ARTICLE
Auteur(s) : Clara Larcher1, Elfriede
Daniel2, Elisabetta Pagani1, Klaus
Maier3, Valentina Pasquetto1, Maria Francesca
Mellina-Bares2, Edna Nemati4, Edoardo
Egarter-Vigl3, Pierfrancesco Zampieri2, Hartwig P
Huemer5
1Laboratory of Microbiology & Virology,
Azienda Sanitaria dell’Alto Adige, Bolzano, Italy
2Dept. Dermatology, Regional Hospital Franz Tappeiner,
Merano, Italy
3Dept. Histopathology, Central Hospital Bolzano,
Bolzano, Italy
4Dept. Physiology & Medical Physics, Innsbruck
Medical University, Innsbruck, Austria
5Dept. Hygiene, Microbiology & Social Medicine,
Innsbruck Medical University, Fritz-Pregl-Str.3, R.301, A-6020
Innsbruck, Austria
accepté le 11 Mars 2009
Orf-virus (Parapoxvirus ovis) causes contagious ecthyma in small
ruminants worldwide. Humans may become infected by contact with
small skin lesions. Therefore, most primary lesions in humans are
found on the hands, presenting as painless pustules that develop a
central necrosis. Although the orf-virus infection is self-limiting
in immunocompetent hosts, a differential diagnosis is important
because the skin lesions can resemble potentially life-threatening
zoonotic infections, including tularemia, cutaneous anthrax, or
erysipeloid [1].
Many studies have reported a broad spectrum of sequelae
associated with human orf-virus infection. The most commonly
reported symptoms are fever, lymphangitis, lymphadenopathy, and
secondary bacterial infection [2]. Others have reported erythema
multiforme, papulovesicular (widespread blistering) eruptions
(rare), bullous pemphigoid-like eruptions several weeks after an
orf infection, and mucous membrane pemphigoid lesions [3-5].
Orf-virus can infect animals repeatedly and an orf-virus infection
does not fully protect against a secondary infection. Thus, it can
severely compromise the immune system of the host [6].
Parapoxviruses have the most impressive spectrum of putative
immunoregulatory proteins, including an interferon resistance
protein, a viral orthologue of mammalian IL-10, and inhibitors of
the cytokines GM-CSF and IL-2. Orf-virus also encodes a virulence
protein homologous to mammalian vascular endothelial growth factor.
Orf-virus influences several different pathways in the host immune
and inflammatory responses; thus, its mechanism of action is very
complex and not fully understood [7].
Due to the lack of a reporting system, the frequency of
occurrence of zoonotic poxvirus infections in humans is not well
established. Many cases are not submitted to a hospital or
laboratory examination due to the benign and self limiting nature
of the skin lesions. The abandonment of smallpox vaccinations
in1977 may have rendered the population more vulnerable to
poxviruses and may have contributed to the numerous reports of
cowpox infections in young Europeans. There is some serological
cross-reactivity between the different genera of poxviruses.
However, it seems rather unlikely that the smallpox vaccine, an
orthopoxvirus, might have a cross-protective effect against
parapoxviruses. A previous parapoxvirus infection does not
even fully protect against a second infection [6]. Jenner noted
this based on empirical evidence in the 18th century and
warned against the use of “spurious pox” (i.e. parapoxvirus)
vaccinations, instead of the cowpoxvirus.
Case report
A 35-year-old man, a veterinarian by profession, presented at the
dermatology outpatient clinic with a pruritic rash that had
developed within the previous five days. He reported that, for the
past two weeks, he had experienced headache, ulcerous lesions on
the lips, and within the past day, joint pain in the right wrist
and the left great toe. Upon clinical examination, umbilicated
vesicles of approximately 7 mm diameter were observed on the
face, neck, head, arms, and symmetrically on both knees (figure 1). The lymph nodes
of the right elbow were palpable. There was some swelling of the
eye lids, but no fever or signs of meningitis.
Two and a half weeks previously, the patient had examined an
anorectic sheep and sustained a slight abrasion on the right hand
from the teeth of the sheep. The sheep showed no lesions on the
mouth or teats; however, some animals in that area had had “scabby
mouth” disease. Three days later, a pustular lesion and associated
perifocal swelling had appeared on the patient’s thumb, supporting
the diagnosis of orf-virus. The patient treated this primary lesion
with birch tar and ichthyol (ammonium bituminosulfonate) as a self
medication. The patient then removed the crust, resulting in some
bleeding and followed by a skin redness of the forearm that
extended up to the elbow. Lymphangitis developed and was regarded
as bacterial superinfection; in contrast to lymphadenopathy,
lymphangitis is not a typical symptom of orf infection. The patient
was treated with amoxicillin-clavulanic acid for six days for
suspected erysipelas; after 3 days of treatment with no response,
the initial standard dose was increased to 6 g/d.
