive for malaria parasite by
microscopy, 12 patients were diagnosed with P. falciparum
infection, 34 with P. vivax infection, 12 with P. malariae
infection, 5 with P. ovale infection, and 5 with mixed P.
falciparum and P. vivax infection. The remaining 53 samples were
negative by microscopy.
[0187] LAMP using the genus-specific primer set detected malaria
parasites in 67 samples out of 68 samples positive by microscopy
(98.5% sensitivity). Among the 53 samples that were negative by
microscopy, genus-specific LAMP detected malaria parasites in 3
samples (94.3% specificity).
[0188] The 3 samples positive by LAMP but negative by microscopy
were re-tested by LAMP.
[0189] All three samples were again positive by genus-specific
LAMP; one was diagnosed with P. falciparum and the other two with
P. vivax by species-specific LAMP.
[0190] It is thus presumed that LAMP is more sensitive than
microscopy and can reduce the false negative diagnosis, which may
clinically cause oversights, to the least possible degree.
[0191] All 12 samples that were positive for P. falciparum by
microscopy were also positive for P. falciparum by species-specific
LAMP. Among 34 samples positive for P. vivax by microscopy, 2
samples were diagnosed with P. ovale infection, and 1 with mixed P.
falciparum and P. vivax infection, by LAMP. Among 12 samples
positive for P. malariae, 1 sample was diagnosed with P. ovale, and
1 sample with mixed P. malariae and P. vivax.
[0192] The above results indicate that the new LAMP malaria
diagnosis method developed by the present inventors has higher
sensitivity and higher specificity than microscopy, and is an
advantageous method for detecting the four species of human malaria
parasites.
[0193] The average detection time by LAMP was as follows.
[0194] Genus-specific LAMP: 25.7.+-.4.9 minutes (mean.+-.SD; 19.4
to 52.9 minutes);
[0195] P. falciparum-specific LAMP: 31.7.+-.4.8 minutes (25.8 to
44.9 minutes);
[0196] P. malariae-specific LAMP: 30.6.+-.5.2 minutes (25.4 to 46.9
minutes);
[0197] P. vivax-specific LAMP: 34.8.+-.4.8 minutes (30.5 to 46.6
minutes); and P. ovale-specific LAMP: 36.1.+-.6.8 minutes (29.9 to
49.8 minutes).
[0198] These results show that LAMP can make diagnosis in a period
of time that is remarkably shorter than the amplification time of
nested PCR.
Example 2
[0199] The primer sets for detecting the genus Plasmodium and
Plasmodium species according to the present invention, and the
method for detecting or identifying the genus Plasmodium, P.
falciparum, P. vivax, P. malariae, and P. ovale separately or
simultaneously using the primer sets, were subjected to comparative
tests with PCR and microscopy in a malaria clinic in Mae Sod,
northwestern Thailand, where malaria is endemic.
[0200] A comparison was also made between the above method carried
out using equipment specially designed for LAMP, and the above
method carried out by a procedure that can be rapidly and easily
performed in a malaria endemic area.
[0201] Eighteen samples that were positive for malaria parasites by
microscopy (the percent parasitemia: 0.04% to 0.31%) were used; and
one sample that appeared to be a technical error was excluded from
the Example beforehand.
[0202] Boiling method was used to extract DNA from patient samples
for the rapid and easy detection and identification of malaria
parasites in a malaria endemic area, according to the present
invention (the genus Plasmodium, P. falciparum, P. vivax, P.
malariae, and P. ovale can be detected simultaneously).
Specifically, 50 .mu.l of distilled water (D.W.) was added to 50
.mu.l of a blood sample, and the resulting mixture was boiled at
99.degree. C. for 5 minutes, followed by centrifugation (5415D,
produced by Eppendorf; 16,000.times.g) to obtain a supernatant.
