Dengue fever among ill-returned travellers and concurrent infection by two dengue virus serotypes Khoa T.D. Thaia,b#, Josta A. Wismeijera, Michèle van Vugta,b, Katja C. Wolthersc and Peter J. de Vriesa,b a
Division of Infectious Diseases, Tropical Medicine & AIDS, Academic Medical Center, Amsterdam, The Netherlands
b
Center for Infection and Immunity (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c
Department of Medical Microbiology, Laboratory of Clinical Virology, Academic Medical Center, Amsterdam, The Netherlands
Abstract A sudden increase in dengue virus (DENV)-infected returned travellers was observed at the outpatient department of Tropical Medicine, Academic Medical Center, Amsterdam, The Netherlands. A descriptive observational study was conducted to analyse the epidemiology, clinical manifestations and laboratory features of imported DENV-infected patients. From September 2008 to June 2009, a total of 45 ill-returned travellers suspected for dengue were prospectively and four ill-returned travellers retrospectively were included. The majority (32 out of 49, 65%) of patients returned after a visit to the Dutch Antilles or Suriname. DENV-1, DENV-2 and DENV-3 were found in 27 viraemic patients. We identified four patients with a concurrent DENV infection with DENV-1 and DENV-2 serotypes and described their clinical and laboratory features. The clinical signs and symptoms in DENV-infected patients were mild and variable. Leukopenia and thrombocytopenia were observed between three to six days after the onset of illness. The majority of the patients had elevated serum transaminases levels between 7 to 10 days after the onset of illness. Within the first six months of 2009, ~10% were diagnosed with dengue infection. DENV infection at our hospital is not a rare imported viral disease. Increased international travel with changing epidemiology and increasing frequency of dengue in the sub-tropics will induce imported DENV infections in Western countries, including The Netherlands. Keywords: Dengue; epidemiology; concurrent; travellers; The Netherlands.
#
E-mail: t.d.thai@amc.uva.nl; Tel.: +31 20 5664380; Fax: +31 20 6972286 Dengue Bulletin – Volume 33, 2009
60
Dengue virus infection in Dutch travellers
Introduction The dengue virus (DENV) transmission takes place primarily through bites by the mosquito vectors, Aedes aegypti and Aedes albopictus. They feed preferentially on human blood and are often found in and around human dwellings.[1,2] Infection with any of the four DENV serotypes does not always cause symptoms. The most common clinical presentations are an undifferentiated febrile illness or more recognizable dengue fever (DF), but life-threatening manifestations such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) may also occur.[3] Dengue has become a major international public health problem due to its increasing geographical distribution. Dengue transition varies between countries, ranging from epidemic transmission with long interepidemic intervals to endemic with seasonal fluctuation.[4,5] Parallel to the global expansion of dengue is an increasingly common diagnosis among travellers who consult the Department of Tropical Medicine of the Academic Medical Center, Amsterdam, The Netherlands. Between 1985 and 1994, 34 travellers, 4% of all travellers with fever, were diagnosed with dengue by serological testing.[6,7] The incidence among short-term travellers to south-east Asia was 30 per 1000 person months. It was also found that not every DENV infection among travellers caused the clinical form of the disease and the majority of DENV infections in travellers remained mild.[8] From September 2008 onwards, a sudden increase in DENV infections was observed in travellers returning to The Netherlands from the Caribbean, mostly the Dutch Antilles and Suriname. Here we present the epidemiology,
clinical manifestations and laboratory findings of imported DENV infections among these travellers.
Methods Study site and study population Returning travellers presenting themselves at the Department of Tropical Medicine of the Academic Medical Center, with a history of ≤14 days of illness and who were suspected for dengue (based on signs, symptoms and travel history), were included in the study. We also included patients diagnosed with dengue retrospectively. (All patients attending our clinic are routinely informed that routinely collected clinical data may be used for scientific reporting. Patients are given the choice to not have their data included. None of the patients objected). Blood samples were collected from each patient for routine diagnostic procedures. All samples were tested by enzyme-linked immunosorbent assay (ELISA) and/or rapid immunochromatographic test (RDT) for antiDENV IgM and IgG antibodies and real-time polymerase chain reaction (RT-PCR). Patients with DENV infection, confirmed by serological and/or RT-PCR testing, were included in this study.
