J. Stephen Dumler, M.D.J. Stephen Dumler, M.D.Division of Medical MicrobiologyDivision of Medical Microbiology

Department of PathologyDepartment of PathologyThe Johns Hopkins University School of MedicineThe Johns Hopkins University School of Medicine

Baltimore, MarylandBaltimore, Maryland

Update on Diagnostic Update on Diagnostic Testing for TickTesting for Tick--borne borne

DiseasesDiseases

* * ≥≥ 147 cases in CT, NY, MA, and RI, 1999147 cases in CT, NY, MA, and RI, 1999** 247 cases in AR, CA, CO, ID, MT, NV, NM, TX, UT, WA, WY, 1990** 247 cases in AR, CA, CO, ID, MT, NV, NM, TX, UT, WA, WY, 1990--2000 2000 **** 11,302 cases in Europe, 1999; rare in US** 11,302 cases in Europe, 1999; rare in US

Tick-borne agents of humandisease of world-wide importance

not reported***not reported***

not reported**not reported**

9595

not reported*not reported*

578578

646646

1,5501,550

14,53614,536

US cases 2005US cases 2005

relapsing feverrelapsing feverBorrelia spp. Borrelia spp. (>10 species)(>10 species)

tularemiatularemiaFrancisella tularensisFrancisella tularensis

human babesiosishuman babesiosisBabesia microtiBabesia microti

Powassan virus Powassan virus encephalitis (TBE, RSSE)encephalitis (TBE, RSSE)

Flavivirus/tickFlavivirus/tick--borne borne encephalitis virusesencephalitis viruses

human monocytic human monocytic ehrlichiosis (HME)ehrlichiosis (HME)Ehrlichia chaffeensisEhrlichia chaffeensis

human granulocytic human granulocytic anaplasmosis (HGA) anaplasmosis (HGA) Anaplasma phagocytophilumAnaplasma phagocytophilum

Rocky Mountain spotted Rocky Mountain spotted feverfeverRickettsia rickettsiiRickettsia rickettsii

Lyme diseaseLyme diseaseBorrelia burgdorferiBorrelia burgdorferi

diseasediseaseagentagent

Tick-borne infections:a clinical dilemma?

blood smearblood smearFever, malaise, myalgia, arthralgiaFever, malaise, myalgia, arthralgiaBabesiosisBabesiosis

* IFA * IFA –– indirect fluorescent antibodyindirect fluorescent antibody

ELISA and ELISA and Western blot Western blot serologyserology

early localized (erythema migrans)early localized (erythema migrans)early disseminated (multiple EM, early disseminated (multiple EM, neuritis, carditis, arthralgis, neuritis, carditis, arthralgis, meningitismeningitis……))Late (arthritis, acrodermatitis, Late (arthritis, acrodermatitis, encephalopathy)encephalopathy)

Lyme diseaseLyme disease

IFA serologyIFA serologyblood smearblood smear

Acute fever, headache; infrequent Acute fever, headache; infrequent rashrash

HME and HGAHME and HGA

IFA* serologyIFA* serologyskin biopsyskin biopsy

Acute fever, headache (d0Acute fever, headache (d0--5), rash 5), rash (median d6)(median d6)

RMSFRMSF

major lab major lab diagnosticsdiagnostics

clinical presentationsclinical presentationsticktick--borne borne diseasedisease

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RMSF HME HGALyme disease ehrlichiosis nos total TBD

Cases of tick-borne disease in the U.S.1920-2006

Ticks and tickTicks and tick--borne diseases in the Midborne diseases in the Mid--AtlanticAtlantic

Amblyomma americanumAmblyomma americanum

Ixodes scapularisIxodes scapularis

Dermacentor variabilisDermacentor variabilis

Ixodes scapularisIxodes scapularis

Tick vectorTick vector

Lone star tickLone star tickHMEHME

Deer tick, blackDeer tick, black--legged ticklegged tickHGAHGA

American dog tickAmerican dog tickRMSFRMSF

Deer tick, blackDeer tick, black--legged ticklegged tickLyme diseaseLyme disease

Common tick nameCommon tick nameDiseaseDisease

Lyme disease – caused by members of theBorrelia burgdorferi sensu lato group

general characteristicsgeneral characteristicsspiralspiral--shaped bacterium, 0.2 x shaped bacterium, 0.2 x 10 10 -- 30 um in size30 um in sizetypical cell wall with thin typical cell wall with thin peptidoglycan layerpeptidoglycan layerflagella in periplasmic space > flagella in periplasmic space > twisting motilitytwisting motilityobligate parasite found in obligate parasite found in association with mammals, association with mammals, birds, ticksbirds, ticks

