Laboratory and surveillance studies following a suspected Dengue case in Greece, 2012

Accepted Manuscript

Title: Laboratory and surveillance studies following asuspected Dengue case in Greece, 2012

Author: Anna Papa Elpida Gavana Marios Detsis EiriniTerzaki Lambrini Veneti Danai Pervanidou TheanoGeorgakopoulou Markos Marangos George KoliopoulosAgoritsa Baka Sotirios Tsiodras Athanasios Tsakris ChristosHadjichristodoulou

PII: S1201-9712(14)01708-1DOI: http://dx.doi.org/doi:10.1016/j.ijid.2014.11.019Reference: IJID 2180

To appear in: International Journal of Infectious Diseases

Received date: 25-8-2014Revised date: 18-9-2014Accepted date: 22-11-2014

Please cite this article as: Papa A, Gavana E, Detsis M, Terzaki E, Veneti L, PervanidouD, Georgakopoulou T, Marangos M, Koliopoulos G, Baka A, Tsiodras S, TsakrisA, Hadjichristodoulou C, Laboratory and surveillance studies following a suspectedDengue case in Greece, 2012, International Journal of Infectious Diseases (2014),http://dx.doi.org/10.1016/j.ijid.2014.11.019

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http://dx.doi.org/doi:10.1016/j.ijid.2014.11.019

http://dx.doi.org/10.1016/j.ijid.2014.11.019

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Laboratory and surveillance studies following a suspected Dengue case in 1

Greece, 2012 2

Anna Papa1*, Elpida Gavana1, Marios Detsis2, Eirini Terzaki2, Lambrini Veneti2, 3

Danai Pervanidou2, Theano Georgakopoulou2, Markos Marangos3, George 4

Koliopoulos4, Agoritsa Baka2, Sotirios Tsiodras2, Athanasios Tsakris5, Christos 5

Hadjichristodoulou2,6 6

7

1 National Reference Centre for Arboviruses, Department of Microbiology, Medical 8

School, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece 9

2 Hellenic Centre for Disease Control and Prevention (HCDCP), Athens 15123, 10

Greece 11

3 Division of Infectious Diseases, Department of Internal Medicine, University 12

Hospital of Patras, 26500 Patras, Greece 13

4 Laboratory of Biological Control of Pesticides, Benaki Phytopathological Institute, 14

Athens 14561, Greece 15

5Department of Microbiology, School of Medicine, University of Athens, 11527 16

Athens, Greece 17

6 Department of Hygiene and Epidemiology, Faculty of Medicine, University of 18

Thessaly, Larissa 41222, Greece 19

20

*Author for Correspondence 21

Prof. Anna Papa, MD, PhD 22

A' Department of Microbiology 23

Medical School, Aristotle University of Thessaloniki 24

54124, Thessaloniki, Greece 25

26

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Abstract 26

Objectives: To describe the laboratory assays applied to investigate a suspected 27

dengue case in summer of 2012 in western Greece and to report the public health 28

response. 29

Design of methods: Samples of the patient were tested for detection of Dengue virus 30

(DENV) antibodies, NS1 antigen and RNA. Public health professionals interviewed 31

residents of the village and blood samples taken from 132 persons were tested for 32

antibodies for DENV and West Nile virus (WNV). Samples from 10 persons who 33

reported symptoms in the 15 days prior (including 3 persons who had fever at the time 34

of sampling) were tested for DENV, WNV and additional arboviruses. Entomological 35

missions were organized in the area of potential exposure to investigate the presence 36

of competent DENV vectors. 37

Results. Based on a combination of serological and molecular methods, DENV 38

infection was excluded, and the problems in the serology, especially in the DENV 39

NS1 antigen, was attributed to interfering factors. A 6.1% WNV seroprevalence was 40

detected in the region, while phlebovirus IgM and IgG antibodies were detected in 41

two of three persons who had fever at the time of sampling. Aedes albopictus adult 42

mosquitoes were present in the region. 43

Conclusions: A multi-disciplinary field and laboratory investigation showed no 44

evidence of DENV infection. There is a need for industries to improve the 45

immunometric assays to avoid interference with rheumatoid or other factors, while 46

increase awareness is needed by laboratorians when evaluating the diagnostic assays. 47

