Transcript Presentation - Armed Forces Pest Management Board

Scrub Typhus Diagnostics:
Which Works Best?
K. Lerdthusnee, W. Leepitakrat, S. Insuan,
T. Monkanna, S. Leepitakrat, W. Chareonsongsermkit,
N. Khlaimanee, W. Akka-graisee, K. Chayapumh,
J. W. Jones, R. E. Coleman, & J. R. Murphy.
Department of Entomology
US Army Medical Component-Armed Forces Research Institute of Medical Sciences
(USAMC-AFRIMS), Bangkok 10400, Thailand
Presented by
CPT (P) Jittawadee R. Murphy, Ph.D.
DoD Tri-Service Pest Management Workshop at the Naval Air Station,
Jacksonville, Florida, USA,
12-16 February 2007
• Armed Forces Research Institute of Medical Sciences
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Scrub Typhus
An important vector-borne disease, first
described in 1899 in Japan.
During World War II, this disease killed
thousands of soldiers who were stationed
in rural or jungle areas of the Pacific
theatre.
The disease occurred and threatened people throughout
Asia & Australia. The range stretches from the Far-east
to the Middle-east(from Japan and Korea, Southeast Asia, Pakistan,
India, to Arab countries and Turkey). There are approx. 1 million
cases each year world-wide, & over 1 billion people at
risk.
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Scrub Typhus: A Rickettsial Disease
Pathogen: Orientia tsutsugamushi
Rickettsial bacteria
An acute febrile, rickettsial disease caused by a
gram-negative, rod-shaped (cocco-bacillus)
bacterium, known as Orientia (Rickettsia)
tsutsugamushi.
Vector: Leptotrombidium
Chigger-Mite
O. tsutsugamushi is transmitted to vertebrate hosts
(rodents-primary host & humans-secondary or accidental
host) by the bite of larval mites (chiggers) of the genus
Leptotrombidium, e. g. L. deliense, L. dimphalum, etc.
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Leptotrombidium Chigger’s Habitats
Dry Habitats
Scrub Area
Wet Habitats
The term scrub of
scrub typhus came
from the type of
vegetations (terrain
between woods &
clearings) that harbor
the vectors.
Rice Field
Areas Around Houses
Moist Areas: Swamp & Bog
Edges of Dense Forest
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Scrub Typhus Symptoms
Humans acquire the disease when infected chiggers bite
them and transmit O. tsutsugamushi.
Eschar = Clinical Sign of Scrub Typhus
Bacteria multiply at the inoculation site and frequently
form a papule that ulcerates & becomes necrotic. This
pathognomic focal lesion is called an eschar. Regional
lymphadenopathy develops & progresses to generalized
lymphadenopathy in a few days.
In the severe cases, it can lead to
:-Pneumonia with adult respiratory distress syndrome
:-Circulatory failure resulting in death.
Mortality rates in untreated patients normally range from 030% but rates as high as 60% have been reported.
Significant morbidity and mortality can be prevented in
patients who receive timely, appropriate treatment with
antibiotic drugs.
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Scrub Typhus
O. tsutsugamushi bacteria are found throughout the mite's body,
but the highest number are present in the salivary glands.
When the mite feeds on rodents & humans, the parasites/bacteria
are transmitted to the host.
In chiggers, O. tsutsugamushi bacteria is transmitted:
:-trans-ovarially (from adult female to eggs) &
:-trans-stadially (from egg to all immature & adult stages)
Reservoir Host :Rattus rattus
Only larval Leptotrombidium mites (chiggers) transmit the disease;
however, the bacteria can be found in all mite developmental
stages (from egg to adult).
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Scrub-typhus (Orientia tsutsugamushi) – Infected
Leptotrombidium (L.) Chigger Colonies
Leptotrombidium Chigger
Vector of Scrub Typhus
Chigger-Mite
•AFRIMS-Ectoparasite Lab in Bangkok is
one of two laboratories in the world
capable of rearing/colonizing scrub
typhus infected Leptotrombidium
mites.
Adult Mite
•Currently, 4 different species of scrub typhus
infected Leptotrombidium chiggers are colonized.
