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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/