1. Spirochetes Margaret Fitzpatrick, MD, MS
Division of Infectious Diseases
September 10, 2018

2. Lecture Objectives
Distinguish the unique structural and morphologic characteristics of spirochetes, including those specific to Treponema pallidum, Borrelia, and Leptospira.
Identify the primary mode of transmission of Treponema pallidum

3. Spirochetes Group of bacteria that share a common spiral morphology
Flexible peptidoglycan cell well
One or more axial fibrils (“internal flagellum”)
Three major genera responsible for human disease: Treponema, Borrelia, Leptospira
: long and slender with helical (spiral) shape

4. Greenwood et al. ed. Medical Microbiology, 18th ed., 2012

5. Treponema pallidum Structural characteristics Thin, tightly coiled
Darkfield microscopy or immunofluorescence
Do not grow well in culture
Outer membrane does not contain lipopolysaccharide
No light microscopy
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

6. Treponema pallidum Culture and growth
Can be cultured but only for a few generations on media with rabbit epithelial cells
Very sensitive to drying and heat
Microaerophilic (3-5% oxygen)
Pathogenicity not well characterized

7. Treponema pallidum: epidemiology
Found worldwide and 3rd most common sexually transmitted infection in U.S.
Steady increase in prevalence since 2005
In 2012, CDC reported > 50,000 new cases
Incidence particularly rising among men who have sex with men (MSM)
Found only in humans

8. 1° and 2° Syphilis. US
MMWR : 402

9. Treponema pallidum: epidemiology
Transmission via direct sexual contact with an infective primary or secondary mucosal lesion
Genitals, anus, lip
Acquisition risk of 30% per sexual contact
Congenital infection
No sexual spread >4 years after acquiring infection
Cannot be spread through contact with inanimate objects
Lesions of primary and secondary syphilis are contagious

10. Lecture Objectives
From a clinical description, recognize the different stages of Treponema pallidum infection, including congenital infection

11. Treponema pallidum: clinical manifestations
Stage
Clinical hallmarks
Timeframe
Primary
Painless ulcer (chancre) at site of inoculation
Painless regional lymphadenopathy
10-90 d after infection
Secondary
Flu-like syndrome
Diffuse lymphadenopathy
Generalized mucocutaneous rash
2-10 weeks after chancre (peaks 3-4 months after infection)
Latent
Asymptomatic
A few years to as many as 25 years
Tertiary
Granulomatous lesions (gummas)
Neurosyphilis
Cardiovascular syphilis
Generally at least 5-10 years since infection
Regional LAD signals dissemination into blood stream as well
Chancre heals spontaneously after 2 months
Rash can be macular, papular, pustular and can involve whole skin surface including palms and soles
1/3 of untreated patients will get tertiary

12. Treponema pallidum: clinical manifestations
Primary syphilis
Chancre has smooth margins and crusted base
Darkfield positive
Firm local adenopathy
No systemic manifestations
Heals spontaneously
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

13. Treponema pallidum: clinical manifestations
Secondary syphilis
Rash – including palms and soles
In moist areaspapules coalesce – condylomata lata
Other sites: hepatitis, aseptic meningitis, periostitis, nephritis
Fever and generalized lymphadenopathy
Heals spontaneously
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

14. Treponema pallidum: clinical manifestations
Tertiary syphilis
1/3 of those untreated will develop tertiary
Gummas: granulomatous mass lesions in many different types of organs (bone, skin, liver, etc)
Neurosyphilis
Cardiovascular – aortic aneurysms, vasculitis
Not infectious at this stage
1/3 cured
1/3 latent syphilis
1/3 progress to tertiary

15. Treponema pallidum: clinical manifestations
Neurosyphilis
Stage
Type
Pathology
Symptoms and signs
Early (secondary)
Asymptomatic
CSF pleocytosis and elevated protein
None
Meningovascular
Meningitis
Vasculitis
Headache, neck stiffness, fever, cranial neuropathy, stroke
Late (tertiary)
General paresis
Chronic meningoencephalitis leading to brain atrophy
Dementia
Aphasia
Muscle weakness
Hallucinations
Tabes dorsalis
Demyelination of spinal cord posterior columns and dorsal roots
Ataxia
Loss of pain and temperature sensation

16. Treponema pallidum: clinical manifestations
Congential syphilis
Most babies born asymptomatic but then develop:
Rhinitis (‘snuffles’) and widespread rash, hepatomegaly
Long-term effects in those who survive:
Bone and teeth malformation – frontal ‘bossing’
Facial abnormalities – ‘saddle nose’
Blindness, deafness, cardiovascular disease
Prevent with routine screening and treatment during 1st trimester of pregnancy
Baby is born to a mother with untreated syphilis
In 2016, the number of CS cases was the highest it’s been since 1998!!