[0203] Subsequently, 2 .mu.l of the above-obtained supernatant
containing DNA was added to 23 .mu.l of a reaction mixture
containing 1 .mu.l of Bst DNA polymerase (Epicentre
Biotechnologies), 12.5 .mu.l of 2.times. reaction mix, 1.6 to 2.4
.mu.M of FIP and BIP in the primer sets obtained in Example 1, 0.2
.mu.M of F3 and B3c in the primer sets obtained in Example 1, and
0.8 .mu.M of LPF and LPB in the primer sets obtained in Example 1,
and reacted at 60.degree. C. for about 60 minutes in a
constant-temperature water bath (Thermominder SM-05R, produced by
TAITEC) capable of containing 96.times.2 (total 192) tubes.
[0204] The presence or absence of turbidity in the reaction product
was visually observed directly to detect the presence or absence of
infection with the genus Plasmodium and identify which of the P.
falciparum, P. vivax, P. malariae, or P. ovale causes the
infection.
[0205] Separately, the reaction mixture containing DNA extracted
from each of the above samples was reacted at 60.degree. C. for
about 60 minutes using a Loopamp real-time turbidimeter (LA-320C,
produced by Eiken Chemical Co., Ltd.) to monitor the formation of
the reaction product in real time.
[0206] Further, for comparison with PCR with respect to the
diagnosis of malaria species, nested PCR was performed on the same
samples. Since strict conditions are required for a PCR reaction,
the PCR reaction may be inhibited when DNA extracted from a
clinical sample by boiling method is used as it is. Thus, dried
blood on filter paper obtained in the malaria clinic was brought to
a well-equipped laboratory in Bangkok, and DNA was extracted using
a DNA extraction kit (QIAamp DNA Mini Kit, produced by QIAGEN) and
subjected to PCR. In the nested PCR, two rounds of PCR were each
performed for 2 hours, for a total of 4 hours. Thereafter, the
obtained sample was subjected to agarose gel electrophoresis and
stained with fluorescence, and detection was performed for a total
of 5 hours.
[0207] As a result, of the 18 samples that were positive for
malaria parasites by microscopy, in the detection of the presence
or absence of infection with the genus Plasmodium using the primers
for the genus Plasmodium, 18 were positive when the samples were
tested using the Loopamp real-time turbidimeter specially designed
for LAMP, and 18 were also positive when the samples were tested by
reacting in a constant-temperature water bath followed by visual
observation. The results of the two tests agreed 100%.
[0208] Table 1 compares the test results for microscopy, nested
PCR, and LAMP for the diagnosis of malaria species using Plasmodium
species-specific primers; and the results using an amplifier
specially designed for LAMP, with the results of the visual
observation after the reaction in a constant-temperature water
bath.
TABLE-US-00001 TABLE 1 LAMP Constant- temperature Sample Specially
water bath + (Percent designed visual parasitemia) PCR amplifier
observation Pf (0.12-0.28%) + 3 (N.D.2) 5 5 Pv (0.04-0.31%) + 12 11
11 Pv + Po Pv+ 1 1 1 (00.8, 0.04%) Po- 1 1 1
[0209] As is apparent in Table 1, with respect to P. falciparum,
only 3 samples were subjected to PCR. In the table, Pf indicates P.
falciparum, Pv indicates P. vivax, and Po indicates P. ovale.
[0210] As a result, in one sample diagnosed by microscopy as being
infected with both P. vivax and P. ovale, both PCR and LAMP (both
the test using an amplifier specially designed for LAMP, and the
test using visual observation after the reaction in a
constant-temperature water bath) detected P. vivax, but not P.
ovale, indicating that the microscopy diagnosis was an error.
[0211] That is, in the comparative test, 17 of the 18 LAMP reaction
results matched the microscopy results (94% agreement). This means
that the LAMP reaction results agreed 100% with the microscopy
results, if the above-mentioned one reaction result in disagreement
with the microscopy result is excluded. These results reveal that
the primer sets for detecting the genus Plasmodium and Plasmodium
species according to the present invention, and the method for
detecting or identifying the genus Plasmodium, P. falciparum, P.
vivax, P. malariae, and P. ovale separately or simultaneously using
these primers, agreed 100% with the microscopy diagnoses in the
detection and identification of Plasmodium parasite infection; and
the test results using an amplifier specially designed for LAMP
agreed 100% with the visual observation results after the reaction
in the constant-temperature water bath. In the diagnoses of malaria
species, the primer sets and method agreed 100%, and achieved
sensitivity equivalent to PCR.