The high WNV seroprevalence in the investigated region prompts for strengthening 48

awareness on vector borne diseases, while the presence of Ae. albopictus suggests 49

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that the possibility of introduction of DENV exists, and preparedness plans are 50

needed. 51

52

Key words: Dengue fever; arbovirus; West Nile virus; Greece 53

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Introduction 54

Dengue virus (DENV) is a flavivirus (family Flaviviridae) transmitted to humans by 55

bites of infected Aedes mosquitoes, mainly Aedes aegypti, while Aedes albopictus is 56

considered a competent although secondary DENV vector. An estimated 50 to 100 57

million DENV infections occur annually in 125 countries and it is considered a major 58

public health threat by the World Health Organization (WHO), as Dengue fever 59

global incidence has increased 30-fold in the last 50 years 1. Main symptoms of 60

Dengue fever include fever, myalgia, arthralgia, rash, while severe cases lead to 61

Dengue hemorrhagic fever or even Dengue shock syndrome. The disease is endemic 62

in tropical and subtropical regions, and several imported cases are being reported 63

annually in travelers returning from these regions. The last large epidemic in Europe 64

was observed during 1927-28 in Greece (>80% of the population in the Athens area 65

was affected), with more than 1,000 reported deaths 2. The possibility of re-66

introduction of DENV to Europe exists, in view of the increasing global travel rates 67

and the wide distribution of its secondary vector, Ae. albopictus, in more than 16 68

European countries 3, also present in the wider region of West Greece since 2003.3 69

Autochthonous cases of Dengue fever have been reported in 2010 in South France 4, 5 70

and Croatia 6, while a large outbreak was detected in 2012 in Madeira, Portugal 7, 71

suggesting that DENV is an emerging public health threat for Europe. 72

Another flavivirus, West Nile virus (WNV), transmitted to humans mainly by bites of 73

infected Culex spp. mosquitoes, is widely distributed in the world, causing sporadic 74

cases and outbreaks. Since 2010, when the virus emerged in Greece 8, up to 2013, 609 75

human cases (432 of them accompanied by neurological symptoms) have been 76

observed 9. Cross-reactivity is often seen among flaviviruses, especially among those 77

belonging to the same serocomplex 10. In the present study we report the laboratory 78

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and field investigation of a suspected autochthonous Dengue case observed in Greece 79

in 2012. 80

81

Case report 82

In late August 2012, an 84-year old male resident of a village in the municipality of 83

Agrinio, western Greece, presented to a local private clinic with fever, fatigue, neck 84

pain and weakness of his lower limbs. Laboratory tests showed elevated bilirubin, 85

thrombocytopenia, and leukocytosis. The patient was transferred to a tertiary care 86

center. His condition deteriorated rapidly and he died on the 10th day of 87

hospitalization, due to Staphylococcus aureus septicemia despite adequate 88

antimicrobial treatment and supportive care. During his hospitalization he developed 89

epistaxis (not thrombocytopenic at the time) that was attributed to a medical history of 90

Rendu-Weber-Osler syndrome. The patient had not received blood transfusions or 91

other blood products during the incubation period, and he did not report any travel 92

beyond the prefecture of his residence. His past medical history included atrial 93

fibrillation for which he was treated with an oral anti-coagulant. 94

95

Laboratory testing 96

Since an outbreak of WNV infection was ongoing at the time in Greece in August 97

2012, blood and cerebrospinal fluid (CSF) samples drawn on the 8th day of illness 98

were sent to the National Reference Laboratory for Arboviruses to be tested for 99

probable WNV infection. Samples were tested for presence of WNV IgM and IgG 100

antibodies using ELISA (WNV IgM capture DxSelect and WNV IgG DxSelect, Focus 101

Diagnostics, Cypress, CA). No WNV IgM and IgG antibodies were detected in the 102

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CSF; a borderline WNV IgM antibody index (1.6 with cut-off 1.5) was seen in the 103

patient’s serum sample, which became negative after subtraction of the background). 104

