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Low serum levels of CCL5 are associated with longer duration of viral shedding in norovirus infection
Journal of Clinical Virology 69 (2015) 133–137
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Low serum levels of CCL5 are associated with longer duration of viral shedding in norovirus infection Lars Gustavsson ∗ , Susann Skovbjerg, Magnus Lindh, Johan Westin, Lars-Magnus Andersson Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Diagnosvägen 21, Gothenburg SE-416 50, Sweden
a r t i c l e
i n f o
Article history: Received 5 March 2015 Received in revised form 12 June 2015 Accepted 15 June 2015 Keywords: Norovirus Cytokines CCL5 Viral shedding Viral load
a b s t r a c t Background: The mechanisms that determine the duration of fecal shedding of norovirus in humans have not been described in detail. Objectives: We investigated serum inflammatory mediator levels in relation to the duration of viral shedding in norovirus infection. Study design: A prospective cohort study of patients hospitalized with acute norovirus genogroup II infection. Rectal swab samples were obtained at inclusion and day 7, 14, 21 and 28. Serum levels of 42 inflammatory mediators were determined with a Luminex-based cytokine assay. Sera from 20 healthy blood donors served as controls. Results: Altogether, 28 patients (54% women, median age 83 years, median duration of symptoms 3 days) were included. Twelve subjects cleared the virus within 14 days and 16 were norovirus-RNA positive for >21 days, constituting the two study groups (“rapid” vs. “slow” clearance). Individuals with norovirus infection had higher levels of IL-18, CXCL9, CXCL10, soluble IL-2 receptor and macrophage migration inhibitory factor (MIF), compared to controls (p < 0.05), with the highest median concentrations in the slow clearance group. In contrast, CCL5 levels were lower in the slow compared to the rapid clearance group (median 54 vs. 134 ng/mL, p < 0.05), and lower in norovirus-infected patients than in controls. Conclusion: Low levels of CCL5 were associated with longer duration of viral shedding, suggesting that CCL5 may influence the clearance of norovirus. © 2015 Elsevier B.V. All rights reserved.
1. Background Norovirus is the most common cause of viral gastroenteritis [1,2] and can be detected in stool for several weeks following symptomatic infection [3]. The duration of shedding varies among individuals [4], but the factors behind this variation are not well
Abbreviations: BLAST, basic local alignment search tool; CCL, chemokine (C Cmotif) ligand; Ct, cycle threshold; CXCL, chemokine (C X C-motif) ligand; IL, interleukin; MIF, macrophage migration inhibitory factor; NoV, norovirus; NoV GII, norovirus genogroup II; PCR, polymerase chain reaction; qPCR, real-time PCR; sIL2R␣, soluble interleukin-2 receptor ␣. ∗ Corresponding author. E-mail addresses: [email protected] (L. Gustavsson), [email protected] (S. Skovbjerg), [email protected] (M. Lindh), [email protected] (J. Westin), [email protected] (L.-M. Andersson). http://dx.doi.org/10.1016/j.jcv.2015.06.088 1386-6532/© 2015 Elsevier B.V. All rights reserved.
understood. Longer duration of shedding is common in infants [5] and immunocompromised patients [6], suggesting that immune mechanisms are involved. In animal models, innate immunity is responsible for initial control of norovirus infection [7], whereas clearance of virus involves both humoral and cell-mediated immune mechanisms [8]. The understanding of the human immune response to norovirus infection is more limited. In other viral infections, studies of systemic cytokine levels have provided insight into how variations in the immune response impact the course of disease [9–11].
2. Objective The aim of this study was to examine the relation between cytokine levels and viral clearance in patients with communityonset norovirus genogroup II gastroenteritis.
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Fig. 1. Overview of the study participants and the basis for the formation of the two study groups with rapid (<14 days) vs. slow (>21 days) clearance of norovirus following acute infection. GII, genogroup II. Inclusion criteria were: age > 18 years, vomiting ≥ 2 times/24 h and/or ≥3 loose stools/24 h for ≤5 days. * 20 patients with rotavirus, 4 with adenovirus, 3 with norovirus GI, 1 with sapovirus.