[0212] It was demonstrated that, for the primer sets for detecting
the genus Plasmodium and Plasmodium species according to the
present invention, and the method for detecting or identifying the
genus Plasmodium, P. falciparum, P. vivax, P. malariae, and P.
ovale separately or simultaneously using the primer sets,
especially in a malaria endemic area, DNA can be extracted by
boiling method, which is rapid, simple, and inexpensive, and a DNA
amplification reaction can be carried out using a
constant-temperature water bath so that a large number of samples
can be treated inexpensively.
[0213] The primer sets for detecting the genus Plasmodium and
Plasmodium species according to the present invention and the
method for detecting or identifying the genus Plasmodium, P.
falciparum, P. vivax, P. malariae, and P. ovale separately or
simultaneously using the primer sets, can be applied more rapidly
and easily than microscopy, and have sensitivity equivalent to PCR.
Therefore, the primer sets and method can be advantageously applied
on-site, especially in a malaria endemic area, for the rapid and
simple detection and identification diagnosis of the Plasmodium
genus and species simultaneously, in a large number of samples.
Example 3
[0214] According to the present invention, from the test results
obtained on-site (in a clinic) in a malaria endemic area in a short
period of time, mefloquine and artesunate can be prescribed for
administration to the five patients diagnosed and identified as
being infected with P. falciparum in Example 2, based on the
information about malaria therapeutic agents and the priority
thereof in the malaria therapeutic agent information stored in the
"treatment guide database". Likewise, chloroquine and primaquine
can be prescribed for administration to the 13 patients diagnosed
and identified as being infected with P. vivax. Thus, an
anti-malaria measure support system can be operated which is
capable, in a malaria endemic area, of rapidly and reliably
detecting the presence or absence of infection with Plasmodium
parasites and providing malaria treatment.
Example 4
[0215] As described above, simple and easy detection of the genus
Plasmodium (in particular, the presence or absence of mixed
infection) is important in malaria endemic areas.
[0216] The present inventors conducted further research on primers
that are capable of detecting Plasmodium species.
[0217] As a result, the inventors found that two types of primer
sets (Pv-7 and Pv-9; Pv-7 is represented by SEQ ID NOs: 37 to 42
and Pv-9 is represented by SEQ ID NOs: 31 to 36) that are useful
for P. vivax diagnosis enable more rapid diagnosis of P. vivax.
[0218] When using the two types of primer sets, it was demonstrated
that these primer sets react none of the DNAs of P. falciparum, P.
malariae, and P. ovale.
[0219] The use of the two types of primer sets (Pv-7 and Pv-9)
useful for P. vivax diagnosis achieved more rapid diagnosis (Pv-7:
27 minutes; Pv-9: 24 minutes) than the previously found primer set
for P. vivax diagnosis (31 minutes). In particular, the use of the
primer set Pv-9 achieved the most rapid diagnosis.
[0220] The invention further provides the following inventions:
[0221] 1A. A primer set for detecting the genus Plasmodium,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 1 to 6, the primer set being capable of
amplifying a particular region of a Plasmodium 18S rRNA gene
sequence.
[0222] 2A. A primer set for detecting Plasmodium vivax, comprising
an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 13 to 18, the primer set being capable
of amplifying a particular region of a Plasmodium vivax 18S rRNA
gene sequence.
[0223] 3A. A primer set for detecting Plasmodium malariae,
comprising an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 19 to 24, the primer set being capable
of amplifying a particular region of a Plasmodium malariae 18S rRNA
gene sequence.
[0224] 4A. A primer set for detecting Plasmodium ovale, comprising
an oligonucleotide set containing nucleic acid sequences
represented by SEQ ID NOs: 25 to 30, the primer set being capable
of detecting a particular region of a Plasmodium ovale 18S rRNA
gene sequence.
[0225] 5A. A primer set for detecting the genus Plasmodium, P.
falciparum, P. vivax, P. malariae or P. ovale, comprising an
oligonucleotide primer set containing any of the nucleic acid
sequences of claims 1A to 4A and nucleic acid sequences represented
by SEQ ID NOs: 7 to 12, the primer set being capable of amplifying
particular regions of a 18S rRNA gene of the genus Plasmodium and
various species of Plasmodium including a particular region of a
Plasmodium falciparum 18S rRNA gene sequence.
