Table XIII-1.

Laboratory Diagnosis of Tickborne Infections

Etiologic Agentsa	Diagnostic Procedures	Optimum Specimens	Transport Issues; Optimal Transport Times
Bacteria
Relapsing fever borreliae (4–6)	Primary testb: Darkfield microscopy or Wright's, Giemsa or Diff-Quik stains of peripheral thin or/and thick blood smears. Can be seen in direct wet preparation of blood in some cases.	Blood, bone marrow	EDTA or citrate blood tube, RT, ≤30 min
Borrelia hermsii (western USA)
Borrelia parkeri (western USA)
Borrelia turicae (southwestern USA)	Others Tests
Borrelia mazzottii (southern USA)	NAAT	Serum, blood, body fluids	Clot tube for serum; sterile tube or citrate tube for body fluids, RT, within 2–4 h
	Culturec
	Serologic testingd	Blood, body fluids
		Serum
Borrelia burgdorferi sensu latocomplex (Lyme borreliosis)e	Early Lyme disease – presence of erythema migrans:	Serum	Clot tube, RT, ≤2 h
Borrelia burgdorferi (USA)	Serologic testing insensitive in the first 2 wk of infectionf
Borrelia garinii (Europe, Asia)	Early/disseminated (weeks through months after tick bite) or late/persistent (months through years after tick bite in untreated patients, almost exclusively seen with B. afzeliig):
Borrelia afzelii (Europe, Asia)
	Primary test: Two-tier testing (acute- and convalescent-phase sera optimal) = EIA IgG and IgM antibody screening. If EIA result is positive or equivocal, confirm with IgG and IgM Western blot (WB).h
	NOTE: A Western blot should NOT be performed unless an initial EIA is reported as positive or equivocal.	Serum and CSF	Clot tube for serum, sterile tube for CSF, RT, ≤1 h
	Neuroborreliosisp
	Paired serum/CSF antibody levels, ie, CSF/serum antibody index.
	NAATi	Blood, biopsy specimens of infected skin, synovial fluid or tissue, CSF, etc.	Transport on ice; ≤1 h
			If DNA not extracted shortly after collection, store frozen at −70°C.
Anaplasma phagocytophilum (human granulocytotropic anaplasmosis)j	Primary Test: Wright or Giemsa stain of peripheral blood or buffy coat leukocytes during week first week of infection.	Blood	EDTA or citrate tube, RT, ≤1 h
	Acute and convalescent IFA titers for Anaplasma antibodies; specificity ranges from 83% to 100% with cross-reactivity among E. chaffeensis and A. phagocytophilium antibodies, as well as a number of clinical conditions such as Rocky Mountain Spotted Fever, typhus, Q fever, Lyme disease, etck	Serum	Clot tube, RT, ≤2 h
	NAAT	Blood	EDTA anticoagulant tube
			Transport on ice; ≤1 h
	Immunohistochemical staining of Anaplasma antigens in formalin-fixed, paraffin-embedded specimens	Bone marrow biopsies or autopsy tissues (spleen, lymph nodes, liver and lung)	Formalin container, RT, ≤2 h
Ehrlichia chaffeensis (human monocytotropic ehrlichiosis)	Primary Test: Wright or Giemsa stain of peripheral blood or buffy coat leukocytes smear during first week of infection.	Blood	EDTA anticoagulant tube, RT, ≤1 h
Ehrlichia ewingiij,k	Serology: acute and convalescent IFA titers for Ehrlichia antibodiesl	Serum	Clot tube, RT, ≤2 h
	NAAT (only definitive diagnostic test for E. ewingii)	Whole blood	Heparin or EDTA anticoagulant tube Transport on ice; ≤1 h
			If DNA not extracted shortly after collection, store frozen.
	Immunohistochemical staining of Ehrlichia antigens in formalin-fixed, paraffin-embedded specimens	Bone marrow biopsies or autopsy tissues (spleen, lymph nodes, liver and lung)	Formalin container, RT, ≤2 h
Rickettsia rickettsii (Rocky Mountain spotted fever)m,n	Serology: acute and convalescent IFA for R. rickettsii IgM and IgG antibodiesl	Serum	Clot tube, RT, ≤2 h
	NAAT	Skin biopsy (preferably a maculopapule containing petechiae or the margin of an eschar) or autopsy tissues (liver, spleen, lung, heart, and brain)	Sterile container
			Transport on ice; ≤1 h
			If DNA not extracted shortly after collection, store frozen.
	Immunohistochemical staining of spotted fever group rickettsiae antigens (up to first 24 h after antibiotic therapy initiated) in formalin-fixed, paraffin-embedded specimens	Skin biopsy (preferably a maculopapule containing petechiae or the margin of an eschar) or autopsy tissues (liver, spleen, lung, heart and brain)	Formalin container, RT, ≤2 h
Protozoa
Babesia microti	Primary Test: Giemsa, Wright's, Wright-Giemsa stains of peripheral thin and thick blood smears (Giemsa preferred)	Whole blood	For whole blood, prepare smears immediately
		Second choice EDTA vacutainer tube	RT, ≤30 min
	NAAT	Blood	EDTA anticoagulant tube, RT, ≤1 h
	Serology: acute and convalescent IFA titers for Babesia antibodies (IgM and IgG)	Serum	Clot tube, RT, ≤2 h
Virus
Colorado Tick Fever Virus	Virus-specific IFA-stained blood smears	Blood	EDTA anticoagulant tube, RT, ≤2 h
	Serology: IFA titers or complement fixationo	Serum	Clot tube, RT, ≤2 h

