Persisting mixed cryoglobulinemia in Chikungunya infection.

Abstract

Chikungunya virus (CHIKV), an arbovirus, is responsible for a two-stage disabling disease, consisting of an acute febrile polyarthritis for the first 10 days, frequently followed by chronic rheumatisms, sometimes lasting for years. Up to now, the pathophysiology of the chronic stage has been elusive. Considering the existence of occasional peripheral vascular disorders and some unexpected seronegativity during the chronic stage of the disease, we hypothesized the role of cryoglobulins.
From April 2005 to May 2007, all travelers with suspected CHIKV infection were prospectively recorded in our hospital department. Demographic, clinical and laboratory findings (anti-CHIKV IgM and IgG, cryoglobulin) were registered at the first consultation or hospitalization and during follow-up.
Among the 66 travelers with clinical suspicion of CHIKV infection, 51 presented anti-CHIKV IgM. There were 45 positive with the serological assay tested at room temperature, and six more, which first tested negative when sera were kept at 4 degrees C until analysis, became positive after a 2-hour incubation of the sera at 37 degrees C. Forty-eight of the 51 CHIKV-seropositive patients were screened for cryoglobulinemia; 94% were positive at least once during their follow-up. Over 90% of the CHIKV-infected patients had concomitant arthralgias and cryoglobulinemia. Cryoglobulin prevalence and level drop with time as patients recover, spontaneously or after short-term corticotherapy. In some patients cryoglobulins remained positive after 1 year.
Prevalence of mixed cryoglobulinemia was high in CHIKV-infected travelers with long-lasting symptoms. No significant association between cryoglobulinemia and clinical manifestations could be evidenced. The exact prognostic value of cryoglobulin levels has yet to be determined. Responsibility of cryoglobulinemia was suspected in unexpected false negativity of serological assays at room temperature, leading us to recommend performing serology on pre-warmed sera.

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Persisting Mixed Cryoglobulinemia in Chikungunya
Infection
Manuela Oliver1, Marc Grandadam2, Catherine Marimoutou3, Christophe Rogier3, Elisabeth
Botelho-Nevers4, Hugues Tolou2, Jean-Luc Moalic1, Philippe Kraemer4, Marc Morillon5, Jean-Jacques
Morand6, Pierre Jeandel3, Philippe Parola7, Fabrice Simon5*
1Laboratoire de Biochimie, Ho ˆpital d’Instruction des Arme ´es Laveran, Marseille, France, 2Unite ´ de Virologie Tropicale, Institut de Me ´decine Tropicale du Service de Sante ´
des Arme ´es, Marseille, France, 3Comite ´ Recherche, HIA Laveran, Marseille, France, 4Service de Pathologie Infectieuse et Tropicale, Ho ˆpital d’Instruction des Arme ´es
Laveran, Marseille, France, 5Laboratoire de Biologie, HIA Laveran, Marseille, France, 6Service de Dermatologie, HIA Laveran, Marseille, France, 7Service des Maladies
Infectieuses et Tropicales, Centre Hospitalo-Universitaire Nord, Assistance Publique Ho ˆpitaux de Marseille, Marseille, France
Abstract
Background: Chikungunya virus (CHIKV), an arbovirus, is responsible for a two-stage disabling disease, consisting of an
acute febrile polyarthritis for the first 10 days, frequently followed by chronic rheumatisms, sometimes lasting for years. Up
to now, the pathophysiology of the chronic stage has been elusive. Considering the existence of occasional peripheral
vascular disorders and some unexpected seronegativity during the chronic stage of the disease, we hypothesized the role of
cryoglobulins.
Methods: From April 2005 to May 2007, all travelers with suspected CHIKV infection were prospectively recorded in our
hospital department. Demographic, clinical and laboratory findings (anti-CHIKV IgM and IgG, cryoglobulin) were registered
at the first consultation or hospitalization and during follow-up.
Results: Among the 66 travelers with clinical suspicion of CHIKV infection, 51 presented anti-CHIKV IgM. There were 45
positive with the serological assay tested at room temperature, and six more, which first tested negative when sera were
kept at 4uC until analysis, became positive after a 2-hour incubation of the sera at 37uC. Forty-eight of the 51 CHIKV-
seropositive patients were screened for cryoglobulinemia; 94% were positive at least once during their follow-up. Over 90%
of the CHIKV-infected patients had concomitant arthralgias and cryoglobulinemia. Cryoglobulin prevalence and level drop
with time as patients recover, spontaneously or after short-term corticotherapy. In some patients cryoglobulins remained
positive after 1 year.