Due to the presence of epistaxis and thrombocytopenia in the patient, samples were 105

additionally tested for DENV IgM and IgG antibodies using ELISA (DENV IgM 106

capture DxSelect and DENV IgG DxSelect, Focus Diagnostics, Cypress, CA). No 107

DENV IgM and IgG antibodies were detected in the CSF; negative were also the 108

results for DENV IgG antibodies in the serum and CSF. A high positive DENV IgM 109

index (out of range) was seen in the patient’s serum, which resulted negative after 110

subtracting the background. 111

Blood and CSF samples were also tested for DENV NS1 antigen (Platelia Dengue 112

NS1 Ag-ELISA (Biorad Laboratories, Marnes-La-Coquette, France); CSF was 113

negative, but a positive result was obtained in the serum sample (3.98, cut-off 1). An 114

NS1 kit was kindly provided by Focus Diagnostics and serum and CSF samples 115

resulted negative. The big difference between indices before and after subtraction of 116

the background in the DENV IgM ELISA, prompted us to repeat the Biorad NS1 117

antigen assay after treatment of the patient's serum with rheumatoid factor (Rf)-118

absorbent (although it is not written in the instructions for use), to investigate any 119

interference with IgM- Rfs, and the result was negative. 120

Two RT-nested PCRs, one using generic flavivirus primers 11, and a second using 121

DENV-specific primers 12, as well as a DENV-specific real time RT-PCR (reagents 122

kindly provided by Ana Vazquez and Antonio Tenorio, Spain) were applied on serum 123

and CSF samples and resulted negative. Based on all the above results, the case was 124

declared as DENV-negative. 125

Serum sample from another WNV-suspected case tested in the Department of 126

Microbiology in the University of Athens gave also borderline WNV IgM and a high 127

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DENV IgM antibody index; in that case the DENV infection was easily excluded, 128

since the DENV IgM was negative after the background subtraction and the NS1 129

antigen was negative. Five serum samples from additional WNV-suspected cases with 130

borderline WNV IgM antibodies were negative after subtraction of the background, 131

and when tested for DENV IgM and IgG antibodies and NS1 antigen were all 132

negative. 133

134

Table 1: Initial laboratory testing results of the suspected Dengue case, Greece, 135

August 2012. CSF: cerebrospinal fluid. 136

Sample WNV IgM

before/after

subtraction

cut off: 1.5

WNV

IgG

DENV

IgM

cut off: 1.0

DENV

IgG

NS1

antigen

cut off: 1.0

PCR

for flaviviruses

and DENV-

specific

Focus Diagnostics BioRad

Serum 1.6/neg neg Out of

range/neg

neg 3.98 neg

CSF neg neg neg neg neg neg

137

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Public health response 139

Immediately after the first suspicion of DENV infection, and before finalization of the 140

laboratory testing, an active case finding mission was organized by the Hellenic 141

Centre for Disease Control and Prevention (HCDCP) in the village in western Greece 142

where the patient resided. Public health (PH) professionals from the HCDCP and the 143

University of Thessaly, in cooperation with regional PH staff, visited the area and 144

offered a physical exam in all residents of the village; the participants were 145

interviewed through a structured questionnaire aiming to identify persons with 146

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possible DENV infection. A total of 132 persons [57 (43.18%) male] were examined, 147

focusing in an area up to 200m radius from the case’s residence including also the 148

patient’s relatives. Their median age was 57 years (range 7-85 years). Ten persons 149

reported fever, headache, arthralgia, myalgia, nausea, vomiting or upper respiratory 150

infection in the last 15 days (3 at the time of examination). Blood samples were 151

obtained from all of them and sent in optimal conditions to the National Reference 152

Laboratory for arboviruses. 153

Serum samples were tested for potential detection of WNV and DENV IgM and IgG 154

antibodies using the same ELISA methods as for the patient's samples. All samples 155

were negative for WNV and DENV IgM antibodies, while WNV IgG antibodies were 156

detected in 8 (8/131, 6.1%) persons, confirmed by plaque reduction neutralization test 157

