Incidence and risk factors of probable dengue virus infection among Dutch travellers to Asia

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1 Tropical Medicine and International Health volume 7 no 4 pp april 2002 Incidence and risk factors of probable dengue virus infection among Dutch travellers to Asia Frank G. J. Cobelens 1, Jan Groen 2, Albert D. M. E. Osterhaus 2, Anne Leentvaar-Kuipers 3, Pauline M. E. Wertheim-van Dillen 4 and Piet A. Kager 1 1 Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, The Netherlands 2 Laboratory of Exotic Viral Infections, Institute of Virology, Erasmus Medical Center, Rotterdam, The Netherlands 3 Department of Infectious Diseases, Section of Public Health and Environment, Municipal Health Service, Amsterdam, The Netherlands 4 Department of Clinical Virology, Academic Medical Center, Amsterdam, The Netherlands Summary We studied the incidence of dengue virus (DEN) infections in a cohort of Dutch short-term travellers to endemic areas in Asia during Sera were collected before and after travel. All post-travel sera were tested for DEN immunoglobulin M (IgM) [IgM capture (MAC)-enzyme-linked immunosorbent assay (ELISA)] and IgG (indirect ELISA). Probable DEN infection was defined as IgM seroconversion or a fourfold rise in IgG ratio in the absence of cross-reaction with antibody to Japanese encephalitis virus (JEV). Infections were considered clinically apparent in case of febrile illness (> 24 H) with headache, myalgia, arthralgia or rash. Probable DEN infection was found in 13 of 447 travellers (incidence rate 30/1000 person-months, 95% CI ). One infection was considered secondary; no haemorrhagic fever occurred. The clinical-to-subclinical infection rate was 1 : 3.3. The risk of infection showed marked seasonal variation. DEN infections are frequent in travellers to endemic areas in Asia; most remain subclinical. keywords dengue, travel, epidemiology correspondence Frank G. J. Cobelens MD PhD, Royal Netherlands Tuberculosis Association (KNCV), Riouwstraat 7, 2585 GP The Hague, The Netherlands. Fax: ; cobelensf@kncvtbc.nl Introduction Dengue is an acute febrile disease caused by infection with any of four serotypes of dengue virus (DEN), a flavivirus. It is a public health problem of growing importance in areas where the insect vector, Aedes aegypti and related species, is abundant (Rigau-Perez et al. 1998). Although most DEN infections present as a benign illness with low mortality, many countries report increasing incidences of severe manifestations, i.e. dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Gubler & Clark 1995). DEN infections have been identified as a cause of febrile illness in travellers. Depending on the population studied and the laboratory methods used, serological evidence of recent DEN infection was found in 7 45% of cases of fever in travellers returning from endemic areas (Lopez-Velez et al. 1996; Schwartz et al. 1996; Yabe et al. 1996; Jelinek et al. 1997). In a retrospective study, 4.3% of German expatriates who had worked in endemic areas for 10 years on average had a DEN IgG response (Jänisch et al. 1997). A prospective study of 104 long-term travellers from Israel to various dengue-endemic countries found a seroconversion rate of 6.7% (Potasman et al. 1999), but there are no data on incidences of DEN infections in short-term travellers. In addition, little is known about the clinical spectrum of DEN infections in this population. In (hyper)endemic areas, an estimated 14 87% of DEN infections were present with few or atypical symptoms (Waterman et al. 1985; Burke et al. 1988; McBride et al. 1998). No data exist on the proportion of subclinical DEN infections in persons from non-endemic areas. The possibility of subclinical infection in travellers seems important, as it has been suggested that infection with DEN predisposes to DHF and DSS when subsequent infection with another serotype occurs (Halstead 1988). A problem in interpreting serologic data on dengue is the broad ª 2002 Blackwell Science Ltd 331