Conclusion: Prevalence of mixed cryoglobulinemia was high in CHIKV-infected travelers with long-lasting symptoms. No
significant association between cryoglobulinemia and clinical manifestations could be evidenced. The exact prognostic
value of cryoglobulin levels has yet to be determined. Responsibility of cryoglobulinemia was suspected in unexpected false
negativity of serological assays at room temperature, leading us to recommend performing serology on pre-warmed sera.
Citation: Oliver M, Grandadam M, Marimoutou C, Rogier C, Botelho-Nevers E, et al. (2009) Persisting Mixed Cryoglobulinemia in Chikungunya Infection. PLoS
Negl Trop Dis 3(2): e374. doi:10.1371/journal.pntd.0000374
Editor: A. Desiree La Beaud, Case Western Reserve University School of Medicine, United States of America
Received May 6, 2008; Accepted January 6, 2009; Published February 3, 2009
Copyright: ? 2009 Oliver et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the French Service de Sante ´ des Arme ´es. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: simon-f@wanadoo.fr
Introduction
Chikungunya fever is an emerging arboviral disease character-
ized by a brief fever, headache and myalgias, occasional
evanescent rash, inflammatory polyarthralgias, arthritides or
tenosynovitis that can last for months to years [1–4]. Chikungunya
virus (CHIKV) was identified in the 1950s in Africa [2], and soon
after in Asia [5]. It can be responsible for major epidemics,
sometimes separated by silent periods [6]. From 2004 to 2006, a
giant CHIKV outbreak successively swept out Kenya and most
islands of western Indian Ocean [7]. In Re ´union Island, the
outbreak was explosive at the beginning of 2006 with a pick of
45,000 cases per week. Up to 2006 June 1st, about one third of the
770,000 residents had been infected [8]. Another huge outbreak
recently stroke India with 2 to 7 million estimated cases [9], and is
currently spreading to Southeastern Asia [10]. During this period,
Chikungunya fever was also identified in more than 1,000 travelers
returning from the epidemic areas to European countries [1,11,12]
and the USA [13,14]. CHIKV-infected travelers included viremic
patients who returned home to countries where competent vectors
are present, raising serious concern for the globalization of the
disease [7,15]. The Italian outbreak in August 2007 has
demonstrated the reality of this threat [16].
During the recent CHIKV outbreaks, previously described
clinical features [3,4] as well as the low rate of asymptomatic
infections were confirmed [7,17]. The disease was also responsible
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for unusual and unfrequent complications, including severe
newborn infections after peripartum mother-to-infant transmis-
sion, meningo-encephalitis, hepatitis, myocarditis, severe epider-
molysis [17,18] and lead to surmortality [8]. Transitory peripheral
vascular disorders (PVD), mostly Raynaud syndrome, were also
observed few weeks after the disease onset [1]. Considering
persistent arthralgias and occasional PVD, we hypothesized that
cryoglobulin could be involved in the pathophysiology of the
disease, as described for hepatitis C infection. In this infection high
level of mixed cryoglobulinemia is commonly detected in patients
with chronic viral replication and is strongly associated with liver
damage and peripheral neuropathies [19,20].
Methods
Patients
From April 2005 throughout May 2007, all patients with
possible imported CHIKV infection (recent travel in Indian
Ocean islands and presence or history of fever and/or arthralgias)
were prospectively recorded at Laveran Military Hospital in
Marseille, France. The criteria for confirmed cases were i)
presence of specific anti-CHIKV IgM and/or positive RT-PCR
and/or isolation of CHIKV from blood and/or specific anti-
CHIKV IgG, ii) recent clinical feature consistent with CHIKV
infection, iii) no other etiology identified. Demographic, clinical
and laboratory findings were registered for all patients at their first
consultation or hospitalization and during follow-up. For patients
seen more than 10 days after the onset of illness, early clinical
features were identified using a retrospective questionnaire. The
clinical status of each patient was actively monitored by the same
physician (FS) during consultations and/or by phone calls every 2–
3 months. The early stage is defined as the first 10 days of clinical
disease while second stage is defined as symptoms and signs
persisting more than 10 days after disease onset [1].
The same physician (FS) orally informed all patients about the
requirement of blood samples to diagnose the aetiology of their
polyarthralgia. The script for obtaining oral consent was accepted
by the Institutional Review Board at Laveran Hospital. All
patients gave their oral consent, which was noted in their
individual medical file. A single blood sample of less than 50 cc
was taken after consultation, when clinically required; no
supplementary blood samples were taken for research.
