PRESENTATION

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  • Eschar = red indurated lesion that eventually enlarges to 8-12 mm in diameter, vesiculates and ruptures, and becomes dark and necrotic in the center
  • 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. 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.
  • SD BioLine-Kit Manufacturer’ Claim - when compared to IFA technique = where this compared in our lab. If not, how were the specimens used against the same serum samples??
  • COULD BE OR ARE CONSIDERED GOLD STANDARDS. There is only one gold standard. This slide doesn’t make any sense. How can you have 3 gold standards but the ELISA and it is also a gold standard.
  • PRESENTATION

    1. 1. CPT (P) Jittawadee R. Murphy, Ph.D. Presented by 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 DoD Tri-Service Pest Management Workshop at the Naval Air Station, Jacksonville, Florida, USA, 12-16 February 2007
    2. 2. An important vector-borne disease, first described in 1899 in Japan . D uring World War II, this disease killed t housands 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.
    3. 3. 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.
    4. 4. Scrub Area Areas Around Houses Dry Habitats Wet Habitats Rice Field The 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
    5. 5. Humans acquire the disease when infected chigger s bite them and transmit O. tsutsugamushi . Scrub Typhus Symptoms B acteria multiply at the inoculation site and frequently form a papule that ulcerates & becomes necro tic. This pathognomic focal lesion is called an eschar. Re gional 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 .
    6. 6. 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 & human s , the parasite s/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). Reservoir Host : Rattus rattus
    7. 7. Scrub-typhus ( Orientia tsutsugamushi ) – Infected Leptotrombidium ( L.) 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. <ul><li>AFRIMS-Ectoparasite Lab in Bangkok is one of two l aborator ies in the world capable of rearing/colonizing scrub </li></ul><ul><li>typhus infected Leptotrombidium </li></ul><ul><li>mites . </li></ul><ul><li>Currently, 4 different species of scrub typhus infected Le ptotrombi dium chiggers are colonized. </li></ul>Adult Mite Chigger-Mite Leptotrombidium Chigger Vector of Scrub Typhus
    8. 8. 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.
    9. 9. <ul><li>Giemsa Staining Technique </li></ul><ul><li>:- utilizes peritoneal scrapings of infected mice. </li></ul>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. Older Techniques :to detect O. tsutsugamushi Weil-Felix Proteus Agglutination Test
    10. 10. 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. Older Techniques :to detect O. tsutsugamushi Complement Fixation Test (CFT)
    11. 11. 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. Older Techniques :to detect O. tsutsugamushi
    12. 12. IFA=is a test used to detect antibodies in serum or other body fluid s . The specific antibodies are labeled with a compound that makes them glow an apple-green colo r when observed microscopically under a fluorescence microscope. Immunological Assays Detection of the presence of antibody against O. tsutsugamushi Using serum samples collected from infected animal & humans 1. Indirect Immuno-Fluorescence (IFA) Newer Techniques :to detect O. tsutsugamushi
    13. 13. 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. Immunological Assays Control Infected Newer Techniques :to detect O. tsutsugamushi
    14. 14. 3. Immuno-cytochemical staining of serial 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. ” 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. Immunological Assays Control Infected Immuno-cytochemical staining Showing positive staining of ovary & brain tissues of infected organs Newer Techniques :to detect O. tsutsugamushi
    15. 15. 4. Enzyme-linked Immunosorbent Assay (ELISA) ELISA test is a technique for detecting & measuring antig en 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. Immunological Assays 1. Add antigens Ag-coated well 3. Add anti-Ab 2. Add mouse serum Ag-Ab complex Optical Density (OD) Reading 4. Add enzyme-substrate mix 5.Let colorize Newer Techniques :to detect O. tsutsugamushi
    16. 16. <ul><li>Pan-Bio ® Rapid diagnostic enzyme dot blot immunoassay </li></ul><ul><li>( PanBio Pty. Ltd, Brisbane, Australia ) </li></ul>Panbio Kit is a rapid diagnostic kit designed for the qualitative detection of IgM or IgG antibod y 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. Immunological Assays (Commercial Kits) Pan-Bio Assay Newer Techniques :to detect O. tsutsugamushi
    17. 17. 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. Immunological Assays (Commercial Kits) Newer Techniques :to detect O. tsutsugamushi SD Bio-Line Assay
    18. 18. Comparison of Sensitivity & Specificity of SD BioLine-Kit vs PanBio-Kit vs ELISA when Tested with the same serum samples 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 95.0% 88.0% PanBio-Kit 94.4% 84.0% SD BioLine-Kit Type of Assay 94.4% 96.0% Specificity Sensitivity ELISA
    19. 19. 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 bacteria Using blood, animal tissue samples and/or chigger specimens Newer Techniques :to detect O. tsutsugamushi
    20. 20. 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 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. 7900 HT Sequence Detection System(Applied Biosystem-ABI Prism TM ) Smart Cycler ® Model (Cepheid Bio-Active) Newer Techniques :to detect O. tsutsugamushi
    21. 21. :-IFA, IIP & Immunocytochemical Staining techniques are for clinic al & laboratory diagnosis , require complicated staining procedures, microscopic equipment & well-trained technicians. In Conclusion : Scrub Typhus Diagnostics: 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.
    22. 22. Recommended Scrub Typhus Diagnostic Techniques ELISA Technique To Detect Scrub Typhus Antibody PCR & rtq-PCR Technique To Detect Scrub Typhus Antigen
    23. 23. THANK YOU Questions Ectoparasites and Systematics Section Department of Entomology, USAMC-AFRIMS Bangkok, Thailand Greetings From Thailand SAWASDEE

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