3. Study design Adult patients admitted with suspected gastroenteritis were screened for inclusion in a prospective cohort study at the Department of Infectious Diseases of a 2000-bed teaching hospital in Gothenburg, Sweden, during three consecutive years (2010–2012). An overview of the study cohort and inclusion criteria is presented in Fig. 1. Age and sex; date of first symptom and symptom resolution; date of admission and discharge; gastroenteritis symptoms,
summarized as Vesikari scores [12]; and Charlson co-morbidity index scores [13] were recorded. Rectal swab samples were obtained at enrollment, and participants were asked to provide follow-up samples on day 7, 14, 21 and 28. Samples were collected in a standardized manner with a flocked swab (Copan Regular Flocked Swab 502CS01; Copan Italia Spa, Brescia, Italy). Samples were stored at −80 ◦ C until analysis, if not processed immediately. Serum samples were drawn on enrollment, and stored at −80 ◦ C until analysis. Rectal swab samples for
Table 1 Characteristics of patients with <14 days (rapid clearance) and >21 days (slow clearance) duration of viral shedding following norovirus infection.
Age, years Women, n (%) Charlson score (co-morbidity) Vesikari score (symptom severity) Disease duration at inclusion, days Fever, n (%) With symptomatic contacts, n (%) CRP (mg/L) Venous lactate (mmol/L) Cycle threshold, day 0 Cycle threshold, day 7 a b
Median (interquartile range). n = 5.
All patients n = 28
Rapid clearance n = 12
Slow clearance n = 16
p-value
83 (51–86)a 15 (54) 1 (0–1) 11 (9–12) 3 (2–4) 6 (21) 17 (61) 18 (7–28) 1.9 (1.5–2.8) 24 (19–28) 27 (25–32)
72 (34–86) 10 (83) 0 (0–1) 10 (9–12) 3 (1–5) 1 (8) 6 (50) 9 (3–22) 1.6 (1.4–2.9) 27 (22–33) 34 (33–39)b
84 (71–89) 5 (31) 1 (0.25–1) 12 (10–13) 3 (2–4) 5 (31) 11 (69) 26 (8–28) 2.0 (1.6–2.8) 21 (19–25) 27 (24–31)
0.21 0.006 0.11 0.12 0.74 0.20 0.32 0.14 0.51 0.03 0.01
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Fig. 2. Serum concentrations of interleukin-18 (IL-18), soluble interleukin-2 receptor (sIL-2R␣), macrophage migration inhibitory factor (MIF), chemokine (C X C-motif) ligand 9 (CXCL9), CXCL10, chemokine (C C motif) ligand 27 (CCL27) and CCL5, in patients admitted with norovirus genogroup II infection. Patients were grouped according to duration of viral shedding: rapid clearance, duration of shedding <14 days (n = 12); slow clearance, duration of shedding >21 days (n = 16). Sera from healthy blood donors (n = 9 for IL-18, IL-2 receptor ␣, CXCL9 and CCL27, n = 20 for CXCL10 and CCL5) were used as control. Horizontal lines denote median values. Stars denote significance level: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.
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bacterial culture were obtained on admission according to clinical routine. An in-house real-time reverse transcriptase qPCR procedure was used for detection of norovirus (NoV) genogroup II (GII), as described previously [14]. Viral genotype was determined by sequencing of approximately 350 base DNA fragments, spanning the polymerase–capsid junction. Cytokines in serum were measured by multiplex bead-based assays (Bio-Plex Pro Human Cytokine 21-Plex and 27-Plex Panels, Bio-Rad Laboratories, Hercules, CA, USA) using xMAP technology (Luminex, Austin, TX, USA), according to the manufacturer’s instructions (for a complete list of mediators, see Supplementary data). De-identified sera from healthy blood donors (aged 18–65 years), frozen at −80 ◦ C, were used as controls (n = 20). Individual age or gender data for the control sera was not available. IBM SPSS statistics 20 software was used for statistical analyses. Proportions were compared with 2 -test and continuous variables with the Mann–Whitney U test. Correlations were investigated using linear regression.
