Vector-Borne and Zoonotic Diseases r ee rP Fo Vector Borne and Zoonotic Diseases: http://mc.manuscriptcentral.com/vbz Re Journal: vi VBZ 2016 1962.R3 Manuscript Type: Guidelines in VBZD ew Manuscript ID Date Submitted by the Author: Complete List of Authors: n/a Keyword: Manuscript Keywords (Search Terms): ot /N ly On Cutler, Sally; University of East London, School of Health and Bioscience Rudenko, Natalia; Biology Centre, Academy of Sciences of the Czech Republic, Institute of Parasitology Golovchenko, Maryna; Institute of Parasitology, Biology Centre CAS Jochum, Wibke; Luxembourg Institute of Health, Department of Infection and Immunity Kirpach, Josiane; Luxembourg Institute of Health, Department of Infection and Immunity Savic, Sara; Scientific Veterinary Institute Novi Sad, Serbia, Rumenački put 20, 21000 Novi Sad, Serbia, Christova, Iva; National Center of Infectious and Parasitic Diseases, Microbiology Amaro, Ana; National Institute for Agrarian and Veterinarian Research (INIAV) Borrelia, Lyme disease, relapsing fever, Diagnostics, Tick(s) Lyme disease, Borrelia species, Diagnostics fo Abstract: tri is rD Borrelia species fall into two groups, the Borrelia burgdorferi sensu lato (Bbsl) complex the cause of Lyme borreliosis (LB; also known as Lyme disease LD) and the relapsing fever group. Both groups exhibit inter and intra species diversity and thus, have variations in both clinical presentation and diagnostic approaches. A further layer of complexity is derived from the fact that ticks may carry multiple infectious agents and are able to transmit them to the host during blood feeding, with potential overlapping clinical manifestations. Besides this, pathogens like Borrelia have developed strategies to evade the host immune system, which allows them to persist within the host, including humans. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 n tio bu Diagnostics can be applied at different times during the clinical course and utilise sample types, each with their own advantages and limitations. These differing methods should always be considered in conjunction with potential exposure and compatible clinical features. Throughout this review, we aim to explore different approaches providing the reader with an overview of methods appropriate for various situations. This review will cover human pathogenic members of Bbsl and relapsing fever borreliae, including newly recognised B. miyamotoi spirochetes. Page 1 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo Re ew vi ot /N ly On Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 n tio bu tri is rD fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 2 of 36 r ee rP Fo 1 2 3 Sally J. Cutler1, Nataliia Rudenko2, Maryna Golovchenko2, Wibke J. Cramaro3, 4 Josiane Kirpach3, Sara Savic4, Iva Christova5, Ana Amaro6. 5 6 7 Re 8 2. Biology Centre CAS, Institute of Parasitology, Ceske Budejovice, 37005, 10 Czech Republic 3. Luxembourg Institute of Health, Department of Infection and Immunity, Esch8 On 11 4LZ, UK. ew 9 1. School of Health, Sport & Bioscience, University of East London, London, E15 vi sur8Alzette, Luxembourg 12 4. Scientific Veterinary Institute “Novi Sad”, Rumenacki put 20, Novi Sad, Serbia 13 5. Department of Microbiology, National Center of Infectious and Parasitic 15 Portugal. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 1 n tio bu tri is rD fo 17 18 19 20 21 6. National Institute for Agrarian and Veterinarian Research (INIAV), Lisboa, ot 16 Diseases, Sofia, Bulgaria. /N 14 ly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 3 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 22 23 species fall into two groups, the sensu lato (Bbsl) 24 complex the cause of Lyme borreliosis (LB; also known as Lyme disease LD) and 25 the relapsing fever group. Both groups exhibit inter8 and intra8species diversity and 26 thus, have variations in both clinical presentation and diagnostic approaches. A 27 further layer of complexity is derived from the fact that ticks may carry multiple 28 infectious agents and are able to transmit them to the host during blood feeding, with 29 potential overlapping clinical manifestations. Besides this, pathogens like 30 have developed strategies to evade the host immune system, which allows them to 31 persist within the host, including humans. ew vi On 32 Re 33 Diagnostics can be applied at different times during the clinical course and utilise 34 sample types, each with their own advantages and limitations. These differing 35 methods should always be considered in conjunction with potential exposure and 36 compatible clinical features. Throughout this review, we aim to explore different 37 approaches providing the reader with an overview of methods appropriate for various 38 situations. This review will cover human pathogenic members of Bbsl and relapsing 39 fever borreliae, including newly recognised ot /N ly fo spirochetes. 40 tri is 41 rD 42 Various methods can be applied to detect the presence of in vectors. Widely 43 used approaches that demonstrate significant sensitivity, specificity and reliability 44 include: multiple formats of PCRs, mostly nested PCR that target different genomic 45 loci, selection of which depends on the sample origin (template); reverse8line blotting Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 2 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases r ee rP Fo 46 (RLB), based on hybridization of amplified selected genes with spirochete8 47 specific probes; multilocus sequences analysis (MLSA) and multilocus sequence 48 typing (MLST), based on the sequence analysis of amplified fragments of spirochete 49 genome or microscopy with stained spirochetes in tick midgut or salivary glands 50 (Aguero8Rosenfeld, et al. 2005, Margos, et al. 2011). The most recently applied 51 techniques include next generation sequencing (NGS) and proteomic approaches. 