1. Chapter 42 Treponema, Borrelia, and Leptospira Chapter 43 Mycoplasma and Ureaplasma Microbiology 2011 May 13, 2011 Yu Chun-Keung DVM, PhD

2. Chapter 42 Treponema, Borrelia, and Leptospira Spirochete 螺旋菌目 : 細長、彎曲、有運動力的細菌 密螺旋體 疏螺旋體 鉤端螺旋體

3. Order Spirochaetales Human diseaseEtiologic agent Treponema 密螺旋體梅毒 Syphilis T. pallidum 貝傑 Bejel T. endemicum 斑病 Yaws T. pertenue 莓疹病 Pinta T. carateum Borrelia 疏螺旋體回歸熱 Relapsing fever B. recurrentis 萊姆氏病 Lyme disease B. burgdorferi Leptospira 鈎端螺旋體 Leptospirosis 鈎端螺旋體症 L. interrogans

4. T. pallidum Strict human pathogen, experimental syphilis only in rabbits, cannot grown in cell-free cultures Syphilis ( 梅毒 ), sexually transmitted disease Syphilis : syn - together, philis - love

5. T. pallidum Virulence factors: Outer membrane protein for adherence, Hyaluronidase for perivascular infiltration, Coated with fibronection for protection against phagocytosis Immune response to infection: tissue destruction Six axial filaments ( 中軸纖維 )

6. Clinical course Primary phase: papule ( 丘疹 ), hard chancre ( 硬 性下疳, painless ulcer) at site of inoculation, lymphadenopathy, endarteritis and periarteritis, heal spontaneously within 2 months, highly infectious, Ab(-). Secondary phase: flu-like syndrome, generalized skin rash and condylomas ( 扁平濕疣 ), meningitis, hepatitis, etc; subside spontaneously week to month, highly infectious, Ab(+). Latent: 2 or 3-10 yr, no clinical sign, non- infectious.

7. Late (tertiary) phase: all tissues may be involved, cardiovascular lesions (aneurysm 主動脈瘤 80-85%), CNS degeneration (5-10%), granulomatous lesions (gummas, 梅毒腫 ) in skin, bone and liver. 30% of cases completely cure after 1 st or 2 nd phase; another 30%, latent, serologic test (+); remainder, progress to tertiary stage. Clinical course

8. Congenital syphilis Treponemas invade fetus at the fifth month of gestation Causes abortion, stillbirth, or death soon after delivery Congenital abnormality: interstitial keratitis, Hutchinson's teeth, saddle nose, and eighth nerve deafness

9. Epidemiology Worldwide; in USA Neisseria gonorrhoeae > Chlamydia > Syphilis Extremely sensitive to drying or disinfectants, cannot be spread through contact with inanimate objects Not highly contagious, 30% change of infection after a single sexual contact Can be acquired congenitally or by transfusion; bacteremia can persist for > 8 years Incidence of late syphilis has markedly decreased, primary and secondary syphilis remain high

10. Diagnosis of syphilis Direct detection of spirochetes : Darkfield microscopy (motile bugs + experience + prompt examination) Silver stain Culture : not used Serology: non-specific and specific tests

11. Non-treponemal tests Antigen: cardiolipin (beef heart) + lecithin + cholesterol Detect nonspecific antibody (Reagin): a mixture of IgM & IgG direct against some normal tissue antigens VDRL (Venereal Disease Research Laboratory) test for serum and CSF samples

12. Advantage of VDRL: cheap, easy to perform quantitative, screen test monitor disease course trace theraputic effect, become “ - ” in 6-18 m after effective treatment.

13. Disadvantage of VDRL Transient false positive: acute febrile disease, vaccination, pregnant women Long-term false positive: chronic autoimmune diseases, liver diseases False negative: progressive tertiary syphilis

14. Treponemal tests Antigen: T. pallidium Detect specific antibody to T. pallidium (1) Fluorescent treponemal antibody (FTA) test Killed organism + patient's serum + labeled anti-human Ig = fluorescence “+” if Ab present (2) Micro-hemagglutination for T. pallidum (MHA-TP) test (RBC + treponemas) + patient's serum = “clumps” if Ab present

15. Treponemal tests Confirmative tests for syphilis Influenced less by therapy False positive: autoimmune diseases Positive serologic test in infants: mean passive transfer of antibody OR congenital infection Re-test 6 months later

16. T/P/C Penicillin: especially for neurosyphilis for pregnant women Tetracycline, doxycyline No vaccine, safe sex

17. Other Treponemes – cause Nonvenereal treponemal diseases Bejel (endemic syphilis) Yaws Pinta

18. Bejel (endemic syphilis) T. pallidum subsp endemicum A highly infectious skin lesion, gummas of skin, bones, and nasopharynx Occur among children in Africa, Asia, and Australia. Person-to-person, contaminated eating utensil

