Beware the black spot BELINDA LIN ID/MICROBIOLOGY REGISTRAR BARWON HEALTH

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Jayson Fletcher
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1 Beware the black spot BELINDA LIN ID/MICROBIOLOGY REGISTRAR BARWON HEALTH

2 Mr MG, 61 Presents unwell 1 week following trekking the Kokoda Headache, arthralgias High fevers to 40 C, drenching sweats Delirium Atovaquone/proguanil (malarone) for malaria prophylaxis

3 Past Medical History Dyslipidemia Gastroesophageal reflux disease Medications Pantoprazole NKDA

4 Examination Blanching rash on back Small lesion Eschar?

5 Bloods from GP Dengue serology and NS1 Ag Negative Malaria thick and thin film No parasites seen Malaria ICT Negative In Hospital WCC 8.8 Neut 3.0 Lymph 4.7x10 9 /L Na 131mmol/L ALT 65, ALP 199, GGT 389IU/L, bilirubin 8 umol/l, albumin 30g/L CRP 10.7mg/L

6 HIV serology Negative HBV serology Immune HCV serology Negative Leptospirosis serology sent Rickettsial serology sent

7 Rickettsial serology 29/5 Spotted fever group Rickettsia australis Negative (<128) Rickettsia honei Negative (<128) Rickettsia conorii Negative (<128) Rickettsia africae Negative (<128) Rickettsia rickettsii Negative (<128) Rickettsia felis Negative (<128) Typhus group Rickettsia prowazekii Negative (<128) Rickettsia typhi Negative (<128) Scrub typhus group R. (Orentia) tsutsugamushi Gilliam POSITIVE (8192) Karp POSITIVE (256) Kato POSITIVE (2048) Cowley Beach POSITIVE (256) Orentia chuto Negative (<128)

8 Rickettsial PCR on skin biopsy Spotted Fever/Typhus group Not detected Scrub Typhus DETECTED

9 Case 2 Ms LZ, 25 Visiting Great Ocean Rd from Melbourne Transferred from a peripheral hospital with Fevers, rigors, meningism & mixed blanching rash on both her legs Proximal DVT, rivaroxaban

History Day 1: fevers and sore throat Day 3: begins trip to Great Ocean Road, develops prominent headache, myalgias, rigors and mild photophobia/neck stiffness

10 History Day 1: fevers and sore throat Day 3: begins trip to Great Ocean Road, develops prominent headache, myalgias, rigors and mild photophobia/neck stiffness Day 2: visit GP in Melbourne, Rx roxithromycin Day 4: visit local hospital, rash noted on her leg and transferred to Geelong. Rx IV dexamethasone and ceftriaxone.

11 Past History Proximal DVT, IVC filter Single functioning kidney Asthma Medications Rivaroxaban Mirena Allergy: penicillin (rash), contrast (rash/itch)

12 Epidemiology Previous travel to Africa and South East Asia 2013 Took malaria prophylaxis Social History Home with parents Stable relationship Vaccination Childhood vaccinations Meningococcal C

13 Examination 38.1 C HR /70 98% on air Photophobia and neck stiffness Rash (see picture)

14 Investigations FBE: WCC 10.5-, Neut 9.4-, Lymph 0.7x10 9 /L UEC: Na 135-, bicarb 19-, urea 4.5mmol/L, Cr 117 umol/l, egfr 56mL/min LFT ALT 18-, ALP 76-, GGT 19U/L, bilirubin 12 umol/l albumin 37g/L CRP 185mg/L INR 1.9, Fibrinogen 5.7g/L B-HCG <1 IU/L CXR no infective changes seen

15 Cultures Urine culture 22/2 WCC <10 Blood culture 22/2 negative LP ideal but not done due to rivaroxaban NPS influenza A, B & RSV PCR negative HIV serology Not done N. meningitidis PCR on serum: negative

16 History Day 1: fevers and sore throat Day 3: begins trip to Great Ocean Road, develops prominent headache, myalgias, rigors and mild photophobia/neck stiffness Day 5: worsening symptoms of meningism Day 2: visit GP in Melbourne, Rx roxithromycin Day 4: visit local hospital, rash noted on her leg and transferred to Geelong. Rx IV dexamethasone and ceftriaxone.