Scrub typhus-infected -Leptotrombidium chigger colonies are
housed in an environmental control chamber, located in our
Bio-Safety Level-III (BSL-3) Room-facility.
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Laboratory Analysis
& Detection of
Orientia tsutsugamushi
Cases are often under reported because scrub typhus
patients show similar symptoms to other fevers of
unknown origin (FUO) and also due to nonspecific clinical
symptoms & lack of presentation of symptoms (e.g. lack of
an eschar).
The difficulty in making a clinical diagnosis of scrub typhus
emphasizes the criticality of developing or improving
laboratory diagnostic methods for O. tsutsugamushi & the
antibody produced in infected animals/humans.
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Older Techniques
:to detect O. tsutsugamushi
1. Giemsa Staining Technique
:- utilizes peritoneal scrapings of infected mice.
2. Weil-Felix Proteus Agglutination Test
:-is a test which relies on the fact that Rickettsia and Proteus OX strains have common
antigens.
:-is a test for the presence & type of rickettsial disease based on the agglutination of Xstrain
Proteus vulgaris with suspected Rickettsia in a patient’s blood serum sample.
:-is commonly used in hospitals & clinics
:-This test is now being replaced by a complement-fixation test.
Weil-Felix Proteus Agglutination Test
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Older Techniques
:to detect O. tsutsugamushi
3. Complement Fixation Test (CFT)
:-is a serological test to detect specific antibody or specific antigen in a patient's serum.
The test utilizes sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with
specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in
serum).
If antibody (or antigen) is present in the patient's serum, then the complement is completely
utilized and SRBC lysis is minimal.
However, if the antibody (or antigen) is not present in the patient's serum, then the complement
binds anti-SRBC antibody and lysis of the SRBCs ensues.
Complement Fixation Test (CFT)
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Older Techniques
:to detect O. tsutsugamushi
All 3 techniques are commonly used to diagnose
rickettsial disease.
However, they often provide false positives &
prove to be non-specific, insensitive, &
inaccurate detection-methods
for scrub typhus diagnosis.
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
Detection of the presence of antibody against O. tsutsugamushi
Using serum samples collected from infected animal & humans
1. Indirect Immuno-Fluorescence
(IFA)
IFA=is a test used to detect antibodies in
serum or other body fluids.
The specific antibodies are labeled with a
compound that makes them glow an applegreen color when observed microscopically
under a fluorescence microscope.
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
2. Indirect Immuno-Peroxidase
(IIP)
IIP= is a modification of IFA technique that
replaces the fluorochrome with peroxidase.
Slide is observed using a bright-field
microscope.
Staining reaction is positive when O.
tsutsugamushi particles stain light brown.
Control
Infected
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
Immuno-cytochemical staining
3. Immuno-cytochemical staining of serial
Infected
paraffin sections of scrub typhus infected
Leptotrombidium chiggers.
Immuno-cytochemical staining is a modification of the IIP
technique by adding peroxidase-label to
detect the positive areas of infected tissues
specimens on the paraffin-embedded serial
section and/or semi-thin plastic sections.”
Control
Showing positive staining of ovary & brain
tissues of infected organs
With the availability of monoclonal antibody to the specific
antigen, this technique provides one of the best
detection methods with its sensitive, specific,
accuracy.
However, it’s difficult, time-consuming & expensive and so
it’s one of the least popular methods.
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
4. Enzyme-linked Immunosorbent Assay (ELISA)
ELISA test is a technique for detecting & measuring antigen or antibody.
:-It is one of the most reliable techniques to detect antibody
against scrub typhus infection.
:-Its procedure is the principal for development of recent
rapid diagnostic kits.
:-This technique is widely used in laboratories & hospitals.
Ag-Ab
complex
Optical
Density
(OD)
Reading
Ag-coated
well
1. Add
antigens
2. Add mouse serum
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3. Add anti-Ab
4. Add enzymesubstrate mix
5.Let colorize
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
(Commercial Kits)
1. Pan-Bio ® Rapid diagnostic enzyme
dot blot immunoassay
(PanBio Pty. Ltd, Brisbane, Australia)
Panbio Kit is a rapid diagnostic kit designed for the qualitative
detection of IgM or IgG antibody to O.
tsutsugamushi. It uses the same principal as the
ELISA technique.