2 cross-reactivity with other flavivirus species (Rigau-Perez et al. 1998). In Asia, the most important is Japanese encephalitis virus (JEV). Clinical JE in travellers is rare: the crude attack rate for travellers to south-east Asia is estimated at 1 in 1 million travel episodes (CDC 1993). However, most JEV infections are non-symptomatic, with an estimated clinical-to-subclinical infection rate of 1 : 60 among non-asians (CDC 1993). Therefore, subclinical or mild JE, or possibly other flavivirus infections, may account for some of the serologic diagnoses of dengue in travellers to Asia. To define incidences and risk factors of clinical and subclinical DEN infections in travellers to Asia, we assessed seroconversion to DEN antibodies and possible cross-reactions with JEV using pre- and post-travel serum samples from a cohort of Dutch short-term travellers to dengue endemic areas in south and south-east Asia. Subjects and methods Study population The study was conducted at the travel immunization clinic of the Amsterdam Municipal Health Service (GG & GD) between February 1991 and February Eligible for inclusion were all travellers of 15 years and older who intended to travel to (sub)tropical destinations in Asia for 1 13 weeks. For practical reasons (sub)tropical destinations were defined as all areas in Asia for which malaria chemoprophylaxis was advised, excluding those west of Pakistan (WHO 1991). The study was approved by the GG & GD s ethical review committee. Questionnaires Written informed consent was obtained from all study participants. They completed a questionnaire and submitted a serum sample shortly before departure and 1 6 weeks after return. The questionnaires addressed, among other issues, travel history, medical history, travel characteristics and febrile episodes. Travel duration was recorded in weeks, and travel destination as the country where most of the travel time was spent. Serological tests Sera were stored at )70 C. All post-travel sera were screened for DEN-specific antibodies with a commercial IgM-capture enzyme-linked immunosorbent assay (MAC- ELISA) and an IgG ELISA (MRL Diagnostics, Cypress, CA, USA). This DEN-specific ELISA system was chosen after extensive evaluation with other commercially available DEN immunoassays (Groen et al. 2000). The OD values for both tests were measured at 450 nm with 620 nm as a reference. Results were expressed as ratios (OD test serum/od calibration serum) and considered positive for ratios >1.0. Of all subjects whose post-travel samples tested positive for IgM or for IgG with an IgG ratio >2, pre-travel samples were tested for DEN-specific IgM and IgG using the same ELISAs. If these pre-travel samples were IgM-negative or a fourfold rise in IgG ratio was observed, the post-travel samples were also tested for IgM and IgG antibodies specific to JEV, using a MAC- ELISA and an indirect IgG ELISA, respectively, as described for DEN, but using JEV instead of DEN antigen (Velzing et al. 1999). Briefly, for the MAC-ELISA, microplates (Costar, Cambridge, MA, USA) were coated with 50 ll cross-reactive anti-igm antibodies for 18 h in 0.1 M carbonate/bicarbonate buffer ph 9.6. After washing three times with phosphate buffered saline (PBS) containing 0.5% Tween-20 (PBS-T), 50 ll of 1 : 100 diluted serum in PBS supplemented with 1% bovine serum albumin (BSA), 0.1% low fat milk powder and 1% foetal calf serum (PBS-BMC) were added and incubated for 2 h at 37 C. The plates were washed three times with PBS-T before 50 ll JEV antigen (Seiken, Tokyo, Japan) was added. After incubation for 1 h at 37 C and washing, antigen-binding antibody was detected by adding 50 ll JEV hyper immune serum 1 : 100 diluted in PBS- BMC. Incubation for 1 h at 37 C and washing was followed by incubation with 50 ll goat antimouse horse radish peroxidase (HRPO) labelled antibody (Dakopatts, Glostrup, Denmark) 1 : 1000 diluted in PBS-BMC. After three additional wash cycles with PBS-T, 100 ll TMB (3,3,5,5 tetramethylbenzidine) was added as a substrate and colour was allowed to develop for 30 min. The reaction was stopped by adding 100 ll 1M H 2 SO 4 to each well. For the IgG ELISA, an optimal concentration of JEV antigen (Seiken, Tokyo, Japan) per well was coated onto a microplate (Costar, Cambridge, MA, USA) in PBS. After incubation overnight at 4 C, the plates were washed three times with PBS-BMC. Serum was diluted 1 : 100 in PBS- BMC. A 100 ll of diluted plasma was incubated for 1 h at 37 C. Three wash cycles with PBS-T were followed by incubation for 1 h at 37 C of 100 ll peroxidase labelled anti-igg (Dakopatts, Glostrup, Denmark) diluted 1 : 4000 in PBS-BMC. After washing three times with PBS-T, 100 ll TMB was added as a substrate and colour was allowed to develop for 30 min. The reaction was stopped by adding 100 ll 1M H 2 SO 4. The OD values for both tests were measured at 450 nm with 620 nm as a reference. Results were expressed as ratios (OD test serum/ OD 3 negative control sample), tests were considered positive for ratios >1.1. All tests were performed on paired samples. 332 ª 2002 Blackwell Science Ltd