Dengue diagnostics Serum samples were tested for dengue with direct IgG enzyme-linked immunosorbent assay (ELISA) and IgM-Capture ELISA and/ or rapid immunochromatographic test (RDT) (Panbio Tech Co., Brisbane, Australia) according to the manufacturer’s instructions. RNA was isolated from plasma or serum as described elsewhere.[9] RNA was reverse transcribed, and DENV viraemia levels were assessed using
Dengue Bulletin – Volume 33, 2009
61
Dengue virus infection in Dutch travellers
an internally controlled, serotype-specific, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay that has been described elsewhere; the results were expressed as cDNA equivalents per ml of serum.[10] The RT-PCR and ELISA results were used for the diagnosis and classification of primary and secondary dengue infections.
Results Study population and travel history From September 2008 to June 2009, a total of 45 ill-returned travellers were prospectively included. In addition, four such travellers were retrospectively included based on their clinical presentation, duration of illness and travel history. The demographic information and travel history in those 49 ill-returned travellers by diagnosis are shown in Table 1. Figure 1A shows a sudden increase in DENVinfected patients. The majority (32 out of 49) had returned from a visit to the Dutch Antilles or Suriname.
Classification of primary and secondary dengue Acute primary DENV infections were confirmed by RT-PCR and/or dengue serum-specific IgM antibodies detection in acute samples, in the absence of both dengue serum-specific IgG antibodies. A negative first acute sample for dengue serum specific IgM and IgG antibodies with seroconversion for dengue serum-specific IgM antibodies within 7 to 14 days was also considered as primary dengue. Acute secondary DENV infections were confirmed on acute samples (≤14 days) with positive IgG results and detection of viral genome, either with or without detectable dengue serum-specific IgM antibodies. Patients who presented with both positive IgM and IgG results in the first acute sample, without viral genome detection by PCR, were assessed by the IgM/IgG ratio. A ratio of anti-dengue IgM to IgG ≥1.8 was the criterion for primary dengue infection and a ratio of IgM to IgG <1.8 was considered as secondary infection.[11]
Diagnosis of DENV infection parity and virological features Viral genomes were detectable in 27 out of 40 patients, of which 18 were classified as primary and 9 as secondary DENV infection. The viraemia levels ranged from 2.4 × 102/ mL to 1.6 × 109/mL (mean of 8.7 x 107/mL). Figure 1B shows the distribution of viraemia levels in ill-returned travellers.
Clinical and laboratory features The clinical manifestations of DENV infection in returned travellers are summarized in Table 2. Figures 2A and 2B show the laboratory features in the acute phase of illness. Leukopenia (<3.1 x 103/µL) and thrombocytopenia (<1.0 x 105/µL) was observed in 12 and 13 of the 40 DENV-infected patients in the acute phase (≤10 days of illness), respectively, and two patients had marked thrombocytopenia with a platelet count <5.0 x 104/µL. Among those patients tested, most had increased liver enzyme levels. Dengue Bulletin – Volume 33, 2009
Statistical analysis All calculations were performed using SPSS (version 17.0, SPSS Inc., Illinois) and SigmaPlot (version 11.0, Systat Software Inc., San Jose). The results were summarized in terms of means for continuous variables. For dichotomous variables, Fisher’s exact test was performed. A P -value of <0.05 was considered as statistically significant. 62
Dengue virus infection in Dutch travellers
Table 1: Demographic data of ill-returned travellers DF (n = 40) Male/female Age* Days ill at presentation* Dengue classification Primary Secondary DENV-1 DENV-2 DENV-3 DENV-1/DENV-2 Africa1 Asia2 Central America/Caribbean3 South America4 17/23 38.2 (17.4-73.6) 6.1 (0-13) 24 16 17 4 2 4 — 10 19 11 OFI (n = 9) 6/3 40.8 (26.3-64.0) 4.0 (0-9) — — — — — — 2 2 3 2 P NS NS NS
Serotype
Travel history
* mean (range) DF: Dengue fever; OFI: Other febrile illness; NS: not significant 1 Mauritius; 2Indonesia, Thailand, Cambodia; 3Aruba, Bonaire, Curacao, Dutch Antilles, Panama; 4Suriname
Figure 1: DENV-infected returned travellers with their virological features [(A) Number of confirmed DENV-infected patients by month presented at the Academic Medical Center, Amsterdam, The Netherlands, February 2008 – June 2009. (B) Distribution of viral loads of 27 DENV-infected patients with a single serotype by DENV infection parity. Four with dual DENV infection with DENV-1 and DENV-2, shown as DENV-2 as , respectively] and
Dengue Bulletin – Volume 33, 2009
63
Dengue virus infection in Dutch travellers
Table 2: Clinical presentations in 36 returned travellers with dengue fever Signs/symptoms Symptoms Fever Headache Muscle pain Retro-orbital pain Arthralgia Anorexia Nausea Diarrhoea Vomiting Fatigue Cough Signs Rash Petechiae Swollen red hands Hepatomegaly Frequency (%) (n=36*) 100 69 67 53 42 31 28 11 11 17 8 17 17 17 14
The mean WBC and platelet counts over the course of the disease decreased up to six days after the onset of illness and increased rapidly to normal values. The most abnormal values of WBC were observed between three and six days after the onset of illness, whereas the most abnormal aspartate transaminase (ASAT) and alanine transaminase (ALAT) values were found beyond seven days of illness. After 14 days of illness, the ASAT and ALAT values decreased, although the majority of values still exceeded the reference values.