52 Cultivation of 53 MKP (modified Kelly8 Pettenkoffer) media, that for a long time considered to be a 54 gold standard in LB diagnostics, is still widely used, but is rather time consuming and 55 challenging. The culture negative cases do not necessarily mean the absence of 56 spirochetes in a sample. The failure to culture the spirochetes might be caused by 57 multiple vector8, spirochete,8 media8 or cultivation conditions related factors (Cerar, 58 et al., 2008, Ružić8Sabljić, et al. 2014, Rudenko, et al., 2016). Nowadays, the priority 59 of all used techniques is re8directed form simple detection of pathogen in either 60 environmental sample or clinical sample, to simultaneous detection and identification 61 of spirochete species (or possible co8infection agents). 62 possibility of the presence of multiple pathogens in tick vectors, the other question is 63 whether to use singleplex or multiplex formats for their detection/identification. Fluidic 64 microarrays allow the assessment of multiple tick8borne pathogens simultaneously 65 (Vayssier8Taussat, et al. 2013). Re vi in commercial BSK (Barbour8Stoenner8Kelly) or home8made ew ot /N ly On Considering the high tri is 66 rD fo 67 Use of proteomic methods to detect presence of some relapsing fever in the 68 hemolymph of ticks provides additional options for borrelial detection in vectors 69 (Fotso Fotso, et al. 2014). Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 3 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 36 Page 5 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 70 These methods provide invaluable research tools and facilitate epidemiological 71 studies, but their clinical relevance is debatable. Detection of a pathogen in the 72 vector does not imply that it has been successfully transmitted to the host upon 73 which the tick has fed. Transmission dynamics are complex and multi factorial and 74 beyond the scope of this review. Home use diagnostic kits are available and allow 75 individuals to test collected ticks for the presence of Lyme borreliae. The reliability of 76 these tests has been highly debated. Tick bites are frequently unnoticed and might 77 only demonstrate that you have been in a risk environment, but do not necessarily 78 correlate with any infectious consequences. That is why use of such tests is of 79 limited value for diagnosis, but can be useful for epidemiological studies. Re ew vi 80 On 81 Recommendation: 82 Tick testing as supportive data for identification of LB endemic regions; correct 83 selection of PCR target based on the final goal of tests and sample nature; re8 84 analysis of tested sample targeting different genomic loci; consider the presence of 85 co8infection with multiple pathogens as highly possible. ot rD fo 87 /N 86 ly 88 A reliable clinical diagnosis of LB is only evident to the non8expert physician when a 89 typical erythema migrans (EM) is present (Stanek and Strle 2003). Since the large 90 majority of LB symptoms have minimal diagnostic value because of their lack of 91 specificity, diagnosis of LB might be challenging for general practicioners in patients 92 without EM (Strle and Stanek 2009). Generally there exists a tendency towards 93 overdiagnosis of chronic Lyme disease (Czupryna, et al. 2016,Koedel, et al. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 4 n tio bu tri is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 6 of 36 r ee rP Fo 94 2015,Sigal 1996). Although different diagnostic approaches (mentioned later) have 95 been explored, to date the only recommended supportive tests used are serological 96 confirmation. Serological results alone are insufficient to distinguish whether the 97 patient suffers from an acute or re8infection that needs treatment, or is only 98 seropositive because of a past infection. This might be especially problematic for 99 individuals that are frequently exposed to ticks and therefore have at high risk of re8 100 infection. However even in low risk areas, the positive predictive value of serological 101 tests can be very low (Lantos, et al. 2015), meaning that clinical manifestations still 102 remain crucially important criteria for a reliable diagnosis of the disease. Factors that 103 need to be integrated for a reliable diagnosis are therefore the occurrence of 104 compatible symptoms, serological results and risk of tick exposure. Figure 1 provides 105 a diagnostic overview for LB. ot /N ly On 108 ew 107 vi 106 Re 109 In cases where EM is clearly evident, serological tests are not needed and treatment 110 should start immediately (Stanek, et al. 2012). In patients who do not develop EM, 111 serological tests are recommended to support the diagnosis (Aguero8Rosenfeld, et 112 al. 2005). Initial problems with the specificity and sensitivity of serological tests have 113 resulted in controversial statements on their efficacy to support diagnosis of acute 114 LB. Recently, serological tests have been optimized switching from a single 115 strain cell extract to a use of combination of more precisely chosen recombinant 116 antigens or synthetic peptides (Fang Ting, et al. 2000,Goettner, et al. 2005). 117 Previously a two8tier test approach, in which the presence of antibodies is first tested Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 5 n tio bu tri is rD fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 7 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 118 by a highly sensitive ELISA and, in case of a positive result, further confirmed by a 119 highly specific immunoblot, was recommended (Branda, et al. 2010,Koedel, et al. 120 2015). Noteworthy, the reported accuracy of ELISAs and immunoblots varies 121 throughout Europe and a recent study revealed no overall benefit of two8tiered tests 122 over single tests (Leeflang, et al. 2016). Only early stage patients (symptoms <6 123 weeks) might still be seronegative, as they have not developed antibodies yet. 124 Therefore, diagnosis of LB should be re8evaluated in seronegative late stage 125 patients (Stanek, et al. 2012). Low antibody titers have been observed after antibiotic 126 treatment indicating that the induced B8cell immune response is probably not very 127 long8lived and robust. Especially patients where 128 seem to develop long8lived antibody titers less efficiently (Aguero8Rosenfeld, et al. 129 1996,Elsner, et al. 2015,Hammers8Berggren, et al. 1994,Nowakowski, et al. 2003). 