Abbreviations: IFA, indirect fluorescent antibody; IgG, immunoglobulin G; IgM, immunoglobulin M; NAAT, nucleic acid amplification test; RT, room temperature.

^a Other tick-borne diseases should be considered if patients have traveled to international destinations. Since travel between North America and Europe is common, Lyme borreliosis caused by Borrelia garinii and Borrelia afzelii have been included in the table. Tick-borne rickettsial diseases such as African tick-bite fever (ATBF) or Mediterranean spotted fever (MSF), occur world-wide and might have epidemiologic, seasonal and clinical features that differ from those observed in the U.S. [215]. Of note, tick-borne disease caused by Rickettsia parkeri is emerging; this organism has a similar clinical presentation as ATBF and MSF with fever, headache, eschars, and regional lymphadenopathy [217].

^b Organisms are best detected in blood while a patient is febrile. With subsequent febrile epidsodes, the number of circulating spirochetes decreases. Even during initial episodes, organisms are seen only 70% of the time.

^c Special media and technical expertise is required for culture of Borrelia species that cause relapsing fever. A centrifugation-based enrichment method followed by Giemsa staining is a rapid and viable approach [218].

^d Not valuable for an immediate diagnosis, however, serologic testing is available through public health and some private laboratories. An acute serum (obtained within 7 days of the onset of symptoms) and convalescent serum (obtained at least 21 days after the onset of symptoms) should be submitted for testing. Of significance, early antibiotic treatment can blunt the antibody response and antibody levels may fall quickly during the months after exposure.

^e To date, 18 genomic species are reported in the literature, three are confirmed agents of localized, disseminated and late manifestations of Lyme disease and are listed in the table. Another 9 species have been described with possible pathogenic potential [219]. A “chronic” or “post” Lyme disease syndrome after initial short-course antibiotic treatment has not been supported in a rigorous scientific study. Treatment of “chronic Lyme disease” is a controversial issue that has been addressed by IDSA in a guideline available on its website (http://cid.oxfordjournals.org/content/43/9/1089.full#sec-36).

^f Erythema migrans (EM) is the only manifestation of Lyme disease in the United States that is sufficiently distinctive to allow clinical diagnosis in the absence of laboratory confirmation. Positive culture rates for secondary EM lesions, primary EM lesions, and large volume (≥9 mL) blood or plasma specimens are 90%, 60%, and 48%, respectively [220]. If skin is biopsied, more than 1 biopsy sample should be taken for culture due to uneven distribution of spirochetes; disinfect the skin prior to collection and submit tissues in sterile saline. Culture is rarely performed outside of research settings.

