• 沒有找到結果。

Slower Rates of Clearance of Viral Load and Virus?Containing Immune Complexes in Patients with Dengue Hemorrhagic Fever

N/A
N/A
Protected

Academic year: 2021

Share "Slower Rates of Clearance of Viral Load and Virus?Containing Immune Complexes in Patients with Dengue Hemorrhagic Fever"

Copied!
8
0
0

加載中.... (立即查看全文)

全文

(1)

M A J O R A R T I C L E

Slower Rates of Clearance of Viral Load and

Virus-Containing Immune Complexes in Patients

with Dengue Hemorrhagic Fever

Wei-Kung Wang,1,3Hui-Ling Chen,1Chao-Fu Yang,1Szu-Chia Hsieh,1Chung-Chou Juan,5Shu-Mei Chang,5 Cheng-Ching Yu,6Li-Hui Lin,6Jyh-Hsiung Huang,4and Chwan-Chuen King2

1Institute of Microbiology, College of Medicine, and2Institute of Epidemiology, College of Public Health, National Taiwan University,3Department of Internal Medicine, National Taiwan University Hospital, and4Center for Disease Control, Department of Health, Taipei, and5Yuan General Hospital and6Hui-Te Hospital, Kaohsiung, Taiwan

Background. Although previous studies have revealed the contribution of an initial high level of dengue virus replication to the severe and potentially life-threatening diseases dengue hemorrhagic fever (DHF) and dengue shock syndrome, the involvement of dengue virus in the immuopathological processes during the transition from fever to defervescence, which is a critical stage in determining the progression to DHF, has not been appreciated. Previously, we reported that dengue virus can be detected in the immune complexes of patients with DHF during this period.

Methods. We investigated plasma dengue viral load, virus in immune complexes, antibody response, comple-ments, and cytokines for 54 patients with dengue fever (a relatively mild form of disease) and 49 patients with DHF. The patients had confirmed secondary infection with dengue virus type 2 from a large outbreak in southern Taiwan in 2002.

Results. Patients with DHF had a significantly higher viral load and a slower rate of clearance than patients with dengue fever. For viral loads15.7 log RNA copies/mL on the day of defervescence, the positive and negative

predictive values for DHF are 0.88 and 0.95, respectively. A higher level and slower decline of dengue virus– containing immune complexes (and a subsequently higher elevation of C5a and soluble interleukin 2 receptor) were found in patients with DHF, compared with patients with dengue fever.

Conclusions. These findings indicate that slower rates of clearance of viral load and virus-containing immune complexes are associated with subsequent immune activation and contribute to the progression of DHF at this critical stage. Moreover, viral load on the day of defervescence can predict cases of DHF.

The 4 serotypes of dengue virus (dengue virus serotypes [DEN] 1–4) cause the most important arboviral disease in tropical and subtropical areas [1–3]. It has been es-timated that150 million dengue virus infections occur

annually worldwide [1–3]. The clinical presentations range from asymptomatic, to a relatively mild illness (dengue fever [DF]), to severe and potentially life-threatening diseases (dengue hemorrhagic fever [DHF] and dengue shock syndrome [DSS]) [2–5].

The pathogenesis of DHF and DSS has been one of

Received 22 March 2006; accepted 5 July 2006; electronically published 14 September 2006.

Reprints or correspondence: Dr. Wei-Kung Wang, Institute of Microbiology, College of Medicine, National Taiwan University, No.1 Sec.1 Jen-Ai Rd., Taipei, 100, Taiwan (wwang60@yahoo.com).

Clinical Infectious Diseases 2006; 43:1023–30

 2006 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2006/4308-0012$15.00

the major issues in dengue virus research. Along with the risk factors reported for DHF and DSS, 2 major factors—the viral strain and the immune status of the host—have been identified [2, 3, 5]. Based on the ob-servation that individuals experiencing secondary in-fection had a higher risk of developing DHF and/or DSS, the immune hypothesis states that cross-reactive, nonneutralizing antibodies from previous infection may enhance dengue virus infection [5]. This was supported by the antibody-dependent enhancement in vitro [6, 7] and confirmed by several cohort studies [8–10]. Be-cause different dengue viral strains have been reported to link to outbreaks of very mild or very severe disease [11–13], the viral hypothesis contends that severe den-gue disease is the result of infection with a more virulent strain [14]. Both hypotheses predict that a higher mag-nitude of viral replication will be seen in patients with more severe disease, as has been demonstrated by a

(2)

positive correlation between peak viral load and disease severity in studies focusing on the early stage of infection [15–17].