(PRNT90). The age of these 8 persons ranged from 74 to 84 years (median 79 years). 158

Sera from the ten persons who reported symptoms in the 15 days prior, were also 159

tested for IgM and IgG antibodies against Chikungunya virus (CHIKV) and 160

phleboviruses by indirect immunofluorescence assays (Euroimmun, Lübeck, 161

Germany). As regards phleboviruses, samples were tested for antibodies against 162

Toscana virus (TOSV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian 163

virus (SFSV) and Cyprus virus (CYPV) (Sandfly fever virus Mosaic 1, Euroimmun, 164

Lübeck, Germany). CHIKV IgM and IgG antibodies were not detected. However, 165

phlebovirus IgM and IgG antibodies were detected in two of the three persons who 166

had fever at the time of sampling (Table 2), suggesting an acute phlebovirus infection. 167

An RT-nested PCR using phlebovirus generic primers 13 applied on the patients' blood 168

samples was negative. 169

PH staff raised awareness of local clinicians and PH professionals on the clinical 170

presentation of DENV infection. Information and communication material was also 171

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provided to health professionals and the public on personal protection measures 172

against mosquitoes, although this activity was on-going due to the recent outbreaks of 173

WNV infections (since 2010 8, 14, 15]) and the report of autochthonous malaria cases 16. 174

175

Table 2. IgM and IgG antibodies against four phleboviruses: Toscana virus (TOSV), 176

sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFNV) and Cyprus 177

virus (CYPV) in two febrile persons detected during the epidemiological 178

investigation. Serum dilution 1:100. T: body temperature; neg: negative; F: female. 179

180

No Age Sex T

(oC)

IgM antibodies IgG antibodies

TOSV SFNV SFSV CYPV TOSV SFNV SFSV CYPV

1 24 F 37.5 - - ++ +++ - - + ++

2 12 F 37.2 - - + + - - + -

181

182

Entomological investigation 183

Two entomological missions were organized in the area of potential exposure to 184

investigate the presence of competent DENV vectors. A team of experts from Benaki 185

Phytopathological Institute and the HCDCP carried out mosquito field surveys in the 186

patient’s village residential area, and in nearby villages as per his reported movement 187

history. Entomological surveillance detected a variety of potential mosquito breeding 188

sites, which yielded only Culex spp. larvae and pupae. In addition, three octenol 189

baited adult traps (2 Biogents Mosquitaire Plus & 1 BG Sentinel) were placed in the 190

area surrounding the patient’s residence and a nearby village, where the patient 191

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reportedly spent some of his summer vacation. A total of 3 Ae. albopictus adult 192

mosquitoes were captured during the mission, which tested DENV-negative at the 193

Department of Parasitology, Entomology and Tropical Diseases at the National 194

School of Public Health. Furthermore, a number of oviposition traps were placed in 195

the immediate surroundings. Eggs were collected from all three locations, which after 196

hatching in the laboratory, were identified as Ae. albopictus. 197

198

Discussion 199

One of the priorities of PH preparedness for emerging pathogens is the capacity for 200

prompt and accurate diagnosis. The present study illustrates the need for a thorough 201

interdisciplinary investigation of a first reported case of an emerging pathogen, as 202

well as the value of a Reference Centre, which, being a member of the European 203

Network of Imported Viral Diseases (ENIVD), participated in several external quality 204

control studies, including those on diagnostics on DENV 17, 18 and WNV 19. 205

The main problem in flavivirus diagnostics is the extensive cross-reactivity among 206

members of the family, and, especially, among members of the same serocomplex. 207

The present report shows an additional problem: the interference of various factors, 208

like the Rf or the heterophilic antibodies, in the performance of the assays for the 209

detection of WNV and DENV antibodies and the DENV NS1 antigen. The extremely 210

high index (out of range) of DENV IgM antibodies observed without the subtraction 211

method was exceptional. Previous studies on WNV cases have showed no interfering 212

factors in high IgM positive samples (index value of >3.5) tested by Focus 213

Diagnostics capture ELISA (MAC-ELISA) 20 (which applies also for DENV ELISA). 214

The negative result after application of the subtraction method (which is suggested by 215