Lyme diseaseBorrelia burgdorferi sensu lato group

ecological characteristicsecological characteristicsmaintained in a small maintained in a small mammal mammal -- IxodesIxodes species species tick cycletick cycleeastern U.S. eastern U.S. -- Peromyscus Peromyscus leucopusleucopus and and I. scapularisI. scapulariswestern U.S. ? reservoir, western U.S. ? reservoir, I. I. pacificuspacificusEurope and Asia Europe and Asia -- small small mammals, mammals, I. ricinus I. ricinus andand I. I. persulcatuspersulcatus

Lyme disease Lyme disease -- seasonalityseasonality

bimodal distributionbimodal distributionmost cases occur in most cases occur in May through JulyMay through Julyassociated with emergence associated with emergence and activity of nymphal deer and activity of nymphal deer ticksticks

secondary peak in secondary peak in late Falllate Fallassociated with emergence associated with emergence and activity of adult deer ticksand activity of adult deer ticks

adults

nymphs

adultslarvae

Number of Cases of Lyme disease, by yearUnited States, 1983-2006

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15000

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25000

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1983

1984

1985

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1988

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1990

1991

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1999

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6Georgia10District of Columbia15South Carolina39Rhode Island47Florida49North Carolina54Vermont61West Virginia

247Maine265New Hampshire274Virginia400New York City646Delaware

1,2351,235MarylandMaryland1,810Connecticut2,336Massachusetts3,363New Jersey4,287Pennsylvania5,165New York (Upstate)

23,305United StatesLyme diseaseArea

Reported cases of Lyme disease by Division of Geographic region, 2005

Adult Ixodes scapularis and Borrelia burgdorferiin the Mid-Atlantic States, 1997-98JE Bunnell, PhD dissertation thesis, JHU School of Hygiene & Public Health

Number of ticks examined 649

Number of B. burgdorferi-infected ticks 151 (23%)

Northern Virginia - 0%

Pennsylvania - 14%

New Jersey - 19%Delaware - 19%

Maryland - 27%

Central Maryland - 33%

North Maryland - 23%

Southern Maryland - 11%

Eastern Shore Maryland - 48%

COUNTY population 2000 cases 2000-2006 00-06 avg incid/105

Queen Anne's County 40,563 284 100.0Kent County 19,197 113 84.1Talbot County 33,812 155 65.5Carroll County 150,897 628 59.5Cecil County 85,951 351 58.3Calvert County 74,563 292 55.9Caroline County 29,772 115 55.2Frederick County 195,277 501 36.7Harford County 218,590 557 36.4How ard County 247,842 570 32.9Somerset County 24,747 52 30.0Worcester County 46,543 73 22.4Washington County 131,923 204 22.1Charles County 120,546 162 19.2Anne Arundel County 489,656 652 19.0Wicomico County 84,644 106 17.9St. Mary's County 86,211 83 13.8Dorchester County 30,674 27 12.6Montgomery County 873,341 605 9.9Prince George's County 801,515 381 6.8Allegany County 74,930 13 2.5Baltimore County 754,292 108 2.0Garrett County 29,846 4 1.9Baltimore city 651,154 61 1.3Maryland 5,296,486 6099 16.5

Lyme disease in Maryland 2000-2066, cases and incidence (2000 census) by county or subdivision

020406080

100

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cent

age

of

ticks

infe

cted

Central West TotalBaltimore County region

Percentage of I. scapularis ticks infected withB. burgdorferi in regions of Baltimore County, 2001

NymphFemaleMaleTotal

Courtesy Doug Norris, PhD; JHU Bloomberg School of Public Health

Lyme disease Lyme disease –– early disseminatedearly disseminated

Systemic manifestationsSystemic manifestationsfeverfevermultiple EMsmultiple EMsperipheral nervous systemperipheral nervous system

radiculopathyradiculopathycranial neuropathycranial neuropathymononeuropathymononeuropathy multiplexmultiplex

central nervous systemcentral nervous systemlymphocytic meningitislymphocytic meningitisrarely, encephalomyelitis rarely, encephalomyelitis (parenchymal inflammation of (parenchymal inflammation of brain and/or spinal cord, with brain and/or spinal cord, with focal abnormalities)focal abnormalities)

atrioventricularatrioventricular heart block heart block ±±clinical clinical myopericarditismyopericarditis

Lyme diseaseLyme diseaseclinical aspectsclinical aspectslate infectionlate infection

RheumatologicRheumatologicmonomono-- or oligoarticular arthritisor oligoarticular arthritis

knee, large joints, TMJknee, large joints, TMJsynovial fluid mean 24,250 WBC/mmsynovial fluid mean 24,250 WBC/mm33

neutrophil predominanceneutrophil predominanceantibioticantibiotic--refractory chronic arthritisrefractory chronic arthritis

HLAHLA--DR4DR4

Central nervous systemCentral nervous systemunifocalunifocal or or multifocalmultifocal encephalomyelitisencephalomyelitis

very rare in U.S.; CSF pleocytosisvery rare in U.S.; CSF pleocytosisperipheral neuropathyperipheral neuropathy

