Lyme notifications – USA

• 6th most common notifiable disease in 2011

http://www.cdc.gov/lyme/stats/chartstables/reportedcases_statelocality.htmlhttp://www.cdc.gov/lyme/stats/chartstables/incidencebystate.html

Reservoir competenceHigh –White footed mouseLow – Deer MouseRefractory – Deer; Lizard

Preferred Hosts North – White footed mouseSouth – LizardWest – Lizard

Lyme Disease Europe

• Annual cases - 65,500 – <1 / 100,000 population - 350 / 100,000 population– Highest in residents of northern & central countries – Focal pattern of distribution related to suitable tick

habitat• hotspots where more than 100 / 100,000 population per year

– Parts of Slovenia, Germany, Austria, Baltic coastline of southern Sweden, and some Estonian & Finnish islands

Euro Surveill. 2011;16(27):pii=19906. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19906

Lyme Disease Australia

• Handful of Lyme like cases Hunter, Central NSW Coast, NSW south coast 1-3

• Culture positive case B.burgdorferi & B.garinii however as well being bitten by a tick had travelled to USA & central Europe4-5

• Seroprevalence in blood bank population and non endemic regions – 2-4%

• 2011 NSW – expert panel review of suspected caes 1. MJA 1982;1:139, 2. MJA 1986;145:364; 3. MJA 1986;144;724 4. CDI 16/24;516; 5. MJA 1998;168:500

Human Infection• Early localised (Stage I) - (requires > 24 hours attachment)

– Dermal infection - erythema migrans (80% of patients)– ± Constitutional signs

• Early disseminated (Stage II) - hematogenous +/-lymphatic dissemination (weeks to months)– 2°EM lesions (multiple)– Neurological (15%) – lymphocytic meningitis; VII N palsy, painful meningoradiculitis

(Bannwarths syndrome)

– Cardiac (5%)- high grade AV conduction defects, myopericarditis– Joints – recurrent brief attacks in 1-3 joints

• Late infection (Stage III) - months to several years – 60% rheumatological involvement– 5% chronic neuroborreliosis - PN, encephalopathy– Rarely acrodermatitis chronica atrophicans in US

Clinical Manifestations of Confirmed Lyme Disease Cases US 2001-2010

Laboratory Diagnosis

• Low numbers of Lyme spirochaetes in any clinical sample necessitates amplification for detection

–Culture–PCR–Serology

Borrelia Culture

• Media: Barbour Stoenner-Kelly (BSK) with rabbit serum & albumin

• Incubate 30°C microaerophilically• Slow growth – days to weeks • Cultivable samples: – ticks – infected reservoirs – human (EM skin biopsy, blood, synovial tissue, CSF)

• Cultures positive mainly from early LD (EM & neuroborreliosis)• Limited clinical utility

Molecular Detection

• No. of spirochaetes is low in clinical specimens • Sample types – skin, blood, CSF, synovial fluid • Targets include – flaB, 16SrRNA, recA, p66, ospA,

5SrRNA-23SrRNA gene spacer region• Australia PCR - PaLMs: B Hudson – nested PCR

targeting flagellin gene • Only accepts skin/CSF – need to contact lab

Approximate Yields Culture v PCR* Site Culture PCR

Skin (EM) > 50% > 60%

Blood (EM patient) > 40% 15%

CSF 10% 20-35%

Synovial Fluid ~ 0% 75%

Aguero-Rosenfeld et al Clin Micro Rev 2006:18;484* Over 30 studies reviewed; positive correlation with multiple EM and other evidence of dissemination… ………. < 1 spirochaete/ml whole blood in over 70% of patients

Differential Expression of Borrelial Genes • Complex antigenic

composition • Differential expression of

many genes between tick and mammal environment

• Antigenic differences among the different species of B.burgdorferi sensu lato

Important antigens

• Flagellar protein Flagellin (41kD) - FlaB– Immunodominant– Early IgG / IgM response– BUT – cross reactive • with other bacteria especially when denatured for immunoblots• mammalian tissue –synovium, neural & cardiac tissue

• Flagellar outer sheath protein (37 kD) - FlaA– Also immunodominant especially in early disease

Serology contd Outer surface proteins - Osp A-F • OspC (21–25kD)

– Immunodominant – Expressed with tick feeding– Highly passaged organisms do not express OspC– Heterogeneous amongst and within genospecies – Virulence factor – infectivity and invasiveness– Antibodies bactericidal – pepC10 = synthetic peptide = Conserved C terminal region

• OspA (31kD)– 25°C unfed tick mid gut – Not expressed early – Detected during late infection– Antibodies bactericidal

Lyme Vaccine

• Recombinant OspA (LYMErix; GlaxoSmithKline, NY) 1998

• anti-Borrelia antibodies - taken into the tick’s gut during feeding

• These abs attack the spirochete, thereby ending the cycle of infection

• Effectiveness rivalled that of simple environmental precautions

• Withdrawn 2002 - economic reasons; long-term efficacy doubts, no recommendations for children or arthritis

Serology contd • Early human infection• FlaB (41 kDa)• Osp C (23 kDa)• VlsE = Vmp-like sequence expressed

lipoprotein (34kD)– Surface exposed lipoprotein from

a linear plasmid – Variable and invariable domains – C6 = invariable of VlsE is highly

immunogenic • BmpA - Borrelia Membrane Protein (39kD)• Late: marked adaptive immune response with

an expanding antibody profile

Serological Assays • At least 80 different commercial assays – FDA approved • Most use B31 type strain as a source of antigen– Whole cell sonicates - IgG, IgM, IgG/IgM

• Low passage isolates • Sensitivity if < 1 week = < 50% • Sensitivity increases with time • Invariably positive with late disease (arthritis)• Lack specificity – cross reactions with heat shock proteins, flagellar antigens

– Recombinant antigens: e.g Bmp A (39 kDa)– Purified antigens – flagellar components– Single synthetic peptide derived from VlsE (C6 peptide)• Vmp-like sequence expressed protein (34kD)

Limitations

• Lack of standardisation – Variation in antigen used in EIA– Immunoglobulin class– FDA approved kits - not standardised against a panel of

well characterised sera – Duration of infection– Disease manifestation (disseminated v localised) – Properties of B.burgdorferi strain

False Positives

• Tick-borne relapsing fever • Syphilis • Anaplasmosis: granulocytic ehrlichiosis• Leptospirosis • Some autoimmune disorders (e.g., lupus) • Bacterial endocarditis • Infection with Helicobacter pylori, Epstein Barr virus, or

Treponema denticola (bacteria found in the mouth that can cause gum disease and/or infection after dental procedures)

Incidence/Pretest Likelihood Predictive Value

• Incidence 1%• Assume Sens 98% Spec 98%• NPV = 970/970 =100%• PPV = 10/30 =33% • (67% false positives )

• Incidence 40% • Assume Sens 98% Spec 98%• NPV = 588/596 =99% • PPV = 392/404 =97%

Test+

Test-

Total

Disease 10 0 10No Disease 20 970 990Total 30 970 1000

Test+

Test-

Total

Disease 392 8 400

No Disease 12 588 600

Total 404 596 1000

Even with a good test, false positives occur when pretest likelihood is low

CDC Criteria for WB interpretation • IgM WB

– Engstrom et al - strain 297– 2 of 3 immunoreactive bands – FlaB (41 kDa), BmpA (39 kDa ), and OspC (23/4 kDa )– Can only be used in the first 4 weeks of illness

• IgG WB – Dressler et al – strain G39/40– 5 of 10 bands – OspC (21kD), FlaB (41kD), 93 (P83/100), 66kD, 58kD, 45kD, BmpA (39kD),

30kD, 28kD, 18kD– Band number increases with neuroborreliosis or late LB– Can be used early or late in illness

• Allows detailed examination of the immune response over time MMWR 1995;44:590-591

• IgM WB 1. 41-kDa, 39-kDa protein, OspC 2. early LB with EM3. early disseminated LB with multiple

EM lesions

• IgG WB 1. significant antigens2. early disseminated LB with

neurological involvement3. Lyme arthritis4. 3 doses OspA vaccine (31kD)

IgG WB ICPMR

• Whole cell organism electrophoresed and transferred to nitrocellulose

• B.burgdorferi and B.afzelii• IgM WB not offered; EIA pos samples only tested • Most common bands - 41kDa (flagellin) and 58kDa • Nearly 42% have no bands• 54% 1-4 bands • 4% (71) have ≥ 5 bands (average from 1994 – 2011).

Unorthodox assays • CD57 is on NK cells• Lyme urine antigen tests (LUAT)• Immunofluorescent staining for cell wall--deficient

forms of Borrelia burgdorferi (QRIBb)• Lymphocyte transformation tests (LTT) • PCR on inappropriate specimens such as blood

and urine or • Interpret Western blots using criteria that have not

been validated and published in peer-reviewed scientific literature.

MMWR February 11, 2005 / 54(05);125CVI 2009, 16:1249–1250 Marques A, et al

Challenge: increase sensitivity, maintain specificity, simplify complexity

• EIA - C6 (VlsE) 1

– Results similar to 2 tier testing – More sensitive marker of early disease (I and II)– Less sensitive in chronic disease (III) – a few patients lacked reactivity

with C6 peptide – Less specific than 2 tier testing

• 2- tier approaches increase specificity • IgM WB

– Sensitive in early disease – Subjective and suffers false positives – CDC – illness <1 month (reduces sensitivity in stage 2 acute

neuroborreliosis 1. CID 2008 ;47:188 Steere et al 2. CID 2010;50:20 Branda et al 3. CID 2011;153:541 Branda et al

Alternative 2 –tier strategies • Standard 2 tier: WC EIA followed by IgM / G WB• 2-EIA: WC EIA followed by C6 EIA • C6 alone

2-EIA strategy realises sensitivity benefits of C6 in stages I and II minimizes complexity/subjectivity and maintains specificity of standardised 2 –tier Branda JA et al CID 2011;53:541-547

• Options:– 2 step EIAs – WC EIA followed by C6 EIA – C6 assays could replace 2 step testing altogether because of

increased sensitivity – Add recombinant antigens & peptides to immunoblots

• Need further evaluations using well characterised LB sera at different stages of illness

• Need to ensure state of art testing algorithms – Develop an Australian Laboratory Case Definition

• No Australian QAP programmeCID 2008:47 :196

Independent Appraisal and Review of ILADS

2004 ‘Evidence-based guidelines for the management of Lyme disease’

8 December 2010The ILADS guidelines are poorly constructed and do not provide ascientifically sound evidence-based approach to the diagnosis and care ofpatients with Lyme borreliosis.The ILADS working group does not provide evidence that it used a Cochrane basedor similar approach in developing the guidelines. Some references donot provide evidence to support statements for which they were cited in theguidelines. Some good-quality peer-reviewed articles are selectively quoted,using sub-group analyses without regard for the broader findings of the fullstudies. Some references were published in a n advocacy group-sponsoredjournal that was not Medline -listed, others are available only as conference /symposium abstracts or are unpublished. Some reference citations areinaccurate, demonstrating poor attention to detail

“Evidence-based guidelines for the management of Lyme disease” (Cameron et al. Exp Rev Anti-infect Ther 2004;2:S1-13).