4. Results In total, we included 93 patients with NoV GII infection. Duration of shedding could be determined in 28 participants without immune deficiency (<7 days in 7 patients, 7–14 days in 5 patients, >21 days in 7 patients and >28 days in 9 patients). The twelve patients with duration of shedding <14 days constituted the rapid clearance group, and the 16 patients with duration of shedding >21 days the slow clearance group. Their characteristics are presented in Table 1. The difference in sex distribution did not remain significant when sex was adjusted for age by stratified analysis. No nursing home residents were included. The 65 excluded NoV GII patients were not different in terms of chronic co-morbidity and general health status. The median cycle threshold (Ct) value on enrollment was significantly lower in the slow clearance group, indicating a higher average viral load. Nucleotide sequences for genotyping could be obtained from 23 of the samples. Of these, 20 (87%) belonged to genotype GII.4. Of the 42 inflammatory mediators included in the assay, seven were detected in serum. Five (IL-18, sIL-2R␣, MIF, CXCL9, CXL10) were increased in the norovirus patients compared to controls, with a trend towards more pronounced elevations in the slow clearance group (Fig. 2A–E). The levels of CCL27 were equally high in patients and controls (Fig. 2F). One chemokine (CCL5) was decreased in patients compared to controls, with significantly lower levels in the slow clearance group (p < 0.05, Fig. 2G). Except for CCL27, we did not find any correlations between cytokine levels and age (for detailed results, see Supplementary data). There was no significant correlation between initial Ct value and CCL5 (R2 = 0.09, p = 0.13), but a low Ct value appeared to be related to longer disease duration prior to sampling (R2 = 0.13, p = 0.06).
5. Discussion In this longitudinal study of elderly hospitalized patients, slow clearance of norovirus infection was associated with low serum levels of CCL5 during acute gastroenteritis. In general, levels of lymphocyte stimulating mediators were increased in norovirus patients compared to controls (except for CCL27, a skin-specific mediator [15]). CXCL9 and CXCL10, IL-18, soluble IL-2 receptor ␣ and MIF are all related to a Th1-type response [16–20], and we found no indications that long duration of shedding would be related to a lack of antiviral immune reaction.
Low levels of CCL5, however, were associated with slow clearance. The serum level of CCL5 has been reported to mirror the activity of CD8+ T-cells [21,22] and CCL5 to sustain the cytotoxic response in prolonged viral infections [23]. Therefore, a possible explanation for our finding is that slow clearance may be associated with impaired cytotoxic T-cell activity. Cytotoxic CD8+ T-cells are critical for the clearance of norovirus from the intestine in animal models [24], and CD8+ T-cells have been found in large numbers in duodenal biopsies from norovirus-infected patients [25]. Restricting the analysis to patients with GII.4 infection did not alter the results (data not shown), and the observed differences could not be explained by major viral heterogeneity. An alternative explanation for the lower CCL5 levels in the slow clearance group, where median Ct value was lower, indicating higher viral load, is that CCL5 secretion may be suppressed by norovirus. Such a mechanism has been demonstrated for murine norovirus [26]. This interpretation is partly contradicted by the lack of a direct correlation between initial Ct value and CCL5. Ct values do not directly represent viral load, however, and associations might be overlooked. Moreover, initial Ct value was correlated to duration of symptoms and samples obtained early during the course of disease may be less representative of peak viral excretion. Thus, our results suggest that clearance of norovirus may be dependent of a strong CD8+ T-cell response, although this finding warrants further investigation. Since the included patients were elderly, the findings may not be applicable to other age groups. Also, the control sera were not matched for age, which makes interpretation of the results difficult. Although we did not find any relevant correlations between cytokine levels and age, old age may influence cytokine response. However, chemokine levels, including CCL5, are reported to increase with age [27,28]. This makes old age a less likely explanation for the low levels of CCL5 observed in patients with long duration of shedding in our study. The sample size of our cohort was too limited to allow for multivariate analysis. We used rectal swab samples instead of stool samples. There is now mounting evidence that swab samples are reliable for the diagnosis of gastroenteritis despite the lower content of virus [29,30]. Serum cytokine concentrations do not necessarily mirror the cytokine milieu in infected tissue. Cytokines have multiple and overlapping effects in vivo, and the interpretation of findings from non-experimental settings should be made cautiously. In conclusion, in this cohort study of hospitalized patients with norovirus GII gastroenteritis, we found that low serum levels of CCL5 were associated with a longer duration of viral shedding, indicating that CCL5 is involved in the clearance of norovirus in the elderly. This preliminary finding needs to be corroborated in further studies on patients of all ages, with focus on CCL5 expression in different body compartments, and its relation to other immune mechanisms, during norovirus infection. Funding Grants from the Regional Research Funds for Western Sweden (VGFOUREG 231011, 314211, 482001) and the Swedish Society of Medicine (SLS 249951). Competing interests None declared. Ethical approval Approved by the Regional Ethical Review Board in Gothenburg, Sweden.