17. Lecture Objectives
Distinguish between non-specific non-treponemal and specific treponemal serologic tests for syphilis
Identify the common patterns of non-treponemal and treponemal serologic reactivity for different stages of syphilis

18. Treponema pallidum: diagnosis
Scrapings or tissue from primary or secondary lesions:
Darkfield microscopy
Direct fluourescent antibody
PCR
All are difficult to perform and not readily available
Darkfield has to be done immediately
Fluoroscein labeled antibodies stain bacteria
CDC/NCHSTP/Division of STD Prevention, STD Clinical Slides

19. Treponema pallidum: diagnosis
Serologic diagnosis with non-treponemal tests:
Reaginic antibodies
IgM and IgG against lipids (bovine cardiolipin)
NOT directed specifically against T. pallidum
VDRL (serum & CSF) – Venereal Disease Research Laboratory
RPR (serum) – Rapid Plasma Reagin
Quantitated into titers and used to follow treatment > revert to negative with treatment or enough time
False positive tests common
Preferred diagnosis
Screening tests that are rapid and cheap
IgM and IgG antibodies are produced against lipids that appear on cell surface of treponemes and also that are released from damaged human cells

20. Treponema pallidum: diagnosis
Serologic diagnosis with specific treponemal tests:
Fluorescent treponemal antibody absorption (FTA-Abs)
T. pallidum particle agglutination (TP-PA or TP-HA)
T. pallidum enzyme immunoassay (EIA) or chemiluminescence immunoassay (CIA)
Tests remain positive for life, regardless of treatment
Cheap, automated and now in wide use

21. Serologic Reactivity in Syphilis

22. Diagnostic Algorithms
False positive non-treponemal (RPR) tests:
Viral infection
Drugs
Autoimmune disease
Leprosy, malaria
Pregnancy
Recent immunization
Rheumatic fever
Rheumatoid arthritis
SLE
MMWR 2011; 60: 133; Tong ML, et al. CID 2015; 58: 1116

23. Treponema pallidum: treatment
Benzathine penicillin G (long acting)
Single injection for primary and early secondary
Three injections weekly for congenital, latent, tertiary
IV penicillin G for neurosyphilis
Jarisch-Herxheimer reaction
Fever, chills, headache, hypotension
Release of toxic products from dying spirochetes
Doxycycline is an alternative
Preferred

24. Question 1
A 23 year old man presents to his primary care physician with complaints of a diffuse rash all over his body. On physical examination, a maculopapular red rash is noted, including the palms and soles. The patient confirms he is sexually active and does not regularly use condoms. He thinks he may have had a painless sore on his penis a few months ago but isn’t sure. It went away on its own and he did not seek medical care at that time. His PCP suspects syphilis and sends serologic tests.
Which combination of serologic tests is most consistent with this patient’s current stage of infection?
RPR negative; TP-PA negative
RPR positive; TP-PA negative
RPR positive; TP-PA positive
RPR negative; TP-PA positive

25. Serologic Reactivity in Syphilis

26. Borrelia

27. Lecture Objectives:
Distinguish the unique structural and morphologic characteristics of spirochetes, including those specific to Treponema pallidum, Borrelia, and Leptospira.
Identify the Borrelia species responsible for Lyme disease in the U.S. and for epidemic relapsing fever
Describe how Borrelia escapes immune recognition during clinical relapsing fever

28. Borrelia
Larger spirochete that is spread from a mammalian reservoir to humans by tick or louse vectors resulting in Relapsing fever or Lyme borreliosis (Lyme disease)
Borrelia burgdorferi – Lyme in US and Europe
Borrelia garinii and B. afzelii – Lyme in Europe and Asia
Borrelia recurrentis – epidemic relapsing fever
Many species – endemic relapsing fever

29. Borrelia: structure and culture
Neither gram-pos nor gram-neg
Larger than other spirochetes
Stain with Giemsa or Wright’s stain
B. recurrentis can be seen in blood smear with light microscopy
Microaerophilic with complex nutritional needs = very difficult to culture
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

30. Borrelia: pathogenesis
Little known due to difficulty with isolation and culture
B. recurrentis escapes immune recognition by altering antigenic structure during infection
Gene switch from silent to expression locus (like N. gonorrhoeae) on plasmid
Relapses caused by emergence and multiplication of antigenic variants
Not much known due to difficulty in isolating and culturing these bacteria from clinical specimens

