Embed Size (px)
Citation preview
1
Dengue Virus Immunoglobulin M Detection in a Reference Laboratory Setting during the 1
2010 Dengue Virus Outbreak on Caribbean Islands 2
3
Harry E. Prince*, Jose L. Matud, and Jay M. Lieberman 4
Focus Diagnostics, Inc. 5
Cypress, CA 90630 USA 6
7
*Corresponding author: 8
5785 Corporate Avenue 9
Cypress, CA 90630 10
Telephone: 714 822-2457 11
FAX: 714 822-3839 12
E-mail: [email protected] 13
14
15
Running title: Dengue IgM detection during 2010 outbreak 16
Key words: Dengue, IgM, 2010 outbreak 17 18
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05096-11 CVI Accepts, published online ahead of print on 25 May 2011
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
2
Abstract 19
A large outbreak of dengue virus (DV) infections occurred on Caribbean Islands during 20
2010, with cases peaking during the second half of the year. In conjunction with the 21
outbreak, we observed an unprecedented spike in the number of sera submitted for DV 22
antibody testing between June and December 2010, with a concomitant increase in the 23
number of IgM-positive specimens, indicative of acute DV infection. Analysis of the 24
place of residence of the IgM-positive patients identified June-December 2010 revealed 25
that 58.1% were residents of Caribbean islands (Puerto Rico and the US Virgin Islands), 26
whereas 40.6% were residents of the United States (US) mainland or Hawaii. The US 27
residents represented 42 states plus the District of Columbia, but most (53%) were from 28
just 3 states (CA, FL, NY). When compared to the Caribbean IgM-positive patient 29
group, the US IgM-positive patient group contained proportionately more adults 21-60 30
years old and fewer individuals <21 years old. These findings indicate that the 2010 31
Caribbean DV outbreak affected many US residents (mostly adults, presumably travelers) 32
from diverse geographic areas, and emphasize the potential for a viremic DV-infected 33
returning traveler to spark a local DV outbreak by introducing DV into a community with 34
competent mosquito vectors. 35
36
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
3
Introduction 37
Dengue virus (DV) infections are transmitted among humans by Aedes mosquitoes 38
(mainly Aedes aegypti) in tropical and subtropical areas worldwide; in these areas, 39
dengue fever and dengue hemorrhagic fever are major public health problems (29). The 40
World Health Organization estimates that as many as 50 million DV infections occur 41
annually throughout the world (35); others estimate this number to be 100 million (10, 42
21). A large DV outbreak occurred in the tropics and subtropics of the Americas during 43
the summer and fall of 2010, with more than one million reported cases (8). One of the 44
areas with highest activity was the Caribbean basin, including the United States (US) 45
territories of Puerto Rico and the US Virgin Islands (7), with more than 21,000 reported 46
cases. DV infections are also an important medical concern outside the tropics and 47
subtropics, where cases in travelers returning from endemic areas have been well-48
documented (1, 6, 19, 20, 26, 31, 36). Measurement of DV Immunoglobulin M (IgM) is 49
an important laboratory tool for identifying individuals with recent DV infection; DV 50
IgM develops within 5 days of symptom onset, and then wanes to undetectable levels 51
within a few months (10, 11, 16, 17, 34). 52
53
Although reports (4, 13, 15, 23, 24, 32, 33) have described the role of DV IgM detection 54
by hospital, medical center, and public health laboratories in conjunction with regional 55
DV outbreaks, little information is available on DV IgM detection in a reference 56
laboratory setting during an outbreak. As part of a large commercial reference laboratory 57
system, our laboratory performs DV antibody testing on specimens submitted from across 58