L. Gustavsson et al. / Journal of Clinical Virology 69 (2015) 133–137
Acknowledgements The authors would like to thank Anna Rosnäs, Helen Christiansen, Ingela Kinell and Maria Andersson for expert technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2015.06.088 References [1] C.F. Amar, C.L. East, J. Gray, M. Iturriza-Gomara, E.A. Maclure, J. McLauchlin, Detection by PCR of eight groups of enteric pathogens in 4627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study (1993-1996), Eur. J. Clin. Microbiol. Infect. Dis. 26 (2007) 311–323. [2] E. Scallan, R.M. Hoekstra, F.J. Angulo, R.V. Tauxe, M.A. Widdowson, S.L. Roy, J.L. Jones, P.M. Griffin, Foodborne illness acquired in the United States – major pathogens, Emerg. Infect. Dis. 17 (2011) 7–15. [3] R.L. Atmar, A.R. Opekun, M.A. Gilger, M.K. Estes, S.E. Crawford, F.H. Neill, D.Y. Graham, Norwalk virus shedding after experimental human infection, Emerg. Infect. Dis. 14 (2008) 1553–1557. [4] E.T. Tu, R.A. Bull, M.J. Kim, C.J. McIver, L. Heron, W.D. Rawlinson, P.A. White, Norovirus excretion in an aged-care setting, J. Clin. Microbiol. 46 (2008) 2119–2121. [5] T. Murata, N. Katsushima, K. Mizuta, Y. Muraki, S. Hongo, Y. Matsuzaki, Prolonged norovirus shedding in infants ≤6 months of age with gastroenteritis, Pediatr. Infect. Dis. J. 26 (2007) 46–49. [6] C. Roddie, J.P. Paul, R. Benjamin, C.I. Gallimore, J. Xerry, J.J. Gray, K.S. Peggs, E.C. Morris, K.J. Thomson, K.N. Ward, Allogeneic hematopoietic stem cell transplantation and norovirus gastroenteritis: a previously unrecognized cause of morbidity, Clin. Infect. Dis. 49 (2009) 1061–1068. [7] S.M. Karst, C.E. Wobus, M. Lay, J. Davidson, Virgin HWt, STAT1-dependent innate immunity to a Norwalk-like virus, Science 299 (2003) 1575–1578. [8] K.A. Chachu, D.W. Strong, A.D. LoBue, C.E. Wobus, R.S. Baric, Virgin HWt, Antibody is critical for the clearance of murine norovirus infection, J. Virol. 82 (2008) 6610–6617. [9] T.C. Dawson, M.A. Beck, W.A. Kuziel, F. Henderson, N. Maeda, Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus, Am. J. Pathol. 156 (2000) 1951–1959. [10] B.R. Lane, S.R. King, P.J. Bock, R.M. Strieter, M.J. Coffey, D.M. Markovitz, The CXC chemokine IP-10 stimulates HIV-1 replication, Virology 307 (2003) 122–134. [11] N.L. Tang, P.K. Chan, C.K. Wong, K.F. To, A.K. Wu, Y.M. Sung, D.S. Hui, J.J. Sung, C.W. Lam, Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome, Clin. Chem. 51 (2005) 2333–2340. [12] T. Ruuska, T. Vesikari, Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes, Scand. J. Infect. Dis. 22 (1990) 259–267. [13] M.E. Charlson, P. Pompei, K.L. Ales, C.R. MacKenzie, A new method of classifying prognostic comorbidity in longitudinal studies: development and validation, J. Chronic Dis. 40 (1987) 373–383.