17 Epidemiology Previous travel to Africa and South East Asia 2013 Contact with wallabies and kangaroos on Philip Island 4 days prior to disease onset

18 Differentials

19 Rickettsial Serology 23/2 Spotted fever group Rickettsia australis POSITIVE (512) Rickettsia honei POSITIVE (128) Rickettsia conorii POSITIVE (128) Rickettsia africae POSITIVE (256) Rickettsia rickettsii POSITIVE (256) Rickettsia felis Negative (<128) Typhus group Rickettsia prowazekii Negative (<128) Rickettsia typhi POSITIVE (128) Scrub typhus group R. (Orentia) tsutsugamushi Negative (<128) Orentia chuto Negative (<128)

20 Rickettsial Serology 1/5 23/2 Spotted fever group Rickettsia australis POSITIVE (256) (512) Rickettsia honei POSITIVE (256) (128) Rickettsia conorii POSITIVE (256) (128) Rickettsia africae POSITIVE (256) (256) Rickettsia rickettsii POSITIVE (256) (256) Rickettsia felis Negative (<128) (<128) Typhus group Rickettsia prowazekii Negative (<128) (<128) Rickettsia typhi POSITIVE (128) (128) Scrub typhus group Not tested R. (Orentia) tsutsugamushi Negative (<128) Orentia chuto Negative (<128)

21 Literature Review Epidemiology of Rickettsial disease and their vectors Diagnostic tools Serology PCR MALDI-TOF

22 Rickettsioses Obligate intracellular gram-negative bacteria that invade endothelial cells and induce the formation of vasculitis Triad symptoms: fevers, headache and rash 26 Species SFG Typhus Scrub typhus Parola et al. CMR 2013

23 Transmission Bite from a tick, flea, louse or helminth Salivary excretions Mite Faecal matter Laboratory based aerosol Transfusion and transplant Seasonal most cases diagnosed late spring and summer

24 Rickettsia in Australia Geographic distribution mainly determined by their vectors

25 Rickettsia Vector R. australis Tick - Ixodes holocyclus R. honei Tick - Bothriocroton hydrosauri R. tsutsugamushi Mites (chiggers) - Leptotrombidium deliense R. prowazekii Human body louse (Pediculus R. typhi humanus corporis), some ticks, fleas

26 Ixodes holocyclus R. australis (QLD tick typhus) 3 host tick Larva, nymph & adult Slow feeders Optimal host - bandicoots

27 Bothriocroton hydrosauri R. honei (Flinders Island SF) Southern reptile tick 3 host tick Primary host Tiliqua rugosa (sleepy lizard) Opportunistically feed on humans, cattle and horses

28 Leptotrombidium mites R. tsutsugamushi Only larval forms of Leptotrombidium mites transmit disease Feeds on liquified skin tissue

29 Map of the area where scrub typhus is endemic. Daryl J. Kelly et al. Clin Infect Dis. 2009;48:S203-S by the Infectious Diseases Society of America

30 Eschar Site of bacterial entry sometimes develops into a localised inflammatory and necrotic skin lesion with a black crust, termed an inoculation eschar

31 Eschar Location Kim et al. Am J Trop Med Hyg 2007

32 Diagnosis of Rickettsial infections

33 Weil-Felix 1916 Uses Proteus OX-19 & OX-2 agglutination Poor sensitivity and specificity

34 Cell culture Vero cells African green monkey kidney epithelial cells Picture: Giménez staining of R. rickettsii-infected Vero 76 cells. The black arrow points to an individual bacterium. Ammerman et al. Curr Protoc Microbiol. 2008

35 Immunofluorescence Assay (IFA) 1963 Reference method How does it work?

36 Bozeman and Elisberg. Proc Soc Exp Biol Med. 1963

37 Seroconversion usually 7-15d after onset of illness 25-28d for R. africae infection IgM and IgG titres 128 considered positive result How well does this correlate with infection?

