Download Immune Parameters in Hemorrhagic Fever with Renal Syndrome

43(5):587-590,2002
CASE REPORT
Immune Parameters in Hemorrhagic Fever with Renal Syndrome during the Incubation
and Acute Disease: Case Report
Alemka Markotiæ, Alenka Gagro, Gorana Dašiæ, Ilija Kuzman1, Davorka Lukas2, Stuart Nichol3,
Thomas G. Ksiazek3, Ante Sabioncello, Oktavija Rode4, Sabina Rabatiæ, Dragan Dekaris
Department of Research and Development, Institute of Immunology; Departments of 1Acute Respiratory Infections
and 2Gastrointestinal Infectious Diseases, University Hospital for Infectious Diseases, Zagreb, Croatia; and
3
Department for Viral and Rickettsial Diseases, CDC, Atlanta, GA, USA; and 4Department of Clinical
Microbiology, University Hospital for Infectious Diseases, Zagreb, Croatia
We describe immune parameters in a Croatian soldier who presented with mild flu-like symptoms and interstitial inflammatory infiltrate in the lungs on an X-ray during the incubation phase of hemorrhagic fever with renal syndrome
(HFRS). Enzyme-linked immunosorbent assay (ELISA) IgM and polymerase chain reaction (PCR) were negative. Two
weeks later, he developed HFRS caused by the Puumala virus. We performed two-color immunofluorescence
cytometry with monoclonal antibodies identifying the activation markers on T cells. Serum samples were also examined by enzyme immunoassay (EIA) for the presence of interleukins IL-2 and IL-6 and their soluble receptors (sR). The
analysis of early and late activation markers during the period of incubation revealed a small increase in the percentage
of helper (CD4+CD25+) T cells and no significant increase in total activated (HLA-DR+TCR+) and cytotoxic
(CD8+CD71+) T cells as compared with healthy controls. In the serum, only the concentration of soluble IL-6 receptor
was increased. However, when the patient developed HFRS, all activation markers on T cells increased. Concentrations of sIL-2Ra and IL-6 remained increased two and six days after HFRS onset, respectively, whereas sIL-6R increased
six days after HFRS onset. IL-2 concentration did not change. Our case indicates that rapid, modern diagnostic tools are
necessary in the diagnosis of infectious diseases and their differential diagnosis. Immunological tests, which provide
information on the patient immune status and especially on early changes in immune parameters, may contribute to
the improvement of the diagnosis, prognosis, and therapy of HFRS.
Key words: Croatia; hemorrhagic fever with renal syndrome; interleukin-6; Puumala virus; receptors, interleukin-2; receptors, interleukin-6; T-Lymphocytes; war
Hemorrhagic fever with renal syndrome is a systemic infectious disease caused by hantaviruses (1).
Hemorrhagic fever with renal syndrome is endemic
in Croatia and the greatest outbreak of the disease
was recorded during 1995 (1-5). Recent research confirmed that Puumala and Dobrava viruses were the
main causative agents of hemorrhagic fever with renal syndrome in Croatia (4). Hemorrhagic fever with
renal syndrome encompasses a broad spectrum of
clinical presentations, ranging from inapparent or
mild illness to fulminant hemorrhagic processes (3,4).
Although hantaviruses predominantly affect kidneys,
mild to moderate pulmonary involvement is common
with Puumala virus infection (3,4,6). Several studies
also showed that alterations in the renal function and
blood pressure could be expected in patients who recovered from hemorrhagic fever with renal syndrome
(7,8). Immunoreactions have been suggested to play
important role in the pathogenesis of the disease, but
it has not been thoroughly investigated yet. Clinical
studies have implicated the role of proinflammatory
cytokines (9-11) and peripheral blood mononuclear
cell phenotype changes (12,13) in the pathogenesis
of hemorrhagic fever with renal syndrome. However,
there are no data on clinical, virological or immunological developments in patients with hemorrhagic
fever with renal syndrome during the incubation period. To the best of our knowledge, this is the first report describing clinical findings and parallel virological and immunological examinations during the incubation phase of hemorrhagic fever with renal syndrome.