31. Lyme Disease Species of Borrelia Location Borrelia burgdorferi
US and Europe
B. garinii, B. afzelii
Europe and Asia

32. Lecture Objectives
Recognize the primary insect vectors, animal reservoirs, and geographic distribution for Borrelia burgdorferi
From a clinical description, distinguish between the early and late stages of Lyme disease

33. Borrelia burgdorferi: epidemiology
Leading vector-borne disease in U.S.
95% of cases are in two geographic areas:
Northeast and Mid-Atlantic states (Maine to Virginia)
Upper Midwest (Minnesota and WI)
Disease most common in children un

35. Borrelia burgdorferi: epidemiology
White-tailed deer and white-footed mouse are primary animal reservoirs
Spread by hard ticks
Ixodes scapularis – NE and Midwestern U.S.
Ixodes pacificus -- Western U.S.
Tick bites mouse/deer and then gets infected
Tick goes on to bite humans and spreads Borelia to humans
Different stages of ticks – larva, nymphs, adults – all can transmit but mostly nymphs
Tick needs to be attached and have prolonged feeding for > 48 hours

36. Tick bites mouse/deer and then gets infected
Tick goes on to bite humans and spreads Borelia to humans
Different stages of ticks – larva, nymphs, adults – all can transmit but mostly nymphs
Tick needs to be attached and have prolonged feeding for > 48 hours

37. Lyme disease: clinical manifestations
Early stage (3-30d post bite):
Erythema migrans – Expanding erythematous target-shaped lesion at site of tick bite
Can culture from biopsy
Often accompanied by flu-like illness – fever, chills, malaise, myalgias
Vesicles and central necrosis can be seen
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

38. Wormser, CID, 2006; 43: 1089.

39. Lyme disease: clinical manifestations
Early disseminated
Within days to weeks of primary infection
Fatigue, headache, fever
Arthritis and arthralgia (60%)
Myalgia
Erythematous skin lesions
Cardiac dysfunction (conduction block)
Neurologic: facial nerve paralysis or other cranial neuropathies, radiculopathy, meningitis, and rarely encephalomyelitis
** untreated

40. Lyme disease: clinical manifestations
Late-stage manifestations:
Recurrent/relapsing arthritis in large joints
Acrodermatitis chronica atrophicans (more common in Europe)
** untreated
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

41. Lecture Objectives
Describe the two-tier serologic testing for Lyme disease and recognize limitations of testing for early Lyme disease

42. Lyme disease: diagnosis
Serum serology is a two-tiered testing system:
Initial screening immunofluorescence assay (EIA, ELISA, IFA)
If negative you are done (unless within 4 weeks of tick bite or erythema migrans rash)
High rate of false + so must be confirmed with a second Western Blot test
Specific IgG and IgM antibodies
IgM should only be used if <4 months from exposure
Other spirochetes, RA, SLE, mono, subacute bacterial endocarditis can all lead to false positives
Your immune system continues to make the antibodies for months or years after the infection is gone. This means that once your blood tests positive, it will continue to test positive for months to years even though the bacteria are no longer present

43. Lyme disease: diagnosis
Nervous system Lyme disease diagnosis
Can see lymphocytic pleocytosis and elevated protein in CSF
Specific serology (IgG and IgM) from CSF
Also PCR available from CSF (reference laboratory)
In the absence of concurrent erythema migrans, need laboratory support for diagnosis
Many are serum positive by two-tiered Lyme antibody testing from serum

44. Lyme disease: treatment and prevention
Early: doxycycline, amoxicillin, cefuroxime x 10-14days
Late: oral as above except for CNS Lyme (ceftriaxone IV may be preferred)
Post-Lyme Disease Syndrome
Poorly understood constellation of non-specific symptoms following treatment
Fatigue, subjective cognitive deficits, arthralgias, myalgias
No evidence for chronic infection and antibiotics do not help
Prevention
Insect repellant, clothing, tick checks
Other spirochetes, RA, SLE, mono, subacute bacterial endocarditis can all lead to false positives
PLDS thought to be immune-mediated

45. Lecture Objectives
Distinguish between epidemic and endemic relapsing fever with regard to vectors, animal reservoirs, and geographic distribution.
Recognize the characteristic clinical manifestations and preferred method of diagnosis for relapsing fever

46. Relapsing Fever Organism Vector Reservoir Infection B. recurrentis
Body louse
Humans
Epidemic (louse-borne)
15 other Borrelia
Soft tick Ornithodoros
Rodents, e.g., chipmunks
Endemic (tick-borne)

47. Relapsing fever: epidemiology
Epidemic – Louse-borne
Crushed lice transmit disease through skin/membranes
Crowded, unsanitary conditions (war, famine, natural disaster)
Central and East Africa
Ethiopia, Eritrea, Somalia, Sudan
Endemic – Tick-borne
Tick bites can be very brief and often unrecognized
Worldwide distribution
Mountain U.S. (WA, CA)
Frequent human contact with infected lice

