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TRANSCRIPT
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Tick-borne Diseases in Wisconsin:Current Status, Testing & Reporting
February 21, 2013Anna Schotthoefer
Project ScientistMarshfield Clinic Research Foundation
Marshfield, WI
Outline
• Introduction of tick-borne diseases in WI & their symptoms
• Currently known distributions of ticks & diseases
• Lyme disease testing
• Anaplasmosis testing
• Babesiosis testing
• Reporting requirements
Common Tick-Borne Diseases in WI
• Lyme disease (Borrelia burgdorferi) – Bacteria– Most common
• 3,609 cases reported in WI in 2011• Incidence rate = 63.3 / 100,000
• Anaplasmosis (Anaplasma phagocytophilium)• Originally named Human Granulocytic Ehrlichiosis (HGE)
• Still called Ehrlichiosis here– Bacteria
• 497 cases reported in WI in 2010• Incidence rate = 9.6 / 100,000
• Babesiosis (Babesia microti)– Parasite
• MC: 2/52 (3.7%) in 2002 vs. • 71/1010 tests (6.5%) in 2011
• WI: 46 cases in 2011
Borrelia burgdorferi bacteria
Anaplasma phagocytophilum inside a white blood cell
Babesia microti inside a red blood cell
Rare Tick-Borne Diseases in WI
• Ehrlichiosis (Ehrlichia muris-like organism)• Not to be confused with Anaplasmosis or infections caused by Ehrlichia
chaffeensis– Bacteria– Discovered in MN & WI 2009– ~30 cases identified to date
• Powassan Virus – Flavivirus = same group as West Nile Virus– First cases in WI in 2003– 10-15% of cases fatal
• Rocky Mountain Spotted Fever (Rickettsia rickettsii)– Bacteria– 16 cases, 1980-2003
– Most acquired outside of WI
• Tularemia (Francisella tularensis)– Bacteria– 3 cases, 2001-2010
A Plaque-reduction/neutralization test is used to confirm Powassan virus infections
Discovery of E. muris-like organism received media attention
Symptoms of infections
• Flu-like illnesses
• Asymptomatic � severe– Fever, sweats, chills
– Myalgia
– Fatigue
– Headache
– Joint pain and swelling
– Sore throat, cough
– Abdominal pain
– Vomiting, diarrhea
• Symptoms usually appear 5-10 days after a tick bite
Early stage of Lyme disease
• Erythema migrans(EM)
– Hallmark sign of Lyme disease
– Bull’s eye rash
– ~50-80% of patients
• Typically occurs within 1 week of tick bite (3-30 days)
• Localized infection
• May be accompanied by fever, weakness
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Disseminated stage of Lyme disease (> 2 wks of infection)
• Annular or diffuse rashes
• Migratory pain in joints and muscles
• Bell’s palsy
• Abnormal heart beating
– Palpitations
– Dizziness
• Motor or sensory nerve problems
• Severe fatigue
Souce: WebMD
Late infection (months����years of infection)
• Prolonged arthritis attacks
– ~60% of untreated patients
• Chronic neurological problems
– ~5% of untreated patients
• Numbness or tingling in hands or feet, sharp shooting pains, loss of short term memory
• Chronic fatigue
Post-treatment Lyme disease syndrome?
• About 10-20% of patients treated for Lyme disease will have lingering symptoms of fatigue, pain, or joint and muscle aches
• Causes not well understood
– Tissue damage due to infection?
– Auto-immune responses?
– Persistent infection?