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Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Low serum levels of CCL5 are associated with longer duration of viral shedding in norovirus infection Lars Gustavsson ∗ , Susann Skovbjerg, Magnus Lindh, Johan Westin, Lars-Magnus Andersson Department of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Diagnosvägen 21, Gothenburg SE-416 50, Sweden
a r t i c l e
i n f o
Article history: Received 5 March 2015 Received in revised form 12 June 2015 Accepted 15 June 2015 Keywords: Norovirus Cytokines CCL5 Viral shedding Viral load
a b s t r a c t Background: The mechanisms that determine the duration of fecal shedding of norovirus in humans have not been described in detail. Objectives: We investigated serum inflammatory mediator levels in relation to the duration of viral shedding in norovirus infection. Study design: A prospective cohort study of patients hospitalized with acute norovirus genogroup II infection. Rectal swab samples were obtained at inclusion and day 7, 14, 21 and 28. Serum levels of 42 inflammatory mediators were determined with a Luminex-based cytokine assay. Sera from 20 healthy blood donors served as controls. Results: Altogether, 28 patients (54% women, median age 83 years, median duration of symptoms 3 days) were included. Twelve subjects cleared the virus within 14 days and 16 were norovirus-RNA positive for >21 days, constituting the two study groups (“rapid” vs. “slow” clearance). Individuals with norovirus infection had higher levels of IL-18, CXCL9, CXCL10, soluble IL-2 receptor and macrophage migration inhibitory factor (MIF), compared to controls (p < 0.05), with the highest median concentrations in the slow clearance group. In contrast, CCL5 levels were lower in the slow compared to the rapid clearance group (median 54 vs. 134 ng/mL, p < 0.05), and lower in norovirus-infected patients than in controls. Conclusion: Low levels of CCL5 were associated with longer duration of viral shedding, suggesting that CCL5 may influence the clearance of norovirus. © 2015 Elsevier B.V. All rights reserved.
1. Background Norovirus is the most common cause of viral gastroenteritis [1,2] and can be detected in stool for several weeks following symptomatic infection [3]. The duration of shedding varies among individuals [4], but the factors behind this variation are not well
Abbreviations: BLAST, basic local alignment search tool; CCL, chemokine (C Cmotif) ligand; Ct, cycle threshold; CXCL, chemokine (C X C-motif) ligand; IL, interleukin; MIF, macrophage migration inhibitory factor; NoV, norovirus; NoV GII, norovirus genogroup II; PCR, polymerase chain reaction; qPCR, real-time PCR; sIL2R␣, soluble interleukin-2 receptor ␣. ∗ Corresponding author. E-mail addresses: [email protected] (L. Gustavsson), [email protected] (S. Skovbjerg), [email protected] (M. Lindh), [email protected] (J. Westin), [email protected] (L.-M. Andersson). http://dx.doi.org/10.1016/j.jcv.2015.06.088 1386-6532/© 2015 Elsevier B.V. All rights reserved.
understood. Longer duration of shedding is common in infants [5] and immunocompromised patients [6], suggesting that immune mechanisms are involved. In animal models, innate immunity is responsible for initial control of norovirus infection [7], whereas clearance of virus involves both humoral and cell-mediated immune mechanisms [8]. The understanding of the human immune response to norovirus infection is more limited. In other viral infections, studies of systemic cytokine levels have provided insight into how variations in the immune response impact the course of disease [9–11].
2. Objective The aim of this study was to examine the relation between cytokine levels and viral clearance in patients with communityonset norovirus genogroup II gastroenteritis.
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Fig. 1. Overview of the study participants and the basis for the formation of the two study groups with rapid (<14 days) vs. slow (>21 days) clearance of norovirus following acute infection. GII, genogroup II. Inclusion criteria were: age > 18 years, vomiting ≥ 2 times/24 h and/or ≥3 loose stools/24 h for ≤5 days. * 20 patients with rotavirus, 4 with adenovirus, 3 with norovirus GI, 1 with sapovirus.
3. Study design Adult patients admitted with suspected gastroenteritis were screened for inclusion in a prospective cohort study at the Department of Infectious Diseases of a 2000-bed teaching hospital in Gothenburg, Sweden, during three consecutive years (2010–2012). An overview of the study cohort and inclusion criteria is presented in Fig. 1. Age and sex; date of first symptom and symptom resolution; date of admission and discharge; gastroenteritis symptoms,
summarized as Vesikari scores [12]; and Charlson co-morbidity index scores [13] were recorded. Rectal swab samples were obtained at enrollment, and participants were asked to provide follow-up samples on day 7, 14, 21 and 28. Samples were collected in a standardized manner with a flocked swab (Copan Regular Flocked Swab 502CS01; Copan Italia Spa, Brescia, Italy). Samples were stored at −80 ◦ C until analysis, if not processed immediately. Serum samples were drawn on enrollment, and stored at −80 ◦ C until analysis. Rectal swab samples for
Table 1 Characteristics of patients with <14 days (rapid clearance) and >21 days (slow clearance) duration of viral shedding following norovirus infection.
