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. 2018 Oct 29;8(1):15932.
doi: 10.1038/s41598-018-34393-9.

Evaluating Polymicrobial Immune Responses in Patients Suffering From Tick-Borne Diseases

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Free PMC article

Evaluating Polymicrobial Immune Responses in Patients Suffering From Tick-Borne Diseases

Kunal Garg et al. Sci Rep. .
Free PMC article

Abstract

There is insufficient evidence to support screening of various tick-borne diseases (TBD) related microbes alongside Borrelia in patients suffering from TBD. To evaluate the involvement of multiple microbial immune responses in patients experiencing TBD we utilized enzyme-linked immunosorbent assay. Four hundred and thirty-two human serum samples organized into seven categories followed Centers for Disease Control and Prevention two-tier Lyme disease (LD) diagnosis guidelines and Infectious Disease Society of America guidelines for post-treatment Lyme disease syndrome. All patient categories were tested for their immunoglobulin M (IgM) and G (IgG) responses against 20 microbes associated with TBD. Our findings recognize that microbial infections in patients suffering from TBDs do not follow the one microbe, one disease Germ Theory as 65% of the TBD patients produce immune responses to various microbes. We have established a causal association between TBD patients and TBD associated co-infections and essential opportunistic microbes following Bradford Hill's criteria. This study indicated an 85% probability that a randomly selected TBD patient will respond to Borrelia and other related TBD microbes rather than to Borrelia alone. A paradigm shift is required in current healthcare policies to diagnose TBD so that patients can get tested and treated even for opportunistic infections.

Conflict of interest statement

Yes, the authors have competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Following Nature Research policy authors of this manuscript have the following competing interests, K.G., L.M. and L.G. have a financial and business interest in Te?ted Oy. O.F., H.P., M.Q.-D. and S.C. have no commercial or financial relationships that could be construed as a potential conflict of interest. Funding sources mentioned in the acknowledgment section and Te?ted Oy had no role in experimental design, reporting of the results, or the decision to publish. K.G., L.M., O.F., H.P., M.Q.-D., S.C. and L.G. do not have any non-financial competing interests to disclose.

Figures

Figure 1
Figure 1
Patient flow diagram. In total, 509 human serum samples were received from various clinical laboratories. Patient samples that arrived without information regarding TBD related symptoms, clinical test results or the diagnosis by a healthcare professional were excluded (n = 77). Remaining 432 patients were tested for their IgM and IgG responses against 20 microbes associated with TBDs. Further, included patients were organized into seven categories based on their respective clinical pictures that followed the Centers for Disease Control and Prevention (CDC) two-tier diagnosis guidelines for Lyme disease, Infectious Disease Society of America (IDSA) guidelines for Post-treatment Lyme Disease Syndrome (PTLDS), and literature regarding the use of lymphocyte and low CD57 cell count in diagnosing patients for Lyme disease. Patient categories included CDC acute (n = 43), CDC late (n = 43), CDC negative (n = 46), PTLDS (n = 31), immunocompromised (n = 61), unspecific (n = 31), and healthy (n = 177).
Figure 2
Figure 2
Polymicrobial infections are present at all stages of tick-borne diseases. (A) Overall positive immunoglobulin M (IgM), and immunoglobulin G (IgG) responses by patients to none, one, or multiple microbes. (B) Overall positive IgM and IgG reactions by patients to 20 individual microbes. (C) IgM and IgG responses by individual patient categories to 20 microbes. Patient categories refer to individuals from Centers for Disease Control and Prevention (CDC) acute, CDC late, CDC negative, Post-Treatment Lyme Disease Syndrome (PTLDS), immunocompromised, and unspecific. Additionally, only 2C includes IgM and IgG responses by healthy individuals to 20 microbes. Microbes include Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto persistent form, Borrelia afzelii persistent form, Borrelia garinii persistent form, Babesia microti, Bartonella henselae, Brucella abortus, Ehrlichia chaffeensis, Rickettsia akari, Tick-borne encephalitis virus (TBEV), Chlamydia pneumoniae, Chlamydia trachomatis, Coxsackievirus A16 (CVA16), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma pneumoniae, Mycoplasma fermentans, and Human parvovirus B19 (HB19V).
Figure 3
Figure 3
Lyme disease diagnostic tests should incorporate Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii in spirochetes and persistent forms. (A and B) immunoglobulin M (IgM) and immunoglobulin G (IgG) responses by patients to different forms of Borrelia and other TBD microbes. Patients refer to individuals from categories Centers for Disease Control and Prevention (CDC) acute, CDC late, CDC negative, Post-Treatment Lyme Disease Syndrome (PTLDS), immunocompromised, and unspecific. Other TBD microbes include Babesia microti, Bartonella henselae, Brucella abortus, Ehrlichia chaffeensis, Rickettsia akari, Tick-borne encephalitis virus (TBEV), Chlamydia pneumoniae, Chlamydia trachomatis, Coxsackievirus A16 (CVA16), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma pneumoniae, Mycoplasma fermentans, and Human parvovirus B19 (HB19V). (A.1 and B.1) Distribution of IgM and IgG levels to different species of Borrelia spirochetes and (A.2 and B.2) Borrelia persistent forms. Abbreviations Bb, Ba, and Bg depict Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, respectively.
Figure 4
Figure 4
Response to Borrelia spirochetes and persistent forms increases reaction frequency to other TBD microbes. Immunoglobulin M (IgM) and immunoglobulin G (IgG) responses by patients (A) to different forms of Borrelia alone versus reactions to different forms of Borrelia together with other TBD microbes, (B) to the number of other TBD microbes with distinctive forms of Borrelia, and (C) to other specific TBD microbes together with different forms of Borrelia. Other TBD microbes include Babesia microti, Bartonella henselae, Brucella abortus, Ehrlichia chaffeensis, Rickettsia akari, Tick-borne encephalitis virus (TBEV), Chlamydia pneumoniae, Chlamydia trachomatis, Coxsackievirus A16 (CVA16), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma pneumoniae, Mycoplasma fermentans, and Human parvovirus B19 (HB19V).
Figure 5
Figure 5
Collective immunoglobulin M (IgM) and immunoglobulin G (IgG) responses should be considered for diagnosing Lyme disease. (A) IgM, IgG, and collective IgM/IgG nonparametric receiver operating characteristic (ROC) curves for different forms of Borrelia. AUC and 95% CI denote Area under the curve, and 95% confidence interval, respectively. (B) IgM, IgG, and collective IgM/IgG diagnostic performance characteristics for different forms of Borrelia represented with 95% confidence interval line across the marker. Borrelia species included in the analyses are Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. IgM and IgG responses from healthy individuals and others with previous test results (Fig. 1, Table S2) were included for ROC and diagnostic performance assessments (Figs 5 and S4–S6).

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