[0226] 6A. A method for detecting the genus Plasmodium present in a
specimen, wherein the method targets a particular region of the
genus Plasmodium 18S rRNA gene sequence, and comprises selectively
amplifying the particular region of the genus Plasmodium 18S rRNA
gene sequence by LAMP using a primer set of claim 1A, and
confirming the presence or absence of an amplified product.
[0227] 7A. A method for detecting Plasmodium vivax present in a
specimen, wherein the method targets a particular region of a
Plasmodium vivax 18S rRNA gene sequence, and comprises selectively
amplifying the particular region of the Plasmodium vivax 18S rRNA
gene sequence by LAMP using a primer set of claim 2A, and
confirming the presence or absence of an amplified product.
[0228] 8A. A method for detecting Plasmodium malariae present in a
specimen, wherein the method targets a particular region of a
Plasmodium malariae 18S rRNA gene sequence, and comprises
amplifying the particular region of the Plasmodium malariae 18S
rRNA gene sequence by LAMP using a primer set of claim 3A, and
confirming the presence or absence of an amplified product.
[0229] 9A. A method for detecting Plasmodium ovale present in a
specimen, wherein the method targets a particular region of a
Plasmodium ovale 18S rRNA gene sequence, and comprises selectively
amplifying the particular region of the Plasmodium ovale 18S rRNA
gene sequence by LAMP using a primer set of claim 4A, and
confirming the presence or absence of an amplified product.
[0230] 10A. A method for detecting the genus Plasmodium, Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium
ovale present in a specimen, wherein the method targets a
particular region of the genus Plasmodium 18S rRNA gene sequence,
the genus Plasmodium falciparum 18S rRNA gene sequence, a
Plasmodium vivax 18S rRNA gene sequence, a Plasmodium malariae 18S
rRNA gene sequence, or a Plasmodium ovale 18S rRNA gene
sequence;
and wherein the method comprises selectively amplifying the
particular region of the genus Plasmodium 18S rRNA gene sequence,
P. falciparum 18S rRNA gene sequence, P. vivax 18S rRNA gene
sequence, P. malariae 18S rRNA gene sequence, or P. ovale 18S rRNA
gene sequence respectively; by LAMP using a primer set of claim 5A;
and confirming the presence or absence of an amplified product.
[0231] 11A. A detection method according to claim 10A, wherein the
genus Plasmodium, P. falciparum, P. vivax, P. malariae or P. ovale
are detected simultaneously or separately.
[0232] 12A. A method for identifying the genus Plasmodium,
comprising isolating a DNA sample from a specimen, performing LAMP
amplification of a particular region of the genus Plasmodium 18S
rRNA gene sequence from the DNA sample using a primer set of claim
1A, and confirming the presence or absence of an amplified
product.
[0233] 13A. A method for identifying Plasmodium vivax, comprising
isolating a DNA sample from a specimen, performing LAMP
amplification of a particular region of a Plasmodium vivax 18S rRNA
gene sequence from the DNA sample using a primer set of claim 2A,
and confirming the presence or absence of an amplified product.
[0234] 14A. A method for identifying Plasmodium malariae,
comprising isolating a DNA sample from a specimen, performing LAMP
amplification of a particular region of a P. malariae 18S rRNA gene
sequence from the DNA sample using a primer set of claim 3A, and
confirming the presence or absence of an amplified product.
[0235] 15A. A method for identifying Plasmodium ovale, comprising
isolating a DNA sample from a specimen, performing LAMP
amplification of a particular region of a P. ovale 18S rRNA gene
sequence from the DNA sample using a primer set of claim 4A, and
confirming the presence or absence of an amplified product.
[0236] 16A. A method for identifying the genus Plasmodium, P.
falciparum, P. vivax, P. malariae, or P. ovale, comprising
isolating a DNA sample from a specimen, performing LAMP
amplification of a particular region of the genus Plasmodium 18S
rRNA gene sequences, a P. falciparum 18S rRNA gene sequence, a P.
vivax 18S rRNA gene sequence, a P. malariae 18S rRNA gene sequence,
or a P. ovale 18S rRNA gene sequence, from the DNA sample using a
primer set of claim 5A; and confirming the presence or absence of
an amplified product.