3 Definitions Dengue virus infection was defined as serocoversion for DEN antibodies, i.e. (1) a post-travel sample positive for DEN IgM with a negative pre-travel sample and the DEN IgM ratio exceeding the JEV IgM ratio, or (2) a fourfold rise in DEN IgG titres with a negative post-travel JEV IgM test and the post-travel DEN IgG ratio exceeding the JEV IgG ratio. Infections were not confirmed by virus isolation or plaquereduction neutralization test, and therefore considered probable DEN infections (WHO 1997). Primary infection was defined as a post-travel DEN IgM ratio exceeding the DEN IgG ratio; secondary infection was considered to have occurred if the IgG ratio was the largest (WHO 1997). Dengue-like illness was defined as any episode of illness with (subjective) fever of more than 24 h duration, occurring between departure and the post-travel follow-up visit, and accompanied by headache, myalgia, arthralgia or rash. Infections were considered clinical if at least one episode of dengue-like illness was reported and subclinical if otherwise. This definition of low specificity was adopted as it would result in a conservative estimate of the proportion of subclinical infections. Data analysis Data were analysed with Stata (version 4.0; Stata Corp., College Station, TX, USA). For comparison of categorized variables, chi-squared tests or, for 2 2 tables, two-sided Fisher s exact tests, were used. Incidences were expressed as incidence rates (IRs) per 1000 person-months of travel by dividing the number of seroconversions by the total number of weeks travelled divided by 4.35 minus half of the person-time denominator (PTD) for seroconverted subjects, as infections were assumed to have occurred halfway through the journey on average. For calculation of destination-specific IRs it was assumed that the PTD for each country was on average equal to the total PTD for those travellers who had that country as their primary destination. Seasonal distributions were assessed by calculating attack rates (ARs) and 3-month moving average ARs based on the mid-journey dates. Exact binomial or Poisson 95% confidence intervals (CIs) were calculated. Risk factors for seroconversion were assessed in the first step by univariate analysis of rate ratios based on IRs. Variables showing (near-)significant univariate association with the risk of seroconversion were subsequently assessed by nested matched case control analysis. For this, each seroconverted subject (case) was compared with four randomly selected non-converted subjects (controls), matched for primary country of destination and travel month (±1, based on mid-journey dates) using conditional logistic regression modelling (Clayton & Hills 1993). The models included travel duration as a covariate; significance testing was based on the likelihood ratio (LR) test for exclusion of the covariate from the model. All tests were carried out at a significance level of Results Initially 502 persons were included who intended to travel to Asia. Excluded were 18 subjects for loss to follow-up (3.6%), 28 (5.6%) for changing their travel plans such that they did not meet the inclusion criteria, and 11 for missing post-travel serum samples, thus leaving 447 subjects (89.0%) for the analysis. Table 1 shows their characteristics. None had received yellow fever vaccination within 3 months prior to departure. Of the post-travel samples, 86.1% had been obtained between 15 and 28 days after return, 6.3% earlier and 7.6% later. Table 1 Characteristics of 447 short-term travellers to (sub)-tropical areas in Asia Characteristic n (%) Males 213 (47.6) Females 234 (52.4) Mean age (SD) (years) 39.6 (12.5) Median, range 36 (20 79) Childhood in (sub)tropics 50 (11.2) Previous time in (sub)tropics (months)* None 110 (24.7) <3 156 (35.0) (22.6) >12 79 (17.7) Primary purpose of travel Tourism 418 (93.5) Visits of relatives and friends 15 (3.4) Professional 14 (3.1) Primary country visited Indonesia 233 (52.1) India 71 (15.9) Thailand 66 (14.8) Sri Lanka 21 (4.7) China 15 (3.4) Malaysia 10 (2.2) Other 31 (6.9) Organized travelà No 267 (59.7) Yes 180 (40.3) Mean duration of travel (weeks) 4.3 ± 1.9 Median (range) 4 (1 12) *After age 10 years. Country were most travel time was spent. àmainly group travel. ª 2002 Blackwell Science Ltd 333