130 Recent mouse studies have shown that 131 cell response (Elsner, et al. 2015,Elsner, et al. 2015,Hastey, et al. 2012,Hastey, et 132 al. 2014). However, the underlying mechanism in humans requires further 133 investigation. Showing the induction of strain specific immunity (not non8 134 crossprotective), mouse and human studies together (Khatchikian, et al. 2014) may 135 explain reinfection of LB. Consequently, previous 136 into account when considering serological testing (Nadelman and Wormser 2007). ew vi took longer to disseminate ly On have a direct effect on the mouse B8 ot /N fo infections must be taken tri is rD 137 Re 138 Despite the described improvement of these tests, we still face the problem of non8 139 standardization and inappropriate application of current serological tests (Ang, et al. 140 2011,Leeflang, et al. 2016,Markowicz, et al. 2015,Muller, et al. 2012). Different (in8 141 house) assays and result interpretation remain a major problem (Fallon, et al. 2014) Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 6 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 8 of 36 r ee rP Fo 142 that should be solved in the future by the implementation of a universial and 143 worldwide (or Europe/USA wide) diagnostic standard test, or as a minimum, use of 144 internationally agreed standards and participation in quality control schemes. 145 However, the problem remains (especially amongst high risk groups) to distinguish 146 between an acute and a resolved infection. Future studies should therefore focus on 147 the development of new strategies that would allow a yes or no result. vi 148 Re 149 Noteworthy, serological tests should not be used as a proof of efficacy of the 150 antibiotic treatment, although antibody titers generally decrease after antibiotic 151 treatment, however patients may remain seropositive for years after the infection in 152 the absence of active disease (Aguero8Rosenfeld, et al. 1996,Glatz, et al. 153 2006,Hammers8Berggren, 154 2010,Lomholt, et al. 2000). Instead, the disappearance of symptoms is a more 155 reliable sign of cure. ew On et al. 1994,Kalish, et al. 2001,Kowalski, et al. ot 156 /N ly 157 When neuroborreliosis is suspected, detection of intrathecally produced anti8 158 antibodies significantly supports the diagnosis. However, results might be negative at 159 early stages and more often in children (Christen, et al. 1993). Measurement of 160 8specific antibodies in CSF cannot be used to assess the efficiency of tri is 162 treatment (Koedel, et al. 2015). rD 161 fo 163 Since antibiotic treatment is generally considered efficient, differential diagnosis is 164 crucial in case of a chronic course of the disease (Halperin 2015,Halperin 165 2016,Hjetland, et al. 2015,Markowicz, et al. 2015,Rebman, et al. 2015,Wills, et al. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 7 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 9 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 166 2016). A chronic course has been observed in patients infected by , viral and 167 non8viral pathogens, such as Epstein8Barr virus (glandular fever), 168 (Q fever), or Ross River virus (epidemic polyarthritis) (Aucott, et al. 2013,Galbraith, 169 et al. 2011,Hickie, et al. 2006,Katz and Jason 2013) and the underlying causes are 170 not clear. In this context, also the general health status and/or the lifestyle of the 171 patient should be considered. In general, immunocompromised or otherwise not 172 completely healthy patients might be at higher risk to develop chronic symptoms 173 after treatment. Patients with hematological malignancies for example seem to suffer 174 more often from disseminated disease and more frequently require retreatment 175 (Maraspin, et al. 2015). In non8immunocompromized cases, where symptoms 176 continue to persist even after appropriate antibiotics treatment, it is currently not 177 recommended to prolong the treatment. Clinical studies have shown that the risk of 178 side effects outweighs any potential therapeutic benefits (Klempner, et al. 179 2001,Koedel, et al. 2015,Krupp, et al. 2003). In these cases, co8infections with other 180 tick borne diseases or other possible causes of the symptoms should be excluded 181 (Belongia 2002,Berghoff 2012,Godar, et al. 2015,Swanson, et al. 2006) and 182 symptomatic 183 neuroborreliosis, is prolongation of the antibiotic treatment justifiable in cases of 184 persistent cerebrospinal fluid (CSF) lymphocytic pleocytosis (Koedel, et al. 2015). Re ew vi ot /N ly On treatment considered (Koedel, fo et al. 2015). Only in late 185 tri is rD 186 In rare cases, can cause problems with the heart and vascular system and 187 might be considered as underlying cause of stroke8like symptoms in patients which 188 otherwise have no obvious risk for cardiovascular diseases (Allen and Jungbluth 189 2016,Zajkowska, et al. 2015). Full description of LB clinical manifestations and their Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 8 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 10 of 36 r ee rP Fo 190 diagnosis have been recently reviewed by Stanek and co8workers (Stanek, et al. 191 2011). 