19. T. pallidum subsp pertenue A chronic skin disease among children; destructive lesions of skin, lymph nodes, bones Tropical areas of South America, Central Africa, Southeast Asia Person-to-person, direct contact Yaws Nodules on the elbow resulting from a Treponema pertenue bacterial infection. The Free Dictionary

20. T. pallidum subsp carateum A skin disease causing unsightly pigment changes Central and South America Person-to-person, direct contact Pinta

21. Chapter 42 Treponema, Borrelia, and Leptospira Borrelia recurrentis Relapsing fever ( 回歸熱 ) Borrelia burgdorferi Lyme disease ( 萊姆病 )

22. A bloodstream infection producing 3 to 10 recurrence of febrile and afebrile cycles. Incubation period about 1 week Bacteremic phase (release endotoxin) 3-7 days - fever, chills, muscle aches, headache, splenomegaly, hepatomegaly The bacteria are rapidly removed by specific antibody (agglutination and complement- mediated lysis) Relapsing fever ( 回歸熱 )

23. Afebrile period 1 week Antigenic variation - alter serotype-specific outer envelope proteins through gene rearrangement Relapse

24. Borrelia recurrentis : epidemic relapsing fever Human body louse (no transovarian transmission), short lifespan Single relapse, more severe, mortality 4%-40% Borrelia spp : endemic relapsing fever Soft ticks (transovarian transmission), long lifespan Repeated relapse, mortality <5% 軟蜱 硬蜱 體蝨

25. Lab diagnosis Human blood → blood smear → Giemsas or Wright stain → Borellia are large enough to be detected by microscope Human blood → inject into to the abdomen of laboratory mouse → mouse blood → smear → stain → Detect Borellia

26. It is caused by spirochete Borrelia burgdorferi It is transmitted to humans by the bite of a hard-shelled tick (Ixodes). Reservoir – rodents, deer, ticks (transovarian transmission) Lyme disease ( 萊姆病 ) Kenneth Todar University of Wisconsin-Madison Department of Bacteriology

27. Disease stages of Lyme disease Initial stage Incubation period 3-30 days Erythema chronicum migrans ( 游走性紅斑 ) develop at site of tick bite Flu-like illness – malaise, severe fastigue, headache, fever, chill Lymphadenopathy, >4 weeks

28. Late stage 80% patients develop late manifestation (if untreated) First phase – neurologic symptoms (meningitis, encephalitis … ), cardiac dysfunction Second phase - arthralgia and arthritis Low numbers of organisms present in skin lesion, immunopathology (?)

31. Sensitivity of Diagnostic Tests for Borrelia Infections Test Sensitivity Test Relapsing fever Lyme disease Microscopy Good Poor Culture Poor Poor Serology Not available confirmatory tests* * IFA, ELISA

32. Treatment Relapsing fever: Tetracycline, Erythromycin Lyme disease: doxycycline

33. Chapter 42 Treponema, Borrelia, and Leptospira

34. Leptospira Leptospira interrogans ( 問號型鉤端螺旋體 ) 218 serovars; shaped like a question mark; pathogenic for wild and domestic animals and humans Leptospira biflexa 63 serovars; twice bent, a free-living saprophyte, not associated with disease

35. Grown in medium with rabbit serum or bovine serum albumin (neither treponema nor borrelia can) Natural reservoir: rodents (rats), farm animals Colonize the renal tubules and shed in urine Streams, standing water, moist soil – source of infection, survive > 6 weeks

36. An zoonotic disease Recreational exposure, occupational exposure, flood; no person-to-person spread Penetrate intact mucous membranes or skin through cuts or abrasions, through blood stream, spread to all tissues Multiply rapidly and damage endothelium of small blood vessels

37. Mild leptospirosis (90% of cases) Severe leptospirosis (Weil ’ s disease) Acute stage (septicemic phase) : the first week after organisms enter the bloodstream, leptospira can be cultured from the blood and cerebrospinal fluid. A short asymptomatic period Chronic stage (immune phase) : leptospirae are found only in the urine. Jaundice (organisms invade liver); nephritis (organisms invade kidney). Death results from kidney failure. Leptospirosis

38. Test MethodSensitivity Microscopy Gram stainInsensitivity DarkfieldInsensitivity FAInsensitivity Culture Blood“+” during 1st wk CSF“+” during 1st or 2nd wk Urine“+” after 1st wk Serology Microagglu.*Sensitive, specific *patient ’ s serum to agglutinate live leptospira (reference laboratory test) Diagnostic tests for Leptospirosis

39. T/P/C Penicillin or ampicillin i.v. for severe cases Doxycycline, ampicillin p.o. for less severe cases Doxycycline for prophylaxatic Vaccination of livestocks and pets Rodent control

40. Wash Those Soda Cans This incident happened recently in North Texas. On Sunday, a woman went boating taking with her some cans of coke which she put into the refrigerator of the boat. On Monday she became ill and was taken to the hospital and placed in the Intensive Care Unit. She died on Wednesday. The autopsy concluded she died of Leptospirosis. This was traced to the can of coke she drank from, not using a glass. Tests showed that the can was infected by dried rat urine and hence the disease Leptospirosis. Rat urine contains toxic and deathly substances. It is highly recommended to thoroughly wash the upper part of soda cans before drinking out of them. The cans are typically stocked in warehouses and transported straight to the shops without being cleaned. A study at NYCU showed that the tops of soda cans are more contaminated than public toilets (i.e.).. full of germs and bacteria. So wash them with water before putting them to the mouth to avoid any kind of fatal accident.

