Infection with Anaplasma phagocytophilum in a seronegative patient in Sicily, Italy: case report.

Abstract

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.
Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog.
Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested 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.

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Annals of Clinical Microbiology and
Antimicrobials
Open Access
Case report
Infection with Anaplasma phagocytophilum in a seronegative patient
in Sicily, Italy: Case Report
J de la Fuente*1,2, A Torina3, V Naranjo2, S Caracappa3, V Di Marco4,
A Alongi3, M Russo4, AR Maggio3 and KM Kocan1
Address: 1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA,
2Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain, 3Intituto
Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi n° 3, 90129 Palermo, Italy and 4Istituto Zooprofilattico Sperimentale della Sicilia, Area
Barcellona Pozzo di Gotto, Via S. Andrea n° 96, 98051 Barcellona, Italy
Email: J de la Fuente* - djose@cvm.okstate.edu; A Torina - torina@pa.izs.it; V Naranjo - MVictoria.Naranjo@uclm.es;
S Caracappa - caracappa@pa.izs.it; V Di Marco - dimarco@pa.izs.it; A Alongi - alongi@pa.izs.it; M Russo - russo@pa.izs.it;
AR Maggio - maggio@pa.izs.it; KM Kocan - kmk285@cvm.okstate.edu
* Corresponding author
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.
Case presentation: Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily,
Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA
was amplified by two independent laboratories and sequenced from two separate patient's blood
samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of
the A. phagocytophilum strain USG3 originally obtained from a dog.
Conclusion: Infection with A. phagocytophilum was confirmed in a patient without a detectable
antibody response against the pathogen. The results reported herein documented the first case of
confirmed HGA in Sicily, Italy. These results suggested 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.
Background
Anaplasma phagocytophilum (Rickettsiales: Anaplasmata-
ceae) causes human granulocytic anaplasmosis (HGA) in
humans, which has been recognized as an emerging tick-
Published: 03 October 2005
Annals of Clinical Microbiology and Antimicrobials 2005, 4:15 doi:10.1186/1476-0711-4-15
Received: 10 August 2005
Accepted: 03 October 2005
This article is available from: http://www.ann-clinmicrob.com/content/4/1/15
© 2005 de la Fuente et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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 con-
firmed by identification of morulae in granulocytes, posi-
tive PCR results using whole blood as a substrate, and/or
isolation of A. phagocytophilum from the blood [2]. Sero-
logical 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 south-
ern Italy is high in workers at risk for tick bites [6], only
two cases of HGA have been reported in Italy [7]. How-
ever, the analysis of A. phagocytophilum DNA was not per-
formed 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, myal-
gia, 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, par-
tridges 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 find-
ings, the patient showed a mild splenomegaly, hemor-
rhoids, osteopenia, hiatal
inflammation of duodenum. Systemic inflammatory
pathology and neoplasia were ruled out. Doctors sus-
pected a depressive syndrome and the patient was treated
with antidepressants.
hernia, gastritis and
Serum antibodies were determined for Lyme borreliosis,
Rickettsia conorii, Coxiella burnetii and A. phagocytophilum
(Fuller Laboratories, Fullerton, CA, USA), and Ehrlichia
chaffeensis and A. phagocytophilum (Focus Technologies,
Cypress, CA, USA) following manufacturer's recommen-
dations. FITC-conjugated anti-human IgG (Sigma, St.
Louis, MO, USA) and IgM (Aliphadia S.A., Wavre, Bel-
gium) were used as secondary antibodies. The immun-
ofluorescence tests for A. phagocytophilum use antigens
derived from human HL-60 cells infected with the HGE-1
isolate and samples were considered negative when no
fluoresce was detected at 1:80 (Fuller Laboratories) or
1:64 (Focus Technologies) dilution of the test serum, as
recommended by the manufacturer. DNA was purified
using GenElute Mammalian Genomic DNA Miniprep Kit
(Sigma) or TriReagent (Sigma) from patient's blood sam-
ples collected in September 8 and October 8, 2004. PCR
analysis for R. conorii, C. burnetii, E. chaffeensis, Theileria
spp., Babesia spp. and Anaplasma spp. were conducted on
both DNA samples as previously described [8-11]. 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 MgSO4, 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 addi-
tion of DNA to rule out contaminations during PCR.
Amplified Anaplasma 16S rDNA fragments were resin
purified (Wizard, Promega) and cloned into pGEM-T vec-
tor (Promega) for sequencing both strands by double-
stranded 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 possi-
bility 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 patho-
gens.
Serology for Lyme borreliosis, R. conorii, C. burnetii, E.
chaffeensis and HGA and PCR analyses for R. conorii, C.
burnetii, E. chaffeensis, Theileria spp. and Babesia spp. were
negative. However, positive PCR results were obtained for

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Anaplasma spp. in patient's blood samples collected in
September 8 and October 8, 2004. PCR reactions for Ana-
plasma spp. 16S rDNA were conducted with independent
DNA extractions in two laboratories (in Palermo, Sicily
and in Ciudad Real, Spain) to confirm the results. Control
reactions ruled out PCR contaminations and the sequence
of the 16S rDNA fragment obtained from the patient
(Genbank accession number DQ029028) was identical in
both DNA samples and identical to the sequence of the A.
phagocytophilum strain USG3 originally obtained from a
dog (Genbank accession number AY055469; ref. [12]).
The patient described herein fulfilled the criteria for con-
firmed HGA, including prolonged fever, arthralgia, myal-
gia, 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 differ-
ences have been reported in A. phagocytophilum and may
be associated with variations in pathogenicity and host
tropism [10,12-14], 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 con-
firmatory 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 immu-
noassays. KMK helped to draft the manuscript. All authors
read and approved the final manuscript.
Acknowledgements
This research was supported by The Ministry of Health, Italy, the Instituto
de Ciencias de la Salud (ICS-JCCM), Spain (project 03052-00) and the
Endowed Chair for Food Animal Research and the Oklahoma Agricultural
Experiment Station Project 1669 (K.M. Kocan). V. Naranjo is funded by
Junta de Comunidades de Castilla – La Mancha (JCCM), Spain. We thank S.
Scimeca, R. D'Agostino and A. Corrente (Intituto Zooprofilattico Speri-
mentale della Sicilia, Palermo, Italy) for technical assistance.
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