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

2008; Elsevier BV; Volume: 15; Linguagem: Inglês

10.1111/j.1469-0691.2008.02695.x

1469-0691

Dimosthenis Chochlakis, Anna Psaroulaki, Sofia Kokkini, S. Kostanatis, E. Arkalati, E. Karagrannaki, K. Tsiatis, Yiannis Tselentis, Achilleas Gikas,

Mosquito-borne diseases and control

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 [4Parola P Beati L Cambon M Brouqui P Raoult D Ehrlichial DNA amplified from ixodes ricinus (acari: Ixodidae) in france.J Med Entomol. 1998; 35: 180-183PubMed Google Scholar]. 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.TABLE 1Demographic data, clinical characteristics and laboratory findings upon presentation in six human granulocytic anaplasmosis patients in the city of Chania, Crete, GreecePatient number123456Age535617696217SexMMFMMMDay of illness326NR32Contact with wild animalsRodents, goats, sheepRodents, goats, sheep, pigeonsNRNRRodentsNRHighest temperature3839.54038.539.640RigorNoNoNoYesYesNoMaculopapular rashNoNoNoYesYesYesHeadacheYesYesNoNoNoNoMalaiseYesNoYesNoNoNoSplenomegalyNoNoYesNoYesNoOtherCervical lymphadenopathyNoAbdominal pain, nausea, constipationNoNoNoAST (U/L)anormal value, 0–40 U/L12512439320139115ALT (U/L)anormal value, 0–40 U/L7412120820118104LDH (U/L)bnormal value, <230 U/L745572946264894498CRP (mg/dL)cnormal value, <0.35 mg/dLNRNR8.64.69.56.3Platelets count (X 109/L)dnormal value, 150–450 × 109/L9812314519472192WBC count (X 109/L)enormal value, 5–10 × 109/L.5.13.87.06.94.85.1NR, not reported;a normal value, 0–40 U/Lb normal value, <230 U/Lc normal value, <0.35 mg/dLd normal value, 150–450 × 109/Le normal value, 5–10 × 109/L. Open table in a new tab NR, not reported; 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; EU090185, Anaplasma sp. 5 16S ribosomal RNA gene; EU090184, Anaplasma sp. 4 16S ribosomal RNA gene; DQ648489, Anaplasma phagocytophilum 16S ribosomal RNA gene). Treatment was administered to all patients on the day of admission. Four patients were treated with doxycycline and ciprofloxacin and two patients were treated with doxycycline and amoxicillin/clavulanate. One patient's clinical condition did not improve and ciprofloxacin was replaced by chloramphenicol on day three of treatment. Defervescence was observed on a mean of 5 days after treatment onset (range, 3–7 days). Data concerning the prevalence of HGA in Europe are limited, but increased medical awareness has led to the detection of human cases in several countries [2Brouqui P Bacellar F Baranton G et al.Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.Clin Microbiol Infect. 2004; 10: 1108-1132Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar]. The non-specific clinical manifestations of the disease make the diagnosis difficult, but high suspicion should arise when compatible epidemiological information is available. In Greece, other tick-borne diseases have been reported and in previous studies various tick species have been identified [5Psaroulaki A Ragiadakou D Kouris G Papadopoulos B Chaniotis B Tselentis Y Ticks, tick-borne rickettsiae, and Coxiella burnetii in the Greek Island of Cephalonia.Ann N Y Acad Sci. 2006; 1078: 389-399Crossref PubMed Scopus (62) Google Scholar]. However, the Ixodes ricinus tick [4Parola P Beati L Cambon M Brouqui P Raoult D Ehrlichial DNA amplified from ixodes ricinus (acari: Ixodidae) in france.J Med Entomol. 1998; 35: 180-183PubMed Google Scholar], regarded as the main vector in Europe, is not a common tick species in Crete; moreover, common reservoirs of A. phagocytophilum, such as white-tailed deer, white-footed mice and dusky-footed wood rats [1Dumler JS Madigan JE Pusterla N Bakken JS Ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment.Clin Infect Dis. 2007; 45: S45-S51Crossref PubMed Scopus (245) Google Scholar], are not present in Greece. Therefore, the suggestion that different reservoirs and vectors are involved in the transmission cycle of HGA in the region should be investigated. The diagnosis of HGA is based on serologic evidence and/or a positive PCR assay [1Dumler JS Madigan JE Pusterla N Bakken JS Ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment.Clin Infect Dis. 2007; 45: S45-S51Crossref PubMed Scopus (245) Google Scholar, 2Brouqui P Bacellar F Baranton G et al.Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.Clin Microbiol Infect. 2004; 10: 1108-1132Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar]. In our study, serological and clinical evidence of acute A. phagocytophilum infection were further confirmed by PCR and sequence analysis. To our knowledge, this is the first description of human cases of acute HGA in Greece.