the US, as well as from outside the US. We thus evaluated DV IgM results generated 59
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
4
during the 2010 DV outbreak; our goals were to determine the geographic distribution of 60
patients with DV IgM-positive samples, and gauge the extent of DV exposure among 61
residents of the US mainland, presumably related to international travel. 62
63
64
Materials and Methods 65
Specimens: Sera included in the analysis were submitted by hospital, medical center, and 66
regional reference laboratories for DV IgM testing between January 2008 and February 67
2011, inclusive. No information regarding clinical symptoms, date of disease onset, or 68
travel history accompanied any of the specimens. 69
70
DV IgM measurement: Sera were tested for DV IgM using a laboratory-developed IgM-71
capture background subtraction enzyme-linked immunosorbent assay (ELISA) as 72
previously described (3, 9, 25, 27). Diluted control sera (internally developed), calibrator 73
serum (internally developed) and patient sera were added to duplicate microtiter wells 74
coated with rabbit-anti-human IgM (Focus Diagnostics, Cypress, CA). After 1h at room 75
temperature (RT), wells were washed three times; one well per specimen then received 76
inactivated DV antigen (containing all 4 DV serotypes, internally developed), and the 77
other well received specimen diluent. After incubation for 2h at room temperature (RT) 78
and washing, all wells received horseradish peroxidase-conjugated 6B6C anti-flavivirus 79
monoclonal antibody (Focus Diagnostics). After 30 minutes at RT and washing, all wells 80
received tetramethylbenzidine (enhanced K-blue; Neogen Corp., Lexington, KY); after 81
10 minutes, the color reaction was stopped by adding sulfuric acid (Ricca Chemicals, 82
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
5
Arlington, TX). Absorbance at 450 nM was measured using an ELISA reader (BioTek, 83
Winooski, VT). The absorbance value of the well receiving specimen diluent was 84
subtracted from the absorbance value of the well receiving dengue antigen, and this 85
corrected absorbance value was then used to calculate the index, defined as the specimen 86
absorbance value divided by the calibrator absorbance value; indices >1.10 were 87
considered positive. 88
89
Determination of patient residence: Clients submitting sera that tested positive for DV 90
IgM were informed of the IgM-positive result, and asked to provide the place of 91
residence for the patient supplying the serum specimen. 92
93
Statistics: Differences among proportions were assessed by Chi-squared analysis using 94
MedCalc (Mariakerke, Belgium) software; significance was defined as P<0.01. 95
96
Results 97
98
As shown in Figure 1, we observed a profound spike in the number of specimens 99
submitted for DV antibody testing between June and December 2010, the same time 100
period as the DV outbreak in the Americas (7, 8). Concomitant with this spike in 101
submitted specimens, there was an increase in the number of IgM-positive samples. This 102
2010 seasonal increase in the number of submitted samples and IgM-positive samples 103
was markedly larger than the subtle increases observed during the summer/fall of the 104
prior 2 years. 105
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
6
106
Analysis of the place of residence of IgM-positive patients identified during the June-107
December 2010 period revealed that 58.1% lived on the Caribbean islands of Puerto Rico 108
and the US Virgin Islands, and 40.6% were residents of the US mainland or Hawaii; the 109
remaining 1.3% resided in other countries, including some countries in the Americas 110
(Table 1). The geographic distribution of IgM-positive patients from the US is shown in 111