4 DEN antibodies were detected in 96 post-travel sera (21.5%), including IgG in 79 (17.7%), IgM in 13 (2.9%) and both IgG and IgM in four (0.9%). Of 17 subjects with IgM-positive post-travel sera, 13 (76.5%) had IgM-negative pre-travel sera. All 13 had post-travel JEV IgM ratios that were lower than the DEN IgM ratios. Of the 83 subjects whose post-travel samples were positive for DEN IgG, 43 had IgG ratios >2.0. Of these, pre-travel samples were also tested for DEN IgG; none had a more than fourfold rise in DEN IgG ratio. Thus, 13 of 447 subjects had serological evidence of probable DEN infection (2.9%; 95% CI ). Twelve of these were considered primary infections, and one secondary. Dengue-like illness was reported by three of these 13 subjects (23.1%, 95% CI ). All episodes had started during travel. One had symptoms for 2 7 days, and two for more that 7 days. These included headache (3), myalgia or arthralgia (2), prostration (2), rash (1) and mild diarrhoea (1). The temperature had been taken by two and was above 38.5 C in both. Symptoms compatible with DHF were not reported. Based on this definition for clinical infection, the clinical to subclinical infection rate was 1 : 3.3 (95% CI 1 : : 12.0). Among all 13 seroconverted subjects, the risk of developing dengue-like illness showed no significant association with age, sex, travel destination or season, number of previous journeys, country of childhood or pre-travel DEN IgG titre (data not shown). The overall IRs were 30.0/1000 person-months of travel (pmt) (95% CI ) for probable DEN infection, and 6.9/1000 pmt (95% CI ) for clinical infection. The primary destination of two of the three subjects with clinical infection had been Indonesia, and India for 1. Of 10 subjects with subclinical or atypical infections, the primary destination had been Indonesia for seven, Thailand for two and Sri Lanka for one. Destination-specific IRs for probable DEN infection ranged from 0 to 78 per 1000 pmt (Table 2); differences were not significant. The AR of probable DEN infection, based on the mid-journey date, increased during the last quarter of 1991 (two-sided Fisher s exact test; P ¼ 0.019) compared with the remaining study period. This was nearsignificantly increased among travellers to the Indian subcontinent (P ¼ 0.055) and the Western Pacific (P ¼ 0.051), but not for the south-east Asian region (P ¼ 1.000). A graph of 3 months moving average ARs (Fig. 1) also showed peaks during the periods September November for the Indian subcontinent, and October December for the western Pacific region, but not for southeast Asia. The risk of probable DEN infection, based on the incidence rate ratios (IRRs), was not associated with age, sex, travel purpose, organized vs. self-arranged travel, geographical region and type of accommodation, although the risk was significantly reduced for use of guesthouses (P ¼ 0.016; Table 3). It was also near-significantly reduced among subjects with a history of previous travel to tropical destinations (P ¼ 0.052). The risk reduction was associated with the number of previous journeys (v 2 -test for trend, P ¼ 0.03) and significant if expressed as any history of such travel compared with none (IRR 0.31, P ¼ 0.028). In addition, rates differed near-significantly by season (P ¼ 0.063); the seroconversion rate during the last quarter was nearly fourfold increased compared with all other seasons together (IRR 3.77; P ¼ 0.010). The effect of previous travel and accommodation was further explored by adjustment for potential confounding in a multivariate matched case control analysis, including 13 seronconverted and 52 non-seroconverted subjects, individually matched for country of primary destination and travel month. In a conditional logistic regression model that included travel duration as an additional Table 2 Attack rates and incidence rates of seroconversions for DEN antibodies by primary destination in 447 Dutch travellers to Asia Primary destination Person-months Seroconversions in all travellers Attack rate (%) (95% CI)* Incidence rate per 1000 pmt (95% CI) Indonesia / ( ) 37.4 ( ) India / ( ) 13.9 ( ) Thailand / ( ) 36.1 ( ) Sri Lanka / ( ) 77.6 ( ) China /15 0 (0 21.8) 0 ( ) Malaysia 9.2 0/10 0 (0 30.9) 0 ( ) Other /31 0 (0 11.2) 0 ( ) Total / ( ) 30.0 ( ) Test for unequal incidence rates: v 2 ¼ 3.69 (d.f. ¼ 12), P ¼ *95% Confidence interval. Person-months of travel. 334 ª 2002 Blackwell Science Ltd