Laboratory Procedures
At each consultation, patients were tested for CHIKV serology
using in-house IgM-capture and IgG-sandwich enzyme-linked
immunosorbent assays [6]. Serologies were performed first on sera
kept at 4uC before analysis and second, on sera kept at 37uC until
analysis. IgG and IgM for West Nile, dengue and Rift Valley fever
viruses were also assayed. On acute sera, RT-PCR and isolation of
CHIKV in Vero-E6 cells were attempted [21]. Cryoglobulinemia
was screened using the following procedure: i) collection of 14 ml
of blood in pre-warmed tubes at the hospital’s biochemistry
laboratory, ii) clotting at 37uC for 3 hours, iii) centrifugation at
37uC (3,000 RPM for 10 minutes), iiii) freezing of the pellet at 4uC
for 10 days, to allow generation of cryoprecipitates. Purification,
characterization and quantification of cryoglobulins were per-
formed by the same operator (MO), as previously described [22].
Cryoglobulins were classified as type I for monoclonal component,
type II for one monoclonal component associated with polyclonal
immunoglobulins, type II–III for more than one monoclonal
component associated with polyclonal immunoglobulins, and type
III for polyclonal immunoglobulins [23,24]. Type III cryoglobu-
linemia was considered positive when level was higher than 5 mg/
L [22], whereas type II and type II–III cryoglobulins were
considered positive regardless of concentration level [22]. CHIKV
RNA in cryoprecipitates was searched using RT-PCR, as
described elsewhere [21].
Patients also underwent immunological tests: (i) determination
of C3 and C4 complement components using an immunonephele-
metric method (Dade Behring Paris France), (ii) determination of
total haemolytic complement using the total haemolytic comple-
ment kit (The Binding Site Saint Egreve France), (ii) search for
rheumatoid factors, by latex immunoagglutination (Biome ´rieux
Marcy l’Etoile France), (iv) detection of antinuclear antibodies by
indirect immunofluorescence (Eurobio Paris France). Serological
screening for hepatitis C virus (HCV) infection was systematically
performed using sandwich enzyme-linked immunosorbent assays
(Biorad Marne La Coquette France). After 6 months, blood testing
for asymptomatic patients was restricted to CHIKV serology and
detection of cryoglobulinemia.
Statistical Analysis
Chi 2, Kruskall-Wallis and exact Fisher’s, two-tailed probability
tests were used with a significant p value of 0.05 (Stata 9.0
Software, StataCorp College Station, Texas, USA).
Results
Patients and First Screening for CHIKV Infection
During the 25 month-study, 66 French patients with clinical
suspicion of CHIKV infection were prospectively included.
Among them, 51 presented with anti-CHIKV IgM (see below).
Fifteen patients remained seronegative for both anti-CHIKV IgM
and IgG. No patients had detectable CHIK virus in sera (RT PCR
and CHIK V isolation on Vero cell were negative).
Sex ratio (M/F) and median age in seropositive patients were
respectively 1.04 and 54 years (range: 21 y–78 y), versus 0.67 and
47 years (21 y–75 y) in seronegative patients (no statistical
difference). Six CHIKV-infected patients were lost to follow-up
after the first consultation. The 45 other patients have been
followed in our medical unit for a median duration of 14 months
Author Summary
Chikungunya virus is present in tropical Africa and Asia
and is transmitted by mosquito bites. The disease is
characterized by fever, headache, severe joint pain and
transient skin rash for about a week. Most patients
experience persisting joint pain and/or stiffness for months
to years. In routine practice, diagnosis is based upon
serology. Since 2004 there has been an ongoing giant
outbreak of Chikungunya fever in East Africa, the Indian
Ocean Islands, India and East Asia. In parallel, more than
1,000 travelers were diagnosed with imported Chikungu-
nya infection in most developed countries. Considering
the clinical features of our patients (joint pain), we
hypothesized that cryoglobulins could be involved in the
pathophysiology of the disease as observed in chronic
hepatitis C infection. Cryoglobulins, which are immuno-
globulins that precipitate when temperature is below
37uC, can induce rheumatic and vascular disorders. From
April 2005 through May 2007, we screened all patients
with possible imported Chikungunya infection for cryo-
globulins. They were present in over 90% of patients, and
possibly responsible for the unexpected false negativity of
serological assays. Cryoglobulin frequency and levels
decreased with time in recovering patients.
Cryoglobulinemia in Chikungunya Infection
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(range: 54 days–25 months), through 2 to 6 consultations per
patient; resulting in a total of 138 consultations for CHIKV
patients performed up to May 2007.