Concurrent DENV infection by two serotypes Among the 27 viraemic DENV-infected returned travellers, there were four patients with a dual DENV-1 and DENV-2 infection. All had a primary DENV infection immune response and the viraemia levels of DENV-1 were higher than in DENV-2 (Figure 1B). Clinical and laboratory features are shown in
*four patients with concurrent DENV-1 and DENV-2 infection are presented in Table 3.
Figure 2: DENV-infected returned travellers with laboratory features [(A) WBC counts and platelets counts distribution by days after onset of illness. Data show mean value and standard error with upper and lower limits. (B) Distribution of ASAT and ALAT values by days after onset of illness. Data show mean value and standard error with upper and lower limits, 1 number of samples per time point.]
Platelet1 5 WBC1 5
22 21
A
20 18
9 8
10 13
ALAT
1
5 5
18 21
B
14 15
8 8
11 13
ASAT1
Cells x 103/µL
U/L 0-2 >15
600 500 400 300 200 100 10 5 0 11-14 3-6 7-10 Days of illness (groups) Maximum value Minimum value Upper & lower reference value
103
102
101
3-6 7-10 11-14 Days of illness (groups) Maximum value Minimum value Upper reference value
0-2
>15
64
Dengue Bulletin – Volume 33, 2009
Dengue virus infection in Dutch travellers
Table 3: Clinical and laboratory data of four DENV-infected patients with concurrent DENV-1 and DENV-2 Patients Age/sex Clinical symptoms 1 38/M Fever 2 43/M Fever, headache, myalgia 202 2.9 60.3 23.2 15.4 1.1 42 46 20 9.4 3 45/M Fever, retro-ortibal pain, myalgia, examthema 151 4.1 59.1 22.7 18.0 0.2 22 15 8.2 9.2 4 41/F Fever, retro-ortibal pain, headache, arthralgia, myalgia, erythema 128 1.5 65.4 26.8 7.8 0.0 31 19 4.7 8.1
Platelets (150-400 x 103/µL) White blood cell (2-7.2 x 103/µL) Neutrophil (50-60 %) Lymphocyte (25-30 %) Monocyte (2-10 %) Basophil (<1 %) ASAT (<40 U/L) ALAT (<50 U/L) C-reactive protein (<10 mg/L) Haemaglobulin (M: 8.5-11.0 mmol/L F: 7.5-10.0 mmol/L) Haematocritt (M: 0.41-0.51 L/L F: 0.36-0.47 L/L) Days after of illness
198 4.2 45.4 35.0 19.4 0.2 43 61 7.5 9.9
ND
0.45
0.44
0.38
2
1
1
2
M: male; F: female; ASAT: aspartate aminotransferase; ALAT: alanine aminotransferase.
Dengue Bulletin – Volume 33, 2009
65
Dengue virus infection in Dutch travellers
Table 3. Leukopenia was found in two patients and the WBC count differentials shows monocytosis (>6%) in all patients. Platelet counts were normal and transaminases were slightly elevated in three patients.