Clinically, a critical stage in determining the progression to DHF and/or DSS is the transition from fever to defervescence, during which severe hemorrhage, plasma leakage, and/or cir-culatory failure occur. Studies of immune responses have re-ported circulating immune complexes and complement acti-vation in patients with DHF and/or DSS during this period [18–22]. In addition, several cytokines and cytokine receptors, such as TNF-a, IL-2, IL-10, IFN-g, IL-6, IL-1b, IL-13, IL-18, soluble IL-2 receptor (sIL-2R), and soluble TNF receptor II (sTNFR-II), have been reported to be elevated in patients with DHF and/or DSS [17, 23–26]. However, the involvement of the virus in the immunopathological processes at this critical stage remains unknown. Previously, we reported that dengue virus can be detected in the immune complexes of patients with DHF during defervescence [27]. In the current study, we examined viral load, virus in the immune complexes, antibody responses, complements, and cytokines in both patients with DF and patients with DHF during this period. The patients were from the 2002 outbreak of DEN-2 infection in Kaohsiung, Taiwan, which was the largest outbreak in Taiwan since World War II.

PATIENTS AND METHODS

Study participants. From June to December 2002, during a DEN-2 infection outbreak in Kaohsiung, a metropolis in south-ern Taiwan, 120 adult patients who were admitted to either Yuan General Hospital or Huei-Te Hospital and received a diagnosis of either DF and DHF [4] were included in the study. The severity of disease was classified by the World Health Or-ganization grading system [4, 27]. None of the participants were found to belong to the previously described intermediate category [17]. Illness day 1 was defined as the day of onset of fever (oral temperature, ⭓38C). Fever day 0 was defined as the day of defervescence (oral temperature,!38C), and days prior to and after this time point were designated consecutively [15, 28]. With informed consent, sequential blood samples were collected, and plasma specimens were prepared [27]. All pa-tients were confirmed to have infection due to DEN-2 by a nested RT-PCR assay [27, 29]. An envelope/membrane IgM/ IgG Elisa was performed using convalescent serum samples to determine whether the patient fulfilled the definition primary or secondary infection [30]. A Japanese encephalitis virus non-structural protein 1 IgM was used to exclude Japanese en-cephalitis virus infection [30].

Dengue viral RNA in plasma and in immune complexes.

Dengue viral RNA in plasma or immune complexes was isolated through a modified immunoprecipitation assay [27] and quan-tified by a real-time RT-PCR assay with the sensitivity of 357 RNA copies per mL of plasma [31].

Duration and clearance rate of viral load. The duration of detectable viral load was estimated by assuming that the period during which viral RNA was detectable began on the day prior to onset of illness and ended on the last day that viral RNA was detected [15]. The rate of clearance of viral load was determined by calculating the slope of viral load decrease between fever day⫺2 and fever day +3.

Antidengue IgM and IgG. Antidengue IgM and IgG an-tibodies were detected using a commercial IgM/IgG capture ELISA (Dengue Duo; PanBio) with the levels determined as the percentage reactivity of the positive control samples [32]. Serial dilutions of the IgM positive control sample (100- to 800-fold dilutions) and of IgG positive control sample (25,600-to 409,600-fold dilutions) were subjected (25,600-to IgM and IgG ELISA, respectively, to obtain a linear curve between absorbance (450 nm) and dilutions. Diluted serum samples (100-fold di-lutions for IgM and 25,600-fold didi-lutions for IgG) were sub-jected to IgM and IgG ELISA simultaneously with respective positive control samples and cutoff samples (100-fold dilution). A ratio of the absorbance of serum to that of a cutoff sample of⭓1 was defined as positive [27,33]. The IgM and IgG ELISA scores of positive samples were the percentage of absorbance compared with that of the IgM positive control sample (100-fold dilution) and that of the IgG positive control sample (25600-fold dilution), respectively. The same batch of positive control samples and cutoff samples (provided by manufacturer) was used for all samples tested.

Western blot analysis. Lysates of DEN-2 New Guinea C strain–infected and mock-infected C6/36 cells were electro-phoresed in an SDS–12% polyacrylamide gel. After transfer and blocking, the nitrocellulose membrane was cut into strips and incubated with serum (5000-fold dilution), followed by per-oxidase-conjugated antihuman IgG (Pierce Biotechnology). The signals were detected by chemiluminescence reagent (Perkin-Elmer Lifesciences).

ELISA. IFN-g, IL-10, and sIL-2R in plasma were deter-mined by ELISA kits from Pierce Endogen, sTNFR-II was de-termined by a kit from R&D systems, and C3a and C5a were determined by kits from BD Biosciences. The upper limits of normal values for IFN-g, IL-10, sIL-2R, and sTNFR-II are 2.6 pg/mL, 15.6 pg/mL, 1521 U/mL, and 2262 pg/mL, respectively.