““stocking glovestocking glove”” paresthesiasparesthesias; ; radicularradicular painpainencephalopathyencephalopathy

mild memory and cognitive function mild memory and cognitive function abnormalities abnormalities

Lyme diseaseLyme diseasediagnosisdiagnosis

cultureculturePCRPCRserologyserology-- enzyme immunoassayenzyme immunoassay-- western blotwestern blot

Cultivation yield (%) of Borrelia burgdorferi from clinical samples

>40% (≥9 mL)Blood (plasma)AnecdotalSynovial fluidNo data availableCerebrospinal fluid>50% (max 86%)Erythema migrans

NonNon--chronicchronic

Sensitivity and specificity metaSensitivity and specificity meta--analyses of published assays for the Lyme disease analyses of published assays for the Lyme disease diagnosis by PCR.diagnosis by PCR.

66--76%76%21%21%17%17%

100%100%66--91%91%24%24%19%19%OverallOverallCSFCSF

99%99%2323--100%100%65%65%73%73%Synovial fluidSynovial fluid

100%100%00--52%52%29%29%26%26%Plasma / serumPlasma / serum

100%100%5959--80%80%71%71%67%67%Erythema migransErythema migrans

MetaMeta--Analysis Analysis SpecificitySpecificity

Range of Range of sensitivitysensitivity

MedianMedianSensitivitySensitivity

MetaMeta--Analysis Analysis SensitivitySensitivity

Specimen typeSpecimen type

Laboratory Diagnosis of Lyme diseaseLaboratory Diagnosis of Lyme diseaseSerologySerologyEnzyme immunoassay (EIA) methodsEnzyme immunoassay (EIA) methods

very sensitive, limited specificity (false positives)very sensitive, limited specificity (false positives)whole bacterial cell lysates or individual recombinant whole bacterial cell lysates or individual recombinant

proteinsproteinsgenerally not immunoglobulin class specificgenerally not immunoglobulin class specific

Western blot assays to aid specificity Western blot assays to aid specificity supplemental method for EIA positivessupplemental method for EIA positivesrequires presence of specific antigen bands onrequires presence of specific antigen bands on

IgM or IgG immunoblotsIgM or IgG immunoblots

Pitfalls in serologic diagnosisPitfalls in serologic diagnosislack of early responselack of early responseabrogation by early Rxabrogation by early Rxpersistent antibody in absence of active diseasepersistent antibody in absence of active diseaselack of standardizationlack of standardizationno FDAno FDA--cleared test for detection of CSF antibodiescleared test for detection of CSF antibodies

Serology in Lyme disease; % reactivity in patients with:

ArthritisNeurological involvement

EM, convalescent

phase

EM,acute phaseTest

978729-7829-40Two-tier testing

96-10064-7215-21, 800-13, 43.6IgG IB

168075-8443-44IgM IB

100 (IgG ELISA)79 (IgG ELISA)76-8633-49Whole-cell ELISA

FlgBFlgB

OspCOspC

OspAOspAOspBOspB

BmpABmpA

IgGIgG

(5 of 10)(5 of 10)

18182121--24 OspC24 OspC2828303039 (39 (BmpABmpA))41 (41 (FlaFla))454558 (not GroEL)58 (not GroEL)66669393

IgMIgM

(2 of 3)(2 of 3)

2121--24 OspC24 OspC39 (39 (BmpABmpA))41 (FlgB)41 (FlgB)

IgM

IgG

EM < 7 days EM ≥ 7 days

Recommendations for Two-Step Testing and Interpretation of Lyme borreliosis Serology

later samplecan't exclude LD alonerepeat in 2 - 4 weeksif strongly suspected

early samplecan't exclude LD alonerepeat in 2 - 4 weeks

negative result

no reliable evidence of infectioncan't exclude LD alonerepeat in 2 - 4 weeks

if LD strongly suspected

negative result

serologic evidence of infectionmay not be current infectionsupports clinical diagnosis

positive result

supplemental testimmunoblot (Western blot)

positive or equivocal result

Clinical diagnosis of Lyme diseaseInitial testELISA, EIA, IFA

Appropriate use of ELISA test for Appropriate use of ELISA test for Lyme disease diagnosisLyme disease diagnosis((TugwellTugwell et al Ann Intern Med 1997)et al Ann Intern Med 1997)

Pretest probabilityPretest probabilityincidence of disease in communityincidence of disease in communityconstellation of clinical findingsconstellation of clinical findings

ELISA (and IFA) accurate if:ELISA (and IFA) accurate if:standardizedstandardizedappropriate cutoffsappropriate cutoffsappropriate quality controlappropriate quality control

Only to be used if pretest probability exceeds 0.20 but is Only to be used if pretest probability exceeds 0.20 but is less than 0.80less than 0.80Arthralgia, myalgia, headache, fatigue, palpitations alone Arthralgia, myalgia, headache, fatigue, palpitations alone not sufficient to raise pretest probability to clinically usefulnot sufficient to raise pretest probability to clinically usefulrangerange

Review of laboratory predictive value and likelihood ratios

Sensitivity = test + / true +Sensitivity = test + / true +Specificity = test Specificity = test -- / true / true ––Positive predictive value =Positive predictive value =

true + / (true +) + (false +)true + / (true +) + (false +)

Therefore, a test with sensitivity and specificity of Therefore, a test with sensitivity and specificity of 95% used to test 1000 persons in which prevalence 95% used to test 1000 persons in which prevalence of disease is 1% would identify 60 positives, 10 true of disease is 1% would identify 60 positives, 10 true positives and 50 false positives.positives and 50 false positives.

