Download Human granulocytic anaplasmosis in eastern France

Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 72 (2012) 214 – 218
www.elsevier.com/locate/diagmicrobio
Human granulocytic anaplasmosis in eastern France: clinical presentation
and laboratory diagnosis☆
Christelle Koebela, 1 , Aurélie Kerna, 1 , Sophie Edouardb , Anh Thu Hoanga , Noéline Celestinc ,
Yves Hansmannc , Benoît Jaulhaca,⁎, Philippe Brouquib , Sylvie Josiane De Martinoa
a
Laboratoire de Bactériologie, Hôpitaux Universitaires de Strasbourg, 1, rue Koeberlé, Strasbourg Cedex, France
Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD UMR 6236, Faculté de Médecine, Marseille, France
c
Service de Maladies Infectieuses et Tropicales, Hôpitaux Universitaires de Strasbourg, 1, place de l'hôpital BP 426, 67091 Strasbourg Cedex, France
Received 8 September 2011; accepted 10 December 2011
b
Abstract
Human granulocytic anaplasmosis (HGA) is a tick-borne infection characterised by an acute, nonspecific febrile illness. To date, few
clinical cases have been supported by both a positive polymerase chain reaction (PCR) assay and subsequent seroconversion against
Anaplasma phagocytophilum antigen all over Europe. We report here 3 consecutive cases of HGA that occurred during the summer of
2009 which fulfilled the epidemiologic, clinical, and biological criteria for HGA. These data highlight PCR assay on ethylenediaminetetraacetic acid blood rather than serology as the diagnostic test of choice during the acute phase of the disease. In endemic areas, HGA should
be investigated in patients presenting an undifferentiated febrile illness with cytopenia, elevated rates of liver enzymes, and increased
C-reactive protein values.
© 2012 Elsevier Inc. All rights reserved.
Keywords: Human granulocytic anaplasmosis; Anaplasma phagocytophilum; PCR; Serology; France; Ixodes ricinus
1. Introduction
Eastern France is an endemic area for Lyme borreliosis
(Hubálek, 2009) and tick-borne encephalitis (Hansmann
et al., 2006). The vector of both these diseases is Ixodes
ricinus. Other illnesses occurring after I. ricinus bite—such
as anaplasmosis, rickettsiosis, or babesiosis—have been
reported in Europe. Human granulocytic anaplasmosis is an
acute infectious disease caused by Anaplasma phagocytophilum. It has been detected in I. ricinus ticks by polymerase
chain reaction (PCR) from most European countries including France (Brouqui et al., 2001). The infection rate of
ticks in northeastern France is low: approximately 0.4% in
nymphs and 1.2% in adult ticks (Ferquel et al., 2006).
HGA was first characterised in northern United States in
1994, and the first European case was reported in 1997 in
☆
This work has been supported in part by the PHRC (grant no. 9777).
⁎ Corresponding author. Tel.: +33-369-55-14-53; fax: +33-369-55-16-98.
E-mail address: [email protected] (B. Jaulhac).
1
These authors contributed equally to this work.
0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2011.12.005
Slovenia (Petrovec et al., 1997). Since then, although
around 70 cases have been reported from several European
countries, most of these were only based on the detection of
specific antibodies, since PCR in acute phase was not often
performed. However, according to European guidelines
(Brouqui et al., 2004), laboratory confirmation of HGA
must be based either on seroconversion or on 4-fold increase
in antibody titer, either on the detection of A. phagocytophilum in blood, on culture, or specific PCR. Diagnosis of
HGA currently frequently relies on clinical suspicion only
due to the limited availability of rapid diagnostic tests such
as PCR, and the absence of detectable serum antibodies at
the time of clinical presentation. The largest series of confirmed HGA occurred in Central Europe, in particular in
Slovenia (Lotric-Furlan et al., 2006) and in Scandinavia
(Norway and Sweden) (Bjoërsdorff et al., 1999), but
sporadic cases have also been described in the Netherlands
(van Dobbenburgh et al., 1999), Austria (Walder et al.,
2006), Italy (de la Fuente et al., 2005; Ruscio and Cinco,
2003), and Spain (García et al., 2006; Oteo et al., 2000). In
France, the only confirmed laboratory case has been
C. Koebel et al. / Diagnostic Microbiology and Infectious Disease 72 (2012) 214–218
reported in the eastern region of Alsace in 2003 (Remy
et al., 2003).