Symptoms of other diseases
Anaplasmosis� Fever
� Headaches
� Rashes very rare
� Low white blood cell
counts
� Low platelet counts
� Elevated liver enzymes
� Infections can be fatal
� Little evidence of
neurological involvement or persistent infections
Babesiosis� Recurrent fevers
� Anemia
� Low platelet counts
� Jaundice
� Infections can be fatal in immunocompromised &
those without a spleen
� Infections can last for years
Wisconsin is a “hotspot”
Source: CDC
Cases are on the rise
Case definition & reporting criteria changed in 2008:Cases categorized as Confirmed,
Probable, or Suspected
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Case definition & reporting criteria
changed in 2008 requiring species designation &
cases categorized as Confirmed, Probable, or
Suspected
Cases have spread over a larger area
1979-1982 1996-1998
2008
Marshfield Clinic Service Area
I. scapularis-positive County
I. scapularis-negative County
No reports
Jackson & DeFoliart (1970)
Davis et al. (1984)
Callister et al. (1988)
French et al. (1995)
Riehle & Paskewitz (1996)
Walker et al. (1996)
Caporale et al. (2005)
Guerra et al. (2002)
Diuk-Wasser et al. (2006)
WDPH (unpublished)
1st tick survey - 1968
The tick has spread across the state
Pop Quiz Question: Which of these ticks is the primary vector for infections?
Copyright: Tom Murray 2010
Photo credit: MN Dept Health
Photo credit: BugGuide.com
Photo credit: uwlax.edu
Copyright: Melinda Fawver
Ixodes scapularisAdult male
Ixodes scapularisAdult female
Ixodes scapularisNymphs
Pop Quiz Question: Which of these ticks is the primary vector for infections?
Dermacentor variabilis Adult females
Dermacentor variabilis Adult male
American Dog Tick= Wood Tick
Pop Quiz Question: Which of these ticks is the primary vector for infections?
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The vector
• Ixodes scapularis, blacklegged tick = deer tick = bear tick
Adultfemale
Adultmale
Nymph
Photos: Okstate.edu, Wikipedia
2 year life cycle of Ixodes scapularisThis image cannot currently be displayed.
Seasonal Activity of I. scapularis
• Most infections occur between May-July
– Nymphal stage of tick
• Infections can also occur in fall
– Adult stage of tick
• Some risk throughout year, anytime no snow on ground & temperatures >40°F
Lyme disease testing
• Two-tiered testing algorithm
– First test = enzyme immunoassay (EIA) or immunofluorescent assay (IFA)• Polyvalent, whole-cell sonicate (WCS) EIA
– If positive or equivocal, second test performed = IgM and IgG WCS Western blots
• Interpreted using specified criteria:
• http://www.cdc.gov/mmwr/preview/mmwrhtml/00038469.
htm
• At least 2 of 3 positive bands on IgM blot
• At least 5 of 10 positive band on IgG blot
Time of illness requirement added to case
definition for reporting in 2011
Drawbacks to current Lyme disease testing
• Poor sensitivity
– 30-40% in early patients with EM
• Doesn’t work well on all “types” of Lyme patients
– Best for patients with Lyme arthritis or late neurological
involvement (~80-90% sensitivity)
• Technical difficulties with Western blots
• High percentage of false positives associated with IgM Western blot
• Physicians may inconsistently adhere to algorithm
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Lyme EIA system at Marshfield Labs
Automated sample dilution, washing, & reading of fluorescence by optical scanner
-Reported as Positive or Negative
Lyme western blot system at Marshfield Labs
Striped blots
Density of bands calibrated & read by machine,Checked by lab staff
IgM blot examples
Negative sample Positive sample
At least 2 of 3 bands required for positive
IgG blot example
Negative sample Positive sample
At least 5 of 10 bands required for positive
Other Lyme disease tests
• Borrelia burgdoferi PCR
– Lyme arthritis (synovial fluid) or neuroborreliosis (CSF)
– Negative PCR does not exclude neuroborreliosis
• CSF Lyme Index
– CSF:serum ratio of anti-B. burgdorferi antibodies
– Ratio > 1 suggestive of neuroborreliosis
– FDA approved
– Should be used instead of PCR
• Borrelia culture of skin
– Not generally indicated
– Done when skin lesions are suspicious but not pathognomonic of EM
Determining if a patient is a Lyme disease case
• Patients with EM do not have to be tested
• Patients without EM,
should be tested
– Positive IgM should only be interpreted if
illness < 30 days
• These are surveillance guidelines only:
• If serologic tests are
negative, but there are other indicators of infection, physician
should go ahead and treat
– Retesting after 2
weeks is also advised
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Anaplasmosis/Ehrlichiosis testing
• Blood smears
– Low sensitivity
– Non-specific
• Serology
– Low sensitivity in acute phase
– Some cross-reactivity among species• But not enough to reliably substitute one for another
– Inability to differentiate between active versus previous infections• Acute and convalescent tests are required for confirmed cases, but this is rarely done
• Real-time PCR
– Potentially high sensitivity during acute phase
– High specificity
– Positive test meets requirements for confirmed case definition
IFA slide
Titers < 1:64 = NegativeTiters ≥ 1:64 = PositivePositive titers reported out to ≥ 1:512
Development of real-time PCR test for Anaplasmosis & Ehrlichiosis
• Heat shock protein operon groEL
• FRET probes & LightCycler technologies
• One set of primers that binds
to the related species (A. phagocytophilum, E. chaffeensis, E. ewingii, EML)
• One acceptor probe for all
• Unique donor probes for A. phagocytophilum and Ehrlichia spp.