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the manufacturers) facilitated the conclusion that the patient had not IgM (and IgG) 216

antibodies against WNV and DENV. 217

The remaining problem was the positive result of the DENV NS1 antigen when the 218

Biorad kit was used (the manufacturer reports no interference with heterophilic 219

antibodies). It is of interest that when the test was repeated after treatment of the 220

patient's serum with Rf-absorbent (for the removal of IgM-Rf rheumatoid factors ), it 221

resulted negative, suggesting that further studies are needed on this topic to 222

investigate the role of Rfs in the NS1 antigen performance. The problem was solved 223

by the negative result in NS1 antigen when the Focus kit was used, and by the 224

negative result of the three PCRs (two nested RT-PCRs and one real-time RT-225

PCR).The epidemiological and entomological investigation showed no evidence of 226

DENV infection in the region, neither in humans nor in mosquitoes. However, the 227

high WNV seroprevalence detected in the population sample of the region (6.1%), 228

together with the fact that only two human cases have been reported from the 229

municipality of Agrinio (one in 2010 and one in 2012), suggests that some WNV 230

cases went probably undetected, prompting for continuing need for increased 231

awareness in the region. The detection of two cases with phlebovirus infection was 232

not unexpected, since the phlebovirus seroprevalence in Greece is high (up to 60%), 233

especially in the islands and the coastal areas of the mainland 21, 22; western Greece, 234

where the cases were detected, is among the regions with high seroprevalence. 235

Patients with mild symptoms, as the two cases of the present study, do not ask for 236

medical advice, and remain undetected. However, phlebovirus infections are not 237

usually included in the differential diagnosis of summer febrile cases, even when they 238

present neurological symptoms, and the number of reported cases is currently very 239

limited 23,24,25,26. 240

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The capture of Ae. albopictus, although DENV-negative, suggests that the possibility 241

of re-introduction of DENV to Greece exists (similar to other southern European 242

countries), and preparedness plans have to be in place. The recent autochthonous 243

dengue case reported in southern France on Aug 20, 2014, emphasises the present risk 244

of dengue transmission in Europe in areas where competent mosquito vectors are 245

established 27. 246

In conclusion, the present case showed that the laboratory diagnosis of a flavivirus 247

infection presents certain challenges and that critical evaluation of the results by 248

experts in the field is essential; this applies for all cases, but especially when the 249

emergence of a pathogen in a new area is suspected. There is a need for industries to 250

improve the immunometric assays to avoid interference with RF or heterophilic 251

antibodies, while increase awareness is needed by laboratorians about the 252

vulnerability of diagnostic assays, especially when only one sample is available. The 253

present investigation was the trigger for a comprehensive approach for detection of 254

arboviral diseases in the area, which can serve as basis for future studies, while it was 255

a good opportunity to further explore collaborations already formed for the 256

management of WNV and malaria in Greece, in the prompt mobilization of a multi-257

disciplinary team to a particular area for field and laboratory investigations. 258

259

Acknowledgements 260

The study was financially supported by the MALWEST project through the 261

Operational Programme entitled “Human Resources Development” of National 262

Strategic Reference Framework” (NSRF) 2007–2013; the programme is co-funded by 263

Greece and the European Union—European Regional Development Fund. We thank 264

the local public health authorities for their help during the investigation. 265

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266

Conflict of interest statement 267

No conflict of interest 268

269

References 270

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3099(14)70925-9. 351

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Hightlights 352

353

• Detailed laboratory investigation excluded Dengue virus infection in a case with 354 fatal outcome. 355

• There is a need for industries to improve the immunometric assays to avoid 356 interference with rheumatoid or other factors. 357

• Increase awareness is needed by laboratorians when evaluating the diagnostic 358 assays for flavivirus infections. 359

• Surveillance study in the region showed no evidence of Dengue virus circulation, 360 high seroprevalence to West Nile virus (6.1%) and detection of two acute 361 phlebovirus infections. 362

• Entomological investigations showed the presence of Aedes albopictus mosquitoes. 363

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Laboratory and surveillance studies following a suspected Dengue case in Greece, 2012

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