The kit consists of ready-to-use antigen-coated micro-wells
and reagents.
Color intensity developed by the reaction in the micro-wells
is directly related to the concentration of the
antibody.
Pan-Bio Assay
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Newer Techniques
:to detect O. tsutsugamushi
Immunological Assays
(Commercial Kits)
2. Standard Diagnostics (SD) BioLine
Tsutsugamushi-Assay
(Standard Diagnostics Inc. Yongin-si, Kyonggi-do, Korea)
This new scrub typhus diagnostic kit was developed by
the SD BioLine Company in Korea:
It is a solid phase immuno-chromatographic assay for
rapid, qualitative detection of IgG, IgM, IgA antibodies to
O. tsutsugamushi in human serum, plasma & whole blood.
This test provides only a preliminary test result.
Therefore, other serological tests like IFA & ELISA must be
used in order to confirm the O. tsutsugamushi infection.
SD Bio-Line Assay
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Comparison of Sensitivity & Specificity of
SD BioLine-Kit vs PanBio-Kit vs ELISA
when Tested with the same serum samples
Type of Assay
Sensitivity
Specificity
SD BioLine-Kit
84.0%
94.4%
88.0%
95.0%
96.0%
94.4%
PanBio-Kit
ELISA
PanBio & SD BioLine Kits could not detect positive scrub typhus at low titers
ELISA provided more sensitivity & equal specificity when compared
to the commercial rapid-diagnostic kits for scrub typhus.
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Newer Techniques
:to detect O. tsutsugamushi
Detection of the presence of O. tsutsugamushi bacteria
Using blood, animal tissue samples and/or chigger specimens
PCR (Polymerase Chain Reaction)
DNA of O. tsutsugamushi is extracted from specimens using a
Wizard Genomic DNA Purification Kit (Promega , Wisconsin, USA)
Nested PCR is performed using primers selected from the
DNA sequence of genes encoding the 56 kD protein of
the Karp strain (Horinuouchi et al. 1996).
A real time quantitative PCR (rtq-PCR) method is also
being used as a means to provide quantitative
information on O. tsutsugamushi especially in
chigger specimens.
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Newer Techniques
:to detect O. tsutsugamushi
For detection of the presence of
O. tsutsugamushi from blood, animal tissue
samples & in chigger specimens, the standard
PCR technique provides the most sensitivity,
reliability & accuracy
7900 HT Sequence Detection
System(Applied Biosystem-ABI PrismTM)
A modification of the PCR-procedures by shortening
the time to extract DNA from the original
samples & using a portable/battery-operated
rtq PCR (Smart-Cycler® II system), could
provide results within hours & would give us
a very rapid diagnostic tool.
Smart Cycler ® Model (Cepheid Bio-Active)
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In Conclusion : Scrub Typhus Diagnostics:
Which Works Best?
:-IFA, IIP & Immunocytochemical Staining techniques are for clinical &
laboratory diagnosis, require complicated staining procedures,
microscopic equipment & well-trained technicians.
:-ELISA has proven to be the most sensitive, reliable & specific technique
for routine detection of scrub typhus.
:-ELISA is the most commonly used technique for detection of scrub
typhus antibody.
:-Commercial-Rapid diagnostic kits (PanBio & SD BioLine Kits) for scrub
typhus provide reliable & well-accepted preliminary results,
however, other serological tests (IFA, IIP & ELISA), must be used
in order to obtain a confirmation of O. tsutsugamushi infection.
:-PCR is the most reliable technique for detecting scrub typhus antigen
from blood, animal tissue samples & chigger specimens.
:-A modification of the PCR technique that can be used in the field as a
rapid-diagnostic tool is needed.
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Recommended Scrub Typhus
Diagnostic Techniques
ELISA Technique To Detect Scrub Typhus Antibody
PCR & rtq-PCR Technique To Detect Scrub Typhus Antigen
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THANK YOU
Questions
SAWASDEE
Ectoparasites and Systematics Section
Department of Entomology, USAMC-AFRIMS
Bangkok, Thailand
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