Annals of Clinical Microbiology and Antimicrobials Open Access Infection with Anaplasma Phagocytophilum in a Seronegative Patient in Sicily, Italy: Case Report

Human granulocytic anaplasmosisAnaplasma phagocytophilum16S rDNA sequence Abstract Background: Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also.


Background
Anaplasma phagocytophilum (Rickettsiales: Anaplasmata-ceae) causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe [1,2]. The disease was first described in the United States in 1994 [3,4] and in Slovenia in 1997 [5]. About 65 cases have been reported in Europe although some of them do not fulfill the criteria for confirmed or probable HGA [2]. Human infection with A. phagocytophilum could be confirmed by identification of morulae in granulocytes, positive PCR results using whole blood as a substrate, and/or isolation of A. phagocytophilum from the blood [2]. Serological tests, particularly indirect immunofluorescence assay (IFA), are commonly used but are often negative during the initial phase of the disease. However, high and stable antibody titers to A. phagocytophilum are usually found in patients with probable HGA who show clinical signs and symptoms that most likely are not the result of a recent infection with A. phagocytophilum [2].
Although the seroprevalence of HGA in central and southern Italy is high in workers at risk for tick bites [6], only two cases of HGA have been reported in Italy [7]. However, the analysis of A. phagocytophilum DNA was not performed in these cases. The objective of this study was to characterize the 16S rDNA sequence of a strain of A. phagocytophilum obtained from a seronegative patient with confirmed HGA in Sicily, Italy.

Case Presentation
Patient, materials and methods On May 8, 2004, a 51-year-old man living in Barcellona Pozzo di Gotto, Messina, Sicily, Italy was admitted to the hospital with a 3-month fever (37.2°C), arthralgia, myalgia, malaise, pallor, stiffnes of hands and knee, nausea, low appetite and weight loss. The patient did not recall a tick bite but he visited hunting areas 15 days before the fever started and kept exotic birds (golden pheasants, partridges and guinea fowls) at his house for two years. The patient showed abnormal liver (462 mU/ml alkaline phosphatase, normal range (nr) = 98-280; 1.20 mg/dl total bilirubin, nr = 0.16-1.10; 0.35 mg/dl direct bilirubin, nr = 0.0-0.2) and renal (1.5 mg/dl creatinine, nr = 0.5-1.2) functions and low hemoglobin (9.9 g/dl; nr = 12-18). Pulmonary, cardiac and immunological involvement was absent. Cell counts and serum C-reactive protein levels were normal.
On September 13, 2004 the patient was admitted to the hospital for a second time. In addition to previous findings, the patient showed a mild splenomegaly, hemorrhoids, osteopenia, hiatal hernia, gastritis and inflammation of duodenum. Systemic inflammatory pathology and neoplasia were ruled out. Doctors suspected a depressive syndrome and the patient was treated with antidepressants.  [8][9][10][11]. PCRs were conducted with 1 µl (0.1-10 ng) DNA using 10 pmol of each primer in a 50-µl volume (1.5 mM MgSO 4 , 0.2 mM dNTP, 1 × AMV/Tfl 5 × reaction buffer, 5u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler for 35 cycles. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb DNA Ladder, Promega). Control reactions were done without the addition of DNA to rule out contaminations during PCR.
Amplified Anaplasma 16S rDNA fragments were resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega) for sequencing both strands by doublestranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). Two independent clones were sequenced from each PCR. Multiple sequence alignment was performed with the program AlignX (Vector NTI Suite, version 5.5; InforMax, North Bethesda, MD, USA).

Results and discussion
The analysis of the clinical symptoms and laboratory results of the patient described herein suggested the possibility of an infectious agent as the cause of the illness. Viral infections were ruled out and the investigation was directed towards the identification of tick-borne pathogens.  [12]).
The patient described herein fulfilled the criteria for confirmed HGA, including prolonged fever, arthralgia, myalgia, malaise, nausea, abnormal liver function and positive PCR and sequence analysis for A. phagocytophilum DNA. However, the patient was seronegative for up to six months after detection of pathogen infection by PCR. These results suggested that the patient presented a chronic stage of infection with symptoms produced by a secondary illness related or not to A. phagocytophilum and/ or could has been infected with a strain of the pathogen that could not be detected by existing serological tests or induces a low antibody response. Strain genetic differences have been reported in A. phagocytophilum and may be associated with variations in pathogenicity and host tropism [10,[12][13][14], although the exact relationship between these factors is presently unknown.

Conclusion
The results reported herein demonstrated a prolonged A. phagocytophilum infection in a patient without a detectable antibody response against the pathogen. These results documented the first case of prolonged A. phagocytophilum infection in Sicily, Italy and suggest the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.

Authors' contributions
JF carried out the sequence analysis, designed the study and drafted the manuscript. AT and SC conceived the study, and participated in its design and coordination and helped to draft the manuscript. VN and AA carried out the molecular genetic studies. VDM and MR collected and analyzed the clinical data. ARM carried out the immunoassays. KMK helped to draft the manuscript. All authors read and approved the final manuscript.