5 Figure 1 3-month moving average attack rates of dengue virus infection, per 100 travelers to 3 regions in Asia. covariate, a history of any travel to tropical destinations remained near-significantly associated with a reduced risk of DEN seroconversion (OR 0.27, 95% CI , LR test P ¼ 0.075). This reduction was again associated with the number of previous journeys (three or more journeys 1 2 vs. none: OR 0.40, LR test P ¼ 0.035). The protective effect of previous travel was unaffected by additional adjustment for age, sex, travel purpose and organized vs. self-arranged travel (data not shown). Use of guesthouses showed no association with the risk of seroconversion in the multivariate analysis (OR 0.56, LR test P ¼ 0.408). Discussion This study suggests that incidences of DEN infection in short-term travellers to endemic parts of Asia are substantial (between 17 and 52/1000 pmt) and comparable with incidences of falciparum malaria in travellers to sub- Saharan Africa or hepatitis A in backpackers to India (Steffen et al. 1990, 1994). The incidence was higher than that derived from the 12% DEN IgG prevalence found with an immunofluorescence test among German expatriate workers after an average stay of 10 years in south-east Asia (Jänisch et al. 1997), and from the attack rate of 6.7% found with an ELISA system among Israeli travellers who stayed in dengue-endemic areas for an average of 6.1 months (Potasman et al. 1999). These differences may be the result of differences in test characteristics, exposure and pre-existing immunity. The incidence of clinical dengue (7/1000 pmt) is also higher than the estimated 1/1000 derived from retrospective studies of expatriates in Bangkok and US troops in Somalia and Haiti (Halstead et al. 1969; Sharp et al. 1994; Trofa et al. 1997; Rigau-Perez et al. 1998). This difference could in part be explained by the rather loose definition we used for dengue-like illness, as some of the febrile episodes among DEN seroconverters may in fact have been caused by other pathogens. The IgM-capture ELISA is a suitable tool for identifying recent DEN infections (Kuno et al. 1991) and the test used has shown high sensitivity and specificity (Velzing et al. 1999). As most dengue patients have lost IgM antibody days after the onset of disease (Gubler 1988), we could have missed seroconversions in subjects who were infected early on a long trip had we tested for this class of antibody only. We therefore also looked for fourfold rises in DEN IgG ratio in the absence of DEN IgM, but found none. With the tests used, cross-reactions with antibodies to other flaviviruses may occur (Rigau-Perez et al. 1998). As we did not confirm the serology results with a plaquereduction neutralization test, the seroconversions in this study should be considered probable DEN infections (WHO 1997). Subclinical infections with other flaviviruses endemic in the study area may account for some of the seroconversions, particularly mosquito-borne infections such as with West Nile (Indian subcontinent) and Kunjin (mainly Sarawak) viruses (Price & O Leary 1967; Ching et al. 1970; Kanamitsu et al. 1979). Infections with JEV were excluded as all post-travel DEN IgM ratios exceeded those for JEV IgM (Innis et al. 1989). In addition, none of the participants had been vaccinated against yellow fever (YF) shortly before or after the pre-travel sample was drawn, which excludes cross-reactions with YF vaccineinduced IgM antibodies. Cross-reactivity is certainly involved in the four of 17 IgM-positive pre-travel samples. Although we could not exclude recent exposure to flaviviruses common in Europe, such as Central European encephalitis, this is most likely of ª 2002 Blackwell Science Ltd 335

6 Table 3 Incidence rates and crude rate ratios of seroconversion to DEN antibodies, by selected potential risk factors Potential risk factor n* Person-months Rate per 1000 pmt (95% CI)à Rate ratio (95% CI)à P Age (years) < ( ) 1.36 ( ) ( ) P ( ) 1.24 ( ) Sex Male ( ) Female ( ) 1.47 ( ) Travelled before No ( ) journeys ( ) 0.47 ( ) P3 journeys ( ) 0.17 ( ) Travel purpose** Tourism ( ) 1 Visit ( ) 2.21 ( ) Professional ( ) Organized travelàà Not organized ( ) Organized ( ) 1.24 ( ) Accommodation With locals ( ) 1.20 ( ) Guesthouses ( ) 0.28 ( ) Large hotels ( ) 1.55 ( ) Camped ( ) 1.90 ( ) Travel season January March ( ) 1 April June ( ) 1.51 ( ) July September ( ) 2.75 ( ) October December ( ) 6.99 ( ) Region Indian