48. 504 cases – from
12 western states

49. Relapsing fever: clinical manifestations
Epidemic and endemic present essentially the same
Abrupt onset fever, chills, myalgias, headache with bacteremic phase
Splenomegaly and hepatomegaly
Resolves in 3-7 days and then recurs a week later
Single relapse for epidemic
As many as 10 relapses for endemic
Relapses are less severe
Mortality high for louse-borne epidemic disease
** untreated

51. Relapsing fever: diagnosis, treatment, prevention
Giemsa or Wright stained blood smear
Serology not helpful due to antigenic phase variation
Treatment:
Tetracyclines (preferred) or penicillins
Jarisch Herxheimer reactions common
Prevention:
Insect repellant, clothing
Rodent control, delousing sprays, hygiene
most sensitive (> 70% of infected patients
Goldenberger et al. Eurosurveillance, 2015; 20(32):pii=21204.

52. Question 2
A 24 year old man seeks care in the Emergency Department for recurrent fever. He has noted very high fever to 104 with severe headache and chills. This lasts a few days, then remits for about a week, and then recurs. He’s had three such episodes. About a week prior to fever onset, he was hiking in eastern Washington state and had tick bites noted. An astute ED physician suspects endemic (tick-borne) relapsing fever.
What is the preferred method of diagnosis for this infection?
Spirochetes observed on Wright/Giemsa stained blood smear
Serum serology with immunofluorescence assay
Blood culture
Serum nucleic acid testing (i.e., PCR)

53. Leptospirosis

54. Lecture Objectives
Distinguish the unique structural and morphologic characteristics of spirochetes, including those specific to Treponema pallidum, Borrelia, and Leptospira.
Identify the primary mode of transmission of Leptospirosis
From a clinical description, diagnose Leptospirosis

55. Leptospirosis: structural and culture characteristics
Thin, coiled spirochete with hook
Many different species and serotypes so generally just referred to as “Leptospirosis”
Culture is difficult but possible
Liquid media supplemented with vitamins, fatty acids, salts
obligate aerobes
Best viewed with darkfield microscopy
Murray, Rosenthal, Pfaller. Medical Microbiology, 8th edition

56. Leptospirosis: pathogenesis
Zoonosis affecting a variety of animal species
Humans acquire the organism by contact with infected animal urine usually through contaminated water
Penetrate intact mucous membranes or skin through small cuts or abrasions
Spread via blood to all tissues and damages endothelium of small blood vessels
Meningitis, hepatitis, renal dysfunction, hemorrhage, myocarditis
Vasculitis is main underlying pathology
Immune-mediated

57. Leptospirosis: epidemiology
Worldwide distribution but only US infections/year
Zoonosis with many animal hosts
Rodents; dogs, pigs, cattle, horses
Transmission
Contact with urine (or other body fluids, except saliva) from infected animals
Contact with water, soil, or food contaminated with the urine of infected animals
Recreational exposure to contaminated water (e.g., lakes) or occupational exposure (vets, farmers, hunters, butchers)
50% reported in Hawaii
Many misdiagnosed as viral syndrome
Most through contact of skin or mucous membranes with contaminated water
Drinking contaminated water can also cause infection. Outbreaks of leptospirosis are usually caused by exposure to contaminated water, such as floodwaters. Person to person transmission is rare.

58. Leptospirosis: clinical manifestations
Most infections not clinically apparent or diagnosed
First stage (Bacteremia)
Fever, chills, headache, myalgias, conjunctival suffusion, abdominal pain
Second stage (Immune)
Aseptic meningitis or generalized illness with myalgias, headache, uveitis and rash
Severe – stages blend
Vascular collapse, thrombocytopenia, hepatitis, kidney involvement, hemorrhage
Mortality 5-10%

59. Leptospirosis: clinical manifestations
“Icteric” Leptospirosis is called Weil’s disease
Striking jaundice with hepatitis in severe Leptospriosis
Hepatic necrosis not seen and most do not develop liver failure

62. Leptospirosis – Epidemic Curve

63. Leptospirosis: diagnosis and treatment
Culture blood and CSF (early), urine (late)
Serology – Microscopic agglutination test (MAT) after first week
ELISA and IHA tests less standardized
PCR very sensitive in research laboratories
Treatment
IV Penicillin or doxycycline
Specially formulated media, grow slowly but most cultures positive within 2 weeks
Several specimens need to be collected
Serologic cross reactions can occur with other spirochetal infections