During that time, there had been an outbreak of chickenpox in
the patient’s village, but he reported that he had had chickenpox
during childhood. Furthermore, the skin lesions were three-times
larger than those usually associated with VZV. In addition, the
patient reported that he had had contact with individuals who had
been exposed to a measles outbreak in nearby Austria [8]; measles
had also been observed in the north of Italy [9]. Unfortunately,
serological data for the exposed contacts were not available.
The patient’s past medical history indicated no evidence of an
atopic skin diathesis and listed no known causes of
immunosuppression, including lymphoma, tumors, HIV, or other immune
disorders. There was no evidence of drug-induced immunosuppression,
a condition that has previously been suggested to play a role in
orthopoxvirus infections [10]. The patient had not been vaccinated
against measles, varicella, or smallpox viruses.
Two skin biopsies were performed for microbiological diagnosis
and histopathology. One swab was performed for bacterial culture,
to exclude anthrax. Two serum samples were drawn within two weeks
for serological examination.
Laboratory findings
Clostridium perfringens was cultured from a swab that the patient
had taken himself from the primary pustule, but was regarded as a
colonizer. Swabs from the generalized rash excluded anthrax but
remained otherwise uninformative. Attempts to grow parapoxvirus in
cell culture were unsuccessful. Vesicle homogenates were generated
in a microtube cell grinder and spotted onto slides. Samples were
analysed by electron microscopy using a negative stain technique
with phosphotungstic acid as the contrasting agent. Analysis of the
electron micrographs showed no parapoxvirus particles.
PCR was performed to detect parapoxvirus DNA in the skin biopsy.
The biopsy DNA was digested with proteinase K, extracted with the
Qiagen DNA Mini Kit, and amplified with the Platinum Blue PCR
Supermix (Invitrogen) according to the semi-nested amplification
protocol described by Inoshima et al. [11]. The PCR products were
analysed with an ABI-3130 DNA sequencer and reference sequences
were aligned with Proseq2.91. Homology trees were generated by the
neighbour-joining method using Jukes and Cantor’s calculation of
genetic distance (figure
2). Interestingly, the DNA sequencing analysis showed that
the sequence from the parapoxvirus (IT08PradStJ) detected in the
patient from the region of Prad, Stilfser Joch in the far north of
Italy was most similar to that of the orf-viruses found in Finland.
Contamination of the PCR can be excluded because the strain used as
a positive control originated from Germany (RVB065-Burghessler) and
had a clearly different DNA sequence (figure 2) The B2L gene
sequences of strains IT08PradStJ and RVB065-Burghessler were
deposited in EMBL/Genbank under the submission numbers FM178392 and
FM178391, respectively. A nested PCR for VZV was performed
using an established protocol [12]. No VZV-specific amplification
products were obtained from biopsy material.
Sera were drawn at three and five weeks after the initial
incident and were analysed by two different methods. Antibodies
against ortho- and parapoxvirus were detected by indirect
immunofluorescence performed on RK13 rabbit kidney cells that had
been infected with the vaccinia virus strain WR or the orf-virus
control strain (figure
3). A FITC-labelled anti-human IgG Fab-antibody (Sigma
Co.) was used as secondary antibody. Only a weak antibody response
(most likely a cross-reaction) against orthopoxvirus was detected
(< 1:50); however, antibodies against orf-virus were detected
and serum sample titres were 1:400 at 3 weeks and 1:800 at 5 weeks
after the initial incident. This titre was considerably higher when
compared to other reported Orf-virus infections. Most previous
studies found antibody responses to be rather modest, due to
immuno-regulatory phenomena, especially in the natural hosts; this
might explain the propensity for this virus to re-infect the same
host.
Antibodies against VZV, measles, HSV1/2, and EBV were detected
with a commercial EIA system (Enzygnost, Dade-Behring).
Interestingly, there was a three-fold increase in specific IgG
antibodies to VZV in the two serum samples. Titres against other
herpes viruses, including EBV and HSV, were unremarkable. However,
there was an unusually high EIA IgG titre against measles virus
observed in both samples. Specific IgM antibodies were not
found.
Histopathology of a vesicle/skin biopsy showed an inflammatory
infiltrate that was restricted to the cutis. Deeper parts of the
dermis were not affected. The infiltrate was mixed
lympho-granulocytic with a perivascular location, consistent with
allergic vasculitis (figure 4).
Discussion
Support for a diagnosis of varicella (with superinfection by
orf-virus)
To support the diagnosis of varicella, we found rising antibody
titres, a possibility of exposure in the village, and itching
lesions. In contrast, orf-pustules are rarely reported to be
pruritic [1]. On the other hand, the lesions were too large to be
chickenpox and we did not detect VZV by PCR. This suggests rapid
clearance of the virus by a pre-existing immune response.
A primary infection seems unlikely due to the case history of
childhood chickenpox and the presence of specific IgG and absence
of IgM antibodies in the first serum sample. Re-infections with
varicella occur rather frequently [13]. The increase in antibody
titres in immune subjects after re-exposure to VZV is an
established phenomenon [14]. Adult immigrants whose primary
varicella infections occurred in Africa or Asia showed asymptomatic
superinfections with different strains and establishment of
co-latency. One third experienced reactivation of a strain
molecularly distinct from those normally found in Africa or Asia
[15]. Bacterial superinfections are frequent in chickenpox, but a
parapoxvirus superinfection has not been reported to date. Although
poxviruses have a tendency to infect pre-injured skin, as in eczema
vaccinatum, it is not certain whether damage to the skin
microvascular system by VZV would also favour the extravasation and
haematogenous spread of orf-virus.