subcontinent ( ) 1 South-east Asia ( ) 1.20 ( ) Western Pacific ( ) 1.88 ( ) * Number of probable dengue infections. Person-months of travel. à 95% Confidence interval. Travel to tropical destinations only. v 2 -test for trend, P ¼ ** Purpose on which most of the travel time was spent. One-sided, 97.5% CI. àà Accommodation and transport arranged by others. Type of accommodation used at least once, compared with not used (IRR ¼ 1). Based on mid-journey date. non-specific nature. Such cross-reactivity may account for unexpected findings of recent DEN infection in retrospective studies of travellers of whom no pre-travel samples were tested. The DEN IgG prevalence was much higher, but part of this reflects cross-reaction with antibodies to YF virus induced by vaccination in the past (Hatgi et al. 1966; Jänisch et al. 1997). There were no significant differences in seroconversion rate by travel destination, but numbers were small. We observed marked seasonal variation for the Indian 336 ª 2002 Blackwell Science Ltd

7 subcontinent and the western Pacific region (primarily Indonesia), where incidences peaked during the second and first half, respectively, of the rainy seasons. This reflects the peak periods of DEN transmission in many endemic areas, when vector populations will be at their maximum (Kuno 1995). However, in south-east Asia dengue outbreaks have been described outside the rainy season (Eamchan et al. 1989; Kuno 1995), which may explain the few seroconversions in travellers who visited this region during the rest of the study period. As DEN transmission may vary not only by season, but also from year to year, our data may over- or underestimate the risk for travellers for other years. Gross differences are unlikely, as surveillance data from the United States showed no important differences in numbers of dengue cases imported from Asia for 1991 compared with previous and subsequent years (CDC 1992; Rigau-Perez et al. 1994). Of the other host and travel-related factors studied, none predicted the risk of probable DEN infection, except perhaps previous travel. In a multivariate matched analysis controlling for differences in destination, travel season and duration of the journey, there was an inverse association between risk of seroconversion and previous travel to tropical destinations. Although this protective effect was just short of statistical significance, it was associated with the number of journeys, suggesting a true effect reflecting immunity by previous infection with the homologous DEN serotype or, possibly, short-lasting immunity because of recent infection with heterologous serotypes (Gubler 1988). Our data indicate that at least half (46 95%) of DEN infections in travellers remain subclinical, which is even a conservative estimate, as we used a case definition of dengue-like illness of rather low specificity. Studies among local populations have shown varying apparent-to-inapparent infection rates (AIIR) of 1 : 2 in Puerto Rico, 1 : 7 in Thailand and 1 : 0.2 in northern Australia (Waterman et al. 1985; Burke et al. 1988; McBride et al. 1998). This variation may be because of differences in (cross-)immunity by infection with other DEN serotypes or other flaviviruses, but also in virulence of the infecting DEN strain (Gubler 1988). During the study period several DEN strains circulated in Asia (Gubler & Clark 1995). The AIIR among travellers suggested by our data thus reflects varying underlying rates for the countries visited, and may be different for travellers to other dengue endemic areas during other years. But it does suggest that the proportion of travellers returning from endemic areas with antibodies to dengue infection is much larger than would be expected from incidences of clinical dengue, which is also supported by our finding of one secondary DEN infection in our study population (IR 2.3/1000 pmt, 95% CI ). This may be important, as non-neutralizing antibodies produced upon primary DEN infection may enhance subsequent infection with a heterologous serotype and lead to DHF and DSS (Halstead 1988). Although this immune enhancement theory has never been confirmed in vivo, and other factors, including the infecting strain, are probably decisive in the development of DHF/DSS (Rigau-Perez et al. 1998), increased frequency of travel to areas where various serotypes circulate may put travellers at risk for severe infection. 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