Statistical analysis. The Mann-Whitney U test was used to compare parameters between the 2 groups for all variables except sex, which was compared using Fisher’s exact test.

RESULTS

Patient characteristics. Of the 120 study participants, 17 ful-filled the definition of primary infection, and 103 fulful-filled the definition of secondary infection. Because primary infection is thought to have a different pathogenic mechanism from sec-ondary infection [2, 3], and because the case number was small,

(3)

Table 1. Characteristics of 103 patients with secondary dengue virus serotype 2 infection. Characteristics Patients with dengue fever (n p54) Patients with dengue hemorrhagic fever (n p 49) P

Age, mean years (range) 51.0 (18–82) 52.1 (18–76) .84

Sex, M/F 15/39 19/30 .30

Illness day at study entry, daysa 3.3 1.6 3.4 1.5 .60 AST level at study entry, U/L 99.9 68.9 173.3 127.2 .001 ALT level at study entry, U/L 81.4 54.0 133.3 103.5 .01 WBC count at study entry, 103cells/mm3 4.1

 1.7 4.5 2.0 .38

Platelet count at study entry, 103platelets/mm3 90.7 51.8 80.7 57.8 .21 Platelet count nadir, 103platelets/mm3 42.8 36.4 20.8 9.7 !.001

NOTE. Data are mean SE, unless otherwise indicated. Dengue fever and dengue hemorrhagic fever are classified according to the World Health Organization case definition [4]. ALT, alanine aminotransferase; AST, aspartate aminotransferase.

a

Illness day 1 is defined as the first day of fever.

Figure 1. Plasma dengue viral load in patients with secondary dengue virus serotype 2 infection during the transition from fever to defervescence. Levels of dengue viral RNA in plasma of patients with dengue fever (DF) and patients with dengue hemorrhagic fever (DHF) were determined as described in Patients and Methods. Fever day 0 is the first day of defer-vescence. The dashed line indicates the limit of detection of the assay (i.e., 357 RNA copies per mL of plasma). Error bars indicate SE.

we focused our analysis on the 103 cases of secondary infection, including 54 cases of DF and 49 cases of DHF. As shown in table 1, there were no statistically significant differences in sex (P p .30, by Fisher’s exact 2-tailed test), age, or day of entry (P p .84and P p .60, respectively, by Mann-Whitney U test) between the DF and DHF groups. Although there was no sta-tistically significant difference in WBC and platelet counts at entry between 2 groups, the nadir platelet counts were lower in the DHF group than in the DF group (P!.001, by Mann-Whitney U test). Moreover, aspartate aminotransferase and al-anine aminotransferase levels at entry were higher in the DHF group than in the DF group (P p .001andP p .01, respectively, by Mann-Whitney U test). These findings were generally in agreement with those reported for pediatric cases [17, 34].

Higher dengue viral load in patients with DHF than in patients with DF during the fever-to-defervescence transition.

To investigate the relationship between viral load and disease severity, the levels of dengue viral RNA in plasma from 2 groups were compared for each day from fever day⫺2 to fever day +4 [31] (figure 1). On fever day ⫺2 and ⫺1, the levels of dengue viral RNA in plasma samples from patients with DHF were higher than those in plasma samples from patients with DF (P p .05andP p .009, respectively, by Mann-Whitney U test). A similar trend was observed during defervescence. Pa-tients with DHF had higher dengue viral RNA levels than did patients with DF on fever day 0, +1, +2, and +3 (P!.001, , , and , respectively, by

Mann-Whit-P!.001 P p .001 P p .03

ney U test).

Virus-containing immune complexes persist at deferves-cence for patients with DHF. To further investigate dengue virus in the immune complexes during this period, we focused on 26 patients (12 patients with DF and 14 patients with DHF) who had⭓3 sequential plasma samples examined [27, 31]. The results of 4 representative cases (2 cases of DF and 2 cases of

DHF) are shown in figure 2. Consistent with the cumulative data in figure 1, viral load dropped to a level below 3.7 log RNA copies/mL on fever day +1 for patients with DF and remained13.7 log RNA copies/mL in patients with DHF.

In-terestingly, dengue virus–containing immune complexes were found in both patients with DF and patients with DHF during the late fever period. At defervescence, they dropped to very low or undetectable level in patients with DF, but remained high in patients with DHF.

