Presence or absence of clinical symptoms, including the type of rash or lesion when present, guides the initial differential diagnosis of patients exposed to a tick-endemic area (Figure). This, in turn, guides appropriate test selection, presumably leading to confirmation of the suspected disorder.

Lyme Disease
Test Selection for Early Lyme Disease
Diagnosis of Lyme disease (B burgdorferi infection) is best made on the basis of a history of exposure to the vector tick or tick habitat and clinical findings. Clinical findings, as well as laboratory testing, vary with the stage of the disease (Table 5). Diagnosis of early-stage, localized Lyme disease can be made on the basis of erythema migrans alone without laboratory testing.⁴ When there is diagnostic uncertainty, positive IgG and/or IgM serology results on acute- and convalescent-phase (ie, 2 to 4 weeks after the acute phase) samples can support the diagnosis (Table 5).

Test Selection for Disseminated Lyme Disease
In the absence of erythema migrans, Lyme disease cannot be diagnosed on the basis of disseminated disease manifestations alone, because these symptoms are nonspecific.⁴ Diagnosis requires demonstration of seropositive test results.⁴ EIA testing for IgG is recommended >4 weeks after the tick bite, whereas combined IgG and IgM testing is performed for patients 2 to 4 weeks after the tick bite (Table 5).⁴ Western blot testing is recommended as a confirmatory test for all specimens positive or equivocal by EIA.¹⁴

Immunoassays used to detect Lyme disease antibodies utilize whole spirochete preparations or synthetic C6 peptide antigen. The C6 peptide antigen is a 26-amino acid sequence derived from the VlsE membrane protein of Borrelia. The C6 antibody test is used to detect infection from both American and European Borrelia species and to reduce false positives that may be caused by antibodies cross-reacting with similar organisms or by Lyme disease vaccination.¹⁵

Methods that detect B burgdorferi DNA may be used to support the diagnosis of disseminated Lyme disease. For neurologic Lyme disease, DNA detection in cerebrospinal fluid (CSF) has a sensitivity of 38% (≥93% specificity).¹⁶ Alternatively, intrathecal antibody production using a CSF to serum antibody ratio may be used (sensitivity, 75%; specificity, 97%).¹⁷ Positive DNA findings in synovial fluid supports the diagnosis of Lyme arthritis (sensitivity, 78%; specificity, 100%).¹⁶ However, because spirochetemia is typically transient or absent, detection of B burgdorferi DNA in whole-blood specimens has low clinical sensitivity (14%) rendering negative results non-informative.¹⁶

Test Interpretation
IgM antibodies may be present within a few weeks of disease onset, whereas IgG antibodies are produced later. A positive IgM result as determined by 2-tier testing, in conjunction with a negative IgG result, is presumptive evidence of early infection unless obtained on a specimen collected more than 1 month following onset. In the latter scenario, a positive IgM finding is more likely to represent a false-positive unless IgG is also positive. A positive IgG result by 2-tier testing is required to confirm the diagnosis of disseminated disease.⁴

The interpretation of Western blot antibody assays is based on the number of positive bands: 2 of 3 bands (23, 39, 41kDa) for IgM positivity and 5 of 10 bands (18, 23, 28, 30, 39, 41, 45, 58, 66, or 93 kDa) for IgG positivity. The Western blot is to be used only following initial EIA testing, and positive results confirm B burgdorferi infection.⁴

False positive antibody test results may be due to vaccination, infectious mononucleosis, systemic lupus erythematosus, or other diseases caused by spirochetes such as syphilis, yaws, pinta, leptospirosis, and relapsing fever. The Lyme disease C6 antibody test does not typically yield false-positive results in the above conditions.¹⁵ Additionally, positive antibody test results, including those from the C6 EIA, may be due to active or past B burgdorferi infection.

Negative serology results may indicate lack of infection or lack of seroconversion, which may occur if samples are collected too early after disease onset or when early antibiotic therapy blunts the antibody response. Untreated patients who continue to be symptomatic but are seronegative for 6 to 8 weeks are unlikely to have Lyme disease, and another diagnosis should be considered.⁴

Expression of paired CSF and serum B burgdorferi antibody results as an antibody index can be used as an aid in the diagnosis of neuroborreliosis. An index >1.2 is deemed positive for intrathecal antibody production if accompanied by a control antibody index <1.0 and an albumin ratio <0.0078. Elevation of either the control antibody index, the albumin ratio, or both may indicate leakage of antibody across the blood-brain barrier and a falsely elevated B burgdorferi antibody index.

In a seropositive patient, positive DNA results support the diagnosis of Lyme disease.⁴  However, in a seronegative patient, positive results should be interpreted with caution.⁴

Testing for Tick-borne Rickettsial Diseases (TBRDs)
Ehrlichioses and anaplasmosis are characterized by infection of leukocytes, in which the causative agents multiply in cytoplasmic membrane-bound vacuoles called morulae. E ewingii and A phagocytophilum infect granulocytes, whereas E chaffeensis infects monocytes. Thus, visualization of morulae on a routine blood smear may provide the first clue for diagnosis and help differentiate HME from HGA and E ewingii infection. Positive results may be seen in up to 60% of patients with HGA and to a lesser extent in patients with HME.⁷

In patients with RMSF, increased band neutrophil counts are generally observed on a complete blood count (CBC), and since they are less commonly seen with viral infection, aid in the differential diagnosis. Symptomatic patients have an increased likelihood of HGA when platelets, WBC’s, or both are reduced; the relative risk was 5.2 when the WBC count was <2.5 x 10⁹ cells/L and 10.3 when the platelet count was <100 x 10⁹ cells/L.¹⁸ Laboratory test results associated with the various TBRDs are listed in Table 6.