Discussion This study reports a sudden increase of DENV infection in returning travellers who presented at the Academic Medical Center, which has not been documented before in The Netherlands. The majority (65%) of patients returned after a visit to the Dutch Antilles or Suriname. DENV-1, DENV-2 and DENV-3 were found in travellers returning from this area, which is suggestive of co-circulation of multiple DENV serotypes in those areas. Clinical signs and symptoms in the DENVinfected patients were mild and variable. The majority of them had elevated transaminases (ASAT and ALAT) with the most abnormal levels between 7 to 10 days post onset of illness. Leukopenia and thrombocytopenia were observed between three to six days after onset of illness. Among the 27 viraemic patients, we identified four patients with a concurrent DENV infection with DENV-1 and DENV-2 serotypes and described their clinical and laboratory features. Due to the spread of the vector, the geographical distribution of dengue is expanding rapidly in tropical and subtropical countries. [12] Growing international travel coupled with increasing transmission or reemergence and changing epidemiology of dengue in various sub-tropical countries may have resulted in a steady rise in confirmed DENV infection among ill-returned travellers who presented at our hospital. In this present study, we have shown a sudden increase in DENV infection in travellers returning from the
Caribbean, including Aruba, Bonaire, Curacao, Dutch Antilles, Panama and Suriname. Reports of dengue infection in travellers from southeast Asia are by far more frequent than from the Americas.[13] This difference may reflect different travel patterns and destinations but also differences in local transmission intensities. Depending on the study design, the study population and the destinations, the prevalence of dengue in travellers varies. [14-16] A previous prospective study at our hospital showed that the incidence rate among Dutch short-term travellers with destinations in endemic areas in Asia was 30 per 1000 person-months, with a clinical/subclinical infection ratio of 1:3.3.[8] In long-term travelers, with a median of 5.3 months’ stay in endemic countries, it showed a seroconversion rate of 6.7%.[17] The incidence rates may differ during epidemics. A high attack rate (69%) during a DENV epidemic among aid workers was reported on a Caribbean island.[18] Variable attack rates and clinical/subclinical ratios may occur, possibly reflecting the role of different virulent virus strains, the importance of initial viral load or entomological or host factors. Most of the clinical manifestations and laboratory features presented in our study agree with the previous reports. [14,15,19,20] Fever, headache (mostly frontal or retroorbital), arthralgia, rash and myalgia were important clinical characteristics; however, swollen red hands have never been observed. The most helpful laboratory results were: leukopenia, thrombocytopenia and increased transaminases. All patients were managed as outpatients and elevated transaminases were found in the majority, including a few patients who showed values higher than ten times the upper limit. The rapid spread of dengue, coupled with co-circulation of multiple DENV serotypes,
66
Dengue Bulletin – Volume 33, 2009
Dengue virus infection in Dutch travellers
have been increasingly reported in the last six decades. [5] Despite the geographical expansion, reports of simultaneous infection of patients with more than one DENV serotype remain low in comparison with the frequency of DENV infections. Concurrent infection with multiple DENV serotypes has been reported in several countries, mainly from south-east Asia and South America. [21-25] Among the DENV-infected patients in this study, four patients with concurrent DENV infection with DENV-1 and DENV-2 serotypes were detected by RT-PCR assay. All had a primary immune response against DENV. Clinical presentation and laboratory features (data not shown) were similar to other DENV-infected returned travellers. The RT-PCR assay used in this study detected all four DENV serotypes and is used extensively in many other studies. However, we did not attempt other techniques, e.g. virus isolation, to support our findings because RTPCR techniques have been shown to be the most robust methods and are more specific than any other viral detection method.[26]
Conclusion This descriptive study shows the sudden rise in the number of DENV-infected travellers returning from the Dutch Antilles and Suriname. It also records the co-circulation of multiple DENV by detecting DENV-1, DENV-2 and DENV-3 in the ill-returned travelers. Most interestingly, we showed the concurrent infection with DENV-1 and DENV-2 serotypes. The growth in international travel, with changing epidemiology and increasing incidence of dengue in the tropics, will lead to an increase in imported DENV infections in Western countries, including The Netherlands. DENV infection at our hospital, therefore, is not a rare occurrence of imported viral disease.
Acknowledgements K.T.D. Thai is supported by a ‘Mosaic’ fellowship from The Netherlands Organization for Scientific Research (NWO).
References [1] Harrington LC, Edman JD, Scott TW. Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood? J Med Entomol 2001;38(3):411-422. [2] Scott TW, Amerasinghe PH, Morrison AC, Lorenz LH, Clark GG, Strickman D, Kittayapong P , Edman JD. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) In Thailand and Puerto Rico: Blood feeding frequency. J Med Entomol 2000;37(1):89-101. [3] Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P , Sanders EJ, Vorndam AV. Dengue and dengue haemorrhagic fever. Lancet 1998;352(9132):971-977. [4] Gibbons RV, Vaughn DW. Dengue: an escalating problem. BMJ. 2002;324(7353):1563-1566. [5] Guzman MG, Kouri G. Dengue: An update. Lancet Infect Dis 2002;2(1):33-42. [6] Bakker RC, Veenstra J, Dingemans-Dumas AM, Wetsteyn JCFM, Kager PA. Imported dengue in the Netherlands. J Travel Med 1996;3(4):204-208. [7] Wetsteyn JC, Driessen SO, de Vries PJ, Cobelens FG, Kager PA. [Tropical diseases and imported disorders in 1763 patients seen at the Outpatient Clinic for Tropical Diseases, Academic Medical Center, Amsterdam (1996-1997)]. Ned Tijdschr Geneeskd . 2000;144(45):2152-2156.