Antibody responses. In agreement with the report that IgG antibody responses predominated in cases of secondary infec-tion, antidengue IgG increased more rapidly and to a higher

(4)

Figure 2. Relationships between plasma dengue viral load, virus in the immune complexes, and antidengue antibody responses during the transition from fever to defervescence in representative patients with dengue fever (DF) (A, C) and patients with dengue hemorrhagic fever (DHF) (B, D) who had secondary dengue virus serotype 2 infection. The levels of dengue viral RNA in plasma (open circles) and in the immune complexes (open squares) and the levels of antidengue IgG (closed triangles) and IgM (closed diamonds) antibodies were determined as described in Patients and Methods. Dashed lines indicate the limit of detection of the assay (357 RNA copies per mL of plasma) and of the ELISA score (33%). Hatched bars indicate the fever period. Fever day 0 is the first day of defervescence. The Western blot analysis is described in Patients and Methods, with the fever day shown above each lane and the molecular weight marker shown on the left. For simplicity, IgG ELISA scores1100% were presented as 100%. E,

envelope; NS1, nonstructural protein 1.

level than antidengue IgM in most cases [28] (figure 2). Because these were cases of DEN-2 infection and the ELISA kit con-tained a mixture of 4 dengue viruses as antigen, we used lysates derived from DEN-2 virus–infected cells in Western blot anal-ysis to examine anti–DEN-2 antibody response. As shown in figure 2, detectable bands of anti-envelope antibody, which was the major antibody response [35], correlated with IgG ELISA scores of⭓100%, indicating that IgG ELISA scores of 100% represent significant levels of anti–DEN-2 envelope antibody response. Of note, the absence of anti–nonstructural protein 1 antibody seen in the 2 patients with DF was not seen in other patients with DF; therefore, this was not pursued further.

Slower rate of clearance of viral load in patients with DHF.

The mean levels of viral load, virus in the immune complexes, anti-dengue IgG and IgM antibodies in these 26 patients are summarized in figure 3A and 3B. Compared with patients with DF, patients with DHF had higher levels of viral load and

virus-containing immune complexes (P p .003 and P p .02, re-spectively, by Mann-Whitney U test) (table 2). Further analysis revealed that the estimated duration of detectable viral load was longer for patients with DHF than for patients with DF (mean duration, 7.7 vs. 6.2 days;P p .02, by Mann-Whitney

U test). Moreover, the slope of the decrease in viral load was

less for patients with DHF than for patients with DF, indicating a slower rate of clearance for patients with DHF (mean clear-ance rate,⫺1.30 vs. ⫺2.01 log RNA copies/mL per day; P p , by Mann-Whitney U test) (table 2). This was further .037

supported by a slower increase of antidengue IgG and IgM antibodies for patients with DHF than for patients with DF (P p .002 and P p .02, for IgG and IgM levels, respectively, between fever day 0 and +1, by Mann-Whitney U test).

Complement and cytokine activation. Similar to the de-crease in viral load, the slope of the dede-crease in virus-containing immune complexes was smaller for patients with DHF than

(5)

Figure 3. Relationships between the mean levels of dengue viral load, virus in the immune complexes, antidengue antibodies, C3a, C5a, cytokines, and cytokine receptors during the transition from fever to defervescence in 12 patients with dengue fever (DF) (A, C, E) and 14 patients with dengue hemorrhagic fever (DHF) (B, D, F) who had secondary dengue virus serotype 2 infection. A and B show the levels of dengue viral RNA in plasma (open circles) and in the immune complexes (open squares) and antidengue IgG (closed triangles) and IgM (closed diamonds) antibodies, determined as described in Patients and Methods. Dashed lines indicate the limit of detection of the assay (357 RNA copies per mL of plasma). Hatched bars indicate the fever period. Fever day 0 is the first day of defervescence. C and D show the levels of C3a (pink closed circles) and C5a (purple closed squares) in plasma, which were determined as described in Patients and Methods. The dashed lines indicate the upper limits of normal values for C3a (689.6 ng/mL) and C5a (7.8 ng/mL). E and F show the levels of soluble IL-2 receptor (sIL-2R; red closed triangles), soluble TNF receptor II (sTNFR-II; green open triangles), IL-10 (blue closed diamonds), and IFN-g (yellow crosses) in plasma, which were determined as described in Patients and Methods. The dashed lines indicate the upper limit of normal values for IFN-g (2.6 pg/mL).

for patients with DF, suggesting that circulating immune com-plexes had been present for a longer duration in patients with DHF than in patients with DF (figure 3A and 3B). To further investigate subsequent immunopathological events, 2 impor-tant complement mediators, C3a and C5a, were examined. As shown in figure 3C and 3D, elevation of C3a and C5a were found in both patients with DF and patients with DHF, with the peaks noted after virus-containing immune complexes de-creased. Moreover, C5a was higher in patients with DHF than in patients with DF (P p .009, by Mann-Whitney U test).