Clinical presentation of HGA is nonspecific and usually
consists of fever N38.5 °C, headache, malaise, myalgia,
and/or arthralgia, and is often accompanied by laboratory
abnormalities such as leukopenia, thrombocytopenia, and
increased activity of hepatic enzymes (Brouqui et al., 2004).
However, many clinical suspicions of this disease are not
investigated, have no proven etiology, and are subsequently
not reported.
We report here 3 consecutive HGA cases, confirmed by
PCR, which occurred in the same area (Alsace, France)
during the summer of 2009 and which fulfilled HGA epidemiologic, clinical, and biological criteria.
215
sp., and Chlamydia sp. (data not shown). Genomic DNA
from Rickettsia sp., Wolbachia sp., A. marginale, and
A. platys was not amplified by the PCR assay either (data
not shown). Veterinary blood samples from cattle, dogs, and
sheep infected by A. phagocytophilum were used as positive
control (data not shown).
Serologic diagnosis of infection with A. phagocytophilum
was made by IFA (Focus Diagnostics, Cypress, CA, USA).
Specimens with IgM titers ≥1/40 and IgG titers ≥1/64
were considered positive. Demonstration of seroconversion
or at least 4-fold increase in antibody titre between acute
and convalescent serum was used to confirm HGA (Comer
et al., 1999).
3. Results
2. Materials and methods
2.1. Patients and samples
In Alsace, during the period June to September 2009, 15
patients presenting a febrile syndrome with a recent history
of tick bites or exposure to ticks were tested for HGA at the
Laboratory of Bacteriology, Strasbourg University Hospital.
Ethylenediaminetetraacetic acid (EDTA) whole blood samples were systematically collected for blood smear and
specific PCR assay during the febrile phase of the disease.
For serologic testing, 2 sera were also collected: the first
during the acute phase and the second 2 to 3 weeks afterwards, during the convalescent phase.
2.2. Microscopic examination
Blood smears were obtained from whole blood samples,
stained with May–Grünwald–Giemsa and examined for the
presence of morulae within the cytoplasm of neutrophils.
2.3. Molecular assay
DNA was extracted from whole-blood samples with the
QIAamp® Mini kit (Qiagen®, Hilden, Germany). A Taqman®-based real-time PCR was applied to amplify a 73-bp
fragment from the A. phagocytophilum msp2/p44 gene. We
used Primer Express® software version 2.0 (Applied
Biosystems) to design specific oligonucleotides: forward
primer, 5′-TGT AGC TAT GGA AGG CAG TGT TG-3′;
reverse primer, 5′-GCG CTC GTA ACC AAT CTC AAG3′; and probe, 6-VIC-CGG TAT TGG TGG TGC CAG GGT
TGA-TAMRA. Real-time PCR was performed on ABI
Prism® 7000 SDS (Applied Biosystems) under standard
PCR conditions. All runs included DNA isolation controls
and no-template controls to monitor the presence of
contaminants during DNA extraction and/or in PCR reagents. The specificity of the primer set was confirmed by
testing genomic DNA from a large diversity of bacteria
including Gram-negative and Gram-positive bacteria,
Borrelia sp., Treponema sp., Leptospira sp., Mycoplasma
Molecular testing using the msp2/p44 gene yielded
positive results in the acute-phase blood of 3 of the 15
patients tested. For the 12 patients with negative PCR assay,
microscopic examination was negative and no specific
antibodies were detected in acute-phase serum. Convalescent
sera were available for 5 of these patients and were all
negative. Laboratory findings were available for 6 patients
among the 12 with a negative PCR assay; all these 6 presented leukopenia, thrombocytopenia, and increased levels
of liver enzymes.
For the 3 patients with positive PCR results, examination
of peripheral blood smears revealed cytoplasmic inclusions
suggestive of A. phagocytophilum morulae (Fig. 1). All 3
patients presented undifferentiated illness characterised by
high-degree fever, headache, myalgia and/or arthralgia, and
lymphadenopathy; associated with bicytopenia; and characterised by elevated serum levels of liver enzymes (Table 1).
Although the serologic test result of acute-phase serum was
negative for these 3 patients, specific antibodies were
detected in convalescent serum for each of them (Table 2).
