Serum Levels of Mannan-Binding Lectin in Patients with Crimean-Congo Hemorrhagic Fever

Abstract

Objective:

Mannan-binding lectin (MBL) is a central component of the innate immune response. Genetic variations in the MBL gene that reduce circulating levels and alter functional properties of the MBL are associated with susceptibility for many infectious diseases. Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by an arbovirus in the family Bunyaviridae. We have investigated serum MBL levels in CCHF patients and a possible association between circulating MBL concentrations and the severity of CCHF.

Materials and Methods:

Forty-seven patients found to have CCHF in Cumhuriyet University Hospital and 29 healthy controls were recruited for this study. There were no differences in terms of age and sex between the patients and the healthy controls (p = 0.64 and p = 0.484, respectively). This study was conducted between July 1 and August 31, 2007, in Cumhuriyet University Hospital in Sivas, a city located in the central Anatolian region of Turkey. Patients with CCHF were matched with healthy controls, and serum MBL levels were measured.

Results:

The median serum MBL level was significantly lower in the patient group than in the healthy control group (48.0 ng/mL [inter-quartile range (QR) 30.4–128.0] and 212.0 ng/mL [IQR: 115.8–524.0], respectively; p < 0.001). No significant difference was found between serum MBL levels in CCHF patients with severe and nonsevere form of the disease (p = 0.167).

Conclusion:

MBL levels were significantly lower in patients with CCHF than in healthy controls. There was no meaningful correlation between the serum MBL level and severity of CCHF disease. Low serum MBL level may be associated with the high consumption of MBL in CCHF infection and/or MBL gene polymorphism.

Introduction

C rimean-Congo hemorrhagic fever (CCHF) is a fatal viral infection. The disease is caused by CCHF virus that belongs to the genus Nairovirus of the family Bunyaviridae. CCHF was first described in the 1940s, when more than 200 human cases occurred in the Crimean peninsula of the former Soviet Union. The disease is now present in about 30 countries in Africa, Asia, and Europe. Humans become infected through tick bites, by crushing infected ticks, through contact with a patient with CCHF during the acute phase of infection, or by contact with blood or tissue from viremic livestock. The disease has the largest geographical range of the medically significant tick-borne viruses. Common clinical features are fever and hemorrhage. Despite increasing knowledge concerning hemorrhagic fever viruses, the pathogenesis of CCHF has not been well described (Whitehouse 2004, Ergonul 2006). The innate immune system provides nonspecific protection, and activates adaptive immune mechanisms against a variety of pathogens through specific signaling mechanisms (Medzhitov and Janeway 1997). The complement system is a major mediator of innate immune defense and contributes to many immune system functions, including inflammation, opsonization, and lysis. The complement system can be activated via three pathways (classical, lectin, and alternative) upon recognition of pathogen-associated molecular patterns.

The classical complement pathway is initiated by the binding of the C1 complex (C1q, r, and s) to bound antibody on pathogen surfaces. The alternative pathway is activated slowly and spontaneously by hydrolysis of the internal C3 thioester bond and is further triggered by contact with various proteins, lipids, and carbohydrate structures on microorganisms. The lectin pathway, the third arm of complement activation, is principally activated by mannan-binding lectin (MBL) that interacts with carbohydrate structures on a wide range of microorganisms, including bacteria, viruses, fungi, and parasites. The serum MBL level is variable in healthy population (Gupta et al. 2008). MBL is produced by the liver, and responds as an acute-phase reactant. Serum concentrations of MBL can increase during an acute phase response, but this response differs between individuals according to genotype (Thiel et al. 1992). Previous studies have shown that the genetic polymorphism of MBL was associated with susceptibility to human immunodeficiency virus (HIV), chronic hepatitis B virus (HBV), and chronic hepatitis C virus (HCV); MBL may also be one of the factors influencing the course of those infections (Garred et al. 1997, Yuen et al. 1999, Segat et al. 2007).

In the literature, there is no study related to serum levels of MBL in CCHF infection. We therefore sought to investigate serum MBL levels in CCHF patients and the association between circulating MBL concentrations and severity of CCHF.

Materials and Methods

This study was conducted between July 1 and August 31, 2007, in Cumhuriyet University Hospital in Sivas, a city located in the central Anatolia. Forty-seven patients were found to have CCHF infection and followed up at the Service of Infectious Diseases and Clinical Microbiology at Cumhuriyet University Hospital, and 29 healthy adults as controls were included in this study. The controls were volunteers from the Cumhuriyet University staff. The study protocol was approved by the Human Ethics Committee of Cumhuriyet University School of Medicine. Informed consent was obtained from all participants.