192 193 When encountering a tick bite, correct and early removal of the tick is a good way to 194 reduce probability of infection. In Europe, only about 2% (Wilhelmsson, et al. 2016) 195 and in USA, about 1% (Heymann and Ellis 2012) of patients bitten by a tick develop 196 LB. Detection of spirochete DNA in ticks alone does not necessarily means the 197 succesful pathogen transmission, which is why the value of this test has limited 198 diagnostic value for LB ((ESGBOR) 2013), but is useful for epidemiological studies 199 (Reye, et al. 2010) to define risk areas. In this context, next generation sequencing 200 is a new emerging technique that allows screening of the same tick in parallel for 201 various tick8borne pathogens, with the potential of getting more detailed information 202 about co8infections of ticks and identification of new yet unrecognised pathogens 203 (Michelet, et al. 2014,Vayssier8Taussat, et al. 2013). As transmission of 204 indeed other pathogens) depends on the length of tick attachment, measurement of 205 scutal and coxial indexes can indicate duration of attachment (Crippa, et al. 206 2002,Gray, et al. 2005,Kahl, et al. 1998,Meiners, et al. 2006,Tijsse8Klasen, et al. 207 2011). In the absence of an EM and the presence of other LB related symptoms, 208 seroconversion can be used for supportive diagnosis. However, in the absence of 209 symptoms, seroconversion is no indication for antibiotic treatment as a study in a 210 Swiss risk group demonstrated that only 2% of patients who seroconverted 211 developed clinical LB (Fahrer, et al. 1991). Thus, as tick bite is a poor predictor of 212 disease, treatment is advisable only upon appearance of LB symptoms. Re ew vi (and ot /N ly On 213 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 9 n tio bu tri is rD fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 11 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 214 Recommendation: 215 Clinical diagnosis alone, given a history of potential exposure and presence of EM, 216 can be sufficient, however clinical interpretation should generally be made in 217 conjunction with supporting laboratory findings to reach a reliable diagnosis. 218 219 vi 220 Re !"#$% ew 221 Direct detection of in the peripheral blood, other body fluids or tissues by 222 microscopy or molecular methods can be used as strong additional evidence in the 223 diagnosis of LB, but might have limited significance when used alone (Aguero8 224 Rosenfeld, et al. 2005). The sensitivity of PCR on skin biopsies is significantly higher 225 than some other molecular tools, however, recognition of the EM itself is the best 226 diagnosis for LB (Aguero8Rosenfeld, et al. 2005), nevertheless this provides useful 227 research data regarding strain prevalence, virulence and provides insights into 228 deciphering pathogenesis of LB (Strle, et al. 2013). Cultivation of 229 patient samples might be an alternative method to detect viable 230 time consuming and challenging (Rudenko, et al. 2016). As such, cultivation is best 231 reserved as a research tool. ot /N ly On 232 from , but is both tri is rD fo 233 Lymphocyte transformation tests (LTT) have been explored for their potential to 234 overcome the diagnostic gap in LB patients without EM but before seroconversion 235 and in re8infected seropositive patients. This assay measures lymphocyte 236 proliferation after stimulation with bu specific antigens. Currently, Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 10 n tio 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 12 of 36 r ee rP Fo 237 results are contradictory and consequently LTT is not recommended as a routine 238 diagnostic tool (Dessau, et al. 2014,Mygland, et al. 2010). T8cell ELISPOT is another 239 stimulation assay currently explored and improved (Jin, et al. 2013). More 240 direct methods measuring peripheral blood levels of specific cell subpopulations 241 (CD57+ cells (Marques, et al. 2009) or antigen8reactive cells (Tario, et al. 2015) by 242 flow cytometry, direct measure of CXCL13 levels in the CSF or metabolites within 243 serum (Molins, et al. 2015) are also not at a point yet to be used reliably for clinical 244 diagnosis. CD57 cell counts seem not to be reliable as a validation study found no 245 difference between patients and healthy controls (Marques, et al. 2009). 246 Demonstration of CSF CXCL13 as an activation marker is not specific for LB, its 247 absence is believed to have some value in excluding neuroborreliosis (Rupprecht, et 248 al. 2014) and it might become a valuable supportive tool to estimate treatment 249 efficiency in case of neuroborreliosis (Koedel, et al. 2015,Schmidt, et al. 2011,Senel, 250 et al. 2010). Problems with HLA types and identification of epitopes for antigen8 251 specific T8cell staining are challenges that need to be addressed to validate the 252 potential of 253 LB. Metabolite measurement is a future strategy under investigation but needs 254 further validation. Re ew vi ot /N ly On specific T8cell counts in peripheral blood to support diagnosis of rD 255 fo 256 Generally, the detection of DNA within ticks as well as other methods 257 discussed above should be considered as valuable research tools providing useful 258 information about the epidemiology of tick8borne diseases in general and LB 259 particularly. As with serological methods, their value is lower when used alone. 260 Combination of diagnostics, clinical and molecular tests provides a more robust and Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 11 n tio bu tri is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 13 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 261 timely diagnosis of disease. In any case, interpretation of tests results and clinical 262 diagnosis of LB remains controversial and should currently be restricted to experts. 263 264 Development and application of new molecular tools allow the detection and 265 differentiation among Lyme borreliosis or relapsing fever spirochetes, clearly 266 separating sensu lato spirochetes from recently described vi 267 Re (Margos, et al. 2008,Rudenko, et al. 2009,Venczel, et al. 2015). 