Patients 1 and 2 were living in rural areas: both had
domestic animals at home and were also in contact with wild
animals. Patient 2 was a hunter and a fisherman. Patient 3
was a forest worker: she reported a tick bite 10 days before
the onset of symptoms and cutaneous examination at the
time of the disease revealed tick bite scars on her limbs.
Patient 1 presented a biphasic course of the illness starting
with febrile syndrome. Three days after the onset of
symptoms, he was admitted to hospital. Initial investigations
showed neutropenia, thrombopenia, and elevated C-reactive
protein (CRP) values, but etiologic investigation results
remained negative. As the symptoms spontaneously decreased, the patient returned home with empiric antibiotic
therapy (cefixime per os). The patient underwent a new onset
of symptoms and came back to hospital where the treatment
was stopped. New investigations showed a positive PCR
result for A. phagocytophilum, but no haematologic and
biochemical abnormalities. Doxycycline was started on day
24 following the first onset of symptoms. Patients 2 and 3
216
C. Koebel et al. / Diagnostic Microbiology and Infectious Disease 72 (2012) 214–218
Patient #1
Patient #2
Patient #3
Fig. 1. Peripheral blood smears were stained with May–Grünwald–Giemsa: examination showed the presence of morulae within the cytoplasm of neutrophils.
Arrows indicate the morulae within granulocytes.
received doxycycline 3 days after onset of fever, which led to
prompt improvement of the clinical manifestations: the fever
dropped within 24 h, the other symptoms resolved
afterwards, and biological markers gradually returned to
normal values. Only a marked fatigue persisted in both
patients through to the third week after hospitalisation.
Further clinical evaluation showed that all patients completely
recovered. Overall, the course of illness was mild for these
3 patients.
4. Discussion
Eastern France, especially Alsace, is an area where
I. ricinus is highly prevalent and Lyme borreliosis is very
common (Hubálek, 2009). In the meantime, many febrile
illnesses that occur after tick bites have no proven etiology
and other pathogens such as A. phagocytophilum, the
agent of HGA, share the same epidemiologic features as
B. burgdorferi, since they are transmitted by the same vector.
Table 1
Summary of clinical and laboratory findings from the 3 patients with confirmed HGA
Patient
Patient 1
Patient 2
Patient 3
Age (years)
Tick bite or exposure to ticks
Clinical symptoms
Fever
Chills
Sweats
Headache
Malaise/fatigue
Arthralgias/myalgias
Nausea/vomiting/abdominal pain
Cough
Lymphadenopathy/hepato-splenomegalia
Cutaneous rash
Laboratory abnormalities
CRP (b4 mg/L)
Platelet count (N: 150–500 × 10 9/L)
Leukocyte count (N: 4–10 × 10 9/L)
Polymorphonuclear cells
Presence of activated lymphocytes
ASAT level (N: 15–40 U/L)
ALAT level (N: 10–50 U/L)
LDH level (N: 230–400 U/L)
Treatment
46
+
47
+
67
+
39.5 °C
+
+
+
+
−
+
+
+
−
40 °C
+
+
+
+
+
+
−
+
−
39.5 °C
+
+
+
+
+
+
−
+
−
263
32
1.90
1.35
+
136
191
611
Ceftriaxone + gentamicin 48 h, cefixime 24 h,
doxycycline 200 mg/day for 10 days
Favorable
188
20
1.83
1.39
+
280
274
1200
Doxycycline 200 mg/day
for 10 days
Favorable
157
52
1.40
0.90
+
66
42
565
Doxycycline 200 mg/day
for 15 days
Favorable
Outcome
C. Koebel et al. / Diagnostic Microbiology and Infectious Disease 72 (2012) 214–218
Table 2
Results of microscopic examination, PCR, and serologic assays from the 3
patients with confirmed HGA after onset of symptoms
Patient
no.
1
2
3
Days after
onset of
symptoms
5
24
90
2
50
6
70
Blood smear
examination
(% infected PMN)
ND
+ (b1%)
ND
+ (5%)
ND
+ (2–3%)
ND
PCR assay
msp2/p44
a
+
+
ND
+
ND
+
ND
Serologic
testing (IFA)
IgM
IgG
b1/20
1/160
1/20
1/20
1/20
1/40
1/20
b1/32
1/2048
1/64
b1/32
1/64
1/32
1/64
PMN = polymorphonuclear; ND = Not done.
a
PCR assay performed on serum.
