presentation

• Armed Forces Research Institute of Medical Sciences www.afrims.orgwww.afrims.org

CPT (P) Jittawadee R. Murphy, Ph.D.

Presented by

Scrub Typhus Diagnostics: Which Works Best? Which Works Best?

K. Lerdthusnee,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 EntomologyUS Army Medical Component-Armed Forces Research Institute of Medical Sciences

(USAMC-AFRIMS), Bangkok 10400, Thailand

DoD Tri-Service Pest Management Workshop at the Naval Air Station, Jacksonville, Florida, USA,

12-16 February 2007


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.

Scrub Typhus

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.


Pathogen: Orientia tsutsugamushi

Rickettsial bacteria

Vector: Leptotrombidium

Chigger-Mite

An acute febrile, rickettsial disease caused by a gram-negative, rod-shaped (cocco-bacillus) bacterium, known as Orientia (Rickettsia) tsutsugamushi.

Scrub Typhus: A Rickettsial Disease

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.


Scrub Area

Areas Around Houses

Dry HabitatsDry Habitats Wet HabitatsWet Habitats

Rice FieldThe term scrub of scrub typhus came from the type of vegetations (terrain between woods & clearings) that harbor the vectors.

Moist Areas: Swamp & Bog

Leptotrombidium Chigger’s Habitats

Edges of Dense Forest


Humans acquire the disease when infected chiggers bite them and transmit O. tsutsugamushi.

Scrub Typhus Symptoms

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.

Eschar = Clinical Sign of Scrub Typhus

Mortality rates in untreated patients normally range from 0-30% 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.


Reservoir Host :Rattus rattus

In chiggers, O. tsutsugamushi bacteria is transmitted:

:-trans-ovarially (from adult female to eggs) &

:-trans-stadially (from egg to all immature & adult stages)

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.

Only larval Leptotrombidium mites (chiggers) transmit the disease; however, the bacteria can be found in all mite developmental stages (from egg to adult).


Scrub-typhus (Scrub-typhus (Orientia tsutsugamushiOrientia tsutsugamushi) – Infected ) – Infected LeptotrombidiumLeptotrombidium ( (L.)L.) Chigger Colonies Chigger Colonies

Scrub typhus-infected -Leptotrombidium chigger colonies are housed in an environmental control chamber, located in our Bio-Safety Level-III (BSL-3) Room-facility.

•AFRIMS-Ectoparasite Lab in Bangkok is one of two laboratories in the world capable of rearing/colonizing scrub typhus infected Leptotrombidium mites.

•Currently, 4 different species of scrub typhus infected Leptotrombidium chiggers are colonized.

Adult Mite

Chigger-Mite

Leptotrombidium Leptotrombidium Chigger Chigger Vector of Scrub Typhus Vector of Scrub Typhus


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.


1. Giemsa Staining Technique:- utilizes peritoneal scrapings of infected mice.

Older Techniques:to detect O. tsutsugamushi

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 X-strain 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



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)



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.


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 apple-green color when observed microscopically under a fluorescence microscope.

Immunological Assays

Detection of the presence of antibody against O. tsutsugamushiUsing serum samples collected from infected animal & humans

Newer Techniques:to detect O. tsutsugamushi


2. Indirect Immuno-Peroxidase (IIP)

Control Infected

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.




3. Immuno-cytochemical staining of serial paraffin sections of scrub typhus infected

Leptotrombidium chiggers.

Control

Infected

Immuno-cytochemical staining

Showing positive staining of ovary & brain tissues of infected organs

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.”

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.




4. Enzyme-linked Immunosorbent Assay (ELISA)

1. Add antigens

Ag-coated well

3. Add anti-Ab2. Add mouse serum

Ag-Ab complex

Optical Optical Density Density

(OD) (OD) Reading Reading

4. Add enzyme-4. Add enzyme-substrate mixsubstrate mix

5.Let colorize5.Let colorize

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.




1. Pan-Bio ® Rapid diagnostic enzyme dot blot immunoassay

(PanBio Pty. Ltd, Brisbane, Australia)

Pan-Bio Assay

Panbio Panbio Kit Kit is is a rapid diagnostic kit designed a rapid diagnostic kit designed for the qualitative for the qualitative detection of IgM detection of IgM or IgGor IgG antibod antibody to y to O. O.

tsutsugamushi.tsutsugamushi. It uses the same principal as the It uses the same principal as the ELISA technique. ELISA technique.

The kit consists of ready-to-use antigen-coated micro-wells The kit consists of ready-to-use antigen-coated micro-wells and reagents. and reagents.

Color intensity developed by the reaction in the micro-wells Color intensity developed by the reaction in the micro-wells is directly related to the concentration of the is directly related to the concentration of the antibody.antibody.

Immunological Assays(Commercial Kits)




2. Standard Diagnostics (SD) BioLine Tsutsugamushi-Assay

(Standard Diagnostics Inc. Yongin-si, Kyonggi-do, Korea)

SD Bio-Line Assay

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.

Immunological Assays(Commercial Kits)


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-Kit84.0%

94.4%

PanBio-Kit88.0% 95.0%

ELISA 96.0% 94.4%

ELISA provided more sensitivity & equal specificity when compared to the commercial rapid-diagnostic kits for scrub typhus.

PanBio & SD BioLine Kits could not detect positive scrub typhus at low titers



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).

PCR (Polymerase Chain Reaction)

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.

Detection of the presence of O. tsutsugamushi bacteriaUsing blood, animal tissue samples and/or chigger specimens


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 & accuracy7900 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)



:-IFA, IIP & Immunocytochemical Staining techniques are for clinical & laboratory diagnosis, require complicated staining procedures, microscopic equipment & well-trained technicians.

In Conclusion : Scrub Typhus Diagnostics: Which Works Best? Which Works Best?

:-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.


Recommended Scrub Typhus Recommended Scrub Typhus Diagnostic Techniques Diagnostic Techniques

ELISA Technique To Detect Scrub Typhus Antibody

PCR & rtq-PCR Technique To Detect Scrub Typhus Antigen


THANK YOU THANK YOU

QuestionsQuestions

Ectoparasites and Systematics SectionDepartment of Entomology, USAMC-AFRIMS

Bangkok, Thailand

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presentation

Health & Medicine

sd bioline

standard diagnostics

immunological

scrub typhus

newer techniques

commercial

older techniques

serological