Positive likelihood ratio (LHR) = true + rate / false + Positive likelihood ratio (LHR) = true + rate / false + raterate((used in calculation of post test odds or post test used in calculation of post test odds or post test probabilityprobability))Pretest odds x LHR = post test oddsPretest odds x LHR = post test odds

Selected recombinant and peptide antigens used for detection of Selected recombinant and peptide antigens used for detection of IgM, IgG, or IgM, IgG, or polyvalent antibodies to polyvalent antibodies to B. burgdorferiB. burgdorferi

1545-68FlgA (37 kDa)

83-986470-90Convalescent

94-10060-9545-74Acute

Invariable region 6 (IR-6, C6 peptide)

879739100100736344-6319-40VlsE

35-5243-53P41 internal fragment

95340-53pepC10

35-4341-73OspC

IgIgGIgMIgIgGIgMIgIgGIgM

LateLateNeurologicalNeurologicalEMEMRange % positive in the indicated LB disease stage Range % positive in the indicated LB disease stage

Recombinant antigen or peptide

CSF serology in Lyme disease

ELISA indicated only with prior ELISA indicated only with prior positive serum ELISApositive serum ELISANo FDANo FDA--approved test or cutoff approved test or cutoff criteriacriteriaNeed to implicate intrathecal antibody Need to implicate intrathecal antibody synthesis (IgG index)synthesis (IgG index)

Predictive value of PCR forB. burgdorferi in skin

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PCR on CSF for

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Predictive value of B. burgdorferiPCR on plasma

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Other tests for Lyme diseaseOther tests for Lyme diseaseDetection of Detection of B. burgdorferiB. burgdorferi antigen in urineantigen in urine

Alternative lab testing atAlternative lab testing at““Lyme literateLyme literate”” facilitiesfacilities

TP = true Positive; TN = true Negative; FP = false Positive; FN = false Negative

35 (TN)LDA, PCR, and RWB Negative

6 (TP) PCR only Positive

94 (TN)LDA, PCR, and RWB Negative

4 (TP)LDA, PCR, and RWB Positive

0RWP Positive2 (TP)LDA and RWB Positive

0PCR Positive4 (TP)LDA and PCR Positive

1 (FP)LDA Positive 4 (FP)LDA only Positive

General Population (n=95) Patients with Lyme Disease Like Symptoms (n=55)

• Fatigue• Low grade fevers, ‘hot flashes’ or chills• Night sweats• Sore throat• Swollen glands• Stiff neck• Migrating arthralgias, stiffness and, less

commonly, frank arthritis• Myalgia• Chest pain and palpitations• Abdominal pain, nausea• Diarrhea• Sleep disturbance• Poor concentration and memory loss

• Irritability and mood swings• Depression• Back pain• Blurred vision and eye pain• Jaw pain• Testicular/pelvic pain• Tinnitus• Vertigo• Cranial nerve disturbance (facial

numbness, pain, tingling,palsy or optic neuritis)• Headaches• ‘Lightheadedness’• Dizziness

9. Symptoms of Lyme diseaseFrom International Lyme and Associated Disease Society: Evidence-based guidelines for the management of Lyme disease. 2004. Expert Rev Anti-Infect Ther 2: S1-S13

“Lyme literate” lab 1SpecificitySpecificityAny sample positive by LDA, and confirmed by either PCR or RWB oAny sample positive by LDA, and confirmed by either PCR or RWB or r both, was considered a true positive. Any sample that was negatiboth, was considered a true positive. Any sample that was negative by ve by all 3 methods was considered a all 3 methods was considered a ““true negative.true negative.”” Of the 162 individuals Of the 162 individuals samples, 146 were considered true negatives.samples, 146 were considered true negatives.

141 individuals were negative by LDA. 4 patients suspected of Ly141 individuals were negative by LDA. 4 patients suspected of Lyme me Disease and 1 normal control were positive by LDA, but negative Disease and 1 normal control were positive by LDA, but negative by by either PCR or RWB. Therefore, these were considered false positieither PCR or RWB. Therefore, these were considered false positives. ves. Based on this data, the Based on this data, the specificity of LDA is >96%.specificity of LDA is >96%.