All the serum specimens were stored at −70°C until testing. Acute and convalescent phase serum samples were sent to the Virology Laboratory of Refik Saydam Hygiene Center in Ankara in Turkey for serological and virological analyses. Definitive diagnosis of CCHF infection was based upon typical clinical and epidemiological findings; by detection of CCHF virus–specific immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA); or by detection of genomic segments of the CCHF virus revealed by reverse transcription–polymerase chain reaction (RT-PCR) in either the acute or convalescent phase of the disease. In our study, all CCHF patients were classified into two groups in terms of disease severity (severe and nonsevere), according to the predictive factors for fatal outcome reported by Swanepoel and coworkers (1989). The presence of any one of the following findings defined severe disease: white blood cell count ≥10 × 10⁹ cells/L, platelet count ≤20 × 10⁹/L, aspartate aminotransferase level ≥200 U/L, alanine aminotransferase ≥150 U/L, activated partial thromboplastin time ≥60 s, or fibrinogen levels ≤110 mg/dL, when these occurred during the first 5 days of the disease. MBL serum levels were measured in serum samples from healthy control subjects and patients with CCHF using the human MBL (lectin assay) ELISA kit (Hycult Biotechnology b.v., Uden, The Netherlands). The kit has a minimum detection level of 0.41 ng/mL.

The Statistical Package for the Social Sciences (SPSS) version 13 for Windows (SPSS, Chicago, IL) was used for the statistical analysis. Results were expressed as mean ± standard deviation, median, and interquartile ranges. Data were analyzed with Student's t-test, Mann–Whitney U-test, and chi-square test, as appropriate. In all comparisons a p-value < 0.05 was considered to indicate statistical significance.

Results

A total of 47 patients with CCHF (20 men and 27 women; mean age, 46.94 ± 18.41 years) and 29 control subjects (10 men and 19 women; mean age, 42.5 ± 9.1 years) were recruited for this study. There were no significant differences in age and sex ratio between the two groups (p = 0.64 and p = 0.484, respectively). All of the CCHF patients had positive IgM and/or RT-PCR results for CCHF virus in blood samples (32 patients were IgM positive, 11 patients were IgM and PCR positive, and 4 patients were PCR positive). Forty-four of 47 patients were employed in animal husbandry. Twenty-seven of the patients had a history of tick bite. The clinical and laboratory characteristics of the patients are presented in Tables 1 and 2.

Table 1.

Some Clinical Characteristics of the Patients

Symptoms and signs	Patients with CCHF (n = 47)	%
Fever	28	60
Conctival hyperemia	24	51
Truncal hyperemia	14	30
Bleeding	10	21
Petechial skin lesions	9	19
Maculopapular rash	3	6
Impaired consciousness	3	6

CCHF, Crimean-Congo hemorrhagic fever.

Table 2.

Some Laboratory Findings of Patients with Crimean-Congo Hemorrhagic Fever

Laboratory findings	Median ± IQR (25–75), n = 47
Prothrombin time (s)	12.6 (11.3–15.7)
Platelet count (platelets/mm³)	47000 (27000–103000)
White blood cell (WBC/mm³)	2700 (1800–5600)
Alanine transferase level (U/L)	68.0 (39.0–133.2)
Aspartate transferase level (U/L)	200.0 (68.0–401.2)

IQR, inter-quartile range.

Twenty-four of the patients were assessed as having the clinically severe form of the disease, and 23 of the patients were assessed as having the nonsevere form according to the Swanepoel criteria (Swanepoel et al. 1989). No significant difference was found in serum MBL levels between patients with severe versus nonsevere forms of the disease (median, 152.0 ng/mL [IQR: 40.4–382.0] vs. 128.0 ng/mL [IQR: 34.4–312], respectively; p = 0.167). Ten of 47 patients died during the severe course of the disease (21%). There was no significant difference between the MBL levels in surviving or deceased CCHF patients (median, 50.4 ng/mL [inter-quartile range (QR): 28.0–120.0] vs. 42.4 ng/mL [IQR: 34.9–165.0], respectively; p = 0.547). There was therefore no evident relationship between disease progression and serum MBL levels. There was, however, a significant difference in serum MBL levels between the CCHF patients and controls (median, 48.0 ng/mL [IQR: 30.4–128.0] and 212.0 ng/mL [IQR: 115.8–524.0], respectively; p < 0.001). No difference in terms of mortality was found between patients receiving ribavirin treatment and those who did not receive this treatment (p = 0.230). Nevertheless, there was a significant difference between the serum MBL levels of the 17 patients receiving ribavirin treatment versus those who did not receive this treatment (median, 200 ng/mL [IQR: 74.4–400.0] and 77.6 ng/mL [IQR: 34.0–296.0], respectively; p = 0.017).