268 Combination of multilocus PCR with electro spray ionisation and mass spectrometry 269 has recently been investigated for the detection and genotyping of 270 in whole blood (Eshoo, et al. 2012). ew 271 species On 272 Recommendation: 273 These tests are valuable research tools providing useful information about the 274 patient’s immune response, but interpretation for clinical diagnosis has not been 275 clearly shown and should currently be restricted to specialised laboratories. ot fo 277 /N 276 ly 278 Veterinary infections are less well documented and benefit from laboratory 279 confirmation to ensure correct diagnosis. This is particularly important as EM lesions 280 have not been reported in animals and clinical signs are often common to several 281 pathologies. As for human cases, serology is the primary diagnostic approach used, 282 sometimes supported by use of PCR. Despite the absence of EM, cardiac, 283 neurological signs and lameness have been reported amongst companion animals 284 (Agudelo, et al. 2011,Hovius, et al. 1999,Krupka and Straubinger 2010). Most Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 12 n tio bu tri is rD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 14 of 36 r ee rP Fo 285 veterinary cases have focussed upon lameness in dogs with positive serology, 286 though this does not necessarily establish borrelial causality for this condition. Rapid 287 immunochormatographic tests are often used in veterinary private practice to aid 288 diagnosis, however these assays have not necessarily undergone the rigorous 289 quality control applied to human serodiagnostic tests (Savić, et al. 2010). Re 290 291 ' ew 292 & vi 293 In general, the clinical presentation of relapsing fever borreliosis is significantly 294 distinct from that of LB. The possible exception to this being the appearance of a 295 skin rash that challenges the previously believed “pathognomonic” EM, caused by 296 the borrelial agent carried by 297 known as STARI (Borchers, et al. 2015,Masters, et al. 2008). On ticks in the United States, /N ly 298 299 Human infection by recently described 300 associated flu8like signs (headache, chills, fatigue, myalgia), occasionally with 301 neurological 302 2014,Krause, et al. 2015). such as fo meningoencephalitis (Fonville, et al. rD 303 complications usually results in fever and ot 304 Relapsing fever, as its name suggests, results in relapsing febrile episodes 305 interspersed by afebrile periods. This is often accompanied by jaundice, muscle 306 pain, headaches and sometimes involvement of major organs (Borgnolo, et al. 307 1993). This clinical picture can often be mistaken for other infections such as malaria 308 that tend to overlap geographically in many endemic regions (Lundqvist, et al. 2010). Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 13 n tio bu tri is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 15 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 309 310 311 312 Though for LB, microscopy is not suitably sensitive for detection, this has been the 313 diagnostic gold standard for detection of many relapsing fever spirochetes. Darkfield 314 examination of unstained wet8preparations, Giemsa or silver8stained blood or tissue 315 sections, or immunofluroescence methods have been successfully used. Despite its 316 frequent use, even relapsing fever can be difficult to detect using microscopy with 317 some species such as 318 others, like 319 sample can be beneficial (Larsson and Bergström 2008). Furthermore, detection is 320 restricted to times of febrile episodes when spirochetes are present at detectable 321 levels. On a cautionary note, various artefacts can share the size and helical shape 322 of spirochetes when viewed by darkfield microscopy, but tend not to show the typical 323 gyrating spirochete8characteristic movement. Microscopy will not provide information 324 regarding the infecting species. Re ew vi typically producing lower blood burdens than . For such cases, a centrifugation step to concentrate the fo 325 ot /N ly On 326 Recommendation: 327 Microscopic methods lack both sensitivity and specificity, but can add value when 328 used in conjunction with other methods. Sample concentration can offer distinct 329 benefits. bu tri is 330 rD 331 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 14 n tio 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases r ee rP Fo 332 Cultivation methods for detection of have been particularly challenging and 333 some members of the genus being particularly refractory to cultivation (Cutler, et al. 334 1994) whilst others are cultivable, but only in complex medium. Huge advances were 335 made with the formulation of BSK medium with a commercial variant BSK8H 336 supporting the growth of LB strains (Barbour 1984). Relapsing fever strains appear 337 more diverse in their requirements. 338 MKP medium (Wagemakers, et al. 2014) or high serum concentrations (Margos, et 339 al. 2015). On a cautionary note, these preferences might reflect batch variations of 340 composite ingredients that can vastly influence performance of these “home8made” 341 media (Cutler personal observation). Collectively, cultivation should be considered a 342 low yield procedure, but vital for recovery of much8needed strains for research 343 purposes (Ružić8Sabljić, et al. 2014). Re for instance appears to prefer ew vi ly On 344 /N 345 Animal inoculation or xenodiagnosis (allowing infected ticks to feed upon a test 346 animal) has been used for primary recovery of isolates prior to cultivation in axenic 347 medium (Naddaf, et al. 2015,Schwan, et al. 2012). It must be remembered that some 348 species are refractory to growth in most animal models, such as ot 349 . rD fo 350 Recommendation: 351 Cultivation is low yield, time consuming and expensive and thus poorly suited to 352 support diagnosis. Nevertheless, it still has a vital role for recovery of isolates for 353 research purposes. bu tri is 354 355 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 15 n tio 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 36 Page 17 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 356 For the relapsing fever group, specific serology can be undertaken using GlpQ 357 protein as antigen. GlpQ is absent from LB species, thus facilitating it’s specificity for 358 diagnostic