38 ,603 febrile patients admitted to the district hospital in Peninsular Malaysia Group 1 diseased population defined by isolation of R. tsutsugamushi and no conflicting clinical or serological diagnosis Group 2 control population negative culture for R. tsutsugamushi and definite alternative diagnosis (S. typhi or paratyphi, malaria, leptospirosis or flavivirus) At a cut-off point of >=1:400, the IFA test was 0.96 specific Brown et al. Am J Trop Med Hyg. 1983

Rickettsial Serology Use in diagnosis of Rickettsial infection: 4-fold increase in the titer in paired serum samples OR a single sample titer when there is an adequate local evidence base

39 Rickettsial Serology Use in diagnosis of Rickettsial infection: 4-fold increase in the titer in paired serum samples OR a single sample titer when there is an adequate local evidence base Disadvantage Cross reactivity between assays for different species in Spotted Fever group, Legionella and Proteus species Seroconversion usually 7-15d after onset of illness Inter-observer variability Blacksell et a. Clin Infect Dis Brouqui et al. Clin. Microbiol. Infect Parola et al. Clin. Microbiol. Rev

40 IFA Inter- & intra-observer Variability IFA interpretation subjective Inter-observer agreement (K) K = 0.45 (moderate) Murine typhus (R. typhi) K = 0.32 (fair) Scrub typhus (R. tsutsugamushi) Microscopist inexperience plays a role Phetsouvanh et al. Am J Trop Med Hyg. 2013

41 PCR Specimen Eschar Whole blood Skin biopsy

42 PCR of Eschar vs Blood in Scrub Typhus Nested PCR Eschar more sensitive than blood Sensitivity 86% (95% CI: 78-92%) Remains positive after initiation of treatment Blood Buffy coat used Sensitivity 82% (95% CI: 74-88%) Specificity 100% (95% CI: %) Kim et al. CID 2006

43 Effect of Abx Whole blood specimen

44 Rickettsemia 3 healthy volunteers infected with R. tsutsugamushi via laboratory-reared chiggers Clinical disease 8-10 days post chigger attachments all had eschars All subjects improved dramatically <24h following Abx therapy Rickettsemias detected in all 3, beginning on d7 PCA (bloods taken every 3d) Seroconversion (IFA) on d13-19 Shirai et al. Japan J Med Sci Biol 1982

45 Identification of Ticks Traditionally - morphology MALDI-TOF technology (research only) Specimen Tick legs Hemolymph protein mixture from tick legs Advantages: Requires no expertise in tick ID Inexpensive, rapid & accurate Dual identification of tick species and associated pathogens Can identify damaged ticks Need tick MS library Yssouf et al. J Clin Micro 2013 Yssouf et al. Ticks Tick Borne Dis 2015

46 Consistent and reproducible MS profiles were obtained from analysis of the legs of six tick vectors: Amblyomma variegatum, Rhipicephalus sanguineus, Hyalomma marginatum rufipes, Ixodes ricinus, Dermacentor marginatus, and Dermacentor reticulatus (French ticks) Yssouf et al. J Clin Micro 2013

47 Fig. 1. Photograph of a tick hemolymph drop collected by severing one leg from an adult specimen for the protein extraction used in the MALDI-TOF MS analysis. The hemolymph drop is indicated by the arrow. Yssouf et al. Ticks Tick Borne Dis. 2015

48 Proof of Concept: identification of tick species and presence of Borreliae in a single assay 88.9% sensitivity and a 93.75% specificity for the detection of B. crocidurae in 50 O. sonrai ticks Fotso Fotso et al. PLoS Negl Trop Dis 2014

49 R. conorii conorii or R. slovaca in Rhipicephalus sanguineus and Dermacentor marginatus ticks Using protein extracts prepared from tick legs Yssouf et al. PLoS Negl Trop Dis 2015

50 SUMMARY Consider Rickettsial disease in patients with fevers, headache and rash Eschar important in diagnosis Immunofluorescence current gold standard and readily available in Victoria Rapid diagnostic tests available PCR on blood, eschar and skin biopsy specimen

51 Acknowledgement ID unit, Barwon Health A/Prof Eugene Athan Dr Raquel Cowan Dr Eleanor MacMorran Australian Rickettsial Reference Laboratory A/Prof Jon Stenos Ms Chelsea Nguyen

Three patients with fever and rash after a stay in Morocco: infection with Rickettsia conorii

Three patients with fever and rash after a stay in Morocco: infection with Rickettsia conorii Stylemans D 1, Mertens R 1, Seyler L 1, Piérard D 2, Lacor P 1 1. Department of Internal Medicine, UZ Brussel