We also examined the cytokines and cytokine receptors that have been reported to reach higher levels in patients with DHF than in patients with DF, including sIL-2R, sTNFR-II, IL-10, and IFN-g [17, 23–25]. As shown in figure 3E and 3F, the peaks for these cytokines and cytokine receptors lagged behind the peaks for viral load for both patients with DF and patients with DHF, with the exception of IFN-g, which peaked slightly

earlier for patients with DHF. The level of sIL-2R was higher for patients with DHF than for patients with DF (P p .01, by Mann-Whitney U test), whereas there was no difference in sTNFR-II, IL-10, and IFN-g between the 2 groups (table 2).

DISCUSSION

Previous studies of DHF have focused on the fever period and have reported the correlation of an initial high level of virus replication to DHF [15–17]. In this study, we focused on the fever-to-defervescence transition period and reported that pa-tients with DHF had a higher viral load and slower rate of clearance than patients with DF. Moreover, a higher level and slower decrease of virus-containing immune complexes, as well as a subsequently higher elevation of C5a and sIL-2R, were found in patients with DHF, compared with patients with DF. To our knowledge, this is the first study demonstrating that

(6)

Table 2. Dengue viral load, virus in the immune complexes, and duration and clearance rate of viral load, complements, and cytokines during the transition to defervescence for patients with secondary dengue virus serotype 2 infection.

Variable Patients with dengue ever (n p 12) Patients with dengue hemorrhagic fever (n p 14) P

Viral load,alog RNA copies/mL 5.1 (3.7–6.2) 7.1 (6.5–8.0) .003 Virus in immune complexes,alog RNA

copies/mL 3.5 (2.5–4.0) 4.7 (4.0–5.8) .02

Duration of detectable viral load, days 6.2 (6–7) 7.7 (7–8) .02 Clearance rate of viral load, log RNA

copies/mL per day ⫺2.01 (⫺1.78 to ⫺2.08) ⫺1.3 (⫺0.88 to ⫺1.77) .037 C3a level,bng/mL 3009.5 (2640.2–3014.5) 3205.0 (2242.2–3792.6) .59

C5a level,bng/mL 30.9 (16.8–40.7) 54.8 (36.4–73.1) .009

Soluble IL-2 receptor level,bU/mL 18041.0 (14778.8–20000.0) 25476.2 (19247.8–28539.8) .01 Soluble TNF receptor II level,bpg/mL 3590.7 (3225.0–3926.8) 3866.7 (3660.2–4007.8) .08 IL-10 level,bpg/mL 106.5 (58.1–148.6) 111.2 (54.3–114.3) .75

IFN-g level,bpg/mL 44.1 (20.0–43.3) 50.0 (20.8–49.7) .87

NOTE. Data are mean value (interquartile range). Twenty-six patients with secondary dengue virus serotype 2 infection, including 12 patients with dengue fever and 14 patients with dengue hemorrhagic fever [4] who had⭓3 sequential plasma samples, were included in the analysis.

a

Determined for fever day⫺1 to fever day 0. b

Determined from fever day⫺2 to fever day +2.

Table 3. Positive and negative predictive values of dengue viral load for patients with dengue hemorrhagic fever during the transition from fever to defervescence. Variable Positive predictive value Negative predictive value Fever day⫺1, viral load ⭓7.5 log RNA copies/mL 0.71 0.94 Fever day 0, viral load⭓5.7 log RNA copies/mL 0.88 0.95 Fever day +1, viral load⭓3.7 log RNA copies/mL 0.94 0.86

NOTE. Plasma dengue viral load was determined as described in Patients and Meth-ods. A total of 103 patients with cases of secondary dengue virus serotype 2 infection, including 54 patients with dengue fever and 49 patients with dengue hemorrhagic fever [4], were included in the analysis. Fever day 0 is the first day of defervescence

slower rates of clearance of viral load and virus-containing immune complexes are linked to subsequent immune activa-tion and contribute to DHF. Studies of more cases of secondary infection in the future would validate these findings. Of note, analysis of 17 patients with primary infection also revealed higher viral load in patients with DHF than in patients with DF during this period (data not shown), although a detailed analysis of immune complexes and subsequent activation was not performed because of the small sample size. Future studies involving a larger number of cases of primary infection, such as cases involving infants !1 year old, would shed new light on the pathogenesis of DHF in cases of primary infection.

Since DHF and DSS are the leading causes of hospitalization and death among children in Asia [2, 3], several laboratory parameters, including aspartate aminotransferase, sTNFR (80

kD), and soluble nonstructural protein 1, have been proposed as predictors for DHF [17, 34, 36]. Because patients with DHF had a significantly higher viral load than did patients with DF between fever day⫺2 and fever day +3, we further examined whether viral load during this period could predict for DHF; the positive and negative predictive values are shown in table 3. Compared with the positive and negative predictive values of the other parameters reported thus far (which have ranged from 0.53 to 0.83) [17, 34, 36], the high positive and negative predictive values (0.88 and 0.95, respectively) for viral load15.7

log RNA copies/mL on fever day 0 (the day of defervescence) indicate that this is a good predictor for DHF. Nonetheless, whether this predictor can be employed clinically in the future requires careful monitoring of defervescence and rapid mea-surement of viral load.