purposes (Fritz, et al. 2013). Alternatively, BipA can also serve as a 359 differential antigen present in relapsing fever spirochetes, but absent from the LB 360 group (Lopez, et al. 2010). As acutely presenting patients may not yet have had 361 sufficient time for seroconversion, serology is best reserved for retrospective 362 diagnosis. Re ew 363 vi 364 ( & 365 PCR provides a valuable diagnostic approach in acutely ill patients (Mediannikov, et 366 al. 2014). This overcomes the poor sensitivity of microscopy and can either be used 367 to diagnose relapsing fever borreliosis, or to further characterise the infecting 368 spirochete. The absence of 369 detection of relapsing fever spirochetes (Takano, et al. 2014). Other assays can 370 either speciate specific relapsing fever borreliae or be designed to detect a single 371 member of the relapsing fever clade such as 372 et al. 2015). The limitation of this approach is having an appropriate sample that is 373 likely to contain spirochetal DNA. Blood collected during febrile episodes and CSF 374 samples have given good results (Gugliotta, et al. 2013). Furthermore, in highly 375 relapsing fever endemic areas, it is possible to have positive PCR results unrelated 376 to current clinical pathology (Cutler, et al. 2010). in LB species makes it a specific target for ot /N (Elbir, et al. 2013,Reiter, Recommendation: Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 16 n tio bu tri is rD fo 377 378 ly On 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases r ee rP Fo 379 PCR can provide useful supporting information, but multiple available assays must 380 be properly standardised, and are hampered by sample timing, type and quality. 381 382 ) 383 NGS offers huge potential and data has only recently been forthcoming limiting 384 comprehensive appraisal at this stage. With the exception of dermato8borreliosis, 385 here the challenge is which diagnostic sample type to investigate for LB in the 386 absence of focal lesions. Sensitivity can be further improved, especially amongst 387 high levels of host DNA. Care should be taken to avoid bias when using target 388 enhancement strategies to amplify low copy number targets. Data analysis 389 represents an additional computational challenge. NGS methods combined with 390 bioinformatics tools might overcome the limitations of culture8connected techniques 391 or of some molecular protocols. However, the extreme diversity of spirochetes from 392 sensu lato complex reduce the usefulness of NGS as it doesn’t 393 differentiate between the pathogenic to human spirochete strains from those that 394 were never connected with human LB. Additionally this offers a means of assessing 395 rank abundance, evolving genomic profiles such as those corresponding to vector 396 adaptations (Gatzmann, et al. 2015) and fluctuations over time providing valuable 397 insights into host8microbial interactions (Strandh and Råberg 2015). + , ew vi ot /N ly On tri is rD fo 398 * Re 399 To date enrichment techniques can only partially overcome sensitivity problems 400 caused by the giant excess of host DNA (vector, endosymbiont and other microbial 401 DNA) compared to the low proportion of target DNA (borrelial DNA in ticks is <0.01% 402 of total DNA within field8collected nymphal ticks) (Carpi, et al. 2015). This can impact Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 17 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 36 Page 19 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 403 upon successful detection with only about a third of infected ticks revealing positive NGS data (Carpi, et al. 2015). 404 405 406 Recommendations: 407 NGS offers huge potential and data has only recently been forthcoming limiting 408 comprehensive appraisal at this stage. Sensitivity can be further improved, 409 especially amongst high levels of host DNA. Care should be taken to avoid bias 410 when using target enhancement strategies to amplify low copy number targets. Data 411 analysis represents an additional computational challenge. Re ew vi 412 On 413 - 414 Several excellent fact sheets have been produced by ECDC to provide information 415 on LB and tick8borne relapsing fever. Furthermore, more specific resources can be 416 obtained 417 www.escmid.org/research_projects/study_groups/esgbor/). from European study /N ly group for Lyme borreliosis (ESGBOR; ot 418 fo 419 . / 420 The poor sensitivity of direct detection methods coupled with the poor predictive 421 value 422 presentations, presents a significant diagnostic challenge. Serology is further 423 challenged by the requirement for sufficient time in order for the host to produce 424 antibody responses to enable detection. Detection of the host response to infections 425 provides a particularly attractive prospect for LB where organism loads are typically 426 low. Indeed, levels of CXCL13 have shown promise for neuroborreliosis, but require of indirect serological methods, rD particularly in less typical clinical Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 18 n tio bu tri is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 20 of 36 r ee rP Fo 427 further validation (Schmidt, et al. 2011,Senel, et al. 2010). It is possible that 428 signature biomarker profiles might have value, but whether this would vary too much 429 between individuals or indeed with differing genetic variants of borreliae awaits 430 investigation. Another diagnostic approach under exploration is based on targeted 431 proteomics. By selected reaction monitoring mass spectrometry, specific 432 proteins can be detected and quantified in skin biopsies (Schnell, et al. 2015). The 433 powerful new emerging technologies provide insights into our understanding of the 434 dynamic interactions of borreliae with their vector, host and other organisms, with the 435 possibility of disclosing opportunities for future intervention. Re ew vi 436 0 437 On 438 During these brief guidelines, we have attempted to highlight the strengths and 439 limitations of various diagnostic methods used to diagnose borrelial infection. No 440 single approach is suitably robust for this purpose, thus making interpretation 441 challenging. Furthermore, laboratory diagnostics need to be viewed in conjunction 442 with potential exposure and compatible clinical features. ot /N ly 443 0 / rD 444 fo 445 This article was done under the frame of EurNegVec COST Action TD1303. 