Different species detected by melting curve analysis
Compliments of Kristina McElroy, CDC
E. ewingii
E. chaffeensis
A. phagocytophllumE. muris-like
Importance of time of testing
• PCR & blood smear had
maximal positivity in early acute phase (≤ 4 days ill)
• PCR extended window of
detection to 30 days in
comparison to 14 days for blood smear
• Serology positivity overall
higher & highest later (22-28
days ill)– Detection of previous exposures 0
10
20
30
40
50
60
70
3 7 14 21 30 60
% p
osit
ive
te
sts
Days ill
Serology
Smear
RT-PCR
391 patient specimens included in study
PCR is available at Marshfield Labs
• Validated & brought online summer 2012
• Laboratory News, vol 35 (12), June 25, 2012
– http://srdweb1/lab/Laboratory%20News/Laboratory%20News%20Vol%2035%20No%2012%20June%2025%202012%20Web.pdf
• Sept 1- Jan 31
– 20 of 916 (2.2%) tests positive
PCR detected 29.2% more infections than serology and blood smear
PCR vs. Serology PCR vs. Smear
Early acute = 0-4 days illsLater > 4 days ills
0
20
40
60
80
100
All Early acute Later
% PCR positve
Stage of infection
Smear positve
Smear negative
0
20
40
60
80
100
All Early acute Later
% PCR positve
Stage of infection
Serology positve
Serology negative
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Babesiosis Testing
• Detection on blood smears= current gold standard
– Can be confused with
early forms of
Plasmodium
• Serologic testing (IFA)
• Development of real-time PCR underway
– Teal et al. 2012IFA detection of Babesia microti
Blood smear detection of Babesia microti
Reporting
• Lyme disease, Anaplasmosis/Ehrlichiosis, Babesiosisare Nationally Notifiable Diseases– http://wwwn.cdc.gov/nndss/
• WI has an electronic reporting system (WEDSS)
– Positive laboratory tests automatically reported to public health
• Paper forms also available
• Changes in 2012, WDPH requiring only EM cases & laboratory positives be reported– http://www.dhs.wisconsin.gov/communicable/WiEpiExpress/PDFfiles/2012WEE/2012WEE0628.pdf
Acknowledgements
• Financial support provided by the Marshfield Clinic Research Foundation
– Collaborators: Drs. Jennifer Meece, Tom Fritsche, Tim Uphoff
• Technical support: Marshfield Labs, CORE lab– Phil Bertz, David Connelly, Jean Erbst, Karen Gallion, Josh Hebert, Lynn Ivacic, Greg Simon, Tara Weiler, Kai Qi Zhang
• Regional labs (YOU!!): Thank you for sending specimens
Questions?
• General Info:• http://www.dhs.wisconsin.gov/communicable/TickBorne/index.htm
• http://www.cdc.gov/ticks/