Age, years Women, n (%) Charlson score (co-morbidity) Vesikari score (symptom severity) Disease duration at inclusion, days Fever, n (%) With symptomatic contacts, n (%) CRP (mg/L) Venous lactate (mmol/L) Cycle threshold, day 0 Cycle threshold, day 7 a b
Median (interquartile range). n = 5.
All patients n = 28
Rapid clearance n = 12
Slow clearance n = 16
p-value
83 (51–86)a 15 (54) 1 (0–1) 11 (9–12) 3 (2–4) 6 (21) 17 (61) 18 (7–28) 1.9 (1.5–2.8) 24 (19–28) 27 (25–32)
72 (34–86) 10 (83) 0 (0–1) 10 (9–12) 3 (1–5) 1 (8) 6 (50) 9 (3–22) 1.6 (1.4–2.9) 27 (22–33) 34 (33–39)b
84 (71–89) 5 (31) 1 (0.25–1) 12 (10–13) 3 (2–4) 5 (31) 11 (69) 26 (8–28) 2.0 (1.6–2.8) 21 (19–25) 27 (24–31)
0.21 0.006 0.11 0.12 0.74 0.20 0.32 0.14 0.51 0.03 0.01
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Fig. 2. Serum concentrations of interleukin-18 (IL-18), soluble interleukin-2 receptor (sIL-2R␣), macrophage migration inhibitory factor (MIF), chemokine (C X C-motif) ligand 9 (CXCL9), CXCL10, chemokine (C C motif) ligand 27 (CCL27) and CCL5, in patients admitted with norovirus genogroup II infection. Patients were grouped according to duration of viral shedding: rapid clearance, duration of shedding <14 days (n = 12); slow clearance, duration of shedding >21 days (n = 16). Sera from healthy blood donors (n = 9 for IL-18, IL-2 receptor ␣, CXCL9 and CCL27, n = 20 for CXCL10 and CCL5) were used as control. Horizontal lines denote median values. Stars denote significance level: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.
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bacterial culture were obtained on admission according to clinical routine. An in-house real-time reverse transcriptase qPCR procedure was used for detection of norovirus (NoV) genogroup II (GII), as described previously [14]. Viral genotype was determined by sequencing of approximately 350 base DNA fragments, spanning the polymerase–capsid junction. Cytokines in serum were measured by multiplex bead-based assays (Bio-Plex Pro Human Cytokine 21-Plex and 27-Plex Panels, Bio-Rad Laboratories, Hercules, CA, USA) using xMAP technology (Luminex, Austin, TX, USA), according to the manufacturer’s instructions (for a complete list of mediators, see Supplementary data). De-identified sera from healthy blood donors (aged 18–65 years), frozen at −80 ◦ C, were used as controls (n = 20). Individual age or gender data for the control sera was not available. IBM SPSS statistics 20 software was used for statistical analyses. Proportions were compared with 2 -test and continuous variables with the Mann–Whitney U test. Correlations were investigated using linear regression.