446 Wibke J. Cramaro and Josiane Kirpach are funded by the Luxembourgish Ministry of 447 Research (project Marlybor, REC8LNSI820110308). Wibke J. Cramaro and Josiane 448 Kirpach were supported by a fellowship from Aides à la Formation Recherche (grant 449 n°4660265 and n°3986187) of the Fonds National de la Recherche, Luxembourg. bu tri is 450 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 19 n tio 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 21 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 451 & 452 (ESGBOR) ESGfLB. Tick tests for the detection of 453 454 are not recommended by the ESCMID Study Group for Lyme Borreliosis (ESGBOR). 2013. Agudelo CF, Schanilec P, Kybicova K, Kohout P. Cardiac manifestations of 455 borreliosis in a dog: A case report. Veterinarni Medicina 2011;56:85892. 456 Aguero8Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diagnosis of Lyme borreliosis. Clin Microbiol Rev 2005;18:4848509. Aguero8Rosenfeld ME, Nowakowski J, Bittker S, Cooper D et al. Evolution of the ew 458 vi 457 Re 459 serologic response to 460 confirmed erythema migrans. J Clin Microbiol 1996;34:189. 461 462 Allen NM, Jungbluth H. Lyme Neuroborreliosis: A Potentially Preventable Cause of Stroke. J Pediatr 2016;170:3348334 e331. ly 463 in treated patients with culture8 On Ang CW, Notermans DW, Hommes M, Simoons8Smit AM et al. Large differences /N 464 between test strategies for the detection of anti8 465 by comparing eight ELISAs and five immunoblots. European Journal of Clinical 466 Microbiology and Infectious Diseases 2011;30:102781032. fo 467 antibodies are revealed ot Aucott JN, Rebman AW, Crowder LA, Kortte KB. Post8treatment Lyme disease 468 syndrome symptomatology and the impact on life functioning: is there something 469 here? Qual Life Res 2013;22:75884. 471 472 1984;57:5218525. Belongia EA. Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis 2002;2:2658273. 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A Re 479 clinical and epidemiological study of 389 patients in Asella Hospital, Ethiopia. 480 Tropical and Geographical Medicine 1993;45:66869. vi 481 Branda JA, Aguero8Rosenfeld ME, Ferraro MJ, Johnson BJ et al. 28tiered antibody 482 testing for early and late Lyme disease using only an immunoglobulin G blot with 483 the addition of a VlsE band as the second8tier test. Clin Infect Dis 2010;50:20826. On 484 ew Carpi G, Walter KS, Bent SJ, Hoen AG et al. Whole genome capture of vector8borne 485 pathogens from mixed DNA samples: A case study of 486 Genomics 2015;16. /N 487 BMC ly Christen HJ, Hanefeld F, Eiffert H, Thomsen R. Epidemiology and clinical 488 manifestations of Lyme borreliosis in childhood. A prospective multicentre study 489 with special regard to neuroborreliosis. Acta Paediatrica, International Journal of 490 Paediatrics, Supplement 1993;82:1875. 494 495 497 sensu stricto and ticks. Vector Borne Zoonotic Dis 2002;2:389. in Cutler SJ, Margarita Bonilla E, Singh RJ. 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Krupp LB, Hyman LG, Grimson R, Coyle PK et al. Study and treatment of post Lyme tri is 611 sensu rD 612 disease (STOP8LD): a randomized double masked clinical trial. Neurology 613 2003;60:192381930. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 26 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 28 of 36 r ee rP Fo 614 Lantos PM, Branda JA, Boggan JC, Chudgar SM et al. Poor positive predictive value 615 of Lyme disease serologic testing in an area of low disease incidence. Clin Infect 616 Dis 2015;61:137481380. 617 Larsson C, Bergström S. A novel and simple method for laboratory diagnosis of 618 relapsing fever borreliosis. The Open Microbiology Journal 2008;2:10812. 619 Re Leeflang MM, Ang CW, Berkhout J, Bijlmer HA et al. 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EFNS guidelines on the 665 diagnosis and management of European Lyme neuroborreliosis. Eur J Neurol 666 2010;17:8816, e11814. Re vi 667 Naddaf S, Ghazinezhad B, Sedaghat M, Asl H et al. Tickborne relapsing fever in 668 southern Iran, 201182013. Emerging Infectious Diseases 2015;21:107881080. 669 Nadelman RB, Wormser GP. Reinfection in patients with Lyme disease. Clin Infect 670 ew Dis 2007;45:103281038. On 671 Nowakowski J, Nadelman RB, Sell R, McKenna D et al. Long8term follow8up of 672 patients with culture8confirmed Lyme disease. Am J Med 2003;115:91896. 673 ly Rebman AW, Crowder LA, Kirkpatrick A, Aucott JN. Characteristics of /N 674 seroconversion and implications for diagnosis of post8treatment Lyme disease 675 syndrome: acute and convalescent serology among a prospective cohort of early 676 Lyme disease patients. Clin Rheumatol 2015;34:5858589. Reiter M, Schötta AM, Müller A, Stockinger H et al. A newly established real8time PCR for detection of 679 borne Diseases 2015;6:3038308. in Ixodes ricinus ticks. Ticks and Tick8 rD 678 Reye AL, Hubschen JM, Sausy A, Muller CP. Prevalence and seasonality of tick8 681 borne pathogens in questing 682 Microbiol 2010;76:292382931. tri is 680 fo 677 ot ticks from Luxembourg. Appl Environ Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 29 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 683 Rudenko N, Golovchenko M, Lin T, Gao L et al. Delineation of a New Species of the Sensu Lato Complex, 684 685 686 sp. nov. J. Clin. Microbiol. 2009;47:387583880. Rupprecht TA, Lechner C, Tumani H, Fingerle V. [CXCL13: a biomarker for acute 687 Lyme neuroborreliosis: investigation of the predictive value in the clinical routine]. 