SensitivitySensitivityOf the 16 true positives, 10 samples were positive by LDA. BasedOf the 16 true positives, 10 samples were positive by LDA. Based on on the limited data, the limited data, the sensitivity of the LDA is approximately 60%.the sensitivity of the LDA is approximately 60%.The number of The number of ““true positives" increased from 10 to 16 (88%) when true positives" increased from 10 to 16 (88%) when PCR was performed. PCR was performed.

Note that LDA and other unproven lab tests are part of Note that LDA and other unproven lab tests are part of the definition of the definition of ““true positivetrue positive”” and and ““true negativetrue negative””

Sensitivity and specificity of LDA based on alternate gold standards

True positive = clinical diagnosis onlyTrue positive = clinical diagnosis onlySensitivity 25% (14/55)Sensitivity 25% (14/55)Specificity 99% (106/107)Specificity 99% (106/107)

True positive = clinical diagnosis + PCRTrue positive = clinical diagnosis + PCRSensitivity 50% (6/12)Sensitivity 50% (6/12)Specificity 95% (143/150)Specificity 95% (143/150)

True positive = clinical diagnosis + RWBTrue positive = clinical diagnosis + RWBSensitivity 100% (6/6)Sensitivity 100% (6/6)Specificity 95% (148/156)Specificity 95% (148/156)

Other tests for Lyme diseaseT-cell proliferative assay

Suggested for partiallySuggested for partially--treated or seronegative treated or seronegative personspersonsLT positive in 75% of normal, healthy subjectsLT positive in 75% of normal, healthy subjectsAssay not amenable to clinical laboratoryAssay not amenable to clinical laboratory

Prophylaxis after tick bites in Lyme disease

NadelmanNadelman et al. (NEJM 2001):et al. (NEJM 2001):prophylaxis with single 200 mg dose prophylaxis with single 200 mg dose doxycycline prevents Lyme disease after doxycycline prevents Lyme disease after Ixodes scapularisIxodes scapularis bites in 87%bites in 87%adverse reactions occur 3X more often than adverse reactions occur 3X more often than placebosplacebos

Lyme diseaseLyme diseasetreatment and outcome

antibiotic therapyantibiotic therapylocalized infection: localized infection: popo amoxicillin, doxycyclineamoxicillin, doxycyclinedisseminated infection without neurologic or cardiac disseminated infection without neurologic or cardiac involvement: involvement: popo amoxicillin, doxycycline, amoxicillin, doxycycline, cefuroximecefuroximedisseminated neurologic or cardiac infection: IV ceftriaxonedisseminated neurologic or cardiac infection: IV ceftriaxone

vaccine (adults 85% effective)vaccine (adults 85% effective)Withdrawn from market by SKBWithdrawn from market by SKB

chronic Lyme diseasechronic Lyme diseasemisdiagnosesmisdiagnosestrue persistence of infectiontrue persistence of infection

Health-related quality of life is not affected by prolonged antibiotic treatment in patients with persistent manifestations of Lyme disease

antibioticantibiotic: IV ceftriaxone: IV ceftriaxone 2 g daily x 30 days, and PO 2 g daily x 30 days, and PO doxycycline, 200 mgdoxycycline, 200 mg daily for 60 days;daily for 60 days;

placeboplacebo: matching intravenous and oral placebos: matching intravenous and oral placebos

Klempner MS, et al. Two controlled trials of antibiotic treatment in patients with persistent symptoms and a history of Lyme disease. N Engl J Med. 2001; 345:85-92.

Cases and incidence of TBRD in the U.S.,1920-20062006 data extrapolated from MMWR August 5, 2006

0

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1 10 100

1000

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no. cases* TBRDx4 = estimated true cases of TBRD

SARSHantavirus Pulmonary Syndrome

Hemolytic Uremic Syndrome (E. coli O157:H7)Toxic Shock SyndromeMeningococcal disease

LegionellosisInvasive pneumococcal disease

Arbovirus and West Nile VirusTBRD

Hepatitis A/B/CTuberculosis

TBRDx4*AIDS

Comparison of reported life-threateningdomestic infectious diseases in the U.S., 2005

(MMWR 2006; 54:1320)

Rocky Mountain spotted fever (RMSF)Tick vectors of R. rickettsii

• Known tick vectors– Dermacentor variabilis -

American dog tick (eastern U.S.)

– Dermacentor andersoni -Wood tick (western U.S.)