Dengue Bulletin – Volume 33, 2009
67
Dengue virus infection in Dutch travellers
[8] Cobelens FG, Groen J, Osterhaus AD, Leentvaar-Kuipers A, Wertheim-van Dillen PM, Kager PA. Incidence and risk factors of probable dengue virus infection among Dutch travellers to Asia. Trop Med Int Health 2002;7(4):331-338. [9] Boom R, Sol C, Beld M, Weel J, Goudsmit J, Wertheim-van Dillen P . I Improved silicaguanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 1999;37(3):615-619. [10] Laue T, Emmerich P , Schmitz H. Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system. J Clin Microbiol 1999;37(8):2543-2547. [11] Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S, Puttisri P , Hoke CH. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 1989;40(4):418-427. [12] Kroeger A, Nathan MB. Dengue: setting the global research agenda. Lancet 2006;368(9554):2193-2195. [13] Schwartz E, Weld LH, Wilder-Smith A, von Sonnenburg F, Keystone JS, Kain KC, Torresi J, Freedman DO; GeoSentinel Surveillance Network. Seasonality, annual trends, and characteristics of dengue among ill returned travelers, 1997-2006. Emerg Infect Dis 2008;14(7):1081-1088. [14] Jelinek T, Dobler G, Hölscher M, Löscher T, Nothdurft HD. Prevalence of infection with dengue virus among international travelers. Arch Intern Med 1997;157(20):2367-2370. [15] López-Vélez R, Pérez-Casas C, Vorndam AV, Rigau J. Dengue in Spanish travelers returning from the tropics. Eur J Clin Microbiol Infect Dis 1996;15(10):823-826.
[16] S c h w a r t z E , M e n d e l s o n E , S i d i Y. Dengue fever among travelers. Am J Med 1996;101(5):516-520. [17] Potasman I, Srugo I, Schwartz E. Dengue seroconversion among Israeli travelers to tropical countries. Emerg Infect Dis 1999;5(6):824-827. [18] Lyerla R, Rigau-Pérez JG, Vorndam AV, Reiter P , George AM, Potter IM, Gubler DJ. A dengue outbreak among camp participants in a Caribbean island, 1995. J Travel Med 2000;7(2):59-63. [19] Badiaga S, Delmont J, Brouqui P , Janbon F, Durant J, Bosseray A, Malvy D, Bonnet E, Sotto A, Dydymski S, Peyramond D. [Imported dengue: study of 44 cases observed from 1994 to 1997 in 9 university hospital centers. Infectio-Sud-France group]. Pathol Biol (Paris) 1999;47(5):539-542. [20] Badiaga S, Barrau K, Brouqui P , Durant J, Malvy D, Janbon F, Bonnet E, Bosseray A, Sotto A, Peyramont D, Dydymski S, Cazorla C, Tolou H, Durant JP , Delmont J; InfectioSud Group. Imported Dengue in French University Hospitals: a 6-year survey. J Travel Med 2003;10(5):286-289. [21] Gubler DJ, Kuno G, Sather GE, Waterman SH. A case of natural concurrent human infection with two dengue viruses. Am J Trop Med Hyg 1985;34(1):170-173. [22] Kukreti H, Chaudhary A, Rautela RS, Anand R, Mittal V, Chhabra M, Bhattacharya D, Lal S, Rai A. Emergence of an independent lineage of dengue virus type 1 (DENV-1) and its cocirculation with predominant DENV-3 during the 2006 dengue fever outbreak in Delhi. Int J Infect Dis 2008;12(5):542-549. [23] Wang WK, Chao DY, Lin SR, King CC, Chang SC. Concurrent infections by two dengue virus serotypes among dengue patients in Taiwan. J Microbiol Immunol Infect 2003;36(2):89-95.
68
Dengue Bulletin – Volume 33, 2009
Dengue virus infection in Dutch travellers
[24] Wenming P , Man Y, Baochang F, Yongqiang D, Tao J, Hongyuan D, Ede Q. Simultaneous infection with dengue 2 and 3 viruses in a Chinese patient return from Sri Lanka. J Clin Virol 2005;32(3):194-198. [25] Wilder-Smith A, Yoksan S, Earnest A, Subramaniam R, Paton NI. Serological
evidence for the co-circulation of multiple dengue virus serotypes in Singapore. Epidemiol Infect 2005;133(4):667-671. [26] Wilder-Smith A, Schwartz E. Dengue in travelers. N Engl J Med 2005;353(9):924-932.
Dengue Bulletin – Volume 33, 2009
69