688 Nervenarzt 2014;85:4598464. vi 689 Re Ružić8Sabljić E, Maraspin V, Cimperman J, Strle F et al. Comparison of isolation rate 690 of 691 Clinical Microbiology and Infection 2014;20:6368641. 692 694 province of Vojvodina, Serbia. Parasite 2010;17:3578361. Schmidt C, Plate A, Angele B, Pfister HW et al. A prospective study on the role of CXCL13 in Lyme neuroborreliosis. Neurology 2011;76:105181058. /N 696 in ticks and dogs in the ly 695 Savić S, Vidić B, Lazić S, Lako B et al. On 693 sensu lato in two different culture media, MKP and BSK8H. ew Schnell G, Boeuf A, Westermann B, Jaulhac B et al. Discovery and targeted 697 proteomics on cutaneous biopsies infected by 698 disease. Molecular and Cellular Proteomics 2015;14:125481264. fo 699 to investigate Lyme ot Schwan TG, Anderson JM, Lopez JE, Fischer RJ et al. Endemic foci of the tick8 700 borne relapsing fever spirochete 701 potential for human infection. PLoS Neglected Tropical Diseases 2012;6. 703 704 Senel M, Rupprecht TA, Tumani H, Pfister HW et al. The chemokine CXCL13 in tri is 702 in Mali, West Africa, and the rD acute neuroborreliosis. J Neurol Neurosurg Psychiatry 2010;81:9298933. Sigal LH. The Lyme disease controversy. Social and financial costs of misdiagnosis 705 and mismanagement. Arch Intern Med 1996;156:149381500. 706 Stanek G, Strle F. Lyme borreliosis. Lancet 2003;362:163981647. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 30 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Vector-Borne and Zoonotic Diseases Page 32 of 36 r ee rP Fo 707 Stanek G, Wormser GP, Gray J, Strle F. Lyme borreliosis. Lancet 2012;379:4618473. 708 Stanek G, Fingerle V, Hunfeld KP, Jaulhac B et al. Lyme borreliosis: Clinical case 709 definitions for diagnosis and management in Europe. Clinical Microbiology and 710 Infection 2011;17:69879. 711 Strandh M, Råberg L. Within8host competition between ospC strains Re 712 in wild hosts as revealed by massively parallel amplicon sequencing. 713 Philosophical Transactions of the Royal Society B: Biological Sciences 2015;370. 715 716 Strle F, Stanek G. Clinical manifestations and diagnosis of Lyme borreliosis. Curr ew 714 vi Probl Dermatol 2009;37:518110. Strle F, Wormser GP, Mead P, Dhaduvai K et al. Gender Disparity between On 717 Cutaneous and Non8Cutaneous Manifestations of Lyme borreliosis. PLoS ONE 718 2013;8. 719 722 Takano A, Toyomane K, Konnai S, Ohashi K et al. Tick surveillance for relapsing fever spirochete in Hokkaido, Japan. PLoS ONE 2014;9. fo 723 ticks. Clin Microbiol Rev 2006;19:7088727. ot 721 Swanson SJ, Neitzel D, Reed KD, Belongia EA. Coinfections acquired from ixodes /N 720 ly Tario JD, Jr., Chen GL, Hahn TE, Pan D et al. Dextramer reagents are effective tools 724 for quantifying CMV antigen8specific T cells from peripheral blood samples. 725 Cytometry B Clin Cytom 2015;88:6820. Tijsse8Klasen E, Jacobs JJ, Swart A, Fonville M et al. Small risk of developing tri is 726 rD 727 symptomatic tick8borne diseases following a tick bite in The Netherlands. Parasit 728 Vectors 2011;4:17. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 31 n tio bu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo 729 Vayssier8Taussat M, Moutailler S, Michelet L, Devillers E et al. Next generation 730 sequencing uncovers unexpected bacterial pathogens in ticks in western Europe. 731 PLoS One 2013;8. 732 733 734 permits simultaneous detection and differentiation of is cultivable in a modified Kelly8Pettenkofer medium, and is ew 738 sensu lato. Infection 2015:189. Wagemakers A, Oei A, Fikrig MM, Miellet WR et al. The relapsing fever spirochete 736 737 and vi 735 Venczel R, Knoke L, Pavlovic M, Dzaferovic E et al. A novel duplex real8time PCR Re resistant to human complement. Parasites and Vectors 2014;7. Wilhelmsson P, Fryland L, Lindblom P, Sjowall J et al. A prospective study on the On 739 incidence of 740 and on the Aland Islands, Finland (200882009). Ticks Tick Borne Dis 2016;7:718 741 79. /N 742 sensu lato infection after a tick bite in Sweden ly Wills AB, Spaulding AB, Adjemian J, Prevots DR et al. Long8term Follow8up of 743 Patients With Lyme Disease: Longitudinal Analysis of Clinical and quality8of8life 744 measures. Clin Infect Dis 2016. 747 Zajkowska J, Garkowski A, Moniuszko A, Czupryna P et al. Vasculitis and stroke due to Lyme neuroborreliosis 8 a review. Infect Dis (Lond) 2015;47:186. Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 32 n tio bu tri is rD 746 fo 745 ot 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9HFWRU%RUQHDQG=RRQRWLF'LVHDVHV Erythema migrans (1) Tick bite (4) U3 )R Lyme disease symptoms (7) High risk of tick exposure (2) HH HY Seropositive Seronegative QO 2 Neuroborreliosis (CSF analysis) Differential diagnosis: Exclude all other possible diseases or causes of symptoms RW Remaining symptoms after treatment (6) U' IR Further antibiotic treatment is currently not recommended and symptomatic treatment should be considered 0DU\$QQ/LHEHUW,QF+XJXHQRW6WUHHW1HZ5RFKHOOH1< Q LR XW ULE LVW Disease manifestations were probably due to Lyme disease and the patient can be considered healthy again \1 Antibiotic treatment recommended only in case of development of symptoms Symptoms disappeared after treatment Correlation between Timepoint of (probable) tick exposure & timepoint of appearance of symptoms & serological result (5) LHZ Immediate antibiotic treatment without the need of confirmatory serology (3) Symptoms strongly associated with Lyme disease (8) Symptoms U5                                                             3DJHRI Page 35 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo ! 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Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 n tio bu tri is &**>" 6 4 % : rD / , fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 36 of 36 Page 37 of 36 Vector-Borne and Zoonotic Diseases r ee rP Fo ' ! 0 ( % Re 0 0 4 0 1 &*+& 6 &*+." ew vi ot /N ly On Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 n tio bu tri is rD fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60