(7)

By examining the slope of the decline in viral load, we showed that the clearance rate of viral load was slower for patients with DHF than for patients with DF. It is possible that this is an extension of higher peak viral load, resulting from larger num-bers of virus-infected cells in patients with DHF. Because of the ubiquitous presence of RNAse, viral RNA detected in plasma most likely derived from virion, rather than from lysis of virus-infected cells. This finding was supported by a good correlation between the levels of viral RNA and the titers of viremia, determined by plaque assay using serial dilution of plasma for some of our patients (data not shown), as well as by cosedimentation of peak viral RNA and peak core antigen after sucrose-gradient ultracentrifugation of plasma containing another RNA virus, HIV-1 [37]. Alternatively, slower clearance of viral load is related to T cell dysfunction. Impaired T cell proliferation responses to dengue antigen have been reported in patients with acute dengue infection [38]. A recent study of T cell repertoire revealed apoptosis of dengue-specific T cell clones in patients with DHF between fever day⫺1 and fever day +1 and reported the “original antigenic sin” of T-cells [39], which has been shown to impair clearance of viremia [40, 41]. Because the timing of T cell apoptosis is after the peak of viral load, it is conceivable that dysfunction of dengue-specific T cells may account for the slower clearance of viral load observed in patients with DHF. Determining whether T cell apoptosis occurs earlier and contributes to the peak of viral replication requires sophisticated analysis of T cells at the early stage of infection. The finding of slower clearance of viral load in pa-tients with DHF is in contrast to the findings of a previous study in which viremia decreased more quickly for patients with DHF than for patients with DF at defervescence [15]. Of note, the presence of viremia in that study [15] was determined by virus isolation, which could potentially be interfered with by the presence of antibodies or circulating immune complexes [20, 28, 42]. It is likely that greater amounts of immune com-plexes in patients with DHF at defervescence may cause a lower efficiency of virus isolation and, therefore, cause lower levels of viremia. Two recent studies using RT-PCR assay that showed a higher viral load until defervescence in patients with DHF, compared with patients with DF, support this interpretation [16, 17].

Previously, circulating immune complexes were reported in patients with DHF and/or DSS by detecting complement com-ponents associated with immune complexes [18–20]; however, the presence of dengue virus in the immune complexes remains unclear. In our study, we detected dengue virus in the immune complexes directly by a modified immunoprecipitation assay [27]. These virus and antibody complexes may augment virus replication in mononuclear cells through Fc receptor–mediated entry—the so-called antibody-dependent enhancement in vi-vo—which was supported by studies involving humans and

rhesus monkeys [43, 44]. Alternatively, they may be involved in the clearance of viral load, presumably by phagocytic cells in the reticuloendothelial system [45]. Based on the timing of detection (i.e., shortly before or after defervescence) and the association with a decrease in viral load, the virus-containing immune complexes detected were most likely in the process of being cleared. Interestingly, the clearance of virus-containing immune complexes was also slower for patients with DHF than for patients with DF (figure 3A and 3B). This would lead to higher levels and a longer presence of circulating immune plexes, which would subsequently lead to more-profound com-plement activation in patients with DHF. Consistent with this prediction, both C3a and C5a peaked after virus-containing immune complexes decreased, and a higher level of C5a, one of the most potent inflammatory peptides, was found in pa-tients with DHF, compared with papa-tients with DF (table 2).

In agreement with previous reports, IFN-g peaked slightly earlier in patients with DHF (figure 3E and 3F). In addition, sIL-2R and sTNFR-II were elevated in patients with DHF, with the level of sIL-2R higher in patients with DHF than in patients with DF (table 2) [17, 23, 25]. IL-10 and IFN-g levels were also elevated in patients with DHF, but they were not higher in patients with DHF than in patients with DF. This could be due to the small sample size and/or due to the day-to-day variation of IFN-g [26]. The observations that peak levels of cytokines and cytokine receptors lag behind peak levels of viral load (figure 3) and that viral loads are higher in patients with DHF, compared with patients with DF, indicate that higher magnitudes of viral replication lead to stronger T cell activation and greater cytokine release in patients with DHF [26]. Higher levels of T cell activation and cytokines, together with the stron-ger complement activation after sustained formation of im-mune complexes, would contribute to the immunopathoge-nesis of DHF.

Acknowledgments

We thank Su-Ru Lin, Min-Huei Wu, Mei-Ying Liao, I-Jung Liu, and Hsien-Ping Hu for technical assistance.