[0237] 17A. A method of identification according to claim 16A,
wherein the identification of any of the genus Plasmodium, P.
falciparum, P. vivax, P. malariae, or P. ovale is performed
simultaneously or separately.
[0238] 18A. A detection kit for the genus Plasmodium, P.
falciparum, P. vivax; P. malariae, or P. ovale; comprising at least
a primer set of any of claim 1A to 5A, a strand displacement DNA
polymerase, dNTPs and a reaction buffer.
[0239] 19A. A detection kit according to claim 18A, wherein the
detection kit detects the genus Plasmodium, P. falciparum, P.
vivax, P. malariae, or P. ovale, simultaneously or separately.
[0240] 20A. An anti-malaria measure support system comprising:
[0241] means for inputting and storing malaria-infected patient
information including the number positive for the genus Plasmodium
parasites that cause malaria in a malaria endemic area, and the
carrier rate in the area;
[0242] a malaria infection-prevention public health measure guide
database that specifies public health measures for the malaria
endemic area based on the malaria-infected patient information,
into which database public health measure selection information for
a malaria parasite-infected area to be inputted together with the
measure priority indicating which of the public health measures
should be given priority in selection has been inputted;
[0243] a public health measure extraction section that extracts
public health measures for the malaria-infected endemic area and
the priority thereof from the malaria infection-prevention public
health measure guide database, according to malaria-infected
patient information about malaria parasites in a subject; and
[0244] a public health measure display section that displays the
public health measures extracted in the public health measure
extraction section, together with the priority thereof.
[0245] 21A. An anti-malaria measure support system according to
claim 20A, wherein the malaria-infected patient information about
malaria parasites in the malaria endemic area is obtained by
identifying the presence or absence of infection with the genus
Plasmodium using a primer set according to claim 1A and/or a
detection method according to claim 6A, or the genus Plasmodium
detection kit according to claim 19A.
[0246] 22A. An anti-malaria measure support system comprising:
[0247] means for inputting and storing malaria therapeutic agent
information for specifying a malaria therapeutic agent that acts on
infection with one or a plurality of four species of malaria
parasites;
[0248] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria endemic
area;
[0249] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria
non-endemic area;
[0250] a treatment guide database into which, according to indices
of efficacy against malaria parasites detected in a subject,
malaria therapeutic agent selection information to be inputted
together with a priority that indicates which malaria therapeutic
agent should be given priority in selection for use against the
malaria parasites;
[0251] a malaria therapeutic agent extraction section that
extracts, according to the information about the pathogen of
malaria infection in the subject, malaria therapeutic agents to be
administered and the priority thereof, from the treatment guide
database; and
[0252] a malaria therapeutic agent display section that displays
the malaria therapeutic agents extracted in the above malaria
therapeutic agent extraction section, together with the priority
thereof.
[0253] 23A. An anti-malaria measure support system according to
claim 22A, wherein the information about the pathogen of malaria
infection in a patient with a fever is obtained by identifying
infection with one or a plurality of four species of malaria
parasites using a primer set according to any one of claims 2A to
5A and/or a method for identifying the genus Plasmodium, Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium
ovale according to any one of claims 13A to 17A, or a detection kit
according to claim 18A or 19A.