• Possible tick vectors• Rhipicephalus sanguineus

- brown dog tick• Amblyomma cajennense

(Central and South America)

DermacentorDermacentor variabilisvariabilis

DermacentorDermacentorandersoniandersoni

Rhipicephalus sanguineusRhipicephalus sanguineus

Amblyomma cajennenseAmblyomma cajennense

Pathogenesis of Rickettsial infectionsPathogenesis of Rickettsial infections

Rickettsiaendothelial cells

phagsome escape

macrophages and neutrophilsEhrlichia and Anaplasma

phagolysosome fusioninhibition

Clinical manifestations of RMSF

fever, headache, myalgias, fever, headache, myalgias, rashrashrash rash -- macular, macular, maculopapular, petechialmaculopapular, petechialshock and multishock and multi--organ failureorgan failuregastrointestinal systemgastrointestinal systemrenal systemrenal system

acute tubular necrosis 2acute tubular necrosis 2°° to to hypotensionhypotension

cardiopulmonary systemcardiopulmonary systemnonnon--cardiogenic pulmonary cardiogenic pulmonary edemaedema

central nervous system central nervous system ––meningoencephalitismeningoencephalitis

cerebral edema, herniationcerebral edema, herniation

macularmacular maculopapularmaculopapular

petechialpetechial

ecchymosisecchymosis

Lymphocyticvasculitis

Lymphocyticvasculitis

immunohistochemistryimmunohistochemistry

InterstitialpneumonitisInterstitial

pneumonitis meningoencephalitismeningoencephalitis

Leukocytoclastic vasculitisLeukocytoclastic vasculitis

Rocky Mountain spotted feverdiagnosis and risk

risk of death 5 x in patients after day 5 of risk of death 5 x in patients after day 5 of illnessillnessmost patients are initially examined before most patients are initially examined before day 5, but not treated until after day 5day 5, but not treated until after day 5major factors for ineffective diagnosis and major factors for ineffective diagnosis and delayed therapy:delayed therapy:-- absence of typical rashabsence of typical rash-- presentation during nonpresentation during non--peak tick activity seasonpeak tick activity season-- presentation during first 3 days of illnesspresentation during first 3 days of illness

1414Opening pressure Opening pressure ≥≥ 250 mm H250 mm H22OO

44443535Protein Protein ≥≥ 50 mg/50 mg/dLdL

141488Glucose Glucose ≤≤ 50 mg/50 mg/dLdL

rarerare606067673333

484846465050

PleocytosisPleocytosismononuclear cell predominancemononuclear cell predominanceneutrophil predominanceneutrophil predominance

Cerebrospinal fluidCerebrospinal fluid

717176766262ALT or AST ALT or AST ≥≥ 2X normal2X normal

5656Serum sodium < 132 Serum sodium < 132 mEqmEq/L/L

717172725252< 150,000< 150,000

Platelet count/Platelet count/μμLL

76766969> 10% bands> 10% bands

49496666<5,000/<5,000/μμLL

11112828> 10,000/> 10,000/μμLL

White blood cell countWhite blood cell count

HGAHGAHMEHMERMSFRMSFLaboratory findingLaboratory finding

(%)(%)Laboratory abnormalities in TBRDLaboratory abnormalities in TBRD

* IFA = indirect fluorescent antibody; LA = latex agglutination94 – 100%79 –94%LA100%83 – 85%IFA IgM99 – 100%89 – 100%IFA IgGRMSFSpecificitySensitivitySerological assay*Disease

Sensitivity and specificity of serological tests for confirmatioSensitivity and specificity of serological tests for confirmation of RMSFn of RMSF

Laboratory diagnosis in TBRD

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology≥≥ 14 days14 days

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology< 14 days< 14 days

Lab testLab test

++++±±++++++HGAHGA

++++++++++++HMEHME

++++++++++++++RMSFRMSF

Lab test Lab test availabilityavailability

convalescentconvalescentacuteacutePhase of illnessPhase of illness

Laboratory diagnosis in TBRD

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology≥≥ 14 days14 days

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology< 14 days< 14 days

Lab testLab test

++++±±++++++HGAHGA

++++++++++++HMEHME

++++++++++++++RMSFRMSF

±±±±++Lab test Lab test availabilityavailability

convalescentconvalescentacuteacutePhase of illnessPhase of illness

Laboratory diagnosis in TBRD

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology≥≥ 14 days14 days

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology< 14 days< 14 days

Lab testLab test

++++±±++++++HGAHGA

++++++++++++HMEHME

++++++++++++++RMSFRMSF

±±±±++++++++++++++++Lab test Lab test availabilityavailability

convalescentconvalescentacuteacutePhase of illnessPhase of illness

Laboratory diagnosis in TBRD

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology≥≥ 14 days14 days

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology< 14 days< 14 days

Lab testLab test

++++±±++++++++++++HGAHGA

++++++++++++++++HMEHME

++++++++++++++00±±RMSFRMSF

±±±±++++++++++++++++Lab test Lab test availabilityavailability

convalescentconvalescentacuteacutePhase of illnessPhase of illness

Laboratory diagnosis in TBRD

CultureCultureIHCIHCPCRPCRBlood Blood smearsmear

SerologySerology≥≥ 14 days14 days

Lab testLab test

00

00

00

±±

000000++++++++HGAHGA

000000++++++++HMEHME

000000++++++++RMSFRMSF

±±++++++++++++++++Lab test Lab test availabilityavailability

convalescentconvalescentacuteacutePhase of illnessPhase of illness

Rocky Mountain spotted feverRocky Mountain spotted fevertreatmenttreatment

adults: doxycycline, tetracyclineadults: doxycycline, tetracyclinechildren: doxycycline, tetracycline, children: doxycycline, tetracycline, chloramphenicol, rifampin?chloramphenicol, rifampin?chloramphenicol associated with excess chloramphenicol associated with excess mortality as compared with doxycycline or mortality as compared with doxycycline or tetracycline when controlled for all other tetracycline when controlled for all other factorsfactors

