1. General Overview of Spirochaetales Gram-negative spirochetes Spirochete from Greek for “coiled hair” Extremely thin and can be very long Tightly coiled helical cells with tapered ends Motile by Periplasmic flagella (axial fibrils or endoflegalla) Dr.T.V.Rao MD1

2. Human pathogen Treponema Borreilia Leptospira 2

3. How they appear 3

4. GenusSpeciesDisease Treponemapallidum ssp. pallidum pallidum ssp. endemicum pallidum ssp. pertenue carateum Syphilis Bejel Yaws Pinta Borreliaburgdorferi recurrentis Many species Lyme disease (borreliosis) Epidemic relapsing fever Endemic relapsing fever LeptospirainterrogansLeptospirosis (Weil’s Disease) Spirochaetales Associated Human Diseases 4

5. 5 Treponema pallidum

6. 6

7. Morphology ◦ Motile, sluggish in viscous environments ◦ Size: 5 to 20 μm in length & 0.09 to 0.5 μm in diameter, with tapered ends ◦ Can be seen on fresh primary or secondary lesions by dark field microscopy or fluorescent antibody techniques 7

8. Staining with special stains Staining by Giemsa and Fontana 8

9. Dark field Microscopy

10. Cultivation of.. ? Treponemes cannot be cultivated in laboratory media and are maintained by subculture in susceptible animals. 10

11. Primary Syphilis - Chancre 11

12. Secondary Syphilis 12

13. Secondary Syphilis 13

14. Secondary Syphilis 14

15. Secondary syphilis 15

16. 16

17. Diagnosis of syphilis A. Direct detection of spirochetes : Darkfield microscopy Silver stain B. Culture : not used C. Serology: non-specific and specific tests 17

18. Venereal Disease Research Laboratory - VDRL 18

19. Rapid Plasma Reagin Test - RPR - General screening test, can be adapted to automation. - CANNOT be performed on CSF. Antigen ◦ VDRL cardiolipin antigen is modified with choline chloride to make it more stable ◦ attached to charcoal particles to allow macroscopic reading ◦ antigen comes prepared and is very stable. Serum or plasma may be used for testing, serum is not heated. 19

20. Treponema pallidum haemagglutination (TPHA) Adapted to micro techniques (MHA- TP) Tanned sheep RBCs are coated with T. pallidum antigen from Nichol’s strain. Agglutination of the RBCs is a positive result. 20

21. Serologic Tests All positive nontreponemal test results should be confirmed with a specific treponemal test 21

22. Every Pregnant women Needs Screening 22

23. 23 Borrelia burgdorferi and Lyme disease

24. B. burgdorferi - Causes Lyme disease, transmitted by the bite of Ixodid ticks (deer tick) - Diagnosis – serological tests

25. 25 Diagnosis - serum antibodies to B. burgdorferi - laboratory strains grow extremely slowly tissue culture media not bacteriological media. culture media not bacteriological media. - patient body fluids/tissue sample almost never grow grow

26. 26 - B. recurrentis Only detected on blood smear

27. 27 Leptospirosis

28. Leptospira - Very thin, delicate spirochetes with hooked ends - 2 species are recognised : 1.L. interrogans – pathogenic to human (rats are the reservoir) 2.L. biflexa – saprophytic, mainly found in surface water.

29. L. interrogans in silver stained smear, hooked ends resemble umbrella handles

30. - Transmission - Leptospires in water contaminated by the urine of rats

32. Diagnosis - Examination of blood – 1 st week only - Urine – 2 nd week of disease, should be examined immediately after voiding - Serology – Abs appear by the end of 1 st week & increase till 4 th week of disease.

33. Microbiology Obligatory intracellular bacteria Infect columnar epithelial cells Survive by replication that results in the death of the cell Takes on two forms in its life cycle: ◦ Elementary body (EB) ◦ Reticulate body (RB) 33

34. Chlamydiaceae Family (species that cause disease in humans) 34 SpeciesDisease C. trachomatis 2 biovars, non-LGV LGV Trachoma, NGU, MPC, PID, conjunctivitis, Infant pneumonia, LGV C. pneumoniae Pharyngitis, bronchitis, pneumonia C. psittaciPsittacosis

35. 35 Chlamydial inclusion bodies (Giemsa smear)

36. 36 Chlamydial inclusion bodies (Giemsa smear)

37. 37 Chlamydial inclusion bodies (immunofluorescent stain)

38. 38 Chlamydial inclusion bodies (immunofluorescent stain)

39. Culture - Historically the “gold standard” - Variable sensitivity (50%-80%) - High specificity - Use in legal investigations - Not suitable for widespread screening 39

40. NAATs - NAATs amplify and detect organism-specific genomic or plasmid DNA or rRNA - Significantly more sensitivity than other tests 40

41. Non-Amplication Tests - Direct fluorescent antibody (DFA) - Detects intact bacteria with a fluorescent antibody - Variety of specimen sites - Enzyme immunoassay (EIA) - Detects bacterial antigens with an enzyme-labeled antibody - Nucleic acid hybridization (NA probe) - Detects specific DNA or RNA sequences of C. trachomatis and N. gonorrhoeae 41

42. Why Screen for Chlamydia? - Screening can reduce the incidence of PID by more than 50%. - Most infections are asymptomatic. - Screening decreases the prevalence of infection in the population and reduces the transmission of disease. 42