[0254] 24A. An anti-malaria measure support system in which a
public health measure relating to an anti-malaria measure and a
malaria parasite treatment measure relating to an anti-malaria
measure are carried out in combination;
[0255] the public health measure comprising:
[0256] means for inputting and storing malaria-infected patient
information including the number positive for the genus Plasmodium
that causes malaria in a malaria endemic area, and the carrier rate
in the area;
[0257] a malaria infection-prevention public health measure guide
database that specifies public health measures for the malaria
endemic area based on the malaria-infected patient information,
into which database public health measure selection information for
a malaria parasite-infected area to be inputted together with a
priority of measures that indicates which of the public health
measures should be given priority in selection has been
inputted;
[0258] a public health measure extraction section that extracts
public health measures for the malaria-infected endemic area and
the priority thereof, from the malaria infection-prevention public
health measure guide database according to malaria-infected patient
information about malaria parasites in the subject; and
[0259] a public health measure display section that displays the
public health measures extracted in the public health measure
extraction section, together with the priority thereof; and
[0260] the malaria parasite treatment measure comprising:
[0261] means for inputting and storing malaria therapeutic agent
information for specifying a malaria therapeutic agent that acts on
infection with one or a plurality of four species of malaria
parasites;
[0262] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in the malaria endemic
area;
[0263] patient information input means for inputting and storing
patient information including information about the pathogen of
malaria infection in a patient with a fever in a malaria
non-endemic area;
[0264] a treatment guide database into which, according to indices
of efficacy against malaria parasites detected in a specimen,
malaria therapeutic agent selection information to be inputted
together with a priority that indicates which malaria therapeutic
agent should be given priority in selection for use against the
malaria parasites;
[0265] a malaria therapeutic agent extraction section that
extracts, according to the information about the pathogen of
malaria infection in the subject, malaria therapeutic agents to be
administered and the priority thereof, from the treatment guide
database; and
[0266] a malaria therapeutic agent display section that displays
the malaria therapeutic agents extracted in the above malaria
therapeutic agent extraction section, together with the priority
thereof.
[0267] 25A. An anti-malaria measure support system according to
claim 24A, wherein the malaria-infected patient information about
malaria parasites in the malaria endemic area is obtained by
identifying the presence or absence of infection with the genus
Plasmodium using a primer set according to claim 1A and/or a
detection method according to claim 6A, or the genus Plasmodium
detection kit according to claim 19A; and/or information about the
pathogen of malaria infection in a patient with a fever is obtained
by identifying infection with one or a plurality of four species of
malaria parasites using a primer set according to any one of claims
2A to 5A and/or a method for identifying the genus Plasmodium,
Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or
Plasmodium ovale according to any one of claims 13A to 17A, or a
detection kit according to claim 18A or 19A.
[0268] 26A. A malaria infection-prevention measure system for
persons who plan to travel to a malaria endemic area, the system
comprising:
[0269] means for obtaining the state of the implementation of
public health measures in the malaria endemic area, information
about the pathogens of malaria infection in the endemic area, and
the state of the treatment of infected patients, from the
anti-malaria measure support system according to claim 24A;
[0270] means for selecting a malaria prophylactic/therapeutic agent
from a malaria parasite treatment guide database; and
[0271] means for administering the selected agent before
travel.
[0272] 27A. A malaria infection-prevention/treatment measure system
for returnees from a malaria endemic area, the system
comprising:
[0273] means for obtaining the state of the implementation of
public health measures in the malaria endemic area, information
about the pathogens of malaria infection in the endemic area, and
the state of the treatment of infected patients;
[0274] means for selecting a malaria prophylactic/therapeutic agent
from a malaria parasite treatment guide database; and
[0275] means for administering the selected agent after returning
from the malaria endemic area, according to Item 24.
[0276] 28A. A malaria infection-prevention/treatment measure system
according to claim 27A, wherein, when a returnee from the malaria
endemic area has a fever, identification of Plasmodium falciparum,
Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale is
performed to select and administer a malaria therapeutic agent that
should be given priority in selection.
INDUSTRIAL APPLICABILITY
[0277] The method for detecting/identifying the genus Plasmodium
and four species of malaria parasites using LAMP developed by the
present inventors have higher sensitivity and higher specificity
than microscopy, and can detect/identify the Plasmodium parasites
in specimens more easily and in a shorter period of time than PCR.
Further, since the method is inexpensive, it is very useful for
malaria clinical diagnosis and control activity in malaria endemic
areas with poor facilities. It is also possible to construct a new
anti-malaria measure support system (FIG. 3) using the method.
SEQUENCE LISTING FREE TEXT
[0278] The 18S rRNA sequences of four Plasmodium species, P.
falciparum (GenBank Accession No. M19172), P. vivax (GenBank
Accession No. U03079 or X13926), P. malariae (GenBank Accession No.
M54897), and P. ovale (GenBank Accession No. L48987), were aligned
for comparison.
