First evidence of Anaplasma infection in Crete, Greece. Report of six human cases.

In this study we investigate the demographic, clinical and laboratory features of six human cases of human granulocytic anaplasmosis (HGA) in Crete, Greece. Six patients, residents of the city of Chania, Crete, were hospitalised in the General Hospital of Chania with clinical manifestations and laboratory findings suggestive of acute A. phagocytophilum infection. Blood samples were taken from all patients on admission before the administration of antibiotics. Acute-phase serum samples were tested using an indirect immunofluorescence assay (IFA) for the presence of IgG and IgM antibodies against A. phagocytophilum antigen (Focus Diagnostics, CA, USA), according to the manufacturer’s recommendations (cut-off for positivity IgG‡1 ⁄ 64 and IgM‡1 ⁄ 20). Whole blood samples were tested by PCR-sequencing analysis of the 16s rRNA gene. DNA extraction was carried out using the QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). PCR reactions were carried out using the primer set EHR16SD– EHR16SR targeting a fragment of 345 bp of the 16s rRNA gene of Anaplasmataceae using the conditions previously described [4]. Positive PCR products were directly sequenced using the sequencer CEQ 8000 Beckman Coulter. Three consecutive blood samples were taken for culture from each patient. Serological testing for suspected potential pathogens that are endemic in the area was also performed. Six patients were diagnosed as having acute A. phagocytophilum infection. Four infections were acquired in September 2006, one in August 2007 and one in September 2007. The mean age was 46.7 years old, ranging from 17 to 69 years old. Five patients were males. Three out of six patients reported contact with wild animals within 1 month before the onset of symptoms. None of the patients had travelled recently. Demographic characteristics, clinical presentation and laboratory findings are presented in Table 1. Symptoms persisted for 2–6 days before presentation. On admission, fever was present in all of the six patients and was accompanied by chills in two patients. Other symptoms observed were a maculopapular rash of the trunk (3 ⁄ 6), headache (2 ⁄ 6), malaise (2 ⁄ 6), splenomegaly (2 ⁄ 6), cervical lymphadenopathy (1 ⁄ 6), and gastrointestinal disturbances (1 ⁄ 6 patients). Common laboratory abnormalities found were high lactate dehydrogenase titres (all patients), elevated transaminases (5 ⁄ 6), elevated serum C-reactive protein (4 ⁄ 6), thrombocytopenia (4 ⁄ 6), and leucopenia (2 ⁄ 6 patients). All blood cultures were negative for common pathogens. Acute infection by other suspected pathogens included in the differential diagnosis was ruled out. All patients revealed IgM antibodies with titres ranging from 1 ⁄ 20 to 1 ⁄ 40. Only one patient revealed both IgM and IgG antibodies against Anaplasma phagocytophilum (IgG 1 ⁄ 128, IgM 1 ⁄ 40). DNA sequences of the PCR products in all patients (Genbank number EU448142), when compared using nucleotide Blast (National Center for Biotechnology Information) (http:// www.ncbi.nlm.nih.gov/BLAST), revealed 100% identity with each other and 98% identity with Anaplasma sp deposited sequences (EU448141, Anaplasma sp. panagCy 16S ribosomal RNA gene; Corresponding author and reprint requests: A. Psaroulaki, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Voutes, Heraklion, Crete 71100, Greece E-mail: annapsa@med.uoc. gr