Table 1 summarizes the demographic, epidemiological and
clinical events of the 51 confirmed cases. Ninety-eight percent of
the seropositive cases suffered at least once with arthralgia, 71%
with tenosynovitis and 20% with transitory PVD. Thirteen
patients experienced at least one clinical relapse during follow-
up, i.e. became symptomatic again after at least one symptom free
month, mostly with subacute arthralgias in hands and feet.
Thirteen patients developed de novo transitory PVD, mainly in
fingers and sometimes in toes, within the second and third months
after the disease onset. No other aetiology (drug, auto-immune
disorder, Buerger disease, local trauma) than CHIKV was
identified for relapses or PVD. No morphological changes were
observed in the 3 patients for whom a digital capillaroscopy was
performed. PVD was not significantly associated with gender.
Conversely, tenosynovitis was significantly more frequent in
women, both on the day of consultation (p=0.01) or during the
previous month (p=0.04).
Table 1. Characteristics of 51 patients with Chikungunya
infection imported from Indian Ocean Islands between April
2005 and October 2006, to Laveran Military hospital, Marseille,
France.
Demographic and epidemiological data
Median age (ranges)54.0 years (21–78 y)
Sex ratio M/F 1.04 (26/25)
Chronic general or rheumatic disease 37
Year of contamination
2005 12
2006 39
Location of contamination
Reunion32
Comoros archipelago9
Mauritius4
Madagascar3
Seychelles1
Reunion or Mauritius1
Location of disease onset
Western Indian Ocean34
Mainland France 17
Mean duration of stay
Residents in Western Indian Ocean9 days
No residents 54 days
Hospitalization
During early (acute) stage4
During chronic stage8
Acute stage (within the first 10 days)
Fever (mean duration)47 (3.5 days)
Mucous manifestations
Gingival or nasal bleeding3
Conjunctivitis3
Mouth ulcers2
Peripheral arthralgias/ arthritis 50
Number of involved joint groups51
None1
Less than 1020
Ten or more 30
Hands and/or feet involvement48
Symmetry of joint involvement44
Periarticular oedema20
Axial involvement29
Tenosynovitis17
Rash21
Pruritis10
Nervous tunnel syndrome3
Peripheral vascular disorder3 (finger coldness)
Complication1 (acute myocarditis)
Chronic stage (after the 10thday)
Chronic joint pain and/or stiffness48
Hand and/or feet involvement47
Symmetry of joint involvement43
Exacerbation of pre-existing pains14
Axial involvement21
Tenosynovitis/tendonitis/tendon rupture34
Nervous tunnel syndrome17
Peripheral vascular disorder
Raynaud syndrome5
Erythermalgia3
Finger coldness4
Pulpar necrosis1
Short-term general corticotherapy26
Once14
Twice 8
Three times3
Four times1
This table reports the most frequent signs and symptoms, regardless of
duration.
Clinical characteristics of 39 of these patients have been described elsewhere
[1].
doi:10.1371/journal.pntd.0000374.t001
Table 1. Cont.
Figure 1. Left side: Red stage of Raynaud syndrome and
swelling of finger joints in a 36-year-old CHIKV-infected
woman 6 weeks after disease onset; right side: concomitant
cryoprecipitate in her blood.
doi:10.1371/journal.pntd.0000374.g001
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Cryglobulinemia Testing
Among the 51 CHIKV-infected patients, cryoglobulinemia was
screened for 48, i.e. 42 patients on first consultation and 6 later
during follow-up. Cryoglobulinemia screening was repeated 2 to 5
times for 38 patients during the study period. The median delay
between CHIKV acute symptomatology onset and first cryoglob-
ulinemia testing was 45 days (range: 5–640 d). On first
consultation, 37/42 patients (88%) were cryoglobulinemic. Among
the five patients without cryoglobulinemia: i) three symptomatic
patients respectively free of cryoglobulinemia on days 5, 16 and 36
after disease onset, developed cryoglobulinemia on days 27, 45
and 69, respectively; ii) one symptomatic patient free of
cryoglobulinemia on his first test at day 124 (a week after a
short-term corticotherapy) became positive with a type II on day
204; iii) one patient had been symptom-free for weeks and was not
cryoglobulinemic when tested on day 107 after disease onset.
Among the 6 patients first tested at the second consultation, 4
were cryoglobulinemic and two remained negative for cryoglob-
ulin on day 143 and day 355, respectively, while they were both
symptom-free. Finally, 94% of the 48 CHIKV-infected tested
patients were positive for cryoglobulinemia at least once during
their follow-up.