During the summer of 2009, we investigated patients with
febrile illness together with a recent history of tick bites or
exposure to ticks. Three patients fulfilled the microbiological
criteria for confirmed HGA (Brouqui et al., 2004): presence
of morulae within granulocytes, positive PCR assay for
A. phagocytophilum, and subsequent seroconversion against
A. phagocytophilum antigen. All 3 patients had been exposed
to ticks as they lived in rural areas or practiced leisure
activities in wooden areas. Clinical characteristics and laboratory findings were similar to those reported in the United
States (Dumler et al., 2007) and other European countries
(Blanco and Oteo, 2002), including illness characterised by
high-degree fever, headache, myalgia and/or arthralgia, and
lymphadenopathy; associated with bicytopenia; and characterised by elevated levels of liver enzymes. The rapid
clinical improvement observed in 2 patients shortly after
initiation of doxycycline was also typical of this disease.
This early antibiotic treatment may explain the weak antibody titers in convalescent sera, especially as these samples
were obtained 50 to 90 days after the onset of symptoms.
Similar to previous observations (Lotric-Furlan et al., 2006),
the disease appeared to be mild and to resolve quickly, even
in the absence of specific antibiotics: in patient 1, fever
abated before the use of doxycycline.
To date, only a few clinical cases have been supported by
both a positive PCR assay and subsequent seroconversion
against A. phagocytophilum antigen all over Europe, with
the largest series of patients being from Slovenia (LotricFurlan et al., 2006) (15 proven cases) and Scandinavia
(Bjoërsdorff et al., 1999) (10 patients). There were also
individual reports from other European countries. Nevertheless, seroepidemiologic surveys of HGA in the two mentioned countries have found prevalence of specific antibodies
of 0–2.9% in blood donors and 1.5–21% in tick-exposed
persons (Blanco and Oteo, 2002), which may suggest that
HGA is mostly a mild or even an asymptomatic infection.
In European medical literature, HGA diagnosis has most
frequently been based on serologic criteria only. However,
an important limitation for diagnostic purposes is that these
latter tests are often negative during the initial phase of the
217
disease: in the Slovenian cohort (Lotric-Furlan et al., 2006),
specific antibodies were detected at initial examination in only
25% of the positive patients, whereas PCR test results were
positive for 63% of the patients between days 2 and 15
following the onset of symptoms. These results show that PCR
on EDTA anticoagulated blood could be a much more efficient tool for diagnosing the initial stages of HGA as compared
to serology. Presence of morulae has been observed in only 3
European cases (García et al., 2006; Remy et al., 2003; van
Dobbenburgh et al., 1999). Morulae are usually detected in
blood smears only during an acute febrile episode; sensitivity
is at the highest during the first week of infection, but falsepositive results may also occur due to the observation of toxic
granulation or Dohle bodies (Dumler and Brouqui, 2004). This
procedure may therefore lack sensitivity and specificity.
Isolation of A. phagocytophilum in HL-60 cell cultures
inoculated with acute-phase blood has never been reported in
Europe (Blanco and Oteo, 2002). A combination of improved
diagnostic tools and of better awareness of the disease by
physicians and biologists has enabled the description of 3 new
cases of HGA in northeastern France over only 1 year. Specific
PCR assay on EDTA anticoagulated blood may be the
diagnostic test of choice during the first 2 weeks following the
onset of symptoms, allowing a faster diagnosis than serology.
HGA should be included in the differential diagnosis of
patients presenting febrile illness associated with bicytopenia,
elevated rates of liver enzymes, increased CRP values, and
whose medical history reveals recent exposure to ticks.
Acknowledgments
The authors are grateful to D. de Briel, I. Grawey, C.
Hess, P. Kieffer, M. Martinot, and E. Wurtz for providing us
with some of the clinical and biological data reported here.
References
Bjoërsdorff A, Berglund J, Kristiansen BE, Söderström C, Eliasson I (1999)
Varying clinical picture and course of human granulocytic ehrlichiosis.
Twelve Scandinavian cases of the new tick-borne zoonosis are
presented. Lakartidningen 96:4200–4204.
Blanco JR, Oteo JA (2002) Human granulocytic ehrlichiosis in Europe. Clin
Microbiol Infect 8:763–772.