Laboratory confirmation validates the accuracy of the presumptive diagnosis and is important from an epidemiology and public health perspective. Confirmatory laboratory testing for TBRDs  includes serology and highly specific nucleic acid detection (Table 6). Immunofluorescence assays (IFA) are considered the gold standard for TBRD serology testing.⁷ A 4-fold rise in titer of IgG or IgM in paired acute and convalescent samples collected 2 to 3 weeks apart is essential to confirm acute infection. Most patients have positive IgG or IgM antibody by the second week of illness. For RMSF, IgG and IgM increase concurrently; IgM wanes after 3 to 4 months, whereas IgG persists for 7 to 8 months.

Detection of DNA in whole blood is especially useful for confirming HGA, HME, and E. ewingii infection because these organisms infect circulating leukocytes. For RMSF, detection of R rickettsii in blood is more likely in advanced disease or fulminant infection. Whereas positive results confirm TBRD, negative results do not exclude the diagnosis.

Testing for Tick-borne Non-Rickettsial Diseases
Colorado Tick Fever
Leukopenia is characteristically seen in a CBC, and thrombocytopenia may be present. Acute and convalescent serology should be considered for patients with clinically suspected Colorado tick fever (Table 7). A 4-fold rise of IgG or IgM titer in paired acute and convalescent samples confirms the diagnosis; the detection of IgM indicates acute infection.

Q Fever
C burnetii infection exhibits 2 antigenic phases (I and II) that are valuable in diagnosing and staging Q fever. Acute disease is characterized by elevated phase II antibodies, whereas chronic disease is reflected by phase I antibodies. Thus, a ratio of phase II to phase I antibody titers >1 suggests acute Q fever, while a ratio <1 suggests chronic Q fever. Active Q fever infections are diagnosed on the basis of a 4-fold increase in serum IgG titers between acute and convalescent samples and/or elevated IgM antibodies directed against phase II antigens (Table 7). Phase II antibodies may be detected within 2 weeks of infection; if not present at 4 weeks, another diagnosis should be considered.¹¹

Phase I IgG testing at 3 and 6 months is suggested for patients who have yet to be treated.¹¹ If the phase I IgG titer is elevated, treatment should be considered if supported by echocardiography or PCR testing (not currently available).¹¹ Thereafter, IgG titers should be tested at 3-month intervals to monitor therapy¹¹; a reduction in titer is suggestive of therapeutic response. Once therapy has been discontinued (or if therapy was not initiated), a 4-fold increase in IgG titer at 3 and 6 months indicates relapse.¹¹ Persistently elevated phase I IgG titers suggest the presence of endocarditis and chronic disease.¹¹

Tularemia
F tularensis serology testing can confirm a diagnosis of tularemia (Table 7). An antibody titer ≥1:20 is suggestive of acute or past infection and a 4-fold increase in titer between acute and convalescent sera collected 2 to 3 weeks apart is considered diagnostic. Antibody begins to appear 2 to 3 weeks post-onset and peaks at about 5 weeks. However, up to 30% of patients infected for 3 weeks have negative serology results.¹² Because Brucella antibodies cross-react with this test, positive specimens should also be tested for Brucella antibody.

Tick-borne Relapsing Fever (TBRF)
Diagnosis of TBRF is made by the detection of spirochetes in the patient’s blood during periods of high fever (sensitivity ≥70%).³ Diagnosis is confirmed by serology testing. The presence of B hermsii IgM titers ≥1:16 are associated with acute infection, while IgG titers ≥1:64 reflect later stages of disease. Single IgG titers ≥1:64 are considered presumptive evidence of infection and a 4-fold increase in titer between acute and convalescent sera provides evidence of recent or current infection (Table 7). Because other Borrelia and Treponema species cross-react in the IFA test, positive specimens should be tested for antibodies to these organisms.

Babesiosis
Laboratory abnormalities may include hemolytic anemia with an elevated reticulocyte count, thrombocytopenia, proteinuria, and elevated levels of liver enzymes, blood urea nitrogen, and creatinine. Because clinical symptoms are nonspecific, diagnosis requires identification of babesial parasites in blood by microscopy, DNA detection, or positive serology (Table 7).⁴

Because of the resemblance to malarial parasites, microscopic identification of intraerythrocytic parasites using Giemsa-stained thin or thick blood smears may require confirmation by additional laboratory testing. Specific B microti antibodies are usually present by the time the patient exhibits parasitemia and invariably within 4 weeks of onset, unless the patient is immunocompromised. Although most patients with acute illness have titers ≥1024, testing of acute and convalescent sera is recommended to confirm the diagnosis. Elevated titers can persist for months following resolution of symptoms. The IFA serology method is known to cross-react with other Babesia species (at lower titers) as well as with other blood and tissue parasites and various tick-borne organisms. Detection of Babesia DNA in blood can also confirm the diagnosis and is slightly more sensitive than serology or microscopic detection of parasites.⁴

Laboratory test results, combined with clinical symptoms, are used to make treatment decisions.⁴ Treatment is recommended in symptomatic patients when babesial parasites have been identified or DNA results are positive.⁴ Treatment is not recommended in symptomatic patients whose blood is negative for babesial parasites or DNA, even if serology testing is positive. Also, treatment is not recommended for asymptomatic individuals, regardless of laboratory test results. Asymptomatic individuals with positive babesial smears and/or DNA results should have these tests repeated, and treatment should be considered if repeat testing is positive >3 months later.