During the study period, 118 cryoglobulin assays were
performed, 83 were positive. There were 61% (51/83) type II,
30% (25/83) type II–III and 8% (7/83) type III. Monoclonal IgM-
k and IgM-l components were found in 56/83 cryoglobulins
(68%) and 48/83 (58%), respectively; polyclonal IgM components
in 83/83 cryoglobulins (100%). Monoclonal IgA was found in only
2/83 cryoglobulins. Thus, all cryoglobulins associated with
CHIKV infection were mixed cryoglobulins.
The cryoglobulin concentration could be determined for 79/83
cryoglobulinemias. The median cryoglobulin level was 8 mg.L21
Table 2. Cryoglobulin types and levels regarding delay after Chikungunya infection onset, at Laveran Military hospital, Marseille,
France.
Delay
No cryoglobulin
Prevalence (%)
Type IIaPrevalence (%)
[median level]
Type II–IIIbPrevalence (%)
[median level]
Type III Prevalence (%)
[median level]
All typescPrevalence (%)
[median level]
0–30 days2/16 (12.5%)10/16 (62.5%) [14.5 mg.L21]2/16 (12.5%) [2.7 mg.L21]2/16 (12.5%) [6.8 mg.L21] 14/16 (87.5%) [7.7 mg.L21]
31–90 days3/28 (10.7%)14/28 (50.0%) [10.1 mg.L21] 7/28 (25.0%) [25.2 mg.L21]4/28 (14.3%) [35.9 mg.L21] 25/28 (89.3%) [10.4 mg.L21]
91–180 days10/27 (37.0%)10/27 (37.0%) [6.54 mg.L21]7/27 (25.9%) [26.2 mg.L21]0/27 (0%) 17/27 (63.0%) [11.7 mg.L21]
181–747 days 20/47 (42.6%)17/47 (36.2%) [3.1 mg.L21]9/47 (19.1%) [4.1 mg.L21] 1/47 (2.1%) [21.6 mg.L21]27/47 (57.4%) [3.7 mg.L21]
ap=0.008.
bp=0.04.
cp=0.002 (Kruskall-Wallis test for distribution between the different delays).
doi:10.1371/journal.pntd.0000374.t002
Figure 2. Evolution of all types cryoglobulin levels over time in 51 Chikungunya-infected travelers, Laveran Military Hospital,
Marseille, France. The sample results are presented as follows: box represents the interquartile range, the included line figuring the median; lines
out of the box represent range with extreme outliers figured as points.
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(range: 3–165). No CHIKV was detected by RT-PCR in 24 tested
cryoprecipitates.
Cryoglobulinemia and serological assay for CHIKV
infection.
When performed on sera kept at 4uC before
analysis, detection of anti-CHIKV IgM was positive for 45 /66
patients. Among them, cryoglobulinemia was found in 40 patients
(89%). Moreover, cryoglobulins were detected in 11 (52%) of the
21 initially seronegative patients. For these 11 patients new ELISA
were conducted on sera kept at 37uC until analysis, to allow
dissolution of putative cryoprecipitates. Anti-CHIKV IgM were
found in 6 of these patients and anti CHIKV IgG in 4. Pre-
warmed sera were characterized by high levels of specific
antibodies and presence of cryoglobulin in 5 /6 cases. When
summarizing, a total of 51 patients were serologically confirmed
Table 3. Clinical and biological status of 51 patients with Chikungunya infection at successive follow-up delays, Laveran Military
hospital, Marseille, France (M: month).
M1M2 M3M5M7 M10 M13
Patients with available status 515047 47464431
Symptomatic patients 5047 4338 3228 18
Cryoglobulemic/all tested patients 13/1516/17 11/13 10/1511/189/13 10/19a
Cryoglobulinemic patients/symptomatic tested patients 12/14 16/17 10/128/118/135/98/13
Cryoglobulinemic patients/asymptomatic tested patients 1/1- 1/12/4 3/64/42/6
Patients with anti-CHIKV IgM /all tested patients 18/18 17/1712/1212/1512/165/12 2/18
Patients with both anti-CHIKV IgM & cryoglobulinemia/tested patients 13/15 16/1711/129/15 7/165/12 2/18b
Patients with anti-CHIKV IgG/tested patients12/18 16/1711/12 14/1514/1610/12 13/18
aone patient underwent two cryoglobulin tests, the one performed one week after general corticotherapy is not presented here.
bthese two patients remained symptomatic even after 15 months.
doi:10.1371/journal.pntd.0000374.t003
Figure 3. Individual follow up of a 62-year-old female patient with CHIKV infection. X-axis, month of follow-up; Y-axis, Cryoglobulin level
in mg.L-1.
doi:10.1371/journal.pntd.0000374.g003
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