^g Ixodes ticks have a broad host range, thereby increasing the chance of acquiring multiple pathogens from reservoir hosts. Thus, patients with one documented tick-transmitted disease are at increased risk for infection with another tick-transmitted organism. Patients with a diagnosis of Lyme disease have demonstrated immunoserologic evidence of coinfection with Babesia microti, Anaplasma phagocytophilum or Erlichia species; in Europe; coinfection with tick-borne encephalitis virus should also be considered [221].

^h Perform an IgM and an IgG WB during the first 4 weeks of illness on a patient with a positive EIA. An IgM WB is not interpretable after a patient has had symptoms for greater than 1 month's duration because the likelihood of a false-positive test result for a current infection is high in these persons; therefore, in patients with symptoms longer than 4 weeks, only test an IgG WB (http://www.cdc.gov/lyme/healthcare/clinician_twotier.html). In addition, a positive IgM WB is considered positive only if 2 of the following 3 bands are present: 24 kDa, 39 kDa and 41 kDa. Similarly, a positive IgG WB is considered positive only if 5 of the following 10 bands are present: 18 kDa, 21 kDa, 28 kDa, 30 kDa, 39 kDa, 41 kDa, 45 kDa, 58 kDa, 66 kDa, and 93 kDa. Laboratories performing this testing are strongly encouraged to report only the presence/absence of these specified bands since misinterpretation of Lyme disease WBs can otherwise possibly occur.

ⁱ Other Lyme-associated diseases can be diagnosed by NAAT (TAT 24–48 hours) or culture (TAT 3 days to 6–12 weeks). Acceptable specimens for multiple erythemata or borrelial lymphocytoma, Lyme carditis, Lyme arthritis, and acrodermatitis are skin biopsy, endomyocardial biopsy, synovial fluid or biopsy, and skin biopsy, respectively [221, 223]. Although Borrelia can be detected by NAAT in blood, biopsy specimens of infected skin, synovial tissue or fluid, or CSF, its usefulness for the diagnosis of Lyme disease is limited at this time. For example, Borrelia DNA is detected in the blood of fewer than half of patients in the early acute stage of disease when the erythema migrans rash is present, and if symptoms of Lyme disease have been present for a month or more, spirochetes can no longer be found in blood. Similarly, NAAT testing of CSF specimens is positive in only about one-third of US patients with early neuroborreliosis, and is even less sensitive in patients with late neurologic disease. The utility of testing synovial fluid and other specimen types is not well established and should be considered only under special circumstances and skin biopsy is not generally recommended because patients with erythema migrans can be reasonably diagnosed and treated on the basis of history and clinical signs alone.

^j Communication with the laboratory is of paramount importance when ehrlichiosis is suspected to ensure that Wright-stained peripheral blood smears will be carefully examined for intracytoplasmic inclusions (morulae) in either monocytes or neutrophils or bands.

^k A newly discovered Ehrlichia species was reported to cause ehrlichiosis in Minnesota and Wisconsin; this Ehrlichia is closely related to Ehrlichia muris [224].

^l Sensitivity of IFA antibody titers for tick-borne rickettsial diseases (RMSF, ehrlichiosis and anaplasmosis) is dependent on the timing of specimen collection; the IFA is estimated to be 94% to 100% sensitive after 14 days of onset of symptoms and sensitivity is increased if paired samples are tested.

^m Treatment decisions for tick-borne rickettsial diseases for acutely ill patients should not be delayed while waiting for laboratory confirmation of a diagnosis. Fundamental understanding of signs, symptoms, and epidemiology of the disease is crucial in guiding requests for tests and interpretation of test results for ehrlichiosis, anaplasmosis and Rocky Mountain spotted fever (RMSF). Misuse of specialized tests for patients with low probability of disease and in areas with a low prevalence of disease might result in confusion.

ⁿ Antibiotic therapy may diminish the development of convalescent antibodies in RMSF.

^o IgM antibodies develop 2 weeks after symptom onset.

^p Laboratory assays for the diagnosis of neuroborreliosis are of limited clinical value [151, 222 ].