Figure 2; patients were from 42 states and the District of Columbia, with the majority 112
(379/709 = 53%) from just 3 states (CA, FL, NY). 113
114
Marked differences were noted in the age distributions of IgM-positive US residents and 115
IgM-positive Caribbean island residents identified during the June-December 2010 116
period (Table 2). The proportions of patients in age groups <21 years were significantly 117
lower in the US patient group compared to the Caribbean island patient group, whereas 118
the proportions of patients in age groups spanning 21-60 years were significantly higher 119
in the US patient group compared to the Caribbean island patient group. 120
121
Discussion 122
123
In conjunction with the 2010 DV outbreak in the Americas that profoundly affected 124
Caribbean islands (7, 8), we observed a marked increase in the number of specimens 125
submitted to our reference laboratory for DV antibody analysis, as well as the number of 126
specimens testing positive for DV IgM, a marker of recent infection. Although subtle 127
upward shifts in submitted and IgM-positive sample numbers were observed in the 128
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
7
summer/fall of 2008 and 2009, these shifts were paltry compared to the profound upward 129
shift observed in 2010. 130
131
The majority of IgM-positive specimens submitted during the June-December 2010 132
outbreak period were supplied by patients residing in the US territories of Puerto Rico 133
and US Virgin Islands; however, a sizeable proportion (40.6%) of IgM-positive patients 134
resided on the US mainland or Hawaii. The close proximity of Caribbean islands to the 135
US mainland makes it reasonable to assume that a significant proportion of IgM-positive 136
US mainland patients were exposed in conjunction with travel to this area. However, the 137
actual proportion of IgM-positive US patients exposed to DV on Caribbean islands, 138
versus other geographic areas where DV is endemic, remains unknown. Likewise, we 139
cannot rule out the possibility that DV IgM detection in some US patients reflected cross-140
reactive antibodies induced by exposure to other flaviviruses (e.g., Japanese encephalitis 141
virus, yellow fever virus) during international travel (10). We also cannot rule out local 142
exposure to cross-reactive flaviviruses endemic to the US (e.g., West Nile virus, St. Louis 143
encephalitis virus) (10, 12); this possibility seems unlikely, however, assuming DV 144
antibody testing was ordered based on a history of travel outside the US. 145
146
The differences we observed in the age distribution of DV IgM-positive US residents 147
versus IgM-positive Caribbean island residents supports the expected epidemiological 148
differences in DV infections among travelers to endemic areas versus residents of 149
endemic areas. Consistent with published findings (20, 30), more than 80% of IgM-150
positive US residents were adults, presumably recent travelers to destinations outside the 151
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
8
US. In contrast, 37% of IgM-positive residents of Caribbean islands were <21 years old, 152
consistent with local DV transmission among residents of a DV-endemic area (28). 153
154
The DV IgM-positive patients residing in the US exhibited a wide geographic 155
distribution, representing 42 states and the District of Columbia. Many of these states are 156
also included in the habitat range of Aedes sp. mosquitoes capable of transmitting DV (2, 157
22). Patients with DV infection are viremic for approximately 5 days after onset of 158
symptoms (10, 14, 29); thus, the possibility that a recently-infected returning traveler 159
may serve as the point source for a local DV outbreak within the US appears very real. 160
Indeed, this scenario may have already occurred; in 2009/2010, recent DV infections 161