Sequence CWU 1
1
47121DNAartificialPrimer for detecting Plasmodium Genus (F3)
1gtatcaatcg agtttctgac c 21220DNAartificialPrimer for detecting
Plasmodium Genus (B3c) 2cttgtcacta cctctcttct
20345DNAartificialPrimer for detecting Plasmodium Genus
(FIP(F1c-F2)) 3tcgaactcta attccccgtt acctatcagc ttttgatgtt agggt
45441DNAartificialPrimer for detecting Plasmodium Genus
(BIP(B1-B2c)) 4cggagaggga gcctgagaaa tagaattggg taatttacgc g
41520DNAartificialPrimer for detecting Plasmodium Genus (LPF)
5cgtcatagcc atgttaggcc 20624DNAartificialPrimer for detecting
Plasmodium Genus (LPB) 6agctaccaca tctaaggaag gcag
24724DNAartificialPrimer for detecting Plasmodium falciparum (F3)
7tgtaattgga atgataggaa ttta 24823DNAartificialPrimer for detecting
Plasmodium falciparum (B3c) 8gaaaacctta ttttgaacaa agc
23941DNAartificialPrimer for detecting Plasmodium falciparum
(FIP(F1c-F2)) 9agctggaatt accgcggctg ggttcctaga gaaacaattg g
411045DNAartificialPrimer for detecting Plasmodium falciparum
(BIP(B1-B2c)) 10tgttgcagtt aaaacgttcg tagcccaaac cagtttaaat gaaac
451116DNAartificialPrimer for detecting Plasmodium falciparum (LPF)
11gcaccagact tgccct 161215DNAartificialPrimer for detecting
Plasmodium falciparum (LPB) 12ttgaatatta aagaa
151323DNAartificialPrimer for detecting Plasmodium vivax (F3)
13ggaatgatgg gaatttaaaa cct 231422DNAartificialPrimer for detecting
Plasmodium vivax (B3c) 14acgaagtatc agttatgtgg at
221542DNAartificialPrimer for detecting Plasmodium vivax
(FIP(F1c-F2)) 15ctattggagc tggaattacc gctcccaaaa ctcaattgga gg
421641DNAartificialPrimer for detecting Plasmodium vivax
(BIP(B1-B2c)) 16aattgttgca gttaaaacgc tcgtaagcta gaagcgttgc t
411718DNAartificialPrimer for detecting Plasmodium vivax (LPF)
17gctgctggca ccagactt 181820DNAartificialPrimer for detecting
Plasmodium vivax (LPB) 18agttgaattt caaagaatcg
201918DNAartificialPrimer for detecting Plasmodium malariae (F3)
19caaggccaaa ttttggtt 182019DNAartificialPrimer for detecting
Plasmodium malariae (B3c) 20cggttattct taacgtaca
192142DNAartificialPrimer for detecting Plasmodium malariae
(FIP(F1c-F2)) 21tattggagct ggaattaccg cgatgatggg aatttaaaac ct
422245DNAartificialPrimer for detecting Plasmodium malariae
(BIP(B1-B2c)) 22aattgttgca gttaaaacgc ctatgttata aatatacaaa gcatt
452319DNAartificialPrimer for detecting Plasmodium malariae (LPF)
23gccctccaat tgccttctg 192424DNAartificialPrimer for detecting
Plasmodium malariae (LPB) 24tcgtagttga atttcaagga atca
242522DNAartificialPrimer for detecting Plasmodium ovale (F3)
25ggaatgatgg gaatttaaaa cc 222621DNAartificialPrimer for detecting
Plasmodium ovale (B3c) 26gaatgcaaag aacagatacg t
212743DNAartificialPrimer for detecting Plasmodium ovale
(FIP(F1c-F2)) 27tattggagct ggaattaccg cgttcccaaa attcaattgg agg
432847DNAartificialPrimer for detecting Plasmodium ovale
(BIP(B1-B2c)) 28gttgcagtta aaacgctcgt agtgtattgt ctaagcatct tatagca
472918DNAartificialPrimer for detecting Plasmodium ovale (LPF)