Case Report
A 36-year-old Croatian soldier was admitted to
the Zagreb University Hospital for Infectious Diseases
in May 1995, during an outbreak of hemorrhagic fever with renal syndrome in Croatia. He developed fewww.cmj.hr 587
Markotiæ et al: Immune Parameters in HFRS
ver (39.5°C), headache, backache, visual impairment
(confirmed by ophthalmological examination), and
renal failure. Creatinine, aspartate aminotransferase
(AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine phosphokinase
(CPK) were increased. A serum sample was examined
by IgM and IgG enzyme-linked immunosorbent assay
(ELISA) test. Analysis of the RNA by polymerase chain
reaction (PCR) was carried out with primers targeting
the S RNA segment of Murinae- and Arvicolinaeborne hantaviruses. Puumala virus infection was confirmed. Two weeks earlier, several soldiers in the patient’s military unit presented with hemorrhagic fever
with renal syndrome after a contact with rodents in
the endemic region of hemorrhagic fever at the Mala
Kapela Mountain. At that time, the patient was in a
group of 40 soldiers who were free from symptoms of
hemorrhagic fever with renal syndrome but were examined by IgM ELISA test for evidence of inapparent
hantavirus infection. All of them were serologically
negative. At that time, the patient had mild flu-like
symptoms, and chest X-ray showed interstitial inflammatory infiltrate in the left lung. The results of ELISA
IgM, IgG, and PCR analysis for hantaviruses were
negative. ELISA IgM, IgG, and IgA tests for Mycoplasma pneumoniae and Coxiella burnetii (in disease
phases 1 and 2), IgM and IgG for Legionella pneumophila, and indirect immunofluorescent antibody (IFA)
IgM, IgG, and IgA assay for Chlamydia psittaci and
Chlamydia pneumoniae were also performed; all results were negative. After examination, the patient
stayed at home and had no further contact with rodents. The same immune parameters were measured
Croat Med J 2002;43:587-590
in the period of incubation (15 days before the onset)
and after the onset of hemorrhagic fever with renal
syndrome. Two-color immunofluorescence cytometry was performed with monoclonal antibodies identifying T cell activation markers (CD25, CD71, and
HLA-DR) (Fig. 1). Serum samples were also examined
with enzyme immunoassay for the presence of interleukins IL-2 and IL-6 and their soluble receptors
(sR) (Fig. 2). The analysis of early and late activation
markers during the period of incubation revealed a
small increase in the percentage of helper (CD4+CD
25+) T cells and no significant increase in total activated (HLA-DR+TCR+) and cytotoxic (CD8+CD71+)
T cells as compared with healthy controls (Fig. 1A). In
serum, only the concentration of soluble IL-6 receptor
was increased (Fig. 2). However, when the patient developed hemorrhagic fever with renal syndrome, all
activation markers on T cells increased (Fig. 1B). The
sIL-2Ra and IL-6 remained elevated two and six days
after hemorrhagic fever with renal syndrome onset;
sIL-6R increased in the second serum sample (Fig. 2),
whereas IL-2 showed no increase at all (data not
shown). Clinical and laboratory examinations performed three and four years after the hemorrhagic fever with renal syndrome showed no signs of chronic
disease (data not shown).
Discussion
We described immune parameters in a Croatian
soldier during the incubation of hemorrhagic fever
with renal syndrome and after the development of the
disease. He was infected with Puumala virus and developed moderate symptoms of the disease. It is not
Figure 1. T cell activation markers (CD25, CD71, and HLA-DR) in the Croatian soldier: A) on day 15 before the onset of hemorrhagic fever with renal syndrome, when he had flu-like symptoms and unilateral interstitial infiltrate confirmed by chest
X-ray; and B) two days after he had developed hemorrhagic fever with renal syndrome (Puumala virus infection). Normal
ranges for in-house healthy controls were the following: TCR+HLA-DR+ 4-24% and CD4+CD25+ 0.6-2.6%.
588
Several studies published over the last decade
showed that alteration in renal function and blood
pressure could be expected in patients who had recovered from hemorrhagic fever with renal syndrome
(7,8). However, four years after our patient had experienced hemorrhagic fever with renal syndrome, no
signs and symptoms of any chronic sequel that could
be linked to hemorrhagic fever with renal syndrome
were found. Still, it is difficult to make conclusions on
the basis of a single case.