4. Results In total, we included 93 patients with NoV GII infection. Duration of shedding could be determined in 28 participants without immune deficiency (<7 days in 7 patients, 7–14 days in 5 patients, >21 days in 7 patients and >28 days in 9 patients). The twelve patients with duration of shedding <14 days constituted the rapid clearance group, and the 16 patients with duration of shedding >21 days the slow clearance group. Their characteristics are presented in Table 1. The difference in sex distribution did not remain significant when sex was adjusted for age by stratified analysis. No nursing home residents were included. The 65 excluded NoV GII patients were not different in terms of chronic co-morbidity and general health status. The median cycle threshold (Ct) value on enrollment was significantly lower in the slow clearance group, indicating a higher average viral load. Nucleotide sequences for genotyping could be obtained from 23 of the samples. Of these, 20 (87%) belonged to genotype GII.4. Of the 42 inflammatory mediators included in the assay, seven were detected in serum. Five (IL-18, sIL-2R␣, MIF, CXCL9, CXL10) were increased in the norovirus patients compared to controls, with a trend towards more pronounced elevations in the slow clearance group (Fig. 2A–E). The levels of CCL27 were equally high in patients and controls (Fig. 2F). One chemokine (CCL5) was decreased in patients compared to controls, with significantly lower levels in the slow clearance group (p < 0.05, Fig. 2G). Except for CCL27, we did not find any correlations between cytokine levels and age (for detailed results, see Supplementary data). There was no significant correlation between initial Ct value and CCL5 (R2 = 0.09, p = 0.13), but a low Ct value appeared to be related to longer disease duration prior to sampling (R2 = 0.13, p = 0.06).
5. Discussion In this longitudinal study of elderly hospitalized patients, slow clearance of norovirus infection was associated with low serum levels of CCL5 during acute gastroenteritis. In general, levels of lymphocyte stimulating mediators were increased in norovirus patients compared to controls (except for CCL27, a skin-specific mediator [15]). CXCL9 and CXCL10, IL-18, soluble IL-2 receptor ␣ and MIF are all related to a Th1-type response [16–20], and we found no indications that long duration of shedding would be related to a lack of antiviral immune reaction.
Low levels of CCL5, however, were associated with slow clearance. The serum level of CCL5 has been reported to mirror the activity of CD8+ T-cells [21,22] and CCL5 to sustain the cytotoxic response in prolonged viral infections [23]. Therefore, a possible explanation for our finding is that slow clearance may be associated with impaired cytotoxic T-cell activity. Cytotoxic CD8+ T-cells are critical for the clearance of norovirus from the intestine in animal models [24], and CD8+ T-cells have been found in large numbers in duodenal biopsies from norovirus-infected patients [25]. Restricting the analysis to patients with GII.4 infection did not alter the results (data not shown), and the observed differences could not be explained by major viral heterogeneity. An alternative explanation for the lower CCL5 levels in the slow clearance group, where median Ct value was lower, indicating higher viral load, is that CCL5 secretion may be suppressed by norovirus. Such a mechanism has been demonstrated for murine norovirus [26]. This interpretation is partly contradicted by the lack of a direct correlation between initial Ct value and CCL5. Ct values do not directly represent viral load, however, and associations might be overlooked. Moreover, initial Ct value was correlated to duration of symptoms and samples obtained early during the course of disease may be less representative of peak viral excretion. Thus, our results suggest that clearance of norovirus may be dependent of a strong CD8+ T-cell response, although this finding warrants further investigation. Since the included patients were elderly, the findings may not be applicable to other age groups. Also, the control sera were not matched for age, which makes interpretation of the results difficult. Although we did not find any relevant correlations between cytokine levels and age, old age may influence cytokine response. However, chemokine levels, including CCL5, are reported to increase with age [27,28]. This makes old age a less likely explanation for the low levels of CCL5 observed in patients with long duration of shedding in our study. The sample size of our cohort was too limited to allow for multivariate analysis. We used rectal swab samples instead of stool samples. There is now mounting evidence that swab samples are reliable for the diagnosis of gastroenteritis despite the lower content of virus [29,30]. Serum cytokine concentrations do not necessarily mirror the cytokine milieu in infected tissue. Cytokines have multiple and overlapping effects in vivo, and the interpretation of findings from non-experimental settings should be made cautiously. In conclusion, in this cohort study of hospitalized patients with norovirus GII gastroenteritis, we found that low serum levels of CCL5 were associated with a longer duration of viral shedding, indicating that CCL5 is involved in the clearance of norovirus in the elderly. This preliminary finding needs to be corroborated in further studies on patients of all ages, with focus on CCL5 expression in different body compartments, and its relation to other immune mechanisms, during norovirus infection. Funding Grants from the Regional Research Funds for Western Sweden (VGFOUREG 231011, 314211, 482001) and the Swedish Society of Medicine (SLS 249951). Competing interests None declared. Ethical approval Approved by the Regional Ethical Review Board in Gothenburg, Sweden.
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