Discussion

The present study is the first investigation of serum MBL levels in patients with CCHF. After admission to hospital, we found that the serum MBL level was lower in CCHF patients than in controls. Of CCHF patients who had severe form of disease or not and who died or not, MBL levels were comparable. MBL is known to play a major role in activation of the complement; however, it is not yet known whether the complement pathway is involved in defense against CCHF infection. Our data suggest either that complement activation contributes to defense against CCHF or that MBL modulates CCHF pathogenesis through noncomplement pathways. One limitation of this investigation is that the number of CCHF patients studied was insufficient to demonstrate an association between severity of CCHF disease and serum MBL levels. Although there was a trend toward higher MBL levels in patients with more severe disease, the difference (p = 0.167) fell short of statistical significance. It is also possible that serum MBL levels during CCHF infection may be affected by polymorphisms in genes encoding MBL and/or complement activation pathways.

MBL is also known as mannan-binding protein or mannose-binding lectin (Presanis et al. 2003). MBL has several important innate immune functions, including initiation of the lectin complement pathway, opsonization of microbes for uptake by phagocytic cells, and direct neutralization of some viruses (Ji et al. 2005). MBL activates complement in an antibody-independent manner. It may also influence phagocytosis in the absence of complement activation through an interaction with one or more collectin receptors. Complexes of MBL bound to microorganisms can bind directly to phagocytic cells via collectin receptors (Takahashi and Ezekowitz 2005).

Research over the past decade has shown that MBL plays an important role in the first hours/days of the primary immune response to sugar-decorated pathogens. MBL deficiency arises primarily from three single-nucleotide polymorphisms in codons 52, 54, and 57 within exon 1 of the MBL-2 gene, leading to impaired assembly of the functional multimeric protein (Klein and Kilpatrick 2004, Garred 2008). These polymorphisms are associated with decreased MBL plasma concentrations and increased susceptibility to several infectious diseases (Eisen and Minchinton 2003, Turner 2003). The pattern of human MBL synthesis parallels to that of two well-characterized acute phase reactants: C reactive protein and serum amyloid A component. It has been shown that MBL levels can increase between 1.5- and 3-fold during the acute phase of infection, but this response is variable between individuals of different genotypes (Thiel et al. 1992). In our study, MBL levels of the patients with CCHF were significantly lower than in healthy controls, and reduced serum MBL levels may contribute to disease development. Reduced levels of MBL in CCHF patients may reflect genetic polymorphism and disease susceptibility.

In addition to complement interactions, in vitro studies have shown that MBL can modulate the production of cytokines in response to fungi, parasites, and bacteria (Chaka et al. 1997, Jack et al. 2001, Santos et al. 2001). MBL insufficiency is associated with several infectious diseases, including HBV, HCV, HIV, and other recurrent infections (Sumiya et al. 1991, Pastinen et al. 1998, Boniotto et al. 2000, Eisen and Minchinton 2003, Segat et al. 2007). In the case of HIV infection, MBL has been shown to bind to the viral gp120 polypeptide and prevent binding of the virus to its CD4 receptor (Ezekowitz et al. 1989). Boniotto and colleagues (2000) reported that the MBL polymorphism at position −550 influences the risk of HIV infection and AIDS progression.

Some studies have reported an association between reduced serum MBL levels and susceptibility to HBV and HCV infection; however, the literature is conflicting. Cheong et al. (2005) reported that there was no significant association between different MBL polymorphisms and either outcome following hepatitis B infection or the development of chronic liver disease. Kilpatrick et al. (2003) found no relationship between MBL levels and the progression of disease due to hepatitis C infection among European patients. Our study does not address progression because we have no data on patients who came into contact with CCHF-infected ticks but did not progress to clinical CCHF infection. In addition, there was no significant difference in serum MBL levels between the patients with the severe and nonsevere forms of the disease.