Financial support. The National Health Research Institute (NHRI-CN-CL9302P) and the National Science Council, Taiwan (NSC94-2320-B-002-065).

Potential conflicts of interest. All authors: no conflicts.

References

1. Monath TP. Dengue, the risk to developed and developing countries. Proc Natl Acad Sci U S A 1994; 91:2395–400.

2. Gubler DJ. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. TRENDS Microbiol 2002; 10:100–3.

3. Guzman MG, Kouri G. Dengue: an update. Lancet Infect Dis 2002; 2: 33–42.

4. World Health Organization (WHO). Dengue hemorrhagic fever, di-agnosis, treatment and control. 2nd ed. Geneva: WHO, 1997. 5. Halstead SB. Pathogenesis of dengue: challenges to molecular biology.

(8)

6. Halstead SB, O’Rourke EJ. Antibody-enhanced dengue virus infection in primate leukocytes. Nature 1977; 265:739–41.

7. Morens DM, Venkateshan CN, Halstead SB. Dengue 4 virus mono-clonal antibodies identify epitopes that mediate immune enhancement of dengue 2 viruses. J Gen Virol 1987; 68:91–8.

8. Sangkawibha N, Rojanasuphot S, Ahandrik S, et al. Risk factors for dengue shock syndrome: a prospective epidemiologic in Rayong, Thai-land. I. The 1980 outbreak. Am J Epidemiol 1984; 120:653–69. 9. Burke DS, Nisalak A, Johnson DE, Scott RM. A prospective study of

dengue infections in Bangkok. Am J Trop Med Hyg 1988; 38:172–80. 10. Guzman MG, Kouri GP, Bravo J, Soler M, Vazquez S, Morier L. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study. Am J Trop Med Hyg 1990; 42:179–84.

11. Gubler DJ, Reed D, Rosen L, Hitchcock JC. Epidemiologic, clinical and virologic observations on dengue in the kingdom of Tonga. Am J Trop Med Hyg 1978; 27:581–9.

12. Leitmeyer KC, Vaughn DW, Watts DM, et al. Dengue virus structural differences that correlate with pathogenesis. J Virol 1999; 73:4738–47. 13. Messer WB, Gubler DJ, Harris E, Sivananthan K, de Silva AM. Emer-gence and global spread of a dengue serotype 3, subtype III virus. Emerg Infect Dis 2003; 9:880–9.

14. Rosen L. The emperor’s new clothes revisited, or reflections on the pathogenesis of dengue hemorrhagic fever. Am J Trop Med Hyg 1977; 26:337–43.

15. Vaughn DW, Green S, Kalayanarooj S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000; 181:2–9.

16. Murgue B, Roche C, Chungue E, Deparis X. Prospective study of the duration of magnitude of viremia in children hospitalized during the 1996–1997 dengue-2 outbreak in French Polynesia. J Med Virol 2000; 60:432–8.

17. Libraty DH, Endy TP, Houng HS, et al. Differing influence of virus burden and immune activation on disease seveity in secondary dengue-3 virus infection. J Infect Dis 2002; 185:121dengue-3–21.

18. Sobel AT, Bokisch VA, Muller-Eberhand HJ. C1q deviation test for the detection of immune complexes, aggregates of IgG, and bacterial prod-ucts in human serum. J Exp Med 1975; 142:139–50.

19. Theofilopoulos AN, Wilson CB, Dixon FJ. The Raji cell radioimmune assay for detecting immune complexes in human sera. J Clin Invest

1976; 57:169–82.

20. Ruangjirachuporn W, Boonpucknavig S, Nimmanitya S. Circulating immune complexes in serum from patients with dengue hemorrhagic fever. Clin Exp Immunol 1979; 36:46–53.

21. Bokisch VA, Top FH, Russell PK, Dixon FJ, Muller-Eberhard HJ. The potential role of complement in dengue hemorrhagic shock syndrome. N Engl J Med 1973; 289:996–1000.

22. Muller-Eberhard HJ, Dixon FJ, Tuchinda P, Bukkavesa S, Suvatte V. Pathogenic mechanisms in dengue hemorrhagic fever: report of an international collaborative study. Bull WHO 1973; 48:117–33. 23. Kurane I, Innis BL, Nimmannitya S, et al. Activation of T lymphocytes

in dengue virus infection. J Clin Invest 1991; 88:1473–80.

24. Green S, Vaughn DW, Kalayanarooj S, et al. Elevated plasma interleu-kin-10 levels in acute dengue correlate with disease severity. J Med Virol 1999; 59:329–34.

25. Green S, Vaughn DW, Kalayanarooj S, et al. Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity. J Infect Dis 1999; 179:755–62.