Brouqui P, Salvo E, Dumler JS, Raoult D (2001) Diagnosis of granulocytic
ehrlichiosis in humans by immunofluorescence assay. Clin Diagn Lab
Immunol 8:199–202.
Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Blanco JR,
Caruso G, Cinco M, Fournier PE, Francavilla E, Jensenius M, Kazar J,
Laferl H, Lakos A, Lotric Furlan S, Maurin M, Oteo JA, Parola P, PerezEid C, Peter O, Postic D, Raoult D, Tellez A, Tselentis Y, Wilske B,
ESCMID Study Group on Coxiella, Anaplasma, Rickettsia and
Bartonella; European Network for Surveillance of Tick-Borne Diseases.
(2004) Guidelines for the diagnosis of tick-borne bacterial diseases in
Europe. Clin Microbiol Infect 10:1108–1132.
Comer JA, Nicholson WL, Olson JG, Childs JE (1999) Serologic testing for
human granulocytic ehrlichiosis at a national referral center. J Clin
Microbiol 37:558–564.
de la Fuente J, Torina A, Naranjo V, Caracappa S, Di Marco V, Alongi A,
Russo M, Maggio AR, Kocan KM (2005) Infection with Anaplasma
218
C. Koebel et al. / Diagnostic Microbiology and Infectious Disease 72 (2012) 214–218
phagocytophilum in a seronegative patient in Sicily, Italy: case report.
Ann Clin Microbiol Antimicrob 4:15.
Dumler JS, Madigan JE, Pusterla N, Bakken JS (2007) Ehrlichioses in
humans: epidemiology, clinical presentation, diagnosis, and treatment.
Clin Infect Dis 45:S45–51.
Dumler JS, Brouqui P (2004) Molecular diagnosis of human granulocytic
anaplasmosis. Expert Rev Mol Diagn 4:559–569.
Ferquel E, Garnier M, Marie J, Bernède-Bauduin C, Baranton G, Perez-Eid C, Postic
D (2006) Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae
members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis
endemicity in France. Appl Environ Microbiol 72:3074–3078.
García JC, Núñez MJ, Castro B, Fraile FJ, López A, Mella MC, Blanco A,
Sieira C, Loureiro E, Portillo A, Oteo JA (2006) Human anaplasmosis:
the first Spanish case confirmed by PCR. Ann NY Acad Sci
1078:545–547.
Hansmann Y, Gut JP, Remy V, Martinot M, Allard Witz M, Christmann D (2006)
Tick-borne encephalitis in eastern France. Scand J Infect Dis 38:520–526.
Hubálek Z (2009) Epidemiology of Lyme borreliosis. Curr Probl Dermatol
37:31–50.
Lotric-Furlan S, Rojko T, Petrovec M, Avsic-Zupanc T, Strle F (2006)
Epidemiological, clinical and laboratory characteristics of patients with
human granulocytic anaplasmosis in Slovenia. Wien Klin Wochenschr
118:708–713.
Oteo JA, Blanco JR, Martínez de Artola V, Ibarra V (2000) First report of
human granulocytic ehrlichiosis from southern Europe (Spain). Emerg
Infect Dis 6:430–432.
Petrovec M, Lotric-Furlan S, Avsic-Zupanc T, Strle F, Brouqui P, Roux V,
Dumler JS (1997) Human disease in Europe caused by a granulocytic
Ehrlichia species. J Clin Microbiol 35:1556–1559.
Remy V, Hansmann Y, De Martino S, Christmann D, Brouqui P (2003)
Human anaplasmosis presenting as atypical pneumonitis in France. Clin
Infect Dis 37:846–848.
Ruscio M, Cinco M (2003) Human granulocytic ehrlichiosis in Italy: first
report on two confirmed cases. Ann NY Acad Sci 990:350–352.
van Dobbenburgh A, van Dam AP, Fikrig E (1999) Human
granulocytic ehrlichiosis in Western Europe. N Engl J Med 340:
1214–1216.
Walder G, Fuchs D, Sarcletti M, Berek K, Falkensammer B, Huber K,
Petrovec M, Dierich MP, Würzner R (2006) Human granulocytic
anaplasmosis in Austria: epidemiological, clinical, and laboratory
findings in five consecutive patients from Tyrol, Austria. Int J Med
Microbiol 296(Suppl 40):297–301.