title: Evaluation of an Improved PCR Diagnostic Assay for Human Granulocytic Ehrlichiosis.
abstract: Background: Human granulocytic ehrlichiosis (HGE) is a tick-borne disease caused by an agent similar to Ehrlichia equi. The etiologic agent has only recently been cultivated, and clinical and laboratory findings are nonspecific. Severity and fatal outcome are associated with delays in diagnosis and therapy. Methods and Results: The sensitivity of the polymerase chain reaction (PCR) technique for HGE was enhanced at least 250-fold for identification of the 16S ribosomal RNA gene of the HGE agent in blood by shortening cycle segments, using 35 rounds of amplification, employing eLONGase polymerase, and by using SYBR Green I for detection of amplified products. From 17 patients with suspected HGE, acute-phase blood was tested by PCR and for E. equi antibodies in acute and convalescent sera. The HGE PCR detected ehrlichial DNA in 1 ng of genomic leukocyte DNA and in as little as 0.6 infected leukocyte per microliter of blood. Of the 17 patients with suspected HGE, 7 (41%) were serologically confirmed, and six of these seven had positive PCRs (86% sensitivity). Ten seronegative patients had negative PCRs, and no patient had a positive PCR and negative serology (100% specificity). Conclusions: The polymerase chain reaction for the HGE agent 16S rRNA gene is a sensitive and specific method for the early confirmation of HGE at a time when therapeutic decisions are required.