The occurrence of CCHF closely approximates to the known world distribution of Hyalomma spp. ticks, and exposure to these ticks is thought to be a major risk factor for contracting CCHF (Whitehouse 2004). The major group at-risk is farmers living in endemic areas. Most affected patients are employed in agriculture and/or animal husbandry (Ergonul 2006). Forty-four (93%) of our patients were employed in animal husbandry. Midilli et al. (2007) reported a history of tick bite in 60% of imported CCHF cases in Istanbul. The majority of our patients with CCHF also had a history of tick contact: 27 of 47 CCHF patients (57%) had a history of tick bite and 5 (11%) reported a history of removing ticks from livestock.

Ribavirin is the recommended antiviral agent for patients with CCHF, although its mechanism of action is not clear. In one of in vitro study, ribavirin was shown to inhibit viral replication, though some CCHF viral strains appeared more sensitive than others (Watts et al. 1989). It should be noted that the use of ribavirin to treat human CCHF has not been investigated in randomized clinical trials, and its efficacy has only been described in observational studies (Smego et al. 2004, Ergonul 2006). We prescribed ribavirin for 17 patients. There was no significant difference in terms of mortality between the patients receiving ribavirin and those who did not receive this treatment. However, it is possible that there was a difference in susceptibility between the subgroups, as MBL levels were significantly higher in patients receiving ribavirin than in untreated patients. No adverse events related to ribavirin therapy were noted among the CCHF patients in our study. All patients were given erythrocytes, fresh frozen plasma, or total blood preparations depending on their hemostasis.

Mean serum MBL levels were significantly lower in patients infected with CCHF virus than in healthy controls. Although the difference was not significant, patients who died from the disease had higher mean serum MBL levels than the surviving patients, although their levels were reduced compared with healthy controls. These results are consistent with the results of the study by Prohászka et al. (1997), who investigated serum MBL levels in patients with HIV. Prohászka et al. (1997) reported that MBL levels were lower in asymptomatic HIV-positive individuals than in HIV-negative controls, and patients with higher MBL levels also had significantly lower CD4 counts.

RT-PCR is the method of choice for the diagnosis of CCHF virus infection. The technique is highly specific, sensitive, and rapid (Drosten et al. 2003). The infection was diagnosed in four of our patients by RT-PCR. IgM antibodies can be detected efficiently by ELISA (Ergonul 2006), and specific IgM in patients with CCHF declines to undetectable levels by 4 months postinfection. The infection was diagnosed in 32 of our patients by IgM detection by ELISA. Both tests were used for the diagnosis of the infection in remaining 11 patients.

In conclusion, MBL insufficiency has been associated with many diseases, sometimes in relation to susceptibility, sometimes as a prognostic factor. For some conditions the data are inadequate to confirm an association; for others, conflicting data have been reported (Kilpatrick 2003). Serum MBL levels have been investigated in many studies on infectious diseases, including HIV (Garred et al. 1997), HBV (Yuen et al. 1999), HCV (Segat et al. 2007), and tuberculosis (Soborg et al. 2003). MBL levels have not previously been studied in CCHF; this is the first study that documents serum MBL levels in CCHF patients.

The role of the innate immune response against CCHFV is poorly understood (Andersson et al. 2008). We found that MBL levels were significantly lower in patients with CCHF than in healthy controls; it is possible that MBL insufficiency impairs clearance of CCHF virus by affecting the MBL-dependent complement pathway. MBL serum levels are profoundly reduced by polymorphisms in the coding region of the MBL gene (Madsen et al. 1995). The cause of reduced serum levels in our CCHF patients is unknown and may reflect MBL gene polymorphisms and/or reduced levels as a consequence of infection. Further studies will be required to elucidate whether low MBL levels increase susceptibility to CCHFV infection, or whether viral infection and disease causes a reduction in serum MBL levels.

Footnotes

Acknowledgments

This study was accepted as an oral poster in 14th Congress of Turkish Clinical Microbiology and Infectious Diseases (KLIMIK 2009), 25–29 March 2009, Atalya, Turkey (www.klimik2009.org). The authors thank Refik Saydam Hygiene Center of Ankara, Turkey, for testing the serum samples and also their colleagues at the Turkish Ministry of Health for their contribution.

Disclosure Statement

There are no conflict of interests to declare.

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