were identified in 26 residents of Key West, FL who had no history of recent 162
international travel (5). However, the subtropical climate and presence of competent 163
mosquito vectors raise the possibility that DV is endemic to this area, and unknown 164
factors triggered its recent reemergence (5). 165
166
In addition to the study limitations already mentioned, it must be emphasized that our 167
data only reflect the patient populations served by medical facilities that refer specimens 168
to us for DV antibody testing. There were undoubtedly additional DV IgM-positive 169
individuals, including US residents, identified by other medical center, reference, and 170
public health laboratories during the time period of the 2010 DV outbreak. 171
Approximately 500 cases of DV infections in US residents returning from international 172
travel have been reported to the Centers for Disease Control and Prevention (CDC) for 173
2009-2010 (18), but the number reported for the June-December 2010 time period and 174
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
9
the overlap with patients identified by our testing are unknown. In addition, many DV 175
infections in travelers are likely not diagnosed because the patient either does not present 176
for medical care or the diagnosis is not considered by the treating physician. Greater 177
awareness by clinicians regarding the risk of DV infection in returning travelers with 178
febrile illness, an understanding of appropriate laboratory tests that aid in diagnosing DV 179
infections, and utilization of reporting systems such as ArboNET (6) are needed to 180
accurately ascertain the burden of DV infections among residents of the US and its 181
territories. DV infection was designated a nationally notifiable disease in the US in 2009 182
(5, 6), which should facilitate more accurate tracking of DV-infected patients. 183
184
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
10
References 185
1. Bakker, R. C., J. Veenstra, A. M. Dingemans-Dumas, J. C. F. M. Wetsteyn, and P. 186
A. Kager. 1996. Imported dengue in The Netherlands. J. Travel Med. 3:204-208. 187
2. Benedict, M. Q., R. S. Levine, W. A. Hawley, and L. P. Lounibos. 2007. Spread of 188
the tiger: global risk of invasion by the mosquito Aedes albopictus. Vector Borne 189
Zoonotic Dis. 7:76-85. 190
3. Branch, S. L., and P. N. Levett. 1999. Evaluation of four methods for detection of 191
immunoglobulin M antibodies to dengue virus. Clin. Diagn. Lab. Immunol. 6:555-192
557. 193
4. Buerano, C. C., I. N. Ibrahim, R. C. Contreras, F. Hasabe, R. R. Matias, F. F. 194
Natividad, A. Igarashi, and Dengue Network Philippines. 2000. IgM-capture 195
ELISA of serum samples collected from Filipino dengue patients. Southeast Asian 196
J. Trop. Med. Public Health 31:524-529. 197
5. Centers for Disease Control and Prevention. 2010. Locally acquired Dengue – Key 198
West, Florida, 2009-2010. Morbid. Mortal. Wkly. Rep. 59:577-581. 199
6. Centers for Disease Control and Prevention. 2010. Travel-associated dengue 200
surveillance – United States, 2006-2008. Morb. Mortal. Wkly. Rep. 59:715-719. 201
7. Centers for Disease Control and Prevention. 2011. 2010: Largest dengue outbreak 202
in Puerto Rico history. The Dengue Update 3:1-2. 203
8. Centers for Disease Control and Prevention. 4 April 2011, accession date. 204
Outbreak Notice Update: Dengue, tropical and subtropical regions. Updated 18 205
January 2011. Center for Disease Control and Prevention, Atlanta, GA. 206
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
11
http://wwwnc.cdc.gov/travel/content/outbreak-notice/dengue-tropical-sub-207
tropical.aspx. 208
9. Groen, J., P. Koraka, J. Velzing, C. Copra, and A. D. Osterhaus. 2000. Evaluation 209
of six immunoassays for detection of dengue virus-specific immunoglobulin M and 210