41. Chapter 43 Mycoplasma and Ureaplasma 200 species; 16 colonize humans and 5 associated with diseases Mycoplasma ( 黴漿菌 ) M. pneumoniae M. hominis M. genitalium Ureaplasma ( 尿漿菌 ) U. urealyticum

42. Smallest (0.1-0.3  m) and simplest free-living bacteria (about twice the genome size of certain large viruses) Small, fried-egg-like colonies (except M. pneumoniae)

43. Lack a cell wall Highly pleomorphic shapes Resistant to penicillin, cephalosporins, vancomycin, but sensitive to tetracycline, erythromycin. Cell membrane contains sterols - rigid Anaerobic (except M. pneumoniae) Grow slowly in cell-free media, need sterols, use glucose as a source of energy (ureaplasmas require urea)

44. Epidemiology M. pneumoniae Strict human pathogen Worldwide disease with no seasonal incidence Most common in school-age children and young adults (5-15y), but all age groups are susceptible Spread by respiratory droplets during coughing episodes in close contact among classmate or family members

45. U. urealyticum, M. hominis, and M. genitalium Infants (females) are colonized with the agents Carriage does not persist. Only a small proportion of prepubertal children remains colonized The incidence of genital mycoplasmas is associated with sexual activity Sexually active men and women 15% with M. hominis and 45-75% with Ureaplasma

46. Extracellular pathogen; infect and colonize mucous membrane (nose, throat, trachea, LRT). Pathogenesis - M. pneumoniae Adheres to sialated glycoprotein receptor (1) at the base of cilia, (2) on surface of RBC by means of P1 antigen.

47. Causes ciliostasis, destroy cilia and ciliated epithelial cells; breakdown clearance activity, lead to LRT infection and persistent cough. M. pneumoniae contains superantigen, can attract inflammatory cells and induce cytokine secretion (TNF, IL-1, IL-6). Pathogenesis - M. pneumoniae

48. Clinical disease - M. pneumoniae Mostly asymptomatic carriage Cause mild URT disease (acute pharyngitis), low-grade fever, malaise, headache, dry and nonproductive cough, persist for > 2 weeks Tracheobronchitis with lymphocyte and plasma cell infiltration, and atypical (walking) pneumonia Secondary complication: hemolytic anemia, arthritis, myocarditis, pericarditis, neurologic abnormalities (e.g., meningoencephalitis)

50. Typical pneumonia - bacterial pneumonia Abrupt, rigorous onset Productive cough, purulent sputum High fever, chest pain, stiffness in the neck Chest consolidation and rales. Murray, et.al: Textbook of Respiratory Medicine

51. Atypical (walking) pneumonia Chronic in both onset and recovery Flulike symptomes - generalized aches, discomfort, headache, chill, dry cough, low- grade fever Chest radiographs: patchy broncho- pneumonia, interstitial pattern, not pneumonia Murray, et.al: Textbook of Respiratory Medicine

52. Diseases caused by U. urealyticum and M. genitalium and M. hominis M. genitalium : nongonococcal urethritis (NGU), pelvic inflammatory disease U. urealyticum : NGU, pyelonephritis, abortion, premature birth M. hominis : pyelonephritis, postpartum fever, systemic infection in immunocompromised patients

53. Lab diagnosis Culture of mycoplasmas is not routinely attempted, and relatively insensitive M. pneumoniae can grow in special medium with animal serum (sterols), yeast extract (nucleic acid), glucose, pH indicator, and penicillin. Colonies have a “mulberry-shaped”. M. hominis requires arginine for growth. Colonies have a fried-egg appearance. Ureaplasma requires urea for growth Microscope: no cell wall, stain poorly, no value

54. Serology – for M. pneumoniae only Complement fixation test : high false- positive rate ELISA for detection of IgM and IgG Abs, more sensitive; need dual serum samples Cold agglutinins: Non-specific IgM Abs that bind the I antigen on human RBC at 4°C, develop in 65% of the patients – insensitive and nonspecific.

55. Treatment / Prevention / Control M. pneumoniae: erythromycin, tetracycline (also good for chlamydia) Ureaplasma: use erythromycin, resistant to tetracycline M. hominis: resistant to erythromycin and tetracycline, use clindamycin Avoidance or safe sex for genital mycoplasma No vaccine available