29tgctggcacc agacttgc 183018DNAartificialPrimer for detecting
Plasmodium ovale (LPB) 30tgaatttcaa agaatcaa
183133DNAartificialPrimer for detecting Plasmodium vivax (PvFIP-9
(F1c+F2)) 31cgctattgga gctggaatac tcaattggag ggc
333239DNAartificialPrimer for detecting Plasmodium vivax (PvBIP-9
(B1+B2c)) 32aattgttgca gttaaaacga ttaagctaga agcgttgct
393316DNAartificialPrimer for detecting Plasmodium vivax (PvF3-9)
33tttaaaacct tcccaa 163417DNAartificialPrimer for detecting
Plasmodium vivax (PvB3c-9) 34aagtatcagt tatgtgg
173515DNAartificialPrimer for detecting Plasmodium vivax (PvLPF-9)
35gctggcacca gactt 153620DNAartificialPrimer for detecting
Plasmodium vivax (PvLPB-9) 36ctcgtagttg aatttcaaag
203732DNAartificialPrimer for detecting Plasmodium vivax (PvFIP-7
(F1c+F2)) 37ctggaattac cgcggctcct tcccaaaact ca
323839DNAartificialPrimer for detecting Plasmodium vivax (PvBIP-7
(B1+B2c)) 38ccaatagcgt atattaaaat tgttgctaga agcgttgct
393916DNAartificialPrimer for detecting Plasmodium vivax (PvF3-7)
39tggaatgatg ggaatt 164018DNAartificialPrimer for detecting
Plasmodium vivax (PvB3c-7) 40gtatcagtta tgtggatt
184119DNAartificialPrimer for detecting Plasmodium vivax (PvLPF-7)
41accagacttg ccctccaat 194223DNAartificialPrimer for detecting
Plasmodium vivax (PvLPB-7) 42gcagttaaaa cgctcgtagt tga
2343198DNAPlasmodium 43atttagtgtg tgtatcaatc gagtttctga cctatcagct
tttgatgtta gggtattggc 60ctaacatggc tatgacgggt aacggggaat tagagttcga
ttccggagag ggagcctgag 120aaatagctac cacatctaag gaaggcagca
ggcgcgtaaa ttacccaatt ctaaagaaga 180gaggtagtga caagaaat
19844250DNAPlasmodium falciparum 44caatacaata tcgaaaaatg attttgtaat
tggaatgata ggaatttaca aggttcctag 60agaaacaatt ggagggcaag tctggtgcca
gcagccgcgg taattccagc tccaatagca 120tatattaaaa ttgttgcagt
taaaacgttc gtagttgaat attaaagaat ccgatgtttc 180atttaaactg
gtttgggaaa accaaatata ttatatattt tgctttgttc aaaataaggt
240tttctaataa 25045244DNAPlasmodium vivax 45caatacaagg ccaatctggc
tttgtaattg gaatgatggg aatttaaaac cttcccaaaa 60ctcaattgga gggcaagtct
ggtgccagca gccgcggtaa ttccagctcc aatagcgtat 120attaaaattg
ttgcagttaa aacgctcgta gttgaatttc aaagaatcga tattttaagc
180aacgcttcta gcttaatcca cataactgat acttcgtatc gactttgtgc
gcattttgct 240atta 24446266DNAPlasmodium malariae 46caatgcaagg
ccaaattttg gttttgcaat tggaatgatg ggaatttaaa accttcccag 60aaggcaattg
gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt
120atattaaaat tgttgcagtt aaaacgctcg tagttgaatt tcaaggaatc
aatattttaa 180gtaatgcttt gtatatttat aacatagttg tacgttaaga
ataaccgcca aggcttatat 240tttttctgtt acattttgtt ttatta
26647245DNAPlasmodium ovale 47caatacaagg ccatttcatg gttttgtaat
tggaatgatg ggaatttaaa accttcccaa 60aattcaattg gagggcaagt ctggtgccag
cagccgcggt aattccagct ccaatagcgt 120atattaaaat tgttgcagtt
aaaacgctcg tagttgaatt tcaaagaatc aatattttaa 180gtaatacttt
tgctataaga tgcttagaca atacaacgta tctgttcttt gcattcctta 240tgcaa
245
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