26. Rothman AL, Ennis FA. Immunopathogenesis of dengue hemorrhagic fever. Virology 1999; 257:1–6.

27. Wang WK, Chao DY, Kao CL, et al. High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis. Virology 2003; 305:330–8.

28. Vaughn DW, Green S, Kalayanarooj S, et al. Dengue in the early febrile phase: viremia and antibody responses. J Infect Dis 1997; 176:322–30. 29. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992; 30:545–51.

30. Shu PY, Chen LK, Chang SF, et al. Comparison of capture immu-noglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clin Diagn Lab Immunol 2003; 10:622–30.

31. Wang WK, Sung TL, Tsai YC, Kao CL, Chang SM, King CC. Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2–infected patients by a reverse transcription-real-time PCR assay. J Clin Microbiol 2002; 40:4472–8.

32. Thein S, Aaskov J, Myint TT, Shwe TN, Saw TT, Zaw A. Changes in levels of anti-dengue virus IgG subclasses in patients with disease of varying severity. J Med Virol 1993; 40:102–6.

33. Vaughn DW, Nisalak A, Solomon T, et al. Rapid serologic diagnosis of dengue virus infection using a commercial capture ELISA that dis-tinguishes primary and secondary infections. Am J Trop Med Hyg 1999; 60:693–8.

34. Kalayanarooj S, Vaughn DW, Nimmannitya S, et al. Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 1997; 176: 313–21.

35. Churdboonchart V, Bhamarapravati N, Peampramprecha S, Sirinavin S. Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop Med Hyg 1991; 44:481–93. 36. Libraty DH, Young PR, Pickering D, et al. High circulating levels of

the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002; 186:1165–8.

37. Piatak, M, Saag MS, Yang LC, et al. High levels of HIV-1 in plasma during all stages of infection determined. Science 1993; 259:1749–54. 38. Mathew A, Kurane I, Green S, et al. Impaired T cell proliferation in

acute dengue infection. J Immunol 1999; 162:5609–15.

39. Mongkolsapaya J, Dejnirattisai W, Xu XN, et al. Original antigic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 2003; 9:921–7.

40. Raymond M, Rothman AL. Dengue immune response: low affinity, high febrility. Nat Med 2003; 9:820–2.

41. Klenerman P, Zinkernagel RM. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature

1998; 394:482–5.

42. Gubler DJ, Suharyono W, Tan R, Abidin M, Sie A. Viremia in patients with naturally acquired dengue infection. Bull WHO 1981; 59:623–30. 43. Halstead SB. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J Infect Dis 1979; 140:527–33. 44. Kliks SC, Nimmanitya S, Nisalak A, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hem-orrhagic fever in infants. Am J Trop Med Hyg 1988; 38:411–9. 45. Igarashi T, Brown C, Azadegan A, et al. Human immunodeficiency

virus type 1 neutralizing antibodies accelerate clearance of cell-free virions from blood plasma. Nat Med 1999; 5:211–6.

數據

Table 1. Characteristics of 103 patients with secondary dengue virus serotype 2 infection
Figure 2. Relationships between plasma dengue viral load, virus in the immune complexes, and antidengue antibody responses during the transition from fever to defervescence in representative patients with dengue fever (DF) (A, C) and patients with dengue h
Figure 3. Relationships between the mean levels of dengue viral load, virus in the immune complexes, antidengue antibodies, C3a, C5a, cytokines, and cytokine receptors during the transition from fever to defervescence in 12 patients with dengue fever (DF)
Table 2. Dengue viral load, virus in the immune complexes, and duration and clearance rate of viral load, complements, and cytokines during the transition to defervescence for patients with secondary dengue virus serotype 2 infection.

參考文獻

相關文件

Animal or vegetable fats and oils and their fractiors, boiled, oxidised, dehydrated, sulphurised, blown, polymerised by heat in vacuum or in inert gas or otherwise chemically

Milk and cream, in powder, granule or other solid form, of a fat content, by weight, exceeding 1.5%, not containing added sugar or other sweetening matter.

Although Taiwan stipulates explicit regulations governing the requirements for organic production process, certification management, and the penalties for organic agricultural

Relationships between systolic time intervals and heart rate during initial response to orthostatic manoeuvre in men of different age. Electrophysiological investigation of the

Consistent with the negative price of systematic volatility risk found by the option pricing studies, we see lower average raw returns, CAPM alphas, and FF-3 alphas with higher

6 《中論·觀因緣品》,《佛藏要籍選刊》第 9 冊,上海古籍出版社 1994 年版,第 1

The first row shows the eyespot with white inner ring, black middle ring, and yellow outer ring in Bicyclus anynana.. The second row provides the eyespot with black inner ring

Teachers may consider the school’s aims and conditions or even the language environment to select the most appropriate approach according to students’ need and ability; or develop