Support for an additional recent case
of measles
We observed unusually high antibody titres against the measles
virus in both serum samples. This indicated a recent boost in the
specific IgG-titres and possibly temporary immunosuppression by
measles virus. Secondary infections and subclinical measles
infections are not uncommon. A secondary immune response
against measles in a vaccinated population, presumably due to field
exposure, was observed in approximately 5% of the population in
Luxemburg. The waning of the immune response years after
vaccination is an important factor in subclinical measles
infections [16]. In another study, subclinical measles occurred in
a surprisingly high percentage of vaccinated individuals. The
frequency was inversely related to pre-exposure antibody titres and
the subclinical cases had an average of 45-fold increases in titres
that remained elevated for at least six months [17]. This would fit
the time frame of a potential measles exposure in our patient.
Though he had not been vaccinated, our patient could have
contracted a wild type measles virus infection during childhood,
and that might explain a high titre in response to a subclinical
infection contracted by possible exposure to measles in Austria
within 6 months of the present study.
Support for an immunological origin
of the pustules
Firstly, the histopathological findings were compatible with
allergic vasculitis rather than VZV, comparable to inflammation
following insect stings. We detected orf-virus only by PCR and not
by electron microscopy or cell culture; thus, we presume that
either there were very small amounts of virus present, or they were
sequestered in inactivated immune-complexes. This phenomenon would
be consistent with the findings described for HSV-associated
erythema multiforme, where no intact virus could be detected in the
skin lesions, though viral DNA material was transported to the skin
[18]. In support of an immune-complex sequestration, the joint
symptoms reported by our patient were compatible with reactive
arthritis and resolved rather quickly.
Suggestions of the involvement of an immune
mediated post-infectious syndrome
To explain the unusual lesions, it is possible that the
parapoxvirus infection was associated with a post-infectious
syndrome known as Sweet’s syndrome (SS). Also called acute,
febrile, neutrophilic dermatosis, SS is an inflammatory disorder
that can affect many organ systems [19]. Our patient showed some,
but not all of the symptoms of SS. SS is characterized by pyrexia
(not present in our patient), painful erythematous plaques, and
aseptic neutrophilic infiltrations that spare the epidermis. Signs
of vasculitis are usually absent in SS. In addition, unlike SS, the
lesions of our patient were not painful, but rather itching and the
mixed lymphocytic infiltrates were mostly perivascular. Common
complications of SS include arthralgias/arthritis and
conjunctivitis; both were present in our patient. However, our
patient also had oral ulcers, which seem to be uncommon in
classical SS. Streptococcal infections of the upper respiratory
tract have been especially implicated in initiating SS. Our patient
most likely had a streptococcal skin infection (lymphangitis,
erysipelas) and was exposed to measles and VZV. One third of
patients with SS experience a recurrence of SS. Although our
patient had no history of previous symptoms of SS, we could not
rule out a genetic predisposition. In addition, cytokines play an
important etiological role in SS [19] and parapoxviruses have an
important influence on various cytokine pathways [7]. This is also
reflected in the proliferative character of orf lesions compared to
the more ulcerative lesions caused by orthopoxviruses.
Possible immunoregulatory contributions of orf-virus
The orf-virus has a complex involvement with different pathways of
the immune and inflammatory responses; thus, it is not exclusively
immunosuppressive [7]. Orf-virus has been shown as a potent immune
stimulator in heterologous hosts. In mice, inactivated viral
particles provided protection against unrelated viral agents,
including hepatitis B and HSV [20]. Accordingly, the orf-virus
infection per se could have mitigated the outcome of a concomitant
VZV infection in our patient, suppressing it to a subclinical
level. Orf-virus also mediated immune stimulation in pigs. This
provided evidence for a viral component functioning as a
superantigen capable of stimulating immune cells independent of
their specific antigen receptors [21].
Similar mechanisms, albeit less potent, are observed in normal
immune responses. Many antigens have been shown to activate
“bystander cells”. A tetanus toxoid boost was associated with
an increase in anti-tetanus IgG and increases in other IgG against
unrelated viral antigens. Thus, repeated, non-specific polyclonal
activation of memory B cells might offer a natural means for
maintaining serological memory for long periods of time [22].
Therefore, one could speculate that the strong stimulation of the
immune system by the orf-virus on its own could increase antibody
titres against unrelated antigens (like VZV) even in the absence of
the antigen.
Acknowledgements
The work was supported by the Austrian Federal Health Ministery
(BMGF) as a contribution to the Austrian smallpox emergency
preparedness plan. (Pockenalarmplan). There is no conflict of
interest of any of the authors.
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