G antibodies. Clin. Diagn. Lab. Immunol. 7:867-871. 211
10. Gubler, D. J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 212
11:480-496. 213
11. Guzman, M. G., and G. Kouri. 1996. Advances in dengue diagnosis. Clin. Diagn. 214
Lab. Immunol. 3:621-627. 215
12. Hogrefe, W. R., R. Moore, M. Lapé-Nixon, M. Wagner, and H. E. Prince. 2004. 216
Performance of Immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent 217
assays using a West Nile virus recombinant antigen (preM/E) for detection of West 218
Nile virus- and other flavivirus-specific antibodies. J. Clin. Microbiol. 42:4641-219
4648. 220
13. Kabilan, L., S. Balasubramanian, S. M. Keshava, and K. Satyanarayana. 2005. The 221
2001 dengue epidemic in Chennai. Indian J. Pediatr. 72:919-923. 222
14. Kao, C-L., C-C. King, D-Y. Chao, H-L. Wu, and G-J. J. Chang. 2005. Laboratory 223
diagnosis of dengue virus infection: current and future prospectives in clinical 224
diagnosis and public health. J. Microbiol. Immunol. Infect. 38:5-16. 225
15. Khan, N. A., E. Azhar, S. El-Fiky, H. H. Madani, M. A. Abuljadial, A. M. Ashshi, 226
A. M. Turkistani, and E. A. Hamouh. 2008. Clinical profile and outcome of 227
hospitalized patients during first outbreak of dengue in Makkah, Saudi Arabia. 228
Acta Trop. 105:39-44. 229
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
12
16. Kuno, G., I. Gomez, and D. J. Gubler. 1991. An ELISA procedure for the 230
diagnosis of dengue infections. J. Virol. Methods 33:101-113. 231
17. Lam, S. K., S. Devi, and T. Pang. 1987. Detection of specific IgM in dengue 232
infection. Southease Asian J. Trop. Med. Public Health 18:532-538. 233
18. Margolis, H. 2011. Dengue diagnostic testing for case management and 234
surveillance. Focusevents Webex presentation, April 26, 2011. 235
https://focusevents.webex.com/mw0306lc/mywebex/default.do?siteurl=focusevents 236
19. Masaki, H., F. Hasebe, K. Ahmed, T. Fukuda, A. Furumoto, K. Watanabe, K. Oishi, 237
A. igarashi, and T. Nagatake. 2002. A clinical, serological, and immunological 238
study in a Japanese traveler with dengue fever. J. Infect. Chemother. 8:365-367. 239
20. Mohammed, H. P., M. M. Ramos, A. Rivera, M. Johansson, J. L. Munoz-Jordan, 240
W. Sun, and K. M. Tomashek. 2010. Travel-associated dengue infections in the 241
United States, 1996-2005. J. Travel Med. 17:8-14. 242
21. Monath, T. P. 1994. Dengue: the risk to developed and developing countries. 243
Proc. Natl. Acad. Sci. USA 91:2395-2400. 244
22. Morens, D. M., and A. S. Fauci. 2008. Dengue and hemorrhagic fever. A potential 245
threat to public health in the United States. J. Amer. Med. Assoc. 299:214-216. 246
23. Nogueira, R. M., M. P. Miagostovich, S. M. Cavalcanti, K. B. Marzochi, and H. G. 247
Schatzmayr. 1992. Levels of IgM antibodies against dengue virus in Rio de 248
Janeiro, Brazil. Res. Virol. 143:423-427. 249
24. Osman, O., M. Y. Fong, and S. Devi. 2007. A preliminary study of dengue 250
infection in Brunei. Jpn. J. Infect. Dis. 60:205-208. 251
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
13
25. Porter, K. R., S. Widjaja, H. D. Lohita, S. H Hadiwijaya, C. N. Maroef, W. 252
Suharyono, and R. Tan. 1999. Evaluation of a commercially available 253
immunoglobulin M enzyme-linked immunosorbent assay kit for diagnosing acute 254
dengue infections. Clin. Diagn. Lab. Immunol. 6:741-744. 255
26. Potasman I., I. Scugo, and E. Schwartz. 1999. Dengue seroconversion among 256
Israeli travelers to tropical countries. Emerg. Infect. Dis. 5:824-827. 257
27. Prince, H. E., C. Yeh, and M. Lapé-Nixon. 2008. Development of a more efficient 258
algorithm for identifying false-positive reactivity results in a dengue virus 259
Immunogloublin M screening assay. Clin. Vacc. Immunol. 15:1304-1306. 260
28. Rigau-Perez, J. G., D. J. Gubler, A. V. Vorndam, and G. G. Clark. 1994. Dengue 261
surveillance – United States, 1986-1992. Morbid. Mortal. Wkly. Rep. 43:7-19. 262