We found significant differences between the
immune parameters measured in the incubation period of hemorrhagic fever with renal syndrome and
after the development of the disease. We have previously described increased expression of both early
and late T cell activation antigens, (CD25, CD71, and
HLA-DR) in patients with hemorrhagic fever with renal syndrome (13), but no such findings were observed in our patient during the incubation period. Increased sIL-2Ra, IL-6, and sIL-6R are characteristics of
the developed disease (our unpublished data); however, only sIL-6R concentration was increased in the
serum of our patient during incubation period. Huang
at al (12) found sIL-2Ra to correspond with the degree
of illness in patients infected with Hantaan virus, and
we observed a similar pattern for IL-6 and its receptor
in our patient. Other authors have also pointed to the
role of cytokines in the immunopathogenesis of hemorrhagic fever with renal syndrome (9-11). Recently,
sIL-6R-mediated signaling has been recognized as an
important intermediary in both the resolution of inflammation and support of transition between the
early, predominantly neutrophilic stage of an infection, and the more sustained mononuclear cell influx
(14). Further studies are necessary to clarify whether
IL-6 (pg/mL)
Il-6sR (ng/mL)
always easy to distinguish mild or moderate hemorrhagic fever with renal syndrome from other infectious diseases. Our patient was in a group of soldiers
tested for specific IgM to hantaviruses in search for
some inapparent cases of hemorrhagic fever with renal syndrome. They were all in the same military unit
where several soldiers suffered from hemorrhagic fever with renal syndrome, and they all spent some
time in the known endemic region of the Mala Kapela
Mountain. It is clear that the patient was in the incubation period of the hemorrhagic fever with renal syndrome when we first tested him. Pulmonary disorders
are common in Puumala virus infections, and this virus usually causes mild or moderate hemorrhagic fever with renal syndrome (3,4,6). We supposed that
his flu-like symptoms and unilateral interstitial infiltrate confirmed by chest X-ray two weeks before the
onset of the disease could be a result of Puumala virus
replication and consequent local inflammation of the
lungs. Although the pathogenesis of pulmonary infiltrates is not clear, direct viral cytolytic effect during
local replication, acute local inflammation with consequent leukocyte recruitment and infiltration, vascular permeability, and immune complexes have been
implicated in the pathogenesis of the lung alterations
(3). To the best of our knowledge, this is the first description of patient with hemorrhagic fever with renal
syndrome in the period of disease incubation.
Croat Med J 2002;43:587-590
IL-2sRa (ng/mL)
Markotiæ et al: Immune Parameters in HFRS
Day
Figure 2. The level of interleukin (IL)-2 soluble receptor alpha (sRa) (top), IL-6 (middle), and IL-6sR (bottom) in the patient’s serum on day 15 before the onset of hemorrhagic fever with renal syndrome, and 2 and 6 days after he had developed hemorrhagic fever with a renal syndrome
(Puumala virus infection). Dashed line – normal values for
healthy controls (according to the manufacturer: IL-2sRa –
2132 pg/mL; IL-6 – 10 pg/mL; IL-6sR – 46 ng/mL); arrow indicates the onset of hemorrhagic fever with renal syndrome.
the measurement of sIL-6R in the incubation period of
hemorrhagic fever with renal syndrome in target population could be of prognostic value for subsequent
development of the disease.
The presented case provides evidence that rapid,
modern diagnostic tools are necessary in the diagnosis of infectious diseases and their differential diagnosis. Modern immunologic tests, which provide information on the patient immune status and especially
on early changes in immune parameters, may contribute to the improvement of the diagnosis, progno589
Markotiæ et al: Immune Parameters in HFRS
sis, and therapy of hemorrhagic fever with renal syndrome.
Acknowledgment
The research was supported by the Croatian Ministry of Sciences and Technology grant No. 021001.
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Received: April 21, 2002
Accepted: July 30, 2002
Correspondence to:
Alemka Markotiæ
Cellular Immunology Unit
Department of Research and Development
Institute of Immunology
Rockefellerova 10
10000 Zagreb, Croatia
[email protected]