Comparison of a Real-Time PCR Method with Serology and Blood Smear Analysis for Diagnosis of Human Anaplasmosis: Importance of Infection Time Course for Optimal Test Utilization

Abstract

c Anaplasmosis and ehrlichiosis are emerging tick-borne diseases with clinically similar presentations caused by closely related pathogens. Currently, laboratories rely predominantly on blood smear analysis (for the detection of intracellular morulae) and on serologic tests, both of which have recognized limitations, for diagnostic purposes. We compared the performance of a published real-time PCR assay that incorporates melt curve analysis to differentiate Anaplasma and Ehrlichia species with blood smear and serologic methods in an upper Midwest population. Overall, 38.5% of the specimens selected for evaluation had one or more tests that were positive for anaplasmosis. The PCR positivity for all specimens was maximal (21.2%; 29/137) during the early acute phase of illness (0 to 4 days since illness onset) and significantly less frequent (11.5%; 20/174) during later phases (>4 days since illness onset). All positive specimens were Anaplasma phagocytophilum; no Ehrlichia species were identified. The realtime PCR detected 100% of infections that were detected by blood smear analysis (14/14) and broadened the detection window from a maximum of 14 days for smear positivity to 30 days for PCR. Additional infections were detected by real-time PCR in 12.9% (11/85) of smear-negative patients. There was poor agreement between the real-time PCR assay and serologic test results: 19.8% (19/96) and 13.7% (29/212) of seropositive and -negative patients, respectively, were PCR positive. Seropositivity increased with increasing days of illness, demonstrating that serologic detection methods are best utilized during presumed convalescence. Our results indicate that the optimal performance and utilization of laboratory tests for the diagnosis of anaplasmosis require knowledge regarding time of symptom onset or days of illness.

Canine ehrlichiosis: prevalence and epidemiology in northeast Brazil

“…can parasitize splenic macrophages,and PCR testing is more sensitive with decreased chance of false-negative results when spleen samples are analyzed (HARRUS & WANER, 2011), particularly in cases of chronic infection in which it is not always possible to detect the DNA of the agent in blood samples (HARRUS et al, 1998). Moreover, the sensitivity of molecular tests for Anaplasmataceae species can be increased with the use ofreal-time PCR testing (HARRUS & WANER, 2011;SCHOTTHOEFER et al, 2013); however, it was not possible to perform this technique in this study. On IFA, cross-reactions can occur between species of the genus Ehrlichia, such as E. ewingii and E. chaffeensis.…”

High Prevalence ofAnaplasmaspp. in Small Ruminants in Morocco

“…Alternatively, bacteraemia in A. phagocytophilum infection may be of shorter duration than for A. marginale and A. ovis . In fact, PCR and microscopy give peak sensitivity in the acute stage of disease, between the 4th and 22nd day post‐infection (PI) for PCR (Pusterla et al., ) and between 2nd and 14th day PI for microscopy (Schotthoefer et al., ). It is also possible that the sample size was too small to detect a disease with such a low prevalence, even though there have been up to now no reports of A. phagocytophilum in Morocco.…”

“…Commonly used diagnostic methods include blood smears. The diagnostic performance of this technique is acceptable for detection of A. ovis , A. marginale and A. phagocytophilum in symptomatic (subacute and acute stages) cases of the disease but lacks sensitivity for long course, chronical or subclinical cases (Carelli et al., ; Schotthoefer et al., ). Serology is also used commonly, and unlike microscopy offers a high sensitivity in chronic disease but lacks sensitivity in acute stages (Bakken et al., ).…”

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus

“…Paired serologic testing is intrinsically an imperfect comparator because it audits exposure over a broad time pe- riod while the TAC tests only a single moment (33). Furthermore, these pathogens cause bacteremia only during certain stages of illness due to different pathogeneses.…”