29. Solomon, T., and M. Mallewa. 2001. Dengue and other emerging flaviviruses. J. 263
Infection 42:104-115. 264
30. Sotir, M. J., D. K. Johnson, and J. P. Davis. 2009. Travel-associated dengue 265
illnesses among Wisconsin residents, 2002-2008. Wisconsin Med. J. 108:447-452. 266
31. Stephenson, I., J. Roper, M. Fraser, K. Nicholson, and M. Wiselka. 2003. Dengue 267
fever in febrile returning travelers to a UK regional infectious diseases unit. Travel 268
Med. Infect. Dis. 1:89-93. 269
32. Tang, J. W., M. R. Khanani, A. M. Zubairi, W. Y. Lam, F. Lai, K. Hashmi, A. 270
Hussain, S. Jamal, and P. K. Chan. 2008. A wide spectrum of dengue IgM and 271
PCR positivity post-onset of illness found in a large dengue 3 outbreak in Pakistan. 272
J. Med. Virol. 80:2113-2121. 273
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
14
33. Thomas, L., Y. Brouste, F. Najioullah, P. Hochedez, Y. Hatchuel, V. Moravie, S. 274
Kaidomar, J. P. King, F. Besnier, S. Abel, S. Carmes, S. Schmitt, P. Brihier, C. 275
Meunier, T. Cardoso, J. Rosine, P. Quenel, R. Cesaire, and A. Cable. 2010. 276
Prospective and descriptive study of adult dengue cases in an emergency 277
department, in Martinique. Med. Mal. Infect. 40:480-489. 278
34. Vaughn, D. W., S. Green, S. Kalayanarooj, B. L. Innis, S. Nimmannitya, S. 279
Suntayakorn, A. L. Rothman, F. A. Ennis, and A. Nisalak. 1997. Dengue in the 280
early febrile phase: viremia and antibody responses. J. Infect. Dis. 176:322-330. 281
35. World Health Organization. 6 April 2011, accession date. Dengue and dengue 282
haemorrhagic fever. Fact sheet N0117, March 2009. World Health Organization, 283
Geneva, Switzerland. http://www.who.int/mediacentre/factsheets/fs117/en/ 284
36. Xu, G., H. Dong, N. Shi, S. Liu, A. Zhou, Z. Cheng, G. Chen, J. Liu, T. Fang, H. 285
Zhang, G. Gu, X. Tan, J. Ye, S. Xie, and G Cao. 2007. An outbreak of dengue 286
virus serotype 1 infection in Cixi, Ningbo, People’s Republic of China, 2004, 287
associated with a traveler from Thailand and high density of Aedes albopictus. Am. 288
J. Trop. Med. Hyg. 76:1182-1188. 289
290
291
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
15
Table 1. Place of residence of DV IgM-positive patients identified June-December 2010 292
(N=1747) 293
Geographic area Number of patients Proportion of all patients
United States mainland plus Hawaii 709 40.6%
Puerto Rico 944 54.0%
US Virgin islands 72 4.1%
Other countries of the Americas* 14 0.8%
Countries outside the Americas** 8 0.5%
294
*Bermuda, Mexico, Peru, Venezuela 295
**Guam, France, Japan, Saudi Arabia, United Kingdom 296
297
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
16
298
Table 2. Age distribution of DV IgM-positive patients in relation to place of residence 299
Age group
(years)
Patients residing on Caribbean
__Islands (N=1016)__
Patients residing in the US
______(N=709)______
No. of samples % of N No. of samples % of N
<10 103 10.1 34 4.8*
11-20 275 27.1 97 13.7*
21-40 236 23.3 211 29.8*
41-60 226 22.2 257 36.2*
>60 169 16.6 104 14.7
unknown 7 0.7 6 0.8
*Significantly different (P<0.01) from the comparable value observed for the Caribbean 300
Islands group. 301
302
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
17
303
Figure legends 304
305
Figure 1. Fold increase in number of monthly samples, relative to the number submitted 306
during January 2008 for DV antibody testing, and relative fold increase in number of 307
IgM-positive samples segregated by geographic origin. 308
309
310
311
Figure 2. Geographic distribution of DV IgM-positive patients residing on the US 312
mianland. The key indicates the number of patients from a given state. Although not 313
shown on the map, there were 6 IgM-positive patients from Hawaii, and none from 314
Alaska. 315
on April 6, 2018 by guest
http://cvi.asm.org/
Dow
nloaded from
