Anaplasma. Agent Searching a Disease
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1 Anaplasma Agent Searching a Disease Dag Nyman Labquality 2017
2 Disclosures Partner, clinical and research director Biomedical Laboratory Bimelix Contact - Prof. Dag Nyman MD PhD <dag.nyman@aland.net> Research funding: grants from - Wilhelm and Else Stockmann Foundation - Åland Cultural Foundation
3 Anaplasma phagocytophilum A rickettsial, gram-negative, obligate intracellular pathogen (Rikihisa 1991) Phylogeny reclassified 2001 (Dumler) Transmitted by ticks (Magnarelli 1995) Most widespread infection in animals in Europe since 70 y (Stuen 2007) Seroprevalent in humans in Europe (Broqui 1995) 1 25% Human pathogen recently USA (Chen 1994) total cases European Human Granulocytic Anaplasmosis (HGA) (Petrovec 1997) total 100 cases (58 according to ESCMID GL, only 26 PCR + serology verified)
4 Taxonomy Anaplasmataceae α-proteobacteria Rickettsiales - Anaplasmataceae - Rickettsiaceae 16SrRNA gene groesl (Dumler & al 2001) Rikihisa, Nature Reviews Microbiology, 2010
5 Anaplasma prevalence in ticks Molecular prevalence of A. p. in European questing (Stuen 2013) - I. ricinus % - I. persulcatus % Adults > nymphs, unfed larvae 0 % Geographic, spot and seasonal variation Åland Islands 1.2% (Henningsson (2015) Southwestern Finland 3.5% (Sormunen 2016) A.p. in salivary gland Ueti 2009
6 Transmission Ixodes spp. hard ticks competent vector (mosquitos?) Tick infection rate in Europe: adults 2-44%, nymphs 1-24% (Nieto & Foley, 2009) Transmission vertebrate to tick enterocytes, hemocytes, salivary glands within 24 h (Liu 2011) Transmission by cofeeding (Ogden 2003) Transstadial, not transovarial transmission (Ogden 1998) Transmission tick to vertebrate h (Hodzic 1998) Incubation time to disease 5 21 d (Chapman 2006)
7 Tick factors for transmission Infection of tick hemocytes is mediated by the salivary protein P11 and is required for successful migration of the pathogen from the midgut to salivary glands (Liu 2011) Colonization of salivary gland cells Salp16 dependent (Sukumaran 2006)
8 Alternate transmission routes Blood transfusions, also leukoreduced (CDC 2016) Transplacental mother-child (Horowitz 1998) Direct contact, deer butchers (Bakken 1996) Human human nosocomial (Zhang 2008)
9 EUROPEAN EPIDEMIOLOGICAL CYCLES Dugat et al Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks (Rikihisa 2011)
10 Anaplasmosis case definition Confirmed human anaplasmosis 1. Febrile illness with a history of a tick bite or tick exposure and 2. Demonstration of Anaplasma phagocytophilum infection by seroconversion or 4-fold change in antibody titre or 3. Positive PCR result with subsequent sequencing of the amplicons demonstrating Anaplasma-specific DNA in blood or 4. Isolation of A. phagocytophilum in blood culture Probable human anaplasmosis 1. Febrile illness with a history of a tick bite or tick exposure and 2. Presence of stable titre of A. phagocytophilum antibodies in acute and convalescent sera if titre > 4-fold above the cut-off value or 3. Positive PCR result without sequence confirmation or 4. Presence of intracytoplasmic morulae in a blood smear ESCMID GL 2004
11 Case definition A case definition is set of uniform criteria used to define a disease for public health surveillance. Case definitions enable public health to classify and count cases consistently across reporting jurisdictions, and should not be used by healthcare providers to determine how to meet an individual patient s health needs. CDC 2015
12 Clinical Anaplasmosis Frequency of complaint Symtom or Sign (N) Median % (IQR) Common Fever (794) 100 (90-100) Malaise (391) 97(90-98) BROAD Headache (648) 82 (64-93) DDx Myalgia (789) 76 (67-87) Arthralgia) 56 (27-69) Less common Stiff neck (64) 45 (34-48) Nausea (521) 39 (35-49) Cough (523) 29 (20-30) Uncommon Diarrhea (317) 21 (13-28) Vomiting (312) 20(19-29) Rash (489) 6(3-10) CNS manifestation < 1% In <10% erythematous, nonpruritic rash on the upper chest and neck NOT EM Bakken 2015
13 Laboratory abnormalities Frequency of Finding Laboratory Abnormality Median % (IQR) Common S-ALAT or S-ASAT (397) 83 (63-98) Thrombocytopenia (566) 75 (61-91) Leukopenia (566) 55 (47-71) Less common S-Creatinine (199) 49 (25-71) Anemia (198) 28 (6-44) Bakken 2015 Inflammatory parameters HGA n=24 (%) Median (Range) ESR >20 mm/h 18 (75) 34 (20-128) CRP >10 mg/l 23 (96) 100 (12-314) Lotric-Furlan 2006