Human Human AnaplasmataceaeAnaplasmataceae infectionsinfections(human ehrlichiosis)(human ehrlichiosis)

human monocytic ehrlichiosis (HME) human monocytic ehrlichiosis (HME) --Ehrlichia chaffeensisEhrlichia chaffeensisHuman granulocytic anaplasmosis Human granulocytic anaplasmosis (HGA) (HGA) -- Anaplasma phagocytophilumAnaplasma phagocytophilumehrlichiosis ehrlichiosis ““EwingiiEwingii”” -- caused by caused by E. E. ewingiiewingii, genetically like , genetically like E. chaffeensisE. chaffeensis, , phenotypically like human anaplasmosisphenotypically like human anaplasmosisHME and human anaplasmosis are HME and human anaplasmosis are undifferentiated febrile illnesses with undifferentiated febrile illnesses with typical laboratory findings.typical laboratory findings.

E. chaffeensisE. chaffeensis

A. phagocytophilumA. phagocytophilum

E. ewingiiE. ewingii

0

200

400

600

800

1000

1200

1400

1600

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1999

2000

2001

2002

2003

2004

2005

2006

** no data available for 1998

num

ber o

f cas

es

HME

HGA

not specifiedall ehrlichiosis

Cases of ehrlichiosis and anaplasmosis in the U.S. Cases of ehrlichiosis and anaplasmosis in the U.S. reported to the CDC, 1986 reported to the CDC, 1986 -- 20062006

HME HME -- Ehrlichia chaffeensisEhrlichia chaffeensisepidemiology and ecologyepidemiology and ecology

•• risk for disease increased with risk for disease increased with age, male sexage, male sex

•• south central and southeastern south central and southeastern US; WorldUS; World--wide?wide?

•• transmitted by transmitted by A. americanum A. americanum active in summer monthsactive in summer months

•• reservoir whitereservoir white--tailed deertailed deer

HGA HGA –– Anaplasma phagocytophilumAnaplasma phagocytophilumepidemiology and ecologyepidemiology and ecology

•• risk for disease increased with age, risk for disease increased with age, male sexmale sex

•• upper Midwest and northeast US, upper Midwest and northeast US, northern California, Europenorthern California, Europe

•• transmitted by transmitted by Ixodes Ixodes sppspp. . nymphs nymphs and adultsand adults

•• reservoir whitereservoir white--footed mice footed mice ((PeromyscusPeromyscus leucopusleucopus), deer), deer

Amblyomma americanumAmblyomma americanum Ixodes scapularisIxodes scapularis Ixodes ricinusIxodes ricinus

1414Opening pressure Opening pressure ≥≥ 250 mm H250 mm H22OO

44443535Protein Protein ≥≥ 50 mg/50 mg/dLdL

141488Glucose Glucose ≤≤ 50 mg/50 mg/dLdL

rarerare606067673333

484846465050

PleocytosisPleocytosismononuclear cell predominancemononuclear cell predominanceneutrophil predominanceneutrophil predominance

Cerebrospinal fluidCerebrospinal fluid

717176766262ALT or AST ALT or AST ≥≥ twice normal valuetwice normal value

5656Serum sodium < 132 Serum sodium < 132 mEqmEq/L/L

717172725252< 150,000< 150,000

Platelet count/Platelet count/μμLL

76766969> 10% bands> 10% bands

49496666<5,000/<5,000/μμLL

11112828> 10,000/> 10,000/μμLL

White blood cell countWhite blood cell count

HGAHGAHMEHMERMSFRMSFLaboratory findingLaboratory finding

(%)(%)Laboratory abnormalities in TBRDLaboratory abnormalities in TBRD

HME and HGA clinical and epidemiologic HME and HGA clinical and epidemiologic featuresfeatures

HMEHMEInterval to medical Interval to medical attention attention –– 4 days4 daysIncubation period:Incubation period: 10.6 d 10.6 d mean (9 d median )mean (9 d median )

range: 0 range: 0 -- 34 days34 days

Tick exposure Tick exposure –– 80%80%SeveritySeverity

40 to 62% hospitalized40 to 62% hospitalizedMedian length of illness Median length of illness –– 23 23 daysdaysCase fatality rate Case fatality rate –– 2.7%2.7%

HGAHGA•• Interval to medical attention Interval to medical attention

–– 4 to 8 days4 to 8 days•• Incubation period Incubation period –– median median

6 to 10 d6 to 10 d–– range 1 to 60 daysrange 1 to 60 days

•• Tick exposure Tick exposure –– 75 to 85%75 to 85%•• SeveritySeverity

–– 56% hospitalized56% hospitalized–– 7% require ICU admission7% require ICU admission–– Median hospitalization Median hospitalization –– 6 6

daysdays–– Case fatality rate Case fatality rate –– 0.5 to 1%0.5 to 1%

HME and HGA HME and HGA -- diagnosisdiagnosis

blood smear (acute phase only)blood smear (acute phase only)HME HME –– 2% sensitive2% sensitiveHGA HGA –– 25 to 75% sensitive25 to 75% sensitive

PCR on blood (acute phase only)PCR on blood (acute phase only)Sensitivity ~50Sensitivity ~50--60% during acute phase60% during acute phaseno chronic phase in humans, brief persistence no chronic phase in humans, brief persistence of DNA after therapyof DNA after therapy

SerologySerology (paired acute and convalescent)(paired acute and convalescent)IFA IFA -- preferredpreferredWestern blot Western blot –– rarely usedrarely usedRecombinant protein EIA Recombinant protein EIA –– no proven benefitno proven benefit

Ehrlichioses and Anaplasmosis:Ehrlichioses and Anaplasmosis: ComplicationsComplications

++++myocarditis or heart failuremyocarditis or heart failure

0.5%0.5%3%3%deathdeath

++

++++++

++++

++++

++

++++++

++++

HMEHME

++++opportunistic infectionsopportunistic infections

++meningoencephalitismeningoencephalitis

++++++increased severity with preincreased severity with pre--existing diseaseexisting disease

--fulminant infections in immunocompromisedfulminant infections in immunocompromised

++++Toxic or septic shockToxic or septic shock--like syndromelike syndrome

++++peripheral neuropathiesperipheral neuropathies

++pneumonia/ARDSpneumonia/ARDS

HGAHGAClinical complicationClinical complication

Results of diagnostic laboratory testing for HGA and outcome of tests of 144 patients with HGA in the upper Midwest and from lower New York State

A. phago.

A. phago.

Acute 1 6 12 18 24 30 36 42

Months

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GM

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Early treatment

No treatment

Effect of therapy and kinetics of serologic reactions in HGAEffect of therapy and kinetics of serologic reactions in HGA

Sensitivity and specificity of serological tests for

confirmation of HME and HGA.

8383--100%100%2727--37%37%IFA IgMIFA IgM

8282--100%100%8282--100%100%IFA IgGIFA IgG

HGAHGA

Not determinedNot determined86%86%IFA IgMIFA IgM

Not determinedNot determined88%88%IFA IgGIFA IgG

HMEHME

SpecificitySpecificitySensitivitySensitivitySerological AssaySerological AssayDiseaseDisease

HME and HGA HME and HGA -- therapytherapyDoxycyclineDoxycycline

200 mg 200 mg popo twice daily for 3twice daily for 3--5 days after afebrile5 days after afebrileempirical clinical efficacyempirical clinical efficacygood in vitro activitygood in vitro activity

Rifampin for pregnancy and children?Rifampin for pregnancy and children?scant empirical efficacy datascant empirical efficacy datagood in vitro activitygood in vitro activityPotentially useful in children, pregnancyPotentially useful in children, pregnancy

Fluoroquinolones?Fluoroquinolones?Potentially active against Potentially active against A. phagocytophilumA. phagocytophilum(HGA), but apparently non bactericidal(HGA), but apparently non bactericidalNot active against Not active against E. chaffeensisE. chaffeensis (HME)(HME)

gyrAgyrA QRDR serine 83 (susceptible) QRDR serine 83 (susceptible) →→ alanine (resistant)alanine (resistant)

Prevention of Lyme disease, RMSF, HME, and HGA Prevention of Lyme disease, RMSF, HME, and HGA in humansin humansprompt tick removalprompt tick removaltransmission of transmission of A. phagocytophilumA. phagocytophilum may require as little as 4hmay require as little as 4h

No vaccine currently available for humans

Prophylaxis for HME and HGA after tick bite not investigated

Thanks forlistening.

Questions?

Thanks forlistening.

Questions?

Human monocytic and granulocytic ehrlichiosisHuman monocytic and granulocytic ehrlichiosiswhen and what to testwhen and what to test……

Fever, headache, history of tickFever, headache, history of tick--bite or bite or ––exposure during atexposure during at--risk risk seasonseason

leukopenia, thrombocytopenia, leukopenia, thrombocytopenia, elevated elevated LFTsLFTs

acute phase EDTAacute phase EDTA-- or citrateor citrate--anticoagulated bloodanticoagulated blood

obtain blood prior to or at the obtain blood prior to or at the time of first therapytime of first therapy

do not withdo not with--hold therapy until hold therapy until laboratorylaboratory--confirmedconfirmed

E. chaffeensisE. chaffeensis

A. phagocytophilumA. phagocytophilum

E. ewingiiE. ewingii