14 Serology IFT for IgG-Ab ( IgM not useful) Acute and convalescent testing Seroconversion or 4-fold change of titre Ab s may persist for months or years in tha absence of any evidence of ongoing infection Positive (green) Anaplasma IFT Focus Relative sensitivity of diagnostic test Duration d Smear Culture PCR IFA 0-7 Medium Medium High Low 8-14 Low Low Low Medium Low High High >60 High Bakken 2005
15 PCR, Smear and Serology PCR + PCR - Sample N n (%) Sample N n (%) p-value Total (15.7) (86.4) Serologypositive Smearpositive Controls w/o symptoms (39.6) (29.6) (56.0) 74 0 (0) < (100) Smear Serology (26) DIAGNOSTIC PERFORMANCE Serology - Sens 40% - Spec 70 % = LR Not useful PCR - Sens 80 %? - Spec 100 % - = LR+ 80 Very useful Schotthofer 2013
16 Morulae on smear Weil 2012 Morulae on peripheral blood smears In 100% of verified HGA cases! Illness duration 1 28 d Count ad 200 granulocytes Rand & al. Am J Clin Pathol May 2014;141: Rand 2014 As with other studies, we found that maximum positivity was obtained in the early phase of illness, when morulae were likely to be visible in blood smear analysis (50% of our PCR-positive patients in the early phase were also smear positive) and before an effective antibody response had been mounted by patients (74% of these PCRpositive results were seronegative (Schotthoefer 2013)
17 HGA Severity & Outcome (USA) Mild 55% Hospitalization 36% ICU 7% Mortality <1% US Cases CDC 2016 Confirmation of the diagnosis is based on laboratory testing, but antibiotic therapy should not be delayed. Bakken 2015
18 Treatment Mild cases w/o Tx recover spontaneously within 60 d Doxycyclin 100 mg x 2 adults for 7 10 d Pregnant, allergic patients rifampicin 300 mg x 2 Clinical improvement in h, if not: other Dx! No chronic cases described Bakken 2015
19 Nordberg, M., Forsberg, P., Berglund, J., Bjöersdorff, A., Ernerudh, J., Garpmo, U., Haglund, M., Nilsson, K. and Eliasson, I. (2014) Aetiology of Tick-Borne Infections in an Adult Swedish Population Are Co-Infections with Multiple Agents Common? Open Journal of Clinical Diagnostics, 4, Co-infections (anaplasmosis?) Confirmed a) IgG antibody seroconversion i.e. from negative to positive or b) at least a four-fold rise in IgG antibody titre or c) positive A. phagocytophilum PCR Probable: a) a permanently high IgG antibody titre of 640 or b) at least a four-fold decrease in IgG antibody titre during the investigation period of 12 months. Signs/ Symptoms IgGAb Borrelia IgG Ab Anaplasma PCR EM 39/109 22/109 Neg EM+Unspec 35/65 14/65 Neg Unspec 12/32 6/32 Neg Total 86/206 42/206 Neg (TBE 2/206)
20 Anaplasma in ticks and seroconversion, STING-study Anna J. Henningsson, Peter Wilhelmsson, Paula Gyllemark, Monika Kozak, Andreas Matussek, Dag Nyman, Christina Ekerfelt, Per-Eric Lindgren, Pia Forsberg Low risk of seroconversion or clinical disease in humans after a bite by an Anaplasma phagocytophilum-infected tick Ticks and Tick-borne Diseases, 6, 2015, (800 from the Åland Islands) ticks detached from humans collected Anaplasma phagocytophilum detected by PCR in 31 (1.2 %), equally in Sweden and the Åland Islands Paired sera with 3 mo interval tested for IgG seroprevalence and seroconversion/increased titre by IFT Tick PCR status n Seroprevalence % Seroconversion n Symtoms n PCR pos A/neg Bb / 6 symptomatic PCR neg A/Bb pos / 6 PCR neg A/Bb neg / 5 1 / 0
21 Clonal complex Clonal complexes and host species of the A. phagocytophilum strains typeable by MLST (n = 284). MLST analysis of seven housekeeping genes (phes, glya, fumc, mdh, suca, dnan, atpa) Number Host species n EU(n all) Clonal complex Number Host species (61) dogs (42) humans 32 (42) humans 1 (61) dogs 28 (28) horses (44) sheep 24 (24) hedgehogs (44) sheep 12 (12) wild boars (12) roe deer 3 (44) sheep without (44) sheep 2 (2) cats 10 (12) roe deer 2 (2) red foxes 3 (3) red deer 14 (20) ticks 2 (6) cattle (17) voles 2 (6) Eu-bison 3 (3) shrews 1 (1) chamois 4 (12) cattle 6 (20) ticks Huhn 2014
22 MLST and 16SrRNA gene, AnkA gene HGA is likely to be underdiagnosed in Europe. Wild boars and hedgehogs may serve as reservoir hosts for granulocytic anaplasmosis in humans and domestic animals in Europe. Roe deer, voles and shrews are unlikely to be reservoir hosts for